Proteomic Analysis of Cell Walls of Two Developmental Stages of Alfalfa Stems

Abstract

Cell walls are important for the growth and development of all plants. They are also valuable resources for feed and fiber, and more recently as a potential feedstock for bioenergy production. Cell wall proteins comprise only a fraction of the cell wall, but play important roles in establishing the walls and in the chemical interactions (e.g., crosslinking) of cell wall components. This crosslinking provides structure, but restricts digestibility of cell wall complex carbohydrates, limiting available energy in animal and bioenergy production systems. Manipulation of cell wall proteins could be a strategy to improve digestibility. An analysis of the cell wall proteome of apical alfalfa stems (less mature, more digestible) and basal alfalfa stems (more mature, less digestible) was conducted using a recently developed low-salt/density gradient method for the isolation of cell walls. Walls were subsequently subjected to a modified extraction utilizing EGTA to remove pectins, followed by a LiCl extraction to isolate more tightly bound proteins. Recovered proteins were identified using shotgun proteomics. We identified 272 proteins in the alfalfa stem cell wall proteome, 153 of which had not previously been identified in cell wall proteomic analyses. Nearly 70% of the identified proteins were predicted to be secreted, as would be expected for most cell wall proteins, an improvement over previously published studies using traditional cell wall isolation methods. A comparison of our and several other cell wall proteomic studies indicates little overlap in identified proteins among them, which may be largely due to differences in the tissues used as well as differences in experimental approach.

Plant cell wall proteomics: the leadership of Arabidopsis thaliana

“…In addition, sub-proteomes such as N -glycoproteomes and a GPI-anchored proteome have been analyzed. All the extracellular proteomes have been obtained with different plants like A. thaliana (see Table 1), Cicer arietinum (Bhushan et al, 2006, 2011), Glycine max (Komatsu et al, 2010), Helianthus annuus (Pinedo et al, 2012), O. sativa (Chen et al, 2008a; Jung et al, 2008; Cho et al, 2009; Zhou et al, 2011), Medicago sativa (Watson et al, 2004; Verdonk et al, 2012), Nicotiana benthamiana (Goulet et al, 2010), Nicotiana tabacum (Robertson et al, 1997; Dani et al, 2005; Morel et al, 2006; Delannoy et al, 2008; Millar et al, 2009), Populus deltoides (Pechanova et al, 2010), S. lycopersicum (Robertson et al, 1997; Yeats et al, 2010; Catalá et al, 2011), Solanum tuberosum (Fernández et al, 2012; Lim et al, 2012) and Zea mays (Zhu et al, 2006, 2007). Besides, several xylem sap proteomes have been analyzed and were found to be very close to cell wall proteomes (Kehr et al, 2005; Alvarez et al, 2006; Dafoe and Constabel, 2009; Ligat et al, 2011).…”

Long‐term cadmium exposure influences the abundance of proteins that impact the cell wall structure in Medicago sativa stems

“…Hence M. sativa is often used to study cell wall development and processes (Verdonk et al . ; Printz et al . ).…”

Cell wall proteome analysis of Arabidopsis thaliana mature stems

“…The other 649 proteins (68.2%) have been considered as intracellular proteins contaminating the cell wall preparation. This percentage of intracellular proteins was higher than that observed in other plant stem proteomes obtained using similar methods to extract proteins from purified cell walls, such as Brachypodium distachyon (39.8% and 24.5% of intracellular contamination in apical and basal internodes, respectively) , Saccharum officinarum (44%) , Medicago sativa (27–37%) and Linum usitatissimum (31%) . This percentage of intracellular proteins was also much higher than that observed (12.7%) in a previous N ‐glycoproteome study performed on A. thaliana stems .…”