Characterization of Cauliflower OR Mutant Variants
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 prote...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2020-01-01
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| Series: | Frontiers in Plant Science |
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| Online Access: | https://www.frontiersin.org/article/10.3389/fpls.2019.01716/full |
| _version_ | 1818296024835293184 |
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| author | Ralf Welsch Xiangjun Zhou Xiangjun Zhou Julian Koschmieder Tim Schlossarek Hui Yuan Hui Yuan Tianhu Sun Tianhu Sun Li Li Li Li |
| author_facet | Ralf Welsch Xiangjun Zhou Xiangjun Zhou Julian Koschmieder Tim Schlossarek Hui Yuan Hui Yuan Tianhu Sun Tianhu Sun Li Li Li Li |
| author_sort | Ralf Welsch |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-13T03:56:58Z |
| format | Article |
| id | doaj.art-068a364dba31417a8064b82bca124b42 |
| institution | Directory Open Access Journal |
| issn | 1664-462X |
| language | English |
| last_indexed | 2024-12-13T03:56:58Z |
| publishDate | 2020-01-01 |
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| series | Frontiers in Plant Science |
| spelling | doaj.art-068a364dba31417a8064b82bca124b422022-12-22T00:00:35ZengFrontiers Media S.A.Frontiers in Plant Science1664-462X2020-01-011010.3389/fpls.2019.01716491816Characterization of Cauliflower OR Mutant VariantsRalf Welsch0Xiangjun Zhou1Xiangjun Zhou2Julian Koschmieder3Tim Schlossarek4Hui Yuan5Hui Yuan6Tianhu Sun7Tianhu Sun8Li Li9Li Li10Faculty of Biology II, University of Freiburg, Freiburg, GermanyRobert W. Holley Center for Agriculture and Health, Agricultural Research Service, US Department of Agriculture, Cornell University, Ithaca, NY, United StatesPlant Breeding and Genetics Section, School of Integrative Plant Science, Cornell University, Ithaca, NY, United StatesFaculty of Biology II, University of Freiburg, Freiburg, GermanyFaculty of Biology II, University of Freiburg, Freiburg, GermanyRobert W. Holley Center for Agriculture and Health, Agricultural Research Service, US Department of Agriculture, Cornell University, Ithaca, NY, United StatesPlant Breeding and Genetics Section, School of Integrative Plant Science, Cornell University, Ithaca, NY, United StatesRobert W. Holley Center for Agriculture and Health, Agricultural Research Service, US Department of Agriculture, Cornell University, Ithaca, NY, United StatesPlant Breeding and Genetics Section, School of Integrative Plant Science, Cornell University, Ithaca, NY, United StatesRobert W. Holley Center for Agriculture and Health, Agricultural Research Service, US Department of Agriculture, Cornell University, Ithaca, NY, United StatesPlant Breeding and Genetics Section, School of Integrative Plant Science, Cornell University, Ithaca, NY, United StatesCauliflower 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.https://www.frontiersin.org/article/10.3389/fpls.2019.01716/fullprotein variantscarotenoidscauliflowerphytoene synthase (PSY)protein stability and foldingdimerization |
| spellingShingle | Ralf Welsch Xiangjun Zhou Xiangjun Zhou Julian Koschmieder Tim Schlossarek Hui Yuan Hui Yuan Tianhu Sun Tianhu Sun Li Li Li Li Characterization of Cauliflower OR Mutant Variants Frontiers in Plant Science protein variants carotenoids cauliflower phytoene synthase (PSY) protein stability and folding dimerization |
| title | Characterization of Cauliflower OR Mutant Variants |
| title_full | Characterization of Cauliflower OR Mutant Variants |
| title_fullStr | Characterization of Cauliflower OR Mutant Variants |
| title_full_unstemmed | Characterization of Cauliflower OR Mutant Variants |
| title_short | Characterization of Cauliflower OR Mutant Variants |
| title_sort | characterization of cauliflower or mutant variants |
| topic | protein variants carotenoids cauliflower phytoene synthase (PSY) protein stability and folding dimerization |
| url | https://www.frontiersin.org/article/10.3389/fpls.2019.01716/full |
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