In vitro cross-linking of extensin precursors by mustard extracellular isoforms of peroxidase that respond either to phytochrome or to wounding
1998
Alconada Magliano, T.M. | Casal, J.J.
The activities of extracellular peroxidase isoforms A3 and A4 from mustard (Sinapis alba) are known to respond to wounding treatments and to phytochrome status, respectively. To investigate the affinity of A3 and A4 for extensin precursors in vitro, these acidic isoforms were extracted by low-speed centrifugation of intact mustard internodes infiltrated with CaCl2 and isolated by chromatofocusing. Extensin precursors from carrot (Daucus carota) roots or mustard stems and leaves were isolated by saline extraction followed by purification on carboxymethyl-cellulose ion exchange and gel-filtration chromatography. Cross-linking of extensin precursors in vitro in the presence of peroxidase isoforms and exogenous H2O2, was quantified following Sephacryl S-400 gel-filtration, as a shift of extensins to higher molecular mass values. Isoforms A3 and A4 had similar affinities for natural extensin precursors. A cationic isoform (previously not characterized) was unable to cross-link extensin precursors. Tissue prints of mustard stems indicate that extensin precursors are present in the cell wall of all the tissues, with maximum staining in vascular bundles and epidermis. Isoforms A3 and A4 were detected in extracts from vascular bundles, cortex and pith. Only A3 was detected in epidermal extracts. The observations are consistent with a role of isoforms A3 and A4 in cross-linking of extensin in muro.
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