Monitoring expression profiles of rice genes under cold, drought, and high-salinity stresses and abscisic acid application using cDNA microarray and RNA gel-blot analyses

Abstract

To identify cold-, drought-, high-salinity-, and/or abscisic acid (ABA)-inducible genes in rice (Oryza sativa), we prepared a rice cDNA microarray including about 1700 independent cDNAs derived from cDNA libraries prepared from drought-, cold-, and high-salinity-treated rice plants. We confirmed stress-inducible expression of the candidate genes selected by microarray analysis using RNA gel-blot analysis and finally identified a total of 73 genes as stress inducible including 58 novel unreported genes in rice. Among them, 36, 62, 57, and 43 genes were induced by cold, drought, high salinity, and ABA, respectively. We observed a strong association in the expression of stress-responsive genes and found 15 genes that responded to all four treatments. Venn diagram analysis revealed greater cross talk between signaling pathways for drought, ABA, and high-salinity stresses than between signaling pathways for cold and ABA stresses or cold and high-salinity stresses in rice. The rice genome database search enabled us not only to identify possible known cis-acting elements in the promoter regions of several stress-inducible genes but also to expect the existence of novel cis-acting elements involved in stress-responsive gene expression in rice stress-inducible promoters. Comparative analysis of Arabidopsis and rice showed that among the 73 stress-inducible rice genes, 51 already have been reported in Arabidopsis with similar function or gene name. Transcriptome analysis revealed novel stress-inducible genes, suggesting some differences between Arabidopsis and rice in their response to stress.

Figure 1.

RNA gel-blot analysis of stress-inducible genes. Each lane was loaded with 10 μg of total RNA isolated from 2-week-old rice seedlings that were exposed to water, dehydration, 250 mm NaCl, 100 μm ABA, and 4°C cold treatment for 1, 2, 5, 10, and 24 h. RNA was analyzed by gel-blot hybridization with gene-specific probes of selected stress-inducible clones by rice cDNA microarray. Stress-inducible clones were classified into various groups on the basis of their expression patterns in RNA gel-blot analysis under each stress treatment. Some of the inducible genes were induced by all four stress treatments; some of them were up-regulated by cold, drought, and high salinity; some of them were induced by drought and high salinity; some of them were induced by cold only, etc.

Figure 1.

RNA gel-blot analysis of stress-inducible genes. Each lane was loaded with 10 μg of total RNA isolated from 2-week-old rice seedlings that were exposed to water, dehydration, 250 mm NaCl, 100 μm ABA, and 4°C cold treatment for 1, 2, 5, 10, and 24 h. RNA was analyzed by gel-blot hybridization with gene-specific probes of selected stress-inducible clones by rice cDNA microarray. Stress-inducible clones were classified into various groups on the basis of their expression patterns in RNA gel-blot analysis under each stress treatment. Some of the inducible genes were induced by all four stress treatments; some of them were up-regulated by cold, drought, and high salinity; some of them were induced by drought and high salinity; some of them were induced by cold only, etc.

Figure 1.

RNA gel-blot analysis of stress-inducible genes. Each lane was loaded with 10 μg of total RNA isolated from 2-week-old rice seedlings that were exposed to water, dehydration, 250 mm NaCl, 100 μm ABA, and 4°C cold treatment for 1, 2, 5, 10, and 24 h. RNA was analyzed by gel-blot hybridization with gene-specific probes of selected stress-inducible clones by rice cDNA microarray. Stress-inducible clones were classified into various groups on the basis of their expression patterns in RNA gel-blot analysis under each stress treatment. Some of the inducible genes were induced by all four stress treatments; some of them were up-regulated by cold, drought, and high salinity; some of them were induced by drought and high salinity; some of them were induced by cold only, etc.

Figure 2.

Venn diagrams showing the classification of genes inducible by cold, drought, and high-salinity stresses and by ABA application identified on the basis of microarray and RNA gel-blot analyses: In total, 36 cold-inducible, 62 drought-inducible, 57 high-salinity-inducible, and 43 ABA-inducible genes were identified by cDNA microarray and confirmed by RNA gel-blot analysis. The identified genes were classified into various groups, such as cold-stress-inducible and drought-stress-inducible, genes that were up-regulated by cold, drought, and high-salinity stresses; genes that were induced by cold or drought stress and ABA application; genes that were up-regulated by cold and drought stresses; and genes that were induced by drought and high-salinity stresses. A, Intersection of genes that were up-regulated by cold stress with those that were either up-regulated by drought stress or high-salinity stress. B, Intersection of genes that were up-regulated by cold stress with those that were either up-regulated by drought stress or ABA application. C, Intersection of genes that were up-regulated by high-salinity stress with those that were either up-regulated by drought stress or ABA application.

Figure 3.

Classification of drought-inducible genes on the basis of expression patterns: Stress-inducible genes were divided into four groups on the basis of their expression patterns under drought stress. A, In the first group consisting of LIP9 and WSI724, expression was induced rapidly after drought stress, reached a maximum at 1 to 2 h after stress, and then decreased. B, In the second group consisting of Asr1 and WSI76, expression was induced within 1 to 2 h of drought stress, and the level was kept relatively constant. C, In the third group consisting of CatB, expression was induced after drought stress, reached a maximum at 5 or 10 h, and then declined. D, In the fourth group consisting of salT, expression was induced after drought stress and reached a maximum at 24 h.