Data_Sheet_2_Mutation of the ALBOSTRIANS Ohnologous Gene HvCMF3 Impairs Chloroplast Development and Thylakoid Architecture in Barley.xlsx

Gene pairs resulting from whole genome duplication (WGD), so-called ohnologous genes, are retained if at least one member of the pair undergoes neo- or sub-functionalization. Phylogenetic analyses of the ohnologous genes ALBOSTRIANS (HvAST/HvCMF7) and ALBOSTRIANS-LIKE (HvASL/HvCMF3) of barley (Hordeum vulgare) revealed them as members of a subfamily of genes coding for CCT motif (CONSTANS, CONSTANS-LIKE and TIMING OF CAB1) proteins characterized by a single CCT domain and a putative N-terminal chloroplast transit peptide. Recently, we showed that HvCMF7 is needed for chloroplast ribosome biogenesis. Here we demonstrate that mutations in HvCMF3 lead to seedlings delayed in development. They exhibit a yellowish/light green – xantha – phenotype and successively develop pale green leaves. Compared to wild type, plastids of mutant seedlings show a decreased PSII efficiency, impaired processing and reduced amounts of ribosomal RNAs; they contain less thylakoids and grana with a higher number of more loosely stacked thylakoid membranes. Site-directed mutagenesis of HvCMF3 identified a previously unknown functional domain, which is highly conserved within this subfamily of CCT domain containing proteins. HvCMF3:GFP fusion constructs were localized to plastids and nucleus. Hvcmf3Hvcmf7 double mutants exhibited a xantha-albino or albino phenotype depending on the strength of molecular lesion of the HvCMF7 allele. The chloroplast ribosome deficiency is discussed as the primary observed defect of the Hvcmf3 mutants. Based on our observations, the genes HvCMF3 and HvCMF7 have similar but not identical functions in chloroplast development of barley supporting our hypothesis of neo-/sub-functionalization between both ohnologous genes.

全基因组复制（whole genome duplication, WGD）所产生的基因对，即所谓的ohnolog基因（ohnologous genes），若该基因对中至少一个成员发生新功能化（neo-functionalization）或亚功能化（sub-functionalization），则会被保留。对大麦（Hordeum vulgare）的ohnolog基因ALBOSTRIANS（HvAST/HvCMF7）与ALBOSTRIANS-LIKE（HvASL/HvCMF3）开展系统发育分析，结果显示二者隶属于一类编码CCT基序（CCT motif，含CONSTANS、CONSTANS-LIKE及TIMING OF CAB1）蛋白的基因亚家族，这类蛋白的典型特征为仅含一个CCT结构域，以及一段推定的N端叶绿体转运肽。本团队此前已证实，HvCMF7对叶绿体核糖体生物发生不可或缺。本研究发现，HvCMF3发生突变会导致幼苗发育迟缓，表现为黄化/淡绿表型（xantha），并逐渐长出淡绿色叶片。与野生型植株相比，突变体幼苗的质体光系统II（PSII）效率下降、核糖体RNA加工受损且含量降低；质体内类囊体与基粒的数量更少，且类囊体膜堆叠更为松散、堆叠数量更多。对HvCMF3的定点诱变（site-directed mutagenesis）鉴定出一个此前未被报道的功能结构域，该结构域在这类含CCT结构域的基因亚家族中高度保守。将HvCMF3与绿色荧光蛋白（GFP）融合的构建体可定位于质体与细胞核中。Hvcmf3与Hvcmf7的双突变体表现为黄化白化（xantha-albino）或白化表型，具体表型取决于HvCMF7等位基因的分子损伤程度。本研究讨论认为，叶绿体核糖体缺陷是Hvcmf3突变体最核心的表型缺陷。基于上述研究结果，HvCMF3与HvCMF7在大麦叶绿体发育过程中功能相似但并不完全等同，这支撑了我们关于这两个ohnolog基因之间发生新功能化/亚功能化的假说。