Vacuole-Targeted Proteins: Ins and Outs of Subcellular Localization Studies

Carqueijeiro, Inês; Sepúlveda, Liuda Johana; Mosquera, Ángela; Payne, Richard M. E.; Corbin, Cyrielle; Papon, Nicolas; Bernonville, Thomas Dugé de; Besseau, Sébastien; Lanoue, Arnaud; Glévarec, Gaëlle; Clastre, Marc; St-Pierre, Benoit; Atehortúa, Lucía; Giglioli-Guivarc’h, Nathalie; O’Connor, Sarah E.; Oudin, Audrey; Courdavault, Vincent

doi:10.1007/978-1-4939-7856-4_4

Cited by 4 publications

(4 citation statements)

References 56 publications

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“…To induce plasmolysis, the onion epidermal cells were placed in a saturated sucrose solution (30%) for 15–30 min following the method of Carqueijeiro et al. (2018).…”

Section: Methodsmentioning

confidence: 99%

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Functional allele of a MATE gene selected during domestication modulates seed color in chickpea

Thakro,

Varshney,

Malik

et al. 2023

The Plant Journal

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SUMMARYSeed color is one of the key target traits of domestication and artificial selection in chickpeas due to its implications on consumer preference and market value. The complex seed color trait has been well dissected in several crop species; however, the genetic mechanism underlying seed color variation in chickpea remains poorly understood. Here, we employed an integrated genomics strategy involving QTL mapping, high‐density mapping, map‐based cloning, association analysis, and molecular haplotyping in an inter‐specific RIL mapping population, association panel, wild accessions, and introgression lines (ILs) of Cicer gene pool. This delineated a MATE gene, CaMATE23, encoding a Transparent Testa (TT) and its natural allele (8‐bp insertion) and haplotype underlying a major QTL governing seed color on chickpea chromosome 4. Signatures of selective sweep and a strong purifying selection reflected that CaMATE23, especially its 8‐bp insertion natural allelic variant, underwent selection during chickpea domestication. Functional investigations revealed that the 8‐bp insertion containing the third cis‐regulatory RY‐motif element in the CaMATE23 promoter is critical for enhanced binding of CaFUSCA3 transcription factor, a key regulator of seed development and flavonoid biosynthesis, thereby affecting CaMATE23 expression and proanthocyanidin (PA) accumulation in the seed coat to impart varied seed color in chickpea. Consequently, overexpression of CaMATE23 in Arabidopsis tt12 mutant partially restored the seed color phenotype to brown pigmentation, ascertaining its functional role in PA accumulation in the seed coat. These findings shed new light on the seed color regulation and evolutionary history, and highlight the transcriptional regulation of CaMATE23 by CaFUSCA3 in modulating seed color in chickpea. The functionally relevant InDel variation, natural allele, and haplotype from CaMATE23 are vital for translational genomic research, including marker‐assisted breeding, for developing chickpea cultivars with desirable seed color that appeal to consumers and meet global market demand.

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“…To induce plasmolysis, the onion epidermal cells were placed in a saturated sucrose solution (30%) for 15–30 min following the method of Carqueijeiro et al. (2018).…”

Section: Methodsmentioning

confidence: 99%

“…After a 16-h incubation in the dark, YFP fluorescence signals were visualized using a Leica TCS SP8 confocal laser scanning microscope (Leica, Heidelberg, Germany). To induce plasmolysis, the onion epidermal cells were placed in a saturated sucrose solution (30%) for 15-30 min following the method of Carqueijeiro et al (2018).…”

Section: Subcellular Localizationmentioning

confidence: 99%

Functional allele of a MATE gene selected during domestication modulates seed color in chickpea

Thakro,

Varshney,

Malik

et al. 2023

The Plant Journal

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“…The subcellular localization of C. roseus MEP pathway enzymes was then analyzed through a fluorescent protein (FP) imaging procedure recently developed for C. roseus cell cultures [47,48]. The full-length sequence of each enzyme was fused to the N-terminal end of green FP (GFP) to express a MEP enzyme-GFP fusion maintaining accessibility of the predicted TP.…”

Section: Mep Pathway Enzymes Are Targeted To Plastidsmentioning

confidence: 99%

“…Transient transformation of C. roseus cells by particle bombardment, fluorescence protein imaging and BiFC were performed following the procedures previously described [36,47,48]. Briefly, C. roseus cells plated onto solid culture medium or ditched young leaves were bombarded with gold DNA coated particles (1 µm) and 1100 psi rupture disk at a stopping-screen-to-target distance of 6 cm, using the Bio-Rad PDS1000/He system.…”

Section: Cell Transformations and Epifluorescence Microscopymentioning

confidence: 99%

Cellular and Subcellular Compartmentation of the 2C-Methyl-D-Erythritol 4-Phosphate Pathway in the Madagascar Periwinkle

Guirimand

Guihur

Perello

et al. 2020

Plants

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The Madagascar periwinkle (Catharanthus roseus) synthesizes the highly valuable monoterpene indole alkaloids (MIAs) through a long metabolic route initiated by the 2C-methyl-D-erythritol 4-phosphate (MEP) pathway. In leaves, a complex compartmentation of the MIA biosynthetic pathway occurs at both the cellular and subcellular levels, notably for some gene products of the MEP pathway. To get a complete overview of the pathway organization, we cloned four genes encoding missing enzymes involved in the MEP pathway before conducting a systematic analysis of transcript distribution and protein subcellular localization. RNA in situ hybridization revealed that all MEP pathway genes were coordinately and mainly expressed in internal phloem-associated parenchyma of young leaves, reinforcing the role of this tissue in MIA biosynthesis. At the subcellular level, transient cell transformation and expression of fluorescent protein fusions showed that all MEP pathway enzymes were targeted to plastids. Surprisingly, two isoforms of 1-deoxy-D-xylulose 5-phosphate synthase and 1-deoxy-D-xylulose 5-phosphate reductoisomerase initially exhibited an artifactual aggregated pattern of localization due to high protein accumulation. Immunogold combined with transmission electron microscopy, transient transformations performed with a low amount of transforming DNA and fusion/deletion experiments established that both enzymes were rather diffuse in stroma and stromules of plastids as also observed for the last six enzymes of the pathway. Taken together, these results provide new insights into a potential role of stromules in enhancing MIA precursor exchange with other cell compartments to favor metabolic fluxes towards the MIA biosynthesis.

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