Tomoya Yamaguchi

cDNA Microarray Analysis of Rice Anther Genes under Chilling Stress at the Microsporogenesis Stage Revealed Two Genes with DNA Transposon Castaway in the 5′-Flanking Region

Rice is most chilling sensitive at the onset of microspore release. Chilling treatment at this stage causes male sterility. The gene expression profile during the microspore development process under chilling stress was revealed using a microarray that included 8,987 rice cDNAs. As many as 160 cDNAs were up- or down-regulated by chilling during the microspore release stage. RT-PCR analysis of 5 genes confirmed the microarray results. We identified 3 novel genes whose expression levels were remarkably changed by chilling in rice anther. A new cis element that includes a DNA transposon Castaway sequence was found in the 5′ upstream region of two genes which were conspicuously down-regulated by chilling temperatures in rice anther.

Molecular Cloning and Characterization of a Novel β-1,3-Glucanase Gene from Rice

Beta-1,3-glucanases are referred to as pathogenesis-related proteins and they are also involved in several developmental processes. We isolated a cDNA for β-1,3-glucanase from rice anther and named it Oryza sativa glucanase 1 (Osg1). Phylogenetic analysis showed that Osg1 belonged to monocotyledonous endo-β-1,3-glucanase subgroup A. RT-PCR analysis revealed that Osg1 transcripts were present in leaves, roots, and anthers.

Expression analysis of genes for callose synthases and Rho-type small GTP-binding proteins that are related to callose synthesis in rice anther.

The most chilling-sensitive stage of rice has been found to be at the onset of microspore release. The microsporocytes produce a wall of callose between the primary cell wall and the plasma membrane, and it has been shown that precise regulation of callose synthesis and degradation in anther is essential for fertile pollen formation. In this study, genes for 10 callose synthases in the rice genome were fully annotated and phylogenetically analyzed. Expression analysis of these genes showed that OsGSL5, an ortholog of microsporogenesis-related AtGSL2, was specifically expressed in anthers, and was notably downregulated by cooling treatment. Gene expression profiles of Rho-type small GTP-binding proteins in rice anther were also analyzed. The mechanisms of callose synthesis in rice pollen formation and its relationships with cool tolerance are discussed.

Susceptibility to Coolness at the Young Microspore Stage under High Nitrogen Supply in Rice (Oryza Sativa L.). Proteome Analysis of Mature Anthers

Abstract In vitro pollen germination experiment using agar plates showed that the growth under high nitrogen conditions enhanced the damage to pollen germination ability caused by the cooling at the young microspore stage. To clarify the physiological factors related to this damage to pollen germination, we performed the comparative proteome analysis of mature anthers and identified proteins that were changed by high nitrogen conditions or high nitrogen plus cooling conditions. Proteins were extracted from mature anther samples and separated by two-dimensional polyacrylamide gel electrophoresis. By comparing anther protein maps of the samples collected from the plants grown under standard nitrogen conditions, high nitrogen conditions and high nitrogen plus cooling conditions, we found 11 protein spots, which varied with the treatment. These protein spots were identified based on the rice proteome database and/or peptide mass fingerprinting (PMF) analysis after digestion with trypsin. Digested samples were analyzed by matrix-assisted laser desorption/ionization-time flight mass spectrometry to produce PMF data. Database searches using these PMF data revealed the identities of 9 proteins. Seven of these proteins were polypeptides involved in cell elongation, stress responses and sugar metabolism. The relation between the fluctuations of these proteins and the decrease in pollen germination are discussed.

Effects of High Nitrogen Supply on the Susceptibility to Coolness at the Young Microspore Stage in Rice(Oryza sativa L.)

Abstract The combined effects of high nitrogen supply, cool temperature regimes and shading on factors related to fertility in rice plants were investigated. High nitrogen supply during the period from the spikelet differentiation stage to the young microspore stage caused a decrease in the number of microspores per anther. High nitrogen supply plus cool treatment at the young microspore stage (12°C for 3 or 4 days) resulted in a conspicuous decrease in the number of engorged pollen grains per anther. Shading also decreased the number of microspores and pollen grains. The lowest numbers of microspores and pollen grains were observed in a treatment combining high nitrogen supply with shading and cooling. The number of pollen grains shed on the stigma and germinated pollen grains were decreased by cooling. These results suggested that the lower fertility due to high nitrogen supply combined with cooling resulted from 1) decrease in number of microspores, 2) large decrease in number of pollen grains per anther, and 3) decreases in number of pollen grains and germinated pollen grains on the stigma.

Cleistogamy decreases the effect of high temperature stress at flowering in rice.

Abstract Rice sterility due to a high temperature at flowering is a serious agricultural problem that has been associated with global warming. The flowering stage in rice plants is most vulnerable to high temperature stress. Closed flowering rice plants may better withstand high temperature stress. The aim of this study was to determine the role of cleistogamy (closed flowering) in avoiding high temperature-induced sterility. Cleistogamy was induced by moderate heat treatment at 30°C during the panicle development stage. Both cleistogamous and chasmogamous (ordinary open flowering) rice plants, which possess the same genetic background, were subjected to 38°C or 36°C for 4 h just before flowering, and the percentage of fertility, number of pollen grains on a stigma, number of germinated pollen grains on a stigma, and temperatures inside and outside of the closed spikelets were examined. The cleistogamous rice plants showed a higher fertility percentage and a larger number of germinated pollen grains on a stigma than the chasmogamous rice plants. The temperature inside the closed flowering spikelets was 1.8°C lower than that outside the spikelets. The cleistogamous rice plants thus showed avoidance to high temperature stress at 38°C at flowering. On the basis of these results, we concluded that cleistogamy was advantageous to rice pollination and fertilization at high temperatures because of glume cooling.

Effects of High Nitrogen Supply on the Susceptibility to Coolness at the Young Microspore Stage in Rice (Oryza sativa L.): Gene Expression Analysis in Mature Anthers

abstract Changes in gene expression patterns by high nitrogen (High-N) and High-N plus cooling at the young microspore stage (High-N-cooling) in rice mature anthers were analyzed by semiquantitative RT-PCR with gene specific primers. Gene expression of alpha-expansin 18 (EXPA18) was repressed under High-N-cooling. Beta-expansin 1 (EXPB1), putative aldehyde dehydrogenase (ALDH) and Fructokinase II (FKII) were upregulated under High-N. EXPB1 and FKII were highly expressed under High-N-cooling. Comprehensive examination of gene expression patterns of 26 alpha-expansins (EXPAs) and 16 beta-expansins (EXPB s) showed that all expansins (EXP s)except EXPA12 were expressed in the anthers. Gene expression of EXP s did not change under High-N except EXPA1, EXPB1 and EXPB5 which were upregulated. In total, 18 EXPAs and 6 EXPB s were repressed under High-N-cooling, and among these, EXPA18, EXPA19 and EXPA20 had high similarities in the amino acid sequences, suggesting that these three genes may constitute a distinct functional gene subfamily related to the decrease in the pollen germination ability.

The superwoman1‐cleistogamy2 mutant is a novel resource for gene containment in rice

Summary Outcrossing between cultivated plants and their related wild species may result in the loss of favourable agricultural traits in the progeny or escape of transgenes in the environment. Outcrossing can be physically prevented by using cleistogamous (i.e. closed‐flower) plants. In rice, flower opening is dependent on the mechanical action of fleshy organs called lodicules, which are generally regarded as the grass petal equivalents. Lodicule identity and development are specified by the action of protein complexes involving the SPW1 and OsMADS2 transcription factors. In the superwoman1‐cleistogamy1 (spw1‐cls1) mutant, SPW1 is impaired for heterodimerization with OsMADS2 and consequently spw1‐cls1 shows thin, ineffective lodicules. However, low temperatures help stabilise the mutated SPW1/OsMADS2 heterodimer and lodicule development is restored when spw1‐cls1 is grown in a cold environment, resulting in the loss of the cleistogamous phenotype. To identify a novel, temperature‐stable cleistogamous allele of SPW1, targeted and random mutations were introduced into the SPW1 sequence and their effects over SPW1/OsMADS2 dimer formation were assessed in yeast two‐hybrid experiments. In parallel, a novel cleistogamous allele of SPW1 called spw1‐cls2 was isolated from a forward genetic screen. In spw1‐cls2, a mutation leading to a change of an amino acid involved in DNA binding by the transcription factor was identified. Fertility of spw1‐cls2 is somewhat decreased under low temperatures but unlike for spw1‐cls1, the cleistogamous phenotype is maintained, making the line a safer and valuable genetic resource for gene containment.

The cleistogamy of the superwoman1-cleistogamy1 mutation is sensitive to low temperatures during the lodicule-forming stage

We reported previously that the rice (Oryza sativa L.) cleistogamous mutation superwoman1-cleistogamy1 (spw1-cls1) was applicable to inhibit outcrossing between genetically modified varieties and their relatives, which causes pollen-mediated gene flow or disturbance of line purity. The cleistogamy of spw1-cls1 is caused by decreased protein-protein interactions between the mutant SPW1 and its partner proteins. Importantly, these interactions are restored under low-temperature conditions, but whether the cleistogamy of spw1-cls1 is affected by this phenomenon was unclear. In this study, we cultivated spw1-cls1 in various regions of Japan and confirmed that its flowers opened at low temperatures. Moreover, we compared the morphology of a series of lodicules generated at various temperatures. The results indicated that the cleistogamy of spw1-cls1 is thermosensitive and is gradually disturbed as the temperature decreases. This was correlated with the protein interaction pattern of the mutant SPW1 as reported previously. Then, we revealed the critical period for the low-temperature-induced instability of the phenotype of spw1-cls1 and examined the effect of daily temperature changes on cleistogamy. The results may facilitate simulation of the phenotype of spw1-cls1 at various temperatures and the prediction of regions where the cleistogamy of spw1-cls1 can be stably used to inhibit outcrossing.