Isolation and Functional Analysis of EPHEMERAL1-LIKE (EPH1L) Genes Involved in Flower Senescence in Cultivated Japanese Gentians

The elongation of flower longevity increases the commercial value of ornamental plants, and various genes have been identified as influencing flower senescence. Recently, EPHEMERAL1 (EPH1), encoding a NAC-type transcription factor, was identified in Japanese morning glory as a gene that promotes flower senescence. Here we attempted to identify an EPH1 homolog gene from cultivated Japanese gentians and characterized the same with regard to its flower senescence. Two EPH1-LIKE genes (EPH1La and EPH1Lb), considered as alleles, were isolated from a gentian cultivar (Gentiana scabra × G. triflora). Phylogenetic analyses revealed that EPH1L belongs to the NAM subfamily. The transcript levels of EPH1L increased along with its senescence in the field-grown flowers. Under dark-induced senescence conditions, the gentian-detached flowers showed the peak transcription level of EPH1L earlier than that of SAG12, a senescence marker gene, suggesting the involvement of EPH1L in flower senescence. To reveal the EPH1L function, we produced eph1l-knockout mutant lines using the CRISPR/Cas9 system. When the flower longevity was evaluated using the detached flowers as described above, improved longevity was recorded in all genome-edited lines, with delayed induction of SAG12 transcription. The degradation analysis of genomic DNA matched the elongation of flower longevity, cumulatively indicating the involvement of EPH1L in the regulation of flower senescence in gentians.