3.4 Amino acid, and secondary and tertiary structure modifications
In the current study, Spanish sample of stevia showed 458 amino acids, while Brazilian one 299 (Figure 3). Besides, the secondary structure of SrUGT76G1 evidenced 13, 23 and 20 helices in Brazilian, Spanish and reference samples (GenBank accession no. AAR06912.1), respectively. In the cases of Spanish and Reference, pretty much covered the same number of amino acids (200 and 201), on the other hand Brazilian samples covering 114 amino acids out of 299. Overall, these helices cover about 42% of amino acids of each sequence/sample (Figure 3). Madhav et al., (2012) reported the secondary structure of glycosyltransferases in stevia plants, with 19 helices covering 191 amino acids (41.8%). This percentage is close to the amino acid coverage observed in this study. Moreover, the number of putative domain boundary remains the same in the evaluated samples, however in Brazilian samples it occurs in the amino acid Phe150, while in the others in the amino acid Gln270 (Supplementary material - Figure 2). Another marked difference in the Brazilian samples is related to seven-strand formation, while reference and Spanish ones form 12 and 11, respectively.
According to results, in Brazilian samples there are three insertions and two deletions in relation to the others. In addition, Brazilian samples exhibited a T-to-A substitution at position 299 (leucina amino acid), resulting in a premature stop codon at position 896 of the mRNA (p.896L>x) (Figure 4). In an earlier study, Yang et al., (2014) also discovered a nonsense mutation (premature stop codon) in the SrUGT76G1 gene, resulting in a protein with altered spatial structure and consequently very low levels of Reb-A. Recently, Zhang et al., (2019) identified five stevia genotypes (N01-N05) that accumulated different amounts of SGs because of some mutations (base substitutions, single nucleotide polymorphisms, and amino acid substitutions/insertions) in the SrUGT76G1 gene.
The comparison of the amino acid sequences among Reference/Spanish, Reference/Brazilian and Brazilian/Spanish samples unveil that the similarity between Reference/Spanish ones were higher than 97%. On the hand, in Reference/Brazilian samples this similarity decay to 58%, as expected (Supplementary material - Table 2). Coupled with this, in samples from plants grown in Spain, six amino acid changes were observed in relation to the reference, while in Brazilian plants this number rises to 101 amino acids (Figure 4). According to Petit et al., (2019), most of the time, substitution of one amino acid may be prejudicing the recognition of substrates and regioselectivity, leading to catalytic activity reduction of SrUGT76G1.
In a recent study, Liu et al., (2020) showed that residues Gly87, Pro91, Ile199 and Leu204 define diterpenoids/flavonoid glycosylation, as well as amino acids Leu85, Met88, Ile90, Ile199, Leu200, and Ile203 mutations likely interfere in the substrate preference. Besides, Yang et al., (2019) reported that Reb A and Rubu complexes sites in hydrophobic pocket formed by Leu85, Met88, Ile 90, Asn196, Ile199, Leu200, and Ile203. Among these amino acids, Brazilian samples showed mutations in the Ile199Lys, Leu200Arg and Leu204Phe (Figure 4). In accordance with these authors, changes of Leu204Phe can narrow substrate-binding pocket to favors flavonoids recognition. Likewise, Leu204Phe mutants decreased steviol glycosides synthesis (Liu et al., 2020). In addition, other mutations were observed in Brazilian stevia plants, such as Thr284Leu. Thr284 is considered key for 1,3-glucosylation of SGs, including Rebaudioside-A (Olsson et al., 2016; Liu et al., 2020). These punctual mutations may partly explain the low/undetectable production of Rebaudioside-A by plants stevia grown in Brazil (Table 1).
Another important substitution (Phe281 into a Leu and Gly282 into a Val) of amino acids were observed in stevia plants from Brazil (Figure 4). According to Olsson et al., (2016) Phe281 and Gly282 form part of the hydrophobic core in the C-terminal domain. For this reason they are considered important. Moreover, Brazilian samples showed replacement of the Leu126 to Ile. Lee et al., (2019) recently suggested that Leu126Ile mutations resulted in 750-fold decrease in catalytic function of SrUGT76G1. Change of amino acid residues and activity reduction were also observed in other SrUGTs, i.e., SrUGT91D2 (Zhang et al., 2021). Conversely, some amino acids were common in the evaluated samples, such as His25 and Asp124. His25 is a common in the active site of all SrUGTs, as well as Asp124 (Madhav et al., 2012). In accordance with these authors, histidine-aspartate interacts to forming a hydrogen bond that provides more stability and functionally to SrUGTs. Besides, His25 and Asp124 form a conserved catalytic dyad and appear to be responsible for transferring the sugar from the donor molecule to the acceptor substrate (Olsson et al., 2016, Yang et al., 2019).
Overall, all glycosyltransferases of Stevia rebaudiana (SrUGTs) showed the presence of Val74 (Madhav et al., 2012), while that in SrUGT76G1 is Glu74. The sequence comparison of SrUGT76G1 showed this replacement in Reference and Spanish samples, but not in Brazilian sample that like the other SrUGTs showed Val74. Other amino acid residues that have gained prominence and have been recently target for the production of next-generation sugars are Thr146 and His155, both are present in the evaluated samples. With regard to His155, Liu et al., (2020) showed that substitutions of this amino acid (into Arg, Trp and Ala) result in decreased production of stevioside. The absence of this mutation may partly justify the production of stevioside (Table 1) in both samples of stevia plants (Brazilian and Spanish). Still in relation to these two amino acids, Olsson et al., (2016) showed reduction of unwanted products and increased contents of Reb A and Reb M in variants of UGT76G1 such as Thr146Gly and His155Leu.
According to Lee et al., (2019) residues Thr146, Ser157, Trp359, Asp380, and Gln381 are considered critical for positioning the glucosyl group in SrUGT76G1. Besides, some of these amino acids (Trp359, Asp380, and Gln381) are part of the PSPG (putative secondary plant glycosyltransferase) sequence motif found in all the plant UGTs. Amino acid changes (substitution, insertion and deletion) in PSPG box and other sites are reflected in catalysis efficiency and protein structure (secondary and tertiary), as shown in Figure 3 and 5, where Brazilian samples have a very different conformation in relation to Reference and Spanish ones.
The composition of SGs, as well as their contents are the focus of many researches and several efforts are geared towards production of larger quantities (Yücesan et al., 2016; Kim et al., 2019, Saifi et al., 2019). However, the understanding of the genetic regulation (transcriptional, post-transcriptional, and post-translational) of SGs biosynthesis pathway, mainly regarding the synthesis of the third-generation sweeteners (Reb-A, D, M, I and Q) is largely unknown. Therefore, if we want to understand/unveil the aspects that influence in this regulation, it is essential to expand studies. In this sense, some strategies have been explored and shown great progress, such as specific miRNA and transcription factors (TFs) discoveries.
Concerning the miRNA in stevia plants, Saifi et al., (2019) demonstrated that miRStv_11 up-regulated SrKAH, whereas miR319g showed the repressive action on SrKO, SrKS and SrUGT85C2 genes which results in low SGs accumulation. This study also co-expressed anti-miR319g and miRStv_11 in leaf and triggers an enhancement of expression of four genes (SrKO, SrKS, SrUGT85C2 and SrKAH) and, consequently, noticed a gain in SGs content. Besides, it was firstly reported that WRKY, MYB, bHLH, and NAC TFs may participate in the regulation of secondary metabolites in stevia (Singh et al., 2017). In a recent study, Zhang et al., (2020b) showed that the SrWRKY71 TFs represses the gene expression of SrUGT76G1 in callus of stevia. These results suggested that SrWRKY71 is an upstream regulator of the SGs biosynthesis in stevia.