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.
Baum
K
K
Institute for General Botany, Applied Molecular Biology of Plants I, University of Hamburg, Germany.
Gröning
B
B
Meier
I
I
D000818:Animals; D019470:Biolistics / Q000379:methods; D001517:Coleoptera; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D005966:Glucuronidase / Q000096:biosynthesis; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D018551:Solanum lycopersicum / Q000235:genetics / Q000502:physiology*; D009928:Organ Specificity; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012273:Ribulose-Bisphosphate Carboxylase / Q000096:biosynthesis* / Q000235:genetics*; D014170:Transformation, Genetic
D011993:Recombinant Fusion Proteins; D008156:Luciferases; D005966:Glucuronidase; D012273:Ribulose-Bisphosphate Carboxylase