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CO2‐responsive CCT protein (CRCT) is a positive regulator of starch synthesis–related genes such as ADP‐glucose pyrophosphorylase large subunit 1 and starch branching enzyme I particularly in the leaf sheath of rice (Oryza sativa L.). The promoter GUS analysis revealed that CRCT expressed exclusively in the vascular bundle, whereas starch synthesis–related genes were expressed in different sites such as mesophyll cell and starch storage parenchyma cell. However, the chromatin immunoprecipitation (ChIP) using a FLAG‐CRCT overexpression line and subsequent qPCR analyses showed that the 5′‐flanking regions of these starch synthesis–related genes tended to be enriched by ChIP, suggesting that CRCT can bind to the promoter regions of these genes. The monomer of CRCT is 34.2 kDa; however, CRCT was detected at 270 kDa via gel filtration chromatography, suggesting that CRCT forms a complex in vivo. Immunoprecipitation and subsequent MS analysis pulled down several 14‐3‐3‐like proteins. A yeast two‐hybrid analysis and bimolecular fluorescence complementation assays confirmed the interaction between CRCT and 14‐3‐3‐like proteins. Although there is an inconsistency in the place of expression, this study provides important findings regarding the molecular function of CRCT to control the expression of key starch synthesis–related genes. CO2‐responsive CCT protein (CRCT) is a positive regulator of starch synthesis in vegetative organs of rice. Here we show that CRCT can interact with 14‐3‐3 proteins and controls the transcription of key starch synthesis–related genes such as ADP‐glucose pyrophosphorylase large subunit 1. [ABSTRACT FROM AUTHOR]

The effects of overexpression of Rubisco activase on photosynthesis were studied in transgenic rice expressing barley or maize Rubisco activase. Immunoblot and SDS–PAGE analyses showed that transgenic lines from both gene constructs expressed the foreign Rubisco activase at high levels. The activation state of Rubisco in transgenic lines was slightly higher than that in non-transgenic plants (NT). In addition, light activation of Rubisco was significantly more rapid in transgenic lines compared with NT. These findings indicate that the overexpression of Rubisco activase can enhance Rubisco activation. However, despite enhanced activation of Rubisco in these transgenic plants, the CO2 assimilation rate at ambient CO2 conditions was decreased. This decrease in CO2 assimilation rate was observed in both young developing and mature leaves independent of nitrogen nutrition. The contents of nitrogen and Chl did not differ significantly between transformants and NT; however, Rubisco content was substantially decreased in transgenic lines. There was no evidence for reduced transcription of RbcS or RbcL in these transgenic lines; in fact, transcript levels were marginally increased compared with NT. These results indicate that the overexpression of Rubisco activase leads to a decrease in Rubisco content, possibly due to post-transcriptional mechanisms. [ABSTRACT FROM PUBLISHER]

Abstract: Rising atmospheric CO2 concentration is accompanied by global warming. However, interactive effects of elevated CO2 and soil temperature have not been well studied in plants. Here, we studied the effects of free air CO2 enrichment (FACE, ambient +20Pa), elevated soil temperature (+2°C), or a combination of both on gene expression in developing 10th and fully expanded flag leaves of rice by microarray. Gene expression was significantly affected by FACE and 400–600 up-regulated or down-regulated genes were identified in each experimental condition. Overall, elevated soil temperature was likely to increase the number of differently expressed genes. Among them, only 31 up-regulated and 83 down-regulated genes were common irrespective of the leaf stage and soil temperature. These included up-regulation of sucrose transporter and fructosyltransferase and down-regulation of chlorophyll a/b binding protein, nitrate transporter and aquaporin, though most of differently expressed genes were functionally unknown. Although there were some exceptions, the expression of most of primary metabolism genes was not significantly altered by FACE. The expression of genes related to CO2 fixation such as Rubisco and carbonic anhydrase were down-regulated, whereas a large proportion of genes related to sucrose synthesis, glycolysis, TCA cycle and N fixation were up-regulated under FACE, though changes were small in all cases. These results might reflect the transcriptional adaptation of metabolism to FACE, reducing the CO2 fixation capacity and stimulating the dark respiration. [Copyright &y& Elsevier]

Abstract: Protease activities and its relation to the contents of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and Rubisco activase were investigated in detached leaves of rice (Oryza sativa L.) floated on the solutions containing abscisic acid (ABA) or benzyladenine (BA). Rubisco and Rubisco activase contents were decreased during the time course and the decreases were enhanced by ABA and suppressed by BA. The decrease in Rubisco activase was faster than that in Rubisco. SDS-dependent protease activities at 50–70kDa (rice SDS-dependent protease: RSP) analyzed by the gelatin containing PAGE were significantly enhanced by ABA. RSPs were also increased in attached leaves during senescence. RSPs had the pH optimum of 5.5, suggesting that RSPs are vacuolar protease. Both decrease in Rubisco and Rubisco activase contents and increase in RSPs activities were suppressed by cycloheximide. These findings indicate that the activities of RSPs are well correlated with the decrease in these protein contents. Immunoblotting analysis showed that Rubisco in the leaf extracts was completely degraded by 5h at pH 5.5 with SDS where it was optimal condition for RSPs. However, the degradation of Rubisco did not proceed at pH 7.5 without SDS where it is near physiological condition for stromal proteins. Rubisco activase was degraded at similar rate under both conditions. These results suggest that RSPs can functions in a senescence related degradation system of chloroplast protein in rice leaves. Rubisco activase would be more susceptible to proteolysis than Rubisco under physiological condition and this could affect the contents of these proteins in leaves. [ABSTRACT FROM AUTHOR]

Abstract: Effects of elevated CO2 (68Pa versus ambient 38Pa) on gene expression were studied in rice leaves grown in soil medium with three different nitrogen conditions (0, 0.6 and 1.2g N per 8-L pot) in CO2 controlled chambers. Soluble protein contents were slightly decreased in leaves grown under elevated CO2 regardless of N supplies, whereas the polypeptide profiles of soluble protein analyzed by 2DE using the same amount of protein were totally unchanged between ambient and elevated CO2. In contrast, gene expressions examined by microarray analyses were significantly affected by elevated CO2. Forty-six up-regulated genes (>1.5-fold) and 35 down-regulated genes (<0.68-fold) were identified and these included many signal transduction and transcription regulation related genes. By contrast, the expressions of most of the genes for primary metabolism were not significantly altered. Although changes were small, the expressions of genes for enzymes involved in CO2 fixation (carbonic anhydrase, Rubisco, phosphoglycerate kinase and glyceraldehyde-3-phosphate dehydrogenase) were down-regulated, whereas that of genes encoding enzymes for RuBP regeneration (fructose bisphosphate phosphatase, fructose bisphosphate aldolase, sedoheptulose bisphosphate phosphatase and phosphoribulokinase) and starch synthesis (ADP-glucose pyrophosphorylase and starch synthase) were up-regulated under elevated CO2. These results suggest that some sets of genes involved in primary metabolism pathway in the chloroplast are co-regulated by elevated CO2. [Copyright &y& Elsevier]

Phospho enolpyruvate 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. [ABSTRACT FROM AUTHOR]

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. [Copyright &y& Elsevier]

Global warming threatens food security by decreasing crop yields through damage to photosynthetic systems, especially Rubisco activation. We examined whether co‐overexpression of Rubisco and Rubisco activase improves the photosynthetic and growth performance of rice under high temperatures. We grew three rice lines—the wild‐type (WT), a Rubisco activase–overexpressing line (oxRCA) and a Rubisco‐ and Rubisco activase–co‐overexpressing line (oxRCA‐RBCS)—and analysed photosynthesis and biomass at 25 and 40°C. Compared with the WT, the Rubisco activase content was 153% higher in oxRCA and 138% higher in oxRCA‐RBCS, and the Rubisco content was 27% lower in oxRCA and similar in oxRCA‐RBCS. The CO2 assimilation rate (A) of WT was lower at 40°C than at 25°C, attributable to Rubisco deactivation by heat. On the other hand, that of oxRCA and oxRCA‐RBCS was maintained at 40°C, resulting in higher A than WT. Notably, the dry weight of oxRCA‐RBCS was 26% higher than that of WT at 40°C. These results show that increasing the Rubisco activase content without the reduction of Rubisco content could improve yield and sustainability in rice at high temperature. Co‐overexpression of Rubisco and Rubisco activase maintains Rubisco activation state at high temperatures, contribute to higher CO2 assimilation rate than wild type. Notably, co‐overexpression line showed greater biomass production than WT at high temperatures. [ABSTRACT FROM AUTHOR]

Long-term high CO2 exposure accelerates photosynthetic induction response due to rapid light increase. However, it is unclear whether the acceleration is caused by acclimation of photosynthetic components (long-term CO2 effect) and/or by the sufficient substrate under high CO2 at the measurement (short-term CO2 effect). Populus koreana × trichocarpa cv. Peace has wide-open stomata almost not responding to changes of photon flux density. Using this species, we examined the long- and short-term CO2 effects on photosynthetic induction by focusing on biochemical components. We grew the plants under [CO2] of 380, 700 and 1020 μmol CO2 mol−1 air and measured the photosynthetic induction response under [CO2] of 380 and 1020 μmol CO2 mol−1 air. Despite significant reduction in Rubisco content and light-saturated photosynthetic rate in the leaves from the high growth CO2, the photosynthetic induction time was similar in leaves from different growth CO2 plants when measurement [CO2] was the same. The induction, however, was significantly fast at the higher than at the lower measurement [CO2], regardless of growth CO2 of the plants. These results demonstrate that the acceleration of apparent photosynthetic induction under high CO2 environment was mainly contributed by a short-term CO2 effect rather than by a long-term acclimation effect when stomatal limitation is not the major factor. [ABSTRACT FROM AUTHOR]

Rice is the model C3 crop for investigating the starch biosynthesis mechanism in endosperm because of its importance in grain production. However, little is known about starch biosynthesis in the vegetative organs of rice. In this study, we used novel rice mutants by inserting Tos17 into the starch synthase (SS) IIIb gene, which is mainly expressed in the leaf sheath (LS) and leaf blade (LB), and an ss1 mutant to clarify the differences in roles among SS isozymes during starch biosynthesis. Native polyacrylamide gel electrophoresis (PAGE)/activity staining for SS, using LS and LB of ss mutants, revealed that the lowest migrating SS activity bands on the gel were derived from SSIIIb activity and those of two ss3b mutants were not detected. The apparent amylose content of LS starch of ss3b mutants increased. Moreover, the chain-length distribution and size-exclusion chromatography analysis using ss mutants showed that SSIIIb and SSI synthesize the B2–B3 chain and A–B1 chain of amylopectin in the LS and LB respectively. Interestingly, we also found that starch contents were decreased in the LS and LB of ss3b mutants, although SSI deficiency did not affect the starch levels. All these results indicated that SSIIIb synthesizes the long chain of amylopectin in the LS and LB similar to SSIIIa in the endosperm, while SSI synthesizes the short chain in the vegetative organ as the same in the endosperm. [ABSTRACT FROM AUTHOR]

Under field conditions, the leaves of plants are exposed to fluctuating light, as observed in sunfleck. The duration and frequency of sunfleck, which is caused by the canopy being blown by the wind, are in the ranges from 0.2 to 50 s, and from 0.004 to 1 Hz, respectively. Furthermore, >60% of the sunfleck duration ranges from 0.2 to 0.8 s. In the present research, we analyzed the effects of repetitive illumination by short-pulse (SP) light of sunflower leaves on the photosynthetic electron flow. The duration of SP light was set in the range from 10 to 300 ms. We found that repetitive illumination with SP light did not induce the oxidation of P700 in PSI, and mainly inactivated PSI. Increases in the intensity, duration and frequency of SP light enhanced PSI photoinhibition. PSI photoinhibition required the presence of O2. The inactivation of PSI suppressed the net CO2 assimilation. On the other hand, the increase in the oxidized state of P700 suppressed PSI inactivation. That is, PSI with a reduced reaction center would produce reactive oxygen species (ROS) by SP light, leading to PSI photodamage. This mechanism probably explains the PSI photodamage induced by constant light. [ABSTRACT FROM PUBLISHER]

To enable prediction of future rice production in a changing climate, we need to understand the interactive effects of temperature and elevated [CO2] (E[CO2]). We therefore examined if the effect of E[CO2] on the light-saturated leaf photosynthetic rate (Asat) was affected by soil and water temperature (NT, normal; ET, elevated) under open-field conditions at the rice free-air CO2 enrichment (FACE) facility in Shizukuishi, Japan, in 2007 and 2008. Season-long E[CO2] (+200 µmol mol−1) increased Asat by 26%, when averaged over two years, temperature regimes and growth stages. The effect of ET (+2°C) on Asat was not significant at active tillering and heading, but became negative and significant at mid-grain filling; Asat in E[CO2]–ET was higher than in ambient [CO2] (A[CO2])–NT by only 4%. Photosynthetic down-regulation at E[CO2] also became apparent at mid-grain filling; Asat compared at the same [CO2] in the leaf cuvette was significantly lower in plants grown in E[CO2] than in those grown in A[CO2]. The additive effects of E[CO2] and ET decreased Asat by 23% compared with that of A[CO2]–NT plants. Although total crop nitrogen (N) uptake was increased by ET, N allocation to the leaves and to Rubisco was reduced under ET and E[CO2] at mid-grain filling, which resulted in a significant decrease (32%) in the maximum rate of ribulose-1,5-bisphosphate carboxylation on a leaf area basis. Because the change in N allocation was associated with the accelerated phenology in E[CO2]–ET plants, we conclude that soil and water warming accelerates photosynthetic down-regulation at E[CO2]. [ABSTRACT FROM AUTHOR]

Triacylglycerols (TAGs) are the major component of plant storage lipids such as oils. Acyl-CoA:diacylglycerol acyltransferase (DGAT) catalyzes the final step of the Kennedy pathway, and is mainly responsible for plant oil accumulation. We previously found that the activity of Vernonia DGAT1 was distinctively higher than that of Arabidopsis and soybean DGAT1 in a yeast microsome assay. In this study, the DGAT1 cDNAs of Arabidopsis, Vernonia, soybean, and castor bean were introduced into Arabidopsis. All Vernonia DGAT1 -expressing lines showed a significantly higher oil content (49% mean increase compared with the wild-type) followed by soybean and castor bean. Most Arabidopsis DGAT1 -overexpressing lines did not show a significant increase. In addition to these four DGAT1 genes, sunflower, Jatropha, and sesame DGAT1 genes were introduced into a TAG biosynthesis-defective yeast mutant. In the yeast expression culture, DGAT1s from Arabidopsis, castor bean, and soybean only slightly increased the TAG content; however, DGAT1s from Vernonia, sunflower, Jatropha, and sesame increased TAG content >10-fold more than the former three DGAT1s. Three amino acid residues were characteristically common in the latter four DGAT1s. Using soybean DGAT1 , these amino acid substitutions were created by site-directed mutagenesis and substantially increased the TAG content. [ABSTRACT FROM AUTHOR]

The role of Rubisco activase in steady-state and non-steady-state photosynthesis was analyzed in wild-type ( Oryza sativa) and transgenic rice that expressed different amounts of Rubisco activase. Below 25°C, the Rubisco activation state and steady-state photosynthesis were only affected when Rubisco activase was reduced by more than 70%. However, at 40°C, smaller reductions in Rubisco activase content were linked to a reduced Rubisco activation state and steady-state photosynthesis. As a result, overexpression of maize Rubisco activase in rice did not lead to an increase of the Rubisco activation state, nor to an increase in photosynthetic rate below 25°C, but had a small stimulatory effect at 40°C. On the other hand, the rate at which photosynthesis approached the steady state following an increase in light intensity was rapid in Rubisco activase-overexpressing plants, intermediate in the wild-type, and slowest in antisense plants at any leaf temperature. In Rubisco activase-overexpressing plants, Rubisco activation state at low light was maintained at higher levels than in the wild-type. Thus, rapid regulation by Rubisco activase following an increase in light intensity and/or maintenance of a high Rubisco activation state at low light would result in a rapid increase in Rubisco activation state and photosynthetic rate following an increase in light intensity. It is concluded that Rubisco activase plays an important role in the regulation of non-steady-state photosynthesis at any leaf temperature and, to a lesser extent, of steady-state photosynthesis at high temperature. [ABSTRACT FROM AUTHOR]

Elucidates the molecular engineering of C[sub4] photosynthesis. Metabolic engineering of C[sub4] enzymes in C[sub4] plants; Molecular engineering of C[sub4] enzymes in C[sub3] plants; Implications for research.

The chloroplastic NADP-malic enzyme (NADP-ME) is a key enzyme of the C4 photosynthesis pathway in NADP-ME type C4 plants such as maize. To express the chloroplastic NADP-ME in leaves of a C3 plant, rice, full-length cDNAs encoding the rice C3-specific isoform and the maize C4-specific isoform of the enzyme were expressed under the control of the rice Cab promoter. Transformants carrying the rice cDNA showed the NADP-ME activities in the leaves less than several-fold that of non-transformants, while those carrying the maize cDNA showed activities up to 30-fold that of non-transformants or about 60% of the NADP-ME activity of maize leaves. These results indicate that expression of the rice C3-specific NADP-ME is suppressed at co- and/or post-transcriptional levels by some regulation mechanisms intrinsic to rice, while that of the foreign C4-specific isoform can escape from such suppression. The accumulation of the maize C4-specific NADP-ME led to bleaching of leaf color and growth hindrance in rice plants under natural light. These deteriorative effects resulted from enhanced photoinhibition of photosynthesis due to an increase in the level of NADPH inside the chloroplast by the action of the maize enzyme. [ABSTRACT FROM PUBLISHER]

Photosynthetic rate at the present atmospheric condition is limited by the CO 2 -fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) because of its extremely low catalytic rate (k cat) and poor affinity for CO 2 (K c) and specificity for CO 2 (S c/o). Rubisco in C 4 plants generally shows higher k cat than that in C 3 plants. Rubisco consists of eight large subunits and eight small subunits (RbcS). Previously, the chimeric incorporation of sorghum C 4 -type RbcS significantly increased the k cat of Rubisco in a C 3 plant, rice. In this study, we knocked out rice RbcS multigene family using the CRISPR-Cas9 technology and completely replaced rice RbcS with sorghum RbcS in rice Rubisco. Obtained hybrid Rubisco showed almost C 4 plant-like catalytic properties, i.e., higher k cat , higher K c , and lower S c/o. Transgenic lines expressing the hybrid Rubisco accumulated reduced levels of Rubisco, whereas they showed slightly but significantly higher photosynthetic capacity and similar biomass production under high CO 2 condition compared with wild-type rice. High-resolution crystal structural analysis of the wild-type Rubisco and hybrid Rubisco revealed the structural differences around the central pore of Rubisco and the βC-βD hairpin in RbcS. We propose that such differences, particularly in the βC-βD hairpin, may impact the flexibility of Rubisco catalytic site and change its catalytic properties. Rubisco is a key enzyme of photosynthetic CO 2 fixation. Hybrid Rubisco with replacement of rice Rubisco small subunit with sorghum counterpart expressed in rice exhibited C 4 plant-like high catalytic activity. Transgenic rice expressing the hybrid Rubisco showed slightly higher photosynthetic capacity. Based on crystal structural analysis, we speculate a possible function of βC-βD hairpin of small subunit in determining the catalytic activity. [ABSTRACT FROM AUTHOR]

CO2-responsive CCT protein (CRCT) is suggested to be a positive regulator of starch biosynthesis in the leaf sheaths of rice, regulating the expression levels of starch biosynthesis-related genes. In this study, the effects of CRCT expression levels on the expression of starch biosynthesis-related enzymes and the quality of starch were studied. Using native-PAGE/activity staining and immunoblotting, we found that the protein levels of starch synthase I, branching enzyme I, branching enzyme IIa, isoamylase 1 and phosphorylase 1 were largely correlated with the CRCT expression levels in the leaf sheaths of CRCT transgenic lines. In contrast, the CRCT expression levels largely did not affect the expression levels and/or activities of starch biosynthesis-related enzymes in the leaf blades and endosperm tissues. The analysis of the chain-length distribution of starch in the leaf sheaths showed that short chains with a degree of polymerization from 5 to 14 were increased in the overexpression lines but decreased in the knockdown lines. The amylose content of starch in the leaf sheath was greatly increased in the overexpression lines. In contrast, the molecular weight of the amylopectin of starch in the leaf sheath of overexpression lines did not change compared with those of the non-transgenic rice. These results suggest that CRCT can control the quality and the quantity of starch in the leaf sheath by regulating the expression of particular starch biosynthesis-related enzymes. [ABSTRACT FROM AUTHOR]

Sugar metabolism pathways such as photosynthesis produce dicarbonyls, e.g. methylglyoxal (MG), which can cause cellular damage. The glyoxalase (GLX) system comprises two enzymes GLX1 and GLX2, and detoxifies MG; however, this system is poorly understood in the chloroplast, compared with the cytosol. In the present study, we determined GLX1 and GLX2 activities in spinach chloroplasts, which constituted 40% and 10%, respectively, of the total leaf glyoxalase activity. In Arabidopsis thaliana, five GFP-fusion GLXs were present in the chloroplasts. Under high CO2 concentrations, where increased photosynthesis promotes the MG production, GLX1 and GLX2 activities in A. thaliana increased and the expression of AtGLX1-2 and AtGLX2-5 was enhanced. On the basis of these findings and the phylogeny of GLX in oxygenic phototrophs, we propose that the GLX system scavenges MG produced in chloroplasts during photosynthesis. [ABSTRACT FROM AUTHOR]

The semiaquatic plants, S agittaria trifolia and S agittaria pygmaea, are perennial and troublesome weeds of rice paddy fields. These species mainly reproduce vegetatively via tubers, which sprout after the rice paddy fields are irrigated. Sprouting finally leads to the emergence of the shoots from the water in the paddy fields. In both species, the first internode elongates around the time of emergence and this lifts the shoot meristems in the soil, aiding in the establishment of the young plants. Therefore, elongation of the internode is a factor that determines the weed damage to rice caused by these species. In this study, the first internode elongation of both species was characterized. During emergence, S . trifolia and S . pygmaea showed distinct growth patterns. In S . trifolia, the internodes elongated before the emergence of the shoots from the water; whereas, in S . pygmaea, the internodes started to elongate only after emergence. We examined environmental prerequisites for internode elongation, and found that the internode elongation of S . trifolia was induced by submergence and was independent of the soil cover, while that of S . pygmaea required both submergence and covering. Next, we determined which gaseous factors were responsible for internode elongation. Treatment with ethylene, which enhances growth of several other aquatic and semiaquatic plants, did not stimulate internode elongation in either species under an anaerobic condition. Our results suggested that the gaseous factor stimulating internode elongation in S . trifolia and S . pygmaea may be oxygen depletion and carbon dioxide, respectively. [ABSTRACT FROM AUTHOR]

Rubisco limits photosynthetic CO2 fixation because of its low catalytic turnover rate (kcat) and competing oxygenase reaction. Previous attempts to improve the catalytic efficiency of Rubisco by genetic engineering have gained little progress. Here we demonstrate that the introduction of the small subunit (RbcS) of high kcat Rubisco from the C4 plant sorghum (Sorghum bicolor) significantly enhances kcat of Rubisco in transgenic rice (Oryza sativa). Three independent transgenic lines expressed sorghum RbcS at a high level, accounting for 30%, 44%, and 79% of the total RbcS. Rubisco was likely present as a chimera of sorghum and rice RbcS, and showed 1.32- to 1.50-fold higher kcat than in nontransgenic rice. Rubisco from transgenic lines showed a higher km for CO2 and slightly lower specificity for CO2 than nontransgenic controls. These results suggest that Rubisco in rice transformed with sorghum RbcS partially acquires the catalytic properties of sorghum Rubisco. Rubisco content in transgenic lines was significantly increased over wild-type levels but Rubisco activation was slightly decreased. The expression of sorghum RbcS did not affect CO2 assimilation rates under a range of CO2 partial pressures. The Jmax/Vcmax ratio was significantly lower in transgenic line compared to the nontransgenic plants. These observations suggest that the capacity of electron transport is not sufficient to support the increased Rubisco capacity in transgenic rice. Although the photosynthetic rate was not enhanced, the strategy presented here opens the way to engineering Rubisco for improvement of photosynthesis and productivity in the future. [ABSTRACT FROM AUTHOR]

The transfer of C4 plant traits into C3 plants has long been a strategy for improving the photosynthetic performance of C3 plants. The introduction of a pathway mimicking the C4 photosynthetic pathway into the mesophyll cells of C3 plants was only a realistic approach when transgenic technology was sufficiently well developed and widely adopted. Here an attempt to introduce a single-cell C4-like pathway in which CO2 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 C4 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 C4 rice. [ABSTRACT FROM PUBLISHER]

Phosphoeno!pyruvate 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 Vmax 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 Oiyza 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM PUBLISHER]

Using an Agrobacterium-mediated transformation system, we have introduced the intact gene of maize phosphoenolpyruvate carboxylase (PEPC), which catalyzes the initial fixation of atmospheric CO[SUB2] in C[SUB4] plants into the C[SUB3] crop rice. Most transgenic rice plants showed high-level expression of the maize gene; the activities of PEPC in leaves of some transgenic plants were two- to threefold higher than those in maize, and the enzyme accounted for up to 12% of the total leaf soluble protein. RNA gel blot and Southern blot analyses showed that the level of expression of the maize PEPC in transgenic rice plants correlated with the amount of transcript and the copy number of the inserted maize gene. Physiologically, the transgenic plants exhibited reduced O[SUB2] (inhibition of photosynthesis and photosynthetic rates comparable to those of untransformed plants. The results demonstrate a successful strategy for installing the key biochemical component of the C[SUB4] pathway of photosynthesis in C[SUB3] plants. [ABSTRACT FROM AUTHOR]