Identification of cis-regulatory elements related to water-deficit and low-temperature stress within the promoter of Citrus sinensis APETALA1

Citrus flowering is promoted by water-deficit and low-temperature stress and inhibited by gibberellins, providing a unique system to investigate regulation of floral development. In silico analysis, using plant cis-acting regulatory element databases, PLACE and PlantCARE, was conducted to compare transcriptional regulation of LEAFY (LFY), APETALA1 (AP1), and TERMINAL FLOWER (TFL) by water deficit, low temperature, abscisic acid and gibberellins in C. sinensis, Arabidopsis thaliana and Populus trichocarpa. A striking enrichment of response elements upregulated by water deficit was found in the C. sinensis AP1 (CsAP1) promoter, but neither the A. thaliana AP1 (AtAP1), nor P. trichocarpa AP1-1 (PtAP1-1) promoter. Notably, a tandem array of three response elements, each containing a LFY binding site, coupling element 3 site (CE3), and dehydration-responsive element (DRE), was found within a 100-bp region of the CsAP1 promoter. The CE3 and DRE sites are associated, respectively, with abscisic acid (ABA)-dependent and ABA-independent signaling pathways induced by water deficit. The CE3 sequence is present in the AtAP1, but not PtAP1-1, promoter. The DRE site is lacking in both AtAP1 and PtAP1-1. Three LFY binding sites are located in the CsAP1 and AtAP1 promoters, with only one in PtAP1-1. Multiple C-repeat binding factor (CBF) response elements associated with low-temperature responses through an ABA-independent signaling pathway are present in the CsAP1 and AtAP1 promoters, but not PtAP1-1. The unique 100-bp regulon of the CsAP1 promoter suggests that flower formation in C. sinensis in response to water-deficit and low-temperature stress is mediated at AP1 through ABA-dependent and ABA-independent pathways.