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Genome-wide identification and expression analysis of the class III peroxidase gene (PRXIII) family in Medicago sativa L. and its function in the abiotic stress response

BMC Plant Biology volume 25, Article number: 443 (2025) Cite this article

Abstract

Peroxidase (POD) is a widespread and highly active enzyme in plants that plays an important role in plant growth and development and stress response. No genome-wide analysis and characterization of the POD gene family in alfalfa has been performed yet. In this study, we used bioinformatics techniques to identify 343 members of this family in alfalfa and performed predictive analyses of their physicochemical properties, subcellular localization, phylogenetic relationships and conserved motifs. Expression analysis showed that 58 of the 343 genes were specifically expressed. Expression pattern analysis under different stresses showed that the MsPOD gene family was responsive to salt stress, cold stress, and drought stress, and there were genes responsive to multiple stresses. Among them, 24 MsPOD genes responded to all three stresses. Understanding the expression patterns of alfalfa MsPOD family members can enhance alfalfa's ability to resist abiotic stresses, thereby providing a theoretical basis for increasing alfalfa yield under adverse conditions.

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Introduction

Alfalfa (Medicago sativa L.) is an herbaceous plant of the legume family that has been cultivated in China for nearly 2000 years [1] and has consistently been a very important source of protein feed for animal husbandry [2]. Among other characteristics, alfalfa exhibits high nutritive value, strong adaptability, and easy digestibility by livestock, which has earned it the title of the ‘king of pasture grasses’ [3, 4]. Additionally, its high yield makes it commercially valuable [5].

Peroxidases are a group of microbial or plant oxidoreductases that facilitate numerous reactions by using hydrogen peroxide as the electron acceptor to oxidize substrates. With iron porphyrin as a cofactor, peroxidases are primarily located in the peroxisomes of cells. They catalyze the oxidation of phenolic and amine compounds by hydrogen peroxide, thereby eliminating the toxicity of both hydrogen peroxide and these compounds [6, 7]. Peroxidases are widely found in plants, animals, and microorganisms and are classified into three superfamilies according to their properties. In plants, the structure of peroxidases varies greatly, and they are classified into class I, class II, and class III according to their structure [8,9,10]. Although sequence homology among these three classes is low, five key amino acids critical for catalysis, structural integrity, and helical folding of the polypeptide are strictly conserved across all classes [11]. In plants, there is a specific peroxidase (PRX) III that regulates the hardening and loosening of the cell wall through peroxidation and hydroxyl cycling [12]. Due to its enzymatic properties, PRXIII plays crucial roles in various physiological processes, including germination, growth, development, and pathogen resistance. Additionally, it contributes to lignification by influencing lignin monomers, making it a major factor in lignin development in plants.

The PRXIII family plays an important role in plants, has and members of this family have been identified in several species, such as Arabidopsis thaliana [13], Glycine max [14], Medicago truncatula [15], Oryza sativa L. [16, 17], Betula pendula [18], Triticum aestivum L [19]. and Vitis vinifera L. [20]. Among them, transgenic Arabidopsis plants expressing antisense cDNA (FBP1) encoding a type III peroxidase were used to verify that the genes Atprx33 and Atprx34 are associated with hydrogen peroxide during defense and pathogen resistance [21, 22]. In A. thaliana, three genes, AtPrx4 [23], AtPrx72 [24, 25], and AtPrx52 [26], are associated with lignin synthesis, and mutants of both AtPrx4 and AtPrx72 genes decrease the total amount of lignin, as well as the proportion of the lignin monomer S, whereas AtPrx52 plays an important role in the lignification of angiosperms. The PRXIII family was found to play a similar role during pear fruit growth [27]. Overexpression of AtPrx22, AtPrx39, and AtPrx69 increased cold resistance in Arabidopsis [28], these peroxidase genes are responsible for the increase in cold resistance in Arabidopsis by increasing the level of ROS accumulation and the expression of genes encoding ROS metabolizing enzymes. Moreover, there is a functional redundancy of these genes, and study of one of them may result in non-significant experimental results. With respect to PRXIII, relatively few specific gene functions have been identified in species other than the model plant A. thaliana, but some preliminary studies have shown that the PRXIII family is potentially involved in the stress response in wheat [29]. In citrus, overexpression of the CsPrx25 gene was found to increase H2O2 levels and reverse the pattern of H2O2 induction during pathogen infection. This regulation of ROS homeostasis, along with enhanced cell wall lignification, enhances plant resistance to pathogens [30]. MdPRX34L was found to enhance plant‒pathogen defenses against gray mold (Botryosphaeria dothidea) by regulating the SA and ABA pathways in apple [31].

PRXIII in plants primarily influences lignin-related pathways and resistance to biotic and abiotic stresses. These properties significantly impact the quality, growth, and adaptability of alfalfa. However, research on alfalfa in this area remains limited. To date, only preliminary validation of the sequence and structure of peroxidase (POD) in alfalfa has been conducted [32], Additionally, the POD gene sequence (GenBank Registry Number L36157.1) has been cloned and validated in Arabidopsis thaliana for its role in resistance to H2O2 and NaCl stress [33]. The POD gene in alfalfa was only functionally verified in Arabidopsis thaliana, likely due to limitations in the alfalfa transformation system at the time. Additionally, as a homotetraploid, alfalfa exhibits high heterozygosity and intersubspecies heterozygosity, resulting in a highly repetitive and complex genome. These characteristics significantly increase the difficulty of genome analysis, making the fineness and accuracy of the study relatively insufficient compared with other species. Therefore, identifying PRXIII gene family members and analyzing their expression patterns will advance our understanding of physiological and metabolic processes in alfalfa. This research will provide valuable insights into the role of alfalfa genes in stress response and tolerance, while also guiding future studies. Ultimately, these efforts aim to improve alfalfa quality, ensure the availability of protein-rich feeds, and promote the development of animal husbandry.

Results

Identification, characterization and physicochemical properties of MsPOD genes

Arabidopsis thaliana is a model organism in plant science with a fully sequenced genome, detailed functional annotation, and extensive functional studies on AtPOD genes. Therefore, we used the 73 protein sequences of the Arabidopsis thaliana PRXIII family as a reference for BLASTP comparison with the genome of the alfalfa cultivar ‘Xinjiangdaye’. We subsequently analyzed the structural domains of the identified alfalfa POD gene-encoding proteins via the Pfam and HMMER tools. Following rigorous screening and merging to eliminate redundancies, we successfully identified 343 unique MsPOD genes, which were systematically renamed MsPOD1 through MsPOD343 on the basis of their chromosomal positions.

A comprehensive analysis of the physicochemical properties of these genes was performed, and the results are summarized in Table S1. This table provides a detailed overview of the properties, including the gene number, name, location within the genome, chromosomal localization, subcellular localization, instability coefficient, theoretical isoelectric point, and protein length, of each POD gene. With respect to subcellular localization, our findings revealed that a majority (270 genes) were intracellularly localized, with chloroplasts being the most prevalent destination for 151 genes, followed by the cytoplasm (38 genes), vesicles (36 genes), and the nucleus (22 genes). A minor fraction of the genes were localized to the plasma membrane, endoplasmic reticulum, mitochondria, and peroxisomes.

Notably, 85 of the total POD genes presented protein instability coefficients exceeding 40, a threshold commonly associated with protein instability. The isoelectric point ranged from a low value of 4.38 (MsPOD116) to a high value of 9.65 (MsPOD245), whereas the protein length varied significantly, ranging from a minimum of 250 amino acids (MsPOD15) to a maximum of 1425 amino acids (MsPOD196).

Phylogenetic analysis of POD genes in Medicago sativa

In order to investigate their evolutionary relationships, a phylogenetic tree was constructed from the amino acid sequences of 343 alfalfa POD family members and 73 A. thaliana POD family members using TBtools software. The POD families of the two species were divided into five groups, with group four being the largest, accounting for approximately 50% of the total, and group three being the smallest, with only 38 members, accounting for approximately 10% of the total (Fig. 1).

Fig. 1
figure 1

Phylogenetic tree of POD genes in Medicago sativa and Arabidopsis thaliana

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Analysis of the basic structure and conserved domains of POD genes in Medicago sativa

In order to find functional structural domains in the sequence,using the MEME motif search tool, 10 related conserved motifs were identified (Fig. 2). Among the many MsPOD genes, the differences between the motifs were not obvious, but the lengths of introns and exons varied greatly among the genes (Fig. 3). Some genes were essentially intronless, consisting of a CDS and conserved structural domains, and some genes contained multiple introns. Only one of the genes, MsPOD154, encoded a peroxidase, while none of the other genes had the particular structural domains associated with peroxidases.

Fig. 2
figure 2

Basic information on the MsPOD protein motifs in Medicago sativa

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Fig. 3
figure 3

Analysis of the gene structure and motifs of the MsPOD genes in Medicago sativa

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Chromosomal locations and collinearity analysis of the MsPOD genes in Medicago sativa

Among the 343 alfalfa POD genes identified, 19 genes (MsPOD325-MsPOD343) were not located on chromosomes, whereas the remaining genes were distributed across all 32 chromosomes (Fig. 4). Chr7.3 and chr7.4 had the lowest number of genes, with only two genes each, namely, MsPOD288 and MsPOD289 on chr7.3 and MsPOD290 and MsPOD291 on chr7.4, followed by chr6.3, with three genes, namely, MsPOD270, MsPOD271, and MsPOD272. Additionally, several chromosomes had relatively few genes, with chr6.2, chr6.4, and chr7.1 containing four genes each. Chr2.4 had the greatest number of genes, i.e., 22.

Fig. 4
figure 4

Chromosomal distribution of POD genes in Medicago sativa

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The statistics on gene duplication events within the PRXIII family of alfalfa revealed that 39 tandem duplication events occurred in 115 genes (Table S2). The tandem duplication events involving the most genes were located on chr2.4 (Fig. 4); specifically, these events occurred in MsPOD114, MsPOD115, MsPOD116, MsPOD117, MsPOD118, MsPOD119, and MsPOD120. The next most frequent tandem duplication events were those that occurred on chr2.2 and chr2.3, involving MsPOD74, MsPOD75, MsPOD76, MsPOD77, MsPOD78, and MsPOD79 and MsPOD92, MsPOD93, MsPOD94, MsPOD95, MsPOD96, and MsPOD97, respectively. Furthermore, 234 segmental duplication events were detected (Table S3), affecting 161 MsPOD genes (Fig. 5). Segmental duplication events occurred on all chromosomes except chr1.1, chr1.2, chr1.3, chr1.4, and chr2.1. In the alfalfa POD family, tandem and segmental duplication events are relatively common, as gene duplication events serve as important pathways for gene amplification and functional diversification [34,35,36]. It is therefore presumed that alfalfa underwent significant changes to adapt to environmental variations, with segmental duplication events being the primary evolutionary force.

Fig. 5
figure 5

Regional collinearity of MsPOD genes in Medicago sativa. The red lines represent duplication events of MsPOD genes, the chromosome number is labeled within the gray rectangles

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Joint collinearity analysis revealed the presence of 131 collinear gene pairs between alfalfa and Arabidopsis (Fig. 6), involving 75 alfalfa POD genes and 33 Arabidopsis genes. Notably, alfalfa and Medicago truncatula presented significantly more collinear gene pairs than did alfalfa and Arabidopsis, with 269 collinear gene pairs, involving 195 alfalfa POD genes and 71 Medicago truncatula homologs.

Fig. 6
figure 6

Collinearity analysis of MsPOD genes in Arabidopsis thaliana and Medicago truncatula. Gray lines in the background represent the collinear blocks within Medicago sativa and other species, while the colored lines highlight the collinear POD gene pairs

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Analysis of cis-acting elements in the promoter regions of POD genes in Medicago sativa

To delve deeper into the functionality of the POD family in alfalfa, we conducted an analysis of the 2000 bp cis-regulatory elements located upstream of the POD family genes (Table S4). These elements were subsequently categorized into 19 distinct groups, culminating in the identification of a comprehensive total of 8,022 elements (Fig. 7). Notably, light-responsive cis-acting elements were the most prevalent, occurring 3,972 times across all the alfalfa POD family promoter sequences and constituting approximately 50% of all the identified cis-acting elements. These were followed closely by elements induced by jasmonic acid and anaerobic conditions. These elements are intricately linked to various growth hormones, notably gibberellins, and play pivotal roles in plant growth processes, circadian rhythms, cell cycle regulation, meristematic tissue development, chloroplast function, and wound response mechanisms. Consequently, the abundance of multiple cis-acting elements within the alfalfa POD family underscores the genetic richness and complexity of this family.

Fig. 7
figure 7

Cis-acting elements of the MsPOD gene promoters in Medicago sativa

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Expression profiling of POD genes in different organs of Medicago sativa

To elucidate the expression patterns of diverse genes within the alfalfa POD family across various tissues, we sourced transcriptomic data from six distinct alfalfa tissue types (roots, elongated stems, preelongated stems, leaves, flowers, and nodules) from publicly accessible databases (Table S5). Analysis of tissue transcriptome data for the 343 MsPOD genes revealed that 257 of these genes could be expressed in at least one tissue (Fig. 8), and 86 genes were not expressed in these 6 tissues. Following data analysis and summarization, we discerned that 61 MsPOD genes within the family are ubiquitously expressed across these six tissues, exemplified by MsPOD25 and MsPOD41 (Fig. 8A). Conversely, 86 genes, such as MsPOD3 and MsPOD15, remained entirely unexpressed in any of the examined tissues. The remaining genes presented varying degrees of tissue-specific expression, with at least partial expression observed in one or more tissues. Notably, a subset of these genes, including MsPOD111, MsPOD149, and MsPOD172, exhibited strong tissue specificity and were exclusively expressed in elongating stems (Fig. 8F).

Fig. 8
figure 8

Relative expression patterns of MsPOD genes in different tissues. A MsPOD genes expressed in six tissues. B MsPOD genes expressed in five tissues. C MsPOD genes expressed in four tissues. D MsPOD genes expressed in three tissues. E MsPOD genes expressed in two tissues. F MsPOD genes expressed in only one tissue

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Intriguingly, although certain genes were detected in all tissues, their expression levels were significantly different, such as MsPOD304 and MsPOD305(Fig. 8A), exceeding those of other genome-wide genes, suggesting that they may have potential functional significance.

Expression of POD genes in Medicago sativa under abiotic stress

The POD family is instrumental in numerous chemical reactions and plays a pivotal role in adversity response. Consequently, we investigated the expression profiles of POD family genes in response to drought, salt, and cold stresses (Tables S6-S8), aiming to elucidate their specific contributions to stress adaptation. Our findings revealed that the highest number of POD family genes activated in response to stress was 105, activated under salt stress (Fig. 9A), followed by 82 genes activated under drought stress (Fig. 9B) and 40 genes activated under cold stress (Fig. 9C). Notably, these stress-responsive genes exhibited multifunctionality, with 24 genes, including MsPOD182, MsPOD82, and MsPOD189, responding simultaneously to all three stresses (Fig. 9D).

Fig. 9
figure 9

Relative expression patterns of MsPOD gene family members under three stresses. A Expression of MsPOD genes in Medicago sativa under salt stress. B Expression of MsPOD genes in Medicago sativa under drought stress. C Expression of MsPOD genes in Medicago sativa under cold stress. D Venn diagrams of MsPOD genes responding to the three stresses. From S1-S6 represent 0.5h, 1h, 3h, 6h, 12h and 24h under salt stress, respectively. From M1-M5 represent 1h, 3h,6h, 12h,24h, under drought stress, respectively. From C1-C4 represent 2h, 6h,24h,48h, under drought stress, respectively.0h as the CK

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In addition, we identified 1, 4, and 52 genes in the POD family that were responsive to specific stresses (drought and cold, cold and salt, and salt and drought, respectively). Notably, MsPOD107, although responsive to both drought and cold stresses, presented different expression patterns, with a maximum upregulation of up to 3.5-fold under drought stress and upregulation followed by downregulation under cold stress.

Similarly, the expression patterns of MsPOD281, MsPOD74, MsPOD11, and MsPOD212 under cold and salt stresses were also disparate, suggesting the existence of intricate gene regulatory mechanisms underlying their stress-specific responses. These observations underscore the complex and nuanced roles played by the POD family in mediating plant responses to diverse environmental challenges.

Expression of MsPOD genes in Medicago sativa under abiotic stress

To verify the accuracy of the RNA-seq data under different stresses (salt, drought, and cold), we selected four POD genes that were responsive to all three stresses. The experimental primers used are presented in Table S9. We also performed real-time quantitative PCR (qRT‒PCR) on the four MsPOD genes, and the results revealed that the expression patterns of the four MsPOD genes determined via qRT‒PCR were consistent with those detected by RNA-seq (Fig. 10). As shown in the figure, MsPOD220 showed a large change trend under salt stress, drought stress and cold stress. The expression of MsPOD189 significantly increased only under cold stress. Several other genes also presented different trends under different stresses. Overall, all the results indicated that the RNA-seq expression profiling results were reliable.

Fig. 10
figure 10

MsPOD expression under salt, drought and cold stress conditions according to RT–qPCR. From S1-S6 represent 0.5h, 1h, 3h, 6h, 12h and 24h under salt stress, respectively. From M1-M5 represent 1h, 3h,6h, 12h,24h, under drought stress, respectively. From C1-C4 represent 2h, 6h,24h,48h, under drought stress, respectively.0h as the CK

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Discussion

The multigene family of PODs plays pivotal regulatory roles in diverse physiological processes crucial to plant growth and development, stress tolerance, and photosynthesis. Given their importance, we conducted a thorough analysis of the physicochemical properties, phylogenetic relationships, chromosomal localization, gene collinearity, cis-regulatory elements, and expression patterns of POD family members in alfalfa. Through comparison with the well-established Arabidopsis POD class III family, coupled with structural domain screening, we identified 343 alfalfa POD genes. The results show that many POD genes are directly homologous between alfalfa and Arabidopsis, and PRXIII is functionally conserved across species. Therefore, Arabidopsis serves as a scientifically justified comparative template. However, due to the higher evolutionary level of alfalfa (e.g., genomic complexity or longer evolutionary history), functional divergence may exist, necessitating further experimental validation.

Our findings revealed a nonuniform distribution of POD genes across chromosomes, with a preponderance of genes concentrated on chr 2.1–2.4, chr4.1–4.4, and chr5.1–5.4. The lengths of POD proteins exhibited substantial variation, with the longest sequence being nearly six times longer than the shortest sequence. Moreover, the subcellular localization of POD proteins is broad, encompassing the cell membrane, nucleus, chloroplasts, vesicles, peroxisomes, endoplasmic reticulum, and even extracellular spaces. This broad distribution underscores the paramount importance of PODs in plant cells, emphasizing their multifunctionality and vital roles. Based on the heterogeneous distribution of the POD gene on chromosomes, we noted that, although both chromosomal tandem duplications and segmental duplications are distributed throughout the genome, chromosomal tandem duplications are particularly concentrated on chr2.1–2.4, chr3.1–3.4, and chr4.1–4.4. This suggests that the distribution of these tandem duplications bears some similarity to that of the POD gene. However, chromosomal segmental duplications do not share this feature, and they are abundant on all chromosomes except chr1.1–1.4. In the process of genome evolution, gene duplication was the main factor that led to gene family expansion [37], and considering the abundance of gene duplication within the alfalfa POD family, we posit that the evolutionary dynamics of this family in alfalfa are driven primarily by fragment duplication, as well as tandem duplication. These mechanisms collectively contribute to the ongoing refinement of the genes and functions within this family.The 343 MsPOD proteins possess ten highly conserved motifs. Most MsPODs contain at least eight conserved motifs, so these motifs may be relevant to MsPOD function. The diversity of gene structure plays an important role in gene family evolution [38, 39]. By studying their gene structure, we found that a portion of the MsPOD genes were essentially intronless, which may be associated with specific pathways [40, 41]. This result is the same as that of previous studies [18]. Moreover, evolutionary analysis revealed that the POD family in alfalfa has evolved at a greater level than that in A. thaliana. This can be attributed to the fact that the alfalfa genome analyzed belongs to the Xinjiang macrophyte variety, which is tetraploid and consequently harbors a greater number of genes [42]. In terms of gene count, MsPODs far surpassed that the POD genes from Arabidopsis thaliana. Furthermore, through joint collinearity analysis exploring the phylogenetic relationships among Arabidopsis, alfalfa, and Medicago truncatula, we found that alfalfa and Medicago truncatula are closely related. Specifically, collinearity analysis between alfalfa and Medicago truncatula yielded 269 gene pairs, involving 195 alfalfa genes and 71 Medicago truncatula genes. In contrast, the analysis between alfalfa and Arabidopsis thaliana identified only 131 gene pairs, with a relatively lower number of genes involved: 75 from alfalfa and 33 from Arabidopsis.

To better investigate the physiological activities and functions of the POD family, we analyzed the homeopathic elements in the promoter region and found that the most abundant homeopathic elements were those involved in the light response, which is consistent with the results of previous studies on other POD families. In addition to the homeopathic elements for the light response, we identified elements associated with salicylic acid, jasmonic acid, gibberellins, and growth hormones, which are important hormones and substances in plants, whereas the POD family also includes elements related to expression in meristematic tissues, specific expression in chloroplasts, the seed endosperm, circadian rhythms, low temperatures, protein metabolism, the cell cycle, and the response to traumatic injuries. These are all important physiological and metabolic processes in plants, so the POD family clearly plays an important role in regulating the growth and physiological activities of plants.RNA expression is an important factor influencing biological phenotypes. On the basis of gene expression levels in different tissues, as well as the cis-acting elements and subcellular localization of the gene, gene function can be inferred. As shown in the figure, most of the POD gene expression was concentrated in the root system. This may indicate that most POD genes function in the root system. In addition, MsPOD10 and MsPOD45 were expressed at much higher levels in flowers than were the other genes. There were 27 action elements in the promoter region of MsPOD10, and these were divided into seven categories: 17 were related to light, followed by three elements necessary for the induction of anaerobiosis, two elements involved in circadian rhythms, and elements related to salicylic acid, gibberellins, and erythrocyanic acid. This promoter region also harbored one MYB-binding site. The composition of the cis-acting elements in the promoter sequence of MsPOD45 was similar to that in the promoter sequence of MsPOD10, so it was hypothesized that MsPOD10 and MsPOD45 play similar roles in flowers, possibly related to light responses, anaerobic induction, and circadian rhythms.MsPOD91 was the most highly expressed of the many root-expressed POD genes. An analysis of its promoter sequence revealed that, in addition to a portion of the gene that responds to light, more than one part of the gene is related to the light response, anaerobic induction, and circadian rhythms. The analysis of its promoter sequence also revealed that, in addition to its role in light response, it is closely involved in the regulation of jasmonic acid, salicylic acid, and abscisic acid signaling and in the response to low temperature. There were two MYB binding sites in the promoter sequence, indicating that MsPOD91 might respond to low-temperature stress by participating in the regulation of jasmonic acid, salicylic acid, and abscisic acid signaling. This gene could also form a regulatory network with MYB genes, jointly regulating its adaptive capacity under stress.MsPOD133 was highly expressed in elongating stems, and its action elements were related to growth hormones and gibberellins, so the gene may promote further plant growth through these two hormones. MsPOD261 was highly expressed in internodes, and its action elements were related to light, low temperature, and abscisic acid. Notably, the homeopathic regulatory elements related to meristematic tissues may be involved in lignin regulation, because internodes consist of a large amount of lignin.Adaptation to adversity is particularly important for plant survival and development, and growth under adversity plays an indispensable role in breeding and expanding cultivation. We analyzed the expression of POD genes under adversity (low temperature, drought, and salt stress) to identify relevant genes with regulatory roles in the response to adversity. There were 24 genes in this family that were involved in the response to three types of adversity, and their expression significantly differed from that in the control group under these three types of stress (P < 0.05).

Taken together, the findings show that the POD family is associated with abiotic stress responses [43,44,45], light responses, and lignin synthesis, which is consistent with previous findings [14, 15, 17, 20]. The ability to breed alfalfa to resist abiotic stress, adapt to different planting environments, and respond to light, improve its photosynthetic capacity to increase yield, or reduce its lignin content to extend the harvesting period is crucial for the promotion of alfalfa cultivation and the expansion of economic benefits. Therefore, the identification of POD family genes and further study of their functions can provide theoretical support for the livestock industry to provide better-quality feed protein and promote the further development of the livestock industry.

Conclusion

In summary, we identified 343 POD genes in Medicago sativa. These POD genes were classified into 5 groups according to their phylogenetic relationships. The MsPODs were found to be distributed in different numbers on the 32 chromosomes. In addition, we identified ten conserved domains of MsPOD proteins. Finally, expression pattern analysis revealed that some MsPODs might play significant roles in the root, xylem, leaf and flower. Furthermore, under abiotic stress conditions, some MsPODs showed different expression patterns at different times. Here, a preliminary study was conducted on the POD genes in Medicago sativa, laying a foundation for further research on the function of the POD gene family.In the subsequent experiments, we will further verify the specific function of peroxidase (POD) and investigate its mechanism in plant resistance to abiotic stress through examining POD's role in ROS homeostasis, with the ultimate goals of enhancing alfalfa's stress tolerance and expanding its application potential.

Materials and methods

Identification and basic information of POD genes in Medicago sativa

The amino acid sequence of alfalfa, the alfalfa gene annotation file (gff file), and the genomic nucleic acid sequence were obtained from a database (https://fgshare.com/projects/whole_genome_sequencing_and_assembly_of_Medicago_sativa/66380) [46]. The sequence of the Arabidopsis thaliana AtPOD protein was used as the query sequence for BLASTP comparison (E value < 1E-5) to obtain homologous sequences. The conserved structural domains of MsPODs (PF00141) were downloaded from the Pfam database [47] (http://pfam.xfam.org/), and the conserved structural domains were compared using HMM3.0 (http://hmmer.org/). The alfalfa MsPOD family members containing conserved structural domains were subsequently screened, and MsPOD family members were selected using BLAST and HMMER. The isoelectric point, molecular weight, and instability analyses of MsPOD proteins were performed using the online tool ExPASy (https://web.expasy.org/protparam/). The subcellular localization of the MsPOD gene was predicted via the WoLF PSORT website (https://wolfpsort.hgc.jp/).

Phylogeny, gene structure, and conserved motif analysis of the MsPOD gene family in alfalfa

MEGA11 is a comprehensive tool with efficient and accurate algorithms, suitable for gene sequence comparison and subsequent analysis. Therefore, MsPOD protein sequences were aligned using MEGA11 [48] with the following parameters: Gap Opening Penalty: 10, Gap Extension Penalty: 0.2. A phylogenetic tree was constructed using the neighbor-joining (NJ) method with 1000 bootstrap replicates. Gene structure analysis was performed based on GFF gene annotation files. To identify MsPOD protein motifs, MsPOD amino acid sequences were analyzed using the MEME website [49] with default parameters, and 10 motifs were identified. Visualization was conducted using TBtools [50] software.

Chromosomal localization and collinearity analysis of MsPOD genes in alfalfa

The physical location of the MsPOD genes on the chromosome was identified from the data in the annotation file of the alfalfa genome. The physical map of the genes on the chromosome was constructed using TBtools. Collinearity analysis (E value < 1E-5) was performed using MCScanX [51], and Advanced Circos was used for visualization.

Cis-acting element analysis of the alfalfa MsPOD genes

The DNA sequence 2.0 kb upstream of the CDS of each alfalfa MsPOD gene was extracted and submitted to the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html) for the prediction of promoter cis-acting elements. The cis-acting elements were visualized and further screened.

Expression characteristics of the alfalfa MsPOD genes in different tissues

To identify the expression characteristics of the alfalfa MsPOD genes in different tissues, RNA-seq data from six tissues (roots, elongated stems, preelongated stems, leaves, flowers, and nodules), as well as RNA-seq data from alfalfa under salt stress, drought stress, and cold stress (SRP055547), were downloaded from the NCBI database. The RNA-seq data (SRR7091780-SRR7091794 and SRR7160313-SRR7160357) collected under salt stress, drought stress, and cold stress were analyzed [52, 53].

Plant materials, growth and stress conditions, and RT‒qPCR analysis

The alfalfa variety used in this study was Zhongmu No. 1, which was obtained from the Institute of Animal Science of the Chinese Academy of Agricultural Sciences. Seeds were treated at 4 °C for 3 days and then incubated in a greenhouse (photoperiod of 16/8 h, relative humidity of 70–80%, day/night temperature of 24 °C/20 °C) for 2 weeks. Salt stress was simulated using a 250 mM NaCl solution. Root tip samples were collected at 0 h (as the control) and at 0.5, 1, 3, 6, 12, and 24 h as sampling time points. Mannitol (400 mM) was used to simulate drought stress, and root tip samples were collected at 1, 3, 6, 12, and 24 h. Plants not treated with cold stress (0 h at 4 °C) served as the control, and leaf samples were collected at 2, 6, 24, and 48 h as sampling time points. Three replicates of each stress treatment were used, each containing five seedlings. Untreated control plants were grown under normal conditions.

The primers used in the study were designed using Primer 5.0 software,and the primers involved in this paper are shown in the attached Table S9.Total RNA from all samples was extracted with TRIzol reagent according to the manufacturer's instructions, and the corresponding cDNAs were obtained with the EasyScript First-Strand cDNA Synthesis Kit [random primers (N9)]. RT‒qPCR experiments were performed using SYBR Premix Ex Taq (TaKaRa, Japan) and a 7500 real-time fluorescence quantitative PCR system (Applied Biosystems, Foster City, CA, USA). Three replicate analyses were performed for each sample, and the data were normalized to alfalfa actin gene expression. Relative expression levels were calculated using the 2−ΔΔCT method [54], with Actin used as an internal reference.

Data availability

No datasets were generated or analysed during the current study.

Abbreviations

POD:

Peroxidase

PRX:

Peroxidase

FBP1:

Fructose-Bisphosphatase 1

NJ:

Neighbor-joining

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Funding

This work was supported by the National Natural Science Foundation of China (32371757, 32441018), the major demonstration project “The Open Competition” for Seed Industry Science and Technology Innovation in Inner Mongolia (No. 2022JBGS0016).

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  1. Yuqi Zhang and Hao Liu contributed equally to this work.

Authors and Affiliations

  1. Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, 100193, China

    Yuqi Zhang, Hao Liu, Xinyue Ma, Li Zhao, Fei He, Mingna Li, Xue Wang, Ruicai Long, Junmei Kang, Qingchuan Yang & Lin Chen

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Contributions

Experimental design and planning and first draft writing, Y.Z, L.C.; preparation and modification of the images, H.L., X.M.; data processing, manuscript modification, F.H., L.Z.; data analysis and test data accuracy, Y.Z., X.W. and M.L.; application and analysis of the software used in the experiment, Y.Z., R.L.; data and manuscript review, J.K., Q.Y.; funding acquisition, L.C. All the authors contributed to the article. All authors have read and agreed to the published version of the manuscript.

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Correspondence to Lin Chen.

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Zhang, Y., Liu, H., Ma, X. et al. Genome-wide identification and expression analysis of the class III peroxidase gene (PRXIII) family in Medicago sativa L. and its function in the abiotic stress response. BMC Plant Biol 25, 443 (2025). https://doi.org/10.1186/s12870-025-06470-5

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BMC Plant Biology

ISSN: 1471-2229

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