The researchers: Maria Laura Federico y Federico Iñiguez Luy, UGB CGNA-INIA Carillanca.

Duration: 4 years.

Funding agency: FONDECYT - 1090726

PROPOSAL ABSTRACT:

Carotenoids are organic pigments produced by photosynthetic organisms. They play important roles in human nutrition and are considered nutraceuticals. Additionally, carotenoids are used as food, feed and cosmetic colorants. Commercial production of specialty carotenoids is mainly based on chemical synthesis; however, metabolic manipulation of carotenoid synthesis in plants could provide the industry with a natural alternative. Certainly, the development and production of crops with "added value" will increase their economic value, provide new options for the food industry, and positively impact regional economies in Chile.

The main goal of this proposal is to better understand and manipulate carotenoid synthesis in Brassica napus (canola) seeds in order to generate a vegetable source of pigments (e.g. β-carotene, cantaxanthin and astaxanthin) routinely used as feed additives in the aquaculture, poultry and swine industries. Due to the hydrophobic nature of carotenoids, modified levels of these pigments should be present in the canola oil after extraction. Depending on the profiles and levels obtained, the future use of this enhanced canola oil in feed formulations could reduce the amount of synthetic pigments required to raise salmon, and other farm animals.

Higher plants are capable of synthesizing carotenoids including β-carotene, but most do not possess the ability to synthesize ketocarotenoids like astaxanthin and canthaxanthin. Thus, manipulation of ketocarotenoid synthesis requires the addition of metabolic steps not usually present in the organs of crop plants. Previous studies have shown that it is possible to successfully manipulate the carotenoid biosynthetic pathway in B. napus to dramatically increase carotenoid seed levels, including carotene but no attempt to manipulate ketocarotenoid synthesis has been undertaken. We plan to manipulate the carotenoid biosynthetic pathway in B. napus by overexpressing a bacterial phytoene synthase (CrtB) in a seed-specific manner in conjunction with other genes (CrtW, CrtZ and CrtS) involved in the synthesis of specialty (high-value) ketocarotenoids. In addition, we plan to increase our understanding of carotenoid synthesis regulation in B. napus seeds through the characterization of phytoene synthase (PSY) gene expression. This will facilitate future pathway manipulations using an endogenous B. napus PSY sequence.

Phytoene synthase (PSY) is the first and a key regulatory enzyme in the carotenoid biosynthetic pathway. In species where PSY is encoded by a single copy gene, like in Arabidopsis, the flexibility and response capabilities of the carotenoid pathway are limited to regulating this rate controlling single copy enzyme. In species that contain more than one copy of PSY, however, gene duplication has resulted in the subfunctionalization of gene expression. This provided a mechanism that allowed for PSY overexpression in the fruit and seed tissues without the detrimental effects on photosynthesis that overexpression throughout the plant would have caused. If several PSY genes exist in B. napus, as expected due to the redundant nature of its genome, they may have evolved to better function in a given tissue at a given developmental stage or under a specific stress condition. It is therefore important to estimate the number of PSY genes present in B. napus and characterize their expression patterns. This knowledge will aid in the development of transgenic and conventional B. napus breeding lines. We plan to estimate the number of PSY genes using Southern blot hybridizations, database and SSCP (single stranded conformation polymorphism) analyses. Gene expression patterns will be determined using cDNA-SSCP and/or qRT-PCR. If a particular PSY gene is preferentially expressed in seed tissue, future efforts should be focused on breeding or metabolically manipulating the expression of that particular gene.

GOALS

GENERAL GOAL

To better understand and manipulate carotenoid synthesis in Brassica napus seeds in order to generate a vegetable source of pigments (e.g. β-carotene, cantaxanthin and astaxanthin) routinely used as feed additives in aquaculture, poultry and swine production.

SPECIFIC GOALS

1-To produce transgenic Brassica napus lines with elevated and novel carotenoid content in seeds.

2-To estimate the copy number of phytoene synthase (PSY) genes present in the genome of Brassica napus.

3-To characterize the spatial and temporal expression patterns of PSY genes in order to determine whether one of them is mainly involved in seed carotenogenesis.