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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 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 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.

Background Reduction in rice yield caused by high temperature-induced spikelet sterility has been a serious concern in rice production. To date, several screening methods have been used, although their reproducibility is sometimes poor due to artifacts mainly caused by varietal differences in heading dates and panicle heights (i.e., the distance from the lamps). Methods We have developed a novel assay system for heat-induced spikelet sterility by using artificial rice paddies in phytotrons to conduct a highly reproducible assay throughout a year. Plants restricted to the main culm were treated under a series of heat conditions, and height uniformity of each plant was ensured by using height-adjustable pots. Results Results suggested that a 3-day heat treatment of 35 °C-day/29 °C-night cycles was the most suitable condition. Under the treatment, two distinct groups were identified among nine heat tolerant cultivars, with no varietal difference in panicle temperature, indicating that the system is capable of eliminating the varietal difference in panicle temperature. Conclusions It is concluded that the assay system would be a powerful tool for selecting heat tolerant varieties, as well as the analysis of genetic factors from various cultivars, eliminating potential artifacts.

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.

Foehn-like extreme hot and dry wind conditions (34°C, >2.5 kPa vapor pressure deficit, and 7 m s(-1)) strongly affect grain quality in rice (Oryza sativa L.). This is a current concern because of the increasing frequency and intensity of combined heat and water-deficit stress under climate change. Foehn-induced dry wind conditions during the grain-filling stage increase ring-shaped chalkiness as a result of spatiotemporal reduction in starch accumulation in the endosperm, but kernel growth is sometimes maintained by osmotic adjustment. Here, we assess the effects of dry wind on chalky ring formation in environmentally controlled growth chambers. Our results showed that hot and dry wind conditions that lasted for >24 h dramatically increased chalky ring formation. Hot and dry wind conditions temporarily reduced panicle water potential to -0.65 MPa; however, kernel growth was maintained by osmotic adjustment at control levels with increased transport of assimilate to the growing kernels. Dynamic tracer analysis with a nano-electrospray-ionization Orbitrap mass spectrometer and quantitative polymerase chain reaction analysis revealed that starch degradation was negligible in the short-term treatment. Overall expression of starch synthesis-related genes was found to be down-regulated at moderately low water potential. Because the events observed at low water potential preceded the packing of starch granules in cells, we concluded that reduced rates of starch biosynthesis play a central role in the events of cellular metabolism that are altered at osmotic adjustment, which leads to chalky ring formation under short-term hot and dry wind conditions.

Grain filling ability is mainly affected by the translocation of carbohydrates generated from temporarily stored stem starch in most field crops including rice (Oryza sativa L.). The partitioning of non-structural stem carbohydrates has been recognized as an important trait for raising the yield ceiling, yet we still do not fully understand how carbohydrate partitioning occurs in the stems. In this study, two rice subspecies that exhibit different patterns of non-structural stem carbohydrates partitioning, a japonica-dominant cultivar, Momiroman, and an indica-dominant cultivar, Hokuriku 193, were used as the model system to study the relationship between turgor pressure and metabolic regulation of non-structural stem carbohydrates, by combining the water status measurement with gene expression analysis and a dynamic prefixed 13C tracer analysis using a mass spectrometer. Here, we report a clear varietal difference in turgor-associated starch phosphorylation occurred at the initiation of non-structural carbohydrate partitioning. The data indicated that starch degradation in Hokuriku 193 stems occurred at full-heading, 5 days earlier than in Momiroman, contributing to greater sink filling. Gene expression analysis revealed that expression pattern of the gene encoding α-glucan, water dikinase (GWD1) was similar between two varieties, and the maximum expression level in Hokuriku 193, reached at full heading (4 DAH), was greater than in Momiroman, leading to an earlier increase in a series of amylase-related gene expression in Hokuriku 193. In both varieties, peaks in turgor pressure preceded the increases in GWD1 expression, and changes in GWD1 expression was correlated with turgor pressure. Additionally, a threshold is likely to exist for GWD1 expression to facilitate starch degradation. Taken together, these results raise the possibility that turgor-associated starch phosphorylation in cells is responsible for the metabolism that leads to starch degradation. Because the two cultivars exhibited remarkable varietal differences in the pattern of non-structural carbohydrate partitioning, our findings propose that the observed difference in grain-filling ability originated from turgor-associated regulation of starch phosphorylation in stem parenchyma cells. Further understanding of the molecular mechanism of turgor-regulation may provide a new selection criterion for breaking the yield barriers in crop production.

Foehn-induced dry wind during the grain-filling stage induces shoot water deficit in rice (Oryza sativa L.) plants, which form a ring-shaped chalkiness in their endosperm that degrades milling quality and rice appearance. Air spaces formed in several inner cells cause significant transparency loss due to irregular light reflection. Although starch synthesis was suggested to be retarded by osmotic adjustment at foehn-induced moderately low water potential, the source of these air spaces remains unknown. We hypothesised that the preservation of vacuoles accompanied by a temporary reduction in starch biosynthesis in the inner cells leads to the chalky ring formation. Panicle water status measurement, light and transmission electron microscopic (TEM) observations, and an absolute qPCR analysis were conducted. Most starch synthesis-related genes exhibited temporarily reduced expression in the inner zone in accordance with the decrease in panicle water status. TEM observations provided evidence that vacuolar compartments remained among the loosely packed starch granules in the inner endosperm cells, where a chalky ring appeared after kernel dehydration. Taken together, we propose that vacuolar compartments sustained among the amyloplasts inadequately accumulated in rice endosperm cells and caused air space formation that leads to ring-shaped chalkiness under dry wind conditions.

The activation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), which catalyses CO 2 fixation in photosynthesis, requires the assistance of the regulatory protein Rubisco activase. Rubisco activase promotes carbamylation of Rubisco by releasing inhibitory sugar phosphates bound to the catalytic site of Rubisco in the light. To clarify the effects of Rubisco activase contents on the photosynthesis of rice, we investigated the steady-state photosynthesis and light-induction of photosynthesis in transgenic rice plants, in which leaf Rubisco activase levels were reduced. The reduction in Rubisco activase did not affect steady-state photosynthesis under high light intensity until the Rubisco activase was about 15% of that in control plants. However, light-induction of photosynthesis, namely, increase in photosynthetic rate following a transition from a low to high light intensity, was considerably low in transgenic rice plants with 20−25% Rubisco activase, which was sufficient to support the steady-state photosynthesis. In addition, the Rubisco activase content was highly correlated with the initial rate of Rubisco activation after the increase in light intensity. These results suggest that Rubisco activase in rice leaves largely limits the light-induction of photosynthesis, but not steady-state photosynthesis.

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.

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.

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.