Sequential Development of Cysteine Proteinase Activities and Gene Expression during Somatic Embryogenesis in Carrot.

Three bands of proteinase activity (Rf values of 0.5, 0.6, and 0.7) were detected on activity-stained gels after native gel electrophoresis of carrot (Daucus carota L. cv US-Harumakigosun) suspension cells. After the induction of somatic embryogenesis, one activity band (0.7 band) rapidly disappeared; the 0.6 band was absent at the heart-shaped embryo stage. However, the intensity of the 0.5 band increased during embryogenesis. An additional band (0.25 band) appeared after the torpedo-shaped stage. Three bands (0.25, 0.5, and 0.6) were also detected in zygotic seeds. Two activity bands (0.5 and 0.6) were classified as cysteine proteinases based on sensitivities to N-Ethylmaleimide (NEM) or L-3-trans-Carboxyoxirane-2-Carbonyl-L-Leucyl-Agmatine (E-64). To find candidate genes for the cysteine proteinases, we cloned seven cDNAs encoding putative cysteine proteinases from suspension cells and developing somatic embryos. The expression patterns of the seven genes were categorized into three types (Type A, mRNAs increase concomitantly with somatic embryogenesis; Type B, mRNAs decrease quickly in organized cells; Type C, no significant change in transcript level during somatic embryogenesis).