Stereochemical determination of carbon partitioning between photosynthesis and photorespiration in C3 plants: Use of (3R-d -[3-3H1,3-14C]glyceric acid

ARCHIVES

OF BIOCHEMISTRY

AND BIOPHYSICS

Vol. 232, No. 1, July, pp. 58-75, 1984

Stereochemical Determination of Carbon Partitioning between Photosynthesis and Photorespiration in C3 Plants: Use of (~R)-D-[~-?-I, ,3-14C]Glyceric Acid’ KENNETH Department

R. HANSON

of Biochemistry and Genetics, The Connecticut Agrkultural P.O. Box 1106, New Haven, Comecticut 06504 Received December

Experimt

Station,

27, 1983, and in revised form March 20, 1984

When (3R)-~-[3-~Hi,3-‘~C]glyceric acid is supplied in tracer amounts to illuminated tobacco leaf discs, the acid penetrates to the chloroplasts without loss of 3H, and is phosphorylated there. Subsequent metabolism associated with the reductive photosynthetic cycle fully conserves 3H. Oxidation of ribulose bisphosphate (RuBP) by RuBP carboxylase-oxygenase (EC 4.1.1.39) results in the formation of (2B)-[2-3H1,14Clglycolic acid which, on oxidation by glycolate oxidase (EC 1.1.3.1), releases 3H to water. Loss of 3H from the combined photosynthetic and photorespiratory systems is, therefore, associated with the oxidative photorespiratory loop. Assuming steady-state conditions and a basic metabolic model, the fraction of RuBP oxidized and the photorespiratory carbon flux relative to gross or net COZ fixation can be calculated from the fraction of supplied 3H retained in the triose phosphates exported from the chloroplasts. This retention can be determined from the 3H:‘4C ratio for glucose obtained from isolated sucrose. The dependence of 3H retention upon Oz and COZ concentrations can be deduced by assuming simple competitive kinetics for RuBP carboxylase-oxygenase. The experimental results confirmed the stereochemical assumptions made. Under conditions of negligible photorespiration ‘H retention was essentially complete. The changes in 3H retention with Oz and COz concentrations were investigated. For leaf discs (upper surface up) in normal air, it was estimated that 39% of the RuBP was oxidized, 32% of the fixed COZ was photorespired, and the photorespiration rate was 46% of the net photosynthetic COB fixation rate. These are minimal estimates, as it is assumed that the only source of photorespired COZ is glycine decarboxylation.

In plants with C3 photosynthesis, an appreciable fraction of the CO2 fixed photosynthetically by the carboxylation of RuBP’ is lost through photorespiration be-

cause of the synthesis and metabolism of glycolate (1, 2). A potential route for increasing crop yields is, therefore, to increase net COZ fixation by decreasing pho-

i A preliminary account of this work was presented at the 6th International Congress on Photosynthesis, Brussels, 1983. r Abbreviations used: DHAP, dihydroxyacetone phosphate; E4P, erythrose 4-phosphate, FBP, fructose 1,6-bisphosphate; F6P, fructose 6-phosphate; Ga3P, glyceraldehyde 3-phosphate; G6P, glucose 6-phosphate; M6P, mannose 6-phosphate; PGA, 3-phosphoglyceric acid; R5P, ribose 5-phosphate; RuBP, ribulose 1,5-bisphosphate; Ru5P, ribulose 5-phosphate; TP,

triose phosphates = DHAP + Ga3P; Xu5P, xylulose 5-phosphate. PMS and PMSH$, oxidized and reduced phenazinemethosulfate. Rt, rabbit; Yt, yeast. PR, photorespiration rate; GPS, gross photosynthetic rate; NPS, net photosynthetic rate; Cod, fixed CO,; CO,T, photorespired COr; Statements in the form a (?b) imply that a is the mean and b is the standard error of sampling, not the standard error of the mean; esr, external standard ratio.

0003-9861/84 $3.00 Copyright All rights

0 1984 by Academic Press, Inc. of reproduction in any form reserved.

58

STEREOCHEMICAL

ASSAY

OF PHOTORESPIRATION

torespiration (3, 4). Progress toward this goal requires not only genetic techniques but a detailed and quantitative knowledge of photorespiratory metabolism. There is a considerable amount of agreement concerning the main features of the metabolism involved (5-g), although uncertainty about some critical issues remains. In contrast, there is no one agreed-upon method for measuring photorespiration in leaf tissues, and different methods give estimates of its importance that range from 20 to 60% of net COz assimilation (2). Assays that seek to distinguish directly between respired and net fixed COBencounter the problem that photorespired COzmixes with entering COz and is therefore refixed. The approach used here differs from all previous investigations because it determines the fraction of RuBP that is diverted to photorespiratory metabolism under steady-state conditions. If this partitioning is known, the photorespiration relative to photosynthetic COz fixation may also be deduced. The estimates obtained rest on specific metabolic assumptions which may be evaluated and, if necessary, changed in the light of future experience. In the standard experiment, leaf discs (tobacco) are maintained on a high specific activity solution of (3R)-D-[3-3H1, 3-‘4Clglyceric acid under steady-state photosynthetic conditions. Tracer amounts of this substrate enter photosynthetic metabolism, and double-labeled neutral sugars are ultimately formed. The loss of 3H, as indicated by the change in 3H:‘4C ratio on going from labeled glycerate to labeled glucose, is a function of the carbon flux through the photorespiratory pathway. The detailed assumptions used to calculate relative photorespiration are presented in the following section. THEORY

The metabolic model employed is described in Figs. l-3. These summarize present knowledge about photosynthesis and photorespiratory metabolism (5-9). It is assumed in this basic model that glycolate derives only from RuBP and photorespired COzonly from glycine. Whereas

IN Cs PLANTS

02

59

co2

4

TP -

NEUTRAL SUGARS I’H.“CI

FIG. 1. Relationship between the reductive photosynthetic cycle (Fig. 2) and the oxidative photorespiratory loop (Fig. 3). g, fraction of RuBP oxidized; f; fraction of TP, or their equivalents, leaving the system of cycle plus loop. Tracer quantities of (3@-D+@&.% ‘V]-glyceric acid enter the chloroplasts from the cytoplasm, and are there converted to PGA. A portion of the SH so introduced is lost to water in the loop.

the details of the metabolism are complex, the relevant partitionings are simply presented. In Fig. 1 the unbroken line represents the reductive photosynthetic cycle (Calvin cycle) and the dashed line the oxidative photorespiratory loop. It is termed a loop because the precursor and product are both intermediates of the reductive cycle and photorespiration is parasitic on that cycle. The enzymes of the cycle are confined to the chloroplasts, whereas the loop involves the cooperation of oxidative organelles, namely peroxisomes and mitochondria. Figure 1, however, is concerned with conceptual rather than physical packaging. To simplify the argument, export from the system is viewed only in terms of the passage of TP from chloroplasts to cytoplasm. Partitioning of carbon between the cycle and the loop is determined by RuBP carboxylase-oxygenase and depends upon the internal concentrations of COz and O2 (7, 10, 11). Carboxylation yields 2 mol of PGA; this reaction falls within the cycle. Oxidation yields PGA from C(3-5) plus 2-

60

KENNETH

HdOH

R. HANSON

H&OH

m-i-•

m-i-o bP

AP

-. Gasp ? 1 OUT ‘d, EL

TP

HObH H&OH Aldolore

HdOH

1

H&OH M-0 6P S”“7P

r

;HOCH HCOH

CHO &OH HdOH 8-i-O

L

TK-Ping

l

1

nH&OH e+

LIP

bP

E4P

FBP

I/. 7I

G6p 1

i

STARCH L’ -----.a’

FIG. 2. Reductive photosynthetic cycle (Calvin cycle) showing conservation of the terminal CCH,-0 of PGA. Hydrogens of this substructure are represented as squares: black, pro-R; open, pro-X As long as the C-CH2-0 system remains intact, these Sequence-Rule-based designations apply. If ‘%I is introduced as (3R)-D33-*H1,3-‘“Clglyceric acid, the black squares represent ‘H and the configuration at all the derived centers shown is (R). The stoichiometry indicated by numbers on the arrows is for the steady-state condition with zero photorespiration, i.e., g = 0, hencef = l/ 6. If PGA is exported or starch is formed, these are treated as TP equivalents;fremains the same. The transfer of a C, fragment by transketolase is indicated as TK-Ping for uptake and TK-Pong for donation.

P-glycolate from C(l-2); this reaction initiates the loop. The fraction of RuBP oxidized is designated g, hence the fraction carboxylated is (1 - g). To salvage the carbon of 2-P-glycolate, PGA is formed; but, the costs are the release of 25% of the carbon as COeand the devotion of a significant portion of electron transport to NH: recovery via glutamine synthesis (6). A second partitioning must be considered. Under steady-state conditions the net carbon fixed (gross fixed less photorespired) must equal the carbon leaving the system. The chloroplast envelope membrane contains a phosphate translocator

that exchanges TP for cytoplasmic Pi (12). If TP is treated as the only carbon leaving the system, fraction f is exported (1 mol/ 3 mol net fixed COz) and (1 - f) is further metabolized to regenerate RuBP. Note, however, that carbon leaves in other ways: the phosphate translocator exports some PGA (12) to yield primarily, in the tobacco leaves studied, vacuole-stored malate (see Results), and some F6P is removed internally as a result of starch formation (5,8, 13). Both PGA and F6P will be regarded as TP equivalents. Aromatic biosynthesis (E4P + PEP), although it can be a major function of chloroplast metabolism, is of

STEREOCHEMICAL

ASSAY

OF PHOTORESPIRATION

IN Cs PLANTS

61

TP

GLYCERATE *

GLYCOLATE 9

. *

If

II

GLYCOLATE

COOH

COOH

~ n -4-O

GLYCERATL

HiOH iH0

GLYQXYLATE

1

::I

COOH

SERINE

iHfl

z a

bH

“-.-;

i

0

HP-/-H

. Ser

# 1

FIG. 3. Oxidative photorespiratory loop (basic model) showing the loss to water of the (p?GZ)H of glycolate through the action of glycolate oxidase. The photorespired CO1 is assumed to derive only from glycine. If g mol of RuBP are oxidized, g/2 mol of CO, are photorespired. At the COz compensation point, (1 - g) mol of COz are fixed for every g/2 mol CO, photorespired, hence g=2/3whenf=O.

minor importance in the leaf tissue being studied. It will therefore be ignored. The possibility of distinguishing experimentally between the cycle and loop arises because of a difference in the stereochemistry of their enzymatic reactions. Figure 2 shows that, during the forward operation of the cycle, the terminal C-CHz-0 group of PGA is returned to PGA intact. Figure 3 shows that the same group in glycolate

loses its @o-2@-H to water when glycolate is oxidized by glycolate oxidase (14, 15). (For a summary of the pro-R/gnwS nomenclature and comments on the stereochemistry of some of the reactions mentioned in this paper see Ref. (16).) If tracer (3R)-D-[3-3H]glycerate enters the cells of the leaf disc, it can enter the chloroplast with the aid of a specific lightstimulated glycerate transporter (1’7).

62

KENNETH

There it mixes with the glycerate generated by the loop. Schmitt and Edwards (18) have shown that glycerate kinase is exclusively located in the chloroplasts of several C3 plants, and state that “No mechanism has been found for conversion of glycerate to PGA outside the chloroplast.” It will be assumed, therefore, that there is no route from glycerate to TP (and hence to sugars) other than via the chloroplast reductive cycle. Under conditions of photosynthesis where there is no RuBP oxidation (low Oz, high COz), all of the tracer 3H entering the PGA pool from glycerate ultimately leaves the chloroplasts in TP. Under conditions where a portion of the RuBP is oxidized, any 3H associated with C(1) of the oxidized molecule appears in (2R)-[2-3H1jglycolate. On oxidation by glycolate oxidase, 3H passes to water and is so diluted that it effectively leaves the system. More 3H passes to water and less leaves in TP as the relative flux of carbon to the loop increases. As the COz compensation point is approached (CO2 fixed = COz photorespired) the fraction of 3H leaving in TP approaches zero. A consideration of the overall stoichiometry of the system and the effect of cycling on 3H loss allows the partitioning of RuBP to be calculated from the fraction of entering 3H exported in TP. ‘H Retention and partitioning. The stoichiometry in going from RuBP to TP is (1 - g)RuBP + (1 - g)CO,l - 2(1 - g)TP for carboxylation, and gRuBP - (3g/Z)TP + (g/2)COPt for oxidation. Hence, overall RuBP + (1 - g)COZl (2 - g/Z)TP + (g/Z)COJ. [l] The gross COBfixed per mole RuBP is thus (1 - g), the net CO2 fixed (1 - 3g/2), and the photorespired COz is (g/Z). Photorespiration relative to gross or net COzfixation may be defined in terms of g; if PR = rate of photorespiration, GPS = rate of gross photosynthesis, and NPS = rate of net photosynthesis so that NPS = GPS - PR, then pg = PR/GPS

R. HANSON

The term pg gives a clearer metabolic picture, e.g., 2% of the COz fixed is photorespired, whereas p,, indicates that the rate of photorespiration is y% of the measured rate of net photosynthetic CO2fixation. The steady-state assumption that the carbon atoms exported as TP are equal to the carbon atoms fixed establishes that (1 - 3g/ 2)/3 mol of TP leave for every mol of RuBP metabolized. The remaining TP reforms 1 mol of RuBP. The fraction of TP exported per turn of cycle plus loop is thus (1 - 3g/ 2)/3(2 - g/Z), i.e., f=

(2 - 3g)/3(4 - g).

[41

Let t be the fraction of 3H retained in one turn of the cycle plus loop on going from TP back to TP, and let T be the fraction of imported 3H retained in exported TP. From Fig. 2 the distribution of 3H at C(1) and C(5) of RuBP formed from (3R)[3-3HJCP is 2:3. The fraction of 3H retained in TP via carboxylation is (1 - g) and via oxidation (3/5)g, hence t = 1 - (2/5)g. When 3H first reaches TP from imported PGA, the initial fraction exported is f with (1 -f) recirculated. After the first turn of cycle plus loop, this is reduced to t(1 - f ) of whichft(1 -f ) is exported and t(1 -f )’ recirculated. After the nth turn the export is ft”(l - f )“. A summation for infinite turns gives the total fraction exported, r = f /(l - t + tf). By substituting for t and f, the relationship r = (2 - 3g)/(2 + g) is obtained. Hence, the fraction of RuBP oxidized and the relative photorespiration can be expressed in terms of r, the 3H retention g = 2(1 - T-)/(3 + T) pg = (1 - r)/(l p, = (1 - r)/4r

+ 3r)

[51

Fl m

The dependence of t, ft and these parameters on r is shown in Fig. 4. Double lobe&g. If 3H labeling only is used, it is not possible to deduce how much (3R)-D-[3-3H&lycerate entered the chloroplasts because, inter alia, the difference = Pn/U + PII) = dW - $7) PI between supplied and recovered substrate is small and cannot be determined with p, = PR/NPS accuracy. This dilemma can be overcome 131 with the aid of the 3H:‘4C labeling. = PIJU - P&J = 942 - w.

STEREOCHEMICAL

r:

‘Ii

RETENTION

ASSAY

OF PHOTORESPIRATION

C%)

FIG. 4. Model parameters as a function of r, the fraction of supplied 3H retained in exported TP. r = 0, compensation point; T = 1, zero photorespiration. t = (11 + 9r)/(15 + 5r) = fraction of *H retained in TP per turn of cycle plus loop. f = 8r/3(10 + 6~) = fraction of TP exported. For g, p8, and p” see Eqs.

[W~l. The 3H:14Cratio for the supplied glyceric acid is known, and the ratio determined for any isolated metabolite may be expressed relative to this. The phrase “relative 3H:‘4Cratio” will be used in this paper to imply such a comparison unless otherwise stated. The relative ratio for PGA derived from supplied glycerate is 100%. The value for r may, therefore, be calculated if the relative ratio for TP and the fraction of PGA 14Cthat leaves as TP can be determined. The relative ratio for TP can be calculated from that of glucose obtained from isolated sucrose as follows: Exported TP is labeled with 3H at C(1) in the (R) configuration. F6P formed from TP is equally labeled in its two halves, with C(1) and C(6) both in the (R) configuration. The pro1R specific G6P isomerase converts F6P to G6P with partial loss to water of 3H from C(1) and partial transfer of 3H from C(1) to C(2) of G6P; both processes occur with discrimination against 3H (19-21). On reversing the reaction any 3H at C(2) of G6P is partially lost to water and partially transferred to C(1) of F6P. If there is an abundance of isomerase so that the reaction is fast compared to the steady-state

IN Ca PLANTS

63

flux from F6P to sucrose (5,6), half of the 3H will be washed out from both F6P and G6P. Under such circumstances the 3H:‘4C ratio for TP is twice the 3H:‘4C ratio of glucose isolated from sucrose. If the isomerase is rate limiting so that all of the G6P is converted as formed to sucrose (via GlP, UDP-Glc, and sucrose-P), the 3H content of glucose from isolated sucrose depends on the fraction of 3H transferred to G6P and the isotopic discrimination against such transfer, For rabbit muscle G6P isomerase (20) transfer is about 50% and b/kn = 0.36. If the leaf enzyme is similar, the 3H:‘4C ratio of glucose from sucrose would be 59% of the original TP ratio instead of 50%. In this kinetic situation F6P would be enriched in 3H, hence, the 3H:‘4C ratio of fructose from sucrose would exceed that of the original TP. T.he metabolism in tobacco leaves approximates the former equilibrium situation. It is convenient, therefore, to define an apparent 3H retention in TP as twice the relative ratio for glucose, R, = 2(‘H:14C glucose)/ (3H:‘4C glycerate supplied).

[8]

The true relative ratio for TP is defined as dR,, where d is the fraction of 3H in glucose at C(6). This factor will provisionally be taken as unity. The rate-limiting situation discussed above suggets a lower limit for d of 0.85. The term “apparent retention” is used because R, = r, the true 3H retention, only if d = 1 and all of the 14Cof PGA leaves as TP. A second correction, c, must be defined to allow for 14Cdiversion such that r = CdR,. The conversion of glycine to serine causes 14Cto migrate from C(2) of glycolate to C(2) + C(3) of PGA (Fig. 3). This leads eventually to glycolate labeled at both carbons; hence, 14C02will be released. However, the diffusion barriers to leaving the cell are appreciable, and the pathways from mitochondria to chloroplasts should be favored over that from mitochondria to air. Refixation of 14C02will, therefore, minimize the consequences of photorespiratory release of 14C.If there is no photorespiration there will be no loss of 14C02and c = 1. As photorespiration increases more

64

KENNETH

14COzwill be lost by the system, and the apparent 3H retention will be greater than the true retention, hence, c < 1. The correction may be estimated from the total 14C02released during an experiment and the 14Cin metabolites derived from TP or TP eqivalents (14Crr). If the total metabolized 14C-labeledglyceric acid 14CoA= 14Crr + 14C02,then as, by definition, dR, = (3Hrr./ 14Crr)/(3HoA/14CoJ and r = 3Hrp/3HoA,

3H~~ “Co~ 14GP r=---.-=-----‘dR,=cdR, “C~~ 3Ho~

“CO*

[9]

and c = (14C&(14Cp + 14CC@). [lo] A reasonable value for c may be calculated by taking 14Crr as the sum of the recovered 14C in neutral. sugars, malic acid, and starch. The first two together accounted for about 80% of the 14Cof the supernatant from the homogenized leaf discs after correcting for unmetabolized glycerate (see Results). The starch was not examined in the studies reported here on the grounds that Connecticut shade tobacco varieties accumulate very little starch as compared to malate (22), but 14Cin starch is being determined in current experiments. In deducing the relationship between r and R, certain assumptions have been made which need to be recognized. (a) It is assumed that steady-state labeling is realized. Initially, the export of 3H and 14C as TP reflects the 3H:14Cratio of PGA derived from supplied glycerate, but as all metabolic pools become labeled, 3H loss through the loop becomes significant. The steady-state condition will be met if the R, values are found to be independent of the duration of the experiment. (b) Isotopic discrimination against 3H and 14Cin the cycle plus loop is held to be unimportant. This condition should be met under all circumstances because neither isotope can appreciably alter the partitioning of RuBP. The 3H isotope effect shown by glycolate oxidase cannot influence the results because 3H and 14Care in separate molecules and the enzyme merely acts as a customs post which allows 14Cpassage but confiscates 3H. (c) The R, values are assumed to be independent of the partitioning of TP

R. HANSON

and TP equivalents between sucrose, malic acid, and starch formation. This should be the case because PGA, TP, and F6P are simply associated in the reductive cycle. Their 3H:‘4C ratios must be the same and it is irrelevant in what proportions these compounds leave the system. Dew on 0, and CO, ccmmntrations. In vitro studies show that COPand Oz are competitive substrates for RuBP carboxylase-oxygenase (7, 11). If quasi-equilibrium kinetics apply and if there are no subunit cooperativity effects, the fraction of RuBP oxidized, g, is determined by the COZ and O2 concentrations, i.e.,

Q= vm&wal)/(1

+ Jmlmw),

WI

where K is a composite of V,,,,, terms and enzyme-substrate dissociation constants (23). If the enzyme in vivo behaves similarly, despite its high active site concentration and the possibility of regulatory interactions, the variation of RuBP partitioning with [O;l and [Cod may be predicted. It is convenient to assume that internal concentrations are proportional to the external concentrations of these gasses. The internal [Od probably exceeds the external at low [Od (24); for [CO,] the proportionality constant varies with net photosynthesis. Equation [ll] may be written as g= a/(1 + a); hence, pg = a/2,p* = a/ (2 - a), and r = (2 - a)/(2 + 3a). If [Od is held constant and [CO,] varied, or vice versa, the equation for r is a rectangular hyperbola with an intercept at a = 2. As a contains the unknown constant K, it is useful to reexpress these equations by reference to the compensation point concentration of the gas being varied. At the compensation point r = 0 and a = 2; hence, if b = [O$[Oz camp] for constant [CO,] or b = [CO2 comp]/[COz] for constant [O.J the replacement a = 2b yields the equations g = 2b/(l + 2b),

P21

r = (1 - b)/(l + 3b),

D31

p,z= b

[I41

and p,, = b/(1 - b).

P51 Figure 5 shows these relationships in terms of semilog plots. The curve for g is a stan-

STEREOCHEMICAL

-

-

[co*]/

[CO2

[02]/[O*COMP]

COMP]

ofl

OR

[O*

ASSAY

OF PHOTORESPIRATION

cow]/[o*]

(LW

scale)

[CO*COMP]/[CO~]

(LW

Scald

FIG. 5. Theoretical dependence of model parameters on internal [OJ at constant [CO& and vice versa, assuming simple competitive kinetics for RuBP carboxylase-oxygenase (curve for g, Eqs. [ll] and [12]). For r, pg, and p, see Eqs. [13]-[15]. [Oe camp] and [COe camp] are introduced as empirical anchor points for the log scale.

dard titration curve with a point of rotational symmetry at (b = l/2, g = l/2) and a symmetry-point slope of 17.5% for doubling b. The curve for T is of the same form but displaced, so that the symmetry point is (b = l/3, r = l/3) and the corresponding slope is -23.5%. A plot of r against log [Oz] or log [COZ] is the most direct approach for comparing results with theory; however, the theory relates to the internal gas concentrations. At the compensation point the two are equal but, under conditions of net COz fixation, the internal concentration is less than the external by the product of the net photosynthetic rate and the diffusive resistance of stomata and membranes. If the variation of r with external [Oz] at constant external [CO,] is studied, the internal [CO,] will decrease as [OzJis lowered because net photosynthesis increases. The effective [O,] will therefore be greater than assumed, and the predicted empirical semilog plot will be somewhat flatter than in Fig. 5. Likewise, if the variation of r with [CO,] at constant [OJ is studied, increasing [COJ will increase net photosynthesis and the increase in internal concentration will be

IN Cs PLANTS

65

less than assumed. Again, the empirical curve will be flattened. These complications must be borne in mind when comparing the form of experimental curves for T with those predicted by the model. Alternative models. A full discussion of how r changes its significance on modification of the basic model lies outside the scope of this paper. It should be noted, however, that the basic model does not take into account the contribution made to photorespiration by the nonenzymatic oxidation by HzOz of glyoxylate and keto acids such as hydroxypyruvate. If glyoxylate is oxidized to COz and formate and if this formate is utilized in serine synthesis via the reductive formation of methylene tetrahydrofolate (25), the route requires 1 mol glycine/mol formate. As the stoichiometry is the same as the basic model, the equations already deduced apply. If formate is oxidized to COzby formate dehydrogenase so that all of the glycolate is converted to COz, it can be shown that g = (1 - r)/(3 - T), pg = (1 - r), and p,, = (1 - r)/r. The photorespiration values for a given r are thus higher if this model applies; in the case of p, they are fourfold higher. The dependence of r on internal [Oz] and [CO,] can also be deduced. As r = 1 - 2a; then, if b/2 is substituted for a, r = 1 - b and Eqs. [14] and [15] are unchanged. A plot of r against log b is steeper than that in Fig. 5 (Eq. [13]), but r likewise approaches 100% when b is small. The case in which all glycolate gives CO, may be regarded as a limiting model; thus, the values for relative photorespiration estimated by the equations derived from the basic model are minimal values, with the true values likely to be higher. MATERIALS

AND

METHODS

Chromatography. AGl(XS)-acetate (200-400 mesh) and AG5OW(X8)-H+ (100-200 mesh) ion-exchange resins were purchased from Bio-Rad. QAE-Sephadex (A-25), from Pharmacia, was equilibrated with 0.5 M Na formate (25). Samples for HPLC were centrifugally filtered through a 0.20-p membrane (Schleicher and Schuell, Inc.) and concentrated in a current of air. Other concentrations were usually effected with a Savant Speed Vat concentrator, or a Biichi RotavaporR. HPLC separations of organic acids (15-20 ~1) were performed with 0.015 N H*SO( on a Bio-Rad Aminex HPX-87H column, 300 X 7.8 mm, plus guard column,

66

KENNETH

at 25°C; flow rate, 0.6 ml/min. The liquid chromatograph was a Perkin-Elmer Series 3B, with uv detection at 210 nm. Peak areas plus retention times were recorded on a Hewlett-Packard 3390A integrator. Sugar separations using deionized samples (40 to 86 ~1) were performed with water on a Bio-Rad Aminex HPX87C column, 306 X 7.8 mm, at 80°C plus anion-OH and cation-H guard columns at 25°C; flow rate, 0.8 ml/min. The chromatographic pump and detector were from LDC (centraMetric III, and refractoMonitor) and the column heater was from Bio-Rad. Inversion of sucrose followed by separation into glucose and fructose was effected by placing two cationH guard columns in the heater at 95°C along with the column. Scintillation counting. Standard conditions were adopted for determining Q:“C ratios; 400 ~1 of sample was always mixed with 20 ml of counting solution (12g Omnifluor (New England Nuclear), 1 liter ethanol, 2 liters toluene). Counting efficiencies for 8H and i4C, respectively, were for channel (A) 27.16 and 20.46%, and channel (B) 0.0 and 24.59%. The mixtures were counted for 20 min in polyethylene vials with an Isocap 300 scintillation counter (Tracer Analytic). The 14C radioactivity of the critically important glucose samples was at least 506 dpm and usually about 1006 dpm. Samples were always counted within a few hours of mixing with the counting solution, as counts in channel A were found to increase and those in channel B to decrease slightly when the same samples were counted at 24-h intervals. Very large changes were observed in the external standard ratio with time. This method was thus useless for ‘H, “C counting when polyethylene vials were employed. Even with glass vials, the standard-conditions method was preferable to the esr method as the external standard values introduced additional random variations into the results. Preparation of ($R)- and ($S)-~$-~H*,s-“C]gI acids (3R)- and (3S)-[3-‘HJPGA were prepared from Djl-‘H]mannose (13.2 Ci/mmol) and D-[1-Qjglucose (15.0 Ci/mmol), respectively, by a modification of the single-step glycolytic sequence of Floss et al (26), and converted to the corresponding glyceric acids with potato acid phosphatase. D+b"~lyCeriC acid was prepared from D-[1-“Cjglucose (58 Ci/mol) as for the (3S)-[3-‘Hi]-labeled compound. Mixtures of the glyceric acids were prepared with a 8H:“C ratio of 20. The yields for 14Clabeling were about 80%, but those for the ‘H-labeled compounds were variable for unknown reasons. Appreciable amounts of tritiated water were formed in the reaction sequence subsequent to the formation of FBP. The following conditions minimize the exposure to aldolase of the q-labeled DHAP formed from C(l-3) of FBP and maximize the rates of isomerization of DHAP to Ga3P and of the reduction plus arsenolysis of Ga3P. (It was not established, however, that rabbit muscle aldolase was responsible for the variable yields. The effect of re-

R. HANSON placing this enzyme by yeast aldolase was not investigated.) Radioactive compounds were purchased from New England Nuclear, and the crystalline enzymes from Boehringer Mannheim (Yt = yeast, Rt = rabbit). The following mixtures were dialyzed against 40 rnrd Tris-Clbuffer, pH 8.3, at 4°C for 2 and 3 h, respectively. (A) Yt hexokinase (40 U, 0.3 mg); Yt M6P isomerase (12 U, 0.2 mg) or Rt G6P isomerase (70 U, 0.2 mg); Rt F6P kinase (60 U, 1 mg); and Rt pyruvate kinase (20 U, 0.14 mg). (B) Rt aldolase (0.45 U, 0.5 mg); Yt TP isomerase (1000 U, 1 mg); and Yt Ga3P dehydrogenase (160 U, 2 mg). After dialysis, mixture A (400 ~1) was added to MgCl, (12 amol), ATP (0.5 rmol), Tris-Clbuffer, pH 8.3 (80 pmol), and the radioactive substrate (either n{l-3Hjmannose (2 X log dpm), D-[l-3H@ucose (2 X 10’ dpm), or D-[l“Clglucose (10’ dpm)). The final volume was 1.1 ml and the temperature 21°C. Cyclohexylammonium PEP was added at lo-min intervals (5 rmol; 5 X 20 ~1). After 1 h, Na3AsOl (18 pmol), NAD (9 rmol), and mixture B were added (final vol, 2 ml). Chromatographic examination of a sample from the first stage on an AGl-acetate column verified that all the radioactivity was associated with the FBP fraction; however, additional PEP (4 X 20 ~1) was added during the second stage in case traces of phosphatase were present. After 2.5 h the reaction mixture was heated at 100°C for 5 min and filtered through glass wool, in a Pasteur pipet, directly onto an AGl-acetate column (14 X 0.58 cm). The column was eluted at 1.2 ml/min with water (30 ml) and then with 2 N formic acid (90 ml; G6P, M6P, F6P, and TP), and at 2.3 ml/ min with 6 N formic acid (120 ml; PGA). Finally, it was eluted with 1 N HCl (20 ml; FBP). The PGA fraction was concentrated to near dryness, applied to an AG50-H+ column in a Pasteur pipet (4 X 0.6 cm), and eluted with water (5 ml) to remove phosphatase inhibitors derived from the AGl column. To the eluate was added Na acetate buffer, pH 5 (200 smol), and potato acid phosphatase (1.4 U, 2 mg; Type II from Sigma). After 4 h at 21°C the reaction mixture was heated at 100°C. filtered, and chromatographed column (6 with 4 N acetic acid on an AGl-acetate X 0.6 cm) to yield glyceric acid. This fraction was concentrated, filtered, and purified by HPLC to remove trace contaminants. A further passage through an AGl-acetate column was necessary to remove the 0.015 N HzSOl from the HPLC step. The resultant 4 N acetic acid solutions from the various preparations were stored at -10°C. Prior to use in the leaf disc experiments, a sample from a stock 3H and ‘4c mixture was carefully concentrated to dryness and dissolved in water. Standard experiment, Mature leaves of greenhousegrown tobacco (Nicotiuna tobacum var. Havana Seed) were washed with dilute soap solution and rinsed with tap water. Representative sets (8 or 9) of seven leaf discs (each 16 mm diam., ca 40 mg) were punched from the leaves according to a modified Latin square

STEREOCHEMICAL

ASSAY

OF PHOTORESPIRATION

IN Ca PLANTS

6’7

ids were accompanied by losses of 3H to water, and as the yields were variable, it was necessary to establish that partial racemization at the 3H-labeled carbon had not taken place. The configurational purity of the products was investigated by a new procedure which takes advantage of the ability of glyoxylate reductase to accept Dglycerate as a substrate (29, 30, 31) and the formation of H202 as a by-product of NAD+ regeneration with phenazinemethosulfate and 0, [(32), p. 593; Fig. 61. The 3H-labele d g1y ceric acids were mixed with 14C-labeled glyceric acid and oxidized in one step to glycolate. Table I shows that the 3H:‘4C ratios were unchanged. Glycolate was then oxidized with the pro-2R specific glycolate oxidase (14, 15, 33). Within the limits of 3H:14Ccounting accuracy, the glyoxylic acid formed from presumed (R)labeled glycolate was 3H free, whereas all of the 3H was retained on oxidation of the presumed (S)-labeled glycolate. The expected 3H isotope effect for the (R)-labeled substrate was observed. The configurational purities were thus close to 100%. Tracer incorporation. The theoretical derivations presented assume that the added labeled glyceric acid does not add carbon to the system in any significant amount. This was established as follows: In the standard experiment (see above) sets of seven tobacco leaf discs (280 mg) were supplied for 50 min with high-specific-activity (3R)-D-[3-3H1,3-‘4Clglyceric acid (3.42 nmol). Typical photosynthetic rates for normal-air conditions were 150 pmol COz gFW-’ h-‘, with 40 pmol COz gFW-’ h-’ observed for the least favorable conditions studied. At most, 10% of the supplied substrate was taken up by the leaf discs, i.e., <3.6 natoms C gFW-’ h-l. The carbon flow into the leaf discs from COz was thus at least 11,000 times that from the supplied glycerate. Comparison of glvcerate and PGA inwrporatim The time courses of incorporation of D-[3-14C]glyceric acid and D-[~-‘~C]PGA were compared for 1,21, and 60% Oz (700 ~1 COJliter; 330 PE me2 s-l). In each pair RESULTS of experiments incorporations were terConfigurational purity of (SR)- and (3’S)- minated at 20, 40, 60, and 80 min. In all ~[3-“Hl,3-‘4CJg1~wric acids. As the enzy- cases the 14C incorporation into the sumatic syntheses of 3H-labeled glyceric ac- pernatant fraction from the homogenized

procedure. The discs were floated on water, upper su$b,ce up. Each set of discs was first maintained in air for 100 min under near-saturating light (650 PE m-z 8) and then placed in a Plexiglas l-liter chamber under the same illumination. Humidified gas of the desired composition at 28°C flowed through the vigorously stirred chamber (1 liter/min). The approach to a steady state was followed by closing the inlet and outlet and assaying gas samples for COz depletion (2 or 3 min) with an infrared gas analyzer. After 25 min of gas flow, the set of discs was removed and blotted, and each disc was placed, upper surface up, on a drop of (3R-D-[3-SH,,3-‘*C]glycerate solution on a fresh Petri dish, total vol, 1 ml (3.42 nmol, 200,000 dpm i4C; 3H:‘4C = 20). The discs were returned to the chamber under the same conditions used for equilibration. Steady-state conditions were quickly reestablished. Photosynthetic rates were determined at intervals. After 50 min, portions of water (3 X 6 ml) were added and withdrawn with a syringe while the leaves were still illuminated (2 min). These washings were frozen for later glycerate recovery. Water (10 ml) was then added as a final rinse and the discs were added to boiling 20 mM NaHS03 in 20% ethanol (20 ml). Two carefully matched chambers were available; thus, it was possible to replicate three or four conditions (9 or 8 sets of discs) in 1 day. The discs were homogenized, centrifuged at 45,000 for 7 min, resuspended in water, and again centrifuged to provide a final supernatant volume of 25 ml. Alifor radioactivity quots (3 X 400 ~1) were withdrawn determinations. AG50-H+ (6 X 0.6 cm) and QAE-Sephadex (5 X 1 cm) columns were coupled so that the effluent from the former flowed into the latter. After the supernatant was added, the columns were washed with additional portions of water (‘7 X 5 ml). The eluated fraction containing the neutral sugars (60 ml) was concentrated to near dryness in a rotary evaporator, the concentrate was washed into a weighed test tube, and water was added to a final weight of 2 g (aliquot for radioactivity determination, 400 ~1). The AG50 column was separately eluted with 4 N NH,OH (6 ml) to give the amino ocid-containingfraction. The QAE-Sephadex column (27) was eluted with 4% formic acid (30 ml) to give the organic acids fraction, and then with pH 4 pyridine-formate buffer (15 ml, 5% formic acid and ca 8% pyridine) to give the phosphate esters fraction. The latter fraction contains both oxalate (27) and the a-hydroxysulfonic acid derived from glyoxylate and the NaHS03 solution used to kill the leaf discs (28). All these solutions were concentrated, made up to 2 g with water, and sampled for radioactivity determinations (400 ~1).

68

KENNETH

REDUCTASE D-GLYCERATE

R. HANSON

HYDROXYPYRUVATE

GLVCOLATE

FIG. 6. First step of the sequence for establishing the configurational purity of (R)- and (S)-D[3-8H1,3-1’Clglyceric acid. In the second step (Table I) the (pro-2R)-H of the glycolate produced was removed with glycolate oxidase. HPLC-purified samples of (R)- and (S)-Dj3-*H&lyceric acid were each mixed with 1.3 X lo6 dpm D-[3-14Clglyceric acid to yield the *H:‘“C ratios listed in Table I. To these samples (about 11 nmol) was added carrier n-glyceric acid (10 pmol) to allow the uptake of Ox to be followed in the Warburg apparatus. The Warburg flask contained labeled glyceric acid, pH 8.5 Tris-Clbuffer (200 rmol), NAD (5 amol), and PMS (about 1 mg), final vol., 600 pl; side arm, spinach glyoxylate reductase (Boehringer-Mannheim, 2.7 U, 0.05 pg); center-well wick, 5 M ethanolamine (50 ~1). The oxygen uptake was complete after about 1 h. Residual traces of hydroxypyruvate were oxidized by adding 2 drops of 30% H,O,. The reaction mixture was heated at 100°C for 5 min and centrifuged, the supernatant was passed through an AGSO-H+ column (2 X 0.6 cm) in a Pasteur pipet to remove PMS, and the effluent was chromatographed on an AGl-acetate column (6 X 0.6 cm). The 4 -N acetic acid fraction accounted for 92% of the original radioactivity. HPLC yielded only glycolate (99.5%) and glycerate (0.5%).

and centrifuged discs was linear. For the neutral sugars fraction, the uptake was linear after a lag of 7 to 12 min. The incorporations into the total acids and total amino acids fractions were linear without any significant lag. The rates of incorporation (between 4 and 5%/h) were very similar for the two substrates. Washings from the discs supplied labeled PGA contained both PGA and glyceric acid, i.e., the discs had exuded phosphatases into the medium. Some of the 14Centering the leaf discs in the PGA experiments, therefore, entered as glyceric acid. Since there were no advantages and some theoretical disadvantages in using PGA as the substrate, all subsequent experiments were performed with labeled glyceric acid. Attainment of steady-state “H, 14Clabeling. Figure 7 shows a typical time course for the incorporation of 14Cfrom (3R)-D[3-3H1,3-‘4Clglyceric acid into the supernatant and neutral sugars fractions. The linear rates of incorporation relative to the rate for the supernatant were: neutral sugars, 50%; organic acids (including unmetabolized glycerate), 35%; phosphate

esters, 8%; and amino acids, 7%. The phosphate ester fraction showed a lag period similar to that for the neutral sugars. The 3H:14Cratios for each of these fractions were determined. No trend was apparent for the supernatant or for any fraction except neutral sugars. The slight trend shown in Fig. 7 may not be statistically significant, as the ratios for the shorter times were determined with samples having less total radioactivity. A fall in the 3H:‘4C ratio, however, would be expected during the lag period when the pools in the photosynthesis-photorespiration system are being labeled, because initially the ratio for TP must be the same as the PGA derived exclusively from imported, labeled glyceric acid. The ratios determined after 40 min or more are satisfactory equilibrium values. Cmervatim of stereospeti$c labeling. In preliminary experiments a comparison was made between (3R)- and (3S)-~-[3-~H,,314C]PGA as substrates using leaf discs in normal air under conditions which resembled those later employed in the standard experiment. When the @)-labeled sub-

STEREOCHEMICAL

ASSAY

OF PHOTORESPIRATION TABLE

CONFIGURATIONAL PURITY OF (R)-

69

IN Cs PLANTS

I AND (S)-D-[a-%]GLYCERIC

ACID

Preparation

Configuration: Yield:

Glyceric acid, initial Glycolic acid, derived Glycolate oxidase treatment Glyoxylic acid formed 5 min, 40% 40 min, 96% 90 min, 99% Glycolic acid remaining 5 min, 60% 40 min, 40% 90 min,
(i) 65% (sH:“C)

A 20% (‘H:“C)

A 17% (W’4C)

45% (‘H:“C)

30.6 29.6

30.7 29.9

28.8 28.9

25.5 26.5

0.014 0.011 0.050 46 300 38

0.008 0.020 0.060 45 500 30

0.060 0.060 0.060 50 3000 n-s

26.5 125

Note. For preparation of the labeled glyceric acids see Materials and Methods. Samples were oxidized to glycolic acid as in Fig. 3 and purified by HPLC. To each (in 1 ml HzSOI, 15 Nequiv) was added Ba(OH)z (13 Fequiv). The suspensions were centrifugally filtered and the filtrate was concentrated. To these were added 1 M Tris-Cl- buffer, pH 9 (100 pmol), and glycolate oxidase (23 mu, 1.1 mg protein); final volume, 400 ~1. The enzyme, a generous gift of Dr. Evelyn Havir, had been purified from tobacco leaves through the Al5 column stage (33). Samples withdrawn at the times indicated were added to 4 N HzSOl (10 pl). Later, the precipitated protein was removed by centrifugal filtration and the filtrates were concentrated. Glycolic and glyoxylic acids were separated by HPLC. Under the oxidation conditions used the amount of oxalate formed was negligible (33). ns, not sufficient to determine 8H:‘“C ratio.

strate was supplied and glycolate was isolated by preliminary ion-exchange chromatography and then HPLC, the glycolate showed a relative 3H:‘4C ratio of 40%. The relative ratios for glycine and serine, isolated by ion-exchange chromatography, were less than 3%. Because the amount of radioactivity in the amino acids was small and the separation achieved on the column imperfect, it was not possible to establish that the amino acids were as free of 3H as the initial substrate. The results, however, were consistent with expectation (Fig. 3). For the @)-labeled substrate the relative ratio for glycolate was greater than 30%, that for glycine 70%, and that for serine 44%. As serine transhydroxymethylase removes the (vo2S)-H of glycine, a ratio of 3H contents of 2:l would be expected in going from glycine to serine (16). (This assumes that, even if ‘H randomization occurs in the transferred methylene group (34), there is no loss of 3H or dilution with other pools.)

A more rigorous test of the conservation of stereospecific. labeling is provided by the data shown in Fig. 8. As noted under Theory, if sucrose is formed from exported (3R)-D-[3-3H1:4C]TP, the glucose moiety is labeled with 3H at C(6), to a limited extent at C(2), and is unlabeled with 3H at C(1). This pattern is determined by the pm1R specificity of G6P isomerase toward F6P. If, however, the exported TP contains some 3H in the pc+3Sposition, this will be found at both C(1) and C(6) of glucose. The configurational purity of the exported TP may be tested, therefore, by oxidizing the derived glycose to gluconic acid with glucose oxidase. As this reaction removes all the hydrogen at C(l), the 3H:‘4C ratio for gluconic acid must be less than that for glucose if there were any 3H in the pro3S position of the exported TP. The neutral sugars fraction isolated from leaf discs supplied (3R)-D-[3-%1 ,3-14C]glyceric acid under a variety of Oa and COz concentrations were found to have a wide range of

70

KENNETH

relative 3H:‘4C ratios. Sucrose was isolated from the neutral sugars fraction with the aid of HPLC. A second HPLC step, in which the column at 95°C was preceded by two sulfonic acid resin guard columns at the same temperature, yielded separate glucose and fructose peaks. A representative number of these samples were further oxidized to gluconic acid. Figure 8 shows a comparison of the relative ratios for gluconic acid and glucose. The regression line does not differ appreciably from 1:l proportionality. Stereospecificity is thus fully conserved over the range of physiological conditions studied. Variation of ‘H retention with [OS] and [COJ. Preliminary experiments showed that the relative 3H:‘4Cratio for the neutral sugars fraction decreased significantly with increasing [Oz] at constant [CO,]. A series of standard 50-min experiments at 350 ~1COJliter were therefore carried out in which glucose was isolated from sucrose as above. R,, the apparent 3H retention in exported TP, was calculated as twice the relative ratio for the isolated glucose (Eq. [8]). It varied with [Oz] as shown in Fig. 9.

SUPERNATANT

NEUTRAL SUGARS

2”40

NEUTRAL

SUGARS .

r

. 201 0

’

’ 20

’ TIME

’ 40 (mid

’

’ 60

’

’

FIG. 7. Time course of incorporation of radioactivity from (3R)-~-[3-*Hr,3-‘~C]glyceric acid. Sets of seven tobacco leaf discs were supplied with the substrate as described under Materials and Methods, but for the time intervals indicated. The order of chamber use randomized long and short incorporation times. 330 pE mm2s-r; 21% Oz; 700 pl COJliter; 174,000 dpm “0 qpc = 20.

R. HANSON

CoMP*RlSON RELATIVE %I: “C RATIOS

0

10

20

GLUCOSE

XI

40

YI

(%)

FIG. 8. Comparison of relative 3H:r4C ratios for gluconic acid and glucose. The samples of glucose were oxidized completely in 1 h at 32°C by excess glucose oxidase (from A. n&r, Sigma; 3.3 mg, 90 U) in Na acetate buffer, pH 5.1 (30 pmol). The gluconic acid was isolated by chromatography on an AGl-acetate column. Fitted line: intercept, 2.1% (k1.0); slope, 0.957 (kO.027); SE residuals, 1.5%; r*, 0.985; P < 0.001 (n = 21).

The curve resembled that predicted for the dependence of 3H retention, r, upon [O,] up to about 22% Oz (cf. Fig. 5). Identity of form would be expected only if R, = r, i.e., if all of the 14COzis refixed, so that c = 1, and if 3H is absent from C(2), so that d = 1. The assumption that all 14C02is refixed appears not to be true at high [O.J. If 14COz leaves the system, the 3H:14Cratio for exported TP must be higher than expected and will not approach zero as the compensation point is approached even though the fractions of imported 3H and 14Cexported as TP separately approach zero.3 The release of i4C02 under conditions of high photorespiration was confirmed using an illuminated chamber with a gas-tight O-ring seal and with two 2 M ethanolamine traps in series attached to the gas exit port. The evolution of 14C0, was followed over 314C retention may be analyzed in the same way as ‘H retention for the hypothetical (and unrealistic) limiting case in which all photorespired r4C02 is lost and 14C labeling is uniform. The ratio of ‘H and r4C retentions may then be expressed in terms of g.

STEREOCHEMICAL

% 4

( LOG

ASSAY

OF PHOTORESPIRATION

SCALE 1

FIG. 9. Relationship

between 3H retention and [OJ. (twice the relative aH:i4C ratio of glucose) and r is the retention after correction for i4C02 loss. The squares, triangles, and circles distinguish values from separate experiments and represent means of triplicates. The stars are corrected means of the data points on the dotted R. curve. The solid r curve has been drawn to have the same slope as the theoretical curve in Fig. 5 in the region corresponding to 11% Oz. The curves for the fraction of RuBP oxidized (g) and for relative photorespiration (p,,p,) were calculated from the curve for r using Eqs. [5], [6], and [+I]. A further correction to allow for partial 3H transfer by G6P isomerase is discussed in the text.

R, is the apparent retention

an 80-min period. The rate was linear after a lag of about 15 min (cf. neutral sugars, Fig. 7). The correction terms, estimated from the trapped 14COzand the recovered 14Cin neutral sugars plus malate (Eq. [lo]), were small for 21% Oz:r = 0.92dRs, but large for 61% 0z:r = 0.52dR,. It was also found that at 21% Oz the correction becomes larger as the COz concentration is lowered. Figure 9 shows a curve for r based on these corrections, assuming d = 1 (cf. Fig. 5), together with derived curves for g, pg, and p,. If in normal air r = 37%, then 37% of the RuBP is oxidized, 30% of the gross COz fixed is photorespired, and the photorespiration rate is 43% of the net photosynthetic rate. The assumption that G6P isomerase completely washes out all 3H from C(2) cannot be correct as the 3H:‘4C ratios of glucose and fructose from isolated sucrose are not identical (see below). Periodate ox-

71

IN C, PLANTS

idation of samples of gluconic acid prepared from sucrose-derived glucose (as described in Fig. 8) indicated that over 90% of the 3H was, in fact, at C(6), but a satisfactory analytical method for determining this fraction, d, has not yet been worked out. In the absence of precise information, it is reasonable to adopt a value of d that causes the values for r to match in Figs. 5 and 9 at low [02]. If d = 0.95, the normal air values given above become r = 35%, g = 39%, pg = 32%, and p,, = 46%. The correction thus raises g, pg, and p, by relatively small amounts. Experiments have also been carried out in which [Oz] was maintained at 22% and [Cod increased from 350 to 7600 pi/liter. The calculated R, at the highest COz concentration was only 65%, and the slope of the linear semilog plot was only +9% for a twofold increase in [CO,], instead of the +23.5% predicted from the basic model on the assumption that the internal [COJ is proportional to the external [CO,] (Fig. 5). The results could imply that the relationship between internal and external concentrations is much more complex for COB than for Oz, or that the metabolism departs from that of the basic model in ways which are not fully reciprocal for Oz and COz. Neutral sugars. Figure 10 shows that the

”

I”

I”

NEUTRAL

50

SUGARS

Ll 60

(%I

FIG. 10. Comparison of apparent 8H retentions, R. (twice the relative aH:‘“C ratios for glucose), with the relative ratios for the corresponding neutral sugars fractions. Fitted line: intercept, 0.5% (f1.6); slope 1.678 (f0.046); SE residuals 3.5%; r*, 0.962; P < 0.001 (n

= 55).

72

KENNETH

factor 1.68 can be used to convert a relative 3H:‘4C ratio for the neutral sugars fraction to an approximate R, value. This provides a convenient short cut for preliminary studies, but the scatter about the fitted line is too large for the use of the factor to replace the isolation of glucose from sucrose. An alternative short cut would be to subject the entire neutral sugars fraction to the HPLC splitting procedure and examine the glucose fraction obtained, but this has not been tested. HPLC of the neutral sugars fraction yielded variable amounts of oligomeric material, a major sucrose fraction, and fractions in which different amounts of glucose and fructose were mixed with minor amounts of unidentified sugars. When the sucrose was split into glucose and fructose, the relative ratio for the sucrose agreed with the average of the ratios for the glucose and fructose. The ratio of 14C in these fractions was 1:l; however, the ratio (3H:14C fructose)/(3H:‘4C glucose) varied. The mean for all determinations (n = 53) was 1.21 (fO.lO), but the range for separate experiments involving eight or nine determinations was from 1.08 (f0.04) to 1.32 (f0.0’7). Much higher ratios would be expected if there had not been extensive equilibration between G6P and F6P (see Theory). It appears that washing out of 3H is incomplete and that there are day-to-day differences in the amounts of G6P isomerase in the leaves being studied. The overall mean (n = 53) for the ratio (3H:‘4C neutral sugars)/(3H:‘4C sucrose) was 1.07 (+O.OB),and the means for individual experiments did not differ significantly from this or from each other. The consistency of the ratio suggests that the 3H and 14Cof the neutral sugars fraction are mainly associated with sucrose together with glucose and fructose (derived either directly from G6P and F6P or from sucrose). A slight bias in favor of fructose, or the consistent presence of some other sugar, is indicated. Much of the scatter about the regression line in Fig. 10, therefore, can be explained by the variation in the fructose/glucose 3H:14Cratios. Glgcerate uptake and recovery. In the standard experiments the 14C in the supernatant fractions ranged from 5.5 to

R. HANSON

11.3% of that supplied to the discs. The range was narrow, however, within single experiments; the standard errors were 1.6%, or less, of the supplied 14C. There were no trends discernible with Oa, COz, or R,. Recent experiments suggest that the motion of water through the leaf and out of the stomata is the primary determinant of glycerate uptake. When the humidity of the entering gas mixture was controlled at a level well below saturation, the 14Cuptake was consistently about 30%. At very high CO, concentrations, where stomata1 closure would be expected, lower 14Cuptake was noted. Table II shows the mean distributions of 14Cbetween the various fractions obtained from the supernatant. HPLC examination of the organic acids fraction showed that glyceric and malic acids together accounted for 80% or more of the radioactivity. Small amounts of 14Cwere associated with citric acid. On the average, glyceric acid accounted for about half of the 14C,but the proportions were variable. Examination of the 3H:‘4C ratios for the

TABLE

II

DISTRIBUTION OF “‘C IN CENTRIFUGED EXTRAIXS OF LEAF DISCS

Fractions Neutral sugars Organic acids Phosphate esters Amino acids Total Less unmetabolized glycerate Malate Neutral sugars + malate

Supernatant (a) 51.9 29.5 6.4 12.2

(k7.2) (k4.5) (k4.1) (k4.3)

199.0

Corrected for unmetabolized glycerate (%) 58.4 20.0 6.5 15.1

(k8.2) (&5.7) (22.7) (f6.8)

loo.0

87.0 (k7.0) -

-

18.5 (k4.0) 77.1 (31)

Note The fractionation is described under Materials and Methods. Replicates within individual experiments were averaged before calculating the means and standard errors (la = 20). Corrections for unmetabolized glycerate were applied to the individual averages before recalculating the means and standard errors.

STEREOCHEMICAL

ASSAY

OF PHOTORESPIRATION

glyceric acid samples showed that most of the radioactivity was attributable to unmetabolized substrate. Glycerate associated with the oxidative loop under normal air conditions should be free of 3H (Fig. 3); the observed relative 3H:‘4C ratio for discs in normal air was 93%, hence 93% of the glycerate was unmetabolized. The mean relative ratio for all samples examined was 95%. Such considerations allow the organic acids fraction to be corrected for unmetabolized glycerate and hence the distribution of metabolized 14Cto be calculated (Table II). Partitioning between L-w&ate and neutral sugars. Neutral sugars plus malate accounted for about 80% of the metabolized 14Cin the supernatant fraction, and this figure becomes even higher if one corrects for 14Cin metabolic intermediates (phosphate esters, glycine, serine, etc.). Between 17 and 33% of the 14Cfound in the malate plus neutral sugars fractions was in malate (mean, 25%). Considerable variation was apparent within single experiments. Regression analysis allowed clear trends to be established. In experiments at 22% O2and varied [CO& the fraction in malate decreased from 30 to 18% as [CO4 increased from 150 to 7600 pi/liter; as net photosynthesis increased from 40 to 1500 pmol COz gFW-’ h-l; and as R, increased from 37 to 66%. The (correlation coefficients)2 for regressions against log (net CO, fixation) and R, (n = 8) were, respectively, 0.748 and 0.814 (P < 0.01). In experiments where [CO4 was constant and [O,] varied, the fraction in malate also decreased with log (net COZfixation) and R,. The simplest interpretation of these results is in terms of competition between PGA and TP for the phosphate translocator (12). Malate is thought to derive from PGA (see next paragraph) and the neutral sugars from TP. With increased net CO2 fixation (and therefore R,) the concentrations of both PGA and TP probably both increase, but if [PGA] increases less than [TP] the ratio [PGA]/[TP] decreases. As the fraction of the total PGA + TP leaving as PGA is hyperbolically determined by [PGA]/[TP] (cf. Eq. [ll]), a decrease in [PGA]/[TP] would result in a decrease in the fraction of 14Cas malate.

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73

Clear evidence that malate and the neutral sugars derive from the same labeling pool was obtained by comparing the relative 3H:14Cratios for malate with the corresponding R, values. A simple correlation would not be expected if malate was derived either from glycerate without entering the chloroplasts, or from the ‘H-free compounds of the photorespiration loop. The range in relative ratios for malate was 16 to 35% and in R, 34 to 99% (n = 16). For a linear regression the (correlation coefficient)2 = 0.926 (P < 0.001). These results are also of interest as the details of the pathway from PGA to malate are a subject of speculation. This topic will be discussed in a future publication when experiments on the stereospecificity of 3H labeling in the malate samples are completed. Testing for metabolic dgerences. One purpose of this study was to develop a method for comparing photorespiration in leaf discs from two sources, e.g., from mutant and wild-type plants, or leaf discs from the same plant treated in different ways. From the variances for replication in individual experiments a best estimate for the standard error for R, values was calculated to be f4.4 (scale of 100). A standard experiment would allow the mean R, values for two sets of four replicates to be compared. A difference in means of 7.6 (scale of 100) would be significant (P = 0.05). From Fig. 9, an increase in R, of 7.6 in going from the control to the test discs would be the same decrease in photorespiration produced by switching from 21 to 15% 02. DISCUSSION

The results show that 3H retention, r, as estimated via the apparent retention, R,, provides a reproducible index of relative photorespiration. Under conditions where photorespiration would be expected to be minimal 3H retention is essentially complete, under normal air conditions it has an intermediate value, and as photorespiration approaches equality with fixation T approaches zero. Such trends have been investigated in terms of changing O2 and changing CO2 concentrations. Despite the

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seasonal and day-to-day variations in photosynthetic rates found with leaf discs from greenhouse-grown tobacco plants, similar physiological conditions appear to yield similar T values during both summer and winter. Such consistency could arise because the method depends upon partitioning of intermediates. The corollary that it can only yield estimates of photorespiration relative to net or gross COZ fixation is an important advantage. In searching for plants with lower photorespiration or in trying to understand the regulation of photorespiration, partitioning is of more interest than absolute rates. The reproducibility of the method should allow useful statistical comparisons of relative photorespiration for leaf discs from mutant and wild-type plants. Certain metabolic implications of the apparent completeness of 3H retention observed at low [Od should be noted. It is theoretically possible for 3H at C(1) of the Ru5P derived from Xu5P (Fig. 2) to lose 3H to water by the action of R5P isomerase (19,21). In the chloroplasts the main function of the isomerase is to convert R5P labeled with 3H only at C(5) to Ru5P. Therefore, Ru5P labeled with 3H at C(1) would have to compete for the active site with both R5P and with Ru5P formed from R5P. Any isotopic discrimination against 3H loss would also hinder the back reaction. In addition, at high light intensities where Ru5P kinase is fully activated and a high energy charge is maintained, the Ru5P concentration should be low. All of these factors would minimize washing out of 3H through R5P isomerase equilibration. A second possible source of 3H loss is associated with the diversion of F6P to starch formation. Even if starch is relatively unimportant in the leaves under study, sufficient chloroplast G6P isomerase could be present to wash out some 3H from C(1) of F6P (Fig. 2). Starch formation, however, disrupts chloroplast structure. It must, therefore, be a highly localized activity bounded by thylakoid membranes [(13), p. 2771.It is possible that diffusion or transport across a membrane commits F6P to starch formation so that equilibration does not influence the F6P pool of the reductive cycle, or that G6P is converted as formed

R. HANSON

to ADP-Glc (via GlP) under the high energy-charge conditions prevailing, with the result that F6P is not reformed from G6P. An alternative possibility is that starch formation takes place in specialized chloroplasts (amyloplasts) which also do not export TP for sucrose formation. The results reported here would then apply only to the chloroplasts that do not form starch. The relative photorespiration rate estimated for discs in normal air floating upper surface up, namely 46% of the net fixation rate, is well within the range of reported values (2). It is a minimal value, as the basic model assumes all of the photorespired COa derives from glycine, i.e., that the stoichiometry is l/2 mol of COz/ mol of glycolate oxidized (one carbon in four). In predicting the dependence of 3H retention upon [Od and [CO,], the additional assumption was made that RuBP carboxylase-oxygenase obeys simple competitive kinetics. The results for changing [Oz] at fixed [CO,] (Fig. 9) agree well with theory, but because of the uncertainty about internal [CO4 and [O.J this is not a critical test of the assumed stoichiometry. The lack of mirror-image complementarity between these results and those for changing [CO,] at fixed [Od suggested that some kinetic or metabolic assumptions will have to be modified. Experiments are now in progress, in collaboration with Dr. Richard B. Peterson, to compare the relative rates of photorespiration determined by 3H retention with those calculated from concurrent direct measurements of net photosynthesis and photorespiration (35, 36). The difference between the results should indicate how far the stoichiometry of COZ release departs from that assumed in the basic model. ACKNOWLEDGMENTS I wish to thank Dr. Evelyn Havir, Dr. Richard Peterson, and Dr. Israel Zelitch for many valuable discussions and for advice concerning experimental techniques; and Carol Barbesino and Ellen Hennessey for their skillful technical assistance. REFERENCES 1. HALLIWELL, B. (1981) Chloroplast Metabolism. Structure and Function of Chloroplasts

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2.

3. 4. 5. 6.

7.

8.

9.

10. 11. 12. 13.

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Green Leaf Cells, pp. 146-1’78, Oxford Univ. Press (Clarendon), London/New York. ZELITCH, I. (1979) in Encyclopedia of Plant Physiology (Gibbs, M. and Latzko, E., eds.), Vol. 6, pp. 353-36’7, Springer Verlsg, Berlin/New York. SOMERVILLE, C. R. (1982) What k New Plant Physid 13, 29-32. ZELITCH, I. (1979) Chem Eng. Neu~s 57, 28-48. KELLY, G. J., LATZKO, E., AND GIBBS, M. (1976) Annu. Rev. Plant Physiol 27, 181-205. KEYS, A. J., BIRD, I. F., CORNELIUS, M. J., LEA, P. J., WALLSGROVE, R. M., AND MIFLIN, B. J. (1978) Nature (Londorc) 275, 741-743. LORIMER, G. H., AND ANDREWS, T. J. (1981) in The Biochemistry of Plants (Hatch, M. D., Boardman, N. K., Stumpf, P. K., and Conn, E. E., eds.), Vol. 8, pp. 329-374, Academic Press, New York. ROBINSON, S. P., AND WALKER, D. A. (1981) in The Biochemistry of Plants (Hatch, M. D., Boardman, N. K., Stumpf, P. K., and Conn, E. E., eds.), Vol. 8, pp. 193-236, Academic Press, New York. TOLBERT, N. E. (1979) in Encyclopedia of Plant Physiology (Gibbs, M., and Latzko, E., eds.), Vol. 6, pp. 338-352, Springer Verlag, Berlin/ New York. JORDAN, D. B., AND OGREN, W. L. (1981) Nature (London) 291, 513-515. LAING, W. A., OGREN, W. L., AND HAGEMAN, R. H. (1974) Plant Physiol 54, 678-685. HEBER, U., AND HELDT, H. W. (1981) AnnzL Rw. Plant Physiol. 32, 139-168. JENSEN, R. G. (1980) in The Biochemistry of Plants (Tolbert, N. E., Stumpf, P. K., and Conn, E. E., eds.), Vol. 1, pp. 273-313, Academic Press, New York. ROSE, I. A. (1958) J. Amer. Chew Sm. 80, 58355836.

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