Regulation of Drought and Salt Tolerance by OsSKL2 and OsASR1 in Rice

Abstract

Abiotic stresses such as salinity and drought greatly impact the growth and production of crops worldwide. Here, a shikimate kinase-like 2 (SKL2) gene was cloned from rice and characterized for its regulatory function in salinity and drought tolerance. OsSKL2 was localized in the chloroplast, and its transcripts were significantly induced by drought and salinity stress as well as H₂O₂ and abscisic acid (ABA) treatment. Meanwhile, overexpression of OsSKL2 in rice increased tolerance to salinity, drought and oxidative stress by increasing antioxidant enzyme activity, and reducing levels of H₂O₂, malondialdehyde, and relative electrolyte leakage. In contrast, RNAi-induced suppression of OsSKL2 increased sensitivity to stress treatment. Interestingly, overexpression of OsSKL2 also increased sensitivity to exogenous ABA, with an increase in reactive oxygen species (ROS) accumulation. Moreover, OsSKL2 was found to physically interact with OsASR1, a well-known chaperone-like protein, which also exhibited positive roles in salt and drought tolerance. A reduction in ROS production was also observed in leaves of Nicotiana benthamiana showing transient co-expression of OsSKL2 with OsASR1. Taken together, these findings suggest that OsSKL2 together with OsASR1 act as important regulatory factors that confer salt and drought tolerance in rice via ROS scavenging.

Keywords: Oryza sativa; OsASR1; OsSKL2; Protein interactions; Reactive oxygen species; Salt and drought tolerance.

Fig. 1

OsSKL2 subcellular localization and expression profiles. A Subcellular localization of OsSKL2 using a rice protoplast transient transformation system, showing chlorophyll localization (bar = 10 μm). B Inducible expression profiles of OsSKL2 in response to various abiotic stresses. Two-week-old rice seedlings (Zhonghua11) were treated with 100 mM NaCl, 100 mM mannitol, 5 mM H₂O_2, or 100 µM ABA. Root, stem and leaf samples were then harvested at 0, 1, 3, 9, 12, and 24 h after treatment, respectively. Expression levels of OsSKL2 were then determined with qRT-PCR using rice Actin1 as an internal control

Fig. 2

OsSKL2 positively regulates salt tolerance in rice. A Phenotypes of wild-type (WT), OsSKL2-overexpressing (OE, OE3 and OE6), and RNA interference (RNAi, Ri6 and Ri9) plants before (upper) and after treatment with 120 or 140 mM NaCl (middle and lower images). Two-week-old seedlings were used for salt treatment (bar = 5 cm). Survival rates of WT and OsSKL2 transgenic plants under B 120 and C 140 mM NaCl stress. Data represent means ± SD (n = 30). D Relative ion leakage and E the relative water content of WT and OsSKL2 transgenic plants under 120 and 140 mM NaCl stress. Data represent means ± SD. Three independent experiments were carried out with similar results. All data were analyzed using one-way analysis of variance (ANOVA) based on the Student’s t-test. *P < 0.05, **P < 0.01

Fig. 3

OsSKL2 positively regulates drought tolerance in rice. A Phenotypes of wild-type (WT), OsSKL2-overexpressing (OE, OE3 and OE6), and RNA interference (RNAi, Ri6 and Ri9) plants before (upper) and after treatment with 20 or25% PEG (middle and lower). Two-week-old seedlings were used for drought treatment (bar = 5 cm). Survival rates of WT and OsSKL2 transgenic plants under B 20 and C 25% PEG stress. Data represent means ± SD (n = 30). D Relative water contents and E relative ion leakage in WT and OsSKL2 transgenic plants under 20 and 25% PEG stress. Data represent means ± SD. Three independent experiments were carried out with similar results. All data were analyzed using one-way analysis of variance (ANOVA) based on the Student’s t-test. *P < 0.05, **P < 0.01

Fig. 4

OsSKL2 promotes tolerance to salt and drought stress by regulating H₂O₂ homeostasis in rice. Leaves of four-leaf-stage wild-type (WT) and OsSKL2 transgenic seedlings were treated with 25% PEG for 5 d or 140 mM NaCl for 7 d then measured for A H₂O₂, B MDA, C SOD, D POD and E CAT enzyme activity. Data represent means ± SD. F H₂O₂ histochemical analyses of 10-day-old seedlings treated with 250 mM mannitol or 140 mM NaCl then stained with 1% 3,3’-diaminobenzidine tetrachloride (DAB) (bar = 1 cm). G Phenotypes of WT and OsSKL2 transgenic plants before (upper) and after treatment with 100 mM H₂O₂ for 10 d (lower) (bar = 5 cm). H Seedling height and I the seminal root number of WT and OsSKL2 transgenic plants under 100 mM H₂O₂ stress. Data represent means ± SD (n = 30). Three independent experiments were carried out with similar results. All data were analyzed using one-way analysis of variance (ANOVA) based on the Student’s t-test. *P < 0.05, **P < 0.01

Fig. 5

OsSKL2 increases ABA sensitivity in rice. A Phenotypes of WT and OsSKL2 transgenic seedlings grown in 1/2 MS medium with or without 5/10 μM ABA for 10 d (bar = 5 cm). B Seedling height and C the seminal root number of WT and OsSKL2 transgenic plants under 5 and 10 μM ABA treatment. Data represent means ± SD (n = 30). Three independent experiments were carried out with similar results. All data were analyzed using one-way analysis of variance (ANOVA) based on the Student’s t-test. *P < 0.05, **P < 0.01

Fig. 6

OsSKL2 interacts with OsASR1. A OsSKL2 was found to interact with OsASR1 in a yeast two-hybrid system, using BD-OsSKL2 + AD, AD-OsASR1 + BD as negative controls. Growth on selective QDO plates indicated a positive interaction. DDO: yeast SD/-Trp/-Leu selective medium, QDO: yeast SD/-Trp/-Leu/-His/-Ade selective medium. B A pull-down assay was then performed to confirm the interaction between OsSKL2 and OsASR1 in vivo. GST and GST-OsASE1 proteins coupled to beads were incubated with MBP-OsSKL2 fusion proteins. Bound OsSKL2 was then detected using mouse anti-MBP antibody. C A BIFC assay further revealed that OsSKL2 interacts with OsASR1 in rice leaf protoplasts (bar = 10 μm)

Fig. 7

OsASR1 positively regulates salt and drought tolerance in rice. A Phenotypes of wild-type (WT), OsASR1-overexpressing (OE, OE11 and OE12), and RNA interference (RNAi, Ri5 and Ri7) plants before (upper) and after treatment with 120 or 140 mM NaCl (middle and lower). Two-week-old seedlings were used for salt treatment (bar = 5 cm). B Survival rates of WT and OsASR1 transgenic plants before and after 120 and 140 mM NaCl stress. Data represent means ± SD (n = 30). C Phenotypes of WT and OsASR1 transgenic plants before (upper) and after treatment with 20% PEG (middle), and after rewatering for 2 d (bottom). Two-week-old seedlings were used for drought treatment (bar = 5 cm). D Survival rates of WT and OsASR1 transgenic plants under drought stress. Data represent means ± SD (n = 30). Three independent experiments were carried out with similar results. All data were analyzed using one-way analysis of variance (ANOVA) based on the Student’s t-test. *P < 0.05, **P < 0.01

Fig. 8

Co-expression of OsSKL2 and OsASR1 decreased ROS production. A Transient co-expression of OsSKL2 and OsASR1 in the leaves of Nicotiana benthamiana. DAB-stained tobacco leaves were transiently transformed with an empty vector (P35S), OsSKL2 (P35S-OsSKL2), OsASR1 (P35S-OsASR1), and OsSKL2 + OsASR1, respectively (bar = 1 cm). B H₂O₂ contents of transiently transformed tobacco leaves.Three independent experiments were carried out with similar results. All data were analyzed using one-way analysis of variance (ANOVA) based on the Student’s t-test. **P < 0.01