IDENTIFICATION OF A NEGATIVE FEEDBACK REGULATORY LOOP CONTROLLING VASCULAR DEVELOPMENT

The acaulis5 (acl5) loss-of-function mutant, also known as thickvein, is dwarf and characterized by the formation of an increased number of veins and vascular elements in leaves and stems (Hanzawa et al., 1997, 2000; Clay et al., 2005). The ACL5 gene, encoding a polyamine synthase producing thermospermine, an isomer of spermine, has been proposed to control xylem specification by preventing premature cell death of tracheary elements (Knott et al., 2007; Muniz et al., 2008). Interestingly, we found that higher levels of ACL5, likely resulting in increased thermospermine concentration, significantly delay or completely inhibit the differentiation of procambial cells into tracheary elements in leaf as well as in primary and secondary vasculature of stem and hypocotyl. Consistently with the up-regulation shown in acl5 (Imai et al. 2006; Kakhei et al., 2008), we found a down-regulation of the expression of the HD-ZIPIII transcription factor ATHB8, a marker of preprocambial cell state that accurately predicts sites of leaf vein formation and promotes proliferation and differentiation of vascular precursor cells (Baima et al., 2001; Donner et al., 2009), in plants with increased ACL5 expression. These observations suggest that the vascular defects observed in both mutant and over-expressing plants may, at least in part, depend on the level of HD-ZIPIII activity. Indeed, a careful genetic analysis of acl5 in combination with athb8 and other hd-zipiii mutants indicated that some of the developmental defects caused by the lack of ACL5 activity are abolished by the loss of HD-ZIPIII transcription factors. On the other hand, we could demonstrate by different approaches, including ChIP, that ACL5 is directly regulated in a positive manner by ATHB8. These findings have been integrated in a model in which HD-ZIPIII and ACL5 genes establish a negative feedback loop necessary for proper regulation of vascular development.