RECOMBINANT TYPE-A AND TYPE-B PHYTOCHROMES FROM POTATO - TRANSIENT ABSORPTION-SPECTROSCOPY

The cDNAs encoding full-length type A and B phytochromes (phyA and phy B, respectively) from potato were expressed in inducible yeast systems (Saccharomyces cerevisiae and Pichia pastoris). In addition, a deleti on mutant of phyB (Delta 1-74) was expressed. The apoproteins were rec onstituted into chromoproteins by incorporation of the native chromoph ore, phytochromobilin (P Phi B), and of phycocyanobilin (PCB). The inc orporation Of P Phi B yielded chromoproteins with difference absorptio ns lambda(max) at 660 and 712 nm (P-r and P-fr, respectively) for phyA , and at 665 and 723 nm for phyB. All difference maxima of PCB phytoch romes are blue-shifted by several nanometers with respect to those obt ained with the P Phi B chromophore. The deletion construct with PCB sh ows difference absorption maxima at 652 and 705 nm with the P-fr absor bance considerably reduced. Time-resolved kinetic analysis of a phyB-t ype phytochrome by nanosecond flash photolysis was performed for the f irst time. Recombinant full-length phyB afforded transient absorbance changes similar (but not identical) to those of phyA from Avena, where as the kinetic behavior of these intermediates was very different. Con trary to phyA from Avena, the I-700 intermediate from phyB reconstitut ed with either PCB or P Phi B decayed following single exponential kin etics with a lifetime of 87 or 84 mu s, respectively, at 10 degrees C. The formation of P-fr of PCB-containing recombinant phyB (phyB-PCB) c ould be fitted with three lifetimes of 9, 127, and 728 ms. The corresp onding lifetimes of phyB-P Phi B are 22.5, 343, and 2083 ms. Whereas f or phyB-PCB all three millisecond lifetimes are related to the formati on of P-fr, the 2 s component of phyB-P Phi B is concomitant with a ra pid recovery of P-r. For recombinant potato phyA and Delta 1-74 phyB, no time-resolved data could be obtained due to the limited quantities available. As described for phytochromes of other dicotelydons, the P- fr forms of full-length phyA and phyB of potato underwent rapid dark c onversion to P-r.