Light Activation of Pyruvate, Orthophosphate Dikinase in Maize Mesophyll Chloroplasts: A Role for Adenylate Energy Charge

Pyruvate, orthophosphate dikinase (EC 2.7.9.1 [EC] ) was activatedin the light and inactivated following a dark treatment in intactmaize mesophyll chloroplasts. Addition of catalase (100–250units/ml) to the assay medium was necessary to obtain good activationand to keep the enzyme in an active state during illumination.Arsenate and carbonyl cyanide m-chlorophenyl-hydrazone, uncouplersof photophosphorylation, inhibited the activation. Pyruvate,which has been proposed to have a critical role in supportingthe light activation of pyruvate, orthophosphate dikinase, actuallyinhibited the activation. The pyruvate level in the chloroplastsuspension decreased when the enzyme was light-activated. Measurementsof adenylates and pyruvate in the chloroplasts indicated thatthe energy state of the chloroplasts was more important forthe light activation than was the level of pyruvate. ¹Present address: Department of Biochemistry, Faculty of Science,Saitama University, 255, Shimo-Okubo, Urawa, 338 Japan ²Present address: National Institute of Agrobiological Resources,Yatabe, Tsukuba, Ibaraki, 305 Japan (Received May 2, 1989; Accepted October 2, 1989)     

植物丙酮酸磷酸双激酶（PPDK）研究进展

丙酮酸磷酸双激酶（PPDK）是C4植物和景天科酸代谢（CAM）植物光合作用的关键酶,催化形成固定CO2的初始分子受体磷酸烯醇式丙酮酸（PEP）。本文重点比较了植物的ppDK基因结构及分子进化关系,综述了PPDK在C4植物和C3植物中的功能、PPDK的调控机理、PPDK在胁迫条件下的功能以及转PPDK基因等在近年来的研究进展。     

Structural Basis of Reversible Phosphorylation by Maize Pyruvate Orthophosphate Dikinase Regulatory Protein

Pyruvate orthophosphate dikinase (PPDK) is one of the most important enzymes in C₄ photosynthesis. PPDK regulatory protein (PDRP) regulates the inorganic phosphate-dependent activation and ADP-dependent inactivation of PPDK by reversible phosphorylation. PDRP shares no significant sequence similarity with other protein kinases or phosphatases. To investigate the molecular mechanism by which PDRP carries out its dual and competing activities, we determined the crystal structure of PDRP from maize (Zea mays). PDRP forms a compact homo-dimer in which each protomer contains two separate N-terminal (NTD) and C-terminal (CTD) domains. The CTD includes several key elements for performing both phosphorylation and dephosphorylation activities: the phosphate binding loop (P-loop) for binding the ADP and inorganic phosphate substrates, residues Lys-274 and Lys-299 for neutralizing the negative charge, and residue Asp-277 for protonating and deprotonating the target Thr residue of PPDK to promote nucleophilic attack. Surprisingly, the NTD shares the same protein fold as the CTD and also includes a putative P-loop with AMP bound but lacking enzymatic activities. Structural analysis indicated that this loop may participate in the interaction with and regulation of PPDK. The NTD has conserved intramolecular and intermolecular disulfide bonds for PDRP dimerization. Moreover, PDRP is the first structure of the domain of unknown function 299 enzyme family reported. This study provides a structural basis for understanding the catalytic mechanism of PDRP and offers a foundation for the development of selective activators or inhibitors that may regulate photosynthesis.In C₄ plants, pyruvate orthophosphate dikinase (PPDK), which catalyzes the reversible phosphorylation of pyruvate to phosphoenolpyruvate, is the most important rate-limiting C₄ cycle enzyme (Edwards et al., 1985). Phosphorylation and dephosphorylation of residue Thr-527 of PPDK lead to its inactivation and activation, respectively. This process is accomplished by a single, bifunctional protein, namely PPDK regulatory protein (PDRP; Burnell and Hatch, 1985), and the process is light intensity dependent (Hatch and Slack, 1969; Chen et al., 2014b). PDRP is an unusual enzyme in three respects. First, PDRP is a bifunctional enzyme that catalyzes both phosphorylation and dephosphorylation, and these activities are usually catalyzed by separate kinases and phosphatases. Second, PDRP uses ADP as the phosphoryl donor for kinase activity, while most kinases utilize ATP. Third, unlike most phosphatases that dephosphorylate substrates to yield inorganic phosphate (Pi), PDRP employs a Pi-dependent, pyrophosphate (PPi)-forming dephosphorylation mechanism (Ashton et al., 1984).Owing to the importance in regulating C₄ photosynthetic cycle, the preliminary studies on maize (Zea mays) PDRP carried out in the 1980s were widely considered to represent a significant breakthrough in the field of C₄ photosynthesis research. Burnell and Hatch (1983) identified PDRP as a regulator of PPDK. They used in vitro activity assays to detect the reversible phosphorylation of PPDK by PDRP (Burnell and Hatch, 1985). By selectively substituting Ser or Tyr for the targeted Thr residue in the active site of PPDK, Chastain et al. (2000) discovered that Ser but not Tyr was functionally similar to the Thr residue (Chastain et al., 2000). This led to the conclusion that PDRP was a member of the Ser/Thr family of protein kinases (Scheeff and Bourne, 2005).Unfortunately, extensive studies on PDRP have been hindered by the low protein abundance in vivo and difficult protein purification in vitro, and our understanding of the mechanism of the dual enzymatic activities was not significantly advanced. More recently, PDRP was cloned from both maize and Arabidopsis (Arabidopsis thaliana), and bioinformatics analysis only predicted a phosphate binding loop (P-loop) domain and a conserved domain of unknown function 299 (DUF299) domain (Burnell and Chastain, 2006; Chastain et al., 2008). Moreover, multiple sequence alignment and molecular phylogenetic analysis has indicated that PDRP from many species appears to lack a canonical protein kinase subdomain and defined protein phosphatase motifs, and the PDRPs of plants may belong to the DUF 299 family (Chastain et al., 2008). Surprisingly, a DUF299 of Escherichia coli regulates the reversible phosphorylation of the target Thr residue in the active site of PEP synthetase (PEPS, homolog of PPDK), and the process is also ADP and Pi dependent just as is maize PDRP. This DUF299 gene was subsequently established as PSRP (EC 2.7.11.33 and 2.7.4.28), the PEPS regulatory protein (Burnell, 2010). However, the detailed catalytic mechanism of PSRP, as with PDRP, has remained obscure.It is also unclear whether PDRP uses two separate or one shared active site to perform phosphorylation and dephosphorylation activities. Differential effects on the enzymatic activities in thermolysin studies indicated two separate sites (Burnell and Hatch, 1986). However, the respective inhibition by phosphorylated and nonphosphorylated PPDK suggested that PDRP may contain separate active sites in relatively close proximity (Burnell and Hatch, 1985).In this study, we determined the crystal structure of PDRP and identified clear electron density corresponding to a bound AMP molecule. Combined structural analysis and enzymatic experiments suggest PDRP uses a single active site to perform both phosphorylation and dephosphorylation activities. Structural alignment and activity assays of site-directed mutagenesis provided comprehensive insight into the evolutionary relationships with other bifunctional protein kinase-phosphatases and the catalytic mechanism that may prove useful for the development of selective activators and inhibitors.     

籽粒苋丙酮酸磷酸二激酶(PPDK)基因的密码子偏好性

运用CHIPS、CUSP和CodonW等程序分析了双子叶C₄植物籽粒苋(Amaranthus hypochondriacus)丙酮酸磷酸二激酶(PPDK)基因的密码子偏好性, 并与马铃薯(Solanum tuberosum)和苜蓿(Medicago truncatula)等双子叶植物及水稻(Oryza sativa)和玉米(Zea mays)等单子叶植物进行了比较, 建立了聚类树状图, 以期在作物高光效基因工程中为籽粒苋PPDK基因选择合适的受体植物提供依据。研究结果表明, 籽粒苋PPDK基因偏好于以A或T结尾的密码子, 与其它几种被比较的双子叶作物的PPDK基因密码子偏好性趋势一致, 而玉米和水稻等单子叶植物更偏好使用以G或C结尾的密码子。PPDK基因密码子使用偏好性的系统聚类分析表明, 籽粒苋与马铃薯和苜蓿等双子叶植物聚为一类, 而稗草(Echinochloa crusgalli)、玉米和高粱(Sorghum bicolor)等单子叶植物聚为一类, 与系统进化地位一致。但单子叶植物水稻的密码子偏好性与籽粒苋较为接近, 与玉米和高粱相差较远。为了选择合适的蛋白质表达系统, 比较并分析了籽粒苋PPDK基因的密码子偏好性与大肠杆菌(Escherichia coli)及酵母菌的异同, 发现其与酵母菌的差异小于大肠杆菌, 表明选择酵母菌表达系统更为合适。     

Light Activation of Pyruvate,Pi Dikinase and NADP-Malate Dehydrogenase in Mesophyll Protoplasts of Maize : Effect of DCMU, Antimycin A, CCCP, and Phlorizin

Pyruvate,Pi dikinase (PPDK, EC 2.7.9.1) and NADP-malate dehydrogenase (MDH, EC 1.1.1.82) were activated in the light and inactivated following a dark treatment in mesophyll protoplasts of maize. DCMU (up to 33 micromolar), an inhibitor of noncyclic electron transport, inhibited activation of MDH much more strongly than it did PPDK. Antimycin A (6.6-33 micromolar), an inhibitor of cyclic photophosphorylation, inhibited the activation of PPDK (up to 61%), but had little or no effect on activation of MDH. Carbonyl cyanide m-chlorophenylhydrazone (0.2-2 micromolar) and nigericin (0.4 micromolar), uncouplers of photophosphorylation, inhibited activation of PPDK while stimulating the activation of MDH. Phlorizin (0.33-1.7 millimolar), an inhibitor of the coupling factor for ATP synthesis, strongly inhibited activation of PPDK but only slightly effected light activation of MDH. These results suggest that noncyclic electron flow is required for activation of NADP-MDH and that photophosphorylation is required for activation of PPDK.     

Correlation of the Activities of Phosphoenolpyruvate Carboxylase and Pyruvate,Orthophosphate Dikinase with Biomass in Maize Seedlings

The relations of three carbon-assimilating enzymes in maizeto biomass productivity were studied. There was no significantcorrelation between biomass and the amount of fraction I protein(RuBP carboxylase/oxygenase protein). In contrast, both theactivities of phosphoenolpyruvate carboxylase and pyruvate,P₁dikinase were highly correlated to the biomass. (Received February 7, 1983; Accepted March 26, 1983)     

The Gene for Pyruvate,Orthophosphate Dikinase in C4 Plants: Structure, Regulation and Evolution

Pyruvate, orthophosphate dikinase (PPDK; EC 2.7.9.1 [EC] ) is a keyenzyme in photosynthesis in plants that exploit the C4 photosyntheticpathway for the fixation of CO₂. This review focuses on thestructure, regulation and evolution of the C4-type ppdk genein the maize genome. The C4-ppdk gene in maize consists of 19exons spanning about 12 kbp. The gene is transcribed from twodifferent initiation sites under the control of two promotersto produce two mRNAs of different sizes. The larger one containsthe exon 1 sequence that encodes the chloroplast transit peptideand its product acts as C4-PPDK in chloroplasts, while the smallerone does not contain the sequence and its product may functionas a C3-enzyme in the cytosol. This unusual dual promoter systemis not unique to the maize C4-type ppdk gene since the sameorganization is also observed in the rice (C3 plant) ppdk geneand in Flaveria. Thus, the two-promoter system is common toplant ppdk genes from C3 and C4, monocot and dicot plants. Adiscussion is also presented of the generation of a system forregulation of the expression of the C4-type ppdk gene. A chimericgene consisting of a reporter gene under the control of thepromoter of maize CA-ppdk is exclusively expressed in photosynthetictissues and not in roots or stems of transgenic rice. The expressionof the introduced gene is also regulated by light: it is lowin etiolated leaves and is enhanced by illumination. These resultsindicate that the regulatory system that controls ppdk expressionin maize is not unique to C4 plants. ¹Recipient of the JSPP Young Investigator Award, 1995.     

Influence of Oxygen and Temperature on the Dark Inactivation of Pyruvate, Orthophosphate Dikinase and NADP-Malate Dehydrogenase in Maize

The influence of oxygen and temperature on the inactivation of pyruvate, Pi dikinase and NADP-malate dehydrogenase was studied in Zea mays. O₂ was required for inactivation of both pyruvate, Pi dikinase and NADP-malate dehydrogenase in the dark in vivo. The rate of inactivation under 2% O₂ was only slightly lower than that at 21% O₂. The in vitro inactivation of pyruvate, Pi dikinase, while dependent on adenine nucleotides (ADP + ATP), did not require O₂.

The postillumination inactivation of pyruvate, Pi dikinase in leaves was strongly dependent on temperature. As temperature was decreased in the dark, there was a lag period of increasing length (e.g. at 17°C there was a lag of about 25 minutes) before inactivation proceeded. Following the lag period, the rate of inactivation decreased with decreasing temperature. The half-time for dark inactivation was about 7 minutes at 32°C and 45 minutes at 17°C. The inactivation of pyruvate, Pi dikinase in vitro following extraction from illuminated leaves was also strongly dependent on temperature, but occurred without a lag period. In contrast, NADP-malate dehydrogenase was rapidly inactivated in leaves (half-time of approximately 3 minutes) during the postillumination period without a lag, and there was little effect of temperature between 10 and 32°C. The results are discussed in relation to known differences in the mechanism of activation/inactivation of the two enzymes.

     

High-Level Expression of Cold-Tolerant Pyruvate, Orthophosphate Dikinase from a Genomic Clone with Site-Directed Mutations in Transgenic Maize

Pyruvate, orthophosphate dikinase (PPDK) is a key enzyme in the C₄ photosynthetic pathway of maize. To improve the cold tolerance of the enzyme in maize, we designed two genomic sequence-based constructs in which the carboxy-terminal region of the enzyme was modified to mimic the amino acid sequence of the cold-tolerant PPDK of Flaveria brownii (Asteraceae). A large amount of PPDK was found to have accumulated in the leaves of many of the maize plants transformed with one of these constructs – that which introduced 17 amino acid substitutions without any alteration of the exon-intron structure – although there was a wide range of variation in the amount of PPDK among the separate plants. In contrast, the production was much less in maize transformed with the second construct in which a cDNA fragment for the same carboxy-terminal region was inserted. The specific activity of PPDK in the plants transformed with the gene with the amino acid substitutions was inversely correlated with the amount of enzyme in the leaves. In addition, the activity of the cold-tolerant recombinant enzyme was judged to be regulated by the PPDK regulatory protein, similar to that of the native PPDK. The cold tolerance of PPDK in crude leaf extracts was greatly improved in plants that produced a large amount of the engineered PPDK. The photosynthetic rate at 8°C increased significantly (by 23%, p<0.05), but there was no obvious effect at higher temperatures. These results support the hypothesis that PPDK is one of the limiting factors in the C₄ photosynthesis of maize under cold conditions.     

Posttranslational Modification of Maize Chloroplast Pyruvate Orthophosphate Dikinase Reveals the Precise Regulatory Mechanism of Its Enzymatic Activity

In C₄ plants, pyruvate orthophosphate dikinase (PPDK) activity is tightly dark/light regulated by reversible phosphorylation of an active-site threonine (Thr) residue; this process is catalyzed by PPDK regulatory protein (PDRP). Phosphorylation and dephosphorylation of PPDK lead to its inactivation and activation, respectively. Here, we show that light intensity rather than the light/dark transition regulates PPDK activity by modulating the reversible phosphorylation at Thr-527 (previously termed Thr-456) of PPDK in maize (Zea mays). The amount of PPDK (unphosphorylated) involved in C₄ photosynthesis is indeed strictly controlled by light intensity, despite the high levels of PPDK protein that accumulate in mesophyll chloroplasts. In addition, we identified a transit peptide cleavage site, uncovered partial amino-terminal acetylation, and detected phosphorylation at four serine (Ser)/Thr residues, two of which were previously unknown in maize. In vitro experiments indicated that Thr-527 and Ser-528, but not Thr-309 and Ser-506, are targets of PDRP. Modeling suggests that the two hydrogen bonds between the highly conserved residues Ser-528 and glycine-525 are required for PDRP-mediated phosphorylation of the active-site Thr-527 of PPDK. Taken together, our results suggest that the regulation of maize plastid PPDK isoform (C₄PPDK) activity is much more complex than previously reported. These diverse regulatory pathways may work alone or in combination to fine-tune C₄PPDK activity in response to changes in lighting.Pyruvate orthophosphate dikinase (PPDK) is an abundant mesophyll-chloroplast enzyme involved in C₄ photosynthesis. It plays an essential role in regenerating phosphoenolpyruvate (PEP), the primary cellular CO₂ acceptor molecule. PPDK activity strongly correlates (r = 0.96) with the photosynthetic rate (Edwards et al., 1985). Therefore, PPDK may limit the rate of CO₂ assimilation in the C₄ cycle (Hatch, 1987). PPDK regulatory protein (PDRP), a unique bifunctional enzyme, catalyzes this light-dependent regulation by reversible phosphorylation of an active-site Thr in PPDK (Thr-527 in maize [Zea mays] in full amino acid sequence [http://www.maizegdb.org]; previously termed Thr-456; Ashton and Hatch, 1983; Burnell and Hatch, 1985; Roeske and Chollet, 1987; Ashton et al., 1990; Burnell, 1990; Chastain et al., 2000, 2011). PDRP is an unusual regulatory protein for three reasons (Chastain et al., 1997, 2008; Burnell and Chastain, 2006; Astley et al., 2011): (1) it is bifunctional, catalyzing both PPDK activation/dephosphorylation and PPDK inactivation/phosphorylation; (2) it uses ADP instead of ATP as the phosphoryl donor; and (3) it employs an inorganic phosphate-dependent, inorganic pyrophosphate-forming dephosphorylation mechanism as opposed to the simple hydrolysis mechanism common to most protein phosphatases.The functional properties of PDRP have been examined by selective substitutions at His-458 and active-site Thr-456 in the maize plastid PPDK isoform (C₄PPDK; Ashton and Hatch, 1983; Burnell and Hatch, 1984, 1985). These studies confirmed that PDRP is a Ser/Thr kinase that requires a phosphorylated His in the target enzyme (Burnell and Hatch, 1986). This regulatory threonyl phosphorylation of PPDK is a monocyclic cascade (Stadtman and Chock, 1977) in which the covalent modification system is assumed to be a continuous process that allows the extent of PPDK activation to be attuned to the metabolic needs (Roeske and Chollet, 1989). Therefore, PDRP can alter the activation state of its target enzyme, PPDK, according to the concentrations of metabolites (e.g. ADP, inorganic phosphate, pyruvate, and PEP) involved in the regulatory cycle. In addition, PPDK activity also can be modulated by Mg²⁺ and temperature (Hatch and Slack, 1968; Wang et al., 2008).In all plants, PPDK is located in both cytoplasmic and plastid compartments (Chastain and Chollet, 2003). Regulation of the bidirectional activities of C₄PPDK has been proposed to be the consequence of light/dark-mediated changes in the stromal ADP level via its action as a potent competitive inhibitor of the PDRP phospho-PPDK dephosphorylation function (Burnell and Hatch, 1985; Chastain et al., 2011). However, GDP can serve as a substrate for the regulatory phosphorylation of the cytoplasmic PPDK isoform (Chastain et al., 2011). Two genes that encode chloroplastic (RP1) and cytosolic (RP2) isoforms of PDRP have been identified in the C₃ plant Arabidopsis (Arabidopsis thaliana). Both of them have kinase and phosphotransferase activities, although RP2 catalyzes PPDK dephosphorylation at a slower rate than does RP1 (Chastain et al., 2008; Astley et al., 2011). Bacterial genomic databases show that PDRP homologs, referred to as Domain of Unknown Function299 (DUF299) genes, are present in all PPDK-containing bacteria (Burnell, 2010). In Escherichia coli, which lacks PPDK, DUF299 regulates the on/off activity of phosphoenolpyruvate synthetase (PEPS) via reversible phosphorylation of the PEPS active-site Thr (Burnell, 2010). This specific target Thr residue for PDRP in C₄PPDK is highly conserved in all dikinases from C₃ angiosperms and prokaryotes that have been examined (Rosche et al., 1994; Fisslthaler et al., 1995; Agarie et al., 1997; Imaizumi et al., 1997; Wei et al., 2000). Taken together, these results suggest that this regulatory threonyl phosphorylation of the PPDK is a very ancient mechanism. This notion implies a common evolutionary pathway for C₄ photosynthesis facilitated by the preexistence of homologs of C₄ enzymes in C₃ plants (Edwards et al., 2001; Hibberd and Quick, 2002; Wang et al., 2009). The most significant adaptation for the enzyme to be utilized in C₄ photosynthesis may have already occurred well before the emergence of the pathway in modern angiosperms (Chastain et al., 2011).A previous empirical study showed that PPDK activity is insensitive to variations in PPDK level when a cold-tolerant ppdk is inserted into the genome of maize (Ohta et al., 2006). Enzyme activity measurements were performed on 48 strains, each with a different PPDK expression level, showing that there was only about a 20% change in the PEP formation rate despite a 5.7-fold variation in PPDK level. A similar phenomenon was also observed in transgenic rice (Oryza sativa) leaves, in which maize PPDKs accumulated at very high levels but failed to activate fully, even after 14 h of illumination and complete inactivation in darkness (Taniguchi et al., 2008). These findings suggest that the mechanism for regulating PPDK is far more complicated than previously thought. It is not known whether all or only part of the PPDK that accumulates in mesophyll chloroplasts is required for C₄ photosynthesis, because the protein level does not affect its enzyme activity. If it is only a select portion of PPDK that is required, then it is also unknown how light regulates the amount of PPDK involved in C₄ photosynthesis.To address these issues, we created a comprehensive profile of PPDK posttranslational modifications. We identified the cleavage site of the transit peptide, its N-terminal acetylated form, and four phosphorylated residues. We found that it is not the light/dark transition per se but rather a change in light intensity that regulates PPDK activity by modulating reversible phosphorylation at Thr-527. Importantly, we also partially determined the catalytic mechanism of PDRP. Taken together, these results suggest that the mechanisms via which PPDK is regulated are more complex than previously described (Ashton and Hatch, 1983; Chastain et al., 2000) and provide a foundation for studies on the molecular mechanism of PPDK regulation in the C₄ pathway.     

Cold Inactivation of Phosphoenolpyruvate Carboxylase and Pyruvate Orthophosphate Dikinase from the C4 Perennial Plant Atriplex halimus

Phosphoenolpyruvate carboxylase (PEPC) and pyruvate orthophosphate dikinase (PPDK) cold inactivation was studied in leaf extracts from Atriplex halimus L. Both enzyme activities gradually reduced as the temperature and the total soluble protein decreased. Mg²⁺ at a concentration of 10 mM stabilized PEPC and PPDK activities against cold inactivation. At low Mg²⁺ concentration (4 mM), PEPC was strongly protected by phosphoenolpyruvate, glucose-6-phosphate, and, partially, byL-malate, while PPDK was protected by PEP, but not by its substrate, pyruvate. High concentrations of compatible solutes (glycerol, betaine, proline, sorbitol and trehalose) proved to be good protectants for both enzyme activities against cold inactivation. When illuminated leaves were exposed to low temperature, PPDK was partially inactivated, while the activity of PEPC was not altered.     

Regulation of C4 Photosynthesis: Inactivation of Pyruvate,Pi Dikinase in Leaf and Chloroplast Extracts in Relation to Dark/Light Regulation in Vivo

Active pyruvate, P₁ dikinase in leaf or chloroplast extractsisolated from illuminated leaves was inactivated by incubatingwith ADP. With chloroplast extracts neither ATP nor AMP alonewas effective. Half the maximum rate of inactivation was observedwith about 55 µM ADP. The following evidence supportedthe view that ADP-mediated inactivation had a co-requirementfor low concentrations of ATP [Buchanan (1980) Ann. Rev. PlantPhysiol. 31: 341], adding hexokinase and glucose prevented inactivationby ADP [Feldhaus et al. (1975) Eur. J. Biochem. 57: 197], whenGDP and UDP were added in place of ADP they mediated rapid inactivationonly when ATP was also provided; GTP was not effective. ATPwas apparently optimally effective at about 1 µM or less.The rate of inactivation was approximately proportional to thesquare of extract concentration suggesting dependancy on a factorin the extracts in addition to active enzyme. The involvementof one or more heat labile protein factors was confirmed bytrypsin treatment of extracts. Pyruvate, P₁ dikinase inactivatedby treatment with ADP was reactivated by incubating with P₁,a property common to the inactive enzyme extracted from darkenedleaves. Thiol/disulphide interconversion was apparently notcritical in the regulation of pyruvate, P₁ dikinase. ³Present address: Department of Agricultural Chemistry, Facultyof Agriculture, Nagoya University, Chikusa, Nagoya 464, Japan. (Received September 22, 1980; Accepted December 6, 1980)     

Activation of NADP-Malate Dehydrogenase, Pyruvate,Pi Dikinase, and Fructose 1,6-Bisphosphatase in Relation to Photosynthetic Rate in Maize

The activity and extent of light activation of three photosynthetic enzymes, pyruvate,Pi dikinase, NADP-malate dehydrogenase (NADP-MDH), and fructose 1,6-bisphosphatase (FBPase), were examined in maize (Zea mays var Royal Crest) leaves relative to the rate of photosynthesis during induction and under varying light intensities. There was a strong light activation of NADP-MDH and pyruvate,Pi dikinase, and light also activated FBPase 2- to 4-fold. During the induction period for whole leaf photosynthesis at 30°C under high light, the time required to reach half-maximum activation for all three enzymes was only 1 minute or less. After 2.5 minutes of illumination the enzymes were fully activated, while the photosynthetic rate was only at half-maximum activity, indicating that factors other than enzyme activation limit photosynthesis during the induction period in C₄ plants.

Under steady state conditions, the light intensity required to reach half-maximum activation of the three enzymes was similar (300-400 microEinsteins per square meter per second), while the light intensity required for half-maximum rates of photosynthesis was about 550 microEinsteins per square meter per second. The light activated levels of NADP-MDH and FBPase were well in excess of the in vivo activities which would be required during photosynthesis, while maximum activities of pyruvate,Pi dikinase were generally just sufficient to accommodate photosynthesis, suggesting the latter may be a rate limiting enzyme.

There was a large (5-fold) light activation of FBPase in isolated bundle sheath strands of maize, whereas there was little light activation of the enzyme in isolated mesophyll protoplasts. In mesophyll protoplasts the enzyme was largely located in the cytoplasm, although there was a low amount of light-activated enzyme in the mesophyll chloroplasts. The results suggest the chloroplastic FBPase in maize is primarily located in the bundle sheath cells.

     

Light-Enhanced Uptake of Metabolites in Mesophyll Protoplasts: Evidence for a Novel Pyruvate Transport System

3 ) and sorghum (C₄) leaves for the measurements of osmotic volume change and metabolite uptake. We first investigated whether the silicone oil layer filtering centrifugation method could be applied to the protoplasts. The density of the silicone oil was optimized (ρ =1.026) and 0.5M betaine was chosen as an osmoticum in the protoplast suspending medium. By using [¹⁴C] sorbitol and [¹⁴C] inulin as the marker of the medium carried over into the pellet, protoplast osmotic or internal volume was estimated to be 200–300 μl (mg Chl)⁻¹, with the medium space in the pellet of 8–15 μl (mg Chl)⁻¹. Lowering of the osmotic pressure of the medium induced protoplast swelling as expected. Light also induced swelling. Using this system, we could detect light-enhanced uptake of ascorbate, glutamate and pyruvate in both barley and sorghum protoplasts. Pyruvate uptake was far higher in barley than in sorghum and inhibited by various inhibitors, showed saturation kinetics and, therefore, seemed to be mediated by a translocator protein. Received 10 August 1999/ Accepted in revised form 6 December 1999     

Partitioning of Nitrogen among Ribulose-1,5-bisphosphate Carboxylase/Oxygenase, Phosphoenolpyruvate Carboxylase, and Pyruvate Orthophosphate Dikinase as Related to Biomass Productivity in Maize Seedlings

Maize (Zea mays L. cv Golden Cross Bantam T51) seedlings were grown under full sunlight or 50% sunlight in a temperature-controlled glasshouse at the temperatures of near optimum (30/25°C) and suboptimum (17/13°C) with seven levels of nitrate-N (0.4 to 12 millimolars). The contents of phosphoenolpyruvate carboxylase (PEPC), pyruvate orthophosphate dikinase (PPD), and ribulose-1,5-P₂ carboxylase/oxygenase (RuBisCO) were immunochemically determined for each treatment with rabbit antibodies raised against the respective maize leaf proteins (anti-PEPC and anti-PPD) or spinach leaf protein (anti-RuBisCO). The content of each enzymic protein increased with increasing N and raised under reduced temperature. The positive effect of light intensity on their contents was evident only at near optimal temperature. The relative increase in PEPC and PPD content with increasing N was significantly greater than that of RuBisCO irrespective of growth conditions. These enzymic proteins comprised about 8, 6, and 35% of total soluble protein, respectively, at near optimal growth condition. In contrast to significant increase in the proportion of soluble protein allocated to PEPC and PPD seen under certain conditions, the proportion allocated to RuBisCO decreased reciprocally with an increased biomass yield by N supply.

These results indicated that the levels of PEPC and PPD parallel to maize biomass more tightly than that of RuBisCO at least under near optimal growth condition.

     

Comparative Studies of the Thylakoid Proteins of Mesophyll and Bundle Sheath Plastids of Zea mays

The proteins from both grana and stroma lamellae of maize (Zea mays) mesophyll plastids and from maize bundle sheath plastid membranes have been compared by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels using a discontinuous buffer system. Peptide differences between grana and stroma lamellae were essentially quantitative and not qualitative. Bundle sheath plastid membrane peptides more closely resembled those of the ultrastructurally similar stroma lamellae. However, bundle sheath membranes contained several peptides not apparent in the stroma lamellae.     

A Comparative Study of the Cold-Sensitivity of Pyruvate,Pi Dikinase in Flaveria Species

Pyruvate,P_i dikinase (PPDK) was isolated and purified from theleaf tissue of a number of Flaveria species and the cold labilityof the purified enzymes studied. The PPDK from F. brownii (aC₃/C₄ intermediate species) showed a high level of stabilitycompared to other Flaveria species. (Received September 7, 1989; Accepted November 22, 1989)     

玉米冠层内不同层次对光能利用的差异性

将玉米冠层分为上、中、下3个层次,分析其不同层次对光能利用的规律.结果表明:整个冠层吸收的光合有效辐射(RAR)占总入射的87.7%,其冠层的中、上层吸收比例达到75%;冠层不同层次在可见光范围内的吸收率是上层＞中层＞下层;上、中、下3层叶温日变化规律一致,不同层次的差异主要是与冠层内的小气候有关;非光化学猝灭系数(qN)与叶温、光化学猝灭系数(qp)与光合速率(Pn)的变化趋势一致;用于光化学反应的能量与用于热能转化的能量呈此消彼长的趋势.