Genome-wide Computational Analysis of DIR Genes inSolanum lycopersicum

doi:10.21203/rs.3.rs-1617785/v1

Background:

Extensive Dirigent proteins impart a key role in the development of organic products in plants. They confer conformational influence on processes that lack stereoselectivity and regioselectivity through processes that are mostly understood. Dirrigent proteins are required to produce lignans, a unique and widely distributed family of plant secondary metabolites with intriguing pharmacological characteristics and potential use in plant development as well. The dirigent (DIR) proteins, which are implicated in the process of lignification, also protect plants from environmental stresses including Biotic and Abiotic. Nevertheless, there is no research have been done so far on the DIR proteins in Solanum lycopersicum. This study provides a detailed information of DIRs protein in the in tomato.

Results:

The Conserved Domain analysis, Phylogeny, Evolutionary Adaptation, cis-acting Elements, Proteomic analysis, Transmembrane Potential Analysis, Sequence Identity and Similarity Analysis, gene assembly, genomic localization, duplication of gene analysis, and evolutionary linkage of all 31 potential dirigent genes (SlDIRs) were revealed in this investigation.

Conclusion:

This research provides an in - depth and comprehensive explanation of the detailed molecular process and structural characterization of SlDIR genes in the genome of Solanum lycopersicum, laying the groundwork for future plant genetic engineering and crop development exploration. This study will aid future research on the DIR gene family and give helpful information for enabling appropriate DIRs genes in higher plants.

Dirrigent

Solanum Lycopersicum

Genome-wide

Bioinformatics

Lignan

Biotechnology

The name dirigent originates with the Latin word dirigere (align), and for the very first time, discovered in Forsythia intermedia (Gang et al., 1999). DIR proteins from Forsythia suspensa (Davin et al., 1997), mayapple (Xia et al., 2000), and western red cedar (Kim et al., 2002) are engaged in the pharmacological direction of E-coniferyl alcohol stereo-specific interaction to generate the enantiomer pinoresinol. DIRs along with DRR (disease resistance response) gene family possess a dirigent-conserved domain (Culley et al., 1995), and that they are thought to regulate the oxygen radicals bonding of monolignol plant phenolic compounds in plants to produce lignans and lignins (Burlat et al., 2001); hence, DIRs were implicated in disease resistance adaptations (Wang & Fristensky, 2001).

Lignan possesses antifungal effects, both constitutive and inducible, and is considered to be largely engaged in plant defensive reaction (Lewis & Davin, 1994). Lignin accumulation is thought to act as a protective border in the defensive reaction towards microbial infection (Moerschbacher et al., 1990). According to(Moura et al., 2010), lignification increases concerning pathogen assaults. According to (Fang et al., 2016), the thicker morphology in leaf tissue includes lignin, which functions as a protective border against microbial assaults. Lignin protects the recipient by acting as a non-degradable protective lining for pathogens. Lignin is indeed an essential chemical found largely in terminally specialized cells of supporting and water-conducting components, and it is principally involved as an exoskeleton, waterways in xylem, and insect and microbe defense (Zhou et al., 2009).

Determining the roles of the dirigent family in physiological and biological processes might be a useful tool for evaluating and enhancing crop defense responses against environmental stresses. Nevertheless, till recently, no research has been done on the DIR family in Solanum lycopersicum. As a result, the contribution of this work is to identify and analyze the DIR family in tomato using a genome-wide strategy. Following computational analysis, a total of 31 SlDIRs from the Solanum lycopersicum genome were determined. Upon that, we investigated the phylogenetic linkage of the DIR family in Solanum lycopersicum by looking at Promotor analysis, gene structural analysis, unique motifs, tertiary configurations, chromosome dispersal, duplication of the gene, Synteny analysis, and potential membrane analysis.

Retrieval of SlDIRs.

Solanum lycopersicum Dirrigent gene was retrieved by using seed file (PF03018) (https://pfam.sanger.ac.uk/) and Arabidopsis thalian DIR genes that were retrieved from TAIR (The Arabidopsis Information Resource) by the way of BLAST approach in database Phytozome13 (https://phytozome.jgi.doe.gov/), (Lamesch et al., 2012) against Solanum lycopersicum genome with defaults parameters. The protein's conserved domain was validated using the CDD (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) database in an automated way after redundant and repetitious segments were eliminated one at a time (Khan A et al., 2018) (Marchler-Bauer et al., 2007).

Structural characterization, and phylogenetic evaluation.

CLUSTALW tool was employed to conduct multiple sequence alignments of proteins. The proteins sequence were employed in the Expasy ProtoParam tool to determine the CDS length, the total amount of units, Aliphatic index, instability index, GRAVY, protein molecular weight (MW), and hypothetical isoelectric point (pI)(http://web.expasy.org/protparam/) Using the online application GSDS (Gene Structure Display Server), we looked at the exon/intron architecture of specific SlDIR genes in Solanum lycopersicum (Song et al., 2019)(AY et al., 2007)(Hu et al., 2015). Utilizing MEME (http://meme-suite.org/tools/meme) with parameters Zero or one occurrence per sequence (Zoops), having a minimum width of motifs is six and with maximum of 50; and the total number of motifs per sequence is 15, to investigate the motifs of SlDIR proteins. The results were shown using TBtools (TBtools v1.09854). Subcellular localization analysis was assessed using WoLF PSORT (https://wolfpsort.hgc.jp/) (Horton et al., 2007). The WoLF PSORT findings were evaluated using a heatmap plot in the TBtools (v1.09854) (Chen et al., 2020) program. Asn (N-glycosylation sites) of the SlDIRs were find out online via NetNGlyc 1.0 server (http://www.cbs.dtu.dk/services/NetNGlyc/).

To do the phylogenetic analysis of the SlDIRs protein family, MEGA X (http://www.megasoftware.net/) (Kumar et al., 2018) was used. Following their aggregation with separation, the SlDIRs genes were divided into different subclasses, and a full dendrogram encompassing Arabidopsis and Solanum lycopersicum was constructed using MEGA X. ClustalW's (http://www.ebi.ac.uk/clustalw/) (Thompson et al., 2003) general parameters were used to realign all of the segments at first. Since not all SlDIRs family members are exactly equivalent, we removed gaps and built a more cautious phylogenetic tree to improve the study's reliability. MEGA X was used to create both phylogenetic trees, which were created using the Neighbor-joining (Felsenstein, 1985) technique and bootstrapping testing with 1000 repetitions (Gu et al., 2002).

Evolutionary Adaptation and Chromosomal Localization of SlDIRs Genes

Each of the SlDIR proteins was mapped to the respective Solanum lycopersicum chromosomes via using the Phenogram online program. (http://visualization.ritchielab.org/phenograms/plot) (Wolfe et al., 2013). The TBtools program (v1.09854; http://cj-chen.github.io/tbtools/) was pre-owned to retrieve chromosome sequence data, as it was employed to locate all of the SlDIRs genes according to their spatial relationship and chromosomal positions, as well as duplicated regions(Poptsova & Gogarten, 2007). The ratios of Ks and Ka were calculated using the TBtools program's default settings (v1.09854). The divergence period of the gene pairs was calculated using the rate of substitutions per synonymous site per year. (Yuan et al., 2015) T = Ks/2x (x = 6.56 109).

Analysis of cis-acting Elements in the Promoters of SlDIRs.

The promoter characterization through the cis-acting element in the SlDIRs family was investigated using the TBtools program (http://cj-chen.github.io/tbtools/) (Chen et al., 2020). We retrieved the SlDIRs gene upstream region (up to 2 kb) from the Solanum lycopersicum genome and stored it in a FASTA file format. PlantCARE (Lescot et al., 2002) was used to submit and evaluate the data (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/).

Proteomic analysis of SlDIRs.

The linkage association of all DIR genes family genes in Solanum lycopersicum were constructed by integrating all DIR genes of Solanum lycopersicum into the freely accessible tool String (https://string-db.org/) (Szklarczyk et al., 2017), with the following criteria: i) minimum confidence: high (score: 0.07), ii) the maximum number of potential interconnection: 5, with all unconnected sequences being eliminated. To illustrate consensus, all SlDIRs genes in Solanum lycopersicum were mapped using String to evaluate their co-occurrence with others closely related taxa. The web hosting server Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index) (Kelley & Sternberg, 2009) was employed to anticipate the tertiary conformations and homologs of SlDIRs, as reported by (Li et al., 2014)

Transmembrane Potential Analysis and Sequence Identity and Similarity Analysis of SlDIRs

All-amino-acid alignments were evaluated in the TMHMM Server, v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM/) online program for analyzing the transmembrane possible mechanisms implicated in all SlDIRs genes. To examine sequence similarity assessment, all of the amino acids were submitted to the online program SIAS (http://imed.med.ucm.es/Tools/sias.html) with the baseline model of the BLOSUM62 method with gap consequences.

(1) The cost of establishing the gap, Po (0-100), is 10.

(2) The cost of expanding the gap, Pe (0-100), is 50.

Sequence Similarity Analysis

The DIR genes in Arabidopsis thaliana and Solanum lycopersicum were assessed using Circoletto, (http://bat.ina.certh.gr/tools/circoletto/) online program that employs Circos to identify two sequence datasets. DIR genes of Solanum lycopersicum were aligned to Arabidopsis thaliana DIR genes. An E-value of 10 to -40 (Strict) was used to perform genome preservation assessment from total local pairings. The SlDIR and AtDIR protein sequences were utilized to search their respective protein repositories, with the best hits obtained based on E-value.

Identification of Dirigent Genes in Solanum lycopersicum.

DIR sequence from the model organism Arabidopsis thaliana and seed file (PF03018) were utilized as keywords in a BLASTp homology search against the Solanum lycopersicum genome to determine all possible DIR gene close relatives in Phytozome v13. By eliminating other repetitive sequence, a maximum number of 31 DIR genes labeled as SlDIR1 to SlDIR31 were discovered in the Solanum lycopersicum genome. All the SlDIRs genes were belongs to the DIR group, as per the annotation of Phytozome v13. All the SlDIRs genes were matched with their Arabidopsis orthologs, and extensive data was anticipated. Subsequently, the existence of a preserved DIR motif was confirmed utilizing SMART and Pfam screening on SlDIRs genes. TBtool was employed to further confirm the Dirrigent domain in all SlDIRs proteins as shown in Fig. 1.

Gene Structural characterization, Conserved Motifs Analysis, and phylogenetic tree construction

The amino acid contents and stoichiometry of molecules in the SlDIR family are diverse, and the amount of compound proteins varies greatly amongst subclasses. The SlDIR genes' amino acid lengths ranged from 60 (SlDIR23) to 399 (SlDIR11), with an averaging of 189 base pairs. The lowest Molecular weight is 6302.19 KDa and the maximum is 41413.18 KDa, with an average weight of 20715.22 kDa. The mean pi was determined to be 7.75, with scores ranging from 4.47 (SlDIR11) to 9.88 (SlDIR11) (SlDIR3). The pi value is greater than 7 in 58 percent of the SlDIR protein family, while it is less than 7 in the rest. As a direct consequence of these discoveries, there are more basic DIR proteins than acid proteins. The presence of Asn in each DIR sequence can be seen in table. All the data can be visualized in Table 1.

We investigated the genomic and CDS sequences of SlDIR genes and examined the makeup of their introns and exons architecture to learn a lot more about how they work. The SlDIRs GENES intron-exon development was studied using the GSDS 2.0 software program (http://gsds.gao-lab.org/). Merely six of the 31 SlDIR genes (16%) have one intron, as per the findings. It's worth noting that 25 of the 31 proteins don't have introns. The genetic structure of all the genes can be seen in Fig. 2. The WoLF PSORT outcomes were predicted using a heatmap. The highest probability values are emphasized in red and contrasting light blue in Fig. 3.

As per the findings, members of the SlDIR family from the same subfamily have comparable motif types and quantities, however, there are variations in motif configurations across subfamily members. The precision of the phylogenetic analysis is improved by discovering comparable gene architectures and preserved domains within the same subfamily. Structure variations across subfamilies, on the other hand, imply that the SlDIR gene family in Solanum lycopersicum has functional variability. The genes and their respective Motifs can be visualized in Fig. 4

ClustalW was employed to evaluate the amino acid patterns of 31 Solanum lycopersicum DIR proteins against 26 Arabidopsis thaliana DIR proteins in addition to assessing the DIR gene family in model plants and Solanum lycopersicum from an evolutionary perspective, and to investigate the Solanum lycopersicum DIR protein's unique features. MEGA X, minimal evolution and neighbor-joining (NJ) methods were used to build the phylogenies. The Solanum lycopersicum DIR, AtDIR, clustered, suggesting that the Solanum lycopersicum DIR genes that can be categorized with Arabidopsis transcripts are part of the same grouping. The DIR group of all these sequences may be categorized into seven subclass (indicated by different colosr) based on their similarity to DIR sequences in Arabidopsis thaliana (Fig. 5).

Table 1

Physiochemical properties of all tomato DIR genes
Gene Id	Protein Weight Kda	Pi	Instability index	Aliphatic index	GRAVY	N-Glyc (Asn) position
SlDIR1	21482.33	9.7	26.84	105.05	0.268	132, 175
SlDIR2	10564.04	7.89	15.57	65.32	0.055	36
SlDIR3	26850.33	9.88	18.96	82.6	0.047	80, 233
SlDIR4	19875.05	8.41	18.64	95.84	0.138	9
SlDIR5	19662.64	8.89	7.76	100.22	0.237	65, 176
SlDIR6	21217.62	9.72	26.84	91.89	-0.094	69, 130, 174
SlDIR7	21300.6	9.27	26.28	91.36	0.038	70,131,175
SlDIR8	21403.73	8.78	27.9	91.41	0.002	70,87,131,175
SlDIR9	20738.68	8.85	21.46	90.21	0.027	51
SlDIR10	20640.84	9.61	32.67	98.16	0.009	31,79,178
SlDIR11	41413.18	4.47	39.97	86.02	0.037	70
SlDIR12	23782.26	4.98	30.11	88.75	-0.136	-
SlDIR13	20446.46	6.43	39.06	87.45	0.172	87,124
SlDIR14	33023.95	4.66	23.02	88.72	0.246	5
SlDIR15	20477.75	9.42	20.18	100.05	0.212	69,127
SlDIR16	19444.18	5.53	20.82	103.05	0.237	32,45,171
SlDIR17	21570.59	6.58	25.08	75.34	-0.058	52,65,122,140
SlDIR18	21138.1	6.5	24.51	89.26	0.034	20,55,68,125,143
SlDIR19	17623.43	6.54	20.8	98.08	0.207	47,50,53,58
SlDIR20	27770.79	9.19	38.95	87.1	0.073	10
SlDIR21	21448.92	9.58	32.77	90.26	0.047	24,36,59,69,130,173
SlDIR22	6437.34	6.51	7.38	74.67	-0.04	26
SlDIR23	6302.19	5.85	-3.46	82.83	0.16	26
SlDIR24	20785.85	8.76	18.16	83.54	0.029	56,66,92,127,170
SlDIR25	20861.97	6.64	14.86	82.89	0.01	70,126,169
SlDIR26	22663.37	9.59	16.37	80.87	0.019	1,83,109,126,144,187
SlDIR27	20924.08	9.3	15.81	100.05	0.128	74,99,100,135
SlDIR28	12082.81	8.79	21.01	70.46	-0.365	51,77
SlDIR29	20271.72	9.38	22.01	91.09	0.191	89,124,167
SlDIR30	13834.51	5.6	31.12	73.05	-0.006	-
SlDIR31	26132.8	5.07	29.65	92.47	0.2	-

Promotor evaluation of SlDIRs

PlantCARE (Lescot et al. 2002) was used to discover cis-acting regions in the sequence of 2 kb bp upstream from each transcription start site of the SlDIR genes to proceed the investigation of the ostensible biological reactions of SlDIR during signaling, development, and endurance to abiotic and biotic stress feedback. We investigated each gene's responsive elements in further detail and observed that each gene appears to have a varied range of actions in response to environmental stress, together with plant development, growth, and regulation, as shown in Fig. 6.

Chromosomal Location and Gene Duplication of SlDIRs.

The Phytozome dataset v13 provided the chromosomal locations of all SlDIRs genes. Every of the selected SlDIRs genes were physically allocated into their appropriate chromosomes employing the phenogram tool, as shown in Fig. 7. The 31 SlDIRs genes were detected to be highly heterogeneous dispersed throughout the Solanum lycopersicum genome on all 12 chromosomes (excluding chr03), indicating that biological variability evolved during the evolution change. Most SlDIRs genes 9 were found on chromosome 10 (chr10). On the other hand, chromosomes 5,8,9,11,12 contained the fewest number of SlDIRs genes, with only one. Two SlDIRs genes were found on three chromosomes (chr04, chr06, and chr07). In addition, chromosomes 2 and 8 (chr02 and chr08) harbored 3 SlDIRs genes. Furthermore, three SlDIRs genes were found on chromosomes 2 and 8 (chr02 and chr08).

The (synonymous rate) Ks, (non-synonymous rate) Ka, and Ka/Ks for these iterations were calculated, and the values were being used to predict duplication divergence time. Throughout the genome, there is a wide variety of duplication. The ratio of Ka/Ks indicated that all of the values are smaller than one, implying that they were purified.

Ka/Ks = 1 indicates neutrality in the process of selection, while Ka/Ks > 1 indicates positive selection, and Ka/Ks < 1 indicates purification selection. Ka/Ks < 1 ratio in all duplicated SlDIRs gene pairs, indicating purifying selection throughout the evolution. Besides this, duplications events of duplicated gene pairs were predicted to have happened somewhere around 3.75 and 74.36 million years ago (Table 2).

Table 2

Ka, Ks, and *Ka/Ks* computation and divergent time of the duplicated *Solanum lycopersicum* DIR gene pairs
Seq_1	Seq_2	Ka	Ks	Ka/Ks	T (MYA)
SlDIR14	SlDIR30	0.085935924	1.007212224	0.085321	6.549994
SlDIR11	SlDIR12	0.083597852	1.861744502	0.044903	6.371787
SlDIR20	SlDIR31	0.148327205	0.666594299	0.222515	11.30543
SlDIR17	SlDIR18	0.26100604	1.245689901	0.209527	19.89375
SlDIR9	SlDIR10	0.975700884	1.790588485	0.544905	74.36745
SlDIR4	SlDIR16	0.601743932	1.058155143	0.568673	45.86463
SlDIR2	SlDIR3	0.102436322	0.084828357	1.207572	7.807646
SlDIR7	SlDIR8	0.048911391	0.09014931	0.54256	3.728002
SlDIR22	SlDIR23	0.121149241	0.170426547	0.710859	9.233936

Synteny Analysis and Tertiary prediction of SlDIR genes

DIR proteins were analyzed to find orthologous pairs between S. Lycopersicum and A. thaliana that will further deduce the evolutionary connection. SlDIRs genes have collinear gene pairs in Arabidopsis thaliana, according to synteny assessment. The Circos map revealed that SlDIRs genes possess a high evolutionary homology connection with Arabidopsis thaliana, indicating that they could have similar biological activities (Fig. 9). Phyre2 results of protein tertiary structure are shown in Fig. 10.

Transmembrane potential and Sequence Identity and Similarity Analysis of SlDIRs genes

To test if the potential transmembrane outcome is active in all of the perspective studied proteins, TMHMM, a free online tool, was used to look at all of the SlDIR genes. 15 of the 31 SlDIR were discovered to be involved in the cellular membrane's essential functions, as shown in the study. The SIAS assessment was used to determine the sequences' distinctiveness, coherence, and global similarity. The table presents common findings in a tabular arrangement. SlDIR revealed a greater value for their resemblance and identification evaluation than Global similarity, based on the data. (Table 3)

Table 3

Sequence analysis of tomato DIR genes
Analysis	Minimum Value	Maximum Value	Mean Value	Standard Deviation
Identity Analysis	1.66	89.52	7.86	6.77
Similarity Analysis	3.33	100	17.25	16.59
Global Similarity (Blosum62)	-0.6	0.91	-0.15	0.12

Plants are constantly exposed to adverse environmental factors such as salt, drought, and cold, that have a significant influence on geographical distribution, proliferation, growth, and plant production. Plants respond to external stressors through a complex set of tolerance strategies that are activated and incorporated by the expression of thousands of genes (Seki et al., 2002). Such stressors induced the production of genes that were discovered to be important in the plant's stress resistance. These gene products act as regulatory proteins, increasing stress tolerance in the plant.

The study of transcriptional regulators is a heated issue in genetic sciences now. Transcription factors influence a range of activities, including physical, biochemical, and evolutionary activities, as well as the activation routes of downstream genes. Numerous transcription factors that control dehydration, high salt, and other environmental variables have been identified in recent years. In addition, genomic data study may be utilized to develop an interpretative layout for gene transformation technology, that can be employed to generate highly resistant transgenic organisms.

Dirigent molecules are a multigene family in plants that responds to pathogen resistance (Li et al., 2014)(Liao et al., 2016)(Jin-long et al., 2012). They serve an essential function in improving stress tolerance in many crops (S. Ralph et al., 2006). DIRs and their homolog have been found in all vascular plants (Davin & Lewis, 2000), and they are thought to play a role in lignin and lignan production (Kim et al., 2002). Varying plant species have different numbers of DIR genes. There have already been reports of 25 DIRs in Arabidopsis, 19 DIRs in I. indigotica, 54 DIRs in rice, 35 DIRs in Picea glauca, and 29 DIRs in B. Rapa (Thamil Arasan et al., 2013)(S. Ralph et al., 2006) (S. G. Ralph et al., 2007) (Li et al., 2014). There has never been a genome-wide identification and characterization of DIR in Solanum lycopersicum before. In this work, we discovered 31 SlDIRs in the Solanum lycopersicum genome. Five well-conserved motifs were identified in the amino acid sequence alignments of all 31 SlDIRs, which is like earlier studies9,11,27,29. Only 16% of SlDIRs had one intron, according to the strength and function. The amino acid (aa) sequences varied from 60 aa (SlDIR23) to 399 aa, according to the ORFs assessment (SlDIR11).

The chromosomal positions indicated that SlDIRs were detected on all 12 chromosomes of Solanum lycopersicum but 3, with chromosome 10 having the most SlDIRs and chromosomes 5,9,11,12 having only one DIR gene in their loci.

The architecture of the DIR proteins discovered in this work, as well as the described earlier DIR genes of A. thaliana and poplar, are basic, with very few introns (Khan et al. 2018). Nevertheless, 1–5 introns are found in one-third of rice genome (Liao et al. 2017). This implies that after divergence, rice, Solanum lycopersicum, poplar, and Arabidopsis may well have divergent paths.

Our findings support previous research on cis-element analyses, with elements linked to stress and light being discovered in the upstream region, demonstrating that environmental stress and light may have a regulatory function in SlDIRs. Furthermore, components sensitive to SA and MeJA have been discovered upstream of a large number of SlDIRs. Gibberellin responsive domains were discovered in the majority of DIR genes, which was surprising. All of the findings suggest that the rhythms of SlDIR's hormone responses are extremely complicated. Distinct DIRs are considered to imply multiple functions in a diverse setting at various times, nevertheless, they still need to be examined further in lab to demonstrate their functionality.

The genomes of plant species may well be studied using data analysis and evolutionary methods. The majority of the SlDIR genes in the Solanum lycopersicum were not displaced as a result of environmental selection, but they did show excellent conservation throughout evolution. These results will indeed offer a base for future research on tomato DIR family members implicated in a complicated plant's growth and development network.

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No competing interests reported.

Genome-wide Computational Analysis of DIR Genes inSolanum lycopersicum

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