Pyruvate Orthophosphate Dikinase Activity Research Articles

Bromoacetic acid inhibits pyruvate orthophosphate dikinase and reduces photosynthetic activity in maize

Pyruvate orthophosphate dikinase (PPDK) catalyzes the conversion of pyruvate to phosphoenolpyruvate, and its activity is essential to maintain photosynthetic CO2 assimilation in C4 plants. Thus, it has been described as a promising target for the development of selective herbicides for C4 weeds. We evaluated whether bromoacetic acid, a known PPDK inhibitor of Clostridium symbiosum, can also inhibit the PPDK of a C4 plant, maize, in in vitro experiments. We also evaluated the effects of foliar application of 0.5–10 mM bromoacetic acid on the leaf pyruvate content and gas exchange of maize plants. Bromoacetic acid reduced maize PPDK activity in vitro in a dose-dependent manner; the degree of inhibition ranged from 13.6 to 73.6% with 0.5–10 mM bromoacetic acid. The foliar application of bromoacetic acid increased the leaf pyruvate content, suggesting that this compound may also inhibit PPDK activity in vivo. As a consequence, impairments to the gas exchange parameters of maize plants were noted, with a marked reduction in the photosynthetic rate and an increase in the intercellular CO2 concentration. Despite the abrupt decrease in stomatal conductance and transpiration, the plants showed large dehydration and reduction of leaf fresh weight after 48 h of application to bromoacetic acid. Our data allow us to affirm that bromoacetic acid has a potent herbicidal effect on a C4 plant, maize; the most likely mechanism of action is the inhibition of PPDK.

Characterization of maize leaf pyruvate orthophosphate dikinase using high throughput sequencing.

In C4 photosynthesis, pyruvate orthophosphate dikinase (PPDK) catalyzes the regeneration of phosphoenolpyruvate in the carbon shuttle pathway. Although the biochemical function of PPDK in maize is well characterized, a genetic analysis of PPDK has not been reported. In this study, we use the maize transposable elements Mutator and Ds to generate multiple mutant alleles of PPDK. Loss-of-function mutants are seedling lethal, even when plants were grown under 2% CO2 , and they show very low capacity for CO2 assimilation, indicating C4 photosynthesis is essential in maize. Using RNA-seq and GC-MS technologies, we examined the transcriptional and metabolic responses to a deficiency in PPDK activity. These results indicate loss of PPDK results in downregulation of gene expression of enzymes of the C4 cycle, the Calvin cycle, and components of photochemistry. Furthermore, the loss of PPDK did not change Kranz anatomy, indicating that this metabolic defect in the C4 cycle did not impinge on the morphological differentiation of C4 characters. However, sugar metabolism and nitrogen utilization were altered in the mutants. An interaction between light intensity and genotype was also detected from transcriptome profiling, suggesting altered transcriptional and metabolic responses to environmental and endogenous signals in the PPDK mutants.

Photosynthetic responses to chilling in a chilling-tolerant and chilling-sensitive Miscanthus hybrid.

Miscanthus is a C4 perennial grass being developed for bioenergy production in temperate regions where chilling events are common. To evaluate chilling effects on Miscanthus, we assessed the processes controlling net CO2 assimilation rate (A) in Miscanthus x giganteus (M161) and a chilling-sensitive Miscanthus hybrid (M115) before and after a chilling treatment of 12/5 °C. The temperature response of A and maximum Rubisco activity in vitro were identical below 20 °C in chilled and unchilled M161, demonstrating Rubisco capacity limits or co-limits A at cooler temperatures. By contrast, A in M115 decreased at all measurement temperatures after growth at 12/5 °C. Rubisco activity in vitro declined in proportion to the reduction in A in chilled M115 plants, indicating Rubisco capacity is responsible in part for the decline in A. Pyruvate orthophosphate dikinase activities were also reduced by the chilling treatment when assayed at 28 °C, indicating this enzyme may also contribute to the reduction in A in M115. The maximum extractable activities of PEPCase and NADP-ME remained largely unchanged after chilling. The carboxylation efficiency of the C4 cycle was depressed in both genotypes to a similar extent after chilling. ΦP :ΦCO2 remained unchanged in both genotypes indicating the C3 and C4 cycles decline equivalently upon chilling.

Reduction of pyruvate orthophosphate dikinase activity is associated with high temperature-induced chalkiness in rice grains

Global warming affects both rice (Oryza sativa) yields and grain quality. Rice chalkiness due to high temperature during grain filling would lower the grain quality. The biochemical and molecular mechanisms responsible for the increased occurrence of chalkiness under high temperature are not fully understood. Previous research suggested that cytosolic pyruvate orthophosphate dikinase (cyPPDK, EC 2.7.9.1) in rice modulates carbon metabolism. The objective of this study was to determine the relationship between cyPPDK and high temperature-induced chalkiness. High temperature treatments were applied during the grain filling of two rice cultivars (9311 and TXZ-25) which had different sensitivity of chalkiness to high temperature. Chalkiness was increased significantly under high temperature treatment, especially for TXZ-25. A shortened grain filling duration and a decreased grain weight in both cultivars were caused by high temperature treatment. A reduction in PPDK activities due to high temperature was observed during the middle and late grain filling periods, accompanied by down regulated cyPPDK mRNA and protein levels. The temperature effects on the developmental regulation of PPDK activity were confirmed at transcription, translation and post-translational levels. PPDK activities were insensitive to variation in PPDK levels, suggesting the rapid phosphorylation mechanism of this protein. The two varieties showed similar responses to the high temperature treatment in both PPDK activities and chalkiness. We concluded that high temperature-induced chalkiness was associated with the reduction of PPDK activity.

Arbuscular mycorrhizal fungi enhances salinity tolerance of Panicum turgidum Forssk by altering photosynthetic and antioxidant pathways

Present experiments were conducted to assess the response of Panicum turgidum to salinity and possible role of arbuscular mycorrhizal fungi (AMF) in enhancing the salt tolerance. The activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), glutathione reductase (GR) and compatible solutes were increased by salt stress and were further enhanced by AMF inoculation. Hydrogen peroxide and malonaldehyde content increased in salt-stressed plants while a reduction was observed due to AMF inoculation. Salt-stressed plants showed higher activities of pyruvate orthophosphate dikinase (PPDK), phosphoenolpyruvate carboxylase (PEPC) and malate dehydrogenase as compared to control and AMF-inoculated plants. Salt stress caused significant decrease in phosphorous, potassium and calcium uptake but an increase in sodium uptake was observed. AMF alleviate salinity-induced negative impact on the plant growth and nutrient uptake by reducing the oxidative damage through strengthening of the antioxidant system.

Transgenic perturbation of the decarboxylation phase of Crassulacean acid metabolism alters physiology and metabolism but has only a small effect on growth.

Mitochondrial NAD-malic enzyme (ME) and/or cytosolic/plastidic NADP-ME combined with the cytosolic/plastidic pyruvate orthophosphate dikinase (PPDK) catalyze two key steps during light-period malate decarboxylation that underpin secondary CO(2) fixation in some Crassulacean acid metabolism (CAM) species. We report the generation and phenotypic characterization of transgenic RNA interference lines of the obligate CAM species Kalanchoë fedtschenkoi with reduced activities of NAD-ME or PPDK. Transgenic line rNAD-ME1 had 8%, and rPPDK1 had 5% of the wild-type level of activity, and showed dramatic changes in the light/dark cycle of CAM CO(2) fixation. In well-watered conditions, these lines fixed all of their CO(2) in the light; they thus performed C(3) photosynthesis. The alternative malate decarboxylase, NADP-ME, did not appear to compensate for the reduction in NAD-ME, suggesting that NAD-ME was the key decarboxylase for CAM. The activity of other CAM enzymes was reduced as a consequence of knocking out either NAD-ME or PPDK activity, particularly phosphoenolpyruvate carboxylase (PPC) and PPDK in rNAD-ME1. Furthermore, the circadian clock-controlled phosphorylation of PPC in the dark was reduced in both lines, especially in rNAD-ME1. This had the consequence that circadian rhythms of PPC phosphorylation, PPC kinase transcript levels and activity, and the classic circadian rhythm of CAM CO(2) fixation were lost, or dampened toward arrhythmia, under constant light and temperature conditions. Surprisingly, oscillations in the transcript abundance of core circadian clock genes also became arrhythmic in the rNAD-ME1 line, suggesting that perturbing CAM in K. fedtschenkoi feeds back to perturb the central circadian clock.

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.

Pyramiding expression of maize genes encoding phosphoenolpyruvate carboxylase (PEPC) and pyruvate orthophosphate dikinase (PPDK) synergistically improve the photosynthetic characteristics of transgenic wheat

Using particle bombardment transformation, we introduced maize pepc cDNA encoding phosphoenolpyruvate carboxylase (PEPC) and ppdk cDNA encoding pyruvate orthophosphate dikinase (PPDK) into the C3 crop wheat to generate transgenic wheat lines carrying cDNA of pepc (PC lines), ppdk (PK lines) or both (PKC lines). The integration, transcription, and expression of the foreign genes were confirmed by Southern blot, Real-time quantitative reverse transcription PCR (Q-RT-PCR), and Western blot analysis. Q-RT-PCR results indicated that the average relative expression levels of pepc and ppdk in the PKC lines reached 10 and 4.6, respectively, compared to their expressions in untransformed plants (set to 1). The enzyme activities of PEPC and PPDK in the PKC lines were 4.3- and 2.1-fold higher, respectively, than in the untransformed control. The maximum daily net photosynthetic rates of the PKC, PC, and PK lines were enhanced by 26.4, 13.3, and 4.5%, respectively, whereas the diurnal accumulations of photosynthesis were 21.3, 13.9, and 6.9%, respectively, higher than in the control. The Fv/Fm of the transgenic plants decreased less than in the control under high temperature and high light conditions (2 weeks after anthesis), suggesting that the transgenic wheat transports more absorbed light energy into a photochemical reaction. The exogenous maize C4-specific pepc gene was more effective than ppdk at improving the photosynthetic performance and yield characteristics of transgenic wheat, while the two genes showed a synergistic effect when they were transformed into the same genetic background, because the PKC lines exhibited improved photosynthetic and physiological traits.

Activities of principal photosynthetic enzymes in green macroalga Ulva linza: functional implication of C4 pathway in CO2 assimilation

The green-tide-forming macroalga Ulva linza was profiled by transcriptome sequencing to ascertain whether the alga carries both C3 and C4 photosynthesis genes. The key enzymes involved in C4 metabolism including pyruvate orthophosphate dikinase (PPDK), phosphoenolpyruvate carboxylase (PEPC), and phosphoenolpyruvate carboxykinase (PCK) were found. When measured under normal and different stress conditions, expression of rbcL was higher under normal conditions and lower under the adverse conditions, whereas that of PPDK was higher under some adverse conditions, namely desiccation, high salinity, and low salinity. Both ribulose-1, 5-biphosphate carboxylase (RuBPCase) and PPDK were found to play a role in carbon fixation, with significantly higher PPDK activity across the stress conditions. These results suggest that elevated PPDK activity alters carbon metabolism in U. linza leading to partial operation of the C4 carbon metabolism, a pathway that, under stress conditions, probably contributes to the hardy character of U. linza and thus to its wide distribution.

Elucidating the controlling role of the pyruvate phosphate dikinase regulatory protein on C4 photosynthesis

Pyruvate phosphate dikinase (PPDK) is a C4 photosynthetic pathway enzyme that converts Pyr to PEP, the substrate for CO2 fixation in C4 leaves. PPDK activity is regulated in response to light intensity to ensure the PEP synthesis rate is in balance with the amount of light energy incident on the leaf. Light regulation of PPDK occurs by reversible phosphorylation of an active‐site Thr. A bifunctional protein kinase/phosphotransferase, the PPDK regulatory protein (PDRP), catalyzes this reversible phosphorylation. Although the biochemical properties of PDRP are well known, its role in overall C4 cycle regulation in leaves is unclear. Our approach for resolving this question entails creating PDRP‐RNAi transgenic knockdown maize lines for assessing the interdependence of C4 PS and PDRP>;PPDK regulation. The first phase of the study involved isolation of homozygous PDRP knockdown T2 maize lines. This was accomplished using PCR and qPCR based screen. A total of 22/134 T2 plants were found to be homozygous for the PDRP‐RNAi transgene. A subset of these T2 plants was analyzed for leaf PDRP transcript abundance via Taqman based qPCR assay. This revealed a 5–50% suppression of PDRP transcript vs. Wt. Western blot analysis of leaf extracts showed a corresponding decrease in PDRP protein. Lines showing severe PDRP suppression developed chlorotic leaves and failed to reach maturity, suggesting PDRP is required for optimal C4 PS.

Relationships Between C4 Enzyme Activities and Yield in Soybeans (Glycine max (L.) Merr.)

To study the relationships between C4 enzyme activities and yield, C4 enzyme activities (phosphoenolpyruvate carboxylase (PEPCase), NADP-malate dehydrogenase (NADP-MDH), NADP-malic enzyme (NADP-ME), and pyruvate phosphate dikinase (PPDK)) in different organs of ten soybean cultivars with different yields were measured at different growth stages in China. The result showed that four enzyme activities in C4 pathway were obviously different among cultivars, especially PPDK activity was not detected in the leaves of Dongnong 1567 and Dongnong 1068 and the young leaves of Gongjiao 9107-1 and Dongnong 97–172, but there were weak activities in pod coats. The order of C4 enzyme activities is young leaves < old leaves < pod coats. The correlation coefficients between PEPCase activity and yield and between NADP-MDH activity at blooming stage and yield were 0.6979 and 0.6565, respectively, and both reached the significant level (5%), and PEPCase activity kept significant positive correlation with plant photosynthetic rate. There was a negative correlation between NADP-ME activity and yield, and no correlation was found between PPDK activity and yield.

The role of C4 metabolism in the marine diatom Phaeodactylum tricornutum

Summary Diatoms are important players in the global carbon cycle. Their apparent photosynthetic affinity for ambient CO2 is much higher than that of ribulose 1,5‐bisphosphate carboxylase/oxygenase (Rubisco), indicating that a CO2‐concentrating mechanism (CCM) is functioning. However, the nature of the CCM, a biophysical or a biochemical C4, remains elusive. Although 14C labeling experiments and presence of complete sets of genes for C4 metabolism in two diatoms supported the presence of C4, other data and predicted localization of the decarboxylating enzymes, away from Rubisco, makes this unlikely. We used RNA‐interference to silence the single gene encoding pyruvate‐orthophosphate dikinase (PPDK) in Phaeodactylum tricornutum, essential for C4 metabolism, and examined the photosynthetic characteristics. The mutants possess much lower ppdk transcript and PPDK activity but the photosynthetic K1/2 (CO2) was hardly affected, thus clearly indicating that the C4 route does not serve the purpose of raising the CO2 concentration in close proximity of Rubisco in P. tricornutum. The photosynthetic Vmax was slightly reduced in the mutant, possibly reflecting a metabolic constraint that also resulted in a larger lipid accumulation. We propose that the C4 metabolism does not function in net CO2 fixation but helps the cells to dissipate excess light energy and in pH homeostasis.

Evidence of coexistence of C₃ and C₄ photosynthetic pathways in a green-tide-forming alga, Ulva prolifera.

Ulva prolifera, a typical green-tide-forming alga, can accumulate a large biomass in a relatively short time period, suggesting that photosynthesis in this organism, particularly its carbon fixation pathway, must be very efficient. Green algae are known to generally perform C3 photosynthesis, but recent metabolic labeling and genome sequencing data suggest that they may also perform C4 photosynthesis, so C4 photosynthesis might be more wide-spread than previously anticipated. Both C3 and C4 photosynthesis genes were found in U. prolifera by transcriptome sequencing. We also discovered the key enzymes of C4 metabolism based on functional analysis, such as pyruvate orthophosphate dikinase (PPDK), phosphoenolpyruvate carboxylase (PEPC), and phosphoenolpyruvate carboxykinase (PCK). To investigate whether the alga operates a C4-like pathway, the expression of rbcL and PPDK and their enzyme activities were measured under various forms and intensities of stress (differing levels of salinity, light intensity, and temperature). The expression of rbcL and PPDK and their enzyme activities were higher under adverse circumstances. However, under conditions of desiccation, the expression of rbcL and ribulose-1, 5-biphosphate carboxylase (RuBPCase) activity was lower, whereas that of PPDK was higher. These results suggest that elevated PPDK activity may alter carbon metabolism and lead to a partial operation of C4-type carbon metabolism in U. prolifera, probably contributing to its wide distribution and massive, repeated blooms in the Yellow Sea.

Is expression of C4 PPDK and its regulator, the PPDK regulatory protein co‐regulated by light intensity in maize leaves?

Pyruvate phosphate dikinase (PPDK) is a cardinal enzyme of the C4 photosynthetic pathway that catalyzes the regeneration of the primary CO2 acceptor, PEP. In C4 leaves, PPDK activity is regulated to match the level of light incident on the leaf. The bifunctional PPDK regulatory protein (PDRP) catalyzes this up/‐ down regulation by reversible phosphorylation of a PPDK active‐site Thr, with the enzyme inactive in the phosphorylated state. PDRP also functions as the light‐level sensory component of the regulation cycle. To uncover how these two proteins may be co‐regulated at the molecular level in maize leaves we are assessing changes in the transcript and polypeptide abundance during high to low light dynamic (non developmental) acclimation. Initial results indicate that PDRP is a very low abundance mRNA being some eight orders of magnitude lower than the transcript level of its target enzyme. More focused experiments on high light to low light effects on PPDK and PDRP gene transcript level and polypeptide level are currently in progress. The results will be summarized with respect to how molecular expression of the two interrelated C4 pathway genes are coordinated for optimizing photosynthetic response to short‐term changes in light intensity. This research was supported by: US National Science Foundation Grant No. IOS‐ 0642190.

Adaptation responses in C4 photosynthesis of maize under salinity

The effect of salinity on C4 photosynthesis was examined in leaves of maize, a NADP-malic enzyme (NADP-ME) type C4 species. Potted plants with the fourth leaf blade fully developed were treated with 3% NaCl solution for 5d. Under salt treatment, the activities of pyruvate orthophosphate dikinase (PPDK), phosphoenolpyruvate carboxylase (PEPCase), NADP-dependent malate dehydrogenase (NADP-MDH) and NAD-dependent malate dehydrogenase (NAD-MDH), which are derived mainly from mesophyll cells, increased, whereas those of NADP-ME and ribulose-1,5-bisphosphate carboxylase, which are derived mainly from bundle sheath cells (BSCs), decreased. Immunocytochemical studies by electron microscopy revealed that PPDK protein increased, while the content of ribulose-1,5-bisphosphate carboxylase/oxygenase protein decreased under salinity. In salt-treated plants, the photosynthetic metabolites malate, pyruvate and starch decreased by 40, 89 and 81%, respectively. Gas-exchange analysis revealed that the net photosynthetic rate, the transpiration rate, stomatal conductance (gs) and the intercellular CO2 concentration decreased strongly in salt-treated plants. The carbon isotope ratio (δ13C) in these plants was significantly lower than that in control. These findings suggest that the decrease in photosynthetic metabolites under salinity was induced by a reduction in gas-exchange. Moreover, in addition to the decrease in gs, the decrease in enzyme activities in BSCs was responsible for the decline of C4 photosynthesis. The increase of PPDK, PEPCase, NADP-MDH, and NAD-MDH activities and the decrease of NADP-ME activity are interpreted as adaptation responses to salinity.

In silico analysis and molecular dissection of the PPDK:PDRP interaction

Pyruvate phosphate dikinase (PPDK) is a key bidirectional enzyme of the C4/CAM photosynthetic pathway that rejuvenates PEP by transferring a phosphate from ATP to a pyruvate. This enzyme is reversibly phosphorylated at Thr456 by the PPDK regulatory protein (PDRP); mediating light/dark regulation of C4/CAM PPDK activity. Attempts to elucidate the tertiary structure of PDRP have been wholly disappointing although previous research has established the mechanism by which PDRP regulates PPDK. Since nothing is known about the 3-D structure of PDRP, we utilized an in silico analysis of residues surrounding the highly conserved 455–462 region of PPDK to identify potentially interacting residues. Based on this analysis, point mutations of PPDK were performed in vivo and mutant enzyme with functional PPDK activity but inhibited PDRP regulation were recovered. Mutant PPDK was compared to wild-type PPDK for enzymatic efficacy and then assayed for the degree of PPDK phosphorylation by PDRP. Using this approach, we will provide novel insights into the structural basis of PDRP catalysis. This work was supported by U.S. National Science Foundation Grant No. IOS-0642190.

Pyrophosphate as a central energy carrier in the hydrogen-producing extremely thermophilic Caldicellulosiruptor saccharolyticus

The role of inorganic pyrophosphate (PPi) as an energy carrier in the central metabolism of the extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus was investigated. In agreement with its annotated genome sequence, cell extracts were shown to exhibit PPi-dependent phosphofructokinase and pyruvate phosphate dikinase activity. In addition, membrane-bound pyrophosphatase activity was demonstrated, while no significant cytosolic pyrophosphatase activity was detected. During the exponential growth phase, high PPi levels (approximately 4 +/- 2 mM) and relatively low ATP levels (0.43 +/- 0.07 mM) were found, and the PPi/ATP ratio decreased 13-fold when the cells entered the stationary phase. Pyruvate kinase activity appeared to be allosterically affected by PPi. Altogether, these findings suggest an important role for PPi in the central energy metabolism of C. saccharolyticus.

Cool C4 Photosynthesis: Pyruvate Pi Dikinase Expression and Activity Corresponds to the Exceptional Cold Tolerance of Carbon Assimilation inMiscanthus×giganteus

The bioenergy feedstock grass Miscanthus x giganteus is exceptional among C(4) species for its high productivity in cold climates. It can maintain photosynthetically active leaves at temperatures 6 degrees C below the minimum for maize (Zea mays), which allows it a longer growing season in cool climates. Understanding the basis for this difference between these two closely related plants may be critical in adapting maize to colder weather. When M. x giganteus and maize grown at 25 degrees C were transferred to 14 degrees C, light-saturated CO(2) assimilation and quantum yield of photosystem II declined by 30% and 40%, respectively, in the first 48 h in these two species. The decline continued in maize but arrested and then recovered partially in M. x giganteus. Within 24 h of the temperature transition, the pyruvate phosphate dikinase (PPDK) protein content per leaf area transiently declined in M. x giganteus but then steadily increased, such that after 7 d the enzyme content was significantly higher than in leaves growing in 25 degrees C. By contrast it declined throughout the chilling period in maize leaves. Rubisco levels remained constant in M. x giganteus but declined in maize. Consistent with increased PPDK protein content, the extractable PPDK activity per unit leaf area (V(max)(,ppdk)) in cold-grown M. x giganteus leaves was higher than in warm-grown leaves, while V(max,ppdk) was lower in cold-grown than in warm-grown maize. The rate of light activation of PPDK was also slower in cold-grown maize than M. x giganteus. The energy of activation (E(a)) of extracted PPDK was lower in cold-grown than warm-grown M. x giganteus but not in maize. The specific activities and E(a) of purified recombinant PPDK from M. x giganteus and maize cloned into Escherichia coli were similar. The increase in PPDK protein in the M. x giganteus leaves corresponded to an increase in PPDK mRNA level. These results indicate that of the two enzymes known to limit C(4) photosynthesis, increase of PPDK, not Rubisco content, corresponds to the recovery and maintenance of photosynthetic capacity. Functionally, increased enzyme concentration is shown to increase stability of M. x giganteus PPDK at low temperature. The results suggest that increases in either PPDK RNA transcription and/or the stability of this RNA are important for the increase in PPDK protein content and activity in M. x giganteus under chilling conditions relative to maize.

Relationships Between Quantum Yield for CO2 Assimilation, Activity of Key Enzymes and CO2 Leakiness in Amaranthus cruentus, a C4 Dicot, Grown in High or Low Light

In C(4) photosynthesis, a part of CO(2) fixed by phosphoenolpyruvate carboxylase (PEPC) leaks from the bundle-sheath cells. Because the CO(2) leak wastes ATP consumed in the C(4) cycle, the leak may decrease the efficiency of CO(2) assimilation. To examine this possibility, we studied the light dependence of CO(2) leakiness (phi), estimated by the concurrent measurements of gas exchange and carbon isotope discrimination, initial activities of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) and pyruvate, orthophosphate dikinase (PPDK), the phosphorylation state of PEPC and the CO(2) assimilation rate using leaves of Amaranthus cruentus (NAD-malic enzyme subtype, dicot) plants grown in high light (HL) and low light (LL). phi was constant at photon flux densities (PFDs) >200 micromol m(-2) s(-1) and was around 0.3. At PFDs <150 micromol m(-2) s(-1), phi increased markedly as PFD decreased. At 40 micromol m(-2) s(-1), phi was 0.76 in HL and 0.55 in LL leaves, indicating that the efficiency of CO(2) assimilation at low PFD was greater in LL leaves. The activities of Rubisco and PPDK, and the phosphorylated state of PEPC all decreased as PFD decreased. Theoretical calculations with a mathematical model clearly showed that the increase in phi with decreasing PFD contributed to the decrease in the CO(2) assimilation rate. It was also shown that the 'conventional' quantum yield of photosynthesis obtained by fitting the straight line to the light response curve of the CO(2) assimilation rate at the low PFD region is seriously overestimated. Ecological implications of the increase in phi in LL are discussed.

Oxidations of various substrates and effects of the inhibitors on purified mitochondria isolated from Kalanchoë pinnata

KalanchoĂŤ pinnata mitochondria readily oxidized succinate, malate, NADH, and NADPH at high rates and coupling. The highest respiration rates usually were observed in the presence of succinate. The high rate of malate oxidation was observed at pH 6.8 with thiamine pyrophosphate where both malic enzyme (ME) and pyruvate dehydrogenase were activated. In CAM phase III of K. pinnata mitochondria, both ME and malate dehydrogenase (MDH) simultaneously contributed to metabolism of malate. However, ME played a main function: malate was oxidized via ME to produce pyruvate and CO2 rather than via MDH to produce oxalacetate (OAA). Cooperative oxidation of two or three substrates was accompanied with the dramatic increase in the total respiration rates. Our results showed that the alternative (Alt) pathway was more active in malate oxidation at pH 6.8 with CoA and NAD+ where ME operated and was stimulated, indicating that both ME and Alt pathway were related to malate decarboxylation during the light. In K. pinnata mitochondria, NADH and NADPH oxidations were more sensitive with KCN than that with succinate and malate oxidations, suggesting that these oxidations were engaged to cytochrome pathway rather than to Alt pathway and these capacities would be desirable to supply enough energy for cytosol pyruvate orthophosphate dikinase activity.