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.