Specifically Increasing Provitamin A Content In Wheat Endosperm By Simultaneously Expressing CrtB And Silencing TaHYD

Carotenoid is one of the major pigments and determines the nutritional value of wheat. Wheat grain has very low carotenoid content and mainly accumulates lutein, which lacks provitamin A activity. Through the deeper understanding of the benefits of carotenoids for human health, scientists have been prompted to explore effective methods to increase the carotenoid composition and content in plants, especially in staple crops. However, the precise carotenoid metabolic engineering in crop plants has been hindered by the limited data on the endogenous regulation of carotenogenic genes in staple crops. Thus, the first step to understand how carotenoids are biosynthesized is to identify the related key enzymes and to clone the relevant genes. In this study, the TaLCYB was cloned and its function and relationship with ?-carotene biosynthesis in wheat grains was characterized. Then, endosperm-specific overexpression CrtB and silencing of HYD were combined in wheat metabolic engineering to increase ?-carotene content.1) Lycopene cyclization is the first branch point of the carotenoid biosynthetic pathway, and is hypothesized to regulate the proportion of carotenes through two competing lycopene cyclases, lycopene ?-cyclase(LCYB) and lycopene ?-cyclase(LCYE). Recently, we found that endogenous LCYB was up-regulated by the co-expression of CrtB and CrtI in transgenic wheat, which resulted in an increase in the total carotenoid and provitamin A contents. However, there is few researches about this key cyclase gene's function and its relationship with ?-carotene content in wheat. A lycopene ?-cyclase gene, designated TaLCYB, was cloned from the hexaploid wheat cultivar Chinese Spring. The cyclization activity of the encoded protein was demonstrated by heterologous complementation analysis. The TaLCYB gene expressed differentially in different tissues of wheat. Although TaLCYB had a higher expression level in the later stages of grain development, the ?-carotene content still showed a decreasing tendency. The expression of TaLCYB in leaves was dramatically induced by strong light and the ?-carotene content variation corresponded with changes of TaLCYB expression. A post-transcriptional gene silencing strategy was used to down-regulate the expression of TaLCYB in transgenic wheat, resulting in a decrease in the content of ?-carotene and lutein, accompanied by the accumulation of lycopene to partly compensate for the total carotenoid content. In addition, changes in TaLCYB expression also affected the expression of several endogenous carotenogenic genes to varying degrees. Our results suggest that TaLCYB is a genuine lycopene cyclase gene and plays a crucial role in ?-carotene biosynthesis in wheat.2) Carotenoid metabolic engineering is a fast and efficient method to improve the carotenoid content in plant, but there is few studies on carotenoid metabolic engineering to specifically increase ?-carotene content. The analysis of developing wheat seeds showed that ?-carotene was abundantly synthesized in common wheat and its concentration decreased progressively during grain development. This observation was of particular interest to meet the purpose of investigating means to accumulate higher content of ?-carotene in mature seeds. Significant increases in ?-carotene content could be achieved by preventing carotenoid turnover during seed maturation. Carotene hydroxylase catalyses the key steps of depleting provitamin A function by converting provitamin A compounds into non-provitamin A xanthophylls. The low expression of ?-carotene hydroxylase gene accompanies higher ?-carotene accumulation. In the present study, endosperm-specific silencing of the TaHYD gene resulted in increased content of ?-carotene 10.5-fold to 1.76 ?g g–1 in wheat endosperm. Over-expression of CrtB introduced an additional flux in the wheat carotenoid pathway, accompanied by a ?-carotene increase of 14.6-fold to 2.45 ?g g-1. When the "push strategy"(over-expressing CrtB) and "block strategy"(silencing TaHYD) were combined in wheat metabolic engineering, significant levels of ?-carotene accumulation were obtained, corresponding to an increase of up to 31 fold to 5.06 ?g g–1. Over-expression of CrtB and silencing of HYD regulate different endogenous carotenoid biosynthesis genes with different variation, suggesting the accumulation of ?-carotene through different mechanisms. Our results suggest that the silencing of HYD increased the ?-carotene content by changing the metabolic equilibrium. This is also the first example of successful metabolic engineering to specifically improve the content of ?-carotene in wheat endosperm through a combination of silencing and over-expression technology and demonstrates the potential of genetic engineering for specific nutritional enhancement of wheat.In conclusion, our attempts to characterize the key carotenoid biosynthesis gene and enhance the content of provitamin A in wheat through the meatbolic engineering will not only strengthen the knowledge of carotenoid biosynthetic regulation in wheat endosperm, but also provide a new metabolic engineering strategy for breeding the transgenic wheat varieties with high ?-carotene content in future.