Screening of MATE in D. carota
The PHMMER revealed 45 genes encoding the MATE family from the D. carota genome. The genes were named DcMATE1–DcMATE45 based on the PHMMER analysis. The gene details such as sequences, locus name, amino acid length, molecular weight (MW), isoelectric point (pI), and transmembrane helix are listed in Supplementary Table 3.
All the nucleotide and amino acid sequences of DcMATE were retrieved from Phytozome. As presented in Supplementary Table 3, the MATE family encoded proteins composed of 275 to 958 amino acids. The prediction of the transmembrane domain by TMHMM suggested that there are 5–16 putative transmembrane segments in the screened DcMATE. Among 45 DcMATE, 14 DcMATE contains 12 transmembrane segments, and 10 DcMATE contains 10 transmembrane segments in the D. carota genome.
Phylogenetic analysis of DcMATEs
For analysing the phylogenetic relationships among D. carota MATE proteins, we constructed a phylogenetic tree using the full-length proteins of the 45 DcMATE using MEGA X. Based on the phylogenetic analysis, 45 DcMATE proteins are classified into three groups. The first group contains 10 DcMATE proteins, followed by the second group of 16 DcMATE proteins, and finally, the third group with 19 DcMATE. All three groups are represented in Figure 3.
Gene duplication analysis
Forty-five DcMATE genes are present among nine chromosomes, and genes were unevenly distributed among the chromosomes (Fig. 2A). The seventh chromosome contains a maximum of 8 DcMATEs followed by the fifth chromosome containing seven DcMATEs, respectively. The locations of DcMATE segmental duplications in the D. carota genome are shown in the Circos plot (Fig. 2B). There are five pairs of segmental and six pairs of tandem duplications of DcMATE present in the D. carota genome. The synonymous (Ks) and non-synonymous (Ka) substitution rates are used to estimate the DcMATE duplicated divergence times (Supplementary Table 4).
Cis-element analysis of the DcMATE
The upstream promoter region plays an important role in the expression initiation and regulation of genes, and it will give a better understanding of the gene’s functions. The analyses of DcMATE cis-regulatory elements revealed the presence of various stress-responsive elements (temperature-responsive, wound-responsive, defense and stress-responsive), phytohormone responsive elements (auxin, abscisic acid, salicylic acid, methyl jasmonate, and gibberellin), light-responsive elements, circadian control, cell cycle regulation, and phytochrome down-regulation responsive elements in the DcMATE promoters (Supplementary Figure 1). The results disclosed the presence of multiple cis-regulatory elements in the upstream region of all the DcMATEs and basic promoter elements (Supplementary Figure 2).
Cloning of full-length DcMATE from in vitro cultures
Based on the previous studies, we had identified the MATE gene (DcMATE21), which showed significant expression among other MATE under salt stress (Saad et al. 2021). The Sanger sequencing analysis of cloned putative DcMATE21 (Accession number: MG682560) revealed an open reading frame (ORF) of 1536 bp, which encoded a predicted protein of 511 amino acids with a theoretical molecular weight of 56.14 kDa and pI value of 7.52. The DcMATE21 shared 46.36, 43.17, and 42.32% similarities with NtMATE, VvMATE, and AtMATE, respectively (Supplementary Figure 3).
Effect of abscisic acid on callus growth and anthocyanin production
Abscisic acid was added to the medium at 10, 25, 50, and 100 μM concentrations on inoculation to study their effect on cell growth and anthocyanin accumulation in the suspension culture of D. carota. After adding ABA, we achieved maximum cell growth in the medium containing 100 µM ABA (15.13 ± 1.17 g L-1 FCW) on day 15 compared to other concentrations and control. The cell growth with elicitors was low during the lag phase (3 to 6 days), followed by increasing from 6 to 15 days, and further declined after 15 days of inoculation (Fig. 4A).
ABA’s positive effect on anthocyanin content is evident in Figure 5A. The maximum accumulation of anthocyanin was observed on day 9 at all levels of ABA tested. The highest anthocyanin content is 8.03 ± 0.16 mg L-1 FCW, which is 2.7-fold higher observed with 50 µM ABA. However, anthocyanin content decreased after 9 days of inoculation. In control cultures, the maximum anthocyanin accumulation was observed on day 12 (2.98 ± 0.11 mg L-1 FCW). The accumulation of total anthocyanin content was decreased with a further increase in the concentration of ABA in the medium.
Effect of sodium nitroprusside on callus growth and anthocyanin content
Sodium nitroprusside act as nitric oxide (NO) donor, which is involved in defense-related responses. Studies were conducted to analyze the influence of SNP on the production of anthocyanin in in vitro cell cultures. In the present investigation, different concentrations (10, 25, 50, and 100 µM) of SNP were supplemented into the suspension culture of D. carota to examine their effects on cell growth and anthocyanin content (Fig. 4 & 5). The highest biomass achieved was 15.12 ± 1.17 g L-1 FCW with the supplementation of 100 µM SNP in the medium on day 15 (Fig 4B). In contrast, SNP led to enhanced cell growth at all concentrations compared to control cultures (12.54 ± 0.37 g L-1 FCW). As shown in Figure 5B, the anthocyanin production increased relatively with the addition of SNP, but once the concentration of SNP exceeded 25 µM, it had an inhibitory effect. By treating with 10 µM SNP, the content of anthocyanin was enhanced by 2.1-fold (6.22 ± 0.16 mg L-1 FCW) on day 9 (Fig. 5B).
Effect of salicylic acid on callus growth and anthocyanin content
Salicylic acid (SA) was found to be involved in activating defense-associated genes in plants. Different concentrations (10, 25, 50, and 100 µM) of SA were added to the media to determine the optimum concentration for anthocyanin production. Application of SA into suspension culture resulted in significantly higher biomass production. However, the addition of a higher concentration of SA (50 to 100 µM) showed a slight reduction in the growth of cells. The maximum biomass of 21.73 ± 0.39 g L-1 was reached on day 12 in the medium supplemented with 25 µM SA (Fig. 4C). Among all the SA levels, 25 µM concentrations resulted in the maximum anthocyanin content (6.21 ± 0.36 mg L-1 FCW) was acquired in the medium supplemented with 25 µM SA on day 12 of inoculation, and this was significant (P > 0.05) in comparison with control (Fig. 5C). In contrast, higher SA (100 µM) concentration suppressed the anthocyanin production in the culture medium.
Effect of methyl jasmonate on callus growth and anthocyanin content
Methyl jasmonate (MJ) is known as a signal transducer in plants. Several studies on the plant system have proved that exogenously applied MJ profoundly affected in vitro production of secondary metabolites. Maximum biomass was obtained with the addition of 10 µM MJ (15.60 ± 0.28 g L-1 FCW) in the suspension culture at day 12. The addition of MJ at higher concentrations (>25 µM) in the culture medium considerably repressed the growth of the cells (Fig. 4D). The suspension cultures subjected to 10 µM MJ showed a significant increase in anthocyanin content (5.60 ± 0.10 mg L-1 FCW) compared to 25, 50, and 100 µM MJ levels (Fig. 5D). The above results indicate that adding MJ at 10 µM concentration in the medium increase the anthocyanin content by 1.88- fold at day 12 compared to control.
Effect of precursor on callus growth and anthocyanin content
Other than elicitors, precursor feeding is also known to enhance anthocyanin content in in vitro cell suspension cultures. However, an overabundance of the precursor may cause input hindrance to the metabolic pathway. Hence, it is essential to decide the suitable concentration for anthocyanin production in the suspension cultures. The influence of precursor (PHE) on cell growth and anthocyanin content in suspension cultures is presented in Figure 4E. The addition of PHE at concentrations of 0.5, 1.0, and 5 mM into the suspension culture of D. carota achieved an almost similar response on cell growth, i.e., 22.90, 21.54, and 20.14 g L-1 FW, respectively. In our observation, day 9 and 12, PHE caused better growth in the presence of 0.5, 1.0, and 5.0 mM in MS medium (Fig. 5E).
Treatment of suspension cultures with various concentrations of precursor showed augmentation of anthocyanin until day 12. The highest anthocyanin content (4.89-fold) was observed in the suspension culture treated with 0.5 mM PHE on day 12 (14.59 ± 1.41 mg L-1 FCW). The anthocyanin content in the cell suspension cultures treated with 1.0 and 5.0-mM PHE were 4.33 and 1.93-fold, respectively, higher than that of the control group on day 12 (Fig. 5E).
Transcript levels of DcMATE21 and anthocyanin biosynthesis genes by abiotic elicitors and precursor
To investigate the effect of elicitors on the expression of DcMATE21 and anthocyanin biosynthesis genes, media was fortified with optimum concentration of ABA (50 μM), SNP (10 µM), SA (25 µM), MJ (10 µM), and PHE (0.5 mM) separately. To gain insight into the transcript abundance of the MATE (DcMATE21) and eight structural biosynthesis genes of the anthocyanin pathway (PAL, CHS, C4H, CL, F3H, DFR, LDOX, and UFGT) were investigated by qPCR (Fig. 6). The DcMATE21 and anthocyanin biosynthesis genes were differentially expressed in suspension cultures treated with elicitors and precursors. With the addition of ABA in suspension cultures, PAL, CHS, C4H, F3H, DFR, and UFGT were expressed at a high level. In particular, the expression levels of the biosynthesis gene PAL, CHS, C4H, DFR, F3H, LDOX, and UFGT genes increased by approximately 4.3, 5.6, 4.1, 4.7, 6.7, 7.3, and 7.8-fold respectively in response to ABA treatment. An increase in expression of DcMATE21 was observed with ABA treatment as 5.9-fold higher compared to control. The expression profiles of DcMATE21 were linked to the anthocyanin biosynthesis genes. In addition, suspension culture treated with PHE showed the transcript levels of PAL, CHS, F3H, LDOX, and UFGT at 8.4, 6.2, 7.7, 8.5, and 9.2-fold, respectively, while C4H and 4CL were increased at 4.1 and 2.3 -fold respectively (Fig. 6).
In response to PHE, the transcript abundance of the MATE increased by 4.1-fold. The MATE and late biosynthesis genes (LDOX and UFGT) revealed higher transcript abundance in suspension cultures of D. carota treated with ABA and PHE. The transcript abundance of PAL, C4H, CL, and DFR, on the other hand, was marginally increased in response to SNP and SA treatments (Fig. 6).
In response to SNP and SA treatment in cell suspension cultures, the transcript abundance of MATE likewise displayed a correlation with the biosynthetic genes, with low levels of expression of 2.5 and 3.0-fold, respectively. PAL, CHS, C4H, CL, DFR, F3H, LDOX, and UFGT were slightly up-regulated in the suspension culture treated with MJ, correlating with the low expression of DcMATE21(Fig. 6).
In the suspension cultures treated with elicitors and precursors, the expression of DcMATE21 was found to be related to the expression of anthocyanin biosynthetic genes. MATE expression was increased together with late anthocyanin biosynthesis genes in the presence of ABA and PHE. In addition, DcMATE21 expression was low along with late biosynthesis genes in the presence of SNP, SA, and MJ. The MATE transporter can be implicated in the anthocyanin accumulation in D. carota cell suspension cultures. A linear increase in transcript levels was found in the order ABA> PHE> SNP> SA> MJ for anthocyanin biosynthesis genes in suspension culture with various elicitors and precursors. The present study proved that adding ABA and PHE to the suspension culture of D. carota caused an increase in the expression profile of anthocyanin biosynthesis genes and DcMATE21.
3.12. Molecular dynamics of DcMATE21 with anthocyanin
Molecular Dynamics simulation was carried out for the MATE-cyanidin complex for 100ns. Major analysis was carried out with Root Mean Square Deviation (RMSD) to quantify the changes in displacement of atoms for a reference frame. From the plot (Fig. 7A), the RMSD (C-α atom) value is ~ 3.7 Å suggesting the system is equilibrated and lacks changes in MATE structure conformation with cyanidin binding after 100 ns. The Root Mean Square Fluctuation (RMSF) (Fig. 7B) describes the local deviations and the amino acid chain. The peaks in the plots represent the protein areas that fluctuate most during the protein simulation. The plot clearly depicts that the residues in contact with cyanidin are stable and do not fluctuate on induced binding forces. DcMATE21 protein interactions (Fig. 8A) with the cyanidin were monitored throughout the simulation studies. The cyanidin showed different interactions with the DcMATE21, and these can be categorized by type and summarized, as shown in Figure 8. DcMATE21 and cyanidin interactions are categorized into four types: Ionic, Hydrogen bonds (H-bonds), Hydrophobic, and Water bridges.
H-bonds play a significant role in ligand binding in protein interactions. The interactions of H-bonds between protein and ligand can be divided into four subtypes: backbone donor; backbone acceptor; side-chain donor; side-chain acceptor. From Figure 8B, we can visualize Tyr 75, Ser101, Ala104, Gly105, Leu106, Glu294, Thr295, Thr322, Gly326, and Asn412 are involved in H-bond interactions. Since the simulation was implicit by nature, the presence of water molecules mediated hydrogen-bonded protein-ligand interactions. Except for Thr295 and Glu326, the above-mentioned remaining residues in H-bond interactions are water-mediated (Water bridges) with hydrogen available as donor atoms. The H-bond geometry is a little relaxed from the normal H-bond definition.
Hydrophobic interactions can be separated into three subtypes: π-Cation; π-π; and Other, non-specific interactions. These types of interactions involve a hydrophobic, aromatic or aliphatic group of amino acids on the ligand. Gly326, and Val329, Ala322 are involved in hydrophobic interactions and Tyr297 with hydrophobic interactions and partial water-mediated H-bonds. The effect is due to Glu294 and Thr295 with H29, H30, and H18 (Fig. 8B) of cyanidin. Cyanidin had no interaction with the POPE membrane throughout the simulation, and the Radius of Gyration (rGyr) was ~ 4.35 Å. Cyanidin RMSF was < 2.0 Å (Fig. 8C) portraying stable interactions. Cyanidin atom numbers specified are mapped and represented in Figure 8D.