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

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.

Norspermine substitutes for thermospermine in the control of stem elongation in Arabidopsis thaliana.

Thermospermine is a structural isomer of spermine and is required for stem elongation in Arabidopsis thaliana. We noted the C3C3 arrangement of carbon chains in thermospermine (C3C3C4), which is not present in spermine (C3C4C3), and examined if it is functionally replaced with norspermine (C3C3C3) or not. Exogenous application of norspermine to acl5, a mutant defective in the synthesis of thermospermine, partially suppressed its dwarf phenotype, and down-regulated the level of the acl5 transcript which is much higher than that of the ACL5 transcript in the wild type. Furthermore, in the Zinnia culture, differentiation of mesophyll cells into tracheary elements was blocked by thermospermine and norspermine but not by spermine. Our results indicate that norspermine can functionally substitute for thermospermine.

Thermospermine is required for stem elongation in Arabidopsis thaliana.

Loss-of-function mutants of the ACAULIS5 (ACL5) gene in Arabidopsis thaliana have severe defects in stem elongation. ACL5 was previously reported as encoding a spermine synthase. A more recent study, however, showed that the bacterial expressed recombinant ACL5 protein catalyzes the conversion of spermidine to thermospermine, a structural isomer of spermine, rather than to spermine. In the present study, we found that thermospermine was detected in wild-type seedlings but was not detectable in the acl5-1 mutant. We further examined the effect of exogenous application of these isomers on the growth of acl5-1. Daily application of 0.1 mM thermospermine onto the shoot apex partially rescued the dwarf phenotype of acl5-1, while that of spermine had no effects on the morphology of the mutant. The acl5-1 transcript level in acl5-1 seedlings, which is much higher than the ACL5 transcript level in wild-type seedlings, was reduced by exogenous thermospermine. Thus we conclude that thermospermine is indeed produced through the action of ACL5 and required for stem elongation in Arabidopsis.

A semi-dominant mutation in the ribosomal protein L10 gene suppresses the dwarf phenotype of the acl5 mutant in Arabidopsis thaliana.

Disruption of the Arabidopsis thaliana ACAULIS5 (ACL5) gene, which has recently been shown to encode thermospermine synthase, results in a severe dwarf phenotype. A previous study showed that sac51-d, a dominant suppressor mutant of acl5-1, has a premature termination codon in an upstream open reading frame (ORF) of SAC51, which encodes a putative transcription factor, and suggested the involvement of upstream ORF-mediated translational control in ACL5-dependent stem elongation. Here we report the identification of a gene responsible for sac52-d, another semi-dominant suppressor mutant of acl5-1. SAC52 encodes ribosomal protein L10 (RPL10A), which is highly conserved among eukaryotes and implicated in translational regulation. Transformation of acl5-1 mutants with a genomic fragment containing the sac52-d allele rescued the dwarf phenotype of acl5-1. GUS reporter activity under the control of a SAC51 promoter with its upstream ORF was higher in acl5-1 sac52-d than in acl5-1, suggesting that suppression of the acl5-1 phenotype by sac52-d is attributable, in part, to enhanced translation of certain transcripts including SAC51. We also found that a T-DNA insertion allele of SAC52/RPL10A causes lethality in the female gametophyte.