Characterisation and chemical inhibition of a novel regulatory module controlling chloroplast protein translation

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The NOT4 E3 ligase is conserved across eukaryotes and consists of a unique RING and RNA Recognition Motif (RRM) domain pairing that places its function at the interface of proteolysis and RNA biology. Plant genomes encode for multiple NOT4 paralogs, compared to single variants found in yeast and animal genomes, which indicates diversification of function for this protein in the plant kingdom. We recently showed that one of the 3 Arabidopsis NOT4 homologs, NOT4A, has diverged towards a key role in regulating chloroplast function, through promoting the expression of the chloroplast targeted protein PGR3 (Bailey et al 2021 Nature Communications). PGR3 is required for stabilisation and translation of several chloroplast RNAs, including those that encode for core photosynthetic components and subunits of the plastid ribosome. As such, the NOT4A-PGR3 signalling module is important for ensuring chloroplast proteostasis by influencing plastid ribosome biogenesis and effective assembly of photosynthetic machineries in the thylakoids. Despite identifying this NOT4A-PGR3 signalling module, we still do not know the underlying mechanisms that connect NOT4A activity to PGR3 production. Here we will use a range of molecular, cell biology and genetic approaches to decipher the molecular connection between NOT4A and PGR3. We will determine how NOT4A ensures efficient PGR3 expression and explore how this contributes to the dynamics of chloroplast function during growth, development and stress response. Furthermore, in conjunction with industrial partner Syngenta we will explore whether this module can be chemically targeted to disrupt chloroplast protein biogenesis. As such, this project will shed new light onto a mechanism regulating chloroplast form and function, and determine if this mechanism can be exploited for the identification of new herbicide candidates.