Rice OsPUB16 modulates the ‘SAPK9-OsMADS23-OsAOC’ pathway to reduce plant water-deficit tolerance by repressing ABA and JA biosynthesis

Abstract

Ubiquitin-mediated proteolysis plays crucial roles in plant responses to environmental stress. However, the mechanism by which E3 ubiquitin ligases modulate plant stress response still needs to be elucidated. In this study, we found that rice PLANT U-BOX PROTEIN 16 (OsPUB16), a U-box E3 ubiquitin ligase, negatively regulates rice drought response. Loss-of-function mutants of OsPUB16 generated through CRISPR/Cas9 system exhibited the markedly enhanced water-deficit tolerance, while OsPUB16 overexpression lines were hypersensitive to water deficit stress. Moreover, OsPUB16 negatively regulated ABA and JA response, and ospub16 mutants produced more endogenous ABA and JA than wild type when exposed to water deficit. Mechanistic investigations revealed that OsPUB16 mediated the ubiquitination and degradation of OsMADS23, which is the substrate of OSMOTIC STRESS/ABA-ACTIVATED PROTEIN KINASE 9 (SAPK9) and increases rice drought tolerance by promoting ABA biosynthesis. Further, the ChIP-qPCR analysis and transient transactivation activity assays demonstrated that OsMADS23 activated the expression of JA-biosynthetic gene OsAOC by binding to its promoter. Interestingly, SAPK9-mediated phosphorylation on OsMADS23 reduced its ubiquitination level by interfering with the OsPUB16-OsMADS23 interaction, which thus enhanced OsMADS23 stability and promoted OsAOC expression. Collectively, our findings establish that OsPUB16 reduces plant water-deficit tolerance by modulating the ‘SAPK9-OsMADS23-OsAOC’ pathway to repress ABA and JA biosynthesis.

OsNF‐YA3 regulates plant growth and osmotic stress tolerance by interacting with SLR1 and SAPK9 in rice

“…Meanwhile, under water-deficit stress, ya3 mutants had higher ABA levels, while OsNF-YA3-overexpressing lines produced less ABA than the wild type (Figure 4e). Altogether, the OsNF-YA3-inhibited osmotic stress tolerance is ABAdependent, which is in agreement with earlier research that ABA-dependent signaling is involved in various osmotic stress responses (Lv et al, 2022;Nakashima & Yamaguchi-Shinozaki, 2013;Xu et al, 2018;Yoshida, Mogami, & Yamaguchi-Shinozaki, 2014).…”

“…ABA is generally considered to promote stomatal closure, thus avoiding the water loss when plant are subjected to water‐deficit or salt stress (Li et al., 2021; Lv et al., 2022; Watkins et al., 2017; Yao et al., 2018). To elucidate the cellular response influenced by OsNF‐YA3 when plants are subjected to osmotic stress, the stomatal movement induced by ABA in OsNF‐YA3 ‐overexpressing plants and ya3 mutants was compared with that in the wild type.…”

“…OsNF-YA3 represses ABA response and reduces ABA levels in rice ABA is generally considered to promote stomatal closure, thus avoiding the water loss when plant are subjected to water-deficit or salt stress (Li et al, 2021;Lv et al, 2022;Watkins et al, 2017;Yao et al, 2018) 4a). Without ABA, the completely closed stomata proportion differed slightly between various genotypic plants of OsNF-YA3; however, following treatment with ABA, the completely closed stomata proportion in ya3 mutants was significantly higher than the wild type (Figure 4b; Figure S8).…”

“…For transient transcriptional regulation assays, the dual-luciferase reporter system was utilized to generate the effector and reporter, as described previously (Lv et al, 2022). Co-transformation of the reporter and effector into 4-week-old N. benthamiana leaves was carried out through the agroinfiltration expression system (Liu et al, 2010).…”

Knockout Mutants of OsPUB7 Generated Using CRISPR/Cas9 Revealed Abiotic Stress Tolerance in Rice

“…OsPUB15 transgenic rice caused severe growth delays and fatal phenotypes in seedling, and transcription levels of OsPUB15 knockout mutants were shown to increase with H 2 O 2 , salinity, and drought stress [ 27 ]. Knockout mutants of OsPUB16 generated by CRISPR/Cas9 showed improved water resistance by mediating ubiquitination and degradation of OsMADS23 , a substrate of OSMOTIC STRESS/ABA-ACTIVATED PROTEIN KINASE 9 (SAPK9), and OsPUB16 over-expression lines showed an increased water deficiency susceptibility to stress [ 28 ].…”