Chisato Masumoto

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 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.

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.

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.

Lessons from engineering a single-cell C4 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.

Overexpression of Rubisco Activase Decreases the Photosynthetic CO2 Assimilation Rate by Reducing Rubisco Content in Rice Leaves

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 CO(2) assimilation rate at ambient CO(2) conditions was decreased. This decrease in CO(2) 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.

Photosynthetic Characteristics of Antisense Transgenic Rice Expressing Reduced Levels of Rubisco Activase

Abstract The activation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), which catalyses CO2 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.

Mechanism of high photosynthetic capacity in BC2F4 lines derived from a cross between Oryza sativa and wild relatives O. rufipogon.

Abstract We found that several BC2F4 lines had high leaf photosynthetic rates under light-saturated and ambient CO2 conditions. These lines are progenies of BC2F1 plants with high photosynthetic capacities which were generated by backcrossing between Oryza rufipogon (W630) and O. sativa cv. Nipponbare, as a recurrent parent. Some photosynthetic characteristics of the BC2F4 lines were investigated to identify the factors increasing photosynthetic rates. Photosynthetic rates of these lines under light-saturated conditions at 50 to 700 ppm CO2 concentrations were higher than those in Nipponbare. The estimated-maximum photosynthetic rates under light-saturated and CO2-saturated conditions in BC2F4 lines were also higher than that in Nipponbare. The photosynthetic rate under light-saturated and ambient CO2 conditions was positively correlated with the carboxylation efficiency as an indicator of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity in vivo rather than stomatal conductance. Initial and total Rubisco activities in vitro tended to be higher in the BC2F4 lines than in Nipponbare. The content of active Rubisco calculated from the activation state of Rubisco was also higher in the BC2F4 lines than in Nipponbare. These results suggest that high photosynthetic capacities of BC2F1 plants can be maintained high in their progenies and high photosynthetic rates under light-saturated and ambient CO2 conditions in the BC2F4 lines are achieved mainly by the high activity of Rubisco due to the high active Rubisco content.

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. Graphical Abstract Two plastidic enolase genes were identified in rice. These were expressed in non-photosynthetic tissues such as vascular bundle of young leaf blade.