Molecular Basis Of Cold-Induced Pollen Sterility In Rice

Abstract

We have used two approaches to study the molecular basis of cold-induced pollen sterility in rice. Firstly, we studied the effect of cold on sugar metabolism in rice anthers, with the intention to identify genes that are affected by cold. Secondly, we used microarray gene expression profiling to identify rice genes that are affected by cold treatment, and to compare the cold response between a cold-tolerant and a coldsensitive variety. The work on sugar metabolism has shown that cold treatment of rice anthers leads to an absence of starch accumulation and non-viability of pollen. Starch is an essential source of energy for pollen development and pollen fertility. At the same time, we found that sucrose – the building block of starch – is accumulating in cold-stressed anthers at the cold-sensitive young microspore stage. This indicates that sucrose somehow fails to be converted to starch in the pollen grains, and that the supply mechanism of sugar to the tapetum and developing pollen grains is disturbed by cold. The tapetum, the cell layer in the anther that feeds the pollen grains, and the pollen cells are physically isolated from the rest of the anther at the young microspore stage. Supply of sugars from the rest of the anther to the tapetum and pollen grains occurs via a specialised mechanism involving two enzymes: cell wall invertase and monosaccharide transporters. Biochemical analysis indicated that the activity of anther cell wall invertase was significantly repressed by cold, suggesting that the first step in the sugar transport chain is functioning at reduced capacity. We cloned the gene that encodes this enzyme, OSINV4, and found that the expression of this gene is repressed by cold. We subsequently identified two monosaccharide transporter genes: OSMST8 was repressed by cold, while OSMST7 was induced by cold. OSINV4 and OSMST8 function in the same pathway that supplies sucrose to the tapetum and pollen, while OSMST7 functions in a different pathway that may lead to starch accumulation in the anther wall. Studying the cold-tolerant Chinese cultivar R31 revealed that this cultivar did not accumulate sucrose, contained starch-filled fertile pollen grains, and did not repress OSINV4 and OSMST8 expression following cold treatment. Thus, there is a strong correlation between these phenotypes and the cold tolerance phenotype, suggesting that we have now some expression markers for coldtolerance. We have also found that these genes are regulated by the plant hormone ABA; ABA perfectly mimics the effect of cold and it serves as a signal to switch of gene expression, including OSINV4 and OSMST8. ABA-accumulation does not occur to the same extent in R31 than in Doongara, and we have identified an anther ABA biosynthetic gene that is induced by cold (OSNCED3). These findings have improved our understanding of the molecular basis of cold-induced pollen sterility significantly, and we are now in the stage of identifying a marker gene that can be used to follow the cold-tolerance trait in a breeding population. We have also made good progress using the microarray approach. By comparing the cold response of Doongara and two tolerant cultivars (R31 and R32) we identified a non-redundant set of 329 genes that are expressed differently between the different cultivars. The genes were sequenced and their chromosome location was determined. This gave us more information about other cellular processes that are affected by cold and how these processes are affected differently in tolerant and sensitive cultivars. We are now in the stage of spotting these genes on a smaller diagnostic microarray, and this array will be used to screen doubled haploid lines of a Doongara/R31 cross (prepared by Dr. X. Zhao, Sydney Univ.). This will enable us to identify suitable marker genes for cold tolerance in rice.