Hormone-Mediated Gene-Specific Translation Regulation

The central role of translation regulation in the control of critical cellular processes has long been recognized. Yet the systematic exploration of quantitative changes in translation at a genome-wide scale in response to specific stimuli has only recently become technically feasible. Using a genetic approach, we have identified new Arabidopsis weak-ethylene insensitive mutants that also display defects in translation, which suggested the existence of a previously unknown molecular module involved in ethylene-mediated translation regulation of components of this signaling pathway. To explore this link in detail, we implemented for Arabidopsis the ribosome-footprinting technology, which enables the study of translation at a whole-genome level at single codon resolution[1]. Using ribosome-footprinting we examined the effects of short exposure to ethylene on the Arabidopsis translatome looking for ethylene-triggered changes in translation rates that could not be explained by changes in transcript levels. The results of this research, in combination with the characterization of a subset of the aforementioned weak-ethylene insensitive mutants that are defective in the UPF genes (core-components of the nonsense-mediated mRNA decay machinery), uncovered a translation-based branch of the ethylene signaling pathway[2]. In the presence of ethylene, translation of a negative regulator of ethylene signaling EBF2 is repressed, despite induced transcription of this gene. These translational effects of ethylene require the long 3´UTR of EBF2 (3´EBF2), which is recognized by the C-terminal end of the key ethylene-signaling protein EIN2 (EIN2C) in the cytoplasm once EIN2C is released from the ER-membrane by proteolytic cleavage. EIN2C binds the 3´EBF2, recruits the UPF proteins and moves to P-bodies, where the translation of EBF2 in inhibited despite its mRNA accumulation. Once the ethylene signal is withdrawn, the translation of the stored EBF2 mRNAs is resumed, thus rapidly dampening the ethylene response. These findings represent a mechanistic paradigm of gene-specific regulation of translation in response to a key growth regulator. Translation regulatory elements can be located in both 3′ and 5′ UTRs. We are now focusing on the ead1 and ead2 mutants, another set of ethylene-signaling mutants defective in translational regulation. Ribosome-footprinting on the ead1 mutant revealed an accumulation of translating ribosomes in the 5´UTRs of uORF-containing genes and reduction in the levels of ribosomes in the main ORF. The mutant is also impaired in the translation of GFP when this reporter is fused to WT 5´UTR of potential EAD1 targets but not when GFP is fused to the uORF-less versions of the same 5´UTRs. Our hypothesis is that EAD1/2 work as a complex that is required for the efficient translation of mRNAs that have common structural (complex 5´UTR with uORFs) and functional (regulation of key cellular processes) features. We are working towards the identification of the conditions where the EAD1 regulation of translation is required. [1] Ingolia, N. et al. (2009) Genome-Wide Analysis in Vivo of Translation with Nucleotide Resolution Using Ribosome Profiling. Science, 324; 218-222 [2] Merchante, C. et al. (2015) Gene-Specific Translation Regulation Mediated by the Hormone-Signaling Molecule EIN2. Cell, 163(3): 684-697

NAC-MYB basedtranscriptional networkinvolved in the regulation of phenylalanine biosynthesis in P. pinaster

P2-2 NAC-MYB-BASED TRANSCRIPCIONAL NETWORK INVOLVED IN THE REGULATION OF PHENYLALANINE BIOSYNTHESIS IN P. PINASTER Mª Belén Pascual, Rafael A. Cañas, Blanca Craven-Bartle, Francisco M. Cánovas and Concepción Ávila Departamento de Biología Molecular y Bioquímica. Facultad de Ciencias. Universidad de Málaga. Campus de teatinos s/n, Málaga, Spain Email: cavila@uma.es Conifer trees divert large quantities of carbon into the biosynthesis of phenylpropanoids, particularly to generate lignin, an important constituent of wood. Since phenylalanine is the precursor for phenylpropanoid biosynthesis, the precise regulation of phenylalanine synthesis and use should occur simultaneously. This crucial pathway is finely regulated primarily at the transcriptional level. Transcriptome analyses indicate that the transcription factors (TFs) preferentially expressed during wood formation in plants belong to the MYB and NAC families. Craven-Bartle et al. (2013) have shown that Myb8 is a candidate regulator of key genes in phenylalanine biosynthesis involved in the supply of the phenylpropane carbon skeleton necessary for lignin biosynthesis. This TF is able to bind AC elements present in the promoter regions of these genes to activate transcription. In Arabidopsis, the transcriptional network controlling secondary cell wall involves NAC-domain regulators operating upstream Myb transcription factors. We have identified in the P. pinaster genome three NAC proteins as potential candidates to be involved in vascular development. One of them, PpNAC1 is expressed both in xylem and compression wood from adult trees and has been thoroughly characterized. Its role upstream the transcriptional network involving Myb8 will be discussed. The understanding of the transcriptional regulatory network associated to phenylpropanoids and lignin biosynthesis in conifers is crucial for future applications in tree improvement and sustainable forest management.