Abstract
The multicellular nature of plants requires that cells should communicate in order to coordinate essential functions. This is achieved in part by molecular flux through pores in the cell wall, called plasmodesmata. We describe the proteomic analysis of plasmodesmata purified from the walls of Arabidopsis suspension cells. Isolated plasmodesmata were seen as membrane-rich structures largely devoid of immunoreactive markers for the plasma membrane, endoplasmic reticulum and cytoplasmic components. Using nano-liquid chromatography and an Orbitrap ion-trap tandem mass spectrometer, 1341 proteins were identified. We refer to this list as the plasmodesmata- or PD-proteome. Relative to other cell wall proteomes, the PD-proteome is depleted in wall proteins and enriched for membrane proteins, but still has a significant number (35%) of putative cytoplasmic contaminants, probably reflecting the sensitivity of the proteomic detection system. To validate the PD-proteome we searched for known plasmodesmal proteins and used molecular and cell biological techniques to identify novel putative plasmodesmal proteins from a small subset of candidates. The PD-proteome contained known plasmodesmal proteins and some inferred plasmodesmal proteins, based upon sequence or functional homology with examples identified in different plant systems. Many of these had a membrane association reflecting the membranous nature of isolated structures. Exploiting this connection we analysed a sample of the abundant receptor-like class of membrane proteins and a small random selection of other membrane proteins for their ability to target plasmodesmata as fluorescently-tagged fusion proteins. From 15 candidates we identified three receptor-like kinases, a tetraspanin and a protein of unknown function as novel potential plasmodesmal proteins. Together with published work, these data suggest that the membranous elements in plasmodesmata may be rich in receptor-like functions, and they validate the content of the PD-proteome as a valuable resource for the further uncovering of the structure and function of plasmodesmata as key components in cell-to-cell communication in plants.
Highlights
An important goal in plant biology is the identification of the proteome of subcellular components and compartments
Transmission electron microscopy of negatively stained samples of the smaller material revealed vesicle-like structures of 50–100 nm (Fig. 1B), which appeared to be composed of limited numbers of concentric membrane layers
Immunoblot analysis of samples collected sequentially during the PD isolation procedure showed that the membranous sample was substantially free of contaminant proteins representative of the endoplasmic reticulum (BiP), plasma membrane (PMA2), Golgi, and chloroplast, whilst showing a corresponding increase in the abundance of PDLP1 (Fig. 1D)
Summary
An important goal in plant biology is the identification of the proteome of subcellular components and compartments. PD are membrane-rich pores that bridge the relatively rigid cell wall to connect adjacent plants cells. They provide routes for the diffusion of small molecules from cell to cell, and for the specific trafficking of larger proteins and nucleic acids that collectively contribute to the regulation of development, growth and defence [1,2]. Despite their importance in these fundamental processes, PD have remained recalcitrant to structural and functional dissection. Based conceptually upon the number of proteins associated with the nuclear pore complex, which has comparable functions in the translocation of small and large molecules between cellular compartments, we and others [3,4]
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