Rice is severely affected by salt stress - one of the major abiotic environmental stresses encountered by crop plants. Present study was conducted to unravel salt stress-induced change of global gene expression patterns in leaves of rice. Rice (cv. Nipponbare) seedlings were hydroponically-grown for 18-days, and treated with 130 mM NaCl as described by Kim et al. (2005). After 3, 4, and 6 days after treatment (DASS), fully expanded 3rd leaves were harvested for microarray (Agilent 22K custom oligo DNA microarray; Kikuchi et al. 2003) analysis using a dye-swap or reverse labeling with Cy3 and Cy5 dyes procedure, and two biological replicates. Scanning (Gene Pix) of hybridized slides, image analysis and data extraction (Gene Pix 4000), and normalization (GeneSpring Ver. 4) was performed to obtain fold changes (>3.0-fold and <0.4-fold) from normalized log ratios. We detected salt stress-induced expression of 773 genes, among which 438 and 335 genes were either up- or down-regulated at least one time point of salt stress (Table 1). It was found that 374 genes have various known (or putative) functions in plant cells. Identified genes were grouped into 7 categories based on the time-dependent change of their expressions and major functions. Among the identified genes, we show the five representative genes highly up- or down-regulated at each time point of salt stress (Table 2). The largest category at all time points of salt stress was the metabolism containing 118 genes like RRJ1, ADP glucose pyrophosphorylase large subunit and glycosyltransferase.

These genes accounted for 31.3, 28.3 and 27.1% of all the identified genes at 3, 4 and 6 DASS. Genes grouped in cell rescue, defense and virulence category accounted for 15% of all identified genes at each time point of salt stress. Out of 48 genes in this category, 40 genes including blt101, LEA-like protein and dehydrin were up-regulated by salt stress. Dehydrin was strongly up-regulated at 3 DASS showing highest expression level at 6 DASS compared with LEA-like protein most strongly up-regulated at 3 DASS followed by drastic decrease in expression level at 4 and 6 DASS. We think that the LEA protein may function at the initial phase of salt stress, but dehydrin may continue its function by responding to the increasing severity of salt stress condition. In cellular communication/signal transduction mechanism category, expression of 45 genes was differentially regulated by salt stress, which contained protein phosphatase 2C, ABA-responsive protein and light-mediated development protein DET1. Among 53 genes in cellular transport, transport facilitation and transport routes categories, a larger number of genes were down-regulated by salt stress. At 6 DASS, in particular, 18 genes including an amino acid carrier were down-regulated while only 6 genes were up-regulated. In the energy category, expression of 30 genes including isocitrate lyase, chlorophyll a/b-binding protein and PEPC1 were differentially regulated by salt stress. These genes are known to be involved in various energy-related processes such as photosynthesis, photorespiration and respiration. Out of 40 genes grouped under transcription, 29 and 11 genes were either up- or down-regulated by salt stress. These genes were mostly transcription factors such as leucine zipper, zinc finger, myb and NAC. In others category, expression of 40 genes was differentially regulated by salt stress. These genes are known to be involved in various cellular functions like protein modification, protein synthesis and nucleotide binding. In conclusion, functional categorization of salt stress-responsive genes revealed an early regulation of transcription and metabolism at 3 days (early-phase), followed by gene expressions related to the cellular communication/signal transduction and metabolism categories at the mid-phase of the ongoing salt stress response (4 days), culminating in the late-phase of salt stress where most of the genes belonged to the cell rescue, defense and virulence and metabolism categories.

References

Kim D. W., R. Rakwal, G. K. Agrawal, Y. H. Jung, J. Shibato, N. S. Jwa, Y. Iwahashi, H. Iwahashi, D. H. Kim, L. S. Shim, K. Usui, 2005. A hydroponic rice seedling culture model system for investigating proteome of salt stress in rice leaf. Electrophoresis 26: 4521-4539.

Kikuchi S., K. Satoh, T. Nagata, N. Kawagashira, K. Doi, et al., 2003. Collection, mapping, and annotation of over 28,000 cDNA clones from japonica rice. Sci. 301: 376-379.