Genome-Wide Characterization of Dirigent Proteins in Populus: Gene Expression Variation and Expression Pattern in Response to Marssonina brunnea and Phytohormones

Marssonina brunnea causes a major disease that limits poplar growth. Lignin and lignan play essential roles in protecting plants from various biological stresses. Dirigent (DIR) proteins are thought to control the stereoselective coupling of coniferyl alcohol in the formation of lignan and lignin. DIR family members have been well studied in several plant species, but no previous detailed genome-wide analysis has been carried out in forest trees, such as poplar. We identified 40 PtDIR genes in Populus trichocarpa and classified them into three subgroups (DIR-a, DIR-b/d, and DIR-e) based on phylogenetic analyses. These genes are distributed on 11 poplar chromosomes, and 80% of PtDIRs (32/40) are intronless. The cis-element analysis inferred that PtDIRs possess many types of biological and abiotic stress-response cis-elements. We also analyzed intraand inter-specific collinearity, which provided deep insights into the evolutionary characteristics of the poplar DIR genes. Analyses of the protein tertiary structure and critical amino acid residues showed that PtDIR7–10 and PtDIR13–16, which belong to the DIR-a subfamily, might be involved in the regioand stereo-selectivity of bimolecular phenoxy radical coupling in poplars. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis revealed different expression patterns for the PtDIR genes of P. trichocarpa and the PeDIR genes of ‘Nanlin 895’ in various tissues. Additionally, we analyzed responses of PeDIRs to M. brunnea and different phytohormone treatments (abscisic acid, salicylic acid, methyl jasmonate, and ethylene) in ‘Nanlin 895’. The results showed that at least 18 genes responded strongly to M. brunnea, and these PeDIRs also showed significant responses to phytohormones. These results suggest that DIR genes are involved in the poplar defense response against M. brunnea, and this study will provide fundamental insights for future research on poplar


Introduction
Poplar is a perennial deciduous tree with a wide range of uses, such as for wood and industrial timber [1,2]. However, woody plants have a long growth cycle and are vulnerable to diseases, insect pests, and other stresses [3]. Poplar black spot, which is mainly caused by Marssonina brunnea is a major disease that limits poplar growth. In poplars, M. brunnea infection leads to premature defoliation and seriously restricts growth [4][5][6]. Therefore, it is essential to investigate M. brunnea resistance genes related to M. brunnea. Phytohormones, including abscisic acid (ABA) [7,8], salicylic acid (SA) [9], methyl jasmonate (MeJA) [4,6,10], ethylene (ETH) [6], play an important role in the process of poplar disease resistance.

Identification and Characterization of the DIR Proteins in Populus trichocarpa
The complete P. trichocarpa genome and protein sequence database were downloaded from Phytozome v12.1 (https://phytozome.jgi.doe.gov/pz/portal.html, accessed on 8 March 2020). To identify DIR candidates of P. trichocarpa, the hidden Markov model (HMM) file corresponding to the DIR domain (PF03018) was downloaded from the Pfam protein family database (http://pfam.sanger.ac.uk/, accessed on 8 March 2020) and HM-MER 3.0 was used to search for PtDIRs in the P. trichocarpa genome database; the default parameters were adopted, and the cutoff value was set to 1.2e-28. After manual removal of the redundant sequences, the output putative DIR protein sequences were submitted to the National Center for Biotechnology Information Conserved Domain Database (CDD) (https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml, accessed on 15 March 2020), Pfam (http://www.pfam.org/, accessed on 15 March 2020), and SMART (http://smart.embl-heidelberg.de/, accessed on 15 March 2020) to confirm the conserved DIR domain. These selected PtDIRs were named based on their positions on the chromosomes. We used the ExPasy website (http://web.expasy.org/protparam/, accessed on 5 January 2021) to obtain sequence length, molecular weight, isoelectric point (pI), and subcellular localization predictions of the identified DIR proteins. The Plant-mPLoc server (http://www.csbio.sjtu.Edu.cn/bioinf/plant-multi/, accessed on 5 January 2021) was used to predict the subcellular location. N-glycosylation sites (asparagine residues) of the P. trichocarpa DIR proteins were searched online using the NetNGlyc 1.0 server (http://www.cbs.dtu.dk/services/NetNGlyc/, accessed on 10 April 2020).

Gene Structure, Conserved Motif Analysis, and cis-Acting Elements of DIRs in P. trichocarpa
The exon-intron organization of PtDIRs was determined by comparing the predicted coding sequence (CDS) with the corresponding full-length sequence using the Gene Structure Display Server (GSDS: http://gsds.cbi.pku.edu.cn, accessed on 15 May 2020). We used MEME (https://meme-suite.org/meme/tools/meme, accessed on 15 May 2020) for protein sequence analysis to identify conserved motifs in the identified PtDIR proteins. The specific setting parameters were as follows: 6 < motif width <50; 10 motifs were identified. To identify the various cis-acting regulatory elements in the promoters of the DIR genes, PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 20 December 2020) was used to extract the region 1500 bp upstream of the CDS.

Chromosomal Location, Gene Duplication, and Calculation of Ka/Ks
The chromosomal distribution of PtDIRs was obtained from the P. trichocarpa genomic database and location mapping was conducted using MapChart. Calculation rates for synonymous (Ks) and non-synonymous (Ka) substitutions were estimated by Ka/Ks Calculator 2.0. Divergence time (T) was calculated using the formula T = Ks/2λ (λ = 1.5 × 10 −8 ) for millions of years ago (MYA) for dicotyledonous plants [38].

Intraspecific and Interspecific Collinearity Analyses
Collinearity analysis was completed using Circos software. The Multiple Collinearity Scan toolkit [38] was adopted to analyze gene duplication events with the default parameters. The syntenic analysis maps were built using multiple synteny plots to show the synteny relationship of the P. trichocarpa intra and interspecific DIRs.2.6. Plant cul-tivation, treatments, and quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis.
In this study, the experimental plants used were P. trichocarpa and 'Nanlin 895'. P. trichocarpa was cultured on 1/2 standard wood plant medium [39]. 'Nanlin 895' (hybrid of Populus deltoides × Populus euramericana) was cultured on 1/2 Murashige and Skoog medium [40]. After one month of growth, seedlings were transferred to standard pots with a mixture of vermiculite/perlite/peat (1:1:3, v/v/v). M. brunnea was cultured in potato dextrose agar (PDA) at 25 • C [4,41]. After the second month of growth, at the 2-month-old seedling stage (15 cm high of aboveground part, 6 leaves, and about 4cm 2 of a single leaf), the two kinds of poplars were harvested to analyze gene expression in different tissues (root, stem, young leaf, and mature leaf) by RT-qPCR analysis. Twomonth-old 'Nanlin 895' plants were pricked with one leaf (about 4cm 2 ) three punctures by a sterile needle and inoculated by spraying the leaves and stems thoroughly with a conidial suspension (1 × 10 6 conidia per mL) harvested from 10-day-old PDA cultures. These plants were incubated in a greenhouse (60% humidity, 25 • C, 16/8 h day/night photoperiod, 20 µmol m −2 s −1 ). The plants were harvested immediately at eight time points: d 0, 0.5, and 1-6 after inoculation. At the same time, 'Nanlin 895' seedlings were treated with 200 µM abscisic acid (ABA), 10 mM salicylic acid (SA), 1 mM methyl jasmonate (MeJA), and 1 mM ethylene (ETH) before being harvested at 0, 3, 6, 9, 12, and 24 h for RNA extraction and RT-qPCR analysis [4,40]. Each experiment was replicated three times using three healthy plants. The specific primer sequences used for the RT-qPCR are listed in Supplementary Table S1. We use RNAprep Pure Plant Plus Kit (Tiangen, Beijing, China) to extract total RNA of respective tissues (starting 100 mg of each sample) according to the kit instructions. In this process, genomic DNA was removed by RNase-free DNase I. The concentration and purity of total RNA were determined by the spectrophotometer (drop, UK) and its integrity was tested by 1% agarose gel electrophoresis. 1 µg RNA of each sample was used to synthesize first-strand cDNA using HiScript ® III 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China). Then the cDNA was diluted 10-fold and was used for RT-qPCR. The RT-qPCR reactions were carried out by StepOnePlus TM (Applied Biosystems, USA) using a three-step PCR procedure with the following cycle parameters: 95 • C for 10 min, then 95 • C for 15 s and 60 • C for 1 min, for 40 cycles, and a melt cycle from 65 • C to 95 • C. The reaction mixture was 20 µL, including 2 µL cDNA, 0.4 µL primer-F (10 µmol/L), 0.4 µL primer-R (10 µmol/L), 10 µL ChamQ SYBR ® qPCR Master Mix (Vazyme, Nanjing, China), 0.4 µL ROX, and 6.8 µL ddH 2 O. The relative gene expression was calculated using 2 -∆∆ CT method.

Multiple Sequence Alignment, Phylogenetic Analysis, and Classification of PtDIRs
To clarify the evolutionary classification of the 40 PtDIRs, we constructed an evolutionary tree. The DIR gene family can be divided into six major subfamilies: DIR-a, b/d, c, e, f, and g ( Figure 1). The DIR gene families of P. trichocarpa and Arabidopsis were only classified into three subfamilies (DIR-a, b/d, and e). Most PtDIRs belong to DIR-b/d (21 in total), followed by the DIR-a and DIR-e subfamilies with 8 and 11 PtDIRs, respectively. To date, studies have shown that the DIR genes, which can specifically control the oxidative coupling of coniferyl alcohol in the formation of (+) or (−) pinoresinol, were mainly concen-trated in the DIR-a subfamily [11,12,35], and these functional genes are highlighted with a red background in Figure 1. However, GhDIR3 and GhDIR4 from Gossypium hirsutum (G. hirsutum), which can contribute to forming the (P)-(+)-gossypol (dextrorotatory gossypol), belong to the DIR-b/d subfamily [36]. A comparison of PtDIR protein sequences shows that the protein similarity ranges from 3-95%, indicating that some PtDIR proteins may differ significantly and show functional diversity. In the DIR-a subfamily, the sequence similarity of PtDIR proteins is exceptionally high, ranging from 52-95%, followed by the DIR-e and DIR-b/d subfamilies, in which it ranged from 27-88% and 6-85%, respectively ( Figure 2).

Gene Structure and Conserved Motifs of the PtDIR Gene Family
The conserved motifs of PtDIR proteins were identified by MEME, and ten putative motifs were revealed ( Figure 3). The lengths of these predicted motifs ranged from 15-41 amino acids. The PtDIR proteins contained 2-7 conserved motifs. We selected the best possible matching sequences as the motif sequences (Supplementary Materials Table S2) and annotated each motif using CDD and Pfam software. Motif 2 was present in all PtDIR sequences except for PtDIR3. Motifs 1 and 3 were present in most DIR proteins. Motif 9 was only found in the proteins of group I and in the PtDIR2 protein of group V. Motifs 4 and 5 were only present in groups IV and V. Motifs 6 and 10 were present only in group I and motif 8 was found only in group V. The proteins from group II contain only two motifs (motifs 1 and 2). Group I is part of the DIR-a subfamily, groups II and III are part of the DIR-e subfamily, and groups IV and V belong to the DIR-b/d subfamily. The shared common motifs, seen via the distribution patterns of the various motifs across the different groups and within the same groups of PtDIR proteins in the phylogenetic tree, may indicate some functional diversity. The PtDIR protein structures were obtained from GSDS software. The results showed that all proteins belonging to the PtDIR family are stable. Most of the PtDIR genes (32/40, 80%) were intronless. In addition, the remaining eight PtDIR members (PtDIR1, 3, 18, 22, 29, 31, 34, and 37) only contained one intron. Furthermore, approximately 42.5% of PtDIR members did not have untranslated regions (UTRs). The PtDIR members from group I all contained 5 and 3 UTRs. In comparison, the PtDIR members from group IV had no UTRs, except for PtDIR11. The PtDIR members from the same branches often shared similar structures.

Chromosome Distribution and Ka/Ks
To examine the genome distribution of the PtDIRs, chromosomal mapping was performed by MapChart. As shown in Figure 4

Stress-Related cis-Elements in the PtDIR Promoters
To further analyze the potential regulatory mechanisms of PtDIRs involved in plant stress responses, 12 cis-elements were selected and are shown in Figure 5. We found that 17 PtDIRs (PtDIR2, 5, 12, 15, 17, 23-27, 29, 33-35, 37, 39, and 40) contained TC-rich repeats (defense and stress-responsive elements), indicating that the PtDIR gene family is related to responsiveness to biotic and abiotic stresses [42].   11, 15, 16, 23, and 33) contained ERE, ABRE, CGTCA-motif, TCA, and TATC-box cisacting elements, respectively, suggesting that PtDIRs may participate in the regulation and metabolism of hormones in plants. Additionally, 19 drought-responsive elements (MBS), 14 low-temperature responsive elements (LTR), and 13 wound-responsive elements (Wunmotif) are also predicted to be specifically distributed in promoters of PtDIRs. Furthermore, four PtDIRs (29, 31, 34, and 36) have cis-acting MBSI elements, which are the MYB binding sites involved in flavonoid biosynthesis gene regulation. Some cis-acting elements like MRE, which is involved in light responsiveness, and the TGA-element, involved in auxin response, were also predicted, as shown in Supplementary Table S4. The cis-element analysis illustrated that PtDIR genes could respond to different stresses.

Synteny Analysis of PtDIRs in P. trichocarpa and Other Species
We conducted a synteny analysis on PtDIRs to investigate the duplication events occurring in the PtDIR gene family (Figure 6a). We identified 10 gene pairs (PtDIR1/19, PtDIR1/36, PtDIR5/17, PtDIR8/13, PtDIR19/36, PtDIR19/25, PtDIR22/31, PtDIR25/36, Pt-DIR26/33, and PtDIR28/32) that were clustered into 10 segmental duplication event regions on chr1, 3, 4, 6, 8-10 and chr16. Chr1 and chr3 contained three clusters, indicating that PtDIRs distributed on these two chromosomes had more connections. To further analyze the phylogenetic history of the PtDIR gene family in different species, we identified DIR gene family members using Arabidopsis as the dicot representative and rice as the monocot representative (Figure 6b). We identified 13 collinear gene pairs between P. trichocarpa and Arabidopsis and only two pairs between P. trichocarpa and rice. These collinear correlative genes are concentrated on chromosomes 1, 2, 3, 8, 9, and 10. PtDIR26 and PtDIR34 were anchored on both Arabidopsis and rice DIR genes. Four PtDIRs (7,8,15, and 34) were respective collinear with two different AtDIR genes simultaneously, indicating potential paralogous gene pairs, and they may have played an essential role in the DIR gene family during evolution.

Analysis of Protein Tertiary Structures and Sequence Alignments of PtDIR Proteins of the DIR-a Subfamily
Phylogenetic analysis revealed that eight PtDIRs (PtDIR7-10 and PtDIR13-16) were DIR-a subfamily members. In this study, using AtDIR6 associated with (−) pinoresinol and PsDRR206 associated with (+) pinoresinol as references, we predicted the 3D structures of eight PtDIR proteins of the DIR-a subfamily. The 3D structures of PtDIRs 7 and 15 and PtDIRs 8-10, 13, 14, and 16 were compared and merged with AtDIR6 and PsDRR206, respectively, using Chimera software. The 3D structures of the PtDIR proteins were highly similar to AtDIR6 or PsDRR206. Integration analysis of the conserved domains (red) showed that those of the PtDIRs could be integrated with AtDIR6 and PsDRR206 (Figure 7a).  Next, we performed a protein sequence alignment using ClustalX 2.0 (Figure 7b). The DIR proteins included FiDIR1, PsDIR202, TpDIR5, TpDIR8, and LuDIR1, which can contribute to forming (+) pinoresinol, and AtDIR5, AtDIR6, LuDIR5, and LuDIR6, which can contribute to forming (−) pinoresinol. The result showed that they have high similarity, with five conserved motifs. Motif I was located from amino acid positions (aa) 100-105. Motifs II-V were located from aa 123-134, 146-154, 170-184, and 191-215, respectively. All protein sequences contained an eight-stranded antiparallel β-barrel (green arrow), five active sites (His87, Asp88, Thr134, Ser141, and Arg191; green triangles), and two N-glycosylation sites (Asn; blue circles). Six differentially conserved residues at aa 145, 162, 165, 167, 180, and 211 were involved in forming (+) pinoresinol or (−) pinoresinol (yellow star). Residues 145, 167, and 180 of PtDIRs are accordant with other DIR genes; however, the residue 162 of PtDIR13 is Phe instead of Leu, and residue 211 of PtDIR13 and PtDIR10 is Leu, not Ile. The alignment results suggest that PtDIR8-10, 13, 14, and 16 may catalyze the formation of (+) pinoresinol, with PtDIR8, 9, 14, and 16 showing the most potential. Residue 211 of PtDIR15 and PtDIR7 is Phe instead of Tyr, while the other residues are conserved. It is likely that PtDIR7 and PtDIR15 can catalyze the formation of (−) pinoresinol rather than (+) pinoresinol.

Expression Patterns of PeDIRs of 'Nanlin 895 in Response to Different Phytohormones
To further analyze the response patterns of PeDIRs to phytohormones, we selected 19 PeDIRs from three PeDIR subfamilies that did not only respond to M. brunnea stronger than others but also showed a higher expression in tissues, and further we explored their response patterns using four hormones (ABA, SA, MeJA, and ETH; heatmap of Figure 10 and bar chart of Figure S1). We included seven PeDIR genes from DIR-a (7-10, 13-15), ten from DIR-b/d (1, 11, 19-21, 25, 36, and 37-39), and two from the DIR-e subfamily (17 and 23) (Figure 9). Some PeDIRs, such as PeDIR9 and PeDIR21, were upregulated by all four phytohormones, and, except for PeDIR21 and PeDIR37, the remaining PeDIRs showed a strong response (more than 2-fold higher expression at least one time point than the d 0 after treatment) to ABA. PeDIR8 and 39 only showed strong responses to ABA, SA, and MeJA, while PeDIR11, 14, and 25 only showed strong responses to ABA, SA, and ETH. PeDIR20 showed an obvious response to ABA, MeJA, and ETH, and PeDIR15 showed a strong response to ABA and SA, while PeDIR7 and PeDIR19 showed strong responses to ABA and MeJA. PeDIR10, 36, 37, and 38 were downregulated under SA stress but upregulated under ABA, ETH, and MeJA stresses.

Discussion
DIR proteins are widely distributed in terrestrial plants [23,28,35,44,45]. However, there are no reports of DIR proteins in primitive aquatic plants. Therefore, it is speculated that DIR proteins are associated with the development of vascular bundles that occurred during the evolution of aquatic plants to terrestrial plants [46], and they likely play an important role in the formation of lignin and lignan. They are involved in a broad range of plant biological functions such as resistance to various biotic and abiotic stresses. Previous studies identified 19, 49, 24, 29, and 35 DIR genes in I. indigotica, rice, pepper, pear, and brassica, respectively [15,28,35,47,48]. To date, little is known about the DIR family of genes in P. trichocarpa. In this study, we identified 40 DIR genes in the P. trichocarpa gene database. These genes were distributed on 11 chromosomes, and 80% PtDIRs were intronless. Nglycosylation is important for secretory proteins [49] and also is a vital characteristic of DIR proteins [11]. Approximately 95% of PtDIR proteins have at least one N-glycosylation site. The signal peptide can direct the newly-synthesized protein to various subcellular organelles [50]. Around 62.5% of PtDIR proteins contain signal peptide sequences, and our subcellular localization results showed that PtDIR proteins were located in various organelles, indicating that PtDIR proteins can be targeted for extracellular release before finally localizing in various subcellular organelles.
Phylogenetic analysis of DIR genes from multiple species showed that they could be divided into six subfamilies (DIR-a, b/d, c, e, f, and g), but not every species has all of the subfamilies [23]. For example, brassica only has the DIR-a and DIR-g subfamilies [28], DIR-f is exclusively found in spruce [23], and no angiosperm DIR proteins were observed in the DIR-f subfamily [23]. In addition, the DIR-c subfamily is only found in angiosperm monocots. The DIR genes of the DIR-c subfamily always combine with the jacalin-like domain to contribute to the defense against insects [15,51,52]. P. trichocarpa has three subfamilies (DIR-a, b/d, and e). The DIR-a subfamily has eight PtDIRs, and compared to other DIR genes that can control the oxidative coupling of coniferyl alcohol specifically in the formation of (+) or (−) pinoresinol, these eight PtDIRs can be divided into two groups. One group (PtDIR8-10, 13, 14, and 16) possibly contributes to the formation of (+) pinoresinol, while the other (DIR7, 15) possibly contributes to the formation of (−) pinoresinol. This was confirmed following analyses of the tertiary structures, conserved motifs, and protein sequence alignments. This is similar to flax in which LuDIR1, 5, and 6 belong to the DIR -a subfamily. It is known that LuDIR5 and LuDIR6 contribute to forming (−) pinoresinol in seed-coats, while LuDIR1 contributes to forming (+) pinoresinol in the vegetative organs [44,53]. However, not all the DIR genes of DIR-a can form a single chiral pinoresinol [13,44]. Further in vitro and in vivo experiments are needed to verify the ability of PtDIRs (especially the DIR-a subfamily) to form unique chiral pinoresinol.
The cis-elements in the 5' upstream region are involved in the dynamic regulation of gene expression [54]. PtDIRs contain elements related defense and stress responses, indicating that they may play an important role in biological and abiotic stresses, and a large number of elements are responsive to hormones such as ABA, SA, MeJA, and ETH. To be able to respond to various stresses, plants have a complex signal transduction network and synthesize a class of small hormones (SA, MeJA, ETH, and ABA) as signaling molecules in the plant defense response [55][56][57]. This indicates that the expression patterns of PtDIRs closely rely on the regulation of the hormone network. The presence of duplications in the genome helps plants adapt to the changing environment and evolve more smoothly [58]. All tandem duplicated PtDIR gene pairs had a Ka/Ks < 0.5, indicating that the PtDIR gene family might have undergone strong purifying selective pressures during evolution [59]. It is estimated that the divergence time of duplicated PtDIR gene pairs from 3.44 (PtDIR8 and PtDIR9)-35.5 (PtDIR10&PtDIR13) MYA, indicating PtDIRs had undergone duplication events over a long time span. Duplicated genes provide a template for new genes that take on new functions [60]. P. trichocarpa contains 10 segmental duplication event regions distributed on eight chromosomes, indicating that segmental duplication events were crucial in the expansion of the PtDIR gene family. Synteny analysis of P. trichocarpa with Arabidopsis and rice showed that P. trichocarpa might be more closely related to Arabidopsis. PtDIR26 and PtDIR34 were anchored in both rice and Arabidopsis, indicating that these collinear pairs may have existed before the evolution of monocotyledons and dicotyledons.
The different tissue expression patterns of the DIR genes have been described in many species, such as pepper, flax, rice, and brassica [15,28,44,47,61]. There is no unified tissue expression pattern for DIR genes. The 'Nanlin 895' poplar has a mature genetic transformation system and is an ideal model to study gene function in poplar [43,62], thus we used 'Nanlin 895' alongside P. trichocarpa to analyze DIR genes. The expression profile analysis of DIR genes in P. trichocarpa and 'Nanlin 895' in different tissues (root, stem, young leaf, and old leaf) showed similarities and differences. In P. trichocarpa, there were 19, 14, and 4 PtDIRs that were highly expressed in root, stem, and leaf tissues, respectively, while there were 16, 14, and 7 PeDIRs that were highly expressed in 'Nanlin 895' root, stem, and leaf tissues, respectively. Among them, 11, 8, and 2 Pt/PeDIRs had similar expression patterns in roots, stems, and leaves, respectively. The result revealed gene expression diversity among tissues and species. In pepper, the expression of most CaDIR genes was lowest in the roots [47]. The tissue expression in poplar differs from the CaDIR genes of pepper but is consistent with the DIR genes from Brassica rapa and I. indigotica [28,35]. GhDIR3 and GhDIR4 are important for the coupling of galactosyl and contribute to producing (+) gossypol [36]. Based on the analysis of the evolutionary tree, GhDIR3 and GhDIR4 are closely related to PtDIR28-30, 32, and 40, but Pt/PeDIR29, 30, and 40 were not detected in either poplar, and the expression of Pt/PeDIR28 was very low in several tissues. Only Pt/PeDIR32 was highly expressed in various tissues. Based on these results, we inferred that Pt/PeDIR32 has a special function in poplar. In Arabidopsis, AtDIR10, which is highly expressed in roots, is essential for the correct formation of the Casparian strips in the roots [63]. The related genes in poplar are Pt/PeDIR27 and Pt/PeDIR33. Pt/PeDIR33 and PtDIR27 were also specifically expressed in roots, indicating that Pt/PeDIR33 and PtDIR27 also play an important role in root development. Lignification of vegetative organs is essential for healthy plant growth, especially for vascular plants [20]. Previous studies have shown that DIR genes can contribute to the lignification of plant tissues. For example, the seed coat of Arabidopsis [64], the pod wall of soybean [65], the hypocotyl of hemp [66], the seed of flax [53], and the cell stone of pear [48] contain traces of the lignification function of the DIR genes. The DIR genes of P. trichocarpa and 'Nanlin 895' were diversely expressed, indicating the probable function of Pt/PeDIRs in lignification during tissue development or biological and abiotic stresses.
Studies and analyses of plant disease resistance genes have always been essential [67,68]. Poplar is a perennial, tall, woody plant and is more susceptible to pathogen stress than common herbs in both time and space [5,69]. Previous studies have discussed the role of DIR genes in disease resistance in cotton [18], pepper [47], soybean [17], and wheat [16]. In 'Nanlin 895', 19 PeDIRs (7, 10, and 2 PeDIRs belonging to DIR-a, DIR-b/d, and DIR-e, respectively) were able to respond to M. brunnea stress, showing expression levels more than two-fold higher than the treatment at d 0. The M. brunnea-responsive PeDIRs were mainly distributed across the DIR-a and DIR-b/d subfamilies. It is likely that the DIR-e subfamily has other functions. Most PeDIRs showed no significant changes at d 0.5. This result is similar to that of Yuan's study concerning the response of other genes to M. brunnea, which indicated that the gene expression profiles showed slightly increased expression at the early stage, within 12 h after inoculation [5]. Most PeDIR responses to the pathogen occurred rapidly at d 1-4 following inoculation and then remained stable at d 5 and 6. In V. vinifera infected with E. necator, analysis of the coniferyl alcohol content changes at different stages led Borges et al. to hypothesize that low DIR expression in the early stages might initially contribute to lignin biosynthesis, and then high DIR gene expression at later stages could increase lignan biosynthesis to enhance antimicrobial activities of plants [33]. It is possible that 'Nanlin 895' also has a similar disease-resistance pattern. Plant defense responses to pathogens are complex. Several important signaling molecules are involved in disease resistance regulation, including SA, JA, ABA, and ETH [6,10,70]. Usually, the plant genes can respond rapidly following a variety of phytohormone treatments. The plant hormone signal transduction network is a key regulatory system for the interaction between plants and pathogens [71,72]. In 'Nanlin 895' we analyzed the responses of 19 PeDIRs to phytohormones, and those responses were complex. Previous studies have shown that under M. brunnea induction, PdLOX1 and PdLOX2 genes in poplar can respond strongly [4]. The expression of PdLOX1 and PdLOX2 is downregulated under SA stress but upregulated under MeJA stress [4]. It is speculated that PdLOX1 and PdLOX2 genes may confer disease resistance mainly through the MeJA signaling pathway. In addition, other research also showed that the AOX gene could respond to M. brunnea stress. The expression of the AOX gene was downregulated under SA stress but upregulated under MeJA and ETH stresses [6]. However, other studies showed that WRKY genes could respond to stress and were upregulated in response to SA stress [9]. Under M. brunnea induction, poplar PPR gene expression was also upregulated under SA and MeJA [73]. We showed that the plant hormone signaling pathways are not independent, and complex crosstalk takes place. These results suggest that PeDIRs may play important roles in disease resistance in poplar. Further research is needed on the specific mechanisms of disease resistance and their association with various hormone pathways.

Conclusions
We identified 40 PtDIRs from the P. trichocarpa genome. They were characterized and further classified into three subfamilies of DIR-a, DIR-b/d, and DIR-e, and the motifs and protein sequences were conserved within each subgroup. Gene duplication and calculation of Ka/Ks values, alongside synteny analysis of intra-and interspecific collinearity, provided an insight in the evolutionary history of the PtDIR genes. The tertiary structures of the PtDIR proteins from the DIR-a subfamily showed that PtDIR7 and 15 could possibly catalyze the formation of (−)-pinoresinol, while PtDIR8, 9, 10, 14, and 16 could possibly catalyze the formation of (+)-pinoresinol. The cis-acting elements of PtDIRs were involved in the response to biotic stress, abiotic stress, and various phytohormones, including ABA, SA, MeJA, and ETH, implying that PtDIRs might be involved in stress resistance. The expression patterns seen in root, stem, young leaf, and mature leaf tissues in P. trichocarpa and 'Nanlin 895' revealed the diversity of DIR gene expression. The expression patterns of the PeDIRs in response to M. brunnea and various phytohormones (such as ABA, SA, MeJA, and ETH) confirmed the disease resistance roles of the DIR genes and the complex phytohormone signaling pathway. These results provide valuable evidence for better understanding of the biological role of DIR genes in poplar.

Supplementary Materials:
The following are available online at https://www.mdpi.com/article/10 .3390/f12040507/s1, Data S1: Text file of the alignment corresponding to the phylogenetic tree in Figure 1, Table S1: List of primers for the RT-qPCR analyses, Table S2: Sequences of 10 predicted motifs of the PtDIR proteins, Table S3: Ka/Ks analysis of PtDIR gene pair duplications, Table S4: Transcription factors predicted for the binding promotors of the PtDIR genes.

Conflicts of Interest:
The authors declare no conflict of interest.