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.

Transcriptional profiling of genes responsive to abscisic acid and gibberellin in rice: phenotyping and comparative analysis between rice and Arabidopsis.

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 approximately 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.

Genomics approach to abscisic acid- and gibberellin-responsive genes in rice.

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.

Transcriptomic analysis indicates putative metabolic changes caused by manipulation of phosphorus availability in rice leaves.

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.

cDNA microarray analysis of gene expression during Fe-deficiency stress in barley suggests that polar transport of vesicles is implicated in phytosiderophore secretion in Fe-deficient barley roots.

To acquire Fe from soil, graminaceous plants secrete mugineic acid family phytosiderophores (MAs) from their roots. The secretion of MAs increases in response to Fe deficiency, and shows a distinct diurnal rhythm. We used a microarray that included 8987 cDNAs of rice EST clones to examine gene expression profiles in barley roots during Fe-deficiency stress. Approximately 200 clones were identified as Fe-deficiency-inducible genes, of which seven had been identified previously. In order to meet the increased demand for methionine to produce MAs, Fe-deficiency enhances the expression of genes that participate in methionine synthesis, as well as recycling methionine through the Yang cycle. Of these 200 genes, approximately 50 exhibited different transcription levels in Fe-deficient roots at noon and at night. Northern blot analysis of time course experiments confirmed that five of these genes exhibited a diurnal change in their level of expression. The diurnal changes in the expression of these genes suggest that polar vesicle transport is involved in the diurnal secretion of MAs.

Rice gene expression in response to N-acetylchitooligosaccharide elicitor: comprehensive analysis by DNA microarray with randomly selected ESTs.

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.

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 Os-SUR1 gene expression was stimulated by a hexokinase-dependent pathway not mediated by Ca2+.

A comprehensive rice transcript map containing 6591 expressed sequence tag sites.

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 approximately 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.