Pyruvate Phosphate Dikinase Research Articles

Role of C4 photosynthetic enzyme isoforms in C3 plants and their potential applications in improving agronomic traits in crops.

As compared to C3, C4 plants have higher photosynthetic rates and better tolerance to high temperature and drought. These traits are highly beneficial in the current scenario of global warming. Interestingly, all the genes of the C4 photosynthetic pathway are present in C3 plants, although they are involved in diverse non-photosynthetic functions. Non-photosynthetic isoforms of carbonic anhydrase (CA), phosphoenolpyruvate carboxylase (PEPC), malate dehydrogenase (MDH), the decarboxylating enzymes NAD/NADP-malic enzyme (NAD/NADP-ME), and phosphoenolpyruvate carboxykinase (PEPCK), and finally pyruvate orthophosphate dikinase (PPDK) catalyze reactions that are essential for major plant metabolism pathways, such as the tricarboxylic acid (TCA) cycle, maintenance of cellular pH, uptake of nutrients and their assimilation. Consistent with this view differential expression pattern of these non-photosynthetic C3 isoforms has been observed in different tissues across the plant developmental stages, such as germination, grain filling, and leaf senescence. Also abundance of these C3 isoforms is increased considerably in response to environmental fluctuations particularly during abiotic stress. Here we review the vital roles played by C3 isoforms of C4 enzymes and the probable mechanisms by which they help plants in acclimation to adverse growth conditions. Further, their potential applications to increase the agronomic trait value of C3 crops is discussed.

Morphological, photosynthetic, and CAM physiological responses of the submerged macrophyte Ottelia alismoides to light quality

Light quality strongly affect a great many physiological processes in terrestrial plants. Whatever, few studies have investigated how the submerged macrophytes response to different light quality. The present research aimed to understand how light quality affect morphogenesis, photosynthesis, as well as crassulacean acid metabolism (CAM) operation during the growth of Ottelia alismoides , a freshwater facultative CAM macrophyte. The plants were grown under three light qualities of white (W), red (R) and blue (B) lights with same light intensity of 100 μmol m −2 s −1 . Leaf development, leaf anatomic characteristics, photosynthetic traits, as well as CAM metabolism were determined on the newly produced leaves during light treatment. Monochromatic R stimulated leaf blade and petiole elongation, leaf area and dry weight, but reduced leaf thickness, negatively influenced chlorophyll content and Fv/Fm. On the contrary, B- grown plants were dwarf and with compact size as well as lower leaf dry weight. The content of photosynthetic product starch under B was significantly lower as compared with R, moreover, combined with the analysis of ultrastructural characteristics of epidermal and mesophyll chloroplasts, it showed that B inhibited starch to be translocated out of epidermal chloroplasts. Furthermore, both R- and B- grown plants exhibited obvious diurnal acidity fluctuation comparable to W- grown plants, and had high activity of enzymes including ribulose-bisphosphate carboxylase/oxygenase (Rubisco), phosphoenolpyruvate carboxylase (PEPC) and pyruvate phosphate dikinase (PPDK) that required for CAM cycle, indicating that CAM was present in both R- and B- grown plants and monochromatic R and B did not affect CAM activity. In conclusion, monochromatic red and blue light differentially played their special roles in regulating leaf growth and morphogenesis, as well as photosynthetic metabolism in O. alismoides . • O. alismoides displayed strong morphological plasticity to light quality. • Leaf elongation was promoted by red light but inhibited by blue light. • Red light favoured O. alismoides leaf development and photosynthesis. • Blue light inhibited translocation of starch out of epidermal chloroplasts. • O. alismoides exhibited comparable CAM activity under white, red and blue light.

Biochemical analysis of a novel allele of the OsPPDKB gene associated with floury endosperm

Here we isolated and characterized floTR1, a new floury endosperm rice mutant. Whole genome sequence and analysis of a cleaved amplified polymorphic sequence marker found that the FloTR1 gene encodes a pyruvate orthophosphate dikinase B (OsPPDKB). The floTR1 mutant has a single G to A base substitution in the OsPPDKB gene, which results in a missense glycine to aspartate mutation. Although there was little change in the mRNA expression levels of OsPPDKB between floTR1 and the original variety (WT) plants, PPDK activity in the floTR1 developing endosperm was null. Recombinant enzyme analysis also found that aspartate replacement, as found in floTR1, resulted in the complete loss of PPDK activity in vitro. Analysis of other recombinant enzymes suggested that the spatial configuration such as size and direction of the side chain in the replaced amino acids predominantly affected its activity. The rice flour of floTR1 showed not only smaller particle size distribution and lower damaged starch content but also higher specific rice bread loaf volume than WT. Our data provides new insights into effects on OsPPDK activity caused by a SNP mutation and suggests applications for this new floury rice mutant.

The Combined Application of Urea and Fulvic Acid Solution Improved Maize Carbon and Nitrogen Metabolism

It has been reported that fulvic acid (FA) application improves soil structure and nutrient availability. However, the effects of combined application of urea (U) and FA solution on the photosynthesis and nitrogen metabolism in maize (Zea mays L.) have rarely been reported. In this study, pot experiments were conducted in 2017 and 2018, and the effects of combined application of urea and FA solution (U+FA) on soil available nutrient contents, maize endogenous hormone concentrations, carbon and nitrogen metabolism-related enzyme concentrations, maize yield, and nitrogen use efficiency (NUE) were researched. Compared with the U treatment, the maize yield and NUE in the U+FA treatment were significantly increased by 8.31% and 17.09 percentage points in 2017 and by 16.90% and 24.31 percentage points in 2018. At the jointing and 12-leaf (V12) stages of maize, soil NH4+ content increased by 139.32% and 12.08%, separately, in the U+FA treatment. At the V12 stage, the auxin, nitrate reductase, nitrite reductase, and glutamine synthetase concentrations in maize root were increased by 42.31%, 74.17%, 16.61%, and 45.60%, respectively, and the concentrations of pyruvate phosphate dikinase and phosphoenolpyruvate carboxylase in maize leave were increased by 29.40% and 42.96%, respectively, in the U+FA treatment. The combined application of urea and FA solution significantly improved soil nutrient availability, increased the concentrations of endogenous hormones in maize, stimulated the activities of enzymes related to nitrogen metabolism, promoted the photosynthetic carbon assimilation efficiency, and ultimately improved crop yield and NUE.

Exogenous trehalose differently improves photosynthetic carbon assimilation capacities in maize and wheat under heat stress

ABSTRACT High temperature inhibits the growth and development of maize (Zea mays L.) and wheat (Triticum aestivum L.) seedlings. The current study attempts to elucidate the underlying mechanisms of exogenous trehalose for the improvement of maize and wheat responses to heat stress. Results show that, in maize seedlings, exogenously-supplied trehalose was able to effectively elevate the capacity of photosynthetic carbon assimilation and improve the carbohydrate content via up-regulating the transcript levels of key enzymes including phosphoenol pyruvate carboxylase (PEPC), NADP-malate dehydrogenase (NADP-MDH), NADP-malic enzyme (NADP-ME), pyruvate phosphate dikinase (PPDK) and Ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit. Besides, it was also able to improve C4 pathway enzyme activities, including PEPC, NADP-MDH, NADP-ME and Ribulose-1,5-bisphosphate carboxylase (RUBPCase). In wheat seedlings, exogenously-supplied trehalose promoted photosynthetic carbon assimilation by elevating the activity of RUBPCase. These results show that exogenous trehalose improved photosynthetic carbon assimilation capacities in maize and wheat seedlings under heat stress through different mechanisms.

Functional Analysis of H+-Pumping Membrane-Bound Pyrophosphatase, ADP-Glucose Synthase, and Pyruvate Phosphate Dikinase as Pyrophosphate Sources in Clostridium thermocellum.

ABSTRACTThe atypical glycolysis of Clostridium thermocellum is characterized by the use of pyrophosphate (PPi) as a phosphoryl donor for phosphofructokinase (Pfk) and pyruvate phosphate dikinase (Ppdk) reactions. Previously, biosynthetic PPi was calculated to be stoichiometrically insufficient to drive glycolysis. This study investigates the role of a H+-pumping membrane-bound pyrophosphatase, glycogen cycling, a predicted Ppdk–malate shunt cycle, and acetate cycling in generating PPi. Knockout studies and enzyme assays confirmed that clo1313_0823 encodes a membrane-bound pyrophosphatase. Additionally, clo1313_0717-0718 was confirmed to encode ADP-glucose synthase by knockouts, glycogen measurements in C. thermocellum, and heterologous expression in Escherichia coli. Unexpectedly, individually targeted gene deletions of the four putative PPi sources did not have a significant phenotypic effect. Although combinatorial deletion of all four putative PPi sources reduced the growth rate by 22% (0.30 ± 0.01 h−1) and the biomass yield by 38% (0.18 ± 0.00 gbiomass gsubstrate−1), this change was much smaller than what would be expected for stoichiometrically essential PPi-supplying mechanisms. Growth-arrested cells of the quadruple knockout readily fermented cellobiose, indicating that the unknown PPi-supplying mechanisms are independent of biosynthesis. An alternative hypothesis that ATP-dependent Pfk activity circumvents a need for PPi altogether was falsified by enzyme assays, heterologous expression of candidate genes, and whole-genome sequencing. As a secondary outcome, enzymatic assays confirmed functional annotation of clo1313_1832 as ATP- and GTP-dependent fructokinase. These results indicate that the four investigated PPi sources individually and combined play no significant PPi-supplying role, and the true source(s) of PPi, or alternative phosphorylating mechanisms, that drive(s) glycolysis in C. thermocellum remain(s) elusive.IMPORTANCE Increased understanding of the central metabolism of C. thermocellum is important from a fundamental as well as from a sustainability and industrial perspective. In addition to showing that H+-pumping membrane-bound PPase, glycogen cycling, a Ppdk–malate shunt cycle, and acetate cycling are not significant sources of PPi supply, this study adds functional annotation of four genes and availability of an updated PPi stoichiometry from biosynthesis to the scientific domain. Together, this aids future metabolic engineering attempts aimed to improve C. thermocellum as a cell factory for sustainable and efficient production of ethanol from lignocellulosic material through consolidated bioprocessing with minimal pretreatment. Getting closer to elucidating the elusive source of PPi, or alternative phosphorylating mechanisms, for the atypical glycolysis is itself of fundamental importance. Additionally, the findings of this study directly contribute to investigations into trade-offs between thermodynamic driving force versus energy yield of PPi- and ATP-dependent glycolysis.

In vivo evolution of lactic acid hyper-tolerant Clostridium thermocellum

Lactic acid (LA) has several applications in the food, cosmetics and pharmaceutical industries, as well as in the production of biodegradable plastic polymers, namely polylactides. Industrial production of LA is essentially based on microbial fermentation. Recent reports have shown the potential of the cellulolytic bacterium Clostridium thermocellum for direct LA production from inexpensive lignocellulosic biomass. However, C. thermocellum is highly sensitive to acids and does not grow at pH < 6.0. Improvement of LA tolerance of this microorganism is pivotal for its application in cost-efficient production of LA. In the present study, the LA tolerance of C. thermocellum strains LL345 (wild-type fermentation profile) and LL1111 (high LA yield) was increased by adaptive laboratory evolution. At large inoculum size (10 %), the maximum tolerated LA concentration of strain LL1111 was more than doubled, from 15 g/L to 35 g/L, while subcultures evolved from LL345 showed 50–85 % faster growth in medium containing 45 g/L LA. Gene mutations (pyruvate phosphate dikinase, histidine protein kinase/phosphorylase) possibly affecting carbohydrate and/or phosphate metabolism have been detected in most LA-adapted populations. Although improvement of LA tolerance may sometimes also enable higher LA production in microorganisms, C. thermocellum LA-adapted cultures showed a yield of LA, and generally of other organic acids, similar to or lower than parental strains. Based on its improved LA tolerance and LA titer similar to its parent strain (LL1111), mixed adapted culture LL1630 showed the highest performing phenotype and could serve as a framework for improving LA production by further metabolic engineering.

The evolutionarily conserved kinase SnRK1 orchestrates resource mobilization during Arabidopsis seedling establishment.

The onset of plant life is characterized by a major phase transition. During early heterotrophic seedling establishment, seed storage reserves fuel metabolic demands, allowing the plant to switch to autotrophic metabolism. Although metabolic pathways leading to storage compound mobilization are well-described, the regulatory circuits remain largely unresolved. Using an inducible knockdown approach of the evolutionarily conserved energy master regulator Snf1-RELATED-PROTEIN-KINASE1 (SnRK1), phenotypic studies reveal its crucial function in Arabidopsis thaliana seedling establishment. Importantly, glucose feeding largely restores growth defects of the kinase mutant, supporting its major impact in resource mobilization. Detailed metabolite studies reveal sucrose as a primary resource early in seedling establishment, in a SnRK1-independent manner. Later, SnRK1 orchestrates catabolism of triacylglycerols and amino acids. Concurrent transcriptomic studies highlight SnRK1 functions in controlling metabolic hubs fuelling gluconeogenesis, as exemplified by cytosolic PYRUVATE ORTHOPHOSPHATE DIKINASE (cyPPDK). Here, SnRK1 establishes its function via phosphorylation of the transcription factor BASIC LEUCINE ZIPPER63 (bZIP63), which directly targets and activates the cyPPDK promoter. Taken together, our results disclose developmental and catabolic functions of SnRK1 in seed storage mobilization and describe a prototypic gene regulatory mechanism. As seedling establishment is important for plant vigor and crop yield, our findings are of agronomical importance.

Spatial distribution of proteins and metabolites in developing wheat grain and their differential regulatory response during the grain filling process.

SUMMARYGrain filling and grain development are essential biological processes in the plant’s life cycle, eventually contributing to the final seed yield and quality in all cereal crops. Studies of how the different wheat (Triticum aestivum L.) grain components contribute to the overall development of the seed are very scarce. We performed a proteomics and metabolomics analysis in four different developing components of the wheat grain (seed coat, embryo, endosperm, and cavity fluid) to characterize molecular processes during early and late grain development. In‐gel shotgun proteomics analysis at 12, 15, 20, and 26 days after anthesis (DAA) revealed 15 484 identified and quantified proteins, out of which 410 differentially expressed proteins were identified in the seed coat, 815 in the embryo, 372 in the endosperm, and 492 in the cavity fluid. The abundance of selected protein candidates revealed spatially and temporally resolved protein functions associated with development and grain filling. Multiple wheat protein isoforms involved in starch synthesis such as sucrose synthases, starch phosphorylase, granule‐bound and soluble starch synthase, pyruvate phosphate dikinase, 14‐3‐3 proteins as well as sugar precursors undergo a major tissue‐dependent change in abundance during wheat grain development suggesting an intimate interplay of starch biosynthesis control. Different isoforms of the protein disulfide isomerase family as well as glutamine levels, both involved in the glutenin macropolymer pattern, showed distinct spatial and temporal abundance, revealing their specific role as indicators of wheat gluten quality. Proteins binned into the functional category of cell growth/division and protein synthesis/degradation were more abundant in the early stages (12 and 15 DAA). At the metabolome level all tissues and especially the cavity fluid showed highly distinct metabolite profiles. The tissue‐specific data are integrated with biochemical networks to generate a comprehensive map of molecular processes during grain filling and developmental processes.

Morphophysiological alterations in transgenic rice lines expressing PPDK and ME genes from the C4 model Setaria italica

C4 plants are superior to C3 plants in terms of productivity and limited photorespiration. PPDK (pyruvate orthophosphate dikinase) and NADP-ME (NADP-dependent malic enzyme) are two important photosynthetic C4-specific enzymes present in the mesophyll cells of C4 plants. To evaluate the effect of C4 enzymes in rice, we developed transgenic rice lines by separately introducing Setaria italica PPDK [SiPPDK] and S. italica ME [SiME] gene constructs under the control of the green tissue-specific maize PPDK promoter. Rice plant lines for both constructs were screened using the polymerase chain reaction (PCR), Southern hybridization, and expression analysis. The best transgenic plant lines for each case were selected for physiological and biochemical characterization. The results from qRT-PCR and enzyme activity analysis revealed higher expression and activity of both PPDK and NADP-ME genes compared with the nontransformed and empty-vector-transformed plants. The average photosynthetic efficiency of transgenic plant lines carrying the PPDK and NADP-ME genes increased by 18% and 12%, respectively, and was positively correlated with the increased accumulation of photosynthetic pigment. The decrease in Fv/Fm, increased electron transport rate (ETR), and increased photochemical quenching (qP) compared with nontransformed control plants suggest that transgenic rice plants transferred more absorbed light energy to photochemical reactions than wild-type plants. SiME-transgenic plants displayed reduced leaf malate content and superior performance under water deficit conditions. Interestingly, the transgenic plants showed yield enhancement by exhibiting increased plant height, panicle length, panicle weight and thousand grain weight. Overall, the exogenous foxtail millet C4 gene PPDK enhanced photosynthesis and yield to a greater extent than NADP-ME.

Towards a dynamic photosynthesis model to guide yield improvement in C4 crops.

SummaryThe most productive C4 food and biofuel crops, such as Saccharum officinarum (sugarcane), Sorghum bicolor (sorghum) and Zea mays (maize), all use NADP‐ME‐type C4 photosynthesis. Despite high productivities, these crops fall well short of the theoretical maximum solar conversion efficiency of 6%. Understanding the basis of these inefficiencies is key for bioengineering and breeding strategies to increase the sustainable productivity of these major C4 crops. Photosynthesis is studied predominantly at steady state in saturating light. In field stands of these crops light is continually changing, and often with rapid fluctuations. Although light may change in a second, the adjustment of photosynthesis may take many minutes, leading to inefficiencies. We measured the rates of CO2 uptake and stomatal conductance of maize, sorghum and sugarcane under fluctuating light regimes. The gas exchange results were combined with a new dynamic photosynthesis model to infer the limiting factors under non‐steady‐state conditions. The dynamic photosynthesis model was developed from an existing C4 metabolic model for maize and extended to include: (i) post‐translational regulation of key photosynthetic enzymes and their temperature responses; (ii) dynamic stomatal conductance; and (iii) leaf energy balance. Testing the model outputs against measured rates of leaf CO2 uptake and stomatal conductance in the three C4 crops indicated that Rubisco activase, the pyruvate phosphate dikinase regulatory protein and stomatal conductance are the major limitations to the efficiency of NADP‐ME‐type C4 photosynthesis during dark‐to‐high light transitions. We propose that the level of influence of these limiting factors make them targets for bioengineering the improved photosynthetic efficiency of these key crops.

Characterization of full-length transcriptome in Saccharum officinarum and molecular insights into tiller development

BackgroundAlthough extensive breeding efforts are ongoing in sugarcane (Saccharum officinarum L.), the average yield is far below the theoretical potential. Tillering is an important component of sugarcane yield, however, the molecular mechanism underlying tiller development is still elusive. The limited genomic data in sugarcane, particularly due to its complex and large genome, has hindered in-depth molecular studies.ResultsHerein, we generated full-length (FL) transcriptome from developing leaf and tiller bud samples based on PacBio Iso-Seq. In addition, we performed RNA-seq from tiller bud samples at three developmental stages (T0, T1 and T2) to uncover key genes and biological pathways involved in sugarcane tiller development. In total, 30,360 and 20,088 high-quality non-redundant isoforms were identified in leaf and tiller bud samples, respectively, representing 41,109 unique isoforms in sugarcane. Likewise, we identified 1063 and 1037 alternative splicing events identified in leaf and tiller bud samples, respectively. We predicted the presence of coding sequence for 40,343 isoforms, 98% of which was successfully annotated. Comparison with previous FL transcriptomes in sugarcane revealed 2963 unreported isoforms. In addition, we characterized 14,946 SSRs from 11,700 transcripts and 310 lncRNAs. By integrating RNA-seq with the FL transcriptome, 468 and 57 differentially expressed genes (DEG) were identified in T1vsT0 and T2vsT0, respectively. Strong up-regulation of several pyruvate phosphate dikinase and phosphoenolpyruvate carboxylase genes suggests enhanced carbon fixation and protein synthesis to facilitate tiller growth. Similarly, up-regulation of linoleate 9S-lipoxygenase and lipoxygenase genes in the linoleic acid metabolism pathway suggests high synthesis of key oxylipins involved in tiller growth and development.ConclusionsCollectively, we have enriched the genomic data available in sugarcane and provided candidate genes for manipulating tiller formation and development, towards productivity enhancement in sugarcane.

Screening of highly efficient photosynthetic hybrids of Oryza officinalis and analysis of their photosynthetic pathway genes

Hereditary properties of strong growth and huge accumulation of biomass in Oryza officinalis exhibit a great potential; however, the genes that code for its high photosynthesis performance are not established. This study screened eight hybrids, using biomass accumulation and photosynthesis analysis, based on the introgression lines constructed by analyzing distant hybridization patterns between Oryza officinalis and cultivars HY-8. We designed 23 primer pairs using transcriptome sequencing of Oryza officinalis and screened two types of photosynthetic enzymes: phosphoenolpyruvate carboxylase (PEPC) and pyruvate orthophosphate dikinase (PPDK), which are two related proteins of ribulose-1,5-biphosphate carboxylase/oxygenase (Rubisco), its binding protein (rubis-subs-bind), and a small subunit (rbcS). Results showed that C4 photosynthetic pathway enzymes, PEPC and PPDK, were highly expressed in hybrids and the source plant, Oryza officinalis. Homology analysis also indicated that the sequences of those two genes were different from those of the C3 and C4 plants investigated. Therefore, a better understanding of the photosynthetic characteristics of Oryza officinalis would provide clues for further isolation of valuable genes from this plant.

Regulation of carbon assimilation in Skeletonema costatum under diel variations

Here, we explored the molecular responses of photosynthetic carbon fixation in a globally distributed marine diatom, Skeletonema costatum, based on incubation experiments over a 24-h period under natural light. A model of carbon metabolism was proposed based on the transcriptome of S. costatum. We examined the diurnal expression patterns and relative importance of the C4 pathway in S. costatum photosynthesis by analyzing the transcript abundance and key enzyme activities during the day/night cycle. This study suggested that the photosynthetic pathways of S. costatum were diverse and could involve combined CO2-concentrating mechanisms. The analysis at the transcriptional level revealed that the transcripts of the C4 pathway-related genes phosphoenolpyruvate carboxylase (PEPC) and pyruvate-phosphate dikinase (PPDK) were upregulated under the photoperiod and had obvious diurnal rhythms, indicating that light obviously affected photosynthetic carbon fixation. Moreover, we studied the pathway of photosynthetic carbon assimilation in S. costatum with carbon isotope δ13C techniques. The data provided evidence that carbon fixation in S. costatum involves the synergy of C3 and C4 photosynthesis. Under low pCO2 levels in the low light photoperiod, the affinity of inorganic carbon increased, supported by the C4 pathway, to acclimate to variations in the environment. This study promotes a better understanding of the photosynthetic carbon fixation mechanism in S. costatum during the diel cycle.

Dynamic changes of genome sizes and gradual gain of cell-specific distribution of C4 enzymes during C4 evolution in genus Flaveria.

C4 plants are believed to have evolved from C3 plants through various C3 -C4 intermediate stages in which a photorespiration-dependent CO2 concentration system known as C2 photosynthesis operates. Genes involved in the C4 cycle were thought to be recruited from orthologs present in C3 species and developed cell-specific expression during C4 evolution. To understand the process of establishing C4 photosynthesis, we performed whole-genome sequencing and investigated expression and mesophyll- or bundle-sheath-cell-specific localization of phosphoenolpyruvate carboxylase (PEPC), NADP-malic enzyme (NADP-ME), pyruvate, orthophosphate dikinase (PPDK) in C3 , C3 -C4 intermediate, C4 -like, and C4 Flaveria species. While genome sizes vary greatly, the number of predicted protein-coding genes was similar among C3 , C3 -C4 intermediate, C4 -like, and C4 Flaveria species. Cell-specific localization of the PEPC, NADP-ME, and PPDK transcripts was insignificant or weak in C3 -C4 intermediate species, whereas these transcripts were expressed cell-type specific in C4 -like species. These results showed that elevation of gene expression and cell-specific control of pre-existing C4 cycle genes in C3 species was involved in C4 evolution. Gene expression was gradually enhanced during C4 evolution, whereas cell-specific control was gained independently of quantitative transcriptional activation during evolution from C3 -C4 intermediate to C4 photosynthesis in genus Flaveria.

Identification and expression profiling of genes involved in circadian clock regulation in red dragon fruit (Hylocereus polyrhizus) by full-length transcriptome sequencing

ABSTRACT Crassulacean acid metabolism (CAM) plants fix CO2 at night, exhibiting a reversed regulatory pattern of metabolomic pathways compared with most model plants, which have C3 and C4 pathways. In this study, we used a valuable tropic fruit, red dragon fruit (Hylocereus polyrhizus), as model plant to identify and analyze the circadian regulation genes. Due to the absence of red dragon fruit’s whole-genome dataset, we established a full-length transcriptome dataset using single-molecule real-time (SMRT) sequencing method. A 7.66-Gb dataset with 4,552,474 subreads was generated, with an average length of 1,683 bp and an N50 of 2,446 bp. Using this dataset, we identified center oscillator genes: CCA1 (CIRCADIAN CLOCK ASSOCIATED1), ELF3 (EARLY FLOWERING 3), GI (GIGANTEA), LHY (LATE ELONGATED HYPOCOTYL), LNK1 (NIGHT LIGHT-INDUCIBLE AND CLOCK-REGULATED 1), and TOC1 (TIMING OF CAB EXPRESSION 1); a gene for the input pathway: CRY1 (CRYPTOCHROME); a gene for the output pathway: CO (CONSTANS); and genes related to the CAM pathway: MDH (MALATE DEHYDROGENASE), ME (MALIC ENZYMES), and PPDK (PYRUVATE PHOSPHATE DIKINASE). We further established the 24-h rhythmic expression pattern of these genes and classified these into three groups: HpCCA1, HpELF3, HpLHY, HpLNK1, and HpGI have expression peaks during the day; HpTOC1, HpCO, and HpCRY1 have highest expression levels at night; The genes involved in the CAM pathways, namely, HpMDH, HpME1, and HpPPDK, have double expression peaks in the day and night. Comparison of these expression patterns between red dragon fruit and model plants could provide clues in understanding the circadian clock regulation and the activity of the CAM pathways in cactus plants.

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.

Adaptation responses in C4 photosynthesis of sweet maize (Zea mays L.) exposed to nicosulfuron

Nicosulfuron is an ingredient in photosynthesis-inhibiting herbicides and has been widely used in corn post-emergence weed control. In the current study, a pair of sister lines, HK301 (nicosulfuron-tolerence, NT) and HK320 (nicosulfuron-sensitive, NS), was used to study the effect of nicosulfuron in sweet maize seedlings on C4 photosynthetic enzymes and non-enzymatic substances, expression levels of key enzymes, and chloroplast structure. Nicosulfuron was sprayed at the four-leaf stage, and water was sprayed as a control. After nicosulfuron treatment, phosphoenolpyruvate carboxylase (PEPC), NADP-malic dehydrogenase (NADP-MDH), NADP-malic enzyme (NADP-ME), pyruvate orthophosphate dikinase (PPDK), and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activities of NT were significantly higher than those of NS. Compared to NT, malate, oxaloacetic acid, and pyruvic acid significantly decreased as exposure time increased in NS. Compared to NS, nicosulfuron treatment significantly increased the expression levels of PEPC, NADP-MDH, NADP-ME, PPDK, and Rubisco genes in NT. Under nicosulfuron treatment, chloroplast ultrastructure of NS, compared to that of NT, nicosulfuron induced swelling of the chloroplast volume and reduced starch granules in NS. In general, our results indicate that in different resistant sweet maize, C4 photosynthetic enzymes activity and key genes expression play a critical role in enhancing the adaptability of plants to nicosulfuron stress at a photosynthetic physiological level.

Response of transgenic Arabidopsis expressing maize C4 photosynthetic enzyme genes to high light

ABSTRACT This study assessed the responses of wild-type (WT) and transgenic Arabidopsis expressing seven combinations of maize (Zea mays) genes phosphoenolpyruvate carboxylase (pepc), pyruvate phosphate dikinase (ppdk), and NADP-malic enzyme (nadp-me) to high light. Our results showed that the net CO2 assimilation rate (P n) and shoot dry weight of four of the transgenic Arabidopsis genotypes were significantly different from those of WT under high-light treatment, being in the order of Zmpepc+Zmppdk+Zmnadp-me (PC-K-M) > Zmpepc+Zmppdk (PC-K) > Zmpepc (PC), Zmpepc+Zmnadp-me (PC-M) > WT. The other genotypes did not differ from WT. This indicated that Zmpepc was essential for maintaining high photosynthetic performance under high light, Zmppdk had a positive synergistic effect on Zmpepc, and the combination of all three genes had the greatest synergistic effect. These four genotypes also maintained higher photosystem II (PSII) activity (K-phase, J-phase, RC/CSm), electron transfer capacity (J-phase), and photochemical efficiency (TRo/ABS), and accumulated less reactive oxygen species (O2·−, H2O2) and suffered less damage to the membrane system (MDA) than WT under high light. Collectively, PC, PC-K, PC-M, and PC-K-M used most of the absorbed energy for CO2 assimilation through a significantly higher P n, which reduced the generation of excess electrons in the photosynthetic apparatus, thereby reducing damage to the membrane system and PSII. This ultimately resulted in improved high-light tolerance. P n was the main reason for the significant difference in the high-light tolerance of the four genotypes. Joint expression of the three maize genes may be of great value in the genetic improvement of high-light tolerance in C3 crops.

Glucose Oxidation to Pyruvate Is Not Essential for Brucella suis Biovar 5 Virulence in the Mouse Model.

Brucella species cause brucellosis, a worldwide extended zoonosis. The brucellae are related to free-living and plant-associated α2-Proteobacteria and, since they multiply within host cells, their metabolism probably reflects this adaptation. To investigate this, we used the rodent-associated Brucella suis biovar 5, which in contrast to the ruminant-associated Brucella abortus and Brucella melitensis and other B. suis biovars, is fast-growing and conserves the ancestral Entner-Doudoroff pathway (EDP) present in the plant-associated relatives. We constructed mutants in Edd (glucose-6-phosphate dehydratase; first EDP step), PpdK (pyruvate phosphate dikinase; phosphoenolpyruvate ⇌ pyruvate), and Pyk (pyruvate kinase; phosphoenolpyruvate → pyruvate). In a chemically defined medium with glucose as the only C source, the Edd mutant showed reduced growth rates and the triple Edd-PpdK-Pyk mutant did not grow. Moreover, the triple mutant was also unable to grow on ribose or xylose. Therefore, B. suis biovar 5 sugar catabolism proceeds through both the Pentose Phosphate shunt and EDP, and EDP absence and exclusive use of the shunt could explain at least in part the comparatively reduced growth rates of B. melitensis and B. abortus. The triple Edd-PpdK-Pyk mutant was not attenuated in mice. Thus, although an anabolic use is likely, this suggests that hexose/pentose catabolism to pyruvate is not essential for B. suis biovar 5 multiplication within host cells, a hypothesis consistent with the lack of classical glycolysis in all Brucella species and of EDP in B. melitensis and B. abortus. These results and those of previous works suggest that within cells, the brucellae use mostly 3 and 4 C substrates fed into anaplerotic pathways and only a limited supply of 5 and 6 C sugars, thus favoring the EDP loss observed in some species.