Abstract:
Polyamines (PAs) are small cationic metabolites, essential for eukaryotic organisms and many prokaryotes. In plants, PAs are important for growth, development, gene expression, and abiotic and biotic stress responses, and their levels are finetuned through transcriptional and translational regulation of PA synthesis and catabolic enzymes (Chen et al., 2019). Synthesis of the most abundant PAs in plants, putrescine, spermidine, and spermine, predominantly starts with decarboxylation of arginine to agmatine by arginine decarboxylase (ADC, typically encoded by two genes, ADC1 and ADC2). Interestingly, all examined land plant ADC transcripts contain a highly conserved GC-rich ~50 bp sequence in their 5’ untranslated region (5’UTRs), termed the “ADC-box” (Wu et al., 2019). Using transient reporter assays, the ADC- box was found to inhibit translation of the downstream coding sequence, possibly by forming an RNA secondary structure that prevents ribosomal scanning. In addition to the ADC-box, ADC 5’UTRs contain a small uORF ~150 bp 5’ to the ADC-box; however, no clear role for this uORF was found (Wu et al., 2019).
This work aimed to determine if the ADC-box and uORF regulate ADC translation in planta and investigate how the ADC-box translational regulatory mechanism functions. CRISPR/Cas9 mutagenesis of ADC-boxes in tomato, Arabidopsis thaliana, and Marchantia polymorpha (relative of the first land plants) was conducted. As a proxy for measuring in planta ADC protein levels, a previously established liquid chromatography-mass spectrometry (LCMS)- based ADC activity assay (Wu et al., 2019) was further optimised, improving sensitivity ~600- fold and number of measured PA-related metabolites. Increased ADC activity and levels of some PA-related metabolites were found in tomato, A. thaliana, and M. polymorpha adc-box mutants, demonstrating that the ADC-box is a repressor of ADC translation in planta and that its role has likely been conserved throughout land plant evolution. The tomato ADC-box RNA secondary structures were experimentally and computationally determined using SHAPE analysis, finding a triple hairpin ADC-box structure. Through analysing ADC activity levels across adc-box mutants, it seems that the central hairpin, which is conserved across vascular and early non-vascular land plants, is essential for ADC-box functionality. CRISPR/Cas9 mutagenesis of the tomato ADC uORFs resulted in reduced ADC activity compared to wildtype (WT) and a shorter plant phenotype with fewer leaves and flowers, suggesting that this uORF not only positively regulates ADC translation but is also essential for plant development. Overall, these findings suggest that plant ADC translation is both negatively and positively regulated by two highly conserved 5’UTR elements that finetune ADC protein levels and maintain PA homeostasis under various conditions.
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In addition to influencing PA levels, ADC activity also correlated with levels of phenolamides, compounds made from PA conjugation to hydroxycinnamic acids (HCAs), which are both antimicrobial compounds and substrates for the formation of physical barriers in roots (i.e., lignin and suberin). Interestingly, the broad-host bacterial pathogen Ralstonia solanacearum, which infects plants via their roots, injects a conserved effector protein (RipTAL) into host cells to transcriptionally activate plant ADC genes and boost PA levels (Wu et al., 2019). Through this work, we hypothesise that R. solanacearum uses RipTALs to manipulate host phenolamide levels to either alter the plant microbiome or production of physical barriers in the roots. In brief, through expanding our capabilities of PA measurement by LCMS and our understanding of how ADCs influence the PA network, new experimental approaches and hypotheses as to how R. solanacearum benefits from a RipTAL-induced PA boost have been developed.
Polyamine