Comprehensive transcriptional profiling of NaCl-stressed Arabidopsis roots reveals novel classes of responsive genes

Abstract

Background: Roots are an attractive system for genomic and post-genomic studies of NaCl responses, due to their primary importance to agriculture, and because of their relative structural and biochemical simplicity. Excellent genomic resources have been established for the study of Arabidopsis roots, however, a comprehensive microarray analysis of the root transcriptome following NaCl exposure is required to further understand plant responses to abiotic stress and facilitate future, systems-based analyses of the underlying regulatory networks.

Results: We used microarrays of 70-mer oligonucleotide probes representing 23,686 Arabidopsis genes to identify root transcripts that changed in relative abundance following 6 h, 24 h, or 48 h of hydroponic exposure to 150 mM NaCl. Enrichment analysis identified groups of structurally or functionally related genes whose members were statistically over-represented among up- or down-regulated transcripts. Our results are consistent with generally observed stress response themes, and highlight potentially important roles for underappreciated gene families, including: several groups of transporters (e.g. MATE, LeOPT1-like); signalling molecules (e.g. PERK kinases, MLO-like receptors), carbohydrate active enzymes (e.g. XTH18), transcription factors (e.g. members of ZIM, WRKY, NAC), and other proteins (e.g. 4CL-like, COMT-like, LOB-Class 1). We verified the NaCl-inducible expression of selected transcription factors and other genes by qRT-PCR.

Conclusion: Microarray profiling of NaCl-treated Arabidopsis roots revealed dynamic changes in transcript abundance for at least 20% of the genome, including hundreds of transcription factors, kinases/phosphatases, hormone-related genes, and effectors of homeostasis, all of which highlight the complexity of this stress response. Our identification of these transcriptional responses, and groups of evolutionarily related genes with either similar or divergent transcriptional responses to stress, will facilitate mapping of regulatory networks and extend our ability to improve salt tolerance in plants.

Figure 1

Hydroponic growth system and physiological responses to NaCl treatments. a) A raft of Arabidopsis plants at 21 dpi, preceding treatment. This raft has been removed from the hydroponic growth medium for the purpose of this photograph; although individual roots cling together in air, they are well separated when submerged in the growth medium. b) Anthocyanin accumulation in Arabidopsis leaves. Plants were mock treated or exposed to 150 mM NaCl for 6, 24 and 48 h. The means of six experiments ± standard deviation are shown. Anthocyanin concentration is reported as an absorbance ratio: (A₅₃₀-1/4*A₆₅₇)/g fresh weight. c) Proline accumulation in roots and leaves of Arabidopsis plants. Arabidopsis plants were mock treated or exposed to 150 mM NaCl for 6, 24 and 48 h, and proline concentration was determined in both leaves and roots. The means of six experiments ± standard deviation are shown.

Figure 2

NaCl-responsive transcripts grouped according to temporal expression profiles. Microarray expression data that had been previously filtered for significance based on signal intensity and SAM [21] statistical analysis, was divided into 16 distinct temporal profiles, using STEM software [22]. Each of the profiles (labelled a-o) is represented as a different plot, with mean expression ratios (log₂; treated:control) for each of the assigned transcripts at each time point plotted in grey. The median of all assigned expression ratios in each profile is plotted in black. To emphasize that the y-axis range is different between plots, a dashed line corresponding to a log₂expression ratio of 0 is also shown in each plot.