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.     

Monitoring of Gene Expression Profiles and Isolation of Candidate Genes Involved in Pollination and Fertilization in Rice (Oryza Sativa L.) with a 10K cDNA Microarray

To monitor gene expression profiles during pollination and fertilization in rice at a genome scale, we generated 73,424 high-quality expressed sequence tags (ESTs) derived from the green/etiolated shoot and pistil (0-5 h after pollination, 5hP) of rice, which were subsequently used to construct a cDNA microarray containing ca. 10 000 unique rice genes. This microarray was used to analyze gene expression in pistil unpollinated (UP), 5hP and 5DAP(5 days after pollination), anther, shoot, root, 10-day-old embryo (10EM) and 10-day-old endosperm (10EN). Clustering analysis revealed that the anther has a gene-expression profile more similar to root than to pistil and most pistil-preferentially expressed genes respond to pollination and/or fertilization. There are 253 ESTs exhibiting differential expression (e +/- 2-fold changes) during pollination and fertilization, and about 70% of them can be assigned a putative function. We also recovered 20 genes similar to pollination-related and/or fertility-related genes previously identified as well as genes that were not implicated previously. Microarray and real-time PCR analyses showed that the array sensitivity was estimated at 1-5 copies of mRNA per cell, and the differentially expressed genes showed a high correlation between the two methods. Our results indicated that this cDNA microarray constructed here is reliable and can be used for monitoring gene expression profiles in rice. In addition, the genes that differentially expressed during pollination represent candidate genes for dissecting molecular mechanism of this important biological process in rice.     

Expression and molecular cloning of drought-induced genes in the wild tomato Lycopersicon chilense.

Protein synthesis and translatable mRNA population changes induced during water stress were studied in leaves of a drought-resistant wild relative of tomato, Lycopersicon chilense, using one- and two-dimensional polyacrylamide gel electrophoresis. Under our experimental conditions, water deficit did not significantly affect total protein synthesis capacity. However, it induced biphasic synthesis of a new set of proteins. These newly synthesized proteins resumed to control levels upon rehydration of the plants. Certain drought-induced proteins also accumulated in leaves subjected to heat shock (39 degrees C) or exogenous abscisic acid (ABA, 1 mM) treatments. A cDNA library was constructed using poly(A)+ RNA from leaves of plants exposed to drought stress for 4 days. Differential screening of the library identified three groups of clones corresponding to drought- and ABA-induced mRNAs. Northern blot analysis showed that the genes of selected clones respond differently to the different environmental stresses. Our data clearly demonstrate that water stress alters gene expression in L. chilense plants resulting in the synthesis of new proteins, of which several respond to high temperature stress and others to an osmotic effect. These responses are in part modulated by ABA.     

Comparative expression profiles of maize genes from a water stress-specific cDNA macroarray in response to high-salinity, cold or abscisic acid

cDNA macroarray has become a useful tool to analyze expression profiles and compare the similarities and differences of various expression patterns. We have prepared a cDNA macroarray containing 190 maize expressed sequence tags (ESTs) specifically induced by water stress to analyze the expression profiles of maize seedlings under abscisic acid (ABA) treatment, high-salinity and cold stress conditions. The results indicated that 48 ESTs in leaves and 111 ESTs in roots were significantly up-regulated by ABA treatment, 36 ESTs in leaves and 41 ESTs in roots by high-salinity stress, 14 ESTs in leaves and 18 ESTs in roots by cold induction, whereas 22 ESTs were induced under all 3 stresses. Results from the hierarchical cluster analysis suggest that the leaves and roots of maize seedlings had different expression profiles after these stresses. The overlap analysis of different stress-induced ESTs indicated that there is more crosstalk between water stress and ABA and high-salinity stress than between water stress and cold stress. It will be helpful to study the precise function of the corresponding overlapping-induced genes for understanding the relationship and crosstalk between different stress signal pathways.     

cDNA微阵列技术研究干旱胁迫下星星草基因的表达

运用cDNA微阵列技术研究干旱胁迫下星星草基因的表达。制备了载有660条星星草单一基因的cDNA微阵列。分别对干旱胁迫和对照星星草的mRNA进行荧光标记,并与载有星星草基因的cDNA微阵列进行杂交,通过芯片的杂交信号强度分析,共获得22个下调表达和17个上调表达的基因。BLASTX分析表明这些基因按功能可以分为脱水保护、信号转导与调控、活性氧清除、代谢、核糖体蛋白等几大类。发现了一些与干旱胁迫相关的功能未知基因和新基因。     

Leucine-rich repeat receptor-like kinase1 is a key membrane-bound regulator of abscisic acid early signaling in Arabidopsis

Abscisic acid (ABA) is important in seed maturation, seed dormancy, stomatal closure, and stress response. Many genes that function in ABA signal transduction pathways have been identified. However, most important signaling molecules involved in the perception of the ABA signal or with ABA receptors have not been identified yet. Receptor-like kinase1 (RPK1), a Leu-rich repeat (LRR) receptor kinase in the plasma membrane, is upregulated by ABA in Arabidopsis thaliana. Here, we show the phenotypes of T-DNA insertion mutants and RPK1-antisense plants. Repression of RPK1 expression in Arabidopsis decreased sensitivity to ABA during germination, growth, and stomatal closure; microarray and RNA gel analysis showed that many ABA-inducible genes are downregulated in these plants. Furthermore, overexpression of the RPK1 LRR domain alone or fused with the Brassinosteroid-insensitive1 kinase domain in plants resulted in phenotypes indicating ABA sensitivity. RPK1 is involved in the main ABA signaling pathway and in early ABA perception in Arabidopsis.     

Transcriptional profiles of primary metabolism and signal transduction-related genes in response to water stress in field-grown sunflower genotypes using a thematic cDNA microarray

A sunflower cDNA microarray containing about 800 clones covering major metabolic and signal transduction pathways was used to study gene expression profiles in leaves and embryos of drought-tolerant and -sensitive genotypes subjected to water-deficit stress under field conditions. Using two-step ANOVA normalization and analysis models, we identified 409 differentially expressed genes among genotypes, water treatment and organs. The majority of the cDNA clones differentially expressed under water stress was found to display opposite gene expression profiles in drought-tolerant genotype compared to drought-sensitive genotype. These dissimilarities suggest that the difference between tolerant and non-tolerant plants seems to be associated with changes in qualitative but not quantitative mRNA expression. Comparing leaves and embryos, 82 cDNA clones showing organ-specific variation in gene expression levels were identified in response to water stress across genotypes. Genes related to amino acids and carbohydrates metabolisms, and signal transduction were induced in embryos and repressed in leaves; suggesting that vegetative and reproductive organs respond differentially to water stress. Adaptive mechanisms controlling water deficit tolerance are proposed and discussed.     

Characterization of a gene family encoding abscisic acid- and environmental stress-inducible proteins of alfalfa.

The phytohormone abscisic acid (ABA) has been proposed as a common mediator controlling adaptive plant responses to a variety of environmental stresses, including water deficit, salinity, wounding, and low temperature. We have recently isolated three cDNAs, pUM90-1, pUM90-2, and pUM91-4, from a cDNA library of ABA-induced mRNAs of alfalfa. These cDNA clones exhibit a very high degree of sequence homology with one another and sequence similarities with certain regions of several stress- and ABA-inducible genes. The polypeptides encoded by these cDNAs are very rich in glycine (35-40%), histidine (7-15%), asparagine (8-14%), and tyrosine (5-10%) and have no tryptophan and proline. All of the encoded polypeptides contain characteristic tandem repeats comprising glycine residues intercepted with histidine and/or tyrosine. The RNAs corresponding to a representative cDNA, pUM90-1, were induced after treatment of seedlings with low temperature, drought, salt, and wounding stress, but not by heat; the induction was maximal under low temperature treatment. ABA and ABA analog rapidly induced the expression of these genes, whereas gibberellic acid treatment exhibited no induction whatsoever. These genes appear to be specifically induced in the shoot tissues. Analysis of ABA induction of genes corresponding to pUM90-1 in alfalfa seedlings of different age groups demonstrated that these genes were inducible in seedlings/plants of all age groups examined. Taken together these results suggest that these cDNA clones encode a group of proteins that are inducible by ABA and multiple environmental stresses and correspond to a new family of genes of plants, designated as ABA- and environmental stress-inducible genes.     

Tuber on a chip: differential gene expression during potato tuber development

Potato tuber development has proven to be a valuable model system for studying underground sink organ formation. Research on this topic has led to the identification of many genes involved in this complex process and has aided in the unravelling of the mechanisms underlying starch synthesis. However, less attention has been paid to the biochemical pathways of other important metabolites or to the changing metabolic fluxes occurring during potato tuber development. In this paper, we describe the construction of a potato complementary DNA (cDNA) microarray specifically designed for genes involved in processes related to tuber development and tuber quality traits. We present expression profiles of 1315 cDNAs during tuber development where the predominant profiles were strong up- and down-regulation. Gene expression profiles showing transient increases or decreases were less abundantly represented and followed more moderate changes, mainly during tuber initiation. In addition to the confirmation of gene expression patterns during tuber development, many novel differentially expressed genes were identified and are considered as candidate genes for direct involvement in potato tuber development. A detailed analysis of starch metabolism genes provided a unique overview of expression changes during tuber development. Characteristic expression profiles were often clearly different between gene family members. A link between differential gene expression during tuber development and potato tissue specificity is described. This dataset provides a firm basis for the identification of key regulatory genes in a number of metabolic pathways that may provide researchers with new tools to achieve breeding goals for use in industrial applications.