Genome-wide identification of GAD genes in aurantioideae and their expression in Poncirus trifoliata plants

【Objective】γ-Aminobutyric acid (GABA), a four-carbon non-protein amino acid, plays crucial roles in plant growth, development, and stress responses. Its primary biosynthesis pathway in plants involves the irreversible decarboxylation of glutamate, catalyzed by glutamate decarboxylase (GAD), a pyridoxal phosphate-dependent enzyme. Suppression of GAD activity is known to reduce endogenous GABA accumulation, impair reactive oxygen species (ROS) scavenging capacity, disrupt hormone interactions, and consequently restrict plant growth. While the GAD gene family has been characterized in various plant species such as cotton, rice, maize, soybean, and sweet orange, a comprehensive genomic analysis within the aurantioideae subfamily remains limited. This study aimed to systematically identify GAD gene family members across 10 aurantioideae species, Atalantia buxfoliata, Citrus clementina, Citrus grandis L. Osbeck‘Wanbaiyou’, Citrus ichangensis, Citrus medica, Citrus reticulata, Citrus sinensis, Fortunella hindsii, Poncirus trifoliata, and Citrus grandis L. Osbeck cv.‘Cupi Majiayou’, and to investigate their evolutionary relationships, gene structures, and expression patterns, particularly in Poncirus trifoliata in response to exogenous GABA.【Methods】GAD protein sequences from Arabidopsis thaliana were used as queries to identify putative GAD members in the 10 target species using TBtools,based on the hidden Markov model profile of the GAD conserved domain. Identifications were subsequently verified using the NCBI Conserved Domain Database. The physicochemical properties of the identified GAD proteins, including molecular weight, theoretical isoelectric point (pI), instability index, and grand average of hydropathicity (GRAVY), were predicted using ExPASy. Subcellular localization predictions were performed with WoLF PSORT. Protein secondary and tertiary structures were predicted using SOPMA and SWISS-MODEL, respectively. Potential phosphorylation sites were identified using NetPhos- 3.1. A phylogenetic tree was constructed with MEGA11, incorporating GAD sequences from the aurantioideae species and other plants. Gene structure, conserved motifs, promoter cis-acting elements, and collinearity relationships were analyzed using TBtools, MEME, PlantCARE, and Advanced Circos, with results visualized accordingly. The tissue- specific expression patterns of GAD genes in Poncirus trifoliata and their transcriptional responses in roots following exogenous GABA treatment were analyzed using quantitative real-time PCR (qRT- PCR).【Results】A total of 21 GAD genes were identified from the genomes of the 10 aurantioideae species. Notably, Poncirus trifoliata possessed three GAD members, while all other species contained two each. Segmental duplication was inferred to be responsible for the additional gene in P. trifoliata. All 21 deduced GAD proteins contained the conserved Glu-decarbox (GAD) domain. Their protein lengths were 494 or 498 amino acids, with molecular weights ranging from 56.194 to 56.294 kD and theoretical pI values between 5.44 and 5.97. With the exception of PtrGAD1 (instability index 40.27), all proteins were predicted to be stable (instability index ＜ 40). The GRAVY values (-0.296 to -0.236) indicated hydrophilic nature, and the aliphatic indices ranged from 85.68 to 88.38. Subcellular localization predictions suggested a cytoplasmic localization for all GAD proteins. Secondary structure analysis revealed a predominance of alphahelices and random coils, with a minor proportion of beta- sheets. Tertiary structure modeling of the PtrGADs indicated high similarity among the three proteins, all potentially forming homomeric hexamers. Additionally, each GAD protein was predicted to contain approximately 40 potential phosphorylation sites. Phylogenetic analysis classified the 21 GAD genes into three distinct subfamilies, each containing members from multiple species. All genes shared a conserved structure of 6 exons and 5 introns, and 10 GAD- specific conserved motifs were identified. Promoter analysis revealed an abundance of hormone-responsive cis-elements. Abscisic acid (ABA) responsive elements were present in all 21 promoters. Methyl jasmonate (MeJA) and auxin (IAA) responsive elements were widely distributed, found in 19 (all except CmGAD2 and PtrGAD3) and 20 (all except CiGAD2) promoters, respectively. Salicylic acid (SA) and Gibberellin (GA) responsive elements were less common, present in only 11 and 13 genes. Various abiotic stress- responsive elements were also identified, suggesting potential roles for GAD genes in hormone signaling and stress adaptation. qRT-PCR analysis demonstrated differential tissue-specific expression of the three PtrGAD genes in P. trifoliata. PtrGAD1 and PtrGAD3 showed the highest expression in stems, whereas PtrGAD2 was predominantly expressed in leaves. Furthermore, exogenous GABA treatment significantly induced the expression of all PtrGADs in roots, but with distinct kinetic patterns: PtrGAD1 and PtrGAD2 exhibited sustained upregulation in roots, while PtrGAD3 exhibited a fluctuating pattern of an initial decrease, followed by an increase and a subsequent decrease. 【Conclusion】The GAD gene family within the aurantioideae subfamily is relatively conserved in evolution. The expansion and potential functional diversification of the family in Poncirus trifoliata, likely via segmental duplication, may contribute to its adaptation to complex regulatory requirements. The distinct tissue- specific expression patterns and differential transcriptional responses to exogenous GABA suggest that individual PtrGAD genes play specialized roles in the growth, development, and GABA-mediated stress responses of Poncirus trifoliata. This study provides a valuable foundation for further functional characterization of GAD genes in citrus and related species.