Up to now the complete mitochondrial genome (mitogenome) sequences of only three species of clitellate have been available. We have determined the complete mitogenome sequences of the elusive Burmese giant earthworm Tonoscolex birmanicus (Clitellata: Megascolecidae), which is endemic to Myanmar. The 15,170-bp long genome contains the 37 genes typical of metazoan mitogenomes [13 protein-coding genes (PCG), two rRNA genes and 22 tRNA genes] and one major non-coding region. All of the 37 genes are transcribed from the same DNA strand. The arrangement of the T. birmanicus mitogenome is identical to that of two within-ordinal species Lumbricus terrestris and Perionyx excavates. All 13 PCGs start with the ATG. For the stop codon, only six PCGs end with the TAA, whereas the remaining ones ends with the incomplete stop codon, T. Genes overlap in a total of 14 bp in five locations, and harbor a total of 16 bp of intergenic spacer sequences in nine locations.

Tetranychid mites are one of the most diverse group including at least 1,200 species in the world. Species identification is difficult due to the small size and similar morphology within the group. We collected 17 species of spider mites from various host plants in different regions of Korea and determined species identity by the comparison of morphological characters and nucleotide sequences of internal transcribed spacer 2 (ITS2) and the cytochrome oxidase subunit I (COI). In addition, we report three new species that were firstly identified in Korea. Amphitetranychus quercivorus (Ehara and Gotoh) was collected from Mongolian oak plant in Daegu, Schizotetranychus miscanthi Saito was from the common reed plant in Ulleungdo, and S. cercidiphylli Ehara was from Bamboo plant in Jeju. Morphological identification of three species were similar with those of Japanese samples, but the ITS2 and COI sequences of A. quercivorus and the COI sequence of S. miscanthi were different with Japanese species at the rates of 1/419, 2/331 and 3/332 nucleotides, repsectively. S. cercidiphylli can be identified by the aedeagus shape of males but we firstly sequenced ITS2 and COI of this species. Our results can be used for the identification of spider mites which are important in plant quarantine.

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.

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.

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.

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

A male gametophyte, or pollen develops in the anther, and its development plays an important male reproductive process in flowering plants. A properly designed transgene construct can help to tailor transgene expression in plants by altering the expression strength, timing, and location. In this process, the promoter plays a pivotal role in controlling transgene expression. In this research, the promoter regions of rice anther/pollen-specific genes, named as OsMSP1 to OsMSP11,were selected from the microarray data sets covering 4 developmental stage of male gametophyte and then used for the construction of vector by Gateway cloning method and transformed into rice and Arabidopsis. All 11 promoters in rice and 9 in Arabidopsis were displayed as anther/pollen-specific/preferential genes by GUS assay and RT-PCR analysis. Three out of 11 promoters showed consistent results with published data. In this study, we demonstrated on eight new anther/pollen-specific or -preferential promoters (OsMSP1, OsMSP2, OsMSP3, OsMSP4, OsMSP5, OsMSP6, OsMSP8, and OsMSP9, which have not been reported before. Although the expression pattern of different genes active in pollen grains is diverse and complex, these experimental results would be helpful to understand the molecular mechanism of regulatory elements in rice microspore/pollen-specific genes.

Pollen development in flowering plants is regulated by a comprehensive pattern of genes. One way to produce hybrid rice based on nuclear male sterility is to find out firstly the potential promoters that function specifically in anthers since it is a specific site for transcription initiation and play key roles for the spatial and temporal expression of the genes. To implement this objective, we were selected promoter region of 16 genes based on the expression pattern of microarray and then those were introduced into the promoterless final destination vector which containing the GFP and GUS reporters genes. The resulting twelve vectors were transformed into monocotyledonous rice (Oryza sativa L) and a dicotyledonous Arabidopsis as heterologous system. Minimum 20 plants for each vector were analyzed by histochemical GUS assay at the flowering stage in Arabidopsis. 9 vectors out of 12 vectors constructed were expressed exclusively at the anther, especially in pollen, however one vector exhibited expression in stigma. For rice, T-DNA insertion were confirmed with specific primers in each promoter and GFP region. All T0 transgenic plants contained T-DNA insertion in their genome. This study would provide valuable information for biotechnological application for the induction of male sterility in plants.

The transport of nascent messenger RNA from the nucleus to the cytoplasm is mediated by the THO/TREX complex and is evolutionary conserved from yeast, metazoa and humans. However, in plants, it is still yet unclear if the similar mechanism of transport exists. Here we identified and characterized a mutant gene, AtTHO2, a putative Arabidopsis thaliana THO2 component protein, homologous to yeast THO2 of the THO/TREX pathway required for mRNA transport. The mutation from this gene resulted to various developmental defects that include semi-dwarfism and abnormal floral development which further leads to sterility. Gene expression analysis revealed that AtTHO2 is expressed in all organs and pollen developmental stages. In addition, the homozygote progeny of null mutants did not persist until mature stage. These results suggest an indispensable role of AtTHO2 in the development of Arabidopsis. Differential gen expression and silencing were also observed between the null mutants and wild type depending on T-DNA insertion. Furthermore, alternative splicing which was tightly linked with the THO/TREX pathways was also defective on AtTHO2 and null mutants. A similar pattern of defect in SR34a was observed in the AtTHO2 and null mutants. In terms of microRNA biosynthesis, no significant differences were seen on the wild-type and mutant plants; however this data should be validated. Thus this work provides some evidences that a similar THO/TREX complex exist in plants and gave a foundation for further studies on the mechanism of nuclear export in plants.

A diverse number of genes are involved in the floral transition and development to ensure the proper timing on the switch from vegetative to reproductive development in Arabiodopsis. MADS-box genes play a major role in floral development especially in the case of vernalization process, In this study we mapped a mutation in MAF5 encoding a MADS-domain protein which was reported to be up-regulated during vernalization and regulates flowering time. The mutant in MAF5 showed several pleiotropic phenotypes that includes semi-dwarfism, delayed senescence and abnormal pollen phenotype, High percentages of vacuolated and aborted pollen phenotype were observed in the mutant plant. Transmission efficiency showed that mutation from this gene was defective in both male and female gametes. Furthermore, gene expression analysis revealed that this gene was predominantly expressed in reproductive organs and gave a strong expression in the mature pollen which coincides with the defect in pollen phenotype. The results from this study provide some evidences on the additional role of MAF5 in pollen development however more specific approaches should be done to determine the specific stages of pollen development altered in this mutant.

Pollen development in flowering plants is regulated by a comprehensive pattern of genes. One way to produce hybrid rice based on nuclear male sterility is to find out firstly the potential promoters that function specifically in anthers since it is a specific site for transcription initiation and play key roles for the spatial and temporal expression of the genes. To implement this objective, we were selected promoter region of 16 genes based on the expression pattern of microarray and then those were introduced into the promoterless final destination vector which containing the GFP and GUS reporters genes. The resulting twelve vectors were transformed into monocotyledonous rice (Oryza sativa L) and a dicotyledonous Arabidopsis as heterologous system. Minimum 20 plants for each vector were analyzed by histochemical GUS assay at the flowering stage in Arabidopsis. 9 vectors out of 12 vectors constructed were expressed exclusively at the anther, especially in pollen, however one vector exhibited expression in stigma. For rice, T-DNA insertion were confirmed with specific primers in each promoter and GFP region. All T0 transgenic plants contained T-DNA insertion in their genome. This study would provide valuable information for biotechnological application for the induction of male sterility in plants.