Genomic and Functional Analysis of the ALOG Gene Family in Dioscorea alata

Abstract

The ALOG (Arabidopsis LIGHT-DEPENDENT SHORT HYPOCOTYLS 1 (LSH1) and Oryza G1) family play crucial regulatory roles in plant growth and development, spanning both vegetative and reproductive growth. This study presents a comprehensive genomic and functional analysis of the ALOG family in greater yam (Dioscorea alata). Ten non-redundant DaALOG genes were identified and classified into two classes (I and II) based on phylogenetic analysis. These classes share a common origin, and family expansion was primarily driven by segmental duplication events. Comparative genomics across 15 plant species revealed widespread, lineage-specific divergence in ALOG gene family size and composition. Expression profiling highlighted several DaALOG genes, particularly DaALOG1, DaALOG3, and DaALOG6A, with significant upregulation in tuber and bulbil tissues, suggesting a potential role in storage organ development. Co-expression network analysis, coupled with yeast one-hybrid assays, indicated that DaALOG3 likely regulates key genes involved in starch biosynthesis. Subcellular localization confirmed the nuclear predominance of DaALOG proteins. Furthermore, functional validation in Arabidopsis demonstrated that overexpression of DaALOG1 leads to pronounced developmental alterations, including irregular leaf morphology and floral organ abnormalities (such as extra stamens and petals). Collectively, our findings establish the DaALOG gene family as an important regulator in greater yam, linking specific members to both vegetative architecture and storage organ development.

1. Introduction

The ALOG (Arabidopsis LIGHT-DEPENDENT SHORT HYPOCOTYLS 1 (LSH1) and Oryza G1) family is a plant-specific gene family whose members contain a conserved ALOG domain (DUF640). These genes were initially described in Arabidopsis thaliana, which harbors ten members known as LSH genes [1]. ALOG genes play critical roles in the development of various plant organs (meristems, inflorescences, floral organs, and nodules) from bryophytes to higher flowering plants [2,3,4]. In sorghum, the ALOG-domain transcription factor gene DOMINANT AWN INHIBITOR (DAI) represses awn elongation by downregulating expression of associated developmental genes [5]. ALOG genes in rice orchestrate the development of an array of structures, from the panicle to several floral organs [6,7]. In Lotus japonicus, LjALOG1 has been identified as a novel positive regulator of nodulation, a function potentially mediated by suppressing the autoregulation of nodulation (AON) pathway [8]. Furthermore, in Medicago truncatula, the ALOG genes LSH1 and LSH2 function to regulate nodule organogenesis, directing both its initiation and subsequent development [9]. In summary, ALOG proteins serve as fundamental, evolutionarily conserved regulators of plant development. Further characterization of ALOG genes across plant species may therefore elucidate diverse mechanisms underlying plant organ development.

ALOG genes function as central regulators in plant development, modulating various biological processes through their integration into key transcriptional networks [10]. In Arabidopsis, the ALOG family genes AtLSH3 and AtLSH4 execute a boundary-establishing function downstream of CUP-SHAPED COTYLEDON1 (CUC1), thereby integrating into the gene regulatory network that coordinates shoot apical meristem development and organ patterning [11]. In Torenia fournieri, TfALOG3 protein physically interacts with TfBOP2 protein (BLADE-ON-PETIOLE protein2) to form a BOP-ALOG complex, thereby regulating corolla differentiation [12]. The formation of BOP-ALOG complexes has also been observed in Arabidopsis, legumes, and tomato, where it functions to regulate plant development [13,14,15,16]. Moreover, The ALOG domain is characterized as a DNA-binding module, which mediates its regulatory function by recognizing conserved promoter sequences in target genes [5,16,17]. As ALOG genes influence plant development by regulating downstream genes and interacting with partner proteins, this understanding provides valuable insights for elucidating their function in other crop species, like greater yam (D. alata).

The availability of high-quality genome sequences enables a comprehensive dissection of the ALOG gene family and the identification of key members involved in specific developmental control. Among the twenty TtALOG genes identified in tetraploid wheat, TtALOG2-1A was functionally characterized to promote branched spike development via the regulation of key TtMADS genes [18]. Research on the Petunia genome has identified 11 ALOG genes, among which PhLSH7a and PhLSH7b play important roles in both vegetative and reproductive development, particularly in fruit development [19]. In rice, 14 ALOG genes (long sterile lemma1/OsG1-likes, OsG1/G1Ls), which expanded via segmental duplication, play roles in inflorescence development and stem elongation [20]. The evolutionary analysis of the ALOG gene family in Solanaceae, based on 648 ALOG genes from 77 species, reveals that it underwent recurrent duplication and functional diversification during angiosperm evolution [21]. Genome-wide analyses of ALOG genes in Rosa, cotton, and Torenia fournieri further demonstrate their crucial roles in plant development [22,23,24]. Thus, a full-genome characterization of the ALOG family enables the translation of functional insights from model plants toward mining key ALOG genes for specific traits in crops.

D. alata (greater yam) is among the most widely cultivated yam species, providing food security for millions. It functions as both a primary food source and a cash crop in regions such as the yam belt of West Africa, where it also holds significant social, religious, and cultural importance [25,26,27]. Recently, a complete genome sequence—including a telomere-to-telomere (T2T) assembly—has been generated for greater yam [28]. Multiple transcriptome datasets were generated for greater yam, providing important information for gene characterization and candidate gene selection [29,30]. These genomic and transcriptomic resources establish a solid foundation for investigating gene families such as ALOG and elucidating their functional roles. In this study, we conducted a systematic characterization of the ALOG gene family in greater yam, including analyses of gene structure, evolution, and expression patterns. We identified five DaALOG genes with high expression in tuber tissues, suggesting possible functional redundancy related to tuber development. Through co-expression analysis and yeast one hybrid assays, DaALOG3 was implicated in the starch synthesis pathway. Furthermore, overexpression of DaALOG1 in Arabidopsis altered floral development. Together, these findings provide a foundation for future functional studies of the ALOG gene family in greater yam.

2. Results

2.1. Gene Characteristics of the ALOG Gene Family (DaALOGs) in Greater Yam

To identify ALOG genes in greater yam, a hidden Markov model (HMM) search was performed against the greater yam protein database using the conserved DUF640 domain as a query. Candidate genes were further validated by confirming the presence of the ALOG domain in the Pfam database. This process identified ten non-redundant DaALOG proteins with complete domains, which were named based on their highest similarity to Arabidopsis LSH homologs (Supplementary Table S1). According to the evolutionary tree DaALOG can be classified in two classes (Figure 1A).

Phylogenetic analysis revealed a closer evolutionary relationship between Class I and Class II, which was supported by higher similarity in gene structure and conserved protein domains (Figure 1A). In Class II, all members lacked introns in the 5′ untranslated region (5′ UTR). In contrast, three members of Class I (DaALOG4A, DaALOG1, and DaALOG3) contained introns within the 5′ UTR, while DaALOG2 (Class I) and DaALOG5A (Class II) each retained an intron in their respective 3′ untranslated regions (3′ UTR) (Figure 1B). Analysis of conserved protein motifs using MEME showed that Class II contained two to four motifs, whereas corresponding motifs in Class I appeared degenerate (Figure 1C). All DaALOG proteins shared a conserved ALOG domain, which comprises five motifs of varying conservation (Figure 1C,D). This domain is predicted to possess DNA-binding capacity [16].

2.2. The Evolutionary Feature of the ALOG Gene Family

The ALOG family has been subgrouped into three classes in Arabidopsis [20]. The phylogenetic tree constructed using DaALOGs and AtLSHs demonstrated that the gene members in the two species shared the class I and class II subfamily, but without class III in greater yam. The genes from each species clustered together within each class, indicating that the genes from Arabidopsis and greater yam in each class share a common origin but have independently expanded in their respective species (Figure 2A).

Analysis of the expansion of the DaALOG genes revealed that genomic duplication served as the primary driver for the increase in gene copy number (Figure 2B). Despite the family comprising only ten members, segmental duplication events contributed to the expansion within both Class I and Class II. These events included a duplication generating DaALOG4B/4C, a triplication producing DaALOG5A/6A/6B, and a tetraplication giving rise to DaALOG1/2/3/4. Furthermore, homologous segments were identified both within and between classes—such as AtLSH1/2/4 in Class I, AtLSH5/6 in Class II, and AtLSH2/6/8/10 across Classes I, II, and III—suggesting that all three classes originated from a common ancestral gene (Figure 2C).

When examining homologous segments containing DaALOGs or AtALOGs between the two species, pairs of genes from same Class were identified (Figure 2D,E). For tetra segments containing Class I members in greater yam, homologous segments containing AtLSH2/4 were detected, but for other ALOG genes, different Classes containing LSH were detected. This may result from the Arabidopsis segmental duplication, which had the same ancenstor LSH genes for different Classes.

The comparison of ALOG genes across 15 species, including the basal extant flowering plant Amborella trichopoda (Atr), six dicot species, and eight monocot species, revealed a widespread divergence in ALOG gene expansions among these species (Figure 3, middle table). Using the same methodology applied to D. alata, we identified the ALOG gene members from D. alata and the other fourteen plant species, with their gene ID detailed in Table S1. A. trichopoda (Atr) was considered the ancestral status for both monocot and dicot species, with the fewest ALOG genes, four, while it still had two genes that were derived from segmental duplication. Among the other 14 species, Ananas comosus had the lowest gene number and fewest gene derived from duplication events, and Musa acuminata (Mac) having the top number (29) may result from the two WGDs (denoted as α and β) [31]. For Malus domestica, Arabidopsis, and Cocos nucifera, all ALOG genes were detected in duplicated genomic segments, while eight species had more than half of their ALOGs derived from genomic duplication, except for A. comosus (2 out of 7), Citrus sinensis (4 out of 9), and Phoenix dactylifera (5 out of 12).

Further phylogenetic analysis of all ALOG genes across the 15 species revealed a clustering pattern consistent with the monocot–dicot divergence (Figure 3, right and Figure S2). The two A. trichopoda ALOG genes were recovered in mixed monocot–dicot clades, indicating that they may share a common evolutionary origin with genes from both monocot and dicot lineages. Meanwhile, the other two A. trichopoda ALOGs grouped specifically with dicot ALOG members. Compared with the basal extant angiosperm A. trichopoda, multiple divergence events have occurred within the ALOG gene family. Two monocot-specific clades were identified, with DaALOGs from Class II forming one distinct group, whereas DaALOGs from Class I do not cluster together. Additionally, dicot ALOGs separated into three subgroups: Class I members were divided into two distinct groups, and one subgroup consisted solely of Class III members.

2.3. The Expression Profile of DaALOG Genes and Predictions of Their Target Genes

Analysis of the great yam transcriptome across leaf, stem, inflorescence, tuber, and bulbil tissues revealed that half of the DaALOG genes exhibited relatively higher expression in tuber or bulbil tissues (Figure 4). Specifically, DaALOG1 and DaALOG3 (Class I) showed high expression in both tuber and bulbil, while DaALOG6A (Class II) was particularly highly expressed in tuber. We further performed RT-qPCR on nine DaALOG genes (excluding DaALOG4B due to its low FPKM) in leaf, stem, and tuber tissues (Figure 4B). The results were consistent with the transcriptome data, confirming that DaALOG1, DaALOG3, DaALOG5A, and DaALOG6A were significantly upregulated in tubers. This suggests their potential involvement in tuber development, with DaALOG1 showing the most pronounced expression (Figure 4B).

To explore potential regulatory targets of DaALOG, we performed Weighted Gene Co-expression Network Analysis (WGCNA, v1.73) to identify co-expressed gene networks for the DaALOG genes. A distinct module containing DaALOG3 and DaALOG2 was identified (Figure 5A). This module also included genes with relevant biological functions, such as CLV1 (CLAVATA1), involved in meristem maintenance; GOXL4 (galactose oxidase) and an extensin gene related to cell expansion; as well as SS3 (starch synthase 3) and D12UGT (UDP-Glycosyltransferase) within the starch biosynthesis pathway (Figure 5A).

Furthermore, from the co-expressed genes, we selected those whose promoter regions contained the putative ALOG-binding motif—YACTGTW (TAGTTTACTGTTGACGT) [16]. These candidate target genes included DaD11UGP (UDP-Glycosyltransferase 2), DaD16PGM (phosphoglucomutase), DaD14Ald (Aldolase), DaD8ISA (isoamylase), DaD15GPT (glucose 6-phosphate), DaD7DBE (debranching enzyme 1), DaD20APL (ADPGLC-PPase large subunit), and DaSUS (sucrose synthase) (Figure 5A). Yeast one-hybrid (Y1H) assays confirmed that DaALOG3 positively interacts with the promoter regions of these selected genes (Figure 5B), indicating that they are likely direct regulatory targets of DaALOG transcription factors.

2.4. Subcellular Location and Function of DaALOGs

ALOG genes encode transcription factors that are primarily expected to function in the nucleus. To determine the subcellular localization of DaALOG proteins, we transiently expressed DaALOG-GFP fusion constructs in tobacco epidermal cells. GFP signals were compared with those of the nuclear marker OsGhd7-RFP. All DaALOG localized to the nucleus, while DaALOG1, DaALOG3, and DaALOG4A also exhibited cytoplasmic distribution (Figure 6). These results indicate that DaALOGs largely retain conserved nuclear localization.

To validate the function of DaALOG1, transgenic Arabidopsis lines overexpressing DaALOG1 (OE-DaALOG1) were generated (Figure 7). In the T₂ generation, homozygous OE lines driven by the 35S promoter displayed distinct developmental abnormalities. These included irregular leaf morphology, the production of extra stamens, and petals that were either five in number or malformed (Figure 7 and Figure S1). In summary, these results demonstrate that DaALOG1 plays a significant regulatory role in both the vegetative and reproductive phases of plant development.

3. Discussion

ALOG/LSH proteins are evolutionarily conserved transcriptional regulators essential for diverse developmental processes—from meristem identity and organogenesis to stress responses—across land plants; these proteins call for deeper investigation into their molecular mechanisms [33]. In this study, we systematically identified 10 ALOG genes in Dioscorea alata, which were phylogenetically classified into two distinct subfamilies. Members within each subfamily appear to have originated primarily from segmental duplication events and exhibit pronounced conservation in gene structure, protein domain organization, predicted DNA-binding motifs, and expression profiles. Notably, several DaALOG genes displayed elevated expression in tuber and bulbil tissues. Further investigation through co-expression network analysis and yeast one-hybrid assays demonstrated that DaALOG3 directly binds to the promoters of key starch biosynthetic genes, supporting its involvement in starch metabolism regulation. Collectively, this work functionally links a conserved developmental transcription factor family to a critical agronomic trait in an understudied crop species.

3.1. Lineage-Specific Expansion of the ALOG Gene Family Across Angiosperms

Our comparative analysis across 15 angiosperm species reveals notable variation in ALOG gene family size, reflecting distinct evolutionary trajectories following the divergence of monocots and dicots (Figure 3 and Figure S2). The basal extant angiosperm A. trichopoda, which was split before monocot–other dicot divergence, possesses only four ALOG genes. The Amborella genome uniquely exhibits an ancient whole-genome duplication that predates angiosperm diversification but lacks lineage-specific polyploidization events [34]. This suggests that the ancestral core repertoire likely consisted of a limited number of copies, possibly three, before undergoing lineage-specific expansions. In contrast, the examined monocot and dicot species display significantly larger ALOG gene sets, ranging from a minimum of seven to eight copies to substantially expanded families containing 18 to 29 members, as observed in bananas (M. acuminata). This pattern indicates that the ALOG gene family has undergone clear but moderate copy number expansion during angiosperm evolution.

The increase in gene number is evident and likely driven largely by segmental and whole-genome duplications; the ALOG family has not proliferated into an extensively large transcription factor family compared to some other major TF families. Genome-wide analysis of ALOGs in other species not included in this study also showed the same thing, such as in wheat, petunia, cotton, Rosa, and Solanaceae species [18,19,21,22,23]. This moderate expansion may reflect a balance between functional diversification and selective constraints, maintaining core regulatory roles in development while allowing for nuanced, lineage-specific adaptations in plant architecture and organ development.

3.2. Functional Redundancy of ALOG Genes

Our findings suggest that functional redundancy may exist within the DaALOG gene family, particularly among members within the same phylogenetic class. This redundancy is likely rooted in their evolutionary origin, as genomic analysis revealed that the expansion of the DaALOG family was primarily driven by segmental duplication events. Duplicated genes often retain overlapping expression patterns and similar biochemical functions, especially when the duplication is recent and the coding sequences remain highly conserved [35]. In this study, DaALOG genes belonging to the same class not only share closely related protein sequences and conserved DNA-binding motifs but also exhibit notably similar expression profiles across different tissues. Such parallel expression supports the possibility that these genes may perform overlapping or partially redundant roles in regulating development.

In Arabidopsis, studies suggest that ALOG genes exhibit functional redundancy, as knockout or RNA interference (RNAi) of individual genes often fails to produce discernible phenotypic defects in plants [11]. The evolutionary analysis of ALOGs in this study reveals that these genes are highly conserved across different plant species, with their chromosomal regions exhibiting significant collinearity in multiple species. While redundancy can provide genetic robustness, it may also obscure the phenotypic impact of single-gene mutations. Future studies employing higher-order mutants, such as those generated through multiplex gene editing, will be essential to dissect the extent of functional overlap among DaALOG members and to clarify whether sub functionalization or neofunctionalization has occurred following duplication.

3.3. Conserved and Lineage-Specific Regulatory Mechanisms of ALOG Transcription Factors

All DaALOG proteins predominantly localize to the nucleus, consistent with their predicted role as transcription factors and supporting their potential function in regulating gene expression. The mechanism of action of ALOG transcription factors appears to involve the direct transcriptional regulation of downstream gene networks, a function that is likely conserved across species yet exhibits lineage-specific target preferences [8,10,36]. In tetraploid wheat, TtALOG2-1A was functionally characterized to promote branched spike development via the regulation of key TtMADS genes [18]. In this study, DaALOG3 could bind to the promoters of multiple core enzymes in the starch biosynthesis pathway, such as UGP, PGM, ISA, and SUS. This positions DaALOG3 as a key upstream regulator of starch metabolism, potentially linking developmental signals with storage accumulation in tubers. This functional emphasis on starch synthesis may represent a specialized adaptation in tuberous crops. Beyond greater yam, ALOG proteins in other species have similarly been reported to regulate distinct gene networks associated with development. For instance, in Arabidopsis, several LSH genes are known to modulate networks controlling meristem maintenance, organ boundary formation, and floral development [1,11,17,37]. In rice, ALOG members influence panicle architecture and grain number [7,20,38]. These comparative observations suggest that while the core biochemical function as transcriptional regulators is conserved, the specific downstream pathways and biological processes regulated by ALOG genes have diversified during evolution, likely in response to species-specific morphological and physiological demands. Further research comparing the genome-wide target genes of ALOG orthologs across species will help clarify how a conserved transcription factor family has been co-opted to regulate both universal and specialized plant traits.

3.4. Functional Conservation of ALOG Genes Across Plant Species

The overexpression of DaALOG1 in Arabidopsis resulted in pronounced developmental abnormalities, including altered leaf morphology, floral organ defects, and prolonged branching. Notably, these phenotypes closely resemble those previously reported for the overexpression of Arabidopsis endogenous ALOG genes [17,37,39]. This phenotypic convergence strongly suggests that the core molecular function of ALOG transcription factors is conserved across divergent plant lineages. Despite the evolutionary distance between greater yam (a monocot) and Arabidopsis (a eudicot), the ALOG proteins appear to regulate analogous developmental programs, particularly those involving organ boundary specification, meristem activity, and floral architecture. Such functional conservation underscores the fundamental role of the ALOG family in shaping plant form and supports the hypothesis that this gene family represents an ancient and essential regulatory module in land plants. Further investigation into whether downstream target genes are also conserved between species will help clarify the extent of mechanistic overlap and reveal how a conserved transcription factor family can be deployed in both shared and lineage-specific developmental contexts.

4. Materials and Methods

4.1. Plant Materials and Growth Conditions

Arabidopsis thaliana accessions Columbia-0 (Col-0) was used as the wild type. When necessary, seeds of Arabidopsis were screened on 1/2 MS medium (1/2Murashige & Skoog (Hopebio, Qingdao, China), supplemented with 1% sucrose and solidified with 0.8% Plant Agar (BioFroxx, Pfungstadt, Germany)) containing Hygromycin (BioFroxx, Pfungstadt, Germany). The cultivation condition of Arabidopsis was 16 h light/8 h dark, 20–22 °C.

4.2. Data Sources and Sequence Retrieval

The D. alata genome sequence, gene protein sequences, and the transcriptome datasets used in this study were downloaded from the National Center for Biotechnology Information (NCBI) website (https://www.ncbi.nlm.nih.gov/ (accessed on 18 December 2024)) and the Phytozome website (http://www.phytozome.net/ (accessed on 18 December 2024)). The transcriptome dataset includes RNA-seq data for five types of tissues—leaf, stem, inflorescence, tuber, and bulbil—which was listed in Supplementary Table S3. Additionally, the genome sequence, gene model information, transcript, and protein sequences of the other 14 species, Amborella trichopoda, Daucus carota, Solanum tuberosum, Vitis vinifera, Malus domestica, Citrus sinensis, Arabidopsis thaliana, Phoenix dactylifera, Elaeis guineensis, Cocos nucifera, Musa acuminata, Ananas comosus, Brachypodium distachyon, and Oryza sativa, were retrieved from the Phytozome website (http://www.phytozome.net/ (accessed on 18 December 2024)) and NCBI website.

4.3. Genome-Wide Identification of ALOGs Genes

The hidden Markov model (HMMER) profile of the ALOG domain (Pfam accession: PF04852) was obtained from the Pfam database (http://pfam.xfam.org/ (accessed on 18 December 2025)) and used as a query to search for ALOG members in D. alata and 14 other species mentioned above. The peptide sequences of the ALOG domain were extracted based on the results of the HMMsearch analysis (v3.3.2). Multiple alignment of the ALOG sequences was conducted using ClustalW (v2.1) with default parameters. The phylogenetic trees of ALOG genes in Figure 1 (DaALOG proteins) and Figure 2 (conserved protein sequences among DaALOGs and AtLSHs) and Figure 3 (right, based on ALOG domain peptides from the 15 plant species in this study) were constructed using the aligned protein sequences in MEGA7 with the JTT matrix-based model, which is specifically designed to model protein sequence evolution.

ALOG genes from D. alata and 13 other species (excluding Arabidopsis) were then subjected to BLAST (v2.14.1) searches against AtALOGs to identify the best homologous hits. The BLAST results were also utilized to assign ALOG subfamilies. The gene list and subfamily information for the 15 species were included in Table S1.

4.4. Evolutionary Analysis of DaALOG Genes

For the species phylogeny shown in of the right Figure 3, an ML tree of the 15 species was constructed using 140 single-copy orthologous genes detected by OrthoFinder (v2.5.4), following the method described in our previous study [32]. Each set of orthologous genes was aligned at the protein level using MUSCLE, and the alignments were concatenated into a supermatrix. The species tree was inferred using RAxML (v8) with the PROTGAMMAJTT model and 1000 bootstrap replicates.

All protein-coding genes from D. alata were aligned using BLAST against the D. alata protein-coding gene database, with a cutoff of 1 × 10⁻⁵. The BLAST results were processed using the software MCScanX (v1.1) to identify homologous chromosomal regions within D. alata and between species that contain ALOG genes. Duplicated gene pairs of DaALOGs, as well as homologous gene pairs of DaALOGs and AtALOGs within homologous genomic segments, were identified based on the following three criteria: (a) the alignment covered >80% of the longer gene; (b) the aligned region had an identity > 80%; and (c) only one duplication event was counted for the tightly linked genes. The duplicated gene pairs and homologous genomic segments were visualized using TBtools (v1.106) software. The same method was used in our previous research [32].

4.5. WGCNA

Co-expression modules were generated based on the above D. alata transcriptomes. The Reads Per Kilobase Million (RPKM) values were processed to remove genes showing low expression (RPKM ≤ 1 in all samples). The Weighted Gene Co-Expression Network Analysis (WGCNA) package installed in the R environment was used to identify co-expression modules for selected genes with min–max normalized and log2-transformed FPKM values. Modules were defined as clusters of highly interconnected genes, and genes within the same cluster had high correlation coefficients with each other. The gene modules were visualized using Cytoscape (v3.9.0).

4.6. Yeast One-Hybrid Assay

Yeast transformation was performed as previously described [32]. The DNA–protein interaction assays were performed on selective yeast synthetic dropout medium lacking Trp, Leu and His, and supplemented with indicated concentrations of 3-amino-1, 2, 4-triazole (3-AT). Plates were grown for 3d at 28 °C before taking pictures. pHis2-p53 and pGAD53m interaction was used as positive control. Interaction between pGADT7 and pHIS2 was used as negative control.

4.7. Transient Expression of DaALOGs in Tobacco Epidermal Cells for Subcellular Localization

The full-length coding sequences of ten DaALOGs were amplified with primers provided in Supplementary Table S2. Overexpression vectors (pc1300 35S DaALOGx eGFP) were constructed using the Uniclone One Step Seamless Cloning Kit (Genesand Biotech Co., Beijing, China) as previously described [32]. Positive recombinant plasmid clones were selected and amplified in Escherichia coli DH5α following heat shock transformation, then cultured overnight at 37 °C in LB broth containing 50 µg/mL kanamycin.

The recombinant plasmids were introduced into Agrobacterium tumefaciens strain GV3101 by heat shock, following established methods [32]. Agrobacterium cultures carrying 35S::eGFP, 35S::DaALOGx::eGFP, or the nuclear marker 35S::OsGhd7::RFP were pelleted and resuspended in infiltration medium (4.3 g/L Murashige and Skoog salts, 30 g/L sucrose, 50 mM MES pH 5.6, and 100 μM acetosyringone). The bacterial suspension was adjusted to an OD₆₀₀ of 1.0 and infiltrated into the abaxial surface of fully expanded leaves from 4-week-old Nicotiana benthamiana plants. GFP signals were observed 48–72 h post infiltration using a confocal microscope (LMS980 with SESIS system).

4.8. Plant Transformation and Transgenic Plants Phenotype Investigation

The overexpression vector pc1300-35S-DaALOG1-eGFP was used for Arabidopsis transformation. The construct was introduced into Agrobacterium tumefaciens strain GV3101 via the freeze–thaw method, and transgenic plants were generated through floral-dipping. Transformants were selected on half-strength Murashige and Skoog medium containing 50 mg/mL hygromycin, as described previously [32]. Independent transgenic lines were grown under a 16 h light/8 h dark photoperiod at 22 °C for subsequent phenotypic analysis

4.9. RT-qPCR of DaALOGs Genes and Statics Analysis

To examine the expression patterns of DaALOG genes, six-months tissue culture plantlets were selected for sampling. Plant tissues—including leaves, stems, and tubers—were collected from each individual as biological replicates. Total RNA was extracted following an established protocol [40]. First-strand cDNA was synthesized using the HiScript III 1st Strand cDNA Synthesis Kit (R312, Vazyme Biotech Co., Nanjing, China) according to the manufacturer’s instructions.

Real-time qPCR was performed with ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech Co., Nanjing, China) on an ABI 7900HT system (Applied Biosystems, Thermo Fisher Scientific, Foster City, CA, USA). The thermal cycling conditions were as follows: 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, 55 °C for 15 s, and 68 °C for 20 s. All reactions were carried out in 384-well optical plates (Applied Biosystems, Thermo Fisher Scientific, Foster City, CA, USA). Gene-specific primers for the DaALOG family are provided in Supplementary Table S2b. For statistical analysis of the RT-qPCR data, relative expression levels were calculated using the comparative ΔΔCt method, with the UBC gene serving as the internal reference for normalization. Significance of differences was determined using the Student’s t-test.

Data were first assessed for normality and homogeneity of variances to confirm the suitability of this test. Differences with a p-value < 0.05 were deemed statistically significant. All analyses were conducted using SPSS software (v24).

5. Conclusions

In summary, this study clarifies the functions and characteristics of the DaALOG gene family in D. alata, underscoring its pivotal regulatory role in plant growth and development. By classifying the ten DaALOG genes into two subfamilies and correlating their conserved structural features with expression profiles, we reinforce the high evolutionary conservation of these transcription factors. Notably, although all DaALOG proteins contain the conserved ALOG domain, the presence of disordered regions flanking this domain suggests functional diversification among family members. Our findings demonstrate that DaALOG genes are important regulators of plant morphology, floral organ development, and starch biosynthesis, highlighting their central position in developmental regulatory networks. This work not only expands the genomic and functional understanding of DaALOG genes but also provides a foundation for future studies investigating their roles in morphology and starch metabolism in D. alata and related species. Overall, DaALOG proteins act as key transcription factors within the complex circuitry governing plant growth and development, with potential implications for crop improvement and cultivation.