Welsch Ralf, Zhou Xiangjun, Koschmieder Julian, Schlossarek Tim, Yuan Hui, Sun Tianhu, Li Li
Faculty of Biology II, University of Freiburg, Freiburg, Germany.
Robert W. Holley Center for Agriculture and Health, Agricultural Research Service, US Department of Agriculture, Cornell University, Ithaca, NY, United States.
Front Plant Sci. 2020 Jan 21;10:1716. doi: 10.3389/fpls.2019.01716. eCollection 2019.
Cauliflower () mutant is characterized by high level of β-carotene in its curd. mutation affects the OR protein that was shown to be involved in the posttranslational control of phytoene synthase (PSY), a major rate-limiting enzyme of carotenoid biosynthesis, and in maintaining PSY proteostasis with the plastid Clp protease system. A transposon integration into the cauliflower wild-type gene () results in the formation of three differently spliced transcripts. One of them is characterized by insertion (), while the other two have exon-skipping deletions ( and ). We investigated the properties of individual BoOR variants and examined their effects on carotenoid accumulation. Using the yeast split-ubiquitin system, we showed that all variants were able to form OR dimers except BoOR-LD. The deletion in BoOR-LD eliminated the first of two adjacent transmembrane domains and was predicted to result in a misplacement of the C-terminal zinc finger domain to the opposite side of membrane, thus preventing OR dimerization. As interaction with PSY is mediated by the N-terminus of BoOR, which remains unaffected after splicing, all BoOR variants including BoOR-LD maintained interactions with PSY. Expression of individual mutant variants in Arabidopsis revealed that their protein stability varied greatly. While expression of and resulted in increased BoOR protein levels as BoOR-wt, minimal amounts of BoOR-LD protein accumulated. Carotenoid accumulation showed correlated changes in calli of Arabidopsis expressing these variants. Furthermore, we found that OR also functions in to increase the proportion of native, enzymatically active PSY from plants upon co-expression, but not of bacterial phytoene synthase CrtB. Taken together, these results suggest that OR dimerization is required for OR stability and that the simultaneous presence of PSY interaction-domains in both OR and PSY proteins is required for the holdase function of OR. The more pronounced effect of simultaneous expression of all variants in cauliflower mutant compared with individual overexpression on carotenoid accumulation suggests an enhanced activity with possible formation of various BoOR heterodimers.
花椰菜（）突变体的特征是其花球中β-胡萝卜素含量高。突变影响OR蛋白，该蛋白被证明参与八氢番茄红素合酶（PSY）的翻译后调控，PSY是类胡萝卜素生物合成的主要限速酶，并且与质体Clp蛋白酶系统一起维持PSY的蛋白质稳态。转座子整合到花椰菜野生型基因（）中导致形成三种不同剪接的转录本。其中一种的特征是插入（），而另外两种有外显子跳跃缺失（和）。我们研究了各个BoOR变体的特性，并检查了它们对类胡萝卜素积累的影响。使用酵母分裂泛素系统，我们发现除了BoOR-LD外，所有变体都能够形成OR二聚体。BoOR-LD中的缺失消除了两个相邻跨膜结构域中的第一个，预计会导致C末端锌指结构域错位于膜的另一侧，从而阻止OR二聚化。由于与PSY的相互作用是由BoOR的N末端介导的，剪接后该末端不受影响，因此包括BoOR-LD在内的所有BoOR变体都保持与PSY的相互作用。在拟南芥中表达各个突变体变体表明它们的蛋白质稳定性差异很大。虽然和的表达导致BoOR蛋白水平像BoOR-wt一样增加，但积累的BoOR-LD蛋白量极少。在表达这些变体的拟南芥愈伤组织中，类胡萝卜素积累呈现出相关变化。此外，我们发现OR在中也发挥作用，共表达时可增加植物中天然的、具有酶活性的PSY的比例，但对细菌八氢番茄红素合酶CrtB不起作用。综上所述，这些结果表明OR二聚化是OR稳定性所必需的，并且OR和PSY蛋白中同时存在PSY相互作用结构域是OR的保持酶功能所必需的。与单个过表达相比，在花椰菜突变体中同时表达所有变体对类胡萝卜素积累有更明显的影响，这表明可能形成各种BoOR异二聚体从而增强了活性。
