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Identification of a group of XTHs genes responding to heavy metal mercury, salinity and drought stresses in Medicago truncatula
Ecotoxicology and Environmental Safety 132 (2016) 153–163
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Identification of a group of XTHs genes responding to heavy metal mercury, salinity and drought stresses in Medicago truncatula Yun Xuan a,c, Zhao Sheng Zhou b,n, Hai Bo Li a, Zhi Min Yang a,n a
Department of Biochemistry and Molecular Biology, College of Life Science, Nanjing Agricultural University, Nanjing 210095, China Jiangsu Province Key Laboratory of Marine Biology, College of Resources and Environmental Science, Nanjing Agricultural University, Nanjing, China c Agricultural Engineering Research Institute, Anhui Academy of Agricultural Sciences, Hefei 230031, China b
art ic l e i nf o
a b s t r a c t
Article history: Received 3 March 2016 Received in revised form 3 June 2016 Accepted 6 June 2016
Xyloglucan endotransglucosylase/hydrolases (XTH) are one of the key enzymes regulating cell wall construction, extension and metabolism. In the study, 44 XTH protein genes from Medicago truncatula genome were identified using bioinformatics, microarray and RT-PCR. Each XTH was showed to possess a highly conserved domain ((D/N)-E-(I/L/F/V)-D-(F/I/L)-E-(F/L)-L-G), and most of XTHs possess four Cys in the C terminal region, which suggests the potential for generating disulfide bonds. Based on the XTH protein sequences, these XTHscan be classified into three major families and each family can be subdivided into more groups. Examination of the genomic location of XTH genes on M. truncatula chromosomes showed that the evolutional expansion of the genes was possibly attributed to localized gene duplications. To investigate the possible involvement of the XTHs responding to heavy metals and other abiotic stresses, the XTH genes were exposed to heavy metal (Hg or Cu), salt and drought stresses. There were 28, 21 and 21 MtXTH genes found to respond to HgCl2, salt and drought stresses, respectively, but their expression were different under the stresses. Some of the XTH genes were well confirmed by quantitative RT-PCR (qRTPCR). We further specified expression of a XTH gene Medtr4g128580 (MtXTH3) under different environmental stresses, and showed that MtXTH3 was induced by Hg exposure. These results indicated that a group of MtXTHs could be differentially expressed under the environmental stresses. & 2016 Elsevier Inc. All rights reserved.
Keywords: Medicago truncatula XTH Salinity Drought Heavy metal
1. Introduction Plant cell wall is a dynamic network composed of celluloses embedded in a matrix of hemicellulosic, pectic polysaccharides and numerous proteins and enzymes (Hayashi, 1989; Cosgrove, 2005). These celluloses are essential for plant growth, development and protecting cell against biotic and abiotic challenges (Jarvis, 2009). Under environmental challenges (e.g. heavy metals and salinity), plant cell wall undergoes a variety of mechanical, physiological and molecular changes, along with variations of cellular morphology, metabolism and a number of non-protein components (Cosgrove et al., 1997). With regard to non-enzymatic components, xyloglucans are the primary cell wall hemicelluloses that tether adjacent cellulose microfibrils by means of hydrogen bonds and contribute a lot to cell-wall strength in dicotyledonous plants such as Arabidposis thaliana (Fry, 1989). The major enzymes involved in biological processes include xyloglucan endotransglucosylase/hydrolases (XET) (Rose et al., 2002; n
Corresponding authors. E-mail addresses: [email protected] (Z.S. Zhou), [email protected] (Z.M. Yang). http://dx.doi.org/10.1016/j.ecoenv.2016.06.007 0147-6513/& 2016 Elsevier Inc. All rights reserved.
Maldonado-Mendoza et al., 2005), endo-1,4-b-D-endoglucanase (EGase) (del Campillo, 1999), expansins (EXP) (Li et al., 2003) and the plasma membrane proton pump (PM-H þ -ATPase (Duby and Boutry, 2009), all of which are believed to modulate cell wall shape through primary or secondary wall-loosening mechanism (Li et al., 2003). XTH is one of the major enzymes essential for cell wall extension, construction and degradation and cleaves xyloglucan chains through xyloglucan endohydrolase (XEH) (Nishitani, 1995; Yokoyama and Nishitani, 2001; Van Sandt et al., 2007) or rejoins xyloglucan chains through xyloglucan endotransglucosylase (Thompson and Fry, 2001). XTHs belong to a multigene family that can be classified into glycoside hydrolase GH16 (Campbell and Braam, 1999). In Arabidopsis, XTHs are divided into three major subgroups (group I, II and III) based on their activities (Campbell and Braam, 1999; Rose et al., 2002). Group I and II show prominent xyloglucan endotransglucosylase activity; and group III can be further subdivided into group IIIA and group IIIB (Baumann et al., 2007). While group IIIB shows prominent XET activity, group IIIA displays extreme XEH activity. XTHs are involved in many physiological responses such as elongation of plant tissue, formation of the secondary vascular tissue and organ ripening (Campbell and
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Fig. 1. Dendrogram displaying the sequences of xyloglucan endotransglucosylase/hydrolases from the M. truncatula. A: Phylogenetic tree of the 44 XTH gene family proteins from only M. truncatula. B: Phylogenetic tree of the XTH gene family protein from Medicago truncatula and Arabidopsis. The diagram was constructed using MEGA6.
Braam, 1999; Bourquin et al., 2002; Miedes and Lorences, 2009; Harada et al., 2011; Singh et al., 2011). Recently, XTHs have been shown to be particularly interesting because some of genes have been identified to regulate plant response to abiotic stresses (Jan et al., 2004; Yokoyama et al., 2004; Cho et al., 2006; Sasidharan et al., 2010; Yang et al., 2011). For example, AtXTH14, 15, and 31 expressions were significantly down-regulated by Al in Arabidopsis roots (Yang et al., 2011). T-DNA insertion mutants, xth31, xth17 and xth15, were more Al resistant than the wild type in Arabidopsis (Zhu et al., 2012, 2014). Constitutive expression of CaXTH3 improves drought and salt tolerance in transgenic Arabidopsis plants (Cho et al., 2006). Over the last decades, many XTHs protein-coding genes have been identified in plants using genome-wide sequencing (Yokoyama and Nishitani, 2001). In rice, Populus, and tomato, there are 29, 41 and 25 genes have been identified, respectively (Yokoyama et al., 2004; Geisler et al., 2006; Saladie et al., 2006). M. truncatula is a model legume species that contributes to our understanding of mutualistic interaction between M. truncatula and arbuscular mycorrhiza fungi (Zhou et al., 2013; Song et al., 2015). Its unique traits enable it one of the excellent species for studying genetic and molecular complexity of development and acclimation to toxic heavy metals (Zhou et al., 2008; Aloui et al., 2009; Zhou et al., 2012). In comparison with Arabidopsis, the molecular and biochemical mechanisms of M. truncatula XTH genes are poorly understood. In this study, we attempted to perform a genomewide identification and analysis of XTH genes in response to mercury (Hg), salt and drought stress. In silico analysis resulted in identification of 44 potential XTH protein genes from M. truncatula. Using a customer-prepared microarray and qRT-PCR, some of the XTH genes in response to Hg were validated. Thus, the goal of the study is to identify M. truncatula XTH genes in responses to
heavy metal and other abiotic stresses and to figure out the possible mechanism for the XTH genes involved in the environmental stress responses. To our knowledge, this is the first report of global identifying XTH genes from M. truncatula responding to heavy metals and abiotic stress.
2. Materials and methods 2.1. Plant materials Seeds of M. truncatula (cv. Jemlog) were surface-sterilized, rinsed and germinated for 2 d in the culture dish under darkness. After germination, young seedlings were transferred to 1.2 L polyetheylene containers with 1/4- Hoagland nutrient solution and cultured for three weeks under the condition of 227 1 °C for 16 h photoperiod. The pH of culture solutions was adjusted to 5.8. Culture solutions were renewed every 3 d. Three week-old seedlings were exposed to PEG6000 (0%, 10%, 20% and 30%), NaCl (0, 25, 150 and 300 mM), HgCl2 (0, 10, 50 and 100 mM), or CuCl2 (0, 10, 50 and 100 mM) for 6 h. For abscisic acid (ABA) treatment, the leaves of seedlings were sprayed with ABA at 0, 10, 50 and 100 mM. When harvested, shoots and roots were separately collected and quickly frozen in liquid nitrogen, and stored at 80 °C for further analyses. All experiments were repeated in triplicate. 2.2. Microarray hybridization and data analysis Three-week-old seedlings with three replicates were treated with 10 μM HgCl2 for 0, 6, 12, 24 and 48 h and harvested, respectively (Zhou et al., 2013; Song et al., 2015). Total RNA from tissues at each time point for the respective treatment was
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Fig. 2. Conserved primary structural features of the sequences of xyloglucan endotransglucosylase/hydrolases from M. truncatula were identified using MEME (http://meme. nbcr.net/meme/).
extracted using RNA Isolation Kit (Applied Biosystem p/n AM1556). The RNA extract was quantified by NanoDrop ND-2000 (Thermo Scientific). RNA integrity was assessed using Agilent Bioanalyzer 2100 (Agilent Technologies). Four subsets of total RNA were prepared, with two derived from the original RNA pools prepared from Hg-treated roots and leaves (R þHg, L þHg) and the other two from the RNA pools derived from Hg-free roots and leaves (R Hg, L Hg). Total RNA from the four libraries was dephosphorylated, denaturated and labeled with Cyanine-3-CTP. After purification, the labeled RNA was hybridized onto the microarray. The custom probes were designed by Agilent Technologies on the basis of spliced transcript sequence of annotational
Medicago truncatula genes (http://www.medicagohapmap.org/? genome). The arrays were scanned with the Agilent Scanner G2505C (Agilent Technologies). Feature Extraction software (version10.7.1.1, Agilent Technologies) was used to analyze array images to get raw data. The Genespring software (version 12.5; Agilent Technologies) was employed to finish the basic analysis with the raw data. The data were normalized with the quantile algorithm. The probes that at least 1 condition out of 2 conditions has flags in “Detected” were chosen for further data analysis. After normalization, variance stabilization and log transformation of data and the log signal intensity values for M. truncatula probe IDs corresponding to XTH protein-encoding genes were treated as a subset.
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Fig. 3. Hierarchial clustering display of the 28 xyloglucan endotransglucosylase/hydrolase genes differentially expressed in Hg-treated and Hg-free roots [(RþHg) vs (R Hg)]_ and leaves [(L þHg) vs (L Hg)] of Medicago truncatula. The log signal values were used for clustering.
For salt stress response analysis, the raw transcriptional data of M. truncatula treated by 180 mM NaCl for 0, 6, 24 and 48 h were downloaded from GEO (http://www.ncbi.nlm.nih.gov/geo/query/ acc.cgi? acc ¼GSE13921) (Li et al., 2009) and normalized using RMA algorithm with Expression Console software (Affymetrix Technologies) (Irizarry, et al., 2003). The identifiers were updated based on the file “AffyMap-Mt4.0v1 Genes Spliced Transcript Seq” (http://www.medicagohapmap.org/? genome). Differentially expressed XTH protein-encoding genes were identified through the fold change (fold change Z2.0) as well as p-value ( r0.05). 2.3. Quantitative RT-PCR analysis Total RNA was isolated based on the method described previously (Shen et al., 2011). One microgram of total RNA extracted using Trizol (Invitrogen, Carlsbad, CA) was incubated at 37 °C for 30 min with 1 unit of RNase-free DNaseI (Takara) and 1 mL 10 reaction buffer with MgCl2 and incubated with 1 mL of 50 mM EDTA for DnaseI inactivation at 65 °C for 10 min. A 1% agarose gel, stained by ethidium bromide, was run to monitor the RNA integrity. The reverse transcription reaction mixture (20 mL) contained 1 mg RNA, 100 mM oligo (dT) primers (1 mL), 10 mM dNTP (deoxyribonucleotide triphosphate) mixture (1 mL), 40 U mL 1 of RNase inhibitor (0.5 mL), 200 U mL 1 of M-MLV reverse
transcriptase (0.5 mL) and 4 mL of 5 M-MLV buffer. The reaction solution was maintained at 42 °C for 10 min and heated at 95 °C for 2 min. The resultant cDNA was then diluted 5 fold with sterile water and kept at 20 °C for qRT-PCR analysis. Amplification reaction was performed in a 20 mL mixture containing 10 ng of template, 10 mL of SYBR-Green PCR Mastermix (Toyoba, Japan) and 10 pmol of primers. Quantitative RT-PCR was performed using MyIQ Single Color Real time PCR system (Bio-Rad) and IQ5 software (Bio-Rad). The temperature profile was 95 °C for 30 s, followed by 40 cycles at 95 °C for 15 s, 60 °C for 60 s and a melt curve at 65 °C 5 s. Data were analyzed using CFX Data Analysis Manager Software. The relative expression level was normalized to that of the EF1α, which were used as the internal control in M. truncatula (Kenneth and Thomas, 2001). The primers used for qRT-PCR are presented in Supplementary data 1. 2.4. GUS assay Histochemical detection of GUS activities in the transgenic Arabidopsis seedlings or different tissues were analyzed based on the method of Song et al. (2012). Plant tissues were incubated at 37 °C in 0.5 mg/mL 5-bromo-4-chloro-3-indolyl-glucuronic acid (X-Gluc), 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, 0.1% Triton X-100, and 100 mM sodium phosphate
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Fig. 4. Quantitative RT-PCR validation of randomly selected XTH genes from Medicago truncatula. Four-week-old seedlings were exposed 10 μM HgCl for 12 h. After that, total RNA was extracted and qRT-PCR was performed.
buffer, pH 7.0. Samples were bleached several times with 70% ethanol. pBI121 was used as the plant expression vector with the promoter sequences of genes and NOS terminator as transcriptional termination sequences. The vector was transferred into Arabidopsis via Agrobacterium-mediated transformation. GUS staining patterns were verified in two independent homozygous T3 lines, and representative individuals were chosen for photography.
(Version 2.0; http://www.clustal.org/) with all predicted XTH motif. A neighbor joining (NJ) tree was constructed by MEGA6 using the p-distance method with gaps treated by pairwise deletion and a 1000 bootstrap replicate. Chromosomal mapping was performed using the GenomePixelizer (http://niblrrs.ucdavis.edu/ GenomePixelizer/GenomePixelizer_Welcome.html). Hierarchial clustering display performed using Euclidean distance according to expression pattern with the MeV software.
2.5. In silico analysis of putative XTH proteins from M. truncatula
2.6. Sequence alignment and phylogenetic tree analysis
Putative XTH proteins of M. truncatula were searched using M. truncatula genome database (http://www.medicagohapmap.org/ downloads/mt40) (Song et al., 2015). Multiple sequence alignments were performed by the Clustal X
To analyze the evolutionary relationships between MtXTHs and those from other plant species, the amino acid sequences of M. truncatula and Arabidopsis thaliana were retrieved from the websites (http://www.jcvi.org/medicago/) and Tair (http://www.arabi
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Fig. 5. Hierarchial clustering display of xyloglucan endotransglucosylase/hydrolase genes of Medicago truncatula seedlings exposed to salinity (A) and drought (B). A: two week-old seedlings were grown in hydroponics media with 180 mM NaCl for 0, 6, 24 and 48 h (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi? acc ¼GSE13921). B: Normal growth seedlings were subject to water withdrew for 0–14 d (http://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-2681/? query). The log signal values were used for clustering.
dopsis.org/). The phylogenetic tree was constructed by the neighbor joining (NJ) method with MEGA6.
3. Results 3.1. Identification of XTH protein-coding genes from M. truncatula
2.7. Statistical analysis Experiments in the study were independently performed in triplicate. Each result shown in the figures was the mean of at least three replicated treatments and each treatment contained at least 30 plants. The significant differences between treatments were statistically evaluated by standard deviation and one-way analysis of variance (ANOVA). The data between differently treated groups were compared statistically by ANOVA, followed by the least significant difference (LSD) test if the ANOVA result was significant at P o0.05.
We use our recent transcriptome (Zhou et al., 2013) and other publicly available datasets (http://www.megasoftware.net/index. php) (Tamura et al., 2013) to identify XTH genes from M. truncatula. In total, 44 non-redundant potential MtXTH genes have been identified (Data S2). Alignment of the MtXTH gene protein sequences revealed that the amino acid residues within the highly conversed regions showed 80% identity, although the full length sequences of XTH proteins were relatively low in similarity. Each MtXTH possessed a highly conserved catalytic domain ((D/N)-E-(I/ L/F/V)-D-(F/I/L)-E-(F/L)-L-G), one N-linked glycosylation site was identified adjacent to the C-terminal side of the catalytic domain
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Fig. 6. Differential expression of MtXTH3 (Medtr4g128580) in tissues of M. truncatula and MtXTH3 promoter–GUS expression pattern in Arabidopsis transgenic plants. A: qRT-PCR analysis of MtXTH3 expression at different tissues. YEL, young expanding leaf; C, cotyledon; R, radical; H, hypocotyls; ST1, young expanding internode of stem (the second internode); ST2, the third internode of stem from the apex; Y, leaf at the end of expansion; and F, flower. B: MtXTH3 promoter–GUS expression pattern. (a) seed; (b) one-day-old germinating seed; (c) two-day-old germinating seed; (d) three-day-old seedling; (e) four-day-old seedling; (f) seven-day-old seedling; (g) fourteen-day-old seedling; (h) eighteen-day-old seedling; (i) stem; (j) radical; (k) rosette leaf; (l) lateral root; (m) flower; (n) tender silique; (o) mature silique; and (p) carpel and seed scattering.
in 36 of the MtXTH protein; most of MtXTHs possessed four Cys in the C terminal region, suggesting a potential for generating disulfide bonds (Data S2). Using the identified M. truncatula XTH sequences for alignments, a phylogenetic tree with all MtXTH genes was created (Fig. 1A). The divergence between clusters I/II of the MtXTHs was not apparent and the third cluster was obviously distinct from clusters I/II. These MtXTHs could be divided into three major clusters. Clusters I/II and cluster III contain 35 and 9 genes, respectively. To figure out the correlation of the M. truncatula XTHs and those from other dicots, a comparative analysis was performed on the distribution of XTHs between M. truncatula and Arabidopsis.
Mapping the MtXTH sequences to those of Arabidopsis resulted in identification of nearly a hundred matches. After removal of the redundancy, 33 XTH genes from Arabidopsis were retrieved. The overall sequences for the individual pairs varied between the M. truncatula and Arabidopsis homologs. Given the three-subgroup classification for the XTH gene family, the clusters I/II contained 35 MtXTH genes and 26 AtXTH genes, whereas the cluster III consisted of 9 MtXTH and 7AtXTH genes, respectively. While a certain proportion of M. truncatula XTHs were clustered together with their Arabidopsis homologs, some of them had low relations from one another (Fig. 1B). The less clustered XTHs between M. truncatula and Arabidopsis suggest that some of the M. truncatula
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XTHs identified here may not be conserved in Arabidopsis. 3.2. Identification of chromosome locations of XTH genes and specific motifs of XTH proteins from M. truncatula The XTH protein-encoding genes were dispersed across eight chromosomes of M. truncatula (Data S3). Some XTHs were located onto the chromosomes as solitary genes and others as clusters of a few genes were found in tandem. Several highly conserved genes such as Medtr2g089140.1/Medtr2g089140.2 and Medtr5g029100.1/ Medtr5g029100.2 were clustered together on the same chromosomes. Nine sets of XTH gene clusters, which had been generated by extensive genome duplications, may share 8 different ancestral genes. For instance, Medtr6g033055.1/Medtr6g033085.1 and Medtr7g084750.1/Medtr7g084760.1/Medtr7g084770.1 share one ancestral gene. We then examined diverse motifs throughout the regions for the XTH protein genes. Using MEME (http://meme.nbcr.net/ meme/) databases, ten types of motifs were identified from M. truncatula (Data S4; Fig. 2). The different types of motifs that the XTH proteins contain were not defined. The majority of XTHs contain most of the motifs. For example, 17 XTHs contain all types 10 of motifs. Also, 17 XTHs contain 9 types of the motifs. Eight XTHs have 8 types of motifs, and only of XTHs has 5 types of motifs. The differential motifs that XTHs contain suggest that the XTHs may have diverse biochemical features and biological functions. 3.3. A set of XTH genes in response to heavy metal stress To investigate XTH genes that respond to heavy metal stress, we analyzed differential expression of the XTH genes in M. truncatula seedlings exposed to the heavy metal mercury (HgCl2) using a customer-made Agilent microarray. Hg was tested because it is one of the most toxic metals to plants (Zhou et al., 2007; Shen et al., 2011). The probes for the chip were designed based on the update annotational gene version (Mt4.0v1, Agilent) of M. truncatula genome. Following the gene hybridization and chip data processing (e.g. quality controls, normalization and log transformation), 28 XTH genes were probed. The log signal values of the XTH genes were presented in Data S5. Of these, 27 genes were probed in rootsand 10 genes expressed in leaves (Fig. 3). Some of the genes were not probed in roots because of the much low abundance. We then narrowed down the genes to 28 XTH genes passing the criteria of two fold change. Profiling of the gene expression revealed that 11 (40.7%) out of 27 genes were up-regulated, 16 (59.3%) were down-regulated in Hg-treated roots, and all genes were down-regulated in Hg-treated leaves (Fig. 3). We randomly chose 6 XTH genes for qRT-PCR validation under Hg exposure. Our analysis showed that the expression of the genes was well confirmed (Fig. 4). 3.4. A group of XTH genes in response to salt and drought stresses To investigate whether the XTH genes were also responding to abiotic stresses, we used publicly available microarrays generated from M. truncatula (http://www.ncbi.nlm.nih.gov/geo/query/acc. cgi? acc¼ GSE13921). In total, 21 genes were identified in response to salt stress (Fig. 5A, Data S6). These genes responded differently to 180 mM NaCl exposure for 48 h. Profiling of the differentially expressed gene revealed that 8 (38.1%) genes were up-regulated, and 13 (61.9%) genes were down-regulated after salt treatment for 6, 24, 48 h. Of these, Medtr4g128580.1 showed higher transcriptional expression, whereas Medtr7g084750.1 displayed a relatively lower expression following 48 h of salt exposure. We also used publicly data to analyze the response of XTH genes to drought
stress (http://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB2681/?query) (Fig. 5B, Data S7). There were also 21 XTH genes identified for different response to drought stress. Five genes were clearly up-regulated and 9 genes repressed in response to 7–14 d drought. 3.5. Expression analysis of MtXTH3 in M. trancatula under heavy metal and other abiotic stresses To confirm the M. truncatula XTH genes in response to the heavy metal and abiotic stresses, we selected a novel gene Medtr4g128580. As Medtr4g128580 has a high sequence similarity to Arabidopsis AtXTH3, it was herein temporarily assigned as MtXTH3. The full-length cDNA of MtXTH3 was cloned. In silico analysis showed it is a single copy gene, with full-length DNA of 1148 bp containing 3 exons and 2 introns (Data S8). The cDNA was predicted to encode a protein with 288 amino acids and a molecular mass of 32.3 kDa (Data S9). MtXTH3 contains a highly conserved domain (DEIDFEFLG), a putative N-glycosylation motif immediately following the catalytic domain and four Cys, which suggests the potential to form disulfide bonds in the C terminal regions. The SignalP program predicted MtXTH3 has a putative N-terminal signal peptide of 24 amino acids, suggesting that it is a secretory protein. Phylogenetic analysis of MtXTH3 showed that it belonged to group subfamily I/II XTHs with xyloglucan endotransglucosylase (XET) activity. The highest level of sequence homology with AtXTH23 was 78% and 73% identity at the nucleotide and amino acid levels, respectively (Data S10). The pattern of MtXTH3 expression in M. trancatula was analyzed by qRT-PCR using the tissues at different growth and flower stages. MtXTH3 was expressed strongly in cotyledon and hypocotyls, moderately in radical, but lowly in young expanding leaf in 7-day-old seedlings. In 12-week-old plants outdoors, MtXTH3 gene expression was high in fully expanded leaves and the third internode of stem from the apex; meanwhile, low expression was observed in flowers and young expanding internodes of stem. Little was expressed in young expanding leaves (Fig. 6A). To confirm the MtXTH3 expression, a 1992 bp DNA fragments upstream of the MtXTH3 ATG start codon was isolated, fused to the β-glucuronidase (GUS) GUS reporter gene, and transformed into Arabidopsis via Agrobacterium-mediated transformation. Analysis of GUS staining patterns showed that the GUS staining could be detected at nearly all developmental stages, including seeds, seedlings, vegetative tissue, flowers and siliques (Fig. 6B). The expression patterns of MtXTH3 were very similar to those from qRT-PCR. We further analyzed transcriptional responses of MtXTH3 to abiotic stresses M. trancatula using qRT-PCR. As shown in Fig. 7A and B, three week-old seedlings were treated with 0, 25, 150 and 300 mM NaCl. MtXTH3 in roots and shoots was not transcriptionally activated under low levels of NaCl. Only at 150 mM NaCl was MtXTH3 expression significantly upregulated in the tissues. Treatment with 300 mM NaCl increased transcripts up to 42.9 and 70.4 fold over the control in roots and shoots, respectively. We then used PEG6000 (10%, 20% and 30%) to treat the seedlings and showed that strong MtXTH3 expression in roots and shoots was found at 30% PEG6000. Treatment with 10% and 20% PEG6000 induced only a moderate expression of shoot MtXTH3, but no induction was observed in roots (Fig. 7C and D). Abscisic acid (ABA) is a well-known environmental stress-responsive signal molecule. Treatment with ABA at 10, 50 and 100 μM considerably induced MtXTH3 transcription in shoots; however, weak MtXTH3 transcription was detected in roots (Fig. 7E and F). Finally, the effect of the heavy metal Hg(II) and Cu(II) on the expression of MtXTH3 was examined. It is shown that while the low concentrations of Hg and Cu (10 mM) induced a moderate expression of MtXTH3, a very strong induction of MtXTH3 was found at higher
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Fig. 7. Quantitative RT-PCR analysis of MtXTH3 (Medtr4g128580) expression in M. trancatula roots and shoots in response to abiotic stresses. Four week-old seedlings were exposed to NaCl (0, 25, 150 and 300 mM), PEG-6000 (0%, 10%, 20% and 30%), HgCl2 (0, 10, 50 and 200 μM) and CuCl2 (0, 10, 50 and 200 μM) for 6 h or ABA (0, 10, 50 and 100 μM) for 1 h. Vertical bars represent standard deviation (n¼3). Asterisks indicate that mean values are significantly different between the treatments and control (po 0.05).
levels of Hg and Cu (50 and 200 mM) in M. trancatula seedlings (Fig. 7G–J). Taken together, these results indicate that MtXTH3 transcription could be induced by environmental factors including salt, drought and Hg or Cu heavy metals.
4. Discussion A global identification of XTH genes will help to understand gene expression and regulatory mechanisms for plant toxicity or tolerance to environmental stresses such as heavy metals and salinity. XTHs are involved in synthesis of most hemicellulose in the primary cell wall of higher plants and reported to play
important roles in plant response to metals such as Al (Zhu et al., 2013). To date, there are many XTHs genes identified from plants including 33 genes identified in Arabidopsis (Yokoyama and Nishitani, 2001; Rose et al., 2002), 29 genes in rice (Yokoyama et al., 2004), 41 genes in Populus (Geisler et al., 2006) and 25 genes in tomato (Saladie et al., 2006). In this study, 44 genes encoding XTHs were identified from M. trancatula. The number of XTH genes in M. truncatula was a little bit higher those of Arabidopsis and rice, suggesting that it could be the direct result of lineage-specific gains and losses (Song et al., 2015). According to its sequence similarity, Arabidopsis XTHs were classified into three major phylogenetic subgroups (group I, II and III) (Campbell and Braam, 1999; Rose et al., 2002). Group III could
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be further divided into subgroups such as group IIIA and group IIIB; while group I, group II and group IIIB showed prominent xyloglucan endotransglucosylase (XET) activity, group IIIA showed extreme xyloglucan endohydrolase (XEH) activity (Baumann et al., 2007). To analyze correlations between the XTH genes in M. truncatula, we constructed a phylogenetic tree of the entire XTHs by alignment of their sequences. Based on the distribution and relationship of the phylogenetic tree, three distinct families were classified. From the topology and groupings, the M. truncatula XTH proteins with the same or similar domain organization were clustered together. Phylogenetic analysis revealed that they could be classified into three groups. The chromosomal locations of the XTH protein-encoding genes on the M. truncatula genome were analyzed because the gene distribution and the way of gene cluster provide a clue of gene expansion during their evolutional process (Gu et al., 2002). Profiling of the XTHs distribution on eight M. truncatula chromosomes showed the evolvement through duplication events, an observation consistent with other genes from the same species (Song et al., 2015). Based on the definition of gene duplication that the length of the sequence alignment covered Z 80% of the longest gene, and the similarity of the aligned gene regions was Z70% (Gu et al., 2002; Yang et al., 2008), we mapped the XTH protein-encoding genes onto the M. truncatula chromosomes and found many clusters of XTH genes evident on different chromosomes. Several XTH genes were arranged in tandem repeats of genes either in the same or inverse orientation, representative of the localized gene duplications. This finding suggests that tandem duplications of chromosomal regions played an important role in expansion of this XTH protein-encoding genes family. Most of XTH protein-encoding genes are located on Chr 4, although a small amount of the genes are also found on other chromosomes. Plant cell wall plays important roles in the regulation of growth, development and environmental stresses. The plant XTHs is a kind of important cell wall modifying enzymes, participating in not only cell wall extension or degradation but also maintaining the integrity and strength of the cell wall under normal and stressful environments (Nishitani et al., 1995; Cosgrove, 2005; Miedes et al., 2009; Zhu et al., 2012). To investigate the XTH genes that were potentially responding to abiotic stresses, we used microarrays generated from M. truncatula. There were 21 and 21 MtXTH genes found to respond to salt and drought. Furthermore, a costumer-made chip analyzed 28 MtXTH genes in response HgCl2. The MtXTH genes were found to be differentially regulated under the environmental stresses. For example, genes like Medtr4g128400.1 and Medtr4g128410.1were found to be up-regulated under Hg exposure, whereas genes Medtr1g105495.1, Medtr7g102260.1, and Medtr8g064180.1 were down-regulated under the salt stress. Some of the homologue genes were reported to confer drought and salt tolerance in other plant species (Cho et al., 2006). Furthermore, the homologue gene PeXTH was up-regulated by salt or Cd in Populus euphratica plants; PeXTH overexpression in tobacco plants increased capacity for salt and Cd tolerance (Han et al., 2013, 2014). To better understand the possible function of M. truncatula XTH genes, we analyzed in detail expression of a XTH gene MtXTH3 under different environmental stresses. Our analysis showed that MtXTH3 could be freely expressed throughout the whole development stage although its expression was feeble at certain tissues, suggesting that the expression of MtXTH3 might be required for normal plant growth or development. We further analyzed the MtXTH3 transcript response to various environmental stresses. MtXTH3 expression was up-regulated when seedlings were treated with PEG6000, high salt, Hg or Cu treatments. These results implied that MtXTH3 could be involved in the environmental stresses responses possibly through incorporating newly deposited xyloglucan to strengthen cell walls. MtXTH3 showed a high sequence
identity with AtXTH23 in Arabidopsis, which is closely related to XETs in group II (Rose et al., 2002). Further molecular and genetic identification of MtXTH3 will clarify the function of MtXTH3 in mediating the plant response to Hg, salt and drought stresses.
5. Conclusions The present study identified 44 XTH protein genes form M. truncatula by bioinformatics and other biological approaches. Based on their sequence conservation, the MtXTH proteins could be divided into three families. To investigate how these MtXTH genes responded to heavy metal and abiotic stresses, we analyzed microarray-based datasets and some of the MtXTH genes found to be differentially expressed under HgCl2, salt and drought stresses. Of these, several genes were well confirmed by qRT-PCR. One of the MtXTH gene MtXTH3 was further analyzed in detail, confirming that M. truncatula XTH genes are able to respond to the environmental stresses.
Conflict of interest The authors declare that they have no conflict of interest.
Acknowledgements This study was funded by the National Natural Science Foundation of China (31071343 and 31200204) and Colleges and Universities in Jiangsu Province Plans to Graduate Research and Innovation (KYZZ_0181).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ecoenv.2016.06.007.
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Contents lists available at ScienceDirect
Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Identification of a group of XTHs genes responding to heavy metal mercury, salinity and drought stresses in Medicago truncatula Yun Xuan a,c, Zhao Sheng Zhou b,n, Hai Bo Li a, Zhi Min Yang a,n a
Department of Biochemistry and Molecular Biology, College of Life Science, Nanjing Agricultural University, Nanjing 210095, China Jiangsu Province Key Laboratory of Marine Biology, College of Resources and Environmental Science, Nanjing Agricultural University, Nanjing, China c Agricultural Engineering Research Institute, Anhui Academy of Agricultural Sciences, Hefei 230031, China b
art ic l e i nf o
a b s t r a c t
Article history: Received 3 March 2016 Received in revised form 3 June 2016 Accepted 6 June 2016
Xyloglucan endotransglucosylase/hydrolases (XTH) are one of the key enzymes regulating cell wall construction, extension and metabolism. In the study, 44 XTH protein genes from Medicago truncatula genome were identified using bioinformatics, microarray and RT-PCR. Each XTH was showed to possess a highly conserved domain ((D/N)-E-(I/L/F/V)-D-(F/I/L)-E-(F/L)-L-G), and most of XTHs possess four Cys in the C terminal region, which suggests the potential for generating disulfide bonds. Based on the XTH protein sequences, these XTHscan be classified into three major families and each family can be subdivided into more groups. Examination of the genomic location of XTH genes on M. truncatula chromosomes showed that the evolutional expansion of the genes was possibly attributed to localized gene duplications. To investigate the possible involvement of the XTHs responding to heavy metals and other abiotic stresses, the XTH genes were exposed to heavy metal (Hg or Cu), salt and drought stresses. There were 28, 21 and 21 MtXTH genes found to respond to HgCl2, salt and drought stresses, respectively, but their expression were different under the stresses. Some of the XTH genes were well confirmed by quantitative RT-PCR (qRTPCR). We further specified expression of a XTH gene Medtr4g128580 (MtXTH3) under different environmental stresses, and showed that MtXTH3 was induced by Hg exposure. These results indicated that a group of MtXTHs could be differentially expressed under the environmental stresses. & 2016 Elsevier Inc. All rights reserved.
Keywords: Medicago truncatula XTH Salinity Drought Heavy metal
1. Introduction Plant cell wall is a dynamic network composed of celluloses embedded in a matrix of hemicellulosic, pectic polysaccharides and numerous proteins and enzymes (Hayashi, 1989; Cosgrove, 2005). These celluloses are essential for plant growth, development and protecting cell against biotic and abiotic challenges (Jarvis, 2009). Under environmental challenges (e.g. heavy metals and salinity), plant cell wall undergoes a variety of mechanical, physiological and molecular changes, along with variations of cellular morphology, metabolism and a number of non-protein components (Cosgrove et al., 1997). With regard to non-enzymatic components, xyloglucans are the primary cell wall hemicelluloses that tether adjacent cellulose microfibrils by means of hydrogen bonds and contribute a lot to cell-wall strength in dicotyledonous plants such as Arabidposis thaliana (Fry, 1989). The major enzymes involved in biological processes include xyloglucan endotransglucosylase/hydrolases (XET) (Rose et al., 2002; n
Corresponding authors. E-mail addresses: [email protected] (Z.S. Zhou), [email protected] (Z.M. Yang). http://dx.doi.org/10.1016/j.ecoenv.2016.06.007 0147-6513/& 2016 Elsevier Inc. All rights reserved.
Maldonado-Mendoza et al., 2005), endo-1,4-b-D-endoglucanase (EGase) (del Campillo, 1999), expansins (EXP) (Li et al., 2003) and the plasma membrane proton pump (PM-H þ -ATPase (Duby and Boutry, 2009), all of which are believed to modulate cell wall shape through primary or secondary wall-loosening mechanism (Li et al., 2003). XTH is one of the major enzymes essential for cell wall extension, construction and degradation and cleaves xyloglucan chains through xyloglucan endohydrolase (XEH) (Nishitani, 1995; Yokoyama and Nishitani, 2001; Van Sandt et al., 2007) or rejoins xyloglucan chains through xyloglucan endotransglucosylase (Thompson and Fry, 2001). XTHs belong to a multigene family that can be classified into glycoside hydrolase GH16 (Campbell and Braam, 1999). In Arabidopsis, XTHs are divided into three major subgroups (group I, II and III) based on their activities (Campbell and Braam, 1999; Rose et al., 2002). Group I and II show prominent xyloglucan endotransglucosylase activity; and group III can be further subdivided into group IIIA and group IIIB (Baumann et al., 2007). While group IIIB shows prominent XET activity, group IIIA displays extreme XEH activity. XTHs are involved in many physiological responses such as elongation of plant tissue, formation of the secondary vascular tissue and organ ripening (Campbell and
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Fig. 1. Dendrogram displaying the sequences of xyloglucan endotransglucosylase/hydrolases from the M. truncatula. A: Phylogenetic tree of the 44 XTH gene family proteins from only M. truncatula. B: Phylogenetic tree of the XTH gene family protein from Medicago truncatula and Arabidopsis. The diagram was constructed using MEGA6.
Braam, 1999; Bourquin et al., 2002; Miedes and Lorences, 2009; Harada et al., 2011; Singh et al., 2011). Recently, XTHs have been shown to be particularly interesting because some of genes have been identified to regulate plant response to abiotic stresses (Jan et al., 2004; Yokoyama et al., 2004; Cho et al., 2006; Sasidharan et al., 2010; Yang et al., 2011). For example, AtXTH14, 15, and 31 expressions were significantly down-regulated by Al in Arabidopsis roots (Yang et al., 2011). T-DNA insertion mutants, xth31, xth17 and xth15, were more Al resistant than the wild type in Arabidopsis (Zhu et al., 2012, 2014). Constitutive expression of CaXTH3 improves drought and salt tolerance in transgenic Arabidopsis plants (Cho et al., 2006). Over the last decades, many XTHs protein-coding genes have been identified in plants using genome-wide sequencing (Yokoyama and Nishitani, 2001). In rice, Populus, and tomato, there are 29, 41 and 25 genes have been identified, respectively (Yokoyama et al., 2004; Geisler et al., 2006; Saladie et al., 2006). M. truncatula is a model legume species that contributes to our understanding of mutualistic interaction between M. truncatula and arbuscular mycorrhiza fungi (Zhou et al., 2013; Song et al., 2015). Its unique traits enable it one of the excellent species for studying genetic and molecular complexity of development and acclimation to toxic heavy metals (Zhou et al., 2008; Aloui et al., 2009; Zhou et al., 2012). In comparison with Arabidopsis, the molecular and biochemical mechanisms of M. truncatula XTH genes are poorly understood. In this study, we attempted to perform a genomewide identification and analysis of XTH genes in response to mercury (Hg), salt and drought stress. In silico analysis resulted in identification of 44 potential XTH protein genes from M. truncatula. Using a customer-prepared microarray and qRT-PCR, some of the XTH genes in response to Hg were validated. Thus, the goal of the study is to identify M. truncatula XTH genes in responses to
heavy metal and other abiotic stresses and to figure out the possible mechanism for the XTH genes involved in the environmental stress responses. To our knowledge, this is the first report of global identifying XTH genes from M. truncatula responding to heavy metals and abiotic stress.
2. Materials and methods 2.1. Plant materials Seeds of M. truncatula (cv. Jemlog) were surface-sterilized, rinsed and germinated for 2 d in the culture dish under darkness. After germination, young seedlings were transferred to 1.2 L polyetheylene containers with 1/4- Hoagland nutrient solution and cultured for three weeks under the condition of 227 1 °C for 16 h photoperiod. The pH of culture solutions was adjusted to 5.8. Culture solutions were renewed every 3 d. Three week-old seedlings were exposed to PEG6000 (0%, 10%, 20% and 30%), NaCl (0, 25, 150 and 300 mM), HgCl2 (0, 10, 50 and 100 mM), or CuCl2 (0, 10, 50 and 100 mM) for 6 h. For abscisic acid (ABA) treatment, the leaves of seedlings were sprayed with ABA at 0, 10, 50 and 100 mM. When harvested, shoots and roots were separately collected and quickly frozen in liquid nitrogen, and stored at 80 °C for further analyses. All experiments were repeated in triplicate. 2.2. Microarray hybridization and data analysis Three-week-old seedlings with three replicates were treated with 10 μM HgCl2 for 0, 6, 12, 24 and 48 h and harvested, respectively (Zhou et al., 2013; Song et al., 2015). Total RNA from tissues at each time point for the respective treatment was
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Fig. 2. Conserved primary structural features of the sequences of xyloglucan endotransglucosylase/hydrolases from M. truncatula were identified using MEME (http://meme. nbcr.net/meme/).
extracted using RNA Isolation Kit (Applied Biosystem p/n AM1556). The RNA extract was quantified by NanoDrop ND-2000 (Thermo Scientific). RNA integrity was assessed using Agilent Bioanalyzer 2100 (Agilent Technologies). Four subsets of total RNA were prepared, with two derived from the original RNA pools prepared from Hg-treated roots and leaves (R þHg, L þHg) and the other two from the RNA pools derived from Hg-free roots and leaves (R Hg, L Hg). Total RNA from the four libraries was dephosphorylated, denaturated and labeled with Cyanine-3-CTP. After purification, the labeled RNA was hybridized onto the microarray. The custom probes were designed by Agilent Technologies on the basis of spliced transcript sequence of annotational
Medicago truncatula genes (http://www.medicagohapmap.org/? genome). The arrays were scanned with the Agilent Scanner G2505C (Agilent Technologies). Feature Extraction software (version10.7.1.1, Agilent Technologies) was used to analyze array images to get raw data. The Genespring software (version 12.5; Agilent Technologies) was employed to finish the basic analysis with the raw data. The data were normalized with the quantile algorithm. The probes that at least 1 condition out of 2 conditions has flags in “Detected” were chosen for further data analysis. After normalization, variance stabilization and log transformation of data and the log signal intensity values for M. truncatula probe IDs corresponding to XTH protein-encoding genes were treated as a subset.
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Fig. 3. Hierarchial clustering display of the 28 xyloglucan endotransglucosylase/hydrolase genes differentially expressed in Hg-treated and Hg-free roots [(RþHg) vs (R Hg)]_ and leaves [(L þHg) vs (L Hg)] of Medicago truncatula. The log signal values were used for clustering.
For salt stress response analysis, the raw transcriptional data of M. truncatula treated by 180 mM NaCl for 0, 6, 24 and 48 h were downloaded from GEO (http://www.ncbi.nlm.nih.gov/geo/query/ acc.cgi? acc ¼GSE13921) (Li et al., 2009) and normalized using RMA algorithm with Expression Console software (Affymetrix Technologies) (Irizarry, et al., 2003). The identifiers were updated based on the file “AffyMap-Mt4.0v1 Genes Spliced Transcript Seq” (http://www.medicagohapmap.org/? genome). Differentially expressed XTH protein-encoding genes were identified through the fold change (fold change Z2.0) as well as p-value ( r0.05). 2.3. Quantitative RT-PCR analysis Total RNA was isolated based on the method described previously (Shen et al., 2011). One microgram of total RNA extracted using Trizol (Invitrogen, Carlsbad, CA) was incubated at 37 °C for 30 min with 1 unit of RNase-free DNaseI (Takara) and 1 mL 10 reaction buffer with MgCl2 and incubated with 1 mL of 50 mM EDTA for DnaseI inactivation at 65 °C for 10 min. A 1% agarose gel, stained by ethidium bromide, was run to monitor the RNA integrity. The reverse transcription reaction mixture (20 mL) contained 1 mg RNA, 100 mM oligo (dT) primers (1 mL), 10 mM dNTP (deoxyribonucleotide triphosphate) mixture (1 mL), 40 U mL 1 of RNase inhibitor (0.5 mL), 200 U mL 1 of M-MLV reverse
transcriptase (0.5 mL) and 4 mL of 5 M-MLV buffer. The reaction solution was maintained at 42 °C for 10 min and heated at 95 °C for 2 min. The resultant cDNA was then diluted 5 fold with sterile water and kept at 20 °C for qRT-PCR analysis. Amplification reaction was performed in a 20 mL mixture containing 10 ng of template, 10 mL of SYBR-Green PCR Mastermix (Toyoba, Japan) and 10 pmol of primers. Quantitative RT-PCR was performed using MyIQ Single Color Real time PCR system (Bio-Rad) and IQ5 software (Bio-Rad). The temperature profile was 95 °C for 30 s, followed by 40 cycles at 95 °C for 15 s, 60 °C for 60 s and a melt curve at 65 °C 5 s. Data were analyzed using CFX Data Analysis Manager Software. The relative expression level was normalized to that of the EF1α, which were used as the internal control in M. truncatula (Kenneth and Thomas, 2001). The primers used for qRT-PCR are presented in Supplementary data 1. 2.4. GUS assay Histochemical detection of GUS activities in the transgenic Arabidopsis seedlings or different tissues were analyzed based on the method of Song et al. (2012). Plant tissues were incubated at 37 °C in 0.5 mg/mL 5-bromo-4-chloro-3-indolyl-glucuronic acid (X-Gluc), 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, 0.1% Triton X-100, and 100 mM sodium phosphate
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Fig. 4. Quantitative RT-PCR validation of randomly selected XTH genes from Medicago truncatula. Four-week-old seedlings were exposed 10 μM HgCl for 12 h. After that, total RNA was extracted and qRT-PCR was performed.
buffer, pH 7.0. Samples were bleached several times with 70% ethanol. pBI121 was used as the plant expression vector with the promoter sequences of genes and NOS terminator as transcriptional termination sequences. The vector was transferred into Arabidopsis via Agrobacterium-mediated transformation. GUS staining patterns were verified in two independent homozygous T3 lines, and representative individuals were chosen for photography.
(Version 2.0; http://www.clustal.org/) with all predicted XTH motif. A neighbor joining (NJ) tree was constructed by MEGA6 using the p-distance method with gaps treated by pairwise deletion and a 1000 bootstrap replicate. Chromosomal mapping was performed using the GenomePixelizer (http://niblrrs.ucdavis.edu/ GenomePixelizer/GenomePixelizer_Welcome.html). Hierarchial clustering display performed using Euclidean distance according to expression pattern with the MeV software.
2.5. In silico analysis of putative XTH proteins from M. truncatula
2.6. Sequence alignment and phylogenetic tree analysis
Putative XTH proteins of M. truncatula were searched using M. truncatula genome database (http://www.medicagohapmap.org/ downloads/mt40) (Song et al., 2015). Multiple sequence alignments were performed by the Clustal X
To analyze the evolutionary relationships between MtXTHs and those from other plant species, the amino acid sequences of M. truncatula and Arabidopsis thaliana were retrieved from the websites (http://www.jcvi.org/medicago/) and Tair (http://www.arabi
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Fig. 5. Hierarchial clustering display of xyloglucan endotransglucosylase/hydrolase genes of Medicago truncatula seedlings exposed to salinity (A) and drought (B). A: two week-old seedlings were grown in hydroponics media with 180 mM NaCl for 0, 6, 24 and 48 h (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi? acc ¼GSE13921). B: Normal growth seedlings were subject to water withdrew for 0–14 d (http://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-2681/? query). The log signal values were used for clustering.
dopsis.org/). The phylogenetic tree was constructed by the neighbor joining (NJ) method with MEGA6.
3. Results 3.1. Identification of XTH protein-coding genes from M. truncatula
2.7. Statistical analysis Experiments in the study were independently performed in triplicate. Each result shown in the figures was the mean of at least three replicated treatments and each treatment contained at least 30 plants. The significant differences between treatments were statistically evaluated by standard deviation and one-way analysis of variance (ANOVA). The data between differently treated groups were compared statistically by ANOVA, followed by the least significant difference (LSD) test if the ANOVA result was significant at P o0.05.
We use our recent transcriptome (Zhou et al., 2013) and other publicly available datasets (http://www.megasoftware.net/index. php) (Tamura et al., 2013) to identify XTH genes from M. truncatula. In total, 44 non-redundant potential MtXTH genes have been identified (Data S2). Alignment of the MtXTH gene protein sequences revealed that the amino acid residues within the highly conversed regions showed 80% identity, although the full length sequences of XTH proteins were relatively low in similarity. Each MtXTH possessed a highly conserved catalytic domain ((D/N)-E-(I/ L/F/V)-D-(F/I/L)-E-(F/L)-L-G), one N-linked glycosylation site was identified adjacent to the C-terminal side of the catalytic domain
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Fig. 6. Differential expression of MtXTH3 (Medtr4g128580) in tissues of M. truncatula and MtXTH3 promoter–GUS expression pattern in Arabidopsis transgenic plants. A: qRT-PCR analysis of MtXTH3 expression at different tissues. YEL, young expanding leaf; C, cotyledon; R, radical; H, hypocotyls; ST1, young expanding internode of stem (the second internode); ST2, the third internode of stem from the apex; Y, leaf at the end of expansion; and F, flower. B: MtXTH3 promoter–GUS expression pattern. (a) seed; (b) one-day-old germinating seed; (c) two-day-old germinating seed; (d) three-day-old seedling; (e) four-day-old seedling; (f) seven-day-old seedling; (g) fourteen-day-old seedling; (h) eighteen-day-old seedling; (i) stem; (j) radical; (k) rosette leaf; (l) lateral root; (m) flower; (n) tender silique; (o) mature silique; and (p) carpel and seed scattering.
in 36 of the MtXTH protein; most of MtXTHs possessed four Cys in the C terminal region, suggesting a potential for generating disulfide bonds (Data S2). Using the identified M. truncatula XTH sequences for alignments, a phylogenetic tree with all MtXTH genes was created (Fig. 1A). The divergence between clusters I/II of the MtXTHs was not apparent and the third cluster was obviously distinct from clusters I/II. These MtXTHs could be divided into three major clusters. Clusters I/II and cluster III contain 35 and 9 genes, respectively. To figure out the correlation of the M. truncatula XTHs and those from other dicots, a comparative analysis was performed on the distribution of XTHs between M. truncatula and Arabidopsis.
Mapping the MtXTH sequences to those of Arabidopsis resulted in identification of nearly a hundred matches. After removal of the redundancy, 33 XTH genes from Arabidopsis were retrieved. The overall sequences for the individual pairs varied between the M. truncatula and Arabidopsis homologs. Given the three-subgroup classification for the XTH gene family, the clusters I/II contained 35 MtXTH genes and 26 AtXTH genes, whereas the cluster III consisted of 9 MtXTH and 7AtXTH genes, respectively. While a certain proportion of M. truncatula XTHs were clustered together with their Arabidopsis homologs, some of them had low relations from one another (Fig. 1B). The less clustered XTHs between M. truncatula and Arabidopsis suggest that some of the M. truncatula
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XTHs identified here may not be conserved in Arabidopsis. 3.2. Identification of chromosome locations of XTH genes and specific motifs of XTH proteins from M. truncatula The XTH protein-encoding genes were dispersed across eight chromosomes of M. truncatula (Data S3). Some XTHs were located onto the chromosomes as solitary genes and others as clusters of a few genes were found in tandem. Several highly conserved genes such as Medtr2g089140.1/Medtr2g089140.2 and Medtr5g029100.1/ Medtr5g029100.2 were clustered together on the same chromosomes. Nine sets of XTH gene clusters, which had been generated by extensive genome duplications, may share 8 different ancestral genes. For instance, Medtr6g033055.1/Medtr6g033085.1 and Medtr7g084750.1/Medtr7g084760.1/Medtr7g084770.1 share one ancestral gene. We then examined diverse motifs throughout the regions for the XTH protein genes. Using MEME (http://meme.nbcr.net/ meme/) databases, ten types of motifs were identified from M. truncatula (Data S4; Fig. 2). The different types of motifs that the XTH proteins contain were not defined. The majority of XTHs contain most of the motifs. For example, 17 XTHs contain all types 10 of motifs. Also, 17 XTHs contain 9 types of the motifs. Eight XTHs have 8 types of motifs, and only of XTHs has 5 types of motifs. The differential motifs that XTHs contain suggest that the XTHs may have diverse biochemical features and biological functions. 3.3. A set of XTH genes in response to heavy metal stress To investigate XTH genes that respond to heavy metal stress, we analyzed differential expression of the XTH genes in M. truncatula seedlings exposed to the heavy metal mercury (HgCl2) using a customer-made Agilent microarray. Hg was tested because it is one of the most toxic metals to plants (Zhou et al., 2007; Shen et al., 2011). The probes for the chip were designed based on the update annotational gene version (Mt4.0v1, Agilent) of M. truncatula genome. Following the gene hybridization and chip data processing (e.g. quality controls, normalization and log transformation), 28 XTH genes were probed. The log signal values of the XTH genes were presented in Data S5. Of these, 27 genes were probed in rootsand 10 genes expressed in leaves (Fig. 3). Some of the genes were not probed in roots because of the much low abundance. We then narrowed down the genes to 28 XTH genes passing the criteria of two fold change. Profiling of the gene expression revealed that 11 (40.7%) out of 27 genes were up-regulated, 16 (59.3%) were down-regulated in Hg-treated roots, and all genes were down-regulated in Hg-treated leaves (Fig. 3). We randomly chose 6 XTH genes for qRT-PCR validation under Hg exposure. Our analysis showed that the expression of the genes was well confirmed (Fig. 4). 3.4. A group of XTH genes in response to salt and drought stresses To investigate whether the XTH genes were also responding to abiotic stresses, we used publicly available microarrays generated from M. truncatula (http://www.ncbi.nlm.nih.gov/geo/query/acc. cgi? acc¼ GSE13921). In total, 21 genes were identified in response to salt stress (Fig. 5A, Data S6). These genes responded differently to 180 mM NaCl exposure for 48 h. Profiling of the differentially expressed gene revealed that 8 (38.1%) genes were up-regulated, and 13 (61.9%) genes were down-regulated after salt treatment for 6, 24, 48 h. Of these, Medtr4g128580.1 showed higher transcriptional expression, whereas Medtr7g084750.1 displayed a relatively lower expression following 48 h of salt exposure. We also used publicly data to analyze the response of XTH genes to drought
stress (http://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB2681/?query) (Fig. 5B, Data S7). There were also 21 XTH genes identified for different response to drought stress. Five genes were clearly up-regulated and 9 genes repressed in response to 7–14 d drought. 3.5. Expression analysis of MtXTH3 in M. trancatula under heavy metal and other abiotic stresses To confirm the M. truncatula XTH genes in response to the heavy metal and abiotic stresses, we selected a novel gene Medtr4g128580. As Medtr4g128580 has a high sequence similarity to Arabidopsis AtXTH3, it was herein temporarily assigned as MtXTH3. The full-length cDNA of MtXTH3 was cloned. In silico analysis showed it is a single copy gene, with full-length DNA of 1148 bp containing 3 exons and 2 introns (Data S8). The cDNA was predicted to encode a protein with 288 amino acids and a molecular mass of 32.3 kDa (Data S9). MtXTH3 contains a highly conserved domain (DEIDFEFLG), a putative N-glycosylation motif immediately following the catalytic domain and four Cys, which suggests the potential to form disulfide bonds in the C terminal regions. The SignalP program predicted MtXTH3 has a putative N-terminal signal peptide of 24 amino acids, suggesting that it is a secretory protein. Phylogenetic analysis of MtXTH3 showed that it belonged to group subfamily I/II XTHs with xyloglucan endotransglucosylase (XET) activity. The highest level of sequence homology with AtXTH23 was 78% and 73% identity at the nucleotide and amino acid levels, respectively (Data S10). The pattern of MtXTH3 expression in M. trancatula was analyzed by qRT-PCR using the tissues at different growth and flower stages. MtXTH3 was expressed strongly in cotyledon and hypocotyls, moderately in radical, but lowly in young expanding leaf in 7-day-old seedlings. In 12-week-old plants outdoors, MtXTH3 gene expression was high in fully expanded leaves and the third internode of stem from the apex; meanwhile, low expression was observed in flowers and young expanding internodes of stem. Little was expressed in young expanding leaves (Fig. 6A). To confirm the MtXTH3 expression, a 1992 bp DNA fragments upstream of the MtXTH3 ATG start codon was isolated, fused to the β-glucuronidase (GUS) GUS reporter gene, and transformed into Arabidopsis via Agrobacterium-mediated transformation. Analysis of GUS staining patterns showed that the GUS staining could be detected at nearly all developmental stages, including seeds, seedlings, vegetative tissue, flowers and siliques (Fig. 6B). The expression patterns of MtXTH3 were very similar to those from qRT-PCR. We further analyzed transcriptional responses of MtXTH3 to abiotic stresses M. trancatula using qRT-PCR. As shown in Fig. 7A and B, three week-old seedlings were treated with 0, 25, 150 and 300 mM NaCl. MtXTH3 in roots and shoots was not transcriptionally activated under low levels of NaCl. Only at 150 mM NaCl was MtXTH3 expression significantly upregulated in the tissues. Treatment with 300 mM NaCl increased transcripts up to 42.9 and 70.4 fold over the control in roots and shoots, respectively. We then used PEG6000 (10%, 20% and 30%) to treat the seedlings and showed that strong MtXTH3 expression in roots and shoots was found at 30% PEG6000. Treatment with 10% and 20% PEG6000 induced only a moderate expression of shoot MtXTH3, but no induction was observed in roots (Fig. 7C and D). Abscisic acid (ABA) is a well-known environmental stress-responsive signal molecule. Treatment with ABA at 10, 50 and 100 μM considerably induced MtXTH3 transcription in shoots; however, weak MtXTH3 transcription was detected in roots (Fig. 7E and F). Finally, the effect of the heavy metal Hg(II) and Cu(II) on the expression of MtXTH3 was examined. It is shown that while the low concentrations of Hg and Cu (10 mM) induced a moderate expression of MtXTH3, a very strong induction of MtXTH3 was found at higher
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Fig. 7. Quantitative RT-PCR analysis of MtXTH3 (Medtr4g128580) expression in M. trancatula roots and shoots in response to abiotic stresses. Four week-old seedlings were exposed to NaCl (0, 25, 150 and 300 mM), PEG-6000 (0%, 10%, 20% and 30%), HgCl2 (0, 10, 50 and 200 μM) and CuCl2 (0, 10, 50 and 200 μM) for 6 h or ABA (0, 10, 50 and 100 μM) for 1 h. Vertical bars represent standard deviation (n¼3). Asterisks indicate that mean values are significantly different between the treatments and control (po 0.05).
levels of Hg and Cu (50 and 200 mM) in M. trancatula seedlings (Fig. 7G–J). Taken together, these results indicate that MtXTH3 transcription could be induced by environmental factors including salt, drought and Hg or Cu heavy metals.
4. Discussion A global identification of XTH genes will help to understand gene expression and regulatory mechanisms for plant toxicity or tolerance to environmental stresses such as heavy metals and salinity. XTHs are involved in synthesis of most hemicellulose in the primary cell wall of higher plants and reported to play
important roles in plant response to metals such as Al (Zhu et al., 2013). To date, there are many XTHs genes identified from plants including 33 genes identified in Arabidopsis (Yokoyama and Nishitani, 2001; Rose et al., 2002), 29 genes in rice (Yokoyama et al., 2004), 41 genes in Populus (Geisler et al., 2006) and 25 genes in tomato (Saladie et al., 2006). In this study, 44 genes encoding XTHs were identified from M. trancatula. The number of XTH genes in M. truncatula was a little bit higher those of Arabidopsis and rice, suggesting that it could be the direct result of lineage-specific gains and losses (Song et al., 2015). According to its sequence similarity, Arabidopsis XTHs were classified into three major phylogenetic subgroups (group I, II and III) (Campbell and Braam, 1999; Rose et al., 2002). Group III could
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be further divided into subgroups such as group IIIA and group IIIB; while group I, group II and group IIIB showed prominent xyloglucan endotransglucosylase (XET) activity, group IIIA showed extreme xyloglucan endohydrolase (XEH) activity (Baumann et al., 2007). To analyze correlations between the XTH genes in M. truncatula, we constructed a phylogenetic tree of the entire XTHs by alignment of their sequences. Based on the distribution and relationship of the phylogenetic tree, three distinct families were classified. From the topology and groupings, the M. truncatula XTH proteins with the same or similar domain organization were clustered together. Phylogenetic analysis revealed that they could be classified into three groups. The chromosomal locations of the XTH protein-encoding genes on the M. truncatula genome were analyzed because the gene distribution and the way of gene cluster provide a clue of gene expansion during their evolutional process (Gu et al., 2002). Profiling of the XTHs distribution on eight M. truncatula chromosomes showed the evolvement through duplication events, an observation consistent with other genes from the same species (Song et al., 2015). Based on the definition of gene duplication that the length of the sequence alignment covered Z 80% of the longest gene, and the similarity of the aligned gene regions was Z70% (Gu et al., 2002; Yang et al., 2008), we mapped the XTH protein-encoding genes onto the M. truncatula chromosomes and found many clusters of XTH genes evident on different chromosomes. Several XTH genes were arranged in tandem repeats of genes either in the same or inverse orientation, representative of the localized gene duplications. This finding suggests that tandem duplications of chromosomal regions played an important role in expansion of this XTH protein-encoding genes family. Most of XTH protein-encoding genes are located on Chr 4, although a small amount of the genes are also found on other chromosomes. Plant cell wall plays important roles in the regulation of growth, development and environmental stresses. The plant XTHs is a kind of important cell wall modifying enzymes, participating in not only cell wall extension or degradation but also maintaining the integrity and strength of the cell wall under normal and stressful environments (Nishitani et al., 1995; Cosgrove, 2005; Miedes et al., 2009; Zhu et al., 2012). To investigate the XTH genes that were potentially responding to abiotic stresses, we used microarrays generated from M. truncatula. There were 21 and 21 MtXTH genes found to respond to salt and drought. Furthermore, a costumer-made chip analyzed 28 MtXTH genes in response HgCl2. The MtXTH genes were found to be differentially regulated under the environmental stresses. For example, genes like Medtr4g128400.1 and Medtr4g128410.1were found to be up-regulated under Hg exposure, whereas genes Medtr1g105495.1, Medtr7g102260.1, and Medtr8g064180.1 were down-regulated under the salt stress. Some of the homologue genes were reported to confer drought and salt tolerance in other plant species (Cho et al., 2006). Furthermore, the homologue gene PeXTH was up-regulated by salt or Cd in Populus euphratica plants; PeXTH overexpression in tobacco plants increased capacity for salt and Cd tolerance (Han et al., 2013, 2014). To better understand the possible function of M. truncatula XTH genes, we analyzed in detail expression of a XTH gene MtXTH3 under different environmental stresses. Our analysis showed that MtXTH3 could be freely expressed throughout the whole development stage although its expression was feeble at certain tissues, suggesting that the expression of MtXTH3 might be required for normal plant growth or development. We further analyzed the MtXTH3 transcript response to various environmental stresses. MtXTH3 expression was up-regulated when seedlings were treated with PEG6000, high salt, Hg or Cu treatments. These results implied that MtXTH3 could be involved in the environmental stresses responses possibly through incorporating newly deposited xyloglucan to strengthen cell walls. MtXTH3 showed a high sequence
identity with AtXTH23 in Arabidopsis, which is closely related to XETs in group II (Rose et al., 2002). Further molecular and genetic identification of MtXTH3 will clarify the function of MtXTH3 in mediating the plant response to Hg, salt and drought stresses.
5. Conclusions The present study identified 44 XTH protein genes form M. truncatula by bioinformatics and other biological approaches. Based on their sequence conservation, the MtXTH proteins could be divided into three families. To investigate how these MtXTH genes responded to heavy metal and abiotic stresses, we analyzed microarray-based datasets and some of the MtXTH genes found to be differentially expressed under HgCl2, salt and drought stresses. Of these, several genes were well confirmed by qRT-PCR. One of the MtXTH gene MtXTH3 was further analyzed in detail, confirming that M. truncatula XTH genes are able to respond to the environmental stresses.
Conflict of interest The authors declare that they have no conflict of interest.
Acknowledgements This study was funded by the National Natural Science Foundation of China (31071343 and 31200204) and Colleges and Universities in Jiangsu Province Plans to Graduate Research and Innovation (KYZZ_0181).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ecoenv.2016.06.007.
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