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 dev eloping tomato (Lycopersicon esculentum) fruits is reported, which all ows high-resolution cis-analysis of fruit-specific transcriptional act ivation. The tomato RBCS2 promoter fused to the firefly luciferase gen e was used as a model system for this study. Osmotic treatment of frui t slices before, during and after particle bombardment, together with the optimization of bombardment conditions, resulted in a 100-fold inc rease in RBCS2 promoter-driven transient luciferase expression compare d with previously reported protocols. Under these conditions, the tran sformed RBCS2 promoter was shown to be properly regulated in a develop mental fashion. A cis-analysis of the RBCS2 promoter was performed. A 37 bp domain is required for high-level RBCS2 promoter activity both i n leaves and young fruits. Two conserved sequence elements within this domain, an I-box element (GATAAG) and a G-box element (CACGTG), are n ecessary for its activity in leaves. In contrast, in young fruits, the G-box is the single dominant cis-acting element. These findings are d iscussed with respect to the proposed functions of G-box and I-box bin ding factors in regulating plant genes in different organs.