Proteomics of ionically bound and soluble extracellular proteins in Medicago truncatula leaves

A large proportion of the apoplast proteome resides in the intercellular fluid (IF) or is ionically bound (IB) to the wall matrix. A combined analysis of IF and IB proteins of the Medicago truncatula leaf apoplast was performed. 2‐DE analyses demonstrated the reproducible presence of 220 IF and 84 IB proteins in the apoplast. These two protein populations were largely distinct; 22 proteins could be spatially matched, but MALDI‐TOF/TOF analyses suggested a considerably smaller number had common identities. MALDI‐TOF/TOF characterisation identified 81 distinct proteins. Analyses of selected IF proteins (45) indicated 17 distinct proteins with mainly defence‐related functions, whereas analyses of IB proteins (70) identified 63 distinct proteins of diverse natures, including proteins of non‐canonical natures. The presence of non‐canonical proteins in IB extracts is discussed in the light of evidence supporting a low level of contamination of purified walls from symplastic proteins. This work indicates that IB and IF proteins are functionally distinct fractions of the apoplast. The data obtained complements earlier studies of the Medicago proteome and therefore will be useful in future studies investigating the role of apoplastic proteins in plant processes.