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Article

Genome-Wide Identification, Evolution, and Expression Analysis of the Dirigent Gene Family in Cassava (Manihot esculenta Crantz)

by
Mingchao Li
1,2,†,
Kai Luo
1,2,†,
Wenke Zhang
1,2,
Man Liu
1,2,
Yunfei Zhang
3,
Huling Huang
1,2,
Yinhua Chen
1,2,
Shugao Fan
3,* and
Rui Zhang
1,2,*
1
School of Breeding and Multiplication, Sanya Institute of Breeding and Multiplication, Hainan University, Sanya 572025, China
2
School of Tropical Agriculture and Forestry, Hainan University, Haikou 570228, China
3
School of Resources and Environmental Engineering, Ludong University, Yantai 264025, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2024, 14(8), 1758; https://doi.org/10.3390/agronomy14081758
Submission received: 8 July 2024 / Revised: 3 August 2024 / Accepted: 8 August 2024 / Published: 11 August 2024
(This article belongs to the Special Issue Responses of Forage Crops to Environmental Stresses and Stress Alleviating Strategies)
Browse Figures
Versions Notes

Abstract

:
Dirigent (DIR) genes play a pivotal role in plant development and stress adaptation. Manihot esculenta Crantz, commonly known as cassava, is a drought-resistant plant thriving in tropical and subtropical areas. It is extensively utilized for starch production, bioethanol, and animal feed. However, a comprehensive analysis of the DIR family genes remains unexplored in cassava, a crucial cash and forage crop in tropical and subtropical regions. In this study, we characterize a total of 26 cassava DIRs (MeDIRs) within the cassava genome, revealing their uneven distribution across 13 of the 18 chromosomes. Phylogenetic analysis classified these genes into four subfamilies: DIR-a, DIR-b/d, DIR-c, and DIR-e. Comparative synteny analysis with cassava and seven other plant species (Arabidopsis (Arabidopsis thaliana), poplar (Populus trichocarpa), soybean (Glycine max), tomato (Solanum lycopersicum), rice (Oryza sativa), maize (Zea mays), and wheat (Triticum aestivum)) provided insights into their likely evolution. We also predict protein interaction networks and identify cis-acting elements, elucidating the functional differences in MeDIR genes. Notably, MeDIR genes exhibited specific expression patterns across different tissues and in response to various abiotic and biotic stressors, such as pathogenic bacteria, cadmium chloride (CdCl2), and atrazine. Further validation through quantitative real-time PCR (qRT-PCR) confirmed the response of DIR genes to osmotic and salt stress. These findings offer a comprehensive resource for understanding the characteristics and biological functions of MeDIR genes in cassava, enhancing our knowledge of plant stress adaptation mechanisms.

1. Introduction

Plants respond to environmental challenges by activating a variety of genes, including those encoding dirigent proteins (DIR) [1]. The role of DIR proteins was first discovered in Border Forsythia (Forsythia × intermedia) in 1997, where they were found to guide the stereoselective coupling of coniferyl alcohol (CA) radicals, leading to the production of (+)-pinoresinol, a type of lignan [2]. Pinoresinol can be converted into other lignans, such as piperitol, laciresinol, and secoisolaresinol, which have been shown to play significant roles in plant defense against pathogens [3,4]. DIR proteins are also implicated in the formation of lignin, with the dirigent protein model hypothesizing their regulation over the formation of specific chemical bonds during monolignol polymerization to create lignin polymers [5,6,7,8]. Additionally, techniques such as in situ mRNA hybridization and immunolabeling have revealed that DIRs related to lignin synthesis are primarily localized in the secondary cell wall and other lignification tissues [5,9]. This indicates that DIR proteins not only catalyze the polymerization of monolignol (coniferyl alcohol) to form lignans but also share a partial biosynthetic pathway with lignan and lignin. Lignans can be transported to the plant cytoderm, where they are converted into lignin. DIR proteins are part of a relatively conserved multi-gene family, characterized by a significant conserved domain that constitutes a large portion of the protein. Usually, the DIR genes do not contain introns [10]. Ralph et al. [1] reported that DIR family genes can be classified into seven groups, designated as DIR-a to DIR-g, based on their evolutionary relationships. The expansion of DIR proteins has led to the identification of two additional subfamilies, DIR-f and DIR-g, with the DIR-b and DIR-d subfamilies being clustered together [11]. Biochemical experiments have demonstrated that the DIR-a subfamily members can direct the polymerization of monolignols into their proper 3D structure, while the biological roles of members of other DIR subfamily remain unknown. As a result, subfamilies other than DIR-a are often referred to as DIR-like [2,12,13,14]. Recently, the DIR gene family has been characterized on a genome-wide scale in several plant species, including pepper (Capsicum annuum) [15], rice [16,17], and potato (Solanum tuberosum) [18].
The functions of numerous plant DIR proteins have been elucidated. The DIR-a subfamily members are involved in the synthesis of pinoresinol [1,10]. Several DIRb/d subfamily members are involved in aromatic diterpenoid biosynthesis [19,20] and pterocarpan biosynthesis [21,22], while the DIR-e subfamily is believed to play a role in the formation of Casparian band lignin [23]. Additionally, DIR genes have been implicated in plant responses to abiotic and biotic stresses. For instance, transcripts of BhDIR1 have been observed to respond to various exogenous hormones and abiotic stresses, such as ethylene glycol tetraacetic acid (EGTA), abscisic acid (ABA), dehydration, temperature, calcium chloride, and hydrogen peroxide stresses [24]. The loss of function of CaDIR7 in pepper weakened plant defense against both mannitol-induced and sodium chloride (NaCl) stress [15]. The overexpression of OsDIR55 improves the development of root apoplastic diffusion barriers and enhances salt tolerance by restricting the rise of Na+ concentrations and the Na+/K+ ratio [25]. Among the 37 DIR and DIR-like genes of mungbean (Vigna radiata), 24 genes are up-regulated by salinity [26]. Lignin acts as a non-degradable mechanical barrier, making the host plant less susceptible to many microorganisms [27]. In canola, the constitutive expression of the DIR gene observed increased resistance to a broad spectrum of fungal pathogens [28]. The overexpression of GHDIR1 in cotton (Nicotiana tabacum) conferred resistance to the spread of Verticillium dahlia [29]. Similar results were found in wheat [14], soybean [30], peppers [15], and flax (Linum usitatissimum) [10].
Cassava, a vital staple crop in tropical and subtropical regions, serves as a primary food source for over 700 million people worldwide [31]. Its foliage boasts a high protein content (ranging from 16.41% to 22.68%), along with essential minerals and gross energy [32,33]. In many countries, cassava foliage is utilized as animal feed, showing promise in enhancing livestock production [34,35]. Notably, cassava foliage harvests exhibit seasonality, with greater biomass availability during summer or rainy seasons [35]. Despite its significance as a cash and forage crop, our understanding of DIR family members in cassava remains limited. Given that DIR genes are involved in regulating various important physiological processes, it is highly important to systematically investigate DIR family members in cassava. With the release of the whole-genome sequence of cassava [32], we have an opportunity to systematically investigate the evolutionary traits, organization, and expression profiles of the DIR gene family members in cassava at the genome-wide level. In this study, a total of 26 cassava DIR (MeDIR) genes were identified from the genome of cassava. Additionally, we conducted a comprehensive bioinformatic analysis of the MeDIR genes. We also performed global expression analyses to identify the involvement of specific MeDIR genes in various tissues and under different stress conditions. Moreover, the expression patterns in cassava in response to osmotic (polyethylene glycol, PEG) and salt (NaCl solution) stressors were examined using qRT-PCR. This study offers valuable insights into the evolutionary history and functional characterization of MeDIR genes in cassava.

2. Materials and Methods

2.1. Identification and General Characterization Analysis of MeDIR Members in Cassava

To identify DIR family members in cassava, we first obtained the corresponding genomic DNA sequences, coding DNA sequences (CDs), and other related sequences from EnsemblPlants (European Molecular Biology Laboratory’s European Bioinformatics Institute, Cambridge, London, UK) (http://plants.ensembl.org/index.html, accessed on 1 April 2024). We employed the Hidden Markov Model (HMM) profile for DIR-specific domains (PF03018), as defined by the Pfam database (http://pfam.xfam.org/family, accessed on 1 April 2024), as queries [36]. The queries were used to search against the cassava proteome database using HMM3.4 software, setting an E-value of < 0.001 [37]. Further, DIR protein sequences from Arabidopsis [11] and rice [16], downloaded from Uniprot (https://www.uniprot.org/, accessed on 1 April 2024, EMBL-EBI, PIR, and the SIB Swiss Institute of Bioinformatics, April 2024) and Phytozome v13 (https://phytozome-next.jgi.doe.gov/, accessed on 3 April 2024, Energy’s Joint Genome Institute), respectively, served as queries in a BLAST search for cassava DIR proteins using TBtools v2.096 software (South China Agricultural University) [38]. Candidate proteins were retained for further analysis if they contained known conserved domains via the Conserved Domain Database (CDD) (https://www.ncbi.nlm.nih.gov/cdd/, accessed on 3 April 2024, National Center for Biotechnology Information of the National Library of Medicine, National Institutes of Health) [39] and InterProScan (https://www.ebi.ac.uk/interpro/, accessed on 3 April 2024 EMBL-EBI) [40]. This method was similarly applied to identify DIR protein sequences in poplar. The physicochemical properties of the MeDIR proteins were estimated using the ExPASy tool (https://web.expasy.org/protparam/, accessed on 6 April 2024 SIB Swiss Institute of Bioinformatics) [41]. Furthermore, the subcellular localization of MeDIR protein sequences was predicted using the WoLF PSORT online tool (https://wolfpsort.hgc.jp/, accessed on 9 April 2024, developers: Paul HORTON and Keun-Joon PARK at CBRC, Takeshi OBAYASHI, and Kenta NAKAI) [42].

2.2. Sequence Analysis and Structural Characterization

Gene structures were analyzed using the Gene Structure Display Server (GSDS: http://gsds.cbi.pku.edu.cn, accessed on 10 April 2024) [43]. The exon/intron structure was determined by comparing the genomic DNA and CDS sequences of MeDIR genes. The MEME online software v5.5.6 (https://meme-suite.org/meme/, accessed on 10 April 2024, University of Nevada, Reno, NV, USA) was utilized to analyze the protein sequences with the following parameters: the maximum number of motifs was set to ten, with all others settings at default. Identified motifs were annotated according to InterProScan (http://www.ebi.ac.uk/Tools/pfa/iprscan/, accessed on 10 April 2024, EMBL-EBI). Sequence logos for MeDIR were created using the WebLogo website (http://weblogo.berkeley.edu/logo.cgi, accessed on 10 April 2024, Computational Genomics Research Group, Department of Plant and Microbial Biology, University of California, Berkeley, CA, USA) [44]. The Gene Structure View module in TBtools v2.096 was used to visualize the motifs and domains of MeDIR genes [38].

2.3. Phylogenetic Analysis

To investigate the phylogenetic relationships of MeDIR proteins, the protein sequences of DIR proteins in cassava, Arabidopsis, rice, and poplar were aligned using Clustal W software v2.1 (Kyoto University Bioinformatics Center, Kyoto, Japan) under default parameters [45]. A phylogenetic tree was generated using the neighbor-joining (NJ) method with 1000 bootstrap replications in MEGA v7.1 software and visualized with the EvolView v3 online tool (https://www.evolgenius.info/, accessed on 15 April 2024) [46].

2.4. Secondary Structure Prediction and Three-Dimensional (3D) Model Construction

The secondary structure of the deduced polypeptides was predicted using SOPMA (https://npsa.lyon.inserm.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_sopma.html, accessed on 15 April 2024, Pôle Rhône-Alpes de Bio-Informatique). The tertiary structure of MeDIR proteins was predicted using Phyre2 with intensive modeling. Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index, accessed on 15 April 2024, Structural Bioinformatics Group, Imperial College, London, UK) was also employed to search for MeDIR homologs using the amino acid sequences as target sequences.

2.5. Chromosomal Localization and Gene Duplication

MeDIR genes were mapped onto cassava chromosomes by identifying their position in genome version 6.1 from the Phytozome v13 database (https://phytozome.jgi.doe.gov/, 18 April 2024) and visualized by Tbtools v2.096 [38]. Collinear blocks were evaluated by MCScan2 (https://github.com/wyp1125/MCScanX, accessed on 18 April 2024), considering alignments with an E-value ≤ 1 × 10−10 as significant matches [47]. The syntenic relationship between MeDIR genes was determined and displayed using TBtools v2.096 software [38]. The Ka/Ks (non-synonymous substitution rate/synonymous substitution rate) values were calculated to analyze the selection pressure and selection mode after identifying duplicate genes.

2.6. Analysis of Promoter Regions

The 2000 bp upstream DNA sequence of MeDIRs, considered the promoter region, was retrieved from the Phytozome v13 database. The PlantCARE online program (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 20 April 2024, joint lab at University of Pretoria) [48] was used to predict cis-regulatory elements in the promoter regions of MeDIR genes, and TBtools v2.096 was employed to visualize the results [38].

2.7. Protein Interaction Network Diagram and GO Annotation

Interologues of Arabidopsis were used to predict the protein–protein interaction network for MeDIRs using the STRING v12.0 protein interaction database (http://string-db.org/, accessed on 25 April 2024, EMBL-EBI, CPR, and the SIB Swiss Institute of Bioinformatics), with the confidence parameter set at 0.15 [49,50]. GO enrichment analysis, including the molecular function (MF), biological process (BP), and cellular component (CC), was performed using Pannzer2 (http://ekhidna2.biocenter.helsinki.fi/sanspanz/, accessed on 26 April 2024, University of Helsinki Institute of Life Sciences, Helsinki, Finland). All Amino acid sequences were submitted in fasta format. GO terms were annotated using Bioinformatics (http://www.bioinformatics.com.cn/, accessed on 28 April 2024, Chinese Academy of Sciences, Beijing, China, Shanghai Jiaotong University, Shanghai, China, Xiangya School of Medicine, Changsha).

2.8. RNA-Seq Data Analysis

The transcriptome data retrieved from the NCBI database (accession numbers: PRJNA324539, GEO dataset: GSE82279, and cassava variety: TME204) [51] were used to analyze the global expression of MeDIR genes in various cassava tissues. Another set of data about cassava bacterial blight (Xanthomonas phaseoli pv. manihotis str. Xam668) (accession numbers: PRJNA163523, strain origin: Indonesia) [52] was used to investigate the expression profiles of MeDIR genes in response to pathogen invasion. Transcriptome data for CdCl2 [accession numbers: PRJNA1128429, cassava variety: South China 9 (SC9)] and atrazine stress (accession numbers: PRJNA1127687, cassava variety: SC9) were obtained from our group. Subsequently, Log2 Fold Change (Log2 FC) values were calculated to evaluate gene expression using TBtools v2.096 software [38].

2.9. Plant Materials and Osmotic and Salt Stress Treatments

SC9, a typical cassava cultivar derived from the National Cassava Germplasm Nursery, was used. The SC9 stem segments were planted in 1/2 strength Hoagland nutrient solution medium and grown in a greenhouse at Hainan University (Haikou, Hainan, China) under controlled conditions for about 40 days and watered every three days before being subjected to osmotic and salt stress treatments. Seedlings were treated with 1/2 strength Hoagland nutrient solution medium with 20% polyethylene glycol (PEG) 6000 and 400 mM NaCl for 4, 12, and 24 h [53,54], respectively, for the osmotic and salt stress treatments. Untreated seedlings (0 h) served as controls. Samples were immediately frozen in liquid nitrogen for RNA isolation.

2.10. RNA Isolation and qRT-PCR

RNA was isolated from leaves using the RNAprep Pure Plan Plus Kit (TIANGEN Biotech Co., Ltd., Beijing, China) according to the manufacturer’s instructions. Total RNA (1 µg) was used for first-strand cDNA synthesis with a reverse transcriptase kit (M1631, Thermo Fisher Scientific, Waltham, MA, USA). The elongation factor 1 α (EF1α) gene served as an internal control. Primers listed in Table S1 were utilized. Real-time PCR was conducted on a 7500 Real-Time PCR System (Thermo Fisher Scientific) with a 20 µL total reaction volume, containing 2 µL cDNA template, 1 µL forward primer, 1 µL reverse primer, 10 µL qPCR Master Mix, and 6 µL sterilized ddH2O. The amplification protocol was set as follows: 95 °C for 30 s for 40 cycles, 95 °C for 5 s, 55 °C for 30 s, 72 °C for 30 s, and a final extension at 72 °C for 10 min [55]. Each reaction was performed in triplicate. Relative gene expression levels were calculated using the 2−ΔΔCT method [56]. The significance analysis was conducted by using the ANOVA in IBM SPSS Statistics v26 software (International Business Machines Corporation, Armonk, NY, USA). Multiple tests were performed using the Duncan method through variance analysis of different comparison groups.

3. Results

3.1. Identification and Characterization of MeDIRs in Cassava Genome

A total of 26 MeDIR proteins in cassava were identified through stringent bioinformatics analyses (Table S2), with all subsequently undergoing domain analysis using CDD and InterProScan. This confirmed the presence of the conversed DIR domain (PF03018). Based on their chromosome locations, these cassava DIR genes were renamed from MeDIR1 to MeDIR26. The ExPASy online tool facilitated the analysis of the physical and chemical characteristics of the MeDIR proteins (Table S2).
The CDs of the MeDIRs ranged from 399 bp to 1119 bp, encoding peptides of 132 to 372 amino acids. The MW of these peptides varied from 14.44 to 38.48 kDa, with pI ranging from 4.25 to 9.84. Additionally, the GRAVY index of MeDIR proteins varied from −0.176 to 0.289, the instability index ranged from 19.31 to 43.13, and the fatty acid index ranged from 66.72 to 106.49. The subcellular localizations of MeDIRs, predicted using the WoLF PSORT web service, revealed that 13 MeDIR proteins were located in the chloroplast, 5 in the extracellular space, 4 in the cytoskeleton, 2 in the endoplasmic reticulum, 1 in the nucleolus, and 1 in the vacuole (Table S2). Pairwise sequence similarities among the predicted amino acids of the 26 MeDIRs showed a variation in identity from as low as 15.88% (MeDIR3 vs. MeDIR13) to as high as 100% (MeDIR2 vs. MeDIR26) (Table S3). Four pairs of genes (MeDIR2 vs. MeDIR26, MeDIR2 vs. MeDIR6, MeDIR6 vs. MeDIR26, MeDIR19 vs. MeDIR15), sharing an amino acid identity greater than 91%, may represent alleles within the species.

3.2. Secondary and Tertiary Structures and Homologs of MeDIRs

The SOPMA online program was utilized to predict the structural features of 26 MeDIR proteins, aiming to better understand their molecular functions (Table S4). The analysis revealed that the random coil configuration constituted a significant portion of the MeDIR proteins’ secondary structures, ranging from 35.64% to 63.44%. The fraction of the beta turn was less than 9% of the overall structure. The alpha helix and extended strand structure comprised 11.11–25.26% and 22.04–40.15% of the secondary structures, respectively.
The predicted three-dimensional structures for the 26 MeDIR proteins are illustrated in Figure 1. Each query sequence matched four top-scoring proteins. Notably, the hypothetical protein c4revB identified as a disease resistance response protein 206 (drr206) and three other proteins belonging to the allene oxide cyclase-like protein (AOC) family were found. The c4revB shared 25–72% sequence identity with MeDIRs and was predicted as a homolog of DIR with 100% probability. The AOC barrel-like protein d2brja1 exhibited 18–25% sequence identity with MeDIRs, with a probability of being a DIR homolog of 97.3–98.3%. The probabilities for the other two proteins, d1zvca1 and c4h69A, were estimated at 96.8–98.3% and 96.7–98.2%, respectively (Table S5).

3.3. Phylogenetic Relationship Analysis

A phylogenetic analysis was conducted to investigate the relationships and evolutionary history within the MeDIR family, incorporating 139 DIR and DIR-like gene protein sequences from Arabidopsis, rice, and poplar as reference proteins. Based on the classification of Ralph et al. [11], the phylogenetic tree was divided into four distinct subfamilies: DIR-a, DIR-b/d, DIR-c, and DIR-e. The DIR-a subfamily, an ancient and conserved clade, included 4 MeDIRs, 5 AtDIRs (Arabidopsis DIRs), 7 OsDIRs (rice DIRs), and 11 PtDIRs (poplar DIRs). The DIR-b/d subfamily was the largest, comprising 15 MeDIRs, 14 AtDIRs, 11 OsDIRs, and 16 PtDIRs, indicating considerable expansion in cassava. The DIR-c subfamily contained only 13 OsDIRs, suggesting a monocotyledon-specific expansion. As previously reported, the DIR-a family members are capable of directing the vitro stereoselective formation of lignans, so the members of this group are known as dirigent genes [11]. Meanwhile, the members of the DIR-b/d, DIR-c, DIR-d, DIR-e, and DIR-g subfamilies are referred to as dirigent-like genes [1,11]. Thus, MeDIR1, MeDIR3, MeDIR4, and MeDIR5 are classified as dirigent genes, whereas the remaining 22 MeDIR genes are considered DIR-like genes. A separate phylogenetic analysis exclusive to MeDIR genes demonstrated that genes within each group clustered together, reinforcing their categorized relationships (Figure 2).

3.4. Chromosomal Locations, Duplication Events, and Synteny Analysis

To investigate the genomic distribution of the cassava DIR genes, we mapped their chromosomal locations on the cassava plant genome’s pseudochromosome assembly to identify duplicated blocks. The analysis revealed that the 26 MeDIR genes are unevenly distributed across 13 of cassava’s 18 chromosomes (Table S2; Figure 3A). Among these chromosomes, chromosome 2 harbors the highest number of MeDIR genes, totaling four members. Chromosomes 4, 8, and 15 each contain three genes, whereas chromosomes 1, 3, 7, and 9 have two genes each. The remaining chromosomes 6, 10, 11, 13, and 17 each possess one MeDIR gene.
Tandem and segmental duplications within the cassava genome were explored using MCScanX 2 software, identifying seven segmental duplication pairs among the MeDIR genes (Figure 3B). These findings imply that gene duplication events, particularly segmental duplication events, have significantly contributed to the evolution of the MeDIR genes. To further examine the selective pressure acting on these duplicated gene pairs, we calculated the Ka and Ks substitution ratios (Ka/Ks). The Ka/Ks ratios for cassava MeDIR gene pairs were all below 1 (Table S6), suggesting that they evolved under the purifying selection. A comparative analysis with other species, including both dicots (Arabidopsis, soybean, tomato, and poplar) and monocots (rice, wheat, and maize), provides a valuable reference for understanding genetic relationships and gene functions across species. This analysis identified 14, 19, 16, 25, 4, 6, and 3 pairs of homologous genes in Arabidopsis, soybean, tomato, poplar, rice, wheat, and maize, respectively (Figure 4).

3.5. Motifs, Conserved Domains, and Gene Structure in MeDIRs

Phylogenetic analysis, restricted to MeDIR family members from the cassava genome (Figure 5A), unveiled a structure similar to the one derived from comparing DIR sequences across four plant species shown in Figure 2. Utilizing the MEME online program, we identified ten conserved motifs shared by the MeDIR proteins, thereby highlighting their diversity within cassava (Figure 5B). The details of the putative motifs are shown in Figure S1. Notably, motif 2, found in all MeDIR proteins, likely represents the conserved DIR domain (Figure 5C). For instance, motif 8 only exists in MeDIR1, MeDIR3, MeDIR4, and MeDIR5, and motif 9 exists only in MeDIR13, MeDIR14, and MeDIR20, suggesting functional diversification among MeDIR proteins. Additionally, proteins within the same group exhibited similar motifs, illustrating divergence across different groups. Exon–intron composition analysis revealed that 24 of the 26 identified MeDIR genes comprise one exon without any introns, which is consistent with the classical DIR gene structure [16]. The exception is MeDIR3 and MeDIR17, which had one and two exons, respectively (Figure 5D).

3.6. Analysis of Cis-Elements in the Promoters of MeDIR Family Genes

To determine the specific types and distribution of cis-acting elements present in the promoters of MeDIR family genes, an analysis of promoter cis-acting elements was conducted using PlantCARE. From the predicted results, a total of 29 representative cis-acting elements were identified within the 2.0 kb upstream region of the 26 MeDIR genes, highlighting an uneven distribution (Figure 6A, Table S7). Among these, light-responsive regulatory elements were predominant, with eight different types detected across nearly all MeDIR promoters (Figure 6A, Table S7). Hormone response cis-elements spanned seven categories, encompassing responses to auxin (AuxRR-core and TGA-element), gibberellin (P-box and TATC-box), abscisic acid (ABRE), methyl jasmonate (CGTCA-motif), and salicylic acid (TCA-element). Additionally, six stress-responsive elements (TC-rich repeats, W box, LTR, ARE, WUN-motif, and MBS) were identified (Figure 6A). Each of the 26 MeDIR genes featured at least one stress-responsive element, and all except MeDIR9 and MeDIR23 harbored at least one hormone-responsive element. MeDIR15 is expected to have the highest number of cis-elements (Figure 6B). These results indicate that the expanded MeDIRs could be significantly involved in hormone signaling as well as the mechanism of stress adaptation in cassava. Furthermore, eight elements associated with tissue-specific expression were identified in MeDIR promoters, including RY-element, CAT-box, GCN4_motif, HD-Zip 1, O2-site, MBSI, GC-motif, and MSA-like, hinting at their involvement in plant growth and development. This comprehensive analysis of MeDIR gene family promoters significantly enriches our understanding of their intricate biological functions.

3.7. Protein–Protein Interaction Network and GO Annotation Analysis of MeDIRs

Utilizing the STRING database and insights from studies on AtDIR and gene homology, we constructed a protein–protein interaction network for MeDIR proteins (Figure 7). Our analysis revealed 21 DIR functional molecules (DIR1-2, DIR4, DIR5, DIR6, DIR8, DIR9, DIR10, DIR11, DIR12, DIR13, DIR14, DIR16, DIR17, DIR18, DIR19, DIR20, DIR21, DIR22, DIR23, DIR24, and DIR25) and two putative interaction proteins (MXA21.17 and NAC057), statistically linked to 26 MeDIR proteins (Figure 7).
GO functional analysis delineated the BP, MF, and CC characteristics of the MeDIR proteins (Table S8). This analysis revealed their putative molecular and biological functions. In the “biological process” category, most proteins were classified as involved in the phenylpropanoid biosynthetic process (GO:0009699), and only one gene was classified as involved in the jasmonic acid biosynthetic process (GO:0009695). In the “molecular function” category, only one gene was classified as having allene-oxide cyclase activity (GO:0046423). Two GO terms related to cellular components, namely apoplast (GO:0048046) and membrane (GO:0016020), were notably enriched. These results indicate that MeDIR proteins are involved in diverse aspects of cellular metabolism.

3.8. Expression of MeDIR Genes in Different Tissues

Employing publicly available cassava RNA-seq data from Wilson et al. [51], we assessed the expression profiles of MeDIR genes across eleven distinct tissues: friable embryogenic callus (FEC), organized embryogenic structures (OES), fibrous root (FR), lateral buds, shoot apical meristem (SAM), petiole, root apical meristem (RAM), leaf, stem, midvein, and storage root (SR). Hierarchical clustering analysis, encompassing all 26 MeDIR genes (Figure 8), unveiled diverse expression patterns across cassava tissues. Notably, MeDIR7 and MeDIR5 were more prominently expressed across nearly all tissues, hinting at their important roles in cassava development. Certain MeDIR genes displayed tissue-specific expression patterns. MeDIR2, MeDIR12, and MeDIR24 were most expressed in fibrous roots (FR). MeDIR1, MeDIR4, MeDIR18, MeDIR23, and MeDIR24 showed elevated levels in RAM. MeDIR1 was most expressed in the stem and petiole. The expression levels of MeDIR4 and MeDIR19 were the highest in OES and SAM, respectively. These patterns of high expression in specific tissues suggest crucial roles in tissue development or functionality (Figure 8).

3.9. Expression Profile of MeDIR Genes in Response to Pathogen Infextion, CdCl2, and Atrazine Treatments

According to the cassava RNA-seq data from our group, we investigated the expression patterns of MeDIR genes following Xam668 infection, CdCl2 exposure, and atrazine treatment. All 26 MeDIR genes were included in the hierarchical clustering analysis. Our results demonstrated that nearly all DIR genes were induced by Xam668 infection (Figure S1). Specifically, the expression of MeDIR1, MeDIR12, MeDIR18, and MeDIR24 increased significantly at 8 h post-infection (hpi). MeDIR9 and MeDIR18 were markedly inhibited at 24 hpi. Additionally, MeDIR16 exhibited reduced expression at 8 and 24 hpi, followed by a substantial increase at 50 hpi under Xam668 infection. The expression levels of MeDIR1, MeDIR4, MeDIR6, MeDIR7, and MeDIR11 increased significantly at 24 hpi. Notably, certain cassava MeDIR family genes, such as MeDIR1 and MeDIR10, were consistently up-regulated following infection, indicating that these genes may play key roles in regulating common signaling pathways in response to Xam668 infection.
Under atrazine stress, MeDIR family genes exhibited generally higher expression in leaves compared to roots. Seventeen of them (MeDIR1, MeDIR2, MeDIR6, MeDIR7, MeDIR8, MeDIR10, MeDIR11, MeDIR14, MeDIR15, MeDIR16, MeDIR17, MeDIR18, MeDIR19, MeDIR20, MeDIR22, MeDIR23, and MeDIR26) exhibited differential expression patterns when the leaves were stressed by atrazine for 7 days, with MeDIR2 displaying the most significant expression trend (Figure S2). After 14 days of atrazine stress, 12 MeDIR genes were down-regulated, with MeDIR2, MeDIR6, MeDIR10, MeDIR18, and MeDIR22 showing consistent down-regulation at both time points. MeDIR3, MeDIR9, and MeDIR25 were not expressed at any time point under atrazine stress. Most MeDIR genes were down-regulated in the root. In total, twenty-three DIR genes exhibited differential expression patterns in cassava roots following atrazine stress, with 17 genes down-regulated after 7 days and 19 genes down-regulated after 14 days. MeDIR12 was significantly up-regulated after 7 days of stress, whereas MeDIR15 was most significantly down-regulated after 14 days of stress.
Following CdCl2 treatment, the expression of MeDIR1, MeDIR11, and MeDIR15 in leaves increased significantly under low-concentration (10 µmol/L CdCl2, T10) CdCl2 stress but was promptly reduced under high-concentration (100 µmol/L CdCl2, T100) CdCl2 stress (Figure S3). The expression levels of MeDIR20 and MeDIR24 in T100 were significantly higher than in the control and T10 treatments. MeDIR8, MeDIR12, MeDIR22, and MeDIR24 in leaves exhibited an initial decrease followed by an increase. In leaves, MeDIR1, MeDIR11, MeDIR15, MeDIR17, and MeDIR19 exhibited a comparable expression pattern, characterized by a sharp increase following T10, followed by a decline after T100. Under T10 CdCl2 stress, three DIR genes (MeDIR1, MeDIR11, and MeDIR15) were differentially expressed and up-regulated. Six DIR genes (MeDIR4, MeDIR5, MeDIR9, MeDIR13, MeDIR14, and MeDIR25) were not expressed under either cadmium concentration. MeDIR24 was significantly activated under high concentrations (T100) of CdCl2.

3.10. Expression of MeDIR Genes under PEG and NaCl Stresses

To identify DIR genes in the cassava genome that respond to osmotic and salt stress, RNA was extracted from cassava seedlings’ leaves subjected to salt and osmotic stress, and the relative expression levels of 20 MeDIR family members were assessed using qRT-PCR. For 16 out of these genes, expression levels consistently increased in response to osmotic stress (Figure 9). Among them, eleven genes (MeDIR1, MeDIR2, MeDIR8, MeDIR12, MeDIR15, MeDIR17, MeDIR20, MeDIR21, MeDIR22, MeDIR24, and MeDIR26) displayed heightened expression, with MeDIR2 showing the highest level. In contrast, MeDIR3 and MeDIR25 exhibited significantly lower expression levels compared to the control. Six DIR genes (MeDIR1, MeDIR11, MeDIR20, MeDIR22, MeDIR24, and MeDIR26) were progressively suppressed, with their expression down-regulated at 24 h compared to 12 h. The expression of 14 genes (MeDIR1, MeDIR2, MeDIR4, MeDIR6, MeDIR7, MeDIR8, MeDIR14, MeDIR15, MeDIR17, MeDIR19, MeDIR20, MeDIR21, MeDIR24, and MeDIR26) reached a maximum value after 4 h of stress
In comparison to osmotic stress, the MeDIR gene family exhibited greater abundance under salt stress treatment (Figure 10). Following NaCl treatment, the expression of MeDIR1, MeDIR2, MeDIR4, MeDIR7, MeDIR11, MeDIR12, MeDIR15, MeDIR18, MeDIR20, MeDIR22, MeDIR24, and MeDIR26 increased considerably and peaked at 12 h. MeDIR7, MeDIR20, and MeDIR22 demonstrated notably higher expression levels in response to salt stress compared to other genes. The expression levels of MeDIR3, MeDIR19, MeDIR21, and MeDIR25 increased significantly and reached their maximum value at 24 h. Interestingly, some cassava DIR genes, including MeDIR2, MeDIR8, MeDIR12, MeDIR15, MeDIR21, MeDIR22, and MeDIR26, were consistently up-regulated across various stress conditions. This suggests that these cassava DIR family members may be involved in regulating common signaling pathways that manage different stress responses.

4. Discussion

DIR and DIR-like proteins are part of a widespread multi-gene family found in most terrestrial plants. They are believed to have evolved key enzymatic functions for the synthesis of lignin and lignan, crucial for plants’ adaptation from aquatic to terrestrial habitats [9,13]. In plants, the DIR gene family not only plays a pivotal role in the synthesis of structural polymers but also significantly impacts plant growth, development, stress responses, and secondary metabolic processes [10,15,28,29,30,57], making them crucial in molecular breeding for enhanced biotic and abiotic resistance. Lignin is a widespread biopolymer in plants, playing a crucial role in the structure of plant cell walls [58,59]. In cassava, the DIR gene family is significantly linked to lignin synthesis, alongside other gene families such as MePOD, MeRAV, MeCAD, and MePAL. The MePOD12 gene can mitigate excess reactive oxygen species during the initial stages of postharvest physiological deterioration (PPD) and contribute to lignin deposition through its interaction with MeCAD15, thereby postponing PPD onset [58]. Additionally, MeRAV5 interacts with both MePOD and MeCAD (including MeCAD5, MeCAD14, and MeCAD15), enhancing cassava’s drought tolerance by aiding in the removal of hydrogen peroxide and lignin production [59]. According to Yao et al. [60], the PAL gene boosts lignin synthesis in response to the two-spotted spider mite infestation, which is crucial for plant defense against piercing and sucking herbivores and offers potential genes for developing pest-resistant cassava varieties through molecular breeding. These cassava genes are vital for improving resistance to diseases, drought, and insect pests, as well as supporting plant growth. Previous studies have established the functional roles of several subtribe DIR genes, with the DIR-a subfamily being involved in pinoresinol biosynthesis [1,10]. Some members of the DIR-b/d subfamily are associated with the production of aromatic diterpenoids [19,20] and pterocarpan [21,22], while the DIR-e subfamily is thought to play a role in forming Casparian strip lignin [23]. However, a comprehensive genome-wide analysis of DIR family genes in cassava has not yet been conducted. Extensive characterization of DIR family genes has been conducted in various higher plants; 25 in Arabidopsis [11], 24 in pepper [15], 35 in spruce (Picea spp.) [11], 45 in alfalfa (Medicago sativa) [61], 54 in rice [16], and 54 in soybean [57]. Despite the critical economic and nutritional role of cassava as a dual-purpose crop (tuber and forage), little was known about the expression and function of the DIR gene family before our study. Using HMMER search and BLASTp methods, we identified 26 DIR proteins within the cassava genome, distributed across 13 out of 18 chromosomes These proteins vary in amino acid length from 132 a.a. (MeDIR5) to 372 a.a. (MeDIR13), with the DIR-e subfamily members exhibiting the longest sequences, and MW ranging from 14.44 (MeDIR5) to 38.48 kDa (MeDIR13). This indicates that as plants have evolved and their environments have changed over time, their divergence has led to structural variations among species [62]. Subcellular localization studies have indicated that the majority of cassava DIR proteins are located in the chloroplast, suggesting a specialized role in managing a variety of tasks [63,64]. The 26 MeDIR proteins had a pI ranging from 4.25 (MeDIR13) to 9.84 (MeDIR7) and most had pI > 7, indicating that this gene family tends to be enriched in basic amino acids.
DIR genes in cassava were clearly classified into four subfamilies: DIR-a, DIR-b/d, DIR-c, and DIR-e. This classification and the topological structure of genes align with those reported in previous studies [16,65,66], confirming the conserved nature of these subfamilies across different species. Notably, our phylogenetic analysis revealed that certain cassava DIR genes were included in the same clusters as the members from Arabidopsis, suggesting that these genes may not only share a common ancestral DIR gene but also retain similar biological functions, which are crucial for their roles in plant physiology and defense mechanisms [1]. Notably, the DIR-a and DIR-g subfamilies are found only in rice, indicating a distant relationship to cassava and suggesting limited homology between cassava and monocotyledonous plants like rice. This suggests a high degree of conservation among different plant species, implying that homologous genes may exhibit similar functions across species. For example, MeDIR1, MeDIR3, MeDIR4, and MeDIR5, which are closely related to drought, salt, osmotic, trauma, and oxidative stress response genes AtDIR5 [67], may also produce similar stress responses in cassava. Similarly, AtDIR19 and AtDIR7 in response to heat stress may also have similar functions to the cassava DIR genes belonging to the DIRb/d subfamily.
Although the physicochemical properties of the cassava DIR family, such as protein length, MW, and pI, were highly variable, the gene structure and amino acid motifs were relatively conserved. The findings indicate that the various descriptions of the DIR gene structure are interconnected, with the results highlighting the distinct characteristics of this gene family. The prediction of the tertiary structure and domain analysis of MeDIR genes together underscore their high level of conservation. Combined with the gene structure and conserved protein motif analysis of MeDIR family members, we found that members of the same subfamily have similar gene structures and conserved motif distributions among themselves, while different subfamilies have larger differences, suggesting conservation within the same subfamily and similar functions, which is consistent with the results of Zhang et al. [67] and Jia et al. [18]. The majority of cassava DIR family genes exhibit a simple genomic structure, predominantly consisting of one exon, which is consistent with the exon/intron structures of DIR genes in other diverse plants such as rice [16], potato [18], pepper [15], strawberry (Fragaria ananassa) [68], watermelon (Citrullus lanatus), cucumber (Cucumis sativus) [69] and pear (Pyrus spp.) [70], This structural simplicity suggests a possible evolutionary advantage in maintaining a streamlined gene architecture for rapid gene expression. Additionally, the expansion of the DIR gene family in cassava has been influenced by segmental duplication, reflecting a common evolutionary strategy among plants to adapt and diversify their genomic capabilities [71].
Gene duplication is a pivotal mechanism that drives the evolution and diversification of gene families across species. Our analysis of gene duplication events in the 26 MeDIR genes identified seven pairs of segmentally duplicated genes, indicating that the expansion of the cassava DIR family is primarily due to segmental duplications. These findings suggest that both segmental and tandem duplications have played a significant role in the growth of the DIR gene family in cassava. Segmental and tandem duplication has also occurred in DIR genes of other species. In the duplication analysis of CaDIRs in capsicum, ten clusters of segmental duplication and two pairs of tandem duplication events were found [15]. Similar duplication events have been reported in the DIR gene families of rice [16]. Interestingly, the Ka/Ks ratios for these gene pairs were found to be less than one, suggesting that the duplicated MeDIR genes are predominantly under purifying selection pressure after gene duplications. Similar duplication events have been noted in other plants [15,16,70,72,73,74,75]. Furthermore, synteny analysis revealed a significant number of orthologous gene pairs between cassava and four other dicots, while fewer orthologues were observed with three monocots. This difference may be due to the evolutionary divergence between monocots and dicots over extended periods of natural selection. The phylogenetic analysis also supports this finding, showing that cassava is more closely related to dicots than to monocots.
Gene promoter analysis is essential for unraveling the complex regulatory architecture that governs gene expression [74]. The presence of cis-elements in gene promoter regions plays a crucial role in regulating transcription, as these elements respond to a range of environmental and developmental signals [75,76]. In cassava MeDIR genes, cis-elements associated with light responsiveness were predominant, constituting 38% of the total identified cis-elements, followed by elements responsive to phytohormones and biotic and abiotic stresses. The functional differentiation of DIR genes has been revealed at the gene expression level in different plant tissues. It has been reported that the expression of most OsDIR genes is comparatively higher in roots, shoot apical meristem, and panicle development than in seed development and leaves, implying that OsDIR genes play an important role in the development of these tissues [16]. In pepper, CaDIRs showed the highest expression in flowers, followed by the stem, leaf, green fruit, and red fruit, with the lowest expression levels in roots [15]. Most cassava DIR genes displayed relatively higher expression levels in the fibrous root (FR) and root apical meristem (RAM) and lower expression levels in the friable embryogenic callus (FEC) and lateral bud, suggesting specific functions in diverse tissues and developmental stages. Furthermore, we found that MeDIR5 and MeDIR7 were expressed at high levels in all tissues, suggesting that these genes may play important roles in the growth process of cassava. The expression patterns of DIR genes in cassava varied across different tissues, indicating that the expression patterns of MeDIR family members exhibit tissue specificity. Gene expression profiling indicates that the MeDIR gene family may play a crucial role in regulating cassava growth and development, with MeDIR5 and MeDIR7 being particularly significant. The involvement of the DIR gene family in both biotic and abiotic stresses is well-documented across various plant species. Khan et al. [15] found that CaDIR7 plays an important role in the defense response of pepper to Phytophthora capsici infection. Ralph et al. [1] showed that spruce DIR genes play a role in both ongoing and induced phenolic defense response mechanisms against stem-boring insects. Likewise, TaDIR2 in wheat [77] and GbDIR18 in cotton [65] have been linked to resistance against fungal diseases. Moreover, the deposition of lignin is regarded as an effective physical barrier against pathogen invasion [78]. Thus, similar roles are observed in defense against pathogens. For instance, in Arabidopsis, specific DIR genes like AtDIR5 and AtDIR22 regulate pathogen resistance through the salicylic acid (SA)-signaling pathway [8]. Based on transcriptomic data, DIR gene expression was differentially expressed after Xam668 infection. Notably, MeDIR1 and MeDIR10 expression increased at all three time points, indicating its potential role in cassava’s defense response to Xam668 infection. Following low and high concentrations of CdCl2 stress, MeDIR2, MeDIR6, MeDIR22, and MeDIR26 were all down-regulated. Overall, there were significantly more differentially expressed genes in roots than in leaves under atrazine stress, while DIR genes were generally more expressed in leaves than in roots. Both MeDIR2, MeDIR6, MeDIR10, MeDIR18, and MeDIR22 were down-regulated in atrazine-treated leaves at 7 and 14 days, while MeDIR17 was consistently up-regulated. The majority of MeDIR genes exhibited down-regulation in the roots.
Previous studies have confirmed that DIR expression responds to salt and osmotic stress [8,15,79]. Research shows that mungbean DIR genes vary in their responses under these conditions [26]. Gou et al. [80] found that NaCl and PEG treatment induced ScDIR gene expression. Similarly, the transcript levels of CaDIR4, CaDIR7, and CaDIR12 were significantly altered in capsicum under NaCl or mannitol stress [15]. Gong et al. [81] found that the expression levels of SiDIR10, SiDIR19, SiDIR20, SiDIR22, SiDIR27, and SiDIR36 could be induced by NaCl treatment, suggesting their potential roles in coping with salt stress. In this study, we employed qRT-PCR to ascertain the expression levels of MeDIR genes under osmotic and salt stress conditions. Specifically, MeDIR2, MeDIR8, MeDIR12, MeDIR15, MeDIR21, MeDIR22, and MeDIR26 demonstrated consistent up-regulation, while MeDIR25 showed consistent down-regulation in response to treatments with PEG and NaCl. This indicates their roles in key signaling pathways mediating stress responses. The presence of stress-responsive and tissue-specific elements within DIR genes is likely associated with their favorable responses to biotic and abiotic stresses, as observed in gene expression profiles. In particular, most MeDIR genes contain ABRE elements, which are extensively involved in the ABA pathway in response to NaCl stress [82]. Correspondingly, MeDIR genes were found to be extensively and significantly upregulated under NaCl stress in qRT-PCR analysis. In summary, the varying expression patterns of DIR genes under biotic and abiotic stresses suggest a complex response mechanism within this gene family. Consequently, further investigation into the specific functions of these genes in relation to biotic and abiotic stress is warranted, as it could offer new insights into plant stress responses. The DIR genes exhibit positive responses to salt and osmotic stress, indicating their potential as valuable genetic resources for enhancing plant tolerance in challenging environments.

5. Conclusions

In this study, we systematically identified 26 MeDIR genes from the entire cassava genome and categorized them into six subfamilies. Members of each subfamily shared similar gene structures, with some MeDIR genes resulting from gene duplication events. Despite significant variations in the physicochemical properties of the MeDIR family, the gene structures and conserved protein sequences were highly conserved. The 26 MeDIR genes were randomly distributed across thirteen chromosomes and were subject to purifying selection. The promoter regions of MeDIR genes contained cis-acting elements associated with plant growth and development, environmental stress responses, and hormone responses. Notably, all 26 DIR genes had hormone response elements, along with tissue-specific expression elements, suggesting a crucial role for MeDIR genes in cassava growth and development as well as hormone signal transduction. Analysis of existing cassava transcriptome data revealed distinct expression profiles of DIR genes across various tissues, indicating potential divergent roles in cassava development. The varied expression patterns of the MeDIR family in different tissues suggest their involvement in responding to both abiotic and biotic stresses. Specifically, under NaCl and osmotic stress treatments, MeDIR genes were strongly upregulated, indicating a role in cassava’s adaptation to these conditions. This study provides a theoretical foundation and groundwork for future research on the functions and mechanisms of cassava DIR genes in plant growth and development.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy14081758/s1, Figure S1: The expression profiles of MeDIR genes profile cassava were infected with Xam668 for 8, 24, and 50 h. Log2 transformed FPKM value was used to create the heat map. The scale represents the relative signal intensity of FPKM values; Figure S2: The expression of MeDIR gene profile in cassava leaves and roots was observed after 7 and 14 d of atrazine stress. Control roots (RC_7d, RC_14d) and leaves (LC_7d, LC_14d). Treatment roots (RT_7d, RT_14d) and leaves (LT_7d, LT_14d). Log2 FC value was used to create the heat map. Red indicates that the gene is up-regulated, blue indicates that the gene is down-regulated, and gray indicates that the gene has no Log2 FC value in the differential comparison group; Figure S3: The expression of MeDIR gene profile of cassava was observed after 10 µM and 100uM CdCl2 stress for 48 h. 0 μM (CK), 10 μM (T10), and 100 μM (T100). Log2 FC value was used to create the heat map. Red indicates that the gene is up-regulated, blue indicates that the gene is down-regulated, and gray indicates that the gene has no Log2 FC value in the differential comparison group; Table S1: The primer was designed for qRT-PCR; Table S2: Basic information of DIR genes in cassava; Table S3: The sequence relatedness of MeDIRs; Table S4: The secondary structure of MeDIR protein sequences; Table S5: The probability and identity of homologous relationships of MeDIRs; Table S6: Segmental duplications of MeDIR genes and Ka/Ks ratio analysis; Table S7: The function and number of cis-acting regulatory elements in the promoter regions of MeDIR genes; Table S8: Functional annotation (gene ontology) of MeDIR proteins; Table S9: Detailed situation table of expression quantity values in Figure 8 and Figure S1 and Log2FC values in Figures S2 and S3.

Author Contributions

Conceptualization, R.Z. and S.F.; methodology, M.L. (Mingchao Li), K.L., W.Z., M.L. (Man Liu) and H.H.; software, M.L. (Mingchao Li) and K.L.; validation, M.L. (Mingchao Li), W.Z. and M.L. (Man Liu); formal analysis, M.L. (Mingchao Li); investigation, M.L. (Mingchao Li), W.Z., M.L. (Man Liu), H.H. and Y.Z.; visualization, W.Z., M.L. (Man Liu) and H.H.; literature search, M.L. (Man Liu) and H.H.; figures, W.Z.; writing—original draft preparation, M.L. (Mingchao Li) and K.L.; writing—review and editing, M.L. (Mingchao Li), K.L, Y.Z., Y.C., S.F. and R.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (No: 32160324, 42005138) and the China Agriculture Research System (No. CARS-11-hncyh).

Data Availability Statement

The original data presented in the study are openly available in NCBI at https://www.ncbi.nlm.nih.gov/ (accessed on 1 July 2024), reference numbers PRJNA324539, PRJNA163523, PRJNA1128429, and PRJNA1127687; [PRJNA324539] [https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA324539, accessed on 7 July 2024]. [PRJNA163523] [https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA163523, accessed on 7 July 2024]. [PRJNA1128429] [https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA1128429, accessed on 7 July 2024]. [PRJNA1127687] [https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA1127687, accessed on 7 July 2024].

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

AbbreviationFull name
ABAabscisic acid
AOCallene oxide cyclase-like protein
BPbiological process
CAconiferyl alcohol
CaCl2cadmium chloride
CCcellular component
CDDConserved Domain Database
CDscoding DNA sequences
CPRNovo Nordisk Foundation Center for Protein Research
DIRdirigent
drr206disease resistance response protein 206
EGTAethylene glycol tetraacetic acid
EMBL-EBIEuropean Bioinformatics Institute
FECfriable embryogenic callus
FPKMtranscript per million reads mapped
FRfibrous root
GOgene ontology
GRAVYgrand average of hydropathicity
HMMHidden Markov Model
Kanon-synonymous substitution rate
Kssynonymous substitution rate
log2 FClog2 Fold Change
MFmolecular function
MWmolecular weight
NaCl sodium chloride
NJneighbor-joining
OESorganized embryogenic structures
PDDpostharvest physiological deterioration
PEGpolyethylene glycol
pIisoelectric point
PIRProtein Information Resource
qRT-PCRquantitative real-time PCR
RAMroot apical meristem
SAMshoot apical meristem
SC9South China 9
SRstorage root

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Figure 1. Prediction of the three-dimensional structure of MeDIR proteins. The colors indicate the predicted confidence of the model along the sequence. Blue indicates the minimum confidence and red indicates the highest confidence.
Figure 1. Prediction of the three-dimensional structure of MeDIR proteins. The colors indicate the predicted confidence of the model along the sequence. Blue indicates the minimum confidence and red indicates the highest confidence.
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Figure 2. Phylogenetic tree of the DIR proteins predicted in cassava and those previously identified in Arabidopsis, rice, and poplar. The phylogenetic tree was constructed using the Neighbor-joining (NJ) method with 1000 bootstrap replications as implemented in MEGA7.1 from a DIR protein sequence alignment. Different clades are distinguished by different colors. Me, Manihot esculanta; At, Arabidopsis thaliana; Os, Oryza sativa; Pt, Populus trichocarpa.
Figure 2. Phylogenetic tree of the DIR proteins predicted in cassava and those previously identified in Arabidopsis, rice, and poplar. The phylogenetic tree was constructed using the Neighbor-joining (NJ) method with 1000 bootstrap replications as implemented in MEGA7.1 from a DIR protein sequence alignment. Different clades are distinguished by different colors. Me, Manihot esculanta; At, Arabidopsis thaliana; Os, Oryza sativa; Pt, Populus trichocarpa.
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Figure 3. Mapping and synteny analysis of MeDIR genes. (A) Distribution of DIR genes on cassava chromosomes. The chromosome numbers were shown at the left of each chromosome. The genes were listed on the left of the chromosomes. The scale on the left is in million bases (Mb). Yellow to blue represents chromosome gene density from high to low. (B) CIRCOS figure of MeDIR genes. The gray line in the background indicates a collinear block in the genome of cassava, while the red line highlights the isomorphic gene pair. The positions of MeDIR15 and MeDIR16, and MeDIR18 and MeDIR19 genes are very close to each other, and the lines of the two gene pairs MeDIR15 and MeDIR19, and MeDIR16 and MeDIR18 appear to be highly overlapped. The chromosome number is indicated in each chromosome. The number of each chromosome is indicated inside each bar. The scale above the box is in mega bases (Mb). The line and heat map in the outer circle represent gene density on the chromosome. The innermost circle, from pink to blue, represents chromosomes from high to low, followed by the red bar graph, where the high and low of the bar graph represent the high and low of the chromosome density.
Figure 3. Mapping and synteny analysis of MeDIR genes. (A) Distribution of DIR genes on cassava chromosomes. The chromosome numbers were shown at the left of each chromosome. The genes were listed on the left of the chromosomes. The scale on the left is in million bases (Mb). Yellow to blue represents chromosome gene density from high to low. (B) CIRCOS figure of MeDIR genes. The gray line in the background indicates a collinear block in the genome of cassava, while the red line highlights the isomorphic gene pair. The positions of MeDIR15 and MeDIR16, and MeDIR18 and MeDIR19 genes are very close to each other, and the lines of the two gene pairs MeDIR15 and MeDIR19, and MeDIR16 and MeDIR18 appear to be highly overlapped. The chromosome number is indicated in each chromosome. The number of each chromosome is indicated inside each bar. The scale above the box is in mega bases (Mb). The line and heat map in the outer circle represent gene density on the chromosome. The innermost circle, from pink to blue, represents chromosomes from high to low, followed by the red bar graph, where the high and low of the bar graph represent the high and low of the chromosome density.
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Figure 4. Synteny analysis of DIR genes between cassava and seven other plant species. The gray lines in the background indicate all synteny blocks within the cassava and the other genomes, and red lines indicate the duplicated DIR gene pairs.
Figure 4. Synteny analysis of DIR genes between cassava and seven other plant species. The gray lines in the background indicate all synteny blocks within the cassava and the other genomes, and red lines indicate the duplicated DIR gene pairs.
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Figure 5. Phylogenetic relationships, conserved motifs, conserved domains, and gene structure of the predicted cassava DIR proteins. (A) The phylogenetic tree of cassava DIR proteins was constructed using the Neighbor-joining (NJ) method in MEGA 7.1 software, and the bootstrap value of the main branches was set to 1000 replicates. The genes in seven subgroups are marked with different colors. (B) Different motif compositions of cassava DIR proteins were detected using MEME. The boxes with different colors on the right denote 10 motifs. (C) Conserved domain in the cassava DIR proteins, where light green boxes represent domain. (D) Gene structure in the cassava DIR proteins, where bottle green boxes represent exons while black lines represent introns.
Figure 5. Phylogenetic relationships, conserved motifs, conserved domains, and gene structure of the predicted cassava DIR proteins. (A) The phylogenetic tree of cassava DIR proteins was constructed using the Neighbor-joining (NJ) method in MEGA 7.1 software, and the bootstrap value of the main branches was set to 1000 replicates. The genes in seven subgroups are marked with different colors. (B) Different motif compositions of cassava DIR proteins were detected using MEME. The boxes with different colors on the right denote 10 motifs. (C) Conserved domain in the cassava DIR proteins, where light green boxes represent domain. (D) Gene structure in the cassava DIR proteins, where bottle green boxes represent exons while black lines represent introns.
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Figure 6. Analysis of cis-acting elements in MeDIR genes. (A) The grid displays various colors and numbers to represent the quantities of cis-acting elements present in each MeDIR gene. Light orange to red indicates more to less. See Table S7 for details (B) The various colors displayed on the histograms correspond to the cumulative total of cis-acting elements within each category.
Figure 6. Analysis of cis-acting elements in MeDIR genes. (A) The grid displays various colors and numbers to represent the quantities of cis-acting elements present in each MeDIR gene. Light orange to red indicates more to less. See Table S7 for details (B) The various colors displayed on the histograms correspond to the cumulative total of cis-acting elements within each category.
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Figure 7. Protein–protein interaction network of MeDIR proteins based on their orthologs in Arabidopsis. The color scales represent the relative signal intensity scores. Colored nodes: query proteins and first shell of interactors; white nodes: second shell of interactors; filled nodes: some 3D structure is known or predicted.
Figure 7. Protein–protein interaction network of MeDIR proteins based on their orthologs in Arabidopsis. The color scales represent the relative signal intensity scores. Colored nodes: query proteins and first shell of interactors; white nodes: second shell of interactors; filled nodes: some 3D structure is known or predicted.
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Figure 8. The expression patterns of MeDIR genes in different cassava tissues. The color gradient from red to blue represents varying levels of gene expression, with red indicating high expression and blue indicating low expression. FEC, OES, FR, SAM, RAM, and SR represent friable embryogenic callus, organized embryogenic structures, fibrous root, shoot apical meristem, root apical meristem, and storage root, respectively. Log2 transformed FPKM value was used to create the heat map. The scale represents the relative signal intensity of FPKM values.
Figure 8. The expression patterns of MeDIR genes in different cassava tissues. The color gradient from red to blue represents varying levels of gene expression, with red indicating high expression and blue indicating low expression. FEC, OES, FR, SAM, RAM, and SR represent friable embryogenic callus, organized embryogenic structures, fibrous root, shoot apical meristem, root apical meristem, and storage root, respectively. Log2 transformed FPKM value was used to create the heat map. The scale represents the relative signal intensity of FPKM values.
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Figure 9. Expression profiles of MeDIR genes under PEG treatment in cassava as determined by qRT-PCR. The error bars represent the standard error of the means of three independent replicates. Values represented by the same letter showed no significant difference at p < 0.05 based on Duncan’s multiple-range tests.
Figure 9. Expression profiles of MeDIR genes under PEG treatment in cassava as determined by qRT-PCR. The error bars represent the standard error of the means of three independent replicates. Values represented by the same letter showed no significant difference at p < 0.05 based on Duncan’s multiple-range tests.
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Figure 10. Expression profiles of MeDIR genes under NaCl treatment in cassava as determined by qRT-PCR. The error bars represent the standard error of the means of three independent replicates. Values represented by the same letter showed no significant difference at p < 0.05 based on Duncan’s multiple-range tests.
Figure 10. Expression profiles of MeDIR genes under NaCl treatment in cassava as determined by qRT-PCR. The error bars represent the standard error of the means of three independent replicates. Values represented by the same letter showed no significant difference at p < 0.05 based on Duncan’s multiple-range tests.
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Li, M.; Luo, K.; Zhang, W.; Liu, M.; Zhang, Y.; Huang, H.; Chen, Y.; Fan, S.; Zhang, R. Genome-Wide Identification, Evolution, and Expression Analysis of the Dirigent Gene Family in Cassava (Manihot esculenta Crantz). Agronomy 2024, 14, 1758. https://doi.org/10.3390/agronomy14081758

AMA Style

Li M, Luo K, Zhang W, Liu M, Zhang Y, Huang H, Chen Y, Fan S, Zhang R. Genome-Wide Identification, Evolution, and Expression Analysis of the Dirigent Gene Family in Cassava (Manihot esculenta Crantz). Agronomy. 2024; 14(8):1758. https://doi.org/10.3390/agronomy14081758

Chicago/Turabian Style

Li, Mingchao, Kai Luo, Wenke Zhang, Man Liu, Yunfei Zhang, Huling Huang, Yinhua Chen, Shugao Fan, and Rui Zhang. 2024. "Genome-Wide Identification, Evolution, and Expression Analysis of the Dirigent Gene Family in Cassava (Manihot esculenta Crantz)" Agronomy 14, no. 8: 1758. https://doi.org/10.3390/agronomy14081758

APA Style

Li, M., Luo, K., Zhang, W., Liu, M., Zhang, Y., Huang, H., Chen, Y., Fan, S., & Zhang, R. (2024). Genome-Wide Identification, Evolution, and Expression Analysis of the Dirigent Gene Family in Cassava (Manihot esculenta Crantz). Agronomy, 14(8), 1758. https://doi.org/10.3390/agronomy14081758

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