Simone Ribeiro Lucho, Marcelo Nogueira do Amaral, Valmor João Bianchi, Lorena Almagro, María Ángeles Ferrer, Antonio Asensio Calderón, Eugenia Jacira Bolacel Braga
{"title":"SrUGT76G1 的测序分析和酶活性测定揭示了甜叶菊植物中 Rebaudioside-A 的开关生产机制","authors":"Simone Ribeiro Lucho, Marcelo Nogueira do Amaral, Valmor João Bianchi, Lorena Almagro, María Ángeles Ferrer, Antonio Asensio Calderón, Eugenia Jacira Bolacel Braga","doi":"10.1007/s13562-024-00888-y","DOIUrl":null,"url":null,"abstract":"<p>Stevia plants are well-known for their ability to synthesize steviol glycosides (SGs), a natural sweetener blend. The principal SGs include stevioside (STV) and Rebaudioside-A (Reb-A), with the latter exhibiting superior sweetness and organoleptic properties. UDP glucosyltransferase-76G1 (UGT76G1) is responsible for converting STV to Reb-A, determining the intensity of sweetness. A better understanding of the structure/activity of SrUGT76G1 could provide insights into Reb-A production in stevia plants. To this end, a combination of enzymatic assays and sequencing analysis was performed using two stevia genotypes (Brazilian and Spanish) with contrasting Reb-A production capabilities (off/on). Relative expression of <i>SrUGT76G1</i> gene showed remarkably higher expression (~ threefold) in Spanish samples compared to Brazilian ones. Foliar protein fractions (crude or partially purified extract) from Brazil plants were unable to convert STV into Reb-A under in vitro conditions, resulting in undetectable levels of Reb-A by HPLC. Molecular analyses revealed that the Brazilian <i>SrUGT76G1</i> gene not only presents a premature stop codon, resulting in the absence of PSPG motif responsible for the binding of glycosyl groups, but also exhibits mutations affecting key amino acid residues in the acceptor-binding pocket. These alterations provide a plausible explanation for the Brazilian protein inability to catalyze the transformation of STV into Reb-A.</p><h3 data-test=\"abstract-sub-heading\">Graphical abstract</h3>\n","PeriodicalId":16835,"journal":{"name":"Journal of Plant Biochemistry and Biotechnology","volume":"29 1","pages":""},"PeriodicalIF":1.6000,"publicationDate":"2024-05-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Sequencing analysis and enzyme activity assay of SrUGT76G1 revealed the mechanism toward on/off production of Rebaudioside-A in stevia plants\",\"authors\":\"Simone Ribeiro Lucho, Marcelo Nogueira do Amaral, Valmor João Bianchi, Lorena Almagro, María Ángeles Ferrer, Antonio Asensio Calderón, Eugenia Jacira Bolacel Braga\",\"doi\":\"10.1007/s13562-024-00888-y\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p>Stevia plants are well-known for their ability to synthesize steviol glycosides (SGs), a natural sweetener blend. The principal SGs include stevioside (STV) and Rebaudioside-A (Reb-A), with the latter exhibiting superior sweetness and organoleptic properties. UDP glucosyltransferase-76G1 (UGT76G1) is responsible for converting STV to Reb-A, determining the intensity of sweetness. A better understanding of the structure/activity of SrUGT76G1 could provide insights into Reb-A production in stevia plants. To this end, a combination of enzymatic assays and sequencing analysis was performed using two stevia genotypes (Brazilian and Spanish) with contrasting Reb-A production capabilities (off/on). Relative expression of <i>SrUGT76G1</i> gene showed remarkably higher expression (~ threefold) in Spanish samples compared to Brazilian ones. Foliar protein fractions (crude or partially purified extract) from Brazil plants were unable to convert STV into Reb-A under in vitro conditions, resulting in undetectable levels of Reb-A by HPLC. Molecular analyses revealed that the Brazilian <i>SrUGT76G1</i> gene not only presents a premature stop codon, resulting in the absence of PSPG motif responsible for the binding of glycosyl groups, but also exhibits mutations affecting key amino acid residues in the acceptor-binding pocket. 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Sequencing analysis and enzyme activity assay of SrUGT76G1 revealed the mechanism toward on/off production of Rebaudioside-A in stevia plants
Stevia plants are well-known for their ability to synthesize steviol glycosides (SGs), a natural sweetener blend. The principal SGs include stevioside (STV) and Rebaudioside-A (Reb-A), with the latter exhibiting superior sweetness and organoleptic properties. UDP glucosyltransferase-76G1 (UGT76G1) is responsible for converting STV to Reb-A, determining the intensity of sweetness. A better understanding of the structure/activity of SrUGT76G1 could provide insights into Reb-A production in stevia plants. To this end, a combination of enzymatic assays and sequencing analysis was performed using two stevia genotypes (Brazilian and Spanish) with contrasting Reb-A production capabilities (off/on). Relative expression of SrUGT76G1 gene showed remarkably higher expression (~ threefold) in Spanish samples compared to Brazilian ones. Foliar protein fractions (crude or partially purified extract) from Brazil plants were unable to convert STV into Reb-A under in vitro conditions, resulting in undetectable levels of Reb-A by HPLC. Molecular analyses revealed that the Brazilian SrUGT76G1 gene not only presents a premature stop codon, resulting in the absence of PSPG motif responsible for the binding of glycosyl groups, but also exhibits mutations affecting key amino acid residues in the acceptor-binding pocket. These alterations provide a plausible explanation for the Brazilian protein inability to catalyze the transformation of STV into Reb-A.
期刊介绍:
The Journal publishes review articles, research papers, short communications and commentaries in the areas of plant biochemistry, plant molecular biology, microbial and molecular genetics, DNA finger printing, micropropagation, and plant biotechnology including plant genetic engineering, new molecular tools and techniques, genomics & bioinformatics.