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N-acetylchitooligosaccharides are potent elicitors to suspension-cultured rice cells, inducing a set of defense reactions. Expression of defense-related genes is considered to play an important role in defense reactions, and we employed microarray analysis of 8987 randomly selected expressed sequence tags to analyze the changes in gene expression caused by N-acetylchitooctaose. In this experiment, 166 genes were significantly induced and 93 genes were repressed. RNA gel blot analysis of 16 of these genes confirmed the microarray results. Of the 259 ESTs identified as responsive to N-acetylchytooctaose, 18 genes are related to signal transduction, including five calcium-dependent protein kinases (CDPKs). Among these, three novel CDPKs responsive to N-acetylchitooctaose were isolated.

Suspension-cultured cell lines from plant species are useful for genetic engineering. However, maintenance of these lines is laborious, involves routine subculturing and hampers wider use of transgenic lines, especially when many lines are required for a high-throughput functional genomics application. Cryopreservation of these lines may reduce the need for subculturing. Here, we established a simple protocol for cryopreservation of cell lines from five commonly used plant species, Arabidopsis thaliana, Daucus carota, Lotus japonicus, Nicotiana tabacum and Oryza sativa. The LSP solution (2 M glycerol, 0.4 M sucrose and 86.9 mM proline) protected cells from damage during freezing and was only mildly toxic to cells kept at room temperature for at least 2 h. More than 100 samples were processed for freezing simultaneously. Initially, we determined the conditions for cryopreservation using a programmable freezer; we then developed a modified simple protocol that did not require a programmable freezer. In the simple protocol, a thick expanded polystyrene (EPS) container containing the vials with the cell-LSP solution mixtures was kept at -30 °C for 6 h to cool the cells slowly (pre-freezing); samples from the EPS containers were then plunged into liquid nitrogen before long-term storage. Transgenic Arabidopsis cells were subjected to cryopreservation, thawed and then re-grown in culture; transcriptome and metabolome analyses indicated that there was no significant difference in gene expression or metabolism between cryopreserved cells and control cells. The simplicity of the protocol will accelerate the pace of research in functional plant genomics.

We used an 8987-EST collection to construct a cDNA microarray system with various genomics information (full-length cDNA, expression profile, high accuracy genome sequence, phenotype, genetic map, and physical map) in rice. This array was used as a probe to hybridize target RNAs prepared from normally grown callus of rice and from callus treated for 6 hr or 3 days with the hormones abscisic acid (ABA) or gibberellin (GA). We identified 509 clones, including many clones that had never been annotated as ABA-or GA-responsive. These genes included not only ABA- or GA-responsive genes but also genes responsive to other physiological conditions such as pathogen infection, heat shock, and metal ion stress. Comparison of ABA- and GA-responsive genes revealed antagonistic regulation for these genes by both hormones except for one defense-related gene, thionin. The gene for thionin was up-regulated by both hormone treatments for 3 days. The upstream regions of all the genes that were regulated by both hormones had cis-elements for ABA and GA response. We performed a clustering analysis of genes regulated by both hormones and various expression profiles that showed three notable clusters (seed tissues, low temperature and sugar starvation, and thionin-gene related). A comparison of the cis-elements for hormone response genes between rice and Arabidopsis thaliana, we identified cis-elements for dehydration-stress response or for expression of amylase gene as Arabidopsis gene-specific or rice gene-specific, respectively.

A cDNA encoding a predicted 15-kDa protein was earlier isolated from sugar-induced genes in rice embryos (Oryza sativa L.) by cDNA microarray analysis. Here we report that this cDNA encodes a novel Ca2+-binding protein, named OsSUR1 (for Oryza sativa sugar-up-regulated-1). The recombinant OsSUR1 protein expressed in Escherichia coli had 45Ca2+-binding activity. Northern analysis showed that the OsSUR1 gene was expressed mainly in the internodes of mature plants and in embryos at an early stage of germination. Expression of the OsSUR1 gene was induced by sugars that could serve as substrates of hexokinase, but expression was not repressed by Ca2+ signaling inhibitors, calmodulin antagonists and inhibitors of protein kinase or protein phosphatase. These results suggested that Os-SUR1 gene expression was stimulated by a hexokinase-dependent pathway not mediated by Ca2+.

Because rice is the major source of food for half of the world population, a comprehensive understanding of its genome will have a tremendous impact on the status of agriculture in the Twenty-first Century. The Rice Genome Research Program (RGP) has undertaken an extensive rice genome analysis since its initial launching in 1991 that has resulted in the establishment of a catalog of rice genes, a high-density linkage map, and a YAC-based physical map. An enormous collection of expressed sequence tags (ESTs) was generated from large-scale cDNA sequencing using cDNA libraries derived from rice callus cultured in different media and tissues such as root, shoot, leaf and panicle. This included more than 9000 partial cDNA sequences corresponding to unique genes have been identified. Based on the results of large-scale cDNA analysis, microarray can be used to monitor gene expression profiles and to initiate functional analysis of the rice genome. We initiated a rice cDNA microarray project beginning in April 1999 that aims to elucidate the function of all genes in rice using a gene expression monitoring system. The National Institute of Agrobiological Resources (NIAR) and the Institute of the Society for Techno-innovation of Agriculture, Forestry and Fisheries (STAFF) are jointly conducting this project in collaboration with 64 research institutions all over Japan. The research areas and investigator/institutions involved in this collaboration are listed in http://microarray.rice.dna.affrc.go.jp/. Homology search of all ESTs was performed by BLASTN and about over 9000 clones with high similarity were clustered. Clustered clones were analyzed for sequence similarity against public protein and nucleic acid databases. Then BLASTN and BLASTX were performed and clones with high similarity were identified putatively. One

Four stereoisomers of 6-amino-6-deoxyheptonic acid, having the l-glycero-d-galacto (1), d-glycero-d-galacto (2), l-glycero-d-gluco (3), and d-glycero-d-gluco (4) configurations, were synthesized from d-galacto- (8) and d-gluco-dialdose (23) derivatives, respectively. Cyanomesylation of 8 and 23 gave two C-6 epimers, respectively, which were separately converted, via the corresponding 6,7-epimino derivatives, into 6-(benzyloxycarbonyl)amino-6-deoxy derivatives by reduction with lithium aluminum hydride, N-(benzyloxycarbonyl)ation, and acetolysis with acetic acid. After deprotection of each hemiacetal, the stereoisomers were oxidized with bromine, followed by total deprotection, to give 1–4. Among these products, 1 and 3 proved to be identical with the naturally occurring destomic and epi-destomic acid obtained from antibiotic destomycins.