Characterization of post-transcriptional and post-translational regulatory mechanisms of secondary cell wall deposition in the model grass Setaria viridis

• Grant number: 24/10125-0
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: December 01, 2024
• End date: October 31, 2027
• Field of knowledge: Biological Sciences - Botany - Pant Physiology
• Principal Investigator: Igor Cesarino
• Grantee: Gabriel Garon Carvalho
• 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

The deposition of the secondary cell wall in grasses relies on the coordinated activity of metabolic pathways responsible for synthesizing lignin, cellulose, hemicelluloses, and a variety of phenolic compounds derived from the shikimate, phenylpropanoid, and flavonoid pathways. These components provide essential structural and functional properties to plant tissues and ultimately determine biomass recalcitrance in biotechnological applications. In monocots, both the composition and the metabolic organization associated with the secondary wall display distinctive features, such as the incorporation of tricin. Although transcriptional networks related to secondary cell wall deposition have been extensively described, the post-transcriptional and post-translational mechanisms involved in this process remain poorly characterized in grasses. Accordingly, the objective of this project is to characterize post-transcriptional and post-translational regulatory mechanisms associated with secondary cell wall deposition in Setaria viridis. To this end, we will first establish a global landscape of alternative splicing using RNA-seq data generated from eight organs that exhibit distinct patterns of secondary wall deposition. This analysis will enable the identification of event classes, differentially regulated splicing events, and associated metabolic pathways. Events of interest will be experimentally validated by RT-PCR. In parallel, miRNA-mRNA regulatory modules identified through microtranscriptome and degradome analyses will be validated via transient expression in Nicotiana benthamiana, allowing us to determine the contribution of these miRNAs to the post-transcriptional regulation of genes involved in the process. The second stage of the project focuses on investigating post-translational mechanisms by characterizing protein-protein interactions among enzymes of the tricin biosynthetic pathway. These interactions will be assessed using split GAL4 RUBY screening and validated by BiFC, enabling us to determine the functional organization of these enzymes and to investigate the formation of multiprotein complexes related to secondary wall deposition. The results will provide an integrated overview of the post-transcriptional and post-translational mechanisms that regulate secondary cell wall deposition in grasses, offering valuable insights for biotechnological strategies aimed at improving the utilization of plant biomass.