Genome-wide identification and expression analysis of dirigent-jacalin genes from plant chimeric lectins in Moso bamboo (Phyllostachys edulis)

Dirigent-jacalin (D-J) genes belong to the plant chimeric lectin family, and play vital roles in plant growth and resistance to abiotic and biotic stresses. To explore the functions of the D-J family in the growth and development of Moso bamboo (Phyllostachys edulis), their physicochemical properties, phylogenetic relationships, gene and protein structures, and expression patterns were analyzed in detail. Four putative PeD-J genes were identified in the Moso bamboo genome, and microsynteny and phylogenetic analyses indicated that they represent a new branch in the evolution of plant lectins. PeD-J proteins were found to be composed of a dirigent domain and a jacalin-related lectin domain, each of which contained two different motifs. Multiple sequence alignment and homologous modeling analysis indicated that the three-dimensional structure of the PeD-J proteins was significantly different compared to other plant lectins, primarily due to the tandem dirigent and jacalin domains. We surveyed the upstream putative promoter regions of the PeD-Js and found that they mainly contained cis-acting elements related to hormone and abiotic stress response. An analysis of the expression patterns of root, leaf, rhizome and panicle revealed that four PeD-J genes were highly expressed in the panicle, indicating that they may be required during the formation and development of several different tissue types in Moso bamboo. Moreover, PeD-J genes were shown to be involved in the rapid growth and development of bamboo shoots. Quantitative Real-time PCR (qRT PCR) assays further verified that D-J family genes were responsive to hormones and stresses. The results of this study will help to elucidate the biological functions of PeD-Js during bamboo growth, development and stress response.


Introduction
Moso bamboo (Phyllostachys edulis) is a major species of woody bamboo that is widely cultivated in tropical and subtropical regions [1]. It belongs to the grass family Poaceae, subfamily Based on their overall structural characteristics, plant lectins are divided into four categories, including partial lectins, total lectins, super lectins and chimeric lectins [24]. Chimeric lectins have at least one jacalin domain containing glycan binding sites. Most jacalin proteins (JRLs) contain only a jacalin domain, but there are some N-or C-terminal chimeric JRLs that contain other domains such as dirigent, NB-ARC, F-Box, GNA and PAG domains [38]. In Gramineae (maize, sorghum, rice, wheat, sugarcane and others), a unique type of chimeric dirigent-jacalin (D-J) lectin is common [39][40][41][42][43][44][45]. These genes play essential roles in plants, responding to biotic and abiotic stresses, as well as regulating plant growth and development [46,47]. Co-evolutionary analysis has shown that there is a significant linkage between the dirigent and jacalin domains in the D-J family. However, because the D-J genes contain both dirigent and jacalin domains, they appear in both the JRL and DIR families. Thus, the exact categorization of this family is still uncertain.
The dirigent domain is a common sequence feature of DIR family proteins, which primarily function in the biosynthesis of cell wall lignin and lignan in response to pests, diseases and abiotic stresses [48,49]. DIR proteins typically contain several glycosylation sites, and a signal peptide sequence associated with the secretory pathway at the N-terminus. DIR proteins also contain eight reverse parallel chains forming a β-barrel structure, with an inner hydrophobic substrate-binding cavity [50]. The recent identification of a new dirigent domain suggests that such chimeric D-J lectins may be more functionally diverse than previously thought [51,52]. Studies have shown that DIR genes are involved in disease resistance responses and may affect the sugar-binding specificity of the entire lectin protein [41,51]. In sugarcane (Saccharum officinarum), JRL domains affiliated with a dirigent domain (Dirigent-Jacalin or D-J) have been shown to play a part in drought tolerance [53].
Chimeric dimeric-jacalin (D-J) lectins have special structural properties and biological properties compared to other species of plant lectin family. Andrade et al. [53] described the role that the JRL domain plays in conjunction with the dirigent domain (dirigent-jacalin, or DJ) in drought tolerance, thus demonstrating the role of chimeric lectin in abiotic stress. Thus far, genome-wide analysis of SBP-like [53], ARF [54], MADS-box [55], UBP [56], MYB [57] and other gene families has been performed in Moso bamboo, but there is little known about the occurrence of different D-J lectins within bamboo species, despite the sequencing of its genome several years ago [58]. The new assembly of the Moso bamboo genome offers an opportunity to improve our understanding of the abundance, distribution and expansion of bamboo lectins [59]. In this study, we identified four Moso bamboo chimeric D-J lectin genes and investigated the structural domains, expansion patterns and evolutionary relationship among bamboo lectins.

Identification of D-J gene family members in Moso bamboo
To determine the presence of the D-J genes in Moso bamboo, HMMER3 [60] was used to search for the dirigent domain (PF03018) and the jacalin domain (PF01419) in the Moso bamboo genome database (ftp://parrot.genomics.cn/gigadb/pub/10.5524/100001_101000/100498/ ). Only hits with E-values less than or equal to 10 −10 were chosen as candidates. Venn diagram analysis was used to identify sequences that contained both a dirigent domain and a jacalin domain (D-J genes) using online software (http://bioinformatics.psb.ugent.be/webtools/Venn/ ) [61]. Using the above criteria, we also screened D-J genes from the genome databases of other plant species, including barley, wheat, rice and corn, which were downloaded from Ensembl (http://www.ensembl.org/index.html) and Phytozome (https://phytozome.jgi.doe. gov/pz/portal.html). The ExPASy ProtParam (https://web.expasy.org/protparam/) [62] and SignalP 4.1 Server [63] (http://www.cbs.dtu.dk/services/SignalP-4.1/) were used to predict basic physicochemical properties and make predictions about signal peptides for each gene family member. The subcellular localization prediction software ApoplastP was used to predict the localization of PeD-J proteins (http://apoplastp.csiro.au/). The identified D-J genes were named PeD-J01 to PeD-J04, according to their positions on Moso bamboo chromosomes.

Chromosomal location and collinearity analyses
All genes of PeD-Js, PeDIRs and PeJRLs were mapped to the pseudomolecules using TBtools software v1.045 [64]. Gene collinearity analyses were performed using the MCScanX program [65] based on the GFF3 files of Moso bamboo. Genes that lacked placement on major chromosomes (Scaffold1-Scffold24, removing Scaffold17, Scaffold18, Scaffold19 and Scaffold20) were excluded from examination. A schematic diagram of the presumed duplications was generated with the TBtools program.

Phylogenetic analysis
The dirigent, jacalin and D-J protein sequences of bamboo were analyzed by an intraspecific phylogenetic tree. The D-J protein sequences extracted from barley, wheat, rice, maize and bamboo were used to construct an inter-species phylogenetic tree. Geneious software was used to implement multiple alignments of amino acid sequences, and next, the neighbor-joining (NJ) method in MEGA7.0 was used to construct interspecific and intraspecific phylogenetic trees [66]. The confidence coefficient of the resulting trees were measured by 1,000 bootstraps with the pairwise deletion option.

Gene structures, conserved domains and motifs of PeD-Js
Gene structures of PeD-Js were analyzed according to the information in the whole-genome GFF annotation file from Moso bamboo. To identify additional conserved domains of PeD-Js, the protein sequences were searched against the NCBI Conserved Domain (https://www.ncbi. nlm.nih.gov/cdd/), Simple Molecular Agriculture Research Tool (SMART) (http://smart. embl-heidelberg.de/) and EMBL-Pfam (https://pfam.xfam.org/) databases. In addition, the full-length protein sequences of PeD-Js were submitted to Multiple Expectation Maximization for Motif Elicitation (MEME) (http://meme-suite.org/tools/meme) [67] with an optimum of 6-50 residues in width and a maximum of 5 motifs. Schematic domain and motif distribution diagrams of PeD-Js were edited with IBS software (http://ibs.biocuckoo.org/online.php) [68].

Homology modeling
Homology modeling was used to predict the 3D structures of PeD-J proteins based on their similarity to proteins with known structures. The appropriate homologous templates were retrieved from the PDB database (http://www.rcsb.org/). The main chain, side chain and loop region of the tertiary structures of proteins was modeled by EasyModeller software (http:// softwaretopic.informer.com/easy-modeller-for-windows/). The optimum models were generated by the SAVES server (https://servicesn.mbi.ucla.edu/SAVES/). Predicted model structures were visualized and manipulated with Discovery Studio, version 2016 (BIOVIA).

Promoter motif analysis
The 1.5-kbp upstream of the ATG start site from each PeD-J coding sequence was considered to be promoter region and extracted from the Moso bamboo genome. The cis-acting regulatory DNA motifs were predicted by PlantCARE software (http://bioinformatics.psb.ugent.be/ webtools/plantcare/html/) [69] and classified to determine elements with functional relevance. The schematic representation of their promoter motif was visualized by TBtools. Several transcriptome libraries (registration numbers SRR6171235, SRR6171236, SRR6171237,  SRR6171238, SRR6171239, SRR6171240, SRR6171241, SRR6171242, SRR6171243,  SRR6131113, SRR6131114, SRR6131115, SRR6131116, SRR6131117, SRR6131118 [70],  SRR5710702, SRR5710701, SRR5710700, SRR5710699, SRR5710698, SRR5710697,  ERR105067, ERR105069, ERR105073 and ERR105075, accession number at S6 Table) were extracted from the NCBI SRA database to determine the gene expression of the PeD-J genes and related JRL and DIR genes during different hormone treatments and development stages. The two hormone treatments included Gibberellin (GA) and naphthaleneacetic acid (NAA) (5 μM). The tissues included bamboo roots, rhizome, panicles and leaves, which were sampled when the plants' germination was 20 cm, 50 cm and 100 cm tall, respectively. Paired reads was mapped to the Moso bamboo genome with TopHat2 [71]. Then, gene expression was computed by the Cufflinks program [72,73]. Gene expression profiles were calculated by transcripts per million reads (TPM). Logarithm base 2 of each TPM value was utilized to draw the gene expression heat map using the Amazing Heatmap module in TBtools.

Plant materials and stress treatment
Moso bamboo seeds were collected from Guilin, Guangxi Province, China. The seeds were pre-treated for 3 days at 4˚C for vernalization before sowing them directly in sterilized potting soil. Seedlings were then cultivated in an experimental greenhouse at 22˚C with a constant light cycle (16h light/8h dark) for 2 months. Roots, stems, young leaves and mature leaves were sampled. For methyl jasmonate (MeJA) hormone treatment: 100 μM MeJA solution was sprayed on the plant leaf surface [74]. Leaves were then sampled at 0 h, 3 h, 6 h, 12 h, 24 h, 48 h and 72 h after spraying. For abiotic stresses, the seedlings were kept at 4˚C for cold treatment, watered with 20% PEG solution for drought treatment and treated with 200 mM NaCl solution for salt treatment. After 8 hours of treatment, the roots, stems, young leaves and mature leaves were sampled. All samples were immediately frozen in liquid nitrogen and then maintained at -80˚C until RNA extraction and isolation.

qRT-PCR analysis of genes
For quantitative RT-PCR (qRT-PCR), we used RNAprep Pure Plant Kit (Tiangen Company) to extract RNA from Moso bamboo samples, and used Prime-Script™ RT Kit (TaKaRa) to synthesize first strand cDNA from RNA according to the manufacturer's manuals. Gene-specific qRT-PCR primers were designed to amplify each CDS by AllelID7 (http://sd.downxia.com/ down/AlleleIsakdhad.rar). qRT-PCR was performed with SYBR Premix Ex Taq II reagent (TaKaRa) and was repeated at least three times on a CFX96 Real-Time System (BioRad). The bamboo actin gene was used as an internal control for normalization. PCR conditions were as follows: 95˚C for 30s, followed by 39 amplification cycles of 95˚C for 5s, and 60˚C for 30s. The expression level for each gene was determined via the 2 (-ΔΔCT) method. Statistical analyses were performed using GraphPad Prism 8 software (GraphPad Software, Inc., La Jolla, CA), and one-way ANOVA was performed on the data, followed by a Tukey test. Where appropriate, data from individual samples were compared pairwise using the nonparametric Student's t-test. Expression differences were considered significant with P values of less than 0.05.

Sub-cellular localization analysis of PeD-J03
Total RNA from Moso bamboo samples was used for cDNA synthesis. The full-length cDNA of the PeD-J03 gene (without the stop codon) was isolated by reverse transcription PCR (RT-PCR) using sequence-specific primers (S4 Table) and cloned into the pCambia1300 vector using a seamless cloning method. In this vector, the PeD-J03 gene was fused to the green fluorescent protein (GFP) gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter. The resulting 35S::PeD-J03-GFP plasmid was used for subcellular localization analysis and Agrobacterium-mediated transformation. The 35S::GFP of pCambia1300 empty vector was used as a control.
Agrobacterium tumefaciens (GV3101) containing the fusion construct was obtained by continuous incubation of rifampicin and kanamycin (0.05 g/ml) in Luria-Bertani (LB) medium at 28˚C for 24h-48h. The bacterial cells were centrifuged and resuscitated in an invasive solution (10 mm MES, 0.1 mm Acetosyringone, 10 mm MgCl2) to a final OD600 = 0.5. A disposable needleless syringe was used to inject into the underside of tobacco leaves. The plants infiltrated with Agrobacterium tumefaciens were grown for 24 h at 23˚C in the dark and then for an additional 24-72 h under a 16h light:8h dark cycle, after which the leaves were separated and the GFP signal was observed under a confocal microscope (Germany, Zeiss, LSM880).

Identification and characterization of PeD-J genes in Moso bamboo
The Hidden Markov Model (HMM) profile of the jacalin domain (accession no. PF01419.17) was retrieved from the Moso bamboo genome to identify jacalin gene family members in Moso bamboo, and 25 family members were identified which had E-values less than or equal to 10 −10 . Next, the DIR domain (PF03018.11) model was searched and 38 DIR family members were identified with E values less than or equal to 10 −10 . Venn diagram analysis was used to identify four genes that were contained in both lists, which were considered PeD-J genes (S1 Fig). Based on their location on the chromosomes of Moso bamboo, we renamed these genes PeD-J01-PeD-J04. The sequence characteristics, localization and physicochemical properties of the D-J genes were then predicted (Table 1). Of the four PeD-J proteins, PeD-J04 was the smallest, with 304 amino acids (aa), and PeD-J02 was the largest (345 aa). The molecular weight (MV) of the proteins ranged from 32.41 KDa to 37.62 KDa, and the isoelectric points (PIs) were between 5.90 and 9.39. The predicted grand average of hydropathicity (GRAVY < 0) for the majority of the D-J members revealed hydrophilicity. Each protein was predicted to have an alpha helix, beta turn, random coil and extended strand regions. All PeD-J proteins were predicted to lack a signal peptide and therefore were likely located in the cytoplasmic or nuclear compartment. Despite the lack of a predicted signal peptide, ApoplastP did predict that two proteins could be apoplastic.

Chromosome location and collinearity analysis of PeD-Js
Next, PeD-Js, PeJRLs and PeDIRs were mapped to the corresponding chromosome scaffolds according to the GFF3 file ( Fig 1A). Location analysis revealed that these genes were non-randomly located on chromosomes, with PeD-Js located on scaffold 7, 12 and 22. Scaffold 7 contained the largest number of PeD-J genes, and there was only one gene located on scaffold 12 and scaffold 22. PeD-J03 was found to be located on scaffold 12 adjacent to the PeJRL08 gene. There was no apparent correlation between the chromosome scaffold length and PeD-J gene distribution. Moreover, only PeD-J01 and PeD-J02 were found to form gene clusters on scaffold 7. To investigate the homologous loci relationships of DIR genes, JRL genes and PeD-J genes, collinearity analysis was performed ( Fig 1B). This analysis showed that some DIR and JRL genes exhibited tandem and segmental duplications, while PeD-J genes did not have any duplication. This suggests that the PeD-J genes did not result from DIR or JRL gene family duplication.

Gene family classification and phylogenetic tree construction of Moso bamboo
To determine the phylogenetic relationship of D-J proteins from bamboo and other plants (rice, wheat, barley and maize), phylogenetic trees were constructed with the neighbor-joining (NJ) method. The D-J proteins were grouped into four distinct clades, named I to IV (Fig 2A).
Most of the D-J proteins from wheat and barley were clustered together, while rice and bamboo each formed their own branches. Homology modeling showed that the four Moso bamboo genes were related to one barley gene. The phylogenetic tree of PeD-J proteins was constructed by MEGA 7.0. All protein sequences of the gene families were grouped into three subfamilies, with the chimeric D-J clustering into one group, and all classical JRLs and DIRs clustering into another two sets. A phylogenetic analysis ( Fig 2B) indicated that the D-J and JRL gene families were closely related to the PeD-J cluster, which was divided into one subbranch.

The genetic structure, motifs, conserved domains and sequence alignments of PeD-J genes
The evolutionary features and structural diversity of the D-J genes in bamboo were explored by examining their exon-intron structures. All PeD-Js possessed three introns as well as UTRs ( Fig 3A). Additionally, D-J family proteins all contained two conserved domains and four motifs ( Fig 3B). Motif1 and motif2 constituted the 130 amino acid N-terminal dirigent domain, while motif3 and motif4 constituted the 130 amino acid C-terminal jacalin domain. Next, four D-J genes were selected for sequence comparison. Their sequence similarity was found to be more than 65%, and all four proteins contained all four motifs. Notably, the PeD-J04 protein also had unique features, including GG and G-X3-D (G-X2-LD) carbohydrate motifs (Fig 3C).

Homologous modeling of PeD-J protein structures
After searching the PDB database, PeD-J01 and PeD-J03 proteins were found to have the highest similarity to 5gvy. PeD-J02 and PeD-J04 proteins were both found to have the highest degree of similarity to 5xfh. PeJRL12 of the JRL family and PeDIR08 of the DIR family were selected for homology modeling using the structural templates 1xxq, 6ood, 5xfh and 5gvy. As described in Fig 4, the tertiary structures of the PeD-J genes were predicted to be a heterozygous dimer with conserved jacalin (blue) and dirigent (purple) domains. The tertiary structure of the PeJRL12 and PeDIR08 proteins only had one domain (S2 and S3 Figs). Structural analysis revealed that proteins containing a DIR domain possessed a three-dimensional structure consisting of eight reverse-parallel chains in a β barrel-shaped structure, while all jacalin
The DIR domain of the PeD-J family proteins also contained the conserved motif V, which was comprised of PeD-Js. Similar conserved amino acids were found in other members of the PeD-J family (S3 Table). The type and distribution of the substrate-binding regions (orange) of the D-J family genes were significantly different from other families as well. In PeD-J02 and PeD-J04, these regions were predominantly distributed in hydrophobic regions at the junction of dirigent and jacalin domains. These regions were predicted to bind N-glycan, a polysaccharide condensate, which includes N-Acetyl-D-Glucosamine (NAG), Alpha-D-Mannose (MAN), Beta-D-Mannose (BMA) and Beta-D-Galactose (GAL). However, the substrates of PeD-J01 and PeD-J03 were mainly predicted to be Alpha-D-Mannose (MAN).

Promoter analysis of PeD-J genes
Promoters play a critical role in the initiation of gene transcription. To further understand the potential roles of PeD-Js in bamboo and how their expression is regulated, cis-acting elements of D-J gene promoters (1500 bp sequences upstream of ATG) were predicted with PlantCARE software. In total, 23 potential cis-acting elements involved in hormone-responsiveness (such as MeJA, abscisic acid, gibberellin, salicylic acid and auxin), abiotic-responsiveness, light-responsiveness and development regulation were identified in D-J promoters ( Fig  5). Various light-responsive elements, such as a AAAC-motif, GT1-motif, TCCC-motif, G-Box, TCT-motif, Sp1-binding site and AE-box, were also found in D-J promoters. The

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PeD-J02 promoter also harbored four G-Box elements. ABRE, the ABA responsiveness elements, could be found in the promoter regions of three PeD-J genes. There were three ABREs among genes with this element, which were found in PeD-J02, PeD-J03 and PeD-J04, but not The TGA-element, which has been shown to be involved in auxin responsiveness, was present in the PeD-J02 promoter. TC-rich repeats have been associated with defense and stress responsiveness and were found in the promoters of PeD-J01 and PeD-J03. MBS is a droughtinducibility element, which was found in the promoters of PeD-J01 and PeD-J04. Regulatory elements essential for the anaerobic induction (ARE) were also found in two promoters, with PeD-J01 containing seven ARE elements, and PeD-J02 containing two ARE elements. Only PeD-J02 was identified as having an element related to anoxic specific inducibility (GC-motif). The RY-element of PeD-J04 was associated with seed-specific regulation, while the CATbox of PeD-J04 was related to meristem expression. HD-Zip 1 of the PeD-J02 element is predicted to be related to differentiation of the palisade mesophyll cells and the A-box regulatory

Expression patterns of the PeD-J genes
The availability of a relevant transcriptome database makes it possible to study the gene expression of PeD-J genes in a comprehensive manner. To explore the potential functions of D-J genes in Moso bamboo, their expression was assessed in seedlings after treatment with different hormones (GA, NAA), at different growth heights (20, 50, 100 cm) and in different tissues (root, rhizome, panicles, leaf). Expression heat maps were drawn using TBtools. Under GA or NAA treatment ( Fig  6A and 6B), there were no significant differences in the expression levels of PeD-J02 and PeD-J04,  (Fig 6C). In the DIR family, five genes were highly expressed, four of which (PeDIR08, PeDIR22, PeDIR04 and PeDIR03) were most strongly expressed at the top of the 100 cm stem. In the JRL family, all genes had low expression, which suggested that PeD-J and PeDIR genes may play a role in the high growth development of shoots. The expression of PeD-J family genes was also found to be tissue-specific. For example, the PeD-J04 gene was highly expressed only in panicles, while PeD-J02 was highly expressed in panicles, leaves, roots and rhizomes (Fig 6D). However, PeD-J03 and PeD-J01 were not highly expressed in roots but were found in other tissues.
The expression patterns of Moso bamboo D-J genes during stress hormone response were investigated by construction of histograms. As could be seen from the figures, the gene

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expression level of the PeD-J genes was increased in seedlings treated with GA or NAA (Fig 7A  and 7B). PeD-J family genes all had tissue-specific expression, indicating that they may play a role in the development of different tissues (Fig 7C). For example, PeD-J04 was highly expressed in panicles but absent in leaves, while PeD-J02/PeD-J03 were highly expressed in leaves and PeD-J01/PeD-J03 were expressed at relatively low levels in the rhizomes. PeD-J03, on the other hand, was significantly expressed in rhizomes, panicles, roots and leaves.
Additionally, PeD-J genes showed differential expression over the course of shoot development (Fig 7D). PeD-J03 was constitutively highly expressed, while the expression levels of PeD-J01/ PeD-J04 decreased during development, and PeD-J02 fluctuated up and down over the course of development. At 20 cm of height, PeD-J03 had the highest expression, followed by PeD-J04 and PeD-J02, then PeD-J01. At 50 cm, PeD-J03 had the highest expression, followed by PeD-J04 and PeD-J02, while PeD-J01 was not expressed at all. When bamboo reached 100 cm in height, PeD-J01 and PeD-J04 were not expressed, while gene PeD-J03 had the highest expression, followed by PeD-J02. The above results indicated PeD-J genes were under strong developmental regulation.

Differential expression of the PeD-J genes in different tissues and during abiotic stress and phytohormone treatments
The cis-elements predicted to be present in the promoters of PeD-J genes suggested that they are likely involved in growth, drought stress and hormone responses. To determine their expression patterns under different stresses and in different tissues, quantitative real-time PCR analysis was performed (S4 Table) to investigate the response of PeD-Js' expression to these treatments in two-month-old seedlings. This analysis revealed that PeD-J01 and PeD-J04 were highly expressed in young leaves, while PeD-J03 was highly expressed in stems and PeD-J02 was absent from nearly all tissues but expressed at a low level in mature leaves (Fig 8A). The expression levels of the D-J genes were analyzed in different tissues under high salinity, PEG, cold and normal conditions (Fig 8B-8D). The abiotic stress results indicated that PeD-J01 was up-regulated in roots under salt and cold stress, while the other three PeD-J genes were down-  indicates that the level of the gene expression in treatment group was significantly different from that in the control group (t-test, p < 0.05). Double asterisks indicate that there is a significant difference (t-test, p < 0.01). The relative expression levels of the target genes were examined using the 2 -ΔΔCt method. Error bars represent standard deviations of the means of three independent replicates. https://doi.org/10.1371/journal.pone.0248318.g008

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of PeD-J04 was significantly up-regulated under cold stress. For the most part, stressful conditions resulted in reduced expression of PeD-J genes in leaves, with the exception of PeD-J01, which was induced under PEG stress. qRT-PCR was used to determine the expression levels of the four PeD-Js under MeJA treatments (Fig 8E). Two genes (PeD-J01/PeD-J02) were significantly down-regulated at 6h of MeJA treatment, while other genes were up-regulated. Interestingly, the expression levels of two genes (PeD-J01/PeD-J03) were significantly increased after MeJA treatment, and the expressions of most genes exhibited two peaks, with an initial rapid increase, followed by a later peak.

Analysis of subcellular localization of the PeD-J03 gene
Transient expression of the 35S:: PeD-J03-GFP plasmid in tobacco epidermal cells was used to determine subcellular localization using an Agrobacterium infestation method. Compared to control 35S::GFP of pCambia1300 empty vector, 35S:: PeD-J03-GFP localized to cell membrane primarily, with a small amount in the nucleus (Fig 9).

Discussion
Plant lectins are a class of proteins with glycan-binding properties and a wide range of functional diversity [75]. By interacting with various compound polysaccharides, lectins function as central mediators of information transfer in biological systems [76]. In the last few years, lectins have been shown to be mainly involved in binding to foreign glycans produced by plant pathogens, sometimes resulting in anti-nutritional or toxic effects on higher animals and insects [38]. Notably, most plant lectins specifically recognize exogenous polysaccharide compounds and are involved in endogenous regulation [37,42,77].
Dirigent-jacalin genes have been shown to play important roles in plant responses to drought stress in sugarcane. At least four DJ groups were identified in the sugarcane genome, which consists of seven non-redundant sequences [45], but the identity and expression patterns of PeD-J genes have rarely been studied in Moso bamboo at the genome-wide level. In this study, we identified four PeD-J genes in the genome of bamboo. We then performed a comprehensive analysis of the PeD-J gene family, including analysis of phylogeny and gene structures, conserved motifs, chromosomal locations and 3D structure, cis-element analysis and expression profiling. According to the inter-species phylogenetic tree, the D-J family of Moso bamboo was closely related to barley and the D-J family belonged to the same branch as the JRL family in the intra-species phylogenetic model. Studies have shown that a common ancestor of JRLs evolved before the divergence between monocotyledons and dicotyledons. The modern JRLs were formed by the duplication and tandem insertion of ancestral domains, and the formation of monocotyledon chimeric lectins occurred when a dirigent domain was inserted into cytoplasmic lectins [24,46]. Closer examination revealed that chimeric jacarins are present in only a small fraction of monocotyledonous taxa and emerged from 50 to 25 million years ago [74]. The intraspecific evolutionary tree showed that JRLs were closely related to D-J genes, indicating that the two families may come from the same ancestor, which was consistent with previous studies.
All four PeD-Js have three exons and two introns, with an extremely similar overall structure. Additionally, the configurations of motifs recognized by MEME also reflected the conserved nature of PeD-Js, with similar motif structures and numbers. This high degree of conservation indicates a strong selection pressure throughout PeD-J gene family evolution.
The tertiary structure prediction results showed that the PeD-J03 protein and PeD-J04 protein have slightly different structures, but both possess a chimeric conserved hydrophobic cavity consisting of a barrel-shaped domain and β-prismatic folded structure. When Pickel et al.
[50] determined the structure of the AtDIR6 protein of Arabidopsis thaliana, they found it was composed of eight reverse parallel chains in series, with a hydrophobic cavity in the middle. These findings indicated that this hydrophobic binding protein might have evolved from a DIR protein, while the tertiary structure of PeDIR08 predicted in this study was similar to that of AtDIR6. The JRL protein structure consisted of a β-prism structure composed of β-sheets [78], and the D-J protein was a chimeric tertiary structure composed of a DIR structure and a JRL structure. Bourne et al. [78] found a JRL protein structure with twelve residues (Glyl1 Gly14, Gly18, Gly84, Tyr102, Asn103, Gly108, Prol09, Gly111, Phel18, Gly129 and Phel30) that played a key role in the folding of the β-prismatic region in Helianthus tuberosus. Through 3D homologous modeling, these conserved amino acid residues (Glyl9, Gly22, Gly26, Gly93, Ser114, Gly 116, Gly120, Pro121, Gly123, Phel31, Gly142 and Phel43) in the JRL domain of the PeJRL12 protein were predicted to also be involved in β-prismatic folding. These residues were also found in PeD-Js, and the amino acid residues of the jacalin domain of PeD-Js were highly conserved. Ralph et al. [79] found five conserved motifs in DIR genes of spruce (motif I-V). Their modeling results indicated that the conserved amino acid residues of motif V were found in the dirigent protein of PeDIR08 (Val73, Val74, Gly75, Gly76, Thr77, Gly78, Asp79, Phe80, Phe81, Met82, Ala83, Arg84, Gly85, Val86, Ala87, Thr88 and Leu89) and also found in chimeric protein sequences of PeD-Js. It has been speculated that the mutation of a few conserved bases may be the cause of their substrate differences.
During multi-sequence comparison and modeling of PeD-J family proteins, conserved carbohydrate-binding sites were identified in jacalin region (S4-S7 Figs). In the G-X3-D (G-X2-LD) motif, one of the glycan-binding-related sites, the L residue in the X3 region, appears to be the most conserved in the taxon [80]. It was found that the sugar-binding sites of the PeD-J04 protein included a GG and a G-X2-LD motif, which was consistent with the second sugar-binding site found in BanLec of a JRL protein by Meagher et al. [80]. Additionally, the G-X2-LD structure of PeD-J04 binds the NAG in the biantennary N-glycan (S6 and S7 Figs). In general, hydrogen bond equilibrium and hydrophobic interactions are the dominant modes of lectin-carbohydrate interaction [81]. However, the loop structures can also affect the specificity of glycosyl binding [82]. Additionally, the conserved aspartic acid of the BL3 linking loop resides in the G-X3-D motif that controls the activity of the lectin and determines the lectin and carbohydrate recognition characteristics of the protein. In addition, a number of other conserved residues and specific amino acid residues have been identified, some of which are specifically involved in sugar binding and quaternary structure formation [83][84][85]. The other three genes with only the GG motif may have undergone base mutations during evolution, so that the sugar-binding site was abolished. Monocotyledonous chimeric lectins show a degree of diversity in terms of carbohydrate binding specificity. Most of the monocot chimeric lectin proteins studied (TaJA1, Ma et al.,[51]; OsJVC1, Jiang et al., [42]; TaHfr1, Subramanyam et al., [44]) specifically bind mannose. Homology modeling indicated that PeD-J01 and PeD-J03 bind mannose, and may belong to the mannose-specific jacalin lectins (mJRLs). The same modeling indicated that PeD-J02 and PeD-J04 could bind either mannose or galactose of biantennary N-glycan (S6 and S7 Figs). The glycan-binding properties of these genes were similar to the classical jacalin genes, which was presumed to be more closely related to the JRL family, and the dirigent domain may have been inserted during later evolution [46].
PeD-Js with dirigent domains at the N-terminal and JRL domains at the C-terminal are characterized by a structure of highly conserved modular domains, and it has been shown that dirigent and jacalin domains are associated during evolution. The lectin domains in maize BGAF and sorghum SL exhibited significantly different glycan specificity compared with fulllength proteins, since the loss of the N-terminal dirigent domains mainly affected glycan-binding specificity [41]. More recently, Ma et al. [51] found that transgenic plants containing Ta-JA1, which only has an N-terminal dirigent domain, could not exhibit agglutination activity and participate in disease resistance, but did have altered sensitivity to salt stress. However, Ta-JA1 contained a unique dual domain, dirigent domain and jacalin domain, which could effectively agglutinate rabbit erythrocytes, leading to enhanced disease resistance against bacterial, fungal and viral pathogens. Van Damme [38] and Subramanyam et al. [25] found that wheat plants were responsive to infection of first-instar Hessian fly larvae by increasing the expression of the Hfr-1 gene, which encodes a protein with a C-terminal jacalin domain and an N-terminal disease responsive dirigent domain. These results implied that protein fusions were functionally significantly enhanced and positively selected.
JRL fusions to other proteins may have been required for the development of new functions and previous research has found D-J genes which appear to be involved in responses to specific biotic or abiotic stimuli [25,53,76,86]. These genes may therefore have evolved via extension of the jacalin domain in JRLs and the fusion of other functional domains that helped plants adapt to various environmental challenges [85].
A comprehensive expression profile of all PeD-J genes in bamboo suggested that PeD-J03 played a role in the development of shoots and PeD-J03 and PeD-J04 responded to hormone stress. Furthermore, comprehensive analysis of gene expression profiles in different tissues identified numerous tissue-specific expression patterns. Chimeric lectins play an important role in plant responses to biotic and abiotic stresses [46]. In particular, PeD-J genes from D-J lectins were significantly induced under PEG, cold and salt stress. It has previously been shown that treatment with MeJA could induce lectin activity in the leaves of rice, barley, wheat and maize [76]. Studies have also shown that plant growth regulators, including phytohormones, could regulate plant responses to environmental stimuli [87,88]. MeJA activates a series of signal transduction pathways involved in plant responses to abiotic and biotic stresses, thereby enhancing stress tolerance [89]. In this study, it was found that the four PeD-J genes responded differently to MeJA treatment [30,90]. The expression profiles of PeD-Js in different tissues were analyzed by qRT-PCR, and PeD-J02 was found to only be expressed in leaves, while the other three genes were expressed in roots, stems and leaves. Taken together, our results provide useful information for further research into the role that PeD-J genes play in development and stress response.
Supporting information S1 Table. The