Folate profiling in rice

Folate is one of the important B vitamins needed for normal cell function, growth and development. Whilst it is naturally found in rice, the level is low in the grain and the naturally-occurring forms of the vitamin are not well studied. LC-MS/MS based technique was developed to profile the mono- and polyglutamated folates while total folates in rice were initially profiled using the microbiological assay (MA). MA was used to screen 51 rice cultivars for their total folate content, and then LC-MS/MS was employed to measure naturally occurring mono- and polyglutamated forms of the vitamin in selected cultivars. Wide natural variation among rice cultivars in terms of total folate content was measured using MA screening and the validated LC-MS/MS technique of simultaneous profiling of mono- and polyglutamated folates through Me OHAA/PO4 extraction revealed that the naturally-occurring species in wild type rice are 5-CH3-H4Pte Glu, 5/10-CHO-PteGlu 5. The developed and optimized LC-MS/MS technique was also used to characterize folate mono- and polyglutamated forms in a variety of transgenic rice lines. There was a general decrease in polyglutamated folate forms in the folate polyglutamating enzyme (FPgS Os03g02030) knockout rice line. Conversely, there was a dramatic increase in 5-CH3-H4 PteGlu 4, 5/10-CHO-H4PteGlu 5, 5-CH3-H4pTe6. and 510-CHO-H4Pteglu6 levels, resulting in 1 2.5 to 8.8-fold increase in the total folate pool in the unpolished grain of rice either overexpressing FPGS genes, or the folate binding proteins-cFBP and GNMT compared to wild type Nipponbare. This study demonstrates that overexpression of the two rice FPGS genes (Os03g02030 and Os10g35940) or folate binding proteins (cFPB and GNMT) results in improved levels and overall folate profile which can be exploited in breeding programmes designed to enhance folate content in this important staple crop.