Abstract
Key message
The AtOR gene enhances carotenoid levels in corn by promoting the formation of plastoglobuli when the carotenoid pool is limited, but has no further effect when carotenoids are already abundant.
Abstract
The cauliflower orange (or) gene mutation influences carotenoid accumulation in plants by promoting the transition of proplastids into chromoplasts, thus creating intracellular storage compartments that act as metabolic sink. We overexpressed the Arabidopsis OR gene under the control of the endosperm-specific wheat LMW glutenin promoter in a white corn variety that normally accumulates only trace amounts of carotenoids. The total endosperm carotenoid content in the best-performing AtOR transgenic corn line was 32-fold higher than wild-type controls (~25 µg/g DW at 30 days after pollination) but the principal carotenoids remained the same, suggesting that AtOR increases the abundance of existing carotenoids without changing the metabolic composition. We analyzed the expression of endogenous genes representing the carotenoid biosynthesis and MEP pathways, as well as the plastid fusion/translocation factor required for chromoplast formation, but only the DXS1 gene was upregulated in the transgenic corn plants. The line expressing AtOR at the highest level was crossed with four transgenic corn lines expressing different carotenogenic genes and accumulating different carotenoids. The introgression of AtOR increased the carotenoid content of the hybrids when there was a limited carotenoid pool in the parental line, but had no effect when carotenoids were already abundant in the parent. The AtOR gene therefore appears to enhance carotenoid levels by promoting the formation of carotenoid-sequestering plastoglobuli when the carotenoid pool is limited, but has no further effect when carotenoids are already abundant because high levels of carotenoids can induce the formation of carotenoid-sequestering plastoglobuli even in the absence of AtOR.
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Acknowledgements
Research at the Universitat de Lleida is supported by MINECO, Spain (BIO2014-54426-P; BIO2014-54441-P), by the Catalan Government (2014 SGR 1296 Agricultural Biotechnology Research Group), and by European Union Framework 7, European Research Council IDEAS Advanced Grant BIOFORCE and POC Grant (to PC). G. Farré is supported by a J de la C fellowship.
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CZ, GS, TC, and PC designed research; JB, UZ, and GF performed research, analyzed data, and wrote the article; VM did TEM and analyzed these data.
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Communicated by Salim Al-Babili.
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Supplementary material 1 Supplementary Figure 1—RNA blot analysis of AtOR mRNA in wild type (M37W) and two different transgenic lines (OR1 and OR2) transformed with the AtOR gene driven by the wheat low-molecular-weight glutelin promoter. Each lane was loaded with 25 μg total RNA isolated from endosperm tissue Ribosomal RNA stained with ethidium bromide is shown as a loading control. Supplementary Figure 2—Transcript accumulation normalized against ACTIN mRNA in wild-type and transgenic lines presented as means of three technical replicates. Standard error bars were not included due to the use of technical replicates rather than biological replicates (the aim was to confirm transgene expression rather than to compare different transcript profiles). Supplementary Figure 3—Schematic representation of the transgenes expressed in our transgenic plants lines and hybrids. Supplementary Table 1—Carotenoid content and composition in wild-type M37W, transgenic lines OR2, CARO1, CARO2, KETO1 and KETO2, and hybrids ORxCARO1, ORxCARO2, ORxKETO1 and ORxKETO2 (T1 plants) at 30 (*) and 60 DAP (**) (µg/g DW±SE) (n = 3–5 seeds). Abbreviations: Phyt, phytoene; Lyco, lycopene; βcryp, β-cryptoxanthin; βcaro, β-carotene; Lut, lutein; Zea, zeaxanthin; Anthe, antheraxanthin; Viola, violaxanthin; CAROT, carotenoids. Supplementary Table 2—Ketocarotenoid content and composition of wild-type M37W, transgenic lines OR2, CARO1, CARO2, KETO1 and KETO2, and hybrids ORxCARO1, ORxCARO2, ORxKETO1 and ORxKETO2 T1 at 30 (*) and 60 DAP (**) (µg/g DW±SE) (n = 3–5 seeds). Abbreviations: Asta, astaxanthin; Cantha, canthaxanthin; Adonir, adonirubin; Adonix, adonixanthin; 3OHechi, 3-OH-echinenone; KETO, ketocarotenoids. Supplementary Table 3—Oligonucleotide sequences of corn ACTIN, endogenous carotenogenic genes and transgenes for real-time quantitative RT-PCR analysis. (DOCX 114 KB)
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Berman, J., Zorrilla-López, U., Medina, V. et al. The Arabidopsis ORANGE (AtOR) gene promotes carotenoid accumulation in transgenic corn hybrids derived from parental lines with limited carotenoid pools. Plant Cell Rep 36, 933–945 (2017). https://doi.org/10.1007/s00299-017-2126-z
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DOI: https://doi.org/10.1007/s00299-017-2126-z