Cloning of B-1,3-glucanases expressed during Cichorium somatic embryogenesis

Three different β-1,3-glucanase cDNA fragments, CG1, CG2 and CG3, were obtained by RT-PCR from RNA isolated from Cichorium hybrid `474' leaf fragments cultured for 11 days under somatic embryogenesis-inducing conditions. When expressed in Escherichia coli the proteins encoded by the three cDNAs were recognized by antibodies raised against 38 kDa extracellular β-1,3-glucanases studied previously (Helleboid et al., Planta 205 (1998) 56–63). The CG2 and CG3 cDNAs may represent expressed alleles of one gene because their sequences showed a very high identity (98.5€and are only 70␒dentical with CG1. Southern blot analysis revealed the presence of 3–4 genes coding for β-1,3-glucanases in the Cichorium genome. Expression analysis of the genes corresponding to the three clones analysed by semi-quantitative RT-PCR indicated that CG1 mRNAs were only detectable in Cichorium hybrid `474' leaf fragments from day 3 of somatic embryogenesis induction, whereas CG2-CG3 mRNAs were already present in non-induced leaf tissue of both the embryogenic hybrid `474' and a non-embryogenic genotype. The level of CG1 mRNAs was particularly high when embryogenic cells were dividing to produce embryos, and when the amount of callose deposited in cell walls surrounding embryogenic cells and young embryos decreased. These results indicate that expression of the CG1 gene is correlated to the somatic embryogenesis process and that it encodes a 38 kDa β-1,3-glucanase protein that may be involved in the degradation of callose localized around embryogenic cells and young embryos. A full-length CG1 cDNA clone was obtained using 3′ and 5′ RACE-PCR, and its sequence revealed that it encodes a β-1,3-glucanase that is equally homologous to both class III and class IV plant β-1,3-glucanases