Leaf responses to mild drought stress in natural variants of Arabidopsis

Abstract

Although the response of plants exposed to severe drought stress has been studied extensively, little is known about how plants adapt their growth under mild drought stress conditions. Here, we analyzed the leaf and rosette growth response of six Arabidopsis (Arabidopsis thaliana) accessions originating from different geographic regions when exposed to mild drought stress. The automated phenotyping platform WIWAM was used to impose stress early during leaf development, when the third leaf emerges from the shoot apical meristem. Analysis of growth-related phenotypes showed differences in leaf development between the accessions. In all six accessions, mild drought stress reduced both leaf pavement cell area and number without affecting the stomatal index. Genome-wide transcriptome analysis (using RNA sequencing) of early developing leaf tissue identified 354 genes differentially expressed under mild drought stress in the six accessions. Our results indicate the existence of a robust response over different genetic backgrounds to mild drought stress in developing leaves. The processes involved in the overall mild drought stress response comprised abscisic acid signaling, proline metabolism, and cell wall adjustments. In addition to these known severe drought-related responses, 87 genes were found to be specific for the response of young developing leaves to mild drought stress.

Figure 1.

Transfer protocol of seedlings to the WIWAM. A, Seeds were germinated on soil-filled 96-well plates. Average-sized seedlings (54; red circles) were selected using an in-house-developed image analysis algorithm. The seedling selection was done only for phenotyping experiments. B, At 4 DAS, seedlings were manually transferred from 96-well plates to the pretreated soil (drought or control). C, Pots were placed on the WIWAM for automatic phenotyping. D, Control soil water content was maintained at a constant value of 2.2 g water g⁻¹ dry soil (solid line) during the entire experiment. For the mild drought condition (dashed line), the soil water content started at 1.2 g water g⁻¹ dry soil after transfer to pots at 4 DAS. The stress level increased from 11 DAS onward until it reached 0.7 g water g⁻¹ dry soil.

Figure 2.

Measurements of rosette area and leaf size. A, Rosette areas at maturity. B, Leaf 3 areas at maturity. Control conditions (C) are indicated in black, and mild drought conditions (S) are indicated in gray. C, Leaf 3 areas at the transition from proliferation to expansion. D, Projected rosette area over time in control conditions for the six accessions. All values are least-square means ± se estimated from the mixed model, and percentages represent reductions under mild drought relative to the control.

Figure 3.

Projected rosette area over time under control (C) and mild drought (S) conditions for the six accessions. Accession names indicated in the figure in black are significantly different from the reference accession in the time × genotype × treatment interaction at a P value cutoff of 0.05, and those indicated in gray are not significant. Values are least-square means ± se estimated from the mixed model.

Figure 4.

Measurements of cellular parameters of the mature third leaf of six accessions in control and mild drought conditions. A, Averaged cell areas of the pavement cells (µm²). B, Total number of pavement cells in the leaf. C, Pavement cell density (cells mm⁻²). D, Stomatal index as a percentage of stomata on total cell number. Control conditions (C) are indicated in black, and mild drought conditions (S) are indicated in gray. All values are means ± se, and percentages represent reductions under mild drought relative to the control.

Figure 5.

Coexpression and regulatory interaction network of common differentially expressed genes in the six accessions. Edges colored blue connect coexpressed genes, and thickness increases with rising coexpression coefficients. Red, green, and black lines represent regulatory interactions, which can be direct (solid lines) or indirect (dashed lines). Regulatory interactions can be activating (green), repressing (red), or unknown (black). The top 20 most interacting genes in the network are indicated in yellow and can be found in Supplemental Table S8. Squares and diamonds are query genes, and circles are neighbor genes. Visualization is based on the coexpression analysis done in CORNET with a coexpression coefficient of 0.7 and confirmed regulatory interactions from AGRIS, microarray gene-target relations, and EVEX.

Figure 6.

Venn diagram showing the overlap between genes involved in severe drought studies (Huang et al., 2008; Matsui et al., 2008; Harb et al., 2010), genes in mature tissue in mild drought studies (Harb et al., 2010; Baerenfaller et al., 2012; Des Marais et al., 2012), and the common drought genes.