OsPP65 Negatively Regulates Osmotic and Salt Stress Responses Through Regulating Phytohormone and Raffinose Family Oligosaccharide Metabolic Pathways in Rice

Abstract

Although type 2C protein phosphatases (PP2Cs) have been demonstrated to play important roles in regulating plant development and various stress responses, their specific roles in rice abiotic stress tolerance are still largely unknown. In this study, the functions of OsPP65 in rice osmotic and salt stress tolerance were investigated. Here, we report that OsPP65 is responsive to multiple stresses and is remarkably induced by osmotic and salt stress treatments. OsPP65 was highly expressed in rice seedlings and leaves and localized in the nucleus and cytoplasm. OsPP65 knockout rice plants showed enhanced tolerance to osmotic and salt stresses. Significantly higher induction of genes involved in jasmonic acid (JA) and abscisic acid (ABA) biosynthesis or signaling, as well as higher contents of endogenous JA and ABA, were observed in the OsPP65 knockout plants compared with the wild-type plants after osmotic stress treatment. Further analysis indicated that JA and ABA function independently in osmotic stress tolerance conferred by loss of OsPP65. Moreover, metabolomics analysis revealed higher endogenous levels of galactose and galactinol but a lower content of raffinose in the OsPP65 knockout plants than in the wild-type plants after osmotic stress treatment. These results together suggest that OsPP65 negatively regulates osmotic and salt stress tolerance through regulation of the JA and ABA signaling pathways and modulation of the raffinose family oligosaccharide metabolism pathway in rice. OsPP65 is a promising target for improvement of rice stress tolerance using gene editing.

Keywords: ABA; JA; Osmotic stress tolerance; PP2C; Raffinose family oligosaccharide; Rice.

Fig. 1

The change in expression of OsPP65 in response to various stress and chemical treatments. Cold, 8 ℃; NaCl, 150 mM NaCl; PEG, 20% PEG6000; H₂O₂, 1% H₂O₂, ABA, 100 μM ABA; JA, 100 μM JA. Values represent the means ± SD of three biological replicates (5 plants for each replicate), and “Relative expression level” indicates the expression relative to EF1α, which was used as an internal control. The asterisks indicate significant differences compared with the 0 h time point at *P < 0.05 and **P < 0.01 (Dunnett's test)

Fig. 2

The expression patterns of OsPP65 in different rice tissues and its subcellular localization. A Transcription analysis of OsPP65 in different rice tissues by quantitative RT-PCR. Values represent the means ± SD of three biological replicates. B GUS staining analysis of OsPP65 promoter-GUS expression in different rice tissues. a, root; b, the third node; c, the second node; d, the first node; e, panicle of the booting stage; f, panicle of the heading stage; g, leaf; h, 3 day-old seedling. Scale bar = 5 mm. C, Subcellular localization of OsPP65 in rice protoplasts. BF indicates bright field, mCh indicates mCherry, and NLS-mCh indicates nuclear localization signal-tagged mCherry. Scale bar = 10 μm

Fig. 3

OsPP65 knockout rice plants show enhanced osmotic stress tolerance. Data represent means ± SD of three biological replicates (16 plants for each replicate), and the asterisks indicate significant differences compared with the wild-type (WT) plants at *P < 0.05 and **P < 0.01 (Dunnett's test). A Three homozygous transgenic lines used for phenotype analysis. Inserted nucleotides are shown in red, while missing nucleotides are shown in blue. B Phenotypes of the WT and OsPP65 knockout plants before and after osmotic stress treatment. Scale bars = 5 cm. C Survival rates of the osmotic stress-treated plants after 5 days of recovery. D, Relative water loss rate of 2-week-old leaves of WT, ko3, ko6, and ko7. E, Relative ion leakage in rice leaves after osmotic stress for 3 days and 4 days. F, SOD activities in the seedlings of WT and OsPP65 knockout plants before (0 h) and after (4 h) osmotic stress treatment

Fig. 4

Stomatal density and size in the leaves of WT and OsPP65 knockout rice plants. A Photographs showing the stomatal density in WT and OsPP65 knockout plant leaves. Red arrows indicate the stomates in leaves. B Quantification of the stomatal density in leaves of WT and OsPP65 knockout plants. C Quantification of stomatal size in leaves of WT and OsPP65 knockout plants. Data are presented as means ± SD of three biological replicates (5 plants for each replicate) and **P < 0.01 (Dunnett’s test)

Fig. 5

OsPP65 modulates the expression of genes involved in ABA signaling and the accumulation of ABA in rice. A Transcription analysis of the genes involved in ABA signaling in WT and OsPP65 knockout plants before (0 h) and after (4 h) osmotic stress treatment. B Quantification of endogenous ABA levels in the WT and OsPP65 knockout plants. FW, fresh weight. Data represent means ± SD of three biological replicates (5 plants for each replicate), and the asterisks indicate significant differences compared with the WT plants or 0 h treatment at **P < 0.01 and *P < 0.05 (Dunnett's test)

Fig. 6

OsPP65 modulates the expression of genes involved in JA signaling and the accumulation of JA in rice. A Expression analysis of genes associated with JA signaling in WT and OsPP65 knockout plants before (0 h) and after (4 h) osmotic stress treatment. Data represent means ± SD of three biological replicates (5 plants for each replicate). B Quantification of endogenous JA levels in the WT and OsPP65 knockout plants. FW, fresh weight. Data represent means ± SD of three biological replicates (15 plants for each replicate). The asterisks indicate significant differences compared with the WT plants or 0 h treatment at **P < 0.01 and *P < 0.05 (Dunnett's test)

Fig. 7

OsPP65-mediated stress response involves the regulation of RFO metabolism in rice. A The number of primary metabolites and unknown metabolites identified by GC–MS. B The number and proportion of identified metabolites in different classes. C The fold changes in galactinol and raffinose contents in WT and OsPP65 knockout plants at 4 h after osmotic stress treatment relative to the 0 h treatment. D The fold changes in the expression of key enzymes involved in RFO metabolism in WT and OsPP65 knockout plants at 4 h after osmotic stress treatment relative to the 0 h treatment. Data represent means ± SD of three biological replicates (15 plants for each replicate), and the asterisks indicate significant differences compared with the WT plants at *P < 0.05 (Dunnett's test)