Fumiko Fujii

Identification of several rice genes regulated by Si nutrition

Abstract Rice plants were grown for 4 weeks in nutrient solutions containing 0, 0.015, 0.15, and 1.5 mm silicate. Si content in leaves was lower in the plants grown at low silicate concentrations. RNA was extracted from leaves and subjected to microarray analysis. Statistical analysis of the data from two independent hybridizations enabled to identify a number of candidate clones regulated by Si nutritional conditions. Quantitative reverse transcriptionme diated-PCR enabled to identify-five genes whose transcript accumulation was affected by Si nutrition.

High-throughput Cryopreservation of Plant Cell Cultures for Functional Genomics

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.

Upregulation of the genes for ferritin, RNase, and DnaJ in leaves of rice plants in response to sulfur deficiency

Abstract Microarray analysis was carried out with mRNAs isolated from aerial portions of rice plants subjected to sulfur deficiency. Among the candidate clones regulated by sulfur deficiency, three genes were identified by northern analysis that showed altered levels of mRNA accumulation. Genes similar to those for ferritin, S-like ribonuclease, and DnaJ-like protein were down-, down-, and upregulated by sulfate deficiency, respectively. These genes were found for the first time to be responsive to sulfur nutrition in the present study.

Novel Gene Encoding a Ca2+-Binding Protein and under Hexokinase-dependent Sugar Regulation

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 OsSUR1 gene expression was stimulated by a hexokinase-dependent pathway not mediated by Ca2+.