Major isoflavonoid contents and proteomics in leaves and tubers of Pueraria mirifica

Abstract

The monthly variation of major isoflavonoid contents namely puerarin, daidzin, genistin, daidzein and genistein from 3 different cultivars of Pueraria mirifica (PM-III, PM-IV and PM-V) in field trial at Ratchaburi province were investigated by the aid of RP-HPLC analysis. The proportions and net levels of puerarin, daidzin, genistin and daidzein in P. mirifica leaves were found to depend on the plant cultivar and to correlate with cultivation temperature and rainfall amount. The determination of protein pattern in P. mirifica tubers and leaves were performed via proteomics approach to better understanding the isoflavonoid biosynthesis pathway. The phenolic method was the suitable protein extraction due to the high protein yield and clearly separated protein band when comparing with other methods (Tris-HCL and TCA-Acetone extraction). Proteins were separated by using 2D-PAGE and mass spectrometry tedchnique. The novel proteome patterns in tubers and leaves were revealed according to 8 major protein functions; 1) cell structure 2) defense 3) metabolism 4) protein synthesis 5) storage protein 6) stress response 7) transportation and 8) unidentified protein. The seasonal harvesting was expressed in difference protein pattern. Protein expression in tuber was up-regulated in winter whereas, in leaf was up-regulated in summer. Many proteins involved in secondary metabolite also found such as glutathione S-transferase and glutathione reductase. Furthermore, protein involved in pathway of bioactive isoflavonoids biosynthesis were expressed; chalcone isomerase (CHI), isoflavone synthase (IFS), cytochrome p450, UDP-glycosyltransferase (UGT) and isoflavone reductase (IFR). Moreover, these proteins also have significant correlated with isoflavonoid contents. From these results, the more understanding of the plant secondary metabolite production in P. mirifjica tuber were recorded and not only protein expression but also relation of secondary metabolites production in different seasonal harvesting.