Characterisation of cDNAs homologous to Rab5-GTP binding protein expressed during early somatic embryogenesis in chicory ☆

Abstract Transcripts from the Cichorium hybrid ‘474’ ( Cichorium intybus L., var. sativum X C. endivia L., var. latifolia ) leaf tissues were compared by differential display to identify cDNA corresponding to genes that are expressed early during somatic embryogenesis (S.E.). B1.5 , one of the isolated cDNAs had a strong sequence similarity with small Ypt/Rab5 GTP binding proteins. Northern blot analyses showed that transcripts hybridising with this cDNA were present in leaf tissues of the embryogenic responsive genotype ‘474’ submitted to S.E.-inducing conditions, but were absent under these conditions when using a non-embryogenic responsive chicory cultivar. Performing 5′RACE PCR, we were able to identify CHI-GTP1 and CHI-GTP2 , 2 cDNAs that represent two highly homologous genes or two alleles of one gene encoding Rab5-like GTP-binding proteins. Amplification of genomic DNA revealed the presence of 2–4 genes in the chicory ‘474’ hybrid genome. The 2 proteins deduced from CHI-GTP1 and CHI-GTP2 are 200 amino-acids long with sizes of about 22 kDa. They differed only in 3 AA. Comparing the different sequences obtained in chicory with GTP-binding proteins from others plants B1.5 most likely represent a product obtained from CHI-GTP1 . Expressed in Escherichia coli , the CHI-GTP1 protein was able to bind GTP confirming its identity. In view of the proposed roles of Rab5 GTP-binding protein, the possible function of the protein encoded by CHI-GTP1 in S.E. process is discussed.