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Some interrelations of photosynthesis and photorespiration among species Downton, William John Sherwin 1969

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S O B S  SOME INTERRELATIONS OP PHOTOSYNTHESIS AND PHOTORESPIRATION AMONG SPECIES by WILLIAM JOHN SHERWIN DOWNTON B.Sc,  University of B r i t i s h Columbia, 1 9 6 5  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of Botany  We accept t h i s thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA July , 1 9 6 9  In p r e s e n t i n g an  this  thesis  in partial  advanced degree a t the U n i v e r s i t y  the  Library  I further for  shall  make i t f r e e l y  agree that  permission  f u l f i l m e n t of the requirements f o r of B r i t i s h  available  Columbia,  I agree  that  f o r r e f e r e n c e and S t u d y .  f o rextensive  copying of this  thesis  s c h o l a r l y p u r p o s e s may be g r a n t e d b y t h e Head o f my D e p a r t m e n t o r  by  h i s representatives.  of  this  written  thes.is f o r f i n a n c i a l  gain  permission.  Department o f  Botany  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, Canada  Date  It i s understood  J u l y 17,. 1969.  Columbia  shall  that  copying or p u b l i c a t i o n  n o t be a l l o w e d w i t h o u t  my  ii ABSTRACT Photosynthesis,  photorespiration and differences i n t h e i r  interactions among species were studied. . In the f i r s t of two parts of the investigation, the CO2 compensation concentration of members of the Gramineae and other plants was determined with an infrared COg analyzer.  In some cases the I n i t i a l prod-  ucts of ^ C 0 2 f i x a t i o n and l e a f anatomy were a l s o examined. In plants with low compensation values (lacking photorespiration) •the i n i t i a l products of photosynthesis by the Cjj-dlcarboxyllo a c i d pathway.  were formed  High compensation plants  (with photorespiration) produced compounds t y p i c a l of the Calvin cycle.  The l e a f veins of low compensation species were sur-  rounded by a s p e c i a l i z e d parenchyma bundle sheath containing a high concentration of chloroplasts with large quantities of starch.  Low  compensation members of the Gramineae belonged  to the a r l s t i d o i d , chloridoid-eragrostoid and panicoid l i n e s of evolution.  Of the genera confined to these phylogenetic  l i n e s , Panlcum was  found to d i f f e r i n photosynthetlc  and bundle sheath c h a r a c t e r i s t i c s at the subgeneric  physiology level.  This  type of photosynthetlc d i v e r s i t y also occurred within Cyperus (Cyperaceae), A t r l p l e x and Bassla (Chenopodiaceae).  Despite  these differences, the c o r r e l a t i o n between type of photosynt h e t l c pathway, compensation value and l e a f anatomy was  con-  sistent. The l i t e r a t u r e Indicates that low compensation grasses have photosynthetlc  rates that are about double those of plants  with photorespiration correlated with a temperature optimum f o r  ill photosynthesis  of about 3 5 ° .  Species with photoresplratlon  have optima within the range 1 0 - 2 5 ° . Some simple assay procedures proposed on the basis of these correlations allow rapid determination biochemical  of the physiological and  status of plants with respect to photosynthesis.  In the second part of the investigation, some published studies of photoresplratlon and glycolate oxidation were reexamined and correlated by infrared CO2 a n a l y s i s . Photosynthetic  rate data a t d i f f e r e n t O2 tensions f o r  wheat, oat and corn seedlings fed 3 - ( 3 , 4 - d i c h l o r o p h e n y l ) - l , 1 dimethyl urea (DCMU) indicated that dark r e s p i r a t i o n continued i n the l i g h t when photosynthesis  was completely i n h i b i t e d .  Phptorespiration was a l s o Inhibited.  The 0 3 s e n s i t i v i t y of  glycolate-stimulated C0£ production was found to be compatible with the proposal that glycolate i s a substrate of photoresplratlon.  Both ' i n vivo' and ' i n v i t r o ' studies of the alga  N l t e l l a f l e x l l l s revealed a pathway of glycolate oxidation resembling that of higher plants.  DCMU i n h i b i t i o n of photosyn-  thesis by N l t e l l a gave r e s u l t s s i m i l a r to those f o r the monocotyledons  tested.  Under very low l i g h t i n t e n s i t y , CO2 compensation i n corn was measurable but was not sensitive to high O2 concentration. I t appears that the lack of photoresplratlon i n t h i s plant i s not the end r e s u l t of e f f i c i e n t i n t e r n a l r e c y c l i n g of C 0 2 to photosynthesis.  iv  TABLE OP CONTENTS Page P a r t 1-A.  Carbon D i o x i d e Compensation - I t s R e l a t i o n t o P h o t o s y n t h e t l c C a r b o x y l a t i o n R e a c t i o n s , Systema t i c a o f the Gramineae, and L e a f Anatomy  INTRODUCTION  1  MATERIALS  3  METHODS  4  Measurement of Carbon D i o x i d e Compensation Anatomical S t u d i e s RESULTS AND DISCUSSION Carbon D i o x i d e Compensation and P h o t o s y n t h e t l c C a r b o x y l a t i o n Reactions . Carbon D i o x i d e Compensation and P h o t o s y n t h e t l c Rates... Carbon D i o x i d e Compensation and Systematics of the Gramineae Carbon D i o x i d e Compensation i n R e l a t i o n t o L e a f Anatomy and S t a r c h D i s t r i b u t i o n LITERATURE CITED P a r t 1-B.  4 5 5 5 9 11 18 25  I n t e r s p e c i f i c D i f f e r e n c e s i n Carbon D i o x i d e Compensation and the Path o f Carbon i n Photos y n t h e s i s Among Non-graminaceous Genera  INTRODUCTION  29  MATERIALS AND METHODS  29  RESULTS AND DISCUSSION  31  LITERATURE CITED  36  APPENDIX 1  38  S t a n d a r d i z a t i o n of Chromatography P a r t 1-C.  38  P h o t o s y n t h e s i s : Temperate and T r o p i c a l Charact e r i s t i c s W i t h i n a S i n g l e Grass Genus  INTRODUCTION  42  V  TABLE OF CONTENTS, cont'd. Page MATERIALS AND METHODS  43  RESULTS  44  Subgenus Eupanlcum  44  Subgenus D l c h a n t h e l i u m  44  DISCUSSION  45  LITERATURE CITED  4?  APPENDIX-: 1 Part 2 .  49  P h o t o r e s p i r a t i o n and G l y c o l a t e Metabolism: A Reexamination and C o r r e l a t i o n o f Some Previous Studies  INTRODUCTION  50  MATERIALS  51  METHODS  52  DCMU Feedings G l y c o l a t e Feedings Enzyme Assay f o r N l t e l l a Oxygen S e n s i t i v i t y of the Compensation Value Under Low L i g h t I n t e n s i t y RESULTS AND DISCUSSION The The  E f f e c t o f DCMU on Carbon Dioxide P r o d u c t i o n Oxygen S e n s i t i v i t y o f G l y c o l a t e O x i d a t i o n i n Relation to Photorespiration Oxygen S e n s i t i v i t y of the Carbon D i o x i d e Compensation Value a t Low L i g h t I n t e n s i t y  LITERATURE CITED  52 53 5^ 55 55 55 59 67 71  Part 3 . CONCLUSIONS  75  vi  LIST OP TABLES Page P a r t 1-A Table  Table Table Table  I.  II. III. IV.  Carbon d i o x i d e compensation i n r e l a t i o n to l a b e l l i n g data of genera t e s t e d byHatch e t a l  7  Photosynthetic rates i n r e l a t i o n to p h o t o r e s p l r a t l o n among s p e c i e s  10  A survey o f carbon d i o x i d e i n the Gramineae  13  compensation  A survey o f carbon d i o x i d e compensation i n r e l a t i o n t o l e a f anatomy and s t a r c h distribution  19  P a r t 1-B Table  Table  Table  I.  II.  III.  I n t e r s p e c i f i c d i f f e r e n c e s i n carbon d i o x i d e compensation and e a r l y photos y n t h e t i c products f o r s p e c i e s f e d -^C02 f o r s i x seconds  32  C l a s s i f i c a t i o n o f the genus A t r l p l e x ( a c c o r d i n g t o Moser) i n r e l a t i o n t o compensation v a l u e s  3^  Rf v a l u e s f o r some compounds separated by one-dimensional paper chromatography u s i n g l i q u e f i e d phenol ( c a . 90$) - a c e t i c a c i d water - 1M e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d (8^-0:160:10:1) a s the s o l v e n t (Appendix I )  4-0  P a r t 1-C Table  I.  The d i s t r i b u t i o n o f r a d i o a c t i v i t y among compounds f o r Panicum spp. f e d ^C02 f o r s i x seconds (Appendix I )  49  Part 2 Table  I.  E f f e c t o f DCMU and oxygen c o n c e n t r a t i o n on the gas exchange of detached wheat and c o r n shoots  56  vii  LIST OP TABLES, cont'd. Page P a r t 2, Table  Table  Table  Table  II.  III.  IV.  V.  cont'd.  E f f e c t of DCMU and oxygen concent r a t i o n on the gas exchange of Nitella flexllls  60  The e f f e c t of s u b s t r a t e and oxygen c o n c e n t r a t i o n on r a t e s of carbon d i oxide e v o l u t i o n by detached monocotyl e d o n shoots i n darkness  61  E f f e c t of g l y c o l a t e and oxygen conc e n t r a t i o n on carbon d i o x i d e product i o n by N i t e l l a f l e x l l l s i n darkness...  62  E f f e c t of oxygen c o n c e n t r a t i o n on the carbon d i o x i d e compensation conc e n t r a t i o n of wheat and c o r n shoots when l i g h t i s a l i m i t i n g f a c t o r  69  viii LIST OP FIGURES  Part Figure  1.  Figures  Low  2-7.  1-A  Page  Carbon dioxide compensation values of members of the Gramlneae i n r e l a t i o n to a phylogenetic scheme based on the newer c r i t e r i a  15  Surfaoe views of the patterns of starch d i s t r i b u t i o n i n dicotyledon leaves of the two carbon dioxide compensation categories.  22  compensation leaves Figure 2. Amaran thus retroflexus Figure A t r l p l e x rosea Figure Mr. Portulaca oleracea  High compensation leaves Figure 5* Figure o. Figure 7.  Celosla golden plume Chenopodlum album A t r l p l e x hastata Part l'-B  Figure  1.  A standard map f o r some compounds separated by two-dimensional chromatography on thin-layer plates coated with 'Avicel* microcrystalline c e l l u l o s e . Developed i n l i q u e f i e d phenol (ca. 90%) - 0.3$ ammonium hydroxide i n the f i r s t dimension and n-propanol - ethyl acetate - water (7:1:2) twice i n the second dimension.... 41  ix  ACKNOWLEDGEMENTS The author expresses h i s sincere appreciation to Dr. Bruce Tregunna f o r d i r e c t i o n , encouragement and " a c c e s s i b i l i t y " throughout t h i s investigation. Many hours of discussion with his graduate students, p a r t i c u l a r l y Mr. Joseph Berry and Mr. Peter J o l l i f f e , were responsible f o r the author's continuing enthusiasm.  Thanks are a l s o extended to Drs. D.P. Ormrod,  V.C. Brink, K. Cole and G.H.N. Towers f o r h e l p f u l advice. Dr. Thana Bisalputra a s s i s t e d with photography. Seeds were provided by Northrup, King and Co.,  Fresno,  C a l i f o r n i a , Buckerfleld's Ltd., Vancouver, B.C., the International Seed Exchange Program and research o f f i c e r s of v a r i ous U.S.D.A. Regional Plant Introduction Stations.  Dr. V.C.  Brink and Mr. Don Pearce, Faculty of A g r i c u l t u r e , The University of B r i t i s h Columbia, and Dr. W.V. Brown, Department of Botany, The University of Texas a t Austin, supplied seed and leaves of many grass species.  The assistance of Mr. Peter Vlckery i s  g r a t e f u l l y acknowledged.  Dr. Janet S t e i n kindly supplied  Nitella. The author was a r e c i p i e n t of National Research Council of Canada Scholarships during the periods 1966-67, 1967-68 and 1968-69.  Research was supported by grants to Dr. Tregunna  from the National Research Council of Canada and the President's Committees on Research, The University of B r i t i s h Columbia.  X  PREFACE A t the time t h i s work began (February t h a t CO2 p r o d u c t i o n was  during photosynthesis  1967)t I t was  (photoresplratlon)  an 0 3 s e n s i t i v e process d i f f e r e n t from r e s p i r a t i o n i n the  dark.  G l y c o l i c a c i d was  photoresplratlon.  i m p l i c a t e d t o be the s u b s t r a t e  Most h i g h e r p l a n t s t h a t had  produced C 0 2 by p h o t o r e s p l r a t l o n , but a few The an  known  path of carbon i n p h o t o s y n t h e s i s  was  of  been s t u d i e d  grasses  believed to  did  not.  involve  i n i t i a l s y n t h e s i s of phosphorylated compounds v i a the  cycle.  Kortschak, Hatch and  S l a c k l a t e r demonstrated a  pathway f o r sugarcane i n which C j ^ - d i c a r b o x y l i c a c i d s a c e t a t e , malate and fixation.  aspartate)  Calvin new  (oxalo-  were the f i r s t products of  CC»2  P h o s p h o r y l a t e d compounds t y p i c a l of the C a l v i n c y c l e 14  were o n l y minor products of short-term cumulated w i t h time. new  pathway was  grasses.  C0  While t h i s t h e s i s was  f i x a t i o n , but  2  i n progress,  the  shown t o be a c h a r a c t e r i s t i c of many t r o p i c a l  I t then became c l e a r t h a t s p e c i e s l a c k i n g  photorespl-  r a t l o n were those w i t h the C ^ - d l e a r b o x y l i c a c i d pathway. observations  ac-  These  were l a t e r extended t o some d i c o t y l e d o n s .  As a consequence of these f i n d i n g s , other l a b o r a t o r i e s have r e o r i e n t e d t h e i r p r o j e c t s t o i n c l u d e s t u d i e s on the pathway and  photoresplratlon.  Cij,-..  S e v e r a l groups a r e c u r r e n t l y  i n v e s t i g a t i n g enzymatic d e t a i l s of the Ci^-path of carbon, part i c u l a r l y the mode or e x i s t e n c e  of carbon t r a n s f e r from C^-com-  pounds t o phosphorylated compounds t y p i c a l of the C a l v i n c y c l e . The  recent discovery  of the peroxisome In l e a v e s and  c h a r a c t e r i z a t i o n of i t s enzyme complement has t h e r e x p e r i m e n t a t i o n and  the  partial  resulted i n fur-  s p e c u l a t i o n on the mechanism of photo-  xi respiration. time,  Although  the t o t a l p i c t u r e i s u n c l e a r a t  i t seems l i k e l y t h a t the b i o c h e m i c a l d e t a i l s of  p i r a t i o n and p h o t o s y n t h e s i s The  this photores-  w i l l be e l u c i d a t e d s h o r t l y .  s i m i l a r i t y of the c o n c l u s i o n s reached by s e v e r a l i n -  dependent l a b o r a t o r i e s a t about the same time r e g a r d i n g  certain  i n t e r r e l a t i o n s of p h o t o s y n t h e s i s and p h o t o r e s p i r a t i o n , and  the  n e c e s s i t y of r a p i d p u b l i c a t i o n t o ensure the o r i g i n a l i t y of d i s c o v e r i e s r e p o r t e d i n t h i s t h e s i s have c r e a t e d a great d e a l of p r e s s u r e and a n x i e t y a t times. a h i g h l e v e l of excitement  This s i t u a t i o n a l s o created  and enthusiasm, and presented  an  o p p o r t u n i t y r a r e l y encountered by a graduate student f o r a c t i v e p a r t i c i p a t i o n a t the i n t e r n a t i o n a l  level.  1 P a r t 1-A.  Carbon Dioxide Compensation Photosynthetlc  - I t s Relation to  Carboxylation Reactions,  of the Gramineae, and L e a f Anatomy.  Systematics  1  INTRODUCTION A few r e p o r t s have i n d i c a t e d t h a t some t r o p i c a l a r e capable  o f l o w e r i n g the C 0  value of about 300 ppm (29,  3D.  2  grasses  c o n c e n t r a t i o n from the normal  t o about 5 PPm  i n a c l o s e d system  Most p l a n t s which have been t e s t e d , however, do not  possess t h i s h i g h f i x a t i o n c a p a c i t y , and the e q u i l i b r i u m conc e n t r a t i o n u s u a l l y a t t a i n e d i s about 50 ppm depending mainly on l i g h t rium  i n t e n s i t y and temperature (7,  i s c a l l e d the C 0  which normally  2  8 , 19).  This  compensation c o n c e n t r a t i o n .  compensate a t about 50 ppm  p l a n t s ) w i l l compensate a t l e s s than 5 PPm  (high in # 2  equilibi Plants  compensation 0 2 (38,  39).  The O2 s e n s i t i v i t y o f the compensation v a l u e , which g i v e s a measure of CO2 p r o d u c t i o n d u r i n g p h o t o s y n t h e s i s  (photorespira-  t i o n ) , c o n t r a s t s w i t h the e f f e c t on r e s p i r a t i o n In the dark which remains u n a f f e c t e d by 0 I n 1962,  Moss (31)  and Saccharum  2  c o n c e n t r a t i o n s above 2%  r e p o r t e d t h a t two t r o p i c a l g r a s s e s , Zea mays  o f f l c i n a r u m . had low compensation v a l u e s .  r e s t e r e t a l . (16)  (38).  For-  showed t h a t f o r Zea mays t h i s value was  sistent f o r a l l varieties tested.  A few other  con-  monocotyledons  1 T h i s a r t i c l e by W.J.S. Downton and E.B. Tregunna appeared i n Canadian J o u r n a l of Botany, V o l . 46: 207-215 (1968). E.B. Tregunna s u p e r v i s e d the study. Subsequent developments on t h i s t o p i c a r e d i s c u s s e d i n f o o t n o t e s and P a r t s 1-B and 1-C.  2 were tested i n an attempt to a s c e r t a i n i f a low compensation value was a c h a r a c t e r i s t i c of t h i s group, but a l l had high El-Sharkawy et a l . (14)  values.  demonstrated that Sorghum  vulgare and two species of the dicotyledonous  genus Amaranthus  did not produce C 0 when illuminated i n C 0 - f r e e a i r . 2  2  This  suggested that they had low compensation values and therefore lacked photoresplratlon.  This has been confirmed  In the genus  Amaranthus and extended to some other amaranths and chenopods by us  (39).  In a d d i t i o n to the Calvin cycle (3) of C 0 f i x a t i o n , a 2  new carboxylation sequence has been reported by two groups (17» 25).  In a recent publication, Hatch et a l . (18)  presented  a l i s t of plants which had been tested f o r the existence of the proposed photosynthetic carboxylation system as indicated by the early l a b e l l i n g of Cij,-compounds.  I t was shown that some  of the t r o p i c a l grasses such as Zea mays and Saccharum narum have t h i s carboxylation system.  offIcl-  An u n i d e n t i f i e d species  of Cyperus. which i s i n a family c l o s e l y a l l i e d to the Gramineae, a l s o showed early C ^ - l a b e l l i n g .  This new carboxylation system  i s Indicated by the rapid l a b e l l i n g of the Cl».-compounds oxaloacetate, malate, and aspartate when "^CC^ Is fed (17). products  Early  of the Calvin sequence are the C^-compound 3-pnospho-  g l y c e r i c a c i d and hexose phosphates (3).  I t was therefore of  interest to e s t a b l i s h I f a c o r r e l a t i o n existed between high and low compensation values and the respective early l a b e l l i n g of C^- and Cj^-compounds. It was of further interest to survey the gramineae i n some d e t a i l to determine i f the compensation values obtained could  3 be r e l a t e d t o any  proposed taxonomic scheme.  seemed f a v o r a b l e s i n c e form should gree.  T h i s approach  r e f l e c t f u n c t i o n t o some de-  Problems e x i s t i n t h i s approach, however, s i n c e some of  the taxonomic schemes c u r r e n t l y i n use are based only on chara c t e r i s t i c s of the i n f l o r e s c e n c e w h i l e others a r e based on comp a r a t i v e anatomy of other Rhoades and sorghum (both low  structures.  Carvalho (36)  have shown t h a t f o r maize and  compensation p a n i c o i d s p e c i e s )  the  sheath  c e l l s c o n t a i n s p e c i a l i z e d p l a s t i d s concerned w i t h s t a r c h format i o n and chyma. and  storage. On  No  s t a r c h was  formed i n the outer  chloren-  the other hand, the p l a s t i d s of both the mesophyll  p o o r l y developed sheath c e l l s of o a t s , wheat, and  barley  (high compensation members of the c l a s s i c a l subfamily  Festu-  coideae) form s t a r c h but  t o a l e s s e r degree than the p l a s t i d s  of the p a n i c o i d sheath.  El-Sharkawy et a l . (13)  reported  that  the arrangement of c e l l s about the v a s c u l a r bundle of a  species  of Amaranthus was  grasses.  s i m i l a r to t h a t found i n the t r o p i c a l  In view of these o b s e r v a t i o n s , the two  freehand s e c t i o n s of l e a v e s  of  compensation c a t e g o r i e s were s t u d i e d t o determine the  r e l a t i o n s h i p of the gas  exchange data t o l e a f anatomy and  starch  distribution.  MATERIALS P l a n t m a t e r i a l f o r t h i s study o r i g i n a t e d from many  sources.  Seeds of T r l t l c u m sativum. Sorghum sudanense. Seoale c e r e a l e . A vena s a t i v a , Zea mays. Lactuca  satlvgj. Beta v u l g a r i s . C h l o r l s  gayana, Pennlsetum glaucum, Axonopus a r g e n t l n u s . tatum, Cynodon d a o t y l o n ,  and  Paspalum d l l a -  Hordeum v u l g a r e were obtained  from  4 commercial seed s u p p l i e r s . at  Summerland, B.C.  Bromus tectorum seed was  Cyperus albomarglnatus, Cyperus  collected eragrostls.  E r a g r o s t l s p i l o s a . Paspalum d i s t l c h u m . and Beckmannla syzlgachne were grown from seed obtained from herbarium specimens a t the U n i v e r s i t y of B r i t i s h Columbia. and grown a t 2G00 f t - c ,  16 hour day, 21-26/18-22° day/night  temperature i n a growth room. of  The seeds were p l a n t e d i n s o i l  The r e s t of the m a t e r i a l c o n s i s t e d  shoots obtained on the U n i v e r s i t y of B r i t i s h Columbia campus  e i t h e r from f i e l d n u r s e r i e s or from greenhouses. t i o n was  Identifica-  from l a b e l s or by i n d i v i d u a l s f a m i l i a r w i t h the p l a n t  material.  METHODS Measurement of C02  Compensation  Shoots were detached. Immediately r e c u t under water, and placed i n small w a t e r - f i l l e d v i a l s .  These were then lowered  i n t o a g l a s s c y l i n d e r which served as a p h o t o s y n t h e t l c chamber. A c l o s e d system I n c o r p o r a t i n g the p h o t o s y n t h e t l c chamber, a p e r i s t a l t i c pump, and an i n f r a r e d gas a n a l y z e r was used.  A  water bath was used t o s t a b i l i z e the temperature i n the photos y n t h e t i c chamber. was 21.5  ± 0.5°.  The temperature range f o r a l l experiments The l i g h t i n t e n s i t y from a General E l e c t r i c  Cool Beam lamp was a p p r o x i m a t e l y 2500 f t - c as measured by a Weston 756  l i g h t meter.  ,  The f o l l o w i n g sequence was used t o measure C0£ compensat i o n v a l u e s and the e f f e c t on compensation of l o w e r i n g 0 centration.  The shoot was  illuminated i n a closed  2  con-  circulating  5 system and allowed to remove CO2 to the compensation l e v e l . The l i g h t was then turned o f f and a r e s p i r a t i o n rate i n the dark was obtained.  I f the compensation value was greater than  5 ppm the sequence was repeated a f t e r a nitrogen f l u s h to lower the 0  2  concentration and addition of C 0 . 2  The determination  of a dark rate under these conditions was necessary to ensure that the low 0 piration.  2  concentration was not i n h i b i t o r y to dark res-  A l l species, except Eucalyptus sp. and Cymbldium sp.,  were tested at l e a s t twice. Anatomical Studies Only a s u p e r f i c i a l study of l e a f anatomy i n relationship to the gas exchange results was attempted here.  The study i n -  volved an examination of freehand sections of both monocotyledons and dicotyledons of the two compensation categories. The water mount was examined f o r the presence of a c l e a r l y defined parenchyma bundle sheath and f o r the arrangement of mesophyll  cells.  A f t e r t h i s , IKI was applied to determine the presence and d i s t r i b u t i o n of starch i n chlorenchymatous c e l l s .  A uniform l e a f  age was not used i n t h i s study and although i t i s possible that some differences In starch patterns may occur with age, none were detected i n leaves from both young and old maize and Amaranthus plants.  Leaves were studied i n the l a t e afternoon  and evening to assure a high starch l e v e l .  RESULTS AND DISCUSSION Carbon Dioxide Compensation and Photosynthetlc Carboxylation Reactions In Table I, the C 0  2  compensation values obtained are shown  6 i n r e l a t i o n s h i p t o the data of Hatch e t a l . (18)  following  14 4 second feedings of  C0  2  to d i f f e r e n t plants.  In every case a  h i g h compensation v a l u e c o r r e l a t e s with a l a c k o f or n e g l i g i b l e l a b e l i n the C^-compounds and heavy l a b e l l i n g i n the C a l v i n i n termediates  of 3-phosphoglycerate and hexose phosphates.  Low  compensation v a l u e s c o r r e l a t e with e a r l y heavy l a b e l l i n g of the Cjj-compounds o x a l o a c e t a t e , malate, and a s p a r t a t e .  Cyperus a l b o -  marglnatus and C. e r a g r o s t l s . non-graminaceous p l a n t s p r e d i c t e d t o have the newly proposed pathway, a l s o had low  compensation  2 values.  The above c o r r e l a t i o n a l l o w s us t o make r e c i p r o c a l  pre-  d i c t i o n s about the major p h o t o s y n t h e t l c pathway In a p l a n t and the magnitude of C 0 istic.  2  r e l e a s e i n the l i g h t g i v e n e i t h e r c h a r a c t e r -  That i s , measurement of the COg  compensation v a l u e  pro-  v i d e s an immediate assay f o r d e t e r m i n a t i o n of the major c a r b o x y l a t i o n pathway i n o p e r a t i o n . thaceae and  Our p r e v i o u s survey of the Amaran-  Chenopodiaceae would then I n d i c a t e t h a t s p e c i e s of  the low compensation genus Amaranthus have the C^-pathway (39).^ Osmond (34) has r e p o r t e d t h a t A t r l p l e x spongiosa s y n t h e s i z e s C^4 compounds. I t s compensation v a l u e , however, i s not yet known. 2 S i n c e t h i s a r t i c l e was p u b l i s h e d , Johnson e t a l . (23) have r e p o r t e d t h a t some s p e c i e s o f Cyperus u t i l i z e the Cty-photosynt h e t i c pathway, but others do not. The data c i t e d i n Table I , P a r t 1-B, shows t h a t Cyperus albomarglnatus s y n t h e s i z e s C^-compounds• 3 T h i s p r e d i c t i o n based on our e a r l i e r work (39) has s i n c e been v e r i f i e d by Johnson e_t a l . (23) and o u r s e l v e s (41). I t was a l s o found t h a t h i g h compensation A t r l p l e x s p e c i e s s y n t h e s i z e C a l v i n c y c l e i n t e r m e d i a t e s whereas low compensation members form C/j.-compounds d u r i n g s h o r t term p h o t o s y n t h e s i s (34, 40, 41). T h i s type of p h o t o s y n t h e t l c d i v e r s i t y a l s o occurs w i t h i n the genus B a s s l a . See Table I, P a r t 1-B. 4 More r e c e n t s t u d i e s have confirmed Is a low compensation s p e c i e s (40),  that A t r l p l e x  spongiosa  TABLE I. Carbon dioxide compensation i n r e l a t i o n to l a b e l l i n g data of genera tested by Hatch et a l . Family Monocotyledoneae Gramineae  Cyperaceae Musaceae Carina ceae  Genus  Saccharum Sorghum Sorghum Sorghum Sorghum Zea Paspalum Paspalum Axonopus Digitaria Digitaria Chioris Eragrostls Eragrostls Eragrostls Trlticum A vena Bambusa Bambusa Cyperus Cyperus Cyperus Musa Musa Canna  Species  Common Name or Variety  offIcinarum almum halepense hybrid sudanense mays dilatatum distichum argentinus  Sugarcane Columbus grass Johnson grass Sorghum Hidan 35 Maize Paspalum Knotgrass Carpet grass  sanguinalis gayana brownll mexlcana pilosa sativum sativa  Crabgrass Rhodes grass  -  —  -  vulgaris albomarginatus eragrostls  -  velutlna  %™C i n C^,-cpds.  -  -  Mexican lovegrass India lovegrass Wheat Oats Bamboo Bamboo Sedge Sedge Sedge Banana Banana Canna  Data of Hatch et a l . ( 1 8 ) . ^L = comp. less than 5 PPm, H = comp. of 37-50 PPm» * = plant not tested. Data of Moss (31).  a  c  a  76 * 70 70  •  88  72 * 66 72 * 70  100  **  0 2  *  0  79 * *  0  *  0  C0o comp. i n 21$ 0  2  L L « * L L L L L * L L * L L H H H * •  L L * H H  c  D  TABLE I. cont'd. Family  Genus  Species  Liliaceae Orchidaceae  Phoenix Phoenix Lilium Cymbidium  canariensis dactylifera  Dicotyledoneae Compositae Chenopodiaceae Legumlnosae Leguminosae Geraniaceae Rosaceae Myrtaceae Solanaceae  Lactuca Beta Phaseolus Glycine Pelargonium Prunus Eucalyptus Nlcotlana  satlva vulgaris vulgaris soja  Palmae  * = plant not tested. H = comp. of 37-50 ppm. Data of Forrester et a l . (16). d  -  —  -  persica mat —  Common Name or Variety  in C^-cpds.  %*- C k  ,»  Date palm Spider l i l y Cym-doris Lettuce Beet Bean Soya bean Geranium Peach Gumtree Tobacco  2  0  2  7  2 2  0 0 0 0  CO2 comp. i n 2\% O2 H « H H H H H, H* H H H H  9 Carbon Dioxide Compensation and Photosynthetlc Rates El-Sharkawy and h i s colleagues (12,  13,  14) have reported  that species with very high photosynthetlc rates have n e g l i g i b l e photorespiration when measured by magnitude of CO2 evolution Into C 0 - f r e e a i r i n the l i g h t . 2  Table II shows that these high  photosynthetlc rates are also correlated with very low compensation values.  In addition, Murata et a l . (32,  33)  have r e -  ported that Rhodes grass (ChiorIs gayana). Bermuda grass (Cynodon daotylon), barnyard grass (Echlnochloa c r u s g a l l l ) . and Bahia grass (Paspalum notatum) a l s o have photosynthetlc rates nearly twice the value f o r the 'northern type' plants which they tested. As shown i n Table I I I , a l l of these except Paspalum notatum were tested f o r C0 fOompensation, 2  and the values were less than 5  ppm.  The two species of Paspalum that were a v a i l a b l e f o r t e s t i n g showed low compensation values.  Therefore, i t i s probable that  P. notatum has n e g l i g i b l e photorespiration. I t appears that n e g l i g i b l e photorespiration can be used as an index of a high photosynthetlc rate as well as an indicator of the major biochemical pathway involved. Since low compensation value means that n e g l i g i b l e CO2 i s released during photosynthesis, then f o r these plants, gross photosynthesis Is e s s e n t i a l l y equal to net photosynthesis. high compensation plants, however, net or apparent i s considerably less than gross photosynthesis.  For  photosynthesis  Lowering the 0  2  concentration around the high compensation plants l i s t e d i n Table III r e s u l t s i n a compensation value of less than 5 probably by blocking the photorespiratory pathway.  PPm,  Such a block  i n high compensation plants a l s o produces a great increase i n  TABLE I I .  Photosynthetlc  rates In r e l a t i o n to photorespiration among species. C r i t e r i a of photorespiration  Species  Psyn. rate, mg CG2/dm h z  Amaranthus edulls  60  Cynodon dactylon  60  Saccharum offlcinarum  Ref.  C0£ prod, i n C02-free a i r , mg C0 /dm h 2  2  a  C0 comp. value, ppm 2  0.0  5  (13)  0.0  5  60  ( i n 14)  0.0  7  Sorghum vulgare  60  (13)  0.0  -  Zea mays  60  (13)  0.0  5  Avena satlva  30  (13)  Beta vulgaris  30  (13)  2.4  ±  0.03  Hellanthus annus  *5  (13)  3.1  i  0.09  (13,1*)  Prom Table I and text of El-Sharkawy et a l . (14). ^Data of Tregunna and Downton (39). Data of Moss (31). c  -  40 47  b  C  11  the net p h o t o s y n t h e t l c  F o r example, F o r r e s t e r ( 1 6 )  rate.  Is 55»4 jig CO2 ab-  shown t h a t , f o r soybean, net photosynthesis sorbed/minute per l e a f under 1 $ 0  2  S i m i l a r enhance-  2  under low 0  2  c o n c e n t r a t i o n have been  shown f o r many p l a n t s by Downes and Hesketh (9t the 0  2  values  20).  c o n c e n t r a t i o n d i d not enhance p h o t o s y n t h e s i s  t r o p i c a l grasses which we (9).  have found t o have low  Lowering of  those  compensation  I t seems u n l i k e l y t h a t l o w e r i n g the O2  t i o n would convert p l a n t s from one  37.8  compared t o a value of  ^ug CO2 absorbed/minute per l e a f under 2 1 $ o . ments of p h o t o s y n t h e s i s  has  concentra-  carboxylation r e a c t i o n to  another, but i t should be tested.-*  I t i s more l i k e l y t h a t  l o w e r i n g the O2 c o n c e n t r a t i o n only makes the p l a n t s s i m i l a r a t the gas  exchange l e v e l .  Carbon D i o x i d e Compensation and Prat  (35)  recognized  of the Gramlneae  f o u r e v o l u t i o n a r y l i n e s i n the  Gramineae, based on epidermal Bambusoideae, Eupanicoideae, anatomy by Brown ( 2 )  Systematlcs  c h a r a c t e r i s t i c s : the and  Chloridoideae.  A study  added the s m a l l e r a r u n d i n o l d and  groups r e s u l t i n g i n s i x major groups or l i n e s . grasses  Festucoideae, of l e a f  aristidoid  Some of the  such as the Oryzeae and S t i p e a e a r e d i f f i c u l t to p l a c e  t a x o n o m i c a l l y and groups.  The  hence a r e not p l a c e d w i t h i n any  of the above  scheme used here i s a s y n t h e s i s of groupings  posed by Brown and  Stebbins  (2,  37).  pro  r  T h i s modern grouping d i f -  5 P r e l i m i n a r y experiments show t h a t wheat s e e d l i n g s exposed t g 2 $ 0 f o r e i t h e r 1 0 minutes or 47.5 hours p r i o r t o f e e d i n g C 0 f o r about 30 seconds a t low 0 do not produce m a l i c and a s p a r t i c a c i d i n g r e a t e r amounts than p l a n t s f e d under 2 1 $ 0 . 2  2  2  2  12  f e r s s u b s t a n t i a l l y from the c l a s s i c a l system which i s based s o l e l y on i n f l o r e s c e n c e c h a r a c t e r i s t i c s . i s supported by many workers who  I t s v a l i d i t y , however,  have shown t h a t , except f o r  s m a l l v a r i a t i o n s , such a system i s c o n s i s t e n t w i t h the newer c y t o g e n e t l c a l , h i s t o l o g i c a l , and a n a t o m i c a l , and criteria  (6,  i n 37).  morphological  Table I I I c i t e s the compensation v a l u e s  that were obtained f o r members of the Gramineae.  The l i n e s of  e v o l u t i o n are shown a l p h a b e t i c a l l y i n Table I I I because of the d i f f i c u l t y of l i s t i n g i n l i n e a r sequence groups which have supposedly evolved by a d a p t i v e r a d i a t i o n t o v a r i o u s h a b i t a t s from some a n c e s t r a l type.  F i g u r e 1 , m o d i f i e d from P r a t (35).  presents  the CO2 compensation v a l u e s of the Gramineae i n r e l a t i o n t o one k i n d of e v o l u t i o n a r y scheme based  on the newer c r i t e r i a .  With the e x c e p t i o n of Beckmannla. a l l g r a s s e s of the c h l o r i d o i d - e r a g r o s t o l d or p a n i c o i d l i n e s compensation.^  (Table I I I ) have low  The h i g h compensation value of Beckmannla  sug-  gests t h a t i t has been misplaced t a x o n o m l c a l l y and does not belong i n the c h l o r l d o l d group. who  (35).  T h i s i s supported by P r a t  s t a t e d "Parmi l e s genres ranges h a b i t u e l l e m e n t dans l e s  C h l o r i d e e s , l e genre Beckmannla Host, c o n s t i t u t e une  exception  par l a presque t o t a l l t e de ses c a r a c t e r e s : son eplderme, son anatomie, son karyotype, sa p l a n t u l e , sont p a r f a i t e m e n t coides.  Ce genre d o l t e t r e r a t t a c h e en r e a l l t e aux  festu-  Festucees."  The homogeneity of the compensation v a l u e s of the s p e c i e s  now  6 Downton et a l . have s i n c e found t h a t two d i f f e r e n t p a t t e r n s of compensation and C 0 f i x a t i o n occur w i t h i n two subgenera of the genus Panlcum ( 1 1 ) . A summary of t h i s i n v e s t i g a t i o n publ i s h e d i n Science Is g i v e n i n P a r t 1-C. 2  TABLE I I I . A survey of carbon dioxide compensation In the Gramineae. Line Arundinoid Bambusoid Chloridoideragrostoid  Tribe  CO2 comp i n 21$ 0 < 2  Cortaderia Bambusa  selloana  Pampasgrass Bamboo  H H  Chlorideae  Beckmannla Chloris Cynodon Eragrostls Eragrostls Agrostis Cinna Phleum Arrhenatherum Avena Bromus Bromus Dactylis Festuca Festuca Poa Hordeum Secale Triticum Anthoxanthum Phalaris  syzigachne gayana dactylon mexicana pilosa alba latifolla  Sloughgrass Rhodes grass Bermuda grass Mexican lovegrass India lovegrass Redtop Drooping woodreed Timothy erecta T a l l catgrass Rodney oats Smooth brome Downy chess Orchard grass Meadow fescue var. commutata Canada bluegrass Vantage barley Storm rye Spring wheat Sweet vernal Reed canary grass  H L L L L H H H H H H H H H H H H H H H H  Agrostideae Aveneae Festuceae  Hordeae Phalarideae  a  Species  Arundlneae Bambuseae  Eragrosteae Festucoid  Genus  Variety or common name  -  -  elatlus sativa inermis tectorum glomerata elatior rubra compressa vulgare cereale sativum odoratum arundinacea  L = comp. less than 5 PPm» H = comp. of 37-50 PPm  7 A r l s t l d a adscenslonsls and Ar1stIda unlplumls of the t r i b e A r i s t i d e a e ( a r l s t i d o l d l i n e ) , and Arundlnella h l r t a of the t r i b e Arundinelleae (panicold l i n e ) have low compensation values. These results have been added to Figure 1 since p u b l i c a t i o n .  TABLE I I I . cont'd.  Line  Tribe  Oryzoid Panicoid  Oryzeae Andropogoneae  Maydeae Paniceae s  Stlpold a  Genus Oryza Saccharum Sorghum Sorghum Sorghum Zea Axonopus Dlgitaria Echinochloa Panicum Panicum Paspalum Paspalum Pennisetum Setaria Setarla Gryzopsis  Species sativa officinarum almum sudanense vulgare mays argentinus sanguinalis crus-galli capiliare miliaceum dilatatum distlchum glaucum lutescens italica racemosa  L = comp . less than 5 PPm, H a comp. of 37-50 ppm  b  Data of Moss ( 3 D .  c  Derlved from data of El-Sharkawy et a l . (14).  Variety or common name Kangni 27 r i c e Sugarcane Columbus grass Sudan grass Sorghum Maize Carpet grass Crabgrass Barnyard grass Witchgrass Hog m i l l e t D a l l i s grass Knot grass Gahi pearl m i l l e t Yellow b r i s t l e g r a s s Hungarian m i l l e t mm  CO'> comp. i n ll% * 02 a  H L L L LC L L L L L L L L L L L H b  15  PANICOIDEAE  CHLORIDOIDEAE — ERAGROSTOIDEAE  FESTUCOIDEAE  BAMBUSOIDEAE F i g u r e 1. Carbon d i o x i d e compensation values of members of the Gramineae i n r e l a t i o n t o a p h y l o g e n e t i c scheme based on the newer c r i t e r i a . The s i z e of the c i r c l e s i s p r o p o r t i o n a l t o the number of s p e c i e s a t t r i b u t e d t o each. (H = compensat i o n c a . 50 ppm; L * compensation o f l e s s than 5 ppm; ? = r e p r e s e n t a t i v e s of t h i s group not t e s t e d . ) Adapted from P r a t .  16 p l a c e d i n the c h l o r i d o i d - e r a g r o s t o i d l i n e i s i n c o n t r a s t to the h e t e r o g e n e i t y which r e s u l t s under the o l d scheme.  In the o l d  scheme, s p e c i e s of the genus E r a g r o s t l s a r e c o n s i d e r e d as members of the Pestuceae and these a r e p l a c e d w i t h the C h l o r i d e a e i n the s u b f a m i l y Pestucoideae, low compensation v a l u e s Brown (2)  thus mixing p l a n t s w i t h h i g h and  (21).  has c o n s i d e r e d the development of the parenchyma  sheath surrounding the v a s c u l a r bundles tematics of the Gramineae.  i n r e l a t i o n s h i p to sys-  R e l a t i v e l y l i t t l e development  has  o c c u r r e d i n the bambusold and f e s t u c o i d l i n e s , but "extreme s p e c i a l i z a t i o n i n r e s p e c t t o both the parenchyma sheath and  cells  the c h l o r o p l a s t s which they c o n t a i n " has o c c u r r e d i n the  p a n i c o i d and c h l o r i d o i d - e r a g r o s t o i d groups ( q u o t a t i o n from r e f . g 37).  There i s an obvious  c o r r e l a t i o n between sheath  i z a t i o n and the p h y s i o l o g y of low compensation.  special-  Beckmannla  l a c k s such sheath s p e c i a l i z a t i o n , which p r o v i d e s f u r t h e r e v i dence t h a t i t has been misplaced  In the C h l o r i d e a e .  The  high  compensation v a l u e of Oryza would appear t o r e l a t e i t t o the bambusoid or f e s t u c o i d l i n e .  T h i s agrees w i t h the  festucoid  type of l e a f anatomy and the m o d i f i e d bambusoid type of l e a f epidermis  of the Oryzeae (37).  epidermal  c h a r a c t e r i s t i c s , the S t i p e a e a r e c l o s e t o the P e s t u -  coideae, which may  In both l e a f anatomy and  leaf  be r e f l e c t e d i n the h i g h compensation value  obtained f o r O r y z o p s l s  (37).  The o n l y member of the a r u n d i n o i d  8 See B i s a l p u t r a e t a l . (1), Downton et a l . (10) and L a e t s c h et a l . (26, 27, 28) f o r an u l t r a s t r u c t u r a l d e s c r i p t i o n and d i s c u s s i o n of the bundle sheath. These papers c i t e s e v e r a l a d d i t i o n a l references.  17 group t e s t e d , C o r t a d e r l a s e l l o a n a , i s a h i g h compensation  plant.  Although t h i s l i n e has w e l l - d e v e l o p e d parenchyma sheath c e l l s and a p o o r l y developed endodermls which i s common t o members of the low-compensation c h l o r i d o i d - e r a g r o s t o i d and p a n i c o i d l i n e s , t h e r e i s a complete l a c k o f c h l o r o p l a s t s i n the parenchyma sheath c e l l s  (2).  Perhaps t h e presence o f s p e c i a l i z e d  chloro-  p l a s t s i n t h i s sheath i s e s s e n t i a l f o r low compensation p h y s i ology.  F u r t h e r - r e s e a r c h l i n k i n g a n a t o m i c a l and p h y s i o l o g i c a l  c h a r a c t e r i s t i c s may h e l p t o c l a r i f y e v o l u t i o n a r y r e l a t i o n s h i p s , p a r t i c u l a r l y among those groups which a r e c u r r e n t l y  difficult  to place. G e o g r a p h i c a l d i s t r i b u t i o n i n d i c a t e s t h a t the p a n i c o i d and c h l o r i d o l d l i n e s developed p r i m a r i l y i n the t r o p i c s , whereas the Festuceae a r e important i n temperate c l i m a t e s (2, 37)• compensation v a l u e may then be a s s o c i a t e d w i t h t r o p i c a l of c e r t a i n graminaceous groups.  Low  origin  The members o f the low com-  p e n s a t i o n genus Amaranthus supposedly o r i g i n a t e d i n the t r o p i c s (5).  I t should be noted t h a t the one bambusoid  member t e s t e d  had a h i g h compensation v a l u e even though t h i s l i n e supposedly "...became dominant  i n the moist f o r e s t s o f the t r o p i c s . "  (37).  F o r t h e c h l o r i d o l d and p a n i c o i d groups, however, a c a u s a l evol u t i o n a r y r e l a t i o n s h i p may e x i s t between c e r t a i n t r o p i c a l ecol o g i c a l factors way.  (perhaps temperature) and the dominant  F o r i n s t a n c e Cooper (4)  C^-path-  s t a t e d t h a t h i g h w i n t e r temperature  i s important i n d e t e r m i n i n g the d i s t r i b u t i o n o f the low compensat i o n Andropogoneae, Murata e t a l .  Paniceae, and E r a g r o s t e a e .  In addition,  (32, 33) r e p o r t e d t h a t the t r o p i c a l grasses (mem-  18 bers of the low compensation c h l o r i d o i d and p a n i c o i d groups) w i t h very h i g h p h o t o s y n t h e t l c f o r photosynthesis 10-25° f o r those normally  r a t e s have a temperature optimum  of about 35°.  T h i s compares with optima of  s p e c i e s (with lower r a t e s and  found i n c o o l e r c l i m a t e s (32,  photorespiration)  33).  Carbon D i o x i d e Compensation i n R e l a t i o n to L e a f Anatomy and Starch D i s t r i b u t i o n The  purpose of t h i s survey was  t o determine whether the  c o r r e l a t i o n between l e a f anatomy and  CO2 compensation which  found i n the Gramineae would p e r s i s t i n h i g h and t i o n dicotyledons.  The  study by Rhoades and  low  Carvalho  was  compensa(36)  of  s t a r c h d i s t r i b u t i o n i n r e l a t i o n to the anatomy of maize and sorghum was  extended t o the d i c o t y l e d o n s .  Some grasses were  a l s o s t u d i e d t o provide a b a s i s f o r comparison. of t h i s survey a r e shown i n Table IV.  The  low  The  results  compensation  d i c o t y l e d o n s t h a t were t e s t e d c h a r a c t e r i s t i c a l l y possessed well-developed  parenchyma bundle sheath as w e l l as a  mesophyll l a y e r arranged p e n s a t i o n grasses and  r a d i a l l y around the sheath.  a  definite Low  com-  ( a l l members of the c h l o r i d o i d - e r a g r o s t o i d  p a n i c o i d l i n e s ) were s i m i l a r to t h i s , except t h a t the meso-  p h y l l c e l l s were not always as d e f i n i t e l y arranged fashion. sheath  in radial  In the high compensation d i c o t y l e d o n s , the bundle  (although  i t i s r e p o r t e d t o be present around the  smaller  v a s c u l a r bundles i n d i c o t y l e d o n s ) c o u l d not be seen as a cbher-  9 Kawanabe (24) has s i n c e p r o v i d e d a d e t a i l e d d i s c u s s i o n on temperature response and s y s t e m a t l c s of the Gramineae. He found the optimum day/night temperature regime f o r r e l a t i v e growth r a t e f o r c h l o r i d o i d - e r a g r o s t o i d and p a n i c o i d grasses to be 30/2536/31°; i t was 21/16-27/22° or 21/16 -30/25° f o r f e s t u c o i d s p e c i e s .  TABLE IV. A survey of carbon dioxide compensation distribution.  i n r e l a t i o n to l e a f anatomy and starch  IKI s t a i n f o r starch co comp.  Defined bundle sheath  H L L  + +  2  Species Dicotyledons A t r i p l e x hastata A t r l p l e x rosea Amaranthus retroflexus Celosia golden plume Chenopodium album Portulaca oleracea Monocotyledons Beckmannia syzigachne Chloris gayana Cyperus albomarginatus Eragrostis pilosa Panicum c a p i l l a r e Setarla lutescens Sorghum sudanense Triticum sativum Zea mays  Radiating mesophyll  + +  H H L H L L L L L L H L  Mesophyll  Bundle sheath  + n.a. Negligible + Adaxlal part, n e g l i g i b l e ; + abaxial part (+), but less than sheath + n.a. + n.a. Pew f a i n t l y s t a i n i n g grains i n many chloroplasts +  Poor with few chloroplasts  + +  + + + + Poorly defined +  + +  +  + + ? ? ?  (+)  small amounts  (+) small amounts +  Note: + = present, ? = i n d e f i n i t e , H = comp. ca. 50 ppm, L = comp. less than 5 ppm, n.a. s not applicable, - = absent.  + + + +  20  ent s t r u c t u r e (15).  The bundle sheath  h i g h compensation monocotyledons. by Rhoades and Caryalho  i s poorly defined i n  As shown i n maize and sorghum  (36), a deep p u r p l e s t a i n i n d i c a t i v e o f  s t a r c h occurred only i n t h e c h l o r o p l a s t s o f the bundle c e l l s i n a l l o f the low compensation grasses mesophyll c h l o r o p l a s t s d i d not s t a i n .  sheath  sectioned.  The  The mesophyll c h l o r o -  p l a s t s of the h i g h compensation monocotyledons and d i c o t y l e d o n s contained  starch.  F o r i n s t a n c e , i n wheat and Beckmannla. both  the c h l o r o p l a s t s o f the p o o r l y d e f i n e d sheath of the mesophyll s t a i n e d f o r s t a r c h . support  c e l l s and those  These r e s u l t s f u r t h e r  the c o n t e n t i o n t h a t Beckmannla i s not a n a t u r a l member  of the c h l o r i d o l d l i n e and should be r e c l a s s i f i e d .  The s t a i n -  i n g p a t t e r n i n the low compensation p l a n t s o u t s i d e o f the f a m i l y Gramineae was not q u i t e as c l e a r - c u t as t h a t i n the low compensation grasses.  The mesophyll o f the low compensation s p e c i e s ,  P o r t u l a c a o l e r a c e a and A t r l p l e x rosea  (39), when s t a i n e d with  IKI, r e v e a l e d a few f a i n t l y s t a i n i n g g r a i n s i n many c h l o r o p l a s t s . N e g l i g i b l e s t a r c h was v i s i b l e i n the a d a x i a l p a r t o f the r a d i a l l y arranged  p a l i s a d e l a y e r o f Amaranthus r e t r o f l e x u s , but a great  d e a l was present  In t h e a b a x i a l p a r t .  i n the r a d i a l l y arranged  Many o f the c h l o r o p l a s t s  mesophyll c e l l s o f Cyperus albomargln-  a t u s , a low compensation monocotyledon, s t a i n e d i n t e n s e l y with IKI, as d i d i t s sheath. Low compensation p l a n t s a r e o u t s t a n d i n g with r e s p e c t t o the apparent h i g h c h l o r o p l a s t c o n c e n t r a t i o n o f the sheath i n comparison t o the c o n c e n t r a t i o n i n the mesophyll  cells  cells.  T h i s c o n c e n t r a t i o n i s so great t h a t a low compensation d i c o t y l e d o n can be d e t e c t e d w i t h the naked eye by d e t a c h i n g a l e a f  21 and h o l d i n g I t a g a i n s t a l i g h t source.  Such l e a v e s show a  dark green r e t i c u l u m , which r e s u l t s from the g r e a t t i o n of c h l o r o p l a s t s i n the sheath and the v a s c u l a r bundles.  mesophyll  concentrasurrounding  High compensation d i c o t y l e d o n s when h e l d  to the l i g h t appear t o be a uniform  green c o l o r except around  the v a s c u l a r bundles, which a r e t r a n s l u c e n t .  This difference  i s more s t r i k i n g l y i l l u s t r a t e d when such l e a v e s a r e b o i l e d i n e t h a n o l t o remove the c h l o r o p h y l l , f o l l o w e d by s t a i n i n g i n IKI. Low  compensation d i c o t y l e d o n s now  show the v a s c u l a r system of  the l e a f i n d e t a i l because the s t a i n occurs only i n the around the v a s c u l a r bundles.  plastids  High compensation d i c o t y l e d o n s  show a uniform p u r p l e c o l o r throughout the l e a f (except around major v e i n s ) r e s u l t i n g from the s t a i n i n g of the mesophyll c h l o r o p l a s t s ( F i g u r e s 2-7).  Monocotyledon l e a v e s must be  sectioned  to be evaluated because of the c l o s e r e l a t i o n of one  vascular  bundle t o the next.  provide  very simple and  These anatomical  immediate assays f o r the p h o t o s y n t h e t l c  ology and b i o c h e m i s t r y list  methods, then,  of a p l a n t under c o n s i d e r a t i o n .  physiThe  g i v e n by Moser (30) should be t e s t e d . The  sheath  c l o s e p r o x i m i t y of the mesophyll c e l l s and  the bundle  c e l l s t o the v a s c u l a r system i n low compensation p l a n t s  would appear to f a c i l i t a t e a more d i r e c t e x i t of photosynthate from the l e a f than i n h i g h compensation p l a n t s . study by H o f s t r a (22)  A translocation  shows t h a t 24 hours a f t e r a pulse  feeding  14 of  C0 , 2  l e a v e s of com,  millet  (Panlcum mlllaceum).  and  sorghum  (low compensation p a n i c o i d s ) r e t a i n e d 12-15$ of a s s i m i l a t e d i n the area which was  fed.  N l o o t l a n a and  "^C  soybean (high compen-  22  Figures 2-7. Surface views of the patterns of starch d i s t r i bution i n dicotyledon leaves of the two carbon dioxide compensation categories. Figures 2-4. Low compensation leaves. Dark areas represent starch i n chlorenchymatous c e l l s surrounding the vascular bundles. Figure 2. Amaranthus r e t r o flexus, X 90. Figure 3. A t r l p l e x rosea. X 60. Figure 4. Portulaca oleracea, X 45. Figures 5-7. High compensation leaves. Dark areas reveal the presence of starch i n the mesop h y l l . Note the lack of starch along the vascular bundles which appear white. Figure 5. Celosla golden plume, X 90. Figure 6. Chenopodium album. X oTT Figure 7. A t r l p l e x hastata, X 75.  23 s a t i o n p l a n t s ) r e t a i n e d 30-50$ of t h e i r a s s i m i l a t e s a t the of the same time p e r i o d . that i n variegated  Rhoades and  Carvalho (36)  maize, s t a r c h d e p o s i t i o n  concluded  i n the sheath p l a s -  t i d s occurs when the r a t e of t r a n s l o c a t i o n from the  mesophyll  c e l l s exceeds the r a t e of movement from the sheath c e l l s the v a s c u l a r  elements.  They a l s o showed that s t a r c h  into  deposition  i n the mesophyll c h l o r o p l a s t s of maize occurred  i f sucrose  f e d to the l e a v e s  the u s u a l  f o r long periods.  Therefore,  of s t a r c h i n the mesophyll of these p l a n t s was  different  of the mesophyll and Taken together,  Inherent c a p a c i t y of the  a l l these c o r r e l a t i o n s suggest some i n t e r -  translocation.  Invoked.  The  photo-  l e a f anatomy, s t a r c h  following explanation  distrimight  P l a n t s h a v i n g the C ^ - p h o t o s y n t h e t i c pathway have  much h i g h e r r a t e s of p h o t o s y n t h e s i s and synthate p r o d u c t i o n  hence g r e a t e r  a c c o u n t a b l e f o r , i n p a r t , by the  t i o n of carbon through the l a c k of p h o t o r e s p i r a t i o n . of CO2 e v o l u t i o n d u r i n g p h o t o s y n t h e s i s may substrate  cells  sheath c e l l s t o form s t a r c h .  synthetic rate, photorespiration,  be  lack  chloroplasts  r e l a t i o n between C3-/Ci|.-metabolism i n p h o t o s y n t h e s i s ,  b u t i o n , and  was  suggested t o  depend upon t r a n s l o c a t i o n of photosynthate out of these r a t h e r than any  end  of CO2 p r o d u c t i o n  conservaThis  be a r e s u l t of  lack the  b e i n g used In the C^-pathway of  p h o t o s y n t h e s i s i n s t e a d of b e i n g d e c a r b o x y l a t e d . the C^-pathway f r e q u e n t l y  photo-  inhabit a r i d areas.  reasonable t o assume t h a t there has  Species with Thus i t seems  been s e l e c t i o n f o r a  close  r e l a t i o n s h i p of the l e a f a s s i m i l a t o r y t i s s u e s to the water columns.  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Hofstra, G. 1967. An approach to the study of the physiol o g i c a l control of translocation In higher plants. Ph.D. Thesis, Simon Fraser University, Burnaby, B.C.  23.  Johnson, H.S. and M.D. Hatch. 1968. D i s t r i b u t i o n of the Cjj.-dicarboxylic a c i d pathway of photosynthesis and i t s occurrence i n dicotyledonous plants. Phytochemlstry 7: 375-380.  24.  Kawanabe, S. 1968. Temperature responses and systematlcs of the Gramineae. Proceedings Jap. Soc. Plant Taxonomists 2: 17-20.  25.  Kortschak, H.P., C.E. Hart and G.0. Burr. 1965. Carbon d i oxide f i x a t i o n i n sugarcane leaves. Plant Physiol. 40: 209-213.  2  2? 26.  L a e t s c h , W.M., D.A. S t e t i e r and A . J . V l i t o s . 1966. The u l t r a s t r u c t u r e o f sugarcane c h l o r o p l a s t s . Z. P f l a n z e n p h y s l o l . 5^: 472-474.  27.  L a e t s c h , W.M. 1968. C h l o r o p l a s t s p e c i a l i z a t i o n i n d i cotyledons p o s s e s s i n g the C ^ - d i c a r b o x y l i c a c i d pathway of p h o t o s y n t h e t l c C 0 f i x a t i o n . Am. J . Botany 55: 2  875-883.  28.  L a e t s c h , W.M. and I a n P r i c e . 1969. Development o f the d i morphic c h l o r o p l a s t s o f sugarcane. Am. J . Botany 56:  77-87.  29.  Meidner, H. 1962. The minimum i n t e r c e l l u l a r - s p a c e C 0 conc e n t r a t i o n ( T"") o f maize l e a v e s and i t s i n f l u e n c e on stomatal movements. J . E x p t l . Botany 131 284-293. 2  1  30.  Moser, H. 193^. Untersuchungen uber d i e B l a t t s t r u k t u r e von A t r l p l e x . B e i h . Bot. Z b l . 52: 378-388.  31.  Moss, D.N. 1962. The l i m i t i n g carbon d i o x i d e c o n c e n t r a t i o n f o r p h o t o s y n t h e s i s . Nature 193: 587.  32.  Murata, Y. and J . Iyama. 1963. S t u d i e s on the photosynt h e s i s o f forage c r o p s . I I . I n f l u e n c e o f a i r - t e m p e r a t u r e upon the p h o t o s y n t h e s i s o f some forage and g r a i n crops. Proc. Crop S c i . Soc. Japan 31: 315-322.  33.  Murata, Y., J . Iyama and T. Honma. 1965. S t u d i e s on the p h o t o s y n t h e s i s o f forage c r o p s . IV. I n f l u e n c e o f a i r temperature upon the p h o t o s y n t h e s i s and r e s p i r a t i o n of a l f a l f a and s e v e r a l southern type forage c r o p s . Proc. Crop S c i . Soc. Japan 34: 154-158.  34.  Osmond, B. I967. ^ - c a r b o x y l a t i o n d u r i n g p h o t o s y n t h e s i s i n A t r l p l e x . Biochlm. Biophys. A c t a 141: 197-199.  35.  P r a t , H. 1936. La systematique des graminees. Ann. S c i . Nat. (Botan.) Ser. 10, 18: 165-258.  36.  Rhoades, M.M. and A. Carvalho. 1944. The f u n c t i o n and s t r u c t u r e o f the parenchyma sheath p l a s t i d s o f the maize l e a f . B u l l . T o r r e y Botan. Club 71: 335-3^6.  37.  S t e b b i n s , G.L. 1956. C y t o g e n e t i c s and e v o l u t i o n o f t h e grass f a m i l y . Am. J . Botany 43: 890-905.  38.  Tregunna, E.B., G. Krotkov and C D . Nelson. 1966. E f f e c t of oxygen on the r a t e o f p h o t o r e s p i r a t i o n i n detached tobacco l e a v e s . P h y s i o l . Plantarura 19: 723-733.  39.  Tregunna, E.B. and J . Downton. 1967. Carbon d i o x i d e comp e n s a t i o n i n members o f t h e Amaranthaceae and some r e l a t e d f a m i l i e s . Can. J . Botany 45: 2385-2387.  28  40.  Tregunna, E.B., J . Downton and P. J o l l i f f e . 1968. Genetic and environmental control of photorespiration. In "Progress i n Photosynthesis Research". Proceedings of the International Congress of Photosynthesis Research, Freudenstadt, Germany. In press.  41.  Tregunna, E.B., B.N. Smith, W.J.S. Downton and J.A. Berry. 1969. Some methods f o r studying the taxonomy of photosynthesis i n the Angiosperms. In preparation.  29 P a r t 1-B.  I n t e r s p e c i f i c D i f f e r e n c e s i n Carbon D i o x i d e Comp e n s a t i o n and the P&th o f Carbon i n P h o t o s y n t h e s i s Among Non-graminaceous Genera.  INTRODUCTION An e a r l i e r study (12) r e v e a l e d t h a t both h i g h and low comp e n s a t i o n s p e c i e s belonged t o the genus A t r l p l e x  (Chenopodiaceae).  I t was l a t e r found t h a t i n t e r s p e c i f i c d i f f e r e n c e s i n compensat i o n v a l u e e x i s t e d a l s o w i t h i n the genera Cyperus and B a s s l a  (Chenopodiaceae)  (13).  (Cyperaceae)  Biochemical information  p r o v i d e d by Osmond (9) and Johnson e t a l .  (6) showed t h a t A t r l p l e x  spongiosa. A t . semlbaocata. Amaranthus v l r l d u s . Am. p a l m e r l . Gomphrena c e l o s o l d e s . Cyperus rotundus. C. p o l y s t a c h y o s and C. bowma n n l s y n t h e s i z e d Cij-intermediates s i m i l a r t o those found f o r t r o p i c a l g r a s s e s such a s Zea mays (5).  Cyperus g r a c i l i s , on the  o t h e r hand, produced phosphorylated compounds o f the C a l v i n c y c l e . In view o f these f i n d i n g s , experiments were conducted t o d e t e r mine i f the CO2 compensation v a l u e would  serve a s a r e l i a b l e i n -  d i c a t o r of the nature o f the path o f carbon where d i f f e r e n c e s e x i s t e d w i t h i n the same genus.  compensation  A few o t h e r s p e c i e s  were t e s t e d .  MATERIALS AND METHODS P l a n t s were grown from seed a t c a . 2000 f t - c , 16 hour day, 21-26/18-22°  day/night temperature i n a growth room.  Papyrus was o b t a i n e d from greenhouse s t o c k .  Cyperus  30  Carbon d i o x i d e compensation v a l u e s were measured as des c r i b e d i n the "Methods" s e c t i o n o f P a r t 1-A.  Determination  of p h o t o s y n t h e t l c products i n v o l v e d p r e - e q u i l i b r a t i n g detached l e a v e s under 1000-2000 f t - c a t 20-24° b e f o r e exposing them t o 14 C0  2  f o r 6 seconds*  The l e a v e s were then k i l l e d and e x t r a c t e d  i n b o i l i n g 80% e t h a n o l .  The e x t r a c t s o f Zea mays, Amaranthus  e d u l i s , A t r l p l e x h a s t a t a . A t , rosea and Gomphrena globosa were r e s o l v e d by two-dimensional chromatography  on t h i n - l a y e r p l a t e s  coated w i t h " A v i c e l " m i c r o c r y s t a l l i n e c e l l u l o s e .  The s o l v e n t  systems used were those o f S h i r o y a e t a l , (11) f o r paper chromatography: l i q u e f i e d phenol ( c a . 80%) w i t h 0.3% ammonium hydroxide i n the f i r s t dimension f o l l o w e d by n-propanol - e t h y l a c e t a t e water  (7:1:2) developed twice i n the second dimension.  Radio-  a c t i v i t y was l o c a t e d by autoradiography. R a d i o a c t i v e a r e a s were scraped from t h e p l a t e s and a c t i v i t y determined by l i q u i d  scin-  t i l l a t i o n counting. B a s s l a convexula. B. h y s s o p i f o l l a . Cyperus Papyrus and C. albomarglnatus e x t r a c t s were r e s o l v e d on paper s t r i p s  irrigated  w i t h l i q u e f i e d phenol ( c a . 90%) - a c e t i c a c i d - water - 1 M. ethylene-diaminetetraacetic acid  (840:160:10:1) (10).  The p e r -  cent o f t o t a l a c t i v i t y i n the separated compounds was d e t e r mined from chromatogram scanner t r a c i n g s . I.  Chromatography was s t a n d a r d i z e d as d e s c r i b e d I n Appendix  31  RESULTS AND The  DISCUSSION  type of p h o t o s y n t h e t l c pathway t h a t operates w i t h i n  a g i v e n s p e c i e s can be determined dimensional  chromatographic  most r a p i d l y w i t h the  technique d e s c r i b e d above.  though i t does not c l e a r l y separate 3-phosphoglyceric from o t h e r phosphorylated labelled  oneAl-  acid  compounds, the method r e s o l v e s the  i n t e r m e d i a t e s of C a l v i n c y c l e - and C^-plants  s u f f i c i e n t l y d i f f e r e n t patterns to give a r e l i a b l e  into  technique  Ik f o r very short-term  C0  2  f i x a t i o n studies.  T a b l e I shows t h a t a l t h o u g h i n t e r s p e c i f i c d i f f e r e n c e s i n C0  2  compensation occur, t h e r e i s a constant and p r e d i c t a b l e r e -  l a t i o n of the p h o t o s y n t h e t l c pathway type to t h i s v a l u e , i . e . low compensation w i t h C^-compounds and h i g h compensation w i t h phosphorylated  compounds.  The  low compensation s p e c i e s i n  t h i s l i s t a l s o have parenchyma bundle sheaths  (2,  13).  The  p h o t o s y n t h e t l c d i v e r s i t y r e p o r t e d here should c a u t i o n one i n g e n e r a l i z i n g from one s p e c i e s to another. compensation p o i n t or examination  Measurement of the  of the l e a f anatomy p r o v i d e s  a f a s t and r e l i a b l e method f o r a s s a y i n g the path of carbon i n an u n t e s t e d s p e c i e s . Low  compensation v a l u e s among the Gramineae a r e  to c e r t a i n p h y l o g e n e t i c l i n e s (2).  W i t h i n the genus Panlcum,  h i g h and low compensation p o i n t s a r e found genera (3,  8,  P a r t 1-C).  genus Euphorbia  (8).  restricted  in different  Moss et a l . a l s o found  sub-  t h i s f o r the  No d e f i n i t e c o n c l u s i o n s can be drawn f o r  s p e c i e s of Cyperus and B a s s l a a t t h i s time because of the p a u c i t y  TABLE I.  I n t e r s p e c i f i c d i f f e r e n c e s i n carbon d i o x i d e compensation and e a r l y products f o r s p e c i e s f e d * C 0 f o r s i x seconds.  photosynthetlc  2  % o f t o t a l -^C i n : aspartate  Species  C02 comp. in 21$ 0  Atrlplex Atrlplex Atrlplex  hastata hortensis rosea  H H L  Bassia Bassla  convexula hyssopifolia  H L  Cyperus Cyperus  albomarglnatus papyrus  Amaranthus  Genus  malate  3-phosphosugar other g l y c e r a t e phosphates compounds t o t a l phosphates  a  2  0  0 0  6 6 6  89 89  5 5  6 55  38  0  0  17  69  L H  10 0  86  edulis  L  67  25  8  0  0  Gomphrena  globosa  L  23  70  7  0  0  Zea  mays  L  26  60  5  9  0  a  D a t a o f Tregunna and Downton (12)  H = comp. o f approx. 50  ppm.  1  26  74 14  0  4 86  0  and Tregunna e t a l . (13).  0  0  14  L=comp, l e s s than 5 PPm.  33 of a v a i l a b l e compensation d a t a .  Of those s p e c i e s t h a t have been  s t u d i e d , some have not been c o n s i d e r e d i n papers which present taxonomic schemes.  Many s p e c i e s of A t r l p l e x have been t e s t e d ,  however, such t h a t a b r i e f c o n s i d e r a t i o n o f the taxonomy and. compensation p o i n t s o f the genus i s i n order (12, 13, 1 4 ) . A study by H a l l and Clements (4) o f American s p e c i e s o f A t r l p l e x used the p o s i t i o n o f the r a d i c l e i n the seed t o d i v i d e the genus i n t o the subgenera E u a t r i p l e x and Obione.  Moser,  (7)  who s t u d i e d the genus on a world-wide b a s i s , d i v i d e d i t i n t o f i v e subgenera based on embryo p o s i t i o n w i t h i n the seed and f r u i t i n g bract characters.  From T a b l e I I i t i s e v i d e n t t h a t  both low:: and hicgh compensation s p e c i e s (and t h e r e f o r e s p e c i e s w i t h and without a bundle sheath) have been p l a c e d w i t h i n some o f the same subgenera.  Moser c o n s i d e r e d the occurrence o f the bundle  sheath t o be a mutable c h a r a c t e r .  Thus he used the presence o r  absence o f the bundle sheath as a c r i t e r i o n only a t lower t a x onomic l e v e l s .  Had the bundle sheath been a major c r i t e r i o n i n  c l a s s i f i c a t i o n , h i s c o n c l u s i o n might have been t h a t the p o s i t i o n of the r a d i c l e w i t h i n the seed i s h i g h l y v a r i a b l e .  I am i n no  p o s i t i o n , however, t o judge which c r i t e r i a most c l e a r l y the e v o l u t i o n of the genus.  reflect  Since there has been no major r e -  view o f the genus A t r l p l e x s i n c e the 193O's,  1  i t would appear  p r o f i t a b l e t o re-examine the genus i n view o f r e c e n t f i n d i n g s .  1 Dr. Brenan ( p e r s o n a l communication). Dr. Brenan i s the Keeper o f the Herbarium, R o y a l B o t a n i c Garden, Kew, England.  TABLE I I .  C l a s s i f i c a t i o n of the genus A t r l p l e x (according to Moser) i n r e l a t i o n to carbon dioxide compensation values. C0  Subgenus Euatrlplex  Section  Subsection  Dichospermum Teutliopsls  Schizotheca  Obionopsis Obione  Normophyllum Ornophyllum  Pterophyton Teleophyton Spongiocarpus  L = comp. less than 5 PPm, H = comp. about 50 ppm. + = present, - = absent.  J  2  Species  comp.  hortensis nitens  H H  hastata littoralls oblongfolia halimus tartarica rosea  H H H L L L  1  bundle sheath  Hef.  (13) (13)  + +  (2, 12) (7. 14) (12.1*0 (7. 13) (7. 14) (2, 12) (7)  nummalaria sibirica  L L  + + + +  (7. 14) (7. 13) (7) (7)  holocarpa spongiosa  L L  + +  (7. 13) (7. 13)  35 I f the occurrence of the bundle sheath among these i s Indeed a h i g h l y mutable c o n d i t i o n , then the c o n v e r s i o n p l a n t with the C a l v i n c y c l e of CO2 f i x a t i o n and t o one w i t h the h i g h l y e f f i c i e n t C^-pathway may (See  Part  1-C).  species of a  photorespiration be e a s i l y a c h i e v e d  36  LITERATURE CITED 1.  Bandurski, R.S. and B. A x e l r o d . 1951. The chromatographic i d e n t i f i c a t i o n o f some b i o l o g i c a l l y important phosphate e s t e r s . J o u r . B i o l . Chem. 193: 405-410.  2.  Downton, W.J.S. and E.B. Tregunna. 1 9 6 8 . Carbon d i o x i d e compensation - i t s r e l a t i o n to photosynthetlc carboxylation r e a c t i o n s , s y s t e m a t l c s o f the Gramineae, and l e a f anatomy. Can. J . Botany 46: 2 0 7 - 2 1 5 ( P a r t 1 - A ) .  3.  Downton, John, Joseph B e r r y and E. Bruce Tregunna. 1 9 6 9 . P h o t o s y n t h e s i s : Temperate and t r o p i c a l c h a r a c t e r i s t i c s w i t h i n a s i n g l e grass genus. Science 1 6 3 : 7 8 - 7 9 .  4.  H a l l , H.M. and F.E. Clements. 1 9 2 3 . The p h y l o g e n e t i c method i n taxonomy. Carnegie I n s t . Wash. P u b l .  5.  Hatch, M.D., C.R. S l a c k and H.S. Johnson. 1 9 6 7 . F u r t h e r s t u d i e s on a new pathway o f p h o t o s y n t h e t l c carbon d i o x i d e f i x a t i o n i n sugarcane and i t s occurrence i n other p l a n t s p e c i e s . Biochem. J . 1 0 2 : 4 1 7 - 4 2 2 .  6. Johnson, H.S. and M.D. Hatch. I 9 6 8 . D i s t r i b u t i o n of  C4-  d i c a r b o x y l l c a c i d pathway i n p h o t o s y n t h e s i s and i t s occurrence i n d i c o t y l e d o n o u s p l a n t s . Phytochemistry 7:  375-380.  7.  Moser, H. 1 9 3 4 . Untersuchungen uber d i e B l a t t s t r u k t u r e von A t r l p l e x . B e i h . Bot. Z b l . 5 2 : 3 7 8 - 3 8 8 .  8.  Moss, Dale N., Eugene G. Krenzer, J r . and W i l l i a m A. Brun. 1 9 6 9 . Carbon d i o x i d e compensation p o i n t s i n r e l a t e d p l a n t species. Science 1 6 4 : 1 8 7 - 1 8 8 .  9.  Osmond, B. 1 9 6 7 . ^ - c a r b o x y l a t i o n d u r i n g p h o t o s y n t h e s i s i n A t r l p l e x . Biochlm. Biophys. A c t a 141: 197-199.  10.  Pedersen, T.A., M. K i r k and J.A. Bassham. 1 9 6 6 . I n h i b i t i o n of phot©phosphorylation and p h o t o s y n t h e t l c carbon c y c l e r e a c t i o n s by f a t t y a c i d s and e s t e r s . Biochlm. Biophys.  Acta 1 1 2 : 1 8 9 - 2 0 3 .  1 1 . S h i r o y a , T., V. S l a n k l s , G. Krotkov and C D . Nelson.  I962.  The nature o f photosynthate i n Plnus strobus s e e d l i n g s . Can. J . Botany 40: 669-676.  12.  Tregunna, E.B. and J . Downton. 1 9 6 7 . Carbon d i o x i d e comp e n s a t i o n i n members o f the Amaranthaceae and some r e l a t e d f a m i l i e s . Can. J . Botany 4 5 : 2 3 8 5 - 2 3 8 7 .  37 13. Tregunna, E.B., J . Downton and P. J o l l i f f e . 1968. Genetic and environmental control of photoresplratlon. In "Progress In Photosynthesis Research". Proceedings of the International Congress of Photosynthesis Research, Preudenstadt, Germany. In press. 14. Iregunna, E.B., B.N. Smith, W.J.S. Downton and J.A. Berry. 1969. Some methods f o r studying the taxonomy of photosynthesis i n the Angiosperms. In preparation.  38  APPENDIX I S t & n d a r d i z a t l o n of Chromatography 1.  Two-dimensional chromatography. P l a n t e x t r a c t s were s p o t t e d on t h i n - l a y e r p l a t e s coated  w i t h a 0.5  mm t h i c k , 18 cm x 18 cm l a y e r o f ' A v l c e l ' micro-  crystalline cellulose. phenol  ( c a . 80$)  The p l a t e s were developed  with 0.3$  dimension and i n n-propanol  i n liquefied  ammonium hydroxide i n the f i r s t - e t h y l a c e t a t e - water (7*1*2) fe  twice i n the second dimension  (11).  P l a t e s were developed i n  a s o l v e n t mixture u n t i l the f r o n t reached the l i m i t o f the • A v i c e l ' c o a t i n g , and then allowed t o d r y b e f o r e b e i n g p l a c e d i n the next s o l v e n t . The f o l l o w i n g standards were used t o c o n s t r u c t the map i l l u s t r a t e d i n F i g u r e 1: (a)  L - a s p a r t i c a c i d , L-glutamic a c i d , L - a l a n i n e , Lg l y c i n e and L - s e r i n e (Calbiochem).  These were de-  t e c t e d with n i n h y d r i n . (b)  s u c r o s e - ^ U.L. (New England N u c l e a r C o r p . ) , D1  14 glucose-1acid-4-  lZf  C  c (New England Nuclear Corp.), L - m a l i c (Calbiochem), and g l y c o l i c  acid-l-^C  ( I n t e r n a t i o n a l Chemical and Nuclear Corp.).  These  compounds were d e t e c t e d by autoradiography. (c)  3-phospho-D-glyceric  acid  6-phosphate (Calbiochem),  (Calbiochem),  fructose-  glucose-6-phosphate  ( N u t r i t i o n a l B i o c h e m i c a l s Corp.) and r i b o s e - 5 - p h o s phate©(Nutritional B i o c h e m i c a l s C o r p . ) .  These com-  pounds were d e t e c t e d w i t h phosphomolybdate reagent  39 and u l t r a v i o l e t l i g h t ( 1 ) . With the exception of some of the phosphorylated standards, the  above compounds were c l e a r l y resolved when a mixture of a l l  the  standards was chromatographed.  Only 3-phosphoglyceric acid  was resolved i n a mixture of the phosphorylated standards. The sugar phosphates tended to streak i n the second dimension. Shiroya e t a a l . (11) used ethyl acetate - a c e t i c a c i d - water (3:1 si) followed by ethyl ether - acetic a c i d - water (13:3*11 i n the second dimension to separate organic acids on paper. This system was t r i e d on 'Avicel' coated thin-layer plates, but abandoned because the organic a c i d spots were extremely d i f f u s e . 2.  One-dimensional  chromatography.  Strips of Whatman No. 1 chromatography paper (4 cm x 60 cm) were spotted with plant extracts and run descending to the paper's edge i n l i q u e f i e d phenol (ca. 90$) - a c e t i c acid - water ethylenediaminetetraacetic acid (840:160:10:1). This solvent mixture was the same as that used by Pedersen et a l . the  f i r s t dimension of a two-dimensional system.  (10) f o r  This one-dimen-  sional modification was f i r s t used i n our laboratory by Mr. Joseph Berry. The same set of standards described above were used to c a l culate the R  f  values of the compounds l i s t e d In Table I I I . Ex32  tracts from wheat seedlings fed the  P were also used to confirm  l o c a t i o n of phosphorylated compounds.  40  TABLE I I I . Rf. values f o r some compounds separated by onedimensional paper chromatography using l i q u e f i e d phenol (ca. 90$) - a c e t i c acid - water - 1M ethylenediaminetetraacetic a c i d (840:160:10:1) as the solvent. Compound  Rf value  alanine  0.62  aspartic a c i d  0.23  glycine  0.45  glutamic a c i d  0.67  serine  0.39  glucose  0.43  sucrose  0.45  glycolic acid  0.49  malic a c i d  0.32  3-phosphoglyceric a c i d fructose-6-phosphate glucose-6-phosphate ribose-5-phosphate  0.08 - 0.09  P R O P A N O L -  E T H Y L  A C E T A T E  -  W A T E R  (7:1:2)  Figure 1. A standard map f o r some compounds separated by twodimensional chromatography on thin-layer plates coated with 'Avicel' mlcrocrystalline c e l l u l o s e . Developed In QQ% phenol with 0.3# ammonium hydroxide i n the f i r s t dimension and n-propanol - ethyl acetate - water (7:1»2) twice In the second dimension.  42 Part 1-C. Photosynthesis: Temperate and Tropical Characterist i c s Within a Single Grass Genus.  1  INTRODUCTION Tropical grasses can f i x C0 a t a rate almost twice that 2  of temperate species (6, 12, 13).  The lower photosynthetlc  rate of temperate leaves i s probably a r e s u l t of photoresplratlon, because blocking t h i s process by lowering the ambient 0  2  concentration (7, 17) can increase the rate of photosynthesis  to approximately that of t r o p i c a l leaves (9).  Temperate plant  yields can also be increased substantially under these conditions (1).  Consequently  genetic s e l e c t i o n f o r greater carbon  conservation during photosynthesis as a means of increasing dry matter production among temperate grass crops i s p a r t i c u larly attractive.  At the same time, the introduction of tem-  perate or t r o p i c a l characters into species of the opposite type may extend t h e i r range of c u l t i v a t i o n . To date we have been unable to test these p o s s i b i l i t i e s i n the Gramineae because of the lack of v a r i a b i l i t y within a phylogenetic l i n e .  That i s , genera belonging to the same phylo-  genetic group have b a s i c a l l y the same photosynthetlc physiology and internal leaf anatomy.  Therefore we have been interested  i n species of any groups reported to have c h a r a c t e r i s t i c s that are i n apparent disagreement with former c o r r e l a t i o n data (4).  1 This a r t i c l e by John Downton, Joseph Berry and E. Bruce Tregunna appeared i n Science. V o l . 163: 78-79 (1969). Joseph Berry conducted the PEP carboxylase assays; E. Bruce Tregunna supervised the study.  ^3 The report (3)  that parenchyma bundle sheath c e l l s of the  rosette leaves of Panicum llndhelmerl do not contain " s p e c i a l ized starch p l a s t i d s t y p i c a l of the t r i b e " was valuable i n this regard.  This species belongs to the subgenus Dichanthelium,  a  group of more than 100 species confined c h i e f l y to eastern North America (2, 10).  Since our previous survey considered  only members of the l a r g e l y t r o p i c a l subgenus Eupanicum, we have extended i t to include some dichantheloid species.  It is  evident that two d i f f e r e n t functional patterns exist within t h i s economically important genus.  MATERIALS AND METHODS Photorespiration was estimated by measuring the C0  2  com-  pensation point of detached leaves as described i n the "Methods" section of Part 1-A.  To determine i n i t i a l photosynthetlc pro-  ducts, leaves were illuminated at 1000 to  C0  2  f t - c and then exposed  i n a i r f o r 6 seconds; they were then k i l l e d and  tracted i n b o i l i n g 80$ ethanol.  ex-  Compounds were resolved on  paper s t r i p s with a l i q u e f i e d phenol (ca. 90%), a c e t i c a c i d , water, 1M ethylenediaminetetraacetic a c i d system (840:l60:10jl) (14).  A chromatogram scanner was used to measure the amount  14 of  c incorporated into photosynthetlc intermediates.  enolpyruvate  Phospho-  (PEP) carboxylase (E.C. 4.1.1.31) a c t i v i t y was  sayed according to Slack and Hatch (15).  as-  Freehand cross sections  of leaves were examined microscopically f o r anatomical  detail.  Addition of IKI to the water mount indicated the areas of starch accumulation.  44  RESULTS Subgenus Eupanlcum Members of the subgenus Eupanlcum, such as Panlcum b u l bosum, P. c a p l l l a r e . and P. millaceum, l i k e other species of the panicoid and chloridoid-eragrostoid l i n e s of phylogeny, formed C^-dicarboxylic acids (malate and aspartate) as i n i t i a l photosynthetlc  products, but d i d not evolve C0  t i o n (4, 8 ) ,  The amount of  2  2  by photorespira-  incorporated into aspartic and 14 ?  malic acids ranged from 85 to 92$ of the t o t a l  C fixed.'' Con-  s i s t e n t with t h i s , leaves of Panicum millaceum contained much PEP carboxylase a c t i v i t y (28.15 jimole of C0  fixed per m i l l i -  2  gram of extractable chlorophyll per minute).  This enzyme i s  used f o r the synthesis of C^-compounds (15).  The parenchyma  bundle sheath was extensively developed and contained large starch-laden chloroplasts.  The mesophyll c e l l s accumulated  l i t t l e starch. Subgenus Dichanthelium The dichantheloid species, Panlcum commutatum. P. l l n d helmerl. and P. paclflcum. l i k e the previously studied temperate grasses, synthesized phosphorylated  compounds t y p i c a l  of the Calvin cycle, as major products of C0  ?  f i x a t i o n (8).  2 D. Moss, personal communication. D. Moss (University of Minnesota) has Independently found the same d i s t r i b u t i o n of C0 compensation values i n Panlcum. (See Science 164: 187-188). 3 A more detailed account of the pounds i s given In Appendix I.  14 C d i s t r i b u t i o n among com-  2  *5  Phosphorylated compounds accounted f o r 86 to 95$  of the t o t a l  14  C fixed. way  The leaves a l s o had an active photorespiratory path-  manifested by the evolution of CO2 during As expected (15),  (4).^ low PEP  carboxylase  photosynthesis  l e a f extracts of Panicum paclflcum  a c t i v i t y (3.16  had  umole of CO2 f i x e d per m i l l i -  gram of extractable chlorophyll per minute).  This value  v i r t u a l l y i d e n t i c a l to that found f o r wheat leaves.  was  Unlike many  temperate grasses, however, leaves of the subgenus Dichanthelium contained  extensively developed parenchyma bundle sheaths.  De-  spite t h i s elaboration, chloroplasts were absent from the tissue. A mestom sheath was also present.  Leaves of the rosette, vernal,  and autumnal growth phases were s i m i l a r i n structure and photosynthetlc  physiology, DISCUSSION  The absence of detectable photorespiration i n plants of t r o p i c a l o r i g i n could r e s u l t from a deficiency of the pathway found i n temperate species (5, that prevents C 0 f o r C0  2  2  loss.  i s well known (18,  11,  16),  or from a mechanism  The great a f f i n i t y of PEP 19).  carboxylase  The high a c t i v i t y of t h i s en-  4 Moss, personal communication, op....clt. 5 I t has since become apparent that t h i n sections of dichant h e l o i d leaves r e a d i l y lose the contents of t h e i r bundle sheath c e l l s . Thicker sections of these leaves Indicate that some chloroplasts are present i n the bundle sheath c e l l s . These chloroplasts - few i n number, peripherally located, somewhat smaller and paler than those of the mesophyll and containing very l i t t l e starch - are not comparable to the specialized chloroplasts of bundle sheath cells" i n eupanicoid species.  46 zyme In t r o p i c a l plants might preclude the release of C0 the outside of the l e a f .  to  2  The s p e c i a l i z e d chloroplasts within  the parenchyma bundle sheath could a l s o be a determinant. Whatever the correct explanation may  be, the important point  i s that t r o p i c a l grass species do not lose carbon during photosynthesis and temperate species do.  The discovery of d i f f e r e n t  physiological and c y t o l o g l c a l phenotypes may  now  within the same genus  permit genetic analysis of photoresplratlon and  assess-  ment of i t s importance to the carbon budget of the plant.  6 W.V. Brown, personal communication. As a consequence of these findings, W.V. Brown (University of Texas) has suggested that a separate genus be proposed f o r the subgenus Dichanthelium.  47  LITERATURE CITED 1.  Bjorkman:-, 0. 1967. E f f e c t of oxygen concentration on dry matter production i n higher plants. Carnegie Inst. Wash. Year Book 66: 228-232.  2.  Brown, W.V. 1948. A c y t o l o g l c a l study i n the Gramineae. Amer. J . Botany 35: 382-395.  3.  Brown, W.V. 1958. Leaf anatomy i n grass systematica. Botan. Gaz. 119: 170-178.  4.  Downton, W.J.S. and E.B. Tregunna. 1968. Carbon dioxide compensation - i t s r e l a t i o n to photosynthetlc carboxyl a t i o n reactions, systematics of the Gramineae, and l e a f anatomy. Can. J . Botany 46: 207-215. (Part 1-A).  5.  Downton, W.J.S. and E.B. Tregunna. 1968. Photorespiration and glycolate metabolism: A re-examlnation and corr e l a t i o n of some previous studies. Plant Physiol. 43: 923-929. (Part 2).  6.  El-Sharkawy, M.A., R.S. Loomis and W.A. Williams. I967. Apparent reassimilation of respiratory carbon dioxide by d i f f e r e n t plant species. Physiol. Plantarum 20: 171-186.  7.  Forrester, M.L., G. Krotkov and C D . Nelson. 1966. E f f e c t of oxygen on photosynthesis, photorespiration and resp i r a t i o n i n detached leaves. I. Soybean. I I . Corn and other monocotyledons. Plant Physiol. 41: 422-431.  8.  Hatch, M.D., C R . Slack and H.S. Johnson. 1967. Further studies on a new pathway of photosynthetlc carbon dioxide f i x a t i o n i n sugar-cane and i t s occurrence i n other plant species. Biochem. J . 102: 417-422.  9.  Hesketh, J . I967. Enhancement of photosynthetlc C02-ass i m i l a t i o n i n the absence of oxygen, as affected by species and temperature. Planta 76: 371-374.  10.  Hitchcock, A.S. 1951. Manual of the grasses of the United States. 2nd ed. U.S.D.A. Misc. Pub. No. 200, pp. 626706.  11.  Meidner, H. 1967. Further observations on the minimum i n t e r c e l l u l a r space carbon-dioxide concentration ( P ) of maize leaves and the postulated roles of "photor e s p i r a t i o n " and glycolate metabolism. J . Exptl. Botany 18: 177-185.  48 12.  Murata, Y. and J . Iyama. 1963. Studies on the photosynthesis of forage crops. I I . Influence of air-temperature upon the photosynthesis of some forage and grain crops. Proc. Crop S c i . Soc. Japan 31: 315-322.  13.  Murata, Y., J . Iyama and T. Honma. 1965. Studies on the photosynthesis of forage crops. IV. Influence of a i r temperature upon the photosynthesis and r e s p i r a t i o n of a l f a l f a and several southern type forage crops. Proc. Crop S c i . Soc. Japan 34: 154-158.  14.  Pedersen, T.A., M. Kirk and J.A. Bassham. 1966. I n h i b i t i o n of photophosphorylation and photosynthetlc carbon cycle reactions by f a t t y acids and esters. Biochlm. Biophys. Acta 112: 189-203.  15.  Slack, C.R. and M.D. Hatch. 1967. Comparative studies on the a c t i v i t y of carboxylases and other enzymes i n r e l a t i o n to the new pathway of photosynthetlc carbon dioxide f i x a t i o n i n t r o p i c a l grasses. Biochem. J . 103: 66O-665.  16.  Tregunna, E.B., G. Krotkov and C D . Nelson. 1964. Further evidence on the effects of l i g h t on r e s p i r a t i o n during photosynthesis. Can. J . Botany 42: 989-997.  17.  Tregunna, E.B., G. Krotkov and C D . Nelson. 1966. E f f e c t of oxygen on the rate of photoresplratlon i n detached tobacco leaves. Physiol. Plantarum 19: 723-733.  18.  Walker, D.A. and J.M.A. Brown. 1957. Physiological studies on a c i d metabolism. 5. E f f e c t s of carbon dioxide concentration on phosphoenolpyruvic carboxylase a c t i v i t y . Biochem. J . 67: 79-83.  19.  Walker, D.A. 1966. Carboxylation i n plants. Endeavour 25: 21-26.  APPENDIX I TABLE I.  The d i s t r i b u t i o n of r a d i o a c t i v i t y among compounds f o r Panicum spp. fed f o r s i x seconds.  CO  % of t o t a l ^ c i n :  Subgenus  malate  aspartate  P. bulbosum  59  26  15  0  P. c a p l l l a r e  25  64  5  6  P. mlliaceum  19  73  6  2  P. commutatum (rosette phase)  0  0  78  22  (vernal + autumnal phase)  0  0  93  7  P. lindheimeri (rosette phase)  0  0  95  5  (autumnal phase)  0  0  94  6  P. paclficum (rosette phase)  0  0  86  14  Species  phosphorylated compounds  other compounds  Eupanicum  Dichanthelium  50 Part 2.  Photoresplratlon and Glycolate Metabolism: A Re-examinat l o n and Correlation of Some Previous S t u d i e s .  1  INTRODUCTION Photorespiration has been described as a C0  producing  2  process that operates during photosynthesis i n some leaves and algae.  The newer evidence supporting t h i s concept i s based on  the response of photorespiration to 0  2  concentration.  I t has  been shown that dark r e s p i r a t i o n i n leaves i s saturated by about 2% 0  2  whereas photorespiration has a much higher 0  quirement (8,  35)•  Much recent l i t e r a t u r e a l s o implicates  g l y c o l i c a c i d as a substrate of photorespiration. t i o n which we have studied i s the 0 stimulated C0  2  re-  2  production.  2  requirement  The  correla-  f o r glycolate-  These r e s u l t s l e d to another  of whether corn leaves produce C0  2  study  by photorespiration during  photosynthesis. A recent paper by El-Sharkawy et a l . (7) data on C0  2  has provided  production i n the l i g h t and dark when C0  sink r e l a t i o n s h i p s were changed by i n h i b i t i n g with DCMU.  2  source-  photosynthesis  Under these conditions the rates i n l i g h t and dark  were i d e n t i c a l and these data were used as evidence f o r a common  source of C0  dark.  2  evolved both during photosynthesis and i n the  We re-examined t h i s work and extended i t with information  about the e f f e c t of 0  2  tension on rates of C0  2  production.  As  1 This a r t i c l e by W.J.S. Downton and E.B. Tregunna was publ i s h e d i n Plant Physiology, Vol. k j i 923-929. (1968). E.B. Tregunna supervised t h i s study. Subsequent developments on t h i s topic are discussed i n footnotes.  51 a check against the p o s s i b i l i t y that the gas exchange results from the DCMU-treated  monocotyledons were an a r t i f a c t of stomatal  behavior, the gas exchange of a DCMU-treated f l e x l l l s , was studied.  alga, N i t e l l a  This alga has been shown to have a photo(4).  respiratory mechanism s i m i l a r to that of some land plants  Results obtained by i n h i b i t i n g photosynthesis by non-chemical means have also been useful i n determining whether photorespiratory C0  2  i s being produced during photosynthesis by leaves with  very low compensation values (19, 20).  The term, "dark r e s p i r a -  t i o n " , i s used i n t h i s paper to denote that process of C0  2  pro-  duction which normally occurs i n the dark, and whose rate i s not influenced by changes i n 0  2  concentration between 2% and  21%.  MATERIALS Seedlings of wheat (Trltlcum vulgare L . ) , oats (Avena satlva L . ) , and corn (Zea mays L.) were grown at a 16 hour photoperlod with a 2k° day/19 environment chamber.  0  night temperature In a controlled  Illumination was supplied by banks of  Cool White Fluorescent tubes supplemented with incandescent bulbs.  The intensity as measured with a Weston 756 Illuminom-  eter was 1200 - 100 f t - c .  A l l seeds were planted i n f l a t s of  vermlcullte and watered d a i l y .  Corn seeds were pretreated with  75% Captan powder to control a seed and root fungus.  Seedling  age f o r experimental t r i a l s ranged from 2 to 3 weeks. N i t e l l a f l e x l l l s L. (C. Agardh), a macroscopic green alga, was grown i n an aquarium i n tapwater at room l i g h t and temperature.  52  METHODS DCMU Feedings. Detached shoots of corn and wheat were submerged i n d i s t i l l e d water or a saturated aqueous s o l u t i o n of DCMU [3-(3»4d l c h l o r o p h e n y l ) - l , l dimethyl urea] , ca 45 mg/1 (7), and placed i n a vacuum desiccator.  The system was evacuated f o r 1 hour  with a water a s p i r a t o r .  Then the cut ends of the shoots were  placed i n small v i a l s containing the same solution that they were i n f i l t r a t e d with, and set under a fluorescent l i g h t an i n t e n s i t y of 700 f t - c .  having  Shoots were tested 3 hours l a t e r by  which time photosynthesis had been inhibited completely.in the DCMU-infiltrated  shoots.  For the test, a v i a l containing shoots was placed i n a glass cylinder which served as a leaf chamber.  The CO2 concen-  t r a t i o n was measured i n a closed system consisting of a diaphragm pump, the l e a f chamber and a Beckman Model IR 215 infra-red C 0 analyzer.  2  The volume of the system was 215 ml; the flow rate,  1.1 l i t e r s per minute.  The l e a f chamber was submerged i n a  water bath, which s t a b i l i z e d the temperature a t 22.5 - 1»5°» The i n t e n s i t y from a General E l e c t r i c "Cool Beam" lamp was 2000 ft-c.  Changes i n the C 0 concentration were measured under the 2  following conditions.  Rates of photosynthesis were determined  between 325 and 275 ppm C 0 . 2  dark was then measured.  The rate of r e s p i r a t i o n i n the  Following t h i s , the chamber was r e -  illuminated and flushed with N  2  to lower the 0  2  concentration  to between 1 to 3$ as monitored with a Clark Polarographic oxygen electrode.  C 0 was introduced to raise the C 0 concen2  2  53 t r a t l o n to the former l e v e l and another set of rates i n l i g h t and dark was determined.  The system was again flushed with  a i r and rates taken to determine the r e v e r s i b i l i t y of the 0  2  effect. The apparatus used to study N i t e l l a was almost Identical to that described above.  The chamber was again a glass column  but with a side-arm near the sintered base which allowed rapid drainage of solutions.  The sintered disc broke up the a i r -  stream which bubbled through the l i q u i d i n which the alga was suspended.  The flow rate was 0.475 l i t e r s per minute.  l i g h t intensity was 1250 t 250 f t - c .  The  Temperature ranged from  21.5 to 2 3 . 0 ° . Rates of photosynthesis and r e s p i r a t i o n were determined under.21% and 1 to 3% 0  2  concentrations with the alga i n 11 mM  phosphate buffer (KH^o^ and NaH P0/ 1:1) adjusted to pH 4.9 2  to 5»0.  l>  Following these determinations, the buffer was removed  via the side-arm and a solution of 11 mM phosphate buffer saturated with DCMU was introduced.  Photosynthesis was inhibited  within 3 minutes and at this time changes i n the C0 concentra2  t i o n were measured i n l i g h t and dark at the two 0  2  concentrations.  Glycolate Feedings. Detached shoots of corn, wheat, and oats were placed i n v i a l s containing solutions of 0.05 M g l y c o l i c acid or 0.05 M a c e t i c a c i d adjusted to pH 5»0 with s o l i d sodium bicarbonate, or d i s t i l l e d water adjusted to the same pH with 0.1 N hydrochloric acid.  A minimum feeding period of 3 to 4 hours under  a fluorescent l i g h t of 700 f t - c intensity ensued before samples  54 were tested.  Individual v i a l s containing 3 - 8  then placed i n a darkened l e a f chamber.  shoots were  Rates of C0  2  evolution  were calculated according to the time required f o r the shoots to raise the C0 system.  concentration from 62 to 100 ppm i n the closed  2  Two d i f f e r e n t 0  2  tensions were applied randomly, N  2  being used to lower the concentration from atmospheric to 1 to 3%.  Tabulated rates represent r e p l i c a t e means.  The system used to feed glycolate to N l t e l l a was the same as that described f o r the DCMU feedings except that a l l rate determinations were carried out i n darkness. termined under the two 0 phate buffer.  2  Rates were de-  tensions with the alga i n 11 mM phos-  Following t h i s , the solution was replaced by 25  mM g l y c o l i c a c i d i n 11 mM phosphate buffer at pH 4.9 to 5.1. To insure that handling had not damaged the alga, i t s a b i l i t y to assimilate C0  2  by photosynthesis was determined before rates  of r e s p i r a t i o n were determined under the two d i f f e r e n t 0  2  ten-  sions. Enzyme Assay f o r N l t e l l a . Crude preparations of g l y c o l i c acid oxidase were made by grinding 15 grams fresh weight of N l t e l l a i n 15 ml of 0.05 M K HP0^ i n a Waring Blendor f o r 1 minute. 2  The brei was ground  further i n a mortar, f i l t e r e d through cheesecloth, centrifuged at 200 X g f o r 5 minutes and the supernatant aerated at room temperature f o r 10 minutes to lower endogenous oxidation.  A  sample of the crude preparation was placed i n a 1.55 ml react i o n vessel and s t i r r e d by a magnet.  Rates of 0  2  consumption  were measured with the Clark Polarographlc oxygen electrode at  55 20°.  The a c t i v i t y of g l y c o l i c a c i d oxidase was determined by  the change i n rate of 0  2  consumption upon addition of 150 yl of  0.05 M g l y c o l i c a c i d . Oxygen S e n s i t i v i t y of the Compensation Intensity.  Value Under Low Light  Two-week-old wheat seedlings were detached and placed i n the l e a f chamber at a l i g h t intensity of 60 - 5 f t - c as measured with a Gossen T r i - l u x lightmeter.  The l i g h t was attenu-  ated with sheets of Whatman No. 1 chromatography  paper. The  system was scrubbed free of C0 with Ascarite, closed and the 2  C0  2  compensation of the wheat shoots i n 21$ 0  The system was then flushed with N  2  2  was determined.  to lower the 0 concentra2  t i o n to 1 to 3$ and C0 compensation measured again. The 2  temperature during these experiments was 21.5°.  Twenty-day-old  corn seedlings were subjected to the above treatment but the l i g h t Intensity required to produce a measurable  compensation  was 18 - 2 f t - c a t 22°.  RESULTS AND DISCUSSION The E f f e c t of DCMU on C0 Production. 2  Prom Table I i t i s evident that DCMU treatment inhibited photosynthesis i n wheat and corn shoots allowing C0 production 2  to be measured i n the l i g h t .  The s i m i l a r i t y of the rates i n  both l i g h t and dark indicate the complete suppression of photosynthesis.  The results are i n agreement with those of E l -  Sharkawy et a l . (7) f o r corn, sugarbeet and Amaranthus edulls under conditions where photosynthesis appears to be completely  TABLE I.  Plant  E f f e c t of DCMU and oxygen concentration on the gas exchange of detached wheat and corn shoots. Infiltration solution  Rate a t 21% O2 Light Dark  Rate at 2% 0 Light Dark  2  Ug CC-2/min X g f r wt Wheat  Corn  a  +3.2 +3.5  -37.5  +3.2  -28.1  -29.2  +3.4 +3.5  -36.5  +3.3  DCMU  +3.6 +3.2  +3.4 +3.2  +3.6  +3.2  it  +2.2 +2.2  +2.7 +2.9  +2.2  Water  r31.2 -34.4  +3.3 +3.2  -31.2  +3.3  n  -42.0 -42.0  +3.4 +3.5  -42.0  +3.3  DCMU  +2.9  +2.7  +3.2  +2.7  it  +3.4 +3.2  +3.4 +3.0  +3.6  +3.2  Water  -24.6 -26.2  11  C02 produced (+) or absorbed (-)  a  ...  57 inhibited.  They have suggested that the C0 released i n the 2  l i g h t i n the presence of DCMU i s that which i s normally comp l e t e l y recycled to photosynthesis  i n plants which do not pro-  duce C0 into C0 -free a i r , and implied a common source of C0 2  2  production both during normal photosynthesis  2  and i n darkness.  The lack of s e n s i t i v i t y of rates of C0 production to 0 2  2  ten-  sions between 2 and 21$ by wheat shoots i n DCMU (Table I) i n dicates that the usual pathway of C0 production which occurs 2  during photosynthesis another process.  (photorespiration) has been replaced by  The observation of C0 production i n the l i g h t 2  by DCMU-treated corn shoots which normally do not produce C0 during photosynthesis  2  suggests the switching on of a C0 -evolv2  ing pathway when photosynthesis  i s completely  inhibited. The  control experiments support these conclusions. seedlings whengplaced under low 0  Control wheat  tension exhibit a great  2  enhancement i n photosynthetlc rate.  This effect can be ex-  plained as a reduction i n C0 release during photosynthesis by 2  suppressed photorespiratory a c t i v i t y under low 0 . 2  The lack  of such an enhancement f o r the corn controls i s consistent with i t s apparent lack of photorespiration (5.  6» 8).  The s i m i l a r i t y of the rates of C0 production i n l i g h t 2  and dark by DCMU-inhibited plants to those of the dark cont r o l s indicates that the lack of 0  2  response by shoots fed DCMU  was not a r e s u l t of stomatal closure brought about by the chemical.  As a check, however, N l t e l l a f i e x l l i s was subjected to  DCMU treatment.  Brown and Tregunna (4) have shown that N l t e l l a  i n a c i d i c medium had a high compensation value which was lowered  58 by reducing the 0  concentration.  2  fect the dark respiratory rates.  Such a reduction did not a f They concluded that r e s p i r a -  t i o n was i n h i b i t e d during photosynthesis  and replaced by another  pathway of C0 evolution (photorespiration) having a high 0 2  requirement.  Rates of photosynthesis  with the alga i n buffer  (Table II) show an enhancement under low 0  2  s i m i l a r to that r e -  ported f o r temperate plants with photorespiration (5). experiment, a l l photosynthetlc  2  In this  a c t i v i t y was i n h i b i t e d within 3  minutes a f t e r addition of DCMU compared to the 3 hours required for such i n h i b i t i o n i n the monocotyledons. photosynthesis  The I n h i b i t i o n of  of N l t e l l a results i n C0 production 2  which i s equal to the rate i n the dark and not 0 tween 2 and 21% oxygen (Table I I ) .  2  in light  sensitive be-  This r e s u l t i s i d e n t i c a l to  that from the DCMU-inhibited monocotyledons and indicates that photorespiration has been replaced by the pathway which i s saturated a t a lower 0  2  concentration.  The DCMU data of Poskuta et a l . (2?) were based on rates of C0 production 2  photosynthesis  i n l i g h t and dark when the apparent rate of  was zero.  That i s , the C0 compensation con2  centration was approximately 300 ppm. Therefore t h e t l c a c t i v i t y was occurring.  some photosyn-  Under these conditions, C0  2  evolution i n l i g h t was found to be greatly i n h i b i t e d compared 2 to the controls. There was a n e g l i g i b l e e f f e c t of DCMU on 2 While this paper was i n press, Poskuta (28) reported that the portion of C0 evolution i n the l i g h t that remained unaffected by DCMU was stimulated by increased 0 tension. 2  2  59 dark respiratory r a t e s .  J  S i m i l a r l y , they have shown that dur2,4-dinitrophenol,  ing i n h i b i t i o n of apparent photosynthesis by  increasing the Cv> concentration from atmospheric to 100$ increased C 0  2  production i n the l i g h t , but had a n e g l i g i b l e ef-  fect on dark r e s p i r a t i o n . photosynthesis  greatly  Hence under conditions where some  i s occurring the process of C 0  l i g h t i s d i f f e r e n t from dark r e s p i r a t i o n .  2  evolution i n (3*0  Tregunna et a l .  have shown that albino corn, which lacks a functional photosynt h e t l c apparatus and therefore photosynthesis, produces C 0  2  at  the same rate In l i g h t and dark indicating a common source of C0  2  production when photosynthetlc a c t i v i t y i s absent.**  corn, on the other hand, does not produce C 0  2  i n the l i g h t as  indicated by i t s low compensation point and lack of C 0 duction into C 0 - f r e e a i r (7, 2  Normal  2  pro-  8).  The Oxygen S e n s i t i v i t y of Glycolate Oxidation i n Relation to Photorespiration. The e f f e c t of 0  2  concentration on g l y c o l i c acid oxidation  i n darkness i s recorded i n Tables III and IV. colate exhibited a great increase i n C 0 0  2  2  2  evolution under  compared to the rates f o r water and acetate feedings.  stimulation i n wheat and corn shoots was 0  Shoots fed gly-  suppressed  to the l e v e l of the shoots fed water or acetate.  21$ This  under 2$ Oats d i f -  fered to some degree i n i t s response i n that an increase i n 0 concentration had a stimulatory effect on C 0  2  2  production f o r  3 Z e l i t c h (4$) found s i m i l a r effects f o r leaf discs treated with CMU [3-(4-chlorophenyl)-l, 1-dimethylurea ] , ant Inhibitor of photosynthetlc electron transport. 4 Hew and Krotkov (14) have reported s i m i l a r results f o r nongreen leaves of other plant species.  TABLE I I . E f f e c t of DCMU and oxygen concentration flexilis.  Sample  Solution  on the gas exchange of N l t e l l a  Rate a t 2% Oo Light Dark  Rate a t 21$ 0 Light Dark 2  ;ig C0 /min X g f r wt 2  1  2  C0  2  Buffer  -4.08  DCMU  +2.16  -6.26  +1.95  +2.14  +2.10  +2.10  +2.10  Buffer  -4.41  +2.94  -6.01  +2.94  DCMU  +2.60  +2.48  +2.48  +2.44  produced (+) or absorbed (-).  a  TABLE I I I .  Plant  The e f f e c t of s u b s t r a t e and oxygen c o n c e n t r a t i o n on r a t e s o f carbon d i o x i d e e v o l u t i o n by detached monocotyledon shoots In darkness.  Replicates  Substrate  Rate a t 2$ 0 ( A ) 2  Rate a t 21$ 0 ( B ) 2  jig C0 /mln X g f r wt 2  Wheat  Water A c e t a t e 0.05  M  G l y c o l a t e 0.05 Oats  Water A c e t a t e 0.05  M  G l y c o l a t e 0.05 Corn  M  M  Water A c e t a t e 0.05  M  G l y c o l a t e 0.05  M  Oxygen stimulation (B/A)  S u b s t r a t e rate, water r a t e i n 21$ 0  Ratio  Ratio  2  6  4.06  4.23  1.05  6  4.92  5.30  1.08  1.25  10  5.14  9.02  1.75  2.13  4  5.50  5.44  0.99  6  4.98  5.86  1.18  1.08  6  7.46  11.34  1.51  2.08  6  4.28  *.55  1.06  6  3.96  4.47  1.13  0.98  8  4.11  6.61  1.61  1.45  TABLE IV.  Sample  E f f e c t of glycolate and oxygen concentration on carbon dioxide by N i t e l l a f l e x l l l s i n darkness.  Solution  Rate at 2% Oo (A)  Rate a t 21$ (B)  »g C0 /min X g f r wt 2  1  2  02  production  Oxygen stimulation (B/A) Ratio  Buffer  2.67  3.07  1.15  Glycolate  3.02  5.33  1.77  Buffer  2.86  3.15  1.10  Glycolate  3.70  5.46  1.48  63 leaves fed acetate. ever, was  much l e s s than that f o r the glycolate feedings.  glycolate was of C0  The magnitude of t h i s stimulation, how-  fed to N i t e l l a (Table IV), a s i m i l a r stimulation  evolution under 21% 0  2  When  2  was  The 0  observed.  2  e f f e c t was  completely r e v e r s i b l e f o r a l l shoots which were fed glycolate. Further support f o r the existence of g l y c o l i c a c i d oxidase act i v i t y i n N i t e l l a came from crude enzyme preparations.  Upon  addition of g l y c o l i c a c i d to the preparation, the endogenous rates of oxygen consumption f o r 2 samples increased from and 9.3  ul 0  2  6.8  per hour per gram fresh weight of tissue extracted  to values of 22.3  a  nd  21.7  The measurement of C0  respectively. 2  evolution In the dark allowed gly-  c o l i c a c i d oxidation (photorespiration?) to be studied i n the The d i f f e r i n g 0  absence of photosynthesis.  2  requirements f o r  g l y c o l i c a c i d oxidation and dark r e s p i r a t i o n allowed separation of the processes. the C0  2  Under low 0  2  both glycolate oxidation and  compensation value become n e g l i g i b l e .  i s also r e a d i l y reversible f o r both. compatible with the high 0  2  The 0  effect  2  These r e s u l t s , then, are  requirement f o r photorespiration  and the implication that glycolate i s the substrate of t h i s process. G l y c o l i c a c i d oxidase a c t i v i t y i n wheat, corn and has been demonstrated by many workers (24, 30, Tolbert (13,  32)  3D.  oats  Hess and  f a i l e d to detect g l y c o l i c a c i d oxidase a c t i v i t y  i n the u n i c e l l u l a r algae, C h l o r e l l a pyrenoidosa (#393)» C h l o r e l l a pyrenoldosa (Warburg), Chlamydomonas r e i n h a r d t l l (-), Ankistrodesmus braunl1 (#245), n d a  Soenedesmus obllquus  (Gaffron  D-3).  and have generalized to state that "glycolate metabolism rep-  64 resents a major difference between algae and higher plants". Our studies have provided both ' i n vivo' and ' i n v i t r o ' evidence f o r an active glycolate oxidation mechanism i n N i t e l l a . 5 G l y c o l i c a c i d , as well as being ubiquitous i n leaves, i s one of the e a r l i e s t products of photosynthesis p a r t i c u l a r l y at lower C0  2  concentrations and higher 0  12, 29, 42).  2  concentrations (1,  2,  In addition, Ludwig and Krotkov (18) have pro-  vided evidence f o r some early photosynthetlc product being o x l 14 dized to C0  2  in light.  flower demonstrated  Measurements of  C0  2  uptake by sun-  that the i n i t i a l rate of uptake decreased  soon a f t e r the introduction of -^COg, indicating that was being evolved.  S i m i l a r l y , Goldsworthy  (9)  l2,  C0  2  has shown that  tobacco segments when subjected to a stream of C0 -free a i r 14 2  following a period of  C0  2  incorporation, produced C0  2  l i g h t with a higher s p e c i f i c a c t i v i t y than i n the dark. rate of C0 high 0  2  2  i n the The  production i n the l i g h t was greatly stimulated by  concentration whereas the dark rate was not.**  The use  of purported i n h i b i t o r s of glycolate metabolism reduced the 5 While this paper was i n press. Lord and Merrett (17) reported the presence of g l y c o l i c acid oxidase i n C h l o r e l l a pyrenoidosa ( s t r a i n 211/8P Cambridge culture c o l l e c t i o n ) . About t h i s same time, Z e l i t c h and Day (44) confirmed i t s presence i n Chlamydomonas relnhardl ( s t r a i n 137c mt ) and Chlorella pryenoldosa ( s t r a i n Tx 71105). +  6 Since t h i s a r t i c l e was released, Z e l i t c h (45, 46) has shown a s i m i l a r e f f e c t . He has used these observations to develop an assay method f o r measuring the magnitude of photorespiration i n leaf discs.  65 s p e c i f i c a c t i v i t y of C0  2  released i n the l i g h t to a l e v e l com-  parable to that released i n the dark. The  investigations of Z e l i t c h and Moss have been a major  support f o r the tenet that the source of C0  2  evolved  l i g h t i s glycolate (21, 22, 23, 40, 41, 42, 43). work has involved the use of a competitive  i n the  Much of t h i s  i n h i b i t o r of gly-  c o l i c a c i d oxidase, <=£-hydroxy-2-pyridinemethane-sulfonic a c i d . 14 The feeding of l a b e l l e d glycolate to tobacco resulted i n production.  C0  2  The source was s p e c i f i c a l l y from the carboxyl car-  bon of glycolate.  Corn was an exception and produced l i t t l e  14 COg compared to tobacco, although i t assimilated as much glyTobacco l e a f discs at 35° i n the presence of the i n 14 h i b i t o r took up at l e a s t 3 times as much C0 as the controls 7 colate.  2  i n water.  There was no e f f e c t on corn.  I t was presumed that  the Inhibitor blocked photorespiration i n tobacco and increased the concentration gradient between the atmosphere and the chloroplast r e s u l t i n g i n a higher rate of photosynthesis  (43).  (21), i n recent studies, has shown that rates of C0  2  Moss  evolution  i n l i g h t into C0 -free a i r exceeded dark rates, despite the 2  l i k e l y occurrence of r e - u t i l i z a t i o n of C0 The  by  2  i n h i b i t o r reduced t h i s evolution of C0  2  photosynthesis.  i n the l i g h t to a  rate lower than that i n darkness, probably by blocking photopublished byglycolate Moss (23) that while t h i s a r t i c lien was r e s7p iExperiments r a t i o n . The amount of accumulated the i n press do not bear t h i s out. Rather i t was found that the i n h i b i t o r caused an almost immediate decline i n photosynthetlc rate. D i f f u s i v e resistance calculations indicate that this i n h i b i t o r also i n h i b i t s photosynthesis d i r e c t l y .  66 presence of the i n h i b i t o r was  s u f f i c i e n t to account f o r the d i f -  ference i n CC>2 evolution between controls and leaves fed i n h i b i tor.  The  Inhibitor had no e f f e c t on r e s p i r a t i o n rates i n the  dark. Contrary to what would be expected from the work of Tregunna (36),  corn was  capable of oxidizing glycolate i n the dark i n the  absence of exogenously added f l a v i n mononucleotide (PMN). results of Moss (22) show a 3 0 $ stimulation i n C0  2  The  production  by corn leaves fed 0.1 M glycolate compared to the water controls. The absence of data f o r corn leaves fed acetate or glucose does not allow comparison of the effects of d i f f e r e n t substrates stimulation of C0 C0  2  2  evolution.  on  Our data of the r a t i o : (rate of  evolution by leaves fed glycolate) : (rate by the leaves i n  water) show that i n t e r s p e c i f i c differences do occur.  Under 21$  03 the rates of CO2 production doubled f o r the wheat and  oat  leaves which were fed glycolate compared to the water controls. For corn shoots, however, the rate increased only by 4 5 $ . . T h i s lower stimulation i n corn may  r e f l e c t a species difference i n  the transport of glycolate to the s i t e of oxidation or i n the a b i l i t y to metabolize i t . The a b i l i t y of corn to produce CO2 from .glycolate i n the-dark but not i n the l i g h t ( 4 3 ) would suggest that either the plant i s extremely e f f i c i e n t i n the i n ternal recycling of photorespiratory does not leak C0  2  CO2  to photosynthesis and  to the outside, or that the pathway of gly-  colate metabolism i s not the same i n the l i g h t as i n the dark. The mechanism proposed by Tregunna ( 3 6 ) appears incorrect i n  67 view of the results of feeding glycolate. Oxygen S e n s i t i v i t y of the CO2 Compensation Value at Low Light Intensity. Complete i n h i b i t i o n of photosynthesis with DCMU resulted i n the replacement of photorespiration by another process. Hence the CO2 source-sink r e l a t i o n s must be changed more subtly to determine whether corn simply recycles the photorespiratory C0 .  The manipulation of a photosynthetlc parameter such as  2  l i g h t intensity provides one approach.  I f corn does possess  the photorespiratory pathway common to wheat and oats (6), under conditions where C0  2  i s not rate l i m i t i n g , such as very  low l i g h t Intensity, some of t h i s photorespiratory C0 leak out. of 0  2  should  This should be detectable by measuring the e f f e c t  concentration on the compensation value.  2  then  Table V shows  the r e s u l t s obtained using wheat and corn shoots.  Wheat as a  control under 65 i 5 f t - c had an average compensation of 170 ppm  i n 21$ 0 .  at C0  2  concentrations above the compensation concentration i n -  dicated that C0 low 0 0  The lack of measurable rates of gas exchange  2  2  2  was not l i m i t i n g at t h i s concentration.  the compensation value was 27 ppm.  Therefore, i n 21$  and low l i g h t i n t e n s i t y , the compensation value was a  2  Under  com-  8 Osmond (26) has since demonstrated a massive stimulation i n the rate of glycolate and glyoxylate decarboxylation upon the a d d i t i o n of PMN to illuminated c e l l - f r e e extracts of corn. The a d d i t i o n of catalase caused a substantial reduction i n C0 release. He suggests that the PMN effect i n intact corn leaves observed by Tregunna (36) was a consequence of the accelerated non-enzymlc destruction of glyoxylate by peroxides whose formation was mediated and stimulated by the photoreduction of FMN. FMN apparently does not stimulate the decarboxylation of glyoxylate by d i r e c t interaction with glyc o l i c a c i d oxidase. 2  68 posite of dark r e s p i r a t i o n , photorespiration, and photosynthesis, Photorespiration was the major source of CO2 i n 21$ 03. The l i g h t i n t e n s i t y required to produce a measurable compensation value i n corn was very low (18 i 2 f t - c ) and narrow i n range.  The compensation point was not 0  2  sensitive between  2 and 21$ 0 3 , and therefore represented equilibrium between photosynthesis and dark r e s p i r a t i o n .  Under conditions s u f f i c i e n t  to produce a measurable compensation value i n corn, there was no loss of C 0  2  produced by photorespiration. This indicates  that corn lacks photorespiration and that the n e g l i g i b l e compensation value at higher l i g h t i n t e n s i t i e s i s not a result of e f f i c i e n t internal r e c y c l i n g of photorespiratory C 0  2  to photo-  synthesis by decreased d i f f u s i o n resistances. Meidner (19t 20) has demonstrated that corn leaves placed under water s t r a i n had an elevated compensation value which was e s s e n t i a l l y unaffected by Q  2  concentrations between 21 and 100$.  also lacked a post-illumination burst.  Such plants  These observations are  compatible with the absence of an operational photorespiratory mechanism i n corn. Although corn does have an active g l y c o l i c a c i d oxidase pathway, i t appears u n l i k e l y that the pathway or reaction products are the same i n the l i g h t during photosynthesis as i n the dark.  The lack of a compensation point, lack of C 0  2  production into C 0 - f r e e a i r and n e g l i g i b l e production of 2  ll  ^C0  2  from l a b e l l e d glycolate i n l i g h t support t h i s (7,  43).  The studies of Hatch etYal. ( 1 0 , 11) have shown that wheat and oats form intermediates of the Calvin cycle as the e a r l i e s t  69  TABLE V.  Plant  E f f e c t of oxygen concentration on the carbon dioxide compensation concentration of wheat and corn shoots when l i g h t i s a l i m i t i n g f a c t o r .  Sample  Wheat  Corn  2  2  2  Light intensity  ppm  ppm  1  157  25  65 *  5  2  183  29  65 ±  5  1  43  40  18 * 2  2  37  38  18 ± 2  products of photosynthesis. tion ( 6 ) ,  C0 compensation 21% 0 2% 0  ft-c'  These plants a l s o have photorespira-  Corn and many t r o p i c a l grasses, which lack photores-  p i r a t i o n , have a d i f f e r e n t carboxylation sequence i n which C^compounds are l a b e l l e d f i r s t  (6, 11).  This appears to be true  also f o r low compensation members of the Amaranthaceae and Q Chenopodiaceae (2-5, 37, unpublished data).  Further studies are  necessary to c l a r i f y the r e l a t i o n s h i p of glycolate to these paths of carbon i n photosynthesis.  10  The opposite nature of the gas exchange processes of photor e s p i r a t i o n to those of the concomitant photosynthesis  consti-  tutes a d i f f i c u l t y i n the study of photorespiration by the i n f r a 9 Since t h i s a r t i c l e went to press, Johnson et a l . (15) and Tregunna et a l . ( 3 8 , 39) have provided further experimental e v i dence i n support of t h i s . 10 Comparative studies reported i n early 1969 have shown that g l y c o l i c a c i d oxidase a c t i v i t y i s much lower i n plants lacking photorespiration (3t 2 6 , 33)• Much of the a c t i v i t y of t h i s enzyme appears confined to the peroxisome ( 3 , 33)• Chloroplasts, but not peroxisomes, are capable of o x i d i z i n g glyoxylate to C0 but only at very low rates ( 1 6 ) . The s i g n i f i c a n c e of these findings to photorespiration i s not yet c l e a r (May 1 9 6 9 ) . 2  70  red method.  I t s apparent dependence upon photosynthesis does  not allow f o r i t s independent pletely blocked. 0  2  study when photosynthesis i s com-  Hence the s e n s i t i v i t y of photorespiration to  concentration provides a powerful means of manipulation i n  the further elucidation of i t s mechanism and i t s importance i n terms of the carbon balance and dry matter production i n plants.  71  LITERATURE CITED 1.  Bassham, J.A. and M. Kirk. 1962. The e f f e c t of oxygen on the reduction of CO2 to g l y c o l i c acid and other products during photosynthesis by C h i o r e l l a . Biochem. Biophys. Acta 9: 375-380.  2.  Benson, A.A. and M. Calvin. 1950. The path of carbon i n photosynthesis. VII. Respiration and photosynthesis. J. Exptl. Botany 1: 63-68.  3.  Breidenbach, R.W. 1969. Personal communication. Department of Agronomy, University of C a l i f o r n i a , Davis, California.  4.  Brown, D.L. and E.B. Tregunna. I967. I n h i b i t i o n of r e s p i r a t i o n during photosynthesis by some algae. Can. J . Botany 45: 1135-1143.  5.  Downes, R.W. and J.D. Hesketh. 1968. Enhanced photosynthesis at low oxygen concentrations: D i f f e r e n t i a l response of temperate and t r o p i c a l grasses. Planta 78: 79-84.  6.  Downton, W.J.S. and E.B. Tregunna. 1968. Carbon dioxide compensation - i t s r e l a t i o n to photosynthetlc carboxyl a t i o n reactions, systematics of the Gramineae, and l e a f anatomy. Can. J . Botany 46: 207-215. (Part 1-A).  7.  El-Sharkawy, M.A., R.S. Loomis and W.A. Williams. I967. Apparent reasslmilation of respiratory carbon dioxide by d i f f e r e n t plant species. P h y s i o l . Plantarum 20: 171-186.  8.  Forrester, M.L., G. Krotkov and CD. Nelson. 1966. Effect of oxygen on photosynthesis, photorespiration and r e s p i r a t i o n i n detached leaves. I. Soybean. I I . Corn and other monocotyledons. Plant Physiol. 41: 422-431.  9.  Goldsworthy, A. 1966. Experiments on the o r i g i n of C0 released by tobacco l e a f segments i n the l i g h t , Phytochemistry 5: 1013-1019.  2  10.  Hatch, M.D. and C.R. Slack, 1966. cane leaves. Biochem. J . 101:  11.  Hatch, M.D., C.R. Slack and H.S. Johnson. 1967. Further studies on a new pathway of photosynthetlc carbon dioxide f i x a t i o n i n sugarcane and i t s occurrence i n other plant species. Biochem. J . 102: 417-422.  Photosynthesis by sugar103-111.  72  12.  Hess, J.L. and N.E. Tolbert. 1966. Glycolate, glycine, serine and glycerate formation during photosynthesis by tobacco leaves. J . B i o l . Chem. 241: 5705-5711.  13.  Hess, J.L. and N.E. Tolbert. 1967. Glycolate pathway i n algae. Plant Physiol. 42: 371-379.  14.  Hew, C.S. and G. Krotkov. 1968. E f f e c t of oxygen on the rates of CO2 evolution i n l i g h t and i n darkness by photosyntheslzing and non^photosynthesizing leaves. Plant P h y s i o l . 43: 467-469.  15.  Johnson, H.S. and M.D. Hatch. 1968. D i s t r i b u t i o n of C^dlcarboxyllc a c i d pathway i n photosynthesis and i t s occurrence i n dicotyledonous plants. Phytochemistry 7: 375-380.  16.  K i s a k i , T. and N.E. Tolbert. 1969. Glycolate and glyoxylate metabolism by i s o l a t e d peroxisomes or chlorop l a s t s . Plant Physiol. 44: 242-250.  17.  Lord, M.J. and M.J. Merrett. 1968. Glycolate oxidase i n C h l o r e l l a pyrenoldosa. Biochim. Blophys. Acta 159: 543-544.  18.  Ludwig, L.J. and G. Krotkov. 1967. The k i n e t i c s of l a b e l ing of the substrates f o r CO2 evolution by sunflower leaves i n the l i g h t . Plant Physiol. 42: S-47.  19.  Meidner, H. 1962. The minimum i n t e r c e l l u l a r - s p a c e C 0 concentration ( F ) of maize leaves and i t s influence on stomatal movement. J . E x p t l . Botany 13s 284-293.  20.  Meidner, H. 1967. Further observations on the minimum i n tercellular-space carbon-dioxide concentration ( P ) of maize leaves and the postulated roles of "Photor e s p i r a t i o n " and g l y c o l l a t e metabolism. J . E x p t l . Botany 18: 177-185.  21.  Moss, D.N. 1966. Glycolate as a substrate f o r photoresp i r a t i o n . Plant Physiol. 4 1 : x x x v i l i .  22.  Moss, D.N. 1967. High a c t i v i t y of the g l y c o l i c a c i d o x i dase system i n tobacco leaves. Plant Physiol. 42:  2  1463-1464.  23.  Moss, D.N. 1968. Photorespiration and glycolate metabolism i n tobacco leaves. Crop Science 8: 71-76.  24.  N o l l , C.R., J r . and R.H. B u r r i s . 1959. Nature and d i s t r i bution of g l y c o l i c a c i d oxidase i n plants. Plant Physiol. 2 9 : 261-265.  73 25.  Osmond, B. I 9 6 7 . ,3-carboxylation during photosynthesis i n A t r l p l e x . Biochim. Biophys. Acta 141: 197-199.  26.  Osmond, C.B. 1 9 6 9 . B-carboxylation photosynthesis and photor e s p i r a t i o n In higher plants. Biochim. Biophys. Acta 172: 144-149.  27.  Poskuta, G., C D . Nelson and G. Krotkov. I 9 6 7 . E f f e c t s of metabolic i n h i b i t o r s on the rates of CO2 evolution i n l i g h t and In darkness by detached spruce twigs, wheat and soybean leaves. Plant Physiol. 42: 1187-1190.  28.  Poskuta, J . 1 9 6 8 . Photosynthesis and r e s p i r a t i o n . I I . E f fect of 3 - ( 3 , 4 - d i c h l o r o p h e n y l ) - l , 1-dimethylurea and of p a r t i a l pressure of oxygen on the rates of carbon dioxide exchange i n l i g h t and darkness of detached wheat leaves. Experentia 24: 344-345.  29.  Shou, L., A.A. Benson, J.A. Bassham and M. Calvin. 1 9 5 0 . The path of carbon i n photosynthesis. XI. The role of g l y c o l i c a c i d . Physiol. Plantarum 3s 487-495.  30.  Tolbert, N.E. and R.H. Burris. 1950. Light a c t i v a t i o n of the plant enzyme which oxidizes g l y c o l i c a c i d . J . B i o l . 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Science 1 5 1 : 1239-1241.  37.  Tregunna, E.B. and W.J.S. Downton. 1 9 6 7 . Carbon dioxide compensation i n members of the Amaranthaceae and some related f a m i l i e s . Can. J . Botany 4 5 : 2385-2387.  74 38.  Tregunna, E.B., J . Downton and P. J o l l i f f e . 1 9 6 8 . Genetic and environmental control of photorespiration. In "Progress i n Photosynthesis Research". Proceedings of the International Congress of Photosynthesis Research, Freudenstadt, Germany. In press.  39.  Tregunna, E.B., BivN. Smith, W.J.S. Downton and J.A. Berry. 1969. Some methods f o r studying the taxonomy of photosynthesis i n the Angiosperms. In preparation.  40.  Z e l i t c h , I. 1958. The role of g l y c o l i c acid oxidase i n the r e s p i r a t i o n of leaves. J . B i o l . Chem. 2 3 3 : 1 2 9 9 -  1303.  41.  Z e l i t c h , I. 1 9 5 9 . The r e l a t i o n s h i p of g l y c o l i c a c i d to r e s p i r a t i o n and photosynthesis i n tobacco leaves. J . B i o l . Chem. 2 3 4 : 3077-3081.  42.  Z e l i t c h , I. I 9 6 5 . The r e l a t i o n of g l y c o l i c a c i d synthesis to the primary photosynthetlc carboxylation reaction i n leaves. J . B i o l . Chem. 240: I 8 6 9 - I 8 7 6 .  43.  Z e l i t c h , I. 1966. Increased rate of net photosynthetlc carbon dioxide uptake caused by the i n h i b i t i o n of glycolate oxidase. Plant Physiol. 41: 1623-1631.  44.  Z e l i t c h , I. and P.R. Day. 1 9 6 8 . Glycolate oxidase i n algae. Plant Physiol. 4 3 : 2 8 9 - 2 9 1 .  45.  Z e l i t c h , I. 1 9 6 8 . Investigations of photorespiration with a sensitive ^ c - a s s a y . Plant P h y s i o l . 4 3 : 1829-1837.  46.  Z e l i t c h , I. and P.R. Day. 1968. V a r i a t i o n i n photorespirat i o n . The e f f e c t of genetic differences i n photoresp i r a t i o n on net photosynthesis i n tobacco. Plant Physiol.  4 3 : 1838-1844.  activity  75 Part  3.  CONCLUSIONS 1.  Plants that form C^-dicarboxylic acids as major products of short term photosynthesis have low compensation values (lack photorespiration) i n a i r ; those that form phosphorylated compounds t y p i c a l of the C a l v i n cycle as major prodducts of short term photosynthesis have high compensation values (photorespiration) i n 21% 0 . 2  2.  Plants with high compensation values i n 21$ 02 have low compensation values at low 0  3.  2  tensions.  Plants with the C^-pathway have higher photosynthetlc rates at high l i g h t intensity than those with the Calvin cycle and photorespiration.  4.  Graminaceous species with low compensation belong to the a r l s t i d o i d , chloridoid-eragrostoid and panicoid l i n e s of evolution.  5.  Within these phylogenetic l i n e s , members of the genus Panicum d i f f e r i n photosynthetlc physiology. Species of the subgenus Eupanlcum photosynthesize v i a the C^-pathway whereas species of the subgenus Dichanthelium u t i l i z e the Calvin cycle of photosynthesis.  6.  This kind of photosynthetlc d i v e r s i t y also occurs within the genera Cyperus (Cyperaceae), A t r l p l e x and Bassia (Chenopodiaceae).  7.  Species with the Cij,-pathway possess a unique type of leaf anatomy. The vascular bundles are surrounded  by large  76  bundle sheath c e l l s containing s p e c i a l i z e d chloroplasts active i n starch formation.  The mesophyll i s arranged  more or l e s s concentrically about the bundle sheath. 8.  Anatomical and compensation data are i n agreement with some e a r l i e r reports that Beckmannla and Eragrostls have been taxonomically  9.  Complete i n h i b i t i o n of photosynthesis photorespiration. which continues  10.  misplaced within the Gramineae. with DCMU Inhibits  There i s no e f f e c t on dark r e s p i r a t i o n  i n the l i g h t .  The O2 s e n s i t i v i t y of glycolate-stimulated CO2  production  supports the implication that glycolate i s a substrate of photorespiration. 11.  Filaments and c e l l - f r e e extracts of the alga N l t e l l a f l e x i l l s can oxidize glycolate.  This organism i s useful for  gas exchange studies as measurements are not confounded by stomatal e f f e c t s . 12.  Corn shoots subjected to a very low l i g h t i n t e n s i t y have an elevated compensation point that lacks 0  2  sensitivity.  The lack of photorespiration i n corn does not appear to be a consequence of complete i n t e r n a l r e c y c l i n g of photorespiratory C 0  2  to  photosynthesis.  

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