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Plants have developed several strategies for coping with phosphorus (P) deficiency. However, the details of the regulation of gene expression of adaptations to low P are still unclear. Using a cDNA microarray, transcriptomic analyses were carried out of the rice genes regulated by P deficiency and P re-supply to P-deficient plants. The OsPI1 gene, which was isolated as the most significant up-regulated gene under -P conditions, was also the most significant down-regulated gene following P re-supply. Many starch metabolism-related genes, as well as several genes for P(i)-liberating enzymes, were up-regulated by -P treatment, suggesting a homeostatic contribution to the P(i) concentration in leaf tissues. mRNAs for glucanases were also induced by P re-supply: these are suspected to play a role in loosening the cell wall compounds. Most of the genes up-regulated by -P treatment were down-regulated by P re-supply, suggesting that their responses were specific to -P conditions. Conversely, the number of genes up-regulated by P re-supply was also larger following P re-supply than in the -P condition. It is proposed that the genes up-regulated by P re-supply play an important role in P acquisition by P-deficient plants.

Near-isogenic lines (NILs) were applied for the first time to microarray analysis of 8,987 randomly selected expressed sequence tags (ESTs) aiming at screening rice genes associated with seed callus proliferation. Callus proliferation of the variety Koshihikari is poor, since its callus tends to become brown with time. We developed ten NILs that each had a chromosomal segment of the variety Kasalath on chromosome 1 in a Koshihikari background, to cover a region that contains two QTLs for subcultured callus color, qSc1-1 and qSc1-2. The existence of QTLs was verified through tissue culture of the seed calli of these NILs. mRNAs from the calli of three NILs that had the Kasalath allele in qSc1-1 and/or qSc1-2 were applied to microarray analysis. A comparison of expression profiles between Koshihikari and each NIL followed by Northern hybridization showed that 22 unique genes were induced and 15 unique genes were repressed in the calli of these Kasalath allele-containing NILs on chromosome 1. The results of a cis-element search using 500 bp of genomic sequence upstream of each gene suggested that the expression profiles of GA- and/or sugar-responsive genes were different between each NIL and Koshihikari, and that defense-related genes and genes that act negatively in metabolism were repressed in the NILs compared with Koshihikari.

In rice (Oryza sativa L.) plants, the leaf sheaths of the upper leaves accumulate a large amount of starch before heading, and the accumulated starch is converted to sucrose and translocated to the panicles after heading. To analyze the regulation of sink-source transition, we carried out large-scale monitoring of gene expression by microarray analysis using 8987 rice expressed sequence tags (ESTs), and identified 102 developmentally regulated genes in the leaf sheaths of the first leaves below the flag leaves during the heading period. Identified genes included multiple genes related to starch biosynthesis, cell division and expansion, and photosynthesis. All of them showed early stage-preferential expression, probably reflecting the decrease of starch biosynthesis, end of elongation and decrease of photosynthesis in the leaf sheaths during the heading period, respectively. The expression patterns of the genes for starch biosynthesis enzymes and α-tubulin suggest that the leaf sheaths displayed both accumulating- and consuming-sink functions at the very early stage of the heading period. Northern blot analysis of the genes for starch degradation and sucrose biosynthesis enzymes (α-amylase3D and sucrose phosphate synthase) revealed that the induction of the genes did not occur when the starch amount began to decrease, suggesting that the mechanisms of starch degradation and sucrose re-synthesis in the leaf sheaths during the heading period were different from those in germinating seeds. Furthermore, we identified 18 developmentally regulated genes whose functions in the leaf sheaths are unknown. They included seven genes those were preferentially expressed in the bottom part of the leaf sheaths, implying that they were involved in functions related to the starch metabolism. The results obtained showed the effectiveness of the microarray technique to analyze complex and uncharacterized phenomena.

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.

To isolate genes responsive to N-acetylchitooligosaccharide elicitor in suspension-cultured rice cells, we screened rice ESTs by DNA microarray analysis, and were able to identify novel elicitor-responsive genes. Sequencing analysis of three ESTs revealed that the up-regulated genes include a transcription factor, Myb, small G-protein, Rac, and calmodulin. The results indicated the genome-wide change of gene expression in response to N-acetylchitooligosaccharide elicitor.

現在の清酒に比べグルタチオン含有量の高い清酒を開発するため,液胞生理機能欠損変異酵母を用い検討を行った. (1) 液胞生理機能欠損変異酵母において,グルタチオンを菌体外に漏出することが認められた. (2) 液胞生理機能欠損変異酵母に,醸造適性を付与することによりアルコール16%,グルタチオン20ppm以上の清酒を製造することが可能となった. (3) さらに,酵素剤を併用し仕込方法を改良した結果,香り,酒質など品質面において,従来の製品と同等の清酒が製造可能となった. 終わりに臨み,液胞生理機能欠損変異酵母を分譲していただいた東京大学大隅良典教授,国税庁醸造試験所北本勝ひこ博士,ならびに終始ご指導を賜りました弊社常務取締役総合研究所所長布川弥太郎博士に深く感謝いたします.

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.

We collected and completely sequenced 32,127 full-length complementary DNA clones from Oryza sativa L. ssp. japonica cv. “Nipponbare.” Mapping of these clones to genomic DNA revealed ∼20,500 transcriptional units (TUs) in the rice genome. For each TU, we selected 60-mers using an algorithm that took into account some DNA conditions such as base composition and sequence complexity. Using in situ synthesis technology, we constructed oligonucleotide arrays with these TUs on glass slides. We targeted RNAs prepared from normally grown rice callus and from callus treated with abscisic acid (ABA) or gibberellin (GA). We identified 200 ABA-responsive and 301 GA-responsive genes, many of which had never before been annotated as ABA or GA responsive in other expression analysis. Comparison of these genes revealed antagonistic regulation of almost all by both hormones; these had previously been annotated as being responsible for protein storage and defense against pathogens. Comparison of the cis-elements of genes responsive to one or antagonistic to both hormones revealed that the antagonistic genes had cis-elements related to ABA and GA responses. The genes responsive to only one hormone were rich in cis-elements that supported ABA and GA responses. In a search for the phenotypes of mutants in which a retrotransposon was inserted in these hormone-responsive genes, we identified phenotypes related to seed formation or plant height, including sterility, vivipary, and dwarfism. In comparison of cis-elements for hormone response genes between rice and Arabidopsis thaliana, we identified cis-elements for dehydration-stress response as Arabidopsis specific and for protein storage as rice specific.

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+.

To determine the chromosomal positions of expressed rice genes, we have performed an expressed sequence tag (EST) mapping project by polymerase chain reaction–based yeast artificial chromosome (YAC) screening. Specific primers designed from 6713 unique EST sequences derived from 19 cDNA libraries were screened on 4387 YAC clones and used for map construction in combination with genetic analysis. Here, we describe the establishment of a comprehensive YAC-based rice transcript map that contains 6591 EST sites and covers 80.8% of the rice genome. Chromosomes 1, 2, and 3 have relatively high EST densities, approximately twice those of chromosomes 11 and 12, and contain 41% of the total EST sites on the map. Most of the EST-dense regions are distributed on the distal regions of each chromosome arm. Genomic regions flanking the centromeres for most of the chromosomes have lower EST density. Recombination frequency in these regions is suppressed significantly. Our EST mapping also shows that 40% of the assigned ESTs occupy only ∼21% of the entire genome. The rice transcript map has been a valuable resource for genetic study, gene isolation, and genome sequencing at the Rice Genome Research Program and should become an important tool for comparative analysis of chromosome structure and evolution among the cereals.

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