Uncovering the evolution of anthocyanin-related glutathione S-transferase (arGSTs) across plant lineages

Najnin Khatun ¹, Milan Borchert ², Boas Pucker ³

Institute of plant biology, Technische Universität Braunschweig , Germany, ¹, Doctoral student ², Assistant Professor ³

Water-soluble anthocyanins are an important subclass of flavonoids that contribute to the vibrant color observed in fruits, flowers, leaves and stems. They serve many functions in plants such as protection from UV radiation, pigmentation, stress response, pollination, and defense against various pathogens. Anthocyanins are also known to exhibit antioxidant properties, which are beneficial for human health. Previously, anthocyanin-related glutathione S-transferases (arGSTs) were thought to play a role in anthocyanin intracellular transport, but a recent study demonstrated that arGSTs have enzymatic functions in anthocyanin biosynthesis instead. The involvement of GST in anthocyanin transport was initially established in Zea mays through the study of bronze-2 (bz2) mutant. Numerous arGST proteins have been identified across diverse plant species, including TT19 in Arabidopsis thaliana, RsGST1 in Raphanus sativus, GST1 in Perilla frutescens, VvGST1 and VvGST4 in Vitis vinifera, AcGST1 in Actinidia chinensis. Our study aims to clarify the evolutionary relationships of arGSTs across diverse plant species involved in the anthocyanin biosynthesis pathway. To achieve this, We began our study by identifying and analyzing arGST sequences from diverse plant species using gene identification through KIPEs, transcriptomic analysis using kallisto, functional annotation based on Arabidopsis thaliana, and co-expression analysis. After performing the co-expression analysis, it was observed that TT19 and BZ2 are co-expressed with key anthocyanin biosynthetic genes such as ANS (anthocyanidin synthase) and DFR (dihydroflavonol 4-reductase). To understand the evolutionary relationships among multiple arGSTs sequences, a phylogenetic tree based on arGSTs protein-encoding sequences from diverse plant species was constructed. Phylogenetic analysis revealed two distinct and independently evolved arGST lineages, named after their representative sequences as the BZ2 group and the TT19/AN9 group. These lineages are not orthologous, indicating they arose through separate evolutionary events to perform analogous functions in the anthocyanin biosynthesis pathway. Instead, they evolved independently, representing separate evolutionary adaptations to fulfill similar roles within the anthocyanin biosynthesis pathway. Interestingly, BZ2 sequences are predominantly found in the Poaceae family but absent in other Poales order members. The Poaceae family divides species into two major subclades: PACMAD and BOP. The BZ2 sequences from the BOP and PACMAD clades clustered together within a Poaceae-specific clade, exhibiting more than 85% conservation in functionally important amino acid residues. Keywords: Anthocyanin biosynthesis; Anthocyanin-related glutathione S-transferases (arGST); Evolution

Main author career stage: Master student

Contribution type: Poster

First choice session: 1. Systematics, phylogenetics, biogeography and evolution

Second choice session: 5. Genetics, genomics, and bioinformatics