Improved ballistic transient transformation conditions for tomato fruit allow identification of organ‐specific contributions of I‐box and G‐box to the RBCS2 promoter activity

An improved protocol for the ballistic transient transformation of developing tomato (Lycopersicon esculentum) fruits is reported, which allows high-resolution cis-analysis of fruit-specific transcriptional activation. The tomato RBCS2 promoter fused to the firefly luciferase gene was used as a model system for this study. Osmotic treatment of fruit slices before, during and after particle bombardment, together with the optimization of bombardment conditions, resulted in a 100-fold increase in RBCS2 promoter-driven transient luciferase expression compared with previously reported protocols. Under these conditions, the transformed RBCS2 promoter was shown to be properly regulated in a developmental fashion. A cis-analysis of the RBCS2 promoter was performed. A 37 bp domain is required for high-level RBCS2 promoter activity both in leaves and young fruits. Two conserved sequence elements within this domain, an I-box element (GATAAG) and a G-box element (CACGTG), are necessary for its activity in leaves. In contrast, in young fruits, the G-box is the single dominant cis-acting element. These findings are discussed with respect to the proposed functions of G-box and I-box binding factors in regulating plant genes in different organs.