CDNA CLONING OF ECP40, AN EMBRYOGENIC-CELL PROTEIN IN CARROT, AND ITSEXPRESSION DURING SOMATIC AND ZYGOTIC EMBRYOGENESIS

A cDNA of 1.2 kb was isolated from a cDNA library of embryogenic cells of carrot (Daucus carota L.) by use of nucleotide sequences that enco de two internal amino-acid sequences of ECP40 (an embryogenic-cell pro tein wit a relative molecular mass of 40000). A genomic Southern blot using the cDNA as probe suggested that there are at least two genes fo r ECP40 in the carrot genome. The cDNA encoded an open reading frame o f 306 amino acids, and the deduced amino-acid sequence was found to sh are two motifs, namely SSSSSSEDDGXGGRRKKGXXXKIKEKLXGG and EKKXXXD-KIKX KLPG, with rabl6 protein from rice and dehydrins from barley and maize . The level of expression of these proteins has been reported to be hi gh during late embryogenesis and to be induced by a plant hormone, ABA . Accumulation of ECP40-specific transcripts started 18 days after flo wering and continued until maturation of seeds, but the levels decreas ed within 24 h after imbibition. ECP40 and its mRNAs were detected in the endosperm and zygotic embryos of mature seeds by immunohistochemis try and in situ hybridization. Exogenous application of 0.1 mM ABA to carrot seedlings did not ind uce expression of the gene for ECP40, whi le drought treatment induced the accumulation of low levels of the mRN As. During somatic embryogenesis, the mRNAs were found at high levels in embryogenic cells and at low levels in somatic embryos at the torpe do stage. Immunohistochemical analysis and in situ hybridization showe d that both ECP40 and its transcripts were preferentially localized in the peripheral cells of the clusters of embryogenic cells. In somatic embryos, application of ABA resulted in increases in levels of mRNAs for ECP40 up to the levels in embryogenic cells, but no such increases were observed in ABA-treated embryogenic cells. The pattern of expres sion of the gene for ECP40 during somatic embryogenesis was basically the same as that of ECP31, another ABA-regulable embryogenic-cell prot ein of carrot, the presence of which has been correlated with the embr yogenic competence of cultured cells (T. Kiyosue, S. Satoh, H. Kamada and H. Harada, Plant Physiol 95 (1991) 1077-1083). The various results together imply that a group of ABA-inducible genes is expressed in th ese embryogenic cells.