Genome-Wide Identification and Expression Analysis of the AS2/LOB Transcription Factor Family in Asparagus officinalis

Abstract

Background: AS2/LOB transcription factors are central regulators of plant organ development and stress responses, yet their characteristics in the monocot crop Asparagus officinalis remain uncharacterized. Methods: In this study, we leveraged the A. officinalis genome to perform a genome-wide identification and comprehensive characterization of the AS2/LOB family. We identified 20 AoAS genes (AoAS01–AoAS20) and analyzed their physicochemical properties, chromosomal localization, conserved domains and motifs, phylogenetic relationships, gene structures, cis-regulatory elements, duplication history, syntenic relationships, protein–protein interaction networks and expression profiles. Results: Phylogenetic analysis divided the AoAS proteins into two major clades (Class I and Class II), while chromosomal mapping revealed their uneven distribution across eight chromosomes. Analysis of publicly available RNA-seq data showed that 14 AoAS genes exhibit dynamic expression across four developmental stages of the stem (10, 25, 40 and 60 cm), with AoAS11 and AoAS14 consistently displaying high transcript levels. Under drought stress, 12 AoAS genes showed significant transcriptional changes, with AoAS04 and AoAS14 exhibiting the most pronounced expression responses. Conclusions: Together, these results provide a genome-wide portrait of the AS2/LOB family in asparagus, reveal their potential roles in development and drought response, nominate candidate genes for breeding stress-tolerant cultivars, and offer a useful benchmark for molecular breeding in economically important species including peony (Paeonia lactiflora).

1. Introduction

To build their complex structures, plants rely on master-switch genes that control development. A key group of these genes acts like architectural surveyors, defining the precise boundaries where organs like leaves and flowers will form. This critical group is known as the ASYMMETRIC LEAVES2/LATERAL ORGAN BOUNDARIES (AS2/LOB) gene family. The AS2/LOB family was first identified by enhancer trapping in Arabidopsis thaliana and represents a group of plant-specific transcription factors [1]. AS2/LOB proteins share a conserved ~100–amino-acid domain composed of three hallmark features: (1) a C-motif (CX₂CX₆CX₃C) containing four cysteines essential for DNA binding; (2) a glycine–alanine–serine (GAS) block that contributes to maintaining structural and functional integrity; and (3) a leucine-zipper-like coiled-coil motif with an LX₆LX₃LX₆L spacing that facilitates dimerization [2,3,4,5,6]. Phylogenetic analysis of these conserved domains divides the family into two major classes—Class I (further subdivided into Ia and Ib) and Class II. Class I members possess AS2/LOB domains closely resembling the canonical LOB proteins, whereas Class II members retain the conserved N-terminal region but diverge more extensively outside the core domain [7]. Genome-wide surveys have since cataloged AS2/LOB family members across diverse plant species, including A. thaliana (42 members), barley (Hordeum vulgare, 24), potato (Solanum tuberosum, 43), moso bamboo (Phyllostachys edulis, 55), turnip rape (Brassica rapa, 62), physic nut (Jatropha curcas, 28), and oilseed rape (Brassica napus, 126) [8,9,10,11,12,13,14]. Despite this diversity of identified family members and emerging functional insights, the identity, evolution, and stress-related roles of AS2/LOB genes in asparagus (Asparagus officinalis L.) remain uncharacterized.

Functional divergence within the AS2/LOB family is largely driven by variation in their C-terminal regions, as revealed in multiple species. In A. thaliana, LBD37/38/39 act as conserved repressors of anthocyanin biosynthesis and nitrogen signaling [15]. In moso bamboo, overexpression of PheLBD29 in A. thaliana reduces leaf size and induces abaxial curling while enhancing drought tolerance via increased soluble sugars and decreased malondialdehyde [16]. In lily, LaLBD37 promotes bulb organ primordia initiation through carbohydrate–hormone crosstalk [17]. In chrysanthemum, CmLBD2 regulates sporopollenin biosynthesis by binding the CmACOS5 promoter; its suppression disrupts tapetum degradation and pollen development [18]. In tea (Camellia sinensis), CsLBD37 causes dwarfism, early flowering, and reduced nitrate accumulation when expressed in A. thaliana [19]. Stress-related functional divergence is also evident: MdLBD3 from apple enhances salinity and drought tolerance and accelerates flowering in A. thaliana [20], whereas VvLBD39 from grape acts as a negative regulator, increasing sensitivity to PEG6000/NaCl and reducing drought/salinity tolerance and ABA responsiveness in heterologous systems [21]. These examples underscore both conserved and context-dependent roles of AS2/LOB members in development and stress adaptation.

Asparagus is a perennial herbaceous plant cultivated for over 2000 years as both a vegetable and a medicinal crop. It is rich in bioactive compounds—including saponins, flavonoids, and polysaccharides—and exhibits antioxidant, anticancer, anti-inflammatory, immunomodulatory, hypoglycemic, and hypolipidemic activities [22,23]. With accelerating global climate change and intensifying abiotic stresses such as drought, soil salinization, and high temperature, elucidating the molecular mechanisms of stress response and breeding stress-resilient asparagus has become increasingly urgent. Several gene families, including NAC, AUX/IAA, HSF and bZIP, have been implicated in asparagus abiotic stress responses [24,25,26]. However, despite extensive characterization of AS2/LOB genes in other species and their established links to stress adaptation, their roles in asparagus remain poorly documented.

In this study, we conducted a genome-wide identification and characterization of the AS2/LOB gene family in asparagus, including analyses of physicochemical properties, chromosomal localization, conserved domains and motifs, phylogenetic relationships, gene structures, cis-regulatory elements, duplication history, syntenic relationships, protein–protein interaction networks and expression profiles. We also leveraged publicly available RNA-seq data to profile AoAS gene expression in asparagus stems across four developmental stages and under drought stress. The resulting dataset provides a theoretical framework and resource for cloning and functional studies and offers candidate genes for breeding asparagus cultivars with improved quality and drought tolerance.

2. Materials and Methods

2.1. Identification and Analysis of the Physicochemical Properties of AoAS Members

The genome sequence, annotation file, protein sequences, and coding sequences (CDS) of asparagus used in this study were sourced from the National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/) under the accession number GCF_001876935.1 (accessed on 4 December 2024). The A. thaliana AS2/LOB protein sequences from Araport11 were downloaded from TAIR (https://www.arabidopsis.org/) (accessed on 1 March 2025) and the AS2/LOB protein domain Hidden Markov Model (HMM) PF03195 was obtained from the Pfam database (http://pfam.xfam.org/) (accessed on 1 March 2025). To screen candidate AS2/LOB sequences of asparagus, both HMMER 3.3 and the local BLAST+ 2.15.0 were utilized with the ‘10 × 10⁻⁵’ E-value parameter to search for sequences containing the AS2/LOB protein domain [27,28]. Following this, the identified protein sequences were uploaded to the Conserved Domain Database (CDD) (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) (accessed on 1 March 2025) and SMART tool (http://smart.embl-heidelberg.de) (accessed on 1 March 2025) for sequence alignment and analysis. A total of 20 candidate AS2/LOB gene family members were identified in asparagus. Basic gene information, such as locus, strand, number of mRNA, number of exons, number of CDSs, and gene length, was compiled using TBtools-II [29]. The ExPASy Proteomics Server (https://www.expasy.org/) (accessed on 3 March 2025) was utilized to analyze the amino acid sequences and calculate their lengths, isoelectric points, and molecular weights [30]. Transmembrane helices were examined using TMHMM 2.0 (https://services.healthtech.dtu.dk/services/TMHMM-2.0/) (accessed on 3 March 2025) [31], while subcellular localization was predicted by DeepLoc-2.1 (https://services.healthtech.dtu.dk/services/DeepLoc-2.1/) (accessed on 3 March 2025) [32].

2.2. Structural Analysis and Chromosomal Localization of AoAS Genes

Information on 20 AoAS genes was extracted from the asparagus genome annotation file using TBtools-II, and gene structure diagrams were generated via the GSDS 2.0 online platform (https://gsds.gao-lab.org/) (accessed on 10 March 2025) [33]. The chromosomal distribution of the 20 AoAS genes across 10 chromosomes was visualized using the MG2C V2.1 online platform (http://mg2c.iask.in/mg2c_v2.1/index_cn.html) (accessed on 13 March 2025) [34].

2.3. Phylogenetic Analysis and Conserved Motif Characterization of AoAS Proteins

The amino acid sequences of 20 asparagus and 42 A. thaliana AS2/LOB family members were aligned using the ClustalW method implemented in MEGA-X software (Version 10.2.2) [35]. A phylogenetic tree was constructed using the Neighbor-Joining (NJ) method with a bootstrap value of 1000 and default parameters. Conserved motifs of AoAS proteins were predicted using the MEME online platform (https://meme-suite.org/meme/) (accessed on 12 March 2025) with the following parameters: site distribution set to zero or one occurrence per sequence (zoops), number of motifs to 15, and motif width ranging from 6 to 50 amino acids [36]. Visualization was performed using TBtools-II, employing different colors to represent the 15 conserved motifs.

2.4. Cis-Acting Element Analysis of AoAS Gene Promoter Regions

Based on the CDS of AS2/LOB genes, TBtools-II was employed to align these sequences against the asparagus genome to retrieve the 2000 bp promoter sequences upstream of each gene’s start codon (ATG). Subsequently, the PlantCARE online tool (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) (accessed on 18 March 2025) was utilized to predict cis-acting elements within the AoAS gene promoter regions [37]. The results were visualized using TBtools-II.

2.5. Analysis of Duplication Events and Synteny of AoAS Genes

The duplication events of AoAS genes were predicted using the “One Step MCScanX—Super Fast” plugin in TBtools-II (E-value = 1 × 10⁻³, Number of Blast Hits = 10). The duplication type of each AoAS gene was subsequently classified. Among the 20 AoAS genes, three types of duplication were identified: dispersed, whole-genome duplication (WGD) or segmental, and tandem. The Ka/Ks ratios of tandemly duplicated gene pairs were calculated using the “Simple Ka/Ks Calculator” in TBtools-II based on the Nei-Gojobori method. The duplication patterns of AoAS genes were visualized with TBtools-II.

The genome sequences and annotation files of A. thaliana, Dioscorea zingiberensis, and Solanum lycopersicum were obtained from NCBI, while Panax ginseng data were downloaded from the National Genomics Data Center (https://ngdc.cncb.ac.cn/). The accession numbers were GCA_000001735.2 (accessed on 21 November 2024) for A. thaliana, GCA_014060945.1 (accessed on 14 November 2024) for D. zingiberensis, GCF_036512215.1 (accessed on 23 November 2024) for S. lycopersicum and GWHBEIL00000000.1 (accessed on 14 November 2024) for P. ginseng. Collinearity analysis between AS2/LOB genes in asparagus and these species was predicted using the “One Step MCScanX—Super Fast” plugin in TBtools-II (E-value = 1 × 10⁻³, Number of Blast Hits = 10). Results were visualized through TBtools-II’s graphic interface.

2.6. Analysis of AoAS Protein Interaction Network

A protein–protein interaction analysis was conducted for 20 AoAS proteins using the STRING database (version 12.0; https://cn.string-db.org/) (accessed on 24 March 2025). A. thaliana was employed as the reference organism, with the required confidence score set to medium (0.400) and the false discovery rate (FDR) stringency set to medium (5 percent). These parameters were chosen to balance the inclusion of relevant interactions while minimizing potential false positives.

2.7. Expression Pattern Analysis of AoAS Genes

We retrieved RNA-seq data for asparagus stems at various developmental stages and under drought stress from public databases to analyze the expression profiles of AoAS genes. The transcriptomic data of asparagus stems at different developmental stages was obtained from the NCBI Gene Expression Omnibus (GEO) database under accession number GSE252560 (accessed on 9 April 2025). The samples were collected at four distinct developmental stages, corresponding to stem heights of 10, 25, 40 and 60 cm [38]. Each treatment was conducted with three biological replicates to ensure statistical reliability. The resulting data underwent standard bioinformatic processing to generate fragments per kilobase of transcript per million fragments mapped (FPKM) values as a measure of gene expression. The means of each gene’s expression across the three biological replicates were normalized using the log2(FPKM + 1) transformation to facilitate visual representation.

The RNA-seq data of asparagus (Pacific Early and Jilv3) leaves under control and drought stress conditions were obtained from 12 SRA files (SRX17208360-SRX17208371) in Project PRJNA873275 (accessed on 16 April 2025) from NCBI. In the control group, plants were watered every two days to maintain a soil water content of 75–80%, while the drought treatment group received no watering. Three biological replicates were set for each treatment [39]. Fastq paired-end data containing forward and reverse reads were generated from the SRA files. The quality of the reads was initially assessed using FastQC [40] and MultiQC [41], and adapter sequences and low-quality reads were trimmed using Trimmomatic [42]. The reads were then aligned to the reference genome using HISAT2 [43] to generate BAM files, and FPKM values for each gene were calculated using StringTie [44]. The expression means of each gene across the three biological replicates were normalized using min-max normalization and log2(FPKM + 1) transformation to facilitate visualization.

3. Results

3.1. Identification and Chromosome Localization of AS2/LOB Gene Family Members in Asparagus

A total of 20 AS2/LOB gene family members were identified in asparagus and designated AoAS01–AoAS20 based on their sequential chromosomal localization. The amino acid sequences and physicochemical properties of all 20 AoAS proteins are summarized in Supplementary Tables S1 and S2, respectively, including locus, strand, transcript number, exon count, CDS count and gene length, as well as protein length, number of transmembrane helices, theoretical pI, molecular weight and predicted subcellular localization. AoAS17 (632 bp) and AoAS12 (8138 bp) were the shortest and longest genes, respectively. AoAS08 contained the fewest amino acids (139 aa) and showed the lowest molecular weight (15,625.34 Da), in contrast to AoAS06, which had the highest amino acid count (238 aa) and largest molecular weight (26,151.45 Da). Transcript variants differed in mRNA number (1–3; AoAS11 has 3), exon count (1–3; AoAS06 has 3) and CDS number (1–2). Among these proteins, 9 were acidic (pI < 7.0) and 11 were alkaline (pI > 7.0). All AoAS members exhibited zero transmembrane helices, with subcellular localization predictions exclusively indicating nuclear localization. These features of AoAS family members—nuclear localization, absence of transmembrane helices, and a characteristic bimodal pI distribution—are consistent with those typically observed for plant transcription factors.

The chromosomal localization of the 20 AoAS genes was analyzed (Figure 1). The results showed that these genes are unevenly distributed across 8 out of the 10 chromosomes (Chr02 and Chr09 contain no AoAS genes). The highest gene density was observed on Chr05 and Chr07, each harboring five genes: AoAS07–AoAS11 and AoAS14–AoAS18, respectively. Chr01 contains three genes (AoAS01–AoAS03), whereas Chr04 and Chr06 each contain two genes (AoAS05, AoAS06 and AoAS12, AoAS13, respectively). Chr03, Chr08, and Chr10 each contain a single gene (AoAS04, AoAS19 and AoAS20, respectively). Notably, approximately 70% of the genes were located at the chromosomal termini. Furthermore, AoAS07 and AoAS08 on Chr05 were identified as tandemly duplicated genes, with an intergenic distance of 1559 bp.

3.2. Analysis of the Amino Acid Sequence of the Conserved Domain and Motifs in AoAS Proteins

The conserved characteristics of the AS2/LOB domain in 20 AoAS proteins were analyzed using DNAMAN 6.0 software. Based on the amino acid sequences of A. thaliana AS2/LOB [1], these proteins were classified into three subgroups: Class Ia (containing 17 proteins), Class Ib (containing 1 protein), and Class II (containing 2 proteins), as shown in Figure 2A. The CX₂CX₆CX₃C sequence (where X represents non-conserved residues and subscript numbers indicate the number of non-conserved residues), hereafter referred to as the C-motif, was strictly conserved across all identified asparagus AS2/LOB protein domains. In addition to the C-motif, the leucine zipper-like (LZ-like) sequence was also highly conserved among these proteins. More than half of the proteins contained the following conserved sequences: CXACKXLRRXCX₃C within the C-motif, FX₃HKVFG..ASNVXKXL between the C-motif and the LZ-like sequence (“.” denotes a gap in the amino acid sequence), and YGCX₃I and LQXQ within the LZ-like sequence. Additionally, five amino acid residues (P20, G43, E73, A74 and R77, where the numbers indicate the residue positions) were strictly conserved in the AS2/LOB domains of all 20 AoAS proteins.

An analysis of conserved motifs was conducted on the amino acid sequences of 20 AoAS proteins (Figure 2B), with each motif’s primary sequence detailed in Supplementary Table S3. Fifteen distinct motifs were identified, and their distribution patterns diverged markedly between Class I and Class II members. Class I proteins uniformly adopted a Motif 2–Motif 3–Motif 1 topology, whereas Class II proteins consistently exhibited a Motif 7–Motif 15–Motif 10 configuration. On the basis of sequence features, we infer that Motif 2 and Motif 7 represent the C-motifs of Class I and Class II, respectively, while Motif 1 and Motif 10 correspond to leucine-zipper-like regions in their respective classes. Motif 3 was conserved across all Class I proteins and Motif 4 was predominantly present in the Class Ia subgroup. Several motifs displayed branch specificity: Motif 13 occurred exclusively in AoAS16 and AoAS09; Motif 8 in AoAS16, AoAS19, and AoAS14; Motif 9 in AoAS10, AoAS15, and AoAS05; Motif 14 in AoAS02 and AoAS13; and Motif 5 in AoAS02, AoAS05, and AoAS20. Moreover, proteins within individual phylogenetic clades shared unique motif signatures: Motif 11 appeared only in AoAS10 and AoAS15; Motif 6 only in AoAS07 and AoAS08; and Motif 15 only in AoAS12 and AoAS18.

3.3. Phylogenetic Analysis of AS2/LOB Proteins

Based on the phylogenetic trees of AS2/LOB proteins in A. thaliana and physic nut [1,8], we constructed a phylogenetic tree using 42 A. thaliana and 20 asparagus AS2/LOB proteins (Figure 3). These 62 proteins were grouped into two major clades, with Class I further divided into two subgroups. The Class Ia subgroup consisted of 29 AS2/LOB proteins in A. thaliana and 17 AoAS proteins (AoAS01–AoAS11, AoAS14–AoAS17, AoAS19 and AoAS20), while the Class Ib subgroup included 7 AS2/LOB proteins in A. thaliana and 1 AoAS protein (AoAS13). The Class II subgroup comprised 6 AS2/LOB proteins in A. thaliana and 2 AoAS proteins (AoAS12 and AoAS18). Furthermore, a total of seven AoAS proteins formed sister pairs with AS2/LOB proteins in A. thaliana. These sister pairs included ASL32 and AoAS13, ASL2 and AoAS06, ASL4 and AoAS11, ASL24 and AoAS09, ASL18 and AoAS17, ASL11 and AoAS15, and ASL6 and AoAS04.

3.4. Analysis of the Structure of AoAS Genes

The intron-exon structure of the 20 AoAS genes is illustrated in Figure 4. Most members encode two CDSs, while AoAS11 and AoAS13 have only one. The majority of the genes have a single intron, except for AoAS06, which contains two, and AoAS13, which has none. Most introns are relatively short, whereas those of AoAS12 and AoAS08 exceed 3 kb. The 5′ and 3′ untranslated regions (UTRs) are generally short across most members, except for AoAS03, which has an extended 3′ UTR region.

3.5. Analysis of the Promoter Elements of AoAS Genes

The analysis of cis-acting elements within the 2000 bp promoter sequences upstream of the start codon of AoAS genes is presented in Supplementary Table S4 and Figure 5. These elements are categorized into three modules based on their functions: environmental signal responsiveness, phytohormone responsiveness, and tissue and development-specific regulation. Each module encompasses several subcategories. Within the environmental signal responsiveness module, elements associated with light responsiveness—such as ATCT-motif, Box 4, G-Box, GATA-motif, LAMP-element, MRE, and TCT-motif—are present in every AoAS gene and occur in substantial numbers. Additionally, elements related to anaerobic induction (ARE) are commonly found across most AoAS genes, suggesting that this gene family may be regulated by light signals and could have specific expression patterns under hypoxic conditions. This observation aligns with cis-element analyses conducted in B. rapa [9]. Furthermore, drought-responsive MYB site elements (MBS) also appear in more than half of the genes. In the phytohormone responsiveness module, gibberellin-responsive elements (TATC-box, P-box, GARE-motif) and MeJA-related elements (TGACG-motif) are present in over half of the genes, indicating that AoAS genes may play roles in plant defense mechanisms or responses to injury. The tissue and development-specific regulation module contains fewer distributed genes compared to the other two modules, with most subcategories scattered across different genes. Notably, flavonoid biosynthetic-related elements (MBSI) are exclusively found in AoAS13, and zein metabolism regulation-related elements (O2-site) are abundantly present in AoAS18, with up to eight occurrences.

3.6. Duplication Event and Synteny Analysis of AoAS Genes

Duplication events among the 20 AoAS genes were systematically analyzed, as presented in Supplementary Table S5 and Figure 6A. Among these, only one tandemly duplicated gene pair was identified: AoAS07 and AoAS08, both located on Chr05. The non-synonymous to synonymous substitution ratio (Ka/Ks) for this gene pair was calculated to be 0.354513. Since this value is less than 1, the duplicated genes are likely under purifying (negative) selection, indicating that functional constraint was retained post-duplication [45]. Moreover, the relatively low Ks value (0.333101) implies that this tandem duplication event likely occurred in a relatively recent evolutionary period [46]. Five pairs of WGD or segmental duplicated genes were identified: AoAS02–AoAS05, AoAS02–AoAS17, AoAS05–AoAS20, AoAS10–AoAS15 and AoAS16–AoAS19. These tandem and WGD or segmental duplication events were distributed across six chromosomes (Chr01, Chr04, Chr05, Chr07, Chr08 and Chr10). The remaining 10 genes (AoAS01, AoAS03, AoAS04, AoAS06, AoAS09, AoAS11, AoAS12, AoAS13, AoAS14 and AoAS18) were classified as dispersed duplicates.

Further analysis of the syntenic relationships of AS2/LOB genes between asparagus and A. thaliana, D. zingiberensis, P. ginseng and S. lycopersicum was performed to identify homologous gene pairs (Figure 6B). A total of 20 AoAS genes exhibited collinearity with AS2/LOB genes from the four other species: 3 A. thaliana genes with 4 AoAS genes, 18 D. zingiberensis genes with 12 AoAS genes, 22 P. ginseng genes with 9 AoAS genes and 7 S. lycopersicum genes with 7 AoAS genes. These findings suggest that asparagus shares the highest degree of homology with D. zingiberensis and the lowest with A. thaliana. In addition, three conserved AS2/LOB genes were found to be present across all five species, corresponding to AoAS14, AoAS18 and AoAS19 in asparagus.

3.7. Analysis of the Interaction of AoAS Proteins

Using A. thaliana as a model species, the protein–protein interaction (PPI) network of AoAS proteins was predicted via the STRING database, as shown in Figure 7 and Supplementary Table S6. Figure 7A illustrates that a total of 13 potential protein interaction pairs were identified. Among them, AoAS18 appeared to be the central hub, interacting with five other proteins: AoAS01, AoAS07, AoAS04, AoAS19, and AoAS11. AoAS12 also showed extensive interactions with four proteins: AoAS06, AoAS11, AoAS13, and AoAS19. Notably, the interaction between AoAS13 and AoAS12 exhibited the highest confidence, with a combined score of 0.791. In contrast, AoAS05, AoAS14, AoAS15, and AoAS16 were not predicted to interact with any other proteins under the chosen cutoff (combined score ≥ 0.4).

Based on the phylogenetic analysis of AS2/LOB proteins (Figure 7B), the functions of seven asparagus proteins homologous to their A. thaliana counterparts were predicted. AoAS04 interacts with WOX4, WOX14, TDR and CLE41/44, suggesting its role in regulating cell division and differentiation in vascular cambium [47,48,49]. AoAS06 interacts with transcription factors KNAT6, RAX3, NAC077 and NFYB7, implicating its involvement in organ boundary establishment, meristem maintenance, and morphogenesis by coordinating transcriptional activities [50,51]. AoAS09 interacts with auxin signaling components (ARF7/19, IAA14), cell wall modifiers (EXPA14/EXPA17) and meristem regulators (PLT3/PLT5), indicating its potential role in coordinating auxin-mediated vascular development [52,53,54]. AoAS11 interacts with KAN1, KAN2, ARF3, AUX1, AP2 and BZIP8, suggesting its participation in shoot apical meristem maintenance, organ boundary formation, vascular differentiation, and auxin signal integration [55,56]; AoAS13 establishes robust interactions with multiple LBD family proteins (such as LBD26, LBD39, and LBD32), potentially contributing to the development of lateral organ boundaries. AoAS15 interacts with NAC075/NAC030 and GATA5/GATA12, potentially regulating stress responses, cytoskeleton organization and cell morphology [24,57]. AoAS17 interacts with root development-associated proteins WOX11/12, ARF7/19 and IAA14, positioning it as a key node linking auxin signaling and the WOX regulatory network [58,59]. Collectively, these AoAS proteins orchestrate critical physiological processes in asparagus, including vascular development, organ boundary patterning, auxin signaling, and stress response, through interaction networks with diverse regulatory partners.

3.8. Analysis of the Expression Pattern of AoAS Genes in the Stems of Asparagus at Different Growth Stages and Under Drought Stress

To investigate the temporal expression characteristics and potential functions of AoAS genes, we analyzed transcriptome data derived from the stems of asparagus at different developmental stages (stem heights of 10, 25, 40 and 60 cm) [38]. The results revealed substantial variation in the expression of 14 AoAS family members across stem samples of varying heights (Figure 8A; Supplementary Table S7A). Some genes, such as AoAS05, AoAS08, AoAS12, AoAS13, AoAS16 and AoAS20, were barely expressed at any stage, whereas others were active across multiple growth stages. AoAS11 and AoAS14 exhibited consistently high expression levels in all tissues, suggesting their involvement in broad developmental and regulatory processes. AoAS14 displayed the highest overall expression, with a gradual decrease over time, indicating a potential role in early-stage stem development. Similarly, AoAS19 showed progressively reduced expression, ultimately becoming undetectable at later stages. In contrast, AoAS03 and AoAS10 displayed a marked increase in expression over time, implying potential involvement in stem maturation or secondary metabolite accumulation. In addition, AoAS06 was exclusively expressed at the 10 cm stage, while AoAS17 was detected only at 40 cm.

To investigate the expression characteristics of AoAS genes under drought stress, we analyzed RNA-seq data focusing on 20 AoAS genes in the leaves of two asparagus cultivars (Jilv3 and Pacific Early) under control and drought stress conditions [39]. The results revealed that a total of 14 AoAS genes were expressed under drought stress, while six genes (AoAS02, AoAS08, AoAS12, AoAS13, AoAS16 and AoAS20) were not detected in either cultivar under control and drought stress conditions (Figure 8B and Supplementary Table S7B). Conversely, seven genes (AoAS03, AoAS04, AoAS10, AoAS11, AoAS14, AoAS15 and AoAS18) were expressed in both cultivars under control and drought stress conditions, with AoAS04 and AoAS14 exhibiting relatively higher expression levels. Notably, AoAS03, AoAS05 and AoAS06 were upregulated in both cultivars under drought stress, whereas AoAS04, AoAS10, AoAS14 and AoAS18 were downregulated. These results show that approximately 60% of AoAS genes respond to drought stress, with cultivar-specific expression differences, with some family members showing pronounced up- or down-regulation under drought conditions.

4. Discussion

In this study, we identified a total of 20 AoAS genes in the genome of asparagus (Figure 1 and Supplementary Table S2), which is substantially fewer than the numbers reported for the AS2/LOB family in several other plant species—for example, A. thaliana (42), B. rapa (62), P. edulis (55), S. tuberosum (43), and B. napus (126). Such variation in family size likely reflects differences in evolutionary history and the extent of genome duplication events across lineages. Gene family expansion is typically driven by duplication mechanisms including WGD, segmental duplication, and tandem duplication, whose frequency and retention vary among taxa [14,45]. Indeed, numerous studies have attributed the expansion of AS2/LOB families in grasses, rice, maize, and Arabidopsis to such duplication events [60]. In contrast, although asparagus experienced ancient WGD events (Asparagales-α and Asparagales-β) early in its evolution, no recent polyploidization has been documented [61,62]. The absence of recent genome duplications may have constrained expansion of the AS2/LOB family in asparagus, resulting in its relatively small complement of AoAS genes.

Consistent with observations in other plants, nearly all AoAS genes exhibit a conserved structural organization of two coding sequence regions separated by a single short intron, with exceptions such as AoAS06 (which contains two introns) and AoAS12/AoAS08 (which harbor unusually long introns) (Figure 4) [4,8,9,12,13,63]. The streamlined exon–intron architecture of AS2/LOB genes is thought to enable rapid transcriptional responses to developmental and environmental cues by facilitating efficient transcription and splicing [64,65]. The particularly long introns of AoAS12 and AoAS08, coupled with their clustering in promoter element analyses, suggest that they may harbor additional cis-regulatory motifs influencing expression dynamics [66]. In contrast, the presence of two introns in AoAS06 raises the possibility of alternative splicing, potentially generating isoforms with tissue-specific or stress-responsive roles and adding regulatory plasticity [67].

Promoter analysis further revealed that AoAS genes possess a multilayered cis-regulatory architecture integrating environmental (light, oxygen, drought), hormonal (gibberellin, methyl jasmonate), and developmental signals (Figure 5; Supplementary Table S4), consistent with the complexity required for fine-tuning nitrogen metabolism and environmental adaptation [68]. The unique occurrence of the MBSI element in AoAS13 and the enrichment of O2-site elements in AoAS18 point toward potential subfunctionalization within the family, whereby paralogs acquire distinct regulatory features to specialize in particular developmental contexts or organs [69]. Dissecting these individual cis-element contributions will facilitate rational design of synthetic promoters for precise spatial–temporal gene expression, as successfully implemented in crops such as rice and maize [25,70,71,72].

Duplication pattern analysis indicated that 50% of AoAS genes are derived from dispersed duplication, whereas only one tandemly duplicated pair (AoAS07/AoAS08) was identified (Figure 6A; Supplementary Table S5). The asparagus genome is rich in TEs, comprising approximately 53% of its content, with LTR retrotransposons predominant [62,73]. High-density TE landscapes can suppress non-allelic homologous recombination (NAHR), thereby limiting the formation of tandem duplicates and favoring dispersed retention [74,75,76,77]. The low Ka/Ks ratio (0.354513) of the AoAS07/AoAS08 pair suggests strong purifying selection preserving their function post-duplication [78,79]. More broadly, most angiosperm AS2/LOB genes similarly originate from WGDs and dispersed duplications, with tandem clusters being rare [80]. Examples include limited tandem duplication in A. thaliana (three pairs among 42 members), B. rapa (five among 62), and P. edulis (two among 55), whereas B. napus—despite harboring 126 AS2/LOB genes—lacks detectable tandem duplicates [9,13,14,81]. This prevalent dispersed retention may reflect evolutionary constraints to minimize functional redundancy or expression interference, as spatial separation (different chromosomes) enables independent regulation and reduces genomic instability associated with tandem arrays [82,83].

Comparative synteny analysis further clarified evolutionary relationships: asparagus shares the most collinear AS2/LOB gene pairs with the monocot D. zingiberensis and the fewest with the eudicot A. thaliana (Figure 6B), consistent with broader angiosperm phylogenetic relationships [84,85]. Notably, both asparagus and D. zingiberensis predominantly accumulate steroidal saponins, whereas P. ginseng is enriched in triterpenoid saponins and S. lycopersicum and A. thaliana lack steroidal saponins [86,87,88,89,90]. The congruence between metabolic profiles and AS2/LOB collinearity suggests potential involvement of conserved family members in secondary metabolite regulation. Three AoAS genes (AoAS14, AoAS18 and AoAS19) are conserved across all five species, implying preservation of fundamental developmental roles. Phylogenetic placement of these genes alongside their respective orthologs—AoAS14 with ASL7/ASL8, AoAS18 with ASL39/ASL41, and AoAS19 with ASL5—supports functional conservation, predicting roles in vascular cambium proliferation and secondary growth (AoAS14), nitrate responsiveness and anthocyanin regulation (AoAS18), and lateral organ development (AoAS19) [91].

Protein–protein interaction predictions revealed that AoAS proteins assemble into a multilayered, feedback-regulated network coordinating key physiological processes, including vascular development, organ boundary specification, integration of auxin and other hormone signaling, and stress adaptation (Figure 7). This integrative regulatory framework underscores the centrality of the AS2/LOB family in transducing endogenous and environmental cues to shape growth and differentiation. Future experimental validation—such as yeast two-hybrid assays and targeted gene editing—will be instrumental in dissecting specific interaction functions and leveraging these insights for precision improvement of asparagus traits.

Spatiotemporal expression profiling demonstrated that AoAS genes exhibit pronounced developmental stage-specific dynamics (Figure 8A), in line with observations in other species [4,8,11,12,92,93]. AoAS11 and AoAS14 are consistently highly expressed across stem samples, suggesting foundational roles in cambial maintenance and xylem differentiation. AoAS14′s high early expression followed by decline implies a pivotal function in early stem development that is modulated as growth proceeds. By contrast, the upregulation of AoAS03 and AoAS10 with increasing stem length suggests involvement in later developmental processes such as secondary growth or cell wall and metabolite biosynthesis. Under drought stress, roughly 60% of AoAS genes respond with differential expression (Figure 8B), with some (AoAS03, AoAS05, AoAS06) induced—potentially contributing to antioxidant defense, osmotic adjustment, or cell wall remodeling—and others (AoAS04, AoAS10, AoAS14, AoAS18) repressed, reflecting a trade-off between growth and stress resilience. Similar downregulation of developmental AS2/LOB genes under stress has been reported in other systems [8,9].

In summary, the AoAS gene family is tightly integrated into the regulatory networks governing organ development and environmental adaptation in asparagus. Its conserved structural features, clarified evolutionary trajectories, and diversified expression responses make it a robust set of candidates for future functional validation and guiding molecular breeding aimed at improving asparagus growth and stress tolerance. Additionally, for high-value, ultra-large-genome crops such as P. lactiflora and Paris polyphylla, whose genomic resources remain nascent, the asparagus-based AS2/LOB gene-family paradigm provides a tractable route to systematically identify and characterize key families [94,95]. Analyses of membership, gene structure, cis-regulatory elements, duplication/expansion and expression can elucidate the genetic architecture of key traits and furnish candidate targets and a theoretical basis for targeted molecular breeding.

5. Conclusions

We performed a comprehensive genome-wide identification and characterization of the AS2/LOB transcription factor family in asparagus and identified 20 AoAS genes that cluster into Class I and Class II subfamilies. These genes were systematically evaluated across multiple dimensions, including physicochemical properties, chromosomal localization, conserved domains and motifs, phylogenetic relationships, gene structures, cis-regulatory elements, duplication history, syntenic relationships, protein–protein interaction networks and expression profiles. Although the AoAS genes share conserved structural features, their expression profiles are highly variable, implying functional diversification in organ development, environmental adaptation and tissue-specific regulation.

By providing the first genome-wide catalogue and expression atlas of AS2/LOB genes in A. officinalis, this work fills an important gap in the functional genomics of this economically important perennial vegetable. The integrated structural and expression evidence highlights specific AoAS members as promising regulators of spear/stem development and drought response, thereby offering testable candidates for future functional studies. In turn, these findings deliver a foundational resource and concrete gene targets for molecular breeding of stress-resilient asparagus and may also inform the improvement of other high-value crops such as peony through comparative and translational genomics.