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UBC Theses and Dissertations

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 THE REQUIREMENTS DOCTOR OF PARTIAL FULFILLMENT OF FOR THE DEGREE OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Botany) We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH 1983 <g) Terry E l l e n Thomas, COLUMBIA 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 requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the 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 reference and study. I f u r t h e r agree t h a t permission f o r extensive copying of 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 granted by the head of my department or by h i s or her r e p r e s e n t a t i v e s . I t i s understood th a t 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 gain s h a l l not be allowed without my w r i t t e n permission. Department of fl<S>t<**y  The U n i v e r s i t y of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date SO, 11% 3 DE-6 (3/81) i i Abstract A comprehensive f i e l d and lab o r a t o r y study of nitrogen uptake i n i n t e r t i d a l seaweeds was undertaken. Methods for measuring nitrogen uptake rates were evaluated. Short i n i t i a l periods of r a p i d ammonium uptake were common in n i t r o g e n d e f i c i e n t p l a n t s . The presence of ammonium i n h i b i t e d n i t r a t e uptake, but a c e r t a i n degree of nitrogen s t a r v a t i o n overcame t h i s suppression. Laboratory s t u d i e s with Porphyra p e r f o r a t a .showed that nitrogen starved c u l t u r e s maintained r a p i d i n i t i a l ammonium uptake r a t e s . The n i t r a t e uptake system d i d not remain a c t i v a t e d . Nitrogen s t a r v a t i o n a l s o r e s u l t e d i n a general decrease i n solu b l e nitrogen content and a t r a n s i e n t increase 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 reductase assays was i n v e s t i g a t e d . The rate of n i t r i t e production i n the in v i v o assay v a r i e d with incubation time. Therefore, the in  v i t r o assay was used. N i t r a t e grown c u l t u r e s of Porphyra p e r f o r a t a . maintained high ammonium uptake r a t e s . I t was suggested that the rate of n i t r a t e reduction was l i m i t i n g the supply of nitrogen f or f u r t h e r a s s i m i l a t i o n which may c o n t r o l ammonium uptake. Ammonium arid ammonium plus n i t r a t e grown c u l t u r e s had very low nitrogen uptake rates and n i t r a t e reductase a c t i v i t i e s . F i e l d s t u d i e s with G r a c i l a r i a verrucosa confirmed that growth on ammonium i n h i b i t e d n i t r a t e uptake, n i t r a t e accumulation and n i t r a t e reductase a c t i v i t y . The presence of ammonium d i d not i n h i b i t n i t r a t e uptake rates i n severely s t a r v e d populations. A l l populations maintained high ammonium uptake rates suggesting that they were nitrogen l i m i t e d at t h i s time (August). Ammonium and n i t r a t e uptake were saturable i n the high i n t e r t i d a l G. verrucosa population but not i n the low i n t e r t i d a l p o p u l a t i o n . An i n v e s t i g a t i o n was made i n t o the e f f e c t of nitrogen source and p e r i o d i c exposure to a i r on growth, development and nit r o g e n uptake i n Fucus d i s t i c h u s germlings. Gamete r e l e a s e , f e r t i l i z a t i o n , germination and germling growth had no requirement f o r a s p e c i f i c form of n i t r o g e n . P e r i o d i c exposure to a i r increased secondary r h i z o i d development. Ammonium and n i t r a t e uptake rates of the germlings were much higher than f o r the mature t h a l l i , but the a f f i n i t y f o r n i t r a t e was s i m i l a r . The germlings showed satur a b l e uptake k i n e t i c s but the mature t h a l l i d i d not. The presence of ammonium i n h i b i t e d n i t r a t e uptake by the mature p l a n t s but not by the germlings. M i l d 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. This uptake response occurred when growth was l i m i t e d by that p a r t i c u l a r n u t r i e n t and when the t h a l l u s had been exposed to p e r i o d i c d e s i c c a t i o n f o r s e v e r a l weeks. The degree of enhancement, the percent d e s i c c a t i o n producing maximum uptake rates and the tol e r a n c e to higher degrees of d e s i c c a t i o n were r e l a t e d to i n t e r t i d a l l o c a t i o n . This was shown to be an i n t r a s p e c i f i c as w e l l as an i n t e r s p e c i f i c a d aptation. Transplant experiments with G. verrucosa showed that enhanced n u t r i e n t uptake rates a f t e r d e s i c c a t i o n were r e l a t e d to i n t e r t i d a l height and not geographic l o c a t i o n and that t h i s i v response could be induced i n approximately f i v e weeks. I t was suggested that t h i s enhanced uptake response was an adaptation to nitrogen procurement and C/N homeostasis f o l l o w i n g p e r i o d i c exposure when carbon was a s s i m i l a t e d but when other n u t r i e n t s were not a v a i l a b l e . V Table of Contents Page Abstract i i L i s t of Tables v i L i s t of Figures v i i Acknowledgements x i i I n t r o d u c t i o n 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 6 2) The E f f e c t of Nitrogen Supply on Nitrogen Uptake, A s s i m i l a t i o n and Accumulation i n Porphyra p e r f o r a t a 19 3) Nitrogen Uptake and Growth of Fucus d i s t i c h u s Germlings and Mature P l a n t s 37 4) D e s i c c a t i o n Enhanced N u t r i e n t Uptake Rates i n I n t e r t i d a l Seaweeds 61 5) Adaptations of G r a c i l a r i a verrucosa to 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 Conclusions .143 References 148 Appendix 1. Techniques for measuring s o l u b l e n itrogen content 156 Appendix 2. I_n v i v o and in v i t r o n i t r a t e reductase a c t i v i t y 170 Appendix 3. Soluble nitrogen content of Porphyra p e r f o r a t a .204 v i L i s t of Tables Page 1. A time course study of nitrogen uptake rates i n marine macrophytes 14 2. The e f f e c t of nitrogen source on the growth of F. d i s t i c h u s germlings 46 3. Soluble n i t r o g e n content of G. verrucosa 123 Appendix 2. 1. Optimal c o n d i t i o n s for n i t r a t e reductase a c t i v i t y i n three marine macrophytes w 181 L i s t of Figures Page 1. Map of Bamfield c o l l e c t i o n s i t e s 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 rates of preincubated P. p e r f o r a t a 25 4. Ammonium uptake rates of preincubated P. p e r f o r a t a 26 5. N i t r a t e content of preincubated P. p e r f o r a t a 28 6. N i t r a t e reductase a c t i v i t y of preincubated P k p e r f o r a t a .30 7. N i t r a t e uptake k i n e t i c s of F. d i s t i c h u s germlings 47 8. N i t r a t e uptake k i n e t i c s of mature F. d i s t i c h u s t h a l l i ...49 9. Ammonium uptake k i n e t i c s of F. d i s t i c h u s germlings 50 10. Ammonium uptake k i n e t i c s of mature F. d i st ichus t h a l l i .51 11. Ammonium uptake k i n e t i c s of mature F. d i st ichus t h a l l i preincubated on ammonium or nitr o g e n starved 53 12. The r e l a t i o n s h i p between n u t r i e n t uptake ra t e s of F. d i st ichus and % d e s i c c a t i o n 70 .13. The r e l a t i o n s h i p between ammonium uptake rates of F. d i st ichus i n J u l y 1980 and % d e s i c c a t i o n 73 14. The r e l a t i o n s h i p between ammonium uptake rates of F. d i st ichus i n September 1980 and d e s i c c a t i o n 74 15. The r e l a t i o n s h i p between ammonium uptake rates of F. 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 75 16. The r e l a t i o n s h i p between n i t r a t e uptake rates of F. 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 76 y i i i 17. The r e l a t i o n s h i p between ammonium uptake rates of F. d i s t i c h u s preincubated on nitrogen free seawater i n February 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 rates of F. 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 78 19. The r e l a t i o n s h i p between nitrogen uptake rates of G. verrucosa and % d e s i c c a t i o n 80 20. The r e l a t i o n s h i p between nitrogen uptake rates of E. i n t e s t i n a l i s and % d e s i c c a t i o n 81 21. The r e l a t i o n s h i p between nitrogen uptake rates of G. p a p i l l a t a and % d e s i c c a t i o n 82 22. The r e l a t i o n s h i p between nitrogen uptake rates of F. d i s t i c h u s and % d e s i c c a t i o n 83 23. The r e l a t i o n s h i p between nitrogen uptake rates of P. l i m i t a t a and % d e s i c c a t i o n 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 producing maximum enhancement of n i t r a t e uptake r a t e s , the r e l a t i v e degree of enhancement of n i t r a t e uptake, the r a t i o of n i t r a t e uptake rates at 30% d e s i c c a t i o n and hydrated uptake r a t e s , and i n t e r t i d a l l o c a t i o n 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 producing maximum enhancement of ammonium uptake r a t e s , the r e l a t i v e degree of enhancement of ammonium uptake, the r a t i o of ammonium uptake rates at 30% d e s i c c a t i o n and hydrated uptake r a t e s , and i n t e r t i d a l l o c a t i o n 86 26. A summary of G. verrucosa t r a n s p l a n t s 98 27. Low i n t e r t i d a l G. verrucosa 103 28. High i n t e r t i d a l G. verrucosa 104 29. A summary of G. verrucosa morphology 106 30. The r e l a t i o n s h i p between n i t r a t e uptake rates of G. i x verrucosa i n l a t e summer and % d e s i c c a t i o n 109 31. The r e l a t i o n s h i p between n i t r a t e uptake rates of G. verrucosa i n June and % d e s i c c a t i o n 110 32. A summary of nitrogen uptake rates for G. verrucosa ...112 33. The r e l a t i o n s h i p between ammonium uptake rates of G. verrucosa i n l a t e summer and % d e s i c c a t i o n 114 34. The r e l a t i o n s h i p between ammonium uptake rates of G. verrucosa i n June and % d e s i c c a t i o n 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 the so l u b l e nitrogen content of G. verrucosa 1 24 38. A summary of the so l u b l e p r o t e i n content and the n i t r a t e reductase a c t i v i t y of G. verrucosa 126 39. The t o t a l amount of n i t r i t e produced with time and the r e l a t i o n s h i p between n i t r a t e reductase a c t i v i t y and the volume of e x t r a c t used for G. verrucosa 127 40. The r e l a t i o n s h i p between n i t r a t e reductase a c t i v i t y i n G. verrucosa and MgS04 concentration *..128 41. The r e l a t i o n s h i p between n i t r a t e reductase a c t i v i t y i n G. verrucosa and pH 129 42. The r e l a t i o n s h i p between n i t r a t e reductase a c t i v i t y i n G. verrucosa and PVP concentration 130 43. The r e l a t i o n s h i p between n i t r a t e reductase a c t i v i t y i n G. verrucosa and n i t r a t e c o n c e n t r a t i o n 131 44. The r e l a t i o n s h i p between n i t r a t e reductase a c t i v i t y i n G. verrucosa and NADH concentration 131 X Appendix 1. 1. Soluble nitrogen content of three 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 with time (min) and 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 reductase a c t i v i t y and the volume of enzyme e x t r a c t used f o r three marine macrophytes 178 2. The pH optimum of i_n v i t r o n i t r a t e reductase a c t i v i t y i n three marine macrophytes 179 3. The relationship.between in_ v i t r o n i t r a t e reductase a c t i v i t y i n three marine macrophytes and MgSO concentration 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 reductase a c t i v i t y i n three marine macrophytes and PVP concentration 182 5. The r e l a t i o n s h i p between iri v i t r o n i t r a t e reductase a c t i v i t y i n three marine macrophytes and NADH concentration 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 reductase a c t i v i t y i n three marine macrophytes and n i t r a t e c o n c e n t r a t i o n 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 reductase a c t i v i t y i n P. p e r f o r a t a and pe 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 a c t i v i t y of three marine macrophytes incubated on the j j i v i v o medium 188 9. 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 reductase a c t i v i t y of P. p e r f o r a t a incubated i n the i_n v i v o medium 190 10. A c t u a l I_n vi v o n i t r a t e reductase a c t i v i t y of P. p e r f o r a t a incubated i n the v i v o medium 191 11. I_n v i t r o and in v i v o n i t r a t e reductase a c t i v i t y of P. pe r f o r a t a incubated in the i_n v i v o medium 193 Appendix 3. 1. Ammonium content of preincubated P. p e r f o r a t a 205 2. Soluble amino a c i d content of preincubated P. p e r f o r a t a 206 3. Soluble p r o t e i n content of preincubated P. p e r f o r a t a ...207 x i i Acknowledgements I would l i k e to thank my graduate s u p e r v i s o r , Dr. P.J. Harrison f o r h i s generous a s s i s t a n c e and i n v a l u a b l e guidance throughout my graduate s t u d i e s . I would a l s o l i k e to thank Dr. D.H. Turpin f o r h i s c r e a t i v e input and unwavering enthusiasm. E r i c B. Taylor's a s s i s t a n c e i n c u l t u r i n g Fucus d i s t i c h u s germlings was a l s o g r e a t l y appreciated. Thanks i s a l s o extended to my supervisory committee members; Dr. R. DeWreede, Dr. T.R. Parsons, and Dr. I.E.P. Ta y l o r . This work was supported by a Na t u r a l Sciences and Engineering Research C o u n c i l of Canada graduate s c h o l a r s h i p . 1 Introduct ion Tides create a unique environment in c o a s t a l regions, where i n t e r t i d a l organisms must be adapted to p e r i o d i c exposure to a i r and submersion. In the Northeast P a c i f i c Ocean, i n t e r t i d a l seaweeds are abundant and t h e i r d i s t r i b u t i o n i s l a r g e l y determined by substrate type, temperature, s a l i n i t y , water motion, and n u t r i e n t s (Druehl 1981). Rocky and sandy c o a s t a l areas support very 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 fr e q u e n t l y c h a r a c t e r i s e d by d i s t i n c t 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 species or group of species. Researchers have a t t r i b u t e d these zones to both b i o l o g i c a l (Jones and Kain 1967; Connell 1972; Mann 1972; Chapman 1973) and p h y s i c a l f a c t o r s (Zaneveld 1937; Doty 1946; Lewis 1964). In some l o c a t i o n s there are maximum changes i n the p h y s i c a l s t r e s s at the l i m i t s of these bands. These l o c a t i o n s are' r e f e r r e d to as a " c r i t i c a l " t i d a l heights (Doty 1946) and are c h a r a c t e r i z e d by a large increase i n the frequency and/or d u r a t i o n of exposure to a i r . Exposure and subsequent submersion cause d r a s t i c changes i n temperature, l i g h t , s a l i n i t y , n u t r i e n t supply, and humidity. Success i n such an environment requires s p e c i f i c morphological, p h y s i o l o g i c a l , and biochemical adaptations a l l o w i n g for s u r v i v a l and p o s s i b l y growth during these environmental f l u c t u a t i o n s . Numerous st u d i e s have examined the e f f e c t s of i r r a d i a n c e , temperature (Schonbeck and Norton 1978; Gordon et a l . 1981; Thorhaug and Marcus 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 et a l . 1981) on macroalgal growth. The most obvious 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 d e s i c c a t i o n r e s i s t a n c e have been i n v e s t i g a t e d (Isaac 1935; Feldman 1951; Schonbeck and Norton 1979 a,b,c; Dromgoole 1980), but l i t t l e i s known about 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 . Recent i n v e s t i g a t i o n s showed that the photosynthetic rates of i n t e r t i d a l macrophytes were maintained or even enhanced over a l i m i t e d range of d e s i c c a t i o n (Johnson'et a l . 1974; Quadir et a l . 1979). Increased carbon procurement i n the absence of e x t e r n a l n u t r i e n t supply should c o n t i n u a l l y increase the C/N r a t i o of the p l a n t , however, f o r growth to occur the C/N r a t i o must be maintained below a c e r t a i n species s p e c i f i c value (Hanisak 1979). I t appears that high i n t e r t i d a l seaweeds are capable of increased nitrogen procurement f o l l o w i n g periods 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 to date. Nitrogen commonly l i m i t s the growth of marine macrophytes (Chapman and C r a i g i e 1977; Hanisak 1979; Rosenberg and Ramus 1982). Their a b i l i t y to compete for t h i s l i m i t e d resource i s important f o r s u r v i v a l . Ion uptake i s the f i r s t step i n nit r o g e n procurement. Seasonal f l u c t u a t i o n s i n nitrogen uptake r a t e s occur i n marine macrophytes (Rosenberg and Ramus 1982). This study examines how nitrogen uptake i n i n t e r t i d a l seaweeds i s adapted to ensure s u r v i v a l i n an environment of f l u c t u a t i n g n i t r o g e n supply and p e r i d o d i c d e s i c c a t i o n . There are two other re p o r t s i n which nitrogen s u p p l i e s to l a b o r a t o r y c u l t u r e s were c o n t r o l l e d before uptake rates were measured ( D ' E l i a and DeBoer 3 1978; Probyn and Chapman 1982). These st u d i e s suggest that i n t e r t i d a l seaweeds may be adapted for r a p i d recovery 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 . This adaptation of nitrogen procurement was i n v e s t i g a t e d . A survey was conducted on n i t r a t e and ammonium uptake r a t e s i n f i v e species of i n t e r t i d a l seaweeds" which were taken d i r e c t l y from the 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; Gerard 1982a). A time course study of the e f f e c t of nitrogen s t a r v a t i o n on nit r o g e n uptake rates i n l a b o r a t o r y c u l t u r e s of P. p e r f o r a t a was a l s o undertaken. " D i f f e r e n c e s i n nit r o g e n uptake k i n e t i c s and the e f f e c t of nitrogen supply on uptake rates were measured i n two populations of G r a c i l a r i a verrucosa and two l i f e h i s t o r y stages (germlings and mature t h a l l i ) of Fucus d i s t i c h u s . A f t e r n i t r o g e n i s taken up, i t i s e i t h e r stored or a s s i m i l a t e d . Marine macrophytes can store large q u a n t i t i e s of inorg a n i c n i t r o g e n and t h i s can be u t i l i z e d during periods of nit 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; Gerard 1982a; Rosenberg and Ramus 1982). Chapman and C r a i g i e (1977) found that Laminaria l o n q i c r u r i s s t o r e d n i t r a t e i n the winter (a p e r i o d of abundant nitrogen supply) and that i t was u t i l i z e d i n the s p r i n g when nitrogen s u p p l i e s were diminished. The l i t e r a t u r e contains i s o l a t e d r e p o r t s of the e f f e c t of ni t r o g e n supply on nitrogen uptake rates ( D ' E l i a and DeBoer 1978; Probyn and Chapman 1982), i n t e r n a l nitrogen pools ( B i r d et a l . 1982b) and nitrogen a s s i m i l a t i o n rates (Weidner and K i e f e r 1981). A comprehensive study of the e f f e c t of nitrogen supply on s e v e r a l aspects of nitrogen u t i l i z a t i o n was undertaken with l a b o r a t o r y c u l t u r e s of Porphyra p e r f o r a t a . The i n t e r a c t i o n 4 between nit r o g e n supply, uptake, accumulation, and a s s i m i l a t i o n was i n v e s t i g a t e d . A p h y s i o l o g i c a l 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 i n the f i e l d . 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. p e r f o r a t a were t e s t e d i n the f i e l d . The nitrogen uptake r a t e s , the s o l u b l e n i t r o g e n content and the n i t r a t e reductase a c t i v i t y of three populations of G. verrucosa growing at d i f f e r e n t i n t e r t i d a l l e v e l s and s l i g h t l y d i f f e r e n t n u t r i e n t regimes were a l s o s t u d i e d . The major d i f f i c u l t y with f i e l d s t u d i e s i s that environmental f a c t o r s cannot be c o n t r o l l e d and may not always be a c c u r a t e l y described. The e f f e c t s of one f a c t o r (e.g., i n t e r t i d a l l o c a t i o n ) may be i s o l a t e d to some extent from others (e.g., 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 given geographic l o c a t i o n ) by t r a n s p l a n t i n g . This was done with the G. verrucosa p o p u l a t i o n s . V e r t i c a l p o s i t i o n i n the i n t e r t i d a l region was changed but geographical l o c a t i o n and n u t r i e n t supply were not c o n s c i o u s l y a l t e r e d . Nitrogen uptake r a t e s , s o l u b l e n i t r o g e n content and n i t r a t e reductase a c t i v i t i e s were monitored. The study of n i t r o g e n uptake and a s s i m i l a t i o n i n marine macrophytes lags f a r behind s i m i l a r work with phytoplankton. A great deal can be learned from s t u d i e s on phytoplankton, but p h y s i o l o g i c a l s t u d i e s with marine macrophytes in v o l v e p a r t i c u l a r c o n s i d e r a t i o n s such as t i s s u e age, appreciable and v a r i a b l e water and polysaccharide content, a l a r g e apoplast, non-uniform exposure to l i g h t , carbon and n u t r i e n t sources, and a l a r g e 5 enough biomass to sto r e s i g n i f i c a n t amounts of organic and inor g a n i c compounds. P h y s i o l o g i c a l s t u d i e s have so f a r been l i m i t e d to a few commonly occu r r i n g s p e c i e s . 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 s p e c i e s . The i n t e r a c t i o n between p h y s i c a l and chemical f a c t o r s and s e v e r a l stages of n i t r o g e n u t i l i z a t i o n were i n v e s t i g a t e d to demonstrate that i n t e r t i d a l seaweeds are uniquely adapted to p e r i o d i c exposure and nitrogen s t a r v a t i o n i n terms of nitrogen procurement. The r e s u l t s give i n s i g h t i n t o the c o n t r o l of n i t r o g e n metabolism and may enhance our understanding of the p h y s i o l o g i c a l adaptations of i n t e r t i d a l seaweeds to growth i n the i n t e r t i d a l environment. 6 Chapter 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 I n t r o d u c t i o n Nitrogen i s thought to be the 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 the marine environment (Ryther and Dunstan 1971; Chapman and C r a i g i e 1977; Hanisak 1979; Topinka and Robbins 1976). Several s t u d i e s have shown that an a d d i t i o n of n i t r o g e n to seawater g r e a t l y enhances the summer growth of marine macrophytes (Chapman and C r a i g i e 1977; Gerard 1982a). Marine macrophytes commonly obta i n nitrogen as inorganic ions such as ammonium, n i t r a t e and o c c a s i o n a l l y as n i t r i t e from seawater. Macrophytes a l s o u t i l i z e organic nitrogen such as urea and amino acid s (Moshen et a l . 1974; DeBoer 1981), but these sources are u s u a l l y l e s s abundant than inorganic sources (Parsons et a l . 1977). Uptake i s the f i r s t step i n the u t i l i z a t i o n of nitrogen for growth. The four basic mechanisms of ion uptake i n seaweeds are: 1) d i f f u s i o n i n t o the apparent free space, 2) passive 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 , and 4) a c t i v e t r a n s p o r t (DeBoer 1981). Solute entering by passive d i f f u s i o n , f a c i l i t a t e d 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 , i s transported across the c e l l membrane and a s s i m i l a t e d . D i f f u s i o n i n t o apparent free space does not in c l u d e t r a n s p o r t of the n u t r i e n t i n t o the c e l l . Transport i n t o the apparent free space does not only c o n s i s t of "simple d i f f u s i o n " , but ion exchange occurs as w e l l (DeBoer 1981). Standard methods of measuring uptake cannot 7 d i f f e r e n t i a t e between t h i s ion exchange and a c t i v e uptake i n t o the c e l l s . N u t r i e n t uptake can be determined by monitoring 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 surrounding seawater or the accumulation of isotope i n the t h a l l u s (Harrison and Druehl 1982). Measurement of n u t r i e n t d e p l e t i o n i s more commonly used and may be undertaken using e i t h e r the "time course" or the "batch" methods (Harrison and Druehl 1982). The decrease i n nitrogen c o n c e n t r a t i o n of the e x t e r n a l medium can be continuously monitored (the time course method), or i t can be determined from the d i f f e r e n c e i n the c o n c e n t r a t i o n at the beginning and end of a set incubation p e r i o d (the batch method). Most researchers 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; Gerard 1982b) and they have employed a v a r i e t y of incubation times from minutes ( H a r l i n and C r a i g i e 1978; Wheeler 1979) to hours (Topinka 1978). I t i s important whether or not the uptake r a t e i s constant .over the i n c u b a t i o n p e r i o d . This can only be determined from a time course study of uptake r a t e . A t r a n s i e n t increase i n ni t r o g e n uptake rate 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 to a n i t r o g e n l i m i t e d phytoplankton c u l t u r e (Conway et a l . 1976; McCarthy and Goldman 1979). An enhanced uptake rate f o r ammonium has been observed under c e r t a i n c o n d i t i o n s f o r 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 1978) and Macrocystis  p y r i f e r a (Haines and Wheeler 1978). Ammonium uptake was i n i t i a l l y enhanced but i t dropped to a lower sustained r a t e w i t h i n a few minutes i n these s t u d i e s . The present study was to determine whether t h i s uptake response commonly occurred i n the 8 f i e l d . Time course s t u d i e s were determined f o r ammonium and n i t r a t e uptake i n f i v e species of i n t e r t i d a l seaweeds ( P e l v e t i o p s i s l i m i t a t a (Setch)Gard., Fucus d i s t i c h u s L., G i g a r t i n a p a p i l l a t a (C.Ag.), Grac i l a r i a verrucosa (Huds.) Papenf., and Enteromorpha i n t e s t i n a l i s (L.)Grev.). The e f f e c t s of ammonium on the rate of n i t r a t e uptake and n i t r a t e on ammonium uptake are a l s o reported. 9 M a t e r i a l s and Methods Species and C o l l e c t i o n S i t e s G i q a r t i n a p a p i l l a t a (C.Ag.), and P e l v e t i o p s i s l i m i t a t a (Setch) Gard. were c o l l e c t e d from the northern shore of Diana I s l a n d , Barkley Sound, B.C. ( F i g . 1) at 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 qardneri by S l i v a see Conomos 1979), Enteromorpha i n t e s t i n a l i s (L.)Grev. and G r a c i l a r i a verrucosa (Huds.) Papenf., were c o l l e c t e d from Wiseman's Bay, Bamfield, B.C., ( F i g . 1). Their heights above Canadian datum were 2.6, 2.0 and 1.0 m, r e s p e c t i v e l y . A l l t h a l l i were c o l l e c t e d from rock faces except G r a c i l a r i a  verrucosa which grew on a rocky beach. Specimens from a second high i n t e r t i d a l (1.8 m) population of G. verrucosa were a l s o s t u d i e d . This population grew on a mudflat at the head of Bamfield I n l e t ( F i g . 1). The study was conducted i n August 1981. P e r i o d i c sampling revealed that n i t r a t e , ammonium and n i t r i t e was not detected (<0.1^iM) i n the surface water at the c o l l e c t i o n s i t e s i n e a r l y s p r i n g and remained at t h i s l e v e l u n t i l l a t e September (L. Druehl pers. comm.) However, during August, when d a i l y measurements of surface n i t r a t e and ammonium concentrations were made there were two days when elevated ammonium concentrations (2-3 /uM) were found at the head of Bamfield I n l e t . Time Course Experiments The general experimental p r o t o c o l for measuring nitrogen 10 F i g . 1. Map of Bamfield I n l e t Bamfield, B.C., Canada, showing the c o l l e c t i o n s i t e s . 11 uptake rates was as f o l l o w s : 1) the medium used was n a t u r a l , f i l t e r e d (0.45 jum), nitrogen d e f i c i e n t ( n i t r a t e , ammonium and n i t r i t e <0.1 juM) seawater, enriched with f/2 phosphate, t r a c e metals, and vitamins ( G u i l l a r d and Ryther 1962), 2) 0-60 pM n i t r a t e and/or ammonium was added depending on the experiment, 3) epiphytes were removed from the p l a n t s by vigorous brushing under running seawater, 4) the incubation v e s s e l s were 1 1 Erlenmeyer f l a s k s (time course experiments) or 500 ml Mason j a r s , 5) the t h a l l i were free f l o a t i n g , 6) the i r r a d i a n c e was 500—150 uE.m" 2.s _ 1, and uptake experiments were always done at approximately the same time each day (1100-1400), 7) the medium was continuously s t i r r e d w i t h a 1 inch s t i r r i n g bar a 120 rpm producing an average current v e l o c i t y of approximately 10 cm.s" 1, 8) the plan t biomass:water volume r a t i o was 0.3—0.2 g dry w t . l " 1 , 9) the temperature was held constant at 11-16°C depending on the experiment, 10) f o r batch experiments incubation times were set at a per i o d over which the uptake rate was constant. This was u s u a l l y 10 min f o r ammonium uptake and 15—30 min for n i t r a t e uptake, and 11) f o r the time course s t u d i e s , uptake rates were monitored continuously f o r 30—60 min. Ammonium and n i t r a t e c o n c e n t r a t i o n s were measured using a Technicon AutoAnalyzer. In t h i s study n i t r a t e and ammonium uptake rates were monitored continuously at 16°C with n i t r o g e n concentrations of: 30 jaM n i t r a t e , or 15 yuM ammonium, or 30 yuM n i t r a t e plus 15 u^M ammonium f o r 30 min. Standard d e v i a t i o n s were determined from batch uptake experiments (Chapters 5,6) which were done immediatly f o l l o w i n g the time course experiments for the s p e c i f i c time periods when uptake was constant and under 12 the same p h y s i c a l c o n d i t i o n s . General trends are discussed. Dry weights were determined by drying the p l a n t s to a constant weight on aluminum f o i l t r a y s i n a 60°C oven. C/N r a t i o s were determined using a CHN analyser (Carlo Erba). 13 R e s u l t s and Di s c u s s i o n A l l species took up both ammonium and n i t r a t e simultaneously (Table 1). E. i n t e s t i n a l i s and high i n t e r t i d a l G. verrucosa were the only populations i n which there was a s u b s t a n t i a l decrease (50%) i n n i t r a t e uptake rate due to the presence of ammonium. They a l s o had the lowest C/N r a t i o s and were probably under l e s s severe n i t r o g e n s t r e s s . I n h i b i t i o n of n i t r a t e uptake by ammonium i n E. i n t e s t i n a l i s l a s t e d only 12 min a f t e r which the ammonium con c e n t r a t i o n dropped below 5 u^M. m A f t e r 30 min the ammonium con c e n t r a t i o n of the medium i n the high i n t e r t i d a l G. verrucosa was s t i l l above 10 yuM and n i t r a t e uptake was i n h i b i t e d . Suppression of n i t r a t e uptake by ammonium has been recorded i n higher p l a n t s (MacKown et a l . 1982), phytoplankton ( S y r e t t and M o r r i s 1963; Conway 1977), and fungi (Goldsmith et a l . 1973). Such p r e f e r e n t i a l uptake has been reported f o r the three major macroalgal d i v i s i o n s (Hanisak and H a r l i n 1978; D' E l i a and DeBoer 1978; Haines and Wheeler 1978; Gordon et a l . 1981), but the r e s u l t s of t h i s study show that t h i s i s not the case f o r a l l marine macrophytes and that the degree of suppression appears to vary with the degree of nitr o g e n l i m i t a t i o n . The low i n t e r t i d a l G. verrucosa with a higher C/N r a t i o than the high i n t e r t i d a l G. verrucosa, d i d not e x h i b i t i n h i b i t i o n of n i t r a t e uptake by ammonium. I t appeared that a c e r t a i n degree of nitr o g e n d e f i c i e n c y prevented the i n h i b i t i o n of n i t r a t e uptake by ammonium. I t i s e n e r g e t i c a l l y favorable for the p l a n t to u t i l i z e ammonium rather than n i t r a t e ( S y r e t t 1962); however, under nitrogen l i m i t a t i o n , the immediate Table 1. A time course study of n i t r a t e and ammonium uptake rates (V) (/jmol.g wet wt" 1.h" 1 or jjmol.g dry wt" 1.h" 1 ± 1 standard d e v i a t i o n , n=3 or 4) f o r high and low i n t e r t i d a l Grac i l a r i a verrucosa, Enteromorpha i n t e s t i n a l i s , Fucus  d i s t i c h u s , G i g a r t i n a p a p i l l a t a , and P e l v e t i o p s i s l i m i t a t a . %=uptake rate as a % of c o n t r o l rate ( i n N0 3~ or NH^+ o n l y ) , d = duration of constant uptake rate (min), and C/N r a t i o i s by atoms. Species 30 fiH NO ; addition 15 pM N H \ addition 30 nM NO" and 15 pM NH+ addition C / N V wet V dry d ¥ wet V dry d V N0- v N H ; V wet ¥ dry d i ¥ wet ¥ dry d f Low Intertidal 13 1.82±0.20 11.5+1.3 15-20 4.58±0.52 29.4 30 2.34±0.25 15.8±1.7 10 85 7.53+1.1 50.9+6.8 30 170 6. verrucosa 3.80 23.9 >20 3.95 26.8 >10 High Intertidal 9 1.28±0.14 6.04+0.66 30 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 10 110 G. verrucosa E. Intestinalis 10 9.77±1.20 46.5+5.7 15-20 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 90 4.40 26.0 >10 9.77 56.6 >12 4.00 23.2 >10 F. distichus 28 3.23+0.61 11.5+2.2 30 2.8 +0.3 10.5 15 2.8 ±0.40 10.4±1.4 30 85 2.75±0.43 10.2±1.6 25 100 1.80 6.8 >15 1.87 6.96 >25 G. papillata 13 2.84+0.44 8.0+1.2 30 6.15tl.60 13.2 12 2.95±0.43 11.7±1.7 20 150 3.9 ±0.8 15.4±3.2 20 85 3.35 7.21 >12 P. 11n1tata 30 1.0 ±0.2 4.19+0.84 20 2.77±0.31 10.2 30 1.17+0.18 4.86±0.75 20 115 2.32±0.28 9.65*1.16 20 95 0.61 2.56 >20 15 requirement f o r nitrogen may outweigh t h i s advantage. When maximum rat e s of n i t r a t e and ammonium uptake are maintained simultaneously, a plant can aquire a greater amount of nitr o g e n per u n i t time. This would be an advantage i n a nitr o g e n d e f i c i e n t environment. P. p e r f o r a t a was grown under va r i o u s degrees of nitrogen d e f i c i e n c y to c l a r i f y the e f f e c t of nitrogen 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 rates i n E. i n t e s t i n a l i s i n the presence of ammonium suggest that a concentration of >5 yuM NH4* i s required to i n h i b i t n i t r a t e uptake. D ' E l i a and DeBoer (1978) found the same response 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 t h r e s h o l d l e v e l has been o c c a s i o n a l l y reported i n some phytoplankton ( M a e s t r i n i et 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 phytoplankton (Conway 1976). E. i n t e s t i n a l i s , F. d i s t i c h u s , G. p a p i l l a t a , and high i n t e r t i d a l G. verrucosa showed rapid" i n i t i a l ammonium uptake ra t e s f o r the f i r s t 15 min of exposure to a s a t u r a t i n g ammonium co n c e n t r a t i o n . This 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 pulse of ammonium to the incubation medium has been reported f o r marine macrophytes by D ' E l i a and DeBoer (1978), Haines and Wheeler (1978), and Probyn and Chapman (1982). The r e s u l t s of t h i s study show that t h i s i s very 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 that t h i s only occurred i f the t h a l l u s was nitr o g e n starved. This i s a l s o the case f o r marine phytoplankton (Conway 1977; McCarthy et a l . 1977; Dortch et a l . 1982; Goldman and G l i b e r t 1982; Wheeler et a l . 1982). P r e c o n d i t i o n i n g one species under v a r y i n g degrees of ni t r o g e n 1 6 l i m i t a t i o n was required to confirm t h i s suggestion (Chapter 2 ) . Wheeler et a l . ( 1 9 8 2 ) have shown that t h i s r a p i d i n i t i a l ammonium uptake i n phytoplankton does not f o l l o w t y p i c a l Michaelis-Menten k i n e t i c s ; where V=(Vmax[S])/(Ks+[S]) when S=substrate c o n c e n t r a t i o n , V=uptake r a t e , Vmax=maximum uptake r a t e , and Ks=the h a l f - s a t u r a t i o n constant. However, i n i t i a l r a p i d uptake of ammonium i n the marine macrophyte Chordaria  f l a g e l l i f o r m i s was saturable 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 1 0 . 6 / J M (Probyn and Chapman 1 9 8 2 ) . When ammonium was s u p p l i e d continuously r a p i d i n i t i a l uptake d i d not occur, and the h a l f — s a t u r a t i o n constant for uptake was 0 . 6 yuM (Probyn and Chapman 1 9 8 2 ) . They suggested that most batch uptake experiments lead to an over es t i m a t i o n of Vmax and Ks and that more c a u t i o n should be taken when e x t r a p o l a t i n g the r e s u l t s of these short term uptake experiments to the p r e d i c t i o n of growth requirements of seaweeds. N i t r a t e uptake rates were g e n e r a l l y lower than uptake rates of ammonium. The exceptions were E. i n t e s t i n a l i s and F. d i s t i c h u s where n i t r a t e and ammonium uptake r a t e s were s i m i l a r . E. i n t e s t i n a l i s had the highest n i t r a t e uptake r a t e ( 4 6 . 5 pmol N 0 3 " . g dry wt~ 1.h~ 1) which was maintained f o r 1 5 — 2 0 min. This was the only example of t r a n s i e n t high i n i t i a l n i t r a t e uptake r a t e s . U n l i k e i n i t i a l high ammonium uptake r a t e s , enhanced n i t r a t e uptake rates d i d not appear to occur with nitrogen s t a r v a t i o n . Dortch et a l . ( 1 9 8 2 ) suggest that n i t r a t e uptake i s suppressed i n nitrogen d e f i c i e n t phytoplankton. N i t r a t e uptake had to be induced i n the low i n t e r t i d a l G. verrucosa by incubation i n n i t r a t e ( 2 0 min) or both n i t r a t e and ammonium ( 1 0 17 min). This G. verrucosa population was more nitrogen d e f i c i e n t than the high 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 study are w i t h i n the range of rates for other marine macrophytes ( 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 1981). H a r l i n (1978) a l s o found that E. i n t e s t i n a l i s has a high 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 time course s t u d i e s of nitrogen uptake. Uptake cannot be assumed to be constant over a given incubation time without t e s t i n g i t . Ammonium uptake by E. 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 with time. A f t e r 5 min the uptake ra t e was 40 /umol.g dry wt" 1.h~ 1 and a f t e r 30 min i t was 26 yumol.g dry w f ' . h " 1 . Wheeler et a l . (1982) recorded d i f f e r e n c e s i n ammonium uptake rates f o r phytoplankton of 0.09 and 0.02 h" 1 when ra t e s were measured at 1 and 15 min, r e s p e c t i v e l y . The time p e r i o d over which uptake i s constant v a r i e s . with the spe c i e s , the form of n i t r o g e n , and the n u t r i t i o n a l past h i s t o r y of the t h a l l u s . The major disadvantage of the "time course" method i s that u s u a l l y only one experiment can be performed at a time. I f uptake r a t e s are req u i r e d at a number of n u t r i e n t c o ncentrations ( f o r example when studying uptake k i n e t i c s ) , a "time course" f o r uptake must be repeated at each n u t r i e n t c o n c e n t r a t i o n . The "batch" method in v o l v e s a n a l y s i s of only two n u t r i e n t c o n c e n t r a t i o n s per experiment and i s l e s s time consuming than the "time course" method. I t i s becoming i n c r e a s i n g l y apparent that marine macrophytes e x h i b i t high i n t e r - p l a n t p h y s i o l o g i c a l 18 v a r i a b i l i t y . With the "batch" method the necessary r e p l i c a t e s can be run simultaneously. In a l l subsequent s t u d i e s , a "time course" study of uptake was conducted to determine the length of time during which uptake was constant and was followed by "batch" experiments conducted f o r t h i s incubation p e r i o d . I n t e r — p l a n t v a r i a b i l i t y i n uptake rates was often very high but was low ( u s u a l l y ± 2 min) f o r the length of time during which uptake was constant. Batch experiments were a l s o run f o r s e v e r a l minutes l e s s than the p e r i o d of constant uptake. The length of time during which uptake i s constant depends on the s p e c i e s , 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; Haines and Wheeler 1978). A time course study of uptake was conducted each time these parameters changed. 19 Chapter 2. The E f f e c t of Nitrogen Supply on Nitrogen Uptake, A s s i m i l a t i o n and I n t r a c e l l u l a r Storage i n Porphyra p e r f o r a t a I n t r o d u c t i o n Seasonal f l u c t u a t i o n s i n nitrogen supply i n c o a s t a l environments impose n u t r i e n t dependent growth patterns on most marine macrophytes (Chapman and C r a i g i e 1977; Rosenberg and Ramus 1982; Gagne et a l . 1982). The study of the r e l a t i o n s h i p between growth rate and n i t r o g e n supply has been complicated by the f a c t that 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 during times of n i t r o g e n d e f i c i e n c y (Buggeln 1974; Chapman and C r a i g i e 1977; Gerard 1982a; Rosenberg and Ramus 1982). I t has a l s o been shown that n i t r o g e n uptake rates ( D ' E l i a and DeBoer 1978) and nitrogen reserves ( B i r d et a l . 1982b) i n marine macrophytes can be i n f l u e n c e d by past n i t r o g e n supply ( D ' E l i a and DeBoer 1978; Probyn and Chapman 1982). This study with P. p e r f o r a t a i s the f i r s t comprehensive e v a l u a t i o n of the e f f e c t of v a r i o u s forms of inorganic nitrogen and n i t r o g e n s t a r v a t i o n on more than one stage of n i t r o g e n u t i l i z a t i o n (uptake, storage and a s s i m i l a t i o n ) . Therefore, i t gives a more complete understanding of the c o n t r o l of n i t r o g e n procurement. Regulation of n i t r o g e n procurement and metabolism i n response to a f l u c t u a t i n g n i t r o g e n supply could be an adaptive advantage i n an environment where the growth of macroalgae i s nitrogen l i m i t e d . 20 M a t e r i a l s and Methods Species and Culture Conditions Young non-reproductive Porphyra p e r f o r a t a J . Ag. (Rhodophyta) t h a l l i were c o l l e c t e d i n e a r l y March from a rocky -land f i l l s i t e on E n g l i s h Bay, K i t s i l a n o , Vancouver, B.C., Canada ( F i g . 2 ) . They were immediately transported to the la b o r a t o r y and r i n s e d with f i l t e r e d (0.45 pm) seawater. For t y grams wet weight of p l a n t m a t e r i a l was added to 10 l i t e r s of f i l t e r e d ' (0.45 pm) enriched (f/20 concentration of phosphate, trace metals and vi t a m i n s ; G u i l l a r d and Ryther 1962) nitro g e n d e f i c i e n t n a t u r a l seawater i n 12 l i t e r flat—bottomed b o i l i n g f l a s k s . Eight c u l t u r e s were stored i n a 10°C c o l d room under an i r r a d i a n c e of 150 juE.m~ 2.s~ 1 provided by f l u o r e s c e n t l i g h t i n g ( 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 . The c u l t u r e s were s t i r r e d continuously with a 2 inch magnetic s t i r r i n g bars at 120 rpm. One c u l t u r e was enriched with 50 pM n i t r a t e , another with 50 pM ammonium and a t h i r d with 50 pM ammonium plus 50 pM n i t r a t e . No nitrogen was added to the f o u r t h c u l t u r e . These nitrogen concentrations were chosen because they were high enough to allow maximum growth, and 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 . Lower concentrations would a l s o have supported maximum growth r a t e s , but the 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 with a high biomass. Du p l i c a t e c u l t u r e s were maintained i n order to produce s u f f i c i e n t biomass f o r subsequent t e s t s . The n i t r a t e and ammonium concentrations i n the f l a s k s were monitored and p e r i o d i c a d d i t i o n s were made to maintain 21 1^8° 1&1-F i g . 2. Map of Stanley Park, Vancouver, B.C., Canada, showing c o l l e c t i o n s i t e . 22 concentrations at 50 yuM. Five umol NaHC03 was added for every umol of nitrogen i n the medium to ensure that the c u l t u r e s d i d not approach carbon l i m i t a t i o n . Uptake r a t e s , n i t r a t e reductase l e v e l s , and i n t e r n a l s o l u b l e nitrogen content were measured each day at 1100 hours. Ammonium and N i t r a t e Uptake Experiments Two grams wet weight of plant m a t e r i a l was placed i n 400 ml f i l t e r e d enriched seawater (see above) with e i t h e r 25 /uM ammonium, 25 pM n i t r a t e , or 25 /UM ammonium plus 25 /uM n i t r a t e . The n i t r a t e and ammonium uptake rates were monitored continuously for 30 min (see chapter 1). An " i n i t i a l " rate l a s t i n g the f i r s t 10—20 min of exposure to the uptake medium was often followed by an increased uptake rate (an "induced" r a t e ) . Both rates were determined. The uptake medium was kept at 12°C with a water—jacketed c o o l i n g system. At the end of the incubation p e r i o d , the t h a l l i were removed and d r i e d to a constant weight (24-48 h) at 60°C. I n t e r n a l N i t r a t e Content One gram wet weight of p l a n t m a t e r i a l was ground i n a mortar with 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 for 6 min and analysed for n i t r a t e as o u t l i n e d i n Appendix 1. A l l 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 . 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 determined (Appendix 1). 23 N i t r a t e Reductase A c t i v i t y The jjri v i t r o n i t r a t e reductase assay o u t l i n e d i n Appendix 2 was used with the optimum assay c o n d i t i o n s for P. p e r f o r a t a n i t r a t e reductase a c t i v i t y (Appendix 2). One gram wet weight of plan t m a t e r i a l was used per assay. These e x t r a c t s were stored at -10°C p r i o r to a n a l y s i s for 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 done i n t r i p l i c a t e . The complexity of methods and the absolute requirement to do a l l the t e s t s at the same time of day made a d d i t i o n a l sampling impossible. Since patterns were complex, a l l curves were f i t t e d by eye i n order to show general trends. The e r r o r bars on the graphs are ± 1 standard d e v i a t i o n . 9 24 Results N i t r a t e Uptake Rates The n i t r a t e uptake rate of P. p e r f o r a t a p l a n t s taken d i r e c t l y from the f i e l d was 13—18 pmol.g dry wt" 1.h~ 1 ( F i g . 3). When p l a n t s were grown on s a t u r a t i n g concentrations of n i t r a t e f o r 48 h n i t r a t e uptake increased i f ammonium was not present i n the uptake medium ( F i g . 3B). N i t r a t e uptake i n the starved p l a n t s was not constant over the 30 min p e r i o d when n i t r a t e uptake was being monitored. I n i t i a l l y , n i t r a t e uptake rates were low ( i n i t i a l uptake r a t e ) , but a f t e r 10—20 min i n the uptake medium n i t r a t e uptake r a t e s increased (induced uptake r a t e ) . The presence of ammonium i n the uptake medium i n h i b i t e d the i n i t i a l n i t r a t e uptake rate of the starved t h a l l i f o r the f i r s t 10-20 min of the uptake experiment. A f t e r 8 days of nitrogen s t a r v a t i o n t h i s i n h i b i t i o n appeared to be overcome ( F i g . 3A). The f i e l d p l a n t s and i n some cases the starved l a b o r a t o r y c u l t u r e d p l a n t s 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 to the uptake medium. Ammonium Uptake Rates The ammonium uptake rates f o r the p l a n t s d i r e c t l y from the f i e l d ranged from 40—50 /umol.g dry w f ' . h " 1 ( F i g . 4). The ammonium uptake rates of the c u l t u r e s grown f o r 0-4 days on ammonium or ammonium p l u s n i t r a t e , decreased 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) i n i t i a l p e r i o d of ammonium uptake (approximately 10 /jmol.g dry wt" 1 .h" 1) at the s t a r t of the ammonium uptake experiments. The 2 5 s ^ 20' N O , N H ; ^starved - induced " " ~ starved initial 3 4 5 Time (days) starved - induced F i g . 3. N i t r a t e uptake rates (umol NO^.g dry wt~ 1 . h _ 1 ) of Porphyra p e r f o r a t a preconditioned 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 plus 50 pM ammonium ( { — x — ) , or nitrogen starved ( — • — • ) ; uptake rates were measured i n the presence of 25 /jM ammonium plus 25 JL\M n i t r a t e (A), or 25 uM n i t r a t e only (B). Uptake rates of the starved t h a l l i were oft e n induced (—•—) during the uptake experiment a f t e r 10-20 min. 2 6 A NH * A 'c 60-1 Time (days) B N O " • N H * 3 4 starved Time (days) F i g . 4. Ammonium uptake rates (tmiol NH4*.g dry wt' ^ h " 1 ) of Porphyra p e r f o r a t a preconditioned f o r 0 - 1 0 days on 5 0 pW n i t r a t e ( - - - • - - - ) , 5 0 pM ammonium (----A - - ) , 50 pM n i t r a t e p l u s 5 0 pM ammonium ( — ~ * — ) , or nitrogen s t a r v e d ; { — • — ) , uptake rates were measured i n the presence of 2 5 pM ammonium (A), or 2 5 pM ammonium plus 2 5 J J M n i t r a t e ( B ) . The ammonium and ammonium pl 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) unsustained uptake rates at the s t a r t of the uptake experiment. These r a t e s are i n d i c a t e d by ( ® ) and ( <g) ) . 2 7 c u l t u r e s grown on n i t r a t e alone maintained an ammonium uptake rate of approximately 30 /umol.g dry w f ' . h ' 1 . Ammonium uptake rates of the starved t h a l l i dropped approximately 30% during the f i r s t two days of s t a r v a t i o n and then recovered to 40-60 jumol.g dry wf ' . h " ' , Ammonium uptake rat e i n the starved c u l t u r e was r a p i d , but t h i s high rate was often s h o r t - l i v e d and decreased a f t e r 10-15 min i n the ammonium uptake medium. This was opposite to n i t r a t e uptake i n the n i t r a t e uptake medium. N i t r a t e had no appreciable e f f e c t on ammonium uptake. Generally ammonium uptake rates were 50% higher 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 content of a l l p l a n t s decreased with time and the i n t e r n a l n i t r a t e content of the starved p l a n t s reached zero a f t e r three days ( F i g . 5). The n i t r a t e grown p l a n t s and the n i t r a t e plus ammonium grown p l a n t s maintained the highest i n t e r n a l n i t r a t e l e v e l s . The e x t r a c t s were 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 described i n Appendix 1. Due to the p o s s i b i l i t y of incomplete e x t r a c t i o n and i n t e r f e r e n c e from other compounds during the 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 are not q u a n t i t a t i v e but i n c r e a s i n g or decreasing trends can be d i s t i n g u i s h e d . The ammonium content of a l l e x t r a c t s decreased by 50% during the f i r s t two days of c u l t u r i n g (Appendix 3). There was an i n i t i a l increase i n the ni n h y d r i n p o s i t i v e m a t e r i a l (assumed to be p r i m a r i l y amino a c i d s ) i n the t i s s u e e x t r a c t s of 28 ~ 12H T i m e (dc iys) F i g . 5. The n i t r a t e content (umol NO" .g wet wt" 1) of Porphyra  p e r f o r a t a preconditioned f o r 0-10 days on n i t r a t e '(---»---), ammonium (-.-.A-, n i t r a t e plus ammonium (—*—) or nitrogen starved ( — • — ) . E r r o r bars represent one standard d e v i a t i o n , 29 a l l p l a n t s except f o r those that had been nit r o g e n starved (Appendix 3). The s o l u b l e p r o t e i n contents of the e x t r a c t s were r e l a t i v e l y constant and s i m i l a r i n magnitude f o r a l l treatments(Appendix 3). N i t r a t e Reductase A c t i v i t y The i n i t i a l n i t r a t e reductase a c t i v i t y of the p l a n t s from the f i e l d was 72 pmol N0 2.g p r o t e i n " 1 . h " 1 ( F i g . 6 ) . Both nitrogen s t a r v a t i o n and the presence of n i t r a t e increased a c t i v i t y up to day 3, and then i t decreased r a p i d l y . This decrease i n a c t i v i t y i n the starved t h a l l i continued u n t i l day 6. Growth on ammonium r e s u l t e d i n a r a p i d decrease i n n i t r a t e reductase a c t i v i t y . The p l a n t s grown on n i t r a t e p l u s ammonium maintained a n i t r a t e reductase a c t i v i t y of approximately 60 /umol N02 .g p r o t e i n " 1 . h " 1 f o r the f i r s t three days. In the next 24 h, n i t r a t e reductase a c t i v i t y dropped by 50%. • 1 1 1 1 2 4 6 8 T i m e ( c l a y s ) 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 - 1.h" 1) of Porphyra pe r f o r a t a preconditioned for 0-10 days on n i t r a t e (—•---), ammonium I- -A- •-) T n i t r a t e plus ammonium ( — x — ) , or nitrogen starved ( — • — ) . Error bars represent one standard d e v i a t i o n , n=3. 31 Disc u s s i o n I t has been shown that nitrogen s t a r v a t i o n causes a t r a n s i e n t increase i n ammonium uptake by marine phytoplankton (Conway 1976) and by 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 1978). Starved P. 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 uptake. In c o n t r a s t , nitrogen r e p l e t e c u l t u r e s of P. p e r f o r a t a e x h i b i t e d very low n i t r a t e and ammonium uptake r a t e s . The moderate uptake r a t e s of the f i e l d p l a n t s suggest that these p l a n t s were p a r t i a l l y n itrogen l i m i t e d . This was unexpected c o n s i d e r i n g the r e l a t i v e l y high n i t r a t e and ammonium concentrations (17 and 4 fM, r e s p e c t i v e l y ) i n the surface water at the time of c o l l e c t i o n . The nitrate-grown c u l t u r e showed r e l a t i v e l y high ammonium uptake r a t e s . This suggested that the mechanism to maintain high ammonium uptake rates that i s normally a s s o c i a t e d with 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 present i n p l a n t s grown only on n i t r a t e . This has a l s o been observed for marine phytoplankton (Horrigan and McCarthy 1981). I t i s p o s s i b l e that n i t r a t e reduction i s the r a t e l i m i t i n g process (Dortch et a l . 1982) and that the supply of a s s i m i l a t e d nitrogen i s much slower f o r a nitrate—grown p l a n t than f o r an ammonium—grown p l a n t . One of the l a t e r steps i n nitrogen metabolism which i s not a c t i v a t e d when n i t r a t e r eduction i s slow, may be the f a c t o r c o n t r o l l i n g ammonium uptake. I t has been p o s t u l a t e d that ammonium uptake may be c o n t r o l l e d by the l e v e l of c e r t a i n amino a c i d s (Dortch 1980). Neither s o l u b l e p r o t e i n or t o t a l amino a c i d content appeared to be the c r i t i c a l c o n t r o l l i n g f a c t o r (Appendix 3). Ammonium i s apparently a b e t t e r nitrogen source 32 f o r growth of marine macrophytes than n i t r a t e (Yamada 1961; DeBoer et a l . 1978) because n i t r a t e must be converted to ammonium before i t i s a s s i m i l a t e d and t h i s reduction requires energy (Nicholas 1959). I f n i t r a t e reduction i s the rate l i m i t i n g step, then n i t r a t e pools should f i l l and n i t r a t e uptake should be reduced to match the rat e of n i t r a t e r e d u c t i o n , assuming that the 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 . The f a c t that the n i t r a t e content of the nitrate-grown c u l t u r e was l a r g e r and that n i t r a t e uptake was d r a m a t i c a l l y reduced a f t e r 24 h, supports t h i s suggestion. The N—starved P. p e r f o r a t a maintained high i n i t i a l ammonium uptake r a t e s . I n i t i a l n i t r a t e uptake r a t e s were low but were increased i n 10-20 min wit h exposure to n i t r a t e . I t appears that the uptake system f o r n i t r a t e does not remain f u l l y a c t i v e i n a N—starved t h a l l u s , but i t can be r a p i d l y r e a c t i v a t e d by a pulse of n i t r a t e . This has been observed i n phytoplankton (Dortch et a l . 1982) and i t i s assumed to be due to a r e a l l o c a t i o n of metabolic energy during s t a r v a t i o n . I t i s c e r t a i n l y an adaptive advantage fo r a nitrogen starved p l a n t to have n i t r a t e uptake r e a d i l y i n d u c i b l e . This i n d u c t i o n of n i t r a t e uptake rate was not seen i n nitrogen r e p l e t e p l a n t s . The delay (48 h) i n the i n d u c t i o n of n i t r a t e uptake i n nitrate—grown c u l t u r e s could be l i n k e d to the time required to induce n i t r a t e reductase a c t i v i t y which was a l s o approximately 48 h. U n t i l t h i s time n i t r a t e taken up would not be a s s i m i l a t e d and n i t r a t e pools would f i l l . T his could l i m i t n i t r a t e uptake. The enhanced i n i t i a l ammonium uptake i n both the 33 ammonium—grown and the ammonium plus nitrate-grown p l a n t s suggests that ammonium uptake was c o n t r o l l e d by a small i n t e r n a l pool which was r a p i d l y f i l l e d . The d e p l e t i o n of t h i s pool might have occurred during the time a f t e r the t h a l l i were removed from ammonium c u l t u r e medium and before the s t a r t of the uptake experiment. However, t h i s was u s u a l l y only a few minutes, a time which i s considered i n s u f f i c i e n t to deplete nitrogen reserves. This surge uptake of ammonium has a l s o been observed i n phytoplankton (McCarthy and Goldman 1979; Turpin and Harrison 1979) and other macrophytes (Chapter 1), but u s u a l l y i t takes s e v e r a l hours to develop 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 nit r o g e n starved P. p e r f o r a t a u n t i l the eighth day of s t a r v a t i o n ( F i g . 3). P r e f e r e n t i a l uptake of ammonium may reduce the energy required by the plan t because i t i s not necessary to reduce n i t r a t e to ammonium (Syrett 1962). P a r t i a l i n h i b i t i o n of n i t r a t e uptake by ammonium i s common i n phytoplankton (Conway 1977; McCarthy et a l . 1977; M a e s t r i n i et a l . 1982) and marine macrophytes ( D ' E l i a and DeBoer 1978; Haines and Wheeler 1978; Hanisak and H a r l i n 1978; Gordon et a l . 1981). The r e s u l t s of t h i s study suggest that t h i s no longer occurs a f t e r a c e r t a i n p e r i o d of nitrogen s t a r v a t i o n . 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 only 10-20 min and then n i t r a t e uptake rates were independent of the presence or absence of ammonium. Ammonium concentrations i n the uptake f l a s k were approximately 10-15 JUM. Concentrations below t h i s 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 uptake. 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 incubated i n 5 pM ammonium. Even lower ammonium conc e n t r a t i o n s (1 pM) have caused i n h i b i t i o n of n i t r a t e uptake i n phytoplankton (Conway 1977; McCarthy et a l . 1977). Nitrogen s t a r v a t i o n caused a general decrease i n i n t e r n a l ammonium, n i t r a t e , and s o l u b l e amino a c i d content i n P. p e r f o r a t a . The smallest decrease was i n t o t a l amino a c i d s . This c o n t r a d i c t s the f i n d i n g s of B i r d et a l . (1982b) and Rosenberg and Ramus (1982) who suggested that amino a c i d s and p r o t e i n s were a more important 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 pool than n i t r a t e or ammonium. On the other hand, Best (1980) showed that amino a c i d content of Ceratophyllum demersum was not a f f e c t e d by nitrogen supply. I n t e r n a l n i t r a t e content decreased more r a p i d l y i n the starved p l a n t s than i n the ammonium-incubated p l a n t s . I t i s p o s s i b l e that i n the ammonium-incubated p l a n t s , ammonium was taken up and a s s i m i l a t e d rather than the i n t e r n a l n i t r a t e being u t i l i z e d . The n i t r a t e reductase l e v e l s of the s t a r v e d c u l t u r e were s t i l l h i gh, and i n t e r n a l n i t r a t e l e v e l s were reduced. The decrease i n i n t e r n a l n i t r a t e content was dependent on n i t r a t e reductase a c t i v i t y which was i n f l u e n c e d by n i t r o g e n d e f i c i e n c y . Nitrogen s t a r v a t i o n caused a t r a n s i e n t increase i n n i t r a t e reductase i n P. p e r f o r a t a . This a l s o occurs i n phytoplankton (Morris and S y r e t t 1965). The subsequent decrease i n a c t i v i t y w i t h more severe nitrogen s t a r v a t i o n r e s u l t s i n i n a c t i v a t i o n of an enzyme 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 maintain during a time of nitrogen s t r e s s . Yet even under N — s t a r v a t i o n , P. p e r f o r a t a maintained a low l e v e l of n i t r a t e 35 reductase a c t i v i t y . Growth on n i t r a t e caused a t r a n s i e n t increase i n n i t r a t e reductase a c t i v i t y . This has been observed i n 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). Growth on ammonium i n h i b i t e d n i t r a t e reductase a c t i v i t y i n P. p e r f o r a t a . Even the c u l t u r e s u p p l i e d with both n i t r a t e and ammonium showed a decrease i n n i t r a t e reductase a c t i v i t y . N i t r a t e reduction was not "turned o f f " as r a p i d l y as n i t r a t e uptake by the presence of ammonium. Eppley et a l . (1969a) found that ammonium i n h i b i t e d n i t r a t e reductase a c t i v i t y i n phytoplankton. This was a l s o true for higher p l a n t s (MacKown et 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 s t u d i e s have examined the i n t e r a c t i o n between nitrogen supply, uptake r a t e s , a s s i m i l a t i o n , and content. Nitrogen uptake and a s s i m i l a t i o n were maintained even under s t a r v a t i o n c o n d i t i o n s i n P. p e r f o r a t a . I n t e r n a l sources of n i t r a t e and ammonium were u t i l i z e d during n i t r o g e n d e p r i v a t i o n , but amino ac 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 . The i n t e r a c t i o n between n i t r a t e uptake and ammonium uptake was dependent on the n u t r i t i o n a l s t a t e of the t h a l l u s and the ambient ammonium co n c e n t r a t i o n . A s s i m i l a t i o n of n i t r a t e was slower than that of ammonium and the c o n t r o l of ammonium uptake and a s s i m i l a t i o n was dependent upon ammonium supply. Simultaneous study of the e f f e c t s of ni t r o g e n supply on se v e r a l stages on ni t r o g e n u t i l i z a t i o n has i l l u s t r a t e d the multi-dimensional complexity of the metabolic c o n t r o l s of nitro g e n procurement. This was achieved i n a l a b o r a t o r y study where the n u t r i e n t regimes could be c o n t r o l l e d . The e c o l o g i c a l 3 6 s i g n i f i c a n c e 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 study with G r a c i l a r i a verrucosa (Chapter 5). 37 Chapter 3. Nitrogen Uptake and Growth of Fucus d i s t i c h u s Germlings and Mature T h a l l i I n t r o d u c t i o n A l g a l zonation occurs because of d i f f e r e n t species' responses to b i o t i c f a c t o r s such as competition (Jones and Kain 1967; Connell 1972; Mann 1972; Chapman 1973) and a b i o t i c f a c t o r s such as changing humidity, l i g h t , s a l i n i t y , temperature and n u t r i e n t supply (Zaneveld 1937; Doty 1946; Lewis 1964; Topinka 1978; Rosenberg and Ramus 1982) Recent s t u d i e s i n d i c a t e that some i n t e r t i d a l macrophytes may a c t u a l l y f l o u r i s h under extreemes i n s e v e r a l environmental f a c t o r s (Johnson et a l . 1974; Quadir et a l . 1979; Thomas and Turpin 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 though i t may be that the s e n s i t i v i t i e s of other l i f e h i s t o r y stages that determine the d i s t r i b u t i o n of the mature p l a n t s (Subbaraju et a l . 1982). McLachlan (1974) suggested that the s e n s i t i v i t y of Fucus edentatus and F. d i s t i c h u s germlings to temperature and l i g h t was important i n c o n t r o l l i n g a d u l t d i s t r i b u t i o n . In a d d i t i o n , d e s i c c a t i o n and s a l i n i t y changes a f f e c t the s u r v i v a l of Phaeostrophion i r r e g u l a r e germlings (Mathieson 1982). Release of gametes, f e r t i l i z a t i o n , and germination are c r i t i c a l phases i n the f u c o i d l i f e h i s t o r y and they have the p o t e n t i a l to l i m i t a l g a l d i s t r i b u t i o n . Terry and Moss (1981) showed that zygotes of four species from the Fucaceae germinated under a wide range of l i g h t and temperature c o n d i t i o n s . A low tole r a n c e to one of the many i n t e r t i d a l environmental s t r e s s e s i n any stage i n the l i f e h i s t o r y of a marine macrophyte may 38 determine a l g a l zonation i n an i n t e r t i d a l h a b i t a t . This study i n v e s t i g a t e d the e f f e c t of nitrogen source and p e r i o d i c exposure to a i r on growth, r h i z o i d development, and nitrogen uptake i n the germlings of the mid— to high— i n t e r t i d a l alga Fucus d i s t i c h u s . The n i t r o g e n uptake rates of the germlings and mature t h a l l i were compared to i l l u s t r a t e the d i f f e r e n t adaptations of these two l i f e h i s t o r y stages. 39 M a t e r i a l s and Methods Organisms and C o l l e c t i o n S i t e F e r t i l e and n o n - f e r t i l e specimens of Fucus d i s t i c h u s L. , were c o l l e c t e d from the i n t e r t i d a l seawall at Brockton P o i n t , Stanley Park, Vancouver, B.C. ( F i g . 2). Their height above Canadian datum was approximately 3.5 m. P l a n t f e r t i l i t y was determined by the presence of w e l l developed conceptacles, dark brown c o l o r a t i o n , and i n f l a t e d r e c e p t a c l e s ( P o l l a c k 1970). C o l l e c t i o n s were made from November to January. Specimens were picked from the w a l l with h o l d f a s t s i n t a c t , placed i n p l a s t i c bags and transported on i c e to the l a b o r a t o r y w i t h i n an hour. The f e r t i l e t h a l l i were arranged on f l a t t r a y s and stored f o r 24 h at 4° C, and 50 jjE.m^.s" 1 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 under a 16:8 l i g h t : d a r k c y c l e . Nitrogen uptake experiments were conducted immediately on the n o n — f e r t i l e t h a l l i . Gamete Discharge The discharge of gametes was accomplished using the dehydration-rehydration technique of P o l l a c k (1970) and McLachlan et a l . (1971). Reproductive receptacles were excised from the t h a l l i and gently scrubbed by hand under a gentle stream of c o l d tap water to remove epiphytes, extruded gametes, or newly formed zygotes. A f t e r scrubbing, r e c e p t a c l e s were placed on paper towels, gently b l o t t e d dry and allowed to stand fo r 45 min at room temperature. Receptacles were then placed i n 90x20 mm disposable p e t r i dishes c o n t a i n i n g 20 ml f i l t e r e d (0.45 jam) enriched n a t u r a l seawater. This seawater had been p r e v i o u s l y depleted of nitrogen by incubating 1 g wet wt of Ulva 40 sp. per l i t r e f o r 12-24 h at 10°C and 12 p E . n r 2 . s " 1 . The seawater was f i l t e r e d and enriched with f/2 concentrations of phosphate, t r a c e metals and vitamins ( G u i l l a r d and Ryther 1962) and 10 pM n i t r a t e , or 10 pM ammonium, or 5 pM (=10 pg—at N . l " 1 ) urea and used as the c u l t u r e medium. The r e c e p t a c l e s were incubated overnight at 15°C and 150 pE.m^.s' 1 w i t h a 16:8 l i g h t : d a r k c y c l e provided by f l u o r e s c e n t l i g h t s ( V i t a - l i t e ) . The spent r e c e p t a c l e s were discarded and the seawater medium re p l e n i s h e d . C u l t u r e 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) at 15°C. Discharge of gametes was suppressed at i r r a d i a n c e s below 35 pE.m~ 2.s~ 1. Germination was independent of i r r a d i a n c e and growth became l i g h t saturated at i r r a d i a n c e s greater than 85 pE.m" 2.s _ 1. Therefore an i r r a d i a n c e of 150 /uE.m^.s"1 on a 16:8 l i g h t : d a r k c y c l e was used to ensure l i g h t s a t u r a t i o n . Germlings were c u l t u r e d f o r three weeks on 20 uM n i t r a t e , 20 pM ammonium, 10 pM (=20 pg.at N . l " 1 ) urea, or 10 pM ammonium pl u s 10 pM n i t r a t e . Night—time low t i d e s that occur i n winter i n Vancouver were simulated by removal of medium from one set of ammonium c u l t u r e s once every 24 h f o r 10 h i n c l u d i n g 8 h of darkness and then r e p l a c i n g the same medium. Fresh medium was provided every three days. Determination of Growth Rates and Morphology Growth ra t e s were determined each week except i n the 41 exposed c u l t u r e s , where a s i n g l e measurement was made a f t e r three weeks. The i n i t i a l lengths of 40 randomly s e l e c t e d zygotes per d i s h were determined using a b i n o c u l a r microscope wi t h a c a l i b r a t e d eyepiece. Length measurements were made at weekly i n t e r v a l s . The percentage of 100 randomly s e l e c t e d germlings which d i s p l a y e d secondary r h i z o i d development a f t e r three weeks of incubation was recorded. Three week o l d c u l t u r e s which had experienced d a i l y exposure were examined and compared to three week o l d ammonium- and nitrate-grown c u l t u r e s that were continuously submerged. Nitrogen Uptake Experiments A time course of nitrogen uptake was determined. A p e t r i d i s h c o n t a i n i n g three week o l d germlings was submerged i n a beaker with 200 ml of f i l t e r e d , enriched seawater (Chapter 1) c o n t a i n i n g a known nit r o g e n c o n c e n t r a t i o n ; e i t h e r -15 pM ammonium, or 30 pM n i t r a t e , or 15 pM ammonium plus 30 pM n i t r a t e . The ammonium and n i t r a t e concentrations were monitored c o n t i n u o u s l y f o r one hour using a Technicon AutoAnalyzer (Davis et a l . 1973). The c u l t u r e v e s s e l was placed on a shaker t r a y (40 rpm) at 15°C and an i r r a d i a n c e of 150 pE.m^.s' 1. Uptake ra t e s were expressed as the change i n nitrogen concentration per u n i t time per gram dry weight of t i s s u e . Each p e t r i d i s h was used i n only one uptake experiment. Nitrogen uptake k i n e t i c s were determined using the p e t r i dishes as the incubation v e s s e l s . The o l d medium was poured o f f and the three week o l d germlings, which remained attached to the bottom of the p e t r i d i s h , were q u i c k l y r i n s e d with f i l t e r e d , 42 enriched seawater c o n t a i n i n g n i t r a t e (0-50 pM) or ammonium (0-50 jjM) depending on the experiment. This medium (35 ml) was then poured i n t o the d i s h . D u p l i c a t e experiments were run at each n i t r a t e or ammonium c o n c e n t r a t i o n . The dishes were placed on a shaker t r a y at 15°C and an i r r a d i a n c e of 150 pE.m~ 2.s~ 1 and incubated f o r 30 min f o r ammonium and one hour f o r n i t r a t e . The medium was then removed and immediately analysed for n i t r a t e and ammonium using the AutoAnalyzer. The average uptake rates over the i n c u bation p e r i o d were determined from the d i f f e r e n c e between the f i n a l c o ncentration of the medium and the t=0 co n c e n t r a t i o n , and then normalized to the dry weight of the germlings i n the incubation d i s h . Nitrogen uptake k i n e t i c experiments were conducted as o u t l i n e d above for n i t r a t e , ammonium, and n i t r a t e — plus ammonium—grown germlings. Some germlings which were p r e v i o u s l y grown on ammonium plus n i t r a t e , were starved of nitrogen by incubating them i n nitrogen f r e e medium f o r 24 h p r i o r to determining uptake r a t e s . N i t r a t e and ammonium uptake rates were determined f o r exposed c u l t u r e s immmediately f o l l o w i n g a 10 h per i o d of exposure by per t u r b i n g them with enriched seawater with 30 pM n i t r a t e or 15 pM ammonium. A l l germlings were c a r e f u l l y loosened from the p e t r i d i s h and placed on a pre-weighed 0.45 pm M i l l i p o r e f i l t e r . The f i l t e r and germlings were then r i n s e d with sodium formate (3% w/v) to remove the s a l t c r y s t a l s which would otherwise form when the f i l t e r was d r i e d . The f i l t e r and germlings were d r i e d at 60°C to a constant weight (1 h ) . Any sodium formate which was l e f t on the f i l t e r , sublimed when i t was heated. There was no 43 increase i n the weight of f i l t e r s without germlings that were t r e a t e d with sodium formate and d r i e d . The germlings u s u a l l y accounted f o r 20-50% of the t o t a l weight ( f i l t e r and germlings). D u p l i c a t e uptake experiments (one d i s h of germlings per experiment) were run. In the 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 placed i n 450 ml of f i l t e r e d enriched seawater (Chapter 1) of known nitrogen 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 , or 15 pM ammonium, or 30 pM n i t r a t e plus w 15 pM ammonium and incubated under an i r r a d i a n c e of 150 pE.m^.s" 1 at 15°C f o r 30 min. The medium was s t i r r e d c o n tinuously (Chapter 1) and the n i t r a t e and ammonium concentrations were monitored continuously using a Technicon AutoAnalyzer (Davis et a l . 1973). A f t e r the experiment, the t h a l l i were d r i e d to a constant weight i n a 60°C oven. The uptake k i n e t i c s s t u d i e s w i t h mature t h a l l i i n v o lved "batch" type uptake experiments. Non-reproductive t h a l l i (2—3 g wet wt) were placed i n 300 ml of enriched f i l t e r e d seawater with a known n i t r a t e or ammonium co n c e n t r a t i o n . These were incubated at 15°C, under an i r r a d i a n c e of 150 pE.m~ 2.s~ 1 f o r 30 min i n the n i t r a t e experiments and f o r 10 min i f ammonium was used. The medium was s t i r r e d c ontinuously w i t h a magnetic s t i r r i n g bar (Chapter 1). A f t e r the experiment the 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 to a constant weight i n a 60°C oven. Each treatment was performed i n t r i p l i c a t e . The n i t r a t e and ammonium concent r a t i o n i n the incubation medium were determined at the end of the experiment. Patterns were often complex and ther e f o r e a l l curves were t t e d by eye to show only general trends i n the data. 45 Results Gamete release was p r o l i f i c and there was a high l e v e l of f e r t i l i z a t i o n and germination i n the presence of the nitrogen sources t e s t e d . The form of nitrogen ( n i t r a t e , ammonium, urea, or n i t r a t e plus ammonium) had no e f f e c t on the germling growth r a t e s (Table 2). P e r i o d i c exposure to a i r had no e f f e c t on the increase i n germling length, but d i d r e s u l t i n a two-fold increase i n secondary r h i z o i d development a f t e r three weeks of in c u b a t i o n . There were no obvious d i f f e r e n c e s among the nit r o g e n treatments with regard to secondary r h i z o i d development. F. d i s t i c h u s germlings maintained i n i t i a l uptake rates of n i t r a t e and ammonium f o r at l e a s t one hour. They could take up n i t r a t e and ammonium simultaneously. The presence of ammonium d i d not i n h i b i t n i t r a t e uptake. N i t r a t e uptake was constant f o r 30 min i n non-reproductive mature F. d i s t i c h u s t h a l l i . Ammonium uptake r a t e s were constant f or the f i r s t 15-20 min a f t e r which they decreased. The presence of ammonium i n h i b i t e d the r a t e of n i t r a t e uptake by approximately 30%. Nitrogen uptake rates per gram dry weight were much higher i n the germlings than the adul t p l a n t s . Ammonium uptake rates at 15 yuM ammonium were approximately eight times g r e a t e r , and n i t r a t e uptake rates at 30 /uM n i t r a t e were 20-40 times greater i n the mature p l a n t s . N i t r a t e uptake i n F. d i s t i c h u s germlings as a f u n c t i o n of n i t r a t e concentration followed s a t u r a t i o n k i n e t i c s ( F i g . 7 ) . Uptake was saturated at 15-20 /uM n i t r a t e . The maximum n i t r a t e uptake ra t e (Vmax) was s i m i l a r i n germlings starved of nitrogen 46 Table 2. The e f f e c t of nitrogen source on average growth rate (pm.week"1) and secondary r h i z o i d development of F. d i s t i c h u s germlings over a three week p e r i o d , and the percent germlings e x h i b i t i n g secondary r h i z o i d development from 100 randomly s e l e c t e d germlings. Values include ± 1 standard e r r o r , n=l00. Nitrogen S o u r c e Growth Rate (Lim.week - 1) % 2° rhi z o i d development NO; 173±14 14 ±5 NH+ 176±26 20±2 H U r e a 183 ±40 25±2 NH+ + NO: * 218±59 19+4 NH+ + Emerged 248±55 57 ±5 0J * 3 3 (D •->• 3 3 (D iQ o o ?r • 3 3 O - J C c • 3 3 a n- *q c z CD o M -3 I C rr • cn it 3 i 0) i D J r f n- M • n> i-« •» cn o r r C i n CD 3 O r r 3 n-cn i-t cn fo rr D) 0) r r i£) ?r fD fD M -< n 3 3 (t» r r 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 N i t r a t e Uptake R a t e ( j umo l N O " • g d r y w t " 1 • h"1 ) a. 2. o 1_ o • 3 lO . : 3 n • : r r • n * —-0) r r O r t ' r-l n -iQ --. r-t O 3 O 3 - CO 48 for 24 h and those grown on n i t r a t e . Germlings grown on ammonium as t h e i r only n i t r o g e n source had a 50% lower maximum n i t r a t e uptake rate than i n other c u l t u r e s . N i t r a t e uptake i n mature F. d i s t i c h u s t h a l l i was not saturated up to 50 pM ( F i g . 8 ) . Uptake rates at low n i t r a t e c oncentrations appeared to f o l l o w s a t u r a t i o n k i n e t i c s , but a non—saturable component became important at n i t r a t e c oncentrations greater than 10 pM. The c o n t r i b u t i o n due to t h i s low a f f i n i t y non—saturable component increased l i n e a r l y with n i t r a t e c o n c e n t r a t i o n . I f the uptake due to t h i s component was subtracted from the uptake rate ( D ' E l i a and DeBoer 1978), the r e s u l t i n g high a f f i n i t y uptake curve was sat u r a b l e and the h a l f — s a t u r a t i o n constant (Ks) for the uptake was c a l c u l a t e d to be approximately 1—3 pM. Ammonium uptake i n F. d i s t i c h u s germlings appeared to f o l l o w s a t u r a t i o n k i n e t i c s up to 20 pM ammonium ( F i g . 9). At ammonium concentrations greater than 20 pM, uptake rates increased l i n e a r l y with ammonium co n c e n t r a t i o n . There were no apprec i a b l e d i f f e r e n c e s i n ammonium uptake rates among the four p r e c o n d i t i o n i n g nitrogen treatments. Maximum uptake rates were approximately 60 pmol NH4*.g dry w f ' . h " 1 and Ks values were approximately 3—5 pM ammonium. Ammonium uptake i n adult F. d i s t i c h u s was p r o p o r t i o n a l to the ambient ammonium concent r a t i o n at c o n c e n t r a t i o n s greater than 15 pM ( F i g . 10). I t i s f e l t that t h i s uptake was due to a low a f f i n i t y d i f f u s i o n uptake component. The existence of a high a f f i n i t y a c t i v e uptake component i s questionable ( F i g . 10). Nevertheless, the c o n t r i b u t i o n due to the low a f f i n i t y p o s s i b l y d i f f u s i o n 1 I I I 10 2 0 3 0 4 0 N i t r a t e (>iM ) F i g . 8. N i t r a t e uptake rates (umol N0 3".g dry w t " 1 . h _ 1 ) a s a func t i o n of n i t r a t e concentration for mature Fucus d i st ichus t h a l l i . Error bars represent one standard d e v i a t i o n , n=3. Uptake rates minus non—saturable component ( — o — ) were determined to assess only the a c t i v e uptake component. A m m o n i u m (juM) F i g . 9. Ammonium uptake rates (umol NH 4 +.g dry wt- 1.h-')as a functio n of ammonium concentration for three week o l d Fucus d i s t i c h u s germlings grown on n i t r a t e ( — • — ) , ammonium (-..-I-.-) f n i t r a t e plus ammonium (••••••) or grown on ammonium then nitrogen starved for 24 h ( — * — ) . 15 3 0 A m m o n i u m ^L IM) 45 F i g . 10. Ammonium uptake rates (pmol NH4 + .g dry w f ' . h - M a s a functio n of ammonium concentration for mature Fucus d i s t i c h u s t h a l l i . Error bars represent one standard d e v i a t i o n , n=3. Uptake rates minus non—saturable component ( — o — ) were determined to assess only the a c t i v e uptake component. 52 component was subtracted to give an estimation of the a c t i v e uptake component ( D ' E l i a and DeBoer 1978). A Ks of approximately 10 uM was c a l c u l a t e d from these data. A minimum concent r a t i o n of 1-2 pM ammonium was required f o r ammonium uptake to take place ( F i g . 10). Incubation for four and a h a l f days i n ammonium enriched or nitrogen d e f i c i e n t seawater r e s u l t e d i n an increased t h r e s h o l d ammonium concent r a t i o n (5 pM) fo r uptake i n mature t h a l l i ( F i g . 11). Exposure to a i r caused a 70% decrease i n n i t r a t e uptake (23.4 to 7.1 pmol.g dry wt~ 1.h~ 1) i n the germlings at 30 pM n i t r a t e but had no e f f e c t on ammonium uptake at 15 pM ammonium (57 pmol.g dry w f ' . h " 1 ) . 2 0 4 0 A m m o n i u m ( / JM ) F i g . 11. Ammonium uptake rates (pmol NH • g dry wt" 1 h'Mas a function of ammonium concentration for mature Fucus d i s t i c h u s t h a l l i preincubated on ammonium for 4.5 d a y l — T ^ h S 1 nitrogen starved for 4.5 days (--o--). ;' or cn CO 54 D i s c u s s i o n The d i s t r i b u t i o n l i m i t s of mature Fucus may be i n f l u e n c e d by the responses of germlings to the environment. Vegetative reproduction i s g e n e r a l l y unimportant i n Fucus spp. (McLachlan 1974). The germling stage of Fucus plays an important r o l e i n population maintenance and d i s p e r s a l . F. d i s t i c h u s i s a very common i n t e r t i d a l seaweed i n the Northeast P a c i f i c Ocean. Gamete r e l e a s e , f e r t i l i z a t i o n , germination, germling growth, and mature t h a l l u s growth have no requirement f o r a s p e c i f i c form of n i t r o g e n . F. s p i r a l i s (Topinka and Robbins 1976) and F. v e s i c u l o s u s (Prince 1974) grew e q u a l l y w e l l on ammonium and n i t r a t e . This i s a l s o true f o r Chondrus (Prince 1974). On the other hand, the mature t h a l l i of s e v e r a l other marine macrophytes do show more ra p i d growth when ammonium i s present than when n i t r a t e i s the n i t r o g e n source (Yamada 1961; DeBoer et a l . 1978). This lower growth rate on n i t r a t e i s apparently due to the u t i l i z a t i o n of energy f o r n i t r a t e reduction (DeBoer 1981). Urea g e n e r a l l y produces lower growth r a t e s than ammonium (Yamada 1961; DeBoer et a l . 1978), although comparable or supe r i o r growth on urea has been reported f o r Porphyra (Iwasaki 1967) and Ulva (DeBoer 1981). The form of nitrogen producing optimal growth may a l s o depend on the conce n t r a t i o n (Iwasaki 1967). Ammonium becomes t o x i c at much lower concentrations than n i t r a t e does (Waite and M i t c h e l l 1972; DeBoer 1981). The concentrations used i n t h i s study were those commonly found under f i e l d c o n d i t i o n s . P e r i o d i c exposure and the s t r e s s e s i t imposes are unique to 55 the i n t e r t i d a l environment. Exposure causes major f l u c t a t i o n s i n t i s s u e water content, s a l i n i t y , i r r a d i a n c e , temperature and n u t r i e n t supply. I n t e r t i d a l macrophytes must be adapted to sur v i v e these changes. There have been s e v e r a l s t u d i e s on these phenomena ( B i e b l 1938; Johnson et a l . 1974; Quadir et a l . 1979; Schonbeck and Norton 1979a; Thomas and Turpin 1980; Chapter 4). The e a r l y l i f e h i s t o r y stages of i n t e r t i d a l macrophytes must a l s o s u r v i v e such s t r e s s e s . F. d i s t i c h u s germlings grew w e l l when they were exposed d a i l y and p e r i o d i c exposure seemed necessary f o r optimum development. Secondary r h i z o i d development was g r e a t l y reduced i f the germlings were not exposed. I t has been shown that p e r i o d i c exposure enhances growth i n mature i n t e r t i d a l fucoids (Schonbeck and Norton 1979a). Nitrogen uptake rates of germlings were much higher than those of mature t h a l l i . This may have been due to the larg e p r o p o r t i o n of storage and support t i s s u e i n the adul t p l a n t s which was n e i t h e r a c t i v e l y t a k i n g up nor r e q u i r i n g n i t r o g e n . The uptake rates of growing a p i c i e s may be higher. I f a large p o r t i o n of the a c t i v e l y growing germling 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 high uptake rates expressed on a dry weight b a s i s , but would not a f f e c t the Ks values. Maximum uptake r a t e s on a dry weight b a s i s were very d i f f e r e n t for germlings and mature t h a l l i , 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 values f o r n i t r a t e and ammonium uptake by the germlings were s i m i l a r to those recorded for Grac i l a r i a  t i k v a h i a e and Neoaqardhiella b a i l e y i ( D ' E l i a and DeBoer 1978), 56 but they are l e s s than most Ks values reported for marine macrophytes (Haines and Wheeler 1978; H a r l i n 1978; H a r l i n and C r a i g i e 1978; Hanisak and H a r l i n 1978; Kautsky 1982), i n c l u d i n g F. s p i r a l i s (Topinka 1978). For F. s p i r a l i s the Ks f o r ammonium and 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). The high a f f i n i t y of F. d i s t i c h u s germlings for n i t r a t e and ammonium may provide a competitive advantage over mature t h a l l i when nitrogen s u p p l i e s i n the i n t e r t i d a l h a b i t a t are low. Ammonium uptake* rates were twice those f o r n i t r a t e by F. d i s t i c h u s germlings and ten times those of the mature t h a l l i . This i s apparently common i n marine macrophytes ( D ' E l i a and DeBoer 1978; Hanisak and H a r l i n 1978; Wheeler 1982), although Topinka (1978) reported that n i t r a t e and ammonium uptake rates i n F. s p i r a l i s were s i m i l a r . The ammonium uptake rates f o r germlings presented i n t h i s study were s i m i l a r to those reported for the mature t h a l l i of other species ( D ' E l i a and DeBoer 1978; Haines and Wheeler 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; Kautsky 1982). The n i t r a t e uptake rates of the adult F. d i s t i c h u s were low i n comparison to other marine macrophytes ( D ' E l i a and DeBoer 1978; H a r l i n 1978; H a r l i n and C r a i g i e 1978). The n i t r a t e concentrations i n the water surrounding the F. d i s t i c h u s bed were probably s a t u r a t i n g (20-25 pM) i n the winter whereas ammonium was undetectable. Temperate seaweeds are ofte n nitrogen saturated i n the winter (Chapman and C r a i g i e 1977; Rosenberg and Ramus 1982). I t appears that t h i s i s not true f o r r a p i d l y growing germlings. Winter nitrogen uptake rates i n the 57 germlings were high and these rates were maintained f o r a long time. Another adaptation to r a p i d nitrogen procurement was that ammonium d i d not i n h i b i t n i t r a t e uptake. This i n h i b i t i o n does occur i n the mature F. d i s t i c h u s and s e v e r a l other macrophytes (Haines and Wheeler 1978; Hanisak and H a r l i n 1978; Chapter 1) but i t seems to be dependent upon n u t r i t i o n a l s t a t u s . Time course uptake experiments with F. d i s t i c h u s i n the 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) c o l l e c t e d F. s p i r a l i s i n the summer and found s i m i l a r r e s u l t s . I t has been p o s t u l a t e d that the i n h i b i t i o n of n i t r a t e uptake by ammonium i s overcome when the t h a l l i are nitrogen l i m i t e d (Chapter 1). The F. d i s t i c h u s germlings were c u l t u r e d at 20 pM n i t r o g e n . I t i s p o s s i b l e they were nitrogen l i m i t e d i n s p i t e of frequent changes of medium. A l t e r n a t i v e l y germlings may possess the a b i l i t y to take up n i t r a t e and ammonium under a l l c o n d i t i o n s . Nitrogen s t a r v a t i o n d i d not a f f e c t ammonium uptake i n F. d i s t i c h u s germlings. The c u l t u r e s s u p p l i e d with nitrogen maintained r a p i d uptake rates which may r e f l e c t the r a p i d growth rate of t h i s j u v e n i l e stage. P e r i o d i c exposure to a i r or growth on ammonium as the only n i t r o g e n source caused a decrease i n n i t r a t e uptake i n F. d i s t i c h u s germlings. The germlings which were nitrogen starved d i d not show t h i s response. The maintenance of a n i t r a t e uptake system i n the absence of an e x t e r n a l n i t r a t e supply must be e n e r g e t i c a l l y expensive, although r e t e n t i o n of the a b i l i t y to take up any form of ni t r o g e n as soon as i t was a v a i l a b l e would 58 be advantageous. F. d i s t i c h u s germlings showed saturable nitrogen uptake k i n e t i c s but the a d u l t s d i d not show t h i s p a t t e r n . Both s a t u r a b l e (Topinka 1978) and non-saturable nitrogen uptake ( D ' E l i a and DeBoer 1978; Haines and Wheeler 1978; Harrison and Druehl 1982) have been found i n marine macrophytes. The r e s u l t s of t h i s study and that reported i n Chapter 5 suggest that t h i s i s not species dependent, but may depend on the n u t r i t i o n a l s t a t e and/or l i f e h i s t o r y stage. Furthermore, the f a c t that non-saturable nitrogen uptake i s common i n macrophytes, but not in phytoplankton, suggests that the non-saturable component may be r e l a t e d to the t h i c k , m u l t i c e l l u l a r t h a l l o i d form. This component c h a r a c t e r i s e s ammonium uptake but not n i t r a t e uptake i n some species ( D ' E l i a and DeBoer 1978). L i t t l e ammonium storage occurs i n marine macrophytes (Rosenberg and Ramus 1982; Appendix 1). I t i s p o s s i b l e that the ammonium was a s s i m i l a t e d and the nitr o g e n stored i n a d i f f e r e n t form which would stop any d i r e c t negative feedback i n h i b i t i o n of ammonium uptake. Rosenberg and Ramus (1982) and B i r d et a l . (1982b) i n d i c a t e that amino a c i d pools are important. This study reports the requirement of a thr e s h o l d ammonium conc e n t r a t i o n for uptake i n mature F. d i s t i c h u s which s u r p r i s i n g l y d i d not decrease a f t e r 4.5 days of ni t r o g e n s t a r v a t i o n . Such a th r e s h o l d would be a severe competitive disadvantage during times of decreased nitrogen supply. Phytoplankton and F. d i s t i c h u s germlings do not show t h i s t h r e s h o l d response. These smaller l i f e forms may out compete the mature t h a l l u s f o r low l e v e l s of nitrogen but they do not 59 have the nitrogen storage c a p a b i l i t i e s of the mature t h a l l u s which s u s t a i n growth during periods of nitrogen s t a r v a t i o n (Chapman and C r a i g i e 1979; Rosenberg and Ramus 1982). I t i s p o s s i b l e that t h i s requirement of a th r e s h o l d ammonium conc e n t r a t i o n f o r the mature F. d i s t i c h u s t h a l l i only occurs when the t h a l l i are nitrogen saturated. Topinka (1978) examined F. s p i r a l i s i n the summer and he d i d not observe t h i s t h r e s h o l d f o r ammonium uptake. F. d i s t i c h u s germlings responded to nitrogen supply i n a s i m i l a r manner to nitrogen l i m i t e d mature t h a l l i . They had high n i t r a t e and ammonium uptake r a t e s , and a f f i n i t i e s , showed no preference f o r the various forms of nitr o g e n and maintained high uptake r a t e s f o r a r e l a t i v e l y long time (> 1 h ) . Further research i s req u i r e d to confirm that these responses are common i n nitrogen s u f f i c i e n t germlings. Mature F. d i s t i c h u s t h a l l i taken d i r e c t l y from the f i e l d d i d not show any of the above responses" and consequently appeared to be nitrogen s u f f i c i e n t . High uptake r a t e s and high uptake a f f i n i t i e s are c h a r a c t e r i s t i c of marine phytoplankton ( S y r e t t 1962). Phytoplankton and germlings appear to be be t t e r adapted for procurement of a l i m i t i n g n u t r i e n t than mature seaweeds. Several s t u d i e s have shown that 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 the i n t e r t i d a l h a b i t a t (Schonbeck and Norton 1979c; Quadir et a l . 1979; Thomas and Turpin 1980). This study shows the F. d i s t i c h u s germlings are a l s o w e l l adapted to 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 st u d i e s on the p h y s i o l o g i c a l 60 adaptations of marine macrophytes to environmental f a c t o r s , yet the c r u c i a l e a r l y l i f e h i s t o r y stages have been v i r t u a l l y ignored. I n v e s t i g a t i o n of the l i f e h i s t o r y stages i s necessary to gain a complete understanding of f a c t o r s c o n t r o l l i n g macroalgal d i s t r i b u t i o n s . 61 Chapter 4. D e s i c c a t i o n Enhanced Nu t r i e n t Uptake Rates i n I n t e r t i d a l Seaweeds I n t r o d u c t i o n The i n t e r t i d a l environment i s one of the most extreme that i s 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 p h y s i c a l and chemical f a c t o r s (e.g., temperature, s a l i n i t y , n u t r i e n t s , humidity, and i r r a d i a n c e ) . Success i n such an environment r e q u i r e s s p e c i f i c p h y s i o l o g i c a l adaptations to these environmental f l u c t u a t i o n s . D e s i c c a t i o n may be the most extreme environmental s t r e s s imposed on i n t e r t i d a l macroalgae by exposure to a i r . (Isacc 1933, 1935; Feldman 1951; Schonbeck and Norton 1978, 1979a). A b i l i t y to t o l e r a t e d e s i c c a t i o n undoubtedly a f f e c t s a l g a l s u r v i v a l i n the i n t e r t i d a l zone, but Dromgoole (1980) showed that there was no d i r e c t c o r r e l a t i o n between rate of dehydration of d i f f e r e n t species and t h e i r i n t e r t i d a l l o c a t i o n . I t has been suggested that tolerance to d e s i c c a t i o n i s more important than avoidance (Schonbeck and Norton I979b,c). 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 on marine macrophytes have been i n v e s t i g a t e d (Isaac 1933,1935; Feldman 1951; Schonbeck and Norton 1978, 1979a) but l i t t l e i s known about 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 . Recent i n v e s t i g a t i o n s revealed that the carbon f i x a t i o n r ates of i n t e r t i d a l algae are maintained or even enhanced when weight l o s s due to d e s i c c a t i o n i s 20-40% (Johnson et a l . 1974; Quadir et a l . 1979). This carbon procurement occurs i n the absence of any new supply of n u t r i e n t s with the exception of carbon. This photosynthetic increase has the 62 p o t e n t i a l 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 the net uptake of such n u t r i e n t s i n t o the t i s s u e i s only p o s s i b l e during submersion. Consequently, one would expect adaptations f o r n u t r i t i o n a l ion uptake i n i n t e r t i d a l algae f o l l o w i n g d e s i c c a t i o n . Schonbeck and Norton (1979a) and Br i n k h u i s et a l . (1976) suggest that n u t r i e n t shortage, compounded by p e r i o d i c exposure when e x t e r n a l s u p p l i e s of n u t r i e n t s are not a v a i l a b l e , i s important i n determining 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 to be the n u t r i e n t that most commonly l i m i t s phytoplankton growth i n the ocean (Ryther and Dunstan 1971). I t has a l s o been shown that marine macrophytes s t o r e n i t r o g e n which i s used during periods of nitrogen l i m i t a t i o n (Chapman and C r a i g i e 1977). Nitrogen storage and nitr o g e n uptake p o t e n t i a l f l u c t u a t e with seasonal nitrogen supply (Chapman and C r a i g i e 1977), and nitrogen upwelling during p e r i o d i c storms (Rosenberg and Ramus 1982). Thomas and Turpin (1980) suggested that p e r i o d i c exposure r e s u l t s i n an i n t e r m i t t e n t supply of n i t r o g e n . The enhancement of n u t r i e n t uptake rates i n response to d e s i c c a t i o n i n the i n t e r t i d a l alga Fucus d i s t i c h u s was s t u d i e d and the r e l a t i o n s h i p s between t h i s response, 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 to determine whether t h i s i s a common response. The e f f e c t of d e s i c c a t i o n on n i t r a t e and ammonium uptake r a t e s i n four a d d i t i o n a l i n t e r t i d a l species ( G r a c i l a r i a verrucosa, Enteromorpha i n t e s t i n a l i s , G i g a r t i n a p a p i l l a t a , and P e l v e t i o p s i s l i m i t a t a ) was st u d i e d to determine i f t h i s 6 3 uptake response to 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 adaptation to i n t e r t i d a l l o c a t i o n . 64 M a t e r i a l s and Methods Species and C o l l e c t i o n S i t e G i g a r t i n a p a p i l l a t a (C.Ag.), and Pelvet i o p s i s l i m i t a t a (Setch) Gardn. were c o l l e c t e d from the northern shore of Diana I s l a n d , Barkley Sound, B.C. ( F i g . 1), at 2.2 and 3.8 m, r e s p e c t i v e l y , above Canadian datum. Specimens of Fucus  d i s t i c h u s L., Enteromorpha i n t e s t i n a l i s (L.)Grev. and G r a c i l a r i a verrucosa (Huds.) Papenf. were c o l l e c t e d from Wiseman's Bay, Bamfield I n l e t , Barkley Sound, B.C. ( F i g . 1). Their heights above Canadian datum were 2.6, 2.0, and 1.0 m, r e s p e c t i v e l y . A l l specimens were non—reproductive and were c o l l e c t e d i n l a t e summer, i n the morning, s h o r t l y a f t e r exposure, and they were rehydrated by submersion i n ambient n a t u r a l seawater f o r 2—6 h before the experiments began. Specimens of F. d i s t i c h u s c o l l e c t e d at Wiseman's Bay only i n the summer, have been c a l l e d F. d i s t i c h u s (Bamfield) to 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 winter near Vancouver ( s i t e described below) and henceforth r e f e r r e d to as F. d i s t i c h u s (Vancouver). Both of the Barkley Sound s i t e s were rocky, with a southwest exposure, and were surrounded by t a l l t r e e s . A l l t h a l l i were c o l l e c t e d from rock faces, except the G. verrucosa, which grew on a rocky beach area which was s l i g h t l y below and 100 m to the east of the F. d i s t i c h u s and E. i n t e s t i n a l i s c o l l e c t i o n s i t e . There was no detectable (< 0.1 /uM) n i t r a t e , ammonium, or n i t r i t e i n the surface water of both Wiseman's Bay and Diana I s l a n d from February u n t i l l a t e September (L. Druehl pe r s. c omm.). 65 Non-reproductive t h a l l i of F. d i s t i c h u s were c o l l e c t e d from the seawall at the south end of Second Beach p o o l , Stanley Park, 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. Experiments were conducted e a r l y i n J u l y , September and February. These p l a n t s were c o l l e c t e d i n the morning a f t e r exposure and were rehydrated by submersion i n f i l t e r e d (0.45 pm) n a t u r a l seawater (with ambient n u t r i e n t concentrations) for 2-6 h p r i o r to experimental d e s i c c a t i o n . D e s i c c a t i o n Procedure P l a n t s were u s u a l l y 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 impossible due to weather c o n d i t i o n s , p l a n t s were de s i c c a t e d at 18°C under a r t i f i c i a l l i g h t , at an i r r a d i a n c e of 600 pE.m~ 2.s~ 1 i n a chamber with d r i e d c i r c u l a t i n g a i r . F. d i s t i c h u s t h a l l i were desic c a t e d at both 6 and 18°C i n the winter experiments. In a l l cases, the c o n t r o l s (hydrated p l a n t s ) were kept i n a humid chamber at the same i r r a d i a n c e and temperature for the same length of time as the p l a n t s that were d e s i c c a t i n g . This allowed a l l p l a n t s to be exposed and starved of n u t r i e n t s for the same length of time (1-3 h ) , thereby a s s u r i n g that any e f f e c t on n u t r i e n t uptake rates could be a t t r i b u t e d s o l e l y to d e s i c c a t i o n and not to 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 obtained from the water absorbed during rehydration was determined from the weight increase which was equated to a volume of water with the predetermined n u t r i e n t content. For example: weight gain = 2 g = 2 ml medium which contains 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 n i t r a t e was obtained v i a water absorb t i o n . 66 Dry weights were obtained by l e a v i n g the t h a l l i i n a 60°C oven u n t i l they reached a constant weight (48-72 h ) . C/N r a t i o s of some samples were determined with a CHN analyser (Carlo Erba). Nitrogen Uptake Experiments Batch uptake experiments were conducted as o u t l i n e d i n Chapter 1. P l a n t s of s i m i l a r s i z e (1-5 g wet weight, depending on the species) were d e s i c c a t e d to 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 placed i n 400 ml of f i l t e r e d (0.45 pm) seawater enriched with 30 pM n i t r a t e or 15 pM ammonium and w i t h a l l other n u t r i e n t s , t r a c e metals and vitamins at a s a t u r a t i n g concentration of f/2 ( G u i l l a r d and Ryther 1962). Each poi n t on the d e s i c c a t i o n versus uptake p l o t s i s therefore the uptake rate of one p l a n t . Although n i t r a t e and ammonium uptake rates i n some macroalgae are not saturated, even at 30 uM ( D ' E l i a and DeBoer 1978; Haines and Wheeler 1978; Topinka 1978; Har r i s o n and Druehl 1982), these 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 oncentrations commonly observed i n the f i e l d i n winter and f o l l o w i n g storms and they were w i t h i n the s e n s i t i v i t y range of the a n a l y t i c a l technique. The use of higher concentrations a l s o seemed unwarranted because of r e p o r t s that s a t u r a t i o n of the uptake system i n some macroalgae i s prevented by the existence of a la r g e d i f f u s i o n component ( D ' E l i a and DeBoer 1978; Topinka 1978; Harri s o n and Druehl 1982; Chapter 2). During the summer a l l p l a n t s were incubated i n an outdoor water bath at 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 ( B r i n k h u i s et 67 a l . 1976; Hanisak and H a r l i n 1978). Winter uptake experiments were conducted i n a 6°C c o l d room with an i r r a d i a n c e of 150 juE.m~ 2.s~ 1 (Chapter 2). Uptake rates ( i n i t i a l c oncentration minus f i n a l n i t r a t e or ammonium concentration i n the incubation medium d i v i d e d by the time taken) were determined during an incubation p e r i o d when uptake rates were constant; 10 min f o r ammonium and 15—30 min fo r the n i t r a t e experiments (Chapter 1). At the end of the incubation p e r i o d , the p l a n t s were removed and the f i n a l n i t r a t e c o n c e n t r a t i o n was determined with a Technicon AutoAnalyzer using methods p r e v i o u s l y described (Davis et a l . 1973). Uptake rates were c a l c u l a t e d from the changes i n n i t r a t e or ammonium concentration during the incubation p e r i o d and were normalized to dry weight. These rates represent the uptake r a t e obtained by a p l a n t when i t i s suddenly submerged i n high n u t r i e n t seawater and thus are measures of p o t e n t i a l uptake r a t e s . There was a great deal 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 data. When general trends were obvious curves were f i t t e d v i s u a l l y . A d d i t i o n a l s t u d i e s were done on F. d i s t i c h u s . In August, the e f f e c t of d e s i c c a t i o n on phosphate uptake was t e s t e d using the method o u t l i n e d above. In February, F. d i s t i c h u s t h a l l i were c o l l e c t e d and preconditioned i n nitrogen f r e e , f i l t e r e d , enriched (Chapter 1) n a t u r a l seawater for 24 h. Twenty grams wet weight of plant m a t e r i a l was placed i n 10 l i t e r s of medium maintained at 6° C, under an i r r a d i a n c e of 150 /uE.nr 2 . s " 1 . The e f f e c t of d e s i c c a t i o n on the nitrogen uptake r a t e s of these p l a n t s was t e s t e d . 68 N a t u r a l D e s i c c a t i o n Levels In s i t u d e s i c c a t i o n l e v e l s were determined f o r ten F. d i s t i c h u s t h a l l i on a hot (23-24°C), sunny (1500-1700 juE .m~2. s " 1 ) , moderately windy J u l y day i n Vancouver. P l a n t s were weighed at 12:00 and again at 13:30, j u s t p r i o r to submersion. Pl a n t s and h o l d f a s t p o s i t i o n s were marked. P l a n t s were removed, with t h e i r h o l d f a s t s i n t a c t , weighed and returned to t h e i r exact l o c a t i o n . At 13:30 the t h a l l i were placed i n f i l t e r e d seawater to hydrate f o r 12 h and wet weights were recorded. Percent " d e s i c c a t i o n was determined using the f o l l o w i n g formula: % D e s i c c a t i o n = [(wet wt - de s i c c a t e d wt)/wet wt] x 100 69 Results The p o t e n t i a l n i t r a t e uptake rate for F. d i s t i c h u s (Bamfield) i n l a t e August was enhanced d r a m a t i c a l l y between 0 and 30% d e s i c c a t i o n ( F i g . 12A). Uptake ra t e s were always greater f o r the desicc a t e d p l a n t s than the non-desiccated p l a n t s (0% d e s i c c a t i o n ) . The maximum enhancement i n uptake rate occurred at approximately 30% d e s i c c a t i o n (14.5 jumol N0 3~.g dry wt"1.h"l) and was greater than twice the p o t e n t i a l uptake rate of non-desiccated p l a n t s (6 jumol N0 3".g dry wt " 1 . h " l ) . Ammonium showed a trend s i m i l a r to that of n i t r a t e . There was more than a two-fold enhancement of p o t e n t i a l ammonium uptake rate at approximately 30% d e s i c c a t i o n ( F i g . 12B). At >50% d e s i c c a t i o n , ammonium uptake appeared to be s i m i l a r to or l e s s than the non-desiccated p l a n t s . The r e l a t i o n s h i p between phosphate uptake and d e s i c c a t i o n was d i f f e r e n t from those observed w i t h n i t r a t e and ammonium. When phosphate uptake experiments were conducted on the day that the p l a n t s were c o l l e c t e d , there was no enhancement of phosphate uptake r a t e a f t e r d e s i c c a t i o n ( F i g . 12C). The p o t e n t i a l phosphate uptake rate i n f a c t decreased a f t e r d e s i c c a t i o n . The n u t r i e n t concentrations i n Bamfield I n l e t and C/N r a t i o s of the pl a n t s (approximately 30 by atoms), suggest that n i t r o g e n was the g r o w t h - l i m i t i n g n u t r i e n t rather than phosphorus. Thus, d e s i c c a t i o n enhanced uptake rates may only occur when the n u t r i e n t i s l i m i t i n g growth. P l a n t s were c o l l e c t e d and incubated f o r 24 h i n Bamfield I n l e t water enriched with a l l other n u t r i e n t s to f/2 concentrations ( G u i l l a r d and Ryther 1962) with the exception of - I - T 20 4 0 20 40 6 0 %> Desiccation •/o D e s i c c a t i o n F i g . 12. Nutrient uptake rate (pmol.g dry w f ' . h " 1 ) f o r Fucus  d i s t i c h u s (Bamfield) 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 for p l a n t s p r e v i o u s l y incubated i n 30 uM n i t r a t e medium for 24 h i n an attempt to produce phosphate d e f i c i e n t p l a n t s (D). Phosphate Uptake Rate (/.imol PC^ • g d r y w t rAV 10 p 8 p C7> h" 1 ) o D n) n c +». .^ O O 3 Phosphate Uptake Rate ( j jmo l PO^ . g dr y w t " l h"1) a 5 o S-1 ro 01 IL 72 phosphate. This enrichment was used to overcome any p o s s i b l e nitrogen d e f i c i e n c y and make phosphate the l i m i t i n g resource. A f t e r the 24 h in c u b a t i o n , an increase i n the uptake rate of phosphate with d e s i c c a t i o n was observed ( F i g . 12D). In c o n t r a s t to the above observations, F. d i s t i c h u s (Vancouver) d i d not show enhancement of ammonium uptake f o l l o w i n g d e s i c c a t i o n 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 rates ( F i g . 13). N i t r a t e was not detected (< 0.1 uM) i n the surface water at t h i s time and the concentr a t i o n of ammonium was low (1.1 juM). In e a r l y September, 20—25% d e s i c c a t i o n r e s u l t e d i n enhanced ammonium uptake rates ( F i g . 14). The n i t r a t e and ammonium concentrations i n the surface water at t h i s time were undetectable (< 0.1 pM) and 5.4 uM, r e s p e c t i v e l y . By February, the water was 6° C and contained 5.0 /uM ammonium and 20-25 /uM n i t r a t e . When t h a l l i were des i c c a t e d <at 6°C (a mild winter temperature), there were no observable trends i n e i t h e r ammonium or n i t r a t e uptake r a t e s ( F i g s . 15,16). Winter t h a l l i were incubated i n nitrogen free seawater f o r 24 h to e s t a b l i s h nitrogen d e f i c i e n c y , but t h i s treatment d i d not produce d e s i c c a t i o n enhanced ammonium uptake ra t e s e i t h e r ( F i g . 17). Drying winter p l a n t s at 18°C to speed up the rate 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 rat e and i n most cases n i t r a t e was excreted i n t o the medium ( F i g . 18). Ammonium and n i t r a t e uptake rates f o r the hydrated p l a n t s were approximately 6 and 1.5 yumol.g dry w f ' . h " 1 , r e s p e c t i v e l y . Three of the four other species t e s t e d showed enhanced p o t e n t i a l n i t r a t e and ammonium uptake rates a f t e r d e s i c c a t i o n 73 12H > o E 3 0) 4J E Z3 C o E E < — i — 20 4 0 Desiccation — i — 6 0 F i g . 13. 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 " 1 . h " 1 ) 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 (Vancouver) i n J u l y 1980. 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 rate (jjmol.g dry wt" 1.h" 1) and % d e s i c c a t i o n for Fucus d i s t i c h u s i n September 1980. 75 I , , 1 ~ 10 20 3 0 % D e s i c c a t i o n F i g . 15. The r e l a t i o n s h i p between ammonium uptake rate (pmol.g dry wt" 1.h" 1) and % d e s i c c a t i o n for Fucus d i s t i c h u s i n February 1981. T h a l l i were desiccated at 6°C and uptake experiments were conducted at 6°C. 76 o E o O — I — 10 i 20 3 0 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 ra t e (pmol.g dry w f ' . h " 1 ) and % d e s i c c a t i o n for Fucus d i s t i c h u s i n February 1981. T h a l l i were de s i c c a t e d at 6 PC and uptake experiments were conducted at 6°C. 77 1 20 D e s i c c a t i on 10 30 F i g . 17. The r e l a t i o n s h i p between ammonium uptake rate (urnol.g dry w f ' . h " 1 ) and % d e s i c c a t i o n f o r Fucus d i st ichus i n February 1981. T h a l l i were preincubated i n nitrogen free seawater for 24 h and then d e s i c c a t e d at 6°C, uptake experiments were concucted at 6°C. 78 * , , 2 0 4 0 6 0 % D e s i c c a t i o n 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 ra t e (pmol.g dry w f .h" 1) and % d e s i c c a t i o n for Fucus d i s t i c h u s i n February 1981. T h a l l i were desic c a t e d at 18°C and uptake experiments were conducted at 6°C. 79 ( F i g s . 19-23). The exception was G. verrucosa, the lowest i n t e r t i d a l species t e s t e d . In some cases the data were v a r i a b l e and consequently no curves could be f i t t e d to the data. When general trends, such as an increase or decrease i n uptake rates 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 of the % d e s i c c a t i o n producing maximum uptake r a t e s and the r a t i o of maximum uptake rates to c o n t r o l uptake r a t e s were made. These r e s u l t s are summarized i n Figures 24,25. The degree of enhancement and the % d e s i c c a t i o n that produced maximum enhancement of n i t r a t e or ammonium uptake were r e l a t e d to i n t e r t i d a l l o c a t i o n ( F i g s . 24,25). The high i n t e r t i d a l s p e c i e s , P. l i m i t a t a , F. d i s t i c h u s and G. p a p i l l a t a showed the greatest enhancement of n i t r a t e and ammonium uptake f o l l o w i n g d e s i c c a t i o n ( F i g s . 24B,25B) and the optimum % d e s i c c a t i o n producing t h i s enhancement was a l s o the highest ( F i g s . 24A,25A). The degree of enhancement of n i t r a t e uptake v a r i e d from 1979 to 1981, but the % d e s i c c a t i o n r e s u l t i n g i n maximum uptake was r e l a t i v e l y constant. There were few d i f f e r e n c e s i n these two parameters, between n i t r a t e and ammonium uptake. One exception was P. l i m i t a t a where the degree of enhancement of n i t r a t e uptake (2.5-3.0 times r e l a t i v e to the uptake rates of the hydrated p l a n t s ) was much lower than f o r ammonium (approximately 10)(Fi g s . 24B,25B). This species a l s o showed an unusual n i t r a t e uptake response i n 1979 and the degree of enhancement could not be estimated. Degrees of d e s i c c a t i o n up to 52% continued to enhance uptake; no plat e a u i n uptake was observed ( F i g . 23). The high i n t e r t i d a l species were a l s o more r e s i s t a n t to A B • • 304 30H O " 20-| z 20 o E E 3 • * * • v \ » • 10 20 "U Desiccation 30 10 r — 20 Desiccation 30 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 rates (pmol.g d r y . wt-'.h" 1). and % d e s i c c a t i o n f or G r a c i l a r i a verrucosa in August 1980 ( — • — ) , and 1981 ( - — ) . Incubation time was 15 min for n i t r a t e and 10 min for ammonium. 00 o B —i— 2 0 4 0 Desiccation 6 0 " T — 1 0 1— 2 0 Desiccation — I — 3 0 F i g . 20. The r e l a t i o n s h i p uptake rates (yumol.g dry Enteromorpha i n t e s t i n a l i s (-+--). between n i t r a t e (A) wf' . h - 1 ) and % in August 1979 and ammonium (B) d e s i c c a t i o n f o r (—A—), and 1981 00 I* z o E \ 1H 9" • e-T V o E E < 20 4 0 Desiccation 60 —I 1 20 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 rates Uimol.g dry w t ^ . h - 1 ) and % d e s i c c a t i o n G i g a r t i n a p a p i l l a t a i n August 1979 ( — * — ) , and 1981 (—-•---) I I— 20 40 •/. Desiccation i 60 20 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 rates (pmol.g dry wt _ 1.h~ 1) and % d e s i c c a t i o n f or Fucus  d i s t i c h u s i n August 1 979 ( — * — ) , and 1981 (—•---). - r — 60 00 y • • —i— 2 0 —I— 4 0 6 0 1' Desiccation 14 oi 12 z 3 E I 4 E E < • • • • / / I l I I • 2 0 r— 4 0 •A. Desiccation Fult»d3'r* + Vle y , e l a t i o n s h i p between n i t r a t e (A) and ammonium SS?SiJ- r a ^ e s U«nol.g dry wf'.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 (-->--pSoH G r o c Gifl Em-Pel Fuc Tidal Height (m) Tidol Height (m) Tidal Height (m) 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 producing the maximum enhancement of n i t r a t e uptake rate (A), the r e l a t i v e degree of enhancement of n i t r a t e uptake rate (B),and the r a t i o of n i t r a t e uptake rate at 30% d e s i c c a t i o n and the rat e for hydrated p l a n t s (C), f o r G r a c i l a r i a verrucosa (Gra), Enteromorpha i n t e s t i n a l i s (Ent), 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 (Fuc), and P e l v e t i o p s i s l i m i t a t a (Pel) i n 1979 ( ) f 1 980 (- -) , and 1981 ( ). Error bars represent the range estimated v i s u a l l y from Figs.12,19-23. CO cn Des icca t ion at Maximal Enhancement of Ammonium Uptake Rate 1-1 PJ 1 c 3 1 o 03 , c D ^ cn CD - a cn rt cn I-I- —• n-3 UD PJ 00 O r t o nr CD c Q j -—- CO < I . . M - | w • c C «L o CJ -I—1 l_i PJ •< 3 PJ Q J 3 i -h a f-t -> O ID t3 3 oo CD —» M 0) 3 3 O 3 3 O rr t-| a» f-1 O 3-3 > < C o a g fo PJ 3 * f t C PJ fD ' O Cu r t 0) 3 ' O fD r t i - * re 0) 3 PJ 3 PJ M - ?. PJ PJ • 3 Co f n h - W -ro PJ cn 3 3 • 3 a rt- PJ r r -3 C 3 ro o — PJ ro 3 h-" Co PJ -If Bl f t H CO cn < Da o ro 3 ft rr r t CO I n N> i-t (a) O • n tr PJ 01 ro TJ ro cn ro 3 cn O M PJ I-t rt-M" 3 PJ oo o QJ ro cn M -O o PJ c •a r t PJ ?r ro Q i tr ro ro c CO *0 r t O PJ o 7T PJ ro r t rt * ro ro 3 PJ ro o 3 <-l ro i—• PJ 3 r t ro < ro r t O •-«• I-t r t voiQ pj ro vo- -r t sr ro »i PJ r t ro CO o 3 PJ rtTD ro i-t o a Q J PJ • C I—1 > o "—' I-1- h-1 - 3 O ia n PJ r t r t tr r t M -ro tr o ro 3 i-t ro 3 t—• pj PJ X r t M-M - 3 < C ro 3 PJ tr o < ro 4T Relative Enhancement of Uptake R a t e r t M -3 - O ro 3 cn tr -a H ? Relat ive Uptake R a t e at 30 ' / . Des icca t i . 8 - <u - J — l - f 1 i I"' -•—'II — I ? 98 87 higher 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 to low i n t e r t i d a l species. Uptake rates greater than those rates for hydrated p l a n t s (0% d e s i c c a t i o n ) were often maintained a f t e r 30-40% d e s i c c a t i o n . A r a t i o of nitrogen uptake rat e at 30% to the uptake rate of hydrated p l a n t s of l e s s than 1 ( F i g s . 24C,25C) i n d i c a t e s a reduced ni 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 high degree of d e s i c c a t i o n . The maintenance of high uptake rates a f t e r high l e v e l s of d e s i c c a t i o n was d i r e c t l y r e l a t e d to 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 i n uptake r a t e s were s i m i l a r when uptake, expressed on a dry weight b a s i s , was r e l a t e d to p a r t i c u l a t e nitrogen or wet weight. Nitrogen uptake v i a water absorption during rehydration was approximately 1% of the t o t a l uptake. At noon on a sunny day i n e a r l y J u l y , a f t e r 6 h of exposure, the F. d i s t i c h u s (Vancouver) averaged 31% d e s i c c a t i o n and j u s t before submersion at 13:30 they averaged 57% d e s i c c a t i o n . Even' on a cloudy summer day 20—30% d e s i c c a t i o n was common. These r e s u l t s show that 30% d e s i c c a t i o n i s an e c o l o g i c a l l y r e a l i s t i c value and i s probably a conservative estimate of the % d e s i c c a t i o n on a dry summer day. 88 D i s c u s s i o n Surface water n u t r i e n t analyses i n d i c a t e that both F. d i s t i c h u s populations were only nitrogen l i m i t e d during the summer. As the p l a n t s became more nitrogen l i m i t e d during l a t e s p r i n g and summer, they may have reached a l e v e l of nitrogen 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 r e s u l t s suggest that b r i e f s t a r v a t i o n , even overnight, was s u f f i c i e n t to induce d e s i c c a t i o n enhanced uptake r a t e s . The Bamfield p l a n t s had been nitrogen l i m i t e d f or s e v e r a l weeks, but" phosphate reserves may have been low a l s o , although they were not measured. In c o n t r a s t , d e s i c c a t i o n d i d not enhance the ammonium uptakes of F. d i s t i c h u s (Vancouver) even though a n a l y s i s of surface n u t r i e n t concentrations suggested that the p l a n t s were nitrogen l i m i t e d i n J u l y and September. Ammonium uptake r a t e s of hydrated p l a n t s were greater 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 nit r o g e n free 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 . F. d i s t i c h u s does accumulate ni t r o g e n (Appendix 1). Such accumulations may be s u f f i c i e n t to s u s t a i n growth and weeks may elapse before they are depleted (Ryther et a l . 1981). E i t h e r s e v e r a l weeks of nitrogen l i m i t a t i o n may be required to invoke d e s i c c a t i o n enhanced uptake or there may be another environmental f a c t o r which a c t s i n conjunction with nitrogen l i m i t a t i o n . Twenty-four hours of phosphate s t a r v a t i o n produced enhanced phosphate uptake rates f o l l o w i n g d e s i c c a t i o n i n summer p l a n t s , but 24 h of nitrogen s t a r v a t i o n d i d not produce enhanced n i t r a t e uptake rates 89 f o l l o w i n g d e s i c c a t i o n with winter p l a n t s . This a d d i t i o n a l c o n t r o l l i n g f a c t o r i s apparently r e l a t e d to the p h y s i o l o g i c a l s t a t e of the plant i n the summer. Enhanced uptake rates f o l l o w i n g d e s i c c a t i o n may be a p h y s i o l o g i c a l response to frequent dryin g over an extended p e r i o d of time (weeks). Schonbeck and Norton (1979c) found a s i m i l a r "hardening" e f f e c t i n terms of drought—tolerance i n F. s p i r a l i s . They a l s o found a "dehardening" e f f e c t 6-8 weeks a f t e r p l a n t s were moved to a lower t i d a l zone or kept permanently submerged. I t i s p o s s i b l e that the F. d i s t i c h u s was not s u f f i c i e n t l y "hardened" to d r y i n g by e a r l y J u l y to show enhanced nitrogen 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 . I f a h i s t o r y of frequent d e s i c c a t i o n i s necessary to t r i g g e r enhanced n u t r i e n t uptake f o l l o w i n g d e s i c c a t i o n , the high i n t e r t i d a l seaweeds should show greater enhancement of uptake rates f o l l o w i n g d e s i c c a t i o n than the low i n t e r t i d a l species. The r e s u l t s of t h i s study support t h i s p r e d i c t i o n . I f the high i n t e r t i d a l h a b i t a t simply s e l e c t s f o r t h a l l i w ith the adaptation 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 d e s i c c a t i o n rather than "hardening" the t h a l l i there would have been some adapted t h a l l i i n the J u l y experiment or a large percentage of the high i n t e r t i d a l population would have died between J u l y and e a r l y September. Neither of these p o s s i b i l i t i e s were observed. At winter temperatures (6°C) i t i s d i f f i c u l t to dry F. 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 . Winter p l a n t s d r i e d at 18°C to 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 i n most cases n i t r a t e was a c t u a l l y excreted. This response was not due to the temperature increase because 90 the hydrated p l a n t s were kept at 18°C and s t i l l showed normal uptake r a t e s . I t appears that the p l a n t s were not adapted to r a p i d d e s i c c a t i o n and were probably "dehardened" over the winte r . F. d i s t i c h u s showed very low n i t r a t e reductase a c t i v i t i e s and high n i t r a t e content i n February (Appendix 2,b), thus n i t r a t e not n i t r i t e could be excreted under s t r e s s . The r e s u l t s of t h i s study suggest that exposure and subsequent d e s i c c a t i o n enhanced nitrogen uptake rates i n F. d i s t i c h u s i n l a t e summer when the p l a n t s were nit r o g e n l i m i t e d and "hardened" to frequent d r y i n g . This response to d e s i c c a t i o n appears to be under s t r i c t c o n t r o l , since i t occurs only when 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 d e s i c c a t i o n occurs d a i l y . Maintenance of the p h y s i o l o g i c a l and biochemical mechanisms required f o r such a response must be expensive e n e r g e t i c a l l y and would only be an advantage when growth i s l i m i t e d by nitrogen supply and when the p r o b a b i l i t y of frequent d e s i c c a t i o n i s high. F. d i s t i c h u s appears to be w e l l adapted to n u t r i e n t procurement i n the r a p i d l y changing i n t e r t i d a l 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 the degree of uptake enhancement f o l l o w i n g d e s i c c a t i o n , the optimum % d e s i c c a t i o n f or uptake, and the uptake rates at 30% d e s i c c a t i o n r e l a t i v e to those at 0% d e s i c c a t i o n support the hypothesis that enhanced n u t r i e n t uptake ra t e s f o l l o w i n g d e s i c c a t i o n are an adaptation to p e r i o d i c exposure to a i r . These r e l a t i o n s h i p s are not e x a c t l y l i n e a r , but n e i t h e r i s the 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 l o c a t i o n . I f the degree of enhancement and the optimum percent d e s i c c a t i o n 91 had been p l o t t e d against t o t a l hours exposed to a i r per day the trends may have been more l i n e a r . The degree of enhancement was more v a r i a b l e than the % d e s i c c a t i o n producing the maximum enhanced uptake r a t e . There was a c l o s e 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 the i n i t i a t i o n of enhanced uptake, but the magnitude of the enhancement was in f l u e n c e d by other f a c t o r s , such as the degree of nitrogen l i m i t a t i o n . A l l species growing i n c l o s e proximity might be expected to experience s i m i l a r n itrogen l i m i t a t i o n because they have been exposed to very s i m i l a r n itrogen regimes, but species requirements f o r ni t r o g e n can vary (Kornfeldt 1982). The i n t e r - p l a n t v a r i a b i l i t y i n nitrogen uptake rates i n t h i s study was high. A n a l y t i c a l f a c t o r s such as the determination of percent d e s i c c a t i o n undoubtedly c o n t r i b u t e d . This parameter required the determination of wet weight, which v a r i e d for a s i n g l e p l a n t . In a d d i t i o n , since the p l a n t s were not c l o n a l , genetic v a r i a b i l i t y was probably important. Other p o s s i b l e sources of v a r i a b i l i t y i n uptake r a t e s were the surface area to biomass r a t i o s w i t h i n a population and the age of the given p l a n t . G. p a p i l l a t a showed the greatest degree of i n t e r — p l a n t v a r i a b i l i t y as w e l l as greatest morphological v a r i a b i l i t y even though an attempt was made to s e l e c t s i m i l a r p l a n t s . The r e l a t i o n s h i p s between the degree of enhancement of uptake r a t e , % d e s i c c a t i o n producing 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 that p l a n t s higher i n the i n t e r t i d a l zone were adapted to higher l e v e l s of d e s i c c a t i o n and shorter p e r i o d i c immersion i n n u t r i e n t s . This i s 92 i l l u s t r a t e d by; a) an increase i n the l e v e l of d e s i c c a t i o n that r e s u l t e d i n maximum enhancement of n u t r i e n t uptake, b) an increase i n the degree of enhancement of uptake at the optimum % d e s i c c a t i o n , and c) maintenance of high uptake rates f o l l o w i n g more severe l e v e l s of d e s i c c a t i o n . Nitrogen, u n l i k e inorganic carbon, can only be obtained from the environment during submersion. Previous s t u d i e s have shown that net photosynthesis i n i n t e r t i d a l algae i s a c t u a l l y enhanced during periods of exposure and d e s i c c a t i o n (Johnson et a l . 1974; Quadir et a l . 1979). I t i s p o s s i b l e that the enhanced photosynthetic r a t e s observed with d e s i c c a t i o n are 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 part by the presence or absence of n u t r i e n t s . As a r e s u l t , during exposure, energy normally used f o r n u t r i e n t uptake may be shunted to carbon f i x a t i o n , r e s u l t i n g i n an increase i n the C/N r a t i o during t h i s p e r i o d . Work by D ' E l i a and DeBoer (1978) i n d i c a t e d that measurable changes i n t h i s r a t i o a f f e c t the ni t r o g e n uptake ra t e s i n some macroalgae. The c o n t r o l p l a n t s were exposed to the atmosphere (removed from e x t e r n a l nitrogen supply) but not d e s i c c a t e d . Therefore i t appears that enhancement of n u t r i e n t 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 i n C/N, but nevertheless enhanced uptake may be important i n long term C/N homeostasis. The observation that enhanced uptake rates f o l l o w i n g d e s i c c a t i o n only occur 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 adaptive s i g n i f i c a n c e of t h i s response. This study gives i n s i g h t i n t o the complexity of p h y s i o l o g i c a l c o n t r o l s governing the i n t e r a c t i o n between environmental 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 n u t r i e n t uptake 93 f o l l o w i n g d e s i c c a t i o n . This uptake response i s dependent on the dur a t i o n 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 recent d e s i c c a t i o n l e v e l . 94 Chapter 5. Adaptations of G r a c i l a r i a verrucosa to N u t r i e n t Procurement i n an I n t e r t i d a l Habitat I n t r o d u c t i o n The p h y s i o l o g i c a l adaptations of marine macrophytes to the i n t e r t i d a l environment have r e c e n t l y been i n v e s t i g a t e d . Adaptative responses i n osmoregulation ( B i e b l 1938), photosynthetic r a t e (Johnson et a l . 1974; Quadir et a l . 1979) and n u t r i e n t uptake rate (Thomas and Turpin 1980; Chapter 4) have been observed during m i l d d e s i c c a t i o n . The r e l a t i o n s h i p between n u t r i e n t 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 important i n understanding a l g a l d i s t r i b u t i o n . Several s t u d i e s have examined the e f f e c t of n u t r i t i o n a l past h i s t o r y on n u t r i e n t uptake rates i n seaweeds ( D ' E l i a and DeBoer 1978; Hanisak and H a r l i n 1978; Topinka 1978; Morgan and Simpson 1981; Rosenberg and Ramus 1982). A high 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 supply as w e l l as c r e a t i n g other environmental .stresses that may a f f e c t the p h y s i o l o g i c a l s t a t e of seaweeds. P e r i o d i c 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 i n t e r t i d a l seaweeds are presumed to be adapted p h y s i o l o g i c a l l y . 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 rates i n s e v e r a l mid— to high— i n t e r t i d a l seaweeds (Chapter 4). The 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 uptake i n three populations of G. verrucosa 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 s t u d i e d to determine i f t h i s response v a r i e d w i t h i n a species. These populations were chosen because they were i s o l a t e d from each 95 other, yet geo g r a p h i c a l l y c l o s e together and they were at d i f f e r e n t , d i s t i n c t i n t e r t i d a l l e v e l s . Morphology, uptake, a s s i m i l a t i o n , and accumulation of nitrogen were stud i e d i n the i n t e r t i d a l p opulations. G. verrucosa p l a n t s were t r a n s p l a n t e d from one s i t e to another at 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 to d i f f e r e n t i a t e between the 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 and geographic l o c a t i o n . This study i s the f i r s t to i n v e s t i g a t e whether c e r t a i n morphological and p h y s i o l o g i c a l adaptations to the high i n t e r t i d a l h a b i t a t are phenotypic or genotypic adaptations. This i s important to the understanding of the a d a p t a b i l i t y of i n t e r t i d a l seaweeds and a l g a l zonation. 96 M a t e r i a l s and Methods Populations Three populations of G r a c i l a r i a verrucosa (Huds.) Papenf. were used i n t h i s study. Their taxonomy i s p r e s e n t l y under i n v e s t i g a t i o n ( B i r d et a l . 1982a). One population was l o c a t e d at 1.8 m above Canadian datum at the head of Bamfield I n l e t , Bamfield, B.C., Canada ( F i g . 1). The other i n t e r t i d a l p opulation was i n Wiseman's Bay i n Bamfield I n l e t and was growing 1.0 m above Canadian datum ( F i g . 1). The t h i r d p opulation was s u b t i d a l and i t was obtained from large f l o a t i n g c u l t u r e bags which were anchored i n Wiseman's Bay ( F i g . 1)(Lindsay and Saunders 1979). These c u l t u r e s were s t a r t e d with 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. verrucosa populations continued over three summers, 1979-1981. In August and e a r l y September 1979, nitrogen uptake s t u d i e s were conducted on a l l three p o p u l a t i o n s . In June and August 1980, nitrogen uptake s t u d i e s were c a r r i e d out on only the two i n t e r t i d a l populations and work with the s u b t i d a l c u l t u r e was di s c o n t i n u e d . In June and August of 1981, nitr o g e n uptake r a t e s , s o l u b l e n i t r o g e n content and n i t r a t e reductase a c t i v i t y were monitored. Transplant Experiments Transplant experiments were conducted with the two i n t e r t i d a l G. verrucosa p o p u l a t i o n s . T h a l l i were t r a n s p l a n t e d i n June of 1980 and 1981. I n d i v i d u a l p l a n t s were d i f f i c u l t to d i s t i n g u i s h i n both i n t e r t i d a l beds where the t h a l l i tended to 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, the procedure was as f o l l o w s : ten bunches, each weighing approximately 6 g were t i e d w i t h cotton s t r i n g to a 3 m galvanized chain which was anchored at each end. P l a n t s were tr a n s p l a n t e d from the head of Bamfield I n l e t (1.8 m) to two d i f f e r e n t 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 1980 ( F i g . 26A). The 1.0 m s i t e was i n the n a t u r a l G. verrucosa bed at Wiseman's Bay. 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 t r a n s p l a n t e d to 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 , 1.0 or 1.8 m, at the head of Bamfield I n l e t . The 1.8 m s i t e was i n the n a t u r a l G. verrucosa bed ( F i g . 26A). These t r a n s p l a n t s were made between June 23 and June 25, 1980. Approximately f i v e weeks l a t e r , morphological changes were recorded and uptake experiments were conducted. In 1981, t h i s t r a n s p l a n t procedure was expanded. Quadrat frames (0.5 m by 3 m) were constructed of PVC p i p i n g . Nylon f i s h n e t t i n g was s t r e t c h e d across the quadrats. Clumps of p l a n t s were t i e d to each square of the net with cotton s t r i n g and the quadrats were anchored at each corner with stakes. The use of quadrats allowed more biomass t o be tr 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 the head of Bamfield I n l e t at 1.8 to 1.0 m i n the same geographical l o c a t i o n as w e l l as to two other s i t e s i n Wiseman's Bay, one at 1.0 m and the other at 1.8 m ( F i g . 26B). P l a n t s from the n a t u r a l bed (1.0 m) i n Wiseman's Bay were t r a n s p l a n t e d higher up i n the i n t e r t i d a l region to 1.8 m as w e l l as to 1.8 m and 1.0 m at the head of Bamfield I n l e t . These .transplants were made between June 6 and June 9. The p l a n t s were observed every two weeks. Six to eight 98 GraciI a r i a verrucosa t ransplants 1980 (A) T I D A L H E I G H T WISEMAN'S BAY 1.8 M « 1.0 M HEAD OF B A M F I E L D I N L E T — NATURAL P O P U L A T I O N / NATURAL P O P U L A T I O N Graci l a r i a verrucosa t ransplants 1981 (B) T I D A L H E I G H T WISEMAN'S BAY 1.8 M HEAD OF B A M F I E L D I N L E T 1.0 M NATURAL P O P U L A T I O N / / / / NATURAL P O P U L A T I O N F i g . 26. A summary of G r a c i l a r i a verrucosa t r a n s p l a n t s made i n June 1980 (A) and 1981 (B). 99 weeks l a t e r 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 reductase a c t i v i t i e s determined. Nitrogen Uptake Experiments Time course stud i e s of n i t r a t e and ammonium uptake r a t e s were conducted (see procedure o u t l i n e d i n Chapter 1). Simultaneous uptake of n i t r a t e and ammonium was t e s t e d i n medium co n t a i n i n g 30 pM n i t r a t e or 15 jaM ammonium. These concentrations were used because they are s i m i l a r i n magnitude and r a t i o to the ammonium and n i t r a t e concentrations found i n the f i e l d during periods of v e r t i c a l mixing (winter and storms). The incubation chamber was kept at 16°C under s a t u r a t i n g l i g h t (300 pE.m~ 2.s~ 1 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 ) . In a second type of uptake experiment t h a l l i were incubated i n a known concentration of the l i m i t i n g n u t r i e n t f or a predetermined time (see procedure for "batch" experiments, Chapter 1). These incubations were conducted outdoors under s a t u r a t i n g n a t u r a l l i g h t (Hanisak and H a r l i n 1978) at 16°C. Pl a n t s were incubated f o r 10 or 15 min during the ammonium or n i t r a t e uptake experiments, r e s p e c t i v e l y . P l a n t s were removed and d r i e d to a constant dry weight i n a 60°C oven. Uptake rates were expressed on a per gram dry weight b a s i s and, f o r the 1979 experiments, on a per 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 ( s p e c i f i c uptake r a t e ) . T o t a l nitrogen 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 using a CHN analyzer (Carlo Erba). N i t r a t e and ammonium uptake rates were t e s t e d at a range of 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 and n i t r a t e uptake rates was t e s t e d . P l a n t s were d r i e d to various degrees of d e s i c c a t i o n , submersed i n medium and uptake rates were determined by the method described above. There was a great deal 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 data. When general trends were obvious curves were f i t t e d v i s u a l l y . D e s i c c a t i o n Procedure The t h a l l i were d e s i c c a t e d outside under n a t u r a l c o n d i t i o n s as o u t l i n e d i n Chapter 4. A n a l y s i s of Soluble Nitrogen Content Complete t h a l l i were ground in hot water to e x t r a c t n i t r a t e , ammonium, ninhydrin p o s i t i v e m a t e r i a l (assumed to be p r i m a r i l y amino a c i d s ) and f r e e p r o t e i n . Plant m a t e r i a l (2 g wet wt) was ground with 1 g of washed, i g n i t e d sand and 25 ml hot d i s t i l l e d d eionized water (Appendix 1). . T r i p l i c a t e e x t r a c t i o n s were conducted to 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 stored at -10°C. N i t r a t e , ammonium, t o t a l free amino a c i d s and free p r o t e i n were analysed as described i n Appendix 1. B o i l i n g ethanol e x t r a c t i o n s were a l s o performed to determine i f ethanol was a supe r i o r e x t r a c t a n t to water. Plant m a t e r i a l (1 g) was b o i l e d for 10 min i n 80% ethanol and then for 10 min i n 95% ethanol. E x t r a c t s were analysed as described i n Appendix 1. 101 N i t r a t e Reductase Assay In v i t r o n i t r a t e reductase a c t i v i t y was assayed as o u t l i n e d i n Appendix 2. Plant m a t e r i a l (2 g) was ex t r a c t e d . w i t h sand (1 g) and 25 ml of phosphate b u f f e r . The optimum pH, MgS04 , and p o l y v i n y l p y r r o l i d o n e (PVP) concen t r a t i o n for a c t i v i t y were e s t a b l i s h e d . The e f f e c t of incubation time, e x t r a c t volume, and NADH, and n i t r a t e concentrations on n i t r a t e reductase a c t i v i t y was t e s t e d for both i n t e r t i d a l p o pulations. A rectangular hyperbola was not a good f i t for the enzyme m k i n e t i c s . Therefore, curves were f i t t e d by eye. The h a l f - s a t u r a t i o n constants (Km) and the standard e r r o r of these estimates were determined by a l i n e a r regression of a p l o t of V/S against V. 102 Res u l t s Morphology P l a n t s from the three G. verrucosa populations d i f f e r e d g r e a t l y i n t h e i r morphology. The s u b t i d a l p l a n t s were very robust and dark red. The branches were sparse but sturdy and larg e i n diameter i n comparison to the i n t e r t i d a l p l a n t s . The average t h a l l u s weighed approximately 10 g. 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 l'arge, weighing about 5-10 g ( F i g . 27). These p l a n t s were p r o f u s e l y branched, and feathery i n appearance. H o l d f a s t s were reduced or non-existent. Wiseman's Bay had a rock and sand bottom; i t was shallow, moderately pro 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 the p l a n t s r e c e i v i n g reduced l i g h t . The head of Bamfield I n l e t was a mudflat and i t was w e l l p r o t e c t e d . The surrounding areas were grass or marshland and t h e r e f o r e , the mudflat r e c e i v e d d i r e c t s u n l i g h t . The G. verrucosa p l a n t s which grew on t h i s mudflat were ye l l o w , and the t i p s of the branches were oft e n dark brown. They had a stunted, contorted appearance ( F i g . 28), were sp a r s e l y branched, and these few branches were bent and tangled. I n d i v i d u a l t h a l l i were very s m a l l . Unbroken segments were r a r e l y l a r g e r than 1—2 g. There were no apparent h o l d f a s t s and the p l a n t s formed a s t i f f coarse mat on top of the mud and s h e l l bottom. The p l a n t s at the head of Bamfield I n l e t were covered with numerous epiphytes, p r i m a r i l y pennate diatoms. The G. verrucosa bed at the head of Bamfield I n l e t was 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, Bamfield, B.C., Canada, i n August 1981. 104 F i g . 28. Morphology of high 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 at the head of Bamfield I n l e t , Bamfield, 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 i n June. I t was d i f f i c u l t to d i s t i n g u i s h new growth. The quadrat method of t r a n s p l a n t i n g was very e f f e c t i v e . There were few l o s s e s due to wave a c t i o n . Transplanted G. verrucosa t h a l l i underwent d e f i n i t e morphological changes ( F i g . 29). By e a r l y August of 1980, the morphology of the p l a n t s which had been t r a n s p l a n t e d i n June from Wiseman's Bay (1.0 m) to the head of Bamfield I n l e t (1.8 m) was s i m i l a r to the p l a n t s growing n a t u r a l l y at the head of Bamfield I n l e t ( F i g . 29A). The p l a n t s were ye l l o w , t w i s t e d , sparsely branched and very coarse. They tended to be l a r g e r than the surrounding r e s i d e n t p l a n t s and appeared to have fewer epiphytes. These p l a n t s increased 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 p l a n t s that were t r a n s p l a n t e d from Wiseman's Bay (1.0 m) to the low i n t e r t i d a l s i t e at the head of Bamfield I n l e t (1.0 m) d i d not s u r v i v e the f i v e week observation p e r i o d . The new s i t e was 0.8 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 bed at the head of Bamfield I n l e t . I t s l o c a t i o n i n an eelgrass bed l e d to excessive epiphytism. The p l a n t s that were t r a n s p l a n t e d from the head of Bamfield I n l e t (1.8 m) to the lower i n t e r t i d a l s i t e i n Wiseman's Bay (1.0 m) a l s o changed mor p h o l o g i c a l l y i n f i v e weeks. The t h a l l i became dark red, very s o f t and feathery. In general, they were s i m i l a r to the surrounding r e s i d e n t Wiseman's Bay p l a n t s , except they were smaller and covered with more epiphytes. The p l a n t s t r a n s p l a n t e d from the head of Bamfield I n l e t (1.8 m) to the upper i n t e r t i d a l s i t e (1.8 m) i n Wiseman's Bay s u r v i v e d w e l l . They became b r i g h t red, very l a r g e (greater than 106 GraciI a r i a verrucosa morphology 1980 (A) T I D A L H E I G H T 1.8 M WISEMAN'S BAY large, red — profusely branched HEAD OF B A M F I E L D I N L E T 1.0 M large, red, profusely branched NATURAL P O P U L A T I O N , ( l a rge , red , profusely branched) NATURAL P O P U L A T I O N smal l , yel low, twisted medium s i z e d , ye I low, twisted very s m a l l , dark red, f r a g i l e (a f ter 3 weeks) GraciI a r i a verrucosa morphology 1981 (B) T I D A L H E I G H T 1.8 M WISEMAN'S BAY medium s i z e d , red profusely branched large, red, profusely branched 1.0 M HEAD OF B A M F I E L D I N L E T large, red , profuse Iy branched NATURAL P O P U L A T I O N . NATURAL P O P U L A T I O N ' sma l l , yel low, twisted / med i um s i zed, ye I Iow, tw i sted 1 ( l a rge , red profusely branched) I very smal l , dark red, f r a g i l e very smal l , dark red, f r a g i l e F i g . 29. A summary of G r a c i l a r i a verrucosa morphology f o r vari 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 weight), p r o f u s e l y branched and had few epiphytes ( F i g . 29A). These robust p l a n t s were growing above the n a t u r a l G. verrucosa bed i n an area that was devoid of any major plant growth. In February 1981, the surface water of Bamfield I n l e t was depleted of n i t r a t e and ammonium. This was e a r l i e r than normal (L. Druehl, pers. comm.). The n a t u r a l G. verrucosa bed i n Wiseman's Bay was approximately o n e - t h i r d smaller than i n previous years, and epiphytism was more ex t e n s i v e . The s i z e of the high i n t e r t i d a l f e d at the head of Bamfield I n l e t was not a l t e r e d . Transplant experiments i n 1981 produced s i m i l a r morphological changes to those of 1980 ( F i g . 29B). The p l a n t s t r a n s p l a n t e d from Wiseman's Bay (1.0 m) to the head of Bamfield I n l e t at 1.8 m were yellow, medium, and t w i s t e d , but more robust than the resident G. verrucosa p l a n t s . Branches were short, convoluted and possessed brown t i p s . The p l a n t s that were tr a n s p l a n t e d from Wiseman's Bay (1.0 m) to the head of Bamfield I n l e t at 1.0 m, remained dark red but had numerous epiphytes and were p a r t i a l l y decayed. No new growth was apparent. The t h a l l i t r a n s p l a n t e d down the i n t e r t i d a l zone from 1.8 m at the head of Bamfield I n l e t to 1.0 m were a l s o decayed. Branches were reduced i n s i z e 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 Bamfield I n l e t (1.8 m) to the n a t u r a l bed i n Wiseman's Bay (1.0 m) became red, r e l a t i v e l y robust, and p r o f u s e l y branched. In general, they 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 p l a n t s t r a n s p l a n t e d from the head of Bamfield I n l e t 108 (1.8 m) to Wiseman's Bay high i n the i n t e r t i d a l (1.8 m) were medium s i z e d , red, with few epiphytes, and were r e l a t i v e l y robust with long s t r a i g h t branches. Some of the p l a n t s were tor n away. The p l a n t s which were moved up i n the i n t e r t i d a l i n Wiseman's Bay from 1.0 t o 1.8 m were l a r g e , dark red, and possessed few epiphytes. Most of the p l a n t s that were tr a n s p l a n t e d 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 various i n t e r t i d a l l o c a t i o n s i s presented i n Figure 30. The high i n t e r t i d a l population showed a response to d e s i c c a t i o n not apparent i n the other populations; at 10—15% d e s i c c a t i o n the p o t e n t i a l n i t r a t e uptake rate was enhanced one to f i v e times depending on the year ( F i g . 30). On the other hand, i n the s u b t i d a l population n i t r a t e uptake dropped to zero a f t e r only 10% d e s i c c a t i o n and n i t r a t e , i n s t e a d of n i t r i t e was excreted at greater d e s i c c a t i o n . The response of the low i n t e r t i d a l p opulation was intermediate between the other two populations. There was no enhancement of uptake rate 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 high i n t e r t i d a l p l a n t s during l a t e summer ( F i g . 30) but not i n June ( F i g . 31). In June 1981, the uptake r a t e s of p l a n t s from both i n t e r t i d a l populations were l e s s i n f l u e n c e d by higher degrees of d e s i c c a t i o n than i n the previous year ( F i g . 31). Uptake rates s i m i l a r to those f o r f u l l y hydrated ( c o n t r o l ) 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 rates (pmol.g dry wt" 1.h" 1) 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), for the n a t u r a l populations of hiqh 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. o A B 1 1 r 10 20 30 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 rates (pmol.g dry wf'.h"') and % d e s i c c a t i o n in June of 1980 (A), and' 1981 (B) for the n a t u r a l populations of high 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 verrucosa. — i 1 1— 10 20 30 Desiccation 111 p l a n t s were maintained up to 15% d e s i c c a t i o n . The uptake rates of the c o n t r o l p l a n t s during l a t e summer v a r i e d from year to year. N i t r a t e uptake rates were very low i n 1979 i n comparison to 1980 and 1981 ( F i g . 30). In 1980 and 1981 the n i t r a t e uptake r a t e s of the n a t u r a l high i n t e r t i d a l p opulation were much lower than those of the n a t u r a l low i n t e r t i d a l p o p u l a t i o n . The n i t r a t e uptake rates of the hydrated ( 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 populations i n June 1980 and 1981 ( F i g s . 31). The p l a n t s t r a n s p l a n t e d from Wiseman's Bay (1.0 m) to the head of Bamfield I n l e t (1.8 m) responded to m i l d d e s i c c a t i o n i n a manner s i m i l a r to the p l a n t s growing n a t u r a l l y at 1.8 m at the head of Bamfield I n l e t . The r e l a t i v e enhancement of n i t r a t e uptake f o r these p l a n t s was 1.5 and 4, i n 1980 and 1981, r e s p e c t i v e l y ( F i g . 32). Their t o l e r a n c e to higher d e s i c c a t i o n (20-60%) was l e s s than that of the res i d e n t p l a n t s at the head of Bamfield I n l e t . The uptake rates of the res i d e n t p l a n t s at 30% d e s i c c a t i o n were s i m i l a r to the uptake rates of the hydrated ( c o n t r o l ) p l a n t s , but the uptake rate of the tra n s p l a n t e d p l a n t s was much l e s s at 30% d e s i c c a t i o n than at 0%. The p l a n t s which were t r a n s p l a n t e d from the head of Bamfield I n l e t (1.8 m) to the high i n t e r t i d a l s i t e i n Wiseman's Bay (1.8 m) maintained an enhanced n i t r a t e uptake i n response to d e s i c c a t i o n ( F i g . 32A). These p l a n t s a l s o r e t a i n e d t h e i r t o l e r a n c e to higher d e s i c c a t i o n . Uptake rates at 30% d e s i c c a t i o n were equal t o those of the hydrated ( c o n t r o l ) p l a n t s . The p l a n t s t r a n s p l a n t e d to the lower i n t e r t i d a l s i t e i n Wiseman's Bay (1.0 m) showed no enhancement of n i t r a t e uptake 1 12 T I D A L H E I G H T 1.8 M 1.0 M. 1980 (A) WISEMAN'S BAY 2 3 . 0 X 1 . 2 HEAD OF B A M F I E L D I N L E T NATURAL POPULATION 5 . 0 X 2 . 0 [ 9 . 0 X 1 . 2 ] 7 " 7 . 0 X 1 . 5 [ 7 . 0 X 1 . 5 ] NATURAL PO P U L A T I O N 2 6 . 0 [ 1 2 . 0 ] did not survive T I D A L H E I G H T 1.8 M 1.0 M 1981 (B) WISEMAN'S BAY HEAD OF B A M F I E L D I N L E T 6 . 0 X 2 1 4 . 5 X 2 [ 2 3 . 0 ] m  [ 3 0 . 8 ] ^ NATURAL P O P U L A T I O N | 1 . 6 X 5 . 0 [ 1 4 . 0 X 1 . 2 ] 4 . Oj [ 1 0 . 0 X 2 . 0 ] NATURAL P O P U L A T I O N 1 0 . 2 1 2 . 1 [ 2 8 . 5 ] 4 . 5 [ 1 6 . 0 ] F i g . 32. A summary of hydrated n i t r a t e and ammonium uptake ra t e s (pmol.g dry wf'.h"' 1) for G r a c i l a r i a verrucosa i n August 1980 (A), and 1981 (B). Square brackets f o r ammonium. X i n d i c a t e s enhanced n i t r a t e uptake rates that occurred at the optimal % d e s i c c a t i o n . The numbers to the r i g h t of the x gives the r e l a t i v e degree of enhancement. The arrows i n d i c a t e the o r i g i n of the tr a n s p l a n t e d p o p u l a t i o n s . 1 13 f o l l o w i n g m i l d d e s i c c a t i o n . However, these p l a n t s r e t a i n e d t h e i r t o lerance to higher d e s i c c a t i o n : p l a n t s d r i e d to 30% d e s i c c a t i o n showed only s l i g h t l y lower n i t r a t e uptake rates than the hydrated p l a n t s . 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 to 1.0 m at the head of Bamfield I n l e t showed no 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 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 at the head of Bamfield I n l e t from 1.8 to 1.0 m showed no enhancement of ni 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). Transplanting up the i n t e r t i d a l zone i n Wiseman's Bay from 1.0 to 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 rates 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 times greater than the p l a n t s growing at the head of Bamfield I n l e t ( F i g . 32). The n i t r a t e uptake rates of the p l a n t s t r a n s p l a n t e d from Wiseman's Bay to the n a t u r a l bed at the head of Bamfield I n l e t were higher than the uptake rates of the re s i d e n t p l a n t s ( F i g . 32). The n i t r a t e uptake rates of the t h a l l i t r a n s p l a n t e d from Wiseman's Bay to the low i n t e r t i d a l s i t e (1.0 m) at the head of Bamfield 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 from the head of Bamfield I n l e t (1.8 m) to 1.8 m i n Wiseman's Bay were intermediate between the two n a t u r a l populations ( F i g . 32). In 1981, moving up the i n t e r t i d a l zone i n Wiseman's Bay increased the n i t r a t e uptake r a t e s of hydrated p l a n t s from 10.2 to 14.5 pmol.g dry w t " 1 . h " 1 ( F i g . 32B). Moving down the i n t e r t i d a l zone at the head of Bamfield I n l e t had l i t t l e e f f e c t on n i t r a t e uptake r a t e s . B 30H 1* D e s i c c a t i o n *r. D e s i c c a t i o n F i g . 33. The r e l a t i o n s h i p between ammonium'uptake rates (jamol.g dry w t _ 1 . h _ 1 ) 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), for the n a t u r a l populations of high 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 verrucosa. 115 Ammonium Uptake In l a t e summer the high i n t e r t i d a l G. verrucosa showed s l i g h t l y enhanced 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 (10-15%). This enhancement was not as pronounced as i t was wit h n i t r a t e ( F i g . 33). The low i n t e r t i d a l G. verrucosa showed no enhancement of ammonium uptake f o l l o w i n g d e s i c c a t i o n . Uptake decreased r a p i d l y a f t e r d e s i c c a t i o n i n the s u b t i d a l and low i n t e r t i d a l p l a n t s i n 1979 ( F i g . 33A). The s u b t i d a l p l a n t s a c t u a l l y excreted n i t r a t e when d e s i c c a t i o n was greater than 10% ( F i g . 30A). Enhancement of ammonium uptake i n the high i n t e r t i d a l 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 . 34). Enhancement was observed i n 1981, but not i n 1980. The low i n t e r t i d a l p l a n t s showed no enhancement of ammonium uptake a f t e r d e s i c c a t i o n ( F i g . 34). In June, the ammonium uptake rates for the hydrated high 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 true l a t e r i n the summer ( F i g s . 33,34). The.high i n t e r t i d a l p l a n t s had lower uptake rates than the 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 . 33). In 1979, the ammonium uptake rate f o r the hydrated high i n t e r t i d a l p l a n t s was much greater than i n 1980 and 1981 and greater than the uptake rates of the hydrated low i n t e r t i d a l p l a n t s ( F i g . 33). In 1980, l o s s of pl a n t m a t e r i a l due to wave a c t i o n and epiphytism prevented determination of the r e l a t i o n s h i p between uptake r a t e and % d e s i c c a t i o n f o r the p l a n t s t r a n s p l a n t e d to 1.0 m at the head of Bamfield I n l e t and to 1.8 m i n Wiseman's Bay ( F i g . 32A). Ammonium uptake rates were higher i n 1981 than i n 1980 F i g . 34. The r e l a t i o n s h i p between ammonium uptake rates (ptnol.g dry w f ' . h - 1 ) and % d e s i c c a t i o n i n June of 1980 (A) and 1981 (B) for the n a t u r a l populations of high (-—•---) and low (—• ) i n t e r t i d a l G r a c i l a r i a verrucosa. 117 ( F i g . 32). The p l a n t s t r a n s p l a n t e d down the i n t e r t i d a l zone at the head of Bamfield I n l e t showed an increase i n ammonium uptake rate ( F i g . 32B). In 1980 and 1981, the re s i d e n t p l a n t s at the head of Bamfield I n l e t as w e l l as the p l a n t s t r a n s p l a n t e d to the n a t u r a l bed (1.8 m) at the head of Bamfield 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 t h i s enhancement f o r the n a t u r a l and tra n s p l a n t e d populations i n 1980 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 . The enhancement of ammonium uptake i n the tra n s p l a n t e d p l a n t s was greater than i n the res i d e n t p l a n t s . The opposite was true for n i t r a t e uptake where the % d e s i c c a t i o n producing maximum n i t r a t e uptake rates i n the tra n s p l a n t e d t h a l l i was a l s o s l i g h t l y l e s s (5-10%). The ammonium uptake rates for tra n s p l a n t e d or resident hydrated p l a n t s were s i m i l a r ( F i g . 32). In 1980 and 1981, the re 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 a f t e r m i l d d e s i c c a t i o n ( F i g . 32). In 1980, t h i s p o pulation showed a s l i g h t t o l e r a n c e to higher d e s i c c a t i o n by maintaining uptake rates equal to the hydrated uptake rates at ^  30% d e s i c c a t i o n . In 1981, t h i s n a t u r a l population i n Wiseman's Bay as w e l l as a l l populations t r a n s p l a n t e d to the low i n t e r t i d a l s i t e i n Wiseman's Bay or the head of Bamfield I n l e t , had a low tolerance to higher d e s i c c a t i o n . At 30% d e s i c c a t i o n ammonium uptake rates were l e s s than h a l f the rates at 0% d e s i c c a t i o n . None of these low i n t e r t i d a l populations showed enhancement of ammonium uptake a f t e r d e s i c c a t i o n , nor d i d the p l a n t s t r a n s p l a n t e d from Wiseman's Bay or from 1.8 to 1.0 m at the head of Bamfield I n l e t 118 ( F i g . 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). Although no enhancement of ammonium uptake was found, the p l a n t s transplanted from the head of Bamfield I n l e t to 1.8 m i n Wiseman's Bay showed a greater t o l e r a n c e to d e s i c c a t i o n than the low i n t e r t i d a l G. verrucosa p l a n t s ; ammonium uptake rates only decreased by approximately o n e - f i f t h when p l a n t s were de s i c c a t e d to 30%. Time Courses of Nitrogen Uptake Time course stud i e s of ammonium and n i t r a t e uptake rates were conducted i n June and August f o r both n a t u r a l i n t e r t i d a l beds of G. verrucosa. The data are presented i n Table 1, Chapter 1. The rate of n i t r a t e uptake was constant for the f i r s t 30 min of incubation i n 30 /uM n i t r a t e . N i t r a t e uptake i n low i n t e r t i d a l G. verrucosa was sti m u l a t e d a f t e r 15 min of inc u b a t i o n on 30 yuM n i t r a t e . Ammonium uptake r a t e s i n high i n t e r t i d a l G. verrucosa decreased a f t e r 10—12 min. The low i n t e r t i d a l p l a n t s f r e q u e n t l y maintained a constant ammonium uptake rate for a longer p e r i o d of time, often up to 30 min. When exposed to both n i t r a t e and ammonium, both i n t e r t i d a l G. verrucosa populations took up n i t r a t e and ammonium simultaneously (Table 1). The high i n t e r t i d a l p l a n t s showed a decrease (50%) i n n i t r a t e uptake i n the 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 the low i n t e r t i d a l G. verrucosa. 119 Nitrogen Uptake K i n e t i c s N i t r a t e uptake i n the low i n t e r t i d a l G. verrucosa appeared to have a hyperbolic component at low n i t r a t e concentrations and a l i n e a r component at n i t r a t e concentrations between 15 and 50 pM ( F i g . 35A). The k i n e t i c s curve at low n i t r a t e concentrations suggests that the Ks was approximately 1 pM i f the l i n e a r ( p o s s i b l y d i f f u s i o n ) component was substracted from the uptake rates ( D ' E l i a and DeBoer 1978) to give an e s t i m a t i o n of the a c t i v e uptake component. N i t r a t e uptake rates i n high i n t e r t i d a l G. verrucosa followed saturable k i n e t i c s 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). The Vmax was approximately 4 pmol.g dry w t _ 1 . h " 1 and the Ks was 6 uM. N i t r a t e uptake r a t e s at 30 uM n i t r a t e , f o r the low i n t e r t i d a l p l a n t s were twice the r a t e s of the high i n t e r t i d a l p l a n t s . There were a l s o d i f f e r e n c e s i n the ammonium uptake k i n e t i c s between the two i n t e r t i d a l p o p u l a t i o n s . The ammonium uptake ra t e s f o r the low i n t e r t i d a l G. verrucosa population were approximately p r o p o r t i o n a l to the ambient ammonium concentration up to 40 pM ( F i g . 36A). Ammonium uptake k i n e t i c s f o r the high i n t e r t i d a l G. verrucosa population was saturable but d i d not f i t a rectangular hyperbola ( F i g . 36B). The Vmax was approximately 30 pmol.g dry wt~ 1.h~ 1 and the Ks was about 10 uM. Ammonium uptake rates at ammonium concentrations 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 . Soluble Nitrogen Content In June, both i n t e r t i d a l populations of G. verrucosa had a 120 s , e S o 6 4 8 , , - I 1 10 20 30 40 NO" U M ) 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 dry w f ' . h - 1 ) 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 populations of low ( — • — ) ( A ) , and high (--o---)(B) i n t e r t i d a l G r a c i l a r i a verrucosa, i n August 1981. Uptake r a t e s minus the non-saturable component ( ) were determined to assessthe a c t i v e uptake component. 1 2 1 20 40 60 80 NH* (/iM ) F i g . 36. Ammonium uptake k i n e t i c s (umol.g dry w f 1 . h _ 1 ) for the n a t u r a l populations of low ( — • — ) ( A ) , and high f — o — ) ( B ) i n t e r t i d a l G r a c i l a r i a verrucosa. 122 very low n i t r a t e content, approximately 0.04 pmol.g wet wt" 1or 0.02 % of the t o t a l n itrogen i n the plan t (Table 3). The p r e c i s e 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 to the crude nature of the e x t r a c t s which may contain compounds that i n t e r f e r e with the c o l o r i m e t r i c a n a l y s i s (Appendix 1). Nevertheless, general trends rather than absolute amounts can be discussed. The high i n t e r t i d a l p l a n t s a l s o had a low ammonium content i n June while the low i n t e r t i d a l p l a n t s had an ammonium content s i m i l a r to those e x t r a c t e d l a t e r i n the summer. In August, a l l n a t u r a l or tran s p l a n t e d populations at the head of Bamfield I n l e t had a n i t r a t e content of about 0.5 pmol.g wet wt" 1 or approximately 0.14 % of the t o t a l n itrogen i n the plant ( F i g . 37). This was much lower than that e x t r a c t e d from the p l a n t s i n Wiseman's Bay which ranged from 2.0 /umol.g wet wt" 1 i n the high i n t e r t i d a l s i t e to approximately 4.0 yumol.g wet wt" 1 or 0.5 % of the t o t a l n itrogen i n the plan t i n the low i n t e r t i d a l s i t e . The lower i n t e r t i d a l p l a n t s i n Wiseman's Bay had a higher n i t r a t e content than the high i n t e r t i d a l p l a n t s at the same l o c a t i o n . There was l i t t l e v a r i a t i o n i n the ammonium content ( F i g . 37). Grin d i n g i n hot water e x t r a c t e d greater amounts of ni n h y d r i n p o s i t i v e m a t e r i a l (assumed to be p r i m a r i l y amino ac i d s ) than b o i l i n g i n ethanol ( F i g . 37). Three populations showed a higher amino a c i d content. They were the t h a l l i t r a n s p l a n t e d from the head of Bamfield I n l e t to 1.8 m i n Wiseman's Bay, and both populations t r a n s p l a n t e d to 1.0 m at the head of Bamfield I n l e t ( F i g . 37). Table 3. Soluble n i t r a t e , ammonium, amino a c i d s and p r o t e i n content i n the n a t u r a l populations of high and low i n t e r t i d a l G r a c i l a r i a verrucosa i n June and August 1981, ± 1 standard d e v i a t i o n , n=3. Soluble nitrogen content (u.mol.g wet w t - 1 ) , % total nitrogen Population Time Nitrate % Ammonium % Amino Acids protein mg.g wet wt" 1 Water % Ethanol % High Int e r t i d a l June 0.4+0.14 0.2 0.82+0.22 0.14 4.09 1.5 7.7 25 August 0.57+0.18 0.14 2.23+0.12 0.56 6.85±0.85 2.6 Low Inte r t i d a l June 0.03+0.02 0.003 2.27+0.72 0.38 0.0 9.5 45 August 4.41+1.30 0.5 4.94±0.95 1.65 5.47±1.3 2.7 0.0 ro to 124 N i t r a t e and [Ammonium] Pools ( /omol • g wet wt ) T I D A L H E I G H T 1.8 M 1.0 M WISEMAN S BAY HEAD OF B A M F I E L D I N L E T 2 . 1 5 + 0 . 1 5 1.38 ± 0.16 [ 3 . 3 9 ] • 3.21 ± 6V8n NATURAL POPULATION 0.57 ± 0 . 1 8 [ 2 . 2 3 + 0.12] 23 + 0.07 [ 1 . 7 6 ± 0.26] 3.47 ± 0.25 [ 2 . 9 8 + 0 . 2 3 ] NATURAL P O P U L A T I O N 4.41 + 1.3 [ 4 . 9 4 ± 0 . 95] 0.61 + 0.17 [ 2 . 8 5 + 0.18] 0.37 + 0.16 [ 3 . 1 3 + 0.50] Amino A c i d Pools (jimol g wet wt ) WISEMAN'S BAY extracted by g r i n d i n q i n b o i l i n g water []extracted by b o i l i n g i n EtOH T I D A L H E I G H T 1.8 M HEAD OF B A M F I E L D I N L E T 1.0 M 12.4 8.93 + 1.81 [ 1 1 . 4 ] « [ 0 . 0 ] 6.75 ± 0.79 NATURAL POPULATION 5.47 + 1.28 [ 0 . 0 ] 17.3 ± 2.6 [ 0 . 1 8 ] 16.0 [ 4 . 9 8 ] F i g . 37. Soluble n i t r a t e , ammonium, and amino a c i d content (jjmol.g wet wt"'1) of G r a c i l a r i a verrucosa i n August 1981, ± one standard 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 of the tran s p l a n t e d populations. 125 The Wiseman's Bay n a t u r a l population had a higher s o l u b l e p r o t e i n content than the other G. verrucosa populations t e s t e d ( F i g . 38). A l l the populations t r a n s p l a n t e d to the head of Bamfield I n l e t had low solu b l e p r o t e i n content. 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 reductase c h a r a c t e r i s t i c s suggests that the two n a t u r a l G. verrucosa populations have the same n i t r a t e reductase enzyme. N i t r i t e production was l i n e a r * with time and a c t i v i t y was p r o p o r t i o n a l to the volume of e x t r a c t used ( F i g . 39). The n i t r a t e reductase enzyme was i n s e n s i t i v e to Mg + + concentration ( F i g . 40), and pH change ( F i g . 41). No c l e a r r e l a t i o n s h i p between PVP concentration and n i t r a t e reductase a c t i v i t y was detected ( F i g . 42). N i t r a t e and NADH supply had a s i m i l a r e f f e c t on the n i t r a t e reductase 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 populations ( F i g s . 43,44). The Km values for n i t r a t e and NADH were 70 ± 7.0 and 75 ± 46 /utM, r e s p e c t i v e l y . In June, the Vmax for the n i t r a t e reductase a c t i v i t y of both n a t u r a l populations was about 150 pmol N02 .g p r o t e i n " 1 . h " 1 . In August, the n a t u r a l population of G. verrucosa i n Wiseman's Bay had twice the n i t r a t e reductase a c t i v i t y of the n a t u r a l population at the head of Bamfield I n l e t ( F i g . 38). Transpla n t i n g from Wiseman's Bay to the head of Bamfield I n l e t r e s u l t e d i n a decrease i n n i t r a t e reductase a c t i v i t y . 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) at the head of Bamfield I n l e t had low n i t r a t e reductase a c t i v i t i e s a l s o ( F i g . 38). T r a n s p l a n t i n g up the i n t e r t i d a l zone i n Wiseman's Bay increased the already high n i t r a t e reductase a c t i v i t y of the Wiseman's Bay G. 126 N i t r a t e Reductase A c t i v i t y (umol NO" • g Protein" [Protein Pools] ' 2 ~' M T I D A L H E I G H T 1.8 M (mg . g wet wt _ 1) WISEMAN'S BAY 43.8 h"1) HEAD OF B A M F I E L D I N L E T [ 8 . 3 1 ] „ NATURAL POPULATION 44 . 2 1 4 1 . 6 [ 8 . 3 1 ] 60.6 [ 6 . 3 5 ] i * / / / / / / / / / / 50.9 [7.95] 1.0 M NATURAL POPULATION 85.0 [ 9 . 4 7 1 7.691 2 6 . 9 [ 7 . 2 5 ] 1 4 . 2 [ 6 . 7 0 ] F i g . 38. Soluble p r o t e i n content (mg p r o t e i n . g wet wt" 1) and n i t r a t e reductase a c t i v i t y (pmol NOj.g p r o t e i n " 1 . h " 1 ) of G r a c i l a r i a verrucosa i n August 1981. The arrows i n d i c a t e the o r i g i n of the tra n s p l a n t e d populations. 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 - 1 ) with time (minT (A),and the r e l a t i o n s h i p between n i t r a t e reductase a c t i v i t y (^ umol NO* .g p r o t e i n " 1 . h " 1 and the volume of enzyme e x t r a c t used (ml) (B), for the n a t u r a l populations of high (-—+—) and low (—•—) i n t e r t i d a l G r a c i l a r i a verrucosa. Error bars represent one standard d e v i a t i o n , n=3. .£ 165-$ 145-> 12H T J. T I 1 1 1 " 1 • 2 4 6 8 M g S 0 4 ( m M ) The r e l a t i o n s h i p between n i t r a t e reductase a c t i v i t y g p r o t e i n " 1 . h " 1 ) and MgSO^ con c e n t r a t i o n (mM) f o r the »—) i n t e r t i d a l one standard F i g . 40. (ujnol NO~ na t u r a l populations of high (---o1-) and low ( G r a c i l a r i a verrucosa. Error bars represent d e v i a t i o n , n=3, 1 29 120i 4) E 0. O) ' Q C M 110' z o E 3 < tn O u D or 10OH 9 0 ' T I I I 8.1 ~ I — 8.2 T i i i —\— 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 reductase a c t i v i t y (umol NOg .g p r o t e i n " 1 . h " 1 ) and pH f o r the n a t u r a l populations of high (-—o —-) and low (—• ) 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. p CL Ol ' Q C N J z o E 3 120H 100J < u or 8 0 -0.1 —1— 0.2 T I I I I - f — 0.3 0.4 P V P ( g P V P . g w e t w t " 1 ) F i g . 42. The r e l a t i o n s h i p (umol NOj.g p r o t e i n - 1 , h " 1 ) p y r r o l i d i n e (PVP) added populations of high (-—o G r a c i l a r i a verrucosa, d e v i a t i o n , n=3. between n i t r a t e reductase a c t i v i t y and the amount of p o l y v i n y l (g.g wet wt" 1) f o r the n a t u r a l -) and low (—•—) i n t e r t i d a l E r r or bars represent one standard 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 a c t i v i t y (umol NOj.g p r o t e i n " 1 . h " 1 ) and n i t r a t e c oncentration (mM) fo r tiie n a t u r a l populations of high (—•-—) 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 standard d e v i a t i o n , n=3. ) one 100 200 300 400 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 a c t i v i t y (umol NO" .g p r o t e i n " 1 . h " 1 ) and NADH concent r a t i o n (uM) fo r the na t u r a l populations of high (---•—") and low (—» G r a c i l a r i a verrucosa. E r r o r bars represent d e v i a t i o n , n=3. ) i n t e r t i d a l one standard 132 verrucosa p o p u l a t i o n . T r a n s p l a n t i n g from the head of Bamfield I n l e t to 1.0 m i n Wiseman's Bay increased n i t r a t e reductase a c t i v i t y . I f p l a n t s were tr a n s p l a n t e d to 1.8 m i n Wiseman's Bay from the head of Bamfield I n l e t the n i t r a t e reductase a c t i v i t y remained constant at about 45 pmol N0 2.g p r o t e i n " 1 . h ' 1 . 133 D i s c u s s i o n The morphologies of the two n a t u r a l populations of G. verrucosa were so d i f f e r e n t that they appeared to be d i f f e r e n t species. Recent s t u d i e s have revealed morphological p l a s t i c i t y i n G r a c i l a r i a ( B i r d et a l . 1982a). Biochemical c h a r a c t e r i s t i c s such as chromosome number and enzyme type are often more u s e f u l i n taxonomic s t u d i e s than morphological d i f f e r e n c e s ( B i r d et a l . 1982a). The r e s u l t s of the t r a n s p l a n t experiments i n t h i s study suggest that the observed morphological d i f f e r e n c e s were phenotypic rather than genotypic. Transplanted p l a n t s e x h i b i t e d a l t e r e d morphologies s i m i l a r to those of the r e s i d e n t p l a n t s . The s u r v i v a l rate of the 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 lower l i m i t s of the n a t u r a l beds. Many of the p l a n t s that were tr a n s p l a n t e d to the high i n t e r t i d a l s i t e i n Wiseman's Bay 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 grazing imposes the upper l i m i t on the n a t u r a l bed found at 1.0 m i n Wiseman's Bay. The t r a n s p l a n t s which were not broken o f f , grew w e l l i n the absence of competition f o r l i g h t and n u t r i e n t s . These tr a n s p l a n t e d p l a n t s had fewer epiphytes than the lower i n t e r t i d a l p l a n t s i n Wiseman's Bay (1.0 m). Lindsay and Saunders (1977) made the same observation i n t h e i r i n t e r t i d a l c u l t u r e pens. The p l a n t s t r a n s p l a n t e d from the head of Bamfield I n l e t (1.8 m) to the high i n t e r t i d a l s i t e i n Wiseman's Bay (1.8 m) d i d not r e t a i n t h e i r yellow c o l o r a t i o n and stunted appearance. This suggests that these c h a r a c t e r i s t i c s were not c o n t r o l l e d by 134 i n t e r t i d a l l o c a t i o n . The exposure to d i r e c t s u n l i g h t and other environmental c o n d i t i o n s such as d i f f e r e n t s u b s t r a t e , wave a c t i o n , or v a r i e d n u t r i e n t supply which were d i f f e r e n t i n Wiseman's Bay and at the head of Bamfield I n l e t , and not p e r i o d i c exposure, may be r e s p o n s i b l e f o r t h i s morphological type. The p l a n t s t r a n s p l a n t e d from 1.8 m to 1.0 m at the head of Bamfield I n l e t a l s o became dark red. This i s f u r t h e r evidence that 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 (1.0) to 1.8 m at the head of Bamfield I n l e t r a p i d l y (3 weeks) changed from red to yellow and from a feathery to a matted appearance, resembling the r e s i d e n t p l a n t s . The G. verrucosa bed at the head of Bamfield I n l e t had a d e f i n i t e lower l i m i t . An e e l g r a s s bed predominated at about 1.2 m above Canadian datum. The t h a l l i t r a n s p l a n t e d to 1.0 m i n t h i s l o c a t i o n grew p o o r l y , whether they were o r i g i n a l l y from a high or low i n t e r t i d a l bed. A f t e r f i v e weeks, the t h a l l i were very small and f r a g i l e . I t i s p o s s i b l e that the eelgrass was 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. verrucosa t h a l l i i n the competition for l i g h t , substrate and/or n u t r i e n t s . There was l i t t l e or no eelgrass i n Wiseman's Bay to compete with the G r a c i l a r i a at 1.0 m. The p l a n t s that were tran s p l a n t e d from the head of Bamfield I n l e t to 1.0 m i n Wiseman's Bay d i d 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 surrounding them. I t i s p o s s i b l e that t h e i r o r i g i n a l growth form, which was adapted fo r growth at 1.8 m at the head of Bamfield I n l e t was a disadvantage i n the new low i n t e r t i d a l s i t e during the important 135 growing p e r i o d i n l a t e June and e a r l y J u l y . Lindsay and Saunders (1977) a l s o found that l o c a l G. verrucosa grew f a s t e r than those t r a n s p l a n t e d to the c u l t u r e l o c a t i o n . Gagne* et a l . (1982) conducted t r a n s p l a n t experiments with Laminaria  l o n g i c r u r i s and they found that i n some cases the p l a n t s p a r t i a l l y r e t a i n e d the 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 population even a f t e r one year. Time courses of uptake are e s s e n t i a l f o r the i n t e r p r e t a t i o n of any uptake rate determined over a p a r t i c u l a r time. Both phytoplankton and macrophytes have high i n i t i a l n i t r o g e n uptake rates which often decrease w i t h i n minutes or hours (Conway et a l . 1976; D ' E l i a and DeBoer 1978). The two i n t e r t i d a l G. verrucosa populations i n t h i s study had t r a n s i e n t i n i t i a l n i t r o g e n uptake r a t e s . The sudden drop i n ammonium uptake by 50 % a f t e r 10—30 min was too la r g e to be accounted for by the decrease i n ammonium conc e n t r a t i o n i n the medium due to uptake. I n i t i a l n i t r a t e uptake rates remained constant f o r a longer p e r i o d than ammonium uptake r a t e s . This was true f o r other i n t e r t i d a l macrophytes (Chapter 1). I t i s p o s s i b l e that t h i s i n i t i a l r a p i d uptake rate i s c o n t r o l l e d by an i n t e r n a l mechanism such as the s i z e of an i n t r a c e l l u l a r p o o l . I n h i b i t i o n of n i t r a t e uptake by ammonium has been observed i n other marine macrophytes (Haines and Wheeler 1978; Hanisak and H a r l i n 1978; Chapter 1). N i t r a t e uptake was i n h i b i t e d by ammonium i n high i n t e r t i d a l G. verrucosa but not i n the low i n t e r t i d a l G. verrucosa. Other species 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 l o n q i c r u r i s ( H a r l i n and C r a i g i e 1978). Since Fucus i s found i n the high i n t e r t i d a l zone and Laminaria i s l a r g e l y s u b t i d a l , i t would appear that the maintenance of a normal n i t r a t e uptake rate i n the presence of ammonium i s not an adaptation to i n t e r t i d a l l o c a t i o n . More species must be t e s t e d , but i t may be that i n h i b i t i o n of n i t r a t e uptake by ammonium i s dependent on n u t r i t i o n a l past h i s t o r y rather than i n t e r t i d a l l o c a t i o n . I t i s e n e r g e t i c a l l y more favorable f o r the plant to take up ammonium rather than n i t r a t e . The ammonium supply at the head of Bamfield " i n l e t was probably due to ground water runoff and was greater than i n Wiseman's Bay. C/N r a t i o s suggest that G. verrrucosa i n Wiseman's Bay was more severely n i t r o g e n l i m i t e d than at the head of Bamfield I n l e t . C/N r a t i o s were approximately 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 that the high i n t e r t i d a l G. verrucosa on the mud f l a t at the head of Bamfield 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 nitr o g e n from the mud and from epiphytes (Capone and Taylor 1977). Nevertheless, the C/N r a t i o of 9 s t i l l suggests that these p l a n t s were nitrogen d e f i c i e n t ( N i e l l 1976). The ammonium and n i t r a t e uptake r a t e s of the various G. verrucosa populations give some i n s i g h t i n t o the n u t r i e n t supply i n the various s i t e s . In August, n i t r a t e uptake r a t e s were much higher i n Wiseman's Bay than at the head of Bamfield I n l e t . This may be due to the presence of ammonium at the head of Bamfield I n l e t and the p o s s i b l e i n t e r m i t t e n t supply of n i t r a t e to Wiseman's Bay through v e r t i c a l mixing. Within a few weeks, the ammonium and n i t r a t e uptake rates of t r a n s p l a n t e d t h a l l i were s i m i l a r to the rates of the l o c a l t h a l l i . T r a n s p l a n t i n g up 1 37 the i n t e r t i d a l 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 per i o d of exposure; t h i s r e s u l t e d i n increased n i t r a t e uptake r a t e s . Transplanting down the i n t e r t i d a l zone at the head of Bamfield I n l e t increased ammonium uptake r a t e s , although a l l the other populations had s i m i l a r ammonium uptake r a t e s . There was a l s o very l i t t l e d i f f e r e n c e between the ammonium uptake rates i n June and i n August. This suggests that the ammonium uptake system was always " a c t i v e " and l i m i t e d s o l e l y by the immediate ammonium supply. There have been s e v e r a l s t u d i e s of the nitr o g e n uptake k i n e t i c s i n marine macrophytes. D ' E l i a and DeBoer (1978) found that ammonium uptake i n G r a c i l a r i a t i k v a h i a e was not saturated at 40 uM ammonium. Haines and Wheeler (1978) found a s i m i l a r trend i n ammonium uptake f o r Macrocystis p y r i f e r a and Hypnea  muse i f o r m i s . Other s t u d i e s w i t h d i f f e r e n t species have found that ammonium uptake was satur a b l e (Hanisak and H a r l i n 1978; Topinka 1978; Kautsky 1982), i n d i c a t i n g that 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. verrucosa showed that one po p u l a t i o n , at the head of Bamfield I n l e t , had sat u r a b l e ammonium uptake and another, i n Wiseman's Bay, d i d not. Even the n i t r a t e uptake rates of the Wiseman's Bay popu l a t i o n were not s a t u r a b l e . The e c o l o g i c a l s i g n i f i c a n c e of saturable or non-saturable uptake i s not c l e a r . I t i s suspected that the non—saturable 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 component increases with substrate c o n c e n t r a t i o n s . Any appreciable d i f f e r e n c e i n uptake 138 rate occurs at concentrations of ammonium and n i t r a t e that are often higher than normally found i n the f i e l d . The d i f f e r e n c e between saturable and non-saturable uptake may therefore be of l i t t l e e c o l o g i c a l s i g n i f i c a n c e . The Ks values f o r n i t r a t e uptake of the two n a t u r a l G. verrucosa populations were higher than those recorded f o r G. t i k v a h i a e ( D ' E l i a and DeBoer 1978) and phytoplankton (Eppley et a l . 1969b). However, they are l e s s than most Ks values c i t e d i n the l i t e r a t u r e f o r marine macrophytes (Haines and Wheeler 1978; Hanisak and H a r l i n ; H a r l i n 1978; H a r l i n and C r a i g i e 1978; Topinka 1978; Kautsky 1982). The Ks fo r ammonium uptake (10 pM) i n the high i n t e r t i d a l G. verrucosa was se v e r a l times greater than that of G. t i k v a h i a e (1.6 uM) (D ' E l i a and DeBoer 1978) and phytoplankton (Eppley et a l . 1969b). Other species such as Enteromorpha  compressa (Kautsky 1982) and Macrocystis p y r i f e r a (Haines and Wheeler 1978) have a s i m i l a r lack of a f f i n i t y f or ammonium. Competition f o r low concentrations 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 or these s p e c i e s , at the time they were t e s t e d . The n i t r a t e and ammonium uptake rates observed i n t h i s study were s i m i l a r i n magnitude to those presented i n the l i t e r a t u r e f o r other species of marine macrophytes ( D ' E l i a and DeBoer 1978; Haines and Wheeler 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; Kautsky 1982). Previous s t u d i e s have shown that high i n t e r t i d a l macrophytes have enhanced nitrogen uptake r a t e s f o l l o w i n g m i l d d e s i c c a t i o n (Thomas and Turpin 1980; Chapter 4). This study 139 w i t h G. verrucosa i n d i c a t e d that t h i s was an i n t r a s p e c i f i c as w e l l as an i n t e r s p e c i f i c adaptation to i n t e r t i d a l l o c a t i o n . The t r a n s p l a n t experiments showed that t h i s phenomenon was dependent on i n t e r t i d a l l e v e l and was not dependent on geographical l o c a t i o n or the o r i g i n of the p l a n t . P l a n t s t r a n s p l a n t e d to high i n t e r t i d a l s i t e s developed t h i s p h y s i o l o g i c a l adaptation fo r n i t r o g e n procurement while those t r a n s p l a n t e d to the low i n t e r t i d a l s i t e s d i d not. The only exception was G. verrucosa t r a n s p l a n t e d to 1.8 m i n Wiseman's Bay i n 1981. These p l a n t s showed no enhanced n i t r a t e uptake r a t e s , but they d i d show enhancement of ammonium uptake. In 1980, d e s i c c a t i o n r e s u l t e d i n enhanced n i t r a t e uptake r a t e s : ammonium uptake was not t e s t e d . In 1981, the ammonium uptake rates of hydrated p l a n t s were very high, and s i m i l a r to the Vmax found during the measurement of ammonium uptake k i n e t i c s . I t i s p o s s i b l e that f u r t h e r enhancement of uptake was p h y s i o l o g i c a l l y impossible. I s o l a t i o n of the environmental f a c t o r s causing enhanced ni t r o g e n uptake a f t e r d e s i c c a t i o n w i l l r e q u i r e c u l t i v a t i o n i n c o n t r o l l e d environments. The degree of enhancement and the % d e s i c c a t i o n r e s u l t i n g i n maximum uptake rates might depend upon past h i s t o r y . There was l i t t l e v a r i a t i o n i n the % d e s i c c a t i o n (10-15%) producing maximum uptake r a t e s . The degree of enhancement of uptake r a t e s f o r the tran s p l a n t e d t h a l l i and res i d e n t p l a n t s were s i m i l a r . The degree of enhancement v a r i e d s l i g h t l y with geographical l o c a t i o n and g r e a t l y from year to year (highest i n 1981 and lowest i n 1979). The c o n t r o l s of such a response to d e s i c c a t i o n were a 140 combination of environmental f a c t o r s prompting enhancement of uptake and the p l a n t ' s a b i l i t y to respond to such f a c t o r s . I t i s p o s s i b l e that environmental f a c t o r s such as nit r o g e n l i m i t a t i o n and p e r i o d i c d e s i c c a t i o n were not prominent i n the summer of 1979. I t i s much more l i k e l y that the p l a n t s were so nitrogen s t r e s s e d that nitrogen uptake systems required f o r r a p i d uptake were not maintained. Another p o s s i b l e adaptation to i n t e r t i d a l l o c a t i o n i s the maintenance of nitrogen uptake rates f o l l o w i n g a greater degree of d e s i c c a t i o n (20-30%). This was true of the uptake rates f o r high i n t e r t i d a l G. verrucosa. In the t r a n s p l a n t experiments, a tolerance to d e s i c c a t i o n appeared to depend upon the i n t e r t i d a l l o c a t i o n ; but the tr a n s p l a n t e d p l a n t s often 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 , which were between those of the resident p l a n t s and the population from which they were tr a n s p l a n t e d . This phenomenon may simply depend- upon i n t e r t i d a l l e v e l and not on the o r i g i n of the t h a l l u s , but required more than f i v e weeks to develop or disappear. The s o l u b l e nitrogen content and n i t r a t e reductase l e v e l s of the va r i o u s G. verrucosa populations confirmed most conclusions drawn from the uptake r a t e s . The n i t r a t e content and the n i t r a t e reductase l e v e l s were higher i n the Wiseman's Bay p l a n t s . This i n d i c a t e s that n i t r a t e i s an important source of n i t r o g e n i n Wiseman's Bay, but not at the head of Bamfield I n l e t . There were few d i s s i m i l a r i t i e s i n the ammonium and so l u b l e amino a c i d content. The amounts of n i t r a t e and ammonium ex 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 to those e x t r a c t e d from G. t i k v a h i a e 141 and Ulva by Rosenberg and Ramus (1982). The n i t r a t e reductase a c t i v i t y of the G. verrucosa i n t h i s study showed no unusual c h a r a c t e r i s t i c s although i t was l e s s s e n s i t i v e to pH change, and Mg+* and PVP concentrations than other i n t e r t i d a l seaweeds (Appendix 2 ). Transplanting up the i n t e r t i d a l zone l e d to increased n i t r a t e uptake ra t e s and n i t r a t e reductase a c t i v i t y but decreased i n t e r n a l n i t r a t e l e v e l s , suggesting a c c e l e r a t e d n i t r a t e u t i l i z a t i o n . The most l i k e l y explanation i s that p e r i o d i c exposure r e s u l t s i n i n t e r m i t t e n t n u t r i e n t supply and produces a requirement f o r a c c e l e r a t e d nitrogen procurement when the alga i s submerged. Enhancement of uptake a f t e r d e s i c c a t i o n i s a s i m i l a r adaptation. The t r a n s p l a n t experiments i n t h i s study confirmed the hypothesis that enhanced n u t r i e n t uptake f o l l o w i n g d e s i c c a t i o n i s dependent p r i m a r i l y on attachment height i n the i n t e r t i d a l zone. They have a l s o prompted some suggestions on f a c t o r s c o n t r o l l i n g the morphology, physiology and d i s t r i b u t i o n of G. verrucosa i n the i n t e r t i d a l h a b i t a t . Nitrogen uptake and a s s i m i l a t i o n rates are c o n t r o l l e d by complex i n t e r a c t i o n s between c e r t a i n p h y s i c a l f a c t o r s and the p h y s i o l o g i c a l s t a t e of the t h a l l u s . 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 the t h a l l u s , a h i s t o r y of repeated p e r i o d i c d e s i c c a t i o n , the previous degree of d e s i c c a t i o n , and the type and amount of n u t r i e n t present are some of the v a r i a b l e s i n v o l v e d . These f a c t o r s can be c o n t r o l l e d i n la b o r a t o r y c u l t u r e s but any observed responses are of uncer 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 . F i e l d s t u d i e s must be conducted. 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 various f a c t o r s i n r e l a t i o n to a l g a l zonation. o 143 Summary and Conclusions The study of the n u t r i e n t physiology of marine macrophytes i s i n the e a r l y stages of i t s development and lags f a r behind phytoplankton physiology. There are a few i s o l a t e d r e p o r t s of adaptations to low n u t r i e n t s , s i m i l a r to those for phytoplankton, however, p r i o r to t h i s work not even a l i m i t e d survey of species using c o n s i s t e n t experimental techniques has been undertaken. In.previous s t u d i e s on macrophytes, procedures used i n phytoplankton s t u d i e s have been a p p l i e d but i t i s not c l e a r that they have been p r o p e r l y modified to the unique c h a r a c t e r i s t i c s of macrophytes. This study of n u t r i e n t uptake r a t e s , i n t e r n a l l e v e l s of s o l u b l e nitrogen compounds, and n i t r a t e reductase a c t i v i t i e s i n cludes attempts to optimize current methods fo r use with marine macrophytes. I n i t i a l periods of r a p i d ammonium uptake f o l l o w i n g nitrogen s t a r v a t i o n were observed i n the la 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 p l a n t s . This study was the f i r s t to document that t h i s response i s common i n the f i e l d . Consequently, the popular "batch" method of determining ammonium uptake rates may be in a p p r o p r i a t e . The presence of ammonium ofte n i n h i b i t e d n i t r a t e uptake rates but a c e r t a i n degree of nitr o g e n d e f i c i e n c y prevented t h i s i n h i b i t i o n . These f i n d i n g s are of e c o l o g i c a l s i g n i f i c a n c e because they suggest that uptake of various nitrogen substrates can be r a p i d l y a l t e r e d to allow energy conser v a t i o n ; ammonium i s taken up p r e f e r e n t i a l l y i n times of nitrogen s u f f i c i e n c y whereas simultaneous maximum n i t r a t e and ammonium uptake rates are 144 maintained during times of nitrogen d e f i c i e n c y . The e f f e c t of nitrogen supply on nitrogen uptake, s o l u b l e n i t r o g e n content and n i t r a t e reductase a c t i v i t y was i n v e s t i g a t e d both under c o n t r o l l e d l a t o r a t o r y c o n d i t i o n s and f i e l d c o n d i t i o n s . Nitrogen starved t h a l l i maintained r a p i d ammonium, but low n i t r a t e , uptake r a t e s . N i t r a t e uptake rates were increased w i t h i n 10-20 min of exposure to n i t r a t e . N i t r a t e -grown c u l t u r e s a l s o maintained high ammonium uptake r a t e s . I t was p o s t u l a t e d that n i t r a t e reduction was the rate l i m i t i n g step and the supply of a s s i m i l a t e d nitrogen was slower f o r n i t r a t e -grown t h a l l i than f o r ammonium-grown t h a l l i . A product of the a s s i m i l a t e d n i t r o g e n , which was absent i n nitrate-grown and starved p l a n t s , may c o n t r o l ammonium uptake. A b r i e f (<5 min) per i o d of enhanced ammonium uptake rate was present i n the ammonium s u f f i c i e n t p l a n t s . This i s the f i r s t record of such a response. Ammonium uptake appeared to be c o n t r o l l e d by a small i n t e r n a l pool which was depleted during the t r a n s f e r from the c u l t u r e v e s s e l to the uptake medium (0.5 h). The slow r a t e of decrease i n i n t e r n a l n i t r a t e suggests that ammonium-grown t h a l l i took up and a s s i m i l a t e d ammonium rather than using e x i s t i n g i n t e r n a l n i t r a t e . Nitrogen s t a r v a t i o n caused a general decrease i n a l l i n t e r n a l n itrogen l e v e l s and a t r a n s i e n t increase i n n i t r a t e reductase a c t i v i t y . This study was the f i r s t simultaneous e v a l u a t i o n of the e f f e c t of various forms of inorganic nitrogen supply and nitr o g e n s t a r v a t i o n on s e v e r a l aspects of nitr o g e n metabolism. A comparison of nitrogen uptake i n Fucus d i s t i c h u s 145 germlings showed that there were marked d i f f e r e n c e s i n the n u t r i t i o n a l physiology of d i f f e r e n t l i f e h i s t o r y stages. The germlings showed r a p i d n i t r a t e and ammonium uptake r a t e s , high ammonium uptake a f f i n i t y , and no ammonium i n h i b i t i o n of n i t r a t e uptake which suggests that they could out—compete the mature t h a l l i f o r ni t r o g e n . This was the f i r s t study of the n u t r i e n t physiology of an e a r l y l i f e h i s t o r y stage. Growth rates of germlings suggested that m i l d d e s i c c a t i o n may be a requirement f o r optimal growth. This was a l s o confirmed, on the ba s i s of nitrogen uptake for mature plan t s of sev e r a l species of i n t e r t i d a l macrophytes. M i l d 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 when the 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 the plan t had been exposed to p e r i o d i c d e s i c c a t i o n f o r s e v e r a l weeks (a "hardening" e f f e c t ) . The degree of t h i s enhancement, the percent d e s i c c a t i o n producing maximum uptake rates and the tol e r a n c e to higher d e s i c c a t i o n i n terms of nitr o g e n uptake, depended on i n t e r t i ' d a l l o c a t i o n . This response to d e s i c c a t i o n was an i n t r a s p e c i f i c as w e l l as an i n t e r s p e c i f i c adaptation to i n t e r t i d a l l o c a t i o n and could be induced w i t h i n f i v e weeks. This n u t r i e n t uptake response f o l l o w i n g a period of carbon procurement but no e x t e r n a l n i t r o g e n supply (exposure to a i r ) , may be important i n long term C/N homeostasis. The p h y s i o l o g i c a l c o n t r o l of enhanced n u t r i e n t uptake f o l l o w i n g d e s i c c a t i o n i s c l o s e l y r e l a t e d to at l e a s t two 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 past h i s t o r y . This r e v e a l s the complexity of the i n t e r a c t i o n between p h y s i c a l f a c t o r s and metabolic c o n t r o l s . N u t r i t i o n a l s t a t u s , recent 146 degree of d e s i c c a t i o n and the e f f e c t of s e v e r a l weeks of p e r i o d i c d e s i c c a t i o n are a l l involved i n inducing enhanced nitrogen uptake rates i n i n t e r t i d a l seaweeds. The discovery of t h i s unique response to d e s i c c a t i o n which optimizes uptake gives 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 i n an environment of such p h y s i c a l extremes. The r e s u l t s of t h i s study show that 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 to periods 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 p o t e n t i a l uptake and a s s i m i l a t i o n rates 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 nitrogen supply and the c o n t r o l of nitrogen metabolism has been i l l u s t r a t e d at three stages of nitrogen procurement (uptake, accumulation, and a s s i m i l a t i o n ) . This was achieved i n a l a b o r a t o r y study where the n u t r i e n t regimes could be c o n t r o l l e d . The e c o l o g i c a l s i g n i f i c a n c e was t e s t e d i n the f i e l d . The importance of conducting both f i e l d and l a b o r a t o r y s t u d i e s must not be underestimated since conclusions drawn from l a b o r a t o r y c u l t u r e s can often become divorced from the n a t u r a l environment. In a l a b o r a t o r y study an environmental f a c t o r can be manipulated and a s p e c i f i c response observed. On the other hand, there i s no c o n t r o l over numerous environmental f a c t o r s i n a f i e l d study. In many cases simultaneous environmental and p h y s i o l o g i c a l 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 l a b o r a t o r y . Transplant experiments combine some of the advantages of both l a b o r a t o r y and f i e l d s t u d i e s by manipulating one or more environmental f a c t o r s while maintaining f i e l d 147 c o n d i t i o n s . I t i s only with a combination of l a b o r a t o r y and f i e l d s t u d i e s that p h y s i o l o g i c a l adaptations to f i e l d c o n d i t i o n s can be discussed. This study i s the f i r s t comprehensive l a b o r a t o r y and f i e l d study of nitrogen u t i l i z a t i o n i n marine macrophytes. P h y s i o l o g i c a l adaptations i n terms of n i t r o g e n procurement i n response to nitrogen supply and other 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 f i e l d . An equivalent study on carbon metabolism i s needed. Such s t u d i e s have p r a c t i c a l 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 for the production of economically important compounds such as p h y c o c o l l o i d s . The i n t e r a c t i o n between carbon and nitrogen metabolism i s necessary to understand how growth and p h y c o c o l l o i d content can be manipulated to increase p h y c o c o l l o i d production. The study of p h y s i o l o g i c a l ecology r e l a t e s the knowledge of the p h y s i o l o g i c a l mechanisms maintaining growth and species d i s t r i b u t i o n . The i n t e r t i d a l zone i s an environment of p h y s i c a l extremes where maintenance of p h y s i o l o g i c a l processes i s o f t e n s t r e s s e d and numerous l i f e forms cannot s u r v i v e . 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Ryther, J.H., Corwin, N., DeBusk, T.A. and W i l l i a m s , L.D. 1981. Nitrogen uptake and storage by the red alga G r a c i l a r i a  t i k v a h i a e (McLachlan, 1979). Aquaculture 26:107-15. Ryther, J.H. and Dunstan, W.M. 1971. Nitrogen, phosphorus and e u t r o p h i c a t i o n i n the c o a s t a l marine environment. Science 171:1008-13. Schonbeck, M. . And Norton, T.A. 1978. Factors c o n t r o l l i n g the upper l i m i t s of f u c o i d algae on the shore. J . exp. mar . B i o l . E c o l . 31:303-13. Schonbeck, M. and Norton, T.A. 1979a. The e f f e c t s of b r i e f p e r i o d i c submergence on i n t e r t i d a l f u c o i d algae. E s t . Coast . Mar. Sc_i. 8:205-1 1 . Schonbeck, M.W. and Norton, T.A. 1979b. An i n v e s t i g a t i o n of 154 drought avoidance i n i n t e r t i d a l Fucoid algae. Bot. Mar. 22:133-44. Schonbeck, M.W. and Norton, T.A. 1979c. Drought-hardening i n the upper—shore seaweeds Fucus s p i r a l i s and P e l v e t i a  c a n a l i c u l a t a . J . E c o l . 67:687—96. Subbaraju, D.P., Ramakrishna, T., and Sreedhara Murthy, M. 1982. Influence of changes i n s a l i n i t y , pH, and temperature on the spores and s p o r e l i n g s of Padina t e t r a s t r o m a t i c a Hauck. J . exp. mar. B i o l . E c o l . 58:163-73. 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.) Physiology and Biochemistry of Algae, Academic Press, New York. pp, 171-88. 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 a s s i m i l a t i o n by ammonium i n C h l o r e l l a . Biochim. Biophys. Acta 67:566-75. Terry, L.A. and Moss, B.L. 1981. The e f f e c t of i r r a d i a n c e and temperature on the germination of four species of Fucales. Br. Phycol. J . 16:143-51. Thomas, T.E. and Turpin, D.H. 1980. D e s i c c a t i o n enhanced n u t r i e n t uptake rates i n the i n t e r t i d a l alga Fucus d i s t i c h u s . Bot. Mar. 23:479-81. Thorhaug, A. and Marcus, J.H. 1981. The e f f e c t s of temperature and l i g h t on attached forms of t r o p i c a l and s e m i - t r o p i c a l macroalgae p o t e n t i a l l y a s s o c i a t e d with OTEC (Ocean Thermal Energy Conversion Operation). Bot. Mar. 24:393-8. Topinka, J.A. 1978. Nitrogen uptake by Fucus s p i r a l i s (Phaeophyceae). J . Phycol. 14:241-7. Topinka, J.A. and Robbins, J.V. 197*6. E f f e c t s of n i t r a t e and ammonium enrichment on growth and ni t r o g e n physiology i n Fucus s p i r a l i s . Limnol. Oceanoqr. 21:659—64. Turpin, D.H. and H a r r i s o n , P.J. 1979. L i m i t i n g n u t r i e n t patchiness and i t s r o l e i n phytoplankton ecology. J . exp. mar. B i o l . E c o l . 39:151-66. Waite, T. and M i t c h e l l , R. 1972. The e f f e c t of n u t r i e n t f e r t i l i z a t i o n on the benthic a l g a Ulva l a c t u c a . Bot. Mar. 15:151-6. Wheeler, P.A. 1979. Uptake of methylamine (an ammonium analogue) by Macrocystis p y r i f e r a (Phaeophyta). J . Phycol. 15:12-7. Wheeler, P.A., G l i b e r t , P.M., and McCarthy, J . J . 1982. Ammonium uptake and i n c o r p o r a t i o n by Chesapeake Bay phytoplankton: short term uptake k i n e t i c s . Limnol. Oceanoqr. 155 27:1113-28. Wheeler, W.N. 1982. Nitrogen n u t r i t i o n of M a c r o c y s t i s . In S r i v a s t a v a , L.M. (Ed.) Synthetic and Degradative Processes  i n Marine Macrophytes, Walter de Gruyter, New York. 121—37. Weidner, M. and K i e f e r , H. 1981. N i t r a t e reduction i n the marine brown alga G i f f o r d i a m i t c h e l l a e (Harv.) Ham. Z. P f l a n z e n p h y s i o l . 104:341-51. Wiencke, C. and L a u c h l i , A. 1980. Growth, c e l l volume, and f i n e s t r u c t u r e of Porphyra u m b i l i c a l i s i n r e l a t i o n to osmotic t o l e r a n c e . P l a n t a 150:303-11. Yamada, N. 1961. Studies on the manure f o r seaweed, on the change of nitrogenous component of Gel idiurn c u l t u r e d w i t h d i f f e r e n t nitrogen sources. B u l l . Jap. Soc. S c i . F i s h . 27:953-7. Zaneveld, J.S. 1937. The l i t t o r a l zonation of some Fucaceae i n r e l a t i o n to d e s i c c a t i o n . J . E c o l . 25:431-68. 156 Appendix 1. Techniques for Measuring Soluble Nitrogen Content I n t r o d u c t i o n The a b i l i t y of c e r t a i n macrophytes to s t o r e nitrogen may be important f o r t h e i r s u r v i v a l during periods when nitrogen i s d e p l e t e d i n the ambient water. In some cases, seasonal growth patterns of macrophytes appear more dependent on these i n t e r n a l reserves than on e x t e r n a l n u t r i e n t s u p p l i e s (Buggeln 1974; Chapman and C r a i g i e 1977; Hanisak 1979; Rosenberg and Ramus 1982). E a r l i e r s t u d i e s determined the t o t a l n itrogen content of d r i e d seaweeds ( N i e l l 1976; Chaumont 1978) but, i t i s s t i l l not known what percentage of the t o t a l plant n i t r o g e n i s r e a d i l y a v a i l a b l e f o r growth. Recent studies i n d i c a t e that s o l u b l e t i s s u e nitrogen i s r a p i d l y u t i l i z e d during the onset of growth ( B i r d et a l . 1982b; Gerard 1982a; Rosenberg and Ramus 1982). Several procedures f o r e x t r a c t i o n of s o l u b l e nitrogenous m a t e r i a l s from marine macrophytes have been reported but none has found acceptance and i t i s unusual to f i n d reports of 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 . There i s a c r i t i c a l need f o r an e v a l u a t i o n of the common methods and the development of a simple e f f i c i e n t e x t r a c t i o n method that can be used on numerous macroalgal species. This study compares four methods, using hot water, or hot 80% ethanol with or without g r i n d i n g or hot water f o l l o w i n g f r e e z i n g i n l i q u i d nitrogen f o r e x t r a c t i n g n i t r a t e , ammonium, so 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 from three i n t e r t i d a l seaweeds, one from each of three major macroalgal d i v i s i o n s . 157 M a t e r i a l s and Methods Young, non-reproductive Porphyra p e r f o r a t a J . Ag., Enteromorpha i n t e s t i n a l i s (L.)Grev., and Fucus 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 February and March from the i n t e r t i d a l zone west of K i t s i l a n o Beach, Vancouver, B.C., Canada (Chapter 1; F i g . 2). Their heights above Canadian datum were 1.5, 2.0, and 2.0 m, r e s p e c t i v e l y . They were immediately transported to the l a b o r a t o r y where they were washed with f i l t e r e d seawater and b l o t t e d dry. The e n t i r e p l a n t was used, except i n the case of F. d i s t i c h u s where h o l d f a s t s were removed. A l l p l a n t m a t e r i a l was e x t r a c t e d w i t h i n 2 h a f t e r c o l l e c t i o n . Four d i f f e r e n t e x t r a c t i o n s were employed: hot water e x t r a c t i o n while g r i n d i n g , hot ethanol e x t r a c t i o n while g r i n d i n g , hot ethanol e x t r a c t i o n without g r i n d i n g , and f r e e z i n g i n l i q u i d n itrogen followed by g r i n d i n g i n hot water. 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 . E r r o r bars represent ± 1 standard d e v i a t i o n . Trends w i l l be discussed. G r i n d i n g Procedure Plant m a t e r i a l (2 g) was ground with 1 g sand ( p r e v i o u s l y washed and i g n i t e d ) and 5 ml of hot water (approximately 80°C) or hot 80% ethanol (approximately 60°C). I t was necessary to cut the F. d i s t i c h u s i n t o small pieces (approximately 0.5 cm square) f o r g r i n d i n g . A f u r t h e r 20 ml of hot ethanol or water was added i n 5 ml a l i q u o t s and the g r i n d i n g continued u n t i l a homogeneous s l u r r y was produced. The t i s s u e 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 min. The top 19 ml f o r F. 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 top 158 16 ml f o r P. p e r f o r a t a was decanted. The remaining m a t e r i a l was f u r t h e r e x t r a c t e d three times with 25 ml water or ethanol. The supernatant from each e x t r a c t i o n was analysed f o r n i t r a t e , ammonium, t o t a l 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 to be p r i m a r i l y free amino a c i d s ) and p r o t e i n . An attempt to increase c e l l u l a r d i s r u p t i o n was made by f r e e z i n g i n l i q u i d n i trogen p r i o r to g r i n d i n g . The t i s s u e (2 g) became b r i t t l e and i t was easy to g r i n d i n t o a f i n e powder which was e x t r a c t e d with hot water or ethanol as o u t l i n e d above. Pouring the hot ex'traction medium over the frozen t i s s u e immediately dropped the temperature of the e x t r a c t i o n medium to approximately 20°C. E x t r a c t i o n Without Grinding Two grams of d i c e d t h a l l i was placed i n a stoppered Erlenmeyer f l a s k w i t h 25 ml of 80% ethanol. This was heated i n a water bath at 100° C f o r 10 min. The e x t r a c t (22 ml) was then decanted and analysed f o r n i t r a t e , ammonium, ni n h y d r i n p o s i t i v e m a t e r i a l (assumed to be p r i m a r i l y amino a c i d s ) and p r o t e i n . With E. i n t e s t i n a l i s , only 15 ml could be decanted without removing some plan t m a t e r i a l . Ethanol (80%) was added to the f l a s k to b r i n g the volume up to 25 ml and the mixture was b o i l e d again. This procedure was repeated four times. Blanks, which were e x t r a c t i o n s without t h a l l i , 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 analysed immediately f o r n i t r a t e and w i t h a Technicon AutoAnalyzer (Davis et a l . 1973). ammonium Samples 159 d i s s o l v e d i n ethanol were d i l u t e d with 125 ml deionized d i s t i l l e d water to prevent bubble formation i n the cadmium column which was used i n the n i t r a t e a n a l y s i s . Two sets of n i t r a t e and ammonium standards were used, one prepared i n 80% ethanol d i l u t e d f i v e times, and the other i n d i s t i l l e d d e ionized water. A n a l y s i s of T o t a l Ninhydrin P o s i t i v e M a t e r i a l T o t a l s o l u b l 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 was determined by the 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 primary amines. The absorbance at 570 nm was assumed to be p r i m a r i l y due to amino a c i d s and ammonium. Standard 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 water or i n 80% ethanol were run with every s e r i e s of analyses. Standard s o l u t i o n s of ammonium were a l s o run. The ammonium content was p r e v i o u s l y measured with the AutoAnalyzer. The absorbance due to t h i s ammonium was c a l c u l a t e d , from the ammonium standard curve for the n i n h y d r i n r e a c t i o n and t h i s absorbance was substracted from the t o t a l absorbance to give an estimate of the absorbance due to free amino a c i d s . Soluble P r o t e i n A n a l y s i s Soluble p r o t e i n was determined using the procedure of Lowry et a l . as modified by Eggstein and Kreutz (1955), see Leg g e t t - B a i l e y (1967). I t was assumed that the c o l o r e d product was p r i m a r i l y due to the presence of s o l u b l e p r o t e i n . A l l ext r a c t e d s o l u t i o n s were d i l u t e d t e n - f o l d to b r i n g the conc e n t r a t i o n of F o l i n p o s i t i v e m a t e r i a l i n t o the high 160 s e n s i t i v i t y range of the p r o t e i n a n a l y s i s technique. A c a l i b r a t i o n curve was e s t a b l i s h e d with each s e r i e s of analyses using bovine serum albumin as the standard p r o t e i n . A n a l y t i c a l r e s u l t s were expressed on a wet weight b a s i s because the need to g r i n d t i s s u e before e x t r a c t i o n precluded dry weight determinations. Normalization to p a r t i c u l a t e nitrogen i n v o l v e s instrumentation that 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 to develop a method fo r measuring s o l u b l e n i t r o g e n content that could be c a r r i e d out i n most l a b o r a t o r i e s . E x t r a c t i o n Blanks Blanks (without p l a n t s ) were run i n t r i p l i c a t e with each e x t r a c t i o n method. There was no detectable contamination, except f o r ammonium, which was present at l e s s than 1% of the ammmonium e x t r a c t e d with b o i l i n g water and with hot ethanol without g r i n d i n g . 161 Res u l t s and Discussion E x t r a c t i o n 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 apparently discouraged the i n v e s t i g a t i o n of accumulation of nitrogenous compounds. With p r a c t i c e , g r i n d i n g of even a tough t h a l l u s l i k e F. d i s t i c h u s can be r a p i d and e f f e c t i v e . Approximately 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 combination f o r g r i n d i n g . These analyses were done on crude e x t r a c t s which may contain polysaccharides and phenolic compounds which i n t e r f e r e with the ninhydrin and F o l i n r e a c t i o n s . Therefore the r e s u l t s of the 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 are l i k e l y to be non—quantitative ( F i g . 1C,D). L i t t l e i s known about the extent 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 . More work i s req u i r e d on p u r i f y i n g such e x t r a c t s before p r o t e i n and amino a c i d reserves can be expressed 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 to the use of hot e x t r a c t i o n media was not a p p r e c i a b l e . The use of c o l d water or ethanol (e.g., pouring hot medium over a frozen t h a l l u s ) d i d not increase the amount of ammonium e x t r a c t e d . L i q u i d nitrogen made the g r i n d i n g process much e a s i e r , but i t d i d not n o t i c a b l y increase the t o t a l n i t r a t e or ammonium ex t r a c t e d (see treatment 4 i n F i g . 1). Three f a c t o r s were considered when determining the usefulness of various e x t r a c t i o n methods: the t o t a l amount of ammonium, n i t r a t e , amino a c i d s and p r o t e i n e x t r a c t e d i n four successive 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 t o t a l e x t r a c t e d i n four successive 3CM ° S 2 0 H a 1 'ro I S o a> E I * o if) 10H A N i t r a t e 1 2 3 4 1 2 3 4 F u c u s E n t e r o m o r p h a P o r p h y r a 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" 1 and p r o t e i n (D) i n mg.g wet wt" 1 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 p e r f o r a t a with b o i l i n g water while g r i n d i n g ( 1 ) , hot 80% ethanol while g r i n d i n g (2), b o i l i n g i n 80% ethanol without g r i n d i n g ( 3 ) , and f r e e z i n g i n l i q u i d n i t r o g e n , followed by b o i l i n g water while g r i n d i n g (4). T o t a l amount extracted i n four e x t r a c t i o n s { — • — - ) , f i r s t e x t r a c t i o n 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 represent one standard "d e v i a t i o n , n=3. S o l u b l e ••• A m m o n i u m Conten t (jumol N H ^ . g we t w t "1 ) 1 2 c n Icn 1 i ro C O > 3 on im o 3 o I'M. - I 3 i ro C O O ro C O e9i Soluble Amino Acid Con ten t -1 (>jmol A m i n o A c i d s • g w e t w t ) ro o _JL_ T1 n 1/1 mrnmrnm ro > > o to o m _» (D O d o -I Ml ^ 3 | • - j — i ro CO " 0 o "D * 7 / H i i ro O CO O o CP o o Aimol Amino Ac ids • g wet wt"^ *9t . : Soluble Pro te in Content ( m g P r o t e i n • g w e t w t " 1 ) ro O _J_ 8 IS 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 separate e x t r a c t i o n s . There were no observable d i f f e r e n c e s between the four methods with regard to e i t h e r the t o t a l amount of n i t r a t e e x t r a c t e d or the 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). In the case of P. p e r f o r a t a , g r i n d i n g i n hot water was l e s s v a r i a b l e than e i t h e r of the two ethanol e x t r a c t i o n methods. Gri n d i n g i n hot water was the 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 . G r i n d i n g i n hot water e x t r a c t e d greater amounts of ammonium from P. p e r f o r a t a and E. i n t e s t i n a l i s , but ethanol was much more e f f e c t i v e for F. 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. p e r f o r a t a was e x c e l l e n t and clo s e to 100% i n the 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). The v a r i a b i l i t y was a l s o low i n a l l the P. p e r f o r a t a e x t r a c t i o n s , with one standard d e v i a t i o n of approximately ±6%. The r e s u l t s from F. d i s t i c h u s were more v a r i a b l e . More pigments were e x t r a c t e d by ethanol than by b o i l i n g water. The ethanol e x t r a c t s of E. i n t e s t i n a l i s , F. d i s t i c h u s and P. p e r f o r a t a were dark green, brown, and reddish-brown, r e s p e c t i v e l y . These pigments could have a f f e c t e d 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, since ammonium was estimated by absorbance at 600 nm. Water was ther e f o r e the p r e f e r r e d e x t r a c t a n t but even then the absolute values are questionable. This appears to be another case where the 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 with c a u t i o n . One can ex t r a c t at l e a s t three times the amount of n i t r a t e per g wet wt from P. p e r f o r a t a compared to F. d i s t i c h u s or E. i n t e s t i n a l i s . The t o t a l ammonium ex t r a c t e d from P. p e r f o r a t a 167 was s i m i l a r to that from F. d i s t i c h u s , but three times that from E. i n t e s t i n a l i s ( F i g . 1B). P. p e r f o r a t a and E. i n t e s t i n a l i s have s i m i l a r morphologies: they have t h i n t h a l l i and are only a few c e l l l a y e r s i n t h i c k n e s s . F. d i s t i c h u s , on the other hand, i s very t h i c k , tough and rubbery with blades up to 0.5 cm t h i c k . The so l u b l e nitrogen content per g wet wt w i l l be i n f l u e n c e d by the polysaccharide and water content. CHN a n a l y s i s showed that %C based on dry weight for F. d i s t i c h u s i s approximately 40% and v a r i e d from 20-30% f o r E. i n t e s t i n a l i s and P. p e r f o r a t a . * Therefore i t would be u s e f u l to normalize s o l u b l e n i t r o g e n content to t o t a l n i t r o g e n or t o t a l p r o t e i n as w e l l as per gram wet weight when comparing species. The low s o l u b l e n i t r a t e content of E. i n t e s t i n a l i s and F. d i s t i c h u s suggests that these species r e l y on r a p i d n itrogen uptake rather than nitrogen pools f o r the onset of growth. Rosenberg and Ramus (1982) have r e f e r r e d to t h i s c h a r a c t e r i s t i c as an " o p p o r t u n i s t i c growth s t r a t e g y " . In c o n t r a s t , P. p e r f o r a t a , with i t s high s o l u b l e n i t r a t e content was r e f e r r e d to as a " r e s i s t a n t s p ecies". The n i t r a t e content of E. i n t e s t i n a l i s and F. d i s t i c h u s i n t h i s study were s i m i l a r i n magnitude to those found by Rosenberg and Ramus (1982) i n Ulva and G r a c i l a r i a t i k v a h i a e . The r e s u l t s i n the present study were more reproducible than those presented by Rosenberg and Ramus (1982). The c o e f f i c e n t of variance f o r n i t r a t e was ± 5% i n t h i s study and ± 40% f o r measurements made by Rosenberg and Ramus (1982). In c o n t r a s t to both these s t u d i e s , B i r d et a l . (1982b) have reported the n i t r a t e content of G r a c i l a r i a t i k v a h i a e to be one to two orders 168 of magnitude smaller. The t o t a l amount of n i t r a t e e x t r a c t e d from Laminaria  l o n q i c r u r i s by Chapman and C r a i g i e (1977) ranged from 50-150 pmol.g f r e s h wt- 1. I t would appear that the k e l p s , for example Laminaria l o n g i c r u r i s , have a greater a b i l i t y to store n i t r a t e than other seaweeds such as F. d i s t i c h u s , E. i n t e s t i n a l i s and P. p e r f o r a t a . I n v e s t i g a t i n g changes i n s o l u b l e nitrogen content with time or with d i f f e r e n t n u t r i e n t or p h y s i o l o g i c a l s t r e s s e s i n one species could be ve'ry f r u i t f u l . B i r d et a l . (1982b) have r e c e n t l y completed the f i r s t i n v e s t i g a t i o n of t h i s type. Several authors have i n v e s t i g a t e d seasonal f l u c t u a t i o n s i n s o l u b l e nitrogen content i n marine macrophytes (Chapman and C r a i g i e 1977; Chaumont 1978; B i r c h et a l . 1981). The e c o l o g i c a l 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 a b i l i t y to store nitrogen would be a d e f i n i t e competitive advantage during times of n i t r o g e n l i m i t a t i o n . Marine macrophytes oft e n undergo nitrogen l i m i t a t i o n i n the summer (Chapman and C r a i g i e 1977; Hanisak 1979; Gerard 1982a); however, nit r o g e n l i m i t a t i o n need not be only seasonal. N u t r i e n t supply i n i n t e r t i d a l h a b i t a t s i s i n t e r r u p t e d by exposure to a i r on a d a i l y b a s i s . The determination of changes i n i n t e r n a l s o l u b l e nitrogen content under f l u c t u a t i n g nitrogen regimes a i d s i n g a i n i n g an understanding of how i n t e r t i d a l macrophytes compete fo r n i t r o g e n . 169 References B i r d , K.B., Habig, C. and DeBusk, T. 1982. Nitrogen a l l o c a t i o n and storage patterns 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. Nitrogen and phosphorus n u t r i t i o n of Cladophora i n the Peel—Harvey e s t u a r i n e system, Western A u s t r a l i a . Bot. 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. (Laminariales) I . Elongation of the 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 l o n q i c r u r i s : R e l a t i o n s with d i s s o l v e d inorganic n u t r i e n t s and i n t e r n a l reserves of n i t r o g e n . Mar. B i o l . 40:197-205. Chaumont, J.P. 1978. V a r i a t i o n s de l a teneur en composes azotes du Rhodymenia palmata Grev. Bot. Mar. 21:23—9. Davis, CO., H a r r i s o n , P.J., and Dugdale, R.C 1973. Continuous c u l t u r e of marine diatoms under s i l i c a t e l i m i t a t i o n . I . Synchronized l i f e c y c l e of Skeletonema  costatum. J . Phycol. 9: 17 5—8 0. Gerard, 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 nitrogen reserves by the giant kelp Macrocystis p y r i f e r a i n a low—nitrogen environment. Mar. B i o l . 66:27—35. Hanisak, M.D. 1979. Nitrogen l i m i t a t i o n of Codiurn f r a g i l e ssp. * tomentosoides as determined by t i s s u e a n a l y s i s . Mar. B i o l . 50:333-7. Lee, Y.P. and Takahashi, T. 1966. An improved c o l o r i m e t r i c determination of amino a c i d s w i t h n i n h y d r i n . Anal. Biochem. 14:71-7. L e g g e t t - B a i l e y , J . 1967. Miscellaneous a n a l y t i c a l methods : es 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 reagent. Ir\ Techniques  i n P r o t e i n Chemistry 2nd ed. E l s e v i e r Publ. 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 . Bot. 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 the seaweeds G r a c i l a r i a f o l i i f e r a (Rhodophyceae) and Ulva sp. (Chlorophyceae): s o l u b l e 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 Vivo N i t r a t e Reductase A c t i v i t y I n t r o d u c t i o n R e l a t i v e l y l i t t l e i s known about nitrogen metabolism of marine macrophytes. Most st u d i e s have i n v e s t i g a t e d f a c t o r s 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; H a r l i n 1978; Topinka 1978) or seasonal f l u c t u a t i o n s i n nitrogen storage (Chapman and C r a i g i e 1977; Gagne et a l . 1982; Rosenberg and Ramus 1982). Even l e s s research has 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 to be the rate l i m i t i n g step i n the procurement of nitrogen i n higher p l a n t s and phytoplankton (Beevers and Hageman 1969; Dortch 1982). N i t r a t e i s reduced to ammonium before i t i s incorporated i n t o organic compounds. The o x i d a t i o n / r e d u c t i o n s t a t e of the N atom i n n i t r a t e i s +5 and i n ammonium i t i s -3. The sequence suggested i n e a r l i e r work was as f o l l o w s : +5 +3 +1 -1 -3 NO -—>NO "—>N202" 2—>NH20H—>NH + (Nicholas 1959; S y r e t t 1962; K e s s l e r l 9 6 4 ) Work with higher p l a n t s (Beevers and Hageman 1969; Hewitt 1970) and algae ( H a t t o r i and Myers 1966; H a t t o r i and Myers 1967; Zumft et a l . 1969; A p a r i c i o et a l . 1971; Syrett 1962) suggests that only two enzymes c a t a l y s e the reduction sequence. They are, n i t r a t e reductase (NAD(P)H: n i t r a t e oxidoreductase EC 1.6.6.2), which c a t a l y s e s the reduction of n i t r a t e to n i t r i t e , and n i t r i t e reductase (NAD(P)H: n i t r i t e oxidoreductase EC 1.6.6.4) which c a t a l y s e s the reduction of n i t r i t e to ammonium. Methods for assaying these enzymes have been developed (Hewitt 171 and Nicholas 1964). Both i r i v i v o and ijn v i t r o assays f o r n i t r a t e reductase have been used i n higher p l a n t s , but recent s t u d i e s have favored the in v i v o assay ( S t r e e t e r and Bosler 1972). The f i r s t report of n i t r a t e reductase a c t i v i t y i n algae (Schafer et a l . (1961) was from an e x t r a c t of C h l o r e l l a  pyrenoidosa. The reduction required NADH as an e l e c t r o n donor and d i d not occur when NADPH was used. FAD st i m u l a t e d the a c t i v i t y . Other stud i e s w i t h C h l o r e l l a and Scenedesmus found s i m i l a r requirements"for NADH (Syrett and M o r r i s 1963; Osajima and Yamafuji 1964). A l l these e a r l y s t u d i e s were conducted with crude e x t r a c t s . Trebst and Burba (1976) p a r t i a l l y p u r i f i e d n i t r a t e reductase from C h l o r e l l a pyrenoidosa and more p r e c i s e information on the enzyme from C h l o r e l l a came from the work of Losada and h i s colleagues (Zumft et a l . 1969; A p a r i c i o et a l . 1971; Vega et a l . 1971; Cardenas et a l . 1972). Eppley et a l . (1969) determined that the enzyme from the marine diatom Ditylum b r i g h t w e l l i i and s e v e r a l other marine phytoplankton required NADH as an e l e c t r o n donor, was i n a c t i v e w i t h NADPH, was unaffected by the a d d i t i o n of FAD, and was s t i m u l a t e d by MgSO, . Maximum a c t i v i t y a l s o required phosphate and a reduced sulphur compound such as g l u t a t h i o n e or d i t h i o t h r e i t o l . Later Thacker and S y r e t t (1972) measured n i t r a t e reductase a c t i v i t y i n e x t r a c t s from Chlamydomonas  r e i n h a r d i using reduced benzyl viologen as an e l e c t r o n donor. There are very few r e p o r t s of n i t r a t e reductase 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 assay procedure has been developed. A r a k i et a l . (1979) studied a p a r t i a l l y p u r i f i e d 172 enzyme e x t r a c t from Porphyra yezoensis and they determined optimal c o n d i t i o n s f o r an ijn v i t r o assay. D i p i e r r o et a l . ( 1977) compared i in v i t r o and i j i v i v o a c t i v i t i e s i n Petroglossum nicaeense but they d i d not determine optimal c o n d i t i o n s f o r the in v i t r o assay. Haxen and Lewis (1981) reported an i_n v i t r o assay f o r the enzyme from Macrocystis  a n g u s t i f o l i a but a l s o d i d not optimize assay c o n d i t i o n s . Weidner and K i e f e r (1981) measured in v i v o n i t r a t e reductase a c t i v i t y i n G i f f o r d i a m i t c h e l l a e but could not use an in v i t r o assay. This study compares the n i t r a t e reductase a c t i v i t i e s of three species of marine macrophytes, one from each of the three major macroalgal d i v i s i o n s . The optimum c o n d i t i o n s f o r i_n v i v o and in v i t r o assays were determined and the merits of both assays are discussed. 173 M a t e r i a l s and Methods Species Young non—reproductive Porphyra p e r f o r a t a J . Ag., Fucus  d i s t i c h u s L., and Enteromorpha i n t e s t i n a l i s (L.)Grev. were c o l l e c t e d i n February and March from a rocky l a n d f i l l s i t e at the foot of Balsam S t r e e t , Vancouver, B.C., Canada (Chapter 2; F i g . 2). Their heights above Canadian datum were 1.5, 2.0 and 2.0 m, r e s p e c t i v e l y . P l a n t s were c o l l e c t e d , cleaned by gentle brushing, and r i n s e d .in f i l t e r e d seawater (0.45 pm), wrapped i n Saran Wrap, and stored at 8°C u n t i l they were assayed (2-3 h ) . Preincubations In some experiments p l a n t s (10 g) were preincubated i n 12 1 of f i l t e r e d seawater (0.45 yum) enriched with f/2 concentrations of phosphate, trace metals and vitamins ( G u i l l a r d and Ryther 1962) and 30 pM n i t r a t e or 15 pM ammonium. The c u l t u r e s were kept i n a 12°C c o l d room with an i r r a d i a n c e of 150 j j E . n r 2 . s _ 1 on a 12:12 l i g h t : d a r k c y c l e and each c u l t u r e was s t i r r e d c o n tinuously with a magnetic s t i r r i n g bar at 120 rpm. The F. d i s t i c h u s t h a l l i were preincubated on n i t r a t e before being assayed f o r n i t r a t e reductase a c t i v i t y . In V i t r o Assay The n i t r a t e reductase e x t r a c t i o n procedures and r e a c t i o n mixtures given i n the l i t e r a t u r e vary c o n s i d e r a b l y . Both phosphate and T r i s [Tris(hydroxymethyl)aminomenthane HCl] b u f f e r s have been used for e x t r a c t i o n , u s u a l l y with an added s u l f u r compound 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 (Hewitt and 174 Nicholas 1964; Eppley et a l . 1969). Since phosphate often enhances in v i t r o n i t r a t e reductase a c t i v i t y (Eppley et a l . 1969), a phosphate b u f f e r was used i n t h i s study. P o l y v i n y l p y r r o l i d o n e (PVP) was added to the e x t r a c t i o n mixture to bind phenolic compounds that could 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 the reducing agent. Approximately 2 g (wet wt) of plant t i s s u e was ground i n 25 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 mM d i t h i o t h r e i t o l . The homogenate was c e n t r i f u g e d at 2,000 xg for 5 min. The supernatant (crude enzyme e x t r a c t ) was decanted and kept on i c e . One 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 to the f o l l o w i n g : Concentration i n incubation mixture 0.2 ml of 0.1 M KN03 11.1 mM 0.1 ml of 0.18 M MgS04 10.0 mM 0.5 ml of 720 uM NADH 200.0 uM This r e a c t i o n mixture was kept at room temperature for 30 min. The r e a c t i o n was stopped by a d d i t i o n of 5 ml of i c e c o l d 95% ethanol (v/v) and 0.2 ml of 1.0 M z i n c a c e t a t e . The r e s u l t a n t suspension was c e n t r i f u g e d at 2,000 xg for 5 min and decanted. N i t r i t e was determined i n the supernatant by the a d d i t i o n of 1 ml sulphanilamide (0.2% w/v) to each tube followed w i t h i n 2 min by 1 ml N-(1-napthyl)ethylenediamine (NNDD)(0.05% w/v). The absorbance of the s o l u t i o n at 543 nm was measured. The c o n t r o l samples lacked only NADH. Enzyme a c t i v i t y was expressed as umol n i t r i t e produced per gram wet wt of t i s s u e per hour, and umol n i t r i t e produced per gram p r o t e i n see 175 (Le g g e t t - B a i l e y 1967). The e f f e c t of incubation time and e x t r a c t volume on n i t r a t e reductase a c t i v i t y was determined. The f o l l o w i n g assay c o n d i t i o n s were t e s t e d to determine the optimum; pH (7.0—8.8), MgS04 concentrations of 0-30 mM, PVP concentrations of 0-0.6 g.g vet w t - 1 , NADH concentrations of 0—400 yuM, and n i t r a t e c o n c entrations of 0—10 mM. In Vivo Assay * This assay was based upon the procedure of D i p i e r r o et a l . (1977). A piece of t i s s u e (0.5 g) was placed i n 50 ml of 3% n—propanol i n a r t i f i c i a l seawater (Harrison et a l . 1980) c o n t a i n i n g 0.1 ml chloramphenicol (0.5 mg.ml - 1) and 1 ml of 1.0 M KN0 3, p r o v i d i n g an e x t e r n a l n i t r a t e c oncentration of 20 mM. The v i a l s were stoppered and shaken i n the dark on an automatic shaker. N i t r i t e concentrations i n the medium were monitored by removing a 2 ml sample. This sample was then mixed with 1 ml (0.2% w/v) sulphanilamide s o l u t i o n followed by 1 ml NNDD s o l u t i o n (0.05% w/v). The absorbance at 543 nm was measured. Standard s o l u t i o n s of n i t r i t e i n 3% n-propanol i n a r t i f i c i a l seawater were analysed to obta i n a standard curve. The e f f e c t of 0-8% n-propanol on n i t r a t e reductase a c t i v i t y was t e s t e d to determine the optimal n—propanol c o n c e n t r a t i o n . Assays were conducted on P. 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 to determine " a c t u a l " i_n v i v o n i t r a t e reductase a c t i v i t y rather than " p o t e n t i a l " a c t i v i t y . 176 In Vivo and Tn V i t r o A c t i v i t y In v i t r o n i t r a t e reductase assays were done every 0.5 h on P. p e r f o r a t a t h a l l i which were incubating i n the i_n v i v o assay medium. 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. Patterns were often complex and therefore curves were f i t t e d by eye to show general trends. A rectangular hyperbola was a very poor f i t f o r the enzyme k i n e t i c data and therefore the k i n e t i c s curves were f i t t e d by eye. H a l f — s a t u r a t i o n constants (Km) and the standard e r r o r s associated with them were determined from a l i n e a r regression of V/S versus V p l o t s . 177 Re s u l t s 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 reductase gave reproducible r e s u l t s f o r a l l three s p e c i e s . One standard d e v i a t i o n was u s u a l l y l e s s than ± 5% of the measured a c t i v i t y , based on three samples. The t o t a l amount of n i t r i t e produced was p r o p o r t i o n a l to the incubation time ( F i g . 1). There was a l i n e a r r e l a t i o n s h i p between the n i t r a t e reductase a c t i v i t y 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 n i t r i t e product ion were d i f f e r e n t f o r a l l three species. The amount of PVP added during enzyme e x t r a c t i o n a f f e c t e d n i t r i t e production ( F i g . 4; Table 1). More PVP was required to produce maximum n i t r a t e reductase a c t i v i t y i n F. d i s t i c h u s than i n P. p e r f o r a t a . PVP i n h i b i t e d in v i t r o n i t r a t e reductase a c t i v i t y i n E. i n t e s t i n a l i s . The r e l a t i o n s h i p between enzyme a c t i v i t y and NADH and n i t r a t e supply were determined. The h a l f - s a t u r a t i o n constants (Km) for F. d i s t i c h u s , P. p e r f o r a t a and E. i n t e s t i n a l i s were 33.2 ± 23.0, 32.2 ± 8.2 and 66.5 ± 57.0 yuM, r e s p e c t i v e l y ( F i g . 5; Table 1). The d e t e c t i o n of n i t r a t e reductase a c t i v i t y i n F. d i s t i c h u s and P. p e r f o r a t a when no n i t r a t e was added to the assay made the 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 complicated than those of NADH o x i d a t i o n ( F i g . 6 ). Since t h e i r a c t i v i t i e s were approximately one-half maximum, t h i s suggests the presence of endogenous n i t r a t e of approximately 0.5 and 0.03 mM i n F. 50| 75H o z o e 50H 25-B Enzyme Extract Volume (ml) S 25. T3 O a i f : i : / / Time (min) F i g . 1. T o t a l In v i t r o n i t r i t e produced (jumol N0 2".g p r o t e i n " 1 over time (min) T A ) , the r e l a t i o n s h i p between n i t r a t e reductase a c t i v i t y (jumol N0 2".g p r o t e i n " 1 .h" 1 and volume of enzyme e x t r a c t used (ml) (B), f o r 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 p e r f o r a t a (*). Error bars represent one standard d e v i a t i o n , n=3. C D 1 7 9 8 pH F i g . 2. (pmol N0 2 ' p e r f o r a t a represent for • h- 1) i n for pH optimum .g p r o t e i n " 1 ( A) and Enteromorpha" one standard d e v i a t i o n , v i t r o n i t r a t e reductase a c t i v i t y Fucus d i s t i c h u s (•), Porphyra  i n t e s t i n a l i s (•). E r r o r bars n=3. F i g . 3. 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 reductase a c t i v i t y (pmol N0 2 _.g p r o t e i n * 1 . h " 1 ) and the concent r a t i o n of added MgS04 (mM) for 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 represent one standard d e v i a t i o n , n=3. Table 1. Optimal c o n d i t i o n s for 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 standard e r r o r of estimates of Km and maximal a c t i v i t i e s Cumol NO^'.g p r o t e i n " 1 , h " 1 ) for p l a n t s taken from the f i e l d and incubated i n the iri v i y p assay medium are a l s o given. Optimal Conditions for Nitrate Reductase Activity Maximal Activity In Vitro In Vivo umol NO 2-g protein" 1.h - 1 Species PVP MgSO NADH |iM NO; mM n-propanol (Field) Induction PH g-g - 1 mM Sat'n Km ±SE Sat'n Km +SE % time (h) In Vitro 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. Intestinalis 8.0 0.0 1.0 >100 43 27 >2.0 0.25 0.05 5 1 160 5 25 P. perforata 8.5 0.025 1-4 > 75 40 9 >0.05 0.025 0.009 3-4 1 50 60 100 1 1 1 — 1 — 1 ~7~T 0.1 0.2 0.3 0.4 0.5 0.6 PVP (c) • g wet wt"1 ) F i g . 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 reductase a c t i v i t y (pmol N0 2".g p r o t e i n " 1 .h" 1T~and the amount of p o l y v i n y l pyrrolidone(PVP) added during e x t r a c t i o n (g PVP.g wet wt" 1) f o r Fucus d i s t i c h u s (•), Porphyra p e r f o r a t a ( A) and Enteromorpha  i n t e s t i n a l i s C"). Error bars represent one standard d e v i a t i o n , n=3. for E n t e r o m o r p h a r 1 5 0 MOO ^ 5 0 200 N A D H 4 0 0 (JUM ) r e l a t ionship F i g . 5. The a c t i v i t y (pmol NO NADH (uM) i n the perf o r a t a (*), and Enteromorpha represent one standard d e v i a t i o n , n=3 between in v i t r o n i t r a t e reductase - 1.h" 1) and the concen t r a t i o n of 2".g p r o t e i n assay mixture for Fucus d i s t i c h u s (•), Porphyra i n t e s t i n a l i s (•). E r r o r bars f o r E n t e r o m o r p h a I. * f. , • . r — , 2 A 6 8 10 N O - ( m M ) 3 F i g . 6. 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 reductase a c t i v i t y (/umol N0 2".g p r o t e i n ' 1 .Ir 7 1") and n i t r a t e c o n c e n t r a t i o n (mM) i n the assay mixture f o r Fucus d i s t i c h u s (•), Porphyra  p e r f o r a t a ( A) and Enteromorpha i n t e s t i n a l i s (•). E r r o r bars represent one standard d e v i a t i o n , n=3. 185 d i s t i c h u s and P. p e r f o r a t a , r e s p e c t i v e l y ( F i g . 6). The Km values for n i t r a t e reductase a c t i v i t y f o r F. d i s t i c h u s , P. p e r f o r a t a , and E. i n t e s t i n a l i s were 0.5 ± 0.2, 0.03 ± 0.01, and 0.26 ± 0.05 mM, r e s p e c t i v e l y (Table 1). Maximum a c t i v i t i e s under optimum assay and e x t r a c t i o n c o n d i t i o n s v a r i e d g r e a t l y with previous n u t r i e n t supply (Table 1). This was e s p e c i a l l y true f o r F. d i s t i c h u s . Experiments were done i n February and March when n i t r a t e and ammonium concentrations at the 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 3-10 pM r e s p e c t i v e l y . I f the t h a l l i of P. p e r f o r a t a and E. i n t e s t i n a l i s were examined immediately 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 a c t i v i t i e s of 1—2 and 1.0-1.5 /umol N0 2".g wet w f ' . h " 1 , or 50 and 160 /umol N0 2.g p r o t e i n - 1 . h " 1 , r e s p e c t i v e l y . I f the p l a n t s were stored 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. Fr e s h l y c o l l e c t e d F. d i s t i c h u s showed no i n v i t r o or 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 that a n i t r a t e reductase i n h i b i t o r p r o t e i n was present seemed u n l i k e l y because a d d i t i o n of c a s e i n , which should minimize the e f f e c t of the i n h i b i t o r p r o t e i n (Lewis et a l . 1982) d i d not produce detectable n i t r a t e reductase a c t i v i t y . However, when the p l a n t s were incubated i n ammonium-free n i t r a t e enriched seawater, n i t r a t e reductase 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 for three days induced j j i v i t r o n i t r a t e reductase a c t i v i t y i n P. p e r f o r a t a and then a c t i v i t y s t a r t e d to d e c l i n e ( F i g . 7). Preincubation i n ammonium i n h i b i t e d n i t r a t e reductase a c t i v i t y . A 30 yuM n i t r a t e pulse was added to the ammonium c u l t u r e on day 3 and t h i s h a l 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 ( jumol NCr . g P ro te in " 1 , h"1) W O n e w 1 H O O l O O cn rr • iD S t r — 3 • DJ o i •1 t-h 3 -» 01 M - • rr 3- -< n 3 "I 3 tt> H" OJ T J rt rt — i-l i-t (D < 0> DJ CO rt" O rr t-h 3 rt O O < 1 tJ o 0> O 3 3 cn w- i-t ro T3 rr o 3" t-t 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 rt J2 i-« a» CbO 2 l-h Q J - O C Q J rr »-t o OJ rr < a> 3 rr OJ M - 3 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 —i 3* 3 3 It O O O 10 Qj H1-• rr •"I r r > ^ o C • 3 n C (D o 3 I o > ^ M o 3 Q J Q J OJ QJ pj 2: <^ cn O CA) C O • . £ 3 uQ 981 187 the decrease i n n i t r a t e reductase a c t i v i t y . In Vivo Assay The iri v i v o n i t r a t e reductase a c t i v i t y depended on the time the pl a n t had been i n the in v i v o n-propanol medium ( F i g . 8; Table 1). N i t r a t e reductase a c t i v i t y was induced and reached a maximum a f t e r approximately 1 h f o r P. p e r f o r a t a and E. 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. d i s t i c h u s ( F i g . 8; Table 1). The increase v a r i e d from 0.5 times for P. p e r f o r a t a and E. i n t e s t i n a l i s to 4.0 times f o r F. d i s t i c h u s based on the f i r s t uptake measurement. 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 both P. p e r f o r a t a and F. d i s t i c h u s 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 E. i n t e s t i n a l i s ( F i g . 8; Table 1). In v i v o a c t i v i t y was detected i n P. p e r f o r a t a when n i t r a t e was absent from the incubation medium. This suggests that the t h a l l i had i n t e r n a l n i t r a t e reserves. Figure 9 shows a time course of " a c t u a l " (no n i t r a t e added) and " p o t e n t i a l " ( n i t r a t e added) i j i v i v o n i t r a t e reductase a c t i v i t y i n P. p e r f o r a t a . The " p o t e n t i a l " a c t i v i t y reached a maximum at the same time as the " a c t u a l " a c t i v i t y , but the 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% greater ( F i g . 9). I f the medium without n i t r a t e was perturbed with n i t r a t e (at 2.5 h) a f t e r a c t i v i t y peaked, the a c t i v i t y increased almost to the l e v e l of p o t e n t i a l a c t i v i t y at t h i s time ( F i g s . 9,10). The R e l a t i o n s h i p Between I_n Vivo and Tn V i t r o A c t i v i t y In v i t r o and in v i v o a c t i v i t i e s of P. p e r f o r a t a 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 reductase a c t i v i t y (jumol NCT .g p r o t e i n * 1 .h _ TT and the p e r i o d of time (hours) incubated i n the i_n v i v o assay medium; with 0.0%—n--, 0 . 5 % — • — , 1.0%—x--, 2.0%--o--f 3 . 0 % - ~ 4 . 0 % — A — F 5.0% — 6 . 0 % —•— , or 8.0% v n-propanol, f o r Fucus  d i s t i c h u s (A), Porphyra p e r f o r a t a (B), and Enteromorpha  i n t e s t i n a l i s (C). E r r o r bars represent one standard d e v i a t i o n , n=3. 6 8 1 N i t r a t e R e d u c t a s e A c t i v i t y ( t i m o l N O " - a Pr ot e m - 1 • h - 1 ) 2 I-i a> l-l fD cn fD 3 rt O 3 fD cn r r 0> 3 a OJ l-t a Q J (D < OJ r r SI 3 3 II cn 3* i-i ' w-O O i i Q C r r O . <-! fD 1 cn >-•• • <x> ^ 3 3 311-O. i |3 C -cr — OJ r r fD 3 a o <-l 3 h j rr 3 3^ fD i-t fD < I-pj cr o 3 cn 3" r r TJ r i OJ cr fO r r fD fD fD n-n « fD fD 3 OJ 3 cn Q J cn QJ O n n fD OJ Qj C OJ o o r r rt OJ C in OJ fD I-0J OJ n- o 3" r r fD M -< 3 fD a M - T J r r C fD •< 3 n n o O i-f air OJ 3 a 3 o >a M o fD Z 3 CTrt O rt i-i g . OJ cn fD i Q I—' O 3 fD o c in ro CO- I 0D o ro O H r- -0--I 061 ;80-O cn CvJ O z o £ u <40i OJ u "O 1) or 4J -t-j o N O ci deled-3 F i g . 10. reductase 3 1 T i m e 2 ( h o u r s ) The r e l a t i o n s h i p between a c t u a l i n v i v o n i t r a t e a c t i v i t y (umol NO" .g p r o t e i n ~ 1. h771") i n Porphyra  per f o r a t a and the period of time (hours) incubated i n the i n  vivo assay medium. A pulse of 0.2 M KN03 was added a f t e r 2.5h. Error bars represent one standard d e v i a t i o n , n=5. 192 i n the i^ n v i v o , n—propanol medium f o r 0—4 h were very s i m i l a r , both reaching a maximum a f t e r 0.5-1.0 h, and dropping to zero w i t h i n 2.5 h ( F i g . 11). 3 -1 N i t r a t e R e d u c t a s e A c t i v i t y ( / imo l N O " • g P r o t e m " 1 • h ) fD 3 < 0) rt O 3 - 3 3 II O J < O PJ in CO OJ >< 3 a> a c 3 •f I 3-K n 3 DJ »-•• rt fO n a» QJ i-t r t -3 3" fD ft ro I—• OJ pj i-t rt ro OJ a c o rt DJ 3 Co 0) CO rt fD 3" fD DJ Ci rt O i-t t r i-t CO I-l fD n i-t fD CO fD 3 rt O 3 fD CO rt DJ 3 Co PJ i-t a TJ rt fD n < O rt i-h~n 3 rt O 3 n> z; O O 3 cn 3" •a cr fD rt < fD fD 3 o iQ c rt »0 —' co n —' o PJ rt 3 fD a M - H " 3 3 | i - . O >|3 C -r r • OJ tr < rt i M -fD - < O J - H O U) J * 41 £61 194 D i s c u s s i o n In V i t r o Assay The e x t r a c t i o n and in v i t r o assay procedure used i n t h i s study gave a reproducible and e a s i l y detectable measurement of n i t r a t e reductase a c t i v i t y . The a c t i v i t i e s found i n P. p e r f o r a t a and E. i n t e s t i n a l i s were s i m i l a r to those found f o r Macrocystis a n g u s t i f o l i a by Haxen and Lewis (1981), but higher than the n i t r a t e reductase a c t i v i t i e s found i n t h i s study for F. d i s t i c h u s and i n Petroglossum nicaeense (0.24 yumol N0 2".g~ 1.h" 1) by D i p i e r r o et a l . (1977). The a c t i v i t y l e v e l s i n t h i s study cannot be compared to the data of Ar a k i et a l . (1979) because they used a p a r t i a l l y p u r i f i e d enzyme e x t r a c t and normalized a c t i v i t y to the p r o t e i n content of that e x t r a c t ; nor can they be compared to the work of Weidner and K i e f e r (1981) because they supplied t h e i r c u l t u r e s with very high ( m i l l i m o l a r ) c o n centrations of n i t r o g e n . The n i t r a t e reductase l e v e l s found i n t h i s study f o r P. p e r f o r a t a and E. i n t e s t i n a l i s are comparable to those reported from higher p l a n t s (Nicholas et a l . 1976; Duke and Duke 1978; Heuer and Plaut 1978; Mann et a l . 1978). The p l a n t s te s t e d i n t h i s study were growing i n high ambient concentrations of both ammonium and n i t r a t e (4 and 17 pM r e s p e c t i v e l y ) . The n i t r a t e reductase a c t i v i t y w i l l depend both on previous n i t r a t e supply and the 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 1978; Haines and Wheeler 1978; Hanisak and H a r l i n 1978). The ammonium i n h i b i t i o n of n i t r a t e reductase a c t i v i t y i n P. p e r f o r a t a has a l s o been observed i n higher p l a n t s (MacKown et a l . 1982), 195 phytoplankton (Morris and S y r e t t 1965; Hipk i n and S y r e t t 1977), and the brown macroalga 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). F r e s h l y c o l l e c t e d 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. A c t i v i t y could be induced by incubation i n n i t r a t e enriched, ammonium-free seawater. The 4 /uM ammonium present i n the seawater at the c o l l e c t i o n s i t e was apparently s u f f i c i e n t to i n h i b i t n i t r a t e reductase a c t i v i t y although 17 jaM n i t r a t e was a l s o present. The biochemical mechanism responsible f o r t h i s i n d u c t i o n and repression or i n h i b i t i o n r e quires f u r t h e r study. F. d i s t i c h u s seemed more s e n s i t i v e to ammonium i n h i b i t i o n of n i t r a t e reduction than P. p e r f o r a t a and E. i n t e s t i n a l i s . Ammonium i n h i b i t e d n i t r a t e uptake by 50% i n E. 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 uptake i n F. d i s t i c h u s and P. p e r f o r a t a (Chapters 1 and 2). The r e s u l t s of t h i s study show that the optimum assay c o n d i t i o n s were ' d i f f e r e n t f o r each species t e s t e d (Table 1). The jjn v i t r o pH optima f o r E. i n t e s t i n a l i s and P. p e r f o r a t a were very d i f f e r e n t . When the pH optimum f o r n i t r a t e reductase a c t i v i t y i n P. p e r f o r a t a was used i n the E. i n t e s t i n a l i s assay or v i c e v e r s a , a c t i v i t y was reduced by as much as 50%. The optimum pH found f o r P. p e r f o r a t a i n t h i s study was a l s o the optimum f o r P. yezoensis (Araki et a l . 1979). F. 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 unless PVP was added and r e q u i r e d much more PVP to obtain maximum a c t i v i t y than the e x t r a c t s from P. p e r f o r a t a . On the other hand the E. i n t e s t i n a l i s enzyme was i n h i b i t e d , by PVP. PVP binds phenolic substances and Fucus has been reported to contain l a r g e amounts 196 of these compounds (Ragan 1981). The three species had appreciable i n v i t r o n i t r a t e reductase a c t i v i t y without the a d d i t i o n of MgS04 . The Mg + + requirement of n i t r a t e reductase i s w e l l documented (Eppley et a l . 1969). I t i s p o s s i b l e that the a c t i v i t y detected arose from use of i n t e r n a l Mg + + s u p p l i e s . N i t r a t e reductase a c t i v i t y of P. p e r f o r a t a was severely i n h i b i t e d when 10 mM of MgS04 was added. This was the optimum concen t r a t i o n f o r the assay of the F. d i s t i c h u s enzyme. This might be explained i n two ways: f i r s t l y , n i t r a t e reductase a c t i v i t y i n P. p e r f o r a t a had a greater a f f i n i t y f o r Mg + + and was more s e n s i t i v e to excess Mg + +; or secondly, P. p e r f o r a t a had greater i n t e r n a l reserves of Mg + +. The amount of Mg + + added to the in v i t r o assay was 500 ^ug.g dry wt" 1. 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 v a r i e s 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 to i n f l u e n c e n i t r a t e reductase a c t i v i t y . The NADH h a l f - s a t u r a t i o n constants f o r a l l three species t e s t e d i n t h i s study were s i m i l a r to those found i n other algae (Eppley et a l . 1969; Ar a k i et a l . 1979). The h a l f - s a t u r a t i o n constants f o r n i t r a t e obtained for F. d i s t i c h u s and E. i n t e s t i n a l i s were s i m i l a r to those reported fo r Porphyra yezoensis (Araki et a l . 1979), phytoplankton (Eppley et a l . 1969), and higher p l a n t s (Beevers and Hageman 1969). The h a l f - s a t u r a t i o n constant f o r P. p e r f o r a t a was one order of magnitude lower. Why t h i s species has n i t r a t e reductase with such a high a f f i n i t y f o r n i t r a t e i s unknown. However, p r e l i m i n a r y s t u d i e s (not reported here) have shown that 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 high Ks value. There are s e v e r a l reports of marine macrophytes s t o r i n g n i t r a t e (Chapman and C r a i g i e 1977; Gerard 1982a; Rosenberg and Ramus 1982) I t was not s u r p r i s i n g t herefore to f i n d i n v i v o and i n v i t r o n i t r a t e reductase a c t i v i t i e s i n the absence of added n i t r a t e . The n i t r a t e content of F. d i s t i c h u s , P. p e r f o r a t a and E. i n t e s t i n a l i s i n March were approximately 7.5, 30.0 and 4.0 .umol N0 2".g wet wt" 1, r e s p e c t i v e l y (Appendix 1). The s i z e of the 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 was 0.3 umol N0 2".g wet wt" 1 f o r P. p e r f o r a t a and 10 pmol N0 2".g wet wt" 1 for F. d i s t i c h u s . Only a small f r a c t i o n of the n i t r a t e content of P. p e r f o r a t a appears to be a v a i l a b l e f o r n i t r a t e reduction a f t e r the t h a l l u s has been ground fo r the i n v i t r o assay. I f a l l i n t e r n a l n i t r a t e was a v a i l a b l e , n i t r a t e reductase would be saturated with n i t r a t e and a d d i t i o n s of n i t r a t e would not enhance a c t i v i t y . This was not the case for the in v i t r o or j j i v i v o assay. In Vivo Assay Some workers ( D i p i e r r o et a l . 1977; S t r e e t e r and Bosher 1972) have recommended the rn v i v o enzyme assay because i t preserves a l l systems i n the n a t u r a l s t a t e , and gives a more r e a l i s t i c e s t i m a t i o n of a c t i v i t y . The ir\ v i v o time course data from t h i s study i l l u s t r a t e s a major drawback: a c t i v i t y v a r i e d w i t h time. The i_n v i v o assay c o n d i t i o n s (an n-propanol medium) increased both _in_ v i v o and iri v i t r o n i t r a t e reductase a c t i v i t y . This enhancement was probably due to high concentrations of n i t r a t e and n—propanol, which enhanced d i f f u s i o n of n i t r a t e i n t o 198 the t i s s u e . The p e r i o d of time r e q u i r e d to produce maximum enhancement v a r i e d with the species. A p l a u s i b l e explanation f o r t h i s i s that 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 at d i f f e r e n t r ates with each species. The type and t h i c k n e s s of the t h a l l u s may be very important. F. d i s t i c h u s i s a t h i c k rubbery t h a l l u s , whereas P. p e r f o r a t a and E. i n t e s t i n a l i s are very t h i n blades. Maximum iri v i v o a c t i v i t y was detected i n F. d i s t i c h u s a f t e r 3 h of incubation i n the i j i v i v o medium; the other two species took l e s s than h a l f t h i s time. In v i v o a c t i v i t y was determined from measurement of the r a t e i n which n i t r i t e was released i n t o the medium. This release of n i t r i t e was l i m i t e d not only by n i t r a t e reductase a c t i v i t y but a l s o by the inward d i f f u s i o n of n i t r a t e and the outward d i f f u s i o n of n i t r i t e . The presence of n—propanol a f f e c t e d these d i f f u s i o n r a t e s ( D i p i e r r o et a l . 1977); but at higher c o n c e n t r a t i o n s , i t i n t e r f e r r e d w i t h c e l l processes, i n c l u d i n g n i t r a t e r e d u c t i o n . The optimum balance between these two e f f e c t s was species dependent. E. i n t e s t i n a l i s appeared l e s s susceptable to n—propanol damage. An a d d i t i o n of 3—4% n-propanol maximized a c t i v i t y f o r the other two s p e c i e s . D i p i e r r o et a l . (1977) reported 1% as the p r e f e r a b l e n-propanol c o n c e n t r a t i o n based on t i s s u e bleaching with higher c o n c e n t r a t i o n s , but they gave no i n d i c a t i o n that t h i s was the c o n c e n t r a t i o n producing maximum a c t i v i t y . In v i v o n i t r i t e production i n the absence of added n i t r a t e suggests that there were i n t e r n a l n i t r a t e s u p p l i e s . The 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 " a c t u a l " (no n i t r a t e added) i n v i v o a c t i v i t y has been used as an i n d i c a t i o n 199 of n i t r a t e pool s i z e i n higher p l a n t s ( B r e t e l e r et a l . 1979). The observation of increased a c t i v i t y f o l l o w i n g a n i t r a t e p e r t u r b a t i o n to the assay medium l a c k i n g n i t r a t e (Fig.11) suggests that the amount of n i t r a t e reductase enzyme was the same f o r both assays. However, the " a c t u a l " a c t i v i t y was l i m i t e d by n i t r a t e supply. On the other hand, t h i s incubation i n n itrogen f r e e _iri v i v o medium increased the a c t u a l n i t r a t e reductase a c t i v i t y . I f a c t u a l n i t r a t e reductase a c t i v i t y was l i m i t e d by n i t r a t e supply then incubation i n the in v i v o medium m of n—propanol and nitrogen free seawater must increase the n i t r a t e supply to the n i t r a t e reductase enzyme. N—propanol increases membrane p e r m e a b i l i t y which may cause leakage of i n t r a c e l l u l a r n i t r a t e pools not normally a v a i l a b l e to the n i t r a t e reductase enzyme. The j j i v i t r o assay r e s u l t s f o r P. p e r f o r a t a suggest that only a small 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 pool was a v a i l a b l e f o r r e d u c t i o n . ^ In s p i t e of the d i f f u s i o n problems inherent i n the jLn v i v o assay 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 assay, i t was encouraging to f i n d s i m i l a r a c t i v i t i e s f o r P. p e r f o r a t a using both assays ( F i g . 11). Both assays appeared to be a reasonably accurate measure of the n i t r a t e reductase 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 the p l a n t s are submerged i n the in v i v o n—propanol medium. An accurate assay f o r n i t r a t e reductase a c t i v i t y i s needed fo r the understanding of environmental e f f e c t s on n i t r o g e n uptake and a s s i m i l a t i o n . Marine macrophytes can store 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, the 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 equal t o the amount a s s i m i l a t e d . A d i r e c t measurement of n i t r a t e reductase a c t i v i t y i s necessary to determine the rate of n i t r a t e a s s i m i l a t i o n . The measurement of many p h y s i o l o g i c a l processes i n marine macrophytes i s subject to l a r g e i n t e r - p l a n t v a r i a b i l i t y . N i t r a t e reductase a c t i v i t y i s no exception; one standard d e v i a t i o n was ± 5—15% of the measured a c t i v i t y f o r the in v i t r o assay and ± 20-30% of the measured a c t i v i t y f o r the iri v i v o assay. N i t r a t e reductase a c t i v i t y i n higher p l a n t s can a l s o be v a r i a b l e (Deane-Drummond and Clarkson 1979; Lewis et a l . 1982). This study i s the f i r s t d i r e c t comparison of n i t r a t e reductase a c t i v i t y i n i n t e r t i d a l macrophytes. The r e s u l t s show the need to 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 spe c i e s . The usefulness of the ir 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 the incubation medium on n i t r a t e reductase a c t i v i t y . A c t i v i t y can f l u c t u a t e d r a s t i c a l l y with time. The in v i t r o assay, on the other hand, i s constant with time and can give an e s t i m a t i o n 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 the f i e l d . 201 References A r a k i , S., Ikawa, T., Oohusa, T., and Nisizawa, K. 1979. Some enzymic p r o p e r t i e s of n i t r a t e reductase from Porphyra  yezoensis Ueda f. Narawaensis Miura. B u l l . Jap. Soc. S c i . F i s h . 45:919-24. A p a r i c i o , P.J., Cardenas, J . , Zumft, W.G., Vega, J.M., Herrera, J . , Paneque, A. and Losada, M. 1971. Molybdenum and i r o n as c o n s t i t u e n t s of the enzymes of the n i t r a t e reducing system from C h l o r e l l a . Phytochemistry 10:1487-95. Beevers, L. and Hageman, R.H. 1969. N i t r a t e reduction i n higher p l a n t s . Ann. Rev. Pla n t P h y s i o l . 20:495-522. B r e t e l e r , H., Hanisch Ten Cate, C.H., and Nissen, P. 1979. Time—course of n i t r a t e uptake and n i t r a t e reductase a c t i v i t y i n n i trogen depleted dwarf beans. P h y s i o l • P l a n t . 47:49-55. Cardenas, J . , Rivas, J . , Paneque, A., and Losada, M. 1972. E f f e c t of i r o n supply on the a c t i v i t i e s of the n i t r a t e — r e d u c i n g system from C h l o r e l l a . Arch. M i k r o b i o l . 81:260-3. Chapman, A.R.O. and C r a i g i e , J.S. 1977. Seasonal growth i n Laminaria l o n g i c r u r i s : R e l a t i o n s with d i s s o l v e d inorganic n u t r i e n t s and i n t e r n a l reserves of n i t r o g e n . Mar. B i o l . 40:197-205. D i p i e r r o , S., Perrone, C. and F e l i c i n i , A.P. 1977. In v i t r o n i t r a t e reductase assay i n Petroglossum nicaeense (Duby) Schotter (Rhodophyta, Phyllophoraceae"57 Phycologia 16:179-82. Dortch, Q. 1982. E f f e c t of growth c o n d i t i o n s on accumulation of i n t e r n a l n i t r a t e , ammonium, amino a c i d s , and p r o t e i n i n three marine diatoms. J . exp. mar. B i o l . E c o l . 61:243-64. Duke, S.H. and Duke, S.O. 1978. Iji v i t r o n i t r a t e reductase a c t i v i t y and iri v i v o phytochrome measurements of maize seedlings as a f f e c t e d by var i o u s l i g h t treatments. Plant  C e l l P h y s i o l . 19:481-9. Eppley, R.W., Coatsworth, J.L., and Solorzano, L. 1969. Studies of n i t r a t e reductase i n marine phytoplankton. Limnol. Oceanogr. 14:194-205. Gagnl, J.A., Mann, K.H. and Chapman, A.R.O. 1982. Seasonal p a t t e r n s of growth and storage i n Laminaria l o n g i c r u r i s i n r e l a t i o n to d i f f e r i n g patterns of a v a i l a b i l i t y of nitrogen i n the water. Mar. B i o l . 69:91-101. G u i l l a r d , R.R.L. and Ryther, J.H. 1962. Studies on marine p l a n k t o n i c diatoms. I . C y c l o t e l l a nana (Hustedt) and 202 Detonula confervaceae (Cleve) Gran. Can. J . M i c r o b i o l . 8:229-39. Hanisak, M.D. and H a r l i n , M.M. 1978. Uptake of inorganic n i t r o g e n by Codiurn f r a g i l e subsp. tomentosoides (Chlorophyta). J . Phycol. 14:450-4. H a r l i n , M.M. 1978. N i t r a t e uptake by Enteromorpha spp. (Chlorophyceae): a p p l i c a t i o n s to aquaculture systems. Aquaculture 15:373-6. H a r r i s o n , P.J., Waters, R.E., and Tay l o r , F.J.R. 1980. A broad spectrum a r t i f i c i a l seawater medium f o r c o a s t a l and open ocean phytoplankton. J . Phycol. 16:28-35. H a t t o r i , A. and Myers, J . 1966. Reduction of n i t r a t e and n i t r i t e by s u b c e l l u l a r preparations of Anabaena c y l i n d r i c a . I . Reduction of n i t r i t e to ammonia. Plant P h y s i o l . 41:1031-6. H a t t o r i , A. and Myers, J . 1967. Reduction of n i t r a t e and n i t r i t e by s u b c e l l u l a r preparations of Anabaena c y l i n d r i c a . I I . Reduction of n i t r a t e t o n i t r i t e . . P lant C e l l P h y s i o l . 8:327-37. Haxen, P.G. and Lewis, O.A.M. 1981. N i t r a t e a s s i m i l a t i o n i n the marine kelp, Macrocystis a n g u s t i f o l i a (Phaeophyceae). Bot. Mar. 24:631-5. Heuer, B. and P l a u t , Z. 1978. Reassessment of the in v i y o assay f o r n i t r a t e reductase i n leaves. P h y s i o l . P l a n t . 43:306-12. Hewitt, E.J. 1970. P h y s i o l o g i c a l and biochemical f a c t o r s 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 n i t r o g e n s u p p l i e s by p l a n t s . In K i r k b y , E.A. (Ed.) Nitrogen N u t r i t i o n of the  P l a n t , Univ. Leeds. Leeds. 78-103. Hewitt, E.J. and Ni c h o l a s , D.J.D. 1964. Enzymes of inorganic n i t r o g e n metabolism. Modern Methods of Plant A n a l y s i s 7:67-172. K e s s l e r , 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 . Ann. Rev. Plant P h y s i o l . 15:57-72. L e g g e t t - B a i l e y , J . 1967. Miscellaneous a n a l y t i c a l methods : est 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 reagent. I_n Techniques  i n P r o t e i n Chemistry 2nd ed. E l s e v i e r Publ. Co., New York. 340-2. Lewis, O.A.M., James, D.M., and Hewitt, E.J. 1982. Nitrogen a s s i m i l a t i o n i n barley ( Hordeum vulgare L. Cv. Mazurka ) i n response to n i t r a t e and ammonium n u t r i t i o n . Ann. Bot. 49:39-49. N i c h o l a s , D.J.D. 1959. Metallo-enzymes i n n i t r a t e 203 micro—organisms. Symp. Soc. Exp. B i o l . 13:1—13. N i c h o l a s , J.C., Harper, J.E., and Hageman, R.H. 1976. N i t r a t e reductase a c t i v i t y i n soybeans ( Gl y c i n e max (L.) Merr.) I . E f f e c t s of l i g h t and temperature. Plant P h y s i o l . 58:731-5. Osajima, Y. and Yamafuji, K. 1964. Reduction of n i t r a t e to ammonium by enzymes i s o l a t e d from green algae. Enzymologia 27:129-140. 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 Ulva sp. (Chlorophyceae): s o l u b l e nitrogen and reserve carbohydrates. Mar. B i o l . 66:251-9. Schafer, 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 reductase. Amer. J . Bot. 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 assays for n i t r a t e reductase i n soybean leaves. P l a n t P h y s i o l . 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.) Physiology and Biochemistry of Algae, Academic Press, New York. pp w 171-88. 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 a s s i m i l a t i o n by ammonium i n C h l o r e l l a . Biochim. Biophys. Acta 67:566-75. Thacker, A. and S y r e t t , P.J. -1972. The a s s i m i l a t i o n of n i t r a t e and ammonium by Chlamydomonas r e i n h a r d i . New P h y t o l . 71:423-33. Topinka, J.A. 1978. Nitrogen uptake by Fucus s p i r a l i s (Phaeophyceae). J . Phycol. 14:241-7. Trebst, A. and Burba, M. 1967. Uber d i e lemmung photosynthetischer reaktionen 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 i n C h l o r e l l a durch d i s a l i c y l i d e n propandiamin. Z. P f l a n z e n p h y s i o l . 57:419-33. Vega, J.M., Herrera, J . , A p a r i c i o , P.J., Paneque, A. and Losada, M. 1971. Role of molybdenum i n n i t r a t e reduction by C h l o r e l l a . P l a n t P h y s i o l . 48:294-9. Weidner, M. and K i e f e r , H. 1981. N i t r a t e reduction i n the marine brown alg a G i f f o r d i a m i t c h e l l a e (Harv.) Ham. 2. P f l a n z e n p h y s i o l . 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 reduction i n C h l o r e l l a . Biochem. Biophys. Res. Commun. 36:980-6. 204 Appendix 3. Soluble Nitrogen Content of Porphyra p e r f o r a t a P. 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 analysed 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 subject to i n t e r f e r e n c e from contaminants such as polysaccharides and phenolic compounds (Appendix 1). More work i s required on the extent of t h i s i n t e r f e r e n c e . Therefore, the r e s u l t s of such analyses are not q u a n t i t a t i v e and are presented here to show general increases or .decreases i n these nitrogen compounds. The ammonium content of a l l p l a n t s decreased by approximately 50% during the f i r s t two days ( F i g . 1). A f t e r day 2 the ammonium and ammonium plus n i t r a t e grown p l a n t s showed a marked increase i n ammonium content. There was high v a r i a b i l i t y i n the measurement of the i n t e r n a l l e v e l s of ni n h y d r i n p o s i t i v e m a t e r i a l (assumed to be p r i m a r i l y amino acids) ( F i g . 2). There was an i n i t i a l increase i n amino a c i d s content i n a l l p l a n t s except for those that had been ni t r o g e n starved ( F i g . 2 ) . The amino a c i d content of the starved p l a n t s decreased very s l o w l y , e x h i b i t i n g an observable change only a f t e r eight days. The i n t e r n a l l e v e l s of s o l u b l e p r o t e i n were r e l a t i v e l y constant and s i m i l a r i n magnitude f o r a l l treatments ( F i g . 3). There was a n o t i c a b l e 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. A m m o n i u m Content (jurnol NH _1 g w e t w t ) 3 in OJ II rt 3 fD M -CAJ OJ 3 i-t uo • i-1 o i-h • < 3 O 03 t-" rt -» Qj C OJ • 3 rt DJ r-AV rt •-3 3-i i • QJ 3 fD fD O OJIV> H 3 O rt 3 r| H r| 3 o c 3 O M - 3 3 rt rt fD fD •-t CL n CT OJ o OJ rt 3 rt fD i-ti rt 01 O fD rt 3 i-f Xi rt N . fD I—1 T> C ? ^ >-l in fD - 3 in o O fD OJ t—1 < 3 3 in rt 3 Co Z O OJ X O 3 <^ 3 r " in • • fD C 3 o * in 3 fD 0) -rt rt OJ 3 a QJ rt O Qj l-t fD < 3 OJ rt rt rt O O uQ 3 fD - 3 3 < Sk H - rt rt rt -pj ^ rt fD O O rt K rt OJ in "5 < fD CL 902 I 1 — 1 — • 1 I— 2 T i m e 4 ( c l ays ) 6 Q F i g . 2. Soluble amino a c i d content (>umol amino aci d s . g wet wt" 1) of Porphyra p e r f o r a t a preconditioned f o r 0-10 days on ro n i t r a t e (---•—), ammonium (-—A—J , n i t r a t e plus ammonium §> (—a—), or nitrogen starved ( — • — ) . E r r o r bars represent one standard d e v i a t i o n , n=3. F i g . 3. Soluble Porphyra p e r f o r a t a (—• ) , ammonium nitrogen starved ('. d e v i a t i o n , n=3. l i m e ( d a y s ) p r o t e i n content (mg p r o t e i n . g wet w t - 1 ) of preconditioned for 0-10 days on n i t r a t e (..-.A---), n i t r a t e plus ammonium (—x ) or — • — ) . E r r or bars represent one standard to o -J 

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