Investigating the role of two small uORFs in the translational control of ATHB8 in Arabidopsis

The HD-ZIP III gene family (ATHB-8, CNA, PHB, PHV, REV) regulates fundamental plant processes such as meristem initiation, leaf and vascular development, and organ polarity (Prigge et al., 2005). Comprehensive genetic and molecular analysis in the model plant Arabidopsis have revealed that these processes are regulated through a complex pattern of overlapping, distinct, and antagonistic roles of each member of the HD-ZIP III family. Evolutionary studies suggest that HD- ZIP III transcription factors have been essential components of the developmental tool-kit that has made possible key innovations in land plant evolution (Floyd et al., 2006; Prigge and Clark, 2006; Floyd and Bowman, 2007). The activity of HD-ZIP III family transcription factors is tightly controlled at several different levels by many different mechanisms including sequence complementarity with evolutionary conserved miRNAs, homo- and hetero-dimerisation of the HD- ZIP III factors themself and with LITTLE ZIPPER (ZPR) proteins, protein interaction with AP2- like transcription factors, and by an as yet unidentified lipid ligand. Moreover, two recent studies have suggested that HD-ZIP III genes could also be regulated at translational level (Pinon et al., 2008; Yao et al., 2008). Intriguingly, HD-ZIP III genes transcripts possess two intrinsic characteristics, i.e. the presence of small upstream ORFs (uORFs) in the 5' leader sequence and a binding site for miRNA recognition, that have been suggested to affect the sensitivity of some transcripts to ribosome heterogeneity and influence their translation rate (Byrne, 2009). The potential to control the translational efficiency of HD-ZIP III gene transcripts would be of great agronomical and biotechnological interest due to the fundamental role played by these genes in plant growth and architecture. In particular, as overexpression of ATHB8 in Arabidopsis promotes the proliferation and differentiation of vascular precursor cells and the production of xylem (Baima et al., 2001), enhancement of ATHB8 translation could be used in alternative as a tool to improve vascularisation and wood production in species of agronomical interest. In this study we report the investigation, by reporter-GUS analysis and functional study of transgenic Arabidopsis plants, of the involvement of the two uORFs present in the ATHB8 transcript in the translational control of this gene.