AUTHOR=Ishitsuka Soichi , Yamamoto Mai , Miyamoto Minaho , Kuwashiro Yoshitaka , Imai Akihiro , Motose Hiroyasu , Takahashi Taku 

TITLE=Complexity and Conservation of Thermospermine-Responsive uORFs of SAC51 Family Genes in Angiosperms

JOURNAL=Frontiers in Plant Science

VOLUME=10

YEAR=2019

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2019.00564

DOI=10.3389/fpls.2019.00564

ISSN=1664-462X

ABSTRACT=<p><italic>ACAULIS5</italic> (<italic>ACL5</italic>) encodes thermospermine synthase in Arabidopsis and its loss-of-function mutant <italic>acl5</italic> shows excess xylem differentiation and severe dwarfism. <italic>SAC51</italic> encodes a basic helix-loop-helix (bHLH) protein and was identified from <italic>sac51-d</italic>, a dominant suppressor mutant of <italic>acl5</italic>, which restores the wild-type phenotype without thermospermine. The 5′ leader of the <italic>SAC51</italic> mRNA contains multiple upstream open-reading frames (uORFs) and <italic>sac51-d</italic> has a premature stop codon in the fourth uORF. This uORF is conserved among <italic>SAC51</italic> family genes in vascular plants. According to the GUS reporter assay, the <italic>SAC51</italic> promoter was not responsive to thermospermine but the <italic>SAC51</italic> 5′ leader fused to the constitutive 35S promoter enhanced the GUS activity in response to thermospermine. Disruption experiments of each start codon of the <italic>SAC51</italic> uORFs revealed that uORF4 and uORF6 whose start codon corresponds to the second methionine codon of uORF4 had an inhibitory effect on the main ORF translation while the other four uORFs rather had a stimulatory effect. The response of the 5′ leader to thermospermine was retained after disruption of each one of six start codons of these uORFs but abolished by mutating both uORF4 and uORF6 start codons, suggesting the importance of the C-terminal sequence shared by these uORFs in the action of thermospermine. We introduced GUS fusions with 5′ leaders of <italic>SAC51</italic> family genes from other angiosperm species into Arabidopsis and found that all 5′ leaders responsive to thermospermine, so far examined, contained these two conserved, and overlapping uORFs.</p>