OsASR5 enhances drought tolerance through a stomatal closure pathway associated with ABA and H2 O2 signalling in rice

Abstract

Drought is one of the major abiotic stresses that directly implicate plant growth and crop productivity. Although many genes in response to drought stress have been identified, genetic improvement to drought resistance especially in food crops is showing relatively slow progress worldwide. Here, we reported the isolation of abscisic acid, stress and ripening (ASR) genes from upland rice variety, IRAT109 (Oryza sativa L. ssp. japonica), and demonstrated that overexpression of OsASR5 enhanced osmotic tolerance in Escherichia coli and drought tolerance in Arabidopsis and rice by regulating leaf water status under drought stress conditions. Moreover, overexpression of OsASR5 in rice increased endogenous ABA level and showed hypersensitive to exogenous ABA treatment at both germination and postgermination stages. The production of H₂ O₂ , a second messenger for the induction of stomatal closure in response to ABA, was activated in overexpression plants under drought stress conditions, consequently, increased stomatal closure and decreased stomatal conductance. In contrast, the loss-of-function mutant, osasr5, showed sensitivity to drought stress with lower relative water content under drought stress conditions. Further studies demonstrated that OsASR5 functioned as chaperone-like protein and interacted with stress-related HSP40 and 2OG-Fe (II) oxygenase domain containing proteins in yeast and plants. Taken together, we suggest that OsASR5 plays multiple roles in response to drought stress by regulating ABA biosynthesis, promoting stomatal closure, as well as acting as chaperone-like protein that possibly prevents drought stress-related proteins from inactivation.

Keywords: ABA; Oryza sativa; OsASR5; Drought; stomata; water content.

Figure 1

Expression analysis of the OsASR5 gene. (a) Real‐time PCR analysis of the expression level of OsASR5 in different tissues of LR variety, Nipponbare, and UR variety, IRAT109. (b) Stress‐inducible expression of OsASR5 under PEG, NaCl, cold, ABA and ethylene treatments. Error bars indicate standard error (SE) based on three replicates.

Figure 2

Enhanced osmotic and drought tolerance in E. coli and Arabidopsis. (a) Isopropylb‐D‐thiogalactopyranoside (IPTG)‐inducible expression of GST and GST‐OsASR5 fusion proteins. GST and GST‐OsASR5 were not (−) or were (+) induced by IPTG. Arrows indicate expression proteins. (b) Growth analysis of cells spotted on LB agar plate supplemented with 0.5 m mannitol. (c) Growth analysis of cells cultured in liquid medium supplemented with 0.5 m mannitol (n = 3). Cell growth densities were measured at 600 nm at the indicated time points. (d) Drought stress tolerance assay of OsASR5 overexpression Arabidopsis transgenic lines and WT by stopping irrigation for 3 weeks and recovery with rewatering for 4 days. (e) Fresh leaf numbers of OsASR5 overexpression Arabidopsis transgenic lines and WT before and after drought stress (n = 3, four plants in each repeat). (f) Water loss rate in the detached leaves of OsASR5 overexpression Arabidopsis transgenic lines and WT under normal conditions (n = 3, 12 leaves in each repeat). Data are mean ± SE. ** indicates significant difference at P < 0.01 probability.

Figure 3

Increased osmotic tolerance of OsASR5 overexpression plants. (a, b) Growth performance of OsASR5 overexpression and NT seedlings under high salinity and mannitol treatments at the seventh d after transplanting, respectively (n = 3, five plants in each repeat). (c, d) The relative plant length and fresh weight of OsASR5 overexpression and NT seedlings corresponding to a, b, respectively. Data are mean ± SE. ** indicates significant difference at P < 0.01 probability.

Figure 4

Enhanced drought tolerance of OsASR5 overexpression plants. (a) Physiological dehydration stress tolerance assay of OsASR5 overexpression and NT plants under 15% PEG6000 treatment. Survival rates of OsASR5 overexpression and NT plants after dehydration stress were examined (n = 3, 15 plants in each repeat). (b) Drought stress tolerance assay of OsASR5 overexpression and NT plants by stopping irrigation for 1 week and recovery with rewatering for 2 weeks. Survival rates of OsASR5 overexpression and NT plants after drought stress were examined (n = 3, nine plants in each repeat). (c) Relative water content of OsASR5 overexpression and NT plants under 15% PEG6000 treatment at the indicated time points (n = 3). Data are mean ± SE. ** indicates significant difference at P < 0.01 probability.

Figure 5

Overexpression of OsASR5 increasing stomatal closure. (a) Scanning electron microscopy images of three levels of stomatal apertures. Bar, 5 μm. (b) The percentage of three levels of stomatal apertures in the leaves of OsASR5 overexpression and NT plants under normal and drought stress conditions (n = 300 stomata for NT under normal conditions; n = 248 stomata for OE‐19 under normal conditions; n = 322 stomata for NT under drought stress; n = 272 stomata for OE‐19 under drought stress). (c) Stomatal density of the middle leaves of OsASR5 overexpression and NT plants (n = 3). Three random scopes were used in each repeat. (d) Stomatal conductance of OsASR5 overexpression and NT plants (n = 3). Data are mean ± SE. ** indicates significant difference at P < 0.01 probability.

Figure 6

ABA accumulation and sensitivity of OsASR5 overexpression plants. (a) ABA contents of OsASR5 overexpression and NT plants under normal and drought stress conditions (n = 3). (b) Real‐time PCR analysis of the expression of ABA biosynthesis and responsive genes under normal and drought stress conditions (n = 3). (c) Germination rates of OsASR5 overexpression and NT seeds under ABA treatment (n = 3, 30 seeds in each repeat). (d) Growth performance and relative plant length of OsASR5 overexpression and NT seedlings under ABA treatment (n = 3, five plants in each repeat). Data are mean ± SE. ** indicates significant difference at P < 0.01 probability.

Figure 7

H₂O₂ accumulation in OsASR5 overexpression plants. (a) 3,3φ‐Diaminobenzidine (DAB) staining for H₂O₂ in the leaves of OsASR5 overexpression and NT plants under normal and drought stress conditions. (b) Quantitative measurement of H₂O₂ in the leaves of OsASR5 overexpression and NT plants under normal and drought stress conditions (n = 3, three plants in each repeat). (c) Growth performance and relative plant length of OsASR5 overexpression and NT plants after MV treatment (n = 3, five plants in each repeat). (d) DAB staining for H₂O₂ in the leaves of OsASR5 overexpression and NT plants after MV treatment corresponding to C. (e) Activity of APX and CAT in the leaves of OsASR5 overexpression and NT plants under normal and drought stress conditions (n = 3). (f) Expression of DST and peroxidase 24 precursor in the leaves of OsASR5 overexpression and NT plants under normal and drought stress conditions. Data are mean ± SE. ** indicates significant difference at P < 0.01 probability.

Figure 8

Increased drought and reduced ABA and oxidative sensitivities of the loss‐of‐function osasr5 mutant. (a) Physiological dehydration stress assay of osasr5 mutant and DJ with 15% PEG6000 treatment. (b) Survival rates of osasr5 mutant and DJ after dehydration stress (n = 3). (c) Relative water content of osasr5 mutant and DJ with 15% PEG6000 treatment observed at three different time intervals (0, 3 and 9d). (d) Germination performance of osasr5 mutant and DJ under ABA treatment (n = 3, 30 seeds in each repeat). (e) Germination rates of osasr5 mutant and DJ corresponding to d. (f) Growth performance of osasr5 mutant and DJ after MV treatment (n = 3, five plants in each repeat). (g) Relative plant length of osasr5 mutant and DJ corresponding to f. (h) DAB staining for H₂O₂ in the leaves of osasr5 mutant and DJ corresponding to f. Data are mean ± SE. ** indicates significant difference at P < 0.01 probability.

Figure 9

Interaction proteins and molecular chaperone activity of OsASR5. (a) Y2H assay of OsASR5 interacting proteins. BD‐OsASR5 co‐transformed with AD empty vector is used as negative control. Three different concentrations of yeast cells were grown on control plate (‐Trp/‐Leu) and selective plate (‐Trp/‐Leu/‐Ade/‐His/X‐α‐gal). (b) BiFC assay for the in vivo interaction of OsASR5 with OsHSP40 and with OsFe(II) Oxy in tobacco epidermal cells (upper) and rice protoplast (lower). Bar, 10 μm. (c) OsASR5 and BSA (control) were not (−) or were (+) boiled at 100 °C for 30 min (n = 3). (d) LDH activity in the presence or absence of OsASR5 during cycles of freeze–thaw treatments (n = 3). Data are mean ± SE. ** indicates significant difference at P < 0.01 probability.

Figure 10

A proposed model explaining the function of OsASR5 in the regulation of stomatal status and drought stress tolerance. Under drought stress, the expression of OsASR5 was up‐regulated,resulting in up‐regulation of ABA biosynthesis and responsive genes,such as OsNCED4 and OsNCED5, RAB16A and RAB16C, leading to ABA accumulation and increased sensitivity to exogenous ABA. Up‐regulation of OsASR5 also affected the activity of H₂O₂‐scavenging enzyme, APX, and suppressed DST and its downstream gene, peroxidase 24 precursor, leading to H₂O₂ accumulation. ABA and H₂O₂ accumulation promotes stomatal closure, resulting in increased water content and finally enhancing drought tolerance. Furthermore, OsASR5 functioned as molecular chaperone and interacted with HSP40 and 2OG‐Fe (II) oxygenase family protein, may prevent those drought stress‐related proteins from inactivation under drought stress conditions. However, the function of those interacted proteins for drought stress tolerance remains to be elucidated in future studies.