Genome-Wide Analysis of the Xyloglucan Endotransglucosylase/Hydrolase (XTH) Gene Family: Expression Pattern during Magnesium Stress Treatment in the Mulberry Plant (Morus alba L.) Leaves

Mulberry (Morus alba L.), a significant fruit tree crop, requires magnesium (Mg) for its optimal growth and productivity. Nonetheless, our understanding of the molecular basis underlying magnesium stress tolerance in mulberry plants remains unexplored. In our previous study, we identified several differential candidate genes associated with Mg homeostasis via transcriptome analysis, including the xyloglucan endotransglucosylase/hydrolase (XTH) gene family. The XTH gene family is crucial for plant cell wall reconstruction and stress responses. These genes have been identified and thoroughly investigated in various plant species. However, there is no research pertaining to XTH genes within the M. alba plant. This research systematically examined the M. alba XTH (MaXTH) gene family at the genomic level using a bioinformatic approach. In total, 22 MaXTH genes were discovered and contained the Glyco_hydro_16 and XET_C conserved domains. The MaXTHs were categorized into five distinct groups by their phylogenetic relationships. The gene structure possesses four exons and three introns. Furthermore, the MaXTH gene promoter analysis reveals a plethora of cis-regulatory elements, mainly stress responsiveness, phytohormone responsiveness, and growth and development. GO analysis indicated that MaXTHs encode proteins that exhibit xyloglucan xyloglucosyl transferase and hydrolase activities in addition to cell wall biogenesis as well as xyloglucan and carbohydrate metabolic processes. Moreover, a synteny analysis unveiled an evolutionary relationship between the XTH genes in M. alba and those in three other species: A. thaliana, P. trichocarpa, and Zea mays. Expression profiles from RNA-Seq data displayed distinct expression patterns of XTH genes in M. alba leaf tissue during Mg treatments. Real-time quantitative PCR analysis confirmed the expression of the MaXTH genes in Mg stress response. Overall, this research enhances our understanding of the characteristics of MaXTH gene family members and lays the foundation for future functional genomic study in M. alba.


Introduction
Mulberry (Morus spp.) is an essential plant in many Chinese provinces, primarily cultivated for its leaves and fruits.It holds particular significance in sericulture, as mulberry leaves serve as the exclusive food source for domestic silkworms (Bombyx mori L.) [1,2].Besides its historical role in silkworm rearing, mulberry, especially Morus alba (M.alba), demonstrates potential as a pioneer tree species in marginal environments [3].Moreover, the leaves of M. alba are of high medicinal value [4] and are believed to possess antioxidant, anti-inflammatory, and anti-allergic properties attributed to various bioactive phytochemicals, including polyphenolic compounds, triterpenoids, and anthocyanins.Although M. alba is of significant economic importance, its growth and development are subject to the influence of nutrient concentrations.High magnesium (Mg) levels or Mg deficiency are among the key factors affecting the growth and development of the plant [5].Nevertheless, M. alba's stress response to different Mg concentrations is unclear, especially at the genomic level.Mg deficiency frequently hinders crop yield in sandy or highly acidic soils, primarily attributable to the high leaching susceptibility of Mg.This occurrence is widely observed and has notable implications for agricultural productivity in such soil conditions [6].Extensive investigations have scrutinized and unveiled the consequences of Mg deficiency on plant physiological aspects, including biomass distribution, carbon dioxide (CO 2 ) uptake, and protection against photooxidative stress [7], resulting in yield reduction and poor fruit quality [8,9].In response to these challenges, plants have developed intricate regulatory mechanisms, including the involvement of distinct gene families, such as Xyloglucan endotransglucosylase/hydrolases (XTHs) [10].XTHs genes are classified within the glycoside hydrolase family 16 and are an essential group of enzymes primarily responsible for cleaving and rearranging the xyloglucan backbones within plant cell walls [11][12][13].Specifically, family members of this gene carry out two distinct biochemical processes that are catalyzed by two specific enzymes: xyloglucan endotransglycosylase (XET) and xyloglucan endohydrolase (XEH) [14].XET catalyzes the transfer of one xyloglucan molecule to another, resulting in the elongation of xyloglucan, whereas XEH is characterized by hydrolyzing an individual xyloglucan molecule, causing an irreversible reduction in the length of the xyloglucan chain [13].
Several XTHs exhibit catalytic properties and play an essential role in regulating the extensibility of plant cell walls, root elongation, and plant growth [11,15].Due to the advancement of sequencing technology and data availability, an expanding repertoire of XTH genes has been discovered and characterized in a broader range of species including Ananas comosus (48) [16], Arachis hypogaea L. (58) [17], Glycine max (61) [18] Arabidopsis thaliana [19], lpomoea batatas (36) [20], Oryza sativa (29) [21], Solanum lycopersicum L. (37) [11], Nicotiana tabacum (56) [22] Brassica rapa (53) and Brassica oleracea (35) [23].Nonetheless, the XTH family constituents in mulberry remain undisclosed.Earlier investigations have demonstrated the involvement of XTH genes in numerous crucial processes, particularly the development and growth of plants via the remodelling of plant cell walls.For instance, in Arabidopsis, genes such as AtXTH17, AtXTH18, AtXTH19, and AtXTH20 exhibited specific expression patterns in the root tissues and were significantly involved in the processes of root elongation and the initiation of root hair formation [19,24].GhXTH1 gene overexpressed in cotton, elongated cotton fibers by 15-20% [25].In addition, some XTH genes have been reported to play active roles in fruit softening and ripening.PavXTH14 and PavXTH15 expression in cherry fruits resulted in a significant decrease in fruit firmness and altered the constitution of hemicellulose and pectin in the cell wall of the transgenic fruit [26].Likewise, XTH influenced the softening and ripening of fruits, including tomatoes [27], strawberries [28], kiwi [29], and pears [29].Several other XTH genes are involved in flower development [29] and leaves [30].
Numerous investigations have also suggested that plant hormones play a role in regulating the activity of XTH genes.For instance, the application of abscisic acid increased the expression of Arabidopsis AtXTH23 [19].Similarly, the ethylene application induced the expression of banana MA-XETI, which is involved in the ripening and softening of the peel and pulp [30].Furthermore, under ethylene induction, three CaXTH genes were significantly upregulated in the leaf tissue of hot pepper [31].Members of the XTH gene Plants 2024, 13, 902 3 of 19 family primarily regulate cell wall responses to biotic and abiotic stressors, which affect plant growth.The overexpression of DkXTHI was found to augment the resistance of transgenic Arabidopsis plants to salt, drought-induced stress, and abscisic acid, consequently impacting the development of roots and leaves [32].Similarly, XTH genes in Chinese cabbage (Brassica rapa L.) exhibited an upregulated expression in response to elevated temperatures [33].Furthermore, under low temperatures, the DkXTH6 gene in persimmons decreased in expression, while the DkXTH7 gene showed noticeably high transcription levels [34].A prior proteomic study in maize revealed that XTHs were differently regulated in response to drought stress [35].Moreover, xyloglucan content was decreased in the Arabidopsis AtXTH31 mutant, which lowered the amount of absorbed Al 3+ and increased resistance to aluminum stress [36].The overexpression of the xyloglucan endotransglucosylase/hydrolase gene in Populus euphratica resulted in increased resistance to cadmium tolerance by limiting cadmium absorption in the root system of transgenic tobacco plants.In addition, the transgenic plants had 56-87% more xyloglucan degrading activity (XDA) than the wild type, which resulted in a 25-27% decrease in the amount of xyloglucan in the root cell walls [37].Moreover, in Arabidopsis, aluminum tolerance was imparted by the induction of ZmXTH, a gene encoding xyloglucan endotransglucosylase/hydrolase from maize [38].Similarly, Arabidopsis mutants xth15 and xthI7 exhibited elevated aluminum tolerance in contrast to wild-type plants [36].
These preliminary studies highlight the key role of XTHs in various plants' responses to various stresses.However, to the best of our knowledge, there is no functional characterization of mulberry XTH gene family members.Consequently, there is a necessity for a systemic and comprehensive exploration of the M. alba XTH gene family across the genome.The present investigation conducted an analysis of the XTH gene family within M. alba based on our previous transcriptomic analysis after Mg stress treatment using the available genome data.Subsequently, detailed information, including phylogenetic analysis, gene structure characterization, chromosomal localization, motif analysis, promoter analysis, and syntenic relationships of MaXTH genes, were examined.Furthermore, real-time quantitative PCR (qRT-PCR) was employed to determine the expression patterns of XTH genes in the leaf tissues of M. alba that were subjected to various levels of Mg stress.The findings of this study are poised to offer significant insights into the XTH genes in M. alba, contributing to deeper comprehension and setting the groundwork for the functional analysis of plant XTH genes in mulberry plants.

Growth Conditions and Magnesium Treatment for the Mulberry Plant (Morus alba)
Mulberry (Yu-711), a member of the M. alba species, was obtained from the National Mulberry GenBank at Jiangsu University of Science and Technology in Zhenjiang, Jiangsu, China.Growth of the mulberry plant materials and Mg treatments followed the methods of our previous study [5].In brief, mulberry seedlings that had been grafted were carefully chosen and then planted in pots with a diameter of 35 cm.These pots included a mixture of loamy soil and vermiculite.The optimal circumstances for the plant's growth in the greenhouse consist of 14 h of light, 10 h of darkness, a temperature of 25 • C during the day and 20 • C at night, and a humidity level ranging from 70 to 80%.A total of 18 pots were utilized, with each pot housing three plant seedlings as duplicates.The seedlings were watered daily and provided with a solution of MS culture medium consisting of 4.37 g of MS media dissolved in 1000 mL of water (pH = 7.0) every three days for a total of 7 days.The plants were subjected to a 7-day treatment with deionized water following complete leaf growth.Mg (MgSO 4 ) treatments were then administered.A total of six concentration gradients, 0 mM (T1) as Mg deficiency, 1 mM and 2 mM (T2 and T3) as low Mg, 3 mM as sufficiency (CK), and 6 mM and 9 mM (T4 and T5) as Mg excess, were applied to the mulberry plants for 20 days.On the 20th day, leaves from all experimental groups and control were collected.Leaf samples harvested from the control and treated plants were wrapped separately in plain plastic bags and temporarily preserved (one week) at a temperature of −80 • C for subsequent studies, including transcriptome analysis [39].

Data Collection and Identification of XTH Gene Family Members in Morus alba
Genetic data in the form of genome sequences (fasta) and annotation files (gff) for three plant species, Arabidopsis thaliana, Populus trichocarpa, and Zea mays, were obtained from the official NCBI website (https://www.ncbi.nlm.nih.gov(accessed on 5 July 2023).M. alba genome sequence annotation file (fasta) was downloaded from NCBI (https://www.ncbi.nlm.nih.gov/datasets/genome/GCA_012066045.3/(accessed on 7 July 2023)).However, the gff was obtained from Professor Weiguo Zhao of Jiangsu University of Science and Technology.To determine the XTH gene in the mulberry plant genome, an HMMER search was executed utilizing a hidden Markov model (HMM) profile comprising binding domains PF00722 and PF06955, sourced from the Pfam database (http://Pfam.xfam.org/(accessed on 12 July 2023)).Sequences with E values < 1 were scrutinized, and any short open reading frames (less than 100 in length) were manually sorted out.The filtered sequences with putative of both PF00722 and PF06955 domains or either one of them were screened as primary candidates for M. alba XTH genes (MaXTH).

Gene Structure, Motif Analysis and Sequence Alignment
Each MaXTH gene structure was visualized in TBtools software v1.098769 [40] by utilizing the genome sequence in conjunction with its corresponding annotation file.Employing the online MEME suite (available at https://meme-suite.org/meme/ (accessed on 15 July 2023), the conserved motifs within MaXTHs were identified using specific parameters: a search for 10 motifs, with a minimum width of 6 and a maximum width of 55.The consensus motif sequence was conducted, and a web logo was generated through the utilization of the MEME tool.Subsequently, the individual motifs were searched in motif scan (https://myhits.sib.swiss/cgi-bin/motif_scan(accessed on 15 January 2024) to identify the Glyco_hydro_16 and XET_C domains.Furthermore, the extracted motif sequences of the MaXTH protein sequences exhibiting the Glyco_hydro_16 and XET_C domains were aligned in Bioedit software (v7.2).

Physicochemical Properties of the MaXTH Gene Family
The ExPASy online platform (https://web.expasy.org/protparam/(accessed on 15 July 2023) was used to obtain the molecular weight (Mw), isoelectric point (pI), and grand average of hydropathy (GRAVY) data for individual XTH proteins.Additionally, the subcellular localization of MaXTH proteins was predicted using the CELLO online resource (http://cello.life.nctu.edu.tw/(accessed on 25 July 2023) [41].

Phylogenetic Analysis
In the present investigation, two phylogenetic trees were constructed to categorize the MaXTH gene family.The first tree encompassed only the MaXTH protein sequences from M. alba, and the second tree included M. alba, P. trichocarpa, and A. thaliana XTH genes.The evolutionary relationships of the XTH genes in the different groups were assessed following the alignment of XTH protein sequences in MEGA 7 software using Clustal W [42].A phylogenetic tree, based on the maximum likelihood method, with 1000 bootstrap replications was generated employing the MEGA 7.0 software (v7.0.26).All the phylogeny trees were performed following the same specifications indicated above.

Analysis of Cis-Regulatory Elements of MaXTH Genes and GO Analysis
The promoter regions located 2000 base pairs prior to the initiation codon (ATG) of MaXTH genes were extracted from the genome of M. alba.Utilizing the PlantCARE online tool (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/(accessed on 12 July 2023) [43], the inherent regulatory elements in these promoter regions were predicted.Subsequently, the outcomes of this predictive analysis were rendered visible through the TBtool software.Gene Ontology (GO) analysis was performed using the web tool Shiny GO (http://bioinformatics.sdstate.edu/go74/(accessed on 2 August 2023).

Chromosomal Localization, Circos, and Synteny Analyses
The length of each chromosome and the chromosomal position of all XTH genes were obtained from the annotated dataset of the M. alba genome.The positional mapping of genes on the chromosomes was visualized through the utilization of TBtools software v1.098769 [40].Based on the alignment and further examination of the phylogenetic relationship of the MaXTH genes, paralogous genes were discovered [20], and the interconnections of the paralogous genes according to their locations on the chromosomes were exhibited on the circos map constructed in the Tbtools (v2.026).The Multiple Collinearity Scan toolkit (MCScanX), integrated into the TBtool (v2.026), was applied for the identification of syntenic blocks and specific gene pairs, adhering to its default configurations [44].Additionally, the Multiple Synteny Plotter program within the TBtool (v2.026) was employed to visually represent the synteny associations among orthologous XTH genes across M. alba, A. thaliana, Zea mays, and Populus trichocarp [45].

Gene Expression Analysis and qRT-PCR Gene Validation
By using the RNA-seq data obtained from our transcriptome analysis after Mg application (https://ncbi.nlm.nih.gov;accession number: PRJNA951543), this study examined the expression of the XTH genes in M. alba treated with different levels of Mg after 20 days (about 3 weeks) and compared it to the expression of the gene in optimum Mg supply (the control) according to our previous study [5].To validate the XTH genes identified through high-throughput sequencing, six differentially expressed genes (DEGs) were chosen for real-time quantitative polymerase chain reaction (qRT-PCR) validation, following the established protocol detailed in a prior study [46].Mulberry leaf samples used for the RNA-seq analysis were also used for total RNA and cDNA synthesis for qRT-PCR validation.Furthermore, the 2 −∆∆Ct method [47] was applied to estimate the fold changes in gene expression.The primer sequences and gene names for qRT-PCR validation are enlisted in Supplementary File S1.

Identification and Physiological Features XTH Genes in M. alba
In our previous investigation, RNA-Seq transcriptome analysis of M. alba leaf tissues was conducted to ascertain candidate genes possibly influenced by high magnesium (Mg) levels or magnesium nutrient starvation.A comprehensive examination of the transcriptome data resulted in the identification of 12 XTH genes.Among them, the candidate MaXTH-1 with the gene ID LOC21405692 had a higher expression level and, therefore, was selected and used as a query gene for the search against the Morus genome using BLASTP and HMMER search.Twenty-four putative XTH proteins were obtained after the search and confirmed in the M. alba genome using their transcript ID.After which, the short and redundant sequences underwent manual scrutiny.Subsequently, the identification of conserved domains was carried out utilizing the Pfam and CDD databases.Finally, the 22 XTH proteins obtained were named MaXTH-1 to MaXTH-22 (Table 1 and Supplementary File S2).Coding sequences (CDS) of the 22 MaXTH proteins ranged from 645 bp (LOC21407360; MaXTH-21) to 1509 bp (LOC21403517; MaXTH-18), with an average length of 915 bp.The amino acid sequence length of the MaXTH proteins spanned from 214-502, where MaXTH-18 was the longest sequence (501 aa), and MaXTH-21 (214 aa) had the shortest sequence.The molecular weights of MaXTH proteins varied between 24.071 kDa (MaXTH-21) and 56.843 kDa (MaXTH-18), averaging 34.40 kDa.The GRAVYs for all MaXTH genes were negative, ranging from −1003 to −0.099 (Supplementary File S2), suggesting that MaXTHs are likely hydrophilic.The predicted putative localization of the M. alba XTH was prevalent in the extracellular region, while a small number were located in the plasma membrane, mitochondrion, cytoplasmic, vacuole, and nuclear region (Table 1).

Gene Structure, Conserved Domain, and Motif Analysis
The structural diversity of the 22 MaXTH genes was investigated by determining their exon and intron structures through the alignment of their genomic and CDS sequences using the TBtool software.In addition, a phylogenetic tree was constructed using the complete MaXTH protein sequences, depicted with the distribution of introns and exons (Figure 1).The resulting phylogenetic analysis revealed the categorization of MaXTH genes into five distinct groups: I, II, III, IV, and V, constituting 7, 3, 8, 3, and 1 MaXTH genes, respectively (Figure 1A).In general, genes that are grouped together share similar structures.For instance, all members of group 1 (MaXTH-1, MaXTH-2, MaXTH-3, MaXTH-4, MaXTH-5, MaXTH-6, and MaXTH-9) contained three exons in their coding region, and two introns (Figure 1B).Members of group II also possessed four exons and three introns, except for MaXTH-7, which had three exons.MaXTH group III members constituted four exons, excluding MaXTH-21 and MaXTH-22.Moreover, MaXTH-21 contained no intron.Interestingly, MaXTH-15 had no 5 ′ or 3 ′ UTRs.
The Pfam and CDD databases were employed to investigate the conserved domains of the 22 MaXTH genes.From the results obtained, only four genes, MaXTH-9, MaXTH-18, MaXTH-21, and MaXTH-22, contained a single conserved domain (Glyco_hydro_16).In contrast, the remaining 16 MaXTH genes exhibited the presence of both Glyco_hydro_16 and XET_C conserved domains, as illustrated in Figure 2. MaXTH proteins were further characterized by predicting their potentially conserved motifs employing the MEME online software.The differences between the MaXTH genes were analyzed through multiple sequence alignment.The findings revealed that 21 members of MaXTH proteins exhibited a typical highly conserved Glyco_hydro_16 domain (Figure 3A).This domain comprised 50 amino acid letters, as illustrated by the sequence logo (QGKGNREQRFYL-

Evolutionary Relationship of the MaXTH Proteins
To comprehend the evolution and classification of MaXTH proteins, a phylogenetic tree based on the maximum likelihood method was constructed using the 22 MaXTH protein sequences with other XTH sequences from Arabidopsis thaliana (16) and Populus trichocarpa (15).From the results obtained, MaXTH proteins formed 5 different groups with XTH proteins from A. thaliana and P. trichocarpa (Figure 4).Groups I, II, III, IV, and V contained 7, 3, 9, 2, and 1 member, respectively.Generally, the phylogenetic analysis depicts that MaXTH genes clustered with XTH orthologs from P. trichocarpa and A. thaliana.Among the five groups, group 1 was the largest, which comprised 11 members each from A. thaliana and P. trichocarpa.Group III was the second largest but mainly consisted of MaXTH genes and only one XTH ortholog from P. trichocarpa.In addition, Group V contained only MaXTH 14 and did not cluster with any of the XTHs from A. thaliana and P. trichocarpa.The non-correlation of the Group 5 member and the limited association between MaXTH genes and P. trichocarpa XTH proteins within Group III suggests that the

Evolutionary Relationship of the MaXTH Proteins
To comprehend the evolution and classification of MaXTH proteins, a phylogenetic tree based on the maximum likelihood method was constructed using the 22 MaXTH protein sequences with other XTH sequences from Arabidopsis thaliana (16) and Populus trichocarpa (15).From the results obtained, MaXTH proteins formed 5 different groups with XTH proteins from A. thaliana and P. trichocarpa (Figure 4).Groups I, II, III, IV, and V contained 7, 3, 9, 2, and 1 member, respectively.Generally, the phylogenetic analysis depicts that MaXTH genes clustered with XTH orthologs from P. trichocarpa and A. thaliana.Among the five groups, group 1 was the largest, which comprised 11 members each from A. thaliana and P. trichocarpa.Group III was the second largest but mainly consisted of MaXTH genes and only one XTH ortholog from P. trichocarpa.In addition, Group V contained only MaXTH 14 and did not cluster with any of the XTHs from A. thaliana and P. trichocarpa.The non-correlation of the Group 5 member and the limited association between MaXTH genes and P. trichocarpa XTH proteins within Group III suggests that the MaXTH proteins present in the branch might be less evolutionarily conserved or more primitive.
Drought-inducible response elements were detected in 13 MaXTH genes.The promoter sequences of two MaXTH genes constituting MaXTH-8 and MaXTH-18 exhibited cis-regulatory elements associated with wound responsiveness.The promoter sequences of two MaXTH genes constituting MaXTH-8 and MaXTH-18 exhibited cis-regulatory elements associated with wound responsiveness.Anaerobic induction-responsive elements were abundantly detected across all 22 MaXTH genes.Other MaXTH genes, including MaXTH-5, MaXTH-9, and MaXTH-12, were identified to contain cell cycle response elements that are associated with cell development.These results inferred that MaXTH genes participate

Cis-Acting Regulatory Elements of XTH Genes from Morus alba
To comprehend the transcriptional regulation of MaXTH genes, the 2000 bp promoter sequence of all the MaXTH genes was retrieved from the M. alba genome and analyzed using the PlantCare online database.The data obtained infer that MaXTH promoters pos sessed several cis-regulatory elements involved in processes such as stress response, hor mone regulation, and cell development (Figure 5).6A and Supplementary File S3).In addition, MaXTH genes were observed to play crucial roles in various biological activities, including the organization of the cell wall, the biogenesis of the cell wall, and processes related to xyloglucan metabolism.
Further, GO analysis revealed that certain members of MaXTH genes were localized in the apoplast and cell wall regions.This observed localization aligns with the predictions made through subcellular localization analysis (Table 1).Notably, MaXTH genes are implicated in cellular glucan metabolic processes and carbohydrate metabolic processes.Additionally, the hierarchical clustering of the functional enrichment pathways depicted in Figure 6B reveals that MaXTH genes collaborate to perform various functions.Gene ontology (GO) analysis was conducted to elucidate the roles of MaXTH genes.proteins encoded by MaXTH gene members exhibited both xyloglucan xyloglucosyl transferase and hydrolase activities (Figure 6A and Supplementary File S3).In addition, MaXTH genes were observed to play crucial roles in various biological activities, including the organization of the cell wall, the biogenesis of the cell wall, and processes related to xyloglucan metabolism.Further, GO analysis revealed that certain members of MaXTH genes were localized in the apoplast and cell wall regions.This observed localization aligns with the predictions made through subcellular localization analysis (Table 1).Notably, MaXTH genes are implicated in cellular glucan metabolic processes and carbohydrate metabolic processes.Additionally, the hierarchical clustering of the functional enrichment pathways depicted in Figure 6B reveals that MaXTH genes collaborate to perform various functions.

Chromosomal Localization, Circos, and Synteny Analyses
The chromosomal positions of XTH genes in M. alba were located through genome annotation in the TB tools.As depicted in Figure 7, the 22 MaXTH genes were unevenly distributed among the chromosomes across the genome of M. alba (Figure 7).The most significant MaXTH genes were positioned on chromosome one with six members.Three

Chromosomal Localization, Circos, and Synteny Analyses
The chromosomal positions of XTH genes in M. alba were located through genome annotation in the TB tools.As depicted in Figure 7, the 22 MaXTH genes were unevenly distributed among the chromosomes across the genome of M. alba (Figure 7).The most significant MaXTH genes were positioned on chromosome one with six members.Three MaXTH genes were allotted on chromosomes 7, 9, and 10, and two on chromosome 14.Chromosomes 3, 4, 6, 12, and 13 contained one MaXTH each.Interestingly, no MaXTH genes were found in chromosomes 2, 5, 8, and 11 (Figure 7A).Based on the phylogenetic relationships of the MaXTH protein sequences (Figure 1A), a total of five MaXTH gene pairs were identified, as shown in the circos map (Figure 7B  To delve deeper into the evolutionary correlations and genetic linkage occurrences within the XTH gene family, a systemic map of XTH genes across M. alba and three additional species, encompassing one monocotyledonous (Zea mays) and two dicotyledonous (Populus trichocarpa and Arabidopsis thaliana) plants were constructed using TBtools software (Figure 8).To delve deeper into the evolutionary correlations and genetic linkage occurrences within the XTH gene family, a systemic map of XTH genes across M. alba and three additional species, encompassing one monocotyledonous (Zea mays) and two dicotyledonous (Populus trichocarpa and Arabidopsis thaliana) plants were constructed using TBtools software (Figure 8).
According to the results of the collinearity analysis, 12 covariate pairs were generally discovered.Among them, 7 pairs were identified in Populus trichocarpa (represented in blue lines), 4 pairs in A. thaliana (represented in green lines), and only one pair was observed in Zea mays (represented in red lines).Furthermore, MaXTH-14 displayed a significant degree of collinearity with three comparable species (two in P. trichocarpa and one each in A. thaliana and Zea mays), inferring that MaXTH genes displayed greater evolutionary differences in P. trichocarpa in contrast to A. thaliana, and Zea mays.

Expression Profiling of MaXTH Genes under Different Magnesium Treatments and qRT-PCR Validation
Several studies have reported that the XTH gene family plays an important role in plant response to abiotic stresses [17,18,20].To validate these accessions, the expression patterns of M. alba XTH genes responding to different treatments of magnesium concentrations were investigated by RNA-seq.RNA from M. alba leaf tissues was sampled on day 20.The XTH genes with expression changes according to the significant differential expression standard (|log2 (Fold change)| ≥ 1 and false discovery rate (FDR) < 0.05) were analyzed.The results from our investigation exhibited different expression levels of XTH genes at various concentrations (Figure 9A-E).At the least concentration of 0 mM (T1), 10 MaXTHs were identified, among which six were significantly downregulated.Four XTHs genes, including LOC21410403, LOC21405693, and LOC21401284, showed higher expression concentrations (Figure 9A).The collinearity analysis of MaXTH genes is represented via a circos map, exhibiting the relationships among the gene pairs.Gene pairs are represented by pink, cyan, and blue lines, while distinct colored labels outside the chromosomes denote gene names belonging to the same phylogenetic family.
Furthermore, six MaXTH genes, including MaXTH-17 (LOC21410403), MaXTH-13 (LOC21401284), MaXTH-21 (LOC21407360), MaXTH-1 (LOC21405692), MaXTH-6 (LOC21404263) and MaXTH-10 (LOC21404346) in response to Mg stresses, were selected to verify their relative expression level in mulberry leaves through qRT-PCR analysis.The results reveal that the selected genes could be expressed in the mulberry leaves, proving the reliability of the XTH genes identified by the transcriptome data (Figure 10A-E).LOC21410403 (MaXTH-17) exhibited a low expression level at 3 mM (CK; optimum concentration for M. alba growth); however, it was highly expressed at an elevated concentration of 6 mM (Figure 10A).For LOC21401284 (MaXTH 13), the highest expression level was observed at Mg deficiency (0 mM) (Figure 10B).LOC21407360 (MaXTH-21) was moderately expressed at 0 mM but was highly expressed at 3 and 9 mM (Figure 10C).The expression of LOC21404263 (MaXTH-6) was highly expressed in 0. 3 and 9 mM Mg treatments (Figure 10D).For the LOC21405692 (MaXTH-10), the highest expression level was observed at 3 mM, and the expression level was reduced at 6 mM concentration (Figure 10E).Fi-nally, the expression of LOC21404346 (MaXTH-1) was higher in 0 and 2 mM concentrations (Figure 10E).Altogether, our findings confirm that the XTH genes family is highly present in the M. alba genome and expressed in M. alba leaf tissues in response to Mg imbalances.This outcome strongly suggests that the XTH gene family is important in M. alba development and nutritional regulation.
Several studies have reported that the XTH gene family plays an important role in plant response to abiotic stresses [17,18,20].To validate these accessions, the expression pa erns of M. alba XTH genes responding to different treatments of magnesium concentrations were investigated by RNA-seq.RNA from M. alba leaf tissues was sampled on day 20.The XTH genes with expression changes according to the significant differential expression standard (|log2 (Fold change)| ≥ 1 and false discovery rate (FDR) < 0.05) were analyzed.The results from our investigation exhibited different expression levels of XTH genes at various concentrations (Figure 9A-E).At the least concentration of 0 mM (T1), 10 MaXTHs were identified, among which six were significantly downregulated.Four XTHs genes, including LOC21410403, LOC21405693, and LOC21401284, showed higher expression concentrations (Figure 9A).In the 1 mM (T2), we detected 14 MaXTHs, of which LOC21462237 was upregulated and had the highest expression level (Figure 9B).In contrast, the remaining 3 XTH genes showed downregulated expressions (LOC21390860, LOC21404346, and LOC21405696).M. alba treatment with 2 mM (T3) revealed 10 MaXTHs (Figure 9C).For 6 mM treatments (T4), it was observed that 3 XTHs (LOC21404346, LOC21404262 and LOC21407360) were low in expression (Figure 9D).In contrast, four XTHs (LOC21410403, LOC21405693, LOC210401284 and LOC21405696) were upregulated (Figure 9D).At the excess Mg of 9 mM (T5), all 10 MaXTH genes identified were significantly upregulated (Figure 9E), with LOC21387254 and LOC21404346 being down-regulated.Furthermore, six MaXTH genes, including MaXTH-17 (LOC21410403), MaXTH-13 (LOC21401284), MaXTH-21 (LOC21407360), MaXTH-1 (LOC21405692), MaXTH-6 (LOC21404263) and MaXTH-10 (LOC21404346) in response to Mg stresses, were selected to verify their relative expression level in mulberry leaves through qRT-PCR analysis.The results reveal that the selected genes could be expressed in the mulberry leaves, proving the reliability of the XTH genes identified by the transcriptome data (Figure 10A-E).LOC21410403 (MaXTH-17) exhibited a low expression level at 3 mM (CK; optimum concentration for M. alba growth); however, it was highly expressed at an elevated concentration of 6 mM (Figure 10A).For LOC21401284 (MaXTH 13), the highest expression level was observed at Mg deficiency (0 mM) (Figure 10B).LOC21407360 (MaXTH-21) was moderately expressed at 0 mM but was highly expressed at 3 and 9 mM (Figure 10C).The expression of LOC21404263 (MaXTH-6) was highly expressed in 0. 3 and 9 mM Mg treatments (Figure 10D).For the LOC21405692 (MaXTH-10), the highest expression level was observed at 3 mM, and the expression level was reduced at 6 mM concentration (Figure 10E).Finally, the expression of LOC21404346 (MaXTH-1) was higher in 0 and 2 mM concentrations (Figure 10E).Altogether, our findings confirm that the XTH genes family is highly present in the M. alba genome and expressed in M. alba leaf tissues in response to Mg imbalances.This outcome strongly suggests that the XTH gene family is important in M. alba development and nutritional regulation.

Discussion
Mulberry (M.alba) is a plant of considerable economic importance, yet its growth and development are influenced by various abiotic factors such as Mg deficits [5].Mg serves diverse functions in biological systems [48].Consequently, gaining insights into how plants respond to both Mg deficiency and excess at the genomic level is essential for effec-

Discussion
Mulberry (M.alba) is a plant of considerable economic importance, yet its growth and development are influenced by various abiotic factors such as Mg deficits [5].Mg serves diverse functions in biological systems [48].Consequently, gaining insights into how plants respond to both Mg deficiency and excess at the genomic level is essential for effective plant nutrient management.Past research indicates that plants have evolved sophisticated regulatory mechanisms, engaging specific gene families like xyloglucan endotransglucosylase/hydrolases (XTHs) [10] to facilitate their adaptation to Mg stress.
XTHs represent a category of plant enzymes responsible for regulating xyloglucan crosslinking within cell walls, playing a pivotal role in the control of plant growth and development [49,50].The role of XTH genes is not only limited to cell wall elongation but also plays a part in plant responses to various environmental stresses.The XTH gene family has been identified across diverse plant species, such as A. thaliana [51], wheat [52], grapevine [53], rice [21], peanut [17], barley [54], sweet potato [20] and poplar [55].Within the scope of this investigation, we present the discovery and characterization of the XTH gene family within the M. alba genome.This includes exploring their phylogenetic relationships, conserved motifs, gene structures, cis-acting regulatory elements, and gene expression patterns in response to Mg starvation, low or high concentrations.
Based on the M. alba genome, 22 XTH genes were identified based on our strictest identification workflow and labeled as MaXTH-1 to MaXTH-22.The number of identified XTH genes was notably less compared to various other species, including tobacco (56), wheat (71), Solanum lycopersicum (37), and Glycine max (61) [11,18,22,52].It is widely acknowledged that the functional attributes of genes are intricately linked to their structural and physicochemical characteristics [20,56].In this study, the 22 MaXTH protein members displayed significant disparities with respect to protein sequence length, molecular weight, isoelectric point (pI), and intron and exon distributions (Table 1).This variation implicates a high diversity among XTH family members in M. alba.Additionally, most MaXTH genes were predicted to be in the extracellular space, while a few were in the plasma membrane, vacuole, mitochondrion, and nuclear region.This is contrary to previous reports for other XTH protein members in other plant species, where the majority of the XTH proteins were in the plasma membrane rather than the extracellular space and other locations [22,53,55].Further, phylogenetic analysis indicated that MaXTH protein families were clustered into five groups (Figure 1), similar to those observed for XTH proteins from sweet cherry [26].Interestingly, the MaXTH proteins belonging to the same group demonstrated similar gene structures (Figure 1) and conserved sequence expression, which is consistent with previously documented literature [11,20], suggesting that XTH members within the same group may exhibit analogous functionalities.Moreover, most of the MaXTH genes demonstrate the presence of two main conserved domains (Glyco_hydro_16 and XET_C domain) (Figure 2).Nevertheless, MaXTH-9, MaXTH-18, MaXTH-21, and MaXTH-22 lacked the XET_C domain.This absence suggests a potential evolutionary divergence, indicating a loss of the XET_C domain during the evolutionary trajectory of XTH proteins in M. alba.
Phylogenetic distribution of XTH proteins from M. alba, A. thaliana, and P. trichocarpa revealed that MaXTH genes could be categorized into five groups (group I-V) (Figure 4).Earlier studies have documented the categorization of XTH gene families into distinct groups in various plant species.In tobacco, for instance, eight family groups were identified [22], while three groups were observed in peanut [17], barley [54], and sweet potato [20].Poplar, on the other hand, exhibited four distinct groups [55].The MaXTH genes were observed to cluster better with XTH proteins from P. trichocarpa than A. thaliana, implying a closer evolutionary relationship between XTH proteins in M. alba and those of P. trichocarpa rather than A. thaliana.According to chromosomal localization analysis, it was observed that MaXTHs were heterogeneously distributed on 10 out of the 14 chromosomes of M. alba (Figure 7).Further investigation revealed five gene pairs among the XTH gene families in the M. alba genome.Previous research has indicated that a set of gene functions exhibit high conservation across various plant species [57,58].Consequently, it is imperative to identify true orthologs in different plant species through the application of synteny analysis.The results obtained from the synteny analysis depicted a significant degree of synteny between the M. alba genome and those of P. trichocarpa and A. thaliana, exhibiting 7 and 4 synthetic blocks of MaXTH between P. trichocarpa and A. thaliana, respectively.In contrast, one synthetic block was identified between Zea mays (Figure 8).
Cis-regulatory elements are essential for regulating gene expression.The comprehension of cis-regulatory elements within the promoter region of genes has the potential to clarify the roles and regulatory mechanisms of specific genes that engage in collaborative interactions with other genes [59,60].Investigating the cis-regulatory elements of the 22 MaXTH exhibited a number of core promoters involved in hormone responsiveness (Abscisic acid, Salicylic acid, MeJA, Gibberellin), stress responsiveness (stress defense, drought, anoxic, and anaerobic inducibility), growth and development elements (Figure 5).MaXTH promoters contain a variety of elements that respond to environmental and plant hormone stimuli, which might indicate various regulatory or functional mechanisms in response to biotic and abiotic stress factors [20,61].Besides, there were significant variations in terms of type and quantity, and certain elements related to metabolism and gene expression were unique to specific MaXTH genes.The structural variations of MaXTH proteins could result in modifying protein functions.Several studies have demonstrated that plant XTH proteins have essential roles in plant growth, development, and stress resistance.The presence of numerous cis-elements identified in the promoter region of the M. alba XTH genes suggests that the XTH genes within M. alba possess the capability to adapt to diverse modifications in the plant, particularly responsiveness to several hormones and numerous stress response elements (anaerobic and anoxic specific inducibility).
Similar contrasting expression patterns of the XTH gene family were identified in Camellia sinensis under fluorine stress where CsXTH7, CsXTH1, CsXTH6, and CsXTH1 were upregulated, while that of CsXTH3 was down-regulated [63].Additionally, the expression of PeXTH experienced a notable upregulation in the roots and leaves of P. euphratica when exposed to Cd stress [37].Likewise, under Al stress, AtXTH15 and AtXTH14 demonstrated a significant decrease, leading to a reduction in xyloglucan endo transferase (XET) activity and consequently enhancing the aluminum tolerance of A. thaliana [64].In this study, we observed different expression patterns of MaXTH genes.Changes in the expression of MaXTH genes can affect cell wall flexibility and strength, which are important factors in stress adaptation.The increased expression of certain MaXTH genes might contribute to cell wall remodeling, allowing for better flexibility and adaptation to magnesium stress.Conversely, decreased expression could be associated with a more rigid cell wall structure.These findings indicate the capacity of MaXTH genes to provide a defense to the M. alba plant during magnesium starvation, undersupply, and excess application.Future works in M. alba should investigate the functional genomic validation of these identified XTH genes and how they regulate Mg nutrition.
Taken together, the results of this research offer novel insights into the expression of MaXTH genes under different Mg concentrations.It could be inferred that the MaXTHs might exhibit heightened functionalities related to the cell wall in stressful conditions through interaction with xyloglucan.However, additional molecular and genetic research is required to confirm their roles.

Conclusions
In this current investigation, an extensive examination of the M. alba XTH gene family was conducted.The results from the investigation successfully identified and further characterized a total of 22 MaXTH genes.These genes were subsequently categorized into five groups (I to V) based on their phylogenetic relationships.Gene structure and motif composition were observed to be consistent within each group.A thorough analysis of gene synteny uncovered evidence of evolutionary relationships among XTH genes in M. alba and three other species, A. thaliana, P. trichocarpa, and Zea mays.Gene ontology analysis revealed that MaXTHs are responsible for encoding proteins demonstrating both xyloglucan xyloglucosyl transferase and hydrolase activities.Moreover, MaXTHs actively contribute to the processes of cell wall biogenesis, as well as the metabolic pathways associated with xyloglucan and carbohydrates.Furthermore, specific Cis-acting regulatory elements detected in the promoter region of MaXTH genes suggest their potential involvement in various biological processes such as development, phytohormone responses, and stress adaptation.Moreover, investigating the expression profiles of MaXTH genes within leaf tissues exposed to different magnesium concentrations revealed diverse patterns of gene expression.Collectively, the findings from this research provide valuable insights into the functions of XTH genes within M. alba and present a better understanding of how mulberry plants respond to various magnesium treatments.

Figure 1 .
Figure 1.Phylogenetic relationship and gene structure of MaXTH gene family.(A) Phylogen of the 22 Morus alba XTH gene family (B) gene structure of the MaXTH genes.Pink color: CD ing sequence region); Green color: UTR (untranslated region); I-V: MaXTH gene family cl tion.

Figure 1 . 20 Figure 2 .
Figure 1.Phylogenetic relationship and gene structure of MaXTH gene family.(A) Phylogenetic tree of the 22 Morus alba XTH gene family (B) gene structure of the MaXTH genes.Pink color: CDS (coding sequence region); Green color: UTR (untranslated region); I-V: MaXTH gene family classification.Plants 2024, 13, x FOR PEER REVIEW 8 of 20

Figure 2 .
Figure 2. Domain analysis of the MaXTH gene family.

Figure 2 .
Figure 2. Domain analysis of the MaXTH gene family.

Figure 3 .
Figure 3. Multiple sequence alignment of domains from MaXTH proteins.(A) Multiple alignments of Glyco_hydro_16 domains, (B)sequence logo of the Glyco_hydro_16 domains.(C) Multiple alignments of XET_C domains (D) sequence logo of the XET_C domains.

Figure 3 .
Figure 3. Multiple sequence alignment of domains from MaXTH proteins.(A) Multiple alignments of Glyco_hydro_16 domains, (B)sequence logo of the Glyco_hydro_16 domains.(C) Multiple alignments of XET_C domains (D) sequence logo of the XET_C domains.
processes and exhibit responsiveness to different biotic and abiotic stress factors.lants 2024, 13, x FOR PEER REVIEW 9 of 20MaXTH proteins present in the branch might be less evolutionarily conserved or more primitive.

Figure 4 .
Figure 4. Maximum likelihood Phylogenetic relations of XTH protein family in Morus alba (red circles), Populus trichocarpa (blue triangle), and Arabidopsis thaliana (green square).I-V; major clusters.Gene ontology (GO) analysis was conducted to elucidate the roles of MaXTH genes.The proteins encoded by MaXTH gene members exhibited both xyloglucan xyloglucosyl transferase and hydrolase activities (Figure6Aand Supplementary File S3).In addition, MaXTH genes were observed to play crucial roles in various biological activities, including the organization of the cell wall, the biogenesis of the cell wall, and processes related to xyloglucan metabolism.Further, GO analysis revealed that certain members of MaXTH genes were localized in the apoplast and cell wall regions.This observed localization aligns with the predictions made through subcellular localization analysis (Table1).Notably, MaXTH genes are implicated in cellular glucan metabolic processes and carbohydrate metabolic processes.Additionally, the hierarchical clustering of the functional enrichment pathways depicted in Figure6Breveals that MaXTH genes collaborate to perform various functions.

Figure 5 .
Figure 5. Cis-elements predicted within the 2 kb sequences upstream of the M. alba XTH gene promoters.Cis-acting elements, with distinct colors in each box, denote different promoters.Drought-inducible response elements were detected in 13 MaXTH genes.The promoter sequences of two MaXTH genes constituting MaXTH-8 and MaXTH-18 exhibited cis-regulatory elements associated with wound responsiveness.The promoter sequences of two MaXTH genes constituting MaXTH-8 and MaXTH-18 exhibited cis-regulatory elements associated with wound responsiveness.Anaerobic induction-responsive elements were abundantly detected across all 22 MaXTH genes.Other MaXTH genes, including MaXTH-5, MaXTH-9, and MaXTH-12, were identified to contain cell cycle response elements that are associated with cell development.These results inferred that MaXTH genes participate in diverse biological processes and exhibit responsiveness to different biotic and abiotic stress factors.Gene ontology (GO) analysis was conducted to elucidate the roles of MaXTH genes.proteins encoded by MaXTH gene members exhibited both xyloglucan xyloglucosyl transferase and hydrolase activities (Figure6Aand Supplementary File S3).In addition, MaXTH genes were observed to play crucial roles in various biological activities, including the organization of the cell wall, the biogenesis of the cell wall, and processes related to xyloglucan metabolism.

Figure 5 .
Figure 5. Cis-elements predicted within the 2 kb sequences upstream of the M. alba XTH gene promoters.Cis-acting elements, with distinct colors in each box, denote different promoters.Plants 2024, 13, x FOR PEER REVIEW 11 of 20

Figure 6 .
Figure 6.Gene ontology (GO) annotation and functional clustering.(A) GO analysis of the 22 XTH genes from Morus alba and (B) A hierarchical clustering tree summarizing the correlation among significant pathways listed in the enrichment tab.Pathways with many shared genes are clustered together.Bigger dots indicate more significant p-values.

Figure 6 .
Figure 6.Gene ontology (GO) annotation and functional clustering.(A) GO analysis of the 22 XTH genes from Morus alba and (B) A hierarchical clustering tree summarizing the correlation among significant pathways listed in the enrichment tab.Pathways with many shared genes are clustered together.Bigger dots indicate more significant p-values.

Figure 7 .
Figure 7. (A) Chromosomal localization pa ern of MaXTH genes.The scale bar on the left represents the length of the chromosomes.(B) The collinearity analysis of MaXTH genes is represented via a circos map, exhibiting the relationships among the gene pairs.Gene pairs are represented by pink, cyan, and blue lines, while distinct colored labels outside the chromosomes denote gene names belonging to the same phylogenetic family.

Figure 8 .
Figure 8. Collinearity analysis of XTH genes from M. alba and other plant species: Populus tricho-

Figure 7 .
Figure 7. (A) Chromosomal localization pattern of MaXTH genes.The scale bar on the left represents the length of the chromosomes.(B) The collinearity analysis of MaXTH genes is represented via a circos map, exhibiting the relationships among the gene pairs.Gene pairs are represented by pink, cyan, and blue lines, while distinct colored labels outside the chromosomes denote gene names belonging to the same phylogenetic family.

Figure 7 .
Figure 7. (A) Chromosomal localization pa ern of MaXTH genes.The scale bar on the left represents the length of the chromosomes.(B)The collinearity analysis of MaXTH genes is represented via a circos map, exhibiting the relationships among the gene pairs.Gene pairs are represented by pink, cyan, and blue lines, while distinct colored labels outside the chromosomes denote gene names belonging to the same phylogenetic family.

Figure 8 .
Figure 8. Collinearity analysis of XTH genes from M. alba and other plant species: Populus trichocarpa, Arabidopsis thaliana, and Zea mays.The presented data delineate XTH syntenic gene pairs through distinct colored lines: blue lines signify pairs between Morus alba and Populus trichocarpa, green lines denote pairs between M. alba and Arabidopsis thaliana, and a red line signifies pairs between M. alba and Zea mays.Additionally, grey lines elucidate the presence of orthologous genes of Morus alba shared with three other plant species.

Figure 8 .
Figure 8. Collinearity analysis of XTH genes from M. alba and other plant species: Populus trichocarpa, Arabidopsis thaliana, and Zea mays.The presented data delineate XTH syntenic gene pairs through distinct colored lines: blue lines signify pairs between Morus alba and Populus trichocarpa, green lines denote pairs between M. alba and Arabidopsis thaliana, and a red line signifies pairs between M. alba and Zea mays.Additionally, grey lines elucidate the presence of orthologous genes of Morus alba shared with three other plant species.

Figure 9 .
Figure 9. Heatmap of the relative gene expression pa ern of the XTH gene family based on gene relative expression in Morus alba under different magnesium treatments.(A) T1; 0 mM, (B) T2; 1 mM, (C) T3; 2 mM, (D) T4; 6 mM and (E) T5; 9 mM.CK represents the optimum concentration of magnesium for M. alba growth (3 mM).From red to green, show the concentration level of the gene expression.

Figure 9 .
Figure 9. Heatmap of the relative gene expression pattern of the XTH gene family based on gene relative expression in Morus alba under different magnesium treatments.(A) T1; 0 mM, (B) T2; 1 mM, (C) T3; 2 mM, (D) T4; 6 mM and (E) T5; 9 mM.CK represents the optimum concentration of magnesium for M. alba growth (3 mM).From red to green, show the concentration level of the gene expression.

Table 1 .
Physiological characteristics of XTH gene family in Morus alba.