Abscisic Acid Is Required for Root Elongation Associated With Ca2+ Influx in Response to Water Stress

Abstract

Abscisic acid (ABA) is a critical hormone for plant survival under water stress. In this study, large-scale mutants of the Arabidopsis ecotype Columbia-0 (Col-0) were generated by ethyl methanesulfonate (EMS)-mutagenesis, and an improved root elongation under water-stress 1 (irew1) mutant showing significantly enhanced root growth was isolated under a water potential gradient assay. Then, irew1 and ABA-related mutants in Arabidopsis or tomato plants were observed under water potential gradient assay or water-deficient conditions. ABA pathway, Ca²⁺ response, and primary root (PR) elongation rate were monitored in addition to DNA- and RNA-Seq analyses. We found that based on phenotyping and transcriptional analyses, irew1 exhibited enhanced PR growth, ABA, and Ca²⁺ responses, compared to wild type subjected to water stress. Interestingly, exogenous Ca²⁺ application enhanced PR growth of irew1, ABA-biosynthesis deficient mutants in Arabidopsis, and tomato plants, in response to water potential gradients or water-deficient conditions. In combination with other ABA-related mutants and pharmacological studies, our results suggest that ABA is required for root elongation associated with Ca²⁺ influx in response to water stress.

Keywords: Arabidopsis thaliana; abscisic acid; calcium; root; tomato; water stress.

FIGURE 1

Growth phenotype and map-based cloning of the Arabidopsis irew1 mutant. (A) Phenotype of 10 days after germination (dag) Col-0 and irew1 seedlings grown on a normal growth media (NM), and a water stress media (WSM). The water potentials (Ψw) on NM and WSM were measured and labeled. (B) SNP-index graphs of the Short Manhattan Plot. x-axis represents the position of five chromosomes; y-axis represents the SNP-index. Major (quantitative trait loci) QTL is located to chromosome 1. (C) Mapping of irew1 mutant. irew1 was mapped near the molecular marker nga111 on chromosome 1. The chromosomal number, markers used for fine-mapping, map distance (cM), physical distance (Mb) are shown. (D) PCR-SSLP analysis of a SNP present in nga111 (27.3 Mb) of Chr. We amplified from Ler and irew1 (Col-0 background), and five recombinant plants (1–5) of the F₂ recessive (Short-root under WSM) phenotype population.

FIGURE 2

Classification of differentially expressed genes (DEGs) of wild type (WT) Col-0 and irew1 under WSM. (A,B) Scatter plots of transcript abundance of 10 dag irew1 (A) and Col-0 (B) seedlings under NM or WSM for two days. Red, green, and blue dots represent up-regulated, down-regulated, and not differently expressed genes, respectively (Probability ≥ 0.8 and log₂Ratio ≥ 1). (C) Venn diagram showing the number of DEGs of irew1 and Col-0 under NM or WSM. (D) Heatmap of significantly enriched gene ontology (GO) terms for DEGs. False discovery rate (FDR) was –log10 transformed and displayed as colors ranging from red to blue as shown in the key.

FIGURE 3

Root tip morphology and differentially expressed root development-related genes of Col-0 and irew1 under WSM. (A) Root tips of 10 dag Col-0and irew1 seedlings grown on NM and WSM were stained with propidium iodide (PI) and observed by a confocal microscope. (B) Measurement of primary root (PR) length of Col-0 and irew1 grown on NM and WSM [four biological replicates; three independent experiments (n = 12)]. Bars with different lowercase letters represent significant difference at P < 0.05, as determined by a Tukey’s post hoc test. (C) Cortex cell sizes of Col-0 and irew1 grown on NM and WSM [six biological replicates; three independent experiments (n = 18)]. Bars with different lowercase letters represent significant difference at P < 0.05, as determined by a Tukey’s post hoc test. WSM represents seedlings grown on a water stress media (WSM); NM represents seedlings grown on the normal media (NM).

FIGURE 4

ABA-responsive genes and ABA-related differentially expressed genes of Col-0 and irew1 under NM and WSM. (A) Relative expression levels of RAB18 in Col-0 and irew1 mutant seedlings under NM and WSM. (B) Relative expression levels of RD26 in Col-0 and irew1 mutant seedlings under NM and WSM. (C) Relative expression levels of RD29B in Col-0 and irew1 mutant seedlings under NM and WSM. The values are means, and error bars show ± SD of four technical replicates from three independent experiments (n = 12). (D) Hierarchical cluster analysis applied to the ABA-related DEGs of Col-0 and irew1 mutant under NM and WSM (Col-0-WSM vs. Col-0-NM and irew1-NM vs. irew1-WSM). The transcriptional profiles of ABA-related gene expression values (log₂Ratio values) were analyzed using the heat map command of the R language. White means no difference in gene expression, while red and blue colors represent up-regulated and down-regulated genes, respectively. (E) Relative gene expression values of ATHB7 (AT2G46680) in Col-0-NM, Col-0-WSM, irew1-NM and irew1-WSM. (F) Relative gene expression values of COR78 (AT4G34710) in Col-0-NM, Col-0-WSM, irew1-NM and irew1-WSM. (G) Relative gene expression values of AZF2 (AT2G19580) in Col-0-NM, Col-0-WSM, irew1-NM and irew1-WSM. (H) Relative gene expression values of COR47 (AT1G20440) in Col-0-NM, Col-0-WSM, irew1-NM and irew1-WSM. The values are means, and error bars show ± SD of four technical replicates from three independent experiments (n = 12).

FIGURE 5

Involvement of Ca²⁺ signaling in irew1 under NM and WSM. (A) Phenotype of Col-0 and irew1 mutant seedlings grown on NM, WSM, and WSM containing 10 mM EGTA after 10 days germination (dag). (B) Measurement of primary root (PR) elongation of Col-0 and irew1 mutant seedlings shown in panel (A). (C) Ca²⁺ influx in the root tips (600–800 mm from the root cap junction) of Col-0 and irew1 under WSM. (D) Images of UBQ10 promoter-driven GCaMP6s fluorescence on NM at 0.5 h or 24 h, and on WSM at 0.5 h or 24 h, respectively. (E) Fluorescence intensity (AU) shown in panel (D). The values are means, and error bars show ± SD of six biological replicates from two independent experiments (n = 12). Student’s t-test was performed. ***p < 0.001 by Student’s t-test.

FIGURE 6

Characterization of Col-0, irew1, aba1-1, aba2-1, and 112458 mutants’ seedlings grown on NM and WSM in the presence of Ca²⁺ and ABA. (A) Growth phenotype of Col-0, irew1, and aba1-1, aba2-1, 112458 mutants’ seedlings grown on WSM in the absence or presence of 5 mM Ca²⁺ or 5 mM Ca²⁺ and 3 μM ABA at 10 days after germination (dag), for vertical growth lasting 5 days after which germinated for 5 days. (B) Growth phenotype of Col-0, irew1, and aba1-1, aba2-1, 112458 mutants’ seedlings grown on NM and WSM in the presence of 3 μM ABA at 10 days after germination (dag), for vertical growth lasting 5 days after which germinated for 5 days. (C) Measurement of primary root (PR) elongation rate of Col-0, irew1, and aba1-1, aba2-1, 112458 mutants’ seedlings shown in panels (A) and (B). The values are means, and error bars show ± SD of five biological replicates from three independent experiments (n = 15). Different lowercase letters represent significant difference at P < 0.05, as determined by a Tukey’s post hoc test.

FIGURE 7

ABA and Ca²⁺ are important for PR growth under water-deficient condition in tomato plants. Phenotype of WT tomato and ABA mutant not exposed to or grown under well-watered condition with Ca²⁺ treatment or without Ca²⁺ treatment (A) and drought-stressed condition with Ca²⁺ treatment or without Ca²⁺ treatment (B). After 4-day of germination, the homogeneous seedlings were transplanted to pots containing 14 and 5% water for well-watered and drought-stressed treatments, respectively. For the treatment of Ca²⁺, it was supplied daily in the form of 1 mM Ca²⁺ to the bottom of the pot and thus the Ca²⁺ could be absorbed by roots, while the control plants were supplied with equal volume of water. Eighteen-day old of plants were harvested and analyzed by WinRHIZO 2016a (Reagent Instruments Canada Inc.). (C) Measurement of PR length shown in panels (A,B). The values are means, and error bars show ± SD of six biological replicates from three independent experiments (n = 18). Bars with different lowercase letters represent significant difference at P < 0.05, as determined by a Tukey’s post hoc test.

FIGURE 8

ABA and Ca²⁺ are important for Arabidopsis plants under WSM. (A) Ca²⁺ influx in the root tips (600–800 mm from the root cap junction) of Col-0, irew1, 112458 and Qabi2-2 on WSM, WSM with 10 mM EGTA, and WSM with 10 μM FLU. The values are means, and error bars show ± SD of six biological replicates from three independent experiments (n = 18). (B) Relative expression levels of RD29B in Col-0, irew1, 112458 and Qabi2-2 on WSM, WSM with 10 mM EGTA and WSM with 10 μM FLU. The values are means, and error bars show ± SD of four technical replicates from three independent experiments (n = 12). (C) Primary root elongation rates of Col-0, irew1, 112458 and Qabi2-2 on WSM, WSM with 10 mM EGTA and WSM with 10 μM FLU. The values are means, and error bars show ± SD of four biological replicates from three independent experiments (n = 12). Bars with different lowercase letters represent significant difference at P < 0.05, as determined by a Tukey’s post hoc test.