Makiko Kawagishi-Kobayashi

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 Alexanders 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.

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.

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°C day/25°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.

Reduction of Gibberellin by Low Temperature Disrupts Pollen Development in Rice

Gibberellins and expression levels of their biosynthesis genes decrease in developing anthers on exposure to moderate low temperatures, disrupting pollen development and reducing grain yields. 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.

Mutational analysis of the RNase-like domain in subunit 2 of fission yeast RNA polymerase II.

Local sequence similarity exists between the subunit 2 of eukaryotic RNA polymerases II and the barnase-type bacterial RNases. The RNase-like domain from the Rpb2 ofSchizosaccharomyces pombe was expressed inEscherichia coli as a GST fusion protein and examined for its RNase activity. When the GST fusion protein was incubated in vitro with32P-labeled RNA, the RNA degradation activity was less than 0.1%, if any, of the level of synthetic barnase. In order to check the in vivo function of this region, we constructed two mutantrpb2 alleles,rpb2E357A andrpb2H3a6L, each carrying a single amino acid substitution at the site correponding to one of the three essential amino acid residues forming the catalytic site in barnase (mutation of barnase at the corresponding sites results in complete loss of RNase activity) and five other mutantrpb2 alleles, each carrying a single mutation at various positions within the RNase-like domain but outside the putative catalytic site for RNase activity. When these mutantrpb2 alleles were expressed in anrpb2-disruptedS. pombe strain, all the mutants grew as well as the wild-type parent and did not show any clear defective phenotypes. These results suggest either that the RNase-like domain in Rpb2 does not function as an RNase in vivo or that the RNase activity of this domain, if present at all, is not essential for cell growth.