Greening in a virescent mutant of maize II. Enzyme studies

Department of Botany, University of Illinois, Urbana, U.S.A., and U.S. Regional Soybean Laboratory, U.S. Department of Agriculture, Urbana, Illinois, U.s.A.

Greening in a Virescent Mutant of Maize II. Enzyme Studies l ) RAYMOND CHOLLET and WILLIAM L. OGREN With 6 figures Received March 17, 1972

Summary The activities of several enzymes were measured during greening of a virescent mutant (v 18) of Zea mays L. The activities of non-plastid enzymes were not markedly affected by either the mutation or greening. In contrast, the activities of mesophyll and bundle sheath plastid enzymes were substantially reduced in the un greened virescent and increased to nearnormal levels with greening. The activity of ribulose-1,5-diphosphate (RuDP) carboxylase was most affected by the mutation. The extreme deficiency of in vitro RuDP carboxylase activity in ungreened leaves and the subsequent increase in activity during greening were confirmed in vivo by pulse-chase feedings of 14CO Z and 12CO Z• The results indicate that photosynthesis in the ungreened virescent is limited by the reaction catalyzed by RuDP carboxylase and provide new evidence for a major role of this enzyme in the photosynthesis of C 4-plants.

Introduction The greening process in higher plants has been studied mainly in species which fix CO 2 by the Calvin cycle (C 3 -plants). Relatively few reports appear in the literature on chloroplast ontogeny in plants possessing the C 4 -pathway of photosynthetic CO 2 fixation (HATCH et aI., 1971). In C 4 -panicoid grasses such as maize and sugarcane, COz is initially fixed by phosphopyruvate (PEP) carboxylase in the mesophyll plastids, with the resulting oxaloacetate being predominantly reduced to malate by an NADP+specific malate dehydrogenase. The malate is transported to the bundle sheath chloroplasts and decarboxylated to pyruvate and CO 2 by malic enzyme. The CO 2 is presumably refixed by RuDP carboxylase forming 3-phosphoglycerate (3-PGA), which flows through the Calvin cycle to carbohydrate (ANDREWS et aI., 1971). Part I of this study (CHOLLET and PAOLILLO, 1972) indicated that greening in the virescent mutant v 18 of maize was accompanied by a structural normalization of aberrant mesophyll and bundle sheath chloroplasts and by a substantial increase in 1) Mention of a trademark name or a proprietary product does not constitute a guarantee or warranty of the product by the USDA, and does not imply its approval to the exclusion of other products that may also be suitable. Publication No. 714 of the u.s. Regional Soybean Laboratory. Z. PJlanzenphysiol. Ed. 68. S. 45-54. 1972.

46

R. CHOLLET and W. L. OGREN

plastid pigments and photosynthetic capacity. Gas exchange studies suggested that photosynthesis in the immature mutant was probably limited by the dark enzymatic reactions of CO 2 fixation. The present report describes changes in the in vitro activity of several plastid and non-plastid enzymes and in the in vivo labeling patterns of photosynthetic intermediates during greening.

Materials and Methods Plant Material and Sampling Procedures Normal (W23/L317) and virescent maize (Zea mays L.) seedlings were grown and sampled as described in the preceding paper (CHOLLET and PAOLILLO, 1972). In addition, leaf material from distal (Section A), middle (Section B), and basal (Section C) regions of the primary virescent leaf was taken for analysis at all three stages in greening. For some of .the enzyme assays, leaf extracts from the C'l-plant soybean [Glycine max (L.) Merrill, vars. Kent and Waseda] were also prepared. Leaf material was harvested from the youngest fullyexpanded trifoliolate of three-week old plants (grown hydroponically at 48.4 klux) and homo genized as described below for maize. Leaf Extracts Primary leaves of mutant and normal maize were detached, sectioned (excluding midveins), blotted dry and weighed. Leaf material (ca. 200 mg fresh tissue) was ground for 2 min at 0°_4° C with a motor-driven Ten Broeck homogenizer in 10 ml of cold buffer containing 40 mM tris-HCI, pH 8.0, 10 mM MgCl 2 , 5 mM dithiothreitol (DTT), 5 mM D-isoascorbate, and 0.25 mM EDTA (BJORKMAN and GAUHL, 1969). Microscopic examination of the crude leaf extract revealed an essentially complete breakage of bundle sheath and mesophyll cells and vascular tissue, indicating thorough extraction of both mesophyll and bundle sheath plastid enzymes. An aliquot of the crude leaf homogenate was centrifuged at 28,700 X g for 10 min at 0° C and the supernatant used as the source of enzyme for the spectrophotometric assays. Enzyme Assays In all assays, blanks contained extract and all reagents except the substrate used to initiate the reaction. RuDP carboxylase (E.C. 4.1.1.39) was assayed by the incorporation of radioactivity into acid stable products in the presence of NaH14C0 3 and RuDP. Reaction mixtures contained 40 mM tris-HCI, pH 8.0, 5 mM MgCI 2, 0.10 mM EDTA, 2.5 mM DTT, 0.10 mM RuDP, 20 mM NaH14C0 3 (1 ,uCi), and 0.1 ml of uncentrifuged leaf extract in a total volume of 1.0 ml (BOWES et aI., 1972). The reactions were initiated with RuDP, run in sealed vials for 6 min at 30° C in a shaker bath, and stopped with 0.1 ml 6 N acetic acid. The samples were dried for 2 h at 90° C, redissolved in 1 ml of water, and radioactivity determined in 10 ml of a modified BRAY'S (1960) liquid scintillator with a Packard Model 3003 Tri-Carb Scintillation Spectrometer. PEP carboxylase (E.C. 4.1.1.31) was similarly assayed in a reaction medium containing 30mM tris-HCI, pH 8.0, 3 mM MgCI 2, 0.10mM EDTA, 1.5mM DTT, 2 mM PEP,S mM sodium glutamate,S mM NaH14C0 3 (0.25 ,uCi), and 0.1 1111 of uncentrifuged leaf extract in a total volume of 1.0 ml. The following enzymes were assayed spectrophotometrically at 340 nm in the 3.0-ml reaction mixtures outlined below. All reactions were run at 22°-24° C. NAD+ -hydroxypyruvate reductase (E.C. 1.1.1.29): 4 mM sodium phosphate buffer, pH 6.2, 0.14 mM NADH, 1 mM hydroxypyruvate, and 0.10 or 0.15 ml centrifuged leaf extract (TOLBERT et aI., 1970). NADP+-isocitrate dehydrogenase (E.C. 1.1.1.42): 33 mM tris-HCI, pH 8.0, 1 mM MnCI 2, 0.12 mM NADP+, 1 mM dl-isocitrate, and 0.05 or 0.10 ml centrifuged leaf extract (GRAHAM et a!., 1970).

Z. Pflanzenphysiol. Bd. 68. S. 45-54. 1972.

Greening in a Virescent Mutant of Maize II.

47

Malic enzyme (E.C. 1.1.1.40): 100 mM tris-HCI, pH 8.0, 1.7 mM MnCI 2 , 1.7 mM DTT, 0.12 mM NADP+, 3.3 mM L-malate, and 0.03 or 0.05 ml centrifuged leaf extract (BERRY et aI., 1970). NADP+-malate dehydrogenase: 25 mM tris-HCI, pH 8.0, 1.7 mM DTT, 1 mM EDTA, 0.16 mM NADPH, 0.5 mM cis-oxaloacetate, and 0.03 or 0.05 ml centrifuged leaf extract (JOHNSON and HATCH, 1970). Pulse-Chase Experiments with

14CO~

Primary leaves were detached, recut under water, and illuminated at 13.5 klux and 25° C for at least 25 min before exposure to 14C0 2 • A dip method similar to that described by BERRY et ai. (1970) was used for the 14CO z-feedings. The chambers for the pulse and chase feedings consisted of 133-ml snap-cap vials immersed in a circulating water bath at 25 ° ± 1 °C. Light intensity was 13.5 klux at the chamber surface. 14C0 2 was generated in the chamber just prior to feeding a series of leaves by injecting a solution of NaH14C0 3 (100 ,uCi) into 1 N H 2S0 4 in the bottom of the sealed vial (final CO 2 concentration, 0.2 Ofo). After aSs pulse in 14CO z, the leaf material was either immediately killed in boiling 80 Ofo (vjv) ethanol or transferred to an adjacent, uncapped vial for a 30 s chase in 12CO z-air. Leaf material from the individual pulse and pulse-chase feedings was pooled and successively extracted in boiling 80 Ofo (v Iv) ethanol, 50 Ofo ethanol, and water. Chlorophyll and lipids were removed from the combined ethanol fraction with petroleum ether. The combined ethanol/water fraction was evaporated to dryness at 38° C under reduced pressure, made to a standard volume with water, and the radioactivity of aliquots determined in 10 ml of a modified BRAY'S (1960) scintillatOr. Incorporation of radiocarbon intO the petroleum ether .tnd insoluble fractions was less than 1 ~/o of the tOtal 14 C incorporated. Labeled compounds in the combined ethanol/ water fraction were separated by onedimensional descending paper chromatOgraphy in liquefied phenol (ca. 90 Ofo)-water-acetic acid-1 M EDTA (840: 160 : 10 : 1 by volume) on 23-cm X 56-cm sheets of oxalic acidwashed Whatman No.1 filter paper (BERRY et aI., 1970). The chromatograms were developed for 48 h, thoroughly air-dried, and the radioactive areas located with a Packard Model 7201 radiochromatogram scanner. Radioactive compounds were eluted from the paper with water, concentrated, and aliquots counted in liquid scintillator. The HC determined in the individual compounds or groups of compounds was expressed as a percentage of the amount initially applied to the chromatograms. Recovery of total counts from the chromatograms was always greater than 98 Ofo. The identity of labeled compounds was determined by cochromatOgraphy and coincidence with authentic non labeled compounds in the phenol solvent system described above and in n-butanol-formic acid-water (5 : 1 : 4 by volume) (malate, aspartate) or n-butanol-propionic acid-water (12 : 8 : 2 by volume) (3-PGA, sugar phosphates). Amino acids were located with a commercially-prepared ninhydrin spray, sugars with p-anisidine (BLOCK et aI., 1958), phosphorylated compounds with ammonium molybdate (BENSON, 1957), and organic acids with a bromophenol blue acid-base indicatOr (JONES et aI., 1953). No attempt was made to recover radioactive oxaloacetate in these ex periments due to its decomposition during extraction (HATCH and SLACK, 1966).

Results

Enzyme Activities Figs. 1-6 are representative histograms illustrating the changes in in vitro enzyme activity during development of comparable normal and virescent leaves. The activities of representative non-plastid enzymes, as illustrated by NADP+-isocitrate dehydrogenase (Fig. 1) and NAD+ -hydroxypyruvate reductase (Fig. 2), were little affected Z. Pjlanzenphysiol. Bd. 68. S. 45-54. 1972.

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Figs. 1, 2. Changes in NADP+-isocitrate dehydrogenase and NAD+ -hydroxypyruvate reductase activity during development of virescent and normal (W/ L) maize. The three regions of the mutant leaf (A, B, C) were assayed separately and the results are given for each region and as an average for the entire virescent leaf (v 18).

by either the mutation or greening. A similar trend (data not shown) was observed with other non-chloroplastic enzymes, including acid phosphatase, glucose-6-P dehydrogenase, and NAD+-malate dehydrogenase. In contrast, the activities of mesophyll and bundle sheath plastid enzymes were substantially reduced in the ungreened virescent leaf and increased to near-normal levels with greening (Figs. 3-6). Reciprocal

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Greening in a Virescent Mutant of Maize II.

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mixing experiments with deproteinized extracts from normal and ungreened virescent leaves indicated that the reduced activities of plastid enzymes in v 18, ungreened were not caused by the presence of endogenous non-protein enzyme inhibitors or the lack of endogenous enzyme activators. The activities of PEP carboxylase (Fig. 3), NADP+-malate dehydrogenase (Fig. 4), and malic enzyme (Fig. 5) in the un greened virescent were far in excess of the low rate of true photosynthesis observed in these leaves (CHOLLET and PAOLILLO, 1972), These enzymes were also present in all three regions of the immature mutant leaf and Z. PJlanzenphysiol. Bd. 68. S. 45-54. 1972.

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Figs. 3-6. Changes in PEP carboxylase, NADP+ -malate dehydrogenase, malic enzyme and RuDP carboxylase activity during development of virescent and normal (WI L) maize. The three regions of the mutant leaf (A, B, C) were assayed separately and the results are given for each region and as an average for the entire virescent leaf (v 18). The activity in leaf extracts of soybean (SOY), a C 3 -plant, is included for comparison.

the activity in each region was greater than that of the «darb enzymes in soybean. The activities increased 3- to 4-fold with greening. In contrast, RuDP carboxylase activity (Fig. 6) was extremely low in the ungreened virescent, often being undetectable in the basal region of the primary leaf, and greening was accompanied by a 10- to Z. PJlanzenphysiol. Bd. 68. S. 45-54. 1972.

51

Greening in a Virescent Mutant of Maize II.

20-fold increase in activity. The extreme deficiency in RuDP carboxylase activity is further illustrated by the high ratio of PEP carboxylase/RuDP carboxylase activity in the ungreened virescent as compared to wild-type (18.6 ± 5.0 vs. 6.77 ± 1.7). With greening, this ratio declined to a near-normal value, reflecting the substantial increase in RuDP carboxylase activity. Labeling Experiments with uCO~ Following aSs exposure of virescent and normal leaf material to 14C0 2, nearly all the radioactivity was found in the C 4 -acids, malate and aspartate, with only a very small percentage in 3-PGA and sugar phosphates (Table 1). The initial fixation of Table 1 Distribution of HC between C 4 -acids and sugar phosphates in the combined ethanol/water fractions of normal and virescent leaf material following pulse-chase experiments with 14CO~. Treatment

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14C02 into C4-acids in vivo is consistent with the in vitro demonstration of PEP carboxylase and NADP+-malate dehydrogenase activity in the virescent and normal leaves. When un greened virescent leaves were exposed to 12C02-air following 14C02, the proportion of label in the Cracids remained extremely high for at least 30 s of chase (Table 1). In corresponding experiments with v 18, greened and W23/L317, substantial amounts of HC accumulated in 3-PGA and sugar phosphates at the expense of the C4-acids labeled during the pulse, as is characteristic of normal C.-plants (BERRY et aI., 1970; HATCH and SLACK, 1966). The restricted movement of radiocarbon from malate and aspartate into Calvin cycle intermediates in the ungreened mutant was further investigated by performing separate pulse-chase experiments with

z.

PJlanzenphysiol. Bd. 68. S. 45-54. 1972.

52

R.

CHOLLET

and W. L. OGREN

the excised distal region (Section A) and the remainder of the immature leaf (Sections B + C). The phosphorylated intermediates in the distal region accumulated more label from the C-acids during the 30 s chase than those in the middle-basal region of the leaf. Discussion Greening in the virescent mutant of maize was accompanied by a substantial increase in activity of mesophyll and bundle sheath plastid enzymes, whereas typical non-chloroplastic enzymes were not markedly affected. This trend is similar to that observed during the light-induced development of C 3- and C 4 -etioplasts (BRADBEER, 1969; GRAHAM et aI., 1970; HATCH et aI., 1969; JOHNSON and HATCH, 1970). Similarly, the presence of PEP carboxylase, NADP+-malate dehydrogenase, and malic enzyme in the basal, most chlorophyll-depleted region of the ungreened virescent leaf is analogous to the occurrence of photosynthetic carbon metabolizing enzymes in etiolated leaf tissue (BRADBEER, 1969; GRAHAM et aI., 1970). However, the absence of detectable RuDP carboxylase activity in the basal region of v 18, ungreened is in marked contrast to these findings and resembles the reported lack or near-lack of Fraction I protein and RuDP carboxylase activity in light-grown albino mutants (FULLER and GIBBS, 1959; HABIG and RACUSEN, 1969; KLEINHOFS and SHUMWAY, 1969; LYTTLETON, 1956). A morphological comparison of the vesicular plastids which predominate in the basal region of v 18, ungreened (CHOLLET and PAOLILLO, 1972) with normal etioplasts (BACHMANN et aI., 1967; LAETSCH and PRICE, 1969) and the plastids of light-grown albinos (BACHMANN et aI., 1967; WALLES, 1965) indicates that the degree of chloroplast structural development is in the order etiolated normal > ungreened virescent :2 albino. This observation, together with the finding that RuDP carboxylase activity invariably follows the gradient of plastid structural complexity in the ungreened virescent leaf, supports the proposal that the development of RuDP carboxylase activity is greatly limited until the chloroplast has attained a certain critical level of membranous structure (HABIG and RACUSEN, 1969). A possible explanation for the correlation between RuDP carboxylase activity and a minimal plastid structural complexity is suggested by the recent indication of specific binding sites for the enzyme on the surface of chloroplast membranes (KANNANGARA et aI., 1970). The results from the pulse-chase experiments with 14C0 2 indicate a severely restricted movement of carbon from the C 4-acids into the Calvin cycle in the ungreened leaves of v 18, a restriction which is overcome with greening. The reduced rate of radiocarbon transfer in the ungreened tissue could be explained by a larger C racid pool size in the aberrant plastids, an anomalous, rapid accumulation of HC in all four carbons of malate and aspartate, or a limitation in the enzymatic reaction(s) involved in the transfer of the C-4 carboxyl of the Cracids into the Calvin cycle, the reactions presumably catalyzed by malic enzyme and RuDP carboxylase. Although pool sizes and the radioactivity in the individual carbon atoms of malate and aspartate Z. Pjlanzenphysiol. Bd. 68. S. 45-54. 1972.

Greening in a Virescent Mutant of Maize II.

53

were not determined, the strong correlation between the changes in in vitro RuDP carboxylase activity and the in vivo transfer of carbon from the C 4 -acids into Calvin cycle intermediates within the ungreened virescent leaf and during greening makes the two former explanations seem unlikely. Since malic enzyme was present in all three regions of the ungreened virescent leaf and its activity was in excess of PEP carboxylase, NADP+-malate dehydrogenase, and the «darb enzyme in soybean, it is doubtful that the observed deficiency in this enzyme in v 18, ungreened limited the transfer of carbon from the C-4 carboxyl of the C-acids into the Calvin cycle. The combined results of the in vitro enzyme studies and the in vivo labeling experiments with 14 C0 2 substantiate the earlier indication, based on gas exchange data (CHOLLET and PAOLILLO, 1972), that photosynthesis in v 18, ungreened is limited by the dark reactions of photosynthesis. More precisely, the present study indicates that photosynthesis in the ungreened virescent is limited by the reaction catalyzed by RuDP carboxylase. The strong correlation between the changes in in vitro RuDP carboxylase activity and the in vivo movement of carbon from the Cracids into Calvin cycle intermediates within the ungreened virescent leaf and during its development provides new evidence for a major role of this enzyme in the carboxyl transfer step during C 4-photosynthesis. The lack of a marked effect of the virescent gene mutation on non-plastid enzyme activity is consistent with the observed normal rate of mitochondrial respiration (per unit soluble protein) and fine structure of non-plastid organelles in the ungreened leaf (CHOLLET and PAOLILLO, 1972), and suggests that the nuclear gene mutation specifically affects the bundle sheath and mesophyll plastids. The substantial reduction of PEP carboxylase activity in the ungreened leaves of v 18 and the apparent specificity of the genetic lesion for chloroplast development provide supportive evidence for the association of this carboxylating enzyme with the plastids of Cplants (ANDREWS et al., 1971; SLACK, 1971). References ANDREWS, T. ]., H. S. JOHNSON, C. R. SLACK, and M. D. HATCH: Phytochemistry 10, 2005 (1971). BACHMANN, M. D., D. S. ROBERTSON, C. C. BOWEN, and I. C. ANDERSON: ]. Ultrastruct. Res. 21,41 (1967). BENSON, A. A.: Methods in Enzymol. 3, 110 (1957). BERRY, ]. A., W.]. S. DOWNTON, and E. B. TREGUNNA: Can.]. Bot. 48, 777 (1970). BJORKMAN, 0., and E. GAUHL: Planta 88, 197 (1969). BLOCK, R. ]., E. L. DURRUM, and G. ZWEIG: A Manual of Paper Chromatography and Paper Electrophoresis, 2nd edn., p. 182. Academic Press, New York (1958). BOWES, G., W. L .OGREN, and R. H. HAGEMAN: Crop Sci. 12, 77 (1972). BRADBEER, ]. W.: New Phytologist 68, 233 (1969). BRAY, G. A.: Anal. Biochem. 1, 279 (1960). CHOLLET, R., and D.]. PAOLILLO, .Ir.: Z. Pflanzenphysiol. 68, 30 (1972). FULLER, R. c., and M. GIBBS: Plant Physiol. 34, 324 (1959). GRAHAM, D., M. D. HATCH, C. R. SLACK, and R. M. SMILLIE: Phytochemistry 9, 521 (1970). HABIG, W., and D. RACUSEN: Can.]. Bot. 47, 1051 (1969).

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HATCH, M. D., and C. R. SLACK: Biochem. J. 101, 103 (1966) . HATCH, M. D., C. R. SLACK, and T. A. BULL : Phytochemistry 8, 697 (1969). HATCH, M. D., C. B. OSMOND, and R. O. SLATYER (eds): Photosynthesis and Photorespiration. Wiley-Interscience, N ew York (1971). JOHNSON, H. S., and M. D . HATCH: Biochem. J. 119,273 (1970). JONES, A. R., E. J. DOWLING, an d W. J. SKRABA: Anal. Chern. 25, 394 (1953). KANNANGARA, C. G., D. VAN WYK, and W. MENKE: Z . Naturforsch. B 25, 613 (1970). KLEINHOFS, A., and L. K. SHUMWAY: Biochem. Genet. 3, 485 (1969). LAETSCH, W. M., and I. PRICE: Amer. J. Bot. 56, 77 (1969). LYTTLETON, J. W.: Nature (London) 177,283 (1956). SLACK, C. R.: In: Photosynthesis and Photorespiration, p. 297, M. D. HATCH, C. B. OSMOND, and R. O. SLATYER, eds., Wiley-Interscience, New York (1971). TOLBERT, N. E., R. K. YAMAZAKI, and A. OESER: J . BioI. Chern. 245, 5129 (1970). WALLES, B. : Hereditas 53, 247 (1965).

Dr. R. CHOLLET, Department of Agronomy, University of Illinois, Urbana, Illinois 61801, U.s.A . Dr. W. L. OGREN, U.S. Regional Soybean Laboratory, Plant Science Research Division, Agricultural Research Service, U.S. Department of Agriculture, Urbana, Illinois 61801, U.S .A.

Z. Pjlanzenphysiol. Bd. 68. S. 45-54. 1972.