INRAE, Clermont Auvergne University, UMR GDEC, Clermont-Ferrand, France.
INRAE, ONF, UMR0588 BioForA, Paris, France.
PLoS One. 2023 Jun 23;18(6):e0287645. doi: 10.1371/journal.pone.0287645. eCollection 2023.
Grain storage proteins (GSPs) quantity and composition determine the end-use value of wheat flour. GSPs consists of low-molecular-weight glutenins (LMW-GS), high-molecular-weight glutenins (HMW-GS) and gliadins. GSP gene expression is controlled by a complex network of DNA-protein and protein-protein interactions, which coordinate the tissue-specific protein expression during grain development. The regulatory network has been most extensively studied in barley, particularly the two transcription factors (TFs) of the DNA binding with One Finger (DOF) family, barley Prolamin-box Binding Factor (BPBF) and Scutellum and Aleurone-expressed DOF (SAD). They activate hordein synthesis by binding to the Prolamin box, a motif in the hordein promoter. The BPBF ortholog previously identified in wheat, WPBF, has a transcriptional activity in expression of some GSP genes. Here, the wheat ortholog of SAD, named TaSAD, was identified. The binding of TaSAD to GSP gene promoter sequences in vitro and its transcriptional activity in vivo were investigated. In electrophoretic mobility shift assays, recombinant TaSAD and WPBF proteins bound to cis-motifs like those located on HMW-GS and LMW-GS gene promoters known to bind DOF TFs. We showed by transient expression assays in wheat endosperms that TaSAD and WPBF activate GSP gene expression. Moreover, co-bombardment of Storage Protein Activator (SPA) with WPBF or TaSAD had an additive effect on the expression of GSP genes, possibly through conserved cooperative protein-protein interactions.
谷物贮藏蛋白（GSPs）的数量和组成决定了小麦面粉的最终用途价值。GSPs 由低分子量谷蛋白（LMW-GS）、高分子量谷蛋白（HMW-GS）和醇溶蛋白组成。GSP 基因的表达受 DNA-蛋白质和蛋白质-蛋白质相互作用的复杂网络调控，该网络协调谷物发育过程中的组织特异性蛋白质表达。该调控网络在大麦中得到了最广泛的研究，特别是 DNA 结合的一个手指（DOF）家族的两个转录因子（TFs）、大麦醇溶蛋白盒结合因子（BPBF）和盾片和糊粉层表达的 DOF（SAD）。它们通过结合醇溶蛋白启动子中的 Prolamin 盒来激活醇溶蛋白的合成。在小麦中先前鉴定的 BPBF 同源物 WPBF 具有一些 GSP 基因表达的转录活性。在此，鉴定了小麦 SAD 的同源物 TaSAD。研究了 TaSAD 在体外与 GSP 基因启动子序列的结合及其在体内的转录活性。在电泳迁移率变动分析中，重组 TaSAD 和 WPBF 蛋白与顺式基序结合，这些基序位于已知与 DOF TFs 结合的 HMW-GS 和 LMW-GS 基因启动子上。我们通过在小麦胚乳中的瞬时表达试验表明，TaSAD 和 WPBF 激活 GSP 基因的表达。此外，用 SPA 与 WPBF 或 TaSAD 共轰击对 GSP 基因的表达具有相加效应，这可能是通过保守的协同蛋白质-蛋白质相互作用实现的。
