Functional analysis of PpIAA11 in response to auxin involved in peach fruit ripening process

【Objective】Previous studies have established that AUX/IAA genes play critical roles at various stages of fruit development across plant species. In peach (Prunus persica L. Batsch), a total of 23 AUX/IAA family genes have been identified, among which several genes have been functionally characterized. However, emerging evidence suggests substantial functional divergence among members of the AUX/IAA gene family. Transcriptomic data from our previous studies indicated that the expression of PpIAA11 increased significantly during fruit maturation in Ri Chuan Bai Feng peach, peaking at the second rapid expansion phase of fruit development. To further elucidate the biological role of PpIAA11 in peach fruit ripening, the heterologous functional validation was performed by overexpressing this gene in Micro-Tom tomato. This approach enabled a preliminary assessment of the contribution of PpIAA11 to fruit development and maturation.【Methods】In this study, the PpIAA11 gene was isolated from Ri Chuan Bai Feng peach fruit via PCR amplification. Multiple sequence alignment was conducted with DNANMAN, and a phylogenetic tree was reconstructed using MEGA 11.0 to illustrate the evolutionary relationships of PpIAA11 across 10 fruit tree species. The protein sequence architecture of PpIAA11 was investigated using the MEME online suite (http://meme-suite.org/), while conserved domains were identified via the conserved domain database (CDD) search tool on the NCBI platform (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). Additionally, cis- regulatory elements within the PpIAA11 promoter region were predicted using PlantCARE (http://bioinformatics.psb.ugent.be/ webtools/plantcare/html/) and New PLACE (https://www.dna.affrc.go.jp/PLACE/action=newplace). The expression pattern of PpIAA11 at various developmental stages of peach fruit, as well as in response to treatments with different concentrations of NAA and NPA, was analyzed using quantitative real- time PCR (qRT- PCR). An overexpression vector harboring the PpIAA11 gene was constructed through homologous recombination and subsequently introduced into Micro-Tom tomatoes via Agrobacterium tumefaciens- mediated leaf disc transformation. Phenotypic characterization of T1 generation fruits was conducted, and the influence of PpIAA11 overexpression on fruit ripening was further evaluated.【Results】Using cDNA synthesized from peach fruit flesh as the template, PCR amplification was performed with primers designed by Primer Premier 5.0. The resulting amplicon exhibited a band size consistent with that of the target gene. Subsequent sequencing confirmed that the PpIAA11 CDS spans 939 bp, encoding a protein of 312 amino acids. qRT-PCR analysis revealed that the expression of PpIAA11 was developmentally regulated in peach fruit, with significantly higher transcript levels detected during the later stages of fruit development. The highest expression was observed at the second rapid expansion phase (70 days post- anthesis), which is consistent with our previous transcriptomic data. Sequence analysis identified a canonical AUX/IAA conserved domain in PpIAA11, showing more than 95% similarity with PdIAA11 (Prunus dulcis), PaIAA11 (Prunus avium), and MdIAA11 (Malus domestica). Additionally, it shared an average similarity exceeding 80% with six other IAA11 homologs: PbIAA11 (Pyrus bretschneideri), VrIAA11 (Vitis riparia), ZjIAA11 (Hippophae rhamnoides), CaIAA11 (Corylus avellana), CiIAA11 (Carya illinoinensis), and JrIAA11 (Juglans regia). A highly conserved region spanning amino acid positions 111-299 was identified, which largely coincides with the AUX/IAA domain, suggesting that this region represents the functional core characteristic of the AUX/IAA gene family. Phylogenetic analysis of AUX/IAA proteins from the ten species revealed that the homologous sequences could be classified into two major clades. The first clade contains VrIAA11, while the second clade includes all remaining sequences and is further divided into two distinct subclades. SubcladeⅠ consists of PpIAA11, PdIAA11, PaIAA11, MdIAA11, and PbIAA11, whereas Subclade Ⅱ comprises ZjIAA11, CaIAA11, CiIAA11, and JrIAA11. This phylogenetic topology further supports a close evolutionary relationship among PpIAA11, PaIAA11, and PdIAA11. Promoter analysis of PpIAA11 identified multiple cis-acting elements associated with light responsiveness, hormone signaling, and stress adaptation. Notably, the promoter region contains 19 putative hormone-responsive elements, among which nine are specifically associated with abscisic acid (ABA) responsiveness. Expression analysis of PpIAA11 revealed distinct dose- and time- dependent regulatory patterns under NAA and NPA treatments. Following treatment with 0.1 mmol· L- 1 NAA, PpIAA11 expression increased 1.5- fold at 6 hours, then sharply decreased to 40% of the control by 12 hours, reaching its lowest level at 24 hours. Exposure to 1.0 mmol·L-1 NAA induced transient upregulation at both 6 and 12 hours, followed by a decline to subbasal levels by 24 hours and stabilization at the minimum by 48 hours. In contrast, the highest NAA concentration (2.0 mmol·L-1 ) resulted in consistent suppression (30%-80% of control) without temporal variation. Under NPA treatments, 0.1 mmol · L- 1 caused immediate downregulation (40% of control at 6 hours), progressively decreasing to 20% by 24 hours. Higher NPA concentrations (1.0-2.0 mmol·L-1 ) led to stable suppression without clear temporal dynamics. These findings indicate that low auxin levels elicit transient induction followed by strong feedback repression of PpIAA11, whereas inhibition of auxin transport results in sustained downregulation. This suggests a sophisticated, concentration- and time-sensitive regulatory mechanism governing PpIAA11 expression during fruit development. Heterologous overexpression of PpIAA11 in Micro- Tom tomatoes resulted in the formation of a distinctive pointed fruit tip, measuring 2-3 mm in length, which was not observed in wild-type (WT) fruits. Additionally, fruit ripening was accelerated by approximately 5 days in the transgenic lines compared to WT controls.【Conclusion】The qRT-PCR analysis revealed that PpIAA11 expression peaked during the second rapid expansion phase of fruit development. Furthermore, its expression exhibited distinct and concentration-dependent regulatory patterns in response to NAA and NPA treatments. Heterologous overexpression of PpIAA11 in tomato accelerated fruit ripening and induced the formation of a distinct fruit tip phenotype. Collectively, these results suggest that PpIAA11 participates in regulating peach fruit development and ripening by mediating responses to auxin signals.