No abstract

A cDNA clone (6PExt 1.2) encoding a novel extensin was isolated from a cDNA library made from 6 h old mesophyll protoplasts of Nicotiana sylvestris. The screening was performed with a heterologous probe from carrot. The encoded polypeptide showed features characteristic of hydroxyproline-rich glycoproteins such as Ser-(Pro)4 repeats and a high content in Tyr and Lys residues. The presence of four Tyr-X-Tyr-Lys motifs suggests the possibility for intramolecular isodityrosine cross-links whereas three Val-Tyr-Lys motifs may participate in intermolecular cross-links. The analysis of genomic DNA gel blots using both the N. sylvestris and the carrot clones as probes showed that the 6PExt 1.2 gene belongs to a complex multigene family encoding extensin and extensin-related polypeptides in N. sylvestris as well as in related Nicotianeae including a laboratory hybrid. This was confirmed by the analysis of RNA gel blots: a set of mRNAs ranging in size from 0.3 kb to 3.5 kb was found by the carrot extensin probe. The 6PExt 1.2 probe found a 1.2 kb mRNA in protoplasts and in wounded tissues as well as a 0.9 kb mRNA which seemed to be stem-specific. The gene encoding 6PExt 1.2 was induced by wounding in protoplasts, in leaf strips and after Agrobacterium tumefaciens infection of stems.

A genomic clone (Ext 1.4) encoding an extensin was isolated from a Nicotiana tabacum genomic library. The encoded polypeptide showed features characteristic of extensins such as Ser-(Pro)4 repeats and a high content in Tyr and Lys residues. The presence of one Tyr-Leu-Tyr-Lys motif suggests the possibility for one intramolecular isodityrosine cross-link whereas numerous Val-Tyr-Lys motifs may participate in intermolecular cross-links. This extensin appears to be close to an extensin already characterized in N. tabacum but very different from the Ext 1.2 extensin of N. sylvestris. The analysis of genomic DNA gel blots using probes spanning different parts of the gene showed that the Ext 1.4 gene belongs to a complex multigene family having one member originating from N. sylvestris and three members from N. tomentosiformis. The Ext 1.4 specific probe found a 1.4 kb mRNA in stems, roots, ovaries and germinating seeds of healthy plants. The Ext 1.4 gene family is strongly induced in actively dividing cell suspension cultures and after wounding of leaves or stems in conditions where root formation occurs. On the contrary, it is not induced in leaves in response to a hypersensitive reaction to a viral infection or after elicitor treatment.