Genome-wide identification of xyloglucan endotransglucosylase/hydrolase gene family members in peanut and their expression profiles during seed germination

Identification of AhXTH family members and their physicochemical properties

Two methods were used to search for the target proteins. First, conservative domain files containing PF00722 (Glyco_hydro_16) and PF06955 (XET_C) were obtained from the Pfam database (http://pfam.xfam.org/), and then they were used to search for the XTH proteins from Peanutbase (https://www.peanutbase.org/) with HMMER 3.0 software. Second, peanut XTHs were retrieved using “cultivated peanut xyloglucan endoglycosidase/hydrolase” as a keyword by searching the NCBI database (https://www.ncbi.nlm.nih.gov/). Then, all obtained XTHs were confirmed by the CDD program (https://www.ncbi.nlm.nih.gov/cdd/).

The online software ProtParam (http://web.expasy.org/protparam/) was used to predict the physical and chemical properties of the gene family members, including the number of amino acids, theoretical molecular weight (MW), and isoelectric point (PI). The subcellular locations of the XTH proteins were predicted through the online software Euk-mPLoc 2.0 (http://www.csbio.sjtu.edu.cn/bioinf/euk-multi-2/) (Chou & Shen, 2010).

Chromosomal location and collinearity analysis of AhXTHs

The physical position of each XTH gene was downloaded from Peanutbase and the NCBI database, and chromosomal location mapping was performed by MapChart 2.30.

All peanut XTH sequences were compared with each other by MCScanX (Wang et al., 2012). If two sequences shared over 70% identity and covered over 70% of their sequences, then they were considered to be homologous genes and to have collinearity (Gu et al., 2002; Yang et al., 2008). Tandemly duplicated genes were defined as adjacent homologous genes on the same chromosome with no more than one intervening gene (Zhu et al., 2014). The duplication relationship of AhXTHs was drawn by Circos mapping software (Krzywinski et al., 2009) and displayed with interconnections according to the positional information on the chromosomes. The values of nonsynonymous substitutions (Ka) and synonymous substitutions (Ks) between homologous genes were calculated by TBtools software, and the Ka/Ks value was used to predict the evolutionary relationship between homologous gene pairs.

Analysis of evolutionary relationships

The gene IDs of Arabidopsis and soybean XTH family members were obtained from published articles (Song et al., 2018; Yokoyama & Nishitani, 2001), and their amino acid sequences were downloaded from the TAIR (https://www.arabidopsis.org/) and JGI (https://phytozome.jgi.doe.gov/pz/portal.html) websites. The XTH protein sequences derived from Arabidopsis thaliana, soybean and peanut were aligned by the MUSCLE algorithm tool, and the unrooted phylogenetic tree was constructed using the NJ (neighbor-joining) method with 1,000 bootstrap replicates by MEGA-X software (Kumar et al., 2018).

Gene structure and analysis of conserved structures

The gene structure map of every AhXTH was drawn on the GSDS 2.0 (Gene Structure Display Server, http://gsds.cbi.pku.edu.cn/) website (Hu et al., 2015). The secondary structure and the conserved structural elements were predicted by the online tool ESPript (http://espript.ibcp.fr/ESPript/ESPript/) (Robert & Gouet, 2014). Using TmNXG1 (PDB id: 2UWA) (Mark et al., 2009) and PttXET16-34 (PDB id: 1UN1) (Johansson et al., 2004) as the reference sequences, all AhXTH sequences were optimally aligned by ClustalW, and the common elements of XTH secondary structures were displayed in the alignment map.

Expression profiles of AhXTHs and qRT–PCR verification

The raw data for the transcriptomes of seeds at three different stages were collected from NCBI (NCBI, Bioproject accession: PRJNA545858) (Xu et al., 2020). The three seed stages were freshly harvested seeds (FS), dried seeds (DS) that has been exposed to sunshine for two weeks, and newly germinated seeds (GS) in which the radicles broke through the seed coat. The cultivated peanut Tifrunner was used as a reference genome (gnm2. J5K5, https://www.peanutbase.org/data/public/Arachis_hypogaea/), and the RNA-seq data were analyzed by Hisat2, Samtools and Cufflinks to obtain FPKM values. A heatmap displaying gene expression patterns (log2FPKM values) was drawn by TBtools software.

Total RNA from FS, DS and GS of Fenghua No. 1 (FH1) was extracted using the RNAprep Pure Plant Plus Kit (Tiangen Biotech, Beijing, China) according to the manufacturer’s protocol, and the quality and quantity of RNA were determined by agarose gel electrophoresis and ultraviolet spectrophotometry (BioPhotometer Plus. Eppendorf, Germany), respectively. First-strand cDNA was synthesized by the PrimeScript II 1st Strand cDNA Synthesis Kit (TaKaRa, Dalian, Liaoning Province, China). Real-time PCR was performed using TB Green Premix Ex Taq with a 7,500 Fast Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). The ACTIN gene was used as a reference, and the PCR conditions were as follows: 95 °C for 30 s followed by 40 cycles of 95 °C for 5 s and 60 °C for 34 s. The primers (File S1) were designed and verified on NCBI, and the results were analyzed by the 2^−∆∆Ct method. Three biological replicates were performed. Pearson correlation coefficient were used to check the data consistency between RNA-seq and qRT-PCR.

Analysis of cis-acting regulatory elements in promoters

The 2,000 bp 5′-upstream sequences from the start codon (ATG) of all AhXTHs were extracted by TBtools and were used for the prediction of cis-acting regulatory elements according to PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) (Lescot et al., 2002).

Construction of GUS expression vectors and GUS histochemical staining

The promoter fragment of the AhXTH gene was obtained by PCR. To construct the vector, the appropriate restriction sites were introduced into the primers (HindIII at the 5′ end; NcoI at the 3′ end). The PCR-amplified products were then inserted into HindIII/NcoI-digested pCAMBIA3301, replacing the cauliflower mosaic virus (CaMV) 35S promoter. A construct harboring the AhXTH promoter was established and introduced into Agrobacterium tumefaciens strain GV3101 using the freeze–thaw method. Transgenic Arabidopsis plants were generated by the floral dip method. The homozygous T₂ transgenic lines with a single-copy insertion were screened by Basta resistance. All Arabidopsis plants grew in a growth room at 23 °C under a 16 h light/8 h dark photoperiod and 65% relative humidity.

GUS histochemical staining was performed as described by Jefferson, Kavanagh & Bevan (1987). The roots and leaves at the 4-leaf stage, stems at the bolting stage, inflorescence, newly germinated seeds without testa, and etiolated seedlings germinated for 24 h and 48 h in the dark from transgenic T₂ lines were incubated in GUS assay buffer with 50 mM sodium phosphate (7.0), 0.5 mM K₃Fe(CN)₆, 0.5 mM K₄Fe(CN)₆·3H₂O, 0.5% Triton X-100, and 1 mM X-Gluc at 37 °C overnight and then cleared with 70% ethanol. The samples were observed by stereomicroscopy.

Identification of the functions of AhXTH4

AtXTH22 is orthologous to AhXTH4 in peanut. Its T-DNA insertion mutant xth22 (CS860818) from the Arabidopsis Biological Resource Center (ABRC, https://abrc.osu.edu) was used for functional analysis. The xth22 homozygous mutant line was selected by Basta resistance and verified by the tri-prime PCR method (primer sequences in File S2). The seeds from xth22 and Col-0 were sterilized, sown on half-strength MS medium in rows, and grown at 20 °C in the dark for 2 days; the seedlings were transferred to the light for 6 h and 24 h. The pROKII-35S::AhXTH4 vector was constructed and transformed into Agrobacterium tumefaciens strain GV3101 for use in functional complementation experiments. Transgenic Arabidopsis plants were obtained by the floral dip method and selected on 1/2 MS medium supplemented with kanamycin. Three independent homozygous lines carrying the single copy gene AhXTH4 were used for further analysis. The phenotypes of Col-0, xth22 and transgenic lines at germination, etiolated seedling establishment and photomorphogenesis were observed and photographed under an anatomical lens.

Identification of XTH family members in peanut

Seventy-six and sixty-five candidate sequences of AhXTHs were retrieved using two methods, among which some incomplete sequences and sequences lacking the conserved DEIDFEFLG motif were removed. Thus, a total of 58 AhXTH protein sequences were obtained and named AhXTH1 to AhXTH58 according to their location on the chromosome (coding sequences in File S3 and protein sequences in File S4).

The length of the AhXTH genes varied from 1,187 bp to 7,499 bp, and AhXTH40 and AhXTH28 were the shortest and longest genes, respectively. Their encoded proteins ranged from 156 to 361 amino acids with an average of 292 amino acids, among which the shortest and longest proteins were AhXTH31 and AhXTH16/AhXTH45, with MWs of 17.78 and 41.8 kDa, respectively (Table 1). Analysis of the physical and chemical properties also showed that the theoretical PIs of the AhXTHs varied from 4.79 to 9.48. Most of the AhXTH members contained a signal peptide, which consisted of the first 25 amino acids of the N-terminus. Subcellular localization prediction revealed that most AhXTHs were localized on the cell wall, and some were located outside of the cell (Table 1).

Chromosomal locations and duplicated events of AhXTHs

The mapping analysis of AhXTH genes on chromosomes showed that except for chromosome (chr.) 07 and 17, all 58 AhXTH genes were unevenly distributed on the remaining 18 chromosomes and mostly concentrated at both ends of the chromosomes. Additionally, 26 and 32 of them belonged to the A genome (chromosomes 01–10) and B genome (chromosomes 11–20) (Fig. 1), respectively. In detail, there are eight AhXTH genes on chr. 11, which held the largest number of AhXTH gene; six AhXTHs were scattered on chr. 01, 03, and 13, and five AhXTH genes were found on chr. 15. The number of genes distributed on these five chromosomes accounted for approximately 53% of the total number of genes. The remaining 13 chromosomes unequally contained 1 to 4 AhXTH genes, accounting for less than 50% of the genes. The results also showed that five gene clusters, including AhXTH3-XTH6; AhXTH29-XTH34; AhXTH21 and AhXTH22; AhXTH51 and AhXTH52; and AhXTH55 and AhXTH56, were located on chr. 01, 11, 06, 16, and 19 (Fig. 1).

The syntenic relationships among all AhXTH genes were assessed based on sequence homology and positional information. A total of 65 replication events, including 42 pairs of segmental duplications and 23 pairs of tandem duplications from five gene clusters located in an interval of less than 100 kb, were recognized; these are shown on the Circos map (Fig. 2). To explore the evolutionary relationship among AhXTH members, the synonymous (Ks), nonsynonymous (Ka) and Ka/Ks ratio values for each duplication event were calculated (Table 2). The Ka values of the segmental duplications and tandem duplications ranged from 0 to 0.7027 and from 0.1226 to 0.3342, respectively, while the Ks values ranged from 0 to 2.3858 and from 0.5394 to 2.8151, respectively. The Ka/Ks ratio of the AhXTH25/AhXTH57 segmental duplication was more than one, suggesting that this gene pair underwent positive selection, while the other pairs of segmental duplications and all gene pairs with tandem duplications had a Ka/Ks < 1 and were negatively selected during evolution. The results suggested that during the evolution and amplification of the genome, most AhXTH genes may have undergone purification selection on the codons.

Phylogenetic relationship of AhXTH members

A total of 152 XTH sequences derived from Arabidopsis, soybean, and peanut were aligned by ClustalW, and their evolutionary relationship was analyzed by MEGA-X software (Fig. 3). In previous studies, AtXTH members were first classified into three groups (Yokoyama & Nishitani, 2001); however, further analysis showed that the XTHs in Groups I and II have no clear demarcation, and Group III could also be divided into two branches, IIIA and IIIB (Yokoyama, Rose & Nishitani, 2004). Therefore, according to the cluster criterion in Arabidopsis, 58 AhXTH members together with 31 AtXTHs and 61 GmXTHs were divided into three subfamilies I/II, IIIA and IIIB on the unrooted phylogenetic tree, among which 40 AhXTHs belonged to subfamily I/II, and subfamilies IIIA and IIIB contained 8 and 10 AhXTH members, respectively (Fig. 3).

Structural analysis of AhXTH genes and their encoded proteins

The gene structure of each AhXTH gene was drawn using GSDS 2.0 software (Fig. 4B). The prediction results showed that the numbers of AhXTH exons ranged from 2 to 8 across all family genes; genes containing 3–4 exons accounted for 85% of the total genes, and AhXTH42 contained the largest number of exons at 8. Additionally, the numbers and sizes of the exons were significantly different within each AhXTH subfamily. The homologous genes with the higher similarity of sequences had higher structural identity. The majority of the AhXTH genes had normal 5′UTR and 3′UTR structures, whereas AhXTH25, 31, 44, and 50 only had the 5′UTR structure, AhXTH8, 19, 37, 49, and 58 only had the 3′UTR structure, and AhXTH40, 48 and 56 lacked both a 5′UTR and 3′UTR. In addition, all AhXTHs contained the active site situated in front of their second or third exons.

The secondary structure of the AhXTH protein was predicted according to the structure of the endoxyloglucan endoglycosidase PttXET16-34 and endoxyloglucan enzyme TmNXG1, and the conserved domains are shown in Fig. 4C. The results showed that all AhXTH sequences contained the active site ExDxE. Previous studies indicated that the N-glycosylation site (NxT/S/Y) near the active site can be combined with N-glycans and is related to the stability of the protein (Campbell & Braam, 1998; Opazo et al., 2010). Our analysis showed that Group I/II members had consistent N-glycosylation sites (NxS/T) close to the active site, marked with an asterisk, “*” in Fig. 4C. However, in Group III, the N-linked glycosylation sites (NxY) of all AhXTHs were approximately 11 amino acids away from the active site. Almost all AhXTHs also contained three conserved loop structures, loop1, loop2 and loop3, and on average, the amino acid composition of loop3 was more conserved in all AhXTHs, with major differences manifested between loop1 and loop2 (Fig. 4C). Loop1 is a unique extension of the active site; it had the same number of amino acids in the IIIA and IIIB groups, but the amino acid composition was significantly different. In Group I/II, loop1 lacked three amino acids compared with that in the III subfamily, but its function was rarely influenced. Loop2 can regulate the ability to bind the substrate and determine endoxyloglucanase activity (Baumann et al., 2007). The AhXTHs of Groups I/II and IIIB lacked 3–4 amino acids in loop2 compared with most Group IIIA members. Loop3 was detectable in 55 AhXTH proteins except for AhXTH7, AhXTH31, and AhXTH36; this loop had the amino acid sequence D(S/N/A)WATR(D/Q)G(S/W) in I/II subfamily members in peanut, and the sequence SWATEN(D)GG in IIIA, while its structure and amino acid sequence in the IIIB members was more complicated, with no identical sequences (Files S5 and S6).

Expression profiles of AhXTHs at the FS, DS and GS stages

The expression levels of AhXTH genes at three stages of peanut maturation and germination were investigated using transcriptome data, and some of the genes were verified by qRT–PCR (Fig. 5). The heatmap analysis showed that the expression levels of almost half of the genes (25 genes: AhXTH1, 2, 7, 8, 9, 10, 12, 17, 19, 20, 21, 22, 26, 27, 28, 36, 37, 39, 46, 48, 49, 50, 53, 56, and 58) did not change at the FS, DS and GS stages, and 7 genes (AhXTH5, 23, 31, 32, 33, 55, and 57) were expressed at higher levels in FS than in DS and GS. Compared with those in FS and DS, the remaining 26 genes (AhXTH3, 4, 6, 11, 13, 14, 15, 16, 18, 24, 25, 29, 30, 34, 35, 38, 40, 41, 42, 43, 44, 45, 47, 51, 52, and 54) were expressed at the highest level in GS, among which AhXTH6, 11, 25, 41, and 44 were only expressed in GS, and the expression of AhXTH15, 24, 29, 52, and 54 was not detectable in FS and DS. It is worth noting that some highly homologous genes had different expression patterns. AhXTH4 and AhXTH25 had high expression levels in GS, while their orthologs AhXTH33 and AhXTH57 were expressed at a higher level in FS. AhXTH51 and AhXTH52 from one cluster were situated on chr. 16 and had higher expression in GS, while their homologous cluster members located on chr. 06, AhXTH21 and AhXTH22, were found to have consistent expression levels at all three stages. Similarly, AhXTH55 and its cluster member AhXTH56, as well as AhXTH23 and its ortholog AhXTH53, also displayed different expression patterns; the former was highly expressed in FS, while the latter had no expression at all three stages (Fig. 5A, File S7). In this study, the expression patterns of 12 AhXTH genes were verified by qRT–PCR analysis at the DS, FS and GS stages. The consistency analysis of the results revealed a moderate correlation between RNA-seq and qRT-PCR, which may be caused by the different batches of samples (File S8). For ten (AhXTH4, 14, 15, 16, 24, 30, 35, 38, 42 and 52) of them, their expression levels at the GS stage were confirmed to be significantly upregulated (Fig. 5B, Files S9 and S10). The qRT–PCR results also verified that AhXTH5 expression in FS was significantly higher than that in GS and DS, and its expression level was not obviously different between GS and DS. The AhXTH31 gene was expressed at the highest level in FS and the lowest level in DS (Fig. 5B).

Analysis of the cis-acting elements of AhXTH promoter regions

Cis-acting elements play a crucial role in gene function, and the gene sequence itself can possibly influence the final expression level (Zhao et al., 2020). In this study, 2,000 bp promoter regions were analyzed using PlantCARE. The results showed that these regions contain not only common regulatory elements such as CAAT boxes and TATA boxes but also harbor numerous cis-acting elements involved in low temperature and drought responsiveness and responses to multiple hormones. In detail, the 5′ regulatory regions of the majority of AhXTHs (47 genes and 46 genes) have multiple abscisic acid responsive elements (ABREs) and ethylene responsive elements (EREs), and in the regulatory regions of 25, 26, 29 and 42 AhXTH genes, several GA-, auxin-, salicylic acid- and MeJA-related responsive elements exist (Fig. 6, Table 3).

Analysis of AhXTH expression patterns by the GUS expression system

To explore the temporal and spatial expression patterns of AhXTH genes, GUS expression vectors driven by AhXTH promoters were established, and the GUS expression patterns in stable transgenic lines of Arabidopsis harboring the AhXTH4 and AhXTH22 promoters (pAhXTH4:GUS and pAhXTH22:GUS) were investigated. The size of AhXTH4 and AhXTH22 promoter cloned is respectively 1,287 and 1,919 bp (File S11). The results of GUS histochemical staining in different tissues and organs showed that in the seedlings at the 4-leaf stage, weak signals were observable only in the veins of rosette leaves of pAhXTH4:GUS plants, while in pAhXTH22:GUS plants, the vascular tissues in rosette leaves or roots were stained blue. In stems and cauline leaves, stronger staining was found only in the leaf margins and trichomes of pAhXTH4:GUS plants, while it was found in stems and whole leaves of pAhXTH22:GUS plants. The flowers of both pAhXTH4:GUS and pAhXTH22:GUS plants were dyed dark blue, and almost no staining was observed in the siliques of pAhXTH4:GUS plants, while weaker GUS expression was found in pAhXTH22:GUS siliques (Fig. 7A). During seed germination and the establishment of etiolated seedlings, GUS expression analysis showed that both AhXTH4 and AhXTH22 had similar temporal-spatial expression patterns, and only AhXTH4 had a higher expression level. Except for a lack of staining in the radicle or root tips and the apical hook of pAhXTH4:GUS plants, all other tissues and organs in newly germinated seeds without testa and seedlings germinated for 24 h and 48 h in the dark displayed stronger GUS expression, especially the hypocotyls and primary roots of 48 h dark-exposed seedlings (Fig. 7B). This result implied that AhXTH4 might play a major role in hypocotyl elongation during seed germination and etiolated seedling establishment.

Ectopic expression analysis of AhXTH4 in Arabidopsis plants with mutated orthologous genes

To validate the roles of AhXTH4 in promoting seed germination and etiolated seedling establishment, the phenotypes of its corresponding mutant xth22 in Arabidopsis and the transgenic lines constitutively expressing AhXTH4 in xth22 were investigated. The results showed that compared to wild-type Arabidopsis Col-0, xth22 showed a slower germination speed and shorter hypocotyl. When germinating in the dark for 2 days, the hypocotyl and radicle lengths of xth22 were much shorter than those of Col-0, while the overexpression of AhXTH4 in xth22 resulted in phenotypes similar to those of Col-0 during seed germination and etiolated seedling establishment (Fig. 8). After the etiolated seedlings were transferred to light for 6 h, both Col-0 and the mutant showed reduced bending of the apical hook and inhibited elongation of the hypocotyl; a similar phenotype was also found in Arabidopsis transgenic lines (Fig. 8A). Under light for 24 h, the seedlings of Col-0 and the transgenic lines had fully open and greenish cotyledons, while the cotyledons of xth22 were incompletely stretched (Fig. 8A); however, there was no significant difference in hypocotyl length among the wild-type and three transgenic lines (Fig. 8B). This result suggested that AhXTH4 was involved in the regulation of hypocotyl elongation during seed germination in the dark.