Submitted to: Plant Journal
Publication Type: Peer reviewed journal
Publication Acceptance Date: 5/30/2008
Publication Date: 6/14/2008
Citation: Wang, S., Liu, J., Feng, Y., Niu, X., Giovannoni, J.J., Liu, Y. 2008. Altered Plastid Levels and Potential for Improved Fruit Nutrient Content via Down-regulation of the Tomato DDB1 Interacting Protein CUL4. Plant Journal. 55:89-103. Interpretive Summary: Ripening-associated color development in many species is largely dependent on differentiation and function of carotenoid rich chromoplasts, a specialized organelle derived from plastids. One of the best-characterized genetic and molecular systems of carotenogenesis and fruit pigmentation is tomato. The major pigments of ripe tomato fruit are carotenoids, specifically beta-carotene and lycopene. beta-carotene is a vitamin A precursor, and lycopene has been associated through epidemiological studies with reduced incidence of degenerative diseases, including heart disease and cancer. By using fruit-specific promoters combined with RNA interference (RNAi), we show that directed repression of the DDB1/HP1 pigment regulatory gene in fruit tissue results in elevated pigment accumulation by increasing plastid size. We also show that downregulation of the tomato Cul4 gene (which interacts with DDB1/HP1) yields a similar enhanced pigmentation phenotype. This work suggests DDB/HP1 and CUL4 are logical targets for manipulating fruit carotenoid and associated nutritional content.
Technical Abstract: Carotenoids are a principal class of compounds found in many fruits, providing nutritional benefits as both precursors to essential vitamins and as antioxidants. Molecular characterization revealed the tomato high-pigment mutant genes (hp1 and hp2) encode UV-DAMAGED DNA BINDING PROTEIN-1 (DDB1) and DE-ETIOLATED-1 (DET1) homologs, respectively, and both are essential components of the recently identified CUL4-based E3 ligase complex. Here we have isolated a tomato CUL4 homolog and performed yeast two-hybrid assays to suggest possible association of tomato DDB1 with CUL4 and DET1. Real-time RT-PCR analysis indicates that both HP1 and CUL4 are expressed constitutively. Abscisic acid (ABA) is implicated in plastid division control and its application substantially enhances HP1/DDB1 mRNA accumulation. Transformation of constructs expressing the CUL4-YFP and DDB1-YFP fusion proteins are targeted to tomato plastids and nuclei simultaneously. Using fruit-specific promoters combined with RNA interference technology, we show that down-regulated DDB1 expression in transgenic fruits results in a significant increase of plastids and corresponding enhanced pigment accumulation. CUL4-RNAi repression lines provide insight regarding CUL4 function during tomato development and reveal that this tomato cullin is important in regulation of plastid number and pigmentation which in turn have a direct impact on fruit nutrient quality.