Molecular Analysis of the rbcS Multigene Family

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This project will investigate the molecular mechanism involved in the differential expression of the multigene family members for the small subunit (SSU) of ribulose.1, 5.bisphosphate carboxylase (rbcS genes) using tomato as a model system. Tomato has five rbcs genes rbcS.1, rbcS.2, rbcS.3A and rbcS.3C, which encode three iso.variant SSU proteins. The five rbcS genes are differentially transcribed in cotyledons, leaves and fruit. Several 5' DNA sequences which are functionally undefined are conserved among the five tomato rbcS genes which share similar developmental and organ.specific expression pattern. In addition, mRNAs from the individual tomato rbcS genes accumulate to substantially different levels in different tissues, but this pattern is not reflected in the accumulation of the three iso. variant SSU proteins. We will examine rbcS cis.acting DNA regions to define their role in the developmental and organ. specific on.off control of transcription and examine how they interact. We will identify protein.binding domains by DNasel footprint analysis to determine if the differential transcription of the rbcS genes is correlated with differences in the DNA: protein binding pattern of 5' DNA fragments. This will complement our analysis of cis.acting DNA sequences in transgenic plants and transient assays. Furthermore, transcription run.on assays and quantitative hybridizations will be used to assess the extent to which transcriptional regulation of the five rbcS genes establishes the relative levels of their mRNAs. At the translational level, we will test if variations in the length of the rbcS 5' untranslated regions result in the differential accumulation of iso.variant SSU proteins. To attain further information on the role of differential rbcS express in fruit, we will take advantage of a fruit.specific promoter for the production of antisense.RNA to block rbcS.1 and rbcS.2 expression. Finally, we will activate the normally inactive rbcS.3A gene to produce the iso.variant 3 SSU protein in fruit to determine what effect, if any, this has on organ development. Developmental, organ.specific and light.dependent regulation of transcription and translation are important control steps in the expression of nuclear genes encoding photosynthetic proteins found in the chloroplasts of higher plants. The results of this study should provide greater insights into those processes.