Comparative analysis of the LEA gene family in seven Ipomoea species focuses on sweet potato (Ipomoea batatas)

doi:10.21203/rs.3.rs-5297525/v1

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Comparative analysis of the LEA gene family in seven Ipomoea species focuses on sweet potato (Ipomoea batatas)

https://doi.org/10.21203/rs.3.rs-5297525/v1

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Late Embryogenesis Abundant (LEA) proteins are extensively distributed among higher plants and are crucial for regulating growth, development, and abiotic stress resistance. However, comprehensive data regarding the LEA gene family in Ipomoea species remains limited. In this study, we conducted a genome-wide comparative analysis across seven Ipomoea species, including sweet potato (I. batatas), I. trifida, I. triloba, I. nil, I. purpurea, I. cairica, and I. aquatica, identifying 73, 64, 77, 62, 70, 70, and 74 LEA genes, respectively. The LEA genes were divided into eight subgroups: LEA_1, LEA_2, LEA_3, LEA_4, LEA_5, LEA_6, SMP, and Dehydrin according to the classification of the LEA family in Arabidopsis. Gene structure and protein motif analyses revealed that genes within the same phylogenetic group exhibited comparable exon/intron structures and motif patterns. The distribution of LEA genes across chromosomes varied among the different Ipomoea species. Duplication analysis indicated that segmental and tandem duplications significantly contributed to the expansion of the LEA gene family, with segmental duplications being the predominant mechanism. The analysis of the non-synonymous (Ka) to synonymous (Ks) ratio (Ka/Ks) indicated that the duplicated Ipomoea LEA genes predominantly underwent purifying selection. Extensive cis-regulatory elements associated with stress responses were identified in the promoters of LEA genes. Expression analysis revealed that the LEA gene exhibited widespread expression across diverse tissues and showed responsive modulation to various abiotic stressors. Furthermore, we selected 15 LEA genes from sweet potatoes for RT-qPCR analysis, demonstrating that five genes responded to salt stress in roots, while three genes were responsive to drought stress in leaves. Additionally, expression levels of seven genes varied at different stages of sweet potato tuber development. These findings enhanced our understanding of the evolutionary dynamics of LEA genes within the Ipomoea genome and may inform future molecular breeding strategies for sweet potatoes.

Ipomoea species

LEA gene family

gene expression

abiotic stress

sweet potato

Abiotic stressors, such as drought, salinity, and extreme weather, disrupt normal crop growth and development, leading to considerable yield losses and quality degradation [1, 2]. Through ongoing evolutionary processes, plants have developed various mechanistic molecular, physiological, and biochemical adaptations to enhance their resistance to these abiotic challenges [3]. For example, transcription factors (TFs) and protein kinases play crucial roles in regulating downstream signaling pathways that activate and modulate specific stress-responsive genes, resulting in physiological responses such as the synthesis of osmoregulatory compounds [4, 5]. Functional proteins associated with plant stress tolerance, including late embryogenesis abundant (LEA) proteins, are upregulated during late embryogenesis and in nutrient tissues in response to water scarcity. These proteins help eliminate reactive oxygen species (ROS) from cells, thus protecting macromolecules and mitigating damage caused by abiotic stressors [6, 7].

LEA proteins are prevalent across the plant kingdom and serve as osmoprotective agents and facilitators of desiccation damage repair. These glycine-rich proteins, characterized by low molecular weights ranging from 10 to 30 kDa, accumulate significantly during the later stages of embryonic development in response to various abiotic stresses, thereby protecting against extreme environmental conditions, particularly drought stress [8]. Although the classification of LEA proteins varies among different species, they are typically divided into eight groups based on conserved structural domains: LEA_1, LEA_2, LEA_3, LEA_4, LEA_5, LEA_6, Dehydrin, and Seed Maturation Protein (SMP) [9]. Understanding LEA gene expression's regulatory mechanisms is essential for advancing modern plant molecular biology. The first LEA protein was identified in Gossypium hirsutum in 1981 [10]. With the development of whole-genome sequencing technologies, additional LEA proteins have been characterized in model organisms such as Arabidopsis thaliana and Oryza sativa [11, 12], as well as in food crops like Zea mays and Triticum aestivum [13, 14], horticultural crops such as Cucumis sativus and Vitis vinifera [15–17], and other organisms, including microbes and invertebrates [18]. While a minority of LEA proteins are localized in the endoplasmic reticulum of plant cells, most are found distributed throughout the nucleus, cytoplasm, and mitochondria [19].

The essential role of LEA proteins in plant responses to abiotic stressors has been highlighted in several studies. For example, the overexpression of AtLEA14 enhanced the expression of salt stress response marker genes in transgenic Arabidopsis thaliana, indicating increased salt tolerance [20]. Similarly, the upregulation of OsLEA3-2 improved salt and drought tolerance in transgenic A. thaliana and O. sativa [21]. The OsLEA increased rice sensitivity to abscisic acid and enhanced osmotic tolerance under drought conditions [22]. Additionally, elevated levels of CaLEA1 promoted stomatal closure and activated related downstream gene expression in response to drought and salt stress [23]. Both abiotic stress and signaling molecules induced the ZmLEA3 expression in leaves, and its overexpression enhanced the osmotic and oxidative stress tolerance in transgenic Nicotiana tabacum [24]. Furthermore, other genes implicated in stress tolerance include TaLEA3 in Leymus chinensis [25], IpLEA in Ipomoea pes-caprae [26], SmLEA in Salvia miltiorrhiza [27], SlLEA6 in tomato [28], and SiLEA14 in Setaria italica [29].

The sweet potato, an allohexaploid crop, belongs to the Convolvulaceae family within the Ipomoea genus, specifically the Batatas section. This starchy root vegetable is of considerable global economic significance, serving as a food source, industrial material, and bioenergy resource [30–32]. Two diploid relatives, Ipomoea trifida and Ipomoea triloba, are employed as model organisms to facilitate sweet potato breeding [33]. Additionally, Ipomoea nil and Ipomoea purpurea are valuable for research into photoperiodic flowering and flower pigmentation [34]. Ipomoea cairica, a perennial vine that blooms year-round, is widely cultivated as an ornamental plant in subtropical and temperate regions and exhibits high levels of bioactive compounds with potential medicinal applications [35]. Furthermore, Ipomoea aquatica, commonly known as water spinach, is one of Asia's most beloved leafy vegetables, characterized by aquatic and terrestrial growth forms [36, 37].

This study performed a genome-wide comparative analysis of the LEA gene family across seven Ipomoea species. A total of 73, 64, 77, 62, 70, 70, and 74 LEA genes were identified in sweet potato (I. batatas), I. trifida, I. triloba, I. nil, I. purpurea, I. cairica, and I. aquatica, respectively. The identified LEA genes underwent phylogenetic analysis, gene structure characterization, chromosomal localization, syntenic analysis, calculation of the Ka to Ks substitution ratio (Ka/Ks), cis-regulatory element (CRE) identification, and expression profiling. Subsequently, 15 differentially expressed genes (DEGs) from sweet potatoes were selected for quantitative reverse transcription PCR (RT-qPCR) analysis. The results revealed that five IbLEA genes were responsive to salt stress in roots, while three IbLEA genes responded to drought stress in leaves. Additionally, seven IbLEA genes exhibited varying expression levels at different sweet potato tuber development stages. These findings provided new insights into the evolution of the LEA gene family in Ipomoea species and may facilitate future advancements in the molecular breeding of sweet potatoes.

2.1 Identification of LEA genes in Ipomoea species

Genomic data for sweet potato were sourced from the Ipomoea Genome Hub (https://ipomoea-genome.org/). I. trifida and I. triloba data were retrieved from the Sweetpotato Genomics Resource (http://sweetpotato.plantbiology.msu.edu/). Genomic information for I. nil was acquired from the Ipomoea nil database (http://viewer.shigen.info/asagao/). Data for I. purpurea were obtained from the CoGe platform (https://genomevolution.org/coge/GenomeInfo.pl?gid=58735), while genomic data for I. aquatica and I.cairica came from NCBI (PRJCA002216 and PRJNA820303). To identify candidate members of the LEA gene family in Ipomoea species, 51 LEA protein sequences from A. thaliana (https://www.arabidopsis.org/) were utilized as query sequences for a genome-wide BLASTP analysis across seven Ipomoea species [38]. The structural domains of the identified proteins were subsequently analyzed using the Conserved Domain Database (CDD, https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi), Pfam (http://pfam.xfam.org/), and SMART (http://smart.embl.de/) databases. Candidate genes possessing any LEA_1, LEA_2, LEA_3, LEA_4, LEA_5, LEA_6, SMP, or Dehydrin structural domains were classified as members of the LEA gene family.

2.2 Physicochemical properties analysis of Ipomoea LEA proteins

The physicochemical properties of all identified LEA proteins in the seven Ipomoea species were predicted using ExPASy online tools (http://www.expasy.org/tools/protparam.html). Additionally, the subcellular localization of all identified LEA genes in the seven Ipomoea species was predicted using the WoLF PSORT online tool (https://wolfpsort.hgc.jp/).

2.3 Phylogenetic analysis of Ipomoea LEA proteins

Using IQtree software, we constructed a maximum likelihood phylogenetic tree for LEA members across several species, including A. thaliana, sweet potato (I. batatas), I. trifida, I. triloba, I. nil, I. purpurea, I. cairica, and I. aquatica [39]. The WAG + R3 substitution model was the optimal fit based on the Bayesian Information Criterion (BIC) score, with 1000 ultra-fast bootstrap replicates performed for robustness. The phylogenetic tree was visualized using the iTOL online platform (https://itol.embl.de/login.cgi).

2.4 Chromosome distribution and collinearity analysis of Ipomoea LEA genes

Chromosomal localization of all identified LEA genes in sweet potato (I. batatas), I. trifida, I. triloba, I. nil, I. purpurea, I. cairica, and I. aquatica was performed using TBtools software based on gene annotations [40]. The collinearity relationships of LEA genes within species were constructed and visualized using TBtools software.

2.5 Synteny analysis and calculation of Ka/Ks values of Ipomoea LEA genes

The synteny analysis in the genomes of the seven Ipomoea species was performed and visualized using TBtools software. The nucleotide substitution rates (Ka and Ks) and Ka/Ks ratios of duplicated LEA genes were estimated using the TBtools software’s Ka/Ks calculator. The estimated time of detergence (T, Mya: million years ago) was calculated as described previously.

2.6 Cis-acting element analysis of the Ipomoea LEA genes

The cis-acting element prediction was conducted using 1500 bp sequences upstream of the identified LEA genes via PlantCARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/). Then, the TBtools software was used to visualize the cis-regulatory element figure.

2.7 Protein conserved domains and motif and gene structure analyses of Ipomoea LEA genes

LEA protein sequences were grouped to construct an evolutionary tree while coding regions and genomic sequences were compared to examine gene structure. The proteins' conserved structural domains and motifs were identified using the HMMER search and the Multiple Expectation Maximization for Motif Elicitation (MEME) tool (https://meme-suite.org/meme/tools/meme). All analyses were visualized using TB tools [40].

2.8 Expression pattern analysis of sweet potato LEA genes

Six transcriptome bio project datasets were selected to analyze LEA gene expression profiles in sweet potato. Three low potassium, hormonal treatment, and cold stress datasets were obtained from the NCBI BioProject (accession numbers PRJNA1013090, PRJNA511028, and PRJNA511028). The remaining three datasets were derived from our unpublished in-house studies on heat, salt, and drought treatments. Specifically, "Xushu 18" was utilized for hormonal treatment; the cold-tolerant "Liaohanshu 21" and cold-sensitive "Shenshu 28" were employed for cold treatment; the low-potassium tolerant "Xushu 32" and low-potassium sensitive "Ningzishu 1" served for low potassium treatment; and the heat-tolerant "Guangshu 87" and heat-sensitive "Ziluolan" were used for heat treatment, while "Guangshu 87" was also used for both salt and drought treatments. Additionally, the gene expression data in I. trifida and I. triloba were downloaded from the Sweet Potato Genomics Resource (http://sweetpotato.plantbiology.msu.edu/). The dataset of I. aquatica, including MF (salt sensitive) and BG (salt tolerant) for salt stress, was downloaded from NCBI BioProject (accession number PRJNA1119567).

2.9 RT-qPCR analysis of sweet potato LEA genes

This study utilized the sweet potato cultivar "Jishu 26" for RT-qPCR analysis. The plants were cultivated in the experimental field of Guangdong Ocean University, Guangdong, China. For tissue expression analysis, samples were collected from various tissues of 3-month-old "Jishu 26" plants, including flower, flower bud, tender leaf, old leaf, tender stem, old stem, and fibrous root, fibrous root, and tuberous root with a diameter of 4 cm, 8 cm, and 13 cm. For abiotic stress treatments, 25 cm twigs from 3-month-old field-grown "Jishu 26" were cultured in Hoagland solution for 14 days. The twigs were treated in Hoagland solution for salt stress with 0 and 200 mM/L NaCl. They were cultured in Hoagland solution for drought stress with 0 and 300 mM/L mannitol. Fibrous root and leaf samples are collected 0, 2, 4, 8, 16, and 24 hours after treatment. Each sample was analyzed in triplicate using Dingfa’s method (Ding et al., 2017), using the IbARF gene as an internal reference, using the primers (Table S4) generated with NCBI. Relative transcript levels were calculated by the 2^−ΔΔCT method.

2.10 Protein‑protein interaction (PPI) network construction

Using the default parameters, the online STRING database (https://string-db.org/) was utilized to predict and execute potential protein-protein interaction networks using IbLEA proteins based on known Arabidopsis homologs. Cytoscape (V3.10.2) visualized the resulting network [41].

3.1 Genome-wide identification and phylogenetic analysis of Ipomoea LEA genes

Sweet potato (I. batatas) had 73 LEA genes, while its two closely related wild species, I. trifida and I. triloba, contained 64 and 77 LEA genes, respectively. 74, 62, 70, and 70 LEA genes were identified in I. aquatica, I. nil, I. purpurea, and I. cairica, respectively (Table S1). Furthermore, they all were renamed based on their positions on the chromosomes. A maximum likelihood (ML) tree was reconstructed for the LEA members of Arabidopsis and seven Ipomoea species. All the 541 LEA proteins were grouped into eight high bootstrap value (1000) categories based on their corresponding unique structural domains: LEA_1, LEA_2, LEA_3, LEA_4, LEA_5, LEA_6, SMP, and Dehydrin (Fig. 1). The LEA_2 was the largest subgroup including 327 LEA proteins from Ipomoea species, namely, 47 IbLEA, 44 ItfLEA, 52 ItbLEA, 51 IaLEA, 39 InLEA, 49 IpLEA, and 45 IcLEA. The LEA_3 was the next largest subgroup, comprising 47 LEA from Ipomoea species. The remaining LEA proteins from the Ipomoea species were divided into other groups: 29 in the SMP, 24 in the LEA_1, 23 in the Dehydrin, and 13, 14 in the LEA_5 and the LEA_6, with 12 in the LEA_4. In sweet potato, there were 47 IbLEA proteins in the LEA_2 with the most, while 2 IbLEA in the LEA_4 and the LEA_5, with the least. There was no mixed presence in each identified group, indicating the relatively conservative distribution of LEA gene family members from Ipomoea species across different groups.

3.2 Physicochemical properties and subcellular localization of the Ipomoea LEAs

The Ipomoea LEA proteins exhibited a diverse range of lengths, with the shortest proteins (InLEA5, InLEA36, InLEA37) consisting of 77 amino acids and the longest protein (IbLEA65) comprising 1089 amino acids, averaging 216.91 amino acids in length (Table S2). The molecular weights of these proteins varied from 8.18 kDa (InLEA5) to 122.14 kDa (IbLEA65), with an average of 23.84 kDa. The predicted isoelectric points span from 4.18 to 10.63, with an average pI of 8.66. Among the seven Ipomoea LEA proteins, 103 were classified as acidic (pI < 7), and 236 exhibited instability index values exceeding 40. 157 LEA proteins displayed positive average hydrophobicity (GRAVY), while the remaining proteins had negative average hydrophobicity. IaLEA26 demonstrated an average hydrophobicity of 0, indicating predominantly hydrophilic characteristics among the Ipomoea LEA proteins. These LEA members were distributed across various organelles, including mitochondria, chloroplasts, cytoplasm, nuclei, cytoskeletons, endoplasmic reticulum, vacuoles, cell membranes, Golgi bodies, and extracellular matrices, with a predominant localization in chloroplasts and cytoplasm.

3.3 Chromosome location and duplication analysis of the Ipomoea LEA genes

In the Ipomoea species, the distribution of LEA genes across chromosomes was irregular (Fig. 2). The highest number of LEA genes occurred 13 across its nine chromosomes in I. triloba, whereas the lowest number, 1 LEA gene, was present on the ItfChr6 and ItfChr13 in I. trifida, the InChr1 of I. nil, and the IaChr4 of I. aquatica.

Collinearity analysis within species revealed the presence of 16 pairs, 21 pairs, 25 pairs, 18 pairs, 16 pairs, and 24 pairs of homologous genes in sweet potato (I. batatas), I. trifida, I. triloba, I. nil, I. purpurea, I. cairica, and I. aquatica, respectively (Fig. 3). Notably, only sweet potato and I. trifida exhibited tandemly duplicated LEA genes, with two pairs and one pair identified, respectively; no tandem duplications were observed in the other five species. Fragment duplication analysis further indicated that sweet potato (I. batatas), I. trifida, I. triloba, I. nil, I. purpurea, I. cairica, and I. aquatica contain 14, 20, 25, 18, 16, and 24 pairs of fragment duplicated genes, respectively (Table S3). These results implied that segmental and tandem duplications significantly contributed to the expansion of LEA genes in sweet potato and I. trifida, with segmental duplication as the predominant mechanism. In contrast, segmental duplication was the exclusive means of LEA gene amplification in the other five species.

3.4 Syntenic analysis of LEA genes in the genomes of Ipomoea species

Collinearity results showed that 490 LEA genes were detected in these seven Ipomoea species, forming 558 orthologous gene pairs (Fig. 4 and Table S3). Among them, I. cairica and I.aquatica had the most orthologous LEA gene pairs (108 pairs), followed by I.purpurea and I.cairica (98 pairs), I. trifida and I. triloba (98 pairs), sweet potato and I. trifida (87 pairs), I. triloba and I. nil (86 pairs), and I. nil and I.purpurea (81 pairs).

3.5 Ka/Ks analysis of duplicated and syntenic Ipomoea LEA genes

To further investigate the selective pressure on homologous gene pairs in seven Ipomoea species, these homologous gene pairs' Ka/Ks values were analyzed (Table S3). Most LEA homologous gene pairs' Ka/Ks ratios were less than 1 (95.7%), indicating that these gene pairs were subject to purifying selection during evolution. Six pairs of homologous gene pairs, including ItbLEA16 and ItbLEA41, IpLEA47 and IpLEA21, IpLEA47 and IpLEA58, IaLEA4 and IaLEA58, IcLEA5 and IcLEA13, and IcLEA13 and IcLEA68, had Ka/Ks values greater than 1. This suggested that these six pairs of genes underwent positive selection and could have a relatively faster evolutionary rate. There were 16 homologous gene pairs in sweet potato, and the Ka/Ks values of these orthologous gene pairs were all less than 1.

The Ka/Ks analysis of homologous genes among Ipomoea species indicates that the occurrence of Ka/Ks ratios exceeding 1 was more prevalent across the seven species than within individual species (Table S3). Specifically, there are 40 pairs (37%) between I. cairica and I. aquatica, 38 pairs (38.8%) between I. purpurea and I. cairica, 28 pairs (28.6%) between I. trifida and I. triloba, 33 pairs (37.9%) between sweet potato and I. trifida, 30 pairs (34.9%) between I. triloba and I. nil, and 32 pairs (39.5%) between I. nil and I. purpurea.

3.6 Conserved structural domains and gene structure analysis of Ipomoea LEA genes

The LEA protein family members across seven Ipomoea species were categorized into eight distinct subgroups, with phylogenetic trees constructed for each subgroup utilizing maximum likelihood methods (Fig. 5, S1). Closely related LEA gene members within the subgroup typically exhibited similar gene structures, particularly regarding intron number and exon length. For instance, LEA_3, 4, 5, 6, SMP, and Dehydrin contain between 0 to 6 introns, with Dehydrin characterized by a single intron. In contrast, LEA_1 and LEA_2 display a broader range of intron counts, with 0 to 14 and 0 to 19 introns, respectively. Notably, LEA_2 exhibits substantial structural variability, housing the highest number of genes and demonstrating significant differences in exon/intron arrangements. These observations implied that functional differentiation within the LEA protein family has transpired among the seven Ipomoea species.

Distinct motifs were identified within each subgroup, with only three motifs in LEA_5. In comparison, LEA_6 contained eight motifs (Fig. 5). LEA_5 demonstrates a high degree of conservation, encompassing Motif 1 and Motif 2 in all members. In subgroups LEA_1, 3, 4, 6, SMP, and Dehydrin, some genes across different species displayed similarities, and all LEA genes within the same species possessed at least one common motif. Conversely, LEA_2 exhibited notable complexity (Fig. S1). This specific classification suggested that LEAs within the same subgroup may share analogous biological functions. The variability in conserved sequences indicated that LEA proteins likely evolved through gene amplification events within their respective gene families.

3.7 Cis-regulatory elements in putative promoter regions of the Ipomoea LEA genes

Predictive analysis of cis-regulatory elements was performed on the 1500 bp nucleotide sequences upstream of the start codon (ATG) for each LEA gene across the seven Ipomoea species (Fig. S2). The identified elements included light-responsive cis-regulatory elements associated with abiotic stress or external pressures, plant hormone-responsive cis-regulatory elements, and plant growth and development elements. Light-responsive cis-regulatory elements were the most prevalent, accounting for 4,209 occurrences. Each LEA gene within the seven Ipomoea species contained these light-responsive elements, with 650 instances identified in sweet potato (46.9%). Six distinct cis-regulatory elements associated with abiotic stress or external pressures were observed, targeting responses to anaerobic conditions, defense and stress, drought, low temperature, MYB factors, and wounding. These results indicated that LEA proteins were likely crucial for drought stress response and tolerance in the seven Ipomoea species. The plant hormone-responsive cis-regulatory elements identified included those responsive to abscisic acid, auxin, gibberellin, jasmonic acid, and salicylic acid, suggesting that plant hormones may regulate the expression of LEA proteins. Furthermore, cis-regulatory elements implicated in plant growth and development were also detected, specifically linked to sweet potato meristem expression, endosperm expression, and seed-specific regulation.

3.8 Protein Interaction Network of IbLEAs in Sweet Potato

An interaction network was generated using homologous proteins from Arabidopsis to investigate the regulatory network of IbLEAs (Fig. 6). Interactions among IbLEAs were also linked to other proteins. Specifically, IbLEA42 interacted with IbLEA60, IbLEA18, IbLEA46, IbLEA50, IbLEA22, and IbLEA7. Furthermore, both IbLEA42 and IbLEA7 demonstrated the ability to interact with Glutamate racemase (Q8LEN7). IbLEA51, IbLEA7, and IbLEA29 also interacted with the MADS-box protein (AGL81).

3.9 Expression profiles of the Ipomoea LEA genes in various tissues

The expression profiles of IbLEA genes across different tissues of sweet potato were analyzed using transcriptome data, revealing specific expression in flowers, fruits, and various morphological roots (Fig. 7a). Notably, eight genes (IbLEA56, IbLEA39, IbLEA40, IbLEA36, IbLEA7, IbLEA71, IbLEA45, IbLEA44) exhibited exclusive expression in flowers. In comparison, five genes (IbLEA34, IbLEA54, IbLEA53, IbLEA67, IbLEA73) were uniquely expressed in primary roots. Furthermore, IbLEA11 showed high expression in tuberous roots, and 16 genes were exclusively expressed in fruits. IbLEA25, IbLEA41, IbLEA42, and IbLEA15 also demonstrated high expression across multiple sweet potato tissues. We investigated the expression profiles of LEA genes across six tissues (flower buds, flowers, stems, leaves, root 1, and root 2) in I. trifida and I. triloba (Fig. S3a, b). In I. trifida, two LEA genes, ItfLEA6 and ItfLEA48, were exclusively expressed in flowers, while four others exhibit tissue-specific expression in leaves and root 2. Five ItfLEA genes were also uniquely expressed in stems (Fig. S3a). In I. triloba, five ItbLEAs showed elevated expression levels in flowers, and four demonstrated specific expression in root 2 (Fig. S3b).

3.10 Expression profiles of the Ipomoea LEA genes under abiotic stress

The expression of IbLEA genes under salt and drought stress conditions was examined (Fig. 7b). Significant differences in expression levels were observed for all 73 IbLEA genes in tissues subjected to salt or drought stress. Under salt stress, eight genes were significantly up-regulated in root tissues, while eighteen were down-regulated. In stem tissues, twenty-two genes were up-regulated, three were down-regulated, whereas in leaf tissues, one was up-regulated, and six were down-regulated. Under drought stress, three genes in root tissues were up-regulated and six down-regulated; in stem tissues, thirteen genes were up-regulated and five down-regulated, while in leaf tissues, eight genes were up-regulated and four down-regulated. These findings indicated distinct expression patterns of IbLEA genes were in response to salt and drought stress, with the majority responding to varying stress conditions.

To investigate the genetic resources of ItfLEAs, ItbLEAs, and IaLEAs in response to drought and salt stress, the expression profiles of these LEAs were analyzed under these conditions. In I. trifida, 18 LEAs exhibited specific upregulation in response to drought stress, while 15 LEAs were upregulated specifically under salt stress (Fig. S3c). In I. triloba, 22 LEAs were notably upregulated during drought conditions, with 14 LEAs showing specific upregulation in response to salt stress (Fig. S3d). In I. aquatica, the LEAs displayed distinct expression patterns under salt stress in two different salt tolerance varieties. Specifically, after 12 hours of salt exposure in the MF (salt-sensitive), the expression of IaLEA51 increased, whereas IaLEA55 was upregulated at both the 6-hour and 12-hour upon salt conditions. In the BG (salt-tolerant), following 6 hours of salt exposure, the expression levels of IaLEA48 and IaLEA58 increased but subsequently decreased after 12 hours (Fig. S3e).

Under high-temperature stress, the expression of the 73 IbLEA genes was analyzed (Fig. 7d). In the fibrous roots of "Guangshu 87", six genes (IbLEA38, IbLEA41, IbLEA42, IbLEA14, IbLEA15, IbLEA59) were significantly up-regulated. In contrast, in tuberous roots, IbLEA55 and IbLEA52 showed significant up-regulation. In the fibrous roots of "Ziluolan", five genes (IbLEA38, IbLEA41, IbLEA42, IbLEA14, IbLEA15) were similarly up-regulated, with IbLEA55, IbLEA52, and IbLEA5 significantly up-regulated in tuberous roots.

Similar analyses were performed for the expression of the 73 IbLEA genes under cold stress (Fig. 7c). In "Shenshu 28," after three hours of cold stress, IbLEA15, IbLEA41, and IbLEA42 were significantly up-regulated, with further increases after 24 hours. In "Liaohanshu 21," five genes, including IbLEA11, IbLEA15, and IbLEA42, were significantly up-regulated after three hours, but after 24 hours, IbLEA15 transcript level decreased, and IbLEA42 and IbLEA11 expression level increased. Analysis of the expression patterns of ItfLEAs and ItbLEAs under heat and cold stress (Fig. S3f, g). In I. trifida, four LEAs were upregulated under heat stress, while twenty-three LEAs were upregulated under cold stress. Similarly, in I. triloba, eleven LEAs were upregulated under heat stress, and twenty-nine LEAs were upregulated under cold stress. Further analysis indicated that the differentially expressed LEA genes typically responding to abiotic stress primarily belonged in the LEA_2 subgroups (Fig. S4).

Using RNA-seq data from "Xushu 18" obtained from the NCBI database (PRJNA511028), the expression patterns of 73 IbLEA genes exposed to diverse phytohormone treatments were identified (Fig. 7e). In fibrous roots, after ABA treatment, the expression of IbLEA32, IbLEA34, and IbLEA54 significantly increased; after MeJA treatment, 15 IbLEA genes showed significant up-regulation; and after SA treatment, 18 IbLEA genes were significantly up-regulated. In stems, 16 genes were significantly induced by ABA treatment, 15 by SA treatment, and 11 by MeJA treatment. Only IbLEA39 was significantly up-regulated in the leaves after ABA treatment, whereas 14 genes were up-regulated following MeJA treatment and 12 after SA treatment.

The expression profiles of ItfLEAs and ItbLEAs were assessed using RNA-seq data from I. trifida and I. triloba following treatments with GA3, 6-BAP, ABA, and IAA (Fig. S3h, i). GA3 treatment specifically upregulated three ItfLEAs and eight ItbLEAs. In contrast, 6-BAP treatment led to the specific upregulation of twelve ItfLEAs and six ItbLEAs. Under ABA treatment, twenty-one ItfLEAs and twenty ItbLEAs showed specific upregulation. No significant alterations in the expression of ItfLEAs and ItbLEAs were observed following IAA treatment.

Finally, transcriptome data regarding potassium deficiency from the low potassium-tolerant variety "Xushu 32" and the low potassium-sensitive variety "Ningzishu 1" from the NCBI database (PRJNA1013090) were used to analyze the expression of the 73 sweet potato LEA genes (Fig. 7f). Under low potassium treatment, IbLEA64 and IbLEA9 were significantly up-regulated in "Ningzishu 1." In contrast, IbLEA73, IbLEA33, and IbLEA32 were significantly up-regulated in "Xushu 32".

3.11 Expression analysis of sweet potato LEA genes by RT-qPCR

Fifteen IbLEA genes were selected for RT-qPCR validation. Analysis across various sweet potato tissues, including primary roots, pencil roots, and tubers of varying sizes (4 cm diameter - DR4, 8 cm diameter - DR8, and 13 cm diameter - DR13), tender stems, old stems, tender leaves, old leaves, flower buds, flowers, and tuberous roots at different developmental stages, revealed that all 15 genes exhibited high expression levels in flower buds. Except for IbLEA15, the remaining 14 genes have displayed significant expression in old leaves, while IbLEA35 and IbLEA64 demonstrated elevated expression in old stems. Notably, five genes (IbLEA26, IbLEA27, IbLEA43, IbLEA59, and IbLEA63) showed variable expression levels in tuberous roots depending on the growth stage (Fig. 8), suggesting they may be potential candidate genes involved in sweet potato tuber development.

Simultaneously, the expression levels of these 15 IbLEA genes were assessed in roots and leaves during re-watering following salt and drought treatments, specifically at 2, 4, 8, 16, and 24 hours (Fig. 9). The results indicated significant changes in the expression of most IbLEA genes across different tissues under both stress conditions. In roots, IbLEA19 exhibited an 18-fold increase under salt stress at 2 hours, followed by a decline from 4 hours onward, a trend similarly observed under drought stress. IbLEA20 began to rise at 2 hours under salt stress, peaking at 70 times the control level at 8 hours. IbLEA26 reached 15 times the control level at 4 hours under salt stress. IbLEA27 demonstrated an initial increase followed by a decrease, with a subsequent rise to 13 times the control at 16 hours under salt stress and 18 times at 8 hours under drought stress. IbLEA32 consistently increased under both stress conditions, achieving 200 times the control level at 4 hours under salt stress and 50 times at 2 hours under drought stress. In leaves, under drought stress, IbLEA19 and IbLEA20 displayed an initial increase followed by a decrease, reaching 12 times and five times the control levels, respectively, at 8 hours, while IbLEA32 showed an overall increase, peaking at 105 times the control level at 8 hours.

Sweet potato is a significant tuber crop globally [42], characterized by a large and complex genome estimated to range from 2 to 3 Gb [43]. Two diploid species closely related to hexaploidy sweet potato are I. trifida and I. triloba, with genome sizes estimated at 526.4 Mb and 495.9 Mb, respectively [33]. Due to their reduced genome sizes and ploidy levels, these species are frequently utilized as model organisms to enhance sweet potato breeding efforts. I. nil, an annual climbing herbaceous plant known for its self-pollination and blue flowers, has an estimated genome size of 750 Mb and is a model for studying photoperiodic flowering and flower pigmentation [34]. I. purpurea, a common agricultural weed with a haploid genome size of approximately 814 Mb, demonstrates varying levels of herbicide resistance [44]. I. cairica, a perennial climbing plant introduced for ornamental horticulture, has an estimated haploid genome size of 730 Mb [35]. I. aquatica, known as water spinach, possesses an estimated genome size of 550.03 Mb and is recognized as a popular leafy vegetable with both aquatic and terrestrial traits [36, 37]. The LEA gene family is extensively distributed among plants and plays crucial roles in cellular protection against abiotic stress as well as the regulation of growth and developmental processes [6, 45]. The LEA gene family has been identified in various crops, including tomato [28], cotton, rice, maize, and wheat [8, 46], and has also been observed in other organisms such as invertebrates and microbes [47]. However, no literature currently addresses the LEA protein family in Ipomoea species.

In this study, we identified 73, 64, 77, 62, 70, 70, and 74 LEA genes from sweet potato (Ipomoea batatas), I. trifida, I. triloba, I. nil, I. purpurea, I. cairica, and I. aquatica, respectively, totaling 490 LEA genes categorized into eight clusters (Fig. 1 and Table S1). Among these, the LEA_2 cluster represented the largest subfamily in sweet potato, a finding consistent with observations in tomato [28], Sorghum bicolor [48], and G. hirsutum [49]. Conversely, the LEA_4 and LEA_5 clusters contained the fewest members, differing from Brassica napus [50], where LEA_4 had the highest member count. This discrepancy was attributed to the compositional variations of LEA subfamilies across species. Most identified LEA members were localized in the chloroplast and cytoplasm in the seven Ipomoea species studied. At the same time, in sweet potato, a significant number were found in the chloroplast and cell membrane (Table S2).

Chromosomal localization analyses revealed that LEA genes within the Ipomoea genus exhibit uneven distribution across the chromosomes of different species (Fig. 2). Such uneven distribution of LEA genes has also been observed in other species, including Juglans regia and its wild relative Juglans mandshurica [51], as well as in rice [11]. Nevertheless, certain gene clusters demonstrated high similarity and collinearity (Fig. 3). The intraspecific collinearity analysis identified 16 pairs, 21 pairs, 25 pairs, 18 pairs, 16 pairs, and 24 pairs of homologous genes in I. batatas, I. trifida, I. triloba, I. nil, I. purpurea, I. cairica, and I. aquatica, respectively. Gene duplication events, including segmental and tandem duplications, are critical for the expansion and distribution of plant gene families [52–54]. Our findings indicated that segmental and tandem duplications facilitated the expansion of LEA genes in the seven Ipomoea species, with segmental duplications being the predominant mechanism (Table S3). This predominance of segmental duplication as a mechanism for LEA gene expansion aligns with findings in tomato [28], but differs from those in A. thaliana [12, 55], another dicotyledonous plant.

Members of the same LEA protein subgroup typically exhibit one or more conserved structural domains and motifs (Fig. 5, S1), indicating their potential involvement in this group's major and specific functional roles. These conserved domains are integral to the physiological functions of proteins, forming the structural basis for their activities. However, notable structural variances across different subgroups highlight the functional complexity of LEA proteins in Ipomoea species. The distribution of conserved motifs implied that genes sharing identical motifs likely originated from expansions within the same group or taxon. Consequently, since different conserved motifs characterized distinct clusters, it can be preliminarily inferred that they correspond to diverse ancestral lineages with unique conserved motifs [17].

The Ka/Ks analysis of duplicated and homologous LEA genes within and across seven species of Ipomoea revealed that the majority underwent purifying selection during speciation. In contrast, the genes experiencing positive selection were comparatively few (Table S3, S4). Notably, instances of positive selection were more prevalent among homologous LEA genes across Ipomoea species. These findings indicated that LEA genes within Ipomoea have predominantly undergone strong purifying selection throughout evolution, resulting in minimal changes post-duplication. At the same time, functional divergences may arise among LEA genes across different species.

Cis-regulatory elements in gene promoters are crucial for transcription initiation and regulation [56, 57]. In this study, we conducted a cis-regulatory element analysis on the 1.5 kb upstream promoter region of Ipomoea LEA genes. Various CREs associated with plant growth, development, and stress responses were identified (Fig. S2). The identified regulatory elements in Ipomoea species include light-responsive cis-regulatory elements, those related to abiotic stress and external pressure, plant hormone-responsive elements, and elements associated with growth and development. Notably, light-responsive cis-regulatory elements were prevalent within the promoters of Ipomoea LEA genes, alongside widespread elements pertinent to abiotic stress responses and plant hormone regulation. This suggests that LEA family members in Ipomoea species may be involved in various plant hormone responses and play critical roles in mechanisms such as abiotic stress defense, stress response, injury recovery, and anaerobic induction. These findings were consistent with prior research on cis-regulatory elements in LEA family members across tomato, T. aestivum, and G. hirsutum [28, 46, 49]. Additionally, elements associated with meristematic tissue expression were detected in sweet potato, indicating a potential link between LEA genes and the development of sweet potato tubers. Thus, LEA genes in Ipomoea species were pivotal for adversity resistance, warranting further in-depth investigation.

The study utilized transcriptomic data and RT-qPCR results from diverse sweet potato tissues (Fig. 7a, 8). The results suggested that IbLEA genes may play a role in the growth and development of various sweet potato parts, including flower buds, mature leaves, and fruits. Specifically, several IbLEA genes, such as IbLEA27, IbLEA32, and IbLEA59, demonstrated differential expression during distinct developmental stages of sweet potato tubers, indicating their potential involvement in tuber growth and expansion. Additionally, the analysis included transcriptomic data from two closely related wild species, I. trifida and I. triloba, revealing differential expression of LEA genes across tissues in both species (Fig. S3a, b). Notably, the highest number of differentially expressed genes was observed in the flower buds of I. trifida and the roots of I. triloba.

This study investigated the stress resistance of sweet potatoes by analyzing six transcriptome datasets associated with abiotic stress, acquiring the expression patterns of IbLEAs in each dataset (Fig. 7b-f). For comparative analysis, this study also examined and analyzed the LEA genes of I. trifida, I. triloba, and I. aquatica in response to abiotic stress (Fig. S3c-i). In sweet potato, the IbLEAs exhibited significant responsiveness to various abiotic stresses, with a particularly marked reaction to salt, drought, cold, and low potassium conditions. In I. aquatica, IaLEA51 and IaLEA48 exhibit heightened expression at certain time points during salt stress. ItfLEAs and ItbLEAs demonstrated comparable expression patterns under abiotic stress, revealing greater sensitivity of LEA genes to drought stress relative to salt stress and heightened responsiveness to cold stress instead of heat stress. These results offered valuable genetic resources for sweet potato breeding. Similarly, under GA3, 6-BAP, ABA, and IAA treatments, ItfLEAs, and ItbLEAs displayed analogous expression patterns. Notably, ABA treatment resulted in the upregulation of 21 and 20 genes in ItfLEAs and ItbLEAs, respectively, while IAA treatment did not significantly alter the expression levels of either gene group.

Previous studies have established the critical role of LEA genes across multiple species in conferring resistance to abiotic stress. For instance, in soybeans, overexpression of the GmDHN9 gene, a member of the Dehydrin subgroup of LEA proteins, enhanced drought tolerance in transgenic Arabidopsis [58]. In tomatoes, silencing of SlLEA6 diminished drought tolerance [28]. In Ipomoea pes-caprae, accumulating IpLEA proteins improved the capacity to clear reactive oxygen species (ROS), enhancing survival and tolerance under abiotic stress [59]. In our study, we utilized RT-qPCR to investigate the spatiotemporal expression patterns of 15 IbLEA genes under salt and drought stress to elucidate the specific roles of different IbLEAs and attain potential candidate IbLEA genes (Fig. 9). The expression of IbLEA genes was responsive to salt and drought stress, with certain genes exhibiting rapid induction and sustained high expression during specific time intervals, indicating distinct functional roles in various stress contexts. Notably, IbLEA genes in leaves show greater sensitivity to drought stress than high salt stress, whereas in roots, they were more responsive to high salt than drought. Interestingly, some IbLEA genes display similar expression patterns in response to salt and drought stress, supporting findings from multiple studies that underscore the significant role of LEA genes in mediating drought stress responses [8]. In conclusion, the integration of transcriptome data from sweet potato subjected to salt and drought stress, in conjunction with RT-qPCR analysis of 15 IbLEA genes, led to the identification of eight potential candidate genes, IbLEA32, IbLEA64, IbLEA27, IbLEA59, IbLEA26, IbLEA20, IbLEA15, and IbLEA63, that may be crucial for stress resistance in sweet potato.

In this study, we comprehensively identified the LEA gene family members in sweet potato and six closely related species. We identified 73, 64, 77, 62, 70, 70, and 74 LEA genes in sweet potato (I. batatas), I. trifida, I. triloba, I. nil, I. purpurea, I. cairica, and I. aquatica. Phylogenetic analysis revealed that LEA genes in the genus Ipomoea were categorized into eight subgroups, consistent with findings in other plant species. Both phylogenetic and syntenic analyses indicated that the LEA gene family was evolutionarily conserved. Additionally, we explored gene structures, motif organizations, duplication events, homology analysis, and the presence of cis-regulatory elements. Analysis of expression profiles revealed that LEA displayed tissue-specific and varied expression patterns in sweet potato and its two closely related species. Based on the expression profiles of IbLEA genes, we selected 15 DEGs for RT-qPCR analysis under salt and drought stress, indicating that eight IbLEA genes may be involved in sweet potato's resistance mechanisms. This study contributed to our understanding of the evolutionary dynamics of LEA genes within Ipomoea species and provided a foundation for resistance breeding efforts in sweet potato.

Authorship contribution statement

HM, ZL, and XL designed and conducted the experiments and wrote the manuscript. BT and MX performed part of the experiments and analyzed the data, while XL, BT, and HM jointly analyzed the data. XL, ZL, and HM revised the manuscript. ZL, XL, HM, and HZ designed the experiments. All authors have read and approved the final manuscript.

Acknowledgments

Not applicable.

Ethical Approval and Consent to Participate

The sweet potato materials used in this study were obtained from the College of Coastal Agriculture, Guangdong Ocean University, Guangdong Province, China. The collection and utilization of sweet potato materials, as well as the methods employed in this study, adhere to the guidelines and regulations set forth by relevant institutions, and national, and international standards.

Consent to Publication

Not applicable.

Conflict of interest

The authors declare no competing interests.

Author Details

1 Department of Biotechnology, College of Coastal Agricultural Sciences, Guangdong Ocean University, Zhanjiang 524088

Funding

This research was funded by the Natural Science Foundation of China-Guangdong Joint Fund, China, and Studies on Resistance Resources and Molecular Mechanisms of Sweet Potato Weevil in South China (Grant number U1701234).

Availability of data and materials

All datasets supporting the results of this study are included in this article and its Supplementary data files.

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Comparative analysis of the LEA gene family in seven Ipomoea species focuses on sweet potato (Ipomoea batatas)

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Abstract

Figures

1. Introduction

2. Material and methods

2.1 Identification of LEA genes in Ipomoea species

2.2 Physicochemical properties analysis of Ipomoea LEA proteins

2.3 Phylogenetic analysis of Ipomoea LEA proteins

2.4 Chromosome distribution and collinearity analysis of Ipomoea LEA genes

2.5 Synteny analysis and calculation of Ka/Ks values of Ipomoea LEA genes

2.6 Cis-acting element analysis of the Ipomoea LEA genes

2.7 Protein conserved domains and motif and gene structure analyses of Ipomoea LEA genes

2.8 Expression pattern analysis of sweet potato LEA genes

2.9 RT-qPCR analysis of sweet potato LEA genes

2.10 Protein‑protein interaction (PPI) network construction

3. Results

3.1 Genome-wide identification and phylogenetic analysis of Ipomoea LEA genes

3.2 Physicochemical properties and subcellular localization of the Ipomoea LEAs

3.3 Chromosome location and duplication analysis of the Ipomoea LEA genes

3.4 Syntenic analysis of LEA genes in the genomes of Ipomoea species

3.5 Ka/Ks analysis of duplicated and syntenic Ipomoea LEA genes

3.6 Conserved structural domains and gene structure analysis of Ipomoea LEA genes

3.7 Cis-regulatory elements in putative promoter regions of the Ipomoea LEA genes

3.8 Protein Interaction Network of IbLEAs in Sweet Potato

3.9 Expression profiles of the Ipomoea LEA genes in various tissues

3.10 Expression profiles of the Ipomoea LEA genes under abiotic stress

3.11 Expression analysis of sweet potato LEA genes by RT-qPCR

4. Discussion

5. Conclusion

Declarations

References

Additional Declarations

Supplementary Files

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