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

Three different beta-1,3-glucanase cDNA fragments, CG1, CG2 and CG3, were o btained by RT-PCR from RNA isolated from Cichorium hybrid '474' leaf fragme nts cultured for 11 days under somatic embryogenesis-inducing conditions. W hen expressed in Escherichia coli the proteins encoded by the three cDNAs w ere recognized by antibodies raised against 38 kDa extracellular beta-1,3-g lucanases studied previously (Helleboid et al., Planta 205 (1998) 56-63). T he CG2 and CG3 cDNAs may represent expressed alleles of one gene because th eir sequences showed a very high identity (98.5%) and are only 70% identica l with CG1. Southern blot analysis revealed the presence of 3-4 genes codin g for beta-1,3-glucanases in the Cichorium genome. Expression analysis of t he 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 embr yogenic hybrid '474' and a non-embryogenic genotype. The level of CG1 mRNAs was particularly high when embryogenic cells were dividing to produce embr yos, and when the amount of callose deposited in cell walls surrounding emb ryogenic cells and young embryos decreased. These results indicate that exp ression of the CG1 gene is correlated to the somatic embryogenesis process and that it encodes a 38 kDa beta-1,3-glucanase protein that may be involve d in the degradation of callose localized around embryogenic cells and youn g embryos. A full-length CG1 cDNA clone was obtained using 3' and 5' RACE-P CR, and its sequence revealed that it encodes a beta-1,3-glucanase that is equally homologous to both class III and class IV plant beta-1,3-glucanases .