Abstract
Cauliflower Orange (Or) mutant is characterized by high level of β-carotene in its curd. Or 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 Or gene (BoOR-wt) results in the formation of three differently spliced transcripts. One of them is characterized by insertion (BoOR-Ins), while the other two have exon-skipping deletions (BoOR-Del and BoOR-LD). 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 BoOR mutant variants in Arabidopsis revealed that their protein stability varied greatly. While expression of BoOR-Del and BoOR-Ins 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 E. coli 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 in planta 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 BoOR variants in cauliflower Or mutant compared with individual overexpression on carotenoid accumulation suggests an enhanced activity with possible formation of various BoOR heterodimers.
Highlights
Carotenoids are a group of structurally diverse natural pigments, which have multifaceted functions in photosynthesis, phytohormone production, and generation of signaling molecules for plant growth and development (Cazzonelli and Pogson, 2010; Nisar et al, 2015; Yuan et al, 2015b; RodriguezConcepcion et al, 2018; Sun et al, 2018; Wurtzel, 2019)
BoOR variants were predicted by ChloroP (Emanuelsson et al, 1999). cDNAs of BoOR variants without the transit peptide sequences were cloned into pNXgate in THY.AP4 to express fusion proteins with the N-terminal moiety of ubiquitin (Nub) and mated with cDNAs cloned in the vector pmetY-cub for fusion proteins with the C-terminal moiety of ubiquitin (Cub) present in THY.AP5
The analysis revealed two putative transmembrane domains located in the central region of the protein sequence with a DnaJ-like Cys-rich zinc finger domain directly adjacent to the second transmembrane domain at the Cterminal half of the protein (Figure 1)
Summary
Carotenoids are a group of structurally diverse natural pigments, which have multifaceted functions in photosynthesis, phytohormone production, and generation of signaling molecules for plant growth and development (Cazzonelli and Pogson, 2010; Nisar et al, 2015; Yuan et al, 2015b; RodriguezConcepcion et al, 2018; Sun et al, 2018; Wurtzel, 2019). Carotenoids are composed of the common building block isopentenyl diphosphate, like a large array of different isoprenoids, such as tocopherols, gibberellins, and chlorophylls. The initial specific reaction leading to the formation of different carotenoids in plastids is the condensation of two molecules of the C20 geranylgeranyl pyrophosphate to phytoene, catalyzed by the enzyme phytoene synthase (PSY). Subsequent desaturation reactions catalyzed by phytoene desaturase (PDS) and z-carotene desaturase (ZDS), as well as cis-trans-isomerization by carotenoid isomerase (CrtISO) and z-carotene isomerase (Z-ISO), result in the synthesis of the red-colored lycopene. Hydroxylations and epoxidations produce different xanthophylls in the carotenoid biosynthesis pathway
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