AUTHOR=Welsch Ralf , Zhou Xiangjun , Koschmieder Julian , Schlossarek Tim , Yuan Hui , Sun Tianhu , Li Li 

TITLE=Characterization of Cauliflower OR Mutant Variants

JOURNAL=Frontiers in Plant Science

VOLUME=10

YEAR=2020

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2019.01716

DOI=10.3389/fpls.2019.01716

ISSN=1664-462X

ABSTRACT=<p>Cauliflower <italic>Orange</italic> (<italic>Or</italic>) mutant is characterized by high level of β-carotene in its curd. <italic>Or</italic> 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 <italic>Or</italic> gene (<italic>BoOR-wt</italic>) results in the formation of three differently spliced transcripts. One of them is characterized by insertion (<italic>BoOR-Ins</italic>), while the other two have exon-skipping deletions (<italic>BoOR-Del</italic> and <italic>BoOR-LD</italic>). 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 <italic>BoOR</italic> mutant variants in Arabidopsis revealed that their protein stability varied greatly. While expression of <italic>BoOR-Del</italic> and <italic>BoOR-Ins</italic> 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 <italic>E. coli</italic> 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 <italic>in planta</italic> 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 <italic>BoOR</italic> variants in cauliflower <italic>Or</italic> mutant compared with individual overexpression on carotenoid accumulation suggests an enhanced activity with possible formation of various BoOR heterodimers.</p>