ERECTA PANICLE 3 (EP3) and ORYZA SATIVA F-BOX KELCH 1 (OsFBK1) proteins share 57% and 54% sequence identity with the Arabidopsis F-box protein HAWAIIAN SKIRT (HWS). Previously we showed that EP3 is a functional orthologue of HWS. Here we demonstrate that OsFBK1 is another functional orthologue of HWS and show the complexity of interaction between EP3 and OsFBK1 genes at different developmental stages of the plant. qRT-PCR expression analyses and studies of EP3-GFP and OsFBK1-RFP promoter reporter lines demonstrate that although EP3 and OsFBK1 expression can be detected in the same tissues some cells exclusively express EP3 or OsFBK1 whilst others co-express both genes. Loss, reduction or gain-of-function lines for EP3 and OsFBK1, show that EP3 and OsFBK1 affect plant architecture, organ size, floral organ number and size, floral morphology, pollen viability, grain size and weight. We have identified the putative orthologue genes of the rice microRNA pathway for ORYZA SATIVA DAWDLE (OsDDL) and ORYZA SATIVA SERRATE (OsSE), and demonstrated that EP3 and OsFBK1 affect their transcript levels as well as those of CROWN ROOT DEFECT 1/ORYZA SATIVA Exportin-5 HASTY (CRD1/OsHST), ORYZA SATIVA DICER-LIKE 1 (OsDCL) and ORYZA SATIVA WEAVY LEAF1 (OsWAF1). We show that EP3 affects OsPri-MIR164, OsNAM1 and OsNAC1 transcript levels. OsNAC1 transcripts are modified by OsFBK1, suggesting two independent regulatory pathways, one via EP3 and OsMIR164 and the other via OsFBK1. Our data propose that EP3 and OsFBK1 conjointly play similar roles in rice to how HWS does in Arabidopsis.
E3連接酶參與蛋白的泛素化和降解過程，其中水稻EP3（ERECTA PANICLE 3）和OsFBK1（ORYZA SATIVAF-BOX KELCH 1）蛋白與擬南芥F-box蛋白HWS（HAWAIIAN SKIRT）的序列同源性分別為57%和54%。之前，研究人員已經(jīng)證明EP3與HWS具有功能同源性。最近，研究者證實(shí)OsFBK1是HWS的另一個(gè)功能同源基因。
研究者通過轉(zhuǎn)錄水平的表達(dá)分析和EP3-GFP和OsFBK1-RFP啟動(dòng)子熒光報(bào)告系的研究發(fā)現(xiàn)，雖然在同一組織中可以檢測(cè)到EP3和OsFBK1表達(dá)，但有些細(xì)胞只表達(dá)EP3或OsFBK1中的一個(gè)，而另一些細(xì)胞則同時(shí)表達(dá)這兩個(gè)基因。EP3和OsFBK1的敲除或RNAi干擾以及超表達(dá)植株的研究結(jié)果表明，EP3和OsFBK1會(huì)影響植株株型、器官大小、花器官數(shù)量和大小、花形態(tài)、花粉活力、籽粒大小和重量等諸多性狀。
此外，研究者還發(fā)現(xiàn)EP3會(huì)影響OsPri-MIR164、OsNAM1和OsNAC1的轉(zhuǎn)錄水平；而OsFBK1改變OsNAC1的轉(zhuǎn)錄水平，但不改變OsPri-MIR164的轉(zhuǎn)錄水平，這表明OsNAC1可能通過OsMIR164受EP3的間接影響，但也可能通過一個(gè)獨(dú)立的調(diào)控途徑參與OsFBK1的表達(dá)。EP3和OsFBK1也會(huì)改變水稻miRNA生物發(fā)生途徑相關(guān)基因CRD1/OsHST、OsDCL和OsWAF1的轉(zhuǎn)錄本。
OsNAC1轉(zhuǎn)錄本被OsFBK1修飾，提示有兩條獨(dú)立的調(diào)控途徑，一條是通過EP3和OsMIR164，另一條是通過OsFBK1。這表明EP3和OsFBK1在水稻中的共同作用類似于HWS在擬南芥中的作用。這些都說明了EP3和OsFBK1基因在植物不同發(fā)育階段之間相互作用的復(fù)雜性。