NADP-malic Enzyme Research Articles

Regulatory Network of the Late-Recruited Primary Decarboxylase C4NADP-ME in Sugarcane.

In agronomically important C4 grasses, efficient CO2 delivery to Rubisco is facilitated by NADP-malic enzyme (C4NADP-ME), which decarboxylates malate in bundle sheath cells. However, understanding the molecular regulation of the C4NADP-ME gene in sugarcane (Saccharum spp.) is hindered by its complex genetic background. Enzymatic activity assays demonstrated that decarboxylation in sugarcane Saccharum spontaneum predominantly relies on the NADP-ME pathway, similar to sorghum (Sorghum bicolor) and maize (Zea mays). Comparative genomics analysis revealed the recruitment of eight core C4 shuttle genes, including C4NADP-ME (SsC4NADP-ME2), in the C4 pathway of sugarcane. Contrasting to sorghum and maize, the expression of SsC4NADP-ME2 in sugarcane is regulated by different transcription factors (TFs). We propose a gene regulatory network for SsC4NADP-ME2, involving candidate TFs identified through gene co-expression analysis and yeast one-hybrid experiment. Among these, ABA INSENSITIVE5 (ABI5) was validated as the predominant regulator of SsC4NADP-ME2 expression, binding to a G-box within its promoter region. Interestingly, the core element ACGT within the regulatory G-box was conserved in sugarcane, sorghum, maize, and rice (Oryza sativa), suggesting an ancient regulatory code utilized in C4 photosynthesis. This study offers insights into SsC4NADP-ME2 regulation, crucial for optimizing sugarcane as a bioenergy crop.

Stomatal response to VPD in C4 plants with different biochemical sub-pathways.

C4 NAD-malic enzyme (NAD-ME) species occurs in drier regions and exhibit different drought responses compared to C4 NADP-malic enzyme (NADP-ME) species. However, a physiological mechanism explaining the geographical discrepancies remains uncertain. This study examined gas exchange patterns that might explain different distributions observed between two subtypes of C4 photosynthesis. We measured the response of leaf gas exchange to vapour pressure deficit (VPD) and CO2 in plants from six distinct C4 clades having closely related NAD-ME and NADP-ME species using a Li-Cor 6400 gas exchange system. We found that NAD-ME species exhibited greater relative reductions in stomatal conductance with increases in VPD than NADP-ME species but observed no consistent subtype differences in C4 cycle activity as indicated by the initial slope of the A response to intercellular CO2 concentration. Based on these results, we hypothesise the greater response of gs to increasing VPD may enable NAD-ME plants to outperform NADP-ME plants in hot, dry environments where VPD is normally high.

NADP-malic Enzyme OsNADP-ME2 Modulates Plant Height Involving in Gibberellin Signaling in Rice

Plants NADP-malic enzymes (NADP-MEs) act as a class of oxidative decarboxylase to mediate malic acid metabolism in organisms. Despite NADP-MEs have been demonstrated to play pivotal roles in regulating diverse biological processes, the role of NADP-MEs involving in plant growth and development remains rarely known. Here, we characterized the function of rice cytosolic OsNADP-ME2 in regulating plant height. The results showed that RNAi silencing and knock-out of OsNADP-ME2 in rice results in a dwarf plant structure, associating with significant expression inhibition of genes involving in phytohormone Gibberellin (GA) biosynthesis and signaling transduction, but with up-regulation for the expression of GA signaling suppressor SLR1. The accumulation of major bioactive GA1, GA4 and GA7 are evidently altered in RNAi lines, and exogenous GA treatment compromises the dwarf phenotype of OsNADP-ME2 RNAi lines. RNAi silencing of OsNADP-ME2 also causes the reduction of NADP-ME activity associating with decreased production of pyruvate. Thus, our data revealed a novel function of plant NADP-MEs in modulation of rice plant height through regulating bioactive GAs accumulation and GA signaling, and provided a valuable gene resource for rice plant architecture improvement.

The Impact of Lighting Treatments on the Biosynthesis of Phenolic Acids in Black Wheat Seedlings.

Light, as a crucial environmental determinant, profoundly influences the synthesis of secondary metabolites in plant metabolism. This study investigated the impacts of the red light combined with ultraviolet-A (UV-A) and ultraviolet-B (UV-B) treatments on phenolic acid biosynthesis in black wheat seedlings. The results demonstrate that the red light combined with UV-A and UV-B treatments significantly enhanced the levels of phenolic acids in black wheat seedlings, at 220.4 μg/seedling and 241.5 μg/seedling, respectively. The content of bound phenolic acids in black wheat seedlings increased by 36.0% under the UV-B treatment. The application of the UV-A/UV-B treatments markedly enhanced the activities of phenylalanine ammonia-lyase, 4-coumarate CoA ligase, and cinnamate 4-hydroxylase in black wheat seedlings while also promoting the expression levels of genes related to phenolic acid synthesis. The expression levels of fructose-1,6-bisphosphate aldolase and NADP-malic enzyme related to photosynthesis were significantly upregulated. This resulted in an augmentation in the chlorophyll content, thereby enhancing photosynthesis in black wheat seedlings. Nevertheless, the UV-A and UV-B treatments also had a significant constraining effect on the growth and development of black wheat seedlings. In addition, the UV-A and UV-B treatments increased the activity and gene expression levels of antioxidant enzymes while significantly increasing the contents of total flavonoids and anthocyanins, activating the antioxidant system. The findings reveal that light-source radiation serves as an effective method for promoting the biosynthesis of phenolic acids in black wheat seedlings.

Enhancing Photosynthesis and Plant Productivity through Genetic Modification.

Enhancing crop photosynthesis through genetic engineering technologies offers numerous opportunities to increase plant productivity. Key approaches include optimizing light utilization, increasing cytochrome b6f complex levels, and improving carbon fixation. Modifications to Rubisco and the photosynthetic electron transport chain are central to these strategies. Introducing alternative photorespiratory pathways and enhancing carbonic anhydrase activity can further increase the internal CO2 concentration, thereby improving photosynthetic efficiency. The efficient translocation of photosynthetically produced sugars, which are managed by sucrose transporters, is also critical for plant growth. Additionally, incorporating genes from C4 plants, such as phosphoenolpyruvate carboxylase and NADP-malic enzymes, enhances the CO2 concentration around Rubisco, reducing photorespiration. Targeting microRNAs and transcription factors is vital for increasing photosynthesis and plant productivity, especially under stress conditions. This review highlights potential biological targets, the genetic modifications of which are aimed at improving photosynthesis and increasing plant productivity, thereby determining key areas for future research and development.

Modulation of secondary metabolism and redox regulation by exogenously applied glutathione improves the shelf life of Capsicum annuum L. fruit

Role of redox homeostasis in fruit ripening of Capsicum annuum L. with oxidative metabolism was studied. The research aims the ability to reduce agents during postharvest storage on fruit for delayed ripening with the regulation of oxidative stress. Thus, we applied 10 mM reduced glutathione (GSH) to fruit as pretreatment followed by 1 mM hydrogen peroxide (H2O2) as ripening-inducing treatment and observed during 7 days of storage at 25 °C. A decrease in total soluble solid and firmness under H2O2, was increased while dehydration in tissue was decreased by GSH pretreatment. Glutathione regulated the turnover of organic acids to reducing sugars with higher activity of NADP malic enzyme that sustained the fruit coat photosynthesis through chlorophyll fluorescence, pigment composition, and photosystem II activity. Malondialdehyde accumulation was inversely correlated with GSH content and antioxidative enzyme activity that reduced loss of cell viability. Conclusively, regulation of oxidative stress with GSH may be effective in the extension of shelf life under postharvest storage.

The operation of PEPCK increases light harvesting plasticity in C4 NAD-ME and NADP-ME photosynthetic subtypes: A theoretical study.

The repeated emergence of NADP-malic enzyme (ME), NAD-ME and phosphoenolpyruvatecarboxykinase (PEPCK) subtypes of C4 photosynthesis are iconic examples of convergent evolution, which suggests that these biochemistries do not randomly assemble, but are instead specific adaptations resulting from unknown evolutionary drivers. Theoretical studies that are based on the classic biochemical understanding have repeatedly proposed light-use efficiency as a possible benefit of the PEPCK subtype. However, quantum yield measurements do not support this idea. We explore this inconsistency here via an analytical model that features explicit descriptions across a seamless gradient between C4 biochemistries to analyse light harvesting and dark photosynthetic metabolism. Our simulations show that the NADP-ME subtype, operated by the most productive crops, is the most efficient. The NAD-ME subtype has lower efficiency, but has greater light harvesting plasticity (the capacity to assimilate CO2 in the broadest combination of light intensity and spectral qualities). In both NADP-ME and NAD-ME backgrounds, increasing PEPCK activity corresponds to greater light harvesting plasticity but likely imposed a reduction in photosynthetic efficiency. We draw the first mechanistic links between light harvesting and C4 subtypes, providing the theoretical basis for future investigation.

Molecular regulatory mechanisms of staminate strobilus development and dehiscence in Torreya grandis.

Gymnosperms are mostly dioecious, and their staminate strobili undergo a longer developmental period than those of angiosperms. However, the underlying molecular mechanisms remain unclear. This study aimed to identify key genes and pathways involved in staminate strobilus development and dehiscence in Torreya grandis. Through weighted gene co-expression network analysis (WGCNA), we identified fast elongation-related genes enriched in carbon metabolism and auxin signal transduction, whereas dehiscence-related genes were abundant in alpha-linolenic acid metabolism and the phenylpropanoid pathway. Based on WGCNA, we also identified PHYTOCHROME-INTERACTING FACTOR4 (TgPIF4) as a potential regulator for fast elongation of staminate strobilus and 2 WRKY proteins (TgWRKY3 and TgWRKY31) as potential regulators for staminate strobilus dehiscence. Multiple protein-DNA interaction analyses showed that TgPIF4 directly activates the expression of TRANSPORT INHIBITOR RESPONSE2 (TgTIR2) and NADP-MALIC ENZYME (TgNADP-ME). Overexpression of TgPIF4 significantly promoted staminate strobilus elongation by elevating auxin signal transduction and pyruvate content. TgWRKY3 and TgWRKY31 bind to the promoters of the lignin biosynthesis gene PHENYLALANINE AMMONIA-LYASE (TgPAL) and jasmonic acid metabolism gene JASMONATE O-METHYLTRANSFERASE (TgJMT), respectively, and directly activate their transcription. Overexpression of TgWRKY3 and TgWRKY31 in the staminate strobilus led to early dehiscence, accompanied by increased lignin and methyl jasmonate levels, respectively. Collectively, our findings offer a perspective for understanding the growth of staminate strobili in gymnosperms.

Enhanced drought tolerance and photosynthetic efficiency in Arabidopsis by overexpressing phosphoenolpyruvate carboxylase from a single-cell C4 halophyte Suaeda aralocaspica.

In crop genetic improvement, the introduction of C4 plants' characteristics, known for high photosynthetic efficiency and water utilization, into C3 plants has been a significant challenge. This study investigates the effects of the desert halophyte Suaeda aralocaspica SaPEPC1 gene from a single-cell C4 photosythetic pathway, on drought resistance and photosynthetic performance in Arabidopsis. We used transgenic Arabidopsis with Zea mays ZmPEPC1 from C4 plant with classic Kranz anatomical structure and Arabidopsis AtPEPC1 from C3 photosynthetic cycle plants as controls. The results demonstrated that C4 photosynthetic-type PEPCs could improve drought resistance in plants through stomatal closure, promoting antioxidant enzyme accumulation, and reducing reactive oxygen species (ROS) accumulation. Overexpression of SaPEPC1 was significantly more effective than ZmPEPC1 in enhancing drought tolerance. Notably, overexpressed SaPEPC1 significantly improved light saturation intensity, electron transport rate (ETR), photosynthetic rate (Pn), and photoprotection ability under intense light. Furthermore, overexpression SaPEPC1 or ZmPEPC1 enhanced the activity of key C4 photosynthetic enzymes, including phosphoenolpyruvate carboxylase (PEPC), pyruvate orthophosphate dikinase (PPDK) and NADP-malic enzyme (NADP-ME), and promoted photosynthetic product sugar accumulation. However, with AtPEPC1 overexpression showing no obvious improvement effect on drought and photosynthetic performance. Therefore, these results indicated that introducing C4-type PEPC into C3 plants can significantly enhance drought resistance and photosynthetic performance. However, SaPEPC1 from a single-cell C4 cycle plant exhibits more significant effect in ETR and PSII photosynthesis performance than ZmPEPC1 from a classical C4 anatomical structure plant, although the underlying mechanism requires further exploration.

Regulatory NADH dehydrogenase-like complex optimizes C4 photosynthetic carbon flow and cellular redox in maize.

C4 plants typically operate a CO2 concentration mechanism from mesophyll (M) cells into bundle sheath (BS) cells. NADH dehydrogenase-like (NDH) complex is enriched in the BS cells of many NADP-malic enzyme (ME) type C4 plants and is more abundant in C4 than in C3 plants, but to what extent it is involved in the CO2 concentration mechanism remains to be experimentally investigated. We created maize and rice mutants deficient in NDH function and then used a combination of transcriptomic, proteomic, and metabolomic approaches for comparative analysis. Considerable decreases in growth, photosynthetic activities, and levels of key photosynthetic proteins were observed in maize but not rice mutants. However, transcript abundance for many cyclic electron transport (CET) and Calvin-Benson cycle components, as well as BS-specific C4 enzymes, was increased in maize mutants. Metabolite analysis of the maize ndh mutants revealed an increased NADPH : NADP ratio, as well as malate, ribulose 1,5-bisphosphate (RuBP), fructose 1,6-bisphosphate (FBP), and photorespiration intermediates. We suggest that by optimizing NADPH and malate levels and adjusting NADP-ME activity, NDH functions to balance metabolic and redox states in the BS cells of maize (in addition to ATP supply), coordinating photosynthetic transcript abundance and protein content, thus directly regulating the carbon flow in the two-celled C4 system of maize.

Effects of 1-Methylcyclopropene Treatment on the Quality and Malic Acid Metabolism of ‘Xiangjiao’ Plum under Low-Temperature Storage

‘Xiangjiao’ plum (Prunus salicina Lindl.) is a stone fruit that is vulnerable to the chilling injury (CI) that is caused by low-temperature stress. The effects of 1-methylcyclopropene (1-MCP) and ethylene absorbent (EA) treatments on the fruit quality and malic acid metabolism of ‘Xiangjiao’ plum stored at 4 °C were compared in this study. Compared with the control check (CK) and EA treatment, fumigation with 1.0 mg·L−1 of 1-MCP for 24 h could more significantly maintain the sensory and physiological quality of the fruit, increase the activity of antioxidant enzymes, and prolong the storage time of plums. Furthermore, 1-MCP treatment can regulate the high expression of the tonoplast dicarboxylate transporter (tDT) and phosphoenolpyruvate carboxylase (PEPC) gene, regulate the high expression of the NAD-malate dehydrogenase (NAD-MDH) gene at the end of storage, and inhibit the expression of the NADP-malic enzyme (NADP-ME) gene. These changes resulted in increased NAD-MDH enzyme activity and decreased NADP-ME enzyme activity, which inhibited the degradation of malic acid that is caused by CI. As a result, 1-MCP can effectively maintain the storage quality of ‘Xiangjiao’ plum, reduce the loss of pleasant sour taste, and improve the edible flavor and commercial value of the fruit.

Coordination of leaf photosynthetic traits and chloroplast relocation of the C4 plant Setaria palmifolia

Setaria palmifolia (Poaceae), an NADP-malic enzyme type C4 grass, is distributed from the understory to the margins of forests. To reveal how this C4 plant acclimates to habitats with contrasting light regimes, we documented the behavior of chloroplast relocation and determined patterns of chloroplast relocation and photosynthetic characteristics in response to the light of S. palmifolia grown under different light regimes. Through the observation of leaf sections and measurement of leaf transmittance (T), we confirmed that mesophyll (M) cells of S. palmifolia performed avoidance and accumulation responses, resulting in an increase and decrease in T, respectively. Measurements of gas exchange revealed that the leaf photosynthetic characteristics of S. palmifolia were modulated by growth light. Shade-grown plants significantly increased dry-weight-based chlorophyll (Chl) contents and specific leaf area (SLA), indicating increased light capture. These plants, however, displayed a reduced light-saturated photosynthetic rate (Pmax) and light saturation point (LSP) compared to unshaded plants. The patterns of chloroplast relocation were closely aligned with the photosynthetic response. Shade-grown plants displayed significantly faster kinetics, a greater extent of chloroplast relocation, and lower incident light for the induction of the avoidance response than unshaded plants. The coordination patterns of chloroplast relocation and photosynthetic response suggest that chloroplast relocation in S. palmifolia complements its leaf photosynthetic responses to light availability. The ability to adjust its photosynthetic behavior and perform chloroplast relocation may enable this C4 species to thrive in habitats with various light conditions.

Overexpression of chloroplastic Zea mays NADP-malic enzyme (ZmNADP-ME) confers tolerance to salt stress in Arabidopsis thaliana.

The C4 plants photosynthesize better than C3 plants especially in arid environment. As an attempt to genetically convert C3 plant to C4, the cDNA of decarboxylating C4 type NADP-malic enzyme from Zea mays (ZmNADP-ME) that has lower Km for malate and NADP than its C3 isoforms, was overexpressed in Arabidopsis thaliana under the control of 35S promoter. Due to increased activity of NADP-ME in the transgenics the malate decarboxylation increased that resulted in loss of carbon skeletons needed for amino acid and protein synthesis. Consequently, amino acid and protein content of the transgenics declined. Therefore, the Chl content, photosynthetic efficiency (Fv/Fm), electron transport rate (ETR), the quantum yield of photosynthetic CO2 assimilation, rosette diameter, and biomass were lower in the transgenics. However, in salt stress (150mM NaCl), the overexpressers had higher Chl, protein content, Fv/Fm, ETR, and biomass than the vector control. NADPH generated in the transgenics due to increased malate decarboxylation, contributed to augmented synthesis of proline, the osmoprotectant required to alleviate the reactive oxygen species-mediated membrane damage and oxidative stress. Consequently, the glutathione peroxidase activity increased and H2O2 content decreased in the salt-stressed transgenics. The reduced membrane lipid peroxidation and lower malondialdehyde production resulted in better preservation, of thylakoid integrity and membrane architecture in the transgenics under saline environment. Our results clearly demonstrate that overexpression of C4 chloroplastic ZmNADP-ME in the C3 Arabidopsis thaliana, although decrease their photosynthetic efficiency, protects the transgenics from salinity stress.

2,4-D mediated moderation of aluminum tolerance in Salvinia molesta D. Mitch. with regards to bioexclusion and related physiological and metabolic changes

We examined the efficacy of 2,4-dichlorophenoxy acetic acid (2,4-D; 500 µM) in enhancing the potential of Salvinia species for tolerance to aluminum (Al) toxicity (240 and 480 µM, seven days). Salvinia showed better efficacy in removal of toxicity of Al by sorption mechanism with changes of bond energy shifting on cell wall residues and surface structure. Plants recorded tolerance to Al concentration (480 µM) when pretreated with 2,4-D through adjustment of relative water content, proline content, osmotic potential, and improved the pigment fluorescence for energy utilization under Al stress. Photosynthetic activities with regards to NADP-malic enzyme and malic dehydrogenase and sugar metabolism with wall and cytosolic invertase activities were strongly correlated with compatible solutes. A less membrane peroxidation and protein carbonylation had reduced ionic loss over the membrane that was studied with reduced electrolyte leakage with 2,4-D pretreated plants. Membrane stabilization was also recorded with higher ratio of K+ to Na+, thereby suggesting roles of 2,4-D in ionic balance. Better sustenance of enzymatic antioxidation with peroxidase and glutathione metabolism reduced reactive oxygen species accumulation and save the plant for oxidative damages. Moreover, gene polymorphism for antioxidant, induced by 2,4-D varied through Al concentrations would suggest an improved biomarker for tolerance. Collectively, analysis and discussion of plant’s responses assumed that auxin herbicide could be a potential phytoprotectant for Salvinia as well as improving the stability to Al toxicity and its bioremediation efficacy.

A complex network regulating malate contents during fruit ripening in climacteric fruits.

This article is a Commentary on Wang et al. (2023), 239: 1014–1034.

Regulation of NADP-Malic Enzyme Activity in Maize (Zea mays L.) under Salinity with Reference to Light and Darkness.

We revealed the functional characterization of C4-NADP-malic enzyme (NADP-ME), extracted and partially purified from maize (Zea mays L. cv. Kaveri 50). The leaf discs were previously activated under 1000-1200 µE m-2 s-1, incubated in bicarbonate (2.0 mM) solution, and subjected to salt stress (100 mM NaCl). Initially, salt stress was evident from the accumulations of proline, chlorophyll content, carbohydrate profile, and Hill activity influencing the C4 enzyme. Primarily, in illuminated tissues, the activity of the enzyme recorded a reduced trend through salinity irrespective of light and darkness compared to the control. On illumination, the kinetic parameters such as Vmax of the enzyme increased by 1.36-fold compared to in the dark under salinity whereas Km was decreased by 20% under the same condition. The extent of light induction was proportionate to limiting (0.01 mM) and saturated (4.0 mM) malate concentrations for enzyme activity. Moreover, the catalytic properties of the enzyme were also tested on concomitant responses to activator (citrate and succinate) and inhibitor (oxalate and pyruvate) residues. The sensitivity to light and dark effects was also tested for reducing agents such as dithiothreitol, suggesting the effect of the changes in redox on the regulatory properties of the enzyme. The ratio of enzyme activity under light and darkness in the presence or absence of a reducing agent was concomitantly increased with varying malate concentrations. At the molecular level, protein polymorphism of the enzyme represented minor variations in band intensities, however, not in numbers through salinity subjected to light and darkness. Therefore, salinity-induced changes in the decarboxylation reaction, evident by NADP-ME activity, may be based on the redox property of regulatory sites and sensitivity to light and darkness.

Epi-Brassinolide Regulates ZmC4 NADP-ME Expression through the Transcription Factors ZmbHLH157 and ZmNF-YC2

Maize is a main food and feed crop with great production potential and high economic benefits. Improving its photosynthesis efficiency is crucial for increasing yield. Maize photosynthesis occurs mainly through the C4 pathway, and NADP-ME (NADP-malic enzyme) is a key enzyme in the photosynthetic carbon assimilation pathway of C4 plants. ZmC4-NADP-ME catalyzes the release of CO2 from oxaloacetate into the Calvin cycle in the maize bundle sheath. Brassinosteroid (BL) can improve photosynthesis; however, its molecular mechanism of action remains unclear. In this study, transcriptome sequencing of maize seedlings treated with epi-brassinolide (EBL) showed that differentially expressed genes (DEGs) were significantly enriched in photosynthetic antenna proteins, porphyrin and chlorophyll metabolism, and photosynthesis pathways. The DEGs of C4-NADP-ME and pyruvate phosphate dikinase in the C4 pathway were significantly enriched in EBL treatment. Co-expression analysis showed that the transcription level of ZmNF-YC2 and ZmbHLH157 transcription factors was increased under EBL treatment and moderately positively correlated with ZmC4-NADP-ME. Transient overexpression of protoplasts revealed that ZmNF-YC2 and ZmbHLH157 activate C4-NADP-ME promoters. Further experiments showed ZmNF-YC2 and ZmbHLH157 transcription factor binding sites on the -1616 bp and -1118 bp ZmC4 NADP-ME promoter. ZmNF-YC2 and ZmbHLH157 were screened as candidate transcription factors mediating brassinosteroid hormone regulation of the ZmC4 NADP-ME gene. The results provide a theoretical basis for improving maize yield using BR hormones.

Two cyclic electron flows around photosystem I differentially participate in C4 photosynthesis.

C4 plants assimilate CO2 more efficiently than C3 plants because of their C4 cycle that concentrates CO2. However, the C4 cycle requires additional ATP molecules, which may be supplied by cyclic electron flow (CEF) around photosystem I. One CEF route, which depends on a chloroplast NADH dehydrogenase-like (NDH) complex, is suggested to be crucial for C4 plants despite the low activity in C3 plants. The other route depends on proton gradient regulation 5 (PGR5) and PGR5-like photosynthetic phenotype 1 (PGRL1) and is considered a major CEF route to generate the proton gradient across the thylakoid membrane in C3 plants. However, its contribution to C4 photosynthesis is still unclear. In this study, we investigated the contribution of the two CEF routes to the NADP-malic enzyme subtype of C4 photosynthesis in Flaveria bidentis. We observed that suppressing the NDH-dependent route drastically delayed growth and decreased the CO2 assimilation rate to approximately 30% of the wild-type rate. On the other hand, suppressing the PGR5/PGRL1-dependent route did not affect plant growth and resulted in a CO2 assimilation rate that was approximately 80% of the wild-type rate. Our data indicate that the NDH-dependent CEF substantially contributes to the NADP-malic enzyme subtype of C4 photosynthesis and that the PGR5/PGRL1-dependent route cannot complement the NDH-dependent route in F. bidentis. These findings support the fact that during C4 evolution, photosynthetic electron flow may have been optimized to provide the energy required for C4 photosynthesis.

Expression profile of genes associated with the accumulation of triacylglycerol (TAG) in Auxenochlorella protothecoides KP7 under alkaline pH and nitrogen starvation conditions

Identification of target genes related to lipid accumulation in microalgae can increase triacylglycerol production without affecting biomass production through genetic manipulation. This study is part of our previous study and aimed to determine the effects of nitrogen starvation, alkaline pH (pH 10), and a combination of these factors on the transcriptional profiles of eight genes potentially related to triacylglycerol accumulation in Auxenochlorella protothecoides KP7. As shown previously, triacylglycerol content in Auxenochlorella protothecoides KP7 reached the maximum level under combined stress conditions. In this study, the highest upregulation for the gene encoding phosphoenolpyruvate carboxylase (PEPC) was observed under both nitrogen starvation and combined stress. Nitrogen starvation alone resulted in a significant increase in the expression level of all genes except NADP-malic enzyme (MEchl), which is localized in the chloroplast. Compared with nitrogen starvation alone, the upregulation of pyruvate dehydrogenase complex E2 component (PDHC-E2), glycerol-3-phosphate dehydrogenase (GPDH), diacylglycerol acyltransferase 1 (DGAT1), and MEchl genes further increased under the combined stress. These results suggest that genes encoding the enzymes DGAT1, GPDH, ME, PEPC, and PDHC-E2 are associated with triacylglycerol accumulation and could be used as target genes for genetic modification of Auxenochlorella protothecoides and possibly other microalgal species.

Increased bundle-sheath leakiness of CO2 during photosynthetic induction shows a lack of coordination between the C4 and C3 cycles.

Use of a complete dynamic model of NADP-malic enzyme C4 photosynthesis indicated that, during transitions from dark or shade to high light, induction of the C4 pathway was more rapid than that of C3 , resulting in a predicted transient increase in bundle-sheath CO2 leakiness (ϕ). Previously, ϕ has been measured at steady state; here we developed a new method, coupling a tunable diode laser absorption spectroscope with a gas-exchange system to track ϕ in sorghum and maize through the nonsteady-state condition of photosynthetic induction. In both species, ϕ showed a transient increase to > 0.35 before declining to a steady state of 0.2 by 1500 s after illumination. Average ϕ was 60% higher than at steady state over the first 600 s of induction and 30% higher over the first 1500 s. The transient increase in ϕ, which was consistent with model prediction, indicated that capacity to assimilate CO2 into the C3 cycle in the bundle sheath failed to keep pace with the rate of dicarboxylate delivery by the C4 cycle. Because nonsteady-state light conditions are the norm in field canopies, the results suggest that ϕ in these major crops in the field is significantly higher and energy conversion efficiency lower than previous measured values under steady-state conditions.