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Ecological aspects of nitrogen uptake in intertidal macrophytes Thomas, Terry Ellen 1983

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ECOLOGICAL ASPECTS OF NITROGEN UPTAKE IN INTERTIDAL MACROPHYTES by  -  TERRY ELLEN THOMAS B . S c , U n i v e r s i t y of B r i t i s h Columbia, 1978  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE  REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  in THE FACULTY OF GRADUATE STUDIES (Department of Botany)  We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o the r e q u i r e d  standard  THE UNIVERSITY OF BRITISH COLUMBIA 1983 <g) T e r r y E l l e n Thomas, 1983  In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the r e q u i r e m e n t s f o r an advanced degree a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I agree t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by t h e head o f my department o r by h i s o r h e r r e p r e s e n t a t i v e s . It is understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n permission.  Department o f fl<S>t<**y The U n i v e r s i t y o f B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date  DE-6  (3/81)  SO,  11%  3  ii  Abstract A comprehensive f i e l d uptake  in  intertidal  and  laboratory  seaweeds  was  of  undertaken.  measuring n i t r o g e n uptake r a t e s were  in  study  nitrogen  Methods f o r  evaluated.  Short i n i t i a l periods  of r a p i d ammonium uptake were  nitrogen  plants.  deficient  i n h i b i t e d n i t r a t e uptake, starvation Porphyra  overcame perforata  maintained  rapid  a  .showed initial  that  r e s u l t e d i n a general  and a t r a n s i e n t i n c r e a s e  of  ammonium  degree  of  nitrogen  Laboratory  nitrogen  ammonium  remain  presence  certain  t h i s suppression.  uptake system d i d not also  but  The  common  studies  starved  uptake r a t e s .  activated.  with  cultures The n i t r a t e  Nitrogen  starvation  decrease i n s o l u b l e n i t r o g e n  content  i n n i t r a t e reductase a c t i v i t y .  The e f f e c t i v e n e s s of i j i v i t r o and ir\ v i v o n i t r a t e r e d u c t a s e a s s a y s was i n v e s t i g a t e d . in  vivo  assay  The r a t e of n i t r i t e p r o d u c t i o n  v a r i e d w i t h i n c u b a t i o n time.  Therefore,  i n the the i n  v i t r o assay was used. N i t r a t e grown c u l t u r e s high  ammonium  uptake r a t e s .  n i t r a t e reduction further  of  was  which  perforata . maintained  I t was suggested t h a t t h e r a t e of  limiting  assimilation  Porphyra  the may  supply  of  control  nitrogen  ammonium  Ammonium arid ammonium p l u s n i t r a t e grown c u l t u r e s had  for  uptake. very  low  n i t r o g e n uptake r a t e s and n i t r a t e r e d u c t a s e a c t i v i t i e s . Field growth  on  accumulation  studies  with  ammonium and  ammonium d i d not  Gracilaria inhibited  verrucosa nitrate  uptake,  nitrate  reductase a c t i v i t y .  inhibit  nitrate  uptake  confirmed that nitrate  The presence of  rates  in  severely  starved  populations.  A l l populations maintained  uptake r a t e s s u g g e s t i n g time  (August).  t h a t they were n i t r o g e n l i m i t e d a t  Ammonium  the h i g h i n t e r t i d a l G. intertidal An  this  and n i t r a t e uptake were s a t u r a b l e i n  verrucosa  p o p u l a t i o n but not i n t h e low  population.  investigation  was  made  into  t h e e f f e c t of n i t r o g e n  s o u r c e and p e r i o d i c exposure t o a i r on growth, nitrogen  h i g h ammonium  uptake  fertilization, requirement  i n Fucus d i s t i c h u s g e r m l i n g s . germination  and  germling  development  Gamete r e l e a s e , growth  f o r a s p e c i f i c form of n i t r o g e n .  and  had  no  P e r i o d i c exposure  t o a i r i n c r e a s e d secondary r h i z o i d development. Ammonium and n i t r a t e uptake r a t e s much  higher  than  but  the germlings  f o r t h e mature t h a l l i ,  n i t r a t e was s i m i l a r . kinetics  of  The  t h e mature  germlings thalli  were  but t h e a f f i n i t y f o r  showed  saturable  d i d not.  The  uptake  presence of  ammonium i n h i b i t e d n i t r a t e uptake by t h e mature p l a n t s  but not  by t h e g e r m l i n g s . Mild  d e s i c c a t i o n enhanced n u t r i e n t uptake r a t e s i n s e v e r a l  i n t e r t i d a l seaweeds. was been  T h i s uptake response o c c u r r e d when  l i m i t e d by t h a t p a r t i c u l a r n u t r i e n t and when t h e t h a l l u s had exposed  to  periodic  d e s i c c a t i o n f o r s e v e r a l weeks.  degree of enhancement, t h e p e r c e n t  d e s i c c a t i o n producing  uptake r a t e s and t h e t o l e r a n c e t o h i g h e r degrees of were  growth  related  to  intraspecific  as  Transplant nutrient intertidal  intertidal location. well  experiments uptake height  rates and  as  an  w i t h G. after not  maximum  desiccation  T h i s was shown t o be an  interspecific  verrucosa desiccation  geographic  The  adaptation.  showed t h a t enhanced were  related  to  l o c a t i o n and t h a t  this  iv  response c o u l d be i n d u c e d i n a p p r o x i m a t e l y f i v e weeks. suggested  that  t h i s enhanced uptake response was an a d a p t a t i o n  t o n i t r o g e n procurement and C/N homeostasis exposure  when  I t was  carbon  were not a v a i l a b l e .  following  periodic  was a s s i m i l a t e d but when o t h e r n u t r i e n t s  V  Table of C o n t e n t s Page Abstract  i i  L i s t of T a b l e s  vi  L i s t of F i g u r e s  vii  Acknowledgements  xii  Introduction 1)  1  A Time Course Study of the I n t e r a c t i o n Between Ammonium  and  N i t r a t e Uptake Rates 2)  3)  The  Effect  of  6 Nitrogen  Supply  on  Nitrogen  Uptake,  A s s i m i l a t i o n and A c c u m u l a t i o n i n Porphyra p e r f o r a t a  19  N i t r o g e n Uptake and Growth of Fucus d i s t i c h u s G e r m l i n g s  and  Mature P l a n t s 4)  Desiccation  37 Enhanced  Nutrient  Uptake  Rates i n I n t e r t i d a l  Seaweeds 5)  61  A d a p t a t i o n s of G r a c i l a r i a v e r r u c o s a t o N u t r i e n t  Procurement  i n an I n t e r t i d a l H a b i t a t .  94  Summary and C o n c l u s i o n s  .143  References Appendix  1.  148 Techniques  f o r measuring  s o l u b l e n i t r o g e n content 156  Appendix  2.  I_n v i v o and in v i t r o n i t r a t e r e d u c t a s e a c t i v i t y  170  Appendix  3.  S o l u b l e n i t r o g e n c o n t e n t of Porphyra p e r f o r a t a  .204  vi  L i s t of T a b l e s 1. A time macrophytes  course  study  of  Page n i t r o g e n uptake r a t e s i n marine 14  2. The e f f e c t of n i t r o g e n source on the growth of F. germlings 3.  S o l u b l e n i t r o g e n c o n t e n t of G.  Appendix  distichus 46  verrucosa  123  2.  1. O p t i m a l c o n d i t i o n s f o r n i t r a t e r e d u c t a s e a c t i v i t y marine macrophytes w  in  three 181  L i s t of F i g u r e s 1.  Map of B a m f i e l d c o l l e c t i o n s i t e s  Page 10  2.  Map of Vancouver c o l l e c t i o n s i t e s  21  3.  N i t r a t e uptake r a t e s of p r e i n c u b a t e d P.  4.  Ammonium uptake r a t e s of p r e i n c u b a t e d P.  5.  N i t r a t e c o n t e n t of p r e i n c u b a t e d P.  6.  N i t r a t e r e d u c t a s e a c t i v i t y of p r e i n c u b a t e d P  7.  N i t r a t e uptake k i n e t i c s of F.  8.  N i t r a t e uptake k i n e t i c s of mature F.  9.  Ammonium uptake k i n e t i c s of F.  10.  perforata perforata  26  perforata  k  28 perforata  d i s t i c h u s germlings  47  d i s t i c h u s germlings  nutrient  d i st ichus  uptake  uptake  14. The r e l a t i o n s h i p between ammonium d i s t i c h u s i n September 1980 and d e s i c c a t i o n  50  d i s t i c h u s t h a l l i .51  11. Ammonium uptake k i n e t i c s of mature F. p r e i n c u b a t e d on ammonium o r n i t r o g e n s t a r v e d  .13. The r e l a t i o n s h i p between ammonium d i s t i c h u s i n J u l y 1980 and % d e s i c c a t i o n  .30  d i s t i c h u s t h a l l i ...49  Ammonium uptake k i n e t i c s of mature F.  12. The r e l a t i o n s h i p between d i s t i c h u s and % d e s i c c a t i o n  25  uptake  15. The r e l a t i o n s h i p between ammonium uptake d i s t i c h u s i n February 1981 and % d e s i c c a t i o n 16. The r e l a t i o n s h i p between n i t r a t e uptake d i s t i c h u s i n February 1981 (6°C) and % d e s i c c a t i o n  thalli 53  r a t e s of F. 70 rates  of  F. 73  r a t e s of F. 74 rates  rates  of  F. 75  of F. 76  yi ii  17. The r e l a t i o n s h i p between ammonium uptake r a t e s of F. d i s t i c h u s p r e i n c u b a t e d on n i t r o g e n f r e e seawater i n F e b r u a r y 1981 and % d e s i c c a t i o n 77 18. The r e l a t i o n s h i p between n i t r a t e uptake r a t e s d i s t i c h u s i n February 1981 (18°C) and % d e s i c c a t i o n 19. The r e l a t i o n s h i p between v e r r u c o s a and % d e s i c c a t i o n 20. The r e l a t i o n s h i p between i n t e s t i n a l i s and % d e s i c c a t i o n  nitrogen  nitrogen  21. The r e l a t i o n s h i p between p a p i l l a t a and % d e s i c c a t i o n 22. The r e l a t i o n s h i p between d i s t i c h u s and % d e s i c c a t i o n  uptake  uptake  nitrogen  nitrogen  23. The r e l a t i o n s h i p between l i m i t a t a and % d e s i c c a t i o n  uptake  uptake  nitrogen  uptake  of  F. 78  r a t e s of G. 80 rates  of  E. 81  r a t e s of G. 82 rates  of  F. 83  r a t e s of P. 84  24. The r e l a t i o n s h i p between % d e s i c c a t i o n p r o d u c i n g maximum enhancement of n i t r a t e uptake r a t e s , t h e r e l a t i v e degree of enhancement of n i t r a t e u p t a k e , t h e r a t i o of n i t r a t e uptake r a t e s at 30% d e s i c c a t i o n and h y d r a t e d uptake r a t e s , and i n t e r t i d a l location 85 25. The r e l a t i o n s h i p between % d e s i c c a t i o n p r o d u c i n g maximum enhancement of ammonium uptake r a t e s , t h e r e l a t i v e degree of enhancement of ammonium uptake, t h e r a t i o of ammonium uptake rates a t 30% d e s i c c a t i o n and h y d r a t e d uptake r a t e s , and intertidal location 86 26.  A summary of G.  27.  Low i n t e r t i d a l G.  28.  H i g h i n t e r t i d a l G.  29.  A summary of G.  30.  The  verrucosa transplants  98  verrucosa  103  verrucosa  104  v e r r u c o s a morphology  relationship  between  nitrate  106 uptake  rates  of G.  ix  v e r r u c o s a i n l a t e summer and % d e s i c c a t i o n 31. The r e l a t i o n s h i p between n i t r a t e v e r r u c o s a i n June and % d e s i c c a t i o n 32.  109  uptake  A summary of n i t r o g e n uptake r a t e s f o r G.  33. The r e l a t i o n s h i p between ammonium v e r r u c o s a i n l a t e summer and % d e s i c c a t i o n 34. The r e l a t i o n s h i p between ammonium v e r r u c o s a i n June and % d e s i c c a t i o n  rates  verrucosa  uptake  uptake  of  G. 110  ...112  r a t e s of G. 114 rates  of  G. 116  35.  N i t r a t e uptake k i n e t i c s of G.  verrucosa  120  36.  Ammonium uptake k i n e t c s of G.  verrucosa  121  37.  A summary of t h e s o l u b l e n i t r o g e n c o n t e n t of G.  verrucosa 1 24  38. A summary of the s o l u b l e p r o t e i n c o n t e n t and t h e n i t r a t e r e d u c t a s e a c t i v i t y of G. v e r r u c o s a 126 39. The t o t a l amount of n i t r i t e produced w i t h time and the r e l a t i o n s h i p between n i t r a t e r e d u c t a s e a c t i v i t y and t h e volume of e x t r a c t used f o r G. v e r r u c o s a 127 40. The r e l a t i o n s h i p between n i t r a t e r e d u c t a s e a c t i v i t y v e r r u c o s a and MgS0 c o n c e n t r a t i o n 4  i n G. *..128  41. The r e l a t i o n s h i p between n i t r a t e r e d u c t a s e a c t i v i t y v e r r u c o s a and pH  i n G. 129  42. The r e l a t i o n s h i p between n i t r a t e r e d u c t a s e a c t i v i t y v e r r u c o s a and PVP c o n c e n t r a t i o n  i n G. 130  43. The r e l a t i o n s h i p between n i t r a t e r e d u c t a s e a c t i v i t y v e r r u c o s a and n i t r a t e c o n c e n t r a t i o n  i n G. 131  44. The r e l a t i o n s h i p between n i t r a t e r e d u c t a s e a c t i v i t y i n G. v e r r u c o s a and NADH c o n c e n t r a t i o n 131  X  Appendix 1. 1.  Soluble nitrogen content  of t h r e e marine macrophytes ....162  Appendix 2. 1. T o t a l amount of ir\ v i t r o n i t r i t e produced w i t h time (min) and t h e r e l a t i o n s h i p between in v i t r o n i t r a t e r e d u c t a s e a c t i v i t y and t h e volume of enzyme e x t r a c t used f o r t h r e e marine macrophytes 178 2. The pH optimum of i_n v i t r o n i t r a t e r e d u c t a s e t h r e e marine macrophytes  activity in 179  3. The r e l a t i o n s h i p . b e t w e e n in_ v i t r o n i t r a t e r e d u c t a s e i n t h r e e marine macrophytes and MgSO c o n c e n t r a t i o n  activity 180  4. The r e l a t i o n s h i p between i_n v i t r o n i t r a t e r e d u c t a s e i n t h r e e marine macrophytes and PVP c o n c e n t r a t i o n  activity 182  5. The r e l a t i o n s h i p between i r i v i t r o n i t r a t e r e d u c t a s e i n t h r e e marine macrophytes and NADH c o n c e n t r a t i o n  activity 183  6. The r e l a t i o n s h i p between in_ v i t r o n i t r a t e r e d u c t a s e i n t h r e e marine macrophytes and n i t r a t e c o n c e n t r a t i o n  activity 184  7. The r e l a t i o n s h i p between i_n v i t r o n i t r a t e r e d u c t a s e a c t i v i t y i n P. p e r f o r a t a and p e r i o d of time c u l t u r e d on n i t r a t e or ammonium 187 8. I_n v i v o n i t r a t e reductase activity macrophytes i n c u b a t e d on t h e j j i v i v o medium 9. P.  of  A c t u a l and p o t e n t i a l in v i v o n i t r a t e r e d u c t a s e p e r f o r a t a i n c u b a t e d i n t h e i_n v i v o medium  10. A c t u a l I_n v i v o n i t r a t e r e d u c t a s e i n c u b a t e d i n t h e v i v o medium  three  activity  a c t i v i t y of P.  11. I_n v i t r o and in v i v o n i t r a t e reductase p e r f o r a t a i n c u b a t e d i n t h e i_n v i v o medium  marine 188  activity  of 190  perforata 191 of P. 193  Appendix 3. 1.  Ammonium content of p r e i n c u b a t e d P.  perforata  2.  S o l u b l e amino a c i d c o n t e n t of p r e i n c u b a t e d P.  3.  S o l u b l e p r o t e i n c o n t e n t of p r e i n c u b a t e d P.  205 p e r f o r a t a 206  p e r f o r a t a ...207  xi i  Acknowledgements I  would  Harrison  like  to  thank my graduate s u p e r v i s o r , Dr.  f o r h i s generous  throughout  assistance  my graduate s t u d i e s .  D.H.  Turpin  Eric  B.  and  assistance  guidance  I would a l s o l i k e t o thank Dr.  f o r h i s c r e a t i v e i n p u t and  Taylor's  invaluable  P.J.  in  unwavering  culturing  Fucus  enthusiasm. distichus  g e r m l i n g s was a l s o g r e a t l y a p p r e c i a t e d . Thanks members;  is  Dr.  R.  also  extended  DeWreede, Dr.  to  my  T.R.  supervisory  committee  P a r s o n s , and Dr.  I.E.P.  Taylor. This Engineering  work  was  supported  by  a  Natural  Sciences  Research C o u n c i l of Canada graduate s c h o l a r s h i p .  and  1  I n t r o d u c t ion T i d e s c r e a t e a unique environment i n c o a s t a l r e g i o n s , where i n t e r t i d a l organisms must be adapted t o p e r i o d i c exposure t o a i r and  submersion.  seaweeds  In  the Northeast  a r e abundant  determined  by  and  substrate  their  type,  P a c i f i c Ocean,  distribution  temperature,  m o t i o n , and n u t r i e n t s (Druehl 1981).  Rocky  intertidal  is  largely  salinity,  and  water  sandy  coastal  a r e a s support v e r y d i f f e r e n t i n t e r t i d a l communities. Lush  and  diverse  macrophyte  growth  i s common on rocky  shores and t h i s h a b i t a t i s f r e q u e n t l y c h a r a c t e r i s e d by  distinct  h o r i z o n t a l bands dominated by a p a r t i c u l a r i n t e r t i d a l s p e c i e s or group  of  species.  R e s e a r c h e r s have a t t r i b u t e d t h e s e zones t o  both b i o l o g i c a l  (Jones and K a i n 1967; C o n n e l l 1972; Mann  Chapman  and  1973)  L e w i s 1964).  p h y s i c a l f a c t o r s ( Z a n e v e l d 1937; Doty 1946;  In some l o c a t i o n s t h e r e a r e maximum changes  p h y s i c a l s t r e s s a t t h e l i m i t s of these bands. are' r e f e r r e d  to  These  d u r a t i o n of exposure t o a i r .  locations  frequency  Exposure and subsequent  s u p p l y , and h u m i d i t y .  Success i n such an  morphological,  adaptations allowing for survival  and  nutrient  environment  physiological,  and  possibly  and/or  submersion  d r a s t i c changes i n t e m p e r a t u r e , l i g h t , s a l i n i t y ,  specific  i n the  as a " c r i t i c a l " t i d a l h e i g h t s (Doty 1946) and  are c h a r a c t e r i z e d by a l a r g e i n c r e a s e i n t h e  cause  1972;  requires  biochemical  growth  during  these environmental f l u c t u a t i o n s . Numerous  studies  have examined the e f f e c t s of i r r a d i a n c e ,  temperature (Schonbeck and Thorhaug  and  Marcus  Norton  1978; Gordon  et  a l . 1981;  1981) and extremes of pH and s a l i n i t y (Rao  2  and Mehta 1973; Wiencke and L a u c h l i 1980; Gordon e t a l . 1981) on m a c r o a l g a l growth. The most o b v i o u s s t r e s s imposed by exposure i s d e s i c c a t i o n . The p h y s i c a l e f f e c t s of d e s i c c a t i o n and have  desiccation  been i n v e s t i g a t e d ( I s a a c 1935; Feldman  1951; Schonbeck and  Norton 1979 a,b,c; Dromgoole 1980), but l i t t l e the p h y s i o l o g i c a l e f f e c t s of d e s i c c a t i o n . showed  that  i s known  Recent  or  even  enhanced  d e s i c c a t i o n (Johnson'et a l . Increased  carbon  over  a  procurement  macrophytes  limited  1974; Quadir e t a l .  about  investigations  t h e p h o t o s y n t h e t i c r a t e s of i n t e r t i d a l  were m a i n t a i n e d  resistance  range  of  1979).  i n t h e absence of e x t e r n a l  n u t r i e n t s u p p l y s h o u l d c o n t i n u a l l y i n c r e a s e t h e C/N r a t i o of t h e p l a n t , however, maintained 1979).  f o r growth  below  a  to  certain  occur species  t h e C/N  ratio  specific  must  be  v a l u e (Hanisak  I t appears t h a t h i g h i n t e r t i d a l seaweeds a r e c a p a b l e  of  i n c r e a s e d n i t r o g e n procurement f o l l o w i n g p e r i o d s of d e s i c c a t i o n , but t h i s has not been s t u d i e d t o d a t e . Nitrogen  commonly  l i m i t s t h e growth of marine  (Chapman and C r a i g i e 1977; Hanisak 1982).  Their  ability  important f o r s u r v i v a l . nitrogen  procurement.  study  1979; Rosenberg  Ion uptake  i s the  (Rosenberg  first  and  step  in  Ramus  1982).  examines how n i t r o g e n uptake i n i n t e r t i d a l seaweeds  s u p p l y and p e r i d o d i c d e s i c c a t i o n .  b e f o r e uptake r a t e s were measured  of  fluctuating  There a r e two o t h e r  r e p o r t s i n which n i t r o g e n s u p p l i e s t o l a b o r a t o r y controlled  Ramus  S e a s o n a l f l u c t u a t i o n s i n n i t r o g e n uptake  i s adapted t o ensure s u r v i v a l i n an environment nitrogen  and  t o compete f o r t h i s l i m i t e d r e s o u r c e i s  r a t e s o c c u r i n marine macrophytes This  macrophytes  cultures  were  ( D ' E l i a and DeBoer  3  1978;  Probyn and Chapman  intertidal  investigated.  This adaptation A  survey  was  ammonium uptake r a t e s i n f i v e which  These  studies  suggest  seaweeds may be adapted f o r r a p i d r e c o v e r y  nitrogen starvation. was  1982).  of  nitrogen  conducted  species  of  on  that  following  procurement nitrate  intertidal  and  seaweeds"  were t a k e n d i r e c t l y from t h e f i e l d i n l a t e summer (a time  of n i t r o g e n d e f i c i e n c y ; Chapman and C r a i g i e 1977; G e r a r d 1982a). A time c o u r s e study of t h e e f f e c t nitrogen  uptake  rates  of  nitrogen  i n l a b o r a t o r y c u l t u r e s of P.  was a l s o u n d e r t a k e n . " D i f f e r e n c e s i n n i t r o g e n and  on  perforata  uptake  kinetics  t h e e f f e c t of n i t r o g e n s u p p l y on uptake r a t e s were measured  i n two p o p u l a t i o n s stages  (germlings  After  inorganic  of G r a c i l a r i a v e r r u c o s a  i s taken  up,  and  and  that Laminaria  Ramus  1982).  lonqicruris  Craigie  stored  or  quantities  of  1977; G e r a r d  stored  nitrate  i n the winter  (a  of abundant n i t r o g e n s u p p l y ) and t h a t i t was u t i l i z e d i n  The  l i t e r a t u r e contains  nitrogen  supply  on  diminished.  i s o l a t e d r e p o r t s of t h e e f f e c t  nitrogen  uptake  1982b) and n i t r o g e n a s s i m i l a t i o n r a t e s (Weidner A  comprehensive study  on s e v e r a l a s p e c t s laboratory  of  (Bird et  and  Kiefer  of t h e e f f e c t of n i t r o g e n  supply  of n i t r o g e n u t i l i z a t i o n was  cultures  of  r a t e s ( D ' E l i a and DeBoer  Probyn and Chapman 1982), i n t e r n a l n i t r o g e n p o o l s  1981).  1982a;  Chapman and C r a i g i e (1977) found  the s p r i n g when n i t r o g e n s u p p l i e s were  1978;  history  t h i s can be u t i l i z e d d u r i n g p e r i o d s of  n i t r o g e n d e f i c i e n c y (Chapman and  period  i t i s either  M a r i n e macrophytes can s t o r e l a r g e  nitrogen  Rosenberg  and two l i f e  and mature t h a l l i ) of Fucus d i s t i c h u s .  nitrogen  assimilated.  al.  starvation  Porphyra  perforata.  undertaken  with  The i n t e r a c t i o n  4  between n i t r o g e n s u p p l y , u p t a k e , a c c u m u l a t i o n , was  and  assimilation  investigated. A  physiological  response observed i n the l a b o r a t o r y i s of  u n c e r t a i n e c o l o g i c a l s i g n i f i c a n c e u n t i l i t has been observed the  field.  The  conclusions  drawn from t h i s i n v e s t i g a t i o n of  n i t r o g e n metabolism i n l a b o r a t o r y c u l t u r e s of P. t e s t e d i n the f i e l d . nitrogen  content  populations levels studied.  and  of G.  and  The  major  uptake  growing  different difficulty  at  the  field  described.  i n t e r t i d a l l o c a t i o n ) may (e.g.,  The  effects  of  transplanting.  verrucosa  populations.  r e g i o n was  changed but g e o g r a p h i c a l  Vertical  were not c o n s c i o u s l y a l t e r e d . nitrogen  content  and  This  was  done  position  that  in  (e.g.,  from  geographic  with the  others  the  uptake  reductase  G.  intertidal  l o c a t i o n and n u t r i e n t  Nitrogen  nitrate  is  factor  the n u t r i e n t supply c h a r a c t e r i s t i c of a g i v e n by  also  not always be  be i s o l a t e d t o some e x t e n t  location)  were  studies  one  three  intertidal  regimes  e n v i r o n m e n t a l f a c t o r s cannot be c o n t r o l l e d and may accurately  soluble  a c t i v i t y of  different  nutrient with  p e r f o r a t a were  rates,  n i t r a t e reductase  verrucosa  slightly  The  nitrogen  the  in  rates,  supply soluble  activities  were  monitored. The  study of n i t r o g e n uptake  macrophytes  and  assimilation  in  marine  l a g s f a r b e h i n d s i m i l a r work w i t h p h y t o p l a n k t o n .  g r e a t d e a l can be l e a r n e d from  studies  on  phytoplankton,  A but  p h y s i o l o g i c a l s t u d i e s w i t h marine macrophytes i n v o l v e p a r t i c u l a r considerations  such  as  water and p o l y s a c c h a r i d e exposure  to  light,  tissue  age,  a p p r e c i a b l e and v a r i a b l e  content, a large apoplast,  carbon  and  non-uniform  n u t r i e n t s o u r c e s , and a l a r g e  5  enough biomass t o  store  significant  amounts  of  organic  and  i n o r g a n i c compounds. Physiological  studies  have  commonly o c c u r r i n g s p e c i e s .  so  f a r been l i m i t e d t o a few  This t h e s i s includes  comprehensive  p h y s i o l o g i c a l s t u d i e s on s e v e r a l e c o l o g i c a l l y important The  interaction  between  physical  s e v e r a l stages of  nitrogen  demonstrate  intertidal  that  and  utilization seaweeds  chemical were  The  results  factors  investigated  and to  are u n i q u e l y adapted t o  p e r i o d i c exposure and n i t r o g e n s t a r v a t i o n i n terms procurement.  species.  give  insight  n i t r o g e n metabolism and may  enhance  our  physiological  adaptations  of  the i n t e r t i d a l  environment.  into  of  nitrogen  the c o n t r o l of  understanding  of  the  i n t e r t i d a l seaweeds t o growth i n  6  Chapter 1.  A Time  Course  Study  Ammonium and N i t r a t e Uptake  of  the I n t e r a c t i o n  Between  Rates  Introduction Nitrogen  i s thought  to  be  t h e n u t r i e n t most commmonly  l i m i t i n g a l g a l growth i n t h e marine Dunstan  1971; Chapman  and Robbins 1976). of  nitrogen  to  seawater.  commonly nitrate  Macrophytes  sources  s t u d i e s have shown t h a t an  (Chapman and  urea and amino a c i d s these  and  addition  seawater g r e a t l y enhances t h e summer growth of  macrophytes  such as ammonium,  (Ryther  and C r a i g i e 1977; Hanisak 1979; Topinka  Several  marine macrophytes Marine  environment  (Moshen  1977; G e r a r d  1982a).  o b t a i n n i t r o g e n as i n o r g a n i c  and  also  Craigie  occasionally  utilize et  organic  as  nitrite  nitrogen  a l . 1974; DeBoer  a r e u s u a l l y l e s s abundant  ions from  such as  1981),  than i n o r g a n i c  but  sources  (Parsons e t a l . 1977). Uptake growth. are:  i s the f i r s t s t e p i n t h e u t i l i z a t i o n of n i t r o g e n f o r  The f o u r b a s i c mechanisms of  1)  diffusion  into  the apparent  d i f f u s i o n , 3) f a c i l i t a t e d d i f f u s i o n , (DeBoer  in  seaweeds  f r e e space, 2) p a s s i v e  and  4)  active  transport  1981).  Solute  entering  by  passive  d i f f u s i o n , and a c t i v e t r a n s p o r t , membrane  and  assimilated.  does not i n c l u d e Transport  transport  diffusion,  i s transported  facilitated  a c r o s s the  cell  D i f f u s i o n i n t o apparent f r e e space of  the  nutrient  into  the  cell.  i n t o t h e apparent f r e e space does not o n l y c o n s i s t of  " s i m p l e d i f f u s i o n " , but i o n exchange 1981).  i o n uptake  Standard  methods  of  occurs measuring  as  well  (DeBoer  uptake  cannot  7  d i f f e r e n t i a t e between t h i s i o n exchange and a c t i v e  uptake  into  the c e l l s . Nutrient  uptake can be determined by m o n i t o r i n g e i t h e r the  d e p l e t i o n of the n u t r i e n t from the s u r r o u n d i n g seawater accumulation 1982). and  of  isotope  in  the  or  t h a l l u s ( H a r r i s o n and D r u e h l  Measurement of n u t r i e n t d e p l e t i o n i s more commonly  may  be  undertaken  using  1982).  nitrogen  external  of  the  The  decrease  medium  c o n t i n u o u s l y m o n i t o r e d (the time c o u r s e method), or determined  from  the  used  e i t h e r the "time c o u r s e " or the  " b a t c h " methods ( H a r r i s o n and D r u e h l concentration  the  difference  in  the  in  can  be  i t can  be  c o n c e n t r a t i o n a t the  b e g i n n i n g and end of a s e t i n c u b a t i o n p e r i o d ( t h e b a t c h method). Most r e s e a r c h e r s have used the second method (Hanisak and H a r l i n 1978; H a r l i n and C r a i g i e 1978; Topinka 1978; G e r a r d they  have  employed  I t i s i m p o r t a n t whether incubation period.  1979) t o hours (Topinka 1978).  or not t h e uptake r a t e i s c o n s t a n t  .over  T h i s can o n l y be d e t e r m i n e d from a time  c o u r s e study of uptake r a t e . uptake  and  a v a r i e t y of i n c u b a t i o n t i m e s from minutes  ( H a r l i n and C r a i g i e 1978; Wheeler  the  1982b)  A transient increase  in  nitrogen  r a t e has been observed f o l l o w i n g an a d d i t i o n of n i t r o g e n  t o a n i t r o g e n l i m i t e d p h y t o p l a n k t o n c u l t u r e (Conway et a l . 1976; McCarthy ammonium  and has  Goldman been  1979). observed  An  enhanced  under  certain  G r a c i l a r i a t i k v a h i a e ( D ' E l i a and DeBoer pyrifera  (Haines  and  Wheeler  i n i t i a l l y enhanced but i t dropped  1978). to  w i t h i n a few minutes i n t h e s e s t u d i e s . determine  whether  1978)  a  uptake  rate  for  conditions  for  and  Ammonium lower  Macrocystis uptake  sustained  was rate  The p r e s e n t study was  to  t h i s uptake response commonly o c c u r r e d i n the  8  field.  Time c o u r s e s t u d i e s were  nitrate  uptake  Pelvetiopsis Gigartina  in  five  limitata papillata  ammonium  on  the  species  (C.Ag.),  f o r ammonium  of i n t e r t i d a l  (Setch)Gard.,  Papenf., and Enteromorpha of  determined  Fucus  Grac i l a r i a  rate  of  nitrate  ammonium uptake a r e a l s o r e p o r t e d .  seaweeds (  distichus verrucosa  i n t e s t i n a l i s (L.)Grev.). uptake  and  The  L., (Huds.) effects  and n i t r a t e on  9  M a t e r i a l s and Methods  S p e c i e s and C o l l e c t i o n S i t e s Giqartina (Setch)  papillata  G a r d . were  (C.Ag.),  collected  I s l a n d , B a r k l e y Sound, B.C.  and  Pelvetiopsis  limitata  from t h e n o r t h e r n shore of Diana  ( F i g . 1) a t 2.2  and  3.2  m  above  Canadian datum, r e s p e c t i v e l y . Specimens  of  Fucus d i s t i c h u s L.(renamed q a r d n e r i by S l i v a  see Conomos  1979),  Enteromorpha  Gracilaria  verrucosa  (Huds.)  intestinalis Papenf.,  Wiseman's Bay, B a m f i e l d , B.C., ( F i g . 1 ) . Canadian thalli  datum were  verrucosa  were  2.6,  collected  2.0  from  rock  studied.  population  grew  B a m f i e l d I n l e t ( F i g . 1 ) . The Periodic  sampling  n i t r i t e was not d e t e c t e d collection  sites  on  study  revealed  i n early  spring  collected  from  Their  heights  except  above A l l  Gracilaria  Specimens from a second verrucosa  a  were  also  m u d f l a t a t t h e head of  was  conducted  i n August  t h a t n i t r a t e , ammonium and water  a t the  and remained a t t h i s  p e r s . comm.)  However,  level during  when d a i l y measurements of s u r f a c e n i t r a t e and ammonium  c o n c e n t r a t i o n s were made  there  ammonium  (2-3 /uM)  Bamfield  were  (<0.1^iM) i n t h e s u r f a c e  u n t i l l a t e September ( L . D r u e h l August,  faces  (1.8 m) p o p u l a t i o n of G.  This  and  1.0 m, r e s p e c t i v e l y .  which grew on a r o c k y beach.  high i n t e r t i d a l  1981.  and  (L.)Grev.  concentrations  were  two were  days found  when  elevated  a t t h e head of  Inlet.  Time Course E x p e r i m e n t s The  general experimental p r o t o c o l  f o r measuring  nitrogen  10  F i g . 1. Map of B a m f i e l d I n l e t B a m f i e l d , B.C., the c o l l e c t i o n s i t e s .  Canada, showing  11  uptake  rates  was  as  f o l l o w s : 1) t h e medium used was n a t u r a l ,  filtered  (0.45 jum), n i t r o g e n d e f i c i e n t  nitrite  <0.1  juM)  and/or  Ryther  1962),  running  seawater,  4)  the  Erlenmeyer f l a s k s (time c o u r s e jars,  5)  the  2)  vigorous  incubation  experiments)  brushing  v e s s e l s were 1 1 or  500  ml  average  current  velocity  cm.s" , 8) t h e p l a n t biomass:water 1  1  depending  9)  on  the  120  constant.  rpm  approximately  volume r a t i o  temperature  the experiment,  of  a  was  10  0.3—0.2  g  was h e l d c o n s t a n t a t 11-16°C  10)  for  batch  experiments  i n c u b a t i o n t i m e s were s e t a t a p e r i o d over which t h e uptake was  at  t h e same time each day (1100-1400), 7) t h e medium  an  wt.l" ,  Mason  done  _1  was c o n t i n u o u s l y s t i r r e d w i t h a 1 i n c h s t i r r i n g bar  dry  pM  t h a l l i were f r e e f l o a t i n g , 6) t h e i r r a d i a n c e was  2  producing  trace  0-60  500—150 u E . m " . s , and uptake experiments were always approximately  and  ammonium was added depending on t h e e x p e r i m e n t ,  3) e p i p h y t e s were removed from t h e p l a n t s by under  ammonium  seawater, e n r i c h e d w i t h f/2 phosphate,  m e t a l s , and v i t a m i n s ( G u i l l a r d and nitrate  (nitrate,  rate  T h i s was u s u a l l y 10 min f o r ammonium uptake and  15—30 min f o r n i t r a t e  uptake,  and  11)  f o r the  time  course  s t u d i e s , uptake r a t e s were m o n i t o r e d c o n t i n u o u s l y f o r 30—60 min. Ammonium  and  nitrateconcentrations  Technicon AutoAnalyzer. uptake  rates  In  this  study  were  measured  nitrate  and  plus  a  ammonium  were m o n i t o r e d c o n t i n u o u s l y a t 16°C w i t h n i t r o g e n  c o n c e n t r a t i o n s o f : 30 jaM n i t r a t e , or 15 yuM ammonium, nitrate  using  15 ^uM  ammonium f o r 30 min.  were determined from b a t c h  uptake  or  30 yuM  Standard d e v i a t i o n s  experiments  (Chapters  5,6)  which were done i m m e d i a t l y f o l l o w i n g t h e time c o u r s e e x p e r i m e n t s f o r t h e s p e c i f i c time p e r i o d s when uptake was c o n s t a n t and under  12  the same p h y s i c a l c o n d i t i o n s . Dry  weights  were  General trends are discussed.  determined  by  c o n s t a n t weight on aluminum f o i l t r a y s  drying in a  r a t i o s were determined u s i n g a CHN a n a l y s e r  the plants to a 60°C  (Carlo  oven. Erba).  C/N  13  R e s u l t s and D i s c u s s i o n All  species  simultaneously G.  (Table  verrucosa  substantial presence  up  1 ) . E.  were  both  ammonium  the only populations  ammonium.  were p r o b a b l y  and  i n t e s t i n a l i s and h i g h  rate  by  intertidal  due  t o the  They a l s o had t h e lowest C/N r a t i o s and  under l e s s severe n i t r o g e n s t r e s s .  uptake  nitrate  i n which t h e r e was a  decrease (50%) i n n i t r a t e uptake  of  nitrate  took  ammonium i n E.  Inhibition  of  i n t e s t i n a l i s l a s t e d o n l y 12  min a f t e r which t h e ammonium c o n c e n t r a t i o n dropped below  5 ^uM.  m  After  30  min  t h e ammonium c o n c e n t r a t i o n of t h e medium i n t h e  h i g h i n t e r t i d a l G.  verrucosa  was s t i l l above 10 yuM and  nitrate  of n i t r a t e uptake by ammonium has been  recorded  uptake was i n h i b i t e d . Suppression in and al.  higher  p l a n t s (MacKown e t a l .  Morris  1963; Conway  1973).  Such  macroalgal  D'Elia  DeBoer  al. the  1977),  and  fungi  (Goldsmith  divisions  (Hanisak  1978; Haines  and  and  Harlin  case  f o r a l l marine  limitation.  appears  macrophytes  t o vary  with  The low i n t e r t i d a l G.  r a t i o than t h e h i g h i n t e r t i d a l G. inhibition  of  nitrate  uptake  t h e degree  verrucosa,  however,  under  nitrogen  of  nitrogen  d i d not  than  exhibit  I t appeared t h a t a the i n h i b i t i o n  I t i s energetically  f o r t h e p l a n t t o u t i l i z e ammonium r a t h e r 1962);  i s not  v e r r u c o s a w i t h a h i g h e r C/N  uptake by ammonium.  by ammonium.  1978;  and t h a t t h e degree of  c e r t a i n degree of n i t r o g e n d e f i c i e n c y p r e v e n t e d nitrate  et  Wheeler 1978; Gordon e t  1981), but t h e r e s u l t s of t h i s study show t h a t t h i s  suppression  of  (Syrett  p r e f e r e n t i a l uptake has been r e p o r t e d f o r t h e  t h r e e major and  1982), p h y t o p l a n k t o n  nitrate  limitation,  the  favorable (Syrett immediate  Table 1. A time course study o f n i t r a t e and ammonium uptake rates (V) (/jmol.g wet w t " . h " o r jjmol.g d r y w t " . h " ± 1 s t a n d a r d d e v i a t i o n , n=3 o r 4) f o r h i g h and low i n t e r t i d a l Grac i l a r i a verrucosa, Enteromorpha intestinalis , Fucus distichus, Gigartina papillata, and P e l v e t i o p s i s limitata. %=uptake r a t e as a % of c o n t r o l r a t e ( i n N 0 ~ o r NH^ o n l y ) , d = d u r a t i o n o f c o n s t a n t uptake r a t e ( m i n ) , and C/N r a t i o i s by atoms. 1  1  1  1  +  3  15 pM  30 fiH NO; addition C/N  13  V wet 1.82±0.20 3.80  6. verrucosa High Intertidal  30 nM NO" and 15 pM NH+ addition  \ addition  V N0-  Species  Low Intertidal  NH  V dry  d  ¥ wet  11.5+1.3  15-20  4.58±0.52  23.9  >20  9  1.28±0.14  6.04+0.66  10  9.77±1.20  46.5+5.7  30  V dry  d  29.4  30  V wet  ¥ dry  v d  i 85  2.34±0.25  15.8±1.7  10  3.95  26.8  >10  NH;  ¥ dry  d  f  7.53+1.1  50.9+6.8  30  170  10  110  10  90  ¥ wet  4.48±0.50  21.2  12  0.67±0.08  3.07+0.37  30  50  5.15±0.52  23.7±2.4  6.93+1.20  40.9  10  4.12+0.51  23.9±3.0  12  50  6.53+1.60  37.9±.9.3  >10  9.77  56.6  >12  2.8 ±0.40  10.4±1.4  30  G. verrucosa E. Intestinalis  F. distichus  G. papillata  28  13  3.23+0.61  2.84+0.44  11.5+2.2  8.0+1.2  15-20  30  30  4.40  26.0  2.8 +0.3  10.5  15  1.80  6.8  >15  6.15tl.60  13.2  3.35 P. 11n1tata  30  1.0 ±0.2 0.61  4.19+0.84 2.56  20 >20  2.77±0.31  7.21 10.2  12  85  4.00  23.2  >10  2.75±0.43  10.2±1.6  25  1.87  6.96  >25  100  2.95±0.43  11.7±1.7  20  150  3.9 ±0.8  15.4±3.2  20  85  1.17+0.18  4.86±0.75  20  115  2.32±0.28  9.65*1.16  20  95  >12 30  15  requirement f o r n i t r o g e n maximum  rates  of  simultaneously, per  unit  may  nitrate  outweigh and  this  advantage.  ammonium uptake a r e m a i n t a i n e d  a p l a n t can a q u i r e a g r e a t e r amount of  time.  This  would  d e f i c i e n t environment.  P.  When  be  an  advantage  p e r f o r a t a was  grown  nitrogen  i n a nitrogen under  various  degrees of n i t r o g e n d e f i c i e n c y t o c l a r i f y t h e e f f e c t of n i t r o g e n s t a r v a t i o n on p r e f e r e n t i a l uptake (Chapter 2 ) . N i t r a t e uptake r a t e s i n E. ammonium  suggest t h a t a c o n c e n t r a t i o n of >5 yuM NH * i s r e q u i r e d 4  t o i n h i b i t n i t r a t e uptake. same  i n t e s t i n a l i s i n t h e presence of  response  l e v e l has  D ' E l i a and DeBoer (1978)  found  i n G r a c i l a r i a t i k v a h i a e and a s i m i l a r  been  occasionally  reported  the  threshold  i n some  phytoplankton  ( M a e s t r i n i e t a l . 1982), but t y p i c a l l y much lower  concentrations  of 1 ^uM ammonium i n h i b i t n i t r a t e uptake i n p h y t o p l a n k t o n  (Conway  1976). E.  intestinalis,  i n t e r t i d a l G. rates  F.  verrucosa  f o r the f i r s t  concentration.  d i s t i c h u s , G.  showed rapid"  p a p i l l a t a , and h i g h  initial  ammonium  uptake  15 min of exposure t o a s a t u r a t i n g ammonium  T h i s t r a n s i e n t r a p i d ammonium uptake f o l l o w i n g a  p u l s e of ammonium t o t h e i n c u b a t i o n medium has been r e p o r t e d f o r marine macrophytes by Wheeler  (1978),  t h i s study  D'Elia  and  DeBoer  (1978),  and Probyn and Chapman (1982).  thallus  was  marine p h y t o p l a n k t o n et  and  The r e s u l t s of  show t h a t t h i s i s v e r y common under f i e l d c o n d i t i o n s .  D ' E l i a and DeBoer (1978) suggested t h a t t h i s the  Haines  nitrogen  starved.  only  occurred  if  T h i s i s a l s o t h e case f o r  (Conway 1977; McCarthy e t a l . 1977; D o r t c h  a l . 1982; Goldman  and  G l i b e r t 1982; Wheeler e t a l . 1982).  P r e c o n d i t i o n i n g one s p e c i e s under v a r y i n g  degrees  of  nitrogen  16  l i m i t a t i o n was r e q u i r e d t o c o n f i r m t h i s s u g g e s t i o n (Chapter 2 ) . Wheeler ammonium  et  al.  uptake  have shown t h a t t h i s r a p i d  (1982)  i n phytoplankton  Michaelis-Menten  kinetics;  does  where  not  rate,  rate,  constant.  Ks=the  half-saturation  r a p i d uptake of ammonium  follow  typical  V=(Vmax[S])/(Ks+[S])  S = s u b s t r a t e c o n c e n t r a t i o n , V=uptake and  initial  Vmax=maximum  i n t h e marine  However,  macrophyte  when uptake initial  Chordaria  f l a g e l l i f o r m i s was s a t u r a b l e and e x h i b i t e d a h a l f — s a t u r a t i o n ( K s ) constant  of  10.6  /JM  (Probyn and Chapman  1982).  was s u p p l i e d c o n t i n u o u s l y r a p i d i n i t i a l uptake  When ammonium  d i d not o c c u r ,  t h e h a l f — s a t u r a t i o n c o n s t a n t f o r uptake was 0 . 6 yuM (Probyn  and  and Chapman  1982).  experiments  lead  They  suggested  that  most  batch  uptake  t o an over e s t i m a t i o n of Vmax and Ks and t h a t  more c a u t i o n s h o u l d be taken when e x t r a p o l a t i n g t h e r e s u l t s these  of  s h o r t term uptake e x p e r i m e n t s t o t h e p r e d i c t i o n of growth  r e q u i r e m e n t s of seaweeds. N i t r a t e uptake r a t e s were g e n e r a l l y lower than uptake r a t e s of  ammonium.  distichus  The  exceptions  were  E.  intestinalis  and  F.  where n i t r a t e and ammonium uptake r a t e s were s i m i l a r .  i n t e s t i n a l i s had t h e h i g h e s t n i t r a t e uptake r a t e ( 4 6 . 5 pmol  E.  N0 ".g 3  d r y w t ~ . h ~ ) which was m a i n t a i n e d f o r 1 5 — 2 0 min. T h i s 1  1  was t h e o n l y example of t r a n s i e n t h i g h rates.  Unlike  initial  high  ammonium  n i t r a t e uptake r a t e s d i d not appear starvation. suppressed had t o be incubation  Dortch et a l .  initial  (1982)  uptake r a t e s ,  t o occur  i n t h e low  with  uptake enhanced nitrogen  suggest t h a t n i t r a t e uptake i s  i n nitrogen d e f i c i e n t phytoplankton. induced  nitrate  intertidal  G.  N i t r a t e uptake verrucosa  by  i n n i t r a t e ( 2 0 min) o r b o t h n i t r a t e and ammonium ( 1 0  17  min).  T h i s G.  v e r r u c o s a p o p u l a t i o n was more n i t r o g e n  deficient  than t h e h i g h i n t e r t i d a l p o p u l a t i o n . The uptake r a t e s found i n t h i s s t u d y a r e w i t h i n of  rates  f o r o t h e r marine macrophytes  the  ( D ' E l i a and DeBoer 1978;  Hanisak and H a r l i n 1978; H a r l i n and C r a i g i e 1978; DeBoer Harlin  (1978)  also  found  that  E.  range  intestinalis  1981).  has a h i g h  n i t r a t e uptake r a t e . The r e s u l t s of t h i s study emphasize the importance of course  s t u d i e s of n i t r o g e n u p t a k e .  Uptake cannot be assumed t o  be c o n s t a n t over a g i v e n i n c u b a t i o n Ammonium  uptake  by  E.  1  wf'.h" .  Wheeler  1  time  rates  were  time p e r i o d over species,  testing i t .  A f t e r 5 min t h e uptake  et  a l . (1982)  measured which  rate  and a f t e r 30 min i t was 26 yumol.g d r y  1  recorded  ammonium uptake r a t e s f o r p h y t o p l a n k t o n of when  without  i n t e s t i n a l i s i s a good example of how  uptake r a t e s can change w i t h t i m e . was 40 /umol.g d r y w t " . h ~  time  differences  0.09  and  0.02  a t 1 and 15 min, r e s p e c t i v e l y .  uptake  i s constant  varies . with  in h"  1  The the  t h e form o f n i t r o g e n , and t h e n u t r i t i o n a l p a s t h i s t o r y  of t h e t h a l l u s . The major d i s a d v a n t a g e of t h e "time c o u r s e " method i s usually  only  one  experiment  can  that  be performed a t a t i m e . I f  uptake r a t e s a r e r e q u i r e d a t a number of n u t r i e n t c o n c e n t r a t i o n s ( f o r example when s t u d y i n g uptake k i n e t i c s ) , a "time c o u r s e " f o r uptake must be r e p e a t e d a t each n u t r i e n t c o n c e n t r a t i o n . The " b a t c h " method i n v o l v e s a n a l y s i s of o n l y concentrations  per  experiment  the "time c o u r s e " method. that  marine  macrophytes  two  nutrient  and i s l e s s time consuming  I t i s becoming  increasingly  exhibit high inter-plant  than  apparent  physiological  18  variability.  With the " b a t c h " method the  necessary  replicates  can be run s i m u l t a n e o u s l y . In  a l l subsequent s t u d i e s , a "time c o u r s e " study of uptake  was conducted t o determine  the  length  uptake  was  f o l l o w e d by " b a t c h " e x p e r i m e n t s  was  constant  and  conducted f o r t h i s i n c u b a t i o n p e r i o d . in  uptake  rates  was  the  experiments  were  is  constant  during  Inter—plant  variability  uptake  was  constant.  depends  The l e n g t h of time d u r i n g on  the  Wheeler 1978).  A time c o u r s e study of uptake was  each time t h e s e parameters changed.  which  species, i t s n u t r i t i o n a l  s t a t u s , and the n u t r i e n t t e s t e d ( D ' E l i a and DeBoer 1978; and  which  a l s o run f o r s e v e r a l minutes l e s s than  p e r i o d of c o n s t a n t uptake.  uptake  time  o f t e n v e r y h i g h but was low ( u s u a l l y ± 2  min) f o r the l e n g t h of time d u r i n g which Batch  of  Haines  conducted  19  Chapter 2.  The  E f f e c t of N i t r o g e n Supply  A s s i m i l a t i o n and  on  Nitrogen  Uptake,  I n t r a c e l l u l a r S t o r a g e i n Porphyra p e r f o r a t a  Introduction Seasonal  fluctuations  in  nitrogen  supply  in  environments impose n u t r i e n t dependent growth p a t t e r n s marine  macrophytes  Ramus 1982; between  (Chapman  and  Gagne et a l . 1982).  Craigie  The  1977;  study of  coastal on  most  Rosenberg  the  and  relationship  growth r a t e and n i t r o g e n s u p p l y has been c o m p l i c a t e d  by  the f a c t t h a t macroalgae s t o r e i n t r a c e l l u l a r n i t r o g e n , which can be u t i l i z e d d u r i n g times of n i t r o g e n d e f i c i e n c y Chapman  and  Craigie  1982).  I t has  also  (D'Elia  and  DeBoer  al.  been  1982b) i n marine  nitrogen  supply  1977;  Gerard shown  1978)  (D'Elia  and  nitrogen  nitrogen  macrophytes  1974;  1982a; Rosenberg and Ramus  that  and  (Buggeln  can  be  DeBoer  uptake  reserves  (Bird  influenced  1978;  rates  by  et past  Probyn and Chapman  1982). T h i s study w i t h P. evaluation  perforata i s  first  comprehensive  of the e f f e c t of v a r i o u s forms of i n o r g a n i c n i t r o g e n  and n i t r o g e n s t a r v a t i o n on utilization  (uptake,  more  than  procurement. i n response  one  stage  s t o r a g e and a s s i m i l a t i o n ) .  g i v e s a more complete u n d e r s t a n d i n g  adaptive  the  of  Therefore, i t  of the c o n t r o l  of  R e g u l a t i o n of n i t r o g e n procurement and to  a  advantage  fluctuating in  macroalgae i s n i t r o g e n  an  nitrogen  environment  limited.  supply where  nitrogen  nitrogen metabolism  could the  be  an  growth  of  20  M a t e r i a l s and Methods  S p e c i e s and C u l t u r e Young  non-reproductive  (Rhodophyta) -land f i l l Canada  Conditions  thalli  site  on  perforata  They  Bay, K i t s i l a n o ,  were  immediately  l a b o r a t o r y and r i n s e d w i t h f i l t e r e d  of  Ag.  Vancouver,  filtered'  B.C.,  transported  t o the  (0.45 pm) seawater.  F o r t y grams wet weight of p l a n t m a t e r i a l was liters  J.  were c o l l e c t e d i n e a r l y March from a r o c k y English  (Fig. 2).  Porphyra  (0.45 pm) e n r i c h e d  added  to  ( f / 2 0 c o n c e n t r a t i o n of  phosphate, t r a c e m e t a l s and v i t a m i n s ; G u i l l a r d and Ryther nitrogen  deficient  boiling flasks. under  an  lighting were  natural  seawater i n 12 l i t e r  of 150 juE.m~ .s~ 2  1  a t 120 rpm.  flat—bottomed  The  cultures  c o n t i n u o u s l y w i t h a 2 i n c h magnetic s t i r r i n g One  culture  was  enriched  room  p r o v i d e d by f l u o r e s c e n t  ( V i t a - l i t e ) on a 12:12 l i g h t : d a r k c y c l e .  stirred  1962)  E i g h t c u l t u r e s were s t o r e d i n a 10°C c o l d  irradiance  10  with  50  pM  bars  nitrate,  a n o t h e r w i t h 50 pM ammonium and a t h i r d w i t h 50 pM ammonium p l u s 50  pM  nitrate.  No  n i t r o g e n was added t o t h e f o u r t h c u l t u r e .  These n i t r o g e n c o n c e n t r a t i o n s were chosen because they were h i g h enough t o a l l o w maximum growth, and were e c o l o g i c a l l y  realistic  concentrations.  Lower c o n c e n t r a t i o n s would a l s o have  supported  maximum growth r a t e s , but t h e n u t r i e n t would have been  depleted  too q u i c k l y i n c u l t u r e s w i t h a h i g h biomass. were  maintained  i n order  to  produce  Duplicate cultures  s u f f i c i e n t biomass f o r  subsequent t e s t s . The monitored  n i t r a t e and ammonium c o n c e n t r a t i o n s and  periodic  additions  were  i n the f l a s k s made  to  were  maintain  21  ^18° 1&1  F i g . 2. Map of S t a n l e y P a r k , Vancouver, B.C., collection site.  Canada, showing  22  concentrations umol  of  a t 50 yuM.  nitrogen  was added  3  for  every  i n the medium t o ensure t h a t the c u l t u r e s d i d  not approach carbon Uptake  F i v e umol NaHC0  rates,  limitation. nitrate  reductase  levels,  and  internal  s o l u b l e n i t r o g e n c o n t e n t were measured each day a t 1100 hours.  Ammonium and N i t r a t e Uptake E x p e r i m e n t s Two filtered  grams wet weight of p l a n t m a t e r i a l was p l a c e d enriched  seawater  (see  above)  with  either  ammonium, 25 pM n i t r a t e , or 25 /UM ammonium p l u s 25 The  nitrate  continuously  and  f o r 30 min  l a s t i n g the f i r s t often  ammonium (see  chapter  f o l l o w e d by an i n c r e a s e d  a  rates 1).  /uM  were  An  25  /uM  nitrate. monitored  "initial"  rate  10—20 min of exposure t o the uptake medium was  Both r a t e s were d e t e r m i n e d . with  uptake  i n 400 ml  uptake r a t e (an " i n d u c e d " r a t e ) .  The uptake medium was  water—jacketed cooling  i n c u b a t i o n p e r i o d , the  thalli  system. were  At  removed  the and  kept at end  12°C of the  dried  to  a  c o n s t a n t weight (24-48 h) a t 60°C.  I n t e r n a l N i t r a t e Content One  gram  wet  weight  of  plant  m a t e r i a l was ground i n a  mortar w i t h 25 ml hot water and 0.5 g sand (washed and i g n i t e d ) . The s l u r r y was c e n t r i f u g e d at 2,000 xg f o r 6 for  nitrate  as  outlined  done i n t r i p l i c a t e .  i n Appendix  1.  min  and  analysed  A l l e x t r a c t i o n s were  The e f f i c i e n c y of t h i s e x t r a c t i o n procedure  was d e t e r m i n e d (Appendix 1).  23  N i t r a t e Reductase  Activity  The jjri v i t r o n i t r a t e r e d u c t a s e assay o u t l i n e d i n Appendix was used w i t h the optimum assay n i t r a t e reductase a c t i v i t y plant  material  conditions  f o r P.  2  perforata  (Appendix 2 ) . One gram wet weight of  was used per assay.  These e x t r a c t s were s t o r e d  a t -10°C p r i o r t o a n a l y s i s f o r s o l u b l e p r o t e i n (Appendix 1 ) . A l l t e s t s were methods  done  in  triplicate.  The  complexity  of  and the a b s o l u t e requirement t o do a l l the t e s t s a t the  same time of day made  additional  sampling  impossible.  Since  p a t t e r n s were complex, a l l c u r v e s were f i t t e d by eye i n o r d e r t o show  general  trends.  standard d e v i a t i o n .  9  The  error  bars  on the graphs a r e ± 1  24  Results N i t r a t e Uptake  Rates  The n i t r a t e uptake directly  rate  from the f i e l d was  of  P.  perforata  plants  13—18 pmol.g d r y w t " . h ~ 1  When p l a n t s were grown on s a t u r a t i n g c o n c e n t r a t i o n s  taken  (Fig. 3).  1  of  nitrate  f o r 48 h n i t r a t e uptake i n c r e a s e d  i f ammonium was not p r e s e n t i n  the  Nitrate  uptake i n the s t a r v e d  30  period  uptake  medium  ( F i g . 3B).  p l a n t s was not c o n s t a n t over the uptake  was  being  monitored.  min  when  I n i t i a l l y , n i t r a t e uptake  were low ( i n i t i a l uptake r a t e ) ,  but  uptake  medium  rates increased  rate).  The presence of ammonium i n the uptake medium  the  initial  first  nitrate  nitrate  starvation  after  10—20  min  rates  in  (induced  the  uptake  inhibited  uptake r a t e of the s t a r v e d t h a l l i f o r the  10-20 min of the  nitrogen  uptake  nitrate  uptake this  experiment.  inhibition  After  appeared  8  days  of  t o be overcome  ( F i g . 3A). The f i e l d p l a n t s and i n some c a s e s the cultured  plants  starved  laboratory  showed no n i t r a t e uptake f o r the f i r s t 1—2  min  a f t e r n i t r a t e was added t o the uptake medium.  Ammonium Uptake Rates The ammonium uptake r a t e s f o r the p l a n t s d i r e c t l y from field  ranged  from  40—50  /umol.g  dry  ammonium uptake r a t e s of the c u l t u r e s  wf'.h" grown  1  (Fig. 4).  for  0-4  days  the The on  ammonium or ammonium p l u s n i t r a t e , d e c r e a s e d d r a m a t i c a l l y w i t h i n 24  and 48 h, r e s p e c t i v e l y .  Both c u l t u r e s showed b r i e f  (<5  min)  /jmol.g  dry  a t the s t a r t of the ammonium uptake e x p e r i m e n t s .  The  i n i t i a l p e r i o d of ammonium uptake ( a p p r o x i m a t e l y 10 wt"  1  .h" ) 1  25  NO,  NH;  s  ^starved - induced  ^ 20'  " " ~  starved  initial  3  4 Time  5 (days)  starved - induced  F i g . 3. N i t r a t e uptake r a t e s (umol NO^.g d r y w t ~ . h ) o f P o r p h y r a p e r f o r a t a p r e c o n d i t i o n e d f o r 0-10 days on 50 yM n i t r a t e (—•---)T 50 /LXM ammonium (—*•-•), 50 u,M n i t r a t e p l u s 50 pM ammonium ( { — x — ) , or n i t r o g e n s t a r v e d ( — • — • ) ; uptake r a t e s were measured i n t h e presence o f 25 /jM ammonium p l u s 25 JL\M n i t r a t e (A), or 25 uM n i t r a t e o n l y ( B ) . Uptake r a t e s of t h e s t a r v e d t h a l l i were o f t e n induced ( — • — ) d u r i n g t h e uptake experiment a f t e r 10-20 m i n . 1  _ 1  26  A  '  c  NH * A  60-1  Time B  (days)  NO" • N H * 3 4  starved  Time  (days)  F i g . 4. Ammonium uptake r a t e s (tmiol NH *.g d r y w t ' ^ h " ) of P o r p h y r a p e r f o r a t a p r e c o n d i t i o n e d f o r 0 - 1 0 days on 5 0 pW n i t r a t e (---•---), 5 0 pM ammonium (----A - - ) , 5 0 pM n i t r a t e p l u s 5 0 pM ammonium ( — ~ * — ) , o r n i t r o g e n s t a r v e d ; { — • — ) , uptake r a t e s were measured i n t h e presence of 2 5 pM ammonium ( A ) , or 2 5 pM ammonium p l u s 2 5 J J M n i t r a t e ( B ) . The ammonium and ammonium p l u s n i t r a t e grown t h a l l i o c c a s i o n a l l y showed t r a n s i e n t ( < 5 min) u n s u s t a i n e d uptake r a t e s a t t h e s t a r t o f t h e uptake e x p e r i m e n t . These r a t e s a r e i n d i c a t e d by ( ® ) and ( <g) ) . 1  4  27  cultures  grown  on  n i t r a t e alone m a i n t a i n e d  r a t e of a p p r o x i m a t e l y Ammonium  30 /umol.g d r y w f ' . h ' . 1  uptake  rates  of  the starved  approximately  30% d u r i n g  then r e c o v e r e d  t o 40-60 jumol.g d r y w f ' . h " ' ,  rate  an ammonium uptake  the f i r s t  thalli  dropped  two days of s t a r v a t i o n and Ammonium  uptake  i n t h e s t a r v e d c u l t u r e was r a p i d , but t h i s h i g h r a t e was  o f t e n s h o r t - l i v e d and d e c r e a s e d a f t e r uptake  medium.  This  was  10-15 min i n t h e ammonium  opposite  t o n i t r a t e uptake i n t h e  n i t r a t e uptake medium. N i t r a t e had Generally  no  appreciable  ammonium  uptake  effect  rates  on  ammonium  uptake.  were 50% h i g h e r than n i t r a t e  uptake r a t e s .  Soluble Nitrogen  Content  The i n t e r n a l n i t r a t e c o n t e n t time  and  the  internal  reached z e r o a f t e r  three  of a l l p l a n t s  nitrate  content  days  (Fig. 5).  decreased  with  of t h e s t a r v e d p l a n t s The  nitrate  grown  p l a n t s and t h e n i t r a t e p l u s ammonium grown p l a n t s m a i n t a i n e d t h e highest i n t e r n a l n i t r a t e The  extracts  were  levels. analysed  f o r s o l u b l e p r o t e i n , ammonium  and t o t a l amino a c i d s as d e s c r i b e d i n Appendix 1. p o s s i b i l i t y o f incomplete compounds  during  t o the  e x t r a c t i o n and i n t e r f e r e n c e from o t h e r  t h e c o l o r i m e t r i c a n a l y s i s , these r e s u l t s a r e  not q u a n t i t a t i v e but i n c r e a s i n g distinguished.  Due  The  or  decreasing  ammonium c o n t e n t  by 50% d u r i n g t h e f i r s t two  days  of  trends  can be  of a l l e x t r a c t s d e c r e a s e d culturing  (Appendix 3 ) .  There was an i n i t i a l i n c r e a s e i n t h e n i n h y d r i n p o s i t i v e m a t e r i a l (assumed  t o be p r i m a r i l y amino a c i d s ) i n t h e t i s s u e e x t r a c t s of  28  ~ 12H  Time  (dciys)  F i g . 5. The n i t r a t e c o n t e n t (umol NO" .g wet w t " ) of Porphyra perforata preconditioned f o r 0-10 days on n i t r a t e '(---»---), ammonium (-.-.A-, n i t r a t e p l u s ammonium ( — * — ) or n i t r o g e n starved ( — • — ) . E r r o r bars r e p r e s e n t one s t a n d a r d d e v i a t i o n , 1  29  a l l p l a n t s except f o r those  that  had  been  (Appendix 3 ) . The s o l u b l e p r o t e i n c o n t e n t s relatively  constant  and  similar  nitrogen  starved  of t h e e x t r a c t s were  in  magnitude  for  a l l  treatments(Appendix 3 ) .  N i t r a t e Reductase A c t i v i t y The the  i n i t i a l n i t r a t e reductase  field  was  72  nitrogen  starvation  activity  up  to  Growth  reductase maintained N0  3,  N0 .g 2  protein" .h" 1  t h e presence and  of  from  ( F i g . 6 ) . Both  1  nitrate  increased  then i t d e c r e a s e d r a p i d l y .  i n the starved t h a l l i continued  until  This day  on ammonium r e s u l t e d i n a r a p i d d e c r e a s e i n n i t r a t e  activity.  The p l a n t s grown on n i t r a t e  a n i t r a t e reductase  .g p r o t e i n " . h " 1  2  and  day  decrease i n a c t i v i t y 6.  pmol  a c t i v i t y of t h e p l a n t s  n i t r a t e reductase  1  plus  a c t i v i t y of a p p r o x i m a t e l y  f o r t h e f i r s t t h r e e days.  a c t i v i t y dropped by 50%.  ammonium 60 /umol  I n t h e next 24 h,  •  1  1  2  4 Time  1  1  6  8  (clays)  F i g . 6. " N i t r a t e reductase a c t i v i t y (^tmol N0$ .g p r o t e i n - . h " ) of Porphyra p e r f o r a t a p r e c o n d i t i o n e d f o r 0-10 days on n i t r a t e (—•---), ammonium I- -A- •-) n i t r a t e p l u s ammonium ( — x — ) , o r nitrogen starved ( — • — ) . E r r o r bars r e p r e s e n t one standard d e v i a t i o n , n=3. 1  T  1  31  Discussion It  has  been  shown  that  nitrogen  t r a n s i e n t i n c r e a s e i n ammonium uptake (Conway  1976) and  1978).  by  S t a r v e d P.  of  P.  by  marine  phytoplankton  uptake.  In  contrast,  exhibited  nitrogen  very  low  The moderate uptake r a t e s o f  plants  these  nitrate  that  plants  were  T h i s was unexpected c o n s i d e r i n g and ammonium c o n c e n t r a t i o n s  replete  nitrate  ammonium uptake r a t e s .  limited.  a  G r a c i l a r i a t i k v a h i a e ( D ' E l i a and DeBoer  perforata  suggest  causes  p e r f o r a t a showed a s i m i l a r reponse w i t h both  n i t r a t e and ammonium cultures  starvation  the  partially  and field  nitrogen  the r e l a t i v e l y  high  (17 and 4 fM, r e s p e c t i v e l y )  i n t h e s u r f a c e water a t t h e time of c o l l e c t i o n . The n i t r a t e - g r o w n uptake  rates.  This  c u l t u r e showed r e l a t i v e l y suggested  h i g h ammonium uptake r a t e s  nitrate.  This  that  phytoplankton  i s normally  1982) and  slower  i s the rate  that  One  been  observed  limiting  f o r marine  I t i s possible that process  (Dortch  plant  than  of t h e l a t e r s t e p s i n n i t r o g e n metabolism which i s  ammonium  uptake.  I t has  been  postulated  ammonium uptake may be c o n t r o l l e d by t h e l e v e l o f c e r t a i n acids  (Dortch  a c i d content (Appendix  et  f o r an ammonium—grown  not a c t i v a t e d when n i t r a t e r e d u c t i o n i s slow, may be t h e controlling  with  t h e supply of a s s i m i l a t e d n i t r o g e n i s much  f o r a nitrate—grown  plant.  also  associated  i n p l a n t s grown o n l y  ( H o r r i g a n and McCarthy 1981).  n i t r a t e reduction al.  has  ammonium  t h a t t h e mechanism t o m a i n t a i n  n i t r o g e n d e f i c i e n t p l a n t s , was a l s o p r e s e n t on  high  1980).  appeared t o  3).  Neither be  factor that amino  s o l u b l e p r o t e i n o r t o t a l amino  the c r i t i c a l  Ammonium i s a p p a r e n t l y  controlling  a better nitrogen  factor source  32  f o r growth of marine DeBoer  et  a l . 1978) because  ammonium before energy  macrophytes  than  nitrate  i t i s a s s i m i l a t e d and  (Nicholas  1959).  be reduced  assuming The was  that  must this  If nitrate  l i m i t i n g s t e p , then n i t r a t e p o o l s should  nitrate  t o match  reduction  fill  rate  of  i s the rate  and n i t r a t e uptake nitrate  reduction,  t h e maximum l e v e l of i n t e r n a l n i t r a t e i s f i x e d .  and  that  nitrate  a f t e r 24 h, s u p p o r t s t h i s The ammonium  N—starved uptake  P.  culture  uptake was d r a m a t i c a l l y reduced  suggestion. perforata  rates.  but were i n c r e a s e d  maintained  high  initial  I n i t i a l n i t r a t e uptake r a t e s were low  i n 10-20 min w i t h exposure  to  nitrate.  appears t h a t t h e uptake system f o r n i t r a t e does not remain a c t i v e i n a N — s t a r v e d t h a l l u s , but i t can be r a p i d l y by  a p u l s e of n i t r a t e .  (Dortch  et  of  metabolic  c e r t a i n l y an a d a p t i v e have  nitrate  i t i s assumed energy  during  delay  nitrate—grown  h.  Until  uptake  readily  (48 h) i n t h e cultures  this  to  be  due  to a It is  advantage f o r a n i t r o g e n s t a r v e d p l a n t inducible.  This  induction  of  induction  to of  replete plants. nitrate  uptake  in  c o u l d be l i n k e d t o t h e time r e q u i r e d t o  induce n i t r a t e r e d u c t a s e a c t i v i t y which was 48  fully  reactivated  starvation.  n i t r a t e uptake r a t e was not seen i n n i t r o g e n The  It  T h i s has been observed i n p h y t o p l a n k t o n  a l . 1982) and  reallocation  to  requires  fact that the n i t r a t e content of the nitrate-grown larger  1961;  converted  reduction  should  the  be  (Yamada  time  nitrate  a s s i m i l a t e d and n i t r a t e p o o l s would  also  taken fill.  up This  approximately would  not be  could  l i m i t n i t r a t e uptake. The  enhanced  initial  ammonium  uptake  i n both  the  33  ammonium—grown  and  t h e ammonium  plus  nitrate-grown  suggests t h a t ammonium uptake was c o n t r o l l e d by a s m a l l p o o l which was r a p i d l y f i l l e d .  culture  medium  and  before  However, t h i s was u s u a l l y o n l y  time  i s considered  reserves.  were removed from  t h e s t a r t of t h e uptake  experiment. which  internal  The d e p l e t i o n of t h i s p o o l might  have o c c u r r e d d u r i n g t h e time a f t e r t h e t h a l l i ammonium  plants  insufficient  a  few  minutes,  a  to deplete  nitrogen  T h i s surge uptake of ammonium has a l s o been  observed  i n p h y t o p l a n k t o n (McCarthy and Goldman 1979; T u r p i n and H a r r i s o n 1979)  and  other  macrophytes (Chapter 1 ) , but u s u a l l y i t t a k e s  s e v e r a l hours t o d e v e l o p a f t e r ammonium  i s depleted  from  the  medium. The  presence  of  ammonium i n h i b i t e d the i n i t i a l uptake of  n i t r a t e i n n i t r o g e n s t a r v e d P. of  starvation  (Fig. 3).  reduce t h e energy r e q u i r e d necessary  Preferential by  the p l a n t  of  phytoplankton  nitrate (Conway  uptake  by  because  ammonium  i t i s not  is  Partial  common  in  1977; McCarthy e t a l . 1977; M a e s t r i n i e t  1982) and marine macrophytes ( D ' E l i a and DeBoer 1978; H a i n e s  and Wheeler 1978; Hanisak and H a r l i n The  day  uptake of ammonium may  t o reduce n i t r a t e t o ammonium ( S y r e t t 1962).  inhibition  al.  p e r f o r a t a u n t i l the eighth  results  of  this  1978; Gordon e t  a l . 1981).  study suggest t h a t t h i s no l o n g e r o c c u r s  a f t e r a c e r t a i n p e r i o d of n i t r o g e n  starvation.  Ammonium i n h i b i t i o n of n i t r a t e uptake l a s t e d o n l y 10-20 min and then n i t r a t e uptake r a t e s were independent of or  absence  of ammonium.  the  presence  Ammonium c o n c e n t r a t i o n s i n t h e uptake  f l a s k were a p p r o x i m a t e l y 10-15 JUM.  C o n c e n t r a t i o n s below  may not have an i n h i b i t o r y e f f e c t on n i t r a t e u p t a k e .  this  D ' E l i a and  34  DeBoer  (1978)  found a s i m i l a r response i n G r a c i l a r i a t i k v a h i a e  i n c u b a t e d i n 5 pM (1 pM)  Even lower ammonium  concentrations  have caused i n h i b i t i o n of n i t r a t e uptake i n p h y t o p l a n k t o n  (Conway 1977;  McCarthy et a l . 1977).  Nitrogen ammonium,  starvation  nitrate,  perforata. This  ammonium.  The  and  soluble  smallest  contradicts  Rosenberg  and  caused a g e n e r a l amino  decrease  the  findings  of  Ramus  (1982) who  decrease i n i n t e r n a l  acid  was  content  in  P.  i n t o t a l amino a c i d s .  Bird  et  a l . (1982b)  and  suggested t h a t amino a c i d s  p r o t e i n s were a more i m p o r t a n t r a p i d l y u t i l i z a b l e n i t r o g e n than n i t r a t e or ammonium. that  amino  acid  On  content  a f f e c t e d by n i t r o g e n  plants  up and  utilized. were  Ceratophyllum  than  in  decreased  ammonium-incubated  s t i l l h i g h , and  r e d u c t a s e a c t i v i t y which was  enzyme  in  the  plants,  Syrett  It is  ammonium  was  i n t e r n a l n i t r a t e being starved  dependent  i n f l u e n c e d by n i t r o g e n  perforata.  This also occurs  1965).  w i t h more s e v e r e n i t r o g e n an  rapidly  on  culture  The  in  The  nitrate  deficiency.  s t a r v a t i o n caused a t r a n s i e n t i n c r e a s e  r e d u c t a s e i n P. and  not  i n t e r n a l n i t r a t e l e v e l s were reduced.  d e c r e a s e i n i n t e r n a l n i t r a t e c o n t e n t was  (Morris  more  n i t r a t e r e d u c t a s e l e v e l s of the  Nitrogen  was  the ammonium-incubated p l a n t s .  a s s i m i l a t e d r a t h e r than the  The  demersum  supply.  p o s s i b l e t h a t i n the taken  pool  the o t h e r hand, B e s t (1980) showed  of  I n t e r n a l n i t r a t e content starved  and  in nitrate  phytoplankton  subsequent d e c r e a s e i n a c t i v i t y  starvation results in inactivation  of  system which i s m e t a b o l i c a l l y c o s t l y f o r the c e l l  to  m a i n t a i n d u r i n g a time N—starvation,  P.  of  nitrogen  perforata  stress.  m a i n t a i n e d a low  Yet  even  under  l e v e l of n i t r a t e  35  reductase  activity.  Growth on n i t r a t e caused a t r a n s i e n t reductase  activity.  mitchellae  (Weidner  This and  Kiefer  i n h i b i t e d n i t r a t e reductase culture  supplied  with  has  been  decrease i n n i t r a t e reductase  observed  1981).  activity  both  increase  nitrate  nitrate  Giffordia  on  ammonium  perforata.  Even t h e  and  activity.  in  Growth  i n P.  in  ammonium  Nitrate  showed  reduction  a was  not " t u r n e d o f f " as r a p i d l y as n i t r a t e uptake by t h e presence of ammonium.  Eppley  n i t r a t e reductase for  higher  e t a l . (1969a) found t h a t ammonium i n h i b i t e d activity  plants  i n phytoplankton.  (MacKown  et  T h i s was a l s o t r u e  a l . 1982) and  the  marine  macrophyte G i f f o r d i a m i t c h e l l a e (Weidner and K i e f e r 1981). Few supply, uptake  s t u d i e s have examined t h e i n t e r a c t i o n between n i t r o g e n uptake and  rates,  assimilation  c o n d i t i o n s i n P. ammonium  a s s i m i l a t i o n , and  were  were m a i n t a i n e d  perforata. utilized  content.  even under s t a r v a t i o n  I n t e r n a l sources  of  nitrate  and  d u r i n g n i t r o g e n d e p r i v a t i o n , but amino  a c i d s and s o l u b l e p r o t e i n s were not u t i l i z e d . between  Nitrogen  The i n t e r a c t i o n  n i t r a t e uptake and ammonium uptake was dependent on t h e  nutritional state concentration.  of  the t h a l l u s  and  t h e ambient  A s s i m i l a t i o n of n i t r a t e was slower  ammonium  than t h a t of  ammonium and t h e c o n t r o l o f ammonium uptake and a s s i m i l a t i o n was dependent upon ammonium  supply.  S i m u l t a n e o u s study of t h e e f f e c t s several  stages  multi-dimensional nitrogen  on  nitrogen  complexity  procurement.  of  utilization of  nitrogen has  the metabolic  T h i s was a c h i e v e d  supply  i l l u s t r a t e d the controls  i n a laboratory  where t h e n u t r i e n t regimes c o u l d be c o n t r o l l e d .  on  The  of study  ecological  36  significance  of these r e s u l t s were then t e s t e d i n a f i e l d  w i t h G r a c i l a r i a v e r r u c o s a (Chapter 5 ) .  study  37  Chapter 3.  Nitrogen  G e r m l i n g s and Mature  Uptake  and  Growth  of  Fucus  distichus  Thalli  Introduction Algal  zonation  occurs  because  of  different  species'  responses t o b i o t i c f a c t o r s such as c o m p e t i t i o n (Jones and  Kain  1967; C o n n e l l 1972; Mann 1972; Chapman 1973) and a b i o t i c f a c t o r s such  as  changing  humidity,  light,  n u t r i e n t s u p p l y ( Z a n e v e l d 1937; Doty 1978;  Rosenberg  some  intertidal  extreemes  and  Ramus  macrophytes  s a l i n i t y , temperature and 1946; Lewis  1964;  Topinka  1982) Recent s t u d i e s i n d i c a t e t h a t may  actually  flourish  under  i n s e v e r a l e n v i r o n m e n t a l f a c t o r s (Johnson e t a l . 1974;  Quadir e t a l . 1979; Thomas and T u r p i n 1980; Chapter 4 ) . Past  i n v e s t i g a t i o n s have been on mature t h a l l i even  i t may be t h a t t h e s e n s i t i v i t i e s of o t h e r that  history  stages  determine t h e d i s t r i b u t i o n of t h e mature p l a n t s ( S u b b a r a j u  et a l . 1982). of  life  though  Fucus  McLachlan  edentatus  (1974) suggested t h a t  and F.  desiccation  sensitivity  d i s t i c h u s g e r m l i n g s t o temperature  and l i g h t was i m p o r t a n t i n c o n t r o l l i n g a d u l t addition,  the  distribution.  In  and s a l i n i t y changes a f f e c t t h e s u r v i v a l  of P h a e o s t r o p h i o n i r r e g u l a r e g e r m l i n g s (Mathieson 1982). Release of critical  phases  gametes,  fertilization,  i n the  and  germination are  f u c o i d l i f e h i s t o r y and they have t h e  potential to limit algal distribution.  Terry  and  Moss  showed t h a t z y g o t e s of f o u r s p e c i e s from t h e Fucaceae under  a  any  germinated  wide range of l i g h t and temperature c o n d i t i o n s .  t o l e r a n c e t o one of t h e many i n t e r t i d a l in  (1981)  stage  i n the  environmental  A low  stresses  l i f e h i s t o r y of a marine macrophyte  may  38  determine a l g a l z o n a t i o n T h i s study periodic  i n an i n t e r t i d a l h a b i t a t .  i n v e s t i g a t e d the e f f e c t of n i t r o g e n  exposure  to  air  source  on growth, r h i z o i d development,  n i t r o g e n uptake i n the g e r m l i n g s of the mid— t o h i g h — alga  Fucus  germlings  distichus. and  mature  d i f f e r e n t adaptations  The thalli  nitrogen were  of these two  uptake  and and  intertidal  rates  of  compared t o i l l u s t r a t e  l i f e h i s t o r y stages.  the the  39  M a t e r i a l s and Methods Organisms and C o l l e c t i o n  Site  F e r t i l e and n o n - f e r t i l e specimens of were  collected  from  datum  was  ( F i g . 2).  approximately  3.5  Their m.  coloration,  and  inflated  from  the  receptacles  h  fertile at  4°  thalli C, and  under a 16:8  were arranged 50 j j E . m ^ . s "  (Pollack  on f l a t  1970).  Specimens  within  an  hour.  t r a y s and s t o r e d f o r 24  N i t r o g e n uptake e x p e r i m e n t s were  on the n o n — f e r t i l e  discharge  of  gametes  was  thalli.  accomplished  McLachlan et a l . (1971).  Reproductive  the  thalli  and  gently  of  Pollack  using  the  (1970)  and  r e c e p t a c l e s were  scrubbed  newly  formed  zygotes.  After  excised  by hand under a g e n t l e  stream of c o l d t a p water t o remove e p i p h y t e s , e x t r u d e d  gametes,  s c r u b b i n g , r e c e p t a c l e s were  p l a c e d on paper t o w e l s , g e n t l y b l o t t e d dry and a l l o w e d t o 45 min a t room temperature.  90x20 mm jam)  were  light:dark cycle.  technique  for  dark  of d a y l i g h t f l u o r e s c e n t l i g h t i n g  dehydration-rehydration  or  was  Discharge  The  from  above  1  conducted immediately  Gamete  height  w a l l with holdfasts i n t a c t , placed in p l a s t i c  bags and t r a n s p o r t e d on i c e t o the l a b o r a t o r y The  Point,  conceptacles,  C o l l e c t i o n s were made from November t o January. picked  L. ,  Plant f e r t i l i t y  d e t e r m i n e d by the presence of w e l l developed brown  distichus  the i n t e r t i d a l s e a w a l l a t B r o c k t o n  S t a n l e y Park, Vancouver, B.C. Canadian  Fucus  R e c e p t a c l e s were then p l a c e d i n  d i s p o s a b l e p e t r i d i s h e s c o n t a i n i n g 20 ml f i l t e r e d  enriched  natural  stand  seawater.  This  seawater  p r e v i o u s l y d e p l e t e d of n i t r o g e n by i n c u b a t i n g 1 g wet  had  (0.45 been  wt of U l v a  40  sp.  per l i t r e f o r 12-24 h a t  seawater  was  phosphate, and  filtered  10°C  and  12  pE.nr .s" . 2  The  1  and e n r i c h e d w i t h f/2 c o n c e n t r a t i o n s of  t r a c e m e t a l s and v i t a m i n s ( G u i l l a r d  and Ryther  1962)  10 pM n i t r a t e , o r 10 pM ammonium, or 5 pM (=10 pg—at  N.l" ) 1  u r e a and used as t h e c u l t u r e medium. The r e c e p t a c l e s were i n c u b a t e d o v e r n i g h t a t pE.m^.s' lights  15°C  and  150  w i t h a 16:8 l i g h t : d a r k c y c l e p r o v i d e d by f l u o r e s c e n t  1  (Vita-lite).  The spent r e c e p t a c l e s were  d i s c a r d e d and  the seawater medium r e p l e n i s h e d .  Culture Conditions The  germlings  were  c u l t u r e d i n the o r i g i n a l p e t r i dishes  (30 ml of medium) a t 15°C. at  D i s c h a r g e of gametes was  i r r a d i a n c e s below 35 pE.m~ .s~ .  of  2  irradiance  2  on  1  G e r m i n a t i o n was  independent  and growth became l i g h t s a t u r a t e d a t i r r a d i a n c e s  g r e a t e r than 85 p E . m " . s . /uE.m^.s"  1  suppressed  _1  Therefore  an  irradiance  of  a 16:8 l i g h t : d a r k c y c l e was used t o ensure  150 light  saturation. G e r m l i n g s were c u l t u r e d f o r t h r e e weeks on 20  uM  nitrate,  20 pM ammonium, 10 pM (=20 p g . a t N . l " ) u r e a , or 10 pM ammonium 1  plus  10  pM n i t r a t e .  i n Vancouver  N i g h t — t i m e low t i d e s t h a t occur i n w i n t e r  were s i m u l a t e d by removal o f medium from one s e t o f  ammonium c u l t u r e s once every 24 h f o r 10 darkness  and  h  including  then r e p l a c i n g t h e same medium.  8  h  of  F r e s h medium was  p r o v i d e d every t h r e e days.  D e t e r m i n a t i o n o f Growth Rates and Morphology Growth r a t e s  were  determined  each  week  except  i n the  41  exposed  cultures,  t h r e e weeks. zygotes  where  The  per  a  initial  dish  lengths  measurement was made a f t e r of  randomly  intervals.  The  Length measurements  percentage  g e r m l i n g s which d i s p l a y e d secondary  of  100  rhizoid  t h r e e weeks of i n c u b a t i o n was r e c o r d e d . which  40  selected  were d e t e r m i n e d u s i n g a b i n o c u l a r microscope  with a c a l i b r a t e d eyepiece. weekly  single  were  made  at  randomly s e l e c t e d development  after  Three week o l d c u l t u r e s  had e x p e r i e n c e d d a i l y exposure were examined and compared  to t h r e e week o l d ammonium- and n i t r a t e - g r o w n c u l t u r e s t h a t were c o n t i n u o u s l y submerged.  N i t r o g e n Uptake Experiments A time c o u r s e of n i t r o g e n uptake was determined. dish  containing  three  week  a  known  ammonium, or 30 pM nitrate.  nitrogen  nitrate,  seawater  concentration;  or  15  pM  ammonium  (Chapter -15  pM  plus  30  pM  monitored  f o r one hour u s i n g a Technicon A u t o A n a l y z e r  et a l . 1973).  The c u l t u r e v e s s e l was p l a c e d on  (40  15°C and an i r r a d i a n c e of 150 p E . m ^ . s ' .  rpm)  at  tray Uptake  as the change i n n i t r o g e n c o n c e n t r a t i o n per  u n i t time per gram d r y weight of t i s s u e . used i n o n l y one uptake uptake  (Davis  shaker 1  r a t e s were e x p r e s s e d  Nitrogen  a  1)  either  The ammonium and n i t r a t e c o n c e n t r a t i o n s were  continuously  petri  o l d g e r m l i n g s was submerged i n a  beaker w i t h 200 ml of f i l t e r e d , e n r i c h e d containing  A  Each  petri  dish  was  experiment. kinetics  were d e t e r m i n e d u s i n g the p e t r i  d i s h e s as t h e i n c u b a t i o n v e s s e l s .  The o l d medium was poured o f f  and the t h r e e week o l d g e r m l i n g s , which remained a t t a c h e d t o t h e bottom of t h e p e t r i d i s h , were  quickly  rinsed  with  filtered,  42  e n r i c h e d seawater c o n t a i n i n g n i t r a t e (0-50 pM) o r ammonium (0-50 jjM)  depending  on t h e e x p e r i m e n t .  poured i n t o t h e d i s h . nitrate  T h i s medium (35 ml) was then  D u p l i c a t e e x p e r i m e n t s were  or ammonium c o n c e n t r a t i o n .  shaker t r a y a t 15°C and an  run a t  each  The d i s h e s were p l a c e d on a  irradiance  of  150  pE.m~ .s~  1  and  i n c u b a t e d f o r 30 min f o r ammonium and one hour f o r n i t r a t e .  The  2  medium was then removed and i m m e d i a t e l y a n a l y s e d f o r n i t r a t e and ammonium the  using the AutoAnalyzer.  incubation  between  the  period  final  were  The average uptake r a t e s over  determined  concentration  of  c o n c e n t r a t i o n , and then n o r m a l i z e d t o germlings  i n the  experiments ammonium,  were and  incubation conducted  nitrate—  dish. as  plus  from  the  t h e medium  difference and t h e t=0  t h e d r y weight Nitrogen  outlined  uptake k i n e t i c  above  ammonium—grown  of t h e  for nitrate,  germlings.  g e r m l i n g s which were p r e v i o u s l y grown on ammonium p l u s were  starved  of  nitrogen  Some  nitrate,  by i n c u b a t i n g them i n n i t r o g e n  free  medium f o r 24 h p r i o r t o d e t e r m i n i n g uptake r a t e s . N i t r a t e and exposed  ammonium  cultures  uptake  immmediately  rates  following  were a  determined f o r 10  exposure by p e r t u r b i n g them w i t h e n r i c h e d seawater  h  period with  30  of pM  n i t r a t e o r 15 pM ammonium. All  germlings  and p l a c e d on a filter  and  were c a r e f u l l y l o o s e n e d from the p e t r i  pre-weighed  germlings  0.45  pm  Millipore  filter.  dish The  were then r i n s e d w i t h sodium formate ( 3 %  w/v) t o remove t h e s a l t c r y s t a l s which would o t h e r w i s e form when the 60°C  filter to  was d r i e d .  The f i l t e r and g e r m l i n g s  were  dried  at  a c o n s t a n t weight (1 h ) . Any sodium formate which was  l e f t on t h e f i l t e r ,  s u b l i m e d when i t was h e a t e d .  There  was  no  43  increase  i n t h e weight of f i l t e r s w i t h o u t g e r m l i n g s t h a t were  t r e a t e d w i t h sodium formate and d r i e d .  The  germlings  usually  accounted f o r 20-50% of t h e t o t a l  weight ( f i l t e r and g e r m l i n g s ) .  Duplicate  (one d i s h  uptake  experiments  of  g e r m l i n g s per  e x p e r i m e n t ) were r u n . In t h e time  course  uptake  experiments  with  mature  F.  d i s t i c h u s t h a l l i , a 4 g t h a l l u s was p l a c e d i n 450 ml of f i l t e r e d enriched  seawater  (Chapter 1) of known n i t r o g e n c o n c e n t r a t i o n ,  e i t h e r 30 pM n i t r a t e , o r 15 pM ammonium, or 30 pM  nitrate  plus  w  15  pM  ammonium  pE.m^.s"  at  1  continuously  and 15°C  incubated f o r 30  (Chapter  1)  c o n c e n t r a t i o n s were m o n i t o r e d AutoAnalyzer  (Davis  et  under  min. and  an  The  medium  the n i t r a t e  continuously  a l . 1973).  t h a l l i were d r i e d t o a c o n s t a n t  irradiance  using  " b a t c h " t y p e uptake e x p e r i m e n t s .  with  mature  2  (Chapter  and  ammonium  a  Technicon  thalli  involved  1  incubated  f o r 30 min i n t h e  f o r 10 min i f ammonium was used.  continuously with  a  magnetic  stirring  and  The bar  1 ) . A f t e r t h e experiment t h e t h a l l i were removed from  the i n c u b a t i o n medium and d r i e d t o a c o n s t a n t weight i n a oven.  (2—3 g  seawater w i t h  These were  a t 15°C, under an i r r a d i a n c e of 150 pE.m~ .s~  medium was s t i r r e d  stirred  Non-reproductive t h a l l i  a known n i t r a t e o r ammonium c o n c e n t r a t i o n .  and  was  A f t e r the experiment, the  wet wt) were p l a c e d i n 300 ml of e n r i c h e d f i l t e r e d  experiments  150  weight i n a 60°C oven.  The uptake k i n e t i c s s t u d i e s  nitrate  of  Each t r e a t m e n t was performed i n t r i p l i c a t e . ammonium  concentration  i n the  incubation  60°C  The n i t r a t e medium  were  d e t e r m i n e d a t t h e end of t h e e x p e r i m e n t . Patterns  were  o f t e n complex and t h e r e f o r e a l l c u r v e s were  t t e d by eye t o show o n l y g e n e r a l t r e n d s i n the d a t a .  45  Results Gamete r e l e a s e was p r o l i f i c and t h e r e was a h i g h fertilization  and  sources tested. or  nitrate  ( n i t r a t e , ammonium,  p l u s ammonium) had no e f f e c t on t h e g e r m l i n g  length,  but d i d r e s u l t  nitrogen  growth  i n a two-fold  i n secondary r h i z o i d development a f t e r t h r e e  incubation.  urea,  P e r i o d i c exposure t o a i r had no e f f e c t on t h e  i n germling  increase  of  i n t h e presence of t h e n i t r o g e n  The form of n i t r o g e n  r a t e s (Table 2 ) . increase  germination  level  There  were  treatments  no  with  obvious  differences  regard  to  weeks  of  among t h e  secondary  rhizoid  development. F.  distichus  g e r m l i n g s m a i n t a i n e d i n i t i a l uptake r a t e s of  n i t r a t e and ammonium f o r a t l e a s t one hour. nitrate  and  ammonium s i m u l t a n e o u s l y .  d i d n o t i n h i b i t n i t r a t e uptake. 30  min  i n non-reproductive  Ammonium  uptake  rates  a f t e r which they d e c r e a s e d . the  rate  of  nitrate  uptake r a t e s p e r  gram  Nitrate  were  distichus  thalli.  f o r the f i r s t  15-20 min  The presence of ammonium by  a p p r o x i m a t e l y 30%.  d r y weight  g e r m l i n g s than t h e a d u l t p l a n t s . ammonium  uptake was c o n s t a n t f o r  F.  constant  uptake  approximately  up  The presence of ammonium  mature  were  They c o u l d t a k e  were  much  higher  inhibited Nitrogen i n the  Ammonium uptake r a t e s a t 15 yuM  eight  t i m e s g r e a t e r , and n i t r a t e  uptake r a t e s a t 30 /uM n i t r a t e were 20-40 t i m e s  greater  i n the  mature p l a n t s . Nitrate  uptake i n F.  n i t r a t e concentration Uptake  was  distichus  followed  g e r m l i n g s as a f u n c t i o n o f  saturation  s a t u r a t e d a t 15-20 /uM n i t r a t e .  uptake r a t e (Vmax) was s i m i l a r  kinetics  (Fig.7).  The maximum n i t r a t e  i n g e r m l i n g s s t a r v e d of  nitrogen  46  T a b l e 2. The e f f e c t of n i t r o g e n source on average growth rate (pm.week" ) and secondary r h i z o i d development of F. distichus g e r m l i n g s over a t h r e e week p e r i o d , and t h e p e r c e n t g e r m l i n g s exhibiting secondary r h i z o i d development from 100 randomly s e l e c t e d g e r m l i n g s . V a l u e s i n c l u d e ± 1 s t a n d a r d e r r o r , n=l00. 1  Growth Rate (Lim.week )  % 2° r h i z o i d development  NO;  173±14  14 ±5  NH+  176±26  20±2  Urea  183 ±40  25±2  218±59  19+4  248±55  57 ±5  Nitrogen  -1  Source  H  NH+ + NO: NH+ + Emerged  *  3 3 o 3 C 3 n-  CD 3 3  0J * 3 (D •->• 3 (D iQ o ?r • 3 O -J • c 3 a *q c z o MI C rr • cn it i 0) i DJ rf M • n> •» cn rr C  ni-« o in CD 3 O 3 ncn i-t cn r r D) 0) r r i£) fD fD < n 3  rr fo ?r  M-  3 (t» rr  l-h C 3 O o cn iQ cn >-l cn  NJ 0) *C *» 3 iQ 3 3 •-i o 3* O o *-> 3 « 3 Z C o o • 3 lO .  : 3 n • : rr • n * —-0) r r O rt ' r-l n -  iQ --. r-t O 3 O 3 -  CO  Nitrate  Uptake  a.  Rate ( jumol N O " • g d r y  2.  wt"  1  • h" ) 1  o1_  48  for  24  h  and  those  grown  on  nitrate.  G e r m l i n g s grown on  ammonium as t h e i r o n l y n i t r o g e n source had a 50%  lower  maximum  n i t r a t e uptake r a t e than i n o t h e r c u l t u r e s . Nitrate  uptake  i n mature  F.  distichus  s a t u r a t e d up t o 50 pM ( F i g . 8 ) . Uptake concentrations  appeared  to  non—saturable  component  follow  low  affinity  non—saturable  nitrate concentration. subtracted  from  important  at  nitrate  The c o n t r i b u t i o n due t o t h i s  component  uptake  constant  be a p p r o x i m a t e l y  1—3 pM.  increased l i n e a r l y with  saturation  linearly  curve  was  distichus  kinetics  concentrations  saturable  and t h e  germlings  appeared  preconditioning  greater  than  20  pM,  uptake  w i t h ammonium c o n c e n t r a t i o n .  nitrogen treatments.  rates  There were no  and  1  4  3—5  pM  ammonium.  four  Maximum uptake r a t e s were  60 pmol NH *.g d r y w f ' . h "  approximately  to  up t o 20 pM ammonium ( F i g . 9 ) . A t  a p p r e c i a b l e d i f f e r e n c e s i n ammonium uptake r a t e s among t h e  approximately  was  ( K s ) f o r t h e uptake was c a l c u l a t e d t o  Ammonium uptake i n F.  increased  nitrate  t h e uptake r a t e ( D ' E l i a and DeBoer 1978), t h e  half—saturation  ammonium  low  I f t h e uptake due t o t h i s component  r e s u l t i n g high a f f i n i t y  follow  at  s a t u r a t i o n k i n e t i c s , but a  became  c o n c e n t r a t i o n s g r e a t e r than 10 pM.  rates  t h a l l i was not  Ks  values  were  Ammonium uptake i n a d u l t F.  d i s t i c h u s was p r o p o r t i o n a l t o t h e ambient ammonium c o n c e n t r a t i o n a t c o n c e n t r a t i o n s g r e a t e r than 15 pM ( F i g . 1 0 ) . I t i s f e l t this  uptake  component. component  was The  is  contribution  due  to  a  existence  questionable due  to  the  low of  a  affinity high  diffusion  that  uptake  a f f i n i t y a c t i v e uptake  ( F i g . 10). low a f f i n i t y  Nevertheless, possibly  the  diffusion  1  I  10  20  I  I  30 Nitrate  (>iM  40  )  F i g . 8. N i t r a t e uptake r a t e s (umol N0 ".g dry w t " . h ) a s a f u n c t i o n of n i t r a t e c o n c e n t r a t i o n f o r mature Fucus d i s t i c h u s thalli. E r r o r bars represent one standard d e v i a t i o n , n=3. Uptake r a t e s minus n o n — s a t u r a b l e component (—o—) were determined t o assess o n l y the a c t i v e uptake component. 1  3  _ 1  Ammonium  (juM)  F i g . 9. Ammonium uptake r a t e s (umol N H . g d r y w t - . h - ' ) a s a f u n c t i o n of ammonium c o n c e n t r a t i o n f o r t h r e e week o l d Fucus distichus germlings grown on n i t r a t e ( — • — ) , ammonium (-..-I-.-) n i t r a t e p l u s ammonium (••••••) or grown on ammonium then n i t r o g e n s t a r v e d f o r 24 h ( — * — ) . +  1  4  f  15  45  30  Ammonium  ^LIM)  F i g . 10. Ammonium uptake r a t e s (pmol NH .g d r y w f ' . h M a s a f u n c t i o n of ammonium c o n c e n t r a t i o n f o r mature Fucus d i s t i c h u s thalli. E r r o r bars r e p r e s e n t one s t a n d a r d d e v i a t i o n , n=3. Uptake r a t e s minus n o n — s a t u r a b l e component (—o—) were determined t o a s s e s s o n l y the a c t i v e uptake component. +  4  -  52  component was s u b t r a c t e d t o g i v e an  estimation  uptake  DeBoer  component  approximately concentration  (D'Elia  and  the  active  A  Ks of  1978).  10 uM was c a l c u l a t e d from these d a t a . of  1-2  pM  ammonium  was  uptake t o t a k e p l a c e ( F i g . 1 0 ) . I n c u b a t i o n days  of  i n ammonium  enriched  or  nitrogen  A  minimum  r e q u i r e d f o r ammonium f o r f o u r and a deficient  seawater  r e s u l t e d i n an i n c r e a s e d t h r e s h o l d ammonium c o n c e n t r a t i o n f o r uptake i n mature t h a l l i  to  7.1  pmol.g  (5 pM)  ( F i g . 11).  Exposure t o a i r caused a 70% (23.4  half  decrease  in nitrate  uptake  d r y w t ~ . h ~ ) i n t h e g e r m l i n g s a t 30 pM 1  1  n i t r a t e but had no e f f e c t on ammonium uptake a t 15 pM (57 pmol.g d r y w f ' . h " ) . 1  ammonium  20  40 Ammonium  (/JM )  F i g . 11. Ammonium uptake r a t e s (pmol NH • g d r y w t " h ' M a s a f u n c t i o n of ammonium c o n c e n t r a t i o n f o r mature Fucus d i s t i c h u s t h a l l i preincubated on ammonium f o r 4.5 dayl—T^hS or n i t r o g e n s t a r v e d f o r 4.5 days (--o--). ' 1  1  ;  cn CO  54  Discussion The  distribution  l i m i t s of mature Fucus may be  by the responses of g e r m l i n g s t o reproduction 1974).  the  environment.  influenced Vegetative  i s g e n e r a l l y unimportant i n Fucus spp.  (McLachlan  The g e r m l i n g stage of Fucus p l a y s an i m p o r t a n t  role  in  p o p u l a t i o n maintenance and d i s p e r s a l . F.  distichus  Northeast  Pacific  germination,  a v e r y common i n t e r t i d a l seaweed i n the  Ocean.  Gamete  release,  and  specific  Robbins  form  1976)  of  nitrogen.  and F.  Chondrus  (Prince  1974).  On  the  This  when  ammonium  is  present  than  rate  also  1961;  more  nitrate  1978).  DeBoer  et  a l . 1978),  Ulva  (DeBoer  1981).  o p t i m a l growth may a l s o depend 1967). nitrate  rapid i s the  This  rates  although  than  lower  ammonium  comparable  s u p e r i o r growth on urea has been r e p o r t e d f o r P o r p h y r a and  true  1981).  Urea g e n e r a l l y produces lower growth  1967)  1974)  on n i t r a t e i s a p p a r e n t l y due t o the u t i l i z a t i o n of  energy f o r n i t r a t e r e d u c t i o n (DeBoer  (Yamada  is  show  when  n i t r o g e n s o u r c e (Yamada 1961; DeBoer e t a l . growth  spiralis  o t h e r hand, the mature  t h a l l i of s e v e r a l o t h e r marine macrophytes do growth  F.  vesiculosus (Prince  grew e q u a l l y w e l l on ammonium and n i t r a t e . for  fertilization,  g e r m l i n g growth, and mature t h a l l u s growth have no  requirement f o r a (Topinka  is  or  (Iwasaki  The form of n i t r o g e n p r o d u c i n g on  the  concentration  (Iwasaki  Ammonium becomes t o x i c a t much lower c o n c e n t r a t i o n s than does  (Waite  and  c o n c e n t r a t i o n s used i n t h i s under f i e l d  Mitchell study  1972; were  DeBoer  those  1981).  commonly  The found  conditions.  P e r i o d i c exposure and the s t r e s s e s i t imposes a r e unique t o  55  the  intertidal  environment.  Exposure causes major f l u c t a t i o n s  i n t i s s u e water c o n t e n t , s a l i n i t y , i r r a d i a n c e , nutrient  supply.  Intertidal  s u r v i v e t h e s e changes.  macrophytes  temperature  must  be adapted t o  There have been s e v e r a l s t u d i e s on t h e s e  phenomena ( B i e b l 1938; Johnson e t a l . 1974; Quadir e t Schonbeck  and  a l . 1979;  and Norton 1979a; Thomas and T u r p i n 1980; Chapter 4 ) .  The e a r l y l i f e h i s t o r y s t a g e s  of  intertidal  also  F.  d i s t i c h u s g e r m l i n g s grew w e l l  survive  when they  were  necessary development exposed. growth  such s t r e s s e s . exposed  for was  daily  optimum greatly  I t has  periodic  development. reduced  been  i n mature  and  shown  macrophytes  exposure  rhizoid  i f the germlings  fucoids  seemed  Secondary  were  t h a t p e r i o d i c exposure  intertidal  must  (Schonbeck  not  enhances  and  Norton  1979a). Nitrogen  uptake  rates  of g e r m l i n g s were much h i g h e r than  those of mature t h a l l i .  T h i s may have been  proportion  and  of  storage  uptake  to  the  support t i s s u e i n the a d u l t  which was n e i t h e r a c t i v e l y t a k i n g The  due  up  nor  requiring  r a t e s of growing a p i c i e s may be h i g h e r .  large plants  nitrogen. I f a large  p o r t i o n of t h e a c t i v e l y growing g e r m l i n g was t a k i n g up n i t r o g e n , t h i s would be r e f l e c t e d i n h i g h uptake r a t e s e x p r e s s e d on a d r y weight  basis,  but  would  not  uptake r a t e s on a d r y weight germlings  and  mature  thalli,  affect  basis  t h e Ks v a l u e s .  were  very  Maximum  different for  but t h e i r a f f i n i t y  ( i n d i c a t e d by  Ks) f o r n i t r a t e was s i m i l a r . The Ks v a l u e s germlings  were  for nitrate similar  to  and  ammonium  uptake  those  recorded  f o r Grac i l a r i a  t i k v a h i a e and N e o a q a r d h i e l l a b a i l e y i  ( D ' E l i a and  DeBoer  by t h e  1978),  56  but  they  are less  macrophytes Craigie F.  most  (Haines and Wheeler  Ks  v a l u e s r e p o r t e d f o r marine  1978; H a r l i n  1978; H a r l i n  1978; Hanisak and H a r l i n 1978; Kautsky 1982),  spiralis  ammonium  (Topinka  and  nitrate  1978).  F o r F.  spiralis  and  including  t h e Ks f o r  n i t r a t e were 9.6±2.6 and 7.8±1.4 pM, r e s p e c t i v e l y  (Topinka 1978). for  than  The h i g h a f f i n i t y of  and  ammonium  over mature t h a l l i when  F.  distichus  germlings  may p r o v i d e a c o m p e t i t i v e  nitrogen  supplies  advantage  i n the  intertidal  h a b i t a t a r e low. Ammonium  uptake*  r a t e s were t w i c e those f o r n i t r a t e by F.  d i s t i c h u s g e r m l i n g s and t e n times those of This  i s apparently  common  t h e mature  i n marine macrophytes  DeBoer 1978; Hanisak and H a r l i n 1978; Wheeler Topinka i n F.  (1978)  thalli.  ( D ' E l i a and  1982),  although  r e p o r t e d t h a t n i t r a t e and ammonium uptake r a t e s  s p i r a l i s were s i m i l a r .  The  ammonium  uptake  rates f o r  g e r m l i n g s p r e s e n t e d i n t h i s s t u d y were s i m i l a r t o t h o s e r e p o r t e d for  t h e mature t h a l l i of o t h e r s p e c i e s ( D ' E l i a and DeBoer 1978;  Haines and Wheeler  1978; Hanisak and  Harlin  1978; H a r l i n . and  C r a i g i e 1978; Kautsky 1982). The  nitrate  uptake  r a t e s of t h e a d u l t F.  low i n comparison t o o t h e r marine macrophytes 1978; H a r l i n concentrations were p r o b a b l y ammonium  was  1978; H a r l i n  and  ( D ' E l i a and DeBoer  1978).  i n t h e water s u r r o u n d i n g t h e F. saturating  (20-25  undetectable.  nitrogen saturated i n the winter Rosenberg and Ramus 1982). rapidly  Craigie  growing g e r m l i n g s .  pM)  d i s t i c h u s were  The  d i s t i c h u s bed  i n the winter  Temperate (Chapman  seaweeds and  nitrate  whereas  are  Craigie  often 1977;  I t appears t h a t t h i s i s not t r u e f o r W i n t e r n i t r o g e n uptake r a t e s i n t h e  57  g e r m l i n g s were h i g h and t h e s e r a t e s were m a i n t a i n e d f o r a time.  long  Another a d a p t a t i o n t o r a p i d n i t r o g e n procurement was t h a t  ammonium  d i d not i n h i b i t n i t r a t e uptake.  o c c u r i n t h e mature F. (Haines  and  Wheeler  but i t seems t o be course  uptake  T h i s i n h i b i t i o n does  d i s t i c h u s and s e v e r a l o t h e r 1978; Hanisak and H a r l i n  dependent  experiments  upon with  1978; Chapter 1)  nutritional F.  macrophytes  status.  distichus  Time  i n t h e summer  showed no i n h i b i t i o n of n i t r a t e uptake by ammonium (Chapter 1 ) . Topinka  (1978)  collected  similar  results.  I t has been  F.  postulated  uptake  by  ammonium  limited  (Chapter 1 ) . The F.  a t 20 pM n i t r o g e n . spite  of  frequent  s p i r a l i s i n t h e summer and found  that  i s overcome  the  inhibition  nitrate  when t h e t h a l l i a r e n i t r o g e n  d i s t i c h u s g e r m l i n g s were  cultured  I t i s p o s s i b l e they were n i t r o g e n l i m i t e d i n changes of medium.  A l t e r n a t i v e l y germlings  may p o s s e s s t h e a b i l i t y t o take up n i t r a t e all  of  and  ammonium  under  conditions. Nitrogen  distichus  starvation  germlings.  d i d not a f f e c t ammonium uptake i n F.  The  cultures  supplied  with  nitrogen  m a i n t a i n e d r a p i d uptake r a t e s which may r e f l e c t t h e r a p i d growth r a t e of t h i s j u v e n i l e s t a g e . Periodic  exposure t o a i r o r growth on ammonium as t h e o n l y  n i t r o g e n s o u r c e caused a distichus  germlings.  decrease  in nitrate  uptake  i n F.  The g e r m l i n g s which were n i t r o g e n s t a r v e d  d i d not show t h i s r e s p o n s e .  The maintenance of a n i t r a t e uptake  system i n t h e absence of an  external  energetically  expensive,  although  nitrate  supply  must  be  r e t e n t i o n of the a b i l i t y t o  t a k e up any form of n i t r o g e n as soon as i t was  available  would  58  be  advantageous. F.  distichus  k i n e t i c s but saturable  germlings  the adults  (Topinka  showed s a t u r a b l e n i t r o g e n uptake  d i d not  1978) and  show  this  non-saturable  ( D ' E l i a and DeBoer 1978; Haines and Wheeler  pattern. nitrogen  this  and/or  life  history  depend  stage.  on  and  The r e s u l t s  s t u d y and t h a t r e p o r t e d i n Chapter 5 suggest t h a t  i s not s p e c i e s dependent, but may state  uptake  1978; H a r r i s o n  D r u e h l 1982) have been found i n marine macrophytes. of  Both  the  this  nutritional  Furthermore, the f a c t  that  n o n - s a t u r a b l e n i t r o g e n uptake i s common i n macrophytes, but not in  p h y t o p l a n k t o n , s u g g e s t s t h a t t h e n o n - s a t u r a b l e component may  be r e l a t e d t o t h e t h i c k , component in  characterises  some s p e c i e s  (D'Elia  multicellular  form.  This  ammonium uptake but not n i t r a t e  uptake  and  DeBoer  thalloid  1978).  storage  o c c u r s i n marine macrophytes  Appendix  1 ) . I t i s p o s s i b l e t h a t t h e ammonium  Little  ammonium  (Rosenberg and Ramus 1982; was  assimilated  and t h e n i t r o g e n s t o r e d i n a d i f f e r e n t form which would s t o p any direct  negative  feedback  inhibition  of  ammonium  uptake.  Rosenberg and Ramus (1982) and B i r d e t a l . (1982b) i n d i c a t e t h a t amino a c i d p o o l s a r e i m p o r t a n t . T h i s study r e p o r t s t h e requirement of a t h r e s h o l d concentration  for  uptake  i n mature  F.  ammonium  distichus  surprisingly  d i d not d e c r e a s e  after  4.5  starvation.  Such  would  be a severe c o m p e t i t i v e  disadvantage  during  Phytoplankton  and  t h r e s h o l d response. the  mature  thallus  a  threshold times  F.  of  distichus  decreased germlings  These s m a l l e r l i f e  forms  days  of  which  nitrogen do may  nitrogen  supply.  not show t h i s out  compete  f o r low l e v e l s of n i t r o g e n but they do not  59  have t h e n i t r o g e n s t o r a g e c a p a b i l i t i e s which  sustain  growth  during  of  periods  t h e mature  of  nitrogen starvation  (Chapman and C r a i g i e 1979; Rosenberg and  Ramus  possible  a  that  concentration  this  requirement  f o r t h e mature F.  of  distichus  when t h e t h a l l i a r e n i t r o g e n s a t u r a t e d . F.  thallus  1982).  It is  threshold thalli  ammonium  only  occurs  Topinka (1978) examined  s p i r a l i s i n t h e summer and he d i d not observe t h i s  threshold  f o r ammonium u p t a k e . F.  distichus  g e r m l i n g s responded t o n i t r o g e n supply  s i m i l a r manner t o n i t r o g e n l i m i t e d mature t h a l l i . n i t r a t e and ammonium uptake r a t e s , preference uptake  in  for a  relatively  long  affinities,  showed  time  (> 1 h ) .  no high  Further  i s r e q u i r e d t o c o n f i r m t h a t t h e s e responses a r e common  nitrogen  s u f f i c i e n t germlings.  Mature F.  t a k e n d i r e c t l y from t h e f i e l d d i d not responses"  and  show  di stichus t h a l l i  any  marine  of  c o n s e q u e n t l y appeared t o be n i t r o g e n  H i g h uptake r a t e s and h i g h uptake a f f i n i t i e s a r e of  They had h i g h  f o r t h e v a r i o u s forms of n i t r o g e n and m a i n t a i n e d  rates  research  and  ina  phytoplankton  g e r m l i n g s appear t o be  (Syrett  better  1962).  adapted  t h e above sufficient.  characteristic  Phytoplankton  f o r procurement  and of  a  l i m i t i n g n u t r i e n t than mature seaweeds. Several  studies  have  shown t h a t Fucus i s p h y s i o l o g i c a l l y  w e l l adapted f o r growth and s u r v i v a l i n t h e (Schonbeck Turpin also  and  1980). well  Norton  1979c;  T h i s study  Quadir  shows t h e F.  intertidal  habitat  e t a l . 1979; Thomas and d i s t i c h u s germlings  are  adapted t o p e r i o d i c exposure which a c t u a l l y enhances  secondary r h i z o i d development. There have  been  numerous  studies  on  the p h y s i o l o g i c a l  60  adaptations  of marine macrophytes t o e n v i r o n m e n t a l f a c t o r s , y e t  the c r u c i a l  early  ignored. to  gain  life  Investigation a  complete  history  stages  been  virtually  of the l i f e h i s t o r y s t a g e s i s n e c e s s a r y  understanding  macroalgal d i s t r i b u t i o n s .  have  of  factors  controlling  61  Chapter  4.  Intertidal  Desiccation  Enhanced  Nutrient  Uptake  Rates  in  Seaweeds  Introduction The i n t e r t i d a l environment i s one of the most extreme is  i n h a b i t e d by macroalgae.  Exposure and subsequent submersion  impose v a r i a b i l i t y i n numerous (e.g.,  temperature,  irradiance).  physical  salinity,  and  chemical  nutrients,  Success i n such an environment  physiological  factors  humidity, requires  adaptations to these environmental f l u c t u a t i o n s .  on i n t e r t i d a l macroalgae by exposure t o a i r . 1951; Schonbeck  and Norton 1978,  in  the  intertidal  affects  r a t e of d e h y d r a t i o n  suggested t h a t t o l e r a n c e t o d e s i c c a t i o n i s more (Schonbeck and Norton I 9 7 9 b , c ) .  (Isaac  marine  1933,1935;  1979a) but l i t t l e desiccation. fixation enhanced  of  been  Schonbeck  and  physiological  investigations  intertidal  The p h y s i c a l  algae  revealed are  in  e x c e p t i o n of  Quadir the  et  a l . 1979).  absence of any new  carbon.  This  This  than  effects  investigated Norton  1978,  effects  of  t h a t the carbon  maintained  when weight l o s s due t o d e s i c c a t i o n i s 20-40%  et a l . 1974; occurs  1951;  I t has been  important  have  i s known about the  Recent  rates  macrophytes  Feldman  algal  zone, but Dromgoole (1980) showed  of d i f f e r e n t s p e c i e s and t h e i r i n t e r t i d a l l o c a t i o n .  of d e s i c c a t i o n on  1935;  1979a).  t h a t t h e r e was no d i r e c t c o r r e l a t i o n between  avoidance  imposed  ( I s a c c 1933,  A b i l i t y t o t o l e r a t e d e s i c c a t i o n undoubtedly survival  and  specific  D e s i c c a t i o n may be the most extreme e n v i r o n m e n t a l s t r e s s  Feldman  that  carbon  or  even  (Johnson  procurement  s u p p l y of n u t r i e n t s w i t h the  photosynthetic  increase  has  the  62  potential  to  cause a d e f i c i e n c y i n a l l n u t r i e n t s except carbon  because t h e net uptake of such n u t r i e n t s i n t o t h e t i s s u e i s o n l y possible during adaptations following  submersion.  Consequently,  for nutritional desiccation.  Schonbeck  Brinkhuis  et  al.  compounded  by  periodic  nutrients  are  i o n uptake  (1976)  not  and  suggest  exposure  available,  one in  when  expect  i n t e r t i d a l algae  Norton  that  would  (1979a)  nutrient  external  and  shortage,  supplies  i s important i n determining  of the  upper l i m i t i n a l g a l d i s t r i b u t i o n s . Nitrogen  i s b e l i e v e d t o be t h e n u t r i e n t t h a t most  limits  phytoplankton  1971).  I t has a l s o been shown  nitrogen  which  (Chapman and uptake  i s used  Craigie  potential  (Chapman  and  periodic  storms  (1980)  i n t h e ocean that  1977).  Craigie  marine  Nitrogen  that  and Dunstan  macrophytes  1977),  with  storage  seasonal  and  nitrogen  (Rosenberg and Ramus 1982).  suggested  (Ryther  d u r i n g p e r i o d s of n i t r o g e n  fluctuate  i n t e r m i t t e n t supply The  growth  periodic  exposure  commonly  store  limitation  and  nitrogen  nitrogen  supply  upwelling  during  Thomas and T u r p i n results  in  an  of n i t r o g e n .  enhancement  of  nutrient  uptake r a t e s i n response t o  d e s i c c a t i o n i n t h e i n t e r t i d a l a l g a Fucus d i s t i c h u s  was  studied  and t h e r e l a t i o n s h i p s between t h i s r e s p o n s e , n u t r i e n t l i m i t a t i o n and  season  were examined.  F i e l d s t u d i e s of d e s i c c a t i o n l e v e l s  were undertaken t o determine whether t h i s i s a common The  effect  of d e s i c c a t i o n on n i t r a t e and ammonium uptake  in four a d d i t i o n a l i n t e r t i d a l species Enteromorpha Pelvetiopsis  response.  intestinalis limitata  )  was  ,  (  Gracilaria  Gigartina  studied  to  verrucosa,  papillata, determine  rates  if  and this  63  uptake  response  to i n t e r t i d a l  t o d e s i c c a t i o n was an i n t e r s p e c i f i c  location.  adaptation  64  Materials S p e c i e s and C o l l e c t i o n Gigartina (Setch)  Island, Barkley respectively, distichus  Site  papillata  Gardn. were  B.C.  above  Gracilaria  (C.Ag.),  and  ( F i g . 1),  Canadian  Enteromorpha  verrucosa  heights  respectively. collected  Canadian  A l l specimens  in  late  exposure, and they were natural  seawater  Specimens the  of F.  summer,  for  been  Fucus  (L.)Grev.  and  collected  from  B.C.  ( F i g . 1).  in  non—reproductive the  h  morning,  by  the  called  F.  in  after ambient  experiments Bay  distichus  as F.  began. only  (Bamfield)  to  winter  ( s i t e d e s c r i b e d below) and h e n c e f o r t h r e f e r r e d  of  the  Barkley  Sound  southwest exposure, and were  sites  surrounded  were by  rocky,  tall  t h a l l i were c o l l e c t e d from rock f a c e s , except the G. grew  on  collection  site.  There  ammonium, or n i t r i t e Diana  Island  pe r s. c omm.).  with  trees.  to  F.  distichus  and  E.  a  All  verrucosa,  a r o c k y beach a r e a which was s l i g h t l y below  100 m t o the e a s t of the  and  in  d i s t i c h u s (Vancouver). Both  which  m,  were  shortly  submersion  before  and  d i s t i n g u i s h them from p l a n t s c o l l e c t e d i n the summer and near Vancouver  m,  datum were 2.6, 2.0, and 1.0  rehydrated 2—6  Sound,  3.8 of  d i s t i c h u s c o l l e c t e d a t Wiseman's  have  limitata  and  Specimens  Papenf. were  were  summer,  2.2  intestinalis  (Huds.)  above  at  datum.  Wiseman's Bay, B a m f i e l d I n l e t , B a r k l e y Their  Pelvet iopsis  c o l l e c t e d from the n o r t h e r n shore of Diana  Sound,  L.,  and Methods  and  intestinalis  was no d e t e c t a b l e (< 0.1 /uM) n i t r a t e ,  i n the s u r f a c e water of b o t h Wiseman's  Bay  from F e b r u a r y u n t i l l a t e September (L. D r u e h l  65  N o n - r e p r o d u c t i v e t h a l l i of from  F.  distichus  were  collected  t h e s e a w a l l a t t h e south end of Second Beach p o o l ,  Stanley  P a r k , Vancouver, B.C., Canada ( F i g . 2 ) . The c o l l e c t i o n s i t e was 3.5m  above Canadian datum.  E x p e r i m e n t s were conducted e a r l y i n  J u l y , September and F e b r u a r y . the  These p l a n t s  were  morning a f t e r exposure and were r e h y d r a t e d  filtered  (0.45 pm)  concentrations)  natural  seawater  (with  f o r 2-6 h p r i o r t o e x p e r i m e n t a l  collected in  by submersion i n ambient  nutrient  desiccation.  D e s i c c a t i o n Procedure Plants  were  usually  d e s i c c a t e d under n a t u r a l c o n d i t i o n s .  When t h i s was i m p o s s i b l e due t o weather c o n d i t i o n s , p l a n t s desiccated  at  600 pE.m~ .s~ 2  distichus winter plants)  1  18°C under a r t i f i c i a l l i g h t , a t an i r r a d i a n c e of i n a chamber w i t h  thalli  were  experiments.  dried  desiccated In  circulating  at  a l l cases,  both  air.  F.  6 and 18°C i n t h e  the c o n t r o l s  (hydrated  were kept i n a humid chamber a t t h e same i r r a d i a n c e and  temperature f o r t h e same l e n g t h of time as t h e p l a n t s t h a t desiccating.  were  T h i s a l l o w e d a l l p l a n t s t o be exposed and s t a r v e d  of n u t r i e n t s f o r t h e assuring  were  that  any  same  length  effect  on  of  time  nutrient  (1-3 h ) , t h e r e b y  uptake r a t e s c o u l d be  a t t r i b u t e d s o l e l y t o d e s i c c a t i o n and not t o n u t r i e n t s t a r v a t i o n . The amount of n u t r i e n t o b t a i n e d rehydration  was  determined  equated t o a volume o f water content.  For  example:  from t h e water  absorbed  from t h e weight i n c r e a s e which was with  weight  the predetermined  absorbtion.  nutrient  g a i n = 2 g = 2 ml medium which  c o n t a i n s 30 /umol n i t r a t e per l i t e r , t h e r e f o r e 0.06 pmol was o b t a i n e d v i a water  during  nitrate  66  Dry  weights  were o b t a i n e d by l e a v i n g t h e t h a l l i  oven u n t i l they reached a c o n s t a n t weight (48-72 h ) . of some samples were  determined  with  a  CHN  i n a 60°C C/N r a t i o s  analyser  (Carlo  Erba).  N i t r o g e n Uptake Experiments Batch  uptake  Chapter 1. on  the  experiments  were  conducted  as o u t l i n e d i n  P l a n t s of s i m i l a r s i z e (1-5 g wet w e i g h t ,  species)  were  desiccated  depending  t o a range of d e s i c c a t i o n s  (0-60%) and then each t h a l l u s was p l a c e d i n 400 ml  of  filtered  (0.45 pm) seawater e n r i c h e d w i t h 30 pM n i t r a t e o r 15 pM ammonium and  with  a l l other  n u t r i e n t s , t r a c e m e t a l s and v i t a m i n s a t a  s a t u r a t i n g c o n c e n t r a t i o n of Each  point  f/2  (Guillard  and  Ryther  1962).  on t h e d e s i c c a t i o n v e r s u s uptake p l o t s i s t h e r e f o r e  the uptake r a t e of one p l a n t .  Although  nitrate  and  ammonium  uptake r a t e s i n some macroalgae a r e not s a t u r a t e d , even a t 30 uM (D'Elia Harrison  and DeBoer 1978; Haines and Wheeler and  Druehl  1982),  these  1978; Topinka 1978;  concentrations  were  used  because they were e c o l o g i c a l l y r e a l i s t i c c o n c e n t r a t i o n s commonly observed  i n the  f i e l d i n w i n t e r and f o l l o w i n g storms and they  were w i t h i n t h e s e n s i t i v i t y range of t h e a n a l y t i c a l  technique.  The use of h i g h e r c o n c e n t r a t i o n s a l s o seemed unwarranted of  reports  that  saturation  of  t h e uptake  macroalgae i s p r e v e n t e d by t h e e x i s t e n c e of component  (D'Elia  and  a  because  system  i n some  large  diffusion  DeBoer 1978; Topinka 1978; H a r r i s o n and  D r u e h l 1982; Chapter 2 ) . D u r i n g t h e summer a l l p l a n t s were i n c u b a t e d i n an water b a t h a t 16°C under s a t u r a t i n g , n a t u r a l l i g h t  outdoor  (Brinkhuis et  67  al.  1976;  Hanisak  and H a r l i n 1978).  were conducted i n a 6°C c o l d room juE.m~ .s~ 2  W i n t e r uptake e x p e r i m e n t s  with  an  irradiance  of  (Chapter 2 ) .  1  Uptake  r a t e s ( i n i t i a l c o n c e n t r a t i o n minus f i n a l n i t r a t e or  ammonium c o n c e n t r a t i o n i n the i n c u b a t i o n medium d i v i d e d time  taken)  were  determined  uptake r a t e s were c o n s t a n t ; for  the  150  nitrate  by  the  d u r i n g an i n c u b a t i o n p e r i o d when  10 min f o r ammonium  experiments  (Chapter  1).  and  15—30  min  At the end of the  i n c u b a t i o n p e r i o d , the p l a n t s were removed and the f i n a l  nitrate  c o n c e n t r a t i o n was determined w i t h a Technicon A u t o A n a l y z e r u s i n g methods p r e v i o u s l y d e s c r i b e d were  calculated  from  the  ( D a v i s e t a l . 1973). changes  in  nitrate  c o n c e n t r a t i o n d u r i n g the i n c u b a t i o n p e r i o d and to  dry  weight.  by a p l a n t when  Uptake or  were  rates  ammonium normalized  These r a t e s r e p r e s e n t the uptake r a t e o b t a i n e d it  is  suddenly  submerged  in  high  nutrient  seawater and thus a r e measures of p o t e n t i a l uptake r a t e s . was  a g r e a t d e a l of i n t e r — p l a n t v a r i a b i l i t y  i n the uptake d a t a .  When g e n e r a l t r e n d s were o b v i o u s c u r v e s were f i t t e d A d d i t i o n a l s t u d i e s were done on F. the  effect  collected  enriched wet  distichus.  and  In F e b r u a r y ,  preconditioned  F.  In  August,  distichus  h.  Twenty  2  of  grams  of p l a n t m a t e r i a l was p l a c e d i n 10 l i t e r s of medium  m a i n t a i n e d a t 6° C, under an i r r a d i a n c e of 150 /uE.nr . s " . effect  thalli  i n nitrogen free, f i l t e r e d ,  (Chapter 1) n a t u r a l seawater f o r 24  weight  visually.  of d e s i c c a t i o n on phosphate uptake was t e s t e d u s i n g  the method o u t l i n e d above. were  There  desiccation  p l a n t s was t e s t e d .  on  the  1  The  n i t r o g e n uptake r a t e s of these  68  Natural Desiccation  Levels  In s i t u d e s i c c a t i o n l e v e l s distichus  thalli  on  a  were  hot  (23-24°C),  juE .m~ . s " ) , moderately windy J u l y 2  were  submersion. were  day  1  weighed  at  12:00  and  determined  again  at  for  sunny  ten  (1500-1700  i n Vancouver. 13:30,  F.  just  Plants prior to  P l a n t s and h o l d f a s t p o s i t i o n s were marked.  Plants  removed, w i t h t h e i r h o l d f a s t s i n t a c t , weighed and  returned  t o t h e i r exact filtered  location.  seawater  recorded.  Percent  following  formula:  to  At 13:30 t h e  hydrate  "desiccation  for was  thalli 12  were  placed  in  h and wet w e i g h t s were determined  using  % D e s i c c a t i o n = [(wet wt - d e s i c c a t e d wt)/wet wt] x 100  the  69  Results The  potential  (Bamfield) and  30%  greater (0%  nitrate  uptake  rate  i n l a t e August was enhanced desiccation  ( F i g . 12A).  f o r F.  distichus  dramatically  Uptake  rates  between were  0  always  f o r t h e d e s i c c a t e d p l a n t s than t h e n o n - d e s i c c a t e d p l a n t s  desiccation).  occurred  The  maximum  enhancement  i n uptake  rate  a t a p p r o x i m a t e l y 30% d e s i c c a t i o n (14.5 jumol N0 ~.g d r y 3  w t " 1 . h " l ) and was g r e a t e r than t w i c e t h e p o t e n t i a l  uptake  rate  of n o n - d e s i c c a t e d p l a n t s (6 jumol N0 ".g d r y w t " 1 . h " l ) . 3  Ammonium was  showed a t r e n d s i m i l a r t o t h a t of n i t r a t e .  more than  uptake >50%  a  rate  two-fold  at  enhancement  approximately  of  potential  There  ammonium  30% d e s i c c a t i o n ( F i g . 12B). A t  d e s i c c a t i o n , ammonium uptake appeared t o be s i m i l a r  t o or  l e s s than t h e n o n - d e s i c c a t e d p l a n t s . The was  relationship  between phosphate uptake and d e s i c c a t i o n  d i f f e r e n t from those observed  with  nitrate  and  ammonium.  When phosphate uptake e x p e r i m e n t s were conducted on t h e day t h a t the p l a n t s were c o l l e c t e d , t h e r e was no enhancement of phosphate uptake  rate  after  desiccation  ( F i g . 12C).  The  potential  phosphate uptake r a t e i n f a c t d e c r e a s e d a f t e r d e s i c c a t i o n . nutrient  concentrations  i n Bamfield  I n l e t and C/N r a t i o s of t h e  p l a n t s (approximately  30 by atoms), suggest  the  nutrient  growth-limiting  d e s i c c a t i o n enhanced  uptake  rather rates  The  that  than  may  nitrogen  phosphorus.  only  occur  was Thus,  when t h e  n u t r i e n t i s l i m i t i n g growth. Plants Inlet  were  water  concentrations  collected  enriched  with  and i n c u b a t e d  f o r 24 h i n B a m f i e l d  a l l other  nutrients  to  f/2  ( G u i l l a r d and Ryther 1962) w i t h t h e e x c e p t i o n of  -I  - T  20  40 •/o  60  20  40 %> Desiccation  Desiccation  F i g . 12. N u t r i e n t uptake r a t e (pmol.g d r y w f ' . h " ) f o r Fucus distichus ( B a m f i e l d ) over a 30 min i n t e r v a l as a f u n c t i o n of d e s i c c a t i o n . N i t r a t e uptake ( A ) , ammonium uptake ( B ) , phosphate uptake ( C ) , phosphate uptake f o r p l a n t s p r e v i o u s l y i n c u b a t e d i n 30 uM n i t r a t e medium f o r 24 h i n an attempt t o produce phosphate d e f i c i e n t p l a n t s (D). 1  Phosphate  rAV  Uptake Rate  p  10  8  (/.imol PC^ • g d r y w t  p C7>  h" ) 1  o  D n)  n  c +». ^. O O  3  Phosphate Uptake  ro  a  5 o  S-  1  IL  Rate  ( j j m o l P O ^ . g d r y w t " l h" ) 1  01  72  phosphate. nitrogen  T h i s enrichment was used t o overcome deficiency  and  any  possible  make phosphate t h e l i m i t i n g  A f t e r t h e 24 h i n c u b a t i o n , an i n c r e a s e  resource.  i n t h e uptake  rate  of  phosphate w i t h d e s i c c a t i o n was o b s e r v e d ( F i g . 12D). In  contrast  (Vancouver) following  to  t h e above  d i d not  show  desiccation  enhancement  (<  0.1  concentration  The  uptake  Nitrate  was not  uM) i n t h e s u r f a c e water a t t h i s time and t h e  of ammonium was low (1.1 juM).  14).  distichus  ammonium  ( F i g . 13).  20—25% d e s i c c a t i o n r e s u l t e d i n enhanced (Fig.  of  F.  i n e a r l y J u l y : >40% d e s i c c a t i o n caused a  decrease i n ammonium uptake r a t e s detected  observations,  nitrate  and  In e a r l y  ammonium  ammonium  September,  uptake  concentrations  rates i n the  s u r f a c e water a t t h i s time were u n d e t e c t a b l e (< 0.1 pM) and  5.4  uM, r e s p e c t i v e l y .  By F e b r u a r y , t h e water was 6° C and c o n t a i n e d  5.0  and  /uM  ammonium  20-25  /uM  d e s i c c a t e d <at 6°C (a m i l d w i n t e r observable  trends  ( F i g s . 15,16). seawater  i n either  nitrate.  temperature),  h  there  either  were  were  i n nitrogen  no  rates free  t o e s t a b l i s h n i t r o g e n d e f i c i e n c y , but t h i s  t r e a t m e n t d i d not produce d e s i c c a t i o n enhanced rates  thalli  ammonium or n i t r a t e uptake  Winter t h a l l i were i n c u b a t e d  f o r 24  When  ammonium  uptake  ( F i g . 1 7 ) . D r y i n g w i n t e r p l a n t s a t 18°C t o speed  up t h e r a t e of d e s i c c a t i o n caused a r a p i d drop i n n i t r a t e uptake r a t e and i n most c a s e s n i t r a t e (Fig.  18).  plants  Ammonium  were  was  excreted  into  t h e medium  and n i t r a t e uptake r a t e s f o r t h e h y d r a t e d  approximately  6  and  1.5 yumol.g  dry  wf'.h" , 1  respectively. Three  of  the four  other  species  p o t e n t i a l n i t r a t e and ammonium uptake  t e s t e d showed enhanced  rates  after  desiccation  73  12H  >  o E  3  0)  4J  E Z3  C  o E E <  — i —  20  40  Desiccation  — i —  60  F i g . 13. The r e l a t i o n s h i p between ammonium u p t a k e r a t e (pmol.g d r y w t " . h " ) and % d e s i c c a t i o n f o r F u c u s d i s t i c h u s (Vancouver) i n J u l y 1980. 1  1  74  %  Desi.cc cit ion  F i g . 14. The r e l a t i o n s h i p between ammonium uptake r a t e (jjmol.g dry w t " . h " ) and % d e s i c c a t i o n f o r Fucus d i s t i c h u s i n September 1980. 1  1  75  I  ,  ,  10 %  20 Desiccation  1  ~  30  F i g . 15. The r e l a t i o n s h i p between ammonium uptake r a t e (pmol.g dry w t " . h " ) and % d e s i c c a t i o n f o r Fucus d i s t i c h u s i n February 1981. T h a l l i were d e s i c c a t e d a t 6°C and uptake e x p e r i m e n t s were conducted a t 6°C. 1  1  76  o E  o O  i  —I—  20  10  30  F i g . 16. The r e l a t i o n s h i p between n i t r a t e uptake r a t e (pmol.g dry w f ' . h " ) and % d e s i c c a t i o n f o r Fucus d i s t i c h u s i n February 1981. T h a l l i were d e s i c c a t e d a t 6 C and uptake e x p e r i m e n t s were conducted a t 6°C. 1  P  77  10  1  20 Desicc at ion  30  F i g . 17. The r e l a t i o n s h i p between ammonium uptake r a t e (urnol.g dry w f ' . h " ) and % d e s i c c a t i o n f o r Fucus d i s t i c h u s i n F e b r u a r y 1981. T h a l l i were p r e i n c u b a t e d i n n i t r o g e n f r e e seawater f o r 24 h and then d e s i c c a t e d a t 6°C, uptake e x p e r i m e n t s were c o n c u c t e d at 6°C. 1  78  *  ,  ,  20  %  40 Desiccation  60  F i g . 18. The r e l a t i o n s h i p between n i t r a t e uptake r a t e (pmol.g dry w f .h" ) and % d e s i c c a t i o n f o r Fucus d i s t i c h u s i n F e b r u a r y 1981. T h a l l i were d e s i c c a t e d a t 18°C and uptake e x p e r i m e n t s were conducted a t 6°C. 1  79  ( F i g s . 19-23).  The  exception  i n t e r t i d a l species tested.  was  G.  v e r r u c o s a , the lowest  I n some cases t h e d a t a were v a r i a b l e  and c o n s e q u e n t l y no c u r v e s c o u l d be f i t t e d t o general  the data.  When  t r e n d s , such as an i n c r e a s e o r d e c r e a s e i n uptake r a t e s  f o l l o w i n g d e s i c c a t i o n were i d e n t i f i e d , an e s t i m a t i o n desiccation  producing  maximum  uptake  rates  of  the %  and t h e r a t i o of  maximum uptake r a t e s t o c o n t r o l uptake r a t e s were  made.  These  r e s u l t s a r e summarized i n F i g u r e s 24,25. The  degree  of  enhancement  and  the %  desiccation that  produced maximum enhancement of n i t r a t e or ammonium uptake  were  related  to  intertidal  intertidal  species,  papillata  showed  location  P.  limitata,  the greatest  ( F i g s . 24,25).  The  high  F.  and  G.  enhancement  ammonium uptake f o l l o w i n g d e s i c c a t i o n optimum  %  desiccation  distichus of  nitrate  ( F i g s . 24B,25B)  The degree of enhancement  of  uptake v a r i e d from 1979 t o 1981, but t h e % d e s i c c a t i o n maximum  uptake  differences ammonium  was  i n these  uptake.  One  relatively two  constant.  parameters,  exception  was  between P.  resulting  nitrate  and  l i m i t a t a where t h e relative  t h e uptake r a t e s of t h e h y d r a t e d p l a n t s ) was much lower than  f o r ammonium ( a p p r o x i m a t e l y also  nitrate  There were few  degree o f enhancement of n i t r a t e uptake (2.5-3.0 times to  and t h e  p r o d u c i n g t h i s enhancement was a l s o t h e  h i g h e s t ( F i g s . 24A,25A).  in  and  showed  degree of  1 0 ) ( F i g s . 24B,25B).  This  species  an u n u s u a l n i t r a t e uptake response i n 1979 and t h e  enhancement  could  not  be  estimated.  Degrees of  d e s i c c a t i o n up t o 52% c o n t i n u e d t o enhance u p t a k e ; no p l a t e a u i n uptake was observed ( F i g . 2 3 ) . The  high  intertidal  species  were a l s o more r e s i s t a n t t o  A  B  •  •  30H  304  •  O" z  20  20-|  o E  E  3  • * * • v  10 "U  \  »  20 Desiccation  30  10  r — 20 Desiccation  F i g . 19. The r e l a t i o n s h i p between n i t r a t e (A) and ammonium (B) uptake r a t e s (pmol.g d r y . wt-'.h" ). and % d e s i c c a t i o n f o r G r a c i l a r i a v e r r u c o s a i n August 1980 ( — • — ) , and 1981 ( - — ) . I n c u b a t i o n time was 15 min f o r n i t r a t e and 10 min f o r ammonium.  30  1  00 o  B  —i—  20  40  "T—  60  10  Desiccation  1—  20 Desiccation  F i g . 20. The r e l a t i o n s h i p between n i t r a t e (A) and ammonium (B) uptake r a t e s (yumol.g d r y w f ' . h - ) and % d e s i c c a t i o n f o r and 1981 i n August 1979 (—A—), Enteromorpha intestinalis  —I—  30  1  (-+--).  00  I* z o E \  1H  e-T  9" •  o E E <  V  20  40 Desiccation  —I  60  20  1  •/.  40 Desiccation  F i g . 21. The r e l a t i o n s h i p between n i t r a t e (A) and ammonium uptake r a t e s Uimol.g d r y w t ^ . h ) and % desiccation G i g a r t i n a p a p i l l a t a i n August 1979 ( — * — ) , and 1981 (—-•---) - 1  I 20  •/.  I— 40 Desiccation  i  20  60  40 Desiccation  F i g . 22. The r e l a t i o n s h i p between n i t r a t e (A) and ammonium (B) uptake r a t e s (pmol.g d r y w t . h ~ ) and % d e s i c c a t i o n f o r Fucus d i s t i c h u s i n August 1 979 ( — * — ) , and 1981 (—•---). _ 1  -r— 60  1  00  14  oi  12  •  z  •  •  y  •  •  • / /  3 E  I  E E <  —i—  —I— 40 Desiccation  20  1'  60  4  I l I I •  20 •A.  r— 40 Desiccation  ult»d 'r* + Vl y , t h i p between n i t r a t e (A) and ammonium SS?SiJ^ s U«nol.g d r y w f ' . h " ' ) and % d e s i c c a t i o n P e l v e t i o p s i s l i m i t a t a i n August 1979 ( A — ) , and 1981 (-->-F  3  e  r a  e  e l a  i o n s  Pel  Fuc  pSoH  Gifl  Em-  Groc  Tidal  Height  (m)  Tidol  Height  Tidal  (m)  Height  F i g . 24. The r e l a t i o n s h i p between i n t e r t i d a l l o c a t i o n (m above Canadian datum) and the % d e s i c c a t i o n p r o d u c i n g t h e maximum enhancement of n i t r a t e uptake r a t e ( A ) , t h e r e l a t i v e degree of enhancement of n i t r a t e uptake r a t e (B),and t h e r a t i o o f n i t r a t e uptake r a t e a t 30% d e s i c c a t i o n and t h e r a t e f o r h y d r a t e d p l a n t s ( C ) , f o r G r a c i l a r i a v e r r u c o s a ( G r a ) , Enteromorpha i n t e s t i n a l i s ( E n t ) , G i g a r t i n a p a p i l l a t a ( G i g ) , Fucus d i s t i c h u s ( F u c ) , and Pelvetiopsis limitata ( P e l ) i n 1979 ( ) 1 980 (-) , and 1981 ( ). E r r o r bars r e p r e s e n t t h e range e s t i m a t e d v i s u a l l y from Figs.12,19-23.  (m)  f  CO  cn  D e s i c c a t i o n at Maximal E n h a n c e m e n t of A m m o n i u m Uptake  1-1  0)  1 c 3 O 3 3 1 o r r t-| 3 O 03 , c a» 3 D ^ cn f-1 PJ  O  a  CD -  cn  3 Co  n h-W ro PJ cn 3 3 • 3 a  C  a g  rt  I-I- —•  -If  PJ PJ •  3-  3><  cn o n3 UD fo  3 PJ M - ?.  Rate  PJ  rt- PJ PJ 00 O 3 * f t C rr -3 r t o nr PJ fD ' O C c Cu r t C D 3 ro Q j -—- CO 0) o — . . < I PJ ro 3 ' O fD M- | 3 h-" rt i - * w • c re 0) Co PJ PJ o C «L Bl 3 ft H rt MCJ I— 3- O ro 3 PJ l_i PJ •< 3 cn QJ 3 tr  f -  Relative  Enhancement  of  Uptake  Rate  4T H ?  1  a cn  i-h  f-t -> O ID t3 3 oo CD —» M  < Da o ro 3 ft rr r t  CO  c •a  -a  oo r t o PJ  Q i tr ?r ro ro ro c CO r t  QJ *0 ro  cn M-  CO  cn  O M  PJ  I-t  In  rt-  N> i-t (a) O  M"  3  • n tr  PJ  7T PJ  o  ro rot 3  PJ  rt  3  o  ro  PJ  rtTD ro i-t  3  Relative  o  <-l  ro i—•  QJ  a  •C  ro  TJ  ro cn ro 3  < ro rt O rt  sr ro  »i  •-«• I-t PJ rt r t voiQ pj ro  ro vo- -  CO  -  at  -  30'/.  Desiccati.  <u  1  h3 O  1  ia n PJ  rt  Rate  I—  > o  "—' I--  PJ  Uptake  8  PJ  1  01  ro  O  PJ  PJ  *  ro r t O ro PJ o 3  -J—l-f 1  i  I"'  rt  tr r t M ro tr o ro 3 i-t  ro 3  t—• pj  PJ  X  PJ  -•—'II  r t M - tr M-3  o  < C <  ro 3 ro  — I ?  98  87  h i g h e r degrees of d e s i c c a t i o n (>30%) i n terms of n i t r o g e n uptake than the mid  t o low  i n t e r t i d a l species.  than those r a t e s f o r h y d r a t e d maintained  Uptake  A r a t i o of n i t r o g e n uptake  r a t e a t 30% t o the uptake r a t e of h y d r a t e d indicates  high  p l a n t s of l e s s than 1  a reduced n i t r o g e n uptake c a p a b i l i t y  f o l l o w i n g a h i g h degree of d e s i c c a t i o n . uptake r a t e s a f t e r  greater  p l a n t s (0% d e s i c c a t i o n ) were o f t e n  a f t e r 30-40% d e s i c c a t i o n .  ( F i g s . 24C,25C)  rates  levels  of  The  maintenance of  desiccation  was  high  directly  r e l a t e d t o i n t e r t i d a l l o c a t i o n ( F i g s . 24C,25C). The  trends  in  uptake  e x p r e s s e d on a dry weight nitrogen  or  wet  At noon on a exposure, the F. and  just  common.  ecologically estimate  was  Nitrogen  day  in  similar  related  when uptake,  to  particulate  uptake v i a water  absorption  1% of the t o t a l u p t a k e . early  July,  after  6  h  of  d i s t i c h u s (Vancouver) averaged 31% d e s i c c a t i o n submersion  Even' on a c l o u d y  These  were  approximately  sunny  before  desiccation.  basis,  weight.  d u r i n g r e h y d r a t i o n was  rates  results  realistic  show value  at  13:30  summer day that and  30% is  they  averaged  20—30% d e s i c c a t i o n desiccation probably  of the % d e s i c c a t i o n on a dry summer  day.  a  is  57% was an  conservative  88  Discussion S u r f a c e water n u t r i e n t distichus summer. spring  populations  analyses  were  only  indicate  nitrogen  that  both  F.  l i m i t e d during the  As t h e p l a n t s became more n i t r o g e n l i m i t e d d u r i n g and  summer,  they  late  may have reached a l e v e l of n i t r o g e n  l i m i t a t i o n which r e s u l t e d i n a d e s i c c a t i o n enhanced uptake r a t e . The  phosphate  overnight, rates.  results  suggest  The  Bamfield  plants  had  weeks,  although  they were not measured.  even  brief  starvation,  even  was s u f f i c i e n t t o induce d e s i c c a t i o n enhanced uptake  several  not  that  enhance  nitrogen  analysis  that  September.  limited for  b u t " phosphate r e s e r v e s may have been low a l s o , In c o n t r a s t ,  t h e ammonium uptakes of F.  though  suggested  been  of  surface  the plants  Ammonium  were  uptake  desiccation d i d  d i s t i c h u s (Vancouver)  nutrient  concentrations  n i t r o g e n l i m i t e d i n J u l y and  rates  of  hydrated  plants  were  g r e a t e r i n J u l y than i n e a r l y September. Incubation  of  winter  F.  d i s t i c h u s (Vancouver) t h a l l i i n  n i t r o g e n f r e e seawater f o r 24 h a l s o d i d not produce d e s i c c a t i o n enhanced ammonium uptake r a t e s . nitrogen  F.  does  accumulate  (Appendix 1 ) . Such a c c u m u l a t i o n s may be s u f f i c i e n t t o  s u s t a i n growth and weeks may e l a p s e (Ryther  distichus  et  a l . 1981).  Either  before several  they weeks  are  depleted  of  nitrogen  l i m i t a t i o n may be r e q u i r e d t o invoke d e s i c c a t i o n enhanced uptake or t h e r e may be conjunction  with  another  environmental  nitrogen  limitation.  phosphate s t a r v a t i o n produced enhanced following starvation  desiccation  i n summer  d i d not produce  factor  which  Twenty-four phosphate  acts  in  hours of  uptake  rates  p l a n t s , but 24 h of n i t r o g e n  enhanced  nitrate  uptake  rates  89  following  desiccation  with  winter  c o n t r o l l i n g factor i s apparently  plants.  related  This  to  the  additional  physiological  s t a t e of the p l a n t i n the summer. Enhanced  uptake  rates  p h y s i o l o g i c a l response period  of  time  to  following frequent  (weeks).  desiccation  drying  over  may an  be  extended  Schonbeck and Norton (1979c) found a  s i m i l a r " h a r d e n i n g " e f f e c t i n terms of d r o u g h t — t o l e r a n c e i n spiralis. after  They  plants  permanently was  not  also  were  found  moved  submerged.  a  to It  "dehardening" e f f e c t 6-8 a  lower  tidal  zone  or  i s p o s s i b l e t h a t the F.  nitrogen  uptake  rates  following  F. weeks kept  distichus  s u f f i c i e n t l y "hardened" t o d r y i n g by e a r l y J u l y t o  enhanced  a  show  desiccation.  If a  h i s t o r y of f r e q u e n t d e s i c c a t i o n i s n e c e s s a r y t o t r i g g e r enhanced nutrient  uptake  seaweeds  following  should  show  greater  f o l l o w i n g d e s i c c a t i o n than results  of  this  desiccation,  study  the  enhancement low  support  i n t e r t i d a l h a b i t a t simply  the  "hardening"  thalli  intertidal  uptake  rates  species.  this prediction.  The  I f the  s e l e c t s f o r t h a l l i w i t h the  the t h a l l i  population  September.  Neither  At w i n t e r  would  high  adaptation  desiccation  rather  t h e r e would have been some adapted  i n the J u l y experiment or a l a r g e p e r c e n t a g e of the  intertidal  have  d i e d between J u l y and  high early  of these p o s s i b i l i t i e s were o b s e r v e d .  t e m p e r a t u r e s (6°C)  i t is  d i s t i c h u s t h a l l i beyond 10% d e s i c c a t i o n . 18°C  of  intertidal  of enhanced n u t r i e n t uptake r a t e s f o l l o w i n g than  high  difficult  to  dry  F.  Winter p l a n t s d r i e d at  t o more than 10% d e s i c c a t i o n showed a r a p i d drop i n n i t r a t e  uptake  rate  and  T h i s response was  in  most c a s e s n i t r a t e was  not due  t o the  temperature  actually  excreted.  increase  because  90  the  hydrated  plants  uptake r a t e s . rapid  I t appears t h a t t h e p l a n t s were  desiccation  winter.  F.  activities  were kept a t 18°C and s t i l l  and  were  distichus  probably  showed  not  adapted  "dehardened" low  results  subsequent  nitrate  of  this  desiccation  be e x c r e t e d under  study  enhanced  suggest nitrogen  and "hardened" t o f r e q u e n t d r y i n g .  reductase  be  exposure  i n F.  nitrogen  limited  T h i s response t o d e s i c c a t i o n  to  nitrogen  i s c o n t i n u a l l y l i m i t i n g growth and moderate  occurs d a i l y . mechanisms  under s t r i c t c o n t r o l , s i n c e  required  by n i t r o g e n  nutrient  i t o c c u r s o n l y when desiccation  Maintenance of t h e p h y s i o l o g i c a l and b i o c h e m i c a l f o r such  e n e r g e t i c a l l y and would o n l y be  desiccation  and  uptake r a t e s  appears  to  the  stress.  that  d i s t i c h u s i n l a t e summer when t h e p l a n t s were  limited  over  to  and h i g h n i t r a t e c o n t e n t i n February (Appendix 2,b),  t h u s n i t r a t e not n i t r i t e c o u l d The  very  showed normal  i s high.  a  response  an  must  advantage  be e x p e n s i v e  when  growth  is  s u p p l y and when t h e p r o b a b i l i t y of f r e q u e n t F.  procurement  d i s t i c h u s appears t o be i n the  rapidly  well  changing  adapted  intertidal  environment. The d i r e c t r e l a t i o n s h i p between i n t e r t i d a l l o c a t i o n and t h e degree of uptake enhancement f o l l o w i n g d e s i c c a t i o n , %  desiccation  desiccation  and  r e l a t i v e t o those  hypothesis  that  desiccation  a r e an  These  f o r uptake,  relationships  enhanced  at  the  0%  nutrient  adaptation  to  uptake  periodic  a r e not e x a c t l y  optimum  rates  desiccation uptake  the  at  30%  support  the  rates  following  exposure  to  l i n e a r , but n e i t h e r  r e l a t i o n s h i p between t o t a l time exposed and i n t e r t i d a l I f t h e degree of enhancement and t h e optimum p e r c e n t  air.  i s the  location. desiccation  91  had  been p l o t t e d a g a i n s t t o t a l hours exposed t o a i r p e r day t h e  t r e n d s may have been more l i n e a r . The  degree  of  d e s i c c a t i o n producing was  a  close  initiation  of  enhancement  enhancement  was  more v a r i a b l e than t h e %  t h e maximum enhanced uptake  r e l a t i o n s h i p between enhanced  uptake,  %  but  was i n f l u e n c e d by o t h e r  rate.  desiccation  might  experience  to  of  similar nitrogen  proximity limitation  because they have been exposed t o v e r y s i m i l a r n i t r o g e n but s p e c i e s r e q u i r e m e n t s f o r n i t r o g e n can v a r y The this  inter-plant v a r i a b i l i t y i n nitrogen  study  determination This  was  high.  of p e r c e n t  parameter  not  clonal,  Analytical desiccation  regimes,  ( K o r n f e l d t 1982). uptake  factors  rates  such  undoubtedly  required the determination  varied for a single plant.  contributed.  of wet w e i g h t , which  g e n e t i c v a r i a b i l i t y was p r o b a b l y  important.  G.  papillata  inter—plant  variability  variability  even  though  as  were Other  i n uptake r a t e s were t h e s u r f a c e  a r e a t o biomass r a t i o s w i t h i n a p o p u l a t i o n and t h e age plant.  in  as t h e  In a d d i t i o n , since the p l a n t s  p o s s i b l e s o u r c e s of v a r i a b i l i t y  given  the  f a c t o r s , such as t h e degree  A l l s p e c i e s growing i n c l o s e  expected  and t h e  t h e magnitude  of n i t r o g e n l i m i t a t i o n . be  There  showed well  as  the greatest greatest  of t h e  degree of  morphological  an attempt was made t o s e l e c t s i m i l a r  plants. The uptake  r e l a t i o n s h i p s between rate,  %  desiccation  t h e degree producing  of  enhancement  maximum enhancement of  uptake and i n t e r t i d a l l o c a t i o n i n d i c a t e d t h a t p l a n t s the i n t e r t i d a l zone were adapted t o h i g h e r and  shorter  periodic  immersion  of  higher  in  l e v e l s of d e s i c c a t i o n  in nutrients.  This  is  92  i l l u s t r a t e d by; a) an i n c r e a s e i n the l e v e l of d e s i c c a t i o n resulted  in  maximum  enhancement  of  nutrient  that  uptake,  b) an  i n c r e a s e i n the degree of enhancement of uptake a t the optimum % d e s i c c a t i o n , and c) maintenance  of h i g h uptake  rates  following  more severe l e v e l s of d e s i c c a t i o n . Nitrogen,  unlike  inorganic  carbon,  from the environment d u r i n g submersion. shown  that  net  photosynthesis  can o n l y be  Previous  obtained  studies  have  i n i n t e r t i d a l algae i s a c t u a l l y  enhanced d u r i n g p e r i o d s of exposure and d e s i c c a t i o n (Johnson al.  1974; Quadir et a l . 1979).  photosynthetic  rates  I t i s p o s s i b l e t h a t the  et  enhanced  observed w i t h d e s i c c a t i o n a r e a r e s u l t of  v a r i a t i o n s i n energy a l l o c a t i o n mediated i n p a r t by the presence or absence of n u t r i e n t s . normally  used  for  As a r e s u l t , d u r i n g  nutrient  uptake  may  by  D'Elia  measurable changes  in this  rates  Work  in  some  the atmosphere desiccated.  macroalgae.  and  DeBoer  ratio  affect  r a t i o during  (1978) the  nitrogen  nevertheless  C/N  homeostasis.  following  but  study  gives  not  The o b s e r v a t i o n only  in  C/N,  uptake may be i m p o r t a n t i n l o n g term that  enhanced  uptake  rates  o c c u r i f the n u t r i e n t i s l i m i t i n g ,  f u r t h e r emphasizes the a d a p t i v e This  uptake  i t appears t h a t enhancement of n u t r i e n t  enhanced  desiccation  that  The c o n t r o l p l a n t s were exposed t o  uptake f o l l o w i n g d e s i c c a t i o n i s not a r e s u l t of changes but  this  indicated  (removed from e x t e r n a l n i t r o g e n s u p p l y ) Therefore  energy  be shunted t o carbon  f i x a t i o n , r e s u l t i n g i n an i n c r e a s e i n the C/N period.  exposure,  s i g n i f i c a n c e of  i n s i g h t i n t o the c o m p l e x i t y  this  response.  of p h y s i o l o g i c a l  c o n t r o l s g o v e r n i n g the i n t e r a c t i o n between e n v i r o n m e n t a l f a c t o r s and p h y s i o l o g i c a l responses such  as  enhanced  nutrient  uptake  93  following desiccation. duration  T h i s uptake response i s dependent  on the  of p e r i o d i c d e s i c c a t i o n , n u t r i t i o n a l s t a t u s and r e c e n t  desiccation  level.  94  Chapter 5.  Adaptations  of  Gracilaria  Procurement i n an I n t e r t i d a l  verrucosa  to  Nutrient  Habitat  Introduction The  p h y s i o l o g i c a l a d a p t a t i o n s of marine macrophytes t o  intertidal  environment  Adaptative  responses  have  recently  in  r a t e (Johnson et a l . 1974;  and  uptake  rate  relationship  (Biebl  Quadir  (Thomas and T u r p i n  have been observed d u r i n g m i l d The  investigated.  osmoregulation  photosynthetic nutrient  been  the  et  1980;  1938), al.  1979)  Chapter  4)  desiccation.  between  nutrient  uptake  and  the  p h y s i o l o g i c a l s t a t e of macrophytes i s i m p o r t a n t i n u n d e r s t a n d i n g algal distribution. nutritional  past  Several history  ( D ' E l i a and DeBoer 1978; Morgan  and  Simpson  s t u d i e s have examined the e f f e c t of on n u t r i e n t uptake r a t e s i n seaweeds  Hanisak and H a r l i n 1978;  1981;  Rosenberg  and  Topinka  Ramus 1982).  i n t e r t i d a l h a b i t a t imposes major changes i n n u t r i e n t well  as  creating  the p h y s i o l o g i c a l  1978; A  supply  o t h e r e n v i r o n m e n t a l . s t r e s s e s t h a t may state  of  seaweeds.  Periodic  high as  affect  exposure  of  p l a n t s i n the i n t e r t i d a l zone r e s u l t s i n a i n t e r m i t t e n t n u t r i e n t supply  and  intertidal  seaweeds  are  presumed  to  be  adapted  physiologically. M i l d d e s i c c a t i o n r e s u l t s i n enhanced n u t r i e n t uptake in  several  mid— t o h i g h — i n t e r t i d a l seaweeds (Chapter 4 ) .  e f f e c t of d e s i c c a t i o n on n i t r o g e n G.  populations  if  this  response  The  uptake i n t h r e e p o p u l a t i o n s  v e r r u c o s a at d i f f e r e n t i n t e r t i d a l l o c a t i o n s was  determine  rates  varied  were chosen because they  within were  studied  a species.  isolated  from  of to  These each  95  other,  yet  different,  geographically distinct  assimilation,  and  intertidal accumulation  i n t e r t i d a l populations. from  one  close  G.  together levels.  and  they  were a t  Morphology,  uptake,  of n i t r o g e n were s t u d i e d i n the  v e r r u c o s a p l a n t s were  transplanted  s i t e t o another a t two d i f f e r e n t i n t e r t i d a l l o c a t i o n s  t o d i f f e r e n t i a t e between t h e e f f e c t s of i n t e r t i d a l l o c a t i o n geographic This  location. study  morphological intertidal  and  i s the  and  habitat  first  physiological are  t o i n v e s t i g a t e whether c e r t a i n adaptations  phenotypic  or  T h i s i s important t o the u n d e r s t a n d i n g i n t e r t i d a l seaweeds and a l g a l z o n a t i o n .  of  to  the  high  genotypic adaptations. the  adaptability  of  96  M a t e r i a l s and Methods  Populations Three  p o p u l a t i o n s of G r a c i l a r i a v e r r u c o s a  were used i n t h i s s t u d y . investigation  (Bird  Their  taxonomy  e t a l . 1982a).  B.C.,  Canada  growing  1.0  m  above  head  ( F i g . 1).  p o p u l a t i o n was i n Wiseman's  Bay  in  Canadian  i s presently  of The  Bamfield other  Bamfield  datum  Inlet  ( F i g . 1).  culture  in  ( F i g . 1)(Lindsay  and  were  anchored  Saunders  1979).  These  and The  from l a r g e  which  Inlet,  intertidal  p o p u l a t i o n was s u b t i d a l and i t was o b t a i n e d bags  under  One p o p u l a t i o n was l o c a t e d  a t 1.8 m above Canadian datum a t t h e Bamfield,  (Huds.) Papenf.  was third  floating  Wiseman's  Bay  cultures  were  s t a r t e d w i t h s u b t i d a l p l a n t s from N u t t a l Bay, Vancouver I s l a n d . The study of these G. three  summers,  verrucosa populations continued  1979-1981.  In August and e a r l y September 1979,  n i t r o g e n uptake s t u d i e s were conducted on a l l t h r e e In June and August 1980, n i t r o g e n uptake out  on  only  the  nitrogen  uptake  reductase  a c t i v i t y were m o n i t o r e d .  rates,  soluble  G.  experiments  carried  I n June and August of nitrogen  in  were  conducted  verrucosa populations.  i n June of 1980 and 1981. distinguish  were  1981,  c o n t e n t and n i t r a t e  Experiments  Transplant intertidal  studies  populations.  two i n t e r t i d a l p o p u l a t i o n s and work w i t h t h e  s u b t i d a l c u l t u r e was d i s c o n t i n u e d .  Transplant  over  with  the  two  T h a l l i were t r a n s p l a n t e d  I n d i v i d u a l p l a n t s were  difficult  to  both i n t e r t i d a l beds where the t h a l l i tended t o  97  be  i n matted clumps, and were t r a n s p l a n t e d i n t h i s s t a t e . In 1980, t h e p r o c e d u r e was as f o l l o w s :  weighing approximately galvanized  chain  1980  which  was anchored a t each end. P l a n t s were  The  1.0  m  verrucosa  bed  a t Wiseman's  Wiseman's  Bay  (1.0 m)  intertidal  locations,  site  These  or  1.8  transplants  changes were r e c o r d e d  to  two  different  were  G.  verrucosa  was  bed  made between June 23 and  weeks  later,  morphological  and uptake e x p e r i m e n t s were conducted.  1981, t h i s t r a n s p l a n t p r o c e d u r e was expanded.  netting  G.  m, a t t h e head of B a m f i e l d  frames (0.5 m by 3 m) were c o n s t r u c t e d stretched  across  of  PVC  Quadrat  piping.  the quadrats.  p l a n t s were t i e d t o each square of t h e n e t w i t h and  t o two  i n the n a t u r a l  transplanted  June 25, 1980. A p p r o x i m a t e l y f i v e  In  (1.8 m)  Bay. P l a n t s growing n a t u r a l l y i n  were 1.0  was  The 1.8 m s i t e was i n t h e n a t u r a l  ( F i g . 26A).  fish  Inlet  i n t e r t i d a l h e i g h t s , 1.0 or 1.8 m, i n Wiseman's Bay i n  ( F i g . 26A).  Inlet.  each  6 g were t i e d w i t h c o t t o n s t r i n g t o a 3 m  t r a n s p l a n t e d from t h e head of B a m f i e l d different  t e n bunches,  Nylon  Clumps of  cotton  string  t h e q u a d r a t s were anchored a t each c o r n e r w i t h s t a k e s .  The  use of q u a d r a t s a l l o w e d more biomass t o be t r a n p l a n t e d . In 1981, p l a n t s were t r a n s p l a n t e d from t h e head of B a m f i e l d I n l e t a t 1.8 t o 1.0 m i n t h e same g e o g r a p h i c a l as  to  two  other  Wiseman's  P l a n t s from t h e n a t u r a l bed (1.0 m)  Bay were t r a n s p l a n t e d h i g h e r up i n t h e i n t e r t i d a l  r e g i o n t o 1.8 m as w e l l as t o 1.8 m and 1.0 m Bamfield June 9.  well  s i t e s i n Wiseman's Bay, one a t 1.0 m and t h e  o t h e r a t 1.8 m ( F i g . 26B). in  l o c a t i o n as  Inlet.  at  t h e head  of  These . t r a n s p l a n t s were made between June 6 and  The p l a n t s were observed e v e r y two weeks.  S i x to eight  98  GraciI a r i a v e r r u c o s a t r a n s p l a n t s  TIDAL  HEIGHT  1.8  1980  WISEMAN'S  M  (A)  BAY  HEAD OF B A M F I E L D — NATURAL  «  INLET  POPULATION  /  1.0  M  NATURAL  Graci l a r i a v e r r u c o s a t r a n s p l a n t s  TIDAL  HEIGHT  1.8  POPULATION  1981  WISEMAN'S  (B)  BAY  HEAD OF B A M F I E L D NATURAL  M  INLET  POPULATION  /  / / /  1.0  M  NATURAL  POPULATION  F i g . 26. A summary of G r a c i l a r i a v e r r u c o s a t r a n s p l a n t s made i n June 1980 (A) and 1981 ( B ) .  99  weeks  later  uptake  experiments  were  conducted,  photographs  taken,  i n t e r n a l s o l u b l e n i t r o g e n e x t r a c t e d and n i t r a t e r e d u c t a s e  a c t i v i t i e s determined.  Nitrogen  Uptake Experiments  Time were  course  conducted  studies  of n i t r a t e and ammonium uptake  (see procedure  outlined  in  Chapter  rates 1).  S i m u l t a n e o u s uptake of n i t r a t e and ammonium was t e s t e d i n medium containing  30  concentrations and  ratio  pM  nitrate  or  jaM ammonium.  15  were used because they a r e s i m i l a r  to  These  i n magnitude  the ammonium and n i t r a t e c o n c e n t r a t i o n s  found i n  the f i e l d d u r i n g p e r i o d s of v e r t i c a l m i x i n g ( w i n t e r and s t o r m s ) . The i n c u b a t i o n chamber was kept a t 16°C under (300 pE.m~ .s~ 2  1  daylight fluorescent  saturating  lighting).  In a second type of uptake experiment t h a l l i were in  a  known  concentration  predetermined  time  Chapter  These  1).  of  the l i m i t i n g  (see p r o c e d u r e incubations  light  incubated  nutrient  f o r "batch"  for a  experiments,  were conducted o u t d o o r s under  saturating natural light  (Hanisak  Plants  f o r 10 or 15 min d u r i n g t h e ammonium o r  were  incubated  and  Harlin  n i t r a t e uptake e x p e r i m e n t s , r e s p e c t i v e l y . and d r i e d t o a c o n s t a n t were  1978) a t  Plants  d r y weight i n a 60°C oven.  were  removed  Uptake r a t e s  e x p r e s s e d on a per gram d r y weight b a s i s and, f o r t h e 1979  e x p e r i m e n t s , on a p e r u n i t p a r t i c u l a t e n i t r o g e n b a s i s uptake  16°C.  rate).  Total  nitrogen  (specific  was determined from p r e v i o u s l y  d r i e d p l a n t m a t e r i a l u s i n g a CHN a n a l y z e r  (Carlo Erba).  Nitrate  and ammonium uptake r a t e s were t e s t e d a t a range o f ammonium and n i t r a t e concentrations  (0-60 pM).  100  The e f f e c t of d e s i c c a t i o n on ammonium rates  was  tested.  desiccation, determined d e a l of  Plants  submersed by  general  dried  i n medium  the method  inter—plant  were  and  described  variability  and  uptake  t o v a r i o u s degrees of uptake  above.  rates  were  There was a g r e a t  i n the uptake  t r e n d s were o b v i o u s c u r v e s were f i t t e d  Desiccation  nitrate  data.  When  visually.  Procedure  The t h a l l i  were d e s i c c a t e d  o u t s i d e under n a t u r a l  conditions  as o u t l i n e d i n Chapter 4.  A n a l y s i s of S o l u b l e N i t r o g e n Complete  thalli  were  Content ground  i n hot  water  n i t r a t e , ammonium, n i n h y d r i n p o s i t i v e m a t e r i a l primarily  amino  acids)  and f r e e p r o t e i n .  to  extract  (assumed  t o be  Plant material  wet wt) was ground w i t h 1 g of washed, i g n i t e d sand hot  distilled  deionized  water  (Appendix  and  (2 g  25  ml  1). . T r i p l i c a t e  e x t r a c t i o n s were conducted t o determine i n t e r — p l a n t v a r i a b i l i t y . E x t r a c t s were s t o r e d a t -10°C. amino  acids  and  free  Nitrate,  protein  were  ammonium, analysed  total  free  as d e s c r i b e d i n  Appendix 1. Boiling determine material  ethanol  extractions  were  also  performed  i f e t h a n o l was a s u p e r i o r e x t r a c t a n t t o water.  to Plant  (1 g) was b o i l e d f o r 10 min i n 80% e t h a n o l and then f o r  10 min i n 95% e t h a n o l . Appendix 1.  E x t r a c t s were a n a l y s e d  as  described  in  101  N i t r a t e Reductase Assay In v i t r o n i t r a t e reductase in  Appendix 2.  a c t i v i t y was assayed as o u t l i n e d  P l a n t m a t e r i a l (2 g) was e x t r a c t e d . w i t h  g) and 25 ml of phosphate b u f f e r . polyvinyl  pyrrolidone  established.  (PVP)  concentration  pH,  MgS0 , 4  for a c t i v i t y  and were  The e f f e c t of i n c u b a t i o n time, e x t r a c t volume, and  NADH, and n i t r a t e c o n c e n t r a t i o n s was t e s t e d f o r both i n t e r t i d a l A  The optimum  sand (1  rectangular  hyperbola  on n i t r a t e  reductase  activity  populations. was not a good f i t f o r the enzyme  m  kinetics.  Therefore,  half-saturation estimates  curves  constants  were  fitted  by  (Km) and the s t a n d a r d  eye.  e r r o r of these  were determined by a l i n e a r r e g r e s s i o n of  V/S a g a i n s t V.  The  a  plot  of  102  Results  Morphology Plants  from  the  t h r e e G.  g r e a t l y i n t h e i r morphology. robust  and  dark  verrucosa populations d i f f e r e d  The  subtidal  plants  very  r e d . The branches were s p a r s e but s t u r d y and  l a r g e i n d i a m e t e r i n comparison t o the i n t e r t i d a l average  were  t h a l l u s weighed a p p r o x i m a t e l y 10 g.  plants.  The  The low i n t e r t i d a l  p l a n t s growing n a t u r a l l y i n Wiseman's Bay (1.0 m) were a l s o dark red  and r e l a t i v e l y  These  plants  l'arge,  were  weighing  profusely  about  5-10  branched,  g  and  feathery  appearance.  H o l d f a s t s were reduced or n o n - e x i s t e n t .  Bay  rock  had  a  and  sand  ( F i g . 27). in  Wiseman's  bottom; i t was s h a l l o w , m o d e r a t e l y  p r o t e c t e d , and l i n e d by t a l l t r e e s which r e s u l t e d i n t h e  plants  r e c e i v i n g reduced l i g h t . The  head  of  B a m f i e l d I n l e t was a m u d f l a t and i t was w e l l  protected.  The s u r r o u n d i n g a r e a s were g r a s s  therefore,  the  mudflat  received  direct  or  marshland  sunlight.  The  and G.  v e r r u c o s a p l a n t s which grew on t h i s m u d f l a t were y e l l o w , and t h e t i p s of t h e branches were o f t e n dark brown. c o n t o r t e d appearance  ( F i g . 28),  these  were  few  branches  were v e r y s m a l l .  Unbroken  g.  no  There  were  were  They had a s t u n t e d ,  sparsely  bent and t a n g l e d .  branched,  Individual  thalli  segments were r a r e l y l a r g e r than  bottom.  The p l a n t s a t the head of B a m f i e l d I n l e t were c o v e r e d epiphytes,  1—2  apparent h o l d f a s t s and t h e p l a n t s formed a  s t i f f c o a r s e mat on t o p of t h e mud and s h e l l  numerous  and  primarily  pennate  v e r r u c o s a bed a t the head of B a m f i e l d  Inlet  diatoms. was  The  with G.  approximately  103  F i g . 27. Morphology of low i n t e r t i d a l G r a c i l a r i a verrucosa growing n a t u r a l l y i n Wiseman's Bay, B a m f i e l d , B.C., Canada, i n August 1981.  104  F i g . 28. Morphology of h i g h i n t e r t i d a l G r a c i l a r i a verrucosa growing n a t u r a l l y a t the head of B a m f i e l d I n l e t , B a m f i e l d , B.C., Canada, i n August 1981.  105  the  same  s i z e and d e n s i t y i n August as i t was  d i f f i c u l t t o d i s t i n g u i s h new  were  verrucosa (Fig.  few  losses  thalli  29).  By  transplanting due  underwent early  was  plants  definite  August  of  (Fig. and  t o the head of B a m f i e l d growing  29A).  naturally  The  very  t o wave a c t i o n .  at  Transplanted  1980,  the morphology of from  I n l e t (1.8 m) was the  head  of  have  fewer  the  Wiseman's  Bay  s i m i l a r t o the Bamfield  They tended t o be l a r g e r than the  r e s i d e n t p l a n t s and appeared t o  G.  changes  p l a n t s were y e l l o w , t w i s t e d , s p a r s e l y  very c o a r s e .  was  effective.  morphological  p l a n t s which had been t r a n s p l a n t e d i n June (1.0 m)  It  growth.  The quadrat method of There  i n June.  Inlet  branched surrounding  epiphytes.  These  p l a n t s i n c r e a s e d i n s i z e a f t e r they were t r a n s p l a n t e d . The m)  plants  t o the low  t h a t were t r a n s p l a n t e d from Wiseman's Bay  i n t e r t i d a l s i t e a t the head of B a m f i e l d  m) d i d not s u r v i v e the f i v e week o b s e r v a t i o n s i t e was at  0.8  led to excessive  Inlet.  morphologically  dark r e d , v e r y s o f t and  I t s l o c a t i o n i n an e e l g r a s s  in  five  feathery.  p l a n t s t r a n s p l a n t e d from the to  survived well.  bed bed  Bamfield  weeks.  The  (1.0 thalli  In g e n e r a l , they were  r e s i d e n t Wiseman's Bay p l a n t s , except  they were s m a l l e r and c o v e r e d w i t h more  m)  new  epiphytism.  s i m i l a r t o the s u r r o u n d i n g  (1.8  The  period.  t o the lower i n t e r t i d a l s i t e i n Wiseman's Bay  m) a l s o changed  The  (1.0  p l a n t s t h a t were t r a n s p l a n t e d from the head of  I n l e t (1.8 m)  became  Inlet  m lower i n the i n t e r t i d a l zone than the n a t u r a l  the head of B a m f i e l d  The  (1.0  the upper i n t e r t i d a l s i t e  epiphytes. head  of  (1.8 m)  Bamfield  Inlet  i n Wiseman's Bay  They became b r i g h t r e d , very l a r g e ( g r e a t e r than  106 GraciI a r i a  TIDAL  verrucosa morphology  HEIGHT  1.8 M  1980  WISEMAN'S  (A)  BAY  HEAD  l a r g e , red — p r o f u s e l y branched  OF B A M F I E L D  INLET  NATURAL POPULATION small, yellow, twisted medium s i z e d , ye I low, t w i s t e d  large, red, p r o f u s e l y branched  1.0  M  GraciI a r i a  TIDAL  v e r r u c o s a morphology  HEIGHT  1.8  M  M  1981  WISEMAN'S  red,  (B)  BAY  HEAD  medium s i z e d , red p r o f u s e l y branched large,  1.0  very s m a l l , dark fragile ( a f t e r 3 weeks)  NATURAL POPULATION , ( l a r g e , r e d , p r o f u s e l y branched)  red, profusely branched  large, red, profuse Iy branched NATURAL POPULATION ( l a r g e , red p r o f u s e l y branched)  ' /  OF  BAMFIELD  INLET  . NATURAL POPULATION small, yellow, twisted  med i um s i z e d , ye I Iow, tw i sted  1  I  very s m a l l , dark r e d ,  fragile  very s m a l l , dark fragile  red,  F i g . 29. A summary of G r a c i l a r i a v e r r u c o s a morphology f o r v a r i o u s t r a n s p l a n t s i n August 1980 (A) and 1981 ( B ) .  107  10 g wet w e i g h t ) , ( F i g . 29A). G.  profusely  branched  and  had  These r o b u s t p l a n t s were growing  v e r r u c o s a bed i n an a r e a t h a t was  few  epiphytes  above the n a t u r a l  d e v o i d of any major  plant  growth. In  February  1981,  the s u r f a c e water of B a m f i e l d I n l e t  d e p l e t e d of n i t r a t e and ammonium. (L. D r u e h l , Wiseman's  p e r s . comm.). Bay  previous  was  The  T h i s was natural  approximately  e a r l i e r than G.  one-third  smaller  the h i g h i n t e r t i d a l f e d a t the head of B a m f i e l d Transplant  experiments  in  m o r p h o l o g i c a l changes t o those of 1980  1981  of  Bamfield  Inlet  at  1.8  m  The  were  m) t o the head of B a m f i e l d I n l e t a t 1.0 m,  growth  was  intertidal m  were  apparent.  and The  were thalli  not  similar  (1.0 m)  to  the  y e l l o w , medium, and verrucosa p l a n t s . brown t i p s .  The p l a n t s t h a t were t r a n s p l a n t e d from Wiseman's  epiphytes  was  ( F i g . 29B).  Branches were s h o r t , c o n v o l u t e d and possessed  numerous  in  s i z e of  produced  t w i s t e d , but more r o b u s t than the r e s i d e n t G.  had  than  Inlet  The p l a n t s t r a n s p l a n t e d from Wiseman's Bay head  normal  v e r r u c o s a bed i n  y e a r s , and e p i p h y t i s m was more e x t e n s i v e .  altered.  was  Bay  (1.0  remained dark red but  partially  decayed.  transplanted  No  new  down  the  zone from 1.8 m a t the head of B a m f i e l d I n l e t t o  also  decayed.  Branches  were  reduced  in  1.0  size  and  fragmented e a s i l y . P l a n t s t r a n s p l a n t e d from the head of B a m f i e l d I n l e t to  the  natural  bed  in  Wiseman's  r e l a t i v e l y r o b u s t , and p r o f u s e l y  Bay  branched.  (1.0 In  m)  (1.8  m)  became r e d ,  general,  they  the head of B a m f i e l d  Inlet  were not as l a r g e as the r e s i d e n t p l a n t s . The  plants  transplanted  from  108  (1.8 m)  t o Wiseman's Bay  medium  sized,  red,  h i g h i n the  with  few  intertidal  epiphytes,  robust w i t h long s t r a i g h t branches. torn  away.  The  Wiseman's Bay possessed transplanted  m)  and were of  the  were  relatively  plants  were  p l a n t s which were moved up i n the i n t e r t i d a l  from 1.0  few  Some  (1.8  to  1.8  epiphytes.  m  were  Most  large,  of  the  dark  plants  red,  in and  that  were  remained i n t a c t .  N i t r a t e Uptake The  r e l a t i o n s h i p between d e s i c c a t i o n and n i t r a t e uptake  at  the v a r i o u s i n t e r t i d a l l o c a t i o n s i s p r e s e n t e d i n F i g u r e 30.  The  high  not  i n t e r t i d a l population  showed a response t o d e s i c c a t i o n  apparent i n the other p o p u l a t i o n s ; potential  nitrate  uptake  rate  depending on the year ( F i g . 30). subtidal  population  10% d e s i c c a t i o n and greater  was  was no  10—15%  desiccation  was  enhanced one  On  the  other  The  intermediate  the  t o f i v e times hand,  in  the  uptake dropped t o z e r o a f t e r o n l y  n i t r a t e , i n s t e a d of n i t r i t e was  desiccation.  population There  nitrate  at  response  of  the  between the o t h e r  excreted  low two  at  intertidal populations.  enhancement of uptake r a t e f o l l o w i n g d e s i c c a t i o n  but p o s i t i v e n i t r a t e uptake r a t e s  were  maintained  up  to  and  beyond 30% d e s i c c a t i o n . There  was  enhancement of n i t r a t e uptake a f t e r d e s i c c a t i o n  i n the h i g h i n t e r t i d a l p l a n t s d u r i n g l a t e summer not  i n June ( F i g . 31).  from  In June 1981,  both i n t e r t i d a l p o p u l a t i o n s  degrees of d e s i c c a t i o n than Uptake  rates  similar  to  in those  ( F i g . 30)  but  the uptake r a t e s of  plants  were l e s s i n f l u e n c e d by  higher  the for  previous  year  f u l l y hydrated  ( F i g . 31). (control)  F i g . 30. The r e l a t i o n s h i p between n i t r a t e uptake r a t e s (pmol.g dry w t " . h " ) and % d e s i c c a t i o n i n the l a t e summer of 1979 ( A ) , 1980 ( B ) , and 1981 ( C ) , f o r t h e n a t u r a l p o p u l a t i o n s of h i q h i n t e r t i d a l (---•-:—), low i n t e r t i d a l ( — • — ) , and s u b t i d a l ( — A - - - ) G r a c i l a r i a verrucosa. 1  1  o  A  B  1  1  r  10  20 Desiccation  30  —i  1  10  20 Desiccation  F i g . 31. The r e l a t i o n s h i p between n i t r a t e uptake r a t e s (pmol.g dry w f ' . h " ' ) and % d e s i c c a t i o n i n June of 1980 ( A ) , and' 1981 (B) f o r the n a t u r a l p o p u l a t i o n s of h i g h i n t e r t i d a l (—•---). and low i n t e r t i d a l ( — • — ) G r a c i l a r i a v e r r u c o s a .  1—  30  111  p l a n t s were m a i n t a i n e d  up t o 15% d e s i c c a t i o n .  The uptake r a t e s of t h e c o n t r o l p l a n t s d u r i n g v a r i e d from year t o y e a r .  late  summer  N i t r a t e uptake r a t e s were very low i n  1979 i n comparison t o 1980 and 1981 ( F i g . 3 0 ) . I n 1980 and 1981 the  nitrate  uptake  rates  of  much  lower  than  p o p u l a t i o n were  i n t e r t i d a l population.  the n a t u r a l those  of  high  intertidal  the n a t u r a l  low  The n i t r a t e uptake r a t e s of t h e h y d r a t e d  ( c o n t r o l ) p l a n t s were s i m i l a r f o r both i n t e r t i d a l p o p u l a t i o n s i n June 1980 and 1981 ( F i g s . 3 1 ) . The  plants  head of B a m f i e l d  t r a n s p l a n t e d from Wiseman's Bay (1.0 m) t o t h e I n l e t (1.8 m) responded t o m i l d d e s i c c a t i o n  in  a manner s i m i l a r t o t h e p l a n t s growing n a t u r a l l y a t 1.8 m a t t h e head  of  Bamfield  Inlet.  uptake f o r t h e s e p l a n t s respectively  The r e l a t i v e enhancement of n i t r a t e  was  (Fig. 32).  1.5  and  4,  in  1980  and 1981,  Their tolerance t o higher d e s i c c a t i o n  (20-60%) was l e s s than t h a t of t h e r e s i d e n t p l a n t s a t of  Bamfield  Inlet.  t h e head  The uptake r a t e s of t h e r e s i d e n t p l a n t s a t  30% d e s i c c a t i o n were s i m i l a r t o t h e uptake r a t e s of t h e h y d r a t e d ( c o n t r o l ) p l a n t s , but t h e uptake r a t e of t h e t r a n s p l a n t e d  plants  was much l e s s a t 30% d e s i c c a t i o n than a t 0%. The Bamfield  plants Inlet  which  tolerance  d e s i c c a t i o n were plants. Wiseman's  from  t h e head  of  an enhanced n i t r a t e uptake i n response t o  ( F i g . 32A). to  transplanted  (1.8 m) t o t h e h i g h i n t e r t i d a l s i t e i n Wiseman's  Bay (1.8 m) m a i n t a i n e d desiccation  were  higher equal  These  plants  desiccation. to  those  of  also Uptake  retained rates  the hydrated  their a t 30%  (control)  The p l a n t s t r a n s p l a n t e d t o t h e lower i n t e r t i d a l s i t e i n Bay  (1.0 m) showed no enhancement of n i t r a t e uptake  1 12  1980 (A) TIDAL  HEIGHT  1.8  WISEMAN'S  M  23.0  X  HEAD OF  BAY  BAMFIELD  NATURAL  1.2 5.0  X 2.0  POPULATION [9.0  7 7.0  1.0  M.  NATURAL  X  1.2]  " X  1.5  POPULATION  26.0  INLET  [7.0  X  1.5]  did not survive  [12.0]  1981 (B) TIDAL  HEIGHT  1.8  M  WISEMAN'S 6.0 14.5  X 2 X 2  BAY  [23.0] [30.8]  HEAD OF m  ^ |1.6  BAMFIELD  NATURAL X 5.0  INLET  POPULATION [14.0  X  1.2]  4 . Oj [ 1 0 . 0  X  2.0]  1 1.0  M  NATURAL 10.2  POPULATION  2.1 4.5  [28.5] [16.0]  F i g . 32. A summary of h y d r a t e d n i t r a t e and ammonium uptake r a t e s (pmol.g d r y wf'.h"' ) f o r G r a c i l a r i a v e r r u c o s a i n August 1980 ( A ) , and 1981 ( B ) . Square b r a c k e t s f o r ammonium. X i n d i c a t e s enhanced n i t r a t e uptake r a t e s t h a t o c c u r r e d a t the o p t i m a l % d e s i c c a t i o n . The numbers t o the r i g h t of the x g i v e s the r e l a t i v e degree of enhancement. The arrows i n d i c a t e t h e o r i g i n of the t r a n s p l a n t e d p o p u l a t i o n s . 1  1 13  following mild their  desiccation.  tolerance  to  higher  However,  these  desiccation:  plants  plants  retained  d r i e d t o 30%  d e s i c c a t i o n showed o n l y s l i g h t l y lower n i t r a t e uptake r a t e s than the h y d r a t e d  plants.  The p l a n t s t r a n s p l a n t e d from 1.0 m i n Wiseman's Bay t o m a t the head of B a m f i e l d uptake  after  I n l e t showed no enhancement of n i t r a t e  desiccation  in  1981  ( F i g . 32B).  In 1981, the  p l a n t s which were moved down the i n t e r t i d a l zone a t the head Bamfield  Inlet  from  1.8  to  1.0  m  of  showed no enhancement of  n i t r o g e n uptake a f t e r d e s i c c a t i o n ( F i g . 32B). the  1.0  Transplanting  up  i n t e r t i d a l zone i n Wiseman's Bay from 1.0 t o 1.8 m r e s u l t e d  i n a two f o l d enhancement i n n i t r a t e uptake a f t e r d e s i c c a t i o n . The n i t r a t e uptake r a t e s of e i t h e r t r a n s p l a n t e d or r e s i d e n t p l a n t s growing i n Wiseman's Bay were 3-5 t i m e s g r e a t e r than  the  plants  The  growing  at  the  head of B a m f i e l d  Inlet  ( F i g . 32).  n i t r a t e uptake r a t e s of the p l a n t s t r a n s p l a n t e d Bay t o the n a t u r a l bed a t the head of B a m f i e l d than  the  uptake  rates  from  I n l e t were h i g h e r  of the r e s i d e n t p l a n t s ( F i g . 3 2 ) .  n i t r a t e uptake r a t e s of the t h a l l i t r a n s p l a n t e d Bay  to  the low i n t e r t i d a l s i t e  (1.8  m)  to  1.8  m  in  from  14.5  intertidal  head  zone  in  the n i t r a t e uptake r a t e s of h y d r a t e d pmol.g  dry  wt" .h" (Fig. 1  1  32B).  zone a t the head of B a m f i e l d  on n i t r a t e uptake r a t e s .  The  Wiseman's  of  Bamfield Bamfield  intermediate  ( F i g . 32).  In 1981, moving up the i n t e r t i d a l  to  the  Wiseman's Bay were  between the two n a t u r a l p o p u l a t i o n s  increased  from  (1.0 m) a t the head of  I n l e t and the p l a n t s t r a n s p l a n t e d Inlet  Wiseman's  Wiseman's  Bay  p l a n t s from  10.2  Moving  down  the  I n l e t had l i t t l e e f f e c t  B 30H  1*  Desiccation  *r.  Desiccation  F i g . 33. The r e l a t i o n s h i p between ammonium'uptake r a t e s (jamol.g dry w t . h ) and % d e s i c c a t i o n i n t h e l a t e summer of 1979 ( A ) , 1980 ( B ) , and 1981 ( C ) , f o r the n a t u r a l p o p u l a t i o n s of h i g h intertidal (--•--), and low i n t e r t i d a l (—•—) Gracilaria verrucosa. _ 1  _ 1  115  Ammonium Uptake In l a t e summer t h e h i g h slightly  enhanced  (10-15%).  intertidal  ammonium  uptake  G.  verrucosa  showed  following mild desiccation  T h i s enhancement was n o t as pronounced as i t was w i t h  n i t r a t e ( F i g . 3 3 ) . The low i n t e r t i d a l G. enhancement  of  ammonium  plants  a c t u a l l y excreted  in  showed  uptake f o l l o w i n g d e s i c c a t i o n .  decreased r a p i d l y a f t e r d e s i c c a t i o n intertidal  verrucosa  1979  i n the s u b t i d a l  ( F i g . 33A).  The  no  Uptake and low  subtidal plants  n i t r a t e when d e s i c c a t i o n was g r e a t e r  than  10%  of  intertidal  ( F i g . 30A). Enhancement  ammonium  uptake  i n the high  p l a n t s was more i n c o n s i s t e n t i n June ( F i g . 3 4 ) . Enhancement was observed i n 1981, but not i n 1980. The showed  no  enhancement  of  ammonium  low  intertidal  uptake  plants  after desiccation  (Fig. 34). In June, t h e ammonium uptake r a t e s f o r t h e h y d r a t e d and  low i n t e r t i d a l G.  verrucosa  were very  s i m i l a r , . b u t t h i s was  not t r u e l a t e r i n t h e summer ( F i g s . 33,34). plants  high  The.high  intertidal  had lower uptake r a t e s than t h e low i n t e r t i d a l p l a n t s i n  1980 and 1981 ( F i g . 3 3 ) . I n 1979, t h e ammonium uptake r a t e f o r the  hydrated  high  intertidal  p l a n t s was much g r e a t e r than i n  1980 and 1981 and g r e a t e r than t h e uptake r a t e s of t h e low  intertidal  material  due  determination  plants to of  wave  (Fig. 33). action  In and  the r e l a t i o n s h i p  hydrated  1980, l o s s  of  epiphytism  prevented  between  plant  uptake r a t e and %  d e s i c c a t i o n f o r t h e p l a n t s t r a n s p l a n t e d t o 1.0 m a t t h e head Bamfield  Inlet  Ammonium  uptake  and  to  rates  1.8 were  m  i n Wiseman's  higher  in  1981  of  Bay ( F i g . 3 2 A ) . than  in  1980  F i g . 34. The r e l a t i o n s h i p between ammonium uptake r a t e s (ptnol.g dry w f ' . h ) and % d e s i c c a t i o n i n June of 1980 (A) and 1981 (B) f o r the n a t u r a l p o p u l a t i o n s of h i g h (-—•---) and low (—• ) i n t e r t i d a l G r a c i l a r i a verrucosa. - 1  117  (Fig. 32).  The p l a n t s t r a n s p l a n t e d down t h e i n t e r t i d a l zone a t  the head of B a m f i e l d  I n l e t showed an i n c r e a s e i n ammonium uptake  r a t e ( F i g . 32B). In 1980 and 1981, t h e r e s i d e n t Bamfield bed  plants  at  t h e head  I n l e t as w e l l as t h e p l a n t s t r a n s p l a n t e d t o t h e n a t u r a l  (1.8 m) a t t h e head of B a m f i e l d  I n l e t showed enhancement of  ammonium uptake f o l l o w i n g m i l d d e s i c c a t i o n .  The degree of  enhancement f o r the n a t u r a l and t r a n s p l a n t e d p o p u l a t i o n s was  1.2  and  1.5 and i n 1981 i t was 1.2 and 2.0, r e s p e c t i v e l y .  g r e a t e r than i n t h e r e s i d e n t p l a n t s .  f o r n i t r a t e uptake where t h e % nitrate  uptake  rates  less  (5-10%).  slightly  this  i n 1980  The enhancement of ammonium uptake i n t h e t r a n s p l a n t e d was  of  The o p p o s i t e was t r u e  desiccation  producing  i n the transplanted The  plants  ammonium  thalli  uptake  t r a n s p l a n t e d or r e s i d e n t h y d r a t e d p l a n t s were s i m i l a r  maximum was  also  rates  for  ( F i g . 32).  In 1980 and 1981, t h e r e s i d e n t p l a n t s i n Wiseman's Bay (1.0 m)  showed  no  enhancement  of  ammonium  uptake  after  mild  d e s i c c a t i o n ( F i g . 3 2 ) . I n 1980, t h i s p o p u l a t i o n showed a s l i g h t tolerance t o higher equal  to  desiccation  the hydrated  than  half  uptake  rates  i n Wiseman's Bay as  well  as  In a l l  t r a n s p l a n t e d t o t h e low i n t e r t i d a l s i t e i n Wiseman's  Bay o r t h e head of B a m f i e l d desiccation.  maintaining  uptake r a t e s a t ^ 30% d e s i c c a t i o n .  1981, t h i s n a t u r a l p o p u l a t i o n populations  by  I n l e t , had a low t o l e r a n c e t o h i g h e r  At 30% d e s i c c a t i o n ammonium uptake r a t e s were l e s s the rates  at  0%  desiccation.  i n t e r t i d a l populations  showed  after  nor d i d t h e p l a n t s  desiccation,  enhancement  None of t h e s e low of  ammonium  uptake  transplanted  from  Wiseman's Bay o r from 1.8 t o 1.0 m a t t h e head of B a m f i e l d  Inlet  118  (Fig. 32). The two t r a n s p l a n t s  at  1.8  m  i n Wiseman's  Bay  showed  enhanced n i t r a t e uptake a f t e r d e s i c c a t i o n , but no enhancement of ammonium uptake ( F i g . 32B). A l t h o u g h no enhancement of ammonium uptake  was  Bamfield  found,  the p l a n t s  I n l e t t o 1.8  tolerance  m  transplanted  i n Wiseman's  Bay  from t h e head of showed  a  t o d e s i c c a t i o n than t h e low i n t e r t i d a l G.  p l a n t s ; ammonium uptake r a t e s o n l y  decreased  by  greater verrucosa  approximately  o n e - f i f t h when p l a n t s were d e s i c c a t e d t o 30%.  Time Courses o f N i t r o g e n Time  course  Uptake  studies  of ammonium and n i t r a t e uptake r a t e s  were conducted i n June and August f o r both beds  of  G.  Chapter 1. first low  verrucosa.  The  The r a t e of n i t r a t e  data  natural  are presented  uptake  was  incubation  on  i n t e r t i d a l G. intertidal  verrucosa  30 yuM  nitrate.  verrucosa  plants  was  stimulated  f o r the  N i t r a t e uptake i n after  15  min o f  Ammonium uptake r a t e s i n h i g h  decreased a f t e r  frequently  i n T a b l e 1,  constant  30 min of i n c u b a t i o n i n 30 /uM n i t r a t e .  i n t e r t i d a l G.  intertidal  maintained  10—12 min. a  constant  The low ammonium  uptake r a t e f o r a l o n g e r p e r i o d of t i m e , o f t e n up t o 30 min. When exposed t o both n i t r a t e and ammonium, both G.  verrucosa  simultaneously decrease  populations (Table  took  up  nitrate  and  1 ) . The h i g h i n t e r t i d a l p l a n t s  intertidal ammonium showed  a  (50%) i n n i t r a t e uptake i n t h e presence of ammonium.  The presence of n i t r a t e s t i m u l a t e d ammonium uptake by 40% i n t h e low  i n t e r t i d a l G.  verrucosa.  119  N i t r o g e n Uptake Nitrate  Kinetics  uptake i n t h e low i n t e r t i d a l G.  verrucosa  appeared  t o have a h y p e r b o l i c component a t low n i t r a t e c o n c e n t r a t i o n s and a l i n e a r component a t n i t r a t e c o n c e n t r a t i o n s between 15  and  50  pM ( F i g . 35A). The k i n e t i c s curve a t low n i t r a t e c o n c e n t r a t i o n s suggests  that  (possibly  diffusion)  rates  t h e Ks  (D'Elia  active  intertidal  DeBoer  component.  G.  approximately  1 pM i f t h e l i n e a r  component was s u b s t r a c t e d from  and  uptake  was  1978) t o g i v e an e s t i m a t i o n of t h e Nitrate  verrucosa  t h e uptake  uptake  followed  rates  saturable  in  high  kinetics  when  p l o t t e d as a f u n c t i o n of n i t r a t e c o n c e n t r a t i o n ( F i g . 35B). Vmax uM.  was  approximately  Nitrate  intertidal  uptake plants  4 pmol.g d r y w t . h " _ 1  rates were  at  30  uM  1  The  and t h e Ks was 6  nitrate,  f o r t h e low  t w i c e t h e r a t e s of the h i g h  intertidal  plants. There were a l s o d i f f e r e n c e s i n t h e ammonium uptake between t h e two i n t e r t i d a l rates  f o r t h e low  approximately  populations.  intertidal  G.  The  kinetics  ammonium  uptake  v e r r u c o s a p o p u l a t i o n were  p r o p o r t i o n a l t o t h e ambient ammonium c o n c e n t r a t i o n  up t o 40 pM ( F i g . 36A). Ammonium uptake k i n e t i c s f o r t h e intertidal fit  a  G.  verrucosa  rectangular  approximately  high  p o p u l a t i o n was s a t u r a b l e but d i d not  hyperbola  ( F i g . 36B).  30 pmol.g d r y w t ~ . h ~ 1  1  The  Vmax  was  and t h e Ks was about 10 uM.  Ammonium uptake r a t e s a t ammonium c o n c e n t r a t i o n s l e s s than 20 uM were s i m i l a r f o r both p o p u l a t i o n s .  S o l u b l e N i t r o g e n Content In June, both i n t e r t i d a l p o p u l a t i o n s of G.  v e r r u c o s a had a  120  s e S 4  8  , o  6  ,  ,  10  20 NO"  -I  1  30  40  UM)  F i g . 35. The r e l a t i o n s h i p between n i t r a t e uptake r a t e s (/jmol.g d r y w f ' . h ) and n i t r a t e c o n c e n t r a t i o n (pM) f o r the n a t u r a l p o p u l a t i o n s of low ( — • — ) ( A ) , and h i g h (--o---)(B) i n t e r t i d a l G r a c i l a r i a v e r r u c o s a , i n August 1981. Uptake r a t e s minus the n o n - s a t u r a b l e component ( ) were d e t e r m i n e d t o a s s e s s t h e a c t i v e uptake component. - 1  121  20  40  60 NH*  (/iM  80  )  F i g . 36. Ammonium uptake k i n e t i c s (umol.g d r y w f . h n a t u r a l p o p u l a t i o n s of low ( — • — ) ( A ) , and h i g h i n t e r t i d a l G r a c i l a r i a verrucosa. 1  _ 1  ) f o r the f—o—)(B)  122  very  low  n i t r a t e content, approximately  0.02 % of t h e t o t a l precise  nitrogen  0.04 pmol.g wet w t " o r 1  i n the plant  (Table  3).  The  amounts of ammonium, amino a c i d s , and p r o t e i n e x t r a c t e d  were not determined due t o t h e crude  nature  of  the  extracts  which may c o n t a i n compounds t h a t i n t e r f e r e w i t h t h e c o l o r i m e t r i c a n a l y s i s (Appendix 1 ) . N e v e r t h e l e s s ,  g e n e r a l t r e n d s r a t h e r than  absolute  The h i g h i n t e r t i d a l  amounts  can be d i s c u s s e d .  a l s o had a low ammonium content  plants  i n June w h i l e t h e low i n t e r t i d a l  p l a n t s had an ammonium c o n t e n t  s i m i l a r t o those e x t r a c t e d  later  i n t h e summer. In  August,  head of B a m f i e l d wet  a l l n a t u r a l or t r a n s p l a n t e d p o p u l a t i o n s a t t h e I n l e t had a n i t r a t e c o n t e n t  w t " or a p p r o x i m a t e l y  0.14 % of t h e t o t a l  1  plant  (Fig. 37).  1  wt"  1  from  2.0  i n the high i n t e r t i d a l s i t e t o approximately or  0.5  %  of  intertidal site. had  nitrogen  wet  4.0 yumol.g wet  The lower i n t e r t i d a l p l a n t s i n Wiseman's  the same l o c a t i o n .  Bay  than t h e h i g h i n t e r t i d a l p l a n t s a t  There was l i t t l e v a r i a t i o n i n t h e ammonium  (Fig. 37).  Grinding  in  ninhydrin positive acids)  /umol.g  t h e t o t a l n i t r o g e n i n t h e p l a n t i n t h e low  a higher n i t r a t e content  content  i n the  T h i s was much lower than t h a t e x t r a c t e d from  the p l a n t s i n Wiseman's Bay which ranged wt"  of about 0.5 pmol.g  than  amino  from  water  material  boiling  showed a h i g h e r transplanted  hot  extracted  (assumed  i n ethanol acid  t h e head  Wiseman's Bay, and both p o p u l a t i o n s head o f B a m f i e l d  Inlet (Fig. 37).  be  They  Bamfield  amounts  primarily  ( F i g . 3 7 ) . Three  content. of  to  greater  were Inlet  of  amino  populations the to  thalli  1.8 m i n  t r a n s p l a n t e d t o 1.0 m a t t h e  T a b l e 3. S o l u b l e n i t r a t e , ammonium, amino a c i d s and p r o t e i n c o n t e n t i n the n a t u r a l p o p u l a t i o n s of h i g h and low i n t e r t i d a l G r a c i l a r i a v e r r u c o s a i n June and August 1981, ± 1 s t a n d a r d d e v i a t i o n , n=3.  S o l u b l e nitrogen content (u.mol.g wet w t ) , % t o t a l n i t r o g e n - 1  Amino Acids Population  High  Intertidal  Low I n t e r t i d a l  Time  Nitrate  %  Ammonium  % Water  %  Ethanol  %  protein mg.g wet wt"  1  June August  0.4+0.14 0.57+0.18  0.2 0.14  0.82+0.22 2.23+0.12  0.14 0.56  6.85±0.85  2.6  4.09  1.5  7.7  25  June August  0.03+0.02 4.41+1.30  0.003 0.5  2.27+0.72 4.94±0.95  0.38 1.65  5.47±1.3  2.7  0.0  0.0  9.5  45  ro to  124 Nitrate  TIDAL  and  [Ammonium] P o o l s  HEIGHT  1.8  M  ( /omol • g wet wt  WISEMAN S BAY 2.15 1.38  + 0.15 ± 0.16  [3.39]  )  HEAD OF B A M F I E L D •  3 . 2 1 ± 6V8n  NATURAL 0.57  23  3.47  1.0  M  ± 0.25 NATURAL  4.41  Amino A c i d TIDAL  Pools  (jimol  1.8  8.93  6.75  1.0  M  + 0.12]  + 0.07 [1.76 ± 0.26]  0.23]  POPULATION  )  0.61 0.37  + 0.17 [2.85 + 0.18] + 0.16 [ 3 . 1 3 + 0.50]  e x t r a c t e d by g r i n d i n q i n b o i l i n g [ ] e x t r a c t e d by b o i l i n g i n EtOH  WISEMAN'S BAY  M  POPULATION  ± 0.18[2.23  + 1.3 [ 4 . 9 4 ± 0 . 9 5 ]  g wet wt  HEIGHT  [2.98 +  INLET  12.4 + 1.81  [11.4] [0.0]  HEAD OF B A M F I E L D  water INLET  «  ± 0.79  NATURAL POPULATION 5.47 + 1.28 [ 0 . 0 ]  17.3 16.0  ± 2.6 [0.18] [4.98]  F i g . 37. S o l u b l e n i t r a t e , ammonium, and amino a c i d c o n t e n t (jjmol.g wet wt"' ) of G r a c i l a r i a v e r r u c o s a i n August 1981, ± one s t a n d a r d d e v i a t i o n , n=3. The arrows i n d i c a t e the o r i g i n o f t h e transplanted populations. 1  125  The Wiseman's Bay n a t u r a l p o p u l a t i o n had a protein (Fig.  c o n t e n t than t h e o t h e r G.  38).  A l l the populations  higher  soluble  verrucosa populations tested  transplanted  t o t h e head  of  B a m f i e l d I n l e t had low s o l u b l e p r o t e i n c o n t e n t .  N i t r a t e Reductase A c t i v i t y A  comparison  of  the n i t r a t e  suggests t h a t t h e two n a t u r a l G. same n i t r a t e r e d u c t a s e enzyme.  reductase  characteristics  v e r r u c o s a p o p u l a t i o n s have t h e Nitrite  production  was  linear  *  w i t h time and a c t i v i t y was p r o p o r t i o n a l t o t h e volume of e x t r a c t used ( F i g . 39). Mg  ++  The n i t r a t e r e d u c t a s e enzyme was i n s e n s i t i v e t o  c o n c e n t r a t i o n ( F i g . 40), and pH change ( F i g . 41).  relationship  between  PVP  concentration  a c t i v i t y was d e t e c t e d ( F i g . 42).  Nitrate  and n i t r a t e  No c l e a r reductase  and NADH s u p p l y had a  s i m i l a r e f f e c t on t h e n i t r a t e r e d u c t a s e a c t i v i t y of both n a t u r a l i n t e r t i d a l p o p u l a t i o n s ( F i g s . 43,44).  The Km v a l u e s f o r n i t r a t e  and NADH were 70 ± 7.0 and 75 ± 46 /utM, In  June,  t h e Vmax  respectively.  f o r t h e n i t r a t e r e d u c t a s e a c t i v i t y of  both n a t u r a l p o p u l a t i o n s was about 150 pmol N0 .g p r o t e i n " . h " . 1  1  2  In August, t h e n a t u r a l p o p u l a t i o n of G. Bay  had  twice  population  at  Transplanting resulted  the n i t r a t e the from  head  v e r r u c o s a i n Wiseman's  r e d u c t a s e a c t i v i t y of t h e n a t u r a l of  Bamfield  Inlet  ( F i g . 38).  Wiseman's Bay t o t h e head of B a m f i e l d  i n a decrease i n n i t r a t e reductase a c t i v i t y .  Inlet  The lower  i n t e r t i d a l t r a n s p l a n t s (1.0 m) a t t h e head of B a m f i e l d I n l e t had low n i t r a t e r e d u c t a s e a c t i v i t i e s a l s o ( F i g . 38). up high  the i n t e r t i d a l nitrate  Transplanting  zone i n Wiseman's Bay i n c r e a s e d t h e a l r e a d y  reductase  activity  of  t h e Wiseman's  Bay  G.  126  N i t r a t e Reductase A c t i v i t y [Protein Pools] TIDAL  HEIGHT  1.8  M  h" )  (umol NO" • g P r o t e i n " ' (mg . g wet w~' t ) 2  1  M _ 1  WISEMAN'S BAY 43.8  [8.31]  HEAD OF B A M F I E L D „  NATURAL  POPULATION  44.2 141.6 i  [8.31]  60.6  /  /  *  /  / 50.9  1.0  M  NATURAL 85.0  7.691  [6.35]  /  / /  INLET  / /  / [7.95]  POPULATION  26.9 14.2  [7.25] [6.70]  [9.471  F i g . 38. S o l u b l e p r o t e i n c o n t e n t (mg p r o t e i n . g wet w t " ) and nitrate reductase activity (pmol NOj.g p r o t e i n " . h " ) of G r a c i l a r i a v e r r u c o s a i n August 1981. The arrows i n d i c a t e t h e o r i g i n of t h e t r a n s p l a n t e d p o p u l a t i o n s . 1  1  1  I  200-  o CI  Time  (min)  Enzyme  Extract Volume  (ml)  F i g . 39. T o t a l i n v i t r o n i t r i t e produced (ujmol NOj.g p r o t e i n ) with time (minT (A),and t h e r e l a t i o n s h i p between nitrate reductase a c t i v i t y (^umol NO* .g p r o t e i n " . h " and t h e volume of enzyme e x t r a c t used (ml) ( B ) , f o r t h e n a t u r a l p o p u l a t i o n s of high (-—+—) and low ( — • — ) intertidal Gracilaria verrucosa. E r r o r bars r e p r e s e n t one s t a n d a r d d e v i a t i o n , n=3. - 1  1  1  T  J.  .£ 165-  T I $ 145>  12H  1  1  2  1  4  6 MgS0  4  "  1  •  8  (mM)  F i g . 40. The r e l a t i o n s h i p between n i t r a t e r e d u c t a s e a c t i v i t y (ujnol NO~ g p r o t e i n " . h " ) and MgSO^ c o n c e n t r a t i o n (mM) f o r t h e n a t u r a l p o p u l a t i o n s of h i g h (---o -) and low ( » — ) i n t e r t i d a l G r a c i l a r i a verrucosa. E r r o r bars represent one standard d e v i a t i o n , n=3, 1  1  1  1 29  120i  4)  E  0.  O) ' Q C M  110'  z o E  3  <  10OH  tn O  u  D or  90'  T i i  T I I I  i ~I— 8.2  8.1  —\— 8.3  8.4  F i g . 41. The r e l a t i o n s h i p between n i t r a t e r e d u c t a s e a c t i v i t y (umol NOg .g p r o t e i n " . h " ) and pH f o r the n a t u r a l p o p u l a t i o n s of h i g h (-—o —-) and low (—• ) G r a c i l a r i a verrucosa . Error bars r e p r e s e n t one s t a n d a r d d e v i a t i o n , n=3. 1  1  120H  p CL Ol 'QCNJ  z o E  3  100J  < u or  80-  T I I I I  —1—  0.1  0.2 ( g P V P . g wet wt"  PVP  1  -f— 0.3 )  0.4  F i g . 42. The r e l a t i o n s h i p between n i t r a t e r e d u c t a s e a c t i v i t y (umol NOj.g p r o t e i n - , h " ) and the amount of polyvinyl pyrrolidine (PVP) added (g.g wet w t " ) f o r t h e n a t u r a l p o p u l a t i o n s of h i g h (-—o -) and low (—•—) intertidal Gracilaria verrucosa, E r r o r bars represent one standard d e v i a t i o n , n=3. 1  1  1  131  NO"  (mM )  F i g . 43. The r e l a t i o n s h i p between n i t r a t e reductase activity (umol NOj.g p r o t e i n " . h " ) and n i t r a t e c o n c e n t r a t i o n (mM) f o r ) tiie n a t u r a l p o p u l a t i o n s of h i g h (—•-—) and low ( i n t e r t i d a l G r a c i l a r i a verrucosa. E r r o r bars r e p r e s e n t one s t a n d a r d d e v i a t i o n , n=3. 1  1  200  100  300  400  F i g . 44. The r e l a t i o n s h i p between n i t r a t e reductase activity (umol NO" .g p r o t e i n " . h " ) and NADH c o n c e n t r a t i o n (uM) f o r t h e n a t u r a l p o p u l a t i o n s o f h i g h (---•—") and low (—» ) i n t e r t i d a l standard Gracilaria verrucosa. E r r o r bars r e p r e s e n t one d e v i a t i o n , n=3. 1  1  132  verrucosa  population.  I n l e t t o 1.0 m i n  Transplanting  Wiseman's  Bay  from the head of  increased  nitrate  Bamfield reductase  activity.  I f p l a n t s were t r a n s p l a n t e d t o 1.8 m i n Wiseman's Bay  from  head of B a m f i e l d  the  remained c o n s t a n t  I n l e t the n i t r a t e r e d u c t a s e  a t about 45 pmol N0 .g p r o t e i n " . h ' . 1  2  1  activity  133  Discussion The  morphologies of the  verrucosa  were  species. in  two  natural  populations  ( B i r d et a l . 1982a).  plasticity  Biochemical c h a r a c t e r i s t i c s  such as chromosome number and enzyme type are o f t e n more in  taxonomic  al.  1982a). The  studies  than  morphological  r e s u l t s of the t r a n s p l a n t  suggest  that  the  observed  experiments  survival  rate  of  the  in  this  study  differences  Transplanted  a l t e r e d m o r p h o l o g i e s s i m i l a r t o those of  useful  d i f f e r e n c e s ( B i r d et  morphological  p h e n o t y p i c r a t h e r than g e n o t y p i c .  The  G.  so d i f f e r e n t t h a t they appeared t o be d i f f e r e n t  Recent s t u d i e s have r e v e a l e d m o r p h o l o g i c a l  Gracilaria  of  were  plants exhibited  the  resident  plants.  t r a n s p l a n t e d t h a l l i a l s o gave some  i n d i c a t i o n of what f a c t o r s might be c o n t r o l l i n g  the  upper  and  t r a n s p l a n t e d t o the  high  lower l i m i t s of the n a t u r a l beds. Many  of  the  plants  that  i n t e r t i d a l s i t e i n Wiseman's Bay  were  broke o f f .  I t i s p o s s i b l e that  wave a c t i o n or g r a z i n g imposes the upper l i m i t bed  found a t 1.0  not  broken  light  and  epiphytes m).  m i n Wiseman's Bay.  off,  grew  nutrients.  well These  The  on  the  natural  t r a n s p l a n t s which were  i n the absence of c o m p e t i t i o n transplanted  plants  had  for  fewer  than the lower i n t e r t i d a l p l a n t s i n Wiseman's Bay  Lindsay  and  (1.0  Saunders (1977) made the same o b s e r v a t i o n  in  t h e i r i n t e r t i d a l c u l t u r e pens. The (1.8 m)  p l a n t s t r a n s p l a n t e d from the  of  Bamfield  t o the h i g h i n t e r t i d a l s i t e i n Wiseman's Bay  not r e t a i n t h e i r y e l l o w c o l o r a t i o n and suggests  head  that  these  (1.8 m)  s t u n t e d appearance.  c h a r a c t e r i s t i c s were  not  Inlet did This  c o n t r o l l e d by  134  intertidal location. environmental a c t i o n , or  exposure t o d i r e c t s u n l i g h t and  conditions  varied  Wiseman's  The  Bay  such  nutrient  and  at  p e r i o d i c exposure, may  the be  as  different  supply head  which of  substrate,  were  for  wave  different  Bamfield  responsible  other  in  I n l e t , and  this  not  morphological  type. The Bamfield that  p l a n t s t r a n s p l a n t e d from 1.8 I n l e t a l s o became dark r e d .  m t o 1.0 This i s  m at the head of further  evidence  d i r e c t s u n l i g h t might be the cause of the y e l l o w i s h c o l o r .  The  p l a n t s moved from Wiseman's Bay  of  Bamfield  and  from a f e a t h e r y  (1.0)  t o 1.8  m at  the  head  I n l e t r a p i d l y (3 weeks) changed from red t o y e l l o w to  a  matted  appearance,  resembling  the  resident plants. The  G.  verrucosa  d e f i n i t e lower l i m i t .  location  h i g h or low very  small  The  t h a l l i transplanted  and  A f t e r f i v e weeks, the  fragile.  the  head  thalli  verrucosa  l i t t l e or no e e l g r a s s i n Wiseman's Bay m.  of B a m f i e l d  The  at  were was in  t o compete  p l a n t s t h a t were t r a n s p l a n t e d  I n l e t t o 1.0  m i n Wiseman's Bay  i s p o s s i b l e t h a t t h e i r o r i g i n a l growth form, which growth  in  thalli  not grow as r a p i d l y as the r e s i d e n t p l a n t s s u r r o u n d i n g  for  m  f o r l i g h t , s u b s t r a t e and/or n u t r i e n t s .  w i t h the G r a c i l a r i a at 1.0 from  1.0  1.2  I t i s p o s s i b l e t h a t the e e l g r a s s  more s u c c e s s f u l than the t r a n s p l a n t e d G.  There was  to  a  grew p o o r l y , whether they were o r i g i n a l l y from a  i n t e r t i d a l bed.  the c o m p e t i t i o n  I n l e t had  An e e l g r a s s bed predominated a t about  m above Canadian datum. this  bed a t the head of B a m f i e l d  1.8  m  d i s a d v a n t a g e i n the new  at low  the  head  of  Bamfield  was  did  them.  adapted  I n l e t was  i n t e r t i d a l s i t e d u r i n g the  It  a  important  135  growing p e r i o d Saunders  in late  (1982)  conducted  l o n g i c r u r i s and partially  and  early  (1977) a l s o found t h a t l o c a l G.  than those t r a n s p l a n t e d al.  June  they  retained  to  the c u l t u r e  transplant found  July.  v e r r u c o s a grew f a s t e r location.  experiments  that  L i n d s a y and  i n some  Gagne* e t  with  cases  Laminaria the  plants  t h e growth p a t t e r n c h a r a c t e r i s t i c of t h e i r  former p o p u l a t i o n even a f t e r one y e a r . Time c o u r s e s of uptake a r e e s s e n t i a l f o r t h e i n t e r p r e t a t i o n of  any uptake r a t e d e t e r m i n e d  phytoplankton  and macrophytes  over  a  particular  time.  Both  have h i g h i n i t i a l n i t r o g e n uptake  r a t e s which o f t e n d e c r e a s e w i t h i n minutes o r  hours  al.  The  two i n t e r t i d a l G.  had  transient  1976; D ' E l i a  verrucosa  populations  n i t r o g e n uptake The  and  DeBoer  1978).  in this  study  (Conway  et  initial  rates.  sudden drop i n ammonium uptake by 50 % a f t e r 10—30 min  was t o o l a r g e t o be accounted f o r by t h e d e c r e a s e concentration  in  t h e medium  due  t o uptake.  i n ammonium  Initial  nitrate  uptake r a t e s remained c o n s t a n t f o r a l o n g e r p e r i o d than ammonium uptake r a t e s .  T h i s was t r u e f o r o t h e r  intertidal  (Chapter 1 ) . I t i s p o s s i b l e t h a t t h i s i n i t i a l is  controlled  intracellular  by  macrophytes  r a p i d uptake r a t e  an i n t e r n a l mechanism such as t h e s i z e of an  pool.  I n h i b i t i o n of n i t r a t e uptake by ammonium has been in  other  marine  macrophytes  (Haines and Wheeler  and H a r l i n 1978; Chapter 1 ) . N i t r a t e uptake ammonium  i n high  i n t e r t i d a l G.  intertidal  verrucosa.  G.  was  observed  1978; Hanisak inhibited  by  v e r r u c o s a but not i n t h e low  Other s p e c i e s i n which n i t r a t e uptake  was not a f f e c t e d by ammonium were Fucus s p i r a l i s  (Topinka  1978)  136  and  Laminaria  lonqicruris  Fucus i s found i n t h e h i g h largely  subtidal,  (Harlin  and  intertidal  i t would  appear  C r a i g i e 1978).  zone  and  Since  Laminaria  is  t h a t t h e maintenance of a  normal n i t r a t e uptake r a t e i n t h e presence of ammonium i s not an adaptation to i n t e r t i d a l location.  More s p e c i e s must be t e s t e d ,  but i t may be t h a t i n h i b i t i o n of n i t r a t e uptake by dependent  on  location.  I t i s energetically  take  up  nutritional  ammonium  past  and  was  r a t h e r than n i t r a t e .  suggest t h a t G.  greater  r a t h e r than i n t e r t i d a l  more f a v o r a b l e f o r t h e p l a n t  the head of B a m f i e l d " i n l e t was runoff  history  ammonium i s  The ammonium s u p p l y a t  probably  than  due  to  i n Wiseman's  ground  Bay.  v e r r r u c o s a i n Wiseman's Bay was  approximately  water  C/N r a t i o s  more  n i t r o g e n l i m i t e d than a t t h e head of B a m f i e l d I n l e t . were  severely C/N r a t i o s  13 and 9, r e s p e c t i v e l y . I t i s p o s s i b l e t h a t  the h i g h i n t e r t i d a l G.  v e r r u c o s a on t h e mud f l a t a t t h e head of  B a m f i e l d I n l e t was r e c e i v i n g a l i m i t e d amount of  nitrogen  the  Taylor  mud  and  from  epiphytes  N e v e r t h e l e s s , t h e C/N r a t i o  of  p l a n t s were n i t r o g e n d e f i c i e n t The  ammonium  to  and  9  (Capone still  (Niell  and  suggests  that  from 1977). these  1976).  n i t r a t e uptake r a t e s of t h e v a r i o u s G.  v e r r u c o s a p o p u l a t i o n s g i v e some i n s i g h t i n t o t h e n u t r i e n t supply in the various s i t e s .  I n August, n i t r a t e uptake r a t e s were much  h i g h e r i n Wiseman's Bay than a t This  may  be  due  to  t h e head  of  Bamfield  t h e presence of ammonium a t t h e head of  B a m f i e l d I n l e t and t h e p o s s i b l e i n t e r m i t t e n t s u p p l y to  Wiseman's  Inlet.  Bay through v e r t i c a l m i x i n g .  the ammonium and n i t r a t e uptake  rates  of  of  nitrate  W i t h i n a few weeks, transplanted  were s i m i l a r t o t h e r a t e s o f t h e l o c a l t h a l l i .  thalli  T r a n s p l a n t i n g up  1 37  the  intertidal  zone imposed more severe n u t r i e n t l i m i t a t i o n by  i n c r e a s i n g the p e r i o d of exposure; t h i s  resulted  in  increased  n i t r a t e uptake r a t e s . Transplanting Bamfield  down  the  intertidal  zone  at  t h e head of  I n l e t i n c r e a s e d ammonium uptake r a t e s , a l t h o u g h  o t h e r p o p u l a t i o n s had s i m i l a r ammonium uptake r a t e s . a l s o v e r y l i t t l e d i f f e r e n c e between June  and  i n August.  This  s u g g e s t s t h a t t h e ammonium uptake  have  been  several  k i n e t i c s i n marine macrophytes. ammonium  studies  immediate  of t h e n i t r o g e n uptake  D ' E l i a and DeBoer (1978)  Haines and Wheeler  i n ammonium  muse i f o r m i s . that  the  uptake  (1978)  for Macrocystis  Other s t u d i e s w i t h d i f f e r e n t  ammonium  found  uptake i n G r a c i l a r i a t i k v a h i a e was not s a t u r a t e d  a t 40 uM ammonium. trend  was  supply.  There  that  There  t h e ammonium uptake r a t e s i n  system was always " a c t i v e " and l i m i t e d s o l e l y by ammonium  a l l the  uptake  was  saturable  found  a  similar  p y r i f e r a and Hypnea species  have  found  (Hanisak and H a r l i n 1978;  T o p i n k a 1978; Kautsky 1982), i n d i c a t i n g t h a t t h i s phenomenon i s species  dependent.  The r e s u l t s of t h i s study on G.  showed t h a t one p o p u l a t i o n , a t t h e head of B a m f i e l d saturable not.  Even  ammonium  uptake  the n i t r a t e  and  uptake  verrucosa Inlet,  had  a n o t h e r , i n Wiseman's Bay, d i d rates  of  t h e Wiseman's  Bay  p o p u l a t i o n were not s a t u r a b l e . The  ecological  uptake i s not c l e a r .  s i g n i f i c a n c e of s a t u r a b l e or  non-saturable  I t i s suspected  non—saturable  that  the  component i s a d i f f u s i o n component but t h i s has not been proved. The  magnitude  of  the non—saturable  substrate concentrations.  component i n c r e a s e s  with  Any a p p r e c i a b l e d i f f e r e n c e i n uptake  138  rate  occurs  a t c o n c e n t r a t i o n s of ammonium and n i t r a t e t h a t a r e  o f t e n h i g h e r than n o r m a l l y found i n t h e f i e l d . between  saturable  l i t t l e ecological  and n o n - s a t u r a b l e uptake may t h e r e f o r e be of significance.  The Ks v a l u e s f o r n i t r a t e uptake of verrucosa  The d i f f e r e n c e  populations  were  t h e two  natural  h i g h e r than those r e c o r d e d f o r G.  t i k v a h i a e ( D ' E l i a and DeBoer 1978) and p h y t o p l a n k t o n al. the  1969b).  (Eppley  et  However, they a r e l e s s than most Ks v a l u e s c i t e d i n  l i t e r a t u r e f o r marine macrophytes (Haines and Wheeler 1978;  Hanisak  and  Topinka  1978; Kautsky  The G.  G.  Harlin;  Harlin  1978; H a r l i n  and  C r a i g i e 1978;  1982).  Ks f o r ammonium uptake (10 pM) i n t h e h i g h  verrucosa  tikvahiae  was  several  (1.6 uM)  (Eppley e t compressa  Other  than  that  species  such  as  of  G.  Enteromorpha  1982) and M a c r o c y s t i s p y r i f e r a  Wheeler 1978) have a s i m i l a r Competition  greater  ( D ' E l i a and DeBoer 1978) and p h y t o p l a n k t o n  a l . 1969b). (Kautsky  times  intertidal  f o r low  lack  of  concentrations  affinity  (Haines and  f o r ammonium.  of ammonium must not have  been a s t r i n g e n t c r i t e r i o n f o r s u r v i v a l f o r these  species,  at  the time they were t e s t e d . The  nitrate  and  study were s i m i l a r literature  ammonium  i n magnitude  uptake to  r a t e s observed  those  presented  in this i n the  f o r o t h e r s p e c i e s of marine macrophytes ( D ' E l i a and  DeBoer 1978; Haines and Wheeler 1978; Hanisak H a r l i n and C r a i g i e 1978; Kautsky Previous  studies  have  and  Harlin  1978;  1982). shown  that  high  intertidal  macrophytes have enhanced n i t r o g e n uptake r a t e s  following  desiccation  4 ) . T h i s study  (Thomas  and  Turpin  1980; Chapter  mild  139  w i t h G.  verrucosa  i n d i c a t e d t h a t t h i s was an  w e l l as an i n t e r s p e c i f i c a d a p t a t i o n  intraspecific  as  to i n t e r t i d a l location.  The  t r a n s p l a n t experiments showed t h a t t h i s phenomenon was dependent on  intertidal  level  and  was  not dependent  l o c a t i o n o r t h e o r i g i n of t h e p l a n t . high for  intertidal  sites  s i t e s d i d not.  transplanted  no  enhanced  nitrate  uptake  enhancement of ammonium uptake. in  enhanced  tested. were  nitrate  uptake  1981.  rates,  high,  and  rates:  similar  to  enhancement  of  t h e low verrucosa  These  I n 1980, d e s i c c a t i o n ammonium  t h e Vmax  measurement of ammonium uptake k i n e t i c s . further  to  plants  but they d i d show  I n 1981, t h e ammonium uptake r a t e s of  very  to  The o n l y e x c e p t i o n was G.  t r a n s p l a n t e d t o 1.8 m i n Wiseman's Bay i n showed  transplanted  developed t h i s p h y s i o l o g i c a l a d a p t a t i o n  n i t r o g e n procurement w h i l e those  intertidal  Plants  on g e o g r a p h i c a l  resulted  uptake  was not  hydrated  plants  found d u r i n g t h e  I t i s possible  that  uptake was p h y s i o l o g i c a l l y i m p o s s i b l e .  I s o l a t i o n of t h e e n v i r o n m e n t a l f a c t o r s c a u s i n g enhanced uptake a f t e r d e s i c c a t i o n w i l l  nitrogen  require c u l t i v a t i o n i n controlled  environments. The  degree  of enhancement and t h e % d e s i c c a t i o n r e s u l t i n g  i n maximum uptake r a t e s might depend upon past was  little  variation  maximum uptake r a t e s . for The  i n t h e % d e s i c c a t i o n (10-15%)  of  enhancement  varied  plants  were  s l i g h t l y with  l o c a t i o n and g r e a t l y from year t o y e a r lowest  There  producing  The degree of enhancement of uptake r a t e s  t h e t r a n s p l a n t e d t h a l l i and r e s i d e n t degree  history.  (highest  similar.  geographical i n 1981 and  i n 1979).  The  controls  of  such  a  response  t o d e s i c c a t i o n were a  140  c o m b i n a t i o n of e n v i r o n m e n t a l f a c t o r s uptake is  and  possible  limitation  enhancement  of  t h e p l a n t ' s a b i l i t y t o respond t o such f a c t o r s .  It  that and  periodic  summer of 1979. nitrogen  environmental  prompting  factors  such  as  nitrogen  d e s i c c a t i o n were not prominent i n t h e  I t i s much more l i k e l y t h a t t h e p l a n t s were  stressed  that  nitrogen  uptake  so  systems r e q u i r e d f o r  r a p i d uptake were not m a i n t a i n e d . Another p o s s i b l e a d a p t a t i o n maintenance  i s the  of n i t r o g e n uptake r a t e s f o l l o w i n g a g r e a t e r  of d e s i c c a t i o n (20-30%). h i g h i n t e r t i d a l G. tolerance  to intertidal location  degree  T h i s was t r u e of the uptake r a t e s  verrucosa.  for  In t h e t r a n s p l a n t e x p e r i m e n t s , a  t o d e s i c c a t i o n appeared t o depend upon the i n t e r t i d a l  l o c a t i o n ; but t h e t r a n s p l a n t e d p l a n t s o f t e n showed uptake r a t e s , f o l l o w i n g 30% d e s i c c a t i o n , resident  plants  transplanted.  and  which  the  were  population  between from  than f i v e weeks t o d e v e l o p or  of  the  soluble various  conclusions and Bay  Inlet.  they  the were  intertidal  required  more  n i t r o g e n c o n t e n t and n i t r a t e r e d u c t a s e l e v e l s G.  verrucosa  drawn  plants.  but  of  disappear.  from  populations  the uptake r a t e s .  t h e n i t r a t e r e d u c t a s e l e v e l s were h i g h e r  of n i t r o g e n  which  T h i s phenomenon may s i m p l y depend- upon  l e v e l and not on the o r i g i n of t h e t h a l l u s ,  The  those  confirmed  most  The n i t r a t e c o n t e n t in  the  Wiseman's  T h i s i n d i c a t e s t h a t n i t r a t e i s an i m p o r t a n t source i n Wiseman's Bay, but not a t the  There  were  s o l u b l e amino a c i d  few  dissimilarities  head in  of  Bamfield  t h e ammonium and  content.  The amounts of n i t r a t e and ammonium e x t r a c t e d i n t h i s study were s i m i l a r i n magnitude t o those e x t r a c t e d from G.  tikvahiae  141  and U l v a by Rosenberg and Ramus (1982). The study  n i t r a t e r e d u c t a s e a c t i v i t y of t h e G.  showed  no  unusual  verrucosa i n t h i s  c h a r a c t e r i s t i c s a l t h o u g h i t was l e s s  s e n s i t i v e t o pH change, and Mg *  and  +  PVP  concentrations  than  o t h e r i n t e r t i d a l seaweeds (Appendix 2 ) . Transplanting nitrate  uptake  decreased nitrate  up  the  intertidal  rates  and  nitrate  internal  nitrate  utilization.  periodic  exposure  The  is a similar The  transplant  verrucosa  explanation  nitrogen  is  that  procurement when  Enhancement o f uptake a f t e r  experiments  primarily  They have  controlling  accelerated  desiccation  adaptation.  dependent  zone.  but  i n i n t e r m i t t e n t n u t r i e n t s u p p l y and  in this  h y p o t h e s i s t h a t enhanced n u t r i e n t uptake is  activity  suggesting  likely  produces a requirement f o r a c c e l e r a t e d the a l g a i s submerged.  l e d t o increased  reductase  levels,  most  results  zone  also  study c o n f i r m e d t h e  following  desiccation  on attachment h e i g h t i n t h e i n t e r t i d a l prompted  some  suggestions  on  factors  t h e morphology, p h y s i o l o g y and d i s t r i b u t i o n of G. i n the  assimilation  intertidal  rates  habitat.  are c o n t r o l l e d  by  Nitrogen complex  uptake  and  interactions  between c e r t a i n p h y s i c a l f a c t o r s and t h e p h y s i o l o g i c a l s t a t e the  thallus.  Previous n u t r i e n t supply, the n u t r i t i o n a l s t a t u s  of t h e t h a l l u s , a h i s t o r y of r e p e a t e d p e r i o d i c d e s i c c a t i o n , previous  degree  of  desiccation,  n u t r i e n t p r e s e n t a r e some factors  can  of  be  of  controlled  o b s e r v e d responses a r e of  and  the  the v a r i a b l e s i n laboratory  uncertain  F i e l d s t u d i e s must be conducted.  the  type and amount of involved. cultures  ecological  These but any  significance.  By t r a n s p l a n t i n g , some f a c t o r s  142  can  be  defined  and manipulated  and t h i s a i d s i n  understanding  the importance of v a r i o u s f a c t o r s i n r e l a t i o n t o a l g a l  o  zonation.  143  Summary and C o n c l u s i o n s  The is  study of t h e n u t r i e n t p h y s i o l o g y  of marine  macrophytes  i n t h e e a r l y stages of i t s development and l a g s f a r behind  phytoplankton  physiology.  adaptations  to  phytoplankton, survey  of  low  In.previous  nitrate  to  those  they  internal  been  levels  of  for  limited  t e c h n i q u e s has  s t u d i e s have been a p p l i e d but  have  reductase  a  of  s t u d i e s on macrophytes, p r o c e d u r e s  properly  c h a r a c t e r i s t i c s of macrophytes. rates,  similar  species using c o n s i s t e n t experimental  used i n p h y t o p l a n k t o n that  nutrients,  reports  however, p r i o r t o t h i s work not even  been u n d e r t a k e n .  clear  There a r e a few i s o l a t e d  modified  T h i s study of  soluble  activities  t o t h e unique  nutrient  nitrogen  includes  i t i s not  uptake  compounds,  attempts  to  and  optimize  c u r r e n t methods f o r use w i t h marine macrophytes. I n i t i a l p e r i o d s of r a p i d ammonium uptake f o l l o w i n g n i t r o g e n s t a r v a t i o n were observed i n t h e l a b o r a t o r y and f r e s h l y c o l l e c t e d plants. is  T h i s study was the f i r s t t o document t h a t t h i s response  common  method  i n the f i e l d .  of  determining  Consequently, ammonium  uptake  the popular rates  "batch" may  be  inappropriate. The  presence  of  ammonium  o f t e n i n h i b i t e d n i t r a t e uptake  r a t e s but a c e r t a i n degree of n i t r o g e n d e f i c i e n c y p r e v e n t e d t h i s inhibition. because  These  findings  a r e of  ecological  significance  they suggest t h a t uptake of v a r i o u s n i t r o g e n  substrates  can be r a p i d l y a l t e r e d t o a l l o w energy c o n s e r v a t i o n ; ammonium i s t a k e n up p r e f e r e n t i a l l y i n t i m e s of n i t r o g e n s u f f i c i e n c y whereas s i m u l t a n e o u s maximum  nitrate  and  ammonium  uptake  rates  are  144  maintained  d u r i n g t i m e s of n i t r o g e n d e f i c i e n c y .  The  effect  of n i t r o g e n supply on n i t r o g e n u p t a k e , s o l u b l e  n i t r o g e n content both  under  controlled  conditions. but low  and n i t r a t e reductase  Nitrogen  nitrate,  increased  within  latoratory  and  conditions  starved t h a l l i maintained  uptake  rates.  10-20 min  grown c u l t u r e s a l s o m a i n t a i n e d was  a c t i v i t y was i n v e s t i g a t e d  Nitrate  and  field  r a p i d ammonium,  uptake  rates  of exposure t o n i t r a t e . h i g h ammonium uptake  were  Nitrate-  rates.  It  p o s t u l a t e d t h a t n i t r a t e r e d u c t i o n was t h e r a t e l i m i t i n g  step  t h e supply of a s s i m i l a t e d n i t r o g e n was slower  grown t h a l l i  than f o r ammonium-grown t h a l l i .  assimilated  nitrogen,  which  was  absent  for nitrate-  A product  of t h e  i n n i t r a t e - g r o w n and  s t a r v e d p l a n t s , may c o n t r o l ammonium uptake. A brief was  present  (<5 min) p e r i o d of enhanced i n t h e ammonium  rate  This i s the  Ammonium uptake appeared t o be  c o n t r o l l e d by a s m a l l i n t e r n a l p o o l which transfer  uptake  sufficient plants.  f i r s t r e c o r d of such a response.  the  ammonium  was  depleted  during  from t h e c u l t u r e v e s s e l t o t h e uptake medium (0.5  h). The  slow r a t e of d e c r e a s e i n i n t e r n a l n i t r a t e suggests t h a t  ammonium-grown t h a l l i than  using  took up and  existing  internal  assimilated  nitrate.  ammonium  Nitrogen  rather  starvation  caused a g e n e r a l d e c r e a s e i n a l l i n t e r n a l n i t r o g e n l e v e l s and a transient was  increase  i n n i t r a t e reductase  activity.  t h e f i r s t s i m u l t a n e o u s e v a l u a t i o n of t h e e f f e c t  forms  of  inorganic  s e v e r a l aspects A  This of  study  various  n i t r o g e n supply and n i t r o g e n s t a r v a t i o n on  of n i t r o g e n m e t a b o l i s m .  comparison  of  nitrogen  uptake  in  Fucus  distichus  145  germlings  showed  that  n u t r i t i o n a l physiology germlings  showed  there  were  marked  of d i f f e r e n t l i f e  d i f f e r e n c e s i n the  history  stages.  r a p i d n i t r a t e and ammonium uptake r a t e s ,  ammonium uptake a f f i n i t y , and no ammonium i n h i b i t i o n of uptake thalli  which  be  that  rates  a  nitrate  they c o u l d out—compete t h e mature  T h i s was t h e f i r s t  study of  the  of g e r m l i n g s suggested t h a t m i l d  requirement  confirmed,  f o r optimal  growth.  on t h e b a s i s of n i t r o g e n  s e v e r a l s p e c i e s of  intertidal  enhanced  uptake  nutrient  nutrient  desiccation  f o r several  desiccation  This  was  also  uptake f o r mature p l a n t s of  macrophytes.  Mild  desiccation  r a t e s when t h e s p e c i f i c n u t r i e n t was  l i m i t i n g growth and t h e p l a n t  had  weeks  been  (a  exposed  to  "hardening"  degree of t h i s enhancement, t h e p e r c e n t maximum  high  of an e a r l y l i f e h i s t o r y s t a g e .  Growth may  suggests  f o rnitrogen.  physiology  The  uptake r a t e s and t h e t o l e r a n c e  effect).  desiccation t o higher  periodic The  producing  desiccation i n  terms o f n i t r o g e n u p t a k e , depended on i n t e r t i ' d a l l o c a t i o n . response t o d e s i c c a t i o n was interspecific induced following nitrogen  adaptation  within a  five  period  an to  intraspecific intertidal  weeks. of  This  carbon  as  well  This as an  l o c a t i o n and c o u l d be  nutrient  uptake  procurement  but  response  no  external  s u p p l y (exposure t o a i r ) , may be important i n long term  C/N h o m e o s t a s i s . The following  physiological desiccation  control  of  i s closely  enhanced related  nutrient to at  f a c t o r s , n u t r i t i o n a l s t a t e and d e s i c c a t i o n p a s t reveals  the complexity  f a c t o r s and  metabolic  of  the  controls.  interaction Nutritional  uptake  least  history.  two This  between p h y s i c a l status,  recent  146  degree  of  desiccation  periodic desiccation nitrogen  and  are  the  all  effect  of  involved  in  several  weeks of  inducing  enhanced  uptake r a t e s i n i n t e r t i d a l seaweeds.  The  discovery  of  t h i s unique response t o d e s i c c a t i o n which o p t i m i z e s uptake g i v e s i n s i g h t i n t o how  i n t e r t i d a l seaweeds can be very  productive  in  an environment of such p h y s i c a l extremes. The  results  of  this  study  show t h a t n i t r o g e n uptake i n  i n t e r t i d a l seaweeds i s adapted t o p e r i o d s of n i t r o g e n s t a r v a t i o n i n terms  of  the  maintanence  of  high  potential  uptake  and  a s s i m i l a t i o n r a t e s arid c o n t r o l of p r e f e r e n t i a l uptake of c e r t a i n forms of n i t r o g e n . The  i n t e r a c t i o n between n i t r o g e n supply and  n i t r o g e n metabolism has been nitrogen  procurement  T h i s was  achieved  regimes  could  in  be  laboratory  (uptake, a  laboratory  The  c o n c l u s i o n s drawn from  study  The  must  not  laboratory  observed.  On  where  the  conducting  in  environmental  a  field  and  nutrient  be  field  underestimated  since  can  often  become  In a l a b o r a t o r y study  the other hand, t h e r e i s no c o n t r o l over factors  of  both  f a c t o r can be m a n i p u l a t e d and a s p e c i f i c  environmental simultaneous  stages  and a s s i m i l a t i o n ) .  cultures  d i v o r c e d from the n a t u r a l environment. environmental  three  e c o l o g i c a l s i g n i f i c a n c e was  importance of  studies  at  accumulation,  controlled.  t e s t e d i n the f i e l d . and  illustrated  the c o n t r o l of  study.  In  physiological  an  response numerous  many  cases  changes  are  observed but cannot be d i r e c t l y r e l a t e d u n t i l they are t e s t e d i n the  laboratory.  Transplant  experiments  advantages of both l a b o r a t o r y and one  or  more  environmental  combine  f i e l d s t u d i e s by  factors  while  some of  the  manipulating  maintaining  field  147  conditions.  I t i s only with a  combination  of  laboratory  f i e l d studies that p h y s i o l o g i c a l adaptations to f i e l d can be  and  conditions  discussed.  This study  study  of  i s the f i r s t comprehensive l a b o r a t o r y and  nitrogen  Physiological  utilization  adaptations  in  in  marine  field  macrophytes.  terms of n i t r o g e n procurement i n  response t o n i t r o g e n s u p p l y and o t h e r p h y s i c a l f a c t o r s have been i d e n t i f i e d and t e s t e d i n the carbon  metabolism  is  field.  needed.  An  Such  equivalent  studies  a p p l i c a t i o n i n the c u l t u r i n g of seaweed f o r economically  important  compounds  such  study  have  the  practical  production  understand  manipulated The  how  growth  and  phycocolloid  study of p h y s i o l o g i c a l e c o l o g y  distribution. extremes  where  stressed  and  investigation  necessary  content  can  be  The  r e l a t e s the knowledge of  maintaining  growth  and  species  i n t e r t i d a l zone i s an environment of p h y s i c a l  maintenance of p h y s i o l o g i c a l p r o c e s s e s numerous of  the  life  forms  nutritional  cannot physiology  seaweeds r e v e a l s t h a t these organisms are of  The  to increase p h y c o c o l l o i d production.  the p h y s i o l o g i c a l mechanisms  terms  of  as p h y c o c o l l o i d s .  i n t e r a c t i o n between carbon and n i t r o g e n metabolism i s to  on  nitrogen  i s often  survive. of  uniquely  This  intertidal adapted  uptake p o t e n t i a l and energy c o n s e r v a t i o n  p e r i o d s of exposure t o a i r and  reduced n u t r i e n t s u p p l y .  in to  148  References B e s t , E.P.H. 1980. 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Ecol. 25:431-68.  156  Appendix 1.  Techniques f o r M e a s u r i n g S o l u b l e N i t r o g e n  Content  Introduction The  a b i l i t y of c e r t a i n macrophytes t o s t o r e n i t r o g e n may  important depleted  for  their  d u r i n g p e r i o d s when n i t r o g e n i s  i n the ambient water.  patterns  In some c a s e s ,  seasonal  growth  of macrophytes appear more dependent on t h e s e  r e s e r v e s than Chapman  survival  and  on  external  Craigie  nutrient  1977;  be  Hanisak  supplies 1979;  internal  (Buggeln  1974;  Rosenberg and Ramus  1982). E a r l i e r s t u d i e s d e t e r m i n e d the t o t a l dried  seaweeds ( N i e l l  1976;  Chaumont 1978)  known what p e r c e n t a g e of the t o t a l available  for  growth.  nitrogen  Recent  plant studies  content  of  but, i t i s s t i l l  not  nitrogen  is  readily  i n d i c a t e that soluble  t i s s u e n i t r o g e n i s r a p i d l y u t i l i z e d d u r i n g the onset  of  growth  ( B i r d et a l . 1982b; Gerard 1982a; Rosenberg and Ramus 1982). Several  procedures  for  e x t r a c t i o n of s o l u b l e  m a t e r i a l s from marine macrophytes have been has  found  acceptance  and  it  is  reported  unusual  e f f i c i e n c y and v a r i a b i l i t y of e x t r a c t i o n .  a  simple  efficient  numerous m a c r o a l g a l  extraction  but  There i s  a  critical  the development  method t h a t can be used on  species.  T h i s study compares f o u r methods, u s i n g hot w a t e r , 80%  ethanol  with  or  without  freezing in l i q u i d nitrogen for soluble  amino  seaweeds, one  none  t o f i n d r e p o r t s of  need f o r an e v a l u a t i o n of the common methods and of  nitrogenous  acids  and  or  hot  g r i n d i n g or hot water f o l l o w i n g extracting  nitrate,  ammonium,  s o l u b l e p r o t e i n from t h r e e  intertidal  from each of t h r e e major m a c r o a l g a l  divisions.  157  M a t e r i a l s and Young,  non-reproductive  Enteromorpha  intestinalis  Methods  Porphyra  (L.)Grev.,  perforata and  Fucus  J.  d i s t i c h u s L.  P l a n t s were c o l l e c t e d i n F e b r u a r y and March from the zone  west  of K i t s i l a n o Beach, Vancouver, B.C.,  T h e i r h e i g h t s above Canadian datum were  and  respectively.  m,  intertidal  Canada (Chapter  1; F i g . 2 ) . 2.0  Ag.,  1.5,  2.0,  They were immediately t r a n s p o r t e d  to  the l a b o r a t o r y where they were washed w i t h f i l t e r e d seawater  and  b l o t t e d dry. F. was  The  e n t i r e p l a n t was  d i s t i c h u s where h o l d f a s t s were removed.  different  extraction  while  extractions grinding,  g r i n d i n g , hot e t h a n o l liquid  were hot  Grinding  washed and  followed  square)  Trends w i l l be  material  (2 g) was  i g n i t e d ) and  80%  c u t the F.  ethanol  by  material  water  in  while  freezing  hot  water.  E r r o r bars represent  ±  1  discussed.  ground w i t h  5 ml of hot water  for  grinding.  tissue  top 19 ml  (previously  I t was  (approximately  the g r i n d i n g  80°C)  necessary to  A f u r t h e r 20 ml of hot e t h a n o l continued  0.5  cm  or water until  a  produced.  slurry  f o r F.  1 g sand  (approximately  ( a p p r o x i m a t e l y 60°C).  added i n 5 ml a l i q u o t s and  The  hot  extraction  grinding  d i s t i c h u s into small pieces  homogeneous s l u r r y was  The  of  Procedure  Plant  hot  employed:  e x t r a c t i o n w i t h o u t g r i n d i n g , and  nitrogen  standard d e v i a t i o n .  was  A l l plant  ethanol  E x t r a c t i o n s were done i n t r i p l i c a t e .  or  case  extracted within 2 h after c o l l e c t i o n . Four  in  used, except i n the  was  c e n t r i f u g e d a t 2,000 xg f o r 6  d i s t i c h u s and  E.  i n t e s t i n a l i s and  the  min. top  158  16 ml f o r P.  p e r f o r a t a was d e c a n t e d .  was  e x t r a c t e d t h r e e times w i t h 25 ml water or e t h a n o l .  The  further  supernatant  ammonium,  The  from each e x t r a c t i o n was  total  ninhydrin  positive  remaining  analysed  material  for  material  nitrate,  (assumed  t o be  p r i m a r i l y f r e e amino a c i d s ) and p r o t e i n . An attempt t o i n c r e a s e  cellular  disruption  freezing i n l i q u i d nitrogen p r i o r to g r i n d i n g . became b r i t t l e and was  extracted  Pouring  the  immediately  i t was  with hot  was  The  made  by  t i s s u e (2 g)  easy t o g r i n d i n t o a f i n e powder which  hot  water  ex'traction  or  e t h a n o l as o u t l i n e d above.  medium  over  the  frozen  tissue  dropped the temperature of the e x t r a c t i o n medium t o  approximately  20°C.  E x t r a c t i o n Without G r i n d i n g Two  grams  Erlenmeyer  of  diced  C f o r 10 min.  d e c a n t e d and a n a l y s e d  W i t h E. removing  was  placed  f l a s k w i t h 25 ml of 80% e t h a n o l .  a water bath a t 100°  material  thalli  (assumed  to  plant  a  T h i s was  be  primarily ml  material.  stoppered heated i n  e x t r a c t (22 ml) was  f o r n i t r a t e , ammonium, n i n h y d r i n  i n t e s t i n a l i s , o n l y 15 some  The  in  then  positive  amino a c i d s ) and p r o t e i n .  could Ethanol  be  decanted  (80%) was  without  added t o the  f l a s k t o b r i n g the volume up t o 25 ml and the m i x t u r e was b o i l e d again.  T h i s p r o c e d u r e was  were e x t r a c t i o n s w i t h o u t  repeated  thalli,  four times.  Blanks,  which  were run w i t h a l l e x t r a c t i o n s .  N i t r a t e and Ammonium A n a l y s i s Samples  were a n a l y s e d  w i t h a Technicon A u t o A n a l y z e r  immediately (Davis  et  for nitrate al.  and ammonium  1973).  Samples  159  dissolved  i n ethanol  were  d i s t i l l e d water t o p r e v e n t column  which  was  used  diluted bubble  with  125  formation  deionized  i n t h e cadmium  i n the n i t r a t e a n a l y s i s .  n i t r a t e and ammonium s t a n d a r d s were used, one ethanol  ml  Two s e t s of  prepared  d i l u t e d f i v e t i m e s , and t h e o t h e r i n d i s t i l l e d  i n 80% deionized  water.  A n a l y s i s of T o t a l N i n h y d r i n Total soluble ninhydrin the  Positive Material p o s i t i v e m a t e r i a l was determined by  method of Lee and Takahashi (1966), which i s a c o l o r i m e t r i c  r e a c t i o n f o r p r i m a r y amines. assumed  to  be  primarily  The due  absorbance  to  amino  at  acids  570  solutions  were run w i t h e v e r y s e r i e s of  ammonium were a l s o r u n .  of  was  and ammonium.  S t a n d a r d s o l u t i o n s of g l y c i n e i n d i s t i l l e d d e i o n i z e d 80% e t h a n o l  nm  water or i n  analyses.  Standard  The ammonium c o n t e n t was  p r e v i o u s l y measured w i t h t h e A u t o A n a l y z e r .  The  absorbance  due  t o t h i s ammonium was c a l c u l a t e d , from t h e ammonium s t a n d a r d c u r v e for  the ninhydrin  r e a c t i o n and t h i s absorbance was  substracted  from t h e t o t a l absorbance t o g i v e an e s t i m a t e of t h e  absorbance  due t o f r e e amino a c i d s .  Soluble Protein Soluble et  Analysis  p r o t e i n was d e t e r m i n e d u s i n g t h e p r o c e d u r e of Lowry  a l . as  modified  Leggett-Bailey  (1967).  was  primarily  extracted  due  solutions  concentration  of  by  Eggstein  and  Kreutz  (1955),  I t was assumed t h a t t h e c o l o r e d  to  t h e presence  were Folin  diluted positive  see  product  of s o l u b l e p r o t e i n . A l l  ten-fold material  to into  bring the  the high  160  sensitivity  range  of  the  protein  analysis  technique.  calibration  curve  was  e s t a b l i s h e d w i t h each s e r i e s of  u s i n g bovine serum albumin as the s t a n d a r d p r o t e i n . r e s u l t s were expressed grind  tissue  determinations.  on a wet  before  A  analyses  Analytical  weight b a s i s because the need t o  extraction  precluded  dry  Normalization to p a r t i c u l a t e nitrogen  weight involves  i n s t r u m e n t a t i o n t h a t i s u s u a l l y not a v a i l a b l e f o r f i e l d s t u d i e s . The  purpose of t h i s study was  s o l u b l e n i t r o g e n content  that  t o d e v e l o p a method f o r measuring could  be  carried  out  in  most  laboratories.  Extraction Blanks extraction except  for  Blanks (without method.  plants) There  ammonium,  was  which was  ammmonium e x t r a c t e d w i t h b o i l i n g without g r i n d i n g .  were run i n t r i p l i c a t e w i t h each no  detectable  contamination,  p r e s e n t a t l e s s than 1% of water  and  with  hot  the  ethanol  161  R e s u l t s and D i s c u s s i o n Extraction  of  the 'rubbery' t i s s u e c h a r a c t e r i s t i c of many  marine macrophytes, i s d i f f i c u l t and has a p p a r e n t l y the  investigation  of  accumulation  With p r a c t i c e ,  grinding  distichus  be  can  of  rapid  even  of a  discouraged  nitrogenous  tough  and e f f e c t i v e .  compounds.  thallus  like  F.  A p p r o x i m a t e l y 5 ml of  e x t r a c t i o n medium and 1 g sand f o r every 2 g of t i s s u e , was  the  most e f f e c t i v e c o m b i n a t i o n f o r g r i n d i n g . These  analyses  were  contain polysaccharides with  the  done  on  crude  extracts  and p h e n o l i c compounds  n i n h y d r i n and F o l i n r e a c t i o n s .  which  Therefore  of t h e amino a c i d a n a l y s i s and the p r o t e i n a n a l y s i s to  be n o n — q u a n t i t a t i v e  (Fig.  1C,D).  Little  on  purifying  such  interfere the r e s u l t s are  likely  i s known about the  e x t e n t of t h i s i n t e r f e r e n c e i n seaweed e x t r a c t s . required  which may  More  work  is  e x t r a c t s b e f o r e p r o t e i n and amino  a c i d r e s e r v e s can be e x p r e s s e d q u a n t i t a t i v e l y . Ammonium v o l a t i l i z a t i o n due t o the use media  was  not  (e.g., pouring  appreciable. hot  medium  of  hot  extraction  The use of c o l d water or e t h a n o l over  a  frozen  thallus)  d i d not  i n c r e a s e the amount of ammonium e x t r a c t e d .  L i q u i d n i t r o g e n made  the  i t d i d not n o t i c a b l y  grinding  process  much  easier,  but  i n c r e a s e the t o t a l n i t r a t e or ammonium e x t r a c t e d (see  treatment  4 i n F i g . 1). Three  factors  were  considered  when  usefulness  of v a r i o u s e x t r a c t i o n methods: the  ammonium,  nitrate,  amino  acids  determining total  amount  the of  and p r o t e i n e x t r a c t e d i n f o u r  s u c c e s s i v e e x t r a c t i o n s ; the e f f i c i e n c y of the i n i t i a l e x t r a c t i o n as a percentage  of  the  total  extracted  in  four  successive  A  Nitrate  3CM  S 20H  °  a  1  'ro  IS o  a> E  Io *  if)  10H  1  2  3  1  4  Fucus  2  3  4  Enteromorpha  Porphyra  F i g . 1. N i t r a t e ( A ) , ammonium ( B ) , amino a c i d s ( C ) , i n yumol.g wet wt" and p r o t e i n (D) i n mg.g wet wt" e x t r a c t e d from Fucus d i s t i c h u s , Enteromorpha i n t e s t i n a l i s , and Porphyra perforata w i t h b o i l i n g water w h i l e g r i n d i n g ( 1 ) , hot 80% e t h a n o l w h i l e g r i n d i n g ( 2 ) , b o i l i n g i n 80% e t h a n o l w i t h o u t g r i n d i n g (3), and freezing in l i q u i d nitrogen, f o l l o w e d by b o i l i n g water w h i l e grinding (4). T o t a l amount e x t r a c t e d i n four extractions {—•—-), f i r s t extraction C^*vJ3s), second e x t r a c t i o n ( — * — ) , t h i r d e x t r a c t i o n (,..,.x,,.,,), f o u r t h e x t r a c t i o n ( = o = ) . E r r o r bars r e p r e s e n t one s t a n d a r d " d e v i a t i o n , n=3. 1  1  :  ;  S o l u b l e ••• A m m o n i u m Content (jumol N H ^ . g w e t w t " ) 1  1  2  1  c n  on  Icn  i  ro  CO  im o  I'M.  3 - I o  3  i ro  CO  O  ro  CO  e9i  > 3  Soluble A m i n o Acid Content -1 (>jmol A m i n o A c i d s • g w e t w t ) ro  o  _JL_  o  T1 n  1/1  ro  > > o to  mrnmrnm Ml  m _» (D  3  ^  O  ro  d o  | •-j—i  -I  CO  "0  o  "D  *  7 / H i i ro O  CO  O  o  CP  o  o  Aimol Amino A c i d s • g wet wt"^  *9t  . : Soluble (mg  Protein Content  Protein • g wet w t " ro O  _J_  IS  1  )  8  ro "0 - J  o 3  r ro  166  e x t r a c t i o n s ; and the v a r i a b i l i t y of s e p a r a t e e x t r a c t i o n s . There  were  no  observable  methods w i t h regard  to  differences  either  the t o t a l  between amount  t h e four  of  nitrate  e x t r a c t e d o r t h e e f f i c i e n c y of n i t r a t e e x t r a c t i o n ( F i g . 1A). I n the  case  of  P.  perforata,  v a r i a b l e than e i t h e r of Grinding  grinding  t h e two  i n hot water was l e s s  ethanol  extraction  methods.  i n hot water was t h e n i t r a t e e x t r a c t i o n method used i n  subsequent s t u d i e s . Grinding from P. more  i n hot water e x t r a c t e d g r e a t e r amounts of ammonium  p e r f o r a t a and E.  effective  f o r F.  i n t e s t i n a l i s , but  The  much  p e r f o r a t a was e x c e l l e n t and  close  100% i n t h e f i r s t e x t r a c t i o n f o r a l l four methods ( F i g . 1B). variability  extractions,  was  with  also  low  one s t a n d a r d  The r e s u l t s from F.  i n a l l the  water. P.  perforata  d i s t i c h u s were more v a r i a b l e . ethanol  The e t h a n o l e x t r a c t s of E. p e r f o r a t a were dark  respectively.  P.  d e v i a t i o n of a p p r o x i m a t e l y ±6%.  More pigments were e x t r a c t e d by  and  was  d i s t i c h u s ( F i g . 1B). The e f f i c i e n c y of  ammmonium e x t r a c t i o n from P. to  ethanol  These  green,  pigments  than  by  i n t e s t i n a l i s , F. brown, could  and have  boiling  distichus  reddish-brown, affected  the  c o l o r i m e t r i c a n a l y s i s f o r ammonium, s i n c e ammonium was  estimated  by absorbance a t 600 nm.  preferred  extractant  but  Water  was  even then t h e a b s o l u t e  therefore  the  values are questionable.  T h i s appears t o be a n o t h e r case where t h e r e s u l t s of a n a l y s i s of crude e x t r a c t s must be i n t e r p r e t e d w i t h  caution.  One can e x t r a c t a t l e a s t t h r e e t i m e s t h e amount of per g wet wt from P. intestinalis.  The  p e r f o r a t a compared t o F.  nitrate  d i s t i c h u s o r E.  t o t a l ammonium e x t r a c t e d from P.  perforata  167  was  similar  from  E.  t o t h a t from F. intestinalis  d i s t i c h u s , but t h r e e  ( F i g . 1B).  P.  i n t e s t i n a l i s have s i m i l a r m o r p h o l o g i e s : and  a r e o n l y a few c e l l  times  perforata  they  have  layers i n thickness.  and E.  thin  F.  that  thalli  d i s t i c h u s , on  the o t h e r hand, i s v e r y t h i c k , tough and rubbery w i t h b l a d e s to  0.5 cm t h i c k .  be  influenced  The s o l u b l e n i t r o g e n c o n t e n t per g wet wt w i l l by  the polysaccharide  and water c o n t e n t .  a n a l y s i s showed t h a t %C based on d r y weight a p p r o x i m a t e l y 40% and v a r i e d from 20-30% and  P.  up  perforata.*  f o r F.  CHN  distichus i s  f o r E.  intestinalis  T h e r e f o r e i t would be u s e f u l t o n o r m a l i z e  s o l u b l e n i t r o g e n content t o t o t a l n i t r o g e n or t o t a l  protein  as  w e l l as p e r gram wet weight when comparing s p e c i e s . The  low s o l u b l e n i t r a t e c o n t e n t of E.  distichus uptake  suggests  r a t h e r than n i t r o g e n  Rosenberg as  that  an  these  i n t e s t i n a l i s and F.  s p e c i e s r e l y on r a p i d n i t r o g e n  pools  f o r t h e onset  and Ramus (1982) have r e f e r r e d t o t h i s  "opportunistic  growth  strategy".  In  of  growth.  characteristic contrast,  P.  p e r f o r a t a , w i t h i t s h i g h s o l u b l e n i t r a t e c o n t e n t was r e f e r r e d t o as a " r e s i s t a n t s p e c i e s " . The  nitrate  i n t h i s study were Rosenberg The  similar  Ramus  (1982)  presented  i n t e s t i n a l i s and F.  i n magnitude  to  were  more  those  by Rosenberg and Ramus (1982).  studies,  Bird  et  a l . (1982b)  by  tikvahiae. than  The c o e f f i c e n t and  measurements made by Rosenberg and Ramus (1982). these  found  reproducible  v a r i a n c e f o r n i t r a t e was ± 5% i n t h i s study  both  distichus  i n U l v a and G r a c i l a r i a  r e s u l t s i n t h e p r e s e n t study  those of  and  c o n t e n t of E.  have  ±  40% f o r  In contrast to reported the  n i t r a t e c o n t e n t of G r a c i l a r i a t i k v a h i a e t o be one t o two  orders  168  of magnitude s m a l l e r . The  total  amount  of  nitrate  l o n q i c r u r i s by Chapman and C r a i g i e pmol.g  f r e s h wt- .  ranged  l o n g i c r u r i s , have a g r e a t e r a b i l i t y t o  than o t h e r seaweeds such as F. P.  (1977)  from  Laminaria  from  d i s t i c h u s , E.  store  nitrate  intestinalis  and  perforata. I n v e s t i g a t i n g changes i n s o l u b l e n i t r o g e n c o n t e n t  or  50-150  I t would appear t h a t the k e l p s , f o r example  1  Laminaria  extracted  with  different  species could recently  be  nutrient  ve'ry  completed  Several  authors  soluble  nitrogen  C r a i g i e 1977;  physiological stresses in  fruitful.  the  have  or  first  Bird  in  Chaumont 1978;  be  a  limitation  in  the  of  B i r c h et a l . 1981). ability  nitrogen  during  times of  Marine macrophytes o f t e n undergo  nitrogen  competitive  summer  to  advantage  (Chapman  and C r a i g i e 1977;  seasonal.  under  Nutrient exposure of  fluctuating  understanding nitrogen.  of  in  The e c o l o g i c a l  only  determination  type.  macrophytes (Chapman and  G e r a r d 1982a); however, n i t r o g e n l i m i t a t i o n  by  have  fluctuations  1979;  interrupted  this  one  store  definite  nitrogen l i m i t a t i o n .  a l . (1982b)  seasonal  marine  i m p l i c a t i o n s are i n t e r e s t i n g . The would  et  investigation  investigated  content  w i t h time  supply to  changes  intertidal  on  a  daily  not  be  habitats  is  basis.  in internal soluble nitrogen  nitrogen how  air  in  need  regimes  intertidal  aids  in  macrophytes  Hanisak  The content  gaining  an  compete  for  169  References B i r d , K.B., Habig, C. and DeBusk, T. 1982. N i t r o g e n a l l o c a t i o n and s t o r a g e p a t t e r n s i n G r a c i l a r i a t i k v a h i a e (Rhodophyta). J . Phycol. 18:344-8. B i r c h , P.B., Gordon, D.M., and McComb, A . J . 1981. N i t r o g e n and phosphorus nutrition of Cladophora i n t h e Peel—Harvey e s t u a r i n e system, Western A u s t r a l i a . B o t . Mar. 24:381-7. -Buggeln, R.G. 1974. P h y s i o l o g i c a l i n v e s t i g a t i o n s of A l a r i a esculenta ( L . ) Grev. ( L a m i n a r i a l e s ) I . E l o n g a t i o n of t h e blade. J . Phycol. 10:283-8. Chapman, A.R.O. and C r a i g i e , J . S . 1977. Seasonal growth i n Laminaria lonqicruris : Relations with dissolved inorganic n u t r i e n t s and i n t e r n a l r e s e r v e s of n i t r o g e n . Mar. Biol. 40:197-205. Chaumont, J.P. 1978. V a r i a t i o n s de l a teneur en composes a z o t e s du Rhodymenia palmata Grev. B o t . Mar. 21:23—9. D a v i s , C O . , H a r r i s o n , P . J . , and Dugdale, R.C 1973. Continuous culture of marine diatoms under silicate limitation. I. Synchronized life c y c l e of Skeletonema c o s t a t u m . J . P h y c o l . 9: 17 5—8 0. G e r a r d , V.A. 1982. Growth and u t i l i z a t i o n of i n t e r n a l n i t r o g e n reserves by t h e g i a n t k e l p M a c r o c y s t i s p y r i f e r a in a l o w — n i t r o g e n environment. Mar. B i o l . 66:27—35. H a n i s a k , M.D. 1979. Nitrogen l i m i t a t i o n of Codiurn fragile s s p . * tomentosoides as d e t e r m i n e d by t i s s u e a n a l y s i s . Mar. Biol. 50:333-7. Lee, Y.P. and T a k a h a s h i , T. 1966. An improved c o l o r i m e t r i c d e t e r m i n a t i o n of amino a c i d s w i t h n i n h y d r i n . A n a l . Biochem. 14:71-7. Leggett-Bailey, J . 1967. M i s c e l l a n e o u s a n a l y t i c a l methods : e s t i m a t i o n of p r o t e i n F o l i n — C i o c a l t e u r e a g e n t . Ir\ Techniques i n P r o t e i n Chemistry 2nd ed. E l s e v i e r P u b l . Co., New York. 340-2. N i e l l , F.X. 1976. C:N r a t i o i n some marine macrophytes and i t s p o s s i b l e e c o l o g i c a l s i g n i f i c a n c e . B o t . Mar. 19:347-50. Rosenberg, G. and Ramus, J . 1982. E c o l o g i c a l growth s t r a t e g i e s i n t h e seaweeds G r a c i l a r i a f o l i i f e r a (Rhodophyceae) and U l v a sp. (Chlorophyceae): soluble nitrogen and reserve carbohydrates. Mar. B i o l . 66:251-9.  170  Appendix 2.  I_n V i t r o and  I_n V i v o N i t r a t e Reductase A c t i v i t y  Introduction Relatively  little  marine macrophytes.  is  known  Most  about n i t r o g e n metabolism of  studies  have  investigated  factors  a f f e c t i n g n u t r i e n t uptake r a t e s (Hanisak and H a r l i n 1978;  Harlin  1978;  storage  Topinka 1978)  (Chapman  and  Ramus 1982).  or s e a s o n a l  Craigie  1977;  fluctuations in nitrogen Gagne et a l .  Even l e s s r e s e a r c h has  1982;  Rosenberg  been conducted on  nitrogen  a s s i m i l a t i o n even though a s s i m i l a t i o n i s b e l i e v e d t o be the limiting  step  in  the procurement of n i t r o g e n  and p h y t o p l a n k t o n (Beevers and Hageman 1969;  o r g a n i c compounds.  The  atom i n n i t r a t e i s +5 and The  NO  it  +3  +1  -1  incorporated s t a t e of the N  S y r e t t 1962;  as f o l l o w s :  -3  2  1959;  +  Kesslerl964)  Work w i t h h i g h e r p l a n t s (Beevers and Hageman 1970)  and a l g a e ( H a t t o r i and Myers 1966;  Zumft  et  a l . 1969;  t h a t o n l y two are,  nitrate  enzymes c a t a l y s e  the  (NAD(P)H:  S y r e t t 1962)  reduction  nitrite  reductase  (NAD(P)H:  nitrate  nitrite  for  assaying  to  They EC  nitrite,  oxidoreductase to  1967;  suggests  oxidoreductase  1.6.6.4) which c a t a l y s e s the r e d u c t i o n of n i t r i t e Methods  Hewitt  sequence.  1.6.6.2), which c a t a l y s e s the r e d u c t i o n of n i t r a t e and  1969;  H a t t o r i and Myers  A p a r i c i o et a l . 1971;  reductase  plants  i n ammonium i t i s -3.  -—>NO "—>N202" —>NH20H—>NH  (Nicholas  is  oxidation/reduction  sequence suggested i n e a r l i e r work was  +5  i n higher  rate  D o r t c h 1982).  N i t r a t e i s reduced t o ammonium b e f o r e into  and  EC  ammonium.  these enzymes have been developed  (Hewitt  171  and N i c h o l a s 1964). nitrate  reductase  Both  iri vivo  have  and  ijn v i t r o  assays  for  been used i n h i g h e r p l a n t s , but r e c e n t  s t u d i e s have f a v o r e d the in  vivo  assay  (Streeter  and  Bosler  1972). The  first  (Schafer  et  report  of  a l . (1961)  pyrenoidosa.  The  Other  was  from  an  extract  of  NADPH  studies  was  used.  FAD  crude  extracts.  Losada and al.  on his  1963;  Osajima  A l l t h e s e e a r l y s t u d i e s were conducted w i t h Trebst  and  Burba  (1976) p a r t i a l l y and  purified  more  precise  the enzyme from C h l o r e l l a came from the work of colleagues  (Zumft  et  a l . 1969;  Aparicio  et  1971; Vega e t a l . 1971; Cardenas e t a l . 1972). Eppley  et  a l . (1969)  marine d i a t o m D i t y l u m phytoplankton  stimulated a  by  required  MgSO, .  several  other  the  addition  of  FAD,  Maximum a c t i v i t y a l s o r e q u i r e d  sulphur  dithiothreitol.  Later  reductase  and  NADH as an e l e c t r o n donor, was  reduced  nitrate  d e t e r m i n e d t h a t the enzyme from the  brightwellii  w i t h NADPH, was u n a f f e c t e d by  and  the  w i t h C h l o r e l l a and Scenedesmus found  n i t r a t e r e d u c t a s e from C h l o r e l l a p y r e n o i d o s a information  donor  stimulated  s i m i l a r r e q u i r e m e n t s " f o r NADH ( S y r e t t and M o r r i s and Y a m a f u j i 1964).  Chlorella  r e d u c t i o n r e q u i r e d NADH as an e l e c t r o n  and d i d not occur when activity.  n i t r a t e reductase a c t i v i t y i n algae  compound Thacker  activity  in  such and  as  inactive and  from  (1972)  was  phosphate  glutathione  Syrett  extracts  marine  or  measured  Chlamydomonas  r e i n h a r d i u s i n g reduced b e n z y l v i o l o g e n as an e l e c t r o n donor. There a r e v e r y few r e p o r t s of n i t r a t e r e d u c t a s e a c t i v i t y i n macroalgae  and no g e n e r a l l y a p p l i c a b l e a s s a y procedure has been  developed.  Araki et a l .  (1979) s t u d i e d  a  partially  purified  172  enzyme  extract  optimal al.  from  conditions  ( 1977)  Porphyra for iin  compared  Petroglossum conditions  nicaeense for  the  in  r e p o r t e d an i_n v i t r o angustifolia  but  an  ijn v i t r o vitro  but  and  they  vitro  assay also  yezoensis  not  Weidner and K i e f e r (1981) measured activity  in  Giffordia  the  they d e t e r m i n e d  assay.  Dipierro  et  i j i vivo  activities  in  not  assay.  for  did  did  and  determine  optimal  Haxen and Lewis enzyme  (1981)  from  Macrocystis  optimize  assay  conditions.  in  nitrate  vivo  reductase  m i t c h e l l a e but c o u l d not use an in v i t r o  assay. T h i s study compares the three  s p e c i e s of marine macrophytes,  major m a c r o a l g a l d i v i s i o n s . and  nitrate  in  vitro  assays  assays are d i s c u s s e d .  were  reductase  activities  of  one from each of the t h r e e  The optimum c o n d i t i o n s f o r i_n determined  vivo  and the m e r i t s of both  173  M a t e r i a l s and Methods Species Young n o n — r e p r o d u c t i v e Porphyra p e r f o r a t a distichus  L.,  and  Enteromorpha  J.  intestinalis  c o l l e c t e d i n February and March from a rocky  Ag.,  Fucus  (L.)Grev.  landfill  were  site  the  foot  Fig.  2 ) . T h e i r h e i g h t s above Canadian datum were 1.5,  2.0  m,  at  o f Balsam S t r e e t , Vancouver, B.C., Canada (Chapter 2;  respectively.  2.0  and  P l a n t s were c o l l e c t e d , c l e a n e d by g e n t l e  b r u s h i n g , and r i n s e d .in f i l t e r e d seawater  (0.45 pm), wrapped  Saran Wrap, and s t o r e d a t 8°C u n t i l they were assayed  in  (2-3 h ) .  Preincubations In of of  some experiments  f i l t e r e d seawater  p l a n t s (10 g) were p r e i n c u b a t e d i n 12 1  (0.45 yum) e n r i c h e d w i t h f/2 c o n c e n t r a t i o n s  phosphate, t r a c e m e t a l s and  1962)  and  30  vitamins  (Guillard  pM n i t r a t e or 15 pM ammonium.  and  Ryther  The c u l t u r e s were  kept i n a 12°C c o l d room w i t h an i r r a d i a n c e of 150 j j E . n r . s 2  a  12:12  light:dark  continuously  with  distichus t h a l l i assayed  cycle  each  was  were  preincubated  on  nitrate  on  stirred The F.  before  being  activity.  Assay  The  nitrate  mixtures  given  phosphate  and  reductase in  the  Tris  e x t r a c t i o n p r o c e d u r e s and r e a c t i o n  literature  compound  vary  considerably.  [Tris(hydroxymethyl)aminomenthane  b u f f e r s have been used f o r e x t r a c t i o n , sulfur  culture  a magnetic s t i r r i n g bar a t 120 rpm.  f o r n i t r a t e reductase  In V i t r o  and  _ 1  usually  with  such as c y s t e i n e or d i t h i o t h r e i t o l  an  Both HCl] added  ( H e w i t t and  174  N i c h o l a s 1964;  Eppley  enhances  vitro  al.  in  e t a l . 1969). nitrate  Since  reductase  phosphate  activity  (Eppley  1969), a phosphate b u f f e r was used i n t h i s s t u d y .  p y r r o l i d o n e (PVP) was added t o t h e e x t r a c t i o n phenolic  compounds  that  often  mixture  et  Polyvinyl t o bind  c o u l d i n t e r f e r e w i t h enzyme a c t i v i t y .  NADH was used as t h e r e d u c i n g agent. A p p r o x i m a t e l y 2 g (wet wt) of p l a n t t i s s u e was ground ml i c e c o l d phosphate b u f f e r c o n t a i n i n g 1.0 The  homogenate  supernatant One  was  centrifuged at  mM  2,000  i n 25  dithiothreitol.  xg f o r 5 min.  The  (crude enzyme e x t r a c t ) was decanted and kept on i c e .  m i l l i l i t e r of t h i s crude enzyme e x t r a c t  was  added  t o the  following: Concentration i n incubation mixture 0.2 ml of 0.1 M KN0 0.1  11.1 mM  3  ml of 0.18 M MgS0  10.0 mM  4  0.5 ml of 720 uM NADH  200.0 uM  T h i s r e a c t i o n m i x t u r e was kept a t room temperature The  reaction  was  f o r 30 min.  stopped by a d d i t i o n of 5 ml of i c e c o l d  95% e t h a n o l (v/v) and  0.2  ml  of  1.0  M  zinc  acetate.  resultant  suspension  was c e n t r i f u g e d a t 2,000 xg f o r 5 min and  decanted.  N i t r i t e was determined  i n the supernatant  The  by t h e  a d d i t i o n o f 1 ml s u l p h a n i l a m i d e (0.2% w/v) t o each tube f o l l o w e d within w/v). The  2  min by 1 ml N - ( 1 - n a p t h y l ) e t h y l e n e d i a m i n e (NNDD)(0.05%  The absorbance control  samples  of t h e s o l u t i o n a t 543 lacked  only  e x p r e s s e d as umol n i t r i t e produced hour,  and umol  nitrite  produced  NADH.  nm  was  measured.  Enzyme a c t i v i t y was  p e r gram wet wt of t i s s u e per per  gram  protein  see  175  (Leggett-Bailey  1967).  The e f f e c t of i n c u b a t i o n time and e x t r a c t volume on n i t r a t e reductase  activity  was  determined.  The  following  c o n d i t i o n s were t e s t e d t o determine t h e optimum; MgS0  4  vet  pH  assay  (7.0—8.8),  c o n c e n t r a t i o n s of 0-30 mM, PVP c o n c e n t r a t i o n s of 0-0.6 g.g wt  - 1  ,  NADH  c o n c e n t r a t i o n s of  0—400  yuM,  and  nitrate  c o n c e n t r a t i o n s of 0—10 mM.  In V i v o Assay *  T h i s assay was based upon al.  (1977).  n—propanol  t h e procedure  of  Dipierro  et  A p i e c e of t i s s u e (0.5 g) was p l a c e d i n 50 ml of 3% in  artificial  seawater  (Harrison  et  a l . 1980)  c o n t a i n i n g 0.1 ml c h l o r a m p h e n i c o l (0.5 mg.ml ) and 1 ml of  1.0  -1  M  KN0 , 3  providing  an e x t e r n a l n i t r a t e c o n c e n t r a t i o n of 20 mM.  The v i a l s were s t o p p e r e d and shaken shaker.  w/v)  solution Standard  T h i s sample was then mixed  sulphanilamide  solution  followed  (0.05% w/v). The absorbance a t 543 solutions  of  nitrite  0-8%  nm  1  was  ml  1  ml  NNDD  measured.  i n 3% n-propanol i n a r t i f i c i a l The  effect  n-propanol on n i t r a t e r e d u c t a s e a c t i v i t y was t e s t e d t o  determine the o p t i m a l conducted  with  by  seawater were a n a l y s e d t o o b t a i n a s t a n d a r d c u r v e . of  automatic  N i t r i t e c o n c e n t r a t i o n s i n t h e medium were m o n i t o r e d by  removing a 2 ml sample. (0.2%  i n t h e dark on an  on  P.  n—propanol  concentration.  Assays  p e r f o r a t a w i t h no n i t r a t e added i n an attempt  t o d e t e r m i n e " a c t u a l " i_n v i v o n i t r a t e r e d u c t a s e a c t i v i t y than " p o t e n t i a l "  were  activity.  rather  176  In V i v o and Tn V i t r o A c t i v i t y In P.  v i t r o n i t r a t e reductase  a s s a y s were done e v e r y 0.5 h on  p e r f o r a t a t h a l l i which were i n c u b a t i n g i n the i_n v i v o  assay  The i_n v i v o and rn v i t r o a c t i v i t i e s were compared.  medium.  Patterns  were  often  complex  and  f i t t e d by eye t o show g e n e r a l t r e n d s . was  a  very  the  kinetics  constants  A  therefore rectangular  c u r v e s were hyperbola  poor f i t f o r the enzyme k i n e t i c data and t h e r e f o r e curves  were  fitted  (Km) and t h e s t a n d a r d  by  eye.  Half—saturation  e r r o r s a s s o c i a t e d w i t h them were  d e t e r m i n e d from a l i n e a r r e g r e s s i o n of V/S v e r s u s V p l o t s .  177  Results  In V i t r o  Assay  The  ijQ_ v i t r o assay f o r n i t r a t e r e d u c t a s e gave r e p r o d u c i b l e  r e s u l t s f o r a l l three usually  One  standard  deviation  was  l e s s than ± 5% of t h e measured a c t i v i t y , based on t h r e e  samples. to  species.  The t o t a l amount of n i t r i t e produced  the  incubation  time  r e l a t i o n s h i p between the  ( F i g . 1).  nitrate  was  There  reductase  proportional was  a  activity  linear and  the  amount of enzyme e x t r a c t used ( F i g . 1 ) . The pH optimum ( F i g . 2) and  MgSO,  concentration  optimum ( F i g . 3) f o r i n v i t r o  nitrite  p r o d u c t i o n were d i f f e r e n t f o r a l l t h r e e s p e c i e s . The amount of PVP added d u r i n g enzyme nitrite  p r o d u c t i o n ( F i g . 4; T a b l e  extraction  1 ) . More PVP was r e q u i r e d t o  produce maximum n i t r a t e r e d u c t a s e a c t i v i t y i n F. i n P.  perforata.  a c t i v i t y i n E. The  PVP  inhibited  relationship  f o r F.  in  vitro  d i s t i c h u s than  nitrate  reductase  intestinalis. between  n i t r a t e s u p p l y were d e t e r m i n e d . (Km)  affected  d i s t i c h u s , P.  33.2 ± 23.0, 32.2  ±  8.2  detection  of  enzyme The  activity  66.5  NADH and  half-saturation  p e r f o r a t a and E. and  and  ±  57.0  constants  i n t e s t i n a l i s were yuM,  respectively  ( F i g . 5; T a b l e 1 ) . The  d i s t i c h u s and P. assay  nitrate  reductase  p e r f o r a t a when no n i t r a t e  was  added  in  F.  to  the  made t h e k i n e t i c s of n i t r a t e u t i l i z a t i o n more c o m p l i c a t e d  than those of NADH o x i d a t i o n ( F i g . 6 ) . were  activity  Since  their  activities  a p p r o x i m a t e l y o n e - h a l f maximum, t h i s s u g g e s t s t h e presence  of endogenous n i t r a t e of a p p r o x i m a t e l y 0.5 and 0.03  mM  in  F.  50|  B  75H  o z o  S  50H  25.  e T O3 a  i f  : i : /  25-  /  Enzyme Extract Volume  Time  (ml)  (min)  F i g . 1. T o t a l I n v i t r o n i t r i t e produced (jumol N0 ".g p r o t e i n " over time (min) T A ) , t h e r e l a t i o n s h i p between n i t r a t e r e d u c t a s e a c t i v i t y (jumol N0 ".g p r o t e i n " .h" and volume of enzyme e x t r a c t used (ml) ( B ) , f o r Fucus distichus ( • ) , Enteromorpha intestinalis (•) and Porphyra perforata (*). Error bars r e p r e s e n t one s t a n d a r d d e v i a t i o n , n=3. 1  2  1  1  2  CD  179  8  pH  F i g . 2. pH optimum f o r i n v i t r o n i t r a t e r e d u c t a s e a c t i v i t y (pmol N0 ' .g p r o t e i n " • h- ) (•), Porphyra f o r Fucus d i s t i c h u s perforata () and Enteromorpha" i n t e s t i n a l i s (•). E r r o r b a r s r e p r e s e n t one s t a n d a r d d e v i a t i o n , n=3. 1  2  A  1  F i g . 3. The r e l a t i o n s h i p between In v i t r o nitrate reductase activity (pmol N 0 . g p r o t e i n * . h " ) and t h e c o n c e n t r a t i o n o f added MgS0 (mM) f o r Fucus d i s t i c h u s (•), Porphyra p e r f o r a t a (*) and Enteromorpha i n t e s t i n a l i s (•). E r r o r bars r e p r e s e n t one standard d e v i a t i o n , n=3. _  2  4  1  1  Table 1. O p t i m a l c o n d i t i o n s f o r in v i t r o and jln v i v o n i t r a t e reductase a c t i v i t y i n Fucus d i s t i c h u s , Porphyra p e r f o r a t a , and Enteromorpha i n t e s t i n a l i s , one s t a n d a r d e r r o r of e s t i m a t e s of Km and maximal a c t i v i t i e s Cumol NO^'.g p r o t e i n " , h " ) f o r p l a n t s taken from the f i e l d and i n c u b a t e d i n t h e i r i v i y p assay medium are a l s o g i v e n . 1  1  Optimal Conditions for Nitrate Reductase Activity  Maximal Activity  In Vitro PVP  Species PH  g-g  -1  MgSO  In Vivo  NADH |iM  NO; mM  mM  Sat'n  Km  ±SE  Sat'n  Km  n-propanol +SE  %  time (h)  umol NO 2-g protein" .h 1  (Field) In Vitro  -1  Induction  In Vivo  In V1vo  F. distichus  8.2  0.2  3-6  >100  35  23  >1.0  0.5  0.1  3-4  3-4  0  0  30  E.  8.0  0.0  1.0  >100  43  27  >2.0  0.25  0.05  5  1  160  5  25  8.5  0.025  1-4  > 75  40  9  >0.05  0.025  0.009  3-4  1  50  60  100  Intestinalis  P. perforata  1  1  0.1  1  0.2  1—  —  0.3  0.4  ~7~T  1  0.5  PVP (c) • g wet wt" ) F i g . 4. The r e l a t i o n s h i p between in vitro nitrate reductase a c t i v i t y (pmol N0 ".g p r o t e i n " .h" T~and the amount of p o l y v i n y l p y r r o l i d o n e ( P V P ) added d u r i n g e x t r a c t i o n (g PVP.g wet w t " ) f o r Fucus d i s t i c h u s (•), Porphyra perforata ( ) and Enteromorpha intestinalis C"). E r r o r bars r e p r e s e n t one s t a n d a r d d e v i a t i o n , n=3. 1  1  1  2  1  A  0.6  for Enteromorpha  r  150  MOO  ^50  400  200 NADH  (JUM )  F i g . 5. The r e l a t i o n s h i p between in v i t r o n i t r a t e r e d u c t a s e a c t i v i t y (pmol NO " . g p r o t e i n . h " ) and t h e c o n c e n t r a t i o n of NADH (uM) i n the assay m i x t u r e f o r Fucus d i s t i c h u s (•), Porphyra perforata (*), and Enteromorpha i n t e s t i n a l i s (•). E r r o r bars r e p r e s e n t one s t a n d a r d d e v i a t i o n , n=3 - 1  2  1  for Enteromorpha  I.  *  f.  ,  •  2  A  NO  (mM )  -  3  .  r—  ,  6  8  10  F i g . 6. The r e l a t i o n s h i p between in vitro nitrate reductase activity (/umol N0 ".g p r o t e i n ' .Ir ") and n i t r a t e c o n c e n t r a t i o n (mM) i n t h e assay m i x t u r e f o r Fucus d i s t i c h u s ( • ) , Porphyra perforata ( ) and Enteromorpha i n t e s t i n a l i s ( • ) . E r r o r b a r s r e p r e s e n t one s t a n d a r d d e v i a t i o n , n=3. 1  2  A  71  185  distichus values  and  P.  perforata,  respectively  f o r n i t r a t e reductase a c t i v i t y  p e r f o r a t a , and E. 0.26 ± 0.05 mM, Maximum  f o r F.  This  P.  r e s p e c t i v e l y (Table 1 ) .  activities  was  were done i n  distichus,  i n t e s t i n a l i s were 0.5 ± 0.2, 0.03 ± 0.01, and  under  optimum  conditions v a r i e d g r e a t l y with previous 1).  ( F i g . 6 ) . The Km  and  March  and  nutrient  e s p e c i a l l y t r u e f o r F.  February  assay  extraction  supply  distichus.  when  nitrate  (Table  Experiments  and  ammonium  concentrations  a t t h e c o l l e c t i o n s i t e f l u c t u a t e d around 20—40 /uM  and  respectively.  E.  3-10  pM  I f the t h a l l i of P.  i n t e s t i n a l i s were examined i m m e d i a t e l y a f t e r c o l l e c t i o n they  showed f a i r l y c o n s i s t e n t maxima of i_n v i t r o and  p e r f o r a t a and  1.0-1.5  of  1—2  /umol N0 ".g wet w f ' . h " , or 50 and 160 /umol N0 .g 1  2  protein .h" , - 1  activities  1  2  respectively.  I f the p l a n t s were s t o r e d  at  8°C  wrapped i n Saran Wrap, a c t i v i t y dropped by 50% w i t h i n 8 h. Freshly  collected  F.  d i s t i c h u s showed no i n v i t r o o r i n  v i v o n i t r a t e reductase a c t i v i t y .  The p o s s i b i l i t y t h a t a n i t r a t e  r e d u c t a s e i n h i b i t o r p r o t e i n was p r e s e n t seemed u n l i k e l y addition  of  casein,  which  should  m i n i m i z e t h e e f f e c t of t h e  i n h i b i t o r p r o t e i n (Lewis e t a l . 1982) d i d not produce n i t r a t e reductase incubated  in  activity.  ammonium-free  because  However,  when  n i t r a t e enriched  the  detectable  plants  were  seawater, n i t r a t e  r e d u c t a s e a c t i v i t y was induced a f t e r 36—48 h. Preincubation  i n n i t r a t e enriched  seawater f o r  induced j j i v i t r o n i t r a t e r e d u c t a s e a c t i v i t y i n P. then  activity  started  to  decline (Fig. 7).  ammonium i n h i b i t e d n i t r a t e r e d u c t a s e a c t i v i t y .  three  days  p e r f o r a t a and  Preincubation i n A 30 yuM  nitrate  p u l s e was added t o t h e ammonium c u l t u r e on day 3 and t h i s h a l t e d  Nitrate Reductase  WO  n e HwO 1 O cn  O l O rr • iD  S tr — 3 • i DJ o •1 t-h 3 -» 01 M - • rr 3- -< n 3 "I 3 tt> H" OJ — TJ rt rt  i-l i-t (D  0> DJ CO rt" 3 rt  o 3  <  O rr t-h O  O < 1 tOJ 3 0>  cn w- i-t T3 rr 3" t-t o co a> I-i •< PJ rr Q J i-t rr OJ Qj — DJ 0) 3 fl> — Qj Qj Ul T3 OJ ^ n> ft i-l r t J2 i-« a» CbO 2 l-h Q J - O C »-t o Q J rr OJ rr < a> 3 rr OJ M3 OJ CO OJ PJ O fl> rr 3 3 O (-•• 3 »-•- C O O C M PJ 3 3 3 rr o C rr C —• 1 3 3 Qj HIt OO O • rr ro  1  •"I rr>^ C • n  o 3  C  o 3 I o  (D  o  >  QJ  OJ QJ  ^  M  3  QJ  pj ^<  CA)  .  2:  cn O C O • £ 3 uQ  981  —i  3*  10  A c t i v i t y (jumol NCr . g P r o t e i n " , h" ) 1  1  187  the d e c r e a s e i n n i t r a t e r e d u c t a s e a c t i v i t y .  In V i v o Assay The the  i r i v i v o n i t r a t e r e d u c t a s e a c t i v i t y depended on t h e time  plant  had  been  Table 1). Nitrate maximum  after  i n t h e in v i v o n-propanol medium ( F i g . 8;  r e d u c t a s e a c t i v i t y was induced and reached  approximately  1  h  i n t e s t i n a l i s , and a f t e r 3-4 h f o r F. 1). E.  The  increase  f o r P.  first  uptake  p e r f o r a t a and E.  distichus  measurement.  distichus  E.  p e r f o r a t a and based  on t h e  perforata  and  F.  and a 5% n—propanol a d d i t i o n gave maximum a c t i v i t y i n  intestinalis  ( F i g . 8; T a b l e 1 ) .  In v i v o a c t i v i t y was d e t e c t e d i n P. was absent from t h e i n c u b a t i o n medium. thalli  Table  The a d d i t i o n of 3-4% n-propanol by  volume produced maximum a c t i v i t y i n b o t h P. distichus  ( F i g . 8;  v a r i e d from 0.5 t i m e s f o r P.  i n t e s t i n a l i s t o 4.0 t i m e s f o r F.  a  had  internal  c o u r s e of " a c t u a l "  nitrate  p e r f o r a t a when n i t r a t e This suggests  reserves.  (no n i t r a t e added) and  activity  the  F i g u r e 9 shows a time "potential"  added) i j i v i v o n i t r a t e r e d u c t a s e a c t i v i t y i n P. "potential"  that  (nitrate  perforata.  The  reached a maximum a t t h e same time as t h e  " a c t u a l " a c t i v i t y , but t h e l e v e l of " p o t e n t i a l " a c t i v i t y was 30% g r e a t e r ( F i g . 9 ) . I f t h e medium w i t h o u t n i t r a t e with  nitrate  ( a t 2.5  h)  was  perturbed  a f t e r a c t i v i t y peaked, t h e a c t i v i t y  i n c r e a s e d almost t o t h e l e v e l of p o t e n t i a l a c t i v i t y a t t h i s (Figs.  time  9,10).  The R e l a t i o n s h i p Between I_n V i v o and Tn V i t r o In v i t r o and in v i v o a c t i v i t i e s of P.  Activity  perforata  submersed  188  Time  (hours)  F i g . 8. The r e l a t i o n s h i p between ~ i h v i v o n i t r a t e r e d u c t a s e activity (jumol NCT .g p r o t e i n * . h T and the p e r i o d of time (hours) i n c u b a t e d i n the i_n v i v o assay medium; w i t h 0.0%—n--, 0.5%—•—, 1.0%—x--, 2.0%--o-3 . 0 % - ~ 4 . 0 % — A — 5.0% — 6 . 0 % —•— , or 8.0% v n - p r o p a n o l , for Fucus distichus ( A ) , Porphyra perforata ( B ) , and Enteromorpha i n t e s t i n a l i s ( C ) . E r r o r bars r e p r e s e n t one s t a n d a r d d e v i a t i o n , n=3. 1  _T  f  F  681  Nitrate  Reductase  A c t i v i t y (timol  N O " - a P r ot e m  - 1  2 I-i  a> l-l fD cn fD 3 rt  3*  i-i  O  O i i Q  '  O  w-  C rr O .  <-! fD cn >-•• • <x> ^ 3 1  3 311- 3^ 3 O. i|3 fD fD C O  — cn cr OJ rr rr 0>  i-t  fD <  3 fD 3  a a OJ  I-  pj cr  o  o  <-l 3 cn 3hj rr 3 3"  l-t  a  QJ  (D <  r r TJ ri  OJ cr fO r r fD fD fD n-  OJ  rr  n  SI  O  3  n  OJ  3  0J  II  cn  «  fD fD fD 3 n  Qj  C  o o  3 fD  in OJ 3 cn Q J fD cn  C OJ  I-  o  OJ  c  3" r r  in  n- o  3 fD fD  a  M-TJ  M -  < CO-I  rr  C fD •<  3 n  ro  rr rt OJ  QJ  OJ  OJ  3  air a  3 o >a n o M o  O  i-f  fD Z 3 CTrt O rt i-i g . OJ cn fD i Q I—'  061  H  r-  -0--I  0D  o  ro O  •h  - 1  )  ;80O cn O CvJ z  o £  NO  3  ci deled-  u  <40i OJ  u "O 1)  or 4J  -t-j  o  Time 2 (hours ) 3 F i g . 10. The r e l a t i o n s h i p between a c t u a l i n vivo nitrate (umol NO" .g p r o t e i n ~ . h ") i n Porphyra reductase a c t i v i t y p e r f o r a t a and the p e r i o d of time (hours) i n c u b a t e d i n t h e i n v i v o assay medium. A p u l s e of 0.2 M KN0 was added a f t e r 2.5h. E r r o r bars r e p r e s e n t one s t a n d a r d d e v i a t i o n , n=5. 1  1  3  771  192  in  the  i^n v i v o , n—propanol  medium f o r 0—4 h were v e r y s i m i l a r ,  both r e a c h i n g a maximum a f t e r 0.5-1.0 h, and w i t h i n 2.5 h ( F i g . 1 1 ) .  dropping  to  zero  3  Nitrate fD <  3  0) rt  O 3 -  I  •f  3K 3 n 3 DJ  3 II OJ <  O  fO  a»  »-•• -3 r t 3" n fD QJ  i-t r t  PJ  ft  ro  — I• OJ CO pj i-t OJ r t ro >< OJ O  in 3  a c o  3 cn r t 3 3" Co 0) DJ  a> a c rt  CO  fD  •a  3" cr fD DJ fD Ci r t TJ r t < fD fD rt n < fD 3 O i-t O r t  3  tr i-t  i-h~n 3  CO  O  I-l r t fD  n 3 i-t fD  CO  fD 3 rt  O 3 fD  n> z; O  o iQ c  rt »0 —'U) J n  co  —' o  rt fD  M-  PJ  3  a  H "  CO 3 3 | i - .  rt O DJ C 3 Co r r PJ  i-t  a  OJ  rt fD  •  >|3 -  tr < i M - <  OJ-HO  £61  -  * 41  Reductase  Activity  -1  (/imol NO" • g P r o t e m " • h ) 1  194  Discussion In V i t r o  Assay  The  extraction  and  in v i t r o assay procedure used i n t h i s  study gave a r e p r o d u c i b l e and e a s i l y d e t e c t a b l e nitrate  reductase  p e r f o r a t a and E. Macrocystis  activity.  The  measurement  activities  found  i n t e s t i n a l i s were s i m i l a r t o those  angustifolia  distichus  and  by Haxen and Lewis (1981), but h i g h e r  i n Petroglossum  nicaeense  N 0 " . g ~ . h " ) by D i p i e r r o e t a l . (1977). 1  i n P.  found f o r  than t h e n i t r a t e r e d u c t a s e a c t i v i t i e s found i n t h i s F.  of  study f o r (0.24 yumol  The a c t i v i t y l e v e l s i n  1  2  t h i s study cannot be compared t o t h e d a t a of A r a k i e t a l . (1979) because they normalized  used  a  partially  purified  enzyme  extract  and  a c t i v i t y t o t h e p r o t e i n c o n t e n t o f t h a t e x t r a c t ; nor  can they be compared t o t h e work of Weidner  and  Kiefer  because they s u p p l i e d t h e i r c u l t u r e s w i t h v e r y h i g h  (1981)  (millimolar)  c o n c e n t r a t i o n s of n i t r o g e n . The  nitrate  p e r f o r a t a and E. from  higher  reductase  l e v e l s found i n t h i s study f o r P.  i n t e s t i n a l i s a r e comparable t o those  plants  reported  ( N i c h o l a s e t a l . 1976; Duke and Duke 1978;  Heuer and P l a u t 1978; Mann e t a l . 1978). The p l a n t s t e s t e d  in this  study  were  growing  i n high  ambient c o n c e n t r a t i o n s of both ammonium and n i t r a t e (4 and 17 pM respectively).  The n i t r a t e r e d u c t a s e a c t i v i t y w i l l depend both  on p r e v i o u s n i t r a t e s u p p l y and t h e presence of ammonium which i s often u t i l i z e d p r e f e r e n t i a l l y  ( D ' E l i a and  DeBoer  and  and  1978).  Wheeler  1978; Hanisak  Harlin  i n h i b i t i o n o f n i t r a t e r e d u c t a s e a c t i v i t y i n P. also  been  observed  i n higher  plants  1978;  Haines  The ammonium p e r f o r a t a has  (MacKown e t a l . 1982),  195  p h y t o p l a n k t o n ( M o r r i s and S y r e t t 1965; H i p k i n and S y r e t t and  t h e brown macroalga G i f f o r d i a m i t c h e l l a e  1977),  (Weidner and K i e f e r  1981). Freshly  collected  F.  d i s t i c h u s d i d not show any n i t r a t e  reductase a c t i v i t y i n February. incubation  in  A c t i v i t y could  be  induced  n i t r a t e e n r i c h e d , ammonium-free seawater.  /uM ammonium p r e s e n t i n t h e seawater a t the c o l l e c t i o n apparently  sufficient jaM  a l t h o u g h 17 mechanism  to  nitrate  responsible  inhibit  was  also  for this  to  E.  by 50% i n E.  uptake i n F.  intestinalis.  d i s t i c h u s and P. study  were ' d i f f e r e n t  and  biochemical r e p r e s s i o n or  distichus  seemed  more  Ammonium  inhibited nitrate  perforata show  (Chapters 1 and 2 ) .  that  the  optimum  assay  intestinalis  and  P.  perforata  v e r y d i f f e r e n t . When t h e pH optimum f o r n i t r a t e r e d u c t a s e  a c t i v i t y i n P.  p e r f o r a t a was used i n t h e E.  or v i c e v e r s a , a c t i v i t y was reduced by optimum  pH  found  optimum f o r P. F. and  The  f o r each s p e c i e s t e s t e d (Table 1 ) .  The jjn v i t r o pH optima f o r E. were  was  i n t e s t i n a l i s , but had no e f f e c t on n i t r a t e  The r e s u l t s of t h i s conditions  site  ammonium i n h i b i t i o n of n i t r a t e r e d u c t i o n than P.  p e r f o r a t a and uptake  present.  F.  The 4  reductase a c t i v i t y  induction  i n h i b i t i o n requires f u r t h e r study. sensitive  nitrate  by  yezoensis  much  as  from  intestinalis  50%.  The  p e r f o r a t a i n t h i s study was a l s o the ( A r a k i e t a l . 1979).  d i s t i c h u s e x t r a c t s were i n a c t i v e u n l e s s PVP  required  extracts  f o r P.  as  i n t e s t i n a l i s assay  was  added  much more PVP t o o b t a i n maximum a c t i v i t y than t h e P. enzyme  perforata. was  On  the  other  i n h i b i t e d , by PVP.  s u b s t a n c e s and Fucus has been r e p o r t e d  hand  the  E.  PVP b i n d s  phenolic  to contain large  amounts  196  of t h e s e compounds (Ragan 1981). The  three  species  had  appreciable  reductase a c t i v i t y without the a d d i t i o n requirement al.  of  1969).  n i t r a t e reductase  perforata  added. F.  + +  supplies.  was  severely  enzyme.  greater a f f i n i t y for Mg secondly,  P.  ++  activity  of  i n h i b i t e d when 10 mM of MgS0  was  might  activity  reductase  4  be e x p l a i n e d i n two ways: i n P.  perforata  had a  and was more s e n s i t i v e t o e x c e s s  + +  perforata  The amount of M g  + +  Mg  i s w e l l documented (Eppley e t  Nitrate  This  f i r s t l y , n i t r a t e reductase  Mg .  The  4  T h i s was t h e optimum c o n c e n t r a t i o n f o r t h e assay of the  distichus  or  MgS0 .  nitrate  I t i s p o s s i b l e t h a t t h e a c t i v i t y d e t e c t e d a r o s e from  use of i n t e r n a l M g P.  of  in vitro  had  ++  g r e a t e r i n t e r n a l r e s e r v e s of  added t o t h e in v i t r o  + +  Mg ;  assay  was  500  ^ug.g d r y w t " . The amount of Mg o c c u r r i n g n a t u r a l l y i n seaweeds 1  varies  from  1,900-66,000  yug.g  dry wt"  1  (DeBoer 1981) and i s  c e r t a i n l y s u f f i c i e n t t o i n f l u e n c e n i t r a t e reductase The NADH h a l f - s a t u r a t i o n c o n s t a n t s f o r tested  activity.  a l l three  species  i n t h i s study were s i m i l a r t o those found i n o t h e r a l g a e  (Eppley e t a l . 1969; A r a k i e t a l . 1979). The h a l f - s a t u r a t i o n c o n s t a n t s f o r n i t r a t e o b t a i n e d f o r distichus for  and  Porphyra  (Eppley  et  E.  i n t e s t i n a l i s were s i m i l a r t o those  yezoensis a l . 1969),  (Araki and  et  a l . 1979),  The h a l f - s a t u r a t i o n c o n s t a n t f o r P.  order  of  lower.  r e d u c t a s e w i t h such a h i g h  reported  phytoplankton  h i g h e r p l a n t s (Beevers and Hageman  1969).  magnitude  F.  Why  affinity  this  perforata species  for nitrate  has  was one nitrate  i s unknown.  However, p r e l i m i n a r y s t u d i e s (not r e p o r t e d here) have shown t h a t the  n i t r a t e uptake k i n e t i c s of P.  p e r f o r a t a do not have such a  197  h i g h Ks  value.  There are nitrate  (Chapman  Ramus 1982) in  several reports  I t was  vitro  nitrate  of  and  Craigie  not  surprising  marine  1977;  macrophytes  G e r a r d 1982a; Rosenberg  therefore to f i n d i n v i v o  The  and  i n t e s t i n a l i s i n March were a p p r o x i m a t e l y 7.5,  4.0  n i t r a t e c o n t e n t of F.  .umol N0 ".g wet  was  0.3  pmol N0 ".g wet 2  distichus,  respectively  1  the  umol wt"  1  N0 ".g wet  f o r F.  n i t r a t e c o n t e n t of P.  v i t r o assay.  30.0  and  The  size  Only a  10  p e r f o r a t a appears t o be  available  t h a l l u s has  been ground f o r  I f a l l i n t e r n a l n i t r a t e was  enhance a c t i v i t y .  not  p e r f o r a t a and  fraction  nitrate  in  perforata  small  saturated  would  f o r P.  1  distichus.  r e d u c t a s e would be  the  P.  (Appendix 1).  wt"  2  f o r n i t r a t e r e d u c t i o n a f t e r the in  and  apparent i n t e r n a l n i t r a t e supply u t i l i z e d by the i n v i t r o  enzyme  of  wt" ,  2  of the  and  r e d u c t a s e a c t i v i t i e s i n the absence of added  nitrate. E.  storing  with  available,  nitrate  and  T h i s was  the  nitrate  additions  not  the  of  case f o r  v i t r o or j j i v i v o a s s a y .  In V i v o Assay Some workers ( D i p i e r r o 1972)  have  recommended  et  the  p r e s e r v e s a l l systems i n the realistic  a l . 1977;  rn  vivo  e s t i m a t i o n of a c t i v i t y .  The  time.  The  enhancement  n i t r a t e and  Bosher  ir\  and  gives  a  more  v i v o time c o u r s e d a t a  drawback:  activity  varied  i_n v i v o assay c o n d i t i o n s (an n-propanol medium)  i n c r e a s e d b o t h _in_ v i v o and This  and  enzyme assay because i t  natural state,  from t h i s study i l l u s t r a t e s a major with  Streeter  was  iri v i t r o n i t r a t e reductase  probably  due  activity.  to h i g h c o n c e n t r a t i o n s of  n — p r o p a n o l , which enhanced d i f f u s i o n of n i t r a t e  into  198  the t i s s u e .  The  enhancement  varied  for  with  required  the s p e c i e s .  w i t h each s p e c i e s .  be  very  thallus,  whereas  distichus  P.  to  produce  3  A p l a u s i b l e explanation  vivo  which  activity  but  also  these  of  n-propanol  to  it  The  on  there  by  nitrate  they  presence  of  with  cell  E.  added)  the This  reductase the  n—propanol  damage. for  the  between  these  appeared  An a d d i t i o n of other  at  processes,  intestinalis  two  3—4%  species.  1% as the p r e f e r a b l e n-propanol bleaching  with  higher  gave no i n d i c a t i o n t h a t t h i s was  the  maximum a c t i v i t y .  were  i n the absence of added  internal  nitrate  nitrate  supplies.  d i f f e r e n c e between " p o t e n t i a l " ( n i t r a t e added) and nitrate  F.  of  the medium.  optimum b a l a n c e  tissue  In v i v o n i t r i t e p r o d u c t i o n that  in  measurement  into  interferred  activity  c o n c e n t r a t i o n producing  suggests  The  n—propanol  based but  detected  very  r a t e s ( D i p i e r r o e t a l . 1977); but  e t a l . (1977) r e p o r t e d  concentrations,  rubbery  time.  from  only  s p e c i e s dependent.  maximized  concentration  thick  thallus  the inward d i f f u s i o n of n i t r a t e and  diffusion  susceptable  a  was  released  nitrate reduction.  two e f f e c t s was  Dipierro  was  nitrite.  concentrations,  including  different  i n t e s t i n a l i s are  activity  l i m i t e d not  by  diffusion  affected higher  and E.  determined  nitrite  r e l e a s e of n i t r i t e was  outward  is  s p e c i e s took l e s s than h a l f t h i s  in  at  of i n c u b a t i o n i n the i j i v i v o medium; the  In v i v o a c t i v i t y was rate  distichus  perforata  h  maximum  type and t h i c k n e s s of the  F.  Maximum i r i  after  o t h e r two  The  important.  thin blades.  less  time  t h i s i s t h a t n i t r a t e d i f f u s e d i n t o the t i s s u e  rates may  p e r i o d of  "actual"  i n v i v o a c t i v i t y has been used as an  The (no  indication  199  of n i t r a t e p o o l s i z e i n h i g h e r p l a n t s The  observation  perturbation  of  to  increased  the  assay  suggests  that  the  same f o r  both  assays.  limited  by  activity  medium  amount  However,  n i t r a t e supply.  activity.  If  et  a l . 1979).  following  lacking  a  nitrate  nitrate  (Fig.11)  of n i t r a t e r e d u c t a s e enzyme was the  "actual"  activity  the was  On the o t h e r hand, t h i s i n c u b a t i o n  i n n i t r o g e n f r e e _iri v i v o medium reductase  (Breteler  increased  the  actual  nitrate  a c t u a l n i t r a t e reductase a c t i v i t y  l i m i t e d by n i t r a t e supply then i n c u b a t i o n i n the in v i v o  was  medium  m  of  n—propanol  and  n i t r a t e supply to increases  the  membrane  intracellular nitrate  nitrogen nitrate  pools  enzyme.  seawater  reductase  permeability  nitrate  reductase  free  which  not  must i n c r e a s e the  enzyme. may  N—propanol  cause  normally  leakage of  available  The j j i v i t r o assay r e s u l t s  to  the  f o r P.  p e r f o r a t a suggest t h a t o n l y a s m a l l p o r t i o n of the i n t r a c e l l u l a r n i t r a t e p o o l was  available for reduction.  ^  In s p i t e of the d i f f u s i o n problems i n h e r e n t i n the jLn assay  vivo  and the u n c e r t a i n t y of the e f f e c t of e x t r a c t i o n on enzyme  systems i n the i j i v i t r o similar  activities  ( F i g . 11). measure  of  assay,  for  P.  it  nitrate  encouraging  perforata  Both assays appeared t o the  was  reductase  be  a  using  both  reasonably  to  find assays  accurate  a c t i v i t y , but the a c t i v i t y  measured i n the i n v i v o assay depends on the p e r i o d of time p l a n t s a r e submerged i n the in v i v o n—propanol An f o r the uptake  the  medium.  a c c u r a t e assay f o r n i t r a t e r e d u c t a s e a c t i v i t y i s needed understanding and  of  assimilation.  environmental  effects  on  nitrogen  Marine macrophytes can s t o r e n i t r a t e  (Chapman and C r a i g i e 1977; Rosenberg and  Ramus  1982;  Appendix  200  1).  Thus,  equal to  the  the  amount  amount  of n i t r a t e taken up i s not n e c e s s a r i l y  assimilated.  A  direct  measurement  n i t r a t e r e d u c t a s e a c t i v i t y i s n e c e s s a r y t o determine nitrate  the r a t e of  assimilation.  The  measurement  macrophytes Nitrate  is  and  assay.  ± 5—15%  ±  of many p h y s i o l o g i c a l p r o c e s s e s i n marine  subject  reductase  d e v i a t i o n was assay  of  20-30%  to  activity  large is  inter-plant no  exception;  variability. one  standard  of the measured a c t i v i t y f o r the in of  vitro  the measured a c t i v i t y f o r the i r i v i v o  N i t r a t e r e d u c t a s e a c t i v i t y i n h i g h e r p l a n t s can a l s o  be  v a r i a b l e (Deane-Drummond and C l a r k s o n 1979; Lewis e t a l . 1982). This  study  is  the  first  direct  comparison  r e d u c t a s e a c t i v i t y i n i n t e r t i d a l macrophytes.  The  of n i t r a t e  results  show  the need t o e s t a b l i s h optimum assay c o n d i t i o n s f o r each s p e c i e s . The  usefulness  of the i r i v i v o assay i s l i m i t e d , because of the  e f f e c t of t h e i n c u b a t i o n medium on n i t r a t e Activity  can  fluctuate  drastically  with  reductase time.  The  activity. in v i t r o  a s s a y , on the o t h e r hand, i s c o n s t a n t w i t h time and can g i v e estimation field.  of enzyme a c t i v i t y of p l a n t s taken d i r e c t l y  from  an the  201  References A r a k i , S., Ikawa, T., Oohusa, T., and N i s i z a w a , K. 1979. Some enzymic properties of n i t r a t e r e d u c t a s e from Porphyra y e z o e n s i s Ueda f . Narawaensis Miura. Bull. J a p . Soc. Sci. Fish. 45:919-24. A p a r i c i o , P . J . , Cardenas, J . , Zumft, W.G., Vega, J.M., H e r r e r a , J . , Paneque, A. and Losada, M. 1971. 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Reassessment assay f o r n i t r a t e reductase i n leaves. 43:306-12.  o f t h e in i o Physiol. Plant. v  y  H e w i t t , E . J . 1970. P h y s i o l o g i c a l and b i o c h e m i c a l factors c o n t r o l l i n g the a s s i m i l a t i o n of inorganic nitrogen supplies by p l a n t s . In K i r k b y , E.A. (Ed.) N i t r o g e n N u t r i t i o n o f t h e P l a n t , U n i v . Leeds. Leeds. 78-103. H e w i t t , E . J . and N i c h o l a s , D.J.D. 1964. Enzymes o f i n o r g a n i c nitrogen metabolism. Modern Methods o f P l a n t Analysis 7:67-172. Kessler, E. 1964. N i t r a t e a s s i m i l a t i o n by p l a n t s . Plant Physiol. 15:57-72.  Ann. Rev.  L e g g e t t - B a i l e y , J . 1967. M i s c e l l a n e o u s a n a l y t i c a l methods : e s t i m a t i o n o f p r o t e i n F o l i n - C i o c a l t e u r e a g e n t . I_n Techniques i n P r o t e i n C h e m i s t r y 2nd ed. E l s e v i e r P u b l . Co., New York. 340-2. L e w i s , O.A.M., James, D.M., and H e w i t t , E . J . 1982. N i t r o g e n assimilation i n b a r l e y ( Hordeum v u l g a r e L. Cv. Mazurka ) i n response t o n i t r a t e and ammonium n u t r i t i o n . Ann. B o t . 49:39-49. Nicholas,  D.J.D.  1959.  Metallo-enzymes  in  nitrate  203  micro—organisms.  Symp.  Soc.  Exp.  Biol.  13:1—13.  N i c h o l a s , J.C., H a r p e r , J.E., and Hageman, R.H. 1976. Nitrate reductase a c t i v i t y i n soybeans ( G l y c i n e max (L.) Merr.) I . E f f e c t s of l i g h t and t e m p e r a t u r e . P l a n t P h y s i o l . 58:731-5. O s a j i m a , Y. and Y a m a f u j i , K. 1964. R e d u c t i o n of ammonium by enzymes i s o l a t e d from green a l g a e . 27:129-140.  n i t r a t e to Enzymologia  Rosenberg, G. and Ramus, J . 1982. E c o l o g i c a l growth s t r a t e g i e s i n the seaweeds G r a c i l a r i a f o l i i f e r a (Rhodophyceae) and U l v a sp. (Chlorophyceae): soluble nitrogen and reserve c a r b o h y d r a t e s . Mar. B i o l . 66:251-9. S c h a f e r , J . , Baker, J.E., and Thompson, J . F . 1961. A C h l o r e l l a mutant l a c k i n g n i t r a t e r e d u c t a s e . Amer. J . B o t . 48:896—9. S t r e e t e r , J.G. and B o s l e r , M.E. 1972. Comparison of i_n v i t r o and in v i v o a s s a y s f o r n i t r a t e r e d u c t a s e i n soybean l e a v e s . Plant Physiol. 49:448-50. S y r e t t , P . J . 1962. Nitrogen a s s i m i l a t i o n . I_n Lewin, R.A. (Ed.) P h y s i o l o g y and B i o c h e m i s t r y of A l g a e , Academic P r e s s , New York. pp 171-88. w  S y r e t t , P . J . and M o r r i s , I . " 1963. The i n h i b i t i o n of n i t r a t e assimilation by ammonium i n C h l o r e l l a . B i o c h i m . B i o p h y s . A c t a 67:566-75. T h a c k e r , A. and S y r e t t , P . J . -1972. and ammonium by Chlamydomonas 71:423-33.  The a s s i m i l a t i o n of n i t r a t e reinhardi. New Phytol.  Topinka, J.A. 1978. N i t r o g e n uptake (Phaeophyceae). J . P h y c o l . 14:241-7.  by  Fucus  spiralis  Trebst, A. and Burba, M. 1967. Uber die lemmung p h o t o s y n t h e t i s c h e r r e a k t i o n e n i n i s o l i e r t e n c h l o r o p l a s t e n und in Chlorella durch disalicyliden propandiamin. Z. Pflanzenphysiol. 57:419-33. Vega, J.M., H e r r e r a , J . , A p a r i c i o , P . J . , Paneque, A. and Losada, M. 1971. R o l e of molybdenum i n n i t r a t e r e d u c t i o n by C h l o r e l l a . Plant Physiol. 48:294-9. Weidner, M. and K i e f e r , H. 1981. N i t r a t e r e d u c t i o n i n t h e marine brown a l g a G i f f o r d i a m i t c h e l l a e (Harv.) Ham. 2. Pflanzenphysiol. 104:341-51. Zumft, W.G., Paneque, A., A p a r i c i o , P . J . , and Losada, M. 1969. Mechanism of n i t r a t e r e d u c t i o n i n C h l o r e l l a . Biochem. B i o p h y s . Res. Commun. 36:980-6.  204  Appendix 3. P.  Soluble Nitrogen  Content of Porphyra p e r f o r a t a  p e r f o r a t a p l a n t s were c u l t u r e d , e x t r a c t e d and a n a l y s e d  as d e s c r i b e d  i n Chapter 2.  A n a l y s i s of crude a l g a l e x t r a c t s f o r  ammonium, s o l u b l e amino a c i d s and s o l u b l e p r o t e i n i s s u b j e c t interference phenolic extent  from  contaminants  such  as  polysaccharides  compounds (Appendix 1 ) . More work i s r e q u i r e d of  analyses  this  interference.  Therefore,  ammonium  content  of  on t h e  here  i n c r e a s e s or .decreases i n these n i t r o g e n  The  and  t h e r e s u l t s of such  a r e not q u a n t i t a t i v e and a r e p r e s e n t e d  general  to  to  show  compounds.  a l l plants  decreased  by  a p p r o x i m a t e l y 50% d u r i n g t h e f i r s t two days ( F i g . 1 ) . A f t e r day 2 t h e ammonium and ammonium p l u s n i t r a t e grown p l a n t s marked i n c r e a s e There  was  i n ammonium high  variability  amino a c i d s )  i n t h e measurement (assumed  ( F i g . 2 ) . There was an i n i t i a l  i n amino a c i d s c o n t e n t i n a l l p l a n t s except f o r t h o s e been  nitrogen  starved  a  content.  i n t e r n a l l e v e l s of n i n h y d r i n p o s i t i v e m a t e r i a l primarily  showed  of t h e t o be increase  that  had  ( F i g . 2 ) . The amino a c i d c o n t e n t o f t h e  s t a r v e d p l a n t s decreased very  slowly, exhibiting  an  observable  change o n l y a f t e r e i g h t days. The constant There  internal  levels  of  soluble  p r o t e i n were r e l a t i v e l y  and s i m i l a r i n magnitude f o r a l l  was  a  noticable  treatments  (Fig. 3).  decrease i n s o l u b l e p r o t e i n i n starved  p l a n t s between day 8 and 10.  Ammonium  3 II CAJ  •  Content  r-AV  in OJ fD M rt i-t uo OJ i-1 o i-h • < 3 O 03 t-" rt -»  3 3  Qj C OJ  3  •  rt DJ  •-3  i  rt 3fD fD • O OJIV> H  i  3 QJ 3 O  rt  r|  3  H  r| 3 c O M3 rt r t fD •-t CL n  o  3 3 fD  o  C OJTr tOJ  3  rt fD i-ti r t O fD 01 rt 3 rt N . i-f Xi fD I— T> C >-l in fD in o O t— < fD OJ in 3 Co Z rt O OJ X O 3 ^< 3 r " in • • fD C 1  ?^  - 3 1  3 3  in rt  3o * 3  OJ  3  fD 0) rt  3 <  Sk H - r t rt QJ rt rt pj ^ rt O fD O  a  Qj l-t fD  < 3  rt r t rt O O uQ 3 fD OJ  -  3  O rt K rt OJ  in  "5 < fD CL  902  (jurnol N H  _1 g wet wt  )  I  1  2  —  Time  1 4  —  •  (clays)  F i g . 2. S o l u b l e amino a c i d c o n t e n t (>umol amino a c i d s . g wet w t " ) of Porphyra p e r f o r a t a p r e c o n d i t i o n e d f o r 0-10 days on nitrate (---•—), ammonium (-—A—J , n i t r a t e p l u s ammonium (—a—), or n i t r o g e n s t a r v e d ( — • — ) . E r r o r bars r e p r e s e n t one standard d e v i a t i o n , n=3. 1  I—  1 6  Q  ro §>  lime  (days)  F i g . 3. S o l u b l e p r o t e i n content (mg p r o t e i n . g wet w t ) of Porphyra p e r f o r a t a p r e c o n d i t i o n e d f o r 0-10 days on n i t r a t e (—• ) , ammonium (..-.A---), n i t r a t e p l u s ammonium ( — x ) or n i t r o g e n s t a r v e d ('.— • — ) . E r r o r bars represent one s t a n d a r d d e v i a t i o n , n=3. - 1  to o -J  

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