Cross-species translation of genomic information may play a crucial role in applying biological knowledge gained from one species to other genomes. To screen and identify a broad range of abiotic stress-responsive genes, we employed a diverse array of resources, including Arabidopsis databases (http://www.arabidopsis.org), expression profiling data and previously reported literatures. As a result, a total of 1,377 genes were identified and classified into 18 different functional criteria based on biological processes of gene ontology. The gene set was translated into M. truncatula, which is a representative model system in the Fabaceae, by identifying orthologous genes between these two genomes with a combination of tBlastx and BlastP analyses. It is shown that approximately 82% of genes were estimated to be translated between the two genomes below the E-value of 10-30. These orthologous loci were used to construct comparative maps by developing a user-friendly analysis platform, resulting in a total of 52 synteny blocks. Furthermore, to discover central genes by which control responses to the abiotic stresses, a combination of AraNet (http://www.functionanet.org) and the Cytoscape program was used for the gene network analysis. The analysis resulted in the identification of 240 potential key genes. We anticipate that these genes may impact molecular breeding programs by discovering trait-associated SNPs followed by marker development.

In order to cope with the recent global warming and climate change that is projected to have a grave impact on agriculture worldwide, we will direct our focus on developing crops tolerant to multiple abiotic stresses including drought, cold and heat, with the following research activities conducted by three different research groups including an international research team at IRRI. 1) Development of heat/cold tolerant rice variety : Major genes conferring heat tolerance and cold resistance in rice will be identified by comparative transcriptome analyses and new molecular markers will be developed based upon these data. EMS mutagenesis and proteomics analyses will accompany this approach to supplement this gene identification and marker development efforts. Once reliable markers are obtained in this way, new varieties of heat/cold tolerant japonica rice will be bred through introgression of these genes. 2) Development of drought/heat tolerant rice variety : Through QTL mapping conducted on RILs between a drought resistant line and an elite line, genes conferring drought/heat tolerance will be identified and molecular markers will be developed using SNP/GBS genotyping methods. Using these markers, new rice varietis with drought/heat tolerance will be bred by employing marker assisted selection (MAS) as well as marker assisted backcross (MABC). 3) Identification of genes involved in multiple stress responses in ginseng and brassica : Molecular breeding of stress tolerance traits in ginseng and brassica is not well established to date. Taking advantage of the ginseng whole genome sequence data information and other comparative genomics approaches, members of the stress-response transcription factor family CBF/DREB will be identified and their functional analyses will be performed in ginseng and brassica using transcriptome profiling of both wild type and transgenic plants including the adventitious root-derived transgenic ginseng. Major Publications: - Lee, J., W. Jiang, et al. (2011). “Shotgun proteomic analysis for detecting differentially expressed proteins in the reduced culm number rice.” Proteomics 11(3): 455-468. - Ji, H., S. R. Kim, et al. (2010). “Inactivation of the CTD phosphatase-like gene OsCPL1 enhances the development of the abscission layer and seed shattering in rice.” The Plant journal 61(1): 96-106. - Chin JH, Gamuyao R, Dalid C, Bustamam M, Prasetiyono J, Moeljopawiro S, Wissuwa M, Heuer S (2011) Developing rice with high yield under P-deficiency: Pup1 sequence and application. Plant Physiology 156: 1-15. - Hong-Il Choi, Nam Hoon Kim et al. (2011) Development of Reproducible EST-derived SSR Markers and their application for genomics and breeding of Panax ginseng Journal of Ginseng Research 35(4): 399-412.

The molecular responses to various abiotic stresses were investigated by the approaches with transcriptomic analysis based on an ACP system. Here we identified differentially expressed genes under abiotic stresses in alfalfa seedlings and they were mostly unknown genes and a few common stress-related genes. Among them, mitochondrial small HSP23 was responded by the diverse stress treatment such as heat, salt, As stresses and thus it could be a strong candidate that may confer the abiotic stress tolerance to plants. When expressed in bacteria, recombinant MsHSP23 conferred tolerance to salinity and arsenic stress. Furthermore, MsHSP23 was cloned in a plant expressing vector and transformed into tobacco, a eukaryotic model organism. The transgenic plants exhibited enhanced tolerance to salinity and arsenic stress under ex vitro conditions. In comparison to wild type plants, the transgenic plants exhibited significantly lower electrolyte leakage. Moreover, the transgenic plants had superior germination rates when placed on medium containing arsenic. Taken together, these overexpression results imply that MsHSP23 plays an important role in salinity and arsenic stress tolerance in transgenic tobacco. The results of the present study show that overexpression of alfalfa mitochondrial MsHSP23 in both eukaryotic and prokaryotic model systems confers enhanced tolerance to salt and arsenic stress. This indicates that MsHSP23 could be used potentially for the development of stress tolerant transgenic crops, such as forages.

Recently there are many reports that signaling pathways of abiotic stress and biotic stress are correlated. These relations are not only antagonistic but also synergistic. In this project we are searching the common components in abiotic and biotic stress signaling through proteome and transcriptome analysis. In this project, we are profiling the transcriptome under ABA and biotic stress treatment and searching the common genes which were regulated in both treatment. Furthermore, we are analyzing the secretome and proteome induced under C.maydis. It would be expected that integrative analysis of transcriptome and proteome will presents us the candidate genes to develop abiotic/biotic stress tolerant transgenic plants.

Tall fescue (Festuca arundinacea Schreb.) is an important cool season forage plant that is not well suited to extreme heat, salts, or heavy metals. To develop transgenic tall fescue plants with enhanced tolerance to abiotic stress, we introduced a MsHsp23 gene expression vector construct through Agrobacterium-mediated transformation. Integration and expression of the transgene were confirmed by PCR, northern blot, and western blot analyses. Under normal growth conditions, there was no significant difference in the growth of the transgenic plants and the non-transgenic controls. However, when exposed to various stresses such as salt or arsenic, transgenic plants showed a significantly lower accumulation of hydrogen peroxide and thiobarbituric acid reactive substances than control plants. We speculate that the high levels of MsHsp23 proteins in the transgenic plants protect leaves from oxidative damage through chaperon and antioxidant activities. These results suggest that MsHsp23 confers abiotic stress tolerance in transgenic tall fescue and may be useful in developing stress tolerance in other crops. Compared with traditional plant breeding, genetic engineering provides a relatively fast and precise means of achieving improved stress tolerance of forage crops. Development of forage crops that are more tolerant to various abiotic stresses could lead to the use of more new lands for cultivation.

The latest report on draft genome of Brassica rapa sequence has been published. To elucidate the functions of a large population of these genes and to search efficiently for agriculturally useful genes, the Full-length cDNA Over-eXpressor (FOX) gene hunting system was used. The FOX library was transformed into rice by Agrobacteriummediated transformation. Approximately 1,150 FOX-rice lines were generated. Genomic PCR analysis indicated that the average length of FL-cDNAs was 900∼1,200 bp with functional annotation of many unknown function (35.5%). Most of the randomly selected transgenic rice lines showed overexpression (92%) and barely mRNA expression in the wild type Gopum. Moreover, 94% of the 850 transgenic rice lines were moderately tolerant (slightly yellow) to cold and 9 lines were tolerant (seedling light green). For the salinity evaluation, most of the transgenic lines (85%) were highly susceptible whereas seven lines were tolerant. In addition, morphological evaluation of rice lines showed minimal phenotypic alteration (12%). About 25.1 and 22% were earlier in terms of days to heading and chlorophyll contents, respectively. Further, 18% of FOX rice lines showed lower chlorophyll contents. Filled grains, number of tillers, panicle length, culm and plant height were relatively less variable among the lines. These results provided useful genes for functional analyses in the mechanisms of identified transgenic tolerant lines.

Cross-species translation of genomic information may play a pivotal role in applying biological knowledge gained from one species to other genomes. Abiotic stress-responsive genes in Arabidopsis have been translated to a legume model system, Medicago truncatula. A total of 1,370 Arabidopsis genes were identified by searching TAIR database, expression profiling data and literatures. For purposes of cross-genome identification of orthologous genes, tBlastX or BlastP were employed between these two model systems. Candidate genes potentially associated with abiotic stress responses were classified into 18 functional criteria and corresponding genomic locations were analyzed by Circos program. To do this, user-friendly bioinformatic analysis platform was established. In order to discover abiotic stress-associated genes, gene network and/or interactome analyses were conducted using a combination of AraNet web-based platform and CytoScape program. As a result, we could identify 240 key genes that appeared to play an important role within central gene networks. We anticipate that these genes may impact molecular breeding programs by developing them into genetic markers and discovering trait-associated nucleotide variations.

In order to uncover gene regulatory networks clustering of co-expressing genes was performed using a rice micorarray dataset of 155 gene expression omnibus sample (GSM) plates in NCBI, generating a total of 1660 clusters. One cluster with 85 co-expressing genes was measured with the correlation coefficient between pairs, resulting in an average r value of 0.66 with a range of -0.08 to 0.98. This result might support the notion that genes included in each cluster play common functional role(s). We also retrieved 23 Affymetrix GeneChip spots IDs corresponding to each of candidate genes related to abiotic stresses obtained from the P1antQTL-GE database and subsequently detected 23 clusters including co-expressing genes with each of the genes. Expression profiles of co-expressing genes revealed some degree of tissue-specific expression patterns, probably reflecting the existence of, at least partial, parallel versions of stress-related networks with evolutionary process, such as subfuntionalization. The finding that several cis-elements related to abiotic stresses was detected by differences in frequency between co-expressing genes and randomly selected genes. Clustering, expression profiles, and putative cis-acting regulatory elements of co-expressing genes related to abiotic stresses may provide clues to shed further light on the gene regulatory network of stress-responsive pathway.