Identification and expression analysis of the walnut JrAUX/IAA gene family

【Objective】Juglans regia L., commonly known as walnut, is a deciduous perennial fruit tree belonging to the Juglandaceae family and ranks among the world's“four major nuts”. Walnuts are prized not only for their high quality but also for their significant economic, nutritional, and medicinal value. The previous research has indicated that the primary cause of walnut kernel exposure is the relatively low levels of glucose and ferulic acid within the inner fruit peel during the initial hardening stage. This deficiency would inhibit the biosynthesis of lignin, cellulose, and hemicellulose, the principal structural components of the inner peel, thereby compromising its lignification. Consequently, incomplete development of the inner peel would lead to kernel exposure, highlighting its critical role in walnut growth, processing, transport, and storage. Concurrently, lignin accumulation is a pivotal step in the development of the walnut's inner peel. Lignin also serves as a key structural component providing robust mechanical support within plant cell walls. Earlier studies have revealed that cell wall formation is influenced by various factors, with phytohormones playing a particularly significant role. Hormones can regulate the formation of plant secondary cell walls by modulating genes associated with xylem development. Our research team observed that in Xinlu walnuts, the concentration of indole-3-acetic acid (IAA) in the inner fruit peel was significantly lower than in the mesocarp during the early hardening phase, but increased again later. This suggests that IAA (auxin) would play an important regulatory role in the hardening process of the inner peel. The AUX/IAA (Auxin/Indole-3-Acetic Acid) gene family represents one of the earliest responsive gene families to auxin signaling and is involved in regulating numerous aspects of plant growth and development.【Methods】Using the Arabidopsis AUX/IAA gene family as a reference, the AUX/IAA genes were identified within the walnut genome. The identity of these genes as the AUX/IAA family members was subsequently confirmed using a hidden Markov model (HMM) search. The comprehensive bioinformatics analysis of the identified gene family was performed utilizing various online tools, including ExPASy, MEGA 11, DNAMAN, Cytoscape, MEME, PlantCARE, STRING, and iTOL. This analysis encompassed the nucleotide sequence characteristics (chromosomal location, homology identification, gene structure, and promoter cis- elements) as well as the properties of the encoded proteins (physicochemical properties, conserved domains and motifs, proteinprotein interaction networks, and phylogenetic relationships). Furthermore, AUX/IAA gene expression patterns were analyzed using publicly available RNA-Seq databases. To investigate the role of auxin in endocarp development, the quantitative reverse transcription polymerase chain reaction (qRT-PCR) was employed to assess the expression levels of the AUX/IAA genes during walnut endocarp development under different concentrations of exogenous indole-3-acetic acid (IAA) treatment.【Results】A total of 63 walnut AUX/IAA genes were identified, and they were unevenly distributed across 16 chromosomes. The protein lengths ranged from 90 to 1296 amino acids, the isoelectric points (pI) ranged from 4.79 to 10.47, and the molecular weights ranged from 10.24 to 144.92 kilodaltons (kD). All family members were hydrophilic proteins. The prediction results for subcellular localization indicated that 47 JrAUX/ IAA proteins were localized in the nucleus, 12 in the chloroplast, and four in the cytoplasm (JrAUX4/13/ 40/63). The phylogenetic tree analysis grouped the proteins into four subgroups (Ⅰ-Ⅳ), and the members of the same subgroup typically exhibited similar gene structures and conserved domains. The Analysis of conserved domains revealed that all 63 JrAUX proteins possessed the AUX/IAA family domain, the B3 DNA-binding domain, and the auxin response factor domain. Most proteins also contained the AUX/IAA family domain. The promoter cis-acting elements indicated that members of this family were associated with numerous light-, stress-, hormone-, and other responsive elements. The combined transcriptome data and qRT- PCR analysis were used to study the expression patterns of the AUX family members under various hormone treatments at different time points. The results showed that the JrAUX2 andJrAUX27 exhibited significantly higher expression levels than the control after treatment with 150 and 200 mg ·L-1 IAA, respectively, reaching 50.67 and 89.95 times of the level of the control. Meanwhile, the JrAUX16 exhibited the most significant downregulation after treatment with 200 mg·L-1 NPA, reaching 89.74 times of that of the control.【Conclusion】This study identified 63 JrAUX/IAA genes and analyzed their gene structure, chromosomal localization, similarity, and expression patterns at various concentrations and stages of walnut development. The genes were found to be unevenly distributed across 16 chromosomes. The subcellular localization analysis revealed that the genes were primarily localized in the cell nucleus. The phylogenetic analysis grouped the AUX/IAA family members into four subfamilies with similar gene structures and conserved motifs. The collinearity analysis revealed two pairs of tandem repeat genes and three pairs of fragment repeat genes within the AUX/IAA family. Through an analysis of the expression patterns of the 63 identified genes combined with quantitative reverse transcription PCR (qRT-PCR) analysis, it was speculated that the JrAUX2 and JrAUX27 might be the key genes influencing lignin synthesis during walnut endocarp development, auxin signaling potentially coordinates lignin biosynthesis through transcriptional control of pathway genes, with differential expression patterns observed across auxin concentration gradients.