Agrobacterium tumefaciens-Mediated Stable Transformation of Daucus carota

González-Calquín, Christian; Stange, Claudia

doi:10.1007/978-1-4939-9952-1_24

Cited by 8 publications

(6 citation statements)

References 21 publications

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“…Nantaise were surface-sterilized and sown in culture medium Murashige and Skoog (MS; 4.4 g L −1 MS salts; Murashige and Skoog, 1962 , 20 g L −1 sucrose and 0.7% w/v agar) and grown in a culture chamber under 16-h long-day photoperiod at 23°C–25°C (W). Plants of 10- to 20-d postgermination were used for Agrobacterium -mediated transformation as described in Gonzalez-Calquin and Stange (2020) . Briefly, the epicotyls of WT plants grown in vitro, were cut and co-cultivated in presence of A. tumefaciens strain GV3101 carrying the DcPAR1-GFP, DcPAR1as, or AtPAR1:GFP vectors and placed on solidified MS in darkness.…”

Section: Methodsmentioning

confidence: 99%

Development and carotenoid synthesis in dark-grown carrot taproots require PHYTOCHROME RAPIDLY REGULATED1

et al. 2022

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Light stimulates carotenoid synthesis in plants during photomorphogenesis through the expression of PHYTOENE SYNTHASE (PSY), a key gene in carotenoid biosynthesis. The orange carrot (Daucus carota) synthesizes and accumulates high amounts of carotenoids in the taproot that grows underground. Contrary to other organs, light impairs carrot taproot development and represses the expression of carotenogenic genes, such as DcPSY1 and DcPSY2, reducing carotenoid accumulation. By means of RNA-seq, in a previous analysis we observed that carrot PHYTOCHROME RAPIDLY REGULATED1 (DcPAR1) is more highly expressed in the underground grown taproot compared to those grown in light. PAR1 is a transcriptional cofactor with a negative role in shade avoidance syndrome regulation in Arabidopsis (Arabidopsis thaliana) through the dimerization with PHYTOCHROME INTERACTING FACTORs (PIFs), allowing a moderate synthesis of carotenoids. Here, we show that overexpressing AtPAR1 in carrot increases carotenoid production in taproots grown underground as well as DcPSY1 expression. The high expression of AtPAR1 and DcPAR1 led us to hypothesize a functional role of DcPAR1 that was verified through in vivo binding to AtPIF7 and overexpression in Arabidopsis, where AtPSY expression and carotenoid accumulation increased together with a photomorphogenic phenotype. Finally, DcPAR1 antisense carrot lines presented a dramatic decrease in carotenoid levels and in relative expression of key carotenogenic genes as well as impaired taproot development. These results suggest that DcPAR1 is a key factor for secondary root development and carotenoid synthesis in carrot taproot grown underground.

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Section: Methodsmentioning

confidence: 99%

Development and carotenoid synthesis in dark-grown carrot taproots require PHYTOCHROME RAPIDLY REGULATED1

et al. 2022

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“…Nantaise were surface-sterilized and sown in culture medium MS (4.4 g/L MS salts (Murashige and Skoog, 1962), 20 g/L sucrose and 0,7% agar) and grown in a culture chamber under 16 h long day photoperiod at 23-25 °C. Plants of 10-20 days post-germination were used for Agrobacterium -mediated transformation as described in Gonzalez-Calquin and Stange (2020). Briefly, the epicotyls of wild-type plants grown in vitro , were cut and co-cultivated in presence of A. tumefaciens strain GV3101 carrying the DcPAR1-GFP, DcPAR1as or AtPAR1:GFP vectors and placed on solidified MS in darkness.…”

Section: Methodsmentioning

confidence: 99%

DcPAR1 is Required for Development and Carotenoid Synthesis in the Dark-Grown Carrot Taproot

Arias

Ortega

González

et al. 2021

Preprint

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Light stimulates carotenoid synthesis in plants during photomorphogenesis through the expression of PHYTOENE SYNTHASE (PSY), a key gene in carotenoid biosynthesis. The orange Daucus carota (carrot) synthesizes and accumulates high amounts of carotenoids in the taproot that grows underground. Contrary to other organs, light impairs carrot taproot development and represses the expression of carotenogenic genes such as DcPSY1 and DcPSY2 reducing carotenoid accumulation. By means of an RNA-seq, in previous analysis we observed that carrot PHYTOCHROME RAPIDLY REGULATED 1 (DcPAR1) is more expressed in the underground grown taproot respect to those grown in light. PAR1 is a transcriptional cofactor with a negative role in the shade avoidance syndrome regulation in Arabidopsis thaliana through the dimerization with PHYTOCHROME INTERACTING FACTORs (PIFs), allowing a moderate synthesis of carotenoids. Here we show that overexpressing AtPAR1 in carrot produces an increment of carotenoids in taproots grown underground as well as higher DcPSY1 expression. The high identity of AtPAR1 and DcPAR1 let us to suggest a functional role of DcPAR1 that was verified through the in vivo binding to AtPIF7 and the overexpression in Arabidopsis, where it increments AtPSY expression and carotenoid accumulation together with a photomorphogenic phenotype. Finally, DcPAR1 antisense carrot lines presented a dramatic decrease in carotenoids levels and in the relative expression of key carotenogenic genes as well as impairment in taproot development. These results let us to propose that DcPAR1 is a key factor for secondary root development, plastid differentiation and carotenoid synthesis in carrot taproot grown underground.

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“…Carrots have numerous germplasms with various patterns of carotenoid accumulation (Deng et al., 2023; Simon, 2000; Simpson et al., 2016; Wang et al., 2022). Owing to its remarkable differentiation ability and well‐established transgenic techniques, carrot is a suitable model plant for exploring carotenoid accumulation (Baranski, 2008; Gonzalez‐Calquin & Stange, 2019; Li et al., 2021; Xu, Feng, & Xiong, 2019). Research on the function of carrot DcLCYE is helpful in understanding the coloration of the red carrot root and in exploring the flow direction of the carotenoid pathway, accounting for the distribution of carotenoids.…”

Section: Introductionmentioning

confidence: 99%

Lycopene ε‐cyclase mediated transition of α‐carotene and β‐carotene metabolic flow in carrot fleshy root

Wang

Zhang

et al. 2023

The Plant Journal

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SUMMARYThe accumulation of carotenoids, such as xanthophylls, lycopene, and carotenes, is responsible for the color of carrot (Daucus carota subsp. sativus) fleshy roots. The potential role of DcLCYE, encoding a lycopene ε‐cyclase associated with carrot root color, was investigated using cultivars with orange and red roots. The expression of DcLCYE in red carrot varieties was significantly lower than that in orange carrots at the mature stage. Furthermore, red carrots accumulated larger amounts of lycopene and lower levels of α‐carotene. Sequence comparison and prokaryotic expression analysis revealed that amino acid differences in red carrots did not affect the cyclization function of DcLCYE. Analysis of the catalytic activity of DcLCYE revealed that it mainly formed ε‐carotene, while a side activity on α‐carotene and γ‐carotene was also observed. Comparative analysis of the promoter region sequences indicated that differences in the promoter region may affect the transcription of DcLCYE. DcLCYE was overexpressed in the red carrot ‘Benhongjinshi’ under the control of the CaMV35S promoter. Lycopene in transgenic carrot roots was cyclized, resulting in the accumulation of higher levels of α‐carotene and xanthophylls, while the β‐carotene content was significantly decreased. The expression levels of other genes in the carotenoid pathway were simultaneously upregulated. Knockout of DcLCYE in the orange carrot ‘Kurodagosun’ by CRISPR/Cas9 technology resulted in a decrease in the α‐carotene and xanthophyll contents. The relative expression levels of DcPSY1, DcPSY2, and DcCHXE were sharply increased in DcLCYE knockout mutants. The results of this study provide insights into the function of DcLCYE in carrots, which could serve as a basis for creating colorful carrot germplasms.

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Agrobacterium tumefaciens-Mediated Stable Transformation of Daucus carota

Cited by 8 publications

References 21 publications

Development and carotenoid synthesis in dark-grown carrot taproots require PHYTOCHROME RAPIDLY REGULATED1

Development and carotenoid synthesis in dark-grown carrot taproots require PHYTOCHROME RAPIDLY REGULATED1

DcPAR1 is Required for Development and Carotenoid Synthesis in the Dark-Grown Carrot Taproot

Lycopene ε‐cyclase mediated transition of α‐carotene and β‐carotene metabolic flow in carrot fleshy root

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