Search

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.

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.

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.