INVESTIGATIONS INTO THE MOLECULAR-SIZE AND SHAPE OF TOMATO EXTENSIN

The molecular characteristics of soluble extensin from tomato have bee n investigated. An apparent molecular mass greater than 240 kDa has be en previously observed with the shape-dependent method of gel-filtrati on chromatography [Brownleader and Dey (1993) Planta (Berlin) 191, 457 -469]. Tomato extensin is a heavily glycosylated protein that does not migrate into SDS/polyacrylamide gels. This shape-dependent behaviour raises doubts about agreement between the observed apparent mass and t he absolute value. The molecular mass measured with matrix-assisted la ser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) was 72.3 kDa, with no evidence of any other species except a doubl y charged ion. The sample was therefore considered to be monodisperse under the conditions used. Electron microscopy of soluble extensin sho wed the presence of particles 40-50 nm in length and 2.0-2.5 nm in wid th. A minority of these particles showed a central 'kink'. A number of smaller and generally wider particles (20 nm x 2-4 nm) were considere d to be folded monomers and larger particles were thought to be dimers . Sedimentation analysis showed that extensin exists in a rapid monome r-dimer equilibrium in the concentration range and buffer used. Sedime ntation equilibrium data gave a K-d of 8.5 mu M and sedimentation velo city data generated a K-d between 1 and 10 mu M. The concentration dep endence of the measured sedimentation coefficient was used, together w ith hydrodynamic bead modelling, to define plausible shapes for monome r and dimer. This suggests that monomeric extensin is an elongated rod of length 40 nm and width 2 nm, which forms staggered dimers of avera ge length 50 nm and width 3 nm. Extensin is an integral component of t he primary cell wall. The physical characteristics (size, shape and fo rm) of the rod-like extensin have been evaluated in this paper so that the role that extensin plays in primary cell wall architecture and du ring plant disease resistance can be more fully understood.