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An optimized apoplastic fluid extraction method for the simultaneous characterization of the proteome and metabolome of grapevine leaves.

ABSTRACT: The study of different cellular compartments also allows a broader conception of cell dynamics. Plant apoplast is the cellular compartment external to the plasma membrane including the cell wall. Despite our knowledge on apoplast involvement on several processes from cell growth to biotic and abiotic stress response, its dynamics is yet still poorly studied due to the need for adaptation of the extraction process according to the plant system and consequently, the difficulty to perform a comprehensive profiling of proteins, metabolites and lipids within a single experiment. Moreover, when looking at woody plants as grapevine, more challenges are raised leading to only few studies published so far focusing on apoplast characterization. Being grapevine one of the most important fruit crops worldwide, a wider characterization of this compartment may unveil the importance of grapevine’s secretome. Here, we describe an optimized vacuum-infiltration-centrifugation method for the simultaneous extraction of apoplastic proteins and metabolites from the grapevine leaf on a single experiment, compatible with high-throughput mass spectrometry analysis.

INSTRUMENT(S): TripleTOF 6600

ORGANISM(S): Vitis Vinifera (grape)

TISSUE(S): Plant Cell, Leaf

SUBMITTER: Céline LECLERCQ

LAB HEAD: Céline LECLERCQ

PROVIDER: PXD015558 | Pride | 2021-02-17

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	F161678.dat	Other
	F161678_NCBI_Vitisvinifera_Leonor band a.MSMS.pride.mgf.gz	Mgf
	F161678_NCBI_Vitisvinifera_Leonor band a.pride.mztab.gz	Mztab
	F161678_NCBI_Vitisvinifera_Leonorbanda.MSMS.mgf	Mgf
	F161678_NCBI_Vitisvinifera_Leonorbanda.mzid.gz	Mzid

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Project description:Fomitiporia mediterranea (Fmed) is one of the main fungal species found in grapevine wood rot, also called “amadou”, one of the most typical symptoms of grapevine trunk disease Esca. This fungus is functionally classified as a white-rot, able to degrade all wood structure polymers, i.e., hemicelluloses, cellulose, and the most recalcitrant component, lignin. Specific enzymes are secreted by the fungus to degrade those components, namely carbohydrate active enzymes for hemicelluloses and cellulose, which can be highly specific for given polysaccharide, and peroxidases, which enable white-rot to degrade lignin, with specificities relating to lignin composition as well. Furthermore, besides polymers, a highly diverse set of metabolites often associated with antifungal activities is found in wood, this set differing among the various wood species. Wood decayers possess the ability to detoxify these specific extractives and this ability could reflect the adaptation of these fungi to their specific environment. The aim of this study is to better understand the molecular mechanisms used by Fmed to degrade wood structure, and in particular its potential adaptation to grapevine wood. To do so, Fmed was cultivated on sawdust from different origins: grapevine, beech, and spruce. Carbon mineralization rate, mass loss, wood structure polymers contents, targeted metabolites and secreted proteins were measured. We used the well-known white-rot model Trametes versicolor for comparison. Whereas no significant degradation was observed with spruce, a higher mass loss was measured on Fmed grapevine culture compared to beech culture. Moreover, on both substrates, a simultaneous degradation pattern and the degradation of wood extractives were demonstrated, and proteomic analyses identified a relative overproduction of oxidoreductases involved in lignin and extractive degradation on grapevine cultures, and only few differences in carbohydrate active enzymes. These results could explain at least partially the adaptation of Fmed to grapevine wood structural composition compared to other wood species and suggest that other biotic and abiotic factors should be considered to fully understand the potential adaptation of Fmed to its ecological niche.

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An optimized apoplastic fluid extraction method for the simultaneous characterization of the proteome and metabolome of grapevine leaves.

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