Molecular and physiological analysis of drought stress in Arabidopsis reveals early responses leading to acclimation in plant growth

Abstract

Plant drought stress response and resistance are complex biological processes that need to be analyzed at a systems level using genomics and physiological approaches to dissect experimental models that address drought stresses encountered by crops in the field. Toward this goal, a controlled, sublethal, moderate drought (mDr) treatment system was developed in Arabidopsis (Arabidopsis thaliana) as a reproducible assay for the dissection of plant responses to drought. The drought assay was validated using Arabidopsis mutants in abscisic acid (ABA) biosynthesis and signaling displaying drought sensitivity and in jasmonate response mutants showing drought resistance, indicating the crucial role of ABA and jasmonate signaling in drought response and acclimation. A comparative transcriptome analysis of soil water deficit drought stress treatments revealed the similarity of early-stage mDr to progressive drought, identifying common and specific stress-responsive genes and their promoter cis-regulatory elements. The dissection of mDr stress responses using a time-course analysis of biochemical, physiological, and molecular processes revealed early accumulation of ABA and induction of associated signaling genes, coinciding with a decrease in stomatal conductance as an early avoidance response to drought stress. This is accompanied by a peak in the expression of expansin genes involved in cell wall expansion, as a preparatory step toward drought acclimation by the adjustment of the cell wall. The time-course analysis of mDr provides a model with three stages of plant responses: an early priming and preconditioning stage, followed by an intermediate stage preparatory for acclimation, and a late stage of new homeostasis with reduced growth.

Figure 1.

Schematic illustration of the drought treatments and sampling in this study. For drought treatments, water was withheld at 25 DAS, and the progress of drought was monitored by soil moisture, shown here as percentage field capacity (FC). Two pDr treatments were done: wilting and prewilting (1 d before wilting, predicted on soil moisture content). Controlled mDr was used to study plant responses at physiological and molecular levels, with the sampling times indicated (−1, 0, 1, 2, and 3). [See online article for color version of this figure.]

Figure 2.

Growth of Arabidopsis ecotype Columbia in response to mDr. A, Response of different developmental stages to mDr in terms of RB. The experiment was repeated twice (n = 7, P < 0.001). B, Relative growth rate (RGR), shown in biomass, and relative expansion rate (RER) in leaf area during two developmental stages, stage 1 (25–30 DAS) and stage 2 (30–35 DAS). Error bars represent se; asterisks indicate significant differences (n = 16, P < 0.01). C, LRWC (%) at different DMD under well-watered (WW) and drought (DRT) conditions. The experiment was repeated twice (n = 12, P < 0.0001). D, LRWC (%; n = 12) and corresponding soil water content (SWC%; n = 20) at different DMD. The experiments were repeated twice. [See online article for color version of this figure.]

Figure 3.

Responses of ABA and JA mutants to mDr treatment. A, Biomass of ABA mutants under well-watered (WW) and drought (DRT) conditions. B, RB of ABA mutants. C, Biomass of JA mutants under well-watered and drought conditions. D, RB of JA mutants. Error bars represent se (n = 8, P < 0.001); asterisks indicate significant differences from the wild-type (WT) control. Col, Ecotype Columbia; Ler, ecotype Landsberg erecta. [See online article for color version of this figure.]

Figure 4.

Gas-exchange measurements and WUEi in a time course of mDr. A, Photosynthesis (Pn), stomatal conductance (gs), and internal CO₂ (Ci). B, WUEi in a time course of mDr. n = 5 per treatment per time point, three leaves were measured per plant, the experiment repeated twice, and error bars represent se (P < 0.001). DRT, Drought; WW, well watered. [See online article for color version of this figure.]

Figure 5.

Gene expression analysis under mDr and pDr. Venn diagrams comparing up-regulated and down-regulated genes of pDr, mDr D01, and mDr D10 treatments are shown at top. Sequence logos of cis-elements derived by de novo promoter analysis with similarity to the ABRE element in the three drought treatments are shown at bottom. [See online article for color version of this figure.]

Figure 6.

Differentially expressed mDr Day01 genes in relation to other factors. The up-regulated and down-regulated mDr Day01 genes compared with other gene sets, including mDr Day10, pDr, GO biological process, GO cellular components, ABA-related, and cis-element-based gene sets, are shown. Only gene set pairs with significant enrichment have been connected to each other. The key describes the identities of nodes, where colors refer to the type of gene set. Node size corresponds to the number of genes in that gene set (indicated in parentheses on each node), and edge thickness corresponds to the level of overlap between the gene sets connected.

Figure 7.

cis-Regulatory elements identified in the upstream regions of mDr- and pDr-regulated genes. Each element identified along the rows was identified using de novo motif discovery to find short degenerate DNA sequences whose presence or absence in the 1-kb upstream regions of genes is highly informative about the expression of the given gene set (e.g. up-regulated genes in mDr Day01) given the background distribution of the sequence in the upstream sequences of all the genes in the genome. The colored matrix indicates which motifs were identified using genes regulated in which drought treatment, with yellow (reverse diagonal stripe) indicating down-regulation and blue (diagonal crosshatch) indicating up-regulation. Motifs informative about up-regulation and down-regulation together are indicated by green (dots). In the adjoining table, the sequences of the de novo motifs are given in the nucleotide International Union of Pure and Applied Chemistry nomenclature along with the Z-score of the information value of the motif, reflecting how far the observed value is, in number of sds, from the average random information (see “Materials and Methods”). Known elements in the PLACE database with significant matches to each de novo motif are presented in the PLACE motifs table in the form of the database identifier (ID), DNA sequence, and E-value of the sequence match with the de novo motif. Motifs with no match to any known element are novel putative regulatory elements. [See online article for color version of this figure.]

Figure 8.

Drought stress responses in ABA levels and ABA-related genes. A, ABA quantification (in percentage of well-watered control) from day 0 to day 2 of mDr (DMD). B, qRT-PCR analysis of stress signaling genes and stress marker genes under pDr prewilting (PPW) and mDr Day01 (MD). C to E, Time-course responses in DMD (x axis) of stress-related genes using qRT-PCR showing fold change (y axis). C, ABA signaling pathway genes NCED3 and ABF3. D, DREB2A (ABA-independent signaling pathway). E, Stress marker genes. For A to D, three replications with five plants pooled per replication were used; error bars represent se. [See online article for color version of this figure.]

Figure 9.

Gene expression profiles of stomatally related genes during mDr treatment. The y axis shows the fold change using qRT-PCR, and the x axis shows the days of mDr. A, PLDα1, GPA1, and GORK. B, PP2Cs. C, RPK1. D, MYB60 transcription factor. For all panels, three replications with five plants pooled per replication were used; error bars represent se. [See online article for color version of this figure.]

Figure 10.

Gene expression profiles of drought-responsive genes. Time course is in DMD (x axis) showing gene expression fold change (y axis). A, Photosynthesis-related genes. B, Antioxidant enzyme genes. For both panels, three replications with five plants pooled per replication were used; error bars represent se. [See online article for color version of this figure.]

Figure 11.

Gene expression profiles of expansin genes in drought acclimation response. A, Expression of expansin genes under pDr prewilting (PPW) and mDr (MD) showing fold change (y axis). B, Expression profiles of expansin genes shown in fold change (y axis) in a time course of mDr (x axis). For both panels, three replications with five plants pooled per replication were used; error bars represent se. [See online article for color version of this figure.]

Figure 12.

Physiological, biochemical, and molecular plant responses to mDr. Plant responses to mDr are dissected into three stages: early priming (preconditioning) stage, in which all stress signaling and avoidance processes take place; intermediate stage, which is preparatory for acclimation, as plants modify and adjust cell walls for reprogrammed growth responses at later stages; and late stage, in which plants are set to a new homeostasis with altered hormonal signaling and reduction in energy-demanding processes, leading to acclimated plants with reduced growth. FC, Field capacity. [See online article for color version of this figure.]