Characterization of cauliflower OR mutant variants

Authors: WELSCH, RALF - University Of Freiburg; ZHOU, XIANGJUN - Cornell University; KOSCHMIEDER, JULIAN - University Of Freiburg; YUAN, HUI - Cornell University; RIEDINGER, MATTHIAS - University Of Freiburg; SUN, TIANHU - Cornell University; Li, Li

Submitted to: Frontiers in Plant Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/5/2019
Publication Date: 1/21/2020
Citation: Welsch, R., Zhou, X., Koschmieder, J., Yuan, H., Riedinger, M., Sun, T., Li, L. 2020. Characterization of cauliflower OR mutant variants. Frontiers in Plant Science. https://doi.org/10.3389/fpls.2019.01716.
DOI: https://doi.org/10.3389/fpls.2019.01716

Interpretive Summary: Carotenoids are important to human nutrition and health. Cauliflower Orange (Or) mutant curd accumulates high levels of b-carotene, the most potent provitamin A carotenoid. The mutation in the cauliflower Or mutant was caused by a transposon integration into the cauliflower Or gene, which results in the formation of various different OR insertion and deletion variants. While expression of the mutated Or gene results in increased carotenoid amounts in other plants, the contribution of individual OR mutant variants is unclear. In this work, we determined the properties of individual OR mutant variants using protein-protein interaction assays and expression in bacterial and plant systems. While wild-type OR is a transmembrane protein, one of the mutant variants investigated showed altered placement of individual OR protein domains on different sites of the membrane. This alteration dramatically affects OR dimerization and interaction with PSY, the key rate-limiting enzyme in the carotenoid biosynthesis pathway. Moreover, the finding that OR also functions in bacterial system allows conclusions on its chaperone function on PSY. Our findings shed new light on the contributions of OR protein domains on OR function and serve as an example for the effect of altered domain positioning on protein function.

Technical Abstract: Cauliflower Orange (Or) mutant is characterized by high levels of b-carotene in its curd. The mutation affects the OR protein that was shown to be involved in posttranslational control of phytoene synthase (PSY), the rate-limiting enzyme of carotenoid biosynthesis, and maintaining PSY proteostasis with the plastid Clp system. Accordingly, increased OR protein levels in transgenic Arabidopsis lines increase PSY protein as well as carotenoid levels, while OR knockout results in decreased PSY protein and carotenoid levels. However, the mutation in the cauliflower Or mutant was caused by a transposon integration into the cauliflower Or gene (BoOR), resulting in the formation of three differently spliced variants. One of them is characterized by insertion (BoOR-Ins), while the other two have exon-skipping deletions (BoOR-Del and BoOR-LD). We molecularly investigated the properties of individual BoOR mutant versions and examined their effects on carotenoid accumulation. Using the yeast split ubiquitin system, we showed that all variants were able to form OR dimer except BoOR-LD. BoR-LD eliminates the first of two adjacent transmembrane domains and is predicted to result in a misplacement of the zinc finger domain to the extraplasmatic side, thus preventing OR dimerization. As interaction with PSY is mediated by the N-terminal moiety of BoOR and does require neither the zinc finger domain nor dimerization, BoOR-LD maintained and even exhibited increased interaction with PSY as shown by interaction assay. BiFC assay confirmed that all BoOR mutant versions were targeted to plastids. Overexpression of individual BoOR mutant variants in Arabidopsis revealed that the mutations strongly affected BoOR protein stability. While BoOR variant overexpression resulted in greatly increased BoOR-Del and BoOR-Ins protein levels, minimal amounts of BoOR-LD protein was accumulated. Concomitantly, correlated carotenoid accumulation was observed in calli of the Arabidopsis lines expressing these variants. Furthermore, co-expression of wild-type and mutant BoOR variants together with PSY in E. coli system also confirmed the different capabilities of BoOR proteins in promoting carotenoid production. Taken together, these results suggest that OR dimerization is necessary for OR protein stability and BoOR-LD might facilitate increased maintenance of active PSY. The more pronounced effect of simultaneous overexpression of all BoOR variants compared with individual overexpression on carotenoid accumulation suggests the formation of BoOR heterodimers with altered properties.