Relationship between reduction in rice (Nipponbare) leaf blade size under elevated CO2 and miR396-GRF module.

Elevated CO2 (eCO2; 1000 ppm) influences developing rice leaf formation, reducing leaf blade length and width as compared to rice grown under ambient CO2 (aCO2; 400 ppm). Since micro RNAs (miRNAs) are known to play multiple roles in plant development, we hypothesized that miRNAs might be involved in modulating leaf size under eCO2 conditions. To identify miRNAs responding to eCO2, we profiled miRNA levels in developing rice leaves (P4; plastochron number of the fourth-youngest leaf) under eCO2 using small RNA-seq. We detected 18 mature miRNA sequences for which expression levels varied more than two-fold between the eCO2 and aCO2 conditions. Among them, only miR396e and miR396f significantly differed between the two conditions. Additionally, the expression of growth-regulating factors (GRFs), potential target mRNA of miR396s, were repressed under the eCO2 condition. We used an antisense oligonucleotide approach to confirm that single-strand DNA corresponding to the miR396e sequence effectively downregulated GRF expression in developing leaves, reducing the leaf blade length, such as for rice grown under eCO2. These results suggest that the miR396-GRF module is crucially relevant to controlling rice leaf blade length in eCO2 environments.

Acetic-acid-induced jasmonate signaling in root enhances drought avoidance in rice.

Conferring drought resistant traits to crops is one of the major aims of current breeding programs in response to global climate changes. We previously showed that exogenous application of acetic acid to roots of various plants could induce increased survivability under subsequent drought stress conditions, but details of the metabolism of exogenously applied acetic acid, and the nature of signals induced by its application, have not been unveiled. In this study, we show that rice rapidly induces jasmonate signaling upon application of acetic acid, resulting in physiological changes similar to those seen under drought. The major metabolite of the exogenously applied acetic acid in xylem sap was determined as glutamine-a common and abundant component of xylem sap-indicating that acetic acid is not the direct agent inducing the observed physiological responses in shoots. Expression of drought-responsive genes in shoot under subsequent drought conditions was attenuated by acetic acid treatment. These data suggest that acetic acid activates root-to-shoot jasmonate signals that partially overlap with those induced by drought, thereby conferring an acclimated state on shoots prior to subsequent drought.

Gene regulatory network and its constituent transcription factors that control nitrogen-deficiency responses in rice.

Increase in the nitrogen (N)-use efficiency and optimization of N response in crop species are urgently needed. Although transcription factor-based genetic engineering is a promising approach for achieving these goals, transcription factors that play key roles in the response to N deficiency have not been studied extensively. Here, we performed RNA-seq analysis of root samples of 20 Asian rice (Oryza sativa) accessions with differential nutrient uptake. Data obtained from plants exposed to N-replete and N-deficient conditions were subjected to coexpression analysis and machine learning-based pathway inference to dissect the gene regulatory network required for the response to N deficiency. Four transcription factors, including members of the G2-like and bZIP families, were predicted to function as key regulators of gene transcription within the network in response to N deficiency. Cotransfection assays validated inferred novel regulatory pathways, and further analyses using genome-edited knockout lines suggested that these transcription factors are important for N-deficiency responses in planta. Many of the N deficiency-responsive genes, including those encoding key regulators within the network, were coordinately regulated by transcription factors belonging to different families. Transcription factors identified in this study could be valuable for the modification of N response and metabolism.

Low assimilation efficiency of photorespiratory ammonia in conifer leaves.

Glutamine synthetase (GS) localized in the chloroplasts, GS2, is a key enzyme in the assimilation of ammonia (NH3) produced from the photorespiration pathway in angiosperms, but it is absent from some coniferous species belonging to Pinaceae such as Pinus. We examined whether the absence of GS2 is common in conifers (Pinidae) and also addressed the question of whether assimilation efficiency of photorespiratory NH3 differs between conifers that may potentially lack GS2 and angiosperms. Search of the expressed sequence tag database of Cryptomeria japonica, a conifer in Cupressaceae, and immunoblotting analyses of leaf GS proteins of 13 species from all family members in Pinidae revealed that all tested conifers exhibited only GS1 isoforms. We compared leaf NH3 compensation point (γNH3) and the increments in leaf ammonium content per unit photorespiratory activity (NH3 leakiness), i.e. inverse measures of the assimilation efficiency, between conifers (C. japonica and Pinus densiflora) and angiosperms (Phaseolus vulgaris and two Populus species). Both γNH3 and NH3 leakiness were higher in the two conifers than in the three angiosperms tested. Thus, we concluded that the absence of GS2 is common in conifers, and assimilation efficiency of photorespiratory NH3 is intrinsically lower in conifer leaves than in angiosperm leaves. These results imply that acquisition of GS2 in land plants is an adaptive mechanism for efficient NH3 assimilation under photorespiratory environments.

Outgrowth of Rice Tillers Requires Availability of Glutamine in the Basal Portions of Shoots.

BACKGROUND: Our previous studies concluded that metabolic disorder in the basal portions of rice shoots caused by a lack of cytosolic glutamine synthetase1;2 (GS1;2) resulted in a severe reduction in the outgrowth of tillers. Rice mutants lacking GS1;2 (gs1;2 mutants) showed a remarkable reduction in the contents of both glutamine and asparagine in the basal portions of shoots. In the current study, we attempted to reveal the mechanisms for this decrease in asparagine content using rice mutants lacking either GS1;2 or asparagine synthetase 1 (AS1). The contributions of the availability of glutamine and asparagine to the outgrowth of rice tillers were investigated. RESULTS: Rice has two AS genes, and the enzymes catalyse asparagine synthesis from glutamine. In the basal portions of rice shoots, expression of OsAS1, the major species in this tissue, was reduced in gs1;2 mutants, whereas OsAS2 expression was relatively constant. OsAS1 was expressed in phloem companion cells of the nodal vascular anastomoses connected to the axillary bud vasculatures in the basal portions of wild-type shoots, whereas cell-specific expression was markedly reduced in gs1;2 mutants. OsAS1 was up-regulated significantly by NH4+ supply in the wild type but not in gs1;2 mutants. When GS reactions were inhibited by methionine sulfoximine, OsAS1 was up-regulated by glutamine but not by NH4+. The rice mutants lacking AS1 (as1 mutants) showed a decrease in asparagine content in the basal portions of shoots. However, glutamine content and tiller number were less affected by the lack of AS1. CONCLUSION: These results indicate that in phloem companion cells of the nodal vascular anastomoses, asparagine synthesis is largely dependent on glutamine or its related metabolite-responsive AS1. Thus, the decrease in glutamine content caused by a lack of GS1;2 is suggested to result in low expression of OsAS1, decreasing asparagine content. However, the availability of asparagine generated from AS1 reactions is apparently less effective for the outgrowth of tillers. With respect to the tiller number and the contents of glutamine and asparagine in gs1;2 and as1 mutants, the availability of glutamine rather than asparagine in basal portions of rice shoots may be required for the outgrowth of rice tillers.

Characterization and expression analyses of two plastidic enolase genes in rice.

To verify the presence of enolase related to the chloroplastic glycolysis in rice, database search was carried out and identified seven putative enolase genes in the rice genome. Among them, OsEno1 and OsEno3 encode long proteins with N-terminal extensions. GFP protein fusions of these N-terminal extensions were both targeted to plastids of onion epidermal cell. Promoter::GUS analysis showed that OsEno3 was highly expressed in young developing leaves, but its expression was drastically decreased during leaf development and greening. On the other hand, the expression of OsEno1 was low and detected in limited portions such as leaf sheath at the tiller base. Recombinant OsEno1 protein showed enolase activity with a pH optimum at pH 8.0, whereas OsEno3 did not exhibit detectable activity. Although it remains obscure if OsEno3 encodes a functional enolase in vivo, our results demonstrate that the entire glycolytic pathway does not operate in rice chloroplasts.

Comparison of plant-type phosphoenolpyruvate carboxylases from rice: identification of two plant-specific regulatory regions of the allosteric enzyme.

Phosphoenolpyruvate carboxylase (PEPC) is a key enzyme of primary metabolism in bacteria, algae and vascular plants, and it undergoes allosteric regulation by various metabolic effectors. Rice (Oryza sativa) has five plant-type PEPCs, four cytosolic and one chloroplastic. We investigated their kinetic properties using recombinant proteins and found that, like most plant-type PEPCs, rice cytosolic isozymes were activated by glucose 6-phosphate and by alkaline pH. In contrast, no such activation was observed for the chloroplastic isozyme, Osppc4. In addition, Osppc4 showed low affinity for the substrate phosphoenolpyruvate (PEP) and very low sensitivities to allosteric inhibitors aspartate and glutamate. By comparing the isozyme amino acid sequences and three-dimensional structures simulated on the basis of the reported crystal structures, we identified two regions where Osppc4 has unique features that can be expected to affect its kinetic properties. One is the N-terminal extension; replacement of the extension of Osppc2a (cytosolic) with that from Osppc4 reduced the aspartate and glutamate sensitivities to about one-tenth of the wild-type values but left the PEP affinity unaffected. The other is the N-terminal loop, in which a conserved lysine at the N-terminal end is replaced with a glutamate-alanine pair in Osppc4. Replacement of the lysine of Osppc2a with glutamate-alanine lowered the PEP affinity to a quarter of the wild-type level (down to the Osppc4 level), without affecting inhibitor sensitivity. Both the N-terminal extension and the N-terminal loop are specific to plant-type PEPCs, suggesting that plant-type isozymes acquired these regions so that their activity could be regulated properly at the sites where they function.

Nocturnal phosphorylation of phosphoenolpyruvate carboxylase in the leaves of hygrophytic C3 monocots.

Phosphoenolpyruvate carboxylase (PEPC) undergoes activity regulation through reversible phosphorylation. The day/night phosphorylation of leaf PEPC in 27 C3 plant species was analyzed by immunoblotting. PEPC was phosphorylated in the daytime in 12 species, whereas it was phosphorylated at night in three species, rice, Monochoria vaginalis, and Sagittaria trifolia, all of which are hygrophytic monocots. Immunoblot analysis of isolated chloroplasts of M. vaginalis identified a PEPC protein inside the chloroplast in addition to cytosolic isozyme(s) as previously shown in genus Oryza. Using transgenic rice overexpressing the maize PEPC in the cytosol, we confirmed that the cytosolic PEPC underwent the nocturnal phosphorylation. These results suggest the interrelationship between the presence of chloroplastic PEPC and the nocturnal phosphorylation of cytosolic isozyme(s).

Elevated CO2 decreases the Photorespiratory NH3 production but does not decrease the NH3 compensation point in rice leaves.

The exchange of gaseous NH3 between the atmosphere and plants plays a pivotal role in controlling the global NH3 cycle. Photorespiration generates NH3 through oxygenation instead of carboxylation by the CO2-fixing enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). The future increase in the atmospheric CO2 concentration, [CO2], is expected to reduce plant NH3 production by suppressing RuBisCO oxygenation (Vo). We measured the net leaf NH3 uptake rate (FNH3) across NH3 concentrations in the air (na) ranging from 0.2 to 1.6 nmol mol(-1) at three [CO2] values (190, 360 and 750 µmol mol(-1)) using rice plants. We analyzed leaf NH3 gas exchange using a custom-made whole-leaf chamber system, and determined the NH3 compensation point (γ), a measure of potential NH3 emission, as the x-intercept of the linear relationship of FNH3 as a function of na. Our γ values were lower than those reported for other plant species. γ did not decrease under elevated [CO2], although leaf NH4 (+) content decreased with decreasing Vo at higher [CO2]. This was also the case for γ estimated from the pH and NH4 (+) concentration of the leaf apoplast solution (γ'). γ' of rice plants, grown at elevated [CO2] for months in a free-air CO2 enrichment facility, was also not decreased by elevated [CO2]. These results suggest that suppression of RuBisCO oxygenation by elevated [CO2] does not decrease potential leaf NH3 emission in rice plants.

Sites of action of elevated CO2 on leaf development in rice: discrimination between the effects of elevated CO2 and nitrogen deficiency.

Elevated CO2 concentrations (eCO2) trigger various plant responses. Despite intensive studies of these responses, the underlying mechanisms remain obscure. In this work, we investigated when and how leaf physiology and anatomy are affected by eCO2 in rice plants. We analyzed the most recently fully expanded leaves that developed successively after transfer of the plant to eCO2. To discriminate between the effects of eCO2 and those of nitrogen deficiency, we used three different levels of N application. We found that a decline in the leaf soluble protein content (on a leaf area basis) at eCO2 was only observed under N deficiency. The length and width of the leaf blade were reduced by both eCO2 and N deficiency, whereas the blade thickness was increased by eCO2 but was not affected by N deficiency. The change in length by eCO2 became detectable in the secondly fully expanded leaf, and those in width and thickness in the thirdly fully expanded leaf, which were at the leaf developmental stages P4 and P3, respectively, at the onset of the eCO2 treatment. The decreased blade length at eCO2 was associated with a decrease in the epidermal cell number on the adaxial side and a reduction in cell length on the abaxial side. The decreased width resulted from decreased numbers of small vascular bundles and epidermal cell files. The increased thickness was ascribed mainly to enhanced development of bundle sheath extensions at the ridges of vascular bundles. These observations enable us to identify the sites of action of eCO2 on rice leaf development.

Lessons from engineering a single-cell C(4) photosynthetic pathway into rice.

The transfer of C(4) plant traits into C(3) plants has long been a strategy for improving the photosynthetic performance of C(3) plants. The introduction of a pathway mimicking the C(4) photosynthetic pathway into the mesophyll cells of C(3) plants was only a realistic approach when transgenic technology was sufficiently well developed and widely adopted. Here an attempt to introduce a single-cell C(4)-like pathway in which CO(2) capture and release occur in the mesophyll cell, such as the one found in the aquatic plant Hydrilla verticillata (L.f.) Royle, into rice (Oryza sativa L.) is described. Four enzymes involved in this pathway were successfully overproduced in the transgenic rice leaves, and 12 different sets of transgenic rice that overproduce these enzymes independently or in combination were produced and analysed. Although none of these transformants has yet shown dramatic improvements in photosynthesis, these studies nonetheless have important implications for the evolution of C(4) photosynthetic genes and their metabolic regulation, and have shed light on the unique aspects of rice physiology and metabolism. This article summarizes the lessons learned during these attempts to engineer single-cell C(4) rice.

Role of OsNPR1 in rice defense program as revealed by genome-wide expression analysis.

NPR1 is a central regulator of salicylic-acid (SA)-mediated defense signaling in Arabidopsis. Here, we report the characterization of OsNPR1, an Oryzae sativa (rice) ortholog of NPR1, focusing on its role in blast disease resistance and identification of OsNPR1-regulated genes. Blast resistance tests using OsNPR1 knockdown and overexpressing rice lines demonstrated the essential role of OsNPR1 in benzothiadiazole (BTH)-induced blast resistance. Genome-wide transcript profiling using OsNPR1-knockdown lines revealed that 358 genes out of 1,228 BTH-upregulated genes and 724 genes out of 1,069 BTH-downregulated genes were OsNPR1-dependent with respect to BTH responsiveness, thereby indicating that OsNPR1 plays a more vital role in gene downregulation. The OsNPR1-dependently downregulated genes included many of those involved in photosynthesis and in chloroplast translation and transcription. Reduction of photosynthetic activity after BTH treatment and its negation by OsNPR1 knockdown were indeed reflected in the changes in Fv/Fm values in leaves. These results imply the role of OsNPR1 in the reallocation of energy and resources during defense responses. We also examined the OsNPR1-dependence of SA-mediated suppression of ABA-induced genes.

Phosphoenolpyruvate carboxylase intrinsically located in the chloroplast of rice plays a crucial role in ammonium assimilation.

Phosphoenolpyruvate carboxylase (PEPC) is a key enzyme of primary metabolism in bacteria, algae, and vascular plants, and is believed to be cytosolic. Here we show that rice (Oryza sativa L.) has a plant-type PEPC, Osppc4, that is targeted to the chloroplast. Osppc4 was expressed in all organs tested and showed high expression in the leaves. Its expression in the leaves was confined to mesophyll cells, and Osppc4 accounted for approximately one-third of total PEPC protein in the leaf blade. Recombinant Osppc4 was active in the PEPC reaction, showing V(max) comparable to cytosolic isozymes. Knockdown of Osppc4 expression by the RNAi technique resulted in stunting at the vegetative stage, which was much more marked when rice plants were grown with ammonium than with nitrate as the nitrogen source. Comparison of leaf metabolomes of ammonium-grown plants suggested that the knockdown suppressed ammonium assimilation and subsequent amino acid synthesis by reducing levels of organic acids, which are carbon skeleton donors for these processes. We also identified the chloroplastic PEPC gene in other Oryza species, all of which are adapted to waterlogged soil where the major nitrogen source is ammonium. This suggests that, in addition to glycolysis, the genus Oryza has a unique route to provide organic acids for ammonium assimilation that involves a chloroplastic PEPC, and that this route is crucial for growth with ammonium. This work provides evidence for diversity of primary ammonium assimilation in the leaves of vascular plants.

Overproduction of C4 photosynthetic enzymes in transgenic rice plants: an approach to introduce the C4-like photosynthetic pathway into rice.

Four enzymes, namely, the maize C(4)-specific phosphoenolpyruvate carboxylase (PEPC), the maize C(4)-specific pyruvate, orthophosphate dikinase (PPDK), the sorghum NADP-malate dehydrogenase (MDH), and the rice C(3)-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 CO(2) assimilation, while in the case of PEPC it was slightly reduced. The reduction in CO(2) 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 CO(2) assimilation, however, was more marked at higher CO(2) concentrations, an indication that overproduction of the four enzymes in combination did not act to concentrate CO(2) 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 C(4)-like photosynthetic pathway into C(3) plants, are discussed.

Glutamate:glyoxylate aminotransferase modulates amino acid content during photorespiration.

In photorespiration, peroxisomal glutamate:glyoxylate aminotransferase (GGAT) catalyzes the reaction of glutamate and glyoxylate to produce 2-oxoglutarate and glycine. Previous studies demonstrated that alanine aminotransferase-like protein functions as a photorespiratory GGAT. Photorespiratory transamination to glyoxylate, which is mediated by GGAT and serine glyoxylate aminotransferase (SGAT), is believed to play an important role in the biosynthesis and metabolism of major amino acids. To better understand its role in the regulation of amino acid levels, we produced 42 GGAT1 overexpression lines that express different levels of GGAT1 mRNA. The levels of free serine, glycine, and citrulline increased markedly in GGAT1 overexpression lines compared with levels in the wild type, and levels of these amino acids were strongly correlated with levels of GGAT1 mRNA and GGAT activity in the leaves. This accumulation began soon after exposure to light and was repressed under high levels of CO(2). Light and nutrient conditions both affected the amino acid profiles; supplementation with NH(4)NO(3) increased the levels of some amino acids compared with the controls. The results suggest that the photorespiratory aminotransferase reactions catalyzed by GGAT and SGAT are both important regulators of amino acid content.

Characterization and functional analysis of phosphoenolpyruvate carboxylase kinase genes in rice.

Phosphoenolpyruvate carboxylase (PEPC), a key enzyme of primary metabolism of higher plants, is regulated by reversible phosphorylation, which is catalyzed by PEPC kinase (PPCK). Rice has three functional PPCK genes, OsPPCK1, OsPPCK2 and OsPPCK3, all of which have an intron close to the 3' end of the coding region. A novel control mechanism was found for expression of OsPPCK2, namely alternative transcription initiation, and two different transcripts were detected. The four different transcripts of the OsPPCK genes showed different expression patterns. While OsPPCK1 and OsPPCK3 were highly expressed in roots and at low levels in other organs, the two OsPPCK2 transcripts were expressed in all organs. OsPPCK3 was expressed mostly at night, while the long OsPPCK2 transcripts were present in the leaves only in the daytime. Nitrate supplementation of leaves selectively induced expression of both OsPPCK2 transcripts, while phosphate starvation only induced the shorter one. Such diverse expression patterns of OsPPCK genes suggest the importance and variety of strict activity regulation of PEPC in rice. From the correlation between gene expression and the phosphorylation level of PEPC, which was monitored as that of the maize PEPC expressed in transgenic rice plants, it was concluded that the short OsPPCK2 transcripts were expressed in rice leaf mesophyll cells upon nitrogen supplementation and phosphate starvation, whereas OsPPCK3 participated in the nocturnal phosphorylation of PEPC in these cells. Expression of PPCK proteins in rice leaves was detected by immunoblotting using a specific antiserum, and the expression of two different OsPPCK2 proteins derived from alternative transcription initiation was confirmed.

Identification of C4 responsive genes in the facultative C4 plant Hydrilla verticillata.

The aquatic monocot Hydrilla verticillata (L.f.) Royle is a well-documented facultative C4 NADP-malic enzyme species in which the C4 and Calvin cycles operate in the same cell with the specific carboxylases confined to the cytosol and chloroplast, respectively. Several key components had already been characterized at the molecular level, thus the purpose of this study was to begin to identify other, less obvious, elements that may be necessary for a functional single-cell C4 system. Using differential display, mRNA populations from C3 and C4 H. verticillata leaves were screened and expression profiles compared. From this study, 65 clones were isolated and subjected to a customized macroarray analysis; 25 clones were found to be upregulated in C4 leaves. Northern and semi-quantitative RT-PCR analyses were used for confirmation. From these screenings, 13 C4 upregulated genes were identified. Among these one encoded a previously recognized C4 phosphoenolpyruvate carboxylase, and two encoded distinct pyruvate orthophosphate dikinase isoforms, new findings for H. verticillata. Genes that encode a transporter, an aminotransferase and two chaperonins were also upregulated. Twelve false positives, mostly housekeeping genes, were determined from the Northern/semi-quantitative RT-PCR analyses. Sequence data obtained in this study are listed in the dbEST database (DV216698 to DV216767). As a single-cell C4 system that lacks Kranz anatomy, a better understanding of how H. verticillata operates may facilitate the design of a transgenic C4 system in a C3 crop species.

Release and reactive-oxygen-mediated damage of the oxygen-evolving complex subunits of PSII during photoinhibition.

Under photoinhibitory illumination of spinach PSII membranes, the oxygen-evolving complex subunits, OEC33, 24 and 18, were released from PSII. The liberated OEC33 and also OEC24 to a lesser extent were subsequently damaged and then exhibited smeared bands in SDS/urea-PAGE. Once deteriorated, OEC33 could not bind to PSII. The effects of scavengers and chelating reagents on the damage indicated that hydroxyl radicals generated from superoxide in the presence of metal ions were responsible for the damage. These results suggest that, like the D1 protein of the PSII reaction center complex, OEC subunits suffer oxidative damage and turnover under illumination.

Degradation of the D1 protein of photosystem II under illumination in vivo: two different pathways involving cleavage or intermolecular cross-linking.

The D1 protein of the photosystem II reaction center turns over the most rapidly of all the proteins of the thylakoid membrane under illumination in vivo. In vitro, the D1 protein sustained cleavage in a surface-exposed loop (DE loop) or cross-linking with another reaction center protein, the D2 protein or cytochrome b(559), under illumination. We found that the D1 protein was damaged in essentially the same way in vivo, although the resultant fragments and cross-linked adducts barely accumulated due to digestion by proteases. In vitro studies detected a novel stromal protease(s) that digested the adducts but not the monomeric D1 protein. These observations suggest that, in addition to cleavage, the cross-linking reactions themselves are processes involved in complete degradation of the D1 protein in vivo. Peptide mapping experiments located the cross-linking sites with the D2 protein among residues 226-244, which includes the cross-linking site with cytochrome b(559) [Barbato, R., et al. (1995) J. Biol. Chem. 270, 24032-24037], in the N-terminal part of the DE loop, while N-terminal amino acid sequencing of the fragment located the cleavage site around residue 260 in the C-terminal part of the loop. We propose a model explaining the occurrence of simultaneous cleavage and cross-linking and discuss the mechanisms of complete degradation of the D1 protein in vivo.

Metabolic consequences of overproduction of phosphoenolpyruvate carboxylase in C3 plants.

Phosphoenolpyruvate carboxylase (PEPC) has a variety of functions in plants, including a major anaplerotic role in replenishing the tricarboxylic acid cycle with intermediates to meet the demand of carbon skeletons for synthesis of organic acids and amino acids. Various transgenic C3 plants that overproduce PEPC have been produced and analyzed in detail. The results indicate that foreign PEPC is under the control of the regulatory mechanisms intrinsic to the host plant and down-regulated so as not to cause detrimental metabolic effects, although the anaplerotic reaction is slightly enhanced by the foreign PEPC. By use of foreign PEPCs that can avert such regulation, metabolic flow is largely directed toward synthesis of organic acids and amino acids. Observations with transgenic C3 plants also shed light on the interrelation among various metabolic pathways inside the cell.