Transcriptional and physiological study of the response of Burma mangrove (Bruguiera gymnorhiza) to salt and osmotic stress

Abstract

We investigated the transcriptional response of Burma mangrove (Bruguiera gymnorhiza) to high salinity (salt stress; 500 mM NaCl) and hyperosmotic stress (osmotic stress; 1 M sorbitol) by microarray analysis. ANOVA (P < 0.05) and significant analysis of microarray (SAM; FDR < 5%) revealed that 865 of 11,997 genes showed significant differential expression under salt and osmotic stress. Scatter plot analysis revealed that the expression level of genes changed at 6 h after salt stress treatment, but recovered at 24 h, while the change at 6 h after osmotic stress treatment diverged at 24 h. Hierarchical clustering of the 865 genes showed that expression profiles under salt stress were distinctly different from those under osmotic stress. Comparison of gene ontology (GO) categories of differentially expressed genes under the stress conditions revealed that the adaptation of Burma mangrove to salt stress was accompanied by the up-regulation of genes categorized for "cell communication," "signal transduction," "lipid metabolic process," "photosynthesis," "multicellular organismal development," and "transport," and by down-regulation of genes categorized for "catabolic process." Burma mangrove maintained its leaf water potential and recovered from its photosynthesis rate that declined temporarily under salt stress, but not under osmotic stress. These results demonstrated a fundamental difference between the response to salt and osmotic stress. Ion and sugar content analysis suggested that salt tolerance of Burma mangrove might be attributed to their ability to accumulate high concentrations of Na+ and Cl(-), even under non-stressed conditions; to uptake additional Na+ and Cl(-) for use as osmolytes; and to maintain K+ homeostasis under salt stress.

Transcriptome Profiling of the Mangrove PlantBruguiera gymnorhizaand Identification of Salt Tolerance Genes byAgrobacteriumFunctional Screening

“…Cultivation of B. gymnorhiza and stress treatment with 500 mM NaCl were performed as previously described. 16) Main roots and lateral roots were collected from sets of three trees at 0, 1, 3, 6, 12, and 24 h, and 3, 6, and 12 d after NaCl-treatment, comprising a total of 27 trees. At each time point, the treated plants were first pulled from pots, and then the roots were washed briefly in water, wiped with paper, and separated from the rest of the plant.…”

“…A B. gymnorhiza microarray containing 11,997 oligoprobes (35-40 mer) was prepared as previously described. 16) RNA extraction, amplification and labeling of aRNA, hybridization, and quantification of spot intensity were also performed as previously described. 16) Data analysis was performed using ArrayAssist software (Stratagene, La Jolla, CA).…”

“…16) RNA extraction, amplification and labeling of aRNA, hybridization, and quantification of spot intensity were also performed as previously described. 16) Data analysis was performed using ArrayAssist software (Stratagene, La Jolla, CA). Local background substitution, Lowess normalization, and pairwise t-test were applied to identify differentially expressed genes.…”

“…To verify the accuracy of the microarray assays, real-time quantitative PCR was performed as previously described. 16) Briefly, single strand cDNA was synthesized from total RNA using a QuantiTect Reverse Transcription Kit (Qiagen, Tokyo) according to the manufacturer's instructions. Real-time quantitative PCR was performed using a QuantiTect SYBR Green PCR Kit (Qiagen) with LightCycler (Roche Diagnostics, Basel, Switzerland).…”

“…Microarray is a powerful tool for comprehensive examination of gene expression, and it has been applied to monitor the expression profiles of both glycophytes and halophytes under salt stress. [7][8][9][10][11][12][13][14][15][16] Transcriptional profiling of the mangrove plant Bruguiera gymnorhiza to salt stress has been reported, 15) but the salt responsive genes identified contained no regulatory gene. This was presumably due to the low quality of the spotted array.…”