Map-based cloning is a basic method for identifying the mutated gene in plants. We selected the gametophytic mutant, named as AP-26-09, in activation-tagging pool. Mutant plant showed various kinds of pollen phenotype, such as the different number of nucleus or abnormal shapes. For the map-based gene cloning, we conducted phenotypic analysis of F2 mapping population through the screening of DAPI-stained pollen using fluorescence microscopy. Genomic DNA of F2 plants is prepared from leaves of approximately 1000 plants. In order to define chromosomal region where mutation is located, we designed SSLP markers and performed PCR amplification. In this study, we characterized gametophytic mutant and determined the chromosomal location using map-based approach.

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.

To identify genes that play critical roles during male gametogenesis in Arabidopsis, we have isolated several pollen morphological mutants from a mutagenized seed pool generated with a T-DNA activation vector. In this study, we have focused on a mutant plant producing ~50% abnormal pollen grains including high levels of collapsed pollen at maturity. The pollen developmental analysis showed that the mutant pollen phenotype was first observed at tricellular stage. Interestingly, the mutation was only maintained as a heterozygote due to the severely reduced genetic transmission through both sexes. TAIL PCR analysis led to the identification of the responsible gene which encodes a conserved oligomeric golgi complex component-related protein (COGCC). RT-PCR analysis showed predominant expression of the gene in reproductive organs including developing spores. The gene identity was confirmed by the result that mutant plants harboring a T-DNA containing corresponding wild type gene produced less level of mutant pollen grains. Furthermore, confocal laser scanning microscopy using mature pollen expressing COGCC-RFP driven under the native promoter showed small punctate signals, which are likely to be from the Golgi complex. Further experiments for co-localization of the COGCC-RFP with the Golgi markers are underway.

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%.

Tissue-specific promoters are a very useful tool for manipulating gene expression in a target tissue or organ; however, their range of applications in other plant species has not been determined, to date. In this study, we identified two late pollen-specific rice promoters (ProOsLPS10 and ProOsLPS11) via meta-anatomical expression analysis. We then investigated the expression of both promoters in transgenic rice (a homologous system) and Arabidopsis (a heterologous system) using ProOsLPS10 or ProOsLPS11::GFP-GUS constructs. As predicted by microarray data, both promoters triggered strong GUS expression during the late stages of pollen development in rice, with no GUS signals detected in the examined microspores and sporophytic tissues. Interestingly, these promoters exhibited different GUS expression patterns in Arabidopsis. While in Arabidopsis, the OsLPS10 promoter conferred GUS expression at the uni- and bi-cellular macrospore stages, as well as at the shoot apical region during the seedling stage, the OsLPS11 promoter was not active in the pollen at any stage, or in the examined sporophytic tissues. Furthermore, by performing a complementation analysis using a sidecar pollen (scp) mutant that displays developmental defects at the microspore stage, we found evidence that OsLPS10, which can be an applied promoter expressed in Arabidopsis, is useful for directing gene expression in the early stages of pollen development. Our results indicate that the OsLPS10 and OsLPS11 promoters can drive the expression of target genes during the late stages of pollen development in rice, but not in Arabidopsis. Our results also emphasize the necessity of confirming the applicability of an established promoter to heterologous systems.

비동형분열을 포함한 식물화분 발달과정에 관여하는 유전자를 탐색하기 위하여, Activation tagging vector를 이용 하여 조성된 3,500여개의 애기장대 T1세대 약 3,500개의 형질전환체 집단으로부터 4종의 화분 변이형을 보이는 돌 연변이체를 선발하였다. 4종의 변이체들은 20-40% 빈도의 성숙화분 변이표현형을 보였다. 변이체에서는 정상적 인 화분으로 발달하지 않은 aborted pollen type, 한 개의 핵만이 관찰되는 tio type, 비정상적으로 분열된 gemini type 등 다양한 변이표현형들이 관찰되었다. TAIL-PCR을 통하여 T-DNA의 삽입부위의 염색체 부위를 동정하여, co-segregation분석을 수행한 결과, 4종 모두 T-DNA삽입과 무관한 자연발생적 돌연변이로 밝혀졌으며, 현재 map-based cloning방법에 의해 변이유발 유전자를 탐색중이다. 이들 유전자들은 향후 식물의 화분발달에 핵심적 인 역할을 하는 유전자를 발굴하고 이를 이용한 기작을 이해하는데 매우 유용한 정보를 제공할 것으로 판단된다.

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.