Transcription factor RAP2.2 and its interacting partner SINAT2: stable elements in the carotenogenesis of Arabidopsis leaves

The promoter of phytoene synthase, the first specific enzyme of carotenoid biosynthesis, shows two main regulatory regions: a G-box-containing region located near the TATA box, and a TATA box distal region containing the cis-acting element ATCTA, which mediates strong basal promoter activity. This second element was also present in the promoter of phytoene desaturase, the next step of the carotenoid pathway, suggesting a common regulatory mechanism. In this work, we demonstrate that AtRAP2.2, a member of the APETALA2 (AP2)/ethylene-responsive element-binding protein transcription factor family, binds to the ATCTA element. In Arabidopsis (Arabidopsis thaliana) leaves, AtRAP2.2 transcript and protein levels were tightly controlled as indicated by unchanged transcript and protein levels in T-DNA insertion mutants in the AtRAP2.2 promoter and 5' untranslated region and the lack of change in AtRAP2.2 protein levels in lines strongly overexpressing the AtRAP2.2 transcript. Homozygous loss-of-function mutants could not be obtained for the AtRAP2.2 5' untranslated region T-DNA insertion line indicating a lethal phenotype. In AtRAP2.2 overexpression lines, modest changes in phytoene synthase and phytoene desaturase transcripts were only observed in root-derived calli, which consequently showed a reduction in carotenoid content. The RING finger protein SEVEN IN ABSENTIA OF ARABIDOPSIS2 (SINAT2) was identified as an AtRAP2.2 interaction partner using a two-hybrid approach. The structure of SINAT2 and related proteins of Arabidopsis show homology to the SEVEN IN ABSENTIA protein of Drosophila that is involved in proteasome-mediated regulation in a variety of developmental processes. The action of SINAT2 may explain the recalcitrance of AtRAP2.2 protein levels to change by altering AtRAP2.2 transcription.