Reduction of gibberellin by low temperature disrupts pollen development in rice.

Microsporogenesis in rice (Oryza sativa) plants is susceptible to moderate low temperature (LT; approximately 19°C) that disrupts pollen development and causes severe reductions in grain yields. Although considerable research has been invested in the study of cool-temperature injury, a full understanding of the molecular mechanism has not been achieved. Here, we show that endogenous levels of the bioactive gibberellins (GAs) GA4 and GA7, and expression levels of the GA biosynthesis genes GA20ox3 and GA3ox1, decrease in the developing anthers by exposure to LT. By contrast, the levels of precursor GA12 were higher in response to LT. In addition, the expression of the dehydration-responsive element-binding protein DREB2B and SLENDER RICE1 (SLR1)/DELLA was up-regulated in response to LT. Mutants involved in GA biosynthetic and response pathways were hypersensitive to LT stress, including the semidwarf mutants sd1 and d35, the gain-of-function mutant slr1-d, and gibberellin insensitive dwarf1. The reduction in the number of sporogenous cells and the abnormal enlargement of tapetal cells occurred most severely in the GA-insensitive mutant. Application of exogenous GA significantly reversed the male sterility caused by LT, and simultaneous application of exogenous GA with sucrose substantially improved the extent of normal pollen development. Modern rice varieties carrying the sd1 mutation are widely cultivated, and the sd1 mutation is considered one of the greatest achievements of the Green Revolution. The protective strategy achieved by our work may help sustain steady yields of rice under global climate change.

The double-stranded break-forming activity of plant SPO11s and a novel rice SPO11 revealed by a Drosophila bioassay.

BACKGROUND: SPO11 is a key protein for promoting meiotic recombination, by generating chromatin locus- and timing-specific DNA double-strand breaks (DSBs). The DSB activity of SPO11 was shown by genetic analyses, but whether SPO11 exerts DSB-forming activity by itself is still an unanswered question. DSB formation by SPO11 has not been detected by biochemical means, probably because of a lack of proper protein-folding, posttranslational modifications, and/or specific SPO11-interacting proteins required for this activity. In addition, plants have multiple SPO11-homologues. RESULTS: To determine whether SPO11 can cleave DNA by itself, and to identify which plant SPO11 homologue cleaves DNA, we developed a Drosophila bioassay system that detects the DSB signals generated by a plant SPO11 homologue expressed ectopically. We cytologically and genetically demonstrated the DSB activities of Arabidopsis AtSPO11-1 and AtSPO11-2, which are required for meiosis, in the absence of other plant proteins. Using this bioassay, we further found that a novel SPO11-homologue, OsSPO11D, which has no counterpart in Arabidopsis, displays prominent DSB-forming activity. Quantitative analyses of the rice SPO11 transcripts revealed the specific increase in OsSPO11D mRNA in the anthers containing meiotic pollen mother cells. CONCLUSIONS: The Drosophila bioassay system successfully demonstrated that some plant SPO11 orthologues have intrinsic DSB activities. Furthermore, we identified a novel SPO11 homologue, OsSPO11D, with robust DSB activity and a possible meiotic function.

Protein kinase PKR mutants resistant to the poxvirus pseudosubstrate K3L protein.

As part of the mammalian cell innate immune response, the double-stranded RNA activated protein kinase PKR phosphorylates the translation initiation factor eIF2alpha to inhibit protein synthesis and thus block viral replication. Poxviruses including vaccinia and smallpox viruses express PKR inhibitors such as the vaccinia virus K3L protein that resembles the N-terminal substrate-targeting domain of eIF2alpha. Whereas high-level expression of human PKR was toxic in yeast, this growth inhibition was suppressed by coexpression of the K3L protein. We used this yeast assay to screen for PKR mutants that are resistant to K3L inhibition, and we identified 12 mutations mapping to the C-terminal lobe of the PKR kinase domain. The PKR mutations specifically conferred resistance to the K3L protein both in yeast and in vitro. Consistently, the PKR-D486V mutation led to nearly a 15-fold decrease in K3L binding affinity yet did not impair eIF2alpha phosphorylation. Our results support the identification of the eIF2alpha-binding site on an extensive face of the C-terminal lobe of the kinase domain, and they indicate that subtle changes to the PKR kinase domain can drastically impact pseudosubstrate inhibition while leaving substrate phosphorylation intact. We propose that these paradoxical effects of the PKR mutations on pseudosubstrate vs. substrate interactions reflect differences between the rigid K3L protein and the plastic nature of eIF2alpha around the Ser-51 phosphorylation site.

Morphological and gene expression analysis under cool temperature conditions in rice anther development.

Cool temperature conditions are known to lead to pollen sterility in rice. Pollen sterility is an agriculturally important phenomenon because it imparts a large influence directly on rice yield. However, cool temperature stress tolerance varies among rice cultivars and avoidance of cool temperature stress is difficult by practical method of agriculture. In this study using two rice cultivars, Hitomebore (high tolerance) and Sasanishiki (low tolerance), we analyzed morphological features and gene expression profiles, under cool temperature stress, in anther development of rice. Hitomebore was given cool temperature stress (19 degrees C) at flowering stage, and showed 87.3% seed fertility. Meanwhile, the seed fertility decreased to 41.7% in the case of Sasanishiki. A transverse section of Hitomebore anther revealed that the degradation of the tapetum started at the uninucleate microspore stage, and the tapetum had completely vanished at mature stage. The tapetum provides nutrients for pollen development, and its degradation occurs at a late stage in pollen development. In contrast, degradation of the tapetum did not occur at the uninucleate microspore stage in Sasanishiki, and the tapetum was clearly intact at mature stage, suggesting that tapetum degradation is critical for accurate pollen development and cool temperature tolerance correlated with the degree of tapetum degeneration. In gene expression analysis of anther, 356 genes that showed different expression levels between two cultivars at cool temperatures were found. These genes will lead to understanding the mechanism of cool temperature stress response in rice pollen development and the identification of genes involved in accurate tapetum degradation.

High temperatures cause male sterility in rice plants with transcriptional alterations during pollen development.

Plant male reproductive development is highly organized and sensitive to various environmental stressors, including high temperature. We have established an experimental procedure to evaluate high temperature injury in japonica rice plants. High temperature treatment (39 degrees C/30 degrees C) starting at the microspore stage repeatedly reduced spikelet fertility in our system. Morphological observations revealed that pollen viability in plants exposed to high temperatures was lower than that in control plants. Most pollen grains in high temperature-treated plants displayed a normal round shape and stained reddish purple with Alexander's reagent; however, the pollen grains were very poorly attached and displayed limited germination on the stigma. To investigate gene regulatory mechanisms in the anther in high temperature environments, DNA microarray analysis was performed by comparing non-treated samples with samples treated with 2-4 d of high heat. Genes responsive to high temperatures were identified from clustering of microarray data. Among these, at least 13 were designated as high temperature-repressed genes in the anther. Expression analyses revealed that these genes were expressed specifically in the immature anther mainly in the tapetum at the microspore stage and down-regulated after 1 d of high temperature. The expression levels of Osc6, OsRAFTIN and TDR, which are tapetum-specific genes, were unaffected by high temperatures. These results suggest that not all tapetal genes are inhibited by increased temperatures and the tapetum itself is not degraded in such an environment. However, high temperatures may disrupt some of the tapetum functions required for pollen adhesion and germination on the stigma.

A rice gene that confers broad-spectrum resistance to ß-triketone herbicides.

The genetic variation of rice cultivars provides a resource for further varietal improvement through breeding. Some rice varieties are sensitive to benzobicyclon (BBC), a ß-triketone herbicide that inhibits 4-hydroxyphenylpyruvate dioxygenase (HPPD). Here we identify a rice gene, HIS1 (HPPD INHIBITOR SENSITIVE 1), that confers resistance to BBC and other ß-triketone herbicides. We show that HIS1 encodes an Fe(II)/2-oxoglutarate-dependent oxygenase that detoxifies ß-triketone herbicides by catalyzing their hydroxylation. Genealogy analysis revealed that BBC-sensitive rice variants inherited a dysfunctional his1 allele from an indica rice variety. Forced expression of HIS1 in Arabidopsis conferred resistance not only to BBC but also to four additional ß-triketone herbicides. HIS1 may prove useful for breeding herbicide-resistant crops.

Separated transcriptomes of male gametophyte and tapetum in rice: validity of a laser microdissection (LM) microarray.

In flowering plants, the male gametophyte, the pollen, develops in the anther. Complex patterns of gene expression in both the gametophytic and sporophytic tissues of the anther regulate this process. The gene expression profiles of the microspore/pollen and the sporophytic tapetum are of particular interest. In this study, a microarray technique combined with laser microdissection (44K LM-microarray) was developed and used to characterize separately the transcriptomes of the microspore/pollen and tapetum in rice. Expression profiles of 11 known tapetum specific-genes were consistent with previous reports. Based on their spatial and temporal expression patterns, 140 genes which had been previously defined as anther specific were further classified as male gametophyte specific (71 genes, 51%), tapetum-specific (seven genes, 5%) or expressed in both male gametophyte and tapetum (62 genes, 44%). These results indicate that the 44K LM-microarray is a reliable tool to analyze the gene expression profiles of two important cell types in the anther, the microspore/pollen and tapetum.

Expression and developmental function of the 3-ketoacyl-ACP synthase2 gene in Arabidopsis thaliana.

The 3-ketoacyl-ACP synthase (KAS) II is a fatty-acid-related enzyme which catalyzes the elongation of 16:0-acyl carrier protein (ACP) to 18:0-ACP in plastids. The fatty acid biosynthesis 1-1 (fab1-1) mutant of Arabidopsis thaliana is partially deficient in its activity of Arabidopsis thaliana 3-ketoacyl-ACP synthase 2 (AtKAS2), and its phenotype has been intensively studied in connection with the chilling resistance and fatty acid composition. In this study, we used the T-DNA insertion mutant of AtKAS2 to examine its possible role in plant development. Reverse transcription (RT)-PCR showed that the AtKAS2 gene was expressed in various plant organs, except for roots, and was highly expressed in siliques. The fusion of beta-glucuronidase (GUS) to the AtKAS2 promoter demonstrated that the promoter was active in various tissues such as embryos, stomatal guard cells, inflorescences and pollen grains. We were not able to identify atkas2 homozygous mutant adult plants in heterozygous mutant progeny. Phenotypic and genetic analyses showed that disruption of the AtKAS2 by T-DNA insertion caused embryo lethality, and the development of the embryos was arrested at the globular stage. Taken together, our results suggest that AtKAS2 is required for embryo development in Arabidopsis during the transition from the globular to the heart stage.

Identification of small RNAs in late developmental stage of rice anthers.

Small RNAs including microRNA (miRNA) and small interfering RNA (siRNA) are known as repressors of gene expression. There are many plant proteins involved in small RNA-mediated gene silencing, such as Dicer ribonucleases and RNA-dependent RNA polymerases. However, most of these proteins have been reported to be absent in the late developmental stage of the plant male gamete, pollen. In order to clarify the existence of the small RNAs during maturation of pollen, we cloned and sequenced small RNAs from rice anthers including tricellular pollen. From fifty six candidates of small RNAs, we identified two known miRNAs (miR166 and miR167), eight potential miRNAs, and ten putative heterochromatic siRNAs (hc-siRNAs). RNA gel blot analyses clearly showed that miR166 and miR167 were accumulated in the uninuclear pollen stage of anther development and remained until the tricellular pollen stage. Our cloning and RNA gel blot analyses of small RNAs led us to propose a possible function of small RNA-mediated gene regulation for the development of male gametes in rice.

Anther-specific genes, which expressed through microsporogenesis, are temporally and spatially regulated in model legume, Lotus japonicus.

Pollen germination and pollen tube elongation are important for pollination and fertilization in higher plants. To date, several pollen-specific genes have been isolated and characterized. However, there is little information about the precise spatial and temporal expression pattern of pollen-specific genes in higher plants. In our previous study, we identified 132 anther-specific genes in the model legume Lotus japonicus by using cDNA microarray analysis, though their precise expression sites in the anther tissues were not determined. In this study, by using in situ hybridization experiments, we determined the spatial and temporal expression sites of 46 anther-specific genes (ca. 35%), which were derived from two groups, cluster I-a and cluster II-a, according to flower developmental stages. In the case of the genes grouped into cluster I-a, thirteen clones were characterized. The specific hybridized signals were varied among the clones, and were observed in tapetum cells, microspores, and anther walls at the early developmental stage of anther tissues. In the case of the genes classified into cluster II-a, we used thirty three different cDNA clones encoding primary and secondary metabolism-related proteins, cell wall reconstruction-related proteins, actin reorganization-related proteins, and sugar transport-related proteins, etc., as a probe. Interestingly, all genes in these thirty three clones examined were specifically expressed in the bicellular pollen grains, though the signal intensity was varied among clones. From the data of the cluster II-a genes, the mRNAs related to pollen germination and pollen tube elongation were specifically transcribed and preserved in mature pollen grains.

Molecular characterization of two anther-specific genes encoding putative RNA-binding proteins, AtRBP45s, in Arabidopsis thaliana.

RRM (RNA-recognition motif) domain is important for the post-transcriptional regulation of gene expression including RNA processing. In our previous study, we found one anther- and/or pollen-specific gene (LjRRM1, previously named as LjMfb-U93) in model legume, Lotus japonicus. Because of the richness of genomic information of another model plant, Arabidopsis thaliana, for functional analysis, we identified and characterized the orthologous genes in A. thaliana. By comparison of the partial nucleotide sequence of LjRRM1 to the public database, we identified three homologous genes (AtRBP45a, AtRBP45b, and AtRBP45c) in A. thaliana genome. Based on promoter analysis, both AtRBP45a and AtRBP45c were specifically expressed in immature anther tissues (tapetum cells) and mature pollen grains of transgenic plants. This expression pattern of AtRBP45a and AtRBP45c is quite similar to that of LjRRM1, indicating that AtRBP45a and AtRBP45c would be orthologous to LjRRM1. Because in another previous experiment, it was shown that proteins having RRM domains were related to pre-mRNA maturation, and as a conclusion, it is possible that LjRRM1, AtRBP45a, and AtRBP45c genes encoding RNA-binding proteins are functionally involved in the repression of translation in mature pollen grains in L. japonicus and A. thaliana.

Premature progression of anther early developmental programs accompanied by comprehensive alterations in transcription during high-temperature injury in barley plants.

High-temperature stress causes abortive male reproductive development in many plant species. Here, we report a putative mechanism of high-temperature injury during anther early development in barley plants (Hordeum vulgare L). Under high-temperature conditions (30 degrees C day/25 degrees C night), cell-proliferation arrest, increased vacuolization, over-development of chloroplasts, and certain abnormalities of the mitochondria, nuclear membrane, and rough endoplasmic reticulum (RER) were observed in developing anther cells, but not in developing ovule cells. Moreover, premature degradation of tapetum cells and premature progression to meiotic prophase in pollen mother cells (PMCs) were also observed. To monitor transcriptional alterations during high-temperature injury, we performed DNA microarray analysis using the 22K Barley1 GeneChip. Expression profiles were captured at four time points during the early development of panicles, and during vegetative growth of seedlings as a control, with or without high-temperature treatment. Abiotic or biotic stress related genes were equally or more dominantly up-regulated in the seedlings exposed to high temperatures compared with the panicles. In contrast, certain genes associated with histones, DNA replication initiation, mitochondria, and ribosomes were specifically repressed in the exposed panicles. In situ hybridization studies indicated that repression locally occurred on the developing anther cells exposed to high temperatures. Microarray analysis also indicated that a series of genes, including a meiosis-specific gene Asy1 and anther-specific lipid transfer protein genes, was prematurely up-regulated at an earlier stage under high-temperature conditions. Real-time quantitative RT-PCR analyses well confirmed the expression differences of certain key genes predicted by the DNA microarrays. These results suggest that high-temperature causes premature progression of anther early development program and fate, such as progression to meiosis of PMCs, cell-proliferation arrest and degradation in anther wall cells, accompanied by comprehensive alterations in transcription.

Induction of class-1 non-symbiotic hemoglobin genes by nitrate, nitrite and nitric oxide in cultured rice cells.

Non-symbiotic hemoglobins (ns-Hbs) are found in all plants, although their physiological function remains to be determined. The present study was undertaken to explore the mode of induction of ns-Hb genes by metabolites of nitrate assimilation using cultured rice (Oryza sativa L.) cells. Two class-1 ns-Hb genes, ORYsa GLB1a and ORYsa GLB1b, were strongly induced by nitrate, nitrite and nitric oxide (NO) donors, S-nitroso-N-acetylpenicillamine and sodium nitroprusside. The rapid and transient accumulation of ORYsa GLB1a and ORYsa GLB1b transcripts in response to nitrate, nitrite and NO donors was similar to that of nia1, which encodes NADH-nitrate reductase (NR), although repression by glutamine and asparagines was significant only for nia1. In the mutants defective in NR mRNA expression, nitrate, nitrite and NO donors failed to induce not only nia1 but also ORYsa GLB1a and ORYsa GLB1b transcripts, indicating that the induction of ns-Hb genes is closely associated with that of the NR gene. Although the kinetics of induction by nitrate, nitrite and NO donors are similar for the two ns-Hb genes, an inhibitor study demonstrated that de novo synthesis of the protein in cytoplasm is essential for inducing ORYsa GLB1b. In contrast, ORYsa GLB1a, like nia1, can be induced in the primary response to these signals without de novo protein synthesis. The role of nitrate, nitrite and NO in the induction of ns-Hb gene expression in rice cells and the possible cellar function of ns-Hbs were discussed in relation to nitrate reduction pathways.

Differential expression of three plastidial sigma factors, OsSIG1, OsSIG2A, and OsSIG2B, during leaf development in rice.

We isolated and characterized two rice nuclear genes, OsSIG2A and OsSIG2B, encoding the putative sigma-factor of the plastid RNA polymerase. Deduced protein sequences predicted a plastid-localizing signal in the N-terminus and subsequent polypeptides similar to known SIG2 proteins. Gene expression analysis revealed that the OsSIG2A transcript is more abundant than the OsSIG2B transcript in all tissues tested and that both rice SIG2s are expressed from earlier stages of leaf development than that in the case of OsSIG1. These results indicate differential expression of SIG genes in leaf morphogenesis, suggesting the existence of tissue- and stage-specific functions of SIG proteins for transcriptional regulation of chloroplast genes in plant development.