Studies On The Roles Of Riboflavin Binding Protein In Plant Growth And Pathogen Defense

Riboflavin (vitamin B₂) participates in many physiological processes, particularlythose characterized by oxidation and reduction. The vitamin can combine with nucleotidesto form flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), which arecoenzyme of flavoproteins, and which play vital roles in many reactions, especially theproduction of reactive oxygen intermediates (ROIs). In plants, ROIs are essential forhypersensitive cell death, growth, and defense against pathogens and insects, as well asmany aspects of responses to environmental cues. Levels of riboflavin in plants arebelieved to be important to these processes. As such, if riboflavin levels could bemanipulated, effects of the compound on the important processes could be monitored andcontrolled as a consequence. This would result in great improvement of plant defense andproductivity. This also could provide us with a particular insight into studying coordinationof plant defense with growth regulation, which may be affected by levels of riboflavin.Riboflavin carrier protein (RCP) or -binding protein (RfBP), first identified in chickenegg and the serum of egg-laying birds is a monomeric, acidic, phosphoglycoprotein andbinds the free vitamin with high affinity. It has recently been shown that RfBP is amembrane component of ovulated oocytes, early embryo and of acrosomal region of maturesperms of rodents and other mammals. Riboflavin transport to the egg by the laying hen ismediated by a 27,000 Da protein, chicken riboflavin carrier or binding protein RfBP.The gene encoding RfBP from soft-shelled turtle was amplified and cloned into theexpression vector pET30a(+), and the prokaryotic expression vector pET30a(+)::RfBP,called pRfBP, was constructed. Plasmid containing RfBP was transformed into competenceEscherichia. coli BL21 (DE3)pLysS. The bacterium was induced byisopropyl-Beta-D-thiogalactopyranoside (IPTG) and its expressed products were loadeddirectly onto sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE),approximately 35 kD exogenous protein was observed on the SDS-PAGE. His-tagged RfBP fusion protein was expressed well at 37℃for an additional 4 h after 1mM IPTGwas added, and a majority of fusion proteins was soluble. Higher concentration of IPTGand longer induced time did not accelerate the expression of the fusion protein.RfBP was purified using the HisTrap HP Kit. The yield was more than 2 mg purifiedRfBP proteins per liter of culture and the concentration of purified RfBP protein was about1.5-2.0 mg/ml. RfBP could not elicit hypersensitive response on tobacco. The polyclonalantibodies against RfBPs of both chicken and soft-shelled turtle were produced. The titer ofthe antibodies against chicken and soft-shelled turtle RfBP reached 1:64,000 and 1:256,000,respectively. Two antibodies against RfBPs could recognize the samples containing RfBPprotein in the analysis of ELISA and Western blot. It is imaginable that the cross reactionof two antibodies, because the amino acid sequence identity of chicken and soft-shelledturtle RfBPs reaches 71.3%.In conclusion, the exogenous application of RfBP to plants induces pathogen defense,but does not affect plant growth. Defense response is consistent with the increase in theendogenous riboflavin. These results provide a good basis for further study on the role ofRfBP modulating for riboflavin contents in plant growth and development regulation.