AtSAGT1 encodes a salicylic acid (SA) glucosyltransferase enzyme that catalyzes the formation of SA glucoside and SA glucose ester. Here, the AtSAGT1 gene expression patterns were determined in AtSAGT1 promoter::GUS transgenic Arabidopsis plants. As a result, the factors regulating the induction of AtSAGT1 were identified as pathogen defense response, wound response, exogenous application of SA, and jasmonic acid treatment.

새로운 유전자원을 창출하기 위해 풍산나물콩에 EMS를 처리한 후 돌연변이 집단을 육성하고, 돌연변이 계통 중 지방산 함량에 대한 변이체를 찾아 콩 유전육종 재료를 선발하고자 실시한 결과를 요약하면 다음과 같다.M2 3,744개체중 형태적 변이를 보이는 1,000개체를 선발하여 M4 세대에서 난쟁이형 (3.3%), 엽형변이 (2.6%), 엽록소결핍 (1.5%), 꽃색변이 (1.1%), 엽형변이를 보이는 난쟁이형 (0.2%)의 변이가 관찰되었다. 야생형인 풍산나물콩의지방산 함량은 palmitic acid 11.6%, stearic acid 3.4%, oleicacid 25.3%, linoleic acid 52.0%, linolenic acid 8.1%를 나타내었다. M4 892 개체의 종자지방산 함량을 분석한 결과palmitic acid, stearic acid, oleic acid, linoleic acid, linolenicacid 함량은 각각 7.4~19.7%, 2.2~13.0%, 14.7~49.0%, 31.8~63.9%, 3.9~15.9%의 범위를 나타내었고, 10.8%, 3.8%25.3%, 52.0%, 8.1%의 평균을 보였다. 각각의 지방산 별로 선발된 돌연변이 개체들은 다음과 같다. 고 palmitic acid함량을 나타내는 PE1542 (17.1%), PE3058 (17.0%), 고 stearicacid 함량을 나타내는 PE977 (12.7%)와 저 stearic acid 함량을 나타내는 PE2166 (1.9%), 고 oleic acid 함량을 나타내는PE450 (44.4%), PE2742 (47.7%), PE3058 (33.4%), 저 linolenicacid 함량을 나타내는 PE594 (4.6%), PE1690 (3.7%)와 고linolenic acid 함량을 나타내는 PE2166 (12.6%) 등이 선발되었다.

The wild relatives of soybean [Glycine soja Sieb. and Zucc.] have curly/wavy nature whereas cultivated varieties are upright. Such morphological characteristics have agronomic importance too. To investigate the molecular mechanism of development contributing to coiled morphology, screening was carried out to look for Arabidopsis mutants in activation tagging lines obtained by activation T-DNA treatment that have curly/wavy morphology. A mutant named Coiled Branch 1 (cbr1), is found to have a wavy and curly morphology with coiling branches. Plasmid rescue and genomic southern blot analysis revealed the site of T-DNA insertion in the genome. RT-PCR was performed to monitor expression levels of the genes adjacent to the T-DNA integration sites, and showed the activation of an E3 ubiquitin ligase gene. Database search showed that the gene with the RING domain belongs to a family of E3 ubiquitin ligases. Complementation test by overexpression and RNA interference of the gene was also carried out. The complementation test results showed that the novel gene activation tagging affected the cbr1 mutant phenotypes. Ubiquitylation has been linked virtually to every cellular process including plant development. E3 ubiquitin ligase has been reported to recognize target proteins that are to be ubiquinated for further degradation by the proteasome complex. Further, more detailed studies are needed to identify the specific substrate(s) of the novel E3 ubiquitin ligase gene.

Soybean [Glycine max (L.) Merr.] seeds are abundant in high-quality proteins and fats. In addition, soybean seeds are also rich in secondary metabolites, such as isoflavones, lecithin, and saponins. Triterpene saponins are major components of these physiologically active metabolites in soybean seeds. Soybean saponins are classified as group A and DDMP saponins. Among them group A saponins are undesirable component of food products due to bitterness and astringency and also cause foaming in tofu production. Whereas, DDMP saponins and their derivatives are less bitter and astringent and beneficial to human health when consumed as regular diet. Therefore, reducing the group A saponins or increasing the DDMP saponins are required to improve the food quality. The present study focused to identify and characterize the gene which is encoding a protein responsible for biosynthesis of DDMP saponins. EMS mutant lines (sg-7-1 & sg-7-2) which lack DDMP saponins were developed. The breeding cross has been made with these two mutants with two cultivars, Pungsannamul and Wooram to study the segregation and genetic linkage analysis, respectively. The segregation analysis showed that the mutant phenotype is controlled by single recessive gene. TLC analysis for phenotyping F2 population of Wooram X sg-7-1 showed mutant, wild and heterozygous types. To surprise two more patterns were detected and they were named as strange type1 (ST1) and strange type2 (ST2). Further, SSR marker analysis will be carried out to locate the gene which encoding a protein responsible for biosynthesis of DDMP saponins.

Soybean germplasm have diverse accessions with great variation in their ability to survive and reproduce under salt stress conditions. In general, cultivated soybeans are more sensitive to salt stress than their wild relatives, however exceptions are found in both the groups. These variations in response to salt stress makes soybean germplasm an interesting collection of genetic resources to be explored for the identification of salt-tolerance genes, and their mechanism of action. Here, in this report we presented a data showing differential response of selected accessions of both cultivated and wild soybeans to salt stress. Two modes of salt treatment; gradual salt stress (GS) as well as salt shock (SS) were used in this study. The GS was found more effective in finding the difference in response of soybean accessions to salt stress. Various genetic marker based methods are in use to identify and isolate the potential genes contributing to the salt tolerance in soybean. Even then there is a paucity of knowledge on the key genes contributing to the salt tolerance in soybean. We expect that a recently developed functional screen based method, like yeast based functional screen, using cDNA library generated from different salt tolerant accessions of soybean could lead to identification of novel genes responsible for salt tolerance in soybean. Also, we propose for the use of RNA isolated from different stages of GS and SS for making cDNA library to be used for functional screening.

Soybean [Glycine max (L.) Merr.] have a variety of flower colors which are controlled by six different genes (W1,W2,W3,W4,Wm, and Wp). Among these genes, mutation in W3 gene causes near white flowers in the background of w4 genotype whereas the genotype W3w4 does purple throat flowers. Earlier studies showed that dihydroflavonol 4-reductase1 (DFR1) gene was closely linked to the flower color variants for W3 locus. In order to find out the W3 gene responsible for w3 phenotype, we first, studied the candidate gene Glyma14g07940 (DFR1) which is having 100% similarity with DFR probe sequence. Sequence analysis of DFR1 between W3 and w3 soybeans showed one base substitution in exon 6 of w3 mutant soybean resulting in one amino acid change in the amino acid sequence. However, comparison of amino acid sequences of DFR proteins from various crop plants showed that there is no functional change in the protein. Besides, the promoter analysis showed that, 311 bp of indel was traced in 5’-upstream promoter region of DFR1 gene in the w3 mutant. Here, we show that the near white or purple throat phenotypes in G. max is associated with existence or nonexistence of indel at 5’- upstream promoter region and low or high expression of DFR1, respectively. These results suggest that w3 phenotype may be caused by certain regulator of DFR1 gene located near or distant from DFR1 in G. max. In further study, we need to check the correlation between promoter indel with W3 expression level through GUS analysis.

Gene expression is regulated by DNA and histone methylation by DNA and histone methyltransferases, respectively. In animal system, DNA methyltransferase with CG methylation activity is modified by SUMO conjugation and then its activity was increased, which means that the activity of DNA methyltransferase is modulated by posttranslational modification. so Chromatin remodeling is a new concept for expression of controlling of gene function. We thus analyzed the effect of E3 SUMO ligase AtSIZ1 in CMT3 (chromometnylase 3)-mediated genome methylation by next-generation sequencing (NGS), methyl binding domain MeDIP-sequencing and gene analysis using siz1-2 and cmt3 mutants. we carried out CG-enrich analysis by MeDIP sequencing revealed that the methylation level of the genome including transposons was significantly low in siz1-2 mutants compared to wild-type. Result showed the genes regulated by methylation, that genes related of embryo and root development, cellulose metabolism, and post-translational modifications. All of our data indicate that the methyltransfearse activity of CMT3 may be able to be regulated by AtSIZ1 and thereby CMT3-mediated gene expression and plant development also can be controlled by E3 SUMO ligase activity. Besides, our data also suggest that ammonium (NH4+) can stimulate AtSIZ1- and CMT3- mediated DNA methylation.

Flowering time is a important agronomic trait for grain production in rice. So the control of flowering time is a critical step. In Arabidopsis, expression of certain key flowering gene such as FLOWERING LOCUS C (FLC) is known to be epigenetically regulated by chromatin modification through Enhancer of Zeste[E(z)], a histone methyltransferase, that core component of repressive complex, polycomb repressive complex2(PRC2). However, the chromatin mechanism involved in the regulation of rice flowering genes is presently not well known. Here we show that predict coding region of a intronic LncRNA[termed rice COLDAIR(OsCOLDAIR)], which is expected to associate with a component of PRC2, is predicted at rice FLC gene. And additionally we suggest interaction of histone methyltransferase and E3 SUMO ligase that indicate possibility of interaction with rice E(z) gene and rice E3 SUMO ligase. Our study contribute to control of rice flowering time by observing two factor that can regulate expression of related of rice FLC gene.

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.

Seed weight (SW), often expressed as 100-seed weight (HSW), is an important yield component in soybean and has been found to show positive correlation with seed yield. It is shown to behave as a quantitative trait controlled by many loci that are largely unclear. In this study, we represent the identification of chromosomal regions controlling the seed weight in soybean. We used a Recombinant Inbred Line (RIL) population, consisting of 188 lines derived from a cross of a wild soybean PI483463 (HSW: 0.85g) and a cultivated soybean cultivar Hutcheson (HSW: 14.05g) to identify the chromosomal regions controlling the SW trait. The population, along with parental samples and check, William82 (HSW: 21.2g) was grown for four years and phenotype data was recorded postharvest. A total of 535 SNP and 16 SSR markers, polymorphic between the parents were employed to genotype the RILs using Golden gate assay to develop the linkage map. Whole genome QTL scanning identified a total of 17 QTLs, spanning 10 chromosomes for the 100-seed weight. All these QTLs explained phenotypic variation (PV) in the range of 3.77 to 12.33%. Of the 17 QTLs, 2 QTLs qSWA1-1 and qSWD2-1, found to be the consistent QTLs, expressing in all the four environments. The QTL qSWD2-1 explained highest contribution to the total PV with 10.04 -12.23 %. The remaining 15 QTLs were identified in at least one environment with PV ranging up to 10.39%. The findings from this study will provide useful information to understand the genetic and molecular basis of SW and facilitate further genomic research leading to the yield improvements in soybean.

The correct development of male gametophytes (pollen grains) in flowering plants is essential for proliferate in gamete production. Here we have taken a map-based cloning approach using Arabidopsis male gametophytic mutant, named gemini pollen3 (gem3) to identify and characterize key gene that is expressed gametophytically for the completion of microgametogenesis focusing on genes which control cell division and cell fate determination. Previously reported gem1 and gem2 mutants with similar characteristics to gem3 that are disturbed at asymmetric division and cytokinesis at pollen mitosis I (PMI) in Arabidopsis. However, gem3 was mapped to a different genetic locus, and pollen developmental analysis revealed that gem3 exert an effect at meiosis and mitosis causing complete sterility. We also discovered that gem3 homozygous lines produce aberrant pollen grains, arising from incomplete cytokinesis during male meiosis with sporophytic phenotypes of twisted-shape leaves, large flowers. This mutation shows reduced genetic transmission of gem3 allele through male gametophyte. In previous results, the gem3 locus was confirmed by mapping to the region located on chromosome 5. To further confirm strong candidate gene, we performed sequencing and genetic complementation analysis. Currently, we are performing functional studies of the gem3 gene for the better understanding of molecular mechanisms that control asymmetric division at meiosis and mitosis during pollen development.

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.

The composition and distribution of seed storage proteins are important factors for eating quality such as grain flavor and quality in rice (OryzasativaL.) Rice protein disulfide isomerase (OsPDI) and binding protein (OsBIP) regulate synthesis, stability and sorting of storage proteins. We thus have tried to develop a marker protein for selection of rice cultivars which have different eating quality. Immunoblot analysis revealed that protein levels of OsPDI and OsBIP have no direct correlation with eating quality, suggesting that they may indirectly participate in control of eating quality through their-interacting partners or other regulatory mechanism.