VARLQTEGKS {N 3- P‘HflSPEflGLYCEEME PEIfiSPEATéSE AME) PKGSPQfiGLYCUEATE I’EGSWAMSE éCTIVETlES mm fi‘t’SiGLCfiICé-i CEANGES Russia ’50:- i‘fic gamma 0'} M. 5. ‘xL‘Céi" SHE‘S URINEESETY Mary J’oyce Abbate ‘29‘7'2 LIBRARY Michigan State. University Mary Joyce Abbate ABSTRACT Changes in 5-P-glycerate phOSphatase and P-glycolate phos- phatase activities were studied in leaves from different stages of growth and development. From spinach and sugarbeet leaves, 3-P-glycerate phosphatase, found in the pellet after centrifuga- tion at 10,000 x g, was closely associated with chloroplasts isolated on sucrose gradients. The activity was solubilized by 0.25 M.MgC12. Total activity and activity in the pellet increased with growth temperature. PhOSphatase activities were measured in soybean, Spinach, sugarbeet and corn leaves of varying ages. In soybeans, PGA phos- phatase activity was nearly constant, while P-glycolate phosphatase activity increased with leaf age from 12 to 27 units (umoles 1xgm-1 phOSphate x min" tissue). In spinach, PGA phosphatase decreased with age (from 6.7 to l.h units), whereas P-glycolate phosPhatase activity was virtually constant. In sugarbeets, PGA phosPhatase activity decreased with age from #6 to 5.5 units when plants were grown in warm conditions and from 8.6 to 3.5 units in cool conditions. P-glycolate phOSphatase activity decreased with age under warm conditions from 15 to 10 units and under cool con- ditions from 21 to 1h units. In sugarbeet seedlings, PGA phOSpha- tase was also more active in young leaves (7.} units) than in coty- ledons (1.2 units), while the same activity of P-glycolate phos- phatase was found in both. Thus in all these leaves, the ratio of activity of PGA phosphatase to P-glycolate ph03phatase was high in young leaves and decreased in old leaves. This trend was most Mary Joyce Abbate obvious in sugarbeets grown under warm conditions where the ratio in young leaves was 5.2 and in old leaves, 0.56. In corn leaves, however, there was no significant change in phosphatase activities with age. The activities of two other enzymes of the glycolate pathway were also determined in young and old leaves. Glycolate oxidase increased with age in sugarbeet leaves from 11 to 18 units per gram tissue and in spinach from 26 to 120 units. Hydroxy- pyruvate reductase increased with age in sugarbeets from 120 to 520 units, in Spinach from 180 to 700 units, and in soybeans from 100 to 590. Both phOSphatase activities were followed in young and old leaves from field-grown sugarbeets from mid-July to mid—November. In young leaves, the PGA phosphatase to P-glycolate phosphatase ratio was initially 1.5, this peaked with high temperatures in late summer at h, and then decreased to a constant value of about 2.5. In old leaves a reverse ratio was found which slowly increased from 0.1h to 0.5 during the season. In homogenates from young leaves, a large part (up to 60%) of the total protein was pre- cipitated perhaps due to tannin. The portion of the total pro- tein in this precipitate varied with the average daily temperature. Total PGA phosphatase activity in the pellet closely followed this change in the amount of precipitated protein. As an indirect measure of photoreSpiration, changes in compensation points were measured as a function of leaf age. From sugarbeets and soybeans, the average compensation point of mature leaves was 68 ppm 002, of intermediate leaves, 150 ppm C02, and of very young leaves, over 570 ppm C02. Dark respiration Mary Joyce Abbate rates were compared with the compensation points as a measure of physiological age. In sugarbeets, as the compensation point de- creased from 570 to 60 ppm C02 with increasing leaf age, the dark reSpiration rates also decreased from 2.1 to 0.5 ppm C02 x min.1 x cm-2. This trend was also found in leaves of soybeans and Spinach. In corn leaves, no significant variations were found in either parameter. An hypothesis has been proposed for the changing ratios of PGA phOSphatase to P-glycolate phosphatase activity with leaf age. Each enzyme initiates a metabolic sequence for glycine and serine biosynthesis. The P-glycolate phosphatase pathway is inefficient, resulting in photoreSpiration and loss of energy; the PGA phosphatase pathway does not waste C02 or energy. It has been proposed that the latter pathway is used preferentially in young leaves which have little or no photoreSpiration. An increase in the P-glycolate phosphatase pathway occurs during maturation as Shown by the high levels of photoreSpiration in old leaves. The high compensation points in the light in very young leaves were associated with a low level of photosynthesis and a high level of dark reSpiration. VARIATIONS IN 5-PHOSPHOGLYCERATE PHOSPHATASE AND PHOSPHOGLYCOLATE PHOSPHATASE ACTIVITIES WITH PHYSIOLOGICAL CHANGES BY Mary Joyce Abbate A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Biochemistry 1971 ACKNOWLEDGMENTS I would like to thank Dr. N. E. Tolbert (Professor, Dept. of Biochemistry) for his guidance and inSpiration during my graduate training. I also thank Dr. F. W. Snyder (Plant Physiolo- gist, U.S.D.A. and Assoc. Professor, Dept. of Crop and Soil Sciences) for all his help, eSpecially with the compensation point, dark respiration rate and field experiments. Thanks are also due to to Dr. J. W. Hanover and Dr. D. P. White (both Professors, Dept. of Forestry) for the use of their walk-in growth chamber. I would also like to thank the following: Doug Randall, for supplying me with the night-time diurnal samples and for leaving me his pet project; Sandy wardell, for all her technical assistance; Kathy Swann, for providing me with an almost never-ending supply of clean test-tubes; ' Dr. A. J. wood (Professor at the University of Victoria, B. C., Canada) for sending me to Michigan State University; my parents, Mr. and Mrs. H. R. Hallett, for allowing me to come, and for their love and encouragement; and my husband, Joe Abbate, for all his patience and his tireless efforts at proofreading and editing this tome. ii TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS LITERATURE REVIEW Acid Phosphatases, Definition Nonspecific Acid Phosphatases Change in PhOSphatase Activity with Physiological Changes PhOSphatase Isozymes P-Glycolate PhOSphatase P-Glycerate PhOSphatase Proposed Functions of PGA PhOSphatase The Glycolate Pathway PhotoreSpiration and C02 Compensation Points Effect of Age on Photosynthetic Metabolism Effect of Leaf Age on Enzyme Activities MATERIALS AND METHODS Biochemicals Buffer Solutions Standard Grinding Procedure Standard Ultracentrifugation Procedure Assays iii vi vii viii 12 13 15 17 19 21 21 21 22 22 EXPERIMENTATION AND RESULTS A) B) C) D) E) F) G) H) I) J) K) PGA Phosphatase Distribution Among Different Plant Species Diurnal Variations in Levels of PhOSphatase Activity I) Soybean Diurnal II) Sugarbeet Diurnal PGA Phosphatase Pellet Activity in Spinach Leaves Change in Activity and Distribution of PGA Phos- phatase with Growth Conditions in Sugarbeet Leaves Change in Total Activity and Cellular Distribution of PGA Phosphatase with Leaf Age in Sugarbeet Leaves The Effect of Grinding Procedure upon Phosphatase Activity Change in Phosphatase Activities with Leaf Age I) PGA Phosphatase and P-Glycolate PhOSphatase in Soybeans II) PGA Phosphatase and P-Glycolate PhOSphatase in Spinach Leaves III) PGA Phosphatase and P-Glycolate Phosphatase in Sugarbeet Leaves IV) PGA PhOSphatase and P-Glycolate PhOSphatase in Leaves from C4 Plants Field Sugarbeets Changes in Glycolate Oxidase and Hydroxypyruvate Reductase with Age of Leaf C02 Compensation Points Dark Respiration Rates DISCUSSION AND SUMMARY REFERENCES iv 25 27 30 5h 57 hl A7 52 56 59 62 69 72 81 8h 91 98 105 I) II) IIIa) IIIb) IVa) IVb) V) VI) VIIa) VIIb) VIIIa) VIIIb) IX) X) XIa) XIb) XII) LIST OF TABLES Non-specific Acid PhOSphatases PGA PhOSphatase Distribution Among Different Plant Species Diurnal Variations in Total Activity of Phos- phatases in Soybeans Diurnal Variations in Specific Activity of Phos- phatases in Soybeans Diurnal Variations in Total Activity of Phos- phatases in Sugarbeets Diurnal Variations in Specific Activity of Phos- phatases in Sugarbeets Sucrose Gradients of Spinach Pellets Change in Activity and Distribution of PGA PhOSphatase with Growth Conditions Change in Distribution of PGA PhOSphatase with Leaf Age Distribution of PGA PhOSphatase in Fractions of a Sucrose Gradient Determination of Grinding Procedure Conversion Factors (Total Activity) Determination of Grinding Procedure Conversion Factors (Specific Activity) Change in Total Phosphatase Activities with Leaf Age in Soybeans Change in Total PhOSphatase Activities with Leaf Age in Spinach Change in Total Phosphatase Activities with Leaf Age in Mature Sugarbeets Change in Total Phosphatase Activities with Leaf Age in Sugarbeet Seedlings PGA PhOSphatase to P-Glycolate PhOSphatase Activity Ratios ‘1 29 52 33 55 56 ho A6 50 51 51+ 55 58 61 65 65 68 XIII) XIVa) XIVb) XV) XVIa) XVIb) XVIIa) XVIIb) XVIIc) Change in Total Phosphatase Activities with Leaf Age in C4 Plants Sugarbeet Field Experiment: PGA Phosphatase Activities Sugarbeet Field Experiment: Continued Changes in Glycolate Oxidase and Hydroxypyruvate Reductase with Leaf Age 002 Compensation Points C02 Compensation Points: Continued Change in Dark ReSpiration Rates with Leaf Age in Sugarbeets Change in Dark Respiration Rates with Leaf Age in Soybean and Spinach Change in Dark Respiration Rates with Leaf Age in Corn vi 71 77 78 83 89 90 95 96 97 l. 2. LIST OF FIGURES Sugarbeet Field Experiment; PGA PhOSphatase and P-Glycolate Phosphatase Activities Sugarbeet Field Experiment; Distribution vii 79 80 AMP, ADP, ATP CM- pCMB DCPIP DEAE- DHAP EDTA fc. F-l,6-diP ¢£GP 6FGP Hom. NAD(P) pNPP PPm Pel. D-P PEP 2-PGA PGA, 5-PGA P-glycolate P-hydroxypyruvate LIST OF ABBREVIATIONS Adenosine mono-, di-, and triphOSphate Carboxymethyl- p-Chloromercuribenzoate 2,5-Dichlor0phenolindophenol Diethylaminoethyl- Dihydroxyacetone phOSphate Ethylene diamine tetraacetate Flavin mononucleotide FOOt-candles Fructose-1,6-diphOSphate d-Glycerol phosphate fi-GlycerolphOSphate Homogenate Michaelis constant Nicotinamide adenine dinucleotide (phOSphate) p-NitrophenylphOSphate Parts per million Pellet PhenylphOSphate Phosphoenolpyruvate 2-PhOSphoglycerate 5-Phosphog1ycerate PhosPhoglycolate Phosphohydroxypyruvate viii P-serine P PP i3 i RNase RNA RuDP Sup. TCA UTP Phosphoserine Inorganic phosphate, pyrophosphate Ribonuclease Ribonucleic acid Ribulose-l,5-diphosphate Supernatant Trichloroacetic acid Uridine triphosphate ix LITERATURE REVIEW ACID PHOSPHATASES ‘Phosphatases are defined as enzymes that catalyze the hydro- lytic cleavage of phOSphoric acid esters (60). The cleavage always occurs at the P-O bond and the products are an alcohol and ortho- phOSphate. The term acid phosphatase is most often used for ortho- phosphoric monoester phosphohydrolase (5.1.5.2), which acts on a wide range of monoesters of orthophosphate but has no effect on pyrophosphates, metaphOSphates or phosPhoric dieSters (15). How- ever many investigators use this term for any enzyme which has phOSphatase activity and functions optimally at an acid pH. Some plant phosphatases have been found that are very non-substrate Specific and can cleave pyrophOSphates and metaphosphates, as well as phOSphomonoesters, all at an acid pH. Consequently these are known as nonSpecific acid phOSphatases. The degree of nonSpecificity depends on the plant tissue and isolation procedure involved. NONSPECIFIC ACID PHOSPHATASES A survey of the literature on the isolation and purification of acid phosphatases, which are very common in plant tissues, shows a preponderance of work on nonSpecific enzymes. These enzymes have been purified on the basis of their activity for pNPP or 8GP, and then have been found active with many substrates. In some cases, evidence that only one enzyme was reSponsible for the various activities was Shown, usually from mixed substrate studies, mm mm n: on N: am aowumuwmaooua quad “mummanm onwamuoum a.md moumMaSm Sowaoasm «.m.¢ "mom: mcowuwa>ounn< * mundane -zo .asam mzoa oumuoouovoH .mzua dfiom ¢Hnm UUQHNM OZ oumamxo -moaa .-m x . .3. 53 « ++nm ++GN sawm oumuuqu saw: .oscn a m. .302 Pm -moma .-m coauSHav « s+am asao om «Hm 0 + -mOa< . m «a 6sz A OoflN .9030 am an Susanancm mmumsmmonmmuos -auua_.aa< .azm .mo mum .mo .aumae-nrm £3. £3 FHA .42 m-s rmo .ama monogamonamaomauu mo .mna .mea ms< .ma4 .amm .mmza maa .mo .mu ma< .aum ”Ham .mmza ma< .mmm «mm .mnaz .maa mo .mna .mmm .mmza .amm .maa mau-m. -m eon .ma< .mna . mm moaua>auo< can: anomquuSSOHAo use aoau -auuaam aun~m0...uaa .m.< .80uno mmmH ooomnoa mm>moH nomcamm oousom constant purification ratios, same pH optima and similar chromato- graphic characteristics. Table I Summarizes the properties of some of these enzymes. Even in highly purified fractions, the phOSphatase activity was quite nonspecific yet did vary with the tissue used. CHANGE IN PHOSPHATASE ACTIVITY WITH PHYSIOLOGICAL CHANGES Some interesting investigations in plant physiology have involved monitoring the changes of various enzyme activities during physiological changes involved in plant growth. Such studies have followed the increase of nonSpecific acid phOSphatase (in many cases, specificity was not even ascertained) during germination, root growth and senescence. Young and Varner (75) followed the activity of acid phos- phatase in cotyledons of pea seeds during germination and found a 20-fold increase up to the 6th day, followed by a rapid decrease. They purified the phosphatase several fold by ammonium sulfate precipitation and found it quite Specific for ATP and ADP with a pH Optimum of 6.5. It was relatively heat stable, and activated by divalent metal ions. From the use of inhibitors of protein synthesis (chloramphenicol and dinitrophenol at concentrations that did not prevent germination) they concluded that the increase in activity was due to net protein synthesis. An increase in acid phOSphatase activity during germination was also seen by Presley and Fowden (#5), but from their studies with a proline analogue, they concluded that the increase was not dependent on dg_novo biosynthesis from amino acids (since growth stopped but the in- crease in activity was not affected), but due to the conversion of latent preformed precursors. They used pNPP as substrate, with the supernatant after centrifugation at 12,000 x g as enzyme, but substrate specificity was not investigated. A similar increase in acid phOSphatase with BGP as substrate, as well as in acid and alkaline pyrophosphatase activity, was observed in germination of wheat (hl). McGregor and Street (57) found increased activity of acid phOSphatases in the growing apices of roots and shoots of tomato plants. Using 6GP as substrate, they found phOSphatase activity in the meristem region, root caps, and zones of elongation and primary cell differentiation. After leaf excision, various phOSphatase activities have been found to increase (69). From Aygn§_leaves, four peaks of activity were isolated and purified by Sephadex G75 column chrom- atography: alkaline and acid phosphodiesterases, RNAse and phos- phomonoesterase. Seven hours after excision, these activities had increased 80, 80, 170 and h0% respectively. This increase was completely inhibited by cycloheximide. During the senescence involved in banana ripening (11), a large increase in acid phosphatase activity was found; it increased 10- to 25-fold in the pellet of a 500 x g centrifugation and h-fold in the supernatant. The activity with the pellet could be extracted with Triton X-100, Ca012 or NaCl. The increase was dependent on RNA synthesis. The enzyme was most active with UTP, ATP, AMP, ADP, pNPP and glucose-6-ph05phate. Another study of senescence involved Rhodeo leaf sections (12). The increase in acid phOSphatase activity occurred 12 hours after cutting but could be accelerated and enhanced by abscisic acid. This latter effect was suppressed by auxin «Inaphthalene acetic acid). No increase in activity was seen in the presence of inhibitors of RNA and protein synthesis. The phOSphatase was most active with ATP, UTP and pNPP, with 80% of maximal activity for ADP and 50% for PGA. PHOSPHATASE ISOZYMES The presence of isozymes or isodynamic phOSphataseslhas also been studied in plant tissues. They are characterized by differ- ences in pH optima, heat stability and the effect of various inhibitors or metal ions on the activity with various substrates. In some cases, they have been separated by chromatographic or electrophoretic techniques. From wheat germ, three isozymes were separated by ion-exchange chromatography on DEAE-cellulose (70). The three all had molecular weights of approximately 55,000, but varied in pH optima (5.5, h.5 and h.0 for both pNPP and ATP). The isozymes differed in Michaelis constants and heat stabilities. The lack of substrate Specificity was the same for each. At pH 5, the phOSphatases were most active with PPi, pNPP, ATP and PEP. Mixed substrate studies showed the competitive inhibition of a nonSpecific phOSphatase for each of the isozymes. Inhibitors varied slightly in effect, 1 Before 1959, the term isodynamic phOSphatase was used, being defined as two or more different phosphatases catalyzing identical reactions (51). although molybdate, fluoride and citrate inhibited all three. EDTA activated one isozyme but had no effect on the others. A number of isodynamic enzymes were separated from the cyto- plasm of sugarbeet and Spinach leaves(7) by ion exchange on Dowex 50, by dialysis at various pH'S and by Specific antigen-antibody reactions. These varied in optimum pH, and in kinetic constants with pNPP; Substrate Specificity was not studied. These enzymes were inactivated by dialysis against highly acid buffers but the activity was restored by the addition of various divalent metal ions. Isodynamic phOSphatases in wheat leaf homogenates were studied by D.W.A. Roberts (51). At pH 5.7,a large number of phosphate substrates were varied with possible inhibitors and different patterns of inhibition were found for many of the substrates. For example, the activity towards PGA was unique in showing an increase with beryllium and aluminum ions, inactivation by fluoride and molybdate, partial inhibition by arsenate and oxalic acid, and no effect with magnesium, ferrous or metavanadate ions. He concluded that the "nonspecific acid phOSphatase" of wheat leaves may be a complex of closely related but distinct enzymes with rather narrow substrate specificities. In another paper (52), Roberts studied heat stability of the phosphatase activity of wheat leaf juice toward various substrates. He found very large differences for many of the substrates, yet activities which appeared to be caused by the same enzyme, as judged by the inhibition studies of the previous paper (51), showed similar stabilities towards heat treatment. Thus further evidence was provided that more than a single phOSphatase was involved. Roberts also studied the effect of various divalent metal ions at pH 5.9 and 5.7, and of dialysis, on the activity of wheat leaf phOSphatases toward various Substrates (55). He found some dis- tinction: at least one phOSphatase was activated, one not affected, and possibly one inhibited by metal ions (the last showed in- activation only at pH 5.9). His "PGA phOSphatase" was Slightly stimulated by metal ions at pH 5.9, but showed no change at pH 5.7. More recently Roberts has separated an orthophosphoric mono- ester phosphohydrolase (highly active for adenosine-5'-phosphate) from a P-glycerol phOSphatase, and partially purified and character- ized these enzymes (Sh-57). In one paper (55), differences in proportions of the various phosPhatase activities were noted with differences in growth conditions. PHOSPHOGLYCOLATE PHOSPHATASE Most plant phOSphatases have been isolated and purified on the basis of activity towards pNPP or BGP and the substrate Specificity determined later. But a few enzymes have been iso- lated on the basis of activity towards a certain physiological substrate; two of these are P-glycolate phosphatase (1) and PGA phOSphatase (#6). During 14C-labelling photosynthetic experiments, P-glycolate and glycolate are labelled early, and the glycolate rapidly appears outside of isolated chloroplasts (50). The enzyme reSponsible for the hydrolysis of P-glycolate was sought. Richardson and Tolbert (50) first isolated and purified P-glycolate phosphatase, llO-fold from tobacco leaves. They found it to be absolutely specific for P-glycolate; activities for other phOSphates disappeared during purification. Its activity was inhibited by EDTA and restored by any of a number of divalent cations. The degree of activation 1 depended on the cation, its concentration and pH. Other inhibitors were fluoride, p-CMB, cysteine and reduced glutathione. A modified isolation procedure was deve10ped later (7h) and after each purifi- cation step, the pH optimum was 6.5. P-glycolate phosphatase has been studied in wheat, tobacco, Spinach and alfalfa leaves, and its ubiquitous distribution is now established (h8). It is formed during biogenesis of chloro- plasts; little activity was found in roots or etiolated tissue. Only 9% of the activity remained with the chloroplasts isolated in sucrose or NaCl media after centrifugation at 1,000 x g for 10 minutes (7h) but 20% was retained in the chloroplasts of Spinach if isolated in a sorbitol medium. Chloroplasts were prepared from freeze-dried spinach leaves in a nonaqueous density gradient of carbon tetrachloride and hexane. Of the total P-glycolate phosphatase activity, 80 to 97% was found localized in these chloroplasts. Since activity was not detected with chlorOplasts from a glycerol gradient, the authors concluded that the enzyme was probably located in the soluble stroma of the chloroplasts. P-GLYCERATE PHOSPHATASE In short-time photosynthesis experiments with 14C02 (5), soybean leaves incorporated 14C into the primary organic acids glyceric acid, 2-PGA, 5-PGA and 2,5-diPGA. In 5 seconds, under steady state conditions, the primary product was free glycerate; thelgcarbon was completely inactive. In a similar experiment, at 60 seconds (27), 12% of the total label was found incorporated into glycerate with a Similar labelling pattern to that of PGA. The rapid appearance of radioactivity in glycerate was originally thought to be due to phOSphatase action during the killing pro- cedure (#0). Because the ratio of PGA to glycerate is not constant but decreases with increasing time of exposure to 14002 (27), it is considered unlikely that the early free glycerate formation is due to an artifact of the killing procedure. Since the 14C dis— tribution in glycerate was nearly identical to that in PGA, it seemed most likely that glycerate arose from the in_!i!g hydrolysis of PGA; the phosphatase responsible for this hydrolysis was then sought (A6). PGA phosphatase has recently been purified 2500-fold from sugar cane leaves, and studied in some detail (M7). By sucrose density gradient centrifugation in 0.25 M KCl, the enzyme appeared to have an approximate molecular weight of 160,000 but it aggregated at low ionic Strength. The chemical reaction catalyzed by this enzyme was the stoichiometric hydrolysis of 5-P-glycerate to gly- erate and inorganic phOSphate. It was not absolutely Specific, but PGA was the preferred physiological substrate. Of the compounds 10 tested, only PEP and pNPP were hydrolyZed at greater than 50% of the rate with PGA, P-glycolate was hydrolyzed at only 11% of this rate and P-serine at 10%. The enzyme favoured phOSphomonoester bonds and had limited nucleotidase or pyrophosphatase activity and no diesterase activity. Relative specificity did not change with purification. Only one enzyme was found by isoelectric focusing, ion exchange chromatography or kinetic analysis. Purified PGA phOSphatase Showed optimum activity at pH 5.9 while crude enzyme preparations had a pH optimum of 6.5. Above 7.5, the enzyme was irreversibly denatured. The phosphatase activ- ity was not affected by changes in ionic Strength up to 0.6 M. The kinetic plots of enzyme activity were hyperbolic; a Simple first order reaction was thus indicated with an apparent Km of 2.85 x 10'!+ M. Unlike many other acid phosphatases, this enzyme did not show any divalent cation requirements. ’There was no loss of activity after dialysis against EDTA and little change in the presence of cations, except at high concentrations, when some inhibition occurred. anH'and Cula, however, were inhibitory even at 10-h M. The enzyme was found to be sensitive to most of the usual phosphatase inhibitors: p-CMB, fluoride, arsenate, borate and molybdate, as well as L (+)-tartrate. It was irreversibly and competitively inhibited by glycidol phOSphate and stimulated in the presence of aSpartate. The physiological characteristics of this enzyme were also investigated (AB). The distribution of PGA phosphatase and P-glycolate phosphatase in various plants was studied by differ- ll ential grinding procedures. In C3 plants, which fix C02 by the photosynthetic carbon reductive cycle of Calvin, PGA phOSphatase was found in the cytosol and P-glycolate phOSphatase in the chloro- plasts. In C4 plants, which fix C02 by the C4-dicarboxylic acid cycle and which have two main types of photosynthetic tissue, P-glycolate phOSphatase was found mainly in the chloroplast frac- tion in the bundle sheath cells while PGA phOSphatase occurred in the cytosol of the mesophyll cells. In leaf homogenates, the ratio of PGA phOSphatase to P-glycolate phosphatase activity was much greater than unity in leaves from C4 plants and much less than unity in those from C3 plants (#8). Thus, PGA phosphatase was more active than P-glycolate phosphatase in C4 plants, while the reverse was true in 03 plants. In exploratory experiments with mature leaves, P-glycolate phOSphatase activity seemed to correlate positively whereas PGA phOSphatase decreased with in- creasing C02 fixation rates in soybean. PGA phoSphatase activity increased with time of illumination in etiolated sugarcane, paralleling the biosynthesis of chlorophyll and protein. It also Showed diurnal variation in Sugarcane, in- creasing 50 to 60% in the late afternoon as the photosynthetic rate was decreasing. A particulate form of PGA phOSphatase in spinach leaves was also studied (#9) and it was shown that 58% of the total PGA phos- phatase activity was found in the pellet after a 6,000 x g centri- fugation. By isopycnic sucrose density centrifugation, h8% of this activity sedimented through 0.25 M sucrose along with what l2 appeared to be starch grains, the rest of the activity being soluble. This Starch pellet activity could be solubilized by sonification, B-amylase or incubation with 0.55 M MgC12 or NaCl, but not by homogenization, dialysis, changes in pH or freezing and thawing. It was partially purified and found to have pro- perties Similar to those of the soluble sugarcane enzyme. PROPOSED FUNCTIONS OF PGA PHOSPHATASE Various regulatory and transport functions have been pro- posed for PGA phOSphatase. Since PGA is a primary product of photosynthesis and a key intermediate in glucose synthesis, control of its pool Size must be important. The increase of PGA phos- phatase activity late in the day in the diurnal study and the proportional increase of activity with chlorophyll biosynthesis in etiolated tissue after illumination both indicate at least an indirect role of this enzyme in photosynthetic metabolism. A proposed role for the soluble phosphatase is in the carbon tranSport between cells of C4 plants during photosynthesis (#8). PGA is synthesized in the bundle sheath cells where there is inadequate reducing power. But 50% of this PGA (as shown by labelling Studies) has been found in the mesophyll cells during photosynthesis. In the mesophyll cytosol PGA phosphatase is thought to break down the entering PGA to glycerate which would then be rephosphorylated and reduced to triose phosphate and DHAP in the meSOphyll chloroplast. Thus PGA phosphatase would act as a pump for this carbon transport. 15 The particulate form of the enzyme may well have a role in the regulation of starch biosynthesis or degradation since its preferred substrate, PGA, is a positive effector for ADP-glucose pyrophOSphorylase which makes the substrate for starch synthetase (15, uh). Hence, when PGA phOSphatase is most active, starch biosynthesis would be turned off. THE GLYCOLATE PATHWAY Perhaps the most important function of PGA phOSphatase is its role in the glycolate pathway. This pathway is a metabolic sequence for the production of glycine and serine from glycolate and glycerate in green plant tissue (67). The pathway is usually considered to start in the chloroplast with the production of P-glycolate, which is hydrolyzed to glycolate. In the peroxisome this glycolate is oxidized to glyoxylate and then transaminated to glycine which is in turn converted to serine. Some serine returns to the peroxisome where it is converted to hydroxypyruvate and then to glycerate. The glycerate produced is phOSphorylated in the chloroplast by glycerate kinase to PGA which is involved in hexose metabolism.i Thus, through the glycolate pathway, there is a cycle of carbon flow from, and back to, the photosynthetic carbon cycle. Proof for this pathway originated mainly in labelling experi- ments (67, 71) as well as studies of the enzymes involved in the reactions (9, 67, 72). Glycolate was found to be uniformly labelled early during photosynthesis (8) as were serine and glycine. When 1h one of the enzymes, glycolate oxidase, was inhibited (75) up to 50% of the labelled carbon fixed photosynthetically accumulated as glycolate, whereas normally only 5% of the label is found in glycolate. When glycolate 2-140 was fed to wheat leaves (71), the label rapidly appeared in glycine and serine. Also, the delay in glycine and serine synthesis during greening of etiolated tissue followed the development of P-glycolate phosphatase activity (67), the enzyme which is credited with the initiation of the glycolate pathway. The glycolate pathway is also involved in gluconeogenesis (9, 67). Serine has been found to be a precursor of carbohydrate synthesis in wheat where 50% of the total radioactivity of the added serine was found in starch (56). This was consistent with the glycolate pathway ending in the formation of PGA by glycerate kinase (72). In addition, the serine to glycerate portion of the glycolate pathway seems to be reversible (67). In isolated chloroplasts (8), the serine formed during 14C02 fixation was carboxy-labelled, apparently from PGA. PGA phOSphatase would thus be the enzyme involved in initiating the glycolate pathway in the reverse direc- tion. Normally, the pathway from glycolate seems to predominate Since in Short-time labelling eXperiments (67) serine appeared uniformly labelled. However, the enzymes needed for the reverse direction of the pathway (i.e., Starting from PGA) have been found and Studied in connection with serine metabolism in higher plants (9, 72). It has also been reported that serine was synthesized 15 from hydroxypyruvate in wheat and pea seedlings (55), which would be consistent with this pathway. Another pathway to serine, called the phosphorylated pathway, has been prOposed (25) that would also eXplain carboxy-labelling in serine. This path goes from PGA to P-hydroxypyruvate to P-serine to serine. All the necessary enzymes, except the phOSphatase con- trolling the 1ast step, have been studied (9, 72). When P-glycerate dehydrogenase and glycerate dehydrogenase activites were used to monitor respectively the phosphorylated pathway and the glycolate pathway from PGA, the phosphorylated pathway was found predominantly in etiolated tissues associated with rapid cell proliferation, while the nonphosphorylated pathway predominated in green leaves (9). PHOTORESPIRATION AND C02 COMPENSATION POINTS PhotoreSpiration is defined as the light Stimulated uptake of 02 and release of C02 (67). It is thought to be the result of glycolate metabolism since it is Stopped by an inhibitor of glycolate oxidase and is stimulated in the dark by the addition of glycolate (67). The oxygen uptake phase of photoreSpiration can be explained by the action of glycolate oxidase (51). In a labelling experi- ment involving addition of labelled substrates to leaf segments (52), the greatest radioactivity was released as labelled 002 from carboxy-labelled glycine, which is the final product of per- oxisomal metabolism. The reaction involved was presumably the conversion of 2 molecules of glycine to l of serine plus 1 002 which is measured as photoreSpiration. The role of light in photores- piration has been attributed to the photosynthetic production of 16 of glycolate (52, 67), since glycolate oxidation and C02 evo- lution are dark reactions. The C02 compensation point can be defined as that concentra- tion of atmospheric C02 in which the rate of photosynthetic C02 fixation is equal to the C02 loss by respiration. It is an in- direct measure of the rate of photoreSpiration, which is a mani- festation of the carbon flow through the glycolate pathway. The compensation point was found to increase linearly with an increase in photoreSpiration or decrease in photosynthesis. Compensation points, when measured in mature leaves, tend to be either very low (less than 5 ppm 002) in C4 plants or moderately high (50 to 70 ppm 002) in Ca plants (16, 68).. The compensation point is a steady state value which can be influenced by differences in environ- mental conditions (59). Increasing temperature was found to increase the compensation point (65), apparently because of differences in Q10 of photo- synthesis and photoreSpiration. It also changed with water stress, when wheat (26) or palm (58) leaves were placed in varying concen- trations of mannitol in water, or when water was removed, the compensation point Showed an increase proportional to the amount of stress and returned almost to normal when water was restored. This effect was apparently due to a depression of photosynthesis resulting from a change in stomatal aperture. Light intensity above a certain minimum had little effect on the compensation point since while the photosynthetic rate increased dramatically with increasing light, it was matched by an increasing rate in photoreSpiration (6). 17 The compensation point was proportional to 02 concentration and almost disappeared at very low levels of 02 (5, 68). This effect was a result of the increasing inhibition of glycolate oxidase, a necessary enzyme in the glycolate pathway. Thus photo- respiration was blocked. Increasing values of 02 concentration to above normal (20%) stimulated photoreSpiration and inhibited photosynthesis while the compensation point increased propor- tionally (18). This effect was seen only with C3 plants, C4 plants having low and relatively constant compensation points (19). Dark reSpiration, which also occurs in plants, is defined as the C02 production, normally occurring in the dark, which is not influenced by changes in oxygen concentration above 2 to 5% (l6). EFFECT OF AGE ON PHOTOSYNTHETIC METABOLISM The effect of age, both of leaves and whole plants, on photo- synthesis and respiration rates has also been Studied. As early as 1910 (28), the development of chlorophyll in illuminated etio- lated shoots was found to proceed by hours the net photosynthetic ability. C02 output in respiration was measured in alternating light and dark and it was Shown that even in shoots with "quite a considerable depth of green" there was no noticeable difference between the amount of C02 respired in the dark and in the light. Under conditions of Optimum light intensity, 002 concen- tration and temperature, variations in the "rates of assimilation" (i.e., photosynthetic efficiency) due to age and level of develop- ment of individual leaves or whole plants was demonstrated with l8 sugarcane, wheat and flax (65). Efficiency increased with maturity and declined with senescence. Carbon assimilation increased in young leaves as the plant aged, and was usually higher in fully expanded young than in old leaves from the same plant. At normal 002 concentrations, however, the assimilation rate was independent of the age of the plant. There was a lower rate of dark respira- tion in old leaves but also less apparent assimilation even though they had a higher chlorophyll content. Much work has been done on changes in respiration in conifers. ReSpiration rates of young and old needles in low and high light intensities have been studied (62) and a much higher rate was found in young than in old leaves, especially under high light intensities. Seasonal changes in reSpiration per unit needle length were also followed. The rate of respiration was found to be highest when rapid growth was occurring, this increase reflecting the increase in metabolism associated with rapid vegetative growth. Another Study using pine needles (2h) showed a net C02 loss in very young needles shortly after emergence, followed by a rapid increase in photosynthetic rate culminating near the end of the most rapid growth phase. The respiration rate of new needles attained a maximum Shortly after needle elongation began and de- clined rapidly after shoot extension ceased. The data indicated a rapid conversion of carbohydrates to metabolic energy in the elongating Stem and needles. Also, in conifers the effect of age of leaves on the rate of photosynthesis was studied (22). This rate was low in very young 19 leaves, Showed a peak in fully expanded young leaves and there- after decreased. Photochemical activity of isolated chloroplasts also was found to increase as the leaves enlarged and matured, and then to decrease with age. This was also shown in wheat and sugar- beets. The maximum photosynthetic capacity therefore was at the time of apparent leaf maturity. These trends were also seen in studies on Eastern cottonwood (1h). Photochemical activity of chloroplasts was found to depend on the former history of the leaves from which they were isolated (10). The photochemical activity increased as leaves enlarged and matured, and then decreased with senescence. A mild water deficit in leaves also increased photochemical activity of iso- lated chloroplasts. EFFECT OF LEAF AGE 0N ENZYME ACTIVITIES Recently, various enzyme activities have been studied as a function of leaf age. Zelitch (75) studied glycolate oxidase activity in tobacco leaves and found an increase in Specific ac- tivity with leaf maturity. He also found higher reSpiration rates as measured by 02 uptake in young leaves. Using Eastern cottonwood leaves, Dickmann found the develop- ment of RuDP carboxylase activity closely paralleled an increase in photosynthetic rate in older leaves (1h). Hill reaction ac- tivity (i.e., the photoreduction of DCPIP ) in isolated chloro- plasts also increased with increasing leaf age but lagged behind the increase in C02 uptake. 2O Salin and Homann (59) Studied changes in photorespiratory activity with leaf age. They found higher mitochondrial or dark respiration and lower photoreSpiration in young leaves than in older leaves. Young leaves from C3 plants had nearly identical rates of photosynthesis at high and low oxygen tension, as did corn leaves. They also found lower activities of glycolate oxidase and glyoxylate reductase in young C3 leaves. This implied that the photoreSpiratory process is relatively slow in young leaves. Low levels of glycolate were also noticed in young leaves with or without a glycolate oxidase inhibitor, whereas in old leaves a large accumulation of glycolate appeared in the presence of the inhibitor. This also implied a lack of the glycolate pathway in young leaves. Higher levels of PEP carboxylase were seen in young 03 leaves amounting to 2-5 times that found in mature leaves, but still not as high as that seen in C4 leaves. Thus, a change in photosynthetic metabolism with leaf develop- ment has been well documented, eSpecially from the physiological standpoint and to a lesser extent at the enzyme level. This thesis extends this study, using PGA phosphatase and P-glycolate phos- phatase, which are thought to initiate the semi-reversible gly- colate pathway from either end. 21 MATERIALS AND METHODS BIOCHEMICALS: 5-P-glycerate was obtained as the barium salt from Boeh- ringer and Soehne, GmBH, Mannheim, West Germany, and was conver- ted to the hydrogen form by passing through a Dowex resin (Biorad . AG 50W-Xl2). P-glycolate was obtained as the tricyclohexylammonium salt from General Biochemicals and solutions of it were adjusted to pH 6.5 before use. BUFFER SOLUTIONS: Cacodylate Grinding Medium: 20 mM sodium cacodylate, 1 mM EDTA and 20 mM ascorbate, adjusted to final pH 6.5. Cacodylate-Sucrose Grinding Medium: as above, but in 0.8 M sucrose. Glycylglycine Grinding Medium: 20 mM glycylglycine and 2 mM EDTA, adjusted to final pH 7.5. Glycylglycine-Sucrose Grinding Medium: as above but in 0.8 M sucrose. Cacodylate ResuSpension Medium: 20 mM sodium cacodylate and 1 mM EDTA, adjusted to pH 6.5. STANDARD GRINDING PROCEDURE Freshly harvested leaves were deribbed, washed and weighed, then ground in buffer (usually cacodylate-EDTA-ascorbate buffer) in a Waring blendor at top Speed for 50 seconds. The slurry was 22 then Strained through 8 layers of cheesecloth and the homogenate centrifuged at 10,000 x g for 20 minutes. The pellet was re- SUSpended in a glass homogenizer in cacodylate-EDTA buffer or by passage up and down in a Pasteur pipette in 0.8 M sucrose. All fractions, including the original homogenate, were assayed for phOSphatase activity. STANDARD ULTRACENTRIFUGATION PROCEDURE: The 10,000 x g pellet, resuSpended in 0.8 M sucrose, was layered on the following gradient: 8 ml of 2.5 M, 10 ml of 1.8 M, 15 ml of 1.5 M and 20 ml of 1.5 M sucrose. The gradients were centrifuged at 25,000 rpm in the 25.2 swinging bucket rotor for 5 hours in a Beckman L-2 ultracentrifuge. The resulting gradients showed bands of different shades of green; these bands were separ- ated by dripping from the bottom of the tubes. The pellet was resuSpended in 0.8 M sucrose. All fractions were then assayed for phOSphatase activity using 5 different volumes of enzyme in duplicate and averaging the results. ASSAYS: PGA phosphatase: An aliquot of the enzyme mixture was made up to 0.50 ml with 0.20 M sodium cacodylate buffer, pH 5.9 and equilibrated to 50° in a conical centrifuge tube. The reaction was Started by the addition of 0.25 ml of 50 mM PGA (pH 5.9), and after 10 minutes at 500 was terminated by 0.25 ml of 10% TCA. The protein was removed by centrifugation in a clinical centri- fuge and inorganic phosphate was determined on a 0.6 ml aliquot 23 of the supernatant. For each enzyme sample, a blank in which the enzyme was denatured by TCA before the substrate was intro- duced was run to determine initial inorganic phOSphate levels. A unit of activity is a umole Pi released per minute per gram of wet weight of tissue; a unit of Specific activity is a umole Pi released per minute per mg protein or mg chlorophyll. P-glycolate4phosphatasezThis enzyme was assayed in a Similar manner, but in 0.20 M sodium cacodylate, pH 6.5 containing 10 mM M3012, and with 20 mM P-glycolate as substrate. Glycolate oxidase: The reaction was started by the addition of 0.1 ml of 0.125 M sodium glycolate to a reaction mixture con- taining 2.0 m1 of 0.187 mM 2,6-DCPIP and 0.1 M sodium pyrophOSphate at pH 8.7, 0.05 ml of 5 mM FMN, 0.05 ml of 0.5% Triton x-100 and 0.50 ml of enzyme diluted in water. The assay was performed at 25° in a Thunberg cuvette in a nitrogen atmOSphere. Increasing absorbance from the formation of reduced DCPIP was followed at 600 nm on a Gilford Recording Spectrophotometer. Hydroxypyruvate reductase: The reaction was started by the addition of 100 pl of 10 mM hydroxypyruvate to the following reac- tion mixture: MOO ul of 0.2 M phOSphate buffer at pH 6.2, 50 ul of a solution of NADH (5 mg/ml) and th ul of enzyme diluted in water. The assay was performed at 25° in a 1 ml cuvette, the decreasing absorbance being followed at 540 nm. 2h InorganicAphosphate was determined using a modified Fiske- SubbaRow method (17). To a 0.6 ml aliquot was added h.8 ml of molybdate reagent (0.5% ammonium molybdate in 0.625 N H2804) and 0.6 ml of elon reducing solution (5% NaHSOa, 1% p-methylaminophenol) with thorough mixing. Colour was allowed to develop for at least 15 minutes and optical activity read at 660 nm on 3 Coleman Junior Spectrophotometer. The concentration of Pi up to hO ug/ml was calculated from a standard curve. Protein was determined by the Lowry procedure (5h). To 1.0 ml of sample was added 5.0 m1 of a reagent solution containing 100 ml of 2% Na2C03 in 0.1 N NaOH, 1 m1 of 1% Cuso4 and 1 ml of 2% sodium tartrate. After being mixed, the solution was allowed to stand for 10 minutes. Then 0.5 ml of 1.0 N phenol reagent was added with immediate mixing and colour was allowed to develop for at least 50 minutes. Optical density was read at 660 nm and the protein concentration was determined from a standard curve, using aliquots of 0.100 mg/ml bovine serum albumin. Chlorophyll was determined by the Arnon method (2): a 1.0 ml sample was added immediately to 8.0 ml of acetone and 1.0 ml of water. This mixture was centrifuged at top speed in a clinical centrifuge for 20 seconds, then stoppered and kept cold and in the dark until the optical density was read at 652 nm. A conversion factor of 0.29 x O.D.652 was used to give concentration in mg of chlorophyll per ml of homogenate. 25 EXPERIMENTATION AND RESULTS It should be emphasized that in the following experiments only crude homogenates were assayed for enzyme activities and that no attempts were made to purify these activities. These homogenates were a complex mixture of enzymes and it is quite probable that a number of different phosphatases had activity towards both PGA and P-glycolate with varying degrees of Spec- ificity for these substrates. The factor of interest, however, was not the activity of a unique PGA or P-glycolate phOSphatase but rather the magnitude of the activities towards these substrates as they were found to change during development and under differ- ent growth conditions. Most of the activity towards PGA or P-glycolate is believed to correspond with the enzymes purified by earlier investigators (#7), for several reasons. First, the assays were run at pH 5.9 or 6.5, optimal for PGA and P-glycolate phosphatases but high for most nonspecific acid phosphatases found in leaves. Secondly, homogenates were prepared in the presence of EDTA which inhibits cation-requiring acid phOSphatases. Magnesium ions were added to the P-glycolate phosphatase reaction medium but not to that of PGA phosphatase, Since the latter en- zyme does not require covalent ions. Thus the activities of these two enzymes can be distinguished. Also, as previously mentioned, the purified enzymes are quite substrate Specific: purified PGA phosphatase hydrolyzed P-glycolate with or without magnesium ions at only 11% of the rate with PGA while purified P-glycolate 26 phosphatase did not hydrolyze PGA at all (A8). Pellet activity is defined as the activity that sediments upon centrifugation at 10,000 x g for 20 minutes. This term will not be used in any other context. The phosphatase assays were routinely run in duplicate on three different volumes of enzyme sample, whether homogenate, supernatant, resuSpended pellet or fraction from a gradient. Usually a Single leaf sample was used, but care was taken that the sample was selected as randomly as possible; e.g. a large number of leaves were harvested, and the weight of leaf tissue required was taken from a mixture of pieces from these leaves. 27 A) PGA PHOSPHATASE DISTRIBUTION AMONG DIFFERENT PLANT SPECIES Various plants were surveyed for soluble and particulate PGA phosphatase activity in leaves during the month of August, 1970 (Table II). Forty gram samples of washed, deribbed leaf tissue were homogenized in 100 ml of the cacodylate grinding medium, at high Speed in the Waring blendor for M5 seconds (or 2 minutes for corn and sorghum leaves). The resulting suspension was forced through 8 layers of cheesecloth, the volume was meas- ured and an aliquot removed. The residue from corn and sorghum leaves was further extracted in additional grinding media with a mortar and pestle with sand; the resulting homogenate was also forced through cheesecloth. In this way mesophyll cells (from the Waring blendor homogenization) were partially separated from the bundle sheath cells which were only broken using more extreme techniques (M8). The homogenates were then centrifuged at 10,000 x g for 20 minutes. The pellets from the Waring blendor homo- genates were resuSpended using a Potter-Elvejehm homogenizer. The pellets from homogenates ground by mortar and pestle were re- suspended by trituration in a Pasteur pipette. PGA phOSphatase activities were determined for the homogenate, supernatant and pellet fractions on 5 or M different volumes in duplicate and the results averaged. Sugarbeet and soybean leaves were freshly harvested from the field, in the morning after a hot sunny day. Corn (Michigan 500) and sorghum (ACCO R-920) were also field grown. Sunflower and 28' tomato plants (the latter a dwarf variety) were greenhouse-grown and Spinach and fresh swiss chard leaves were purchased locally. As can be seen in Table II, sorghum was found to have the highest total activity (2M units per gram of tissue in the meso- phyll cells), with soybean next (8.5), then sugarbeets (M.5). Tomato leaves were lowest with 0.55 units per gram. The bundle sheath cells were responsible for 56% of the total activity found in corn leaves and 10% of that in sorghum leaves. The percentage distribution of PGA phosphatase also varied but only sugarbeet (M6%), Spinach (56%) and swiss chard (11%) had appreciable act- ivity in the pellet. These results emphasize that a wide variation in total PGA phOSphatase activity exists among plants and in the distribution of this enzyme in the cell. Sorghum had by far the greatest total PGA phosphatase activity but since the original goal was to study the particulate form of this enzyme, sugarbeet and spinach were selected for further study. Soybean leaves also had a high level of total PGA phosphatase activity, twice that of sugarbeet leaves, but little activity in the pellet. 29 TABLE II PGA PHOSPHATASE DISTRIBUTION AMONG DIFFERENT PLANT SPECIES PGA PhOSphatase Recovery %Distribution Plant (umole ng min‘1 x g'l) £21 .EEE; Pel. Hom. _§33; Pel. Sugarbeet M.h6 2.08 1.78 90 55.9 h6.1 Spinach 2.09 1.92 1.08 1A2 6h.0 56.0 Swiss chard 2.22 2.02 0.27 106 88.h 11.6 Tomato 0.55 0.69 0.06 155 92.2 7.8 Corn lst grind 0.58 0.62 0.05 109 95.2 h.7 2nd grind 0.52 0.25 0.01 88 97.6 2.M Sorghum lst grind 25.82 21.52 0.69 92 96.7 5.5 2nd grind 2.77 2.67 0.12 100 95.7 h.5 Soybean 8.51 8.78 0.29 116 96.8 5.2 Sunflower 1.60 1.55 0.05 100 97.1 2.9 50 B) DIURNAL VARIATION IN LEVELS OF PHOSPHATASE ACTIVITY A diurnal change in PGA phosphatase activity has been shown in sugarcane leaves (M8), in which a peak of activity was seen in the late afternoon. This experiment was repeated using both sugar- beet and soybean leaves and extended to demonstrate any diurnal change in the distribution of the PGA phosphatase activity between pellet and supernatant as well as its change in total activity in the crude homogenate. Changes in P-glycolate phOSphatase activity were also measured in the homogenate. I) SOYBEAN DIURNAL The diurnal changes in PGA and P-glycolate phosphatase acti- vities were studied in soybean leaves over a M8 hour period in July, 1970. Healthy, green leaves from top trifoliates were harvested from field-grown plants every three hours and the light intensity recorded. The leaves from each trifoliate were separated into three replicate groups. From each group, 20 grams of leaf tissue were ground and pellet fractions were prepared following the standard procedure. Enzyme samples were kept on ice or at M0 at all times. PGA phosphatase activity was determined in the homogenate, Supernatant and pellet fractions; P-glycolate phos- phatase only in the homogenate. For the determination of protein concentration, enzyme samples were centrifuged for 6 minutes at high Speed in the clinical centrifuge and only the supernatant was used. Thus, Specific activities are reported in terms of soluble protein. 51 The results are summarized in Tables IIIa and IIIb. The average variation from the mean of determinations of total acti- vity for the three replicate groups of leaves was: for PGA phos- phatase in the homogenate 5%, in the supernatant 8%, in the pellet 9%; for P-glycolate phOSphatase in the homogenate 9%. At night, total PGA phosphatase activity was lowest, about 12 to 1M units per gram of tissue. The total activity increased during the day to a peak value of 19 units at noon, representing a 50% increase in activity over this period. The activity decreased slowly or not at all during the latter half of the day and into the evening. These results are similar to the diurnal pattern for PGA phos- phatase in sugar cane leaves reported by Randall and Tolbert (M8), although the changes in activity in soybean leaves were not as great. The variations in activity observed during the day were all in the soluble fraction; the amount in the pellet remained constant at 5%. Total P-glycolate phosphatase activity also showed an increase around noon (over 50% increase on the second day), followed by a slow decrease into the evening hours. The total activity of P-glycolate phosphatase remained approximately twice as high as PGA phosphatase throughout this experiment. However, the total soluble protein in soybean leaf homo- genates also increased to peak around noon and slowly decreased during the rest of the day. Thus, the Specific activities of either phOSphatase did not Show consistent diurnal changes on the basis of soluble protein. This result does not necessarily negate the fact that the total activities of the phosphatases increased 52 TABLE IIIa DIURNAL VARIATIONS IN TOTAL ACTIVITY OF PHOSPHATASES IN SOYBEAN LEAVES PGA PhOSphatase P-Glycolate * activity is in umole Pix min"1 x g' wet weight Harvest Activityf Recovery Distribution PhOSphatase Hom. Sup. Pel. (%l_ %Sup %Pe1 Activity 11:50 PM 12.5 9.1 0.88 97 91.1 8.8 27.7 2:M0 AM 11.6 9.5 0.87 109 91.6 8.h 27.9 5:50 AM 12.0 9.h 0.89 102 91.M 8.6 28.6 8:50 AM 15.7 15.1 0.66 95 95.2 h.8 29.2 (8000 fc) 11:50 AM 18.6 16.5 0.80 97 95.1 h.9 5h.1 (8600 fc) 2:25 PM 18.7 17.0 0.95 101 9h.8 5.2 55.5 (10h00 fc) 5:25 PM 16.7 15.5 0.77 101 95.2 h.8 55.5 (2000 fc) 8:25 PM 17.9 15.2 0.88 97 9h.6 5.h 52.1 11:55 PM 17.0 15.9 0.78 10h 95.5 h.7 50.8 2:h0 AM 1h.0 12.2 0.85 106 95.6 6.h 50.0 8:50 AM 16.0 15.9 0.85 101 9h.2 5.8 51.0 (5600 fc) 11:25 AM 19.5 17.2 0.96 100 9M.7 5.5 h7.5 (9000 fc) 2:50 PM 18.2 15.9 0.91 95 9M.6 5.h h5.2 (10M00 fc) 5:50 PM 17.2 1h.8 0.91 97 9M.5 5.7 h2.8 (5600 fc) 8:20 PM 16.8 1h.h 0.95 101 95.9 6.1 u2.5 35 TABLE IIIb DIURNAL VARIATIONS IN SPECIFIC ACTIVITY OF PHOSPHATASES IN SOYBEAN LEAVES PGA Phosphatase P-Glycolate Total Specific Activity* PhOSphatase Soluble Time of Specific Protein’ .EEEXEEE 592:. Essa. 221; Asiixiizi ii2_281 11:50 PM 0.M8 0.M2 0.52 1.07 511 2:h0 AM 0.h1 0.h5 0.50 1.00 572 5:50 AM. 0.58 0.5M 0.29 0.82 628 8:50 AM 0.h2 0.56 0.29 0.79 759 11:50 AM 0.h7 1.05 0.52 0.8M 861 2:25 PM 0.h8 1.0M 0.29 0.91 787 5:25 PM 0.h5 1.12 0.2h 0.82 77h 8:25 PM 0.M9 1.05 0.28 0.91 759 11:55 PM 0.h6 1.10 0.25 0.8M 7M7 2:h0 AM 0.h8 0.77 0.50 1.10 M67 8:50 AM 0.M5 0.59 0.52 0.85 715 11:25 AM. 0.hh 1.0M 0.50 1.10 869 2:50 PM 0.M1 0.80 0.29 0.97 887 5:50 PM 0.h1 0.80 0.29 1.01 865 8:20 PM 0.h5 0.76 0.5M 1.11 767 * Specific Activity is in umoles P‘x min'1 x mg'lsol- uble protein 1 Calculated for the homogenate 51. during the day, but it suggests that a diurnal fluctuation of these small amounts is not specific to the phosphatases. II) SUGARBEET DIURNAL Because of adverse weather conditions, the diurnal investi- gation of PGA and P-glycolate phosphatases in sugarbeets was discontinuous, divided into one night and two consecutive days. Mature green leaves from the middle of the plant were harvested from the field-grown plants every three hours. Exact time of harvest, light intensity and temperature were recorded. The leaves were washed, deribbed and chopped finely; three groups of leaf tissue, 20 grams each, were separated and fractions were prepared following the Standard grinding procedure. Assays for the phOSphatases and chlorophyll were performed immediately. Total activities are shown in Table IVa and specific activities on the basis of chlorophyll in Table IVb. The average variation from the mean of determinations of total activity for the three replicate groups of leaves was: for PGA phOSphatase in the homo- genate 6%, in the supernatant 10% and in the pellet 7%; for P-glycolate phosphatase in the homogenate 9%. No diurnal variation in total or Specific activity for either phosphatase was found. The activity levels appeared to vary ran- domly, most of the averaged values lying within the natural vari- ation around the overall average. Percentage of PGA phOSphatase activity in the pellet varied between 20 to 55% of the total activity. The average total activity of P-glycolate phosphatase was four times greater than that of PGA phOSphatase. 55 TABLE IVa DIURNAL VARIATIONS IN TOTAL ACTIVITY OF PHOSPHATASES IN SUGARBEET LEAVES PGA PhosPhatase P-Glycolate Harvest Temp Activity* Recovery Distribution PhOSphatase (PC) Hom. Sup. Pel. (%) %Sup %Pel Activityf night: 9 PM - 6.71 5.75 1.56 106 80.7 19.5 15.2 12 PM - M.98 5.79 1.28 101 7M.6 25.M 21.8 5 AM - 5.59 h.55 1.uu 110 75.0 25.0 19.9 6 AM - 5.52 M.M8 1.02 10h 81.6 18.M 2M.h day 1: 9 AM 25 6.50 5.76 2.57 97 61.2 58.8 20.8 (7200 fc) 12 AM 2M 5.25 5.25 1.29 85 71.M 28.6 21.5 (10800 fc) 5 PM 2h 5.75 h.05 1.50 96 75.0 27.0 22.2 (6200 fc) 6 PM 2h 5.79 5.79 2.11 105 ou.u 55.6 20.0 (1200 fc) day 2: 9 AM 16 5.72 5.87 1.15 92 77.M 22.6 20.2 (6800 fc) 12 AM. 25 h.90 5.07 1.h5 95 67.9 52.1 17.1 (8M00 fc) 5 PM 25 5.02 h.50 1.21 112 78.2 21.8 21.5 (9200 fc) 6 PM 25 5.69 2.8M 0.97 106 7M.5 25.5 17.0 (2M00 fc) 9 PM 21 5.28 2.7M 0.55 10h 8h.0 16.0 19.0 * Activity is in umole ka min'1 x g"1 wet weight 56 TABLE IVb DIURNAL VARIATIONS IN SPECIFIC ACTIVITY OF PHOSPHATASES IN SUGARBEET LEAVES PGA Phosphatase P-Glycolate _§pecific Activity* Phosphatase Total Time of Specific Chlorophyllf _Ha_r_v_e_s_t_ Hom. §3p_._ _P_el_._ Activity* (in mg) night: 9 PM 0.M5 0.59 0.25 0.8M 515 12 PM 0.5M 0.26 0.25 1.h5 298 5 AM 0.56 0.51 0.50 1.52 500 6 AM 0.57 0.52 '0.21 1.78 288 day 1: 9 AM 0.55 0.55 0.59 1.6M 251 12 AM 0.h2 0.28 0.51 1.68 2h0 5 PM 0.h8 0.57 0.58 1.87 _ 217 6 PM 0.5M 0.58 0.60 1.87 217 day 2: 9 AM. 0.h9 0.5M 0.51 1.7M 25M 12 AM 0.hh 0.50 0.59 1.52 225 5 PM 0.h0 0.56 0.52 1.6M 257 6 PM 0.52 0.25 0.25 1.59 2M7 9 PM 0.51 0.25 0.1M 1.6M 255 * Specific Activity is in umole P‘x min-1 x mg'1 chlorophyll. f ChlorOphyll was measured in the homogenate only. 57 C) PGA PHOSPHATASE PELLET ACTIVITY IN SPINACH LEAVES The distribution of PGA phosphatase in the particles from Spinach leaves has been found to be variable. In the first group of experiments, the Spinach used was purchased locally and thus was field grown. Forty grams of leaf tissue were homogenized in 100 ml of cacodylate grinding medium in the Waring blendor at top speed for M5 seconds. The homogenate was centrifuged at 10,000 x g for 20 minutes. The pellet was resuSpended in cacodylate-EDTA buffer using a Potter-Elvejehm glass homogenizer. An aliquot was removed and the rest recentrifuged to give a second pellet and second supernatant from the first pellet. PGA phosphatase activity was then determined in each fraction. 0f the original total PGA phosphatase activity, 56% was found in the first pellet (data not Shown). After resuSpension, which should have broken all chloro- plasts and peroxisomes, 29% of the original total activity was still particulate; i.e., only 20% of the activity found in the ori- ginal pellet was solubilized in a glass homogenizer. Since the pellet activity of PGA phOSphatase was quite resistant to solu- bilization, the phosphatase appears tightly particulate bound, and is certainly not loosely contained within chloroplasts or per- oxisomes. These results appear to be consistent with those of Randall and Tolbert (M9) who found PGA phosphatase activity bound to the starch pellet. In a second group of experiments, large mature Spinach leaves were harvested from plants in a growth chamber. Leaf tissue was 58 ground in either cacodylate (no sucrose) or glyclyglycine-sucrose grinding media following the standard grinding procedure. The pellets were resuSpended in 0.8 M sucrose and separated into frac- tions by ultracentrifugation following the standard procedure. All fractions were assayed for PGA phOSphatase activity (Table V). Slightly more total activity was extracted using glycylglycine- sucrose buffer although the cacodylate buffer gave more activity in the pellet fraction. Neither of these differences may be sig- nificant. In both cases, the pellet activity was less than was found in field-grown leaves. This may well be a function of growth temperature as will be shown for sugarbeets (see Table VI). The distribution of PGA phosPhatase in the sucrose gradients varied apparently as a result of the different grinding media. When the cacodylate medium was used, 5M% of the PGA phOSphatase activity in the 10,000 x g pellet was in the broken chlor0p1ast fractions and 10% more in the whole chloroplast fraction of the gradient. There was 29% of the total gradient phoSphatase activ- ity in the supernatant and only 1% in the starch pellet. However, in particles prepared in glycylglycine-sucrose, the PGA phOSphatase activity was more widely distributed on the gradient with 55% in the broken chloroplast fraction, 12% in the whole chloroplast fraction and 25% in the supernatant. The starch pellet fraction which sedimented through 2.5 M sucrose contained 1M% of the original pellet activity. Thus, when particles were prepared in glycylgly- cine-sucrose medium as Randall and Tolbert (M9) had done, PGA phosphatase appeared more tightly bound to the starch grains than 59 when particles were prepared in cacodylate buffer without sucrose. The distribution of these gradients, however, was very different from the pattern that was found using field—grown leaves (M9) where the 10,000 x g pellet activity was equally distributed in sucrose gradients between the supernatant and the starch pellet and with virtually no activity in the chloroplast fractions. Although this particular experiment was done only once, many similar sucrose gradients have been run and in each case over 60% of the activity found in the pellet of a 10,000 x g centrifugation occurs in the chloroplast fractions. The Significance of this difference in distribution is not clear and more definitive eXperi- ments are required. However the results suggest that the distri- bution of PGA phosphatase may vary with the grinding procedure employed as well as the previous history of the leaves." M0 TABLE V SUCROSE GRADIENTS OF SPINACH PELLETS Original Homogenate Supernatant Pellet % Recovery Sucrose Gradient Whole chloroplasts Broken chloroplasts Supernatant Starch pellet Other fractions % Recovery PGA Phosphatase Glycyl- Cacodylate glycine- sucrose (Activity*) 2.52 5.15 2.68 2.71 0.55 0.5M 0.062 0.056 0.556 0.106 0.17M 0.070 0.007 0.0h2 0.0h5 0.0M8 Distribution Glycyl Cacodylate glycine- sucrose (%) (%) 85 89 17 11 128 97 10 12 5M 55 28 25 1 1M 7 16 117 95 * Activity is in umole Pix min"1 x g’1 wet weight. Ml D) CHANGE IN ACTIVITY AND DISTRIBUTION OF PGA PHOSPHATASE WITH GROWTH CONDITIONS IN SUGARBEET LEAVES Variations in the total activity and particulate distribution of PGA phosphatase were noticed in field grown plants harvested at various seasons during the first year of this investigation. In particular, a substantial PGA phosphatase activity in the par- ticulate fraction was found during the summer but when experiments were repeated in October, less total phosphatase was found, with only trace activity in the particulate fraction. Since a tempera- ture effect was suSpected, plants were grown in control environment growth chambers. Although temperature and day length control were thus possible, the resultant artificial light of 2,000 to 5000 foot-candles may have introduced an additional variable. Sugarbeet seedlings were started in the greenhouse at approx- imately 2M-27° with a lM-hour day from a bank of fluorescent lights providing intensities of 1,000 foot-candles. After 52 days, some plants were moved to a "cool" growth chamber with an l8-hour day at 20° and a 16° night, and with a light intensity of approximately 2,000 foot-candles. Leaves were harvested 52 days after planting, they were ground and pellets were prepared and separated by ultra- centrifugation following the standard procedures. All gradient fractions as well as the original homogenate and supernatant were assayed for PGA phosphatase activity. Some of the plants in the cool growth chamber were then moved back into the warm greenhouse, and some greenhouse plants were 1.2 moved into a "warm" growth chamber with 25° days and 200 nights. After 12 days (6M days after planting), leaves were harvested from the three sets of plants: those grown in the greenhouse, those grown in the cool growth chamber, and those moved back into the greenhouse from the cool growth chamber. The preceeding methods of phosphatase analysis were again employed. Ninety-two days after planting, leaves were harvested from the plants in the warm growth chamber, in the cool growth chamber, and those grown continuously in the greenhouse. Equal weights of large and mature leaves were ground in either the cacodylate medium as above, or 0.8 M sucrose. Supernatants and pellets of the first 10,000 x g centrifugation as well as homogenates were assayed for PGA phosphatase; sucrose gradients were not run. The results of these experiments are in Table VI. After 52 days,leaves of the greenhouse—grown plants had slightly higher total PGA phosphatase activity with a greater percentage in the pellet than had plants grown in the cool growth chamber. When these pellets were run on a sucrose gradient, the greenhouse- grown leaves showed a greater percent activity in the whole and broken chloroplasts and less in the supernatant than did the growth chamber leaves. After 6M days, the total activity in greenhouse leaves was Slightly lower than before, but was diStributed evenly between the supernatant and the pellet of the 10,000 x g centrifugation. Plants placed in the cool growth chamber had lost phOSphatase M5 activity, having only l/M the activity found in plants kept in the warmer greenhouse and now only 9% of this activity was found in the pellet of the 10,000 x g centrifugation. When the pellets were run on sucrose gradients, the distribution of activity was approximately the same with over 70% in the chloroplast fractions and very little in the starch pellet. There was, however, a much larger percentage of activity (26% rather than 6%) in the whole chloroplast fraction in the case of greenhouse-grown leaves. The total PGA phosphatase activity and the percentage of this activity in the pellet of plants grown for 20 days in the cool growth cham- ber and then returned to the greenhouse for 12 days were inter— mediate between the low levels in plants kept 52 days in the cool growth chamber and the high levels in plants kept constantly in the greenhouse. In all the sucrose gradients run, the greatest amount of acti- vity (70-80%) coincided with the chloroplasts, as marked by chloro- phyll, with up to 20% in the supernatant and always less than 10% in the heavy starch pellet. After 92 days, the greenhouse leaves again showed the greatest total activity (approximately M times that of the cool growth- chamber leaves) and the greatest percentage of activity in the 10,000 x g pellet. The values for plants in the warm growth cham- ber were intermediate both in total activity and in pellet activity. There was an increase in total activity with the increasing age of the plant in all these environments although in all cases the leaves MM. used were large and mature. When sucrose was used as a grinding medium, somewhat less activity was extracted and a smaller per- centage of activity in the pellet was found each time. Thus there appeared to be a definite correlation between PGA phosphatase levels and temperature. The highest activity was seen in greenhouse-grown plants, where the temperature was held at 2M-27°, the lowest in the cool growth chamber (16-208), with the plants transferred to a warmer growth chamber (20-2M’) having an intermediate activity level. When plants from the cool growth chamber were returned to the greenhouse, the activity also increased to an intermediate level. In addition, there was a correlation between growth conditions and size and colour of the leaves: plants grown in the cool growth chamber had small, dark green leaves, while those in the greenhouse were large and medium-light green, the others being intermediate. Thus there were great physiological as well as enzymatic changes involved. The distribution of the (phOSPhaLase activity also seemed dependent on growth conditions, with the highest percentage found in the pellet (MO-50%) in the greenhouse-grown plants with only a negligible amount in the plants grown in the cool growth chamber. Also, the warm growth chamber plants and the cool growth chamber plants that were returned to warmer conditions had intermediate values. In all cases, only one leaf sample was obtained, since few leaves could be harvested without damaging each plant. However the magnitude of the changes in total activity and in distribution after a 10,000 x g centri- fugation is such that the above conclusions seem to be justified. M5 The sucrose gradients of the various pellets also showed some differences: the activity in the whole chloroplast fraction was highest in the greenhouse-grown plants (2-M times that found in cool growth chamber plants). It is important to note that in all the sucrose gradients run, the greatest amount of the original 10,000 x g pellet activity was found in the whole and broken chloroplast fraction with little in the supernatant or pellet (contrary to the previous findings in Spinach by Randall and T01- bert, M9). Since the activity remained with the chloroplasts even when broken it appears to be bound to the chloroplast mem- brane. The form of binding is not known, but may be due to non- specific adsorption. M6 .Eofivofi wcgvawum omouoom magma vengeano mum mononucmuwa aw moofim> * .umHHom season am mucoumaquSm am “mummHQOHOaao aoxown Aom “mummfioouofino oHonz mg ..03 + .unwwm3 uma Amy oH Away mm Remy m: H OH em 8 m a ma mo Ha ea n m an em 0m m we 6m ea m m m an Hm SH m m um 62 .Hom H “Ema om Away mm Anwv Pm Hm ow 0m mm mm .msm -w x +coHusnwuunwn ucogwmuu & consonanooao $71 A©J.HV w>.H A:>.Hv mm.m .85 one mm.o we.a ma.m mm.m om.m amua>ano¢ Hauoa so mm Ammmwv ow< tows x «m oaoai a“ ma huw>wuow Hauoa * panacea Hooo whom 0d + umpfimno Bums name 0: + omnoncmmuw when 0: + umpfimno Hooo mmmv Na + omsoncoouw ammo Na + onsoncoonw mmmv NH.+ <<1<3 m m < Amy umpfimno nuaoum aooo AHHU< ZH mozmmu H> mammH M7 E) CHANGE IN TOTAL ACTIVITY AND CELLULAR DISTRIBUTION OF PGA PHOSPHATASE WITH LEAF AGE IN SUGARBEET. During the early investigations, it was noticed that young leaves had a significantly different level of PGA phOSphatase activ- ity than did older leaves from the same plant. The following exper- iments were done to elucidate this difference and to investigate changes in cellular distributidn as well. Also, Since magnesium ions solubilize PGA phosphatase activity from starch grains(M9), M3012 was added to the grinding medium in one set of experiments to ascertain its effect. Young and old leaves harvested from greenhouse-grown sugarbeets were ground in cacodylate-sucrose grinding medium, in either a Waring blendor at top Speed for 50 seconds, or an Omnimix for 20 seconds, they were then strained through cheesecloth and the homo- genates were centrifuged at 10,000 x g for 20 minutes. The pellets were resuSpended in 10 ml of 0.8 M sucrose, and layered on the following gradient: 10 ml of 2.5 M and 20 m1 of 1.5 M sucrose. The gradients were centrifuged following the standard procedure. All fractions were assayed for PGA phosphatase (Tables VIIa and VIIb) and activities in gradients for the whole and broken chloroplasts were combined. In these experiments, young leaves were about 1/5 of full size but fully green and unfurled, old leaves were fully eXpanded, and leaves of medium age had almost attained full size. PGA phosphatase activity in young leaves (averaging 18 units per gram of wet weight) was 6 times higher than in the old (averaging 2.9 units). The distribution of activity also varied with age; LB. there were much higher percentages of activity in the pellets in young leaves than in old. From sucrose gradients of these pellets, 96% of the pellet activity was with the chloroplasts from young leaves with extremely little activity being found in the starch pellet or the soluble fraction. From old leaves, only 62% of the remaining 10,000 x g pellet activity was in the chlor0p1ast frac- tion, while almost 20% was found in the supernatant of the gradient and 17% in the starch pellet. Leaves of medium age showed a dis- tribution intermediate between these extremes. AS can be seen in Table VIIa, the total activities found in young leaves varies considerably with the sampling and the grinding procedure, whereas this variation is not as great in old leaves. But each experiment (i.e., A, B or C) was run on different days and no effort was made to follow a strict definition of "young" as was attempted in the following experiments. The gradients shown in Table VIIb were run only once but again variations in distribution found in leaves of varying ages were considerable. The recovery of the activity put on the gradient was close to 100% for all three ages of leaves. A concentrated solution of MgClg (0.25 M) in the grinding medium (see D and E in Table VIIa) solubilized the pellet activity from 17% to 5% and also caused an activation of total phosphatase activity in leaves of medium age. The leaves used for D and E were duplicate samples. Since the variation from the mean among triplicate sam- ples as seen in the experiments on diurnal change (Section B) was never greater than 10%, the almost 50% increase in activity in the sample ground in the presence of MgC12 Should be significant. 1+9 There was so little phOSphatase activity left in the pellet that it could not be detected in the sucrose gradient. The effect of activation and solubilization of activity is consistent with the findings of Randall and Tolbert (M9). The results of these experiments clearly indicate that young sugarbeet leaves have a much higher level of PGA phosphatase per gram of wet weight and have more of this activity in the chloro- plasts than have older leaves. Much more of the total PGA phos- phatase activity in the old leaves was in the soluble fraction and of the remaining particulate activity more was in the starch pellet when compared to that in young leaves. 50 TABLE VIIa CHANGE IN DISTRIBUTION OF PGA PHOSPHATASE WITH LEAF AGE* PGA PhosPhatase Leaf Age Eth. Activity+ Recovery Distribution Hom. Sup. Pel. 3%1_ %Sup %Pel Young A 25.1 1M.2 10.1 106 59 M1 B 19.7 17.1 9.57 120 65 55 c 12.0 5.5M 2.M5 88 69 51 Old A 2.97 2.71 1.29 96 95 5 B 5.59 2.80 0.52 102 8M 16 c 2.h6 2.10 0.16 95 92 8 Medium D 5.98 M.67 0.9M 95 85 17 plus Mg“’ E 8.88 8.69 0.26 101 97 5 * Young and old leaves for each experiment were from the same plants. A,B leaves were ground by Waring blendor, C by Omnimix, D,E leaves were duplicate samples, ground with and without Mg++ +-Activity is in umole Pix min"1 x g'1 wet weight. 51 TABLE VIIb DISTRIBUTION OF PGA PHOSPHATASE IN FRACTIONS OF A SUCROSE GRADIENT % of Original Leaf Age* Gradient Total % of Total Homogenate Fraction Activity* Gradient Activity Young (A) Soluble 0.9M 1.0 0.M Chloroplast 9.01 96.0 59.0 Starch Pellet 1.59 1.5 0.6 Medium (D) Soluble 0.0M5 5.M 0.9 Chloroplast 0.666 79.2 15.5 Starch Pellet 0.115 15.5 2.5 Old (A) Soluble 0.029 19.5 1.0 Chloroplast 0.09M 61.7 5.1 Starch Pellet 0.025 16.8 0.8 * A and D refer to experiment numbers in Table VIIa. + Activity is in umole Pix min"1 x 3‘1 wet weight. 52 F) THE EFFECT OF THE GRINDING PROCEDURE UPON PHOSPHATASE ACTIVITY At this stage in the investigation, it appeared necessary to obtain quantitative data upon the measurement of total phosphatase activity in homogenates of leaves of different ages. Until this time in the program, a Waring blendor extraction for 50 seconds at full speed had been used for several reasons. This method was convenient, quick, applicable to large random samples and had been used previously by Randall and Tolbert (M7,M8) for all their work on PGA phosphatase. The grinding time in the Waring blendor had been kept short in order to recover particles and Study cellular distribution of the phosphatases, but did not break all the tissue. Longer periods of grinding in the Waring blendor had been found to inactivate P-glycolate phOSphatase (M8) and produce such an extreme amount of foaming that good recovery was impossible. To ascertain quantitatively the limitations of the standard grinding procedure, a series of experiments were performed in which duplicate samples of leaf tissue were ground either by mortar and pestle with sand or by the waring blendor and the resulting PGA or P-glycolate phOSphatase activities_were compared (Table VIIIa). In the former, small samples were thoroughly ground at M°, the homogenate filtered through Miracloth, the residue reextracted and the filtrates combined. Much higher total activity was always recovered by grinding with the mortar and pestle; this was eSpecially true in PGA phosphatase activity with young and very young leaves, particularly in Spinach where there was severe foaming. Older leaves usually showed 55 reasonable recoveries (60-90%) of total activity. The recovery of phOSphatases in terms of Specific activity (Table VIIIb) was much greater (except in the case of Spinach), which implied that the difference between these two grinding procedures was due only to the extent of cell rupture. Admittedly it would have been better to grind all samples in a mortar and pestle, but the number and Size of samples needed to ensure randomness were limiting. So a compromise was made. In tissue where the recovery by Waring blendor was reasonably high an appropriate correction factor was used in reporting the results. But in the case of Spinach leaves, mortar and pestle grinding was used instead. Thus all results in the next set of experiments are given in terms of activity released by mortar and pestle grind- ing, assuming that this is the total maximum phosphatase activity present in the leaf cells. 5M TABLE VIIIa DETERMINATION OF GRINDING PROCEDURE CONVERSION FACTORS (FOR TOTAL ACTIVITY) Plant Leaf Age PGA PHOSPHATASE Soybean old young Spinach old young very young Sugarbeet old young very young P-GLYCOLATE PHOSPHATASE Soybean old young Spinach old young very young Sugarbeet old young very young Total Activity Waring Blendor Mortar and Pestle (umole Pix min"1 x g'l) 27. 52. .m. .15 .52 .51 .8 .61 4H4? M40 8 5 .M 15.2 .15 18. .11 1M. 12. .7M 0 6 l \N {DION \OCDO H mm 4=‘\J'IO\ O\\N\l \O nan) (pin paid Idtutd 4mm 42"le U1U'l \Nl'Dh) were: v1 Recovery* ($6) 81 60 27 26 59 5M 61 52 7M 77 1+5 90 80 51 * Recovery is based on mortar and pestle procedure as 100%. Plant 55 TABLE VIIIb DETERMINATION OF GRINDING PROCEDURE CORRECTION FACTORS (FOR SPECIFIC ACTIVITY) Leaf Age PGA PHOSPHATASE Soybean Spinach Sugarbeet old young old young very young old young very young P-GLYCOLATE PHOSPHATASE Soybean Spinach Sugarbeet old young old young very young old young very young Specific Activity* Waring Blendor 629 910 19 208 79 125 2M2 250 5M8 57M 59M M26 197 M00 250 115 Mortar and 2293.13. 561 888 28 819 115 155 37M 552 M15 M52 M71 M96 168 529 251 115 Recovery iii 112 102 67 26 68 79 65 69 8M 86 85 86 117 121 99 98 * Specific Activity is in nmole P‘x min'1 x mg’1 protein. Recovery is based on mortar and pestle procedure as 100%. 56 G) CHANGE IN PHOSPHATASE ACTIVITIES WITH AGE OF LEAF To extend the results found earlier with young and old sugar- beet leaves (in Section E), phosphatase levels in leaves of varying ages from Spinach, soybean and sugarbeet plants (all grown in controlled environment growth chambers) were studied in the follow- ing experiments. Both PGA and P-glycolate phosphatases were determined in very young (unopened), young and old leaves. I) PGA PHOSPHATASE AND P-GLYCOLATE PHOSPHATASE IN SOYBEANS Soybean (Glycine max L. variety Chippewa) were grown with a light intensity of 1500-2000 foot-candles in a growth chamber at 26° during the lM-hour day and at 21° at night. Leaves of differ- ent ages were harvested on the MOth and M6th days after planting and homogenized in cacodylate-sucrose grinding medium in a Waring blendor at top speed for 50 seconds. The resulting slurry was forced through 8 layers of cheesecloth and assayed immediately for phosphatase activity. Leaves were also harvested from soybean plants grown for 5M days in a greenhouse. These plants had shorter internodes and seemed more advanced as indicated by their having more trifoliate leaves than the plants in the growth chamber. These leaves were extracted using a mortar and pestle with sand. In all cases, the old leaves were the oldest trifoliate before senes- cence on each plant, and averaged 1.6 g per trifoliate without Stem. The very young leaves were unopened and immature, light yellow green in colour and weighed approximately 0.05 g per tri- foliate. The young leaves were intermediate in weight, 0.55 g per 57 trifoliate, and were seleCted just after they had unfolded and turned green. The activity of PGA phOSphatase (Table IX) was virtually iden- tical in young and old leaves on the basis of wet weight or protein but Since the old leaves weighed 5 times more than young leaves, the total activity per leaf was much greater. In very young, devel- oping leaves, there was about half as much PGA phosphatase activity by weight or by protein; thus, the total activity per leaf was extremely small when compared to the larger mature leaves. P-glycolate phosphatase activity was twice as high in the old as in the young leaves and was very low in the very young leaves. Thus, the amount of P-glycolate phosphatase per unit weight in soybean leaves varied greatly with leaf age. To analyse these changes, the ratio of total PGA phosphatase to P-glycolate phos- phatase activity in soybean leaves is presented in Table XII. In old leaves, there was about equal amounts of these two phos- phatases, although there was somewhat more PGA phOSphatase than P-glycolate phosphatase. In young green leaves, there was 2-M times more PGA phosphatase than P-glycolate phosphatase, and in very young immature leaves, this ratio increased to M-8 times, as there was little P-glycolate phosphatase present. Thus the ratio of PGA to P-glycolate hydrolysis rates varied with leaf age, becoming smaller as the leaf ages. The high levels of PGA phos- phatase activity in soybean leaves should also be noted; in almost every case this enzyme was found more active than P-glycolate phos- phatase.' This is not characteristic of 03 plants (M8), as is shown below for the cases of mature Spinach and sugarbeet leaves. 58 TABLE IX CHANGE IN TOTAL PHOSPHATASE ACTIVITIES WITH LEAF AGE IN SOYBEANS Age from Leaf Age Ratio of Activities Planting old young very old : young: very (days)? young young PGA PHOSPHATASE (Total Activity*) M0 27.6 26.6 15.1b 1.0M : 1.00 : 0.57 M6 55.1 59.M 25.0b 1.00 : 1.12 : 0.65 5M 25.2 28.M 21.0 1.00 : 1.15 : 0.85 average 29.M 51.6 19.7 1.00 : 1.08 : 0.67 (Specific Activity*) M0 557 M81 2M2d 1.12 : 1.00 : 0.50 M6 615 57M 552d 1.07 : 1.00 : 0.61 5M M16 50M 507 1.57 : 1.00 : 1.01 average 522 M55 510 1.1M : 1.00 : 0.68 P-GLYCOLATE PHOSPHATASE (Total Activity*) M0 19.8 12.0 1.98 1.65 : 1.00 : 0.16 M6 29.2 9.6 5.53 5.06 : 1.00 : 0.56 5M 50.M 15.8 M.8 2.20 : 1.00 : 0.55 average 26.5 11.8 M.0 2.25 : 1.00 : 0.5M (Specific Activity*) M0 585 215 28c 1.80 : 1.00 : 0.1M M6 518 159 7MC 5.72 : 1.00 : 0.55 5M 50M 1M9 71 5.M0 : 1.00 : 0.M8 average M68 168 58 2.78 : 1.00 : 0.55 * Total Activity is in nmole Pix min"1 x g'1 Specific Activity iS in nmole Pix min"1 x mg”1 protein 1 Plants M0 and M6 days old were grown in a growth chamber, those 5M days old were grown in the greenhouse. a corrected for M0% recovery b corrected for 50% recovery c corrected for 85% recovery d represents 100% recovery 59 II) PGA PHOSPHATASE AND P-GLYCOLATE PHOSPHATASE IN SPINACH LEAVES An initial experiment was run on spinach leaves from plants were grown in a greenhouse for 50 days on a short 8-hour day fol- lowed by 15 days with 16 hours of light. Old, young, and very young leaves were ground either by a Waring blendor or by mortar and pestle (Table VIIIa). EXperiments which used grinding with the mortar and pestle showed 50 times more activity on a wet weight basis of PGA phosphatase in young green leaves than in the old leaves. There was also a considerable amount of activity in the very young leaves (still 15 times greater than in the old). Due to shortage of leaves, however, the experiment on greenhouse spinach was not repeated. The rest of the spinach experiments were run on plants (§Pifl' acea oleracea L. Savoy hybrid 612) grown in a growth chamber at 26° during the 11-hour day and with light intensity at 1600- 2M00 foot-candles, and at 21° at night. Old, young, and very young leaves were harvested on the 55rd, 68th, 75th and 87th days after planting. The old leaves were the oldest, most healthy leaves before senescence, weighing usually 2-5 g per leaf; the very young leaves were the smallest central leaves, usually light green to yellow in colour and weighing 0.05-0.06 g per leaf; and the young leaves were intermediate at approximately 0.5 g per leaf and already the characteristic dark green colour. Since recoveries of PGA phosphatase activity in young and very young leaves had been so very low when a Waring blendor was used (Table VIIIa), these experiments were only done by grinding with a mortar and pestle. 60‘ In this series of experiments, it was found that there was 5 times more total PGA phosphatase in young leaves than in old, or in terms of specific activity, over twice as much as that in the old (Table X). Levels in very young leaves were also high. P- glycolate phosphatase levels, in terms of total activity, were also greater in young than in old leaves, but in terms of specific ac- tivity this difference disappeared. When PGA phosphatase to P- glycolate phosphatase activity ratios were determined for different aged leaves (Table XII), there was more PGA phosphatase activity per unit of P-glycolate phosphatase activity in young leaves than in old. This follows the same trend seen in soybeans. This ratio of activities averaged about l:M for 03 plants including Spinach in a survey run by Randall and Tolbert (M8). They used mature but not old leaves. These results indicate, however, that this ratio can vary considerably depending upon leaf age. In old leaves, the ratio was about 1:6 (Table XII) but in young green leaves, this ratio was only about 1:2. Thus, varying ratios reported by Randall and Tolbert may in part be accounted for by the different ages of leaves used. In the case of growth chamber-grown Spinach, there was always more total P-glycolate phOSphatase activity than PGA phOSphatase activity even when the latter was at its peak. It is also inter- esting to note that the greenhouse spinach (ground by mortar and pestle) showed much more phosphatase activity per wet weight and per mg protein in young and very young leaves than had the growth chamber spinach (compare Table VIIIa and VIIIb with X) This is pre- sumably because of growth temperatures or perhaps day length. This trend was also seen in P-glycolate phosphatase activity in old leaves. 61 TABLE X CHANGE IN TOTAL PHOSPHATASE ACTIVITIES WITH LEAF AGE IN SPINACH Age from Leaf Age Ratio of Activities Planting old young very old : young: very (days) young young PGA PHOS PHATAS E (Total Activity)* 55 1.29 5.81 2.69 1.00 : M.M7 : 2.06 68 1.9M 6.10 M.M6 1.00 : 5.15 : 2.50 75 1.16 6.20 5.59 1.00 : 5.29 : 2.89 87 1.26 8.92 M.8M 1.00 : 7.10 : 5.8M average 1.M2 6.75 _ 5.8M 1.00 : M.75 : 2.70 (Specific Activity)* 55 91.5 118 55.5 1.00 : 1.50 : 0.61 68 82.2 156 69.0 1.00 : 1.89 : 0.8M 75 51.2 125 77.5 1.00 : 2.M5 : 1.51 87 62.0 205 122 1.00 : 5.27 : 1.97 average 71.6 151 81.0 1.00 : 2.20 : 1.1M P-GLYCOLATE PHOSPHATASE (Total Activity)* 55 7.M2 11.M 9.65 1.00 : 1.5M : 1.29 68 9.51 18.6 M.98 1.00 : 2.00 : 0.55 75 7.80 15.2 8.80 1.00 : 1.69 : 1.12 87 11.1 19.M 7.15 1.00 : 1.75 : 0.6M average 8.95 15.7 7.6M 1.00 : 1.76 : 0.86 (Specific Activity)* 55 259 19M 165 1.25 : 1.00 : 0.85 68 59M 56M 155 1.08 : 1.00 : 0.M2 75 5M2 287 200 1.19 : 1.00 : 0.70 87 5M9 MM2 181 1.16 : 1.00 : 0.M1 average 581 525 17M 1.18 : 1.00 : 0.5M * Total activity is in nmole Pix min‘1 x g"1 wet weight. Specific activity is in nmole P;x min'1 x mg"1 protein 62 III) PGA PHOSPHATASE AND P-GLYCOLATE PHOSPHATASE IN SUGARBEET LEAVES Sugarbeets (Beta vulgaris L.) were grown in two growth chambers: one warm, at 25° during the lM-hour day with light intensity of 2200-5800 foot-candles and 170 at night, and the other cool, at 15° during the day with 2000-2M00 foot-candles and 5° at night. Young and old leaves from each chamber were harvested between the 5Mth and 89th day after planting, homogenized in cacodylate grinding medium in a Waring blendor at top Speed for 50 seconds, filtered through cheesecloth and assayed immediately for phosphatase acti- vities. From plants grown in the warm growth chamber, the old leaves weighed 5-9 g each and the young leaves 1.0-1.5 g each. From plants grown in the cold chamber, the old leaves varied from 5-8 g, and the young leaves 1.5-5.0 g. In each case, there was an attempt made to pick leaves of the same physiological age when comparing warm and cold growth conditions. The results (Table XIa) show the profound effect of the dif- ferent growth conditions upon total phOSphatase activities. The young leaves from the plants grown in the warm growth chamber had 15 times more PGA phosphatase and 1.5 times more P-glycolate phos- phatase activity, on the basis of wet weight, than had the old leaves. In the plants which grew Slowly in the cool growth cham- ber, there was still more PGA phosphatase activity in the young leaves than in the old, but in this case the activity was only doubled. P-glycolate phosphatase in young leaves was also 1.5 times that in old leaves. Thus in the old leaves, the PGA 65 TABLE XIa CHANGE IN TOTAL PHOSPHATASE ACTIVITIES WITH LEAF AGE IN MATURE SUGARBEETS Growth Ratio of Chamber Expt. Leaf Age Activities Temperature old young old :young PGA PHOSPHATASE “7 (Total Activity*) warm B 5.51 Ml.6 1.00 : 11.8 c 5.55 51.0 1.00 : 15.2 average 5.M5 M6.5 1.00 : 15.5 cool A 2.97 7.0M 1.00 : 2.56 B 5.88 9.52 1.00 : 2.M5 C M.26 7.10 1.00 : 1.66 average 5.71 8.61 1.00 : 2.52 P-GLYCOLATE PHOSPHATASE (Total Activity*) warm A,B 12.0 16.2 1.00 : 1.56 c 8.6 15.5 1.00 : 1.5M average 10.5 1M.8 1.00 : 1.M6 cool A 10.2 19.2 1.00 : 1.88 B 16.9 C 16.8 27.7 1.00 : 1.65 1.50 average 15.5 21.5 1.00 : * Total Activity is in nmole Pix min"l x g'1 wet weight. 6M phosphatase to P-glycolate phosphatase ratio (Table XII) was the 1:5 value typical for 03 plants, regardless of the growth temper- ature. The same was true for young leaves which grew slowly in the cool growth chamber. But for plants which were rapidly growing in the warm growth chamber, the great amount of PGA phOSphatase activity reversed this normal ratio from 1:5 to 5:1. When phosphatase activities were compared between plants grown under cool and warm growth conditions, PGA phOSphatase acti- vity was five times greater in the warm growth chamber in young leaves but was identical in old leaves. P-glycolate phOSphatase activity on the other hand was slightly higher in the cool growth chamber for both young and old leaves. Thus high PGA phosphatase activity seems to be experimentally associated with rapid growth, which in this case was greatest in young leaves grown under warm conditions. In another experiment, cotyledons and young leaves were har- vested from 5 week old sugarbeet seedlings. The seedlings were grown outside in pots and fed a mineral nutrient solution. The cotyledons averaged 0.1M g per leaf and the young leaves 0.07 g per leaf. Both leaf types were quite dark green in colour. Homogenates were prepared following the standard procedure and assayed for both PGA and P-glycolate phosphatase activity. The results are summarized in Table XIb. Only one sample of each leaf type was obtained. The cotyledons, although less than 5 weeks old, acted "physiologically old" with a lower level of PGA phOSphatase activity (1/6 of that found in the young leaves) and approximately the same level of P-glycolate phosphatase was 65 TABLE XIb CHANGE IN TOTAL PHOSPHATASE ACTIVITIES WITH LEAF AGE IN SUGARBEET SEEDLINGS Leaves Usedf Cotyledons True Leaves weight per leaf 0.1M5 g . 0.0728 g (Total Activity*) PGA Phosphatase 1.21 7.50 P-glycolate Phosphatase 7.60 6.12 Ratio of PGA to P-glycolate PhOSphatase 0.185 1.25 (Specific Activity*) PGA Phosphatase 81 261 P-glycolate Phosphatase 512 208 * Total Activity is in nmole x min'1 x g"1 wel weight. Specific Activity is in nmole x min"1 x mg" protein- f leaves were from 5 week old sugarbeet seedlings- 66 found in both types of leaves. Thus, the PGA phOSphatase to P-glycolate phOSphatase ratio was only 0.2 in cotyledons but was 1.5 in the young leaves. When these results are compared to those of the leaves from mature plants grown under warm con- ditions (see Table XIa and XIb), cotyledons had 1/5 the PGA phosphatase activity and approximately the same amount of P-glycolate phosphatase activity per gram of wet weight as had the mature leaves. The young leaves from seedlings also showed much less PGA phOSphatase activity, only 1/7 that of the young leaves from a mature plant. The PGA to P-glycolate phosphatase ratio was much lower for cotyledons, 1/2 that of old leaves from a mature plant, and for young leaves from seedlings was also 1/2 that of the young leaves from the mature plants. Thus, the increase in the ratio from "old" to "young" leaves was approxi- mately the same in the two systems. The variation in the levels of the two phOSphatases in varying ages of leaves from C3 plants becomes more important when it is remembered these enzymes are thought to initiate the two directions of the glycolate pathway which lead to serine biosynthesis. P- glycolate phosphatase initiates the pathway in the direction of photoreSpiration, a molecule of C02 being released for each serine molecule made. In the reverse direction, starting from PGA phos- phatase, there is no carbon loss. Thus, this pathway would seem to be less wasteful to the leaf than that initiated by P-glycolate phOSphatase. It is reasonable to assume that relatively more 67 serine and glycine would be required in a young, actively growing leaf, than in a mature leaf which is mainly concerned with upkeep of the available cells. Also such a young leaf would be more sensitive to the inefficiency of the glycolate pathway starting from P-glycolate. Therefore, it could be expected, assuming both pathways exist and function in the leaf cell, that PGA phosphatase should be more active in young tissue, or at least more active in proportion to P-glycolate phosphatase in young leaves. This was found to be the case in all three 03 plants tested. The effect of temperature could also be explained since in the cold growth was retarded and this phenomenon seems to occur only in rapidly growing leaves. PGA phosphatase activity was lower in very young leaves than in the young leaves, but this may be due to inactivation during the grinding procedure. Homogenates of very young leaves were always extremely foamy, even when ground by mortar and pestle, and they often discoloured quickly with time although kept in ice. Another explanation for the low activity could be the existence of an alternate pathway to serine metabolism in very young leaves, quite possibly the phOSphorylated system of Hanford and Davies (25), which has been investigated and found most active in non- photosynthetic tissue (9). The very young leaves were extremely light green to yellow with presumably little photosynthetic effi- ciency, so that the presence of this alternate pathway is a possi- bility. 68 TABLE XII PGA PHOSPHATASE TO P-GLYCOLATE PHOSPHATASE ACTIVITY RATIOS PGA to P-Glycolate Phosphatase Ratio+ Plant Expt.* Leaf Age 21d oun very young Soybean M0 1.M0 2.25 7.82 M6 1.20 M.12 M.55 5M 0.85 2.06 M.58 average 1.1M 2.80 5.51 Spinach 55 0.17M 0.510 0.279 68 0.208 0.528 0.900 75 0.151 0.M7o 0.588 87 0.11M 0.M59 0.680 average 0.162 0.MM9 0.562 Sugarbeet (warm) A,B 0.525 2.56 c 0.591 5.8M average 0.558 5.20 (cool) A 0.291 0.566 B 0.562 C 0.260 0.256 average 0.276 0.595 * age from planting for soybean and spinach (see Tables IX,X and XIa) f calculated on the basis of total activity 69 IV) PGA PHOSPHATASE AND P-GLYCOLATE PHOSPHATASE IN LEAVES FROM 04 PLANTS Since PGA phosphatase and P-glycolate phOSphatase activity levels varied in developing leaves from C3 plants, it remained to be seen whether this variation in activity occurred in leaves from C4 plants as well. The following experiments investigated this problem. All the corn plants used were field grown with the exception of the 7-day old seedlings which were grown outside in pots and fed a mineral nutrient solution. The 7th or 8th leaf (again with the exception of the seedlings, from which the 2nd or 5rd leaf was used) was harvested from plants of varying ages. The leaves from 27, M7 and 77 day old plants were assayed for activity on the same day, so that variables other than age were eliminated. The other plants used were M1 and 82 days old. Sorghum and the sorghum-Sudan grass hybrid were also field grown; the leaves were from mature plants. The leaves were ground in the Waring blendor for 2 minutes and the homogenates, strained through cheesecloth, were assayed for PGA and P-glycolate phOSphatase activities. Therefore, the activity found was from the meSOphyll cells only and the bundle sheath cells were not homogenized. The results are summarized in Table XIII. PGA phOSphatase and P-glycolate phOSphatase activity levels did Show an increase in leaves from medium aged corn plants, followed by a decrease as the plant matured, but the trend is not consistent, and may 70 not be significant, eSpecially since only a Single sample was obtained from plants of each age. The ratio of PGA to P-glycolate activity also seemed to increase with "leaf age"; this is the reverse of the trend seen in leaves from C3 plants. P-glycolate phosPhatase activity varied from 2 to M times that of PGA phos- phatase. Leaves from mature sorghum plants showed an extremely high level of PGA phosphatase activity: 10 to 20 times more than that found in corn leaves and 8 times more than that of P-glycolate phosphatase. Leaves from the sorghum-sudan grass hybrid showed an intermediate level of PGA phosphatase activity which was still 5 times greater than that found in corn. P-glycolate phOSphatase activity was at approximately the same level in all three C4 plants tested. Levels of PGA phOSphatase higher than those of P-glycolate phOSphatase were common for mesophyll cells of C4 plants with the single exception of corn (M8). It is interesting to note that there was little change in phosphatase levels with leaf age in corn. This may be a characteristic unique to corn, however, Since corn has unusually low levels of PGA phosphatase. Thus it is unwise to extend to all C4 plants any conclusions about phos- phatase activities varying with leaf age which are based on results found only for corn leaves. 71 TABLE XIII CHANGE IN TOTAL PHOSPHATASE ACTIVITIES WITH LEAF AGE IN C4 PLANTS * Age was measured from emergence; the plant, 2nd or 5rd from the 7 day old PGA P-Glycolate PhOSphatasef Phosphatase1 Plant* Total Spec. Total Spec. Act. Act. Act. Act. Corn very young (7 days) 0.M58 l2.M 1.92 M7.1 (27 days) 1.21 51.1 5.16 80.8 medium (Ml days) 1.5M 51.1 2.5M 55.9 (M7 days) 1.5M 5M.2 2.9M 75.0 mature (77 days) 1.16 27.2 2.58 58.0 (82 days) 0.6Ml 15.2 1.52 51.1 Sorghum mature 19.M M10.0 2.29 M8.M Sorghum-Sudan Grass Hybrid mature 7.51 15M.O 2.98 65.0 Ratio of PGA to P-Glycolate Phosphatase 0.259 0.585 0.527 0.M56 0.MM9 0.M2M 8.M8 2.MM 7th or 8th leaf from each seedlings, were used. 1 -1 1 Total activity was in umoles P x min- x g Specific activity was in nmoleS'Pi x min’1 x mg‘1 protein. #14; 72 H) FIELD SUGARBEETS In previous experiments involving sugarbeets grown in warm and cool growth chambers (see Sections D and G111), the results showed that there was a great increase in PGA phosphatase activity in warm growth conditions, eSpecially in young leaves. The fol- lowing experiment is a study over the growing season of sugar- beets in the field. Both young and old leaves were harvested and phosphatase levels were determined to look for any variation in activity with temperature as was previously shown under artificial conditions. Leaves were harvested almost weekly from mid-July to mid- November, 1971 from sugarbeets (SP 6822-P) growing in a nearby field which had been planted May lMth. The young leaves each weighed approximately 2 grams early in the experiment, but de- creased to approximately 1 gram in late September and less than 1/2 grams in November although the physiological age of the leaves was considered to be comparable. In all cases, the leaves were pale green and curled; late fall samples were slightly greener but still much lighter than fully expanded leaves. The old leaves were dark green, fully expanded and mature but not senescent. During summer, they averaged 20 g each, but this weight decreased to about 8 g in November. There was a heavy frost on two nights (low -5. and -7° on Nov. 7th and 8th respectively) which heavily damaged or killed most of the outer leaves. Thus, in the last two harvests, random sampling for old leaves was extremely dif- ficult since few were undamaged. However, each time, at least 75 10 leaves were selected that seemed to be normal (i.e., appeared the same as leaves sampled before the frost). Almost all the young leaves were protected in the centre of the plant and Sur- vived, although growth after the frost was slowed considerably. Thirty grams of both young and old leaf tissue (up to 90 young leaves, and randomly selected pieces of 8-10 old leaves) were deribbed, washed, and ground in the standard grinding medium for 50 seconds at high speed in a Wering blendor. After being strained through 8 layers of cheesecloth, forty ml of the homo- genates were centrifuged at 10,000 x g and the pellets were resus- pended in a glass homogenizer in cacodylate-EDTA buffer. All fractions were assayed for both PGA and P-glycolate phOSphatase activities as well as for protein. The results are summarized in Tables XIVa and XIVb and shown schematically in Figures 1 and 2. The temperatures listed are the averaged values of the high and low temperatures of the three days immediately preceeding harvest. Day length of the day pre- vious to harvest decreased from 15.2 to 9.7 hours over the course of the experiment. The enzyme assays did not give linear results (i.e., the increase in phosphate released was not always propor- tional to an increase in enzyme concentration - data not shown), but 5 concentrations in duplicate were used and the results were averaged. The range of the individual points was always less than 15% of the average value. Over the growing season, the highest activities of PGA phos- phatase in young leaves were found at the beginning of September, 7M after a gradual increase during the early summer months. In the fall, the activity levels followed the low temperatures quite closely. This was particularly obvious on the 25rd and 50th of September, and the lMth and 26th of October, where the activity decreased Sharply when the temperature dropped to M° or below and peaked when the average low was close to 16°. This trend, however, was not seen in the last few harvests; even after the severe frost, PGA phOSphatase activity was Still reasonably high. PGA phOSphatase activity in old leaves also varied over the growing season but not consistently with temperature or any other para- meter measured. However, it was never greater than 1/2 and some- times as little as 1/8 of the activity (on the basis of protein concentration) found in young leaves. P-glycolate phOSphatase activity in both old and young leaves was greatest earlier in the summer, peaking in early August, then decreased into fall. In old leaves the curve of activity vs. date of harvest is very irregular, but the peaks and troughs are not consistent with trends in temperature. P-glycolate phOSphatase activity in young leaves was never greater than 2/5 and often as little as l/M of the activity found in old leaves. PGA phOSphatase to P-glycolate phosphatase activity ratios also varied during the season. In young leaves, the ratio was highest in early September (over M), mirroring the high PGA phos- phatase activity, and then decreased to an almost constant level of 2.5. In old leaves, PGA phosphatase activity was much lower than that of P-glycolate phosphatase, but this ratio showed an 75 almost constant increase to nearly 0.5 by the end of the season. The significance of these trends, especially the latter, is un- known. It was noticed early in this experiment that a great amount of protein (in some cases over 60%) was being precipitated out of the homogenates of young leaves, leaving a clear brown super- natant after centrifugation at 10,000 x g. This also occurred to a lesser extent in old leaves. Twice during the season, Polyclar AT, as used by Randall, Tolbert and Gremel (M8), was added to the grinding solution with duplicate samples of leaves, but the total activity increased no more than 15-20% (data not Shown) and the supernatant was still noticeably brown. The amount of protein that precipitated varied with changes in temperature during the growing season. PGA phOSphatase pellet activity in young leaves correlated very closely with the amount of this pre- cipitated protein, except near theend of the season, when more PGA phOSphatase activity was found in the supernatant while the amount of protein in the precipitate remained constant. P—glyco- late phosphatase as well as PGA phosphatase in old leaves was not affected by this nonSpecific precipitation of protein but remained in the soluble fraction (around 90%) throughout the season (data not shown for P-glycolate phOSphatase). The results from this field experiment were disappointing since variation with temperature did occur but not as dramatically as found in growth chamber experiments, and PGA phosphatase in young leaves never really "shut off" even after a very severe 76 frost. The season was an unusual one; usually the summer temp- eratures are warmer and the fall temperatures cooler than seen this year and a heavy frost occurs by the middle of October fol- lowed by fairly mild temperatures. This year the first real frost was on the 7th of November and was extremely heavy, killing most of the old leaves. By late fall the plants were quite old with large beets and apparently the leaves are then less modified by changes in temperature. Also, in field experiments there are innumerable variables which are controlled in a growth chamber: variations in daily temperature, day length, precipitation, cloud cover, light intensity, etc. Thus, the problem becomes much more complex and it is possible that variations in the other factors are more significant to the plant than high and low temperatures. .fimw vocasumuom mnu mo emoumaa zuo>000u KOCH ou mouoouuoo ma Aufi>fiuom owaooam * H 77 someooeAaoem eon *omMumsomonm ¢om mm>HHU< mmmHmmmmomm Hx mqmauom owmwoomm m Him x Hinge x «m moHosi aw nu mufi>wuom kuoa * mm mm w.m a.om am.m moH mm mm m.ma m.mw c.m nmm >H\HH mm maa m.m H.mm om.a eaa cm em n.em >.m> o.oH emm HH\HH ma ooH 0.0 0.4m sm.n m.mm me om n.am >.m> w.oH New e\HH cm OHM m.m m.om nm.m n.c> me maa e.on w.mm a.oH omm cm\oH am maa m.m a.am eo.m s.am ma Has e.an c.mo n.m sma ea\oH mm mHH m.m s.am me.n a.ws em mm m.mn m.mw m.HH Nam m\oH am maa n.6a m.mm we.m n.6m cm MoH m.mm H.Ha o.>H OHS on\m ma mm e.m m.am we.m o.mw o: emm m.mm >.oe m.m sown nm\m am moH a.e m.mm ca.n o.mm no ooH m.oe s.mn n.na an: ca\m mm moH H.m m.am .ow.m m.mm mm on m.mm m.ee H.mH cm: m\m mm am >.ma m.aw me.n . sea me man s.mo m.en s.ma one wa\m mm mm m.» e.mm mm.e 66H ma cm m.ma m.mm m.sa ma: m\m mm moH m.c m.mm mm.m w.me as mm a.mn e.ec m.ma Hen mm\s mm aoH H.6H m.mw mm.m m.ms em mm w.ma a.am >.m New Hm\e mm mm m.m m.am mm.m m.me mm mm c.ma a.am o.HH man wH\s uoafimm ca ~50m% Asomv umHHom cw Aaomv Amomv cwououm R Nuo>ooom Hmmfi :ma .uo< .uo< camuoum R mum>oowm Hmmfi msmw .uo¢ .uo< Hence * coausnfiwunfin Hmuoa .uomm Hence * coauonawunao Heuoa .oomm mama 78 .ovmuwwucooo a“ ma ounumuomaoa + .awououa Hume x Hucflfi x gm oHofia a« ma mufi>wuow owmflooam m Hum x Huafia x «m oaoan nu ma huw>wuom Houoa * s.m m.e mm.o msa.o oc.m 6mm mm.m m.>m mm.m m 0H >H\aa m.m m.e mm.o mma.o .SH.m mew o.oH wee o>.: n- e HH\HH H.6H H.8H em.o oam.o me.m omm a.HH ooH om.m m ea s\HH m.oH o.ma am.o mmm.o ow.m 6mm om.m m.mm 06.: me ha mm\oH m.aa m.ma am.o mmm.o ma.m nma e>.> o.wm mm.n N ha eH\oH m.HH w.ma ow.o nam.o Hm.n 6mm m.eH o.me mn.n m ma m\oH m.HH w.sa mm.H osm.o mm.n mmm m.ma mam we.a mm mm on\m m.ma m.am mm.a Hmm.o mm.m awn H.ma nma mn.m m ma nm\m m.ma m.mm mH.H mmm.o ma.m mmm m.ma eaH mm.e ma mm wa\m H.ma m.ma oH.H wma.o ea.a sen m.ea mma Hm.e om mm m\m m.na m.ma o>.H msm.o am.H nmm e.ma omm mm.m ma em 6H\m e.aa c.0m mw.a aeH.o em.a ewe m.mm son c.ma m mm e\w s.aH s.mm mw.a Hma.o mm.a mam m.ma cam mm.w oH mm mm\e m.ea m.om wn.m maa.o mm.H 6mm m.ea wee am.w w em Hm\s m.ma m.mm mo.m wma.o we.a ems m.w~ oma mm.m nH em oa\s Aura Amy .Iamql .Immml mmmmm .no< .uo< .uo< .uo< sou mmmu nowaoq 0H0 manor cguwm mmmumammosm .ummm HmuoH .ooam HauoH .o>< .o>< mama moo uswfimz mama muwHoo%HUum cu mam 0H0 mono» +ououwuoaama enouManonm oumaoohaoim QmDZHHZOU “HZMZHmmmxm nqum HummmHN mqm 575 Sugarbeet cotyledon 2.17 109 (seedling) 1.9M 8M 1.60 98 lst and 2nd 10.9 81 true leaves 5.56 95 5.22 90 very small 1.87 162 1.0M 160 Soybean large 61.5 ‘ 65 56.2 58 59:1 65 medium 10.9 151 10.8 155 9-50 157 very small 1.06 520 0.92 515 * using a glass chamber in a closed continuous system 90 TABLE XVIb 002 COMPENSATION POINTS (CONT.)* Compensation Plant Type of Point Leaf (PPm €02) Sugarbeet old 61.2 61.8 62.9 young > 500 Soybean old 81 9M 10M Spinach old 7M 76 young 111 120 * using Mylar bags 91 K) DARK RESPIRATION RATES The study of compensation points in leaves of various ages was extended in this series of experiments to obtain dark res- piration rates for these tissues. The unexpectedly high values of C02 compensation points found for young leaves implied a high total respiration rate in comparison to the rate of photosynthesis. But this rate is most likely a combination of dark respiration and photoreSpiration. In mature leaves, dark respiration in the light is insignificant compared to the high photoreSpiration _: rate, but in very young leaves where there is little photosyn- thesis, dark reSpiration becomes a very significant factor. Also, a large amount of material is known to be translocated to the rapidly growing young leaves from the rest of the mature plant to be used in dark reSpiration as a source of energy and raw materials. In the following experiments, both dark respiration and C02 compensation points were determined in various sizes of leaves to elucidate the effect of leaf age. Again, the closed system in the walk-in growth chamber (as in Section I) was used but this time the photosynthetic chamber was much smaller (only 200 cm3) and water was added directly to the chamber. In this system, the increase in C02 concentration in a control run (i.e., with no leaf tissue) was negligible over 50 minutes. Whole small and very small leaves, and large sections of mature leaves were floated upside down in water. In the first sugarbeet experiment, the leaves were from a 90-day old plant growing outdoors in vermiculite supplied with mineral nutrient 92 solution. All the leaves, of varying ages, were from the same plant. In the second experiment, the leaves were from similarly grown plants which were 125 days old. The soybean plants were greenhouse-grown, less than a month old; the lst, hth and 5th trifoliates of 3 different plants as well as very young leaves from a number of plants were used. The Spinach leaves were from mature growth chamber-grown plants (on a ll—hour day at 25°, 200 night); both the old and young leaves were dark green. Only the tips of various corn leaves were used from h plants grown out- doors in vermiculite supplied with mineral nutrient solution. In one case, the leaf was not yet fully emerged. In another experiment, cotyledons and young leaves were used from 5 day old corn seedlings. The system was again purged of C02 by bubbling through KOH and,after the KGB was removed, was allowed to come up to the compensation point. This process usually took only 10 to 15 minutes. The system was again purged, the KOH removed, and the growth chamber lights were extinquished. The increase in C02 concentration between exactly 2 to h minutes in the dark was then divided by the area of leaf tissue (determined by the weight of photOCOpied images, as in Section J) to give a dark respiration rate. The magnitude of the compensation points were not always found to be a function of size as measured by area (in some cases larger leaves had higher compensation points) but instead may be a function of the "physiological age" of the leaves; i.e., a measure of leaf {E " '71 93 development. In this series of experiments, the compensation point of a leaf or leaf sample was taken to indicate its physio- logical age, and the dark respiration rates were compared accor- dingly. In all the 03 plants tested, there was an obvious trend of higher dark respiration rates with higher compensation points (see Tables XVIIa and XVIIb). When the leaves from one sugarbeet plant were used, the dark respiration rate varied from 0.50 ppm 002 x min”1 x cm- in large leaves to 2.06 in very small leaves, a h-fold difference. The same trend was seen with the older sugar- beet plants, but in this case the compensation points and dark respiration rates were higher for comparatively sized leaves; i.e., the leaves were as large but not as old physiologically as in the preceeding experiment. Soybean leaves showed an even greater range in dark reSpiration rates: from O.h in old to 2.6 in very young leaves, a 6-fold difference. Once again, the compensation points increased with the dark respiration rates, showing at least a 6-fold difference between old (65 ppm 002) and very young (over 570 ppm 002). In spinach the difference in compensation points or dark respiration rates between young and old leaves was not as great, but both groups of leaves were quite dark green and presumably photosynthetically active. Corn leaves (Table XVIIc), however, did not show a significant change in either compensation points or dark reSpiration rates except in the case of a very young leaf that was still in its sheath. Even then, the dark respiration rate was only twice 9h that of other leaves. Leaves from 5 day old seedlings also did not follow the trend seen in leaves from Cs plants. Thus, when there was an increase in compensation points with increasingly young age of leaves of three C3 species, there was also an increase in dark reSpiration rates that approximately matched the compensation point increases in magnitude. 95 TABLE XVIIa CHANGE IN DARK RESPIRATION RATES WITH LEAF AGE IN SUGARBEETS Dark Area per Physiological Compensation Respiration Expt.* Leaf Age Point RateI cm2 (Rpm C022 A 1.12 very young 575 2.06 3.96 young 202 l.h9 7.h2 young 179 1.27 20.57 intermediate 89 l.lh 67.9 mature 65 1.01 175 mature 65 0.50 B 8.08 young 26} 1.76 11.6 215 2.09 57.7 inteimediate 165 1.65 65.1+ 122 1.1+8 hlS mature 61 0-75 607 6+ _ 0.97 626 65 0.79 * in Expt. A, one plant, approximately 90 days old, was used; in B., the plants were 125 days old I Dark ReSpiration Rates are in ppm C02 x min'1 x cm'2 96 TABLE XVIIb CHANCE IN DARK RESPIRATION RATES WITH LEAF AGE IN SOYBEAN AND SPINACH Plant Physiological Plant Area per Compensation Dark Age No. Leaf Point Respiration (cm2) (ppm C02) Rate* Soybean? very young 1.27 575 2.6h young A 29.5 118 0.950 B 19.2 215 1.50 c 52.0 91 0.850 intermediate B 90.5 67 0.650 mature A 57.5 69 0.616 B 57.9 70 o.h00 c 59.8 63 o.h15 Spinach young 1.65 265 1.92 1.66 127 1.8h 5-85 79 2-25 mature 57.7 61 0.852 71.5 67 0.796 * Dark reSpiration rates are in ppm C02 x min'1 x cm.2 I Young leaves are the 5th trifoliate, intermediate the hth and mature the lst trifoliate from each soybean plant. 97 TABLE XVIIc CHANGE IN DARK RESPIRATION RATES WITH LEAF AGE IN CORN Dark Leaf Plant Leaf Compensation ReSpiration Description No. No. Point Rate* (ppm 002) full grown A h 11 0.778 B h 23 0.828 C 2 18 ' 0.755 D 5 1h 0 . 7&6 immature+ A 8 55 - B 8 22 0.626 C 8 17 0.655 C 9 - 1.275 D 9 in 0.728 cotyledons E*’ 1 15 0.605 lst true leaves E 2 11 0.701 * Dark respiration rates are in ppm C02 x min'1 x cm"2 I C9 was still covered, not yet emerged and had a very high compensation point. I Plants "E" were seedlings, 5 days after emergence. _J5 98 DISCUSSION AND SUMMARY It is necessary at this point to mention sample variation. The term percent recovery in the preceeding experiments usually refers to the amount of total activity found originally in the homogenate that is recovered in the supernatant and the pellet of the 10,000 x g centrifugation. Usually, recovery was close to 100% but was subject to change. Whenever identical leaf samples were used with identical grinding conditions, as in the studies (it of diurnal change, percent recoveries were virtually identical. Only when either condition was changed; i.e., when samples were harvested at different times as in the field study or when dif- ferent grinding conditions were used, was there a change in the recoveries. The reasons for this phenomenon are unknown. Re- coveries from sucrose gradients were usually close to 100%. Large deviations from this occurred only when very small amounts of enzyme activity were originally run on the gradient. The effects of growth conditions and stage of leaf develop- ment on PGA phOSphatase and P-glycolate phoSphatase activities have been studied. Early work showed PGA phOSphatase activity in leaves of all the various plants tested, but with varying levels of total activity and activity sedimented at 10,000 x g. Diurnal studies were done on both phosphatases in both soybean and sugarbeet leaves but no significant changes were seen. PGA phosphatase pellet activity was first studied in Spin- ach leaves. A much greater percentage of activity in the pellet as well as a higher total activity was found in field-grown plants. 99 The pellet activity from growth chamber plants was firmly bound to the chloroplasts, as seen in sucrose gradients, with little activity in the other fractions. In spinach leaves little of the pellet activity was solubilized by a Potter—Elvejehm homogenizer, which stresses the particulate nature of the enzyme. In sugarbeets, the study of the pellet activity was extended and the further variable of growth conditions was added. PGA phosphatase activity was higher in plants grown under warm conditions than under cool conditions. Pellet activity was also higher in these plants. The activity found in the 10,000 x g pellet of all these leaves banded with whole and broken chloroplasts when separ- ated on sucrose gradients. Distribution and total activity of PGA phOSphatase was also seen to vary with leaf age in sugarbeets. Young leaves had a much greater percentage of activity in the pellet than had old leaves. Also, this activity was much more firmly bound to the chloroplast fractions than in old leaves where much of the activity was found with the starch pellet and the soluble fraction of the gradient. The pellet activity was solubilized by grinding in a medium containing magnesium ions. Total activity of PGA phos- phatase was also much higher in young leaves. The change in phOSphatase activities with leaf age was studied more carefully with the total activity reported in terms of the amount released by grinding in a mortar and pestle. The results from soybean leaves showed PGA phosphatase activity was almost constant, but P-glycolate phosphatase increased with leaf age 100 (from 12 to 27 units per gram of wet weight). In Spinach leaves, PGA phosphatase activity decreased with age (from 6.7 to l.h) but P-glycolate phOSphatase activity was virtually constant. ' Sugarbeets were grown both in warm and in cool growth chambers; there was a very high level of PGA phOSphatase in young leaves in the warm chamber (h6 units) that decreased in old leaves to a level of activity that was the same (5.5 units) at either growth -temperature. In the cool growth chamber, PGA phosphatase in young leaves (8.6 units) was still higher than that of the old leaves but this difference was not nearly as great as that found in the warm growth chamber leaves. Temperature did not seem to affect P-glycolate phOSphatase activities, which were only 50% greater in the young than the old at both growth temperatures. In sugar- beet seedlings grown outdoors, young leaves had a higher PGA phosphatase activity than had the cotyledons (7.5 to 1.2 units), but had approximately the same level of P-glycolate phOSphatase activity. Thus in the plants tested, there was a much higher ratio of PGA phosphatase to P-glycolate phOSphatase activities in young, actively growing leaves, eSpecially in fully grown plants. This was not true in corn, the only C4 plant tested, where there was no significant change in phOSphatase activity levels with leaf age. One explanation for the change in phoSphatase activities is a change in the predominant pathway to serine biosynthesis. The "normal" pathway in a mature leaf is through the glycolate path- way initiated by P-glycolate phosphatase which also results in photoreSpiration and the loss of energy. The other pathway is 101 initiated by PGA phosphatase, which results in no loss of C02 and a smaller loss of energy. Since PGA phOSphatase activities are so high in young, rapidly growing leaves, the hypothesis pro- posed is that in such leaves serine originates from this second, more efficient pathway, but changes over to the P-glycolate phos- phatase initiated pathway as the leaf ages. Alternatively, it may be that the PGA phosphatase initiated pathway, which predominates but is not the only pathway in young leaves, is suppressed with age and a constant level of serine continues to be produced by the normal direction of the glycolate pathway. The activities of two other enzymes of the glycolate pathway were also determined in young and old leaves. Glycolate oxidase increased greatly with leaf age, which is in agreement with the hypothesis that the glycolate pathway increases in importance with leaf age. Hydroxypyruvate reductase, which is on the PGA phOSphatase initiated pathway to serine, might be expected to decrease with leaf age as does PGA phosphatase, but the reverse was found. However, since this enzyme is reversible, and can thus function in both directions of the glycolate pathway, this does not negate the hypothesis. From the field eXperiment a few important results should be noted. First, it shows that it is extremely difficult to extend the results of growth chamber experiments to field conditions. Each hypothesis should be tested in the field if it is to be sig- nigicant to the grower. Also, the value of the pellet activity of PGA phOSphatase is now uncertain. In this experiment, the pellet activity followed total protein in the pellet very closely; i.e., the phenomenon seems to be due to a nonspecific precipitation of 102 protein. Thus, it is unlikely that the increase in pellet activity is of physiological importance, but it is instead probably an artifact of grinding due to the extremely large amounts of tannin in young tissue. The appearance of brown colour in the supernat— ants from young leaves has been noticed before in field-grown sugarbeets and in very young leaves but, unfortunately, protein concentrations of supernatant fractions were not obtained. This phenomenon was not seen however in the leaves used for the gradient experiments. The nonlinearity of the phosphatase assay seems also 4: to be unique to field plants. PGA phosphatase in plants grown either in the greenhouse or growth chambers always behaved "normally"; i.e., variation from the mean of individual points determined for the 5 aliquots in duplicate, which were routine for all assays, was seldom greater than 10%. It has been shown that PGA phosphatase and P-glycolate phos- phatase exist in leaf cells, and vary differently with physiolo- gical changes, with growth, and with temperature conditions. PGA phosphatase showed greatest activity in young, rapidly growing leaves and it may well be actively involved in serine metabolism by the reverse of the glycolate pathway. However, it is not necessarily that simple. The advantage of the existence of the glycolate path- way, with its characteristic inefficiency, is not yet known. It is tied in with the formation of serine but this may not be the only or even a major route of serine biosynthesis. That some serine is made from PGA through the reverse of the glycolate pathway is known by enzyme activities and labelling experiments. However, the extent of this synthesis is not known; it may or may not be important in 105 in the leaf cell. Also, the initiation of the reverse of the glycolate pathway may not even be a major role of PGA phOSphatase activity in the young leaf; the enzyme may be more involved in transport or starch synthesis as mentioned in the literature re- view. The activity of an enzyme outside the living cell may not be a real measure of its true physiological activity. This is especially true in the case of acid phOSphatases that might be compartmentalized in the cell, far from substrates, or in conditions different from optimum assay conditions, with possible control by effectors. Thus, an increase in activity outside the cell, in crude homogenates, can only indicate what may be occurring physio- logically. That is why it is important to try to correlate enzym~ ology with physiological changes. Therefore, another measure of the glycolate pathway was sought: that of C02 released as photoreSpiration. Presumably, if the glycolate pathway, as initiated by P-glycolate phoSphatase, was functioning at a lower level in young leaves, photoreSpiration would also be low. One measure of photoreSpiration is the 002 compensation point and so a low compensation point would be expected in young leaves, as found in leaves from C4 plants. But the opposite results were found, possibly because of high dark reSpiration rates, which have also been determined and shown here, and which have been reported in the literature. Furthermore, there are the ex- tremely low rates of photosynthesis which occur with the low level of chlorophyll found in young leaves. There are three unknowns in the formula for determining 002 compensation points: the levels of photosynthesis, photoreSpiration and dark respiration (which is 10b. relatively insignificant in mature leaves). The first two are closely related since photoreSpiration is dependent on the pro- duction of P-glycolate which is thought to be an early product of photosynthesis. Thus a low concentration of chlorophyll implies a low level of photosynthesis which limits the level of photo- respiration. But unless direct methods are used to determine photoreSpiration (e.g., by labelling experiments), a knowledge of the photosynthetic rates in young and old leaves is necessary to calculate the levels of photoreSpiration. However, a low level of photoreSpiration in young leaves has been reported in the literature very recently (59). 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