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Starch, as a main energy storage substance, plays an important role in plant growth and human life. Despite the fact that several enzymes and regulators involved in starch biosynthesis have been identified, the regulating mechanism of starch synthesis is still unclear. In this study, we isolated a rice floury endosperm mutant M14 from a mutant pool induced by 60Co. Both total starch content and amylose content in M14 seeds significantly decreased, and starch thermal and pasting properties changed. Compound starch granules were defected in the floury endosperm of M14 seeds. Map-based cloning and a complementation test showed that the floury endosperm phenotype was determined by a gene of OsPPDKB, which encodes pyruvate orthophosphate dikinase (PPDK, EC 2.7.9.1). Subcellular localization analysis demonstrated that PPDK was localized in chloroplast and cytoplasm, the chOsPPDKB highly expressed in leaf and leaf sheath, and the cyOsPPDKB constitutively expressed with a high expression in developing endosperm. Moreover, the expression of starch synthesis-related genes was also obviously altered in M14 developing endosperm. The above results indicated that PPDK played an important role in starch metabolism and structure in rice endosperm.

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 V max 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.

Gluconeogenesis is a fundamental metabolic process that allows organisms to make sugars from non-carbohydrate stores such as lipids and protein. In eukaryotes only one gluconeogenic route has been described from organic acid intermediates and this relies on the enzyme phospho enol pyruvate carboxykinase (PCK). Here we show that two routes exist in Arabidopsis , and that the second uses pyruvate, orthophosphate dikinase (PPDK). Gluconeogenesis is critical to fuel the transition from seed to seedling. Arabidopsis pck1 and ppdk mutants are compromised in seed-storage reserve mobilization and seedling establishment. Radiolabelling studies show that PCK predominantly allows sugars to be made from dicarboxylic acids, which are products of lipid breakdown. However, PPDK also allows sugars to be made from pyruvate, which is a major product of protein breakdown. We propose that both routes have been evolutionarily conserved in plants because, while PCK expends less energy, PPDK is twice as efficient at recovering carbon from pyruvate. During seed germination plants use gluconeogenesis to mobilize noncarbohydrate energy reserves. Here Eastmond et al . show that plants, unlike other eukaryotes, do not solely rely on a gluconeogenic pathway via the enzyme PCK but also use a second pathway relying on PPDK.

Summary In maize, two pyruvate orthophosphate dikinase (PPDK) regulatory proteins, ZmPDRP1 and ZmPDRP2, are respectively specific to the chloroplast of mesophyll cells (MCs) and bundle sheath cells (BSCs). Functionally, ZmPDRP1/2 catalyse both phosphorylation/inactivation and dephosphorylation/activation of ZmPPDK, which is implicated as a major rate‐limiting enzyme in C4 photosynthesis of maize. Our study here showed that maize plants lacking ZmPDRP1 or silencing of ZmPDRP1/2 confer resistance to a prevalent potyvirus sugarcane mosaic virus (SCMV). We verified that the C‐terminal domain (CTD) of ZmPDRP1 plays a key role in promoting viral infection while independent of enzyme activity. Intriguingly, ZmPDRP1 and ZmPDRP2 re‐localize to cytoplasmic viral replication complexes (VRCs) following SCMV infection. We identified that SCMV‐encoded cytoplasmic inclusions protein CI targets directly ZmPDRP1 or ZmPDRP2 or their CTDs, leading to their re‐localization to cytoplasmic VRCs. Moreover, we found that CI could be degraded by the 26S proteasome system, while ZmPDRP1 and ZmPDRP2 could up‐regulate the accumulation level of CI through their CTDs by a yet unknown mechanism. Most importantly, with genetic, cell biological and biochemical approaches, we provide evidence that BSCs‐specific ZmPDRP2 could accumulate in MCs of Zmpdrp1 knockout (KO) lines, revealing a unique regulatory mechanism crossing different cell types to maintain balanced ZmPPDK phosphorylation, thereby to keep maize normal growth. Together, our findings uncover the genetic link of the two cell‐specific maize PDRPs, both of which are co‐opted to VRCs to promote viral protein accumulation for robust virus infection.

Starch biosynthetic enzymes from maize (Zea mays) and wheat (Triticum aestivum) amyloplasts exist in cell extracts in high molecular weight complexes; however, the nature of those assemblies remains to be defined. This study tested the interdependence of the maize enzymes starch synthase IIa (SSIIa), SSIII, starch branching enzyme IIb (SBEIIb), and SBEIIa for assembly into multisubunit complexes. Mutations that eliminated any one of those proteins also prevented the others from assembling into a high molecular mass form of approximately 670 kD, so that SSIII, SSIIa, SBEIIa, and SBEIIb most likely all exist together in the same complex. SSIIa, SBEIIb, and SBEIIa, but not SSIII, were also interdependent for assembly into a complex of approximately 300 kD. SSIII, SSIIa, SBEIIa, and SBEIIb copurified through successive chromatography steps, and SBEIIa, SBEIIb, and SSIIa coimmunoprecipitated with SSIII in a phosphorylation-dependent manner. SBEIIa and SBEIIb also were retained on an affinity column bearing a specific conserved fragment of SSIII located outside of the SS catalytic domain. Additional proteins that copurified with SSIII in multiple biochemical methods included the two known isoforms of pyruvate orthophosphate dikinase (PPDK), large and small subunits of ADP-glucose pyrophosphorylase, and the sucrose synthase isoform SUS-SH1. PPDK and SUS-SH1 required SSIII, SSIIa, SBEIIa, and SBEIIb for assembly into the 670-kD complex. These complexes may function in global regulation of carbon partitioning between metabolic pathways in developing seeds.

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 CO₂ fixation in some Crassulacean acid metabolism (CAM) species. We report the generation and phenotypic characterization of transgenic RNA interference lines of the obligate CAM speciesKalanchoë fedtschenkoiwith reduced activities of NAD-ME or PPDK. Transgenic linerNAD-ME1had 8%, andrPPDK1had 5% of the wild-type level of activity, and showed dramatic changes in the light/dark cycle of CAM CO₂ fixation. In well-watered conditions, these lines fixed all of their CO₂ in the light; they thus performed C₃ 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 inrNAD-ME1. Furthermore, the circadian clock-controlled phosphorylation of PPC in the dark was reduced in both lines, especially inrNAD-ME1. This had the consequence that circadian rhythms of PPC phosphorylation, PPCkinasetranscript levels and activity, and the classic circadian rhythm of CAM CO₂ 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 therNAD-ME1line, suggesting that perturbing CAM inK. fedtschenkoifeeds back to perturb the central circadian clock.

Pyruvate phosphate dikinase (PPDK) is a vital enzyme in cellular energy metabolism catalyzing the ATP- and P i -dependent formation of phosphoenolpyruvate from pyruvate in C 4 -plants, but the reverse reaction forming ATP in bacteria and protozoa. The multi-domain enzyme is considered an efficient molecular machine that performs one of the largest single domain movements in proteins. However, a comprehensive understanding of the proposed swiveling domain motion has been limited by not knowing structural intermediates or molecular dynamics of the catalytic process. Here, we present crystal structures of PPDKs from Flaveria , a model genus for studying the evolution of C 4 -enzymes from phylogenetic ancestors. These structures resolve yet unknown conformational intermediates and provide the first detailed view on the large conformational transitions of the protein in the catalytic cycle. Independently performed unrestrained MD simulations and configurational free energy calculations also identified these intermediates. In all, our experimental and computational data reveal strict coupling of the CD swiveling motion to the conformational state of the NBD. Moreover, structural asymmetries and nucleotide binding states in the PPDK dimer support an alternate binding change mechanism for this intriguing bioenergetic enzyme.

Four enzymes, namely, the maize C4-specific phosphoenolpyruvate carboxylase (PEPC), the maize C4-specific pyruvate, orthophosphate dikinase (PPDK), the sorghum NADP-malate dehydrogenase (MDH), and the rice C3-specific NADP-malic enzyme (ME), were overproduced in the mesophyll cells of rice plants independently or in combination. Overproduction individually of PPDK, MDH or ME did not affect the rate of photosynthetic CO2 assimilation, while in the case of PEPC it was slightly reduced. The reduction in CO2 assimilation in PEPC overproduction lines remained unaffected by overproduction of PPDK, ME or a combination of both, however it was significantly restored by the combined overproduction of PPDK, ME, and MDH to reach levels comparable to or slightly higher than that of non-transgenic rice. The extent of the restoration of CO2 assimilation, however, was more marked at higher CO2 concentrations, an indication that overproduction of the four enzymes in combination did not act to concentrate CO2 inside the chloroplast. Transgenic rice plants overproducing the four enzymes showed slight stunting. Comparison of transformants overproducing different combinations of enzymes indicated that overproduction of PEPC together with ME was responsible for stunting, and that overproduction of MDH had some mitigating effects. Possible mechanisms underlying these phenotypic effects, as well as possibilities and limitations of introducing the C4-like photosynthetic pathway into C3 plants, are discussed.

36 I. 36 II. 37 III. 44 IV. 49 V. 53 54 References 55 SUMMARY: Anoxia tolerance in plants is distinguished by direction of the sparse supply of energy to processes crucial to cell maintenance and sometimes to growth, as in rice seedlings. In anoxic rice coleoptiles energy is used to synthesise proteins, take up K⁺, synthesise cell walls and lipids, and in cell maintenance. Maintenance of electrochemical H⁺gradients across the tonoplast and plasma membrane is crucial for solute compartmentation and thus survival. These gradients sustain some H⁺‐solute cotransport and regulate cytoplasmic pH. Pyrophosphate (PPᵢ), the alternative energy donor to ATP, allows direction of energy to the vacuolar H⁺‐PPᵢase, sustaining H⁺gradients across the tonoplast. When energy production is critically low, operation of a biochemical pHstat allows H⁺‐solute cotransport across plasma membranes to continue for at least for 18 h. In active (e.g. growing) cells, PPᵢproduced during substantial polymer synthesis allows conversion of PPᵢto ATP by PPᵢ‐phosphofructokinase (PFK). In quiescent cells with little polymer synthesis and associated PPᵢformation, the PPᵢrequired by the vacuolar H⁺‐PPᵢase and UDPG pyrophosphorylase involved in sucrose mobilisation via sucrose synthase might be produced by conversion of ATP to PPᵢthrough reversible glycolytic enzymes, presumably pyruvate orthophosphate dikinase. These hypotheses need testing with species characterised by contrasting anoxia tolerance.

Pyruvate orthophosphate dikinase (PPDK) is one of the most important enzymes in C₄ photosynthesis. PPDK regulatory protein (PDRP) regulates the inorganic phosphate-dependent activation and ADP-dependent inactivation of PPDK by reversible phosphorylation. PDRP shares no significant sequence similarity with other protein kinases or phosphatases. To investigate the molecular mechanism by which PDRP carries out its dual and competing activities, we determined the crystal structure of PDRP from maize (Zea mays). PDRP forms a compact homo-dimer in which each protomer contains two separate N-terminal (NTD) and C-terminal (CTD) domains. The CTD includes several key elements for performing both phosphorylation and dephosphorylation activities: the phosphate binding loop (P-loop) for binding the ADP and inorganic phosphate substrates, residues Lys-274 and Lys-299 for neutralizing the negative charge, and residue Asp-277 for protonating and deprotonating the target Thr residue of PPDK to promote nucleophilic attack. Surprisingly, the NTD shares the same protein fold as the CTD and also includes a putative P-loop with AMP bound but lacking enzymatic activities. Structural analysis indicated that this loop may participate in the interaction with and regulation of PPDK. The NTD has conserved intramolecular and intermolecular disulfide bonds for PDRP dimerization. Moreover, PDRP is the first structure of the domain of unknown function 299 enzyme family reported. This study provides a structural basis for understanding the catalytic mechanism of PDRP and offers a foundation for the development of selective activators or inhibitors that may regulate photosynthesis