Euscaphis konishii is an evergreen plant that is widely planted as an industrial crop in Southern China. It produces red fruits with abundant secondary metabolites, giving E. konishii high medicinal and ornamental value. Auxin signaling mediated by members of the AUXIN RESPONSE FACTOR (ARF) and auxin/indole-3-acetic acid (Aux/IAA) protein families plays important roles during plant growth and development. Aux/IAA and ARF genes have been described in many plants but have not yet been described in E. konishii. In this study, we identified 34 EkIAA and 29 EkARF proteins encoded by the E. konishii genome through database searching using HMMER. We also performed a bioinformatic characterization of EkIAA and EkARF genes, including their phylogenetic relationships, gene structures, chromosomal distribution, and cis-element analysis, as well as conserved motifs in the proteins. Our results suggest that EkIAA and EkARF genes have been relatively conserved over evolutionary history. Furthermore, we conducted expression and co-expression analyses of EkIAA and EkARF genes in leaves, branches, and fruits, which identified a subset of seven EkARF genes as potential regulators of triterpenoids and anthocyanin biosynthesis. RT-qPCR, yeast one-hybrid, and transient expression analyses showed that EkARF5.1 can directly interact with auxin response elements and regulate downstream gene expression. Our results may pave the way to elucidating the function of EkIAA and EkARF gene families in E. konishii, laying a foundation for further research on high-yielding industrial products and E. konishii breeding.

Anthocyanins, crucial water-soluble pigments in plants, determine coloration in floral and fruit tissues, while fulfilling essential physiological roles in terms of plant growth, development, and stress adaptation. The biosynthesis of anthocyanins is transcriptionally regulated by WRKY factors, one of the largest plant-specific transcription factor families. Euscaphis japonica is an East Asian species, prized for its exceptionally persistent butterfly-shaped fruits that undergo pericarp dehiscence, overturning, and a color transition to scarlet red. This species represents an ideal system for studying anthocyanin regulation. However, the mechanisms by which WRKY transcription factors orchestrate anthocyanin accumulation during this process remain unknown. In this study, we identified 87 WRKY genes (EjaWRKYs) from the E. japonica genome. Phylogenetic analysis was used to classify these genes into three primary groups, with five subgroups, revealing conserved gene structures and motif compositions, supported by collinearity and comparative synteny analyses. Crucially, ten EjaWRKYs exhibited peak expression during the mature fruit stages, showing positive correlations with key anthocyanin biosynthesis genes. Functional validation through the use of transient transactivation assays in Nicotiana benthamiana confirmed that the five selected EjaWRKYs bind W-box elements and strongly activate reporter gene expression. Our results reveal EjaWRKYs’ regulation of anthocyanin accumulation in E. japonica fruit, provide the first comprehensive WRKY family characterization of this species, and establish a foundation for manipulating ornamental traits in horticultural breeding.

Background/Objectives: This study was based on the joint analysis of transcriptome and metabolome to explore the key genes and metabolic pathways of gardenia single flower petal number variation and to explore the possible mechanism of floral organ variation. Methods: Five, six, and seven petals of single-flower gardenia were selected as test materials for transcriptome and metabolome determination to excavate the key genes in regulating petal number in gardenia. Results: Metabolomic analysis identified triethylamine, succinic acid, succinylaldehyde, 2-phenylethanol, and o-xylene as the top five differentially expressed metabolites affecting petal number variation in gardenia. In the KEGG enrichment analysis, gardenia five, six, and seven DEGs were mainly enriched in amphetamine biosynthesis, the biosynthesis of plant secondary metabolites; transcriptome results showed that the identified differential transcription factors mainly come from NAC, ERF, C2H2, MYB, and MADS-box gene families; the expression of GjMADS50, GjMADS59, and GjERF28 changed with the increase in petal number. The commonality between gardenia five, six, and seven flowers exceeded the difference, and the expression pattern of MADS-box and ERF gene family members was the upregulation of GjERF28, GjERF39, and GjMADS67 and downregulation of GjMADS50, GjMADS59, and GjMADS60. Conclusions: We propose that ERF transcription factors may determine the initial number of petal primordia by mediating gibberellin biosynthesis or signaling, thereby coordinately regulating floral meristem activity and specific metabolic states.