Functional characterization of SvUGTs potentially involved in tricin glycosylation in Setaria viridis

• Grant number: 22/12496-0
• Support Opportunities: Scholarships in Brazil - Doctorate
• Effective date (Start): December 01, 2022
• Effective date (End): April 30, 2026
• Field of knowledge: Biological Sciences - Botany - Pant Physiology
• Principal Investigator: Igor Cesarino
• Grantee: Arthur de Barros Rates
• Host Institution: Instituto de Biociências (IB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated research grant: 21/06142-8 - Steering the shikimate/phenylpropanoid pathway: from gene function discovery to green factories, AP.BIOEN.JP2

Abstract

Lignin is an aromatic polymer abundantly present in plant secondary cell walls. Consequently, it builds up a large portion of plant biomass. Because of its recalcitrant nature, lignin is considered an obstacle to the extraction of fermentable sugars from plant biomass to produce downstream products, which are increasingly regarded as a sustainable/renewable alternative to fossil resources. Recently, the flavone tricin was identified as an authentic lignin monomer present mainly in grass lignin, where it functions as an initial polymerization site. Therefore, tricin may be a prime target for bioengineering plants towards improved delignification. Previously, we observed that leaves and stems of the model C4 grass Setaria viridis accumulate vastly different amounts of tricin incorporated into their lignins even though its biosynthetic genes are similarly expressed in both tissues. Given the role of plant UDP glycosyltransferases (UGTs) in the storage of phenylpropanoids, we hypothesized that these proteins may play a major role in the regulation of tricin incorporation into the lignin polymer. This project aims to identify and characterize SvUGTsinvolved in the glycosylation of tricin in Setaria viridis, a model C4 grass used in biofuel studies, and investigate their roles controlling tricin incorporation into lignin. First, a genomewide characterization of the SvUGT family will be performed. Second, SvUGT genes fromthe UGT706 and UGT707 subfamilies (previously implicated in flavone and flavonol glycosylation) which are preferentially expressed stems will be heterologously expressed in E. coli and the recombinant proteins will be characterized for their ability to glycosylate tricin in vitro. The SvUGT gene whose corresponding enzyme shows the best kinetic parameters towards tricin glycosylation will be further overexpressed to evaluate its function in planta. The resulting transgenic lines will be characterized for biomass parameters, cell wall deposition, tricin incorporation into lignin and changes in the phenolic profile. Finally, the effect of SvUGT overexpression on biomass recalcitrance will be evaluated using saccharification assays. This work will provide not only novel knowledge regarding tricin metabolism and its regulation but also potential targets for future bioengineering strategies.