Identification of the Xyloglucan Endotransglycosylase/Hydrolase (XTH) Gene Family Members Expressed in Boehmeria nivea in Response to Cadmium Stress

Xyloglucan endotransglycosylase/hydrolase (XTH) genes play an important role in plant resistance to abiotic stress. However, systematic studies of the response of Boehmeria nivea (ramie) XTH genes (BnXTHs) to cadmium (Cd) stress are lacking. We sought to identify the BnXTH-family genes in ramie through bioinformatics analyses and to investigate their responses to Cd stress. We identified 19 members of the BnXTH gene family from the ramie genome, referred to as BnXTH1-19, among which BnXTH18 and BnXTH19 were located on no chromosomes and the remaining genes were unevenly distributed across 11 chromosomes. The 19 members were divided into four groups, Groups I/II/IIIA/IIIB, according to their phylogenetic relationships, and these groups were supported by analyses of intron–exon structure and conserved motif composition. A highly conserved catalytic site (HDEIDFEFLG) was observed in all BnXTH proteins. Additionally, three gene pairs (BnXTH6–BnXTH16, BnXTH8–BnXTH9, and BnXTH17–BnXTH18) were obtained with a fragment and tandem-repeat event analysis of the ramie genome. An analysis of cisregulatory elements revealed that BnXTH expression might be regulated by multiple hormones and abiotic and biotic stress responses. In particular, 17 cisregulatory elements related to abiotic and biotic stress responses and 11 cisregulatory elements related to hormone responses were identified. We also found that most BnXTH genes responded to Cd stress, and BnXTH1, BnXTH3, BnXTH6, and BnXTH15 were most likely to contribute to the Cd tolerance of ramie, as evidenced by the substantial increases in expression under Cd treatment. Heterologous expression of BnXTH1, BnXTH6, and BnXTH15 significantly enhanced the Cd tolerance of transgenic yeast cells. These results suggest that the BnXTH gene family is involved in Cd stress responses, laying a theoretical foundation for functional studies of BnXTH genes and the innovative breeding of Cd-tolerant ramie.


Introduction
Cadmium (Cd) is a nonessential trace metal with high mobility and toxicity [1]. It accumulates in plants through polluted farmland, soil, and water sources and enters the human body through the food chain, thus affecting human health [2]. According to statistics, more than 5 million soils worldwide, mainly in developing and underdeveloped countries (India, Bangladesh, Pakistan, etc.), are polluted with heavy metals [3]. China has the most serious heavy-metal pollution among these countries, with several "Cd rice" incidents [4]. A survey of cultivated land in China showed that about 2.79 × 10 9 m 2 of agricultural land, accounting for 20% of the total cultivated land area, is polluted with Cd [5,6]. The southern provinces, such as Hunan, Guizhou, Guangdong, Guangxi, and Fujian, have the most Cd pollution mainly because they are mining areas and smelting-process zones of nonferrous metals; their wastewaters are used for irrigation without treatment, polluting

Identification of Chromosomal Locations and Physicochemical-Property Analysis of the BnXTH Gene Family
A total of 19 BnXTH genes were identified from published ramie genome data. The genes were denoted BnXTH1-19, among which BnXTH6-19 were named based on chromosomal positions ( Figure S1 and Table 1). As illustrated in Figure S1, BnXTH18 and BnXTH19 were located on the Scaffold16 fragment and not on the chromosome, which may have been due to the poor assembly of the ramie genome. The other 17 BnXTH genes were unevenly distributed on chromosomes 2,4,5,6,7,8,9,11,12,13, and 14. Chromosomes 1, 3, and 10 had no XTH genes, and chromosome 6 contained the largest number of BnXTH genes (3; 15.79%), followed by chromosomes 4, 5, and 14, which contained two BnXTH genes each. The remaining chromosomes only had one BnXTH member each. This study also found that the number of family genes mapped in the chromosomes had no correlation with chromosome length. To further understand the physical and chemical properties of the BnXTH-protein family, we evaluated each protein's CDS length, amino acid (aa) number, molecular weight (Mw), isoelectric point (PI), grand average of hydropathicity (GRAVY), and aliphatic index. We also predicted the subcellular locations of these proteins (Table 1). These results showed that out of the 19 BnXTH protein sequences, the shortest was BnXTH10, which was encoded by 264 amino acids, while BnXTH3 was the longest, encoded by 395 amino acids. The Mw of the BnXTHs ranged from 30.812 (BnXTH10) to 40.228 kDa (BnXTH4), while the GRAVY ranged from −0.800 (BnXTH12) to −0.223 (BnXTH5). The aliphatic index of the proteins was between 59.44 (BnXTH12) and 73. 39 (BnXTH7), and the PI ranged from 4.64 (BnXTH17) to 9.47 (BnXTH9). Moreover, subcellular localization prediction showed that BnXTH13/17 might have been located in extracellular regions, while BnXTH3/7/9/15/18/19 may have been located in the cell wall or cytoplasm, and the remaining BnXTH proteins may have played roles in the cell wall.

Phylogenetic Analysis and Multiple Sequence Alignment of BnXTHs
To better understand the evolutionary relationships between BnXTHs and determine their classifications, we used the sequences of the 19 ramie BnXTH genes and the AtXTH family protein sequences of A. thaliana to generate a phylogenetic tree ( Figure 1) using the maximum likelihood (ML) method. We divided the BnXTH-family proteins identified in ramie into four subgroups based on a previous classification of the family: namely, Groups I, II, IIIA, and IIIB. The BnXTHs were mainly clustered in Groups I and II, which had 14 members. Among these members, BnXTH1/2/3/7/10/11/12 belonged to Group I, while BnXTH8/9/13/17/15/18/19 belonged to Group II. The remaining BnXTHs (BnXTH4/5/14/6/16) were included in Groups IIIA and IIIB.
Multiple alignments of the 19 BnXTHs showed that the BnXTH proteins contained a highly conserved catalytic site (HDEIDFEFLG) (Figure 2), with a deviation of one or two amino acids in a few sequences (Figure 2a). Except for the BnXTHs in Group III (BnXTH5/6/16), the active catalytic regions (HDEIDFEFLG) (shown with rectangular purple frames in Figure 2b) of the BnXTHs were adjacent to the N-linked glycosylation site. maximum likelihood (ML) method. We divided the BnXTH-family proteins identified in ramie into four subgroups based on a previous classification of the family: namely Groups I, II, IIIA, and IIIB. The BnXTHs were mainly clustered in Groups I and II, which had 14 members. Among these members, BnXTH1/2/3/7/10/11/12 belonged to Group I while BnXTH8/9/13/17/15/18/19 belonged to Group II. The remaining BnXTH (BnXTH4/5/14/6/16) were included in Groups IIIA and IIIB. Multiple alignments of the 19 BnXTHs showed that the BnXTH proteins contained a highly conserved catalytic site (HDEIDFEFLG) (Figure 2), with a deviation of one or two amino acids in a few sequences (Figure 2a). Except for the BnXTHs in Group II

Structural Analysis of the Conserved Motifs of BnXTHs
To analyze the gene structures and conserved motifs of BnXTHs, we constructed an evolutionary tree using 19 BnXTH proteins (Figure 3a), which were grouped into four subclasses. Structural analysis of the genomic DNA sequence showed that each BnXTH had two or three introns ( Figure 3b) and that the members in Group I, except for BnXTH10 each contained three introns. The Group II members, except for BnXTH13, contained two

Structural Analysis of the Conserved Motifs of BnXTHs
To analyze the gene structures and conserved motifs of BnXTHs, we constructed an evolutionary tree using 19 BnXTH proteins (Figure 3a), which were grouped into four subclasses. Structural analysis of the genomic DNA sequence showed that each BnXTH had two or three introns ( Figure 3b) and that the members in Group I, except for BnXTH10, each contained three introns. The Group II members, except for BnXTH13, contained two introns each, while the Group IIIA and Group IIIB members had three introns each. Furthermore, MEME analysis showed that 10 conserved motifs were found in the 19 BnXTH protein sequences ( Figure 3c). The amino acid sequence that encoded the protein sequences and the SeqLogo of the 10 conserved motifs are shown in Table S1. Motifs 1, 2, 3, 4, 5, and 6 were abundant in BnXTH proteins, among which motif 2 contained the characteristic active site (HDEIDFEFLG), suggesting that it is the specific motif for the enzymatic reaction of this family of proteins and present in all BnXTHs. We also found that motif 8 seemed unique to Group I, while motif 10 mainly existed in Groups I and II. Thus, these results also support the group-classification results of the phylogenetic tree above.

Gene-Duplication Analysis of BnXTHs
To determine the relationships between BnXTH members, we used the MCScanX method for a collinearity analysis. Six genes (BnXTH6-BnXTH16, BnXTH8-BnXTH9, and BnXTH17-BnXTH18) exhibited complex segmental duplication events (Figure 4a), implying that segmentally duplicated genes may have similar functions regulated via the same biological pathways in ramie. To further evaluate the evolution and development of the BnXTH family, we compared the collinearity of XTH genes between B. nivea and four other plants (O. sativa, S. bicolor, A. thaliana, and P. trichocarpa) (Figure 4b-e). These results showed that three pairs of homologous genes existed between B. nivea and O. sativa, while two pairs existed between B. nivea and S. bicolor. Moreover, 18 pairs of homologous genes were detected between B. nivea and A. thaliana, and 21 pairs were identified between B. nivea and P. trichocarpa (Table S2). These results show that ramie XTHs have less homology with monocotyledons than with dicotyledons, and thus, we speculate that these XTH genes may be involved in differentiation of dicotyledons.

Gene-Duplication Analysis of BnXTHs
To determine the relationships between BnXTH members, we used the MCScanX method for a collinearity analysis. Six genes (BnXTH6-BnXTH16, BnXTH8-BnXTH9, and BnXTH17-BnXTH18) exhibited complex segmental duplication events (Figure 4a), implying that segmentally duplicated genes may have similar functions regulated via the same biological pathways in ramie. To further evaluate the evolution and development of the BnXTH family, we compared the collinearity of XTH genes between B. nivea and four other plants (O. sativa, S. bicolor, A. thaliana, and P. trichocarpa) (Figure 4b-e). These results showed that three pairs of homologous genes existed between B. nivea and O. sativa, while two pairs existed between B. nivea and S. bicolor. Moreover, 18 pairs of homologous genes were detected between B. nivea and A. thaliana, and 21 pairs were identified between B. nivea and P. trichocarpa (Table S2). These results show that ramie XTHs have less homology with monocotyledons than with dicotyledons, and thus, we speculate that these XTH genes may be involved in differentiation of dicotyledons.
other plants (O. sativa, S. bicolor, A. thaliana, and P. trichocarpa) (Figure 4b-e). These results showed that three pairs of homologous genes existed between B. nivea and O. sativa, while two pairs existed between B. nivea and S. bicolor. Moreover, 18 pairs of homologous genes were detected between B. nivea and A. thaliana, and 21 pairs were identified between B. nivea and P. trichocarpa (Table S2). These results show that ramie XTHs have less homology with monocotyledons than with dicotyledons, and thus, we speculate that these XTH genes may be involved in differentiation of dicotyledons.

Ciselement Analysis of the BnXTHs
We used the PlantCARE service to analyze the cisregulatory elements of BnXTHs in the upstream sequences (~2000 bp) of their promoters that were associated with response to abiotic and biotic stress, phytohormone signaling, and plant growth and development. We predicted 56 related cisacting elements ( Figure 5 and Table S3), among which 17 were involved in abiotic-and biotic-stress responses. ARE, MYC, STRE, and MBS were the most abundant among the 17 ciselements. Moreover, 11 cisregulatory elements were related to plant hormone responses, including abscisic acid (ABA), gibberellin (GA), salicylic acid (SA), methyl jasmonate (MeJA), and auxin responses. These elements included TGACG motifs, ABREs, TCA elements, TGA elements, and other related ciselements. There were 28 cisregulatory elements related to plant growth and development, among which lightresponsive elements, including GT1 motifs, TCCC motifs, GATA motifs, Sp1, and other related elements, were the most abundant.

Ciselement Analysis of the BnXTHs
We used the PlantCARE service to analyze the cisregulatory elements of BnXTHs in the upstream sequences (~2000 bp) of their promoters that were associated with response to abiotic and biotic stress, phytohormone signaling, and plant growth and development. We predicted 56 related cisacting elements ( Figure 5 and Table S3), among which 17 were involved in abiotic-and biotic-stress responses. ARE, MYC, STRE, and MBS were the most abundant among the 17 ciselements. Moreover, 11 cisregulatory elements were related to plant hormone responses, including abscisic acid (ABA), gibberellin (GA), salicylic acid (SA), methyl jasmonate (MeJA), and auxin responses. These elements included TGACG motifs, ABREs, TCA elements, TGA elements, and other related ciselements. There were 28 cisregulatory elements related to plant growth and development, among which lightresponsive elements, including GT1 motifs, TCCC motifs, GATA motifs, Sp1, and other related elements, were the most abundant.

Expression Analysis of the BnXTH-Family Genes under Cd Treatment
To determine whether BnXTH-family genes are involved in Cd stress responses, we treated ramie seedlings with CdCl2 and sampled the roots at 0, 3, 6, 9, 12, 24, and 48 h after this treatment. The expression levels of the 19 BnXTH genes ( Figure 6) were analyzed with RT-qPCR, and these results showed that expression of BnXTH1/6/15, especially BnXTH1, increased significantly under Cd treatment. The expressions of the BnXTH1 genes at 6 h

Expression Analysis of the BnXTH-Family Genes under Cd Treatment
To determine whether BnXTH-family genes are involved in Cd stress responses, we treated ramie seedlings with CdCl 2 and sampled the roots at 0, 3, 6, 9, 12, 24, and 48 h after this treatment. The expression levels of the 19 BnXTH genes ( Figure 6) were analyzed with RT-qPCR, and these results showed that expression of BnXTH1/6/15, especially BnXTH1, increased significantly under Cd treatment. The expressions of the BnXTH1 genes at 6 h and 9 h after the treatment were 5.78 and 6.34 times higher than that at 0 h, respectively. BnXTH3 was slightly upregulated and had the highest expression at 12 h: 41.22% higher than that at 0 h. Conversely, expression of BnXTH4/9/14/19 genes had no response to the Cd treatment, while the other BnXTH genes exhibited were downregulated under the Cd treatment. In general, the BnXTH-family genes responded differently to Cd stress.   Boehmeria nivea actin (BnActin) was used as the internal control. Data are presented as means ± SD (n = 3). Statistical significance was determined using an LSD test. Different letters indicate significant differences at p ≤ 0.05.

Functional Analysis of BnXTHs in Yeast
Since the expression levels of BnXTH1/3/6/15 genes were upregulated in response to Cd stress, we selected BnXTH1, BnXTH3, BnXTH6, and BnXTH15 as candidate genes and analyzed their roles in Cd tolerance. The full-length CDSs of BnXTH1, BnXTH3, BnXTH6, and BnXTH15 were cloned and ligated into yeast expression vector p426 GPD and introduced into Cd-sensitive yeast mutant ∆yap1. The Cd-tolerance characteristics of transgenic yeast were then analyzed (Figure 7). We found no significant difference in the growth between the BnXTH1/3/6/15 transgenic yeast strains and the empty vector on the SD-URA medium without Cd. However, the growth of the yeast strains that contained the empty vector was significantly lower compared to that in the BnXTH1/6/15-containing yeast strains, particularly the BnXTH1-carrying yeast strains, on the medium that contained 75 µM of Cd. These results suggest that BnXTH1/6/15 genes play important roles in Cd tolerance. However, the growth of the transgenic yeast strain that contained BnXTH3 was not significant under Cd stress, indicating that BnXTH3 had no Cd tolerance. These results show that the BnXTH gene family is related to the Cd tolerance of ramie, and the three Cd-tolerant genes (BnXTH1/6/15) can be used for genetic improvement of Cd tolerance in ramie.
growth between the BnXTH1/3/6/15 transgenic yeast strains and the empty vector on the SD-URA medium without Cd. However, the growth of the yeast strains that contained the empty vector was significantly lower compared to that in the BnXTH1/6/15-containing yeast strains, particularly the BnXTH1-carrying yeast strains, on the medium that contained 75 μM of Cd. These results suggest that BnXTH1/6/15 genes play important roles in Cd tolerance. However, the growth of the transgenic yeast strain that contained BnXTH3 was not significant under Cd stress, indicating that BnXTH3 had no Cd tolerance. These results show that the BnXTH gene family is related to the Cd tolerance of ramie, and the three Cd-tolerant genes (BnXTH1/6/15) can be used for genetic improvement of Cd tolerance in ramie.

Evolutionary Characteristics of the BnXTH Gene Family in Ramie
A total of 19 BnXTH genes were identified in ramie, compared with the 33 family members in A. thaliana and 29 in O. sativa, suggesting that there are many fewer members of the BnXTH gene family in ramie. This may have been due to the incomplete assembly of the genome, resulting in a failure to identify other members of the BnXTH gene family

Evolutionary Characteristics of the BnXTH Gene Family in Ramie
A total of 19 BnXTH genes were identified in ramie, compared with the 33 family members in A. thaliana and 29 in O. sativa, suggesting that there are many fewer members of the BnXTH gene family in ramie. This may have been due to the incomplete assembly of the genome, resulting in a failure to identify other members of the BnXTH gene family or a loss of several BnXTH genes in the genome [32]. This result is consistent with those of a previous study that showed that Brassica oleracea contains fewer XTH family members than those in Brassica rapa [22]. These phylogenetic results showed that BnXTHs were relatively conserved at the DNA and protein levels and could be divided into four subclasses (Figure 1), similar to those of other plants [33,34]. There were 14 BnXTH members in Groups I and II, accounting for 73.68% of all members. When only BnXTH-family proteins were used to construct an evolutionary tree, members of Groups I and II clustered together, making it difficult to distinguish them (Figure 3a). This was similar to the findings of previous studies, which reported no significant difference between members of Groups I and II, suggesting that the two can be combined into a larger group: that is, Group I/II [35,36]. Furthermore, a multiple sequence alignment showed that the members of Groups I, II, and IIIB (except for BnXTH5) were located near the XET catalytic site (HDEIDFEFLG) and had typical Nglycosylation residues, while Group IIIA members lacked a consensual N-glycosylation site. The same phenomenon was reported in A. thaliana [37,38]. Additionally, variation of the conserved catalytic motif (HDEIDFEFLG) was observed in BnXTH proteins, consistently with previous studies involving plants, such as A. thaliana [18] and P. trichocarpa [14]. Variation in this motif may affect its enzyme catalytic activity, which necessitates further studies of the enzyme activity and other functions of these motif-variant genes.

Evolutionary Analysis of the BnXTH Gene Family
It is speculated that A. thaliana experienced genome-wide duplication thrice in the past 250 million years, while O. sativa experienced genome-wide duplication about 70 million years ago [39]. As early as 1970, scholars proposed that gene replication is the basic mechanism through which genes obtain new functions, enabling one gene to maintain its original function and another to change its function [40]. Gene duplications are also considered one of the main forces of evolution and expansion of gene families [41]. Previous studies of XTH-family genes have shown that gene-fragment-repetition events have occurred in tobacco [19], soybeans [30], and other plants. Gene-duplication phenomena also occurred in the BnXTH gene family of ramie, with fragment-repetition events of the three pairs of isogenous genes (BnXTH6-BnXTH16, BnXTH8-BnXTH9, and BnXTH17-BnXTH18) (Figure 4a). This indicates that gene replication plays a certain role in evolution of BnXTH genes but is not the main driver of the expansion of the BnXTH gene family. The sequence similarity between the BnXTH8-BnXTH9 and BnXTH17-BnXTH18 gene pairs was significantly high (Figure 3a), and these two gene pairs exhibited similar expression patterns under Cd stress ( Figure 6). Although the BnXTH6-BnXTH16 gene pair also had high sequence similarity, its expression patterns were completely different under Cd stress. Similar observations were reported in B. rapa, where the BraA.XTH32.a-BraA.XTH32.c pair and the BraA.XTH14.a-BraA.XTH14.b pair showed similar expression patterns, while BraA.XTH23.a and BraA.XTH23.b showed different expression patterns [22]. This may have been because duplicate genes underwent nonfunctionalization, neofunctionalization, or subfunctionalization during evolution, resulting in similar or different gene-expression patterns [42]. There were few collinear gene pairs between ramie and monocotyledonous plants (O. sativa and S. bicolor) compared to the gene pairs detected between ramie and dicotyledonous plants (A. thaliana and P. trichocarpa). This indicates that numerous XTH gene variations and replications may occur in dicotyledonous plants during evolution. This evolution phenomenon was also found in the CAX-family genes of P. trichocarpa [43].

BnXTH Gene Response to Cd Stress
Abiotic stress can lead to transcript-level changes in XTH genes. For example, expression of AtXTH14 and AtXTH15 decreased significantly under Al stress, resulting in reduced XET activity and thus enhancing the Al tolerance of A. thaliana [26]. Additionally, expression of PeXTH was significantly upregulated in the roots and leaves of P. euphratica under Cd stress [44]. The current study found that the BnXTH gene family responded to Cd stress, under which BnXTH1, BnXTH3, BnXTH6, and BnXTH15 were upregulated, while BnXTH5, BnXTH16, BnXTH17, and BnXTH18 were significantly downregulated. Similar contrasting expression patterns of this gene family in response to abiotic stress have been reported in other plants. For example, expression of CsXTH1, CsXTH4, CsXTH6, and CsXTH7 was upregulated, while that of CsXTH3 was downregulated, in Camellia sinensis under fluorine stress [33]. Furthermore, we also found that BnXTH-family proteins have different subcellular localizations; for example, most BnXTH-family proteins were located in the cell wall, while BnXTH13 and BnXTH17 were located in the extracellular region. This may have been due to the expression-pattern diversity of the XTH gene family [18]. The differences in the subcellular localizations and expressions of proteins in the same family lead to differences in gene function [45], indicating that the different members of the BnXTH gene family exhibit different functions.
Expression of a gene often depends on the regulation of its upstream promoter [46]; thus, it is particularly important to analyze the upstream promoter sequences of a gene. The sequence analysis of the upstream promoter sequence of the BnXTH-family gene showed that the promoter of the BnXTH-family gene contained several cisacting elements, such as MYB, ABRE, AS-1, STRE, and MBS, that were involved in biotic and abiotic stress responses. MYB, MBS, and other ciselements are the binding sites of MYB transcription factors, which regulate defense responses by binding to the MBS elements on target genes [47]. Some hormone response elements, such as EREs and ABREs, are also involved in biotic and abiotic stress responses, whereby ABREs play important roles in response to abiotic stress [48,49]. These elements ensure that BnXTH genes are rapidly induced under stressful conditions.
Excessive absorption of heavy metals by plants causes serious toxicity to those plants [50]. Cell walls, especially hemicellulose, are reportedly the key Cd storage areas in plants [12]. In A. thaliana, phosphorus-deficiency tolerance significantly reduced hemicellulose content in the cell wall and alleviated Cd toxicity [51]. Heterologous expression of PeXTH in tobacco increased the root length and fresh weight of transgenic plants by enhancing their tolerance of Cd [44]. Similarly, in this study, the tolerance analysis of the transgenic yeast showed that heterologous expression of BnXTH1, BnXTH6, and BnXTH15 under Cd stress could enhance the Cd tolerance of yeast cells (Figure 7). These results suggest that the BnXTH gene family is involved in Cd stress responses.

Identification and Analysis of BnXTH-Family Genes in the Ramie Genome
There are two conserved domains in XTH proteins: the Glyco_hydro_16 domain (PF00722) and the XET_C domain (PF06955) [22]. We generated a hidden Markov model (HMM) file of these two conserved domains using the Pfam database (https://Pfam.xfam. org/ (accessed on 13 October 2021)) [52]. The ramie genome was analyzed using HMMER v3.3.2 (Howard Hughes Medical Institute, Washington, DC, USA) [53], which identified candidate genes. Redundant sequences were manually removed, and the ramie BnXTH genes were finally obtained.

Sequence Alignment and Phylogenetic Analyses
The XTH protein sequence of A. thaliana was searched for in the NCBI protein database (http://www.ncbi.nlm.nih.gov/protein/ (accessed on 21 September 2022)), and coding sequences (CDSs) of BnXTH-family genes were used to generate BnXTH protein sequences. A neighbor-joining (NJ) phylogenetic tree based on full-length sequences of AtXTHs and BnXTHs was constructed via MEGA 6.0 [57] using 1000 bootstrap replicates. A multiple sequence alignment of all BnXTH proteins was then conducted with Clustal (version:X 2.0, University College Dublin, Dublin, Ireland) [58].

Gene Structure and Motif Composition Analysis
The genomic sequences and CDSs of the BnXTH-family genes were extracted from the ramie genome, and structures of the BnXTH-family genes were constructed using GSDS 2.0 online software (http://gsds.gao-lab.org/ (accessed on 22 September 2022)) [59]. MEME online software (https://meme-suite.org/meme/tools/meme (accessed on 22 September 2022)) [60] was then used to analyze the conserved motifs of the BnXTH proteins, after which the conserved sites were set at 6-50 and the conserved-motif number parameter was set to 10. These structures were then visualized using TBtools (version: v1.098774, South China Agricultural University, Guangzhou, China) [61].

Analysis of Gene Duplication Events and Collinearity of the BnXTHs
We used a multiple-collinearity scanning toolkit (MCScanX) and TBtools software plugins to analyze gene duplication events of BnXTH in the ramie genome and the collinearity of the XTH genes between Boehmeria nivea and O. sativa, Sorghum bicolor, A. thaliana, and Populus trichocarpa. A collinearity graph was then generated using TBtools.

Analysis of the BnXTH Gene Promoter
The 2000 bp sequence upstream of the BnXTH gene was predicted using the Plant-CARE database [62], and its cisregulatory elements were analyzed. These cisregulatory elements were then counted and classified according to the functional effects on the promoter. Thereafter, TBtools was used to visualize results and generate heat maps.

RT-qPCR Analysis of BnXTH Expression under Cd Stress
The "Xiangzhu No. 3" ramie material used in this study was provided by our research group at Hunan Agricultural University. Terminal buds were cultured in a half-strength Hoagland nutrient solution for 3 weeks, after which the roots of ramie seedlings with the same growth rate were collected. After 1 week of culturing in the half-strength Hoagland nutrient solution, the ramie seedlings were treated with 50 µM of CdCl 2 , and the root samples were collected at 0, 3, 6, 9, 12, 24, and 48 h after the treatment. The samples were frozen in liquid nitrogen and preserved at −80 • C. The growth conditions of the ramie were as follows: 14 h day/10 h night photoperiod, day and night temperature of 26/24 • C, relative humidity of 60%, and light intensity of 20,000 lux.
Total RNA was extracted according to the instructions of the plant RNA extraction kit (Vazyme, Nanjing, China). The extracted RNA was reverse-transcribed into cDNA using a reverse transcription kit (Vazyme, Nanjing, China) with primers that were designed using Premier 5.0 and synthesized via Sangon Biotech (Shanghai, China). The primer sequences are presented in Table S4. The synthesized cDNA was used as a template for qPCR analysis on the Bio-Rad CFX96 instrument (Bio-Rad, Hercules, CA, USA) using the AceQ ® Universal SYBR qPCR kit (Vazyme, Nanjing, China) and the BnActin gene as the control. Each sample was quantified in triplicate, and the relative quantitation of each gene was conducted using the 2 −∆∆Ct method [21].

Functional Analysis of Cd-Induced Expression of BnXTHs in Yeast
The BnXTH1, BnXTH3, BnXTH6, and BnXTH15 genes, which were shown to respond to Cd stress, were introduced into the yeast cells for functional analysis. Briefly, the ramie cDNA was used as a template for PCR, using the primers shown in Table S4. The PCR conditions and procedures were as described by Jiang et al. [14]. Thereafter, the PCR products were cloned into the pEASY-blunt vector (TransGen Biotech Company, Beijing, China) and sequenced at Sangon Biotech (Shanghai, China), followed by subsequent cloning into the p426 GPD vector at the SmaI/SalI sites. The p426-BnXTH1/3/6/15 recombinant vector and the empty p426 vector were introduced into a mutant Saccharomyces cerevisiae yeast strain, ∆yap1, which lacked transcriptional regulatory protein YAP-1 for Cd tolerance. For the Cd-tolerance analysis, the transgenic yeast cells were inoculated into a liquid synthetic dropout medium without uracil (SD-URA) and incubated at 30 • C on a shaker at 200 rpm until OD 600 = 1.0 was reached. The precipitate was collected via centrifugation at 10,000 rpm for 1min, followed by suspension in ddH 2 O. The suspension was diluted to 10 −1 , 10 −2 , and 10 −3 times its original state, and 2 µL of the 10 −2 dilution was cultured as droplets on solid SD-URA media that contained 0 and 75 µM of CdCl 2 . The plates were incubated at 30 • C for 3 days, and the cultures were observed and photographed.

Statistical Analysis
All data are presented as means ± SD. The data were analyzed with one-way analysis of variance (ANOVA), followed by an LSD post hoc test using SAS 9.4 (SAS Institute, Cary, NC, USA) at a p ≤ 0.05 significance level.

Conclusions
This study identified 19 BnXTHs and evaluated their evolution, phylogeny, chromosomal locations, gene duplications, and cisregulatory elements. RT-qPCR results showed that most BnXTH genes responded to Cd stress. Many cisregulatory elements in BnXTH gene promoters were related to abiotic and biotic stress responses. In summary, under Cd stress, transcription factors that are located upstream of BnXTHs are triggered to bind to the cisregulatory elements that are upstream of BnXTHs and regulate expression of BnXTH genes to enhance the Cd tolerance of ramie. Additionally, functional analysis of heterologous expression in yeast showed that BnXTH1, BnXTH6, and BnXTH15 may be involved in Cd tolerance. However, further validation, using transgenic methods, in ramie or other plants is needed. These results improved our understanding of the BnXTH gene family and laid a foundation for exploration of the function of BnXTH genes in Cd tolerance and enrichment in ramie.