Ryu Ohsugi

Rice metal-nicotianamine transporter, OsYSL2, is required for the long-distance transport of iron and manganese.

Rice (Oryza sativa) is indispensable in the diet of most of the worlds population. Thus, it is an important target in which to alter iron (Fe) uptake and homeostasis, so as to increase Fe accumulation in the grain. We previously isolated OsYSL2, a functional iron [Fe(II)]- and manganese [Mn(II)]-nicotianamine complex transporter that is expressed in phloem cells and developing seeds. We produced RNAi (OsYSL2i) and overexpression lines (OXOsYSL2) of OsYSL2. At the vegetative stage in an OsYSL2i line, the Fe and Mn concentrations were decreased in the shoots, and the Fe concentration was increased in the roots. At the reproductive stage, positron-emitting tracer imaging system analysis revealed that Fe translocation to the shoots and seeds was suppressed in OsYSL2i. The Fe and Mn concentrations were decreased in the seeds of OsYSL2i, especially in the endosperm. Moreover, the Fe concentration in OXOsYSL2 was lower in the seeds and shoots, but higher in the roots, compared with the wild type. Furthermore, when OsYSL2 expression was driven by the sucrose transporter promoter, the Fe concentration in the polished rice was up to 4.4-fold higher compared with the wild type. These results indicate that the altered expression of OsYSL2 changes the localization of Fe, and that OsYSL2 is a critical Fe-nicotianamine transporter important for Fe translocation, especially in the shoots and endosperm.

A rice calcium‐dependent protein kinase OsCPK12 oppositely modulates salt‐stress tolerance and blast disease resistance

Calcium-dependent protein kinases (CDPKs) regulate the downstream components in calcium signaling pathways. We investigated the effects of overexpression and disruption of an Oryza sativa (rice) CDPK (OsCPK12) on the plants response to abiotic and biotic stresses. OsCPK12-overexpressing (OsCPK12-OX) plants exhibited increased tolerance to salt stress. The accumulation of hydrogen peroxide (H(2) O(2) ) in the leaves was less in OsCPK12-OX plants than in wild-type (WT) plants. Genes encoding reactive oxygen species (ROS) scavenging enzymes (OsAPx2 and OsAPx8) were more highly expressed in OsCPK12-OX plants than in WT plants, whereas the expression of the NADPH oxidase gene, OsrbohI, was decreased in OsCPK12-OX plants compared with WT plants. Conversely, a retrotransposon (Tos17) insertion mutant, oscpk12, and plants transformed with an OsCPK12 RNA interference (RNAi) construct were more sensitive to high salinity than were WT plants. The level of H(2) O(2) accumulation was greater in oscpk12 and OsCPK12 RNAi plants than in the WT. These results suggest that OsCPK12 promotes tolerance to salt stress by reducing the accumulation of ROS. We also observed that OsCPK12-OX seedlings had increased sensitivity to abscisic acid (ABA) and increased susceptibility to blast fungus, probably resulting from the repression of ROS production and/or the involvement of OsCPK12 in the ABA signaling pathway. Collectively, our results suggest that OsCPK12 functions in multiple signaling pathways, positively regulating salt tolerance and negatively modulating blast resistance.

Introduction of the ZmDof1 gene into rice enhances carbon and nitrogen assimilation under low-nitrogen conditions

The excessive application of nitrogen fertilizer to maximize crop yields causes negative environmental effects such as pollution and ecological imbalance. To overcome this problem, researchers have attempted to improve the nitrogen assimilation capacity of crops. Maize Dof1 (ZmDof1) is a plant-specific transcription factor shown to promote nitrogen assimilation in Arabidopsis thaliana (Arabidopsis) even under nitrogen-deficient conditions. The present study examines the effect of the introduction of the ZmDof1 gene on carbon and nitrogen assimilation in rice. ZmDof1 induced the expression of phosphoenolpyruvate carboxylase (PEPC) genes in transgenic rice plants and transactivated the PEPC promoters in protoplast transient assays, showing similar effects in rice as in Arabidopsis. Transgenic rice expressing ZmDof1 and grown in the presence of 360 μm (nitrogen-sufficient) or 90 μm (nitrogen-deficient) of nitrogen concentrations showed modulation of metabolite content and gene expression associated with the anaplerotic pathway for the TCA cycle, suggesting an increased carbon flow towards nitrogen assimilation. Furthermore, increases in carbon and nitrogen amounts per seedling were found in Dof1 rice grown under nitrogen-deficient conditions. Nitrogen deficiency also resulted in the predominant distribution of nitrogen to roots, accompanied by significant increases in root biomass and modification of the shoot-to-root ratio. Measurement of the CO₂ gas exchange rate showed a significant increase in the net photosynthesis rate in Dof1 rice under nitrogen-deficient conditions. Taken these together, the present study displayed that ZmDof1 expression in rice could induce gene expressions such as PEPC genes, modulate carbon and nitrogen metabolites, increase nitrogen assimilation and enhance growth under low-nitrogen conditions.

Functional characterisation of OsCPK21, a calcium-dependent protein kinase that confers salt tolerance in rice

Calcium acts as a messenger in various signal transduction pathways in plants. Calcium-dependent protein kinases (CDPKs) play important roles in regulating downstream components in calcium signaling pathways. In rice, the CDPKs constitute a large multigene family consisting of 29 genes, but the biological functions and functional divergence or redundancy of most of these genes remain unclear. Using a mini-scale full-length cDNA overexpressor (FOX) gene hunting system, we generated 250 independent transgenic rice plants overexpressing individual rice CDPKs (CDPK FOX-rice lines). These CDPK FOX-rice lines were screened for salt stress tolerance. The survival rate of the OsCPK21-FOX plants was higher than that of wild-type (WT) plants grown under high salinity conditions. The inhibition of seedling growth by abscisic acid (ABA) treatment was greater in the OsCPK21-FOX plants than in WT plants. Several ABA- and high salinity-inducible genes were more highly expressed in the OsCPK21-FOX plants than in WT plants. These results suggest that OsCPK21 is involved in the positive regulation of the signaling pathways that are involved in the response to ABA and salt stress.

Morphological development of rice caryopses located at the different positions in a panicle from early to middle stage of grain filling

Rice caryopses show different patterns of grain filling depending on position within a panicle. Caryopses located on the upper primary rachis branches generally accumulate larger amounts of starch at maturity than caryopses located on the secondary rachis branches of the lower primary rachis. In this study, the former and latter types of caryopses were defined as superior and inferior caryopses, respectively. Superior caryopses elongated soon after flowering, whereas inferior caryopses hardly elongated and were morphologically stagnant until the first 4 d after flowering (DAF). However, once inferior caryopses began elongation, their morphological development was the same as superior caryopses until the middle stage of grain filling. Cell division of the inner integument ceased before endosperm cellularization, pericarp functioned as a transient starch storage tissue until endosperm accumulated starch, and endosperm cell number was determined concomitantly with nucellus disintegration. These results implied the coordinated development of the endosperm with maternal tissues. In addition, differences of inner-integument cell number and endosperm cell number were related to a difference of endosperm size between superior and inferior caryopses.

CDPK-mediated abiotic stress signaling

Calcium-dependent protein kinases (CDPKs) constitute a large multigene family in various plant species. CDPKs have been shown to have important roles in various physiological processes, including plant growth and development and abiotic and biotic stress responses in plants. Functional analysis using gain-of-function and loss-of-function mutants has revealed the biological function of CDPKs in planta. Several CDPKs have been shown to be essential factors in abiotic stress tolerance, positively or negatively regulating stress tolerance by modulating ABA signaling and reducing the accumulation of reactive oxygen species (ROS). This review summarizes recent results describing the biological function of CDPKs that are involved in abiotic stress tolerance.

Analysis of a C4 maize pyruvate, orthophosphate dikinase expressed in C3 transgenic Arabidopsis plants

Abstract Pyruvate,orthophosphate dikinase (PPDK) catalyzes the formation of phospho enol pyruvate, the initial acceptor of CO 2 in the C4 photosynthetic pathway. Transgenic C3 Arabidopsis plants expressing the maize C4 PPDK gene under the control of either the Arabidopsis rbcS promoter or the cauliflower mosaic virus 35S promoter were studied. The level of PPDK protein was quite low in contrast to the high steady-state level of PPDK transcripts in several transgenic plants. A PPDK polypeptide with a similar size to that in maize was found exclusively in the chloroplasts of transgenic Arabidopsis plants. This result indicates that the transit peptide of C4 PPDK in the C4 monocot maize is functional in the chloroplast protein import system of the C3 dicot Arabidopsis . The activities of PPDK in leaf extracts of the transgenic plants were up to four times higher than those in the control nontransgenic plants and the transgenic plants with the β -glucuronidase (GUS) gene, although they were still less than 3% of the PPDK activity in maize. The relative PPDK activity per unit PPDK protein in transgenic Arabidopsis was similar to that in maize. These results suggest that the low PPDK activity in transgenic Arabidopsis plants may be attributed to possible regulation at post-transcriptional and/or translational levels. The modestly increased PPDK activity did not influence the activities of ribulose-1,5-bisphosphate carboxylase and other C4-related enzymes (phospho enol pyruvate carboxylase, NAD(P)-malic enzyme), and photosynthetic CO 2 -exchange parameters.

Disruption of a gene for rice sucrose transporter, OsSUT1, impairs pollen function but pollen maturation is unaffected

Sucrose transporters (SUTs) are known to play critical roles in the uptake of sucrose from the apoplast in various steps of sugar translocation. Because developing pollen is symplastically isolated from anther tissues, it is hypothesized that SUTs are active in the uptake of apoplastic sucrose into pollen. To investigate this possibility, a comprehensive expression analysis was performed for members of the SUT gene family in the developing pollen of rice (Oryza sativa L.) using real-time RT-PCR combined with a laser microdissection technique. Among the five SUT genes, OsSUT1 and OsSUT3 were found to be preferentially expressed and had temporal expression patterns that were distinct from each other. Expression of OsSUT1 in pollen was confirmed by a promoter–GUS fusion assay. The physiological function of OsSUT1 in pollen was further investigated using retrotransposon insertion mutant lines. While the homozygote of disrupted OsSUT1 (SUT1–/–) could not be obtained, heterozygote plants (SUT1+/–) showed normal grain filling. Their progeny segregated into SUT1+/– and SUT1+/+ with the ratio of 1:1, suggesting that the pollen disrupted for OsSUT1 is dysfunctional. This hypothesis was reinforced in vivo by a backcross of SUT1+/– plants with wild-type plants and also by in vitro pollen germination on the artificial media. However, starch accumulation during pollen development was not affected by disruption of OsSUT1, suggesting that the sugar(s) required for starch biosynthesis is supplied by other sugar transporters.

Changes in nitrogen assimilation, metabolism, and growth in transgenic rice plants expressing a fungal NADP(H)-dependent glutamate dehydrogenase (gdhA)

In plants, glutamine synthetase (GS) is the enzyme that is mainly responsible for the assimilation of ammonium. Conversely, in microorganisms such as bacteria and Ascomycota, NADP(H)-dependent glutamate dehydrogenase (GDH) and GS both have important roles in ammonium assimilation. Here, we report the changes in nitrogen assimilation, metabolism, growth, and grain yield of rice plants caused by an ectopic expression of NADP(H)-GDH (gdhA) from the fungus Aspergillus niger in the cytoplasm. An investigation of the kinetic properties of purified recombinant protein showed that the fungal gdhA had 5.4–10.2 times higher Vmax value and 15.9–43.1 times higher Km value for NH4+, compared with corresponding values for rice cytosolic GS as reported in the literature. These results suggested that the introduction of fungal GDH into rice could modify its ammonium assimilation pathway. We therefore expressed gdhA in the cytoplasm of rice plants. NADP(H)-GDH activities in the gdhA-transgenic lines were markedly higher than those in a control line. Tracer experiments by feeding with 15NH4+ showed that the introduced gdhA, together with the endogenous GS, directly assimilated NH4+ absorbed from the roots. Furthermore, in comparison with the control line, the transgenic lines showed an increase in dry weight and nitrogen content when sufficient nitrogen was present, but did not do so under low-nitrogen conditions. Under field condition, the transgenic line examined showed a significant increase in grain yield in comparison with the control line. These results suggest that the introduction of fungal gdhA into rice plants could lead to better growth and higher grain yield by enhancing the assimilation of ammonium.

Plant regeneration from suspension culture derived protoplasts of tall fescue (Festuca arundinacea Schreb.) of a single genotype

Abstract Protoplasts were isolated from suspension cultured cells of a single genotype from tall fescue (Festuca arundinacea Schreb.) and cultured by the agarose bead method. Colonies were formed only when nurse cells were added to the liquid medium around the agarose blocks. Plating efficiency was higher in AA medium than in B5 medium. Protoplast-derived colonies were transferred to MS or N6 solid medium supplemented with, or without, 2,4-D and proline. Some of the protoplasts from cultivar Nanryo grew into yellow compact calluses that differentiated into green plantlets. When the protoplast derived colonies were plated directly on hormone-free media, the regerants were less vigorous than when subcultured once or twice on media supplemented with 2,4-D and proline. Albino shoots were regenerated more frequently than green shoots. More than thirty regenerated plants could be transferred to soil.