| In this study,we focus on the function of OsDET1 in rice development,analyzing the stress-induced and spatial expression profiles of OsDET1 in rice. Meanwhile, the OsDET1 deficiency, OsDET1-GFP and OsDET1 overexpression transgenic plants were used to explore the function of OsDET1 in ABA signaling pathwayand ABA biosynthesis in Rice. Finally, we draw the following conclusions.We demonstrated that down-regulation of OsDET1 lead to pleiotropic phenotypes in rice. The extremity of OsDET1 silencing was lethal when it occurred at early vegetative growth stage and seed germination. OsDET1 deficiency also resulted in the marked decrease of overall biomass and seed yield. In addition, the vast majority of spikelets were infertility in OsDET1 RNAi transgenic plants, which demonstrates that OsDET1 is essential for spikelets fertility in rcie. Overall, according to these extreme phenotypes in OsDET1 RNAi transgenic plants, we conclude that the OsDET1 deficiency had strong detrimental effects on basic plant development in rice, indicating that OsDET1 is essential for maintaining normal development in rice and is required throughout the plant life cycle.We observed some phenotypes related to ABA hypersensitivity in OsDET1 RNAi transgenic plants. Such as the changes of pollen grain morphology and stomatal pores were closed in OsDET1 RNAi transgenic plants. But the content of ABA was reduced in OsDET1 RNAi transgenic plant leaves. These results indicated that OsDET1 deficiency enhanced the sensitivity to ABA in rice. Furthermore, OsDET1 deficiency accelerated ABA- and dark-induced leaf senescence, which further confirming that OsDET1 deficiency lead to ABA hypersensitivity in rice. In Arabidopsis, ABA signaling is mediated by PYR/PYL/RCAR family of ABA receptors. DET1 play a negative regulator of ABA signaling by modulating ubiquitination of PYLs ABA receptor. We speculate that a similar ABA signal regulatory mechanism also exists in rice. The results of protein interaction analysis and the abundance of PYR/PYL/RCAR ABA receptors strongly support this model. First, we found that OsDET1 interacts with Os DDB1 and Os COP10 in vivo. Next, we demonstrated that Os DDA1 interacts with Os PYL5 and Os DDB1. Moreover, the degradation of Os PYL5 was impaired in OsDET1 RNAi plants. In addition, Os PYL5 was previously shown to promote the expression of ABA-inducible marker genes(including Os RAB16 A, Os LEA and Os LIP9) during ABA treatment and to play an important role in mediating ABA signaling during seed germination and early seedling growth. In the current study, these ABA-inducible marker genes were significantly upregulated in OsDET1 RNAi plants during ABA treatment compared to WT. These results are consistent with the finding that Os PYL5 accumulated in OsDET1 RNAi plants, which further supports our hypothesis. In addition, the correct structure of DET1 is necessary for maintaining the function of the CDD complex. A C-terminal GFP-tag on DET1 hinders the formation of the CDD complex by DET1, COP10 and DDB1 in Arabidopsis. We found that overexpressing OsDET1-GFP also increased plant sensitivity to ABA signaling and delayed the degradation of Os PYL5 in OsDET1-GFP plants.These findings suggest that structural changes in OsDET1 impair the normal functioning of CDD and may affect ABA receptor stability in rice, further supporting the role of OsDET1 in modulating ABA signaling.Interestingly, OsDET1 also influences ABA biosynthesis in rice. The contents of ABA were decreased in OsDET1 RNAi transgenic plants. Concurrently, the transcription levels of ABA-inactivation genes were significantly increased, and the expression of ABA biosynthesis genes were down-regulated in OsDET1 RNAi transgenic plants compared with WT. On the contrary, the contents of ABA were increased in OsDET1 overexpression transgenic plants. Meanwhile, the transcription levels of ABA-inactivation genes were significantly decreased, and the expression of ABA biosynthesis genes were up-regulated compared with WT. We found that seed germination was delayed in OE-OsDET1 plants and that root and shoot growth was inhibited during ABA treatment. OE-OsDET1 plants also showed premature leaf senescence during dark treatment. These phenotypes are in accordance with the higher ABA levels in OE-OsDET1 plants, and they also support our conclusion that OsDET1 plays a positive role in ABA biosynthesis. In addition, the current study found that there was less wax crystallization on the leaf surface of OsDET1 RNAi plants than WT. On the contrary, more wax crystallization was existed on the OE-OsDET1 plants leaves surface than WT. These results are consistent with the change of ABA contents in transgenic plants, further supporting the notion that OsDET1 modulates ABA biosynthesis.In conclusion, the disruption of OsDET1 expression leads to a range of altered phenotypes. OsDET1 deficiency or structural changes impair the degradation of Os PYL5, stimulating ABA signaling, thereby causing ABA hypersensitivity in rice. OsDET1 also plays a positive role in ABA biosynthesis. Due to these two effects, the OsDET1 RNAi plants exhibited some seemingly contradictory phenotypes related to ABA. In addition, overexpression of OsDET1 also caused ABA hypersensitivity in OE-OsDET1 plants. Overall, our work demonstrates that OsDET1 is not only involved in regulating the ABA signaling pathway, but it also regulates ABA biosynthesis pathway in rice, implying that this protein has diverse effects on the ABA signaling pathway. |
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