Limits...
Mining the surface proteome of tomato (Solanum lycopersicum) fruit for proteins associated with cuticle biogenesis.

Yeats TH, Howe KJ, Matas AJ, Buda GJ, Thannhauser TW, Rose JK - J. Exp. Bot. (2010)

Bottom Line: Of the approximately 200 proteins that were identified, a subset is potentially involved in the transport, deposition, or modification of the cuticle, such as those with predicted lipid-associated protein domains.The epidermal-specific transcript accumulation of several of these candidates was confirmed by laser-capture microdissection and quantitative reverse transcription-PCR (qRT-PCR), together with their expression during various stages of fruit development.This indicated a complex pattern of cuticle deposition, and models for cuticle biogenesis and restructuring are discussed.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Biology, Cornell University, Ithaca, NY 14853, USA.

ABSTRACT
The aerial organs of plants are covered by the cuticle, a polyester matrix of cutin and organic solvent-soluble waxes that is contiguous with the polysaccharide cell wall of the epidermis. The cuticle is an important surface barrier between a plant and its environment, providing protection against desiccation, disease, and pests. However, many aspects of the mechanisms of cuticle biosynthesis, assembly, and restructuring are entirely unknown. To identify candidate proteins with a role in cuticle biogenesis, a surface protein extract was obtained from tomato (Solanum lycopersicum) fruits by dipping in an organic solvent and the constituent proteins were identified by several complementary fractionation strategies and two mass spectrometry techniques. Of the approximately 200 proteins that were identified, a subset is potentially involved in the transport, deposition, or modification of the cuticle, such as those with predicted lipid-associated protein domains. These include several lipid-transfer proteins, GDSL-motif lipase/hydrolase family proteins, and an MD-2-related lipid recognition domain-containing protein. The epidermal-specific transcript accumulation of several of these candidates was confirmed by laser-capture microdissection and quantitative reverse transcription-PCR (qRT-PCR), together with their expression during various stages of fruit development. This indicated a complex pattern of cuticle deposition, and models for cuticle biogenesis and restructuring are discussed.

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Related in: MedlinePlus

Epidermis structure and experimental design. Confocal microscopy of cryosectioned tomato breaker stage fruit epidermis, co-stained with the fluorescent lipid stain Auramine O (A) and the cellulose stain Calcofluor white M2R (B). The merged image (C) illustrates the cuticle and epidermal cell wall in the context of the epidermal cell layer. (D) Schematic representation of the extraction protocol used to isolate proteins from the cuticle and epidermal cell wall.
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fig1: Epidermis structure and experimental design. Confocal microscopy of cryosectioned tomato breaker stage fruit epidermis, co-stained with the fluorescent lipid stain Auramine O (A) and the cellulose stain Calcofluor white M2R (B). The merged image (C) illustrates the cuticle and epidermal cell wall in the context of the epidermal cell layer. (D) Schematic representation of the extraction protocol used to isolate proteins from the cuticle and epidermal cell wall.

Mentions: As illustrated in Fig. 1A–C, the fluorescently stained cuticle (Fig. 1A) covers the surface of the tomato fruit but is separated from the epidermal cells by a subcuticular polysaccharide cell wall (Fig. 1B). Previous studies have indicated that wax, rather than cutin, is the major barrier to the diffusion of polar molecules, including water (Leide et al., 2007; Isaacson et al., 2009), and presumably proteins, across the cuticle. It was reasoned that, despite the relatively low abundance of wax in the tomato fruit cuticle, which is of the order of 5 μg cm−2 compared with 1 mg cm−2 for the cutin polymer (Baker et al., 1982), a brief immersion of the fruits in an organic solvent would allow the isolation of proteins directly associated with cuticular wax, as well as those localized within the subcuticular epidermal cell wall and possibly epidermal intracellular proteins, depending on the degree to which the cells were compromised. A standard protocol was therefore used to remove waxes by immersion of intact plant organs in an organic solvent to obtain extracts for profiling of the fruit surface proteome, as was previously attempted on a smaller scale with Brassica oleracea leaves (Pyee et al., 1994).


Mining the surface proteome of tomato (Solanum lycopersicum) fruit for proteins associated with cuticle biogenesis.

Yeats TH, Howe KJ, Matas AJ, Buda GJ, Thannhauser TW, Rose JK - J. Exp. Bot. (2010)

Epidermis structure and experimental design. Confocal microscopy of cryosectioned tomato breaker stage fruit epidermis, co-stained with the fluorescent lipid stain Auramine O (A) and the cellulose stain Calcofluor white M2R (B). The merged image (C) illustrates the cuticle and epidermal cell wall in the context of the epidermal cell layer. (D) Schematic representation of the extraction protocol used to isolate proteins from the cuticle and epidermal cell wall.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2921210&req=5

fig1: Epidermis structure and experimental design. Confocal microscopy of cryosectioned tomato breaker stage fruit epidermis, co-stained with the fluorescent lipid stain Auramine O (A) and the cellulose stain Calcofluor white M2R (B). The merged image (C) illustrates the cuticle and epidermal cell wall in the context of the epidermal cell layer. (D) Schematic representation of the extraction protocol used to isolate proteins from the cuticle and epidermal cell wall.
Mentions: As illustrated in Fig. 1A–C, the fluorescently stained cuticle (Fig. 1A) covers the surface of the tomato fruit but is separated from the epidermal cells by a subcuticular polysaccharide cell wall (Fig. 1B). Previous studies have indicated that wax, rather than cutin, is the major barrier to the diffusion of polar molecules, including water (Leide et al., 2007; Isaacson et al., 2009), and presumably proteins, across the cuticle. It was reasoned that, despite the relatively low abundance of wax in the tomato fruit cuticle, which is of the order of 5 μg cm−2 compared with 1 mg cm−2 for the cutin polymer (Baker et al., 1982), a brief immersion of the fruits in an organic solvent would allow the isolation of proteins directly associated with cuticular wax, as well as those localized within the subcuticular epidermal cell wall and possibly epidermal intracellular proteins, depending on the degree to which the cells were compromised. A standard protocol was therefore used to remove waxes by immersion of intact plant organs in an organic solvent to obtain extracts for profiling of the fruit surface proteome, as was previously attempted on a smaller scale with Brassica oleracea leaves (Pyee et al., 1994).

Bottom Line: Of the approximately 200 proteins that were identified, a subset is potentially involved in the transport, deposition, or modification of the cuticle, such as those with predicted lipid-associated protein domains.The epidermal-specific transcript accumulation of several of these candidates was confirmed by laser-capture microdissection and quantitative reverse transcription-PCR (qRT-PCR), together with their expression during various stages of fruit development.This indicated a complex pattern of cuticle deposition, and models for cuticle biogenesis and restructuring are discussed.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Biology, Cornell University, Ithaca, NY 14853, USA.

ABSTRACT
The aerial organs of plants are covered by the cuticle, a polyester matrix of cutin and organic solvent-soluble waxes that is contiguous with the polysaccharide cell wall of the epidermis. The cuticle is an important surface barrier between a plant and its environment, providing protection against desiccation, disease, and pests. However, many aspects of the mechanisms of cuticle biosynthesis, assembly, and restructuring are entirely unknown. To identify candidate proteins with a role in cuticle biogenesis, a surface protein extract was obtained from tomato (Solanum lycopersicum) fruits by dipping in an organic solvent and the constituent proteins were identified by several complementary fractionation strategies and two mass spectrometry techniques. Of the approximately 200 proteins that were identified, a subset is potentially involved in the transport, deposition, or modification of the cuticle, such as those with predicted lipid-associated protein domains. These include several lipid-transfer proteins, GDSL-motif lipase/hydrolase family proteins, and an MD-2-related lipid recognition domain-containing protein. The epidermal-specific transcript accumulation of several of these candidates was confirmed by laser-capture microdissection and quantitative reverse transcription-PCR (qRT-PCR), together with their expression during various stages of fruit development. This indicated a complex pattern of cuticle deposition, and models for cuticle biogenesis and restructuring are discussed.

Show MeSH
Related in: MedlinePlus