692  2001 American Society for Photobiology 0031-8655/00 $5.00+0.00 Photochemistry and Photobiology, 2001, 73(6): 692–696 Recombinant Phytochrome A in Yeast Differs by its Spectroscopic and Photochemical Properties from the Major phyA and is Close to the Minor phyA: Evidence for Posttranslational Modification of the Pigment in Plants ¶ V. Sineshchekov* 1 , L. Hennig 2 , T. Lamparter 3 , J. Hughes 3 , W. Ga ¨ rtner 4 and E. Scha ¨ fer 2 1 Department of Biology, M. V. Lomonosov Moscow State University, Moscow, Russian Federation; 2 Biologisches Institut II der Universita ¨ t, Freiburg, Germany; 3 Institut fu ¨ r Pflanzenphysiologie und Mikrobiologie, Freie Universita ¨ t Berlin, Berlin, Germany and 4 Max-Plank Institut fu ¨ r Strahlenchemie, Mu ¨ lheim an der Ruhr, Germany Received 30 November 2000; accepted 10 February 2001 ABSTRACT Previously, two pools of phytochrome A (phyA and phyA) have been detected by in situ low-temperature fluorescence spectroscopy and photochemistry; it was suggested that they might differ in the nature of their posttranslational modification. In order to verify this pos- sibility Arabidopsis and rice (Oryza) phyA were expressed in yeast and the pigments were assembled in vivo with phycocyanobilin (PCB) and phytochromobilin (PB). The resulting recombinant phytochromes in the red- light–absorbing form (Pr) were characterized in the yeast cell by (1) the fluorescence emission spectra; (2) the tem- perature dependence of Pr fluorescence intensity and ac- tivation energy of fluorescence decay; and (3) the extent of photoconversion of Pr into photoproduct lumi-R ( 1 ) or far-red–light absorbing form (Pfr) ( 2 ). Both Arabi- dopsis phyA/PCB and Oryza phyA/PB had low  1 of ca 0.05, allowing their attribution to the Pr phenomenolog- ical type of phytochrome comprising phyA, phyB and cryptogam phytochromes. The spectroscopic properties of Oryza phyA/PB were also very close to phyA. How- ever, both investigated holoproteins differed from phyA, ¶Posted on the website on 28 February 2001. *To whom correspondence should be addressed at: Department of Biology, M. V. Lomonosov Moscow State University, 119899 Moscow, Russian Federation. E-mail: v.sineshchekov@mtu-net.ru Abbreviations: E a , activation energy of fluorescence decay; F, fluo- rescence; F b , background light; F 0 ,F 1 ,F 2 , fluorescence of the red- light–absorbing phytochrome form at 85 K under different con- ditions of light adaptation; FR, far-red light; lumi-R, photoproduct of the phototransformation of the R-absorbing form of phyto- chrome; P, phytochrome; PCB, phycocyanobilin; PB, phyto- chromobilin; phyA, phyB, phytochromes A and B; phyA/PCB, phyA/PB, phyA reconstituted with PCB and PB, respectively; phyA', phyA, subpopulations of phyA; Pfr, FR-light–absorbing phytochrome form; Pr, R-light–absorbing phytochrome form; Pr', Pr, different species of Pr; P tot , total phytochrome; R, red light;  1 ,  2 , extent of the Pr phototransformation into lumi-R at 85 K and into Pfr at 273 K upon R illumination, respectively;  a ,  ex ,  em ,  max , wavelength of the actinic, exciting and emitted light and of the fluorescence maximum. both with respect to the character of temperature depen- dence of the fluorescence yield and activation energy. Thus, recombinant Oryza phyA/PB is similar but not identical to phyA. The data demonstrate that the low- abundance–fraction plant phyA (phyA) comes from the same gene as the major (phyA) fraction. Because both endogenous phyA fractions differ from the phytochrome expressed in yeast, they appear to be posttranslationally modified and/or bound to partner proteins or cellular substructures. However, the character of the presumed chemical modification is different in phyA and phyA and its extent is more profound in the case of the former. INTRODUCTION The plant photoreceptor phytochrome exhibits remarkable structural and functional variabilities in the plant cell. This heterogeneity is a major issue in the current research of this pigment. For example, the family of phytochromes in Ara- bidopsis (phyA–phyE)² show marked differences in primary structure. Accordingly, the dominant types, phyA and phyB, possess physiological functions which in some cases are contrasting and, in others, complementary (1,2). Indeed the situation is likely to be even more complex. Two phyA pools (subpopulations), phyA' and phyA, were detected in the plant cell differing in their spectroscopic and photochemical properties, their abundance and localization pattern in plant tissues and their light stability. Even within phyA' different subspecies (most probably conformers of the chromophore) were distinguished (3–5). The major distinction between phyA' and phyA is their different photochemical activities at low temperatures (Pr' and Pr phenomenological types, respectively). PhyA' is more efficient with respect to the Pr → lumi-R photocon- version ( 1 = 0.5 for phyA' versus  1  0.1 for phyA). It has the emission (absorption) maximum,  max = 687 (673) nm, whereas for phyA max = 682–683 (668) nm. PhyA' is the dominant species but is rapidly lost in red light. Its con- centration is highest in the apical parts of seedlings and in the root tips. In contrast, phyA is a minor species relatively