Zeatin accumulation and misexpression of a class I knox gene are intimately linked in the epiphyllous response of the interspecific hybrid EMB-2 (Helianthus annuus x H. tuberosus)

Epiphylly, occurring in a somaclonal variant (EMB-2) of the interspecific hybrid Helianthus annuus x H. tuberosus, was used to investigate molecular and cyto-physiological mechanisms that underlie cellular fate change. EMB-2 plants are characterized by profuse proliferation of shoot- and embryo-like structures on some leaves. We addressed the putative relationship between cytokinins and knox genes in EMB-2 plants. A class I knox gene, HtKNOT1, was isolated from H. tuberosus. A high level of HtKNOT1 transcripts was detected in EMB-2 epiphyllous leaves compared to non-epiphyllous (NEP) ones. In addition, epiphylly was related to a localized increases in zeatin and N-glycosylated cytokinins. As ectopic morphogenesis proceeded, HtKNOT1 transcripts and zeatin co-localized and showed different patterns in ectopic shoot compared with embryo-like structures, consistent with the differential role of both cytokinin and knox genes in the two morphogenetic events. Notably, a massive shoot/embryo regeneration was induced in EMB-2 NEP leaves by in vitro zeatin treatment. These results clearly indicate that localized cytokinin accumulation and ectopic expression of HtKNOT1 are closely linked in the epiphylly of EMB-2 plants.