In many temperate woody species, dormancy is induced by short photoperiods. Earlier studies have shown that the photoreceptor phytochrome A (phyA) promotes growth. Specifically, Populus plants that over-express the oat PHYA gene (oatPHYAox) show daylength-independent growth and do not become dormant. However, we show that oatPHYAox plants could be induced to set bud and become cold hardy by exposure to a shorter, non-24 h diurnal cycle that significantly alters the relative position between endogenous rhythms and perceived light/dark cycles. Furthermore, we describe studies in which the expression of endogenous Populus tremula x P. tremuloides PHYTOCHROME A (PttPHYA) was reduced in Populus trees by antisense inhibition. The antisense plants showed altered photoperiodic requirements, resulting in earlier growth cessation and bud formation in response to daylength shortening, an effect that was explained by an altered innate period that leads to phase changes of clock-associated genes such as PttCO2. Moreover, gene expression studies following far-red light pulses show a phyA-mediated repression of PttLHY1 and an induction of PttFKF1 and PttFT. We conclude that the level of PttPHYA expression strongly influences seasonally regulated growth in Populus and is central to co-ordination between internal clock-regulated rhythms and external light/dark cycles through its dual effect on the pace of clock rhythms and in light signaling.

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The recombinant 65-kDa polypeptide of phyA oat phytochrome was expressed by yeast Pichia pastoris and assembled into two chromopeptides with the chromophores phytochromobilin (POB) and phycocyanobilin (PCB), respectively. The P r and Pf r states of the two protein variants were characterized by resonance Raman (RR) spectroscopy and compared with native phyA oat phytochrome demonstrating that the deletion of the C-terminal half of phyA does not alter the structure of the chromophore site within the N-terminal half. Most of the RR spectral changes observed upon replacing POB by PCB can be attributed exclusively to altered vibrational mode compositions due to the different ring D substitutions (vinyl vs. ethyl), implying that the chromophore structures are largely the same for POB-and PCB-assembled phytochromes. Only in the P r state may the RR spectral changes also reflect subtle differences of the POB and PCB conformations in the 65-kDa phyA, presumably brought about by the specific steric requirements of the vinyl and ethyl groups.

Two different yeast expression systems, Pichia pastoris and Hansenula polymorpha, are compared for their capability to express in functional form the 65 kDa N-terminal portion of oat phytochrome A (phyA, spanning amino acids 1-595). The front half of phytochrome was selected for this investigation because it exhibits a greater stability than the full-length protein, and it harbors full spectroscopic and kinetic properties of phytochrome, allowing an exact proof of the functional integrity of the recombinant material. In the comparison between the two expression systems used, special emphasis was given to optimizing the yield of the expression and to improving the quality of the expressed material with respect to the proportion of functional protein. From identical volumes of cell culture, H. polymorpha synthesized between 8- and 10-fold more functional protein than P. pastoris. Following the observation by Wu and Lagarias (Proc. Natl. Acad. Sci. USA 93, 8989-8994, 1996) that P. pastoris endogenously produces the chromophore of phytochrome, phytochromobilin (P phi B) in significant amounts that leads to formation of spectrally active phytochrome during expression, the invention of an alternative high-yield expression system was strongly demanded. A His6-tag was attached to the C-terminus of the recombinant protein, which allows for a convenient and efficient purification and selects the full-length proteins over translationally truncated peptides. Fully reconstituted chromoproteins showed an A660/A280 ratio of > 1.2, indicating the high degree of reconstitutable apoprotein obtained by this procedure. The assembly between apoprotein and the chromophore phycocyanobilin when followed time-resolved yielded a time constant (tau obs) of 35 s. The lambda max values of the red-(Pr) and the far red-absorbing (Pfr) forms of phytochrome (665 and 729 nm) of the recombinant 65 kDa chromopeptide, reconstituted with P phi B are nearly identical to those of native full-length oat phytochrome. The kinetic parameters of the affinity-purified 65 kDa phytochrome chromoprotein for the Pr-->I700--> -->Ptr conversion are compared to those of the recombinant 65 kDa chromoprotein, lacking the His-tag and to wild-type oat phytochrome. Referring to wild-type phytochrome allows determination of whether the recombinant material has lost spectral properties during the purification procedure. The decay of the primary intermediate (I700) occurs with nearly the same time constant for the His-tagged chromoprotein and for the reference (110 and 90 microseconds, respectively). The formation of the Ptr form was fitted with three exponentials in both the His-tagged and the reference chromoprotein with the middle component being slightly smaller and the longest component being remarkably larger for the His-tagged protein (1.5, 10 and 300 ms) than for the reference (1.4, 18 and 96 ms). This selective slowing down of the long kinetic component in the millisecond time range may be indicative of stronger interactions between protein domains involving the C...