Physiological, anatomical and transcriptional alterations in a rice mutant leading to enhanced water stress tolerance

Abstract

Water stress is one of the most severe constraints to crop productivity. Plants display a variety of physiological and biochemical responses both at the cellular and whole organism level upon sensing water stress. Leaf rolling, stomatal closure, deeper root penetration, higher relative water content (RWC) and better osmotic adjustment are some of the mechanisms that plants employ to overcome water stress. In the current study, we report a mutant, enhanced water stress tolerant1 (ewst1) with enhanced water stress tolerance, identified from the ethyl methanesulfonate-induced mutant population of rice variety Nagina22 by field screening followed by withdrawal of irrigation in pots and hydroponics (PEG 6000). Though ewst1 was morphologically similar to the wild type (WT) for 35 of the 38 morphological descriptors (except chalky endosperm/expression of white core, decorticated grain colour and grain weight), it showed enhanced germination in polyethylene glycol-infused medium. It exhibited increase in maximum root length without any significant changes in its root weight, root volume and total root number on crown when compared with the WT under stress in PVC tube experiment. It also showed better performance for various physiological parameters such as RWC, cell membrane stability and chlorophyll concentration upon water stress in a pot experiment. Root anatomy and stomatal microscopic studies revealed changes in the number of xylem and phloem cells, size of central meta-xylem and number of closed stomata in ewst1. Comparative genome-wide transcriptome analysis identified genes related to exocytosis, secondary metabolites, tryptophan biosynthesis, protein phosphorylation and other signalling pathways to be playing a role in enhanced response to water stress in ewst1. The possible involvement of a candidate gene with respect to the observed morpho-physiological and transcriptional changes and its role in stress tolerance are discussed. The mutant identified and characterized in this study will be useful for further dissection of water stress tolerance in rice.

Keywords: Differential gene expression; EMS-induced mutant; SSR genotyping; drought tolerance; germination; grain chalkiness; root traits; stomata.

Figure 1.

Identification of a gain-of-function mutant under PEG-induced water stress and soil-water stress. (A) Twenty-one-day-old seedlings of the selected mutant screened in pots by withholding irrigation for 6 days. (B) The extent of recovery of the mutant after 4 days of the recovery period.

Figure 2.

Germination of ewst1 when compared with its WT Nagina22 (NC, Nagina22 control; NS, Nagina22 stress; MC, mutant control; MS, mutant stress). (A) Germination of ewst1 and Nagina22 in PEG-infused media under control and −0.7 MPa osmotic stress conditions. (B) Comparison of plumule development between WT and ewst1 under three different osmotic levels. (C) Comparison of radicle development between WT and ewst1 under three different osmotic levels. (B and C) Values are mean ± SE of three individual replications having 30 seeds in each plate. Statistical significance was determined using the Holm–Sidak method, with α = 5.000 %. Asterisks indicate significant differences between WT and ewst1 (Student's t-test P < 0.05).

Figure 3.

Morpho-physiological changes in the mutant when compared with Nagina22. (A) Comparison of agronomic traits, namely plant height (PH), PL, NP and FL in centimetres. (B) Comparison of 100 grain weight of unhulled and hulled grain. (C) Grain morphology of Nagina22 (left) and mutant (right) showing 100 % grain chalkiness only in the ewst1. (D) Percentage of RWC under control and stress conditions. (E) Total chlorophyll concentration under control and stress conditions. Values are mean ± SE of five individual replications for (A) and three individual replications for (B–D). Statistical significance determined using the Holm–Sidak method, with α = 5.0 %. Asterisks indicate significant differences between Nagina22 and the mutant (Student's t-test: P < 0.05).

Figure 4.

Comparative stomatal behaviour of ewst1 and WT under control and stress conditions showing CO, PO and CC stomata.

Figure 5.

Comparative root study of ewst1 and the WT. (A) Development of root in a PVC tube under control and stress conditions. (B) Comparison of root traits like MRL, RW, RV and root number (RN) under control and stress conditions. (C) Relative values of maximum root length (RMRL), relative root weight (RRW), relative root volume (RRV) and relative total root number (RRN) under control and stress. (D) Anatomy of root magnifying the stele region (en, endodermis; cb, casparian band; pe, pericycle; cmx, central meta-xylem; mx, meta-xylem; ph, phloem; p, pith). Values are mean ± SE of three individual replications. Statistical significance determined using the Holm–Sidak method, with α = 5.0 %. Asterisks indicate significant differences between ewst1 and WT (Student's t-test: P < 0.05).

Figure 6.

Six-way Venn diagram depicting the number of DEGs in six possible combinations of four samples (NC, Nagina22 control; NS, Nagina22 stress; MC, mutant control; MS, mutant stress). (A) The coloured chambers of the six-way Venn diagram representing uniquely up- and down-regulated differentially expressed genes (URDEGs) out of six combinations (specific colour written for MC, MS, NC, NS, MC and MS and also NC and NS represents specific URDEGs for respective samples). (B) Number of DEGs in six combinations. (C) Number of URDEGs in 12 clusters of similar co-expression.

Figure 7.

Heatmaps of different clusters of URDEGs on the basis of their expression pattern (MC, mutant control; MS, mutant stress; NC, Nagina control; NS, Nagina stress).

Figure 8.

Alterations in biological pathways in the mutant revealed by GO analysis (arrows in the upper and lower direction indicate pathways induced by up-regulated and down-regulated URDEGs, respectively; pathways given in the left, right and middle are altered under control, stress and also both control and stress conditions, respectively).