Isolation and characterization of cDNA clones for carrot extensin and a proline-rich 33-kDa protein

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Abstract
Extensins are hydroxyproline-rich glycoprotein associated with most dicotyledonous plant cell walls. To isolate c[complementary]DNA clones encoding extensin, poly(A)+ RNA was isolated from carrot root tissue, and the RNA was translated in vitro, in the presence of 3H-Leu or Pro. A 33 kDa [kilodalton] peptide was identified in the translation products as a putative extensin precursor because: it is rich in proline and poor in Leu, and the message appears to be more abundant when carrot tissue is wounded. From a cDNA library constructed with poly(A)+ RNA from wounded carrots, one cDNA clone (pDC5) was identified that specifically hybridized to poly(A)+ RNA encoding this 33-kDa peptide. Three cDNA clones (pDC11, pDC12 and pDC16) were isolated from another cDNA library using pDC5 as a probe. DNA sequence data, RNA hybridization analysis, and hybrid released in vitro translation indicate that the cDNA clone pDC11 encodes extensin and that cDNA clones pDC12 and pDC16 encode the 33-kDa peptide, which as yet has an unknown identity and function. The assumption that the 33-kDa peptide iwas an extensin precursor was invalid. RNA hybridization and DNA sequence analysis indidcate that pDC5 is a hybrid clone corresponding to 2 RNA species. RNA hybridization analysis showed that RNA encoded by both clone types is accumulated upon wounding.

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