We recently reported rice promoters that are active in late stages of pollen development. However, rice promoters that allow manipulation of gene expression at earlier stages of pollen development are still very limited to date. In this study, we have chosen 10 putative microspore promoters, OsMSP1 through OsMSP10, based on publicly available transcriptomic datasets in rice (Oryza sativa L.). Sequence analysis of these promoter regions revealed some cis regulatory elements involved in pollen-specific expression. We also examined promoter activities using the promoter-GUS reporter constructs in both transgenic rice and Arabidopsis. In rice, all of the 10 promoters directed GUS signals from the microspore stage throughout the all stages of pollen development. In addition, while GUS signals from 4 promoters, OsMSP2, OsMSP7, OsMSP9 and OsMSP10, seem to be expressed preferentially during pollen development, those from other six promoters were observed in vegetative tissues such as leaves, stems, and roots of seedlings. Similarly, in Arabidopsis, all of the 10 promoters directed GUS signals during pollen development. In detail, 8 promoters, OsMSP1 ~ OsMSP8 directed GUS signals from the microspore stage, whereas 2 promoters, OsMSP9 and OsMSP10, exhibited GUS signals from tricellular stage. Furthermore, seven promoters, except for OsMSP1, OsMSP2 and OsMSP10, showed GUS signals in shoot apical region or root tissues of seedlings. Furthermore, we verified microspore activity of four promoters, OsMSP1, OsMSP2, OsMSP3 and OsMSP6, by complementation analysis of the sidecar pollen (scp) mutant which displays microspore-specific defects. Currently, further analyses are underway for GUS expression of T2 generation in transgenic rice and scp complementation with remaining promoters.

In the course of map-based cloning, mutant genes are identified through linkage to specific region on genetic map. Here, we demonstrated gametophytic mutant line, named as AP-28-23, in which mutant gene was mapped on chromosome 2. Based on phenotypic analysis of mature pollen, mutant phenotype of AP-28-23 was classified into three classes, wild-type showing 2-4%, moderate 35-53% and severe type 97-100% on aberrant pollen frequencies, respectively. The severe type is completely sterilized with 100% unfertilized ovules. We also revealed that the transmission was reduced through male gametophyte in the AP-28-23 line. The transmission efficiency (TE) through the male gametophyte is only 0.67%, whereas in the female gametophyte is 89.87%.

OsLPS is pollen specific gene that express at late stage of pollen development in rice. Based on microarray database, promoter region of two genes Os03g0106900 and Os03g0106500 were identified. The sequence of 2287bp and 2468bp upstream region of these genes were amplified and designated as OsLPS10 and OsLPS11. These promoters were fused with GUS-GFP reporter gene in a destination vector, pKGWFS7 and introduced into rice (Dongjin cultivar) and Arabidopsis (Col-0). The results of GUS assay showed different pattern of gene expression in pollen of rice and Arabidopsis. In Arabidopsis, the OsLPS10 gene strongly activated in young anther and not expressed in mature pollen. Pollen development analysis revealed GUS expression was detected at unicellular stage and strongest at the bicellular pollen developmental stage. No GUS signal was recorded in mature pollen. In case of OsLPS11, no GUS signal was detected in during pollen development of inflorescent. By contrast, in rice, the GUS expression pattern of OsLPS10 and OsLPS11 exhibited similar. GUS expression was first detectable in the anthers of spikelets at the bicellular stage and intensity increased in tricellular and mature pollen. The GUS signal was not detected in the anthers in unicellular microspores in both genes, OsLPS10 and OsLPS11. The results suggested that these genes were different activity in heterologous plant system, monocot and dicot. Complementation analysis and Cis-regulatory elements will be examined to illuminate the characteristic of these genes

Based on the results of microarray analysis we selected ten candidate genes that express in pollen at the early pollen developmental stage. By PCR amplification, the promoter region of these genes were amplified from rice genomic DNA (Nipponbare) and cloned into the destination pKGWFS7 vector via an entry vector, pDONR201. The characteristic of promoters were evaluated in Arabidopsis thaliana (Col-0) through GUS expression analysis. Fifty T2 plants respectively from each promoter were tested. Whole inflorescence of individual plant was stained with 1mM X-Gluc solution to observe tissue-specific GUS expression patterns. The results showed that all 10 promoters activated in pollen tissues. Among them six promoters expressed at the early developmental stage (unicellular) of pollen and the others expressed at both early (unicellular) and late pollen developmental stage (mature pollen). The results indicated that these promoters would be potential applicable for the studies of pollen function. Currently, we are performing these promoters analysis in rice transgenic plants as well as molecular characterization.