Abstract
ACAULIS5 (ACL5) encodes thermospermine synthase in Arabidopsis and its loss-of-function mutant acl5 shows excess xylem differentiation and severe dwarfism. SAC51 encodes a basic helix-loop-helix (bHLH) protein and was identified from sac51-d, a dominant suppressor mutant of acl5, which restores the wild-type phenotype without thermospermine. The 5′ leader of the SAC51 mRNA contains multiple upstream open-reading frames (uORFs) and sac51-d has a premature stop codon in the fourth uORF. This uORF is conserved among SAC51 family genes in vascular plants. According to the GUS reporter assay, the SAC51 promoter was not responsive to thermospermine but the SAC51 5′ leader fused to the constitutive 35S promoter enhanced the GUS activity in response to thermospermine. Disruption experiments of each start codon of the SAC51 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 SAC51 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.
Highlights
Thermospermine is a structural isomer of spermine and present in some bacteria and ubiquitously in plants (Minguet et al, 2008; Fuell et al, 2010; Takano et al, 2012)
To confirm whether more upstream regions are responsive to thermospermine or not, we generated transgenic lines carrying the GUS reporter gene under the control of the SAC51 promoter or that followed by the first exon and intron with its splice acceptor site (Figure 1A) and examined the GUS activity
The results showed that the SAC51 promoter directed the GUS expression sharply to vascular cells (Figure 1B) and neither only the promoter nor the promoter followed by the first exon and intron was responsive to thermospermine (Figure 1C)
Summary
Thermospermine is a structural isomer of spermine and present in some bacteria and ubiquitously in plants (Minguet et al, 2008; Fuell et al, 2010; Takano et al, 2012). In Arabidopsis thaliana, the ACAULIS5 (ACL5) gene encodes thermospermine synthase (Knott et al, 2007; Kakehi et al, 2008) and is expressed exclusively in differentiating xylem vessels (Clay and Nelson, 2005). Expression of the ACL5 gene is upregulated by auxin but down-regulated by thermospermine while the excess xylem phenotype of the acl mutant is enhanced by synthetic and persistent auxin analogs but suppressed by thermospermine, indicating that both thermospermine synthesis and auxin-induced xylem differentiation are under negative feedback control by thermospermine (Yoshimoto et al, 2012). We have developed an artificial inhibitor of thermospermine synthase, named xylemin, which showed a potent inducing effect of ectopic xylem formation in tobacco leaves in the presence of a synthetic auxin analog (Yoshimoto et al, 2016). In view of biotechnological applications, the combinatorial use of these plant growth regulators might have a potential for the control of woody biomass
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