Isolation and functional characterization of <it>Lycopene β-cyclase </it>(<it>CYC-B</it>) promoter from <it>Solanum habrochaites</it>

• Main Authors: Chinnusamy Viswanathan, Dalal Monika, Bansal Kailash C
• Format: Article
• Language: English
• Published: BMC 2010-04-01
• Series: BMC Plant Biology
• Online Access: http://www.biomedcentral.com/1471-2229/10/61

<p>Abstract</p> <p>Background</p> <p>Carotenoids are a group of C40 isoprenoid molecules that play diverse biological and ecological roles in plants. Tomato is an important vegetable in human diet and provides the vitamin A precursor <it>β</it>-carotene. Genes encoding enzymes involved in carotenoid biosynthetic pathway have been cloned. However, regulation of genes involved in carotenoid biosynthetic pathway and accumulation of specific carotenoid in chromoplasts are not well understood. One of the approaches to understand regulation of carotenoid metabolism is to characterize the promoters of genes encoding proteins involved in carotenoid metabolism. <it>Lycopene β-cyclase </it>is one of the crucial enzymes in carotenoid biosynthesis pathway in plants. Its activity is required for synthesis of both α-and β-carotenes that are further converted into other carotenoids such as lutein, zeaxanthin, etc. This study describes the isolation and characterization of chromoplast-specific <it>Lycopene β-cyclase </it>(<it>CYC-B</it>) promoter from a green fruited <it>S. habrochaites </it>genotype EC520061.</p> <p>Results</p> <p>A 908 bp region upstream to the initiation codon of the <it>Lycopene β-cyclase </it>gene was cloned and identified as full-length promoter. To identify promoter region necessary for regulating developmental expression of the <it>ShCYC-B </it>gene, the full-length promoter and its three different 5' truncated fragments were cloned upstream to the initiation codon of <it>GUS </it>reporter cDNA in binary vectors. These four plant transformation vectors were separately transformed in to <it>Agrobacterium</it>. <it>Agrobacterium</it>-mediated transient and stable expression systems were used to study the <it>GUS </it>expression driven by the full-length promoter and its 5' deletion fragments in tomato. The full-length promoter showed a basal level activity in leaves, and its expression was upregulated > 5-fold in flowers and fruits in transgenic tomato plants. Deletion of -908 to -577 bp 5' to ATG decreases the <it>ShCYC-B </it>promoter strength, while deletion of -908 to -437 bp 5' to ATG led to significant increase in the activity of GUS in the transgenic plants. Promoter deletion analysis led to the identification of a short promoter region (-436 bp to ATG) that exhibited a higher promoter strength but similar developmental expression pattern as compared with the full-length <it>ShCYC-B </it>promoter.</p> <p>Conclusion</p> <p>Functional characterization of the full-length <it>ShCYC-B </it>promoter and its deletion fragments in transient expression system <it>in fruto </it>as well as in stable transgenic tomato revealed that the promoter is developmentally regulated and its expression is upregulated in chromoplast-rich flowers and fruits. Our study identified a short promoter region with functional activity and developmental expression pattern similar to that of the full-length <it>ShCYC-B </it>promoter. This 436 bp promoter region can be used in promoter::reporter fusion molecular genetic screens to identify mutants impaired in <it>CYC-B </it>expression, and thus can be a valuable tool in understanding carotenoid metabolism in tomato. Moreover, this short promoter region of <it>ShCYC-B </it>may be useful in genetic engineering of carotenoid content and other agronomic traits in tomato fruits.</p>