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A chemical approach to extend flower longevity of Japanese morning glory via inhibition of master senescence regulator EPHEMERAL1

Nature Plants volume 10, pages 1377–1388 (2024)Cite this article

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Abstract

Petal senescence in flowering plants is a type of programmed cell death with highly regulated onset and progression. A NAM/ATAF1,2/CUC2 transcription factor, EPHEMERAL1 (EPH1), has been identified as a key regulator of petal senescence in Japanese morning glory (Ipomoea nil). Here we used a novel chemical approach to delay petal senescence in Japanese morning glory by inhibiting the DNA-binding activity of EPH1. A cell-free high-throughput screening system and subsequent bioassays found two tetrafluorophthalimide-based compounds, Everlastin1 and Everlastin2, that inhibited the EPH1–DNA interaction and delayed petal senescence. The inhibitory mechanism was due to the suppression of EPH1 dimerization. RNA-sequencing analysis revealed that the chemical treatment strongly suppressed the expression of programmed cell death- and autophagy-related genes. These results suggest that a chemical approach targeting a transcription factor can regulate petal senescence.

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Fig. 1: Establishment of a high-throughput assay to detect the EPH1–DNA interaction and screening of EPH1 inhibitors.
Fig. 2: Screening of analogues of the compounds obtained from the first screening.
Fig. 3: Effects of Everlastin1 and Everlastin2 on the DNA interaction and dimerization of EPH1.
Fig. 4: ChIP and transcriptome analysis of Everlastin1- and Everlastin2-treated petals.
Fig. 5: Effects of Everlastin1 and Everlastin2 on petal senescence.

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Data availability

All data generated or analysed in this study are included in this published article and its supplementary information files. Raw RNA-seq data are available at DDBJ Sequence Read Archive (https://www.ddbj.nig.ac.jp/dra/index-e.html, with accession numbers DRR437987–DRR437998, DRR545318–DRR545320). The I. nil genome (RefSeq assembly accession: GCF_001879475.1) was used for the sequencing analysis. Source data are provided with this paper.

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Acknowledgements

We thank the Applied Protein Research Laboratory at Ehime University for protein analysis, the Drug Discovery Initiative at the University of Tokyo for providing the chemical libraries and the National BioResource Project ‘Morning glory’, Japan, for providing seeds of I. nil. We also thank M. Nakayama (National Agriculture and Food Research Organization) for valuable discussions about chemical structures, and K. Maruyama (Ehime University) for assistance with in vitro interaction assays. This work was supported by a grant from a commissioned project study on ‘Development of post-harvest technology in cut flowers’ (JP15650941 to K.S.) from the Ministry of Agriculture, Forestry and Fisheries, Japan.; the Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research) from Japan Agency for Medical Research and Development (AMED) under grant numbers JP21am0101086 (support number 1590 to K.S.) and JP21am0101077 (support number 1144 to K.S.); and a Grant-in-Aid for Scientific Research on Innovative Areas (JP16H06579 to T.S.), Grants-in-Aid for Scientific Research (21K05589 to K.S., 19H03218 to T.S. and 19K05815 to A.N.) from the Japan Society for the Promotion of Science.

Author information

Authors and Affiliations

  1. Institute of Vegetable and Floriculture Science, National Agriculture and Food Research Organization (NARO), Tsukuba, Japan

    Kenichi Shibuya

  2. Proteo-Science Center (PROS), Ehime University, Matsuyama, Japan

    Akira Nozawa, Chikako Takahashi & Tatsuya Sawasaki

Authors
  1. Kenichi Shibuya
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  2. Akira Nozawa
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Contributions

K.S., A.N. and T.S. designed experiments. K.S. and A.N. performed chemical screening. A.N., C.T. and K.S. performed in vitro interaction assays. K.S. performed the experiments with Japanese morning glory plants and RNA-seq analysis. K.S., A.N. and T.S. analysed and interpreted data. K.S. and A.N. wrote the manuscript, and all authors contributed to editing.

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Correspondence to Kenichi Shibuya or Akira Nozawa.

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Nature Plants thanks Junping Gao and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Chemical structures of G1-1 and its analogues.

The inhibition assay of EPH1–DNA interaction activity was performed in the presence of G1-1 analogues at a final concentration of 5 µM.

Extended Data Fig. 2 Inhibition rate of EPH1–DNA interaction activity for fluorine-substituted compounds of G1-5 and G1-6.

a, b, Chemical structures (a) and the inhibition rate of EPH1–DNA interaction activity (b) for fluorine-substituted compounds of G1-5. c, d, Chemical structures (c) and the inhibition rate of EPH1–DNA interaction activity (d) for fluorine-substituted compounds of G1-6. The inhibition assays were performed in the presence of compounds at a final concentration of 10 µM. Values are means ± s.e.m. of n = 3 independent experiments (b, d). Asterisks indicate significant differences. **** P < 0.0001; one-way ANOVA with Dunnett’s test compared with G1-5 (b) or G1-6 (d).

Source data

Extended Data Fig. 3 Effects of Everlastin1 and Everlastin2 on the DNA interaction of NAC TFs other than EPH1.

a, Alignment of EPH1 (GenBank Accession No. AB849126), Arabidopsis ORE1 (NM_123323), and Lilium LhNAP (LC807006) polypeptide sequences. Identical amino acids are shown in reverse type. The overline indicates the conserved DNA-binding domain. b, Phylogenic tree of EPH1, Arabidopsis ORE1 and Lilium LhNAP proteins. Sequence alignment with ClustalW2 and phylogenic tree analysis with the neighbor-joining method were performed using Geneious Prime software (Biomatters, Auckland, New Zealand). c, Interaction assay for NAC TFs and DNA. Interactions between DNA and full-length ORE1 or LhNAP in the presence of Everlastin1 or Everlastin2 (5 µM each) were analyzed. DNA–protein interactions were analyzed using the AlphaScreen (AS) technology. Values are means ± s.e.m. of n = 4 independent experiments. Asterisks indicate significant differences. ns, not significant; **** P < 0.0001; one-way ANOVA with Dunnett’s test compared with DMSO.

Source data

Extended Data Fig. 4 Effects of Everlastin1 and Everlastin2 on petal senescence.

a, Petal segments treated with Everlastin1 or Everlastin2. Petal segments of I. nil were floated adaxial side up in DMSO control or Everlastin1 or Everlastin2 solutions at a final concentration of 5 µM. The pictures show petal segments at 24 h after flower opening (t24). Petal segments in the DMSO control showed water-soaking symptoms. Petal segments were prepared from five different flowers per treatment (Petri dish) and photographs of representative sets from at least three independent experiments are shown (a). b, Gel image of DNA degradation in petal segments treated with Everlastin1 or Everlastin2. A representative photograph from three biological replicates is shown. c, Cut flowers of I. nil treated with Everlastin2. Whole cut flowers were floated in Everlastin2 solution at a final concentration of 5 µM. The picture shows flowers at t24. A representative photograph from six biological replicates is shown.

Extended Data Fig. 5 Effects of Everlastin1 and Everlastin2 on petal senescence and gene expression in EPH1-knockout plants.

a, Time-course of visible senescence of the petal segments in EPH1-knockout (EPH1-KO) plants. Petal segments were floated adaxial side up in the DMSO control, Everlastin1, or Everlastin2 solution at a final concentration of 5 µM. Petal segments were prepared from five different flowers per treatment (Petri dish) and photographs of representative sets from at least three independent experiments are shown. b, Venn diagrams showing the overlap of the up-regulated (log2 fold change >1) and down-regulated (log2 fold change <−1) genes between EPH1-KO and Everlastin1 or Everlastin2 treatment compared to DMSO-treated WT control at t8. c, Volcano plot showing differentially expressed genes between EPH1-KO and Everlastin1-treated petal segments at t8. Red and blue plots indicate up-regulated (log2 fold change >1 and p-value < 0.05) and down-regulated (log2 fold change <−1 and p-value < 0.05) genes, respectively. p-value, Benjamini-Hochberg adjusted p-value. RNA-seq analysis was performed using three biological replicates per condition (b, c).

Supplementary information

Supplementary Information

Supplementary Fig. 1, Table 2 and unprocessed blot of Fig. 1.

Reporting Summary

Supplementary Table 1

Differentially expressed genes between EPH1-KO and Everlastin1-treated petal segments.

Supplementary Table 3

Exact P values.

Source data

Source Data Fig. 1

Statistical source data.

Source Data Fig. 2

Statistical source data.

Source Data Fig. 3

Statistical source data.

Source Data Fig. 4

Statistical source data.

Source Data Fig. 5

Statistical source data.

Source Data Fig. 1

Unprocessed blot.

Source Data Fig. 2

Unprocessed blot.

Source Data Extended Data Fig. 2

Statistical source data.

Source Data Extended Data Fig. 3

Statistical source data.

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Shibuya, K., Nozawa, A., Takahashi, C. et al. A chemical approach to extend flower longevity of Japanese morning glory via inhibition of master senescence regulator EPHEMERAL1. Nat. Plants 10, 1377–1388 (2024). https://doi.org/10.1038/s41477-024-01767-z

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