Folate malnutrition is a major problem in many countries around the world especially in Asia and Africa. Stable foods such as rice contain very low amounts of folate. White rice which is the most popular form for human consumption contains less than 80 µg folate per 100 g. Given that it forms a major part of the South East Asian diet, rice represents an important target for enhancing folate levels.
The objectives of this thesis is study the mechanisms that regulate total folate levels in rice grains and attempt to enhance and stabilise folate in rice endosperm. Three main strategies were adopted. First, the natural variation of folate biosynthesis gene expression was probed using RT- qPCR. Second, functional genomic approaches were used to manipulate the activity of rice folylpolyglutamate synthetase (FPGS), the enzyme which adds glutamate residues to folate. Third, genetic engineering was used to express FPGS enzymes and mammalian folate binding proteins in rice endosperm.
RT-qPCR revealed that the variation in folate biosynthesis transcript abundance was closely correlated with total folate levels among rice varieties. High transcript abundance of all folate biosynthesis genes was associated with high total folate levels in Moroberekan rice mature seed. Comparative genomic studies revealed that rice FPGS is encoded by two distinct genes, FPGS Os03g02030 and FPGS Os10g35940. Transcript abundance of FPGS Os03g02030 appeared higher than that of FPGS Os10g35940 in seed, whilst, transcript abundance of FPGS Os10g35940 was higher in leaf.
To determine the function of the FPGS Os03g02030 gene in rice seed, a TDNA knock out line was characterised. Disrupting Os03g02030 gene expression resulted in delayed seed maturation and decreased mono- and polyglutamylated folate pools in mutant seed. RT-qPCR detected an increase in the transcript abundance of folate biosynthesis genes in seed of the knock out plant, whereas the folate deglutamylating enzyme y-glutamyl hydrolase (GGH) mRNA level was reduced. A potential feedback mechanism to maintain folate abundance during rice development was uncovered through the alternative functional FPGS Os10g35940 activity and reduction of folate breakdown.
Protein-bound folate forms are better protected from oxidative degradation resulting in greater folate stability (Suh et al. 2001). Two rice FPGS and mammalian folate binding proteins was successfully introduced into rice endosperm using Agrobacterium based transformation in an attempt to retain and stabilise folate pool within rice endosperm. Analysis in terms of folate abundance and bioavailability will form part of future studies
