Abstract
Di19 (drought-induced protein 19) family is a novel type of Cys2/His2 zinc-finger proteins. In this study, we demonstrated that cotton Di19-1 and Di19-2 (GhDi19-1/-2) proteins could be phosphorylated in vitro by the calcium-dependent protein kinase (CDPK). Mutation of Ser to Ala in N-terminus of GhDi19-1/-2 led to the altered subcellular localization of the two proteins, but the constitutively activated form (Ser was mutated to Asp) of GhDi19-1/-2 still showed the nuclear localization. GhDi19-1/-2 overexpression transgenic Arabidopsis seedlings displayed the hypersensitivity to high salinity and abscisic acid (ABA). However, Ser site-mutated GhDi19-1(S116A) and GhDi19-2(S114A), and Ser and Thr double sites-mutated GhDi19-1(S/T-A/A) and GhDi19-2(S/T-A/A) transgenic Arabidopsis did not show the salt- and ABA-hypersensitive phenotypes. In contrast, overexpression of Thr site-mutated GhDi19-1(T114A) and GhDi19-2(T112A) in Arabidopsis still resulted in salt- and ABA-hypersensitivity phenotypes, like GhDi19-1/-2 transgenic lines. Overexpression of GhDi19-1/-2 and their constitutively activated forms in Atcpk11 background could recover the salt- and ABA-insensitive phenotype of the mutant. Thus, our results demonstrated that Ser phosphorylation (not Thr phosphorylation) is crucial for functionally activating GhDi19-1/-2 in response to salt stress and ABA signaling during early plant development, and GhDi19-1/-2 proteins may be downstream targets of CDPKs in ABA signal pathway.
Highlights
Drought, high salinity and low temperature are the most common abiotic stresses that limit distribution of plants and affect crop productivity and quality[1,2]
There was no significant difference in root length between GhDi19-1(S/T-A/A) and GhDi19-2(S/T-A/A) transgenic lines and wild type under NaCl and abscisic acid (ABA) treatments (Fig. 6c). These results demonstrated that overexpression of the mutated GhDi19-1(T114A) and GhDi19-2(T112A) genes in Arabidopsis still resulted in the transgenic plants salt- and ABA-hypersensitivity, like GhDi19-1/-2 transgenic lines, suggesting that the Thr site is inessential for GhDi19-1 and GhDi19-2 proteins involving in response to salt stress and ABA signaling during seed germination and early seedling development
Roots of Atcpk[] mutant were longer than those of wild type, and root length of the GhDi19-1/cpk[11] and GhDi19-2/ cpk[11] transgenic lines was similar to wild type, but roots of GhDi19-1(S116D)/cpk[11] and GhDi19-2(S114D)/cpk[11] transgenic seedlings were shorter than those of wild type under ABA and NaCl treatments (Fig. 7e). These results indicated that overexpression of GhDi19-1/-2 in Atcpk[11] background could recover the salt- and ABA-insensitive phenotype of the mutant, implying the other calcium-dependent protein kinase (CDPK) may activate GhDi19-1/-2 in the transgenic Arabidopsis plants, and Ser phosphorylation is important for functionally activating GhDi19-1/-2 in response to salt stress and ABA signaling during seed germination and early seedling development
Summary
High salinity and low temperature are the most common abiotic stresses that limit distribution of plants and affect crop productivity and quality[1,2] Plant adaptation to these stresses is dependent on the activation of cascades of molecular networks involving in stress perception, signal transduction and expression of specific stress-related genes[3,4]. Previous studies revealed that plant transcription factors (TFs) (such as bZIP, HD-ZIP, NAC, MYB, MYC and AP2/ERF) are involved in plant stress response[3,16] These TFs regulate expression of the stress-related genes by binding to the cis-elements in the target promoters[17]. A study revealed that Arabidopsis Di19-1 as a transcription factor participates in plant response to drought stress by binding to the TACA(A/G)T element within the promoters of PR1 (pathogenesis-related 1), PR2 and PR5 genes[31]. The calcium-dependent protein kinase (CDPK) is able to phosphorylate these Di19 proteins at Ser[116] of GhDi19-1 and at Ser[114] of GhDi19-2
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