Background Plant annexins are calcium- and lipid-binding proteins that have multiple functions, and a significant amount of research on plant annexins has been reported in recent years. However, the functions of annexins in diverse biological processes in rice are largely unclear. Results Herein, we report that OsANN4, a calcium-binding rice annexin protein, was induced by abscisic acid (ABA). Under ABA treatment, the plants in which OsANN4 was knocked down by RNA interference showed some visible phenotypic changes compared to the wild type, such as a lower rooting rate and shorter shoot and root lengths. Moreover, the superoxide dismutase (SOD) and catalase (CAT) activities of the RNAi lines were significantly lower and further resulted in higher accumulation of O2.- and H2O2 than those of the wild-type. A Non-invasive Micro-test Technology (NMT) assay showed that ABA-induced net Ca2+ influx was inhibited in OsANN4 knockdown plants. Interestingly, the phenotypic differences caused by ABA were eliminated in the presence of LaCl3 (Ca2+ channel inhibitor). Apart from this, we demonstrated that OsCDPK24 interacted with and phosphorylated OsANN4. When the phosphorylated serine residue of OsANN4 was substituted by alanine, the interaction between OsANN4 and OsCDPK24 was still observed, however, both the conformation of OsANN4 and its binding activity with Ca2+ might be changed. Conclusions OsANN4 plays a crucial role in the ABA response, partially by modulating ROS production, mediating Ca2+ influx or interacting with OsCDPK24.
34Plant annexins are calcium-and lipid-binding proteins that have multiple functions, 35 and a significant amount of research on plant annexins has been reported in recent 36 years. However, the functions of annexins in diverse biological processes in rice are 37 largely unclear. Herein, we report that OsANN4, a calcium-binding rice annexin 38 protein, is a substrate for OsCDPK24, and the OsANN4 phosphorylation site is the 39 13th serine, which is a key site for phosphorylation. Most strikingly, abscisic acid 40 (ABA) promotes the interaction between OsANN4 and OsCDPK24. Moreover, 41 knocking down OsANN4 by RNA interference resulted in visible and invisible 42 phenotypes with exogenous ABA treatment, such as shorter shoots of seedlings, less 43 lateral roots, earlier root aerenchyma formation and so on. The further analyzed 44 results showed that decreased superoxide dismutase (SOD) and catalase (CAT) 45 activity of the RNAi lines, which control H2O2 accumulation for redox homeostasis, 46 and further promoted earlier aerenchyma formation of the root. These results suggest 47 that a proposed molecular mechanism exists between OsANN4 and H 2 O 2 production 48 to response ABA. 49
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