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

Abstract

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.

Keywords: CRISPR/Cas9; EPHEMERAL1; Japanese gentian; NAC transcription factor; corolla; flower longevity; genome editing; senescence.

Figure 1

(A) Alignment of the deduced amino acid sequences of EPH1 and EPH1L proteins. The amino acid sequences of EPH1 from the Japanese morning glory (Ipomoea nil) and EPH1L (EPH1La and EPH1Lb) from the Japanese gentian (“Albireo”) were aligned by using the Clustal X program [32]. The amino acid residues conserved among the proteins are highlighted in black. The amino acid residues that are different between EPH1La and EPH1Lb are indicated with an asterisk *. The black and orange overlines indicate the conserved NAC domain and the putative nuclear localization signal, respectively. Asterisks *** indicate different amino acid residues in EPH1La and EPH1Lb. (B) The molecular phylogenic tree of EPH1, EPH1La, EPH1Lb, and Arabidopsis NAC proteins belonging to the NAM subfamily. The branch lengths were proportional to the genetic distances as calculated by the neighbor-joining method [33]. Amino acid sequences for all NAM subfamilies and outgroups in A. thaliana were obtained via TAIR (http://www.arabidopsis.org/, accessed on 1 April 2022).

Figure 2

Changes in the corolla appearance, genome structure, and gene expression with aging of field-grown Japanese gentians. (A) The appearance of field-grown Japanese gentians (“Albireo”). (B). Image of the agarose gel electrophoresis for the “Albireo” corolla genome at stage 4 and senescence corolla (partially wilted and fully wilted). (C) The relative expression level of EPH1L and SAG12 determined by a quantitative reverse transcription polymerase chain reaction (n = 6). Asterisks indicate statistically significant differences compared with stage 1 as demonstrated by Student’s t-test. (** p < 0.01).

Figure 3

Dark-induced senescence of “Albireo” corolla at stage 4. (A) The state of “Albireo” corolla before the start of dark-induced senescence treatment. The cover is opened for the photograph. (B) Image of the agarose gel electrophoresis of the genomic DNAs of “Albireo” corolla from before the start of the dark-induced senescence treatment until the 20th day after the start of the treatment (every 4 days). (C) The relative expression levels of EPH1L and SAG12 determined by quantitative reverse transcription polymerase chain reaction (n = 6). Asterisks indicate statistically significant differences between 0 d and 4–16 d as demonstrated by Student’s t-test (* p < 0.05, ** p < 0.01).

Figure 4

Schematic diagram of the binary vector and the target sequences in the EPHEMERAL1-LIKE in Japanese gentians. (A) The diagram of the pSALS-35SpCas9-CmYLCVpEPH1Lt1t2. LB, left border of T-DNA; Nos(P), promoter of the nopaline synthase gene of Agrobacterium tumefaciens; AtADH 5′-UTR, 5′—untranslated region of the alcohol dehydrogenase gene of Arabidopsis thaliana; GtmutALS, mutant acetolactate synthase gene of Gentiana triflora; rbc(T), terminator of small subunit 2B of ribulose 1, 5-bisphosphate carboxylase/oxygenase of A. thaliana; 35SP(P), Cauliflower mosaic virus 35S promoter; pcoCas9, plant codon-optimized Cas9 of Streptococcus pyogenes; HSP(T), terminator of heat shock protein 18.2 of A. thaliana; CmYLCV(P), Cestrum yellow leaf curling virus promoter; SC, single-guide RNA scaffold; RB, right border of T-DNA. (B) First exon sequence containing target sites 1 and 2 for genome editing. R, W, and M are marked by IUPAC notation. Single nucleotide polymorphism (SNP) shows different bases between EPH1La and EPH1Lb. PAM, protospacer adjacent motif.

Figure 5

Evaluation of the flower longevity of eph1l genome-edited knockout mutant lines (#6-6, #8-2, and #8-5) lines. (A) Changes in corolla appearance every 4 days. (B) Image of the agarose gel electrophoresis of the genomic DNA from wild type (WT) and three eph1l lines before and after dark-induced senescence treatments. (C) The relative expression level of SAG12 from WT and three eph1l lines determined by quantitative reverse transcription polymerase chain reaction (n = 4–6). Different letters indicate significant differences during the 16 d treatment period in each line (Tukey–Kramer test, p < 0.05).