©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
In Vivo Characterization of Phytochrome-Phycocyanobilin Adducts in Yeast (*)

(Received for publication, May 11, 1995; and in revised form, June 21, 1995)

Tim Kunkel (§) Volker Speth (§) Claudia Büche (§) Eberhard Schäfer(§)(¶)

From the Institut für Biologie II, Albert-Ludwigs-Universität Freiburg, Schänzlestrasse 1, 79104 Freiburg, Federal Republic of Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The in vivo reconstitution of phycocyanobilin with apophytochrome leads to photoreversible adducts in living yeast cells. Investigations with the rice phytochrome A phycocyanobilin adduct (PHYA*) and the tobacco phytochrome B phycocyanobilin adduct (PHYB*) show that the protein stability in yeast is independent of the form of the photoreceptor.

After in vivo assembly and irradiation with red light, 25.6% of the far-red light-absorbing form of PHYB* exhibited dark reversion with a half-life time of approximately 20 min. Control experiments with PHYA* revealed no dark reversion. The data indicate that the molecular basis for this reaction is the formation of heterodimers between the red and the far-red light absorbing form of phytochrome.

Electron microscopic in situ localizations and in vitro sequestering experiments showed that phytochrome A was able to sequester in yeast. On the electron microscopic level, the sequestered areas of phytochrome from etiolated plants and yeast are indistinguishable. The sequestering reaction in yeast is independent of the formation of the far-red light absorbing form of phytochrome. Therefore, we discuss a new model for this reaction in plants.

Since it is unlikely that yeast cells contain elements that distinguish between phytochrome A and B, we conclude that sequestering and dark reversion reflect intrinsic properties of phytochrome.


INTRODUCTION

Phytochrome is one of the most important and best characterized photoreceptors in plants. The ability to purify the photoreceptor from etiolated (dark-grown) seedlings and the molecular cloning of phytochrome genes give insight into structural and functional properties of phytochrome.

The phytochrome dimer is located in the cytosol of the plant cell (1, 2, 3) . The apparent molecular mass of the monomer is approximately 120 kDa. Phytochromobilin, a linear tetrapyrrole that is covalently attached to a cysteine residue of apophytochrome is responsible for light perception. The assembly reaction with the chromophore is an autocatalytic process of the photoreceptor. Phytochrome can absorb light over a wide range (approximately 300-800 nm) of the spectrum (4) . The two forms of the photoreceptor possess two characteristic absorption maxima in red (Pr (^1)form, 660 nm) or far-red light (Pfr form, 730 nm). The bathochrome shift in the red light absorption maximum from Pr to Pfr is due to the photochemical Z/E isomerization of a chromophore double bond(5) . As a consequence, the photoreceptor can change to the physiologically active Pfr form(6) . These changes are photoreversible, and since the absorption spectra of both phytochrome forms overlap, irradiation leads to a wavelength-dependent photoequilibrium between the inactive Pr and the active Pfr form.

Since the genes that encode for the phytochrome family have been characterized and phytochrome mutants become available, it has been possible to assign the members of the family to photoreceptors that differ in their abundance and light regulation(7) .

The predominant photoreceptor in etiolated plants is the light-labile phytochrome A (PHYA). After transition into Pfr, the protein is rapidly degraded (half-life time approximately 2 h), and the expression of the PHYA encoding gene (phyA) is down-regulated by light. Prior to degradation, PHYA is concentrated in distinct regions in the cytosol. The sequestering reaction leading to sequestered areas of phytochrome (SAP) takes place in a few seconds at room temperature, needs ATP, and is dependent on Pfr formation. SAP formation has been discussed as a prerequisite for the ubiquitin-dependent proteolytic degradation of PHYA(8) .

In contrast to PHYA, which accumulates to high levels in etiolated plants, the light-stable phytochromes B and C are photoreceptors with low abundance. With a half-life time of approximately 12 h, PHYB Pfr is relatively stable. Independent of the light treatment, phyB and phyC expression is nearly constitutive(7) .

The rapid destruction, together with the down-regulation of phyA expression, are probably important processes in switching off a given PHYA Pfr signal. Dark reversion of Pfr to physiological inactive Pr may be an additional possibility to prevent constitutive Pfr signals. With the exception of the Caryophyllales, dicotyledonous PHYA exhibits dark reversion, whereas Pfr from monocotyledonous plants seems to be stable. In etiolated mustard seedlings, the percentage of reverting phytochrome is dependent on the reached photoequilibrium between Pr and Pfr and is virtually complete within 10 min(9, 10) . Keeping in mind that phytochrome is a dimer, Brockmann et al.(1) calculated that the amount of dark-reverting phytochrome matches closely with the percentage of conformation heterodimers between Pr and Pfr.

Some examples give indirect evidence that PHYB Pfr dark reversion actually occurs in plants. Investigations with cauliflower florets (11) and artichoke receptacles (12) revealed almost complete reversion from Pfr to Pr with a half-life time of approximately 60 min. Additionally, experiments dealing with the flowering induction of Pharbitis nil and Chrysanthemum indicate the existence of a dark-reverting phytochrome(13) . Since these plants were grown in white light, it is likely that the phytochrome therein is PHYB-like.

Besides the differences in photoreceptor stability and light-dependent expression, PHYA and -B have different physiological roles. Investigations with phytochrome mutants of Arabidopsis revealed that PHYA perceives far-red light(14) , seems to cause the very low fluence response, and mediates the high irradiance response(15) . Contrary to PHYA, PHYB shows low fluence and red light responses(16) . Studies with phytochrome overexpressing plants and phytochrome mutants revealed that PHYA and PHYB have distinct but also overlapping physiological functions(16, 17, 18) . In synopsis, PHYA may be responsible for the rapid deetiolation even after weak light pulses, whereas PHYB regulates light responses in green and adult plants.

The low level of PHYB in plants has so far prevented the analysis of PHYB signal transduction, dark reactions, and photoreceptor destruction. The use of overexpressing systems is one possibility for a direct investigation of this important photoreceptor.

Phytochrome overexpressing yeast systems (19, 20, 21, 22) function as good suppliers of spectral- and biologically active phytochrome. (^2)Yeast cells contain no endogenous chromophores that assemble with phytochrome. This enables us to work with the apoprotein or to use the substitute chromophore, phycocyanobilin(23) , for holophytochrome reconstitution. In vitro investigations with the tobacco PHYB-phycocyanobilin-adduct (PHYB*) revealed no differences compared to phytochrome A regarding the spectral properties, dimerization, and chromophore attachment. On the other hand, since phytochrome types differ in their physiological role, it is likely that the proteins also differ in their molecular reactions.

Therefore, two protocols for the in vivo reconstitution of phytochrome in yeast were established. Using the in vivo reconstitution system, we investigated the following molecular properties of PHYB* and the rice phytochrome A-phycocyanobilin adduct (PHYA*). 1) The in vivo system enabled the first the direct measurement of PHYB* dark reversion. 2) The in vivo system was used to study the sequestering reaction of phytochrome. 3) The functionality of in vivo reconstituted phytochrome in yeast was tested by coexpression with reporter plasmids.


EXPERIMENTAL PROCEDURES

Generation of a Polyclonal Antiserum against the 66-kDa N- terminal Domain of the Tobacco PHYB Apoprotein

For isopropyl-1-thio-beta-D-galactopyranoside-induced expression of a 66-kDa recombinant phytochrome apoprotein fragment with a six-histidine tag at the N terminus, the BamHI-PstI fragment of the full-length tobacco phyB cDNA (24) was inserted in frame into the expression vector pQE9 (Diagen) and transformed into Escherichia coli strain pREP4. Expression of the fusion protein, denaturing preparation of crude extracts, and purification of the tagged protein on nickel-agarose columns (Diagen) was performed according to the manufacturer's instructions. Fractions containing the 66-kDa fusion protein were pooled, and the fusion protein was purified to homogeneity via quantitative SDS-polyacrylamide gel electrophoresis and subsequent electroelution(25) . The homogeneity of the eluted fraction was monitored by silver staining of SDS-gels (not shown). Immunization of a white New Zealand rabbit and preparation of the antiserum were performed as published by Harlow and Lane(36) . The antiserum was tested for specificity by enzyme-linked immunosorbent assay and Western analysis.

Plasmid Construction and Yeast Strains

The generation of the galactose inducible rice PHYA (pARiA) and tobacco PHYB (pATB) expression vectors, based on the plasmid pAA7 (26) is described by Tomizawa et al.(27) and Kunkel et al.(22) . These plasmids were transformed into the yeast strain KN380 (28) in order to investigate the in vivo assembly, dark reversion, and the in vivo and in vitro sequestering of phytochrome in yeast. The resulting phytochrome A- and B-overexpressing strains are referred to as KpARiA and KpATP.

To analyze the expression of chalcone synthase promoter constructs in phytochrome-overexpressing yeast, the BamHI-SalI fragments (encoding complete phytochrome protein sequences) of the rice PHYA (29) and the tobacco PHYB were cloned into the constitutive expression vector pG1(30) , resulting in the expression vector pGRiA and pGATB. To generate a noninducible, uracil, selectable yeast episomal plasmid, the Gal1 promoter of the expression plasmid pYES2 (Invitrogen) was deleted by SpH1 digestion, and the vector was religated. The resulting vector (pYE) was used to generate the chalcone synthase unit 1 reporter plasmid.

A fusion of the U1 (unit 1 promoter region of the chalcone synthase gene from parsley(31) ) with the uidA gene from E. coli(32, 33) was cloned as HindIII-SacI fragment into pYE, resulting in the reporter plasmid pYEU1. In combination with pGRiA or pGTB, pYEU1 was transformed into the yeast strain INVSC1 (Invitrogen). The respective ``reporter strains'' are I1pGRiAU1 and I1pGTBU1.

Expression and Detection of Phytochrome in Yeast

The materials and experimental procedures employed in the expression of phytochrome in yeast are described by Kunkel et al.(22) and Hofmann et al.(34) .

However, the following changes were performed. To culture transgenic yeast cells, synthetic yeast media (35) lacking uracil (CMU) or uracil and tryptophan (CMUT), at a pH of 5.0, containing either 2% (w/w) glucose or galactose were used.

For electron microscopic investigations yeast, spheroplasts were prepared as described by Harlow and Lane(36) . The cells were embedded in Lowicryl K4M and cut into ultrathin sections(37) . Immunocytochemistry was performed by using Immunogold-labeled secondary antibodies (Amersham Corp.) and phytochrome-specific antibodies as described in Speth et al.(3) and Hofmann et al. (34) . Electron microscopy was carried out with a Phillips CM10 microscope.

After assembly with phycocyanobilin (see below), the in vivo phytochrome content was determined with a dual wavelength spectoratiometer (Ratiospect)(38) . Quantification of photoreversible phytochrome was performed using standards of purified, native oat PHYA.

In Vivo Assembly of Phytochrome Apoproteins with Phycocyanobilin

Method 1

Phytochrome A or B overexpressing yeast strains (KpARiA, KpATB) were cultured at 30 °C in 250-ml Fernbach flasks, with 100 ml of galactose-containing CMU, at constant agitation (150 rpm). To prevent phytochrome phototransformation during the assembly procedure, all steps were carried out under green safety light. Yeast overnight cultures were divided into 20-ml aliquots, and phycocyanobilin and Tween 40 (Sigma) were added to a final concentration of 20 µM and 0.9% (v/v), respectively. The cultures were incubated in darkness for 4 to 6 h under constant agitation (150 rpm) at 30 °C. After centrifugation (400 g, 10 min, at 25 °C) the cells were washed twice with 50 ml of potassium phosphate buffer (50 mM potassium phosphate, pH 7, 4). The cells were resuspended either in 2 ml of potassium phosphate buffer (in vivo phytochrome determination, dark reversion, and sequestering experiments) or glucose-containing CMU (destruction analysis).

Method 2

For phytochrome expression in combination with U1 reporter plasmids (I1pGRiAU1 and I1pGTBU1), cells were grown on glucose-containing CMUT until the culture reached an optical density (600 nm) of approximately 3.0. After centrifugation (400 g, 10 min, at 25 °C), the cells were washed twice with 50 ml of potassium phosphate buffer and resuspended in 2 ml of 30% (v/v) Me(2)SO. The chromophore was added to a final concentration of 300-400 µM, and the suspension was incubated (without agitation) for 15 min in darkness. To remove the Me(2)SO, 48 ml of culture medium was added and the yeast cells were precipitated by centrifugation (400 g, 10 min, 25 °C), and resuspended in 10 ml of glucose-containing CMUT.

Determination of Chalcone Synthase U1-Promoter Activity

Starting from the 10-ml culture (see above), 100 ml of glucose-containing CMUT was inoculated to an optical density (600 nm) of 0.5. Culturing was performed at 25 °C with constant agitation at 150 rpm, either in red light or in darkness. Two-ml aliquots were harvested by centrifugation (15,000 g, 5 min, 25 °C) at different optical densities between the starting point and an A of 2. The pellet was frozen in liquid nitrogen and stored at -80 °C.

To disrupt the yeast cells, 400 µl of assay buffer (50 mM NaH(2)PO(4), pH 7, 1 mM EDTA, 0.1% (v/v) Triton X-100, 10 mM mecaptoethanol) was added to the frozen material and whirled with glass beads(36) . Insoluble cell components were removed by centrifugation (15,000 g, 15 min, 4 °C), and the supernatant was used for protein quantification and determination of beta-glucuronidase activity(32, 33, 39) .


RESULTS

In Vivo Phytochrome Assembly

The addition of phycocyanobilin into Tween 40 substituted culture media (see ``Experimental Procedures'' under ``Method 1'') induced the uptake of chromophore into the yeast cells. Excitation of phycocyanobilin with green light leads to the typical red fluorescence of tetrapyrroles in chromophore-treated yeast cells, whereas the untreated control shows no red fluorescence (Fig. 1B). To determine whether the Tween 40 or the Me(2)SO treatment (see ``Experimental Procedures'' under ``Method 2'') kills yeast, the cells were colored with the vital dyes methylene blue and fluorescein diacetate. These stainings indicated that 89-94% of the cells survived the treatment. To test whether the chromophore can cause photooxidative damage in red light, the cells were either irradiated for 6 h or kept in darkness (Table 1). Table 1shows the percentage of living cells 6 h after Me(2)SO treatment and chromophore uptake. Independent of chromophore and light treatment, the percentage of viable cells is above 90%.


Figure 1: Protection of in vivo reconstituted rice PHYA* in yeast. A shows the protection of in vivo reconstituted PHYA* against proteolytic degradation with Proteinase K. The reconstitution of phytochrome with phycocyanobilin (PCB) was performed according to method 1 (see ``Experimental Procedures''). After the addition of phycocyanobilin, the culture was incubated for 5 h in darkness, and the phytochrome content was measured in vivo. B shows the phycocyanobilin-induced red fluorescence in chromophore-treated yeast cultures after excitation with green light (550 nm).





The covalent assembly of apophytochrome with phytochromobilin and phycocyanobilin is an autocatalytic reaction of the photoreceptor (19, 20, 21, 22) . Consequently, the uptake of phycocyanobilin into the yeast cells leads to the formation of photoreversible PHYA* and B* (Table 1). Table 1gives a summary of several in vivo experiments with rice phytochrome A and tobacco phytochrome B. High amounts of spectral active photoreceptor were obtained after in vivo assembly of phytochrome. This is independent of the phytochrome type. The level of in vivo reconstituted phytochrome is significantly higher than in vitro(22) . In control experiments with biliverdin and the corresponding wild-type yeast strain, no photoreversibility could be observed (Table 1).

The location of in vivo formed holophytochrome inside the cell is demonstrated by the protection against proteolytic digestion (Fig. 1A). After assembly with phycocyanobilin, the PHYA* amount was measured in vivo. The signal arising from the photoreversible PHYA* was unaltered until 25 µg of purified oat PHYA was added to the suspension. Two min after adding Proteinase K, the externally applied phytochrome was completely digested. However, the signal arising from the yeast cells remained stable for half of an hour. Afterwards, the PHYA* signal inside the yeast cells declined slowly. This is probably due to the digestion of the yeast cells by the proteinase.

In Vivo Dark Reversion

Even with the most sensitive methods, the PHYB amount in plants is usually too low for direct measurements. Thus, it was so far impossible to find out whether or not phytochrome B from dicotyledonous plants performs dark reversion. As opposed to this, the amount of in vivo reconstituted PHYA* and B* in yeast was relatively high and almost identical (Table 1). The high amount of phytochrome and the possibility to perform in vivo quantification with the Ratiospect (38) enable the direct investigation of PHYB* dark reversion.

Denaturing during phytochrome extraction can lead to artificial Pfr instability and dark reversion. This in vitro reaction is dependent on the presence of divalent cations and reductants(40) . Additionally, PHYA from monocotyledonous plants can dark revert in vitro but not in vivo. To avoid these problems, we exclusively studied the in vivo reaction.

Since both dark reversion and protein destruction lead to the loss of Pfr, protein stability is a prerequisite for the exact quantification of the Pfr reversion. Therefore we investigated the stability of phytochrome. After in vivo reconstitution, the spectral active amount of PHYA* and B* remained stable for at least 1 day (not shown). To see whether Pr and Pfr differ in their stability, yeast cultures were grown in darkness or red light. After PHYA* reconstitution, the cultures were supplemented with fresh growth media, irradiated with far red light (10 min), and transferred to darkness or directly irradiated with red light. After a 12-h incubation, the phytochrome content was spectroscopically measured (Table 1). This light treatment had no influence on the PHYA* level in yeast. In contrast to plants, the Pfr formation caused no rapid destruction of the photoreceptor.

Based on the absolute spectra of PHYB*(22) , we calculated the photoequilibrium between Pfr and Pr. The irradiation with 660-nm red light leads to an equilibrium value of approximately 80% Pfr. In Fig. 280% Pfr is referred to as maximum (100%), and the Pfr amounts are expressed as percentage of the maximum.


Figure 2: Dark reversion of PHYB*. The dark reversion of PHYB* after assembly with phycocyanobilin is shown. After saturating irradiation with red light, the amount of reverting Pfr was spectroscopically determined(38) . The assembly of phytochrome with phycocyanobilin was performed using method 1 as described under ``Experimental Procedures.'' The level of dark-reverting PHYB* was determined in at least four independent experiments each consisting of three to seven measurements. The bars represent the standard deviation of the measured values. The level of Pfr after red light irradiation is 80% of the total phytochrome. This equilibrium value was normalized to 100% Pfr.



Depending on the time in darkness, PHYB* showed a rapid dark reversion. Approximately 30% of the total Pfr was involved, and the reaction was almost completed within 60 min. During the measurements the level of spectral active phytochrome remained unchanged. After dark reversion from Pfr to Pr, the molecule was fully photoreversible (Fig. 2). To exclude the possibility that phycocyanobilin causes dark reversion in phytochrome adducts, we performed control experiments with PHYA*. As in plants, the monocotyledonous PHYA* exhibited no dark reversion (Fig. 2).

Sequestering of Phytochrome in Yeast

Like the dark reversion, the formation of sequestered areas of phytochrome is an in vivo reaction that occurs after the transition of phytochrome into Pfr. This reaction was described for phytochrome from etiolated monocotyledonous plants(3, 34, 41, 42) . After transition into Pfr, phytochrome A is enriched in electron dense particles in the cytosol. This SAP formation is probably ATP-dependent and extremely rapid at room temperature. The temperature and ATP dependence of the SAP formation lead to the hypothesis that the redistribution of phytochrome into defined areas of the cytosol involves an enzymatic process or direct modification of the photoreceptor. However, it was impossible to identify a component that is involved in the SAP formation.

To see whether SAP formation can occur in yeast, we performed electron microscopic investigations after in vivo reconstitution of PHYA*. The ability to perform SAP formation in a fully heterologous system should help to understand this reaction in plants. The experiments with PHYB* were performed to test whether this phytochrome type has the ability to sequester.

Prior to fixation and embedding, the phytochrome content of the spheroplasts was measured, and the cells were either irradiated with red light or kept in darkness. For parallel experiments with yeast cells that contained phytochrome apoprotein, the complete procedure was carried out without chromophore. For the phytochrome A and B localization, we used type-specific antisera (see ``Experimental Procedures'').

The in situ localization of PHYA* (Fig. 3A) shows particles inside the yeast cells. The intensive PHYA* label was clearly limited to the electron dense particles. For comparison, Fig. 3D shows a SAP from etiolated oat(34) . Both particles are very similar with respect to their size and density. In contrast to PHYA*, there were no such particles in PHYB*-containing cells (Fig. 3B). Compared with PHYA*, the density of the immunolabeling was weak in the PHYB*-overexpressing cells (Fig. 3B). On the other hand, the spheroplasts contained almost identical amounts of photoreversible phytochrome A* and B* (104 µg/g fresh weight for PHYA* and 106 µg/g fresh weight for PHYB*).


Figure 3: Immunolocalization of phytochrome in yeast cells. After the in vivo assembly of apophytochrome and phycocyanobilin, yeast spheroplasts were fixed and embedded. The in situ localization was performed with phytochrome-specific antisera, using the immunogold method. The gold particles indicate the localization of PHYA* (A) and PHYB* (B). C shows the control experiment with the corresponding yeast wild-type strain KN380 and phytochrome antisera. For direct comparison of plant- and yeast-derived SAP, a SAP from etiolated oat (3) is shown in D. The bar represents 0.5 µm.



Control experiments with the respective preimmune sera (not shown) and with the corresponding yeast wild-type strain (Fig. 3C) revealed no unspecific background. To test whether the sequestering of PHYA* is due to a simple ``overloading'' effect, we performed further in situ localizations with yeast cultures possessing lower amounts of photoreversible phytochrome (53 µg/g fresh weight PHYA*, 44 µg/g fresh weight PHYB*). The relatively low phytochrome level had no influence on the sequestering of PHYA*.

In parallel experiments, we examined the behavior of the phytochrome apoproteins (not shown). The in situ localizations with apophytochrome A and B revealed no differences to the reconstituted holophytochrome. Whereas apophytochrome B showed no sequestering, the apophytochrome A formed the same particles as the reconstituted phytochrome. Therefore, the ``sequestering reaction'' of phytochrome A in yeast is clearly independent of Pfr formation.

In etiolated plants, the SAP formation leads to the in vitro pelletability of the phytochrome aggregates formed in the cytosol(42) . Easy centrifugation and washing procedures serve to enrich phytochrome particles from crude extracts of etiolated plants(34) . To gain more information about the ``yeast-SAP'' and to compare the in vitro behavior of plant and yeast-SAP, we performed in vitro pelletability assays. Since the chromophore is no prerequisite for the sequestering in yeast, the experiments were carried out with apophytochrome.

For immunolocalizations, yeast-SAP were fixed and embedded(34) . The pellets derived from phytochrome B overexpressing yeast cultures contained only slight traces of phytochrome (not shown). In contrast, the yeast apophytochrome A particles could be enriched in the pellet fraction. Fig. 4contains a comparison between two micrographs of a plant (Fig. 4B) and yeast-derived (Fig. 4A) SAP. The phytochrome particles are virtually indistinguishable with respect to the immunostaining, the size, and the enhanced electron density.


Figure 4: Comparative analysis of phytochrome in yeast- and plant-derived pellet fractions. The pellet fraction of yeast crude extracts containing apophytochrome A were fixed and embedded as described by Hofmann et al(34) . Immunolocalizations were performed with phytochrome-specific antisera using secondary, gold-labeled antibodies. A shows the pellet fraction from yeast and yeast- derived SAP. For a comparative purpose, B shows the pellet fraction from crude extracts of etiolated oat and ``plant-derived SAP''(34) . The bar represents 0.5 µm.



Expression of Chalcone Synthase-uidA Constructs in Phytochrome Overexpression in Yeast

Since yeast is a model system for many signal transduction cascades, we investigated the functionality of in vivo reconstituted phytochrome in yeast.

To test whether phytochrome can influence gene expression in yeast, we performed coexpression experiments of phytochrome with unit 1 reporter plasmids (parsley chalcone synthase unit 1 uidA fusions, see ``Experimental Procedures''). After in vivo reconstitution, the phytochrome level was spectroscopically determined and the cultures were either irradiated for 10 min with far-red light and transferred to darkness or directly irradiated with red light. The cells were harvested at various time points, and the beta-glucuronidase activity was determined in yeast crude extracts. Fig. 5shows a representative experiment with in vivo reconstituted PHYA* and the unit 1 reporter plasmid. Although unit 1 can mediate uidA expression in yeast, the control experiments (bilirubin and Me(2)SO) demonstrate that neither the light treatment nor the presence of phytochrome as apoprotein or holoprotein can influence beta-glucuronidase activity.


Figure 5: beta-Glucuronidase activity in apophytochrome overexpressing yeast. The in vivo reconstitution of phytochrome and coexpression with reporter plasmids for the unit 1 parsley chalcone synthase promoter was performed as described under ``Experimental Procedures.'' Rice apophytochrome A was in vivo reconstituted with phycocyanobilin (PHYA*) or treated with bilirubin (apo-PHYA + bilirubin). Additionally, a control experiment without chromophore was performed (apo-PHYA). The spectroscopically detectable amount of phytochrome in the starting culture was 165 µg/g of fresh weight. After this treatment, the cultures were either irradiated with far-red light and then transferred into darkness or directly irradiated with continuous red light. The cells were harvested at an optical density (600 nm) between 0.85 and 1.1. The beta-glucuronidase activity was determined in yeast crude extracts. The bars represent the standard deviation of four dependent probes. The experiments were repeated several times with PHYA*, and the tobacco phytochrome B-phycocyanobilin adduct. Additionally, yeast cultures of diverse optical densities were analyzed for the expression of unit 1 reporter constructs. In none of these experiments could a significant light regulation be observed.



A similar set of experiments was performed with PHYB*, together with the unit 1 reporter plasmid and with the full-length chalcone synthase promoter (not shown). None of these experiments indicated a phytochrome-dependent expression of the reporter genes.


DISCUSSION

Since PHYA and B have overlapping but distinct physiological roles in plants(17, 18) , it seems likely that the proteins differ not only in their signal transduction chains, but also in their molecular properties. Over the last few years, yeast systems have been very effective for the biochemical study of different phytochrome types (19, 20, 21, 22) . On the other hand, the in vitro characterization of PHYA* and PHYB* in yeast extracts revealed no differences between the labile and stable type of phytochrome. To gain more information about the molecular properties, we started to investigate phytochrome in vivo reactions in yeast. The in vivo experiments with PHYB* enabled for the first time the direct investigation of this type of phytochrome. Using the in vivo system, we described Pfr dark reversion and the sequestering reaction. The data indicate that these reactions are intrinsic properties of the photoreceptor itself. In the following we discuss the physiological relevance of dark reversion and consequences for the sequestering model in plants.

In Vivo Reconstitution of Phytochrome with Phycocyanobilin

The first step in this comparative analysis was to establish two protocols for the in vivo reconstitution of phytochrome. It was shown by Quail and co-workers (43) that the addition of phycocyanobilin to plant culture media leads to chromophore uptake and to the reconstitution of holophytochrome in the chromophore deficient mutants (hy1,2) of Arabidopsis(43) . As in plants, the addition of phycocyanobilin into synthetic yeast culture media leads to the uptake (Fig. 1B) and to the formation of photoreversible phytochrome (Table 1). Since both PHYA and -B assemble autocatalytic with the chromophore, the reaction is independent of the phytochrome type. The location of the apophytochrome inside the yeast cells is demonstrated by the protection against proteolytic degradation (Fig. 1A), and the investigations with vital dyes clearly indicate that the reconstitution occurs in vivo. Our results therefore confirm the results recently published by Li and Lagarias (44) .

To avoid long reconstitution times, we adopted a second method (see ``Method 2'' under ``Experimental Procedures'') published for the uptake of phthalocyanins into yeast cells. Paardekooper and co-workers (45) reported that Me(2)SO treatment of yeast cells leads to chromophore uptake and that the treatment is not toxic. Since the structures of phthalocyanins and phycocyanobilin are similar, it is not surprising that the Me(2)SO treatment induces the uptake of the linear tetrapyrrole. In contrast to Paardekooper et al.(45) who reported a light-induced photooxidative effect after phthaloyanine treatment, our results (Table 1) indicate no differences in viability after phycocyanobilin uptake and irradiation with red light. Simultaneously the red-light experiments confirm the finding of Li and Lagarias (44) that phytochrome is not degraded in a light-specific manner in yeast.

In Vivo Dark Reversion

The molecular properties of PHYA from etiolated plants have been characterized with respect to SAP formation, protein degradation, and dark reversion. The very small amount in plants prevents the direct investigation of PHYB in vivo reactions. In contrast to plants, the phytochrome level in yeast is relatively high and independent of the phytochrome type (Table 1). Additionally, the phytochrome amount remains stable for a long time.

Using the in vivo system, we investigated the dark reversion of phytochrome in yeast. PHYB* performs a rapid dark reversion while retaining full photoreversibility. The dark reversion affects approximately 30% of the total Pfr with a half-life time of approximately 20 min following a logarithmic decay. In a control experiment we also tested PHYA* dark reversion in yeast. As in plants, the monocotyledonous PHYA* exhibits no dark reversion in yeast. This demonstrates that PHYB* dark reversion is not due to the usage of the substitute chromophore phycocyanobilin.

Observations on cauliflower florets and artichoke receptacles(11, 12) gave indirect evidence that the observed PHYB* Pfr reversion also occurs in plants. In these organs, phytochrome is light-stable and exhibits dark reversion with a half-life time of approximately 60 min. Additionally, the existence of a dark reverting, light stable phytochrome was concluded from flowering induction experiments with the Japanese morninglory Pharbitis nil and Chrysanthemum(13) . These examples indicate that PHYB dark reversion is of physiological relevance in light-grown plants. However, light-stable phytochrome is not necessarily PHYB. It is possible that even a light-labile phytochrome behaves as stable protein if the rapid destruction is disabled.

Dark reversion of PHYB* Pfr may be a general physiological mechanism in dicotyledonous plants. Since PHYB turnover is slow and the phyB gene is constitutively expressed, the Pfr-reversion possibly prevents continuous PHYB signals. Another consequence of Pfr reversion may be the inability of PHYB to mediate far-red light and very low fluence responses. Since PHYA and B probably possess identical absorbance spectra(22) , the differences cannot be explained with the spectral properties of the two photoreceptors. Therefore, dark reversion combined with the low PHYB level may prevent the formation of Pfr amounts that are sufficient to mediate very low fluence and far-red light responses.

Assuming that yeast has no system that discriminates between PHYA* and -B*, we conclude that dark reversion is an intrinsic property of the photoreceptor. Based on reversion measurements in etiolated mustard, Brockmann et al.(1) presented a theoretical model to explain phytochrome A Pfr-reversion. Their calculations indicate that the amount of dark-reverting phytochrome correlates with the amount of structural heterodimers between Pr and Pfr. For an 80% photoequilibrium, the theoretical level of those Pr/Pfr heterodimers is 32% of the total phytochrome. The measured amount of PHYB* Pfr dark reversion in yeast was 25.6% of the total phytochrome (at 80% photoequilibrium). Since the theoretical value is similar to the measurable, the heterodimer model may serve as explanation for the intrinsic property to perform Pfr reversion.

Sequestering of Phytochrome

A further in vivo reaction observable in yeast is the formation of phytochrome particles. Investigations in etiolated plants showed that PHYA Pfr forms SAP in the cytosol of the plant cells. SAP formation in plants is extremely rapid at room temperature and leads to the in vitro pelletability of PHYA(42) .

The electron microscopic in situ localization revealed that the rice phytochrome A formed SAP-like particles in yeast (Fig. 3A). From in vitro experiments, it has become clear that this leads, just as in plants, to an enhanced pelletability of the yeast-derived SAP (Fig. 4A). Since PHYB showed no sequestering and SAP formation in yeast, the PHYA sequestering reflects probably another molecular difference between these two types of the photoreceptor.

A simple aggregation of PHYA* caused by the high expression level is unlikely, since both phytochromes were present in identical amounts. Additionally, phytochrome A also sequestered in yeast cells with relatively low levels of photoreversible phytochrome. This indicates that the high phytochrome level is not responsible for the formation of phytochrome particles. The ability to form SAP-like particles in yeast had no influence on the protein stability. The sequestering in yeast is therefore not a sufficient signal for enhanced protein degradation. So far, we have no evidence for a possible ubiquitination of phytochrome in these particles (not shown). This may contrast with the view that the SAP formation in plants is the responsible signal for ubiquitination and rapid destruction of the photoreceptor via the proteasome.

On the level of our investigations, we could not find any differences between plant- and yeast-derived SAP with respect to enhanced electron density, immunolabeling, and size of the particles. In deviation from plants, the sequestering was independent of the form of the photoreceptor.

This finding may have important consequences for the interpretation of the sequestering reaction itself. The ATP dependence(41) , together with the temperature and Pfr dependence, lead to the hypothesis that phytochrome A sequestering is performed by an enzyme-like sequestering machinery. This reaction possibly involves a direct, ATP-dependent modification of phytochrome(41) . According to this hypothesis, Pfr is specifically recognized and transported into distinct regions of the cytosol. At these locations, it first forms smaller particles (approximately 0.3 µm) and later on larger aggregates (>1 µm) of those initial particles(3, 41) . On the other hand, inhibitor studies with cytochalasin B and colchicine excluded the involvement of the cytoskeleton(41, 42) , thus, a directed phytochrome transport seems difficult. Additionally, no other components, except ubiquitin, could be co-located with these particles(3, 34, 42) .

The results in yeast indicate that the sequestering is, as PHYB* dark reversion, an intrinsic property of the photoreceptor. Since apophytochrome A sequestered in yeast the question arises why PHYA in plants shows no spontaneous sequestering. If phytochrome sequestering in plants and yeast reflects the same reaction, Pr sequestering in plants has to be prevented. After transition into Pfr, this block is removed, and this reaction is probably ATP-dependent. As a consequence of this receptor property, PHYA sequesters without any additional components.

In spite of its hypothetical nature, such a model is in good accordance with the behavior of the initial phytochrome particles that would form larger aggregates by diffusion and the well known ability of molecular chaperones to release proteins under ATP hydrolysis.

Regulation of Gene Expression in Phytochrome Overexpressing Yeast

The in vivo experiments in yeast revealed molecular properties of the photoreceptor itself. To test whether phytochrome in yeast also exhibits functionality, we performed coexpression experiments with unit 1 reporter plasmids. The unit 1 is a cis element of the parsley chalcone synthase promoter that is sufficient to mediate light- and tissue-specific expression(46, 47) . This element contains a G box motif that has been shown to be important for the expression of chalcone synthase(31, 48) . Interestingly, yeast cells contain trans acting factors that bind to the G box and can mediate gene expression(49) . Additionally, microinjection experiments revealed wide-spread components like G-proteins and Ca/calmodulin for phytochrome signal transduction(50, 51) . Since related components are likely to be present in yeast cells, it seemed possible that in vivo reconstituted phytochrome may influence gene expression in yeast.

The results showed that the used promoter fragment served to mediate uid A expression in yeast. Again, this indicates the presence of a functional G box binding activity in yeast(49) . But neither PHYA* nor PHYB* can influence the expression of the used reporter plasmids.

Physiological Relevance of the Results

The major problem with the fully heterologous yeast system is to elucidate the physiological relevance of the findings. On the other hand, the in vivo system is excellent for the investigation of the molecular differences between PHYA* and PHYB*. Yeast cells express both phytochrome types in almost identical amounts, and it seems unlikely that they contain elements that distinguish between phytochrome types. Especially for PHYB, this is of great advantage since the level in plants is too low for direct investigations. However, we cannot definitely exclude that the observed differences between phytochrome A and B arise from problems caused by the overexpression in yeast. Because of the following reasons, we believe that this interpretation is unlikely.

1) The in vitro characterization of PHYB* in yeast extracts, concerning the spectral properties, dimerization, chromophore attachment and quantum efficiency,^2 revealed no difference to phytochrome from the A type(19, 20, 21, 22) .

2) Although both types of phytochrome were expressed using identical expression systems, yeast strains, and experimental conditions, the in vivo characterization revealed clear differences between PHYA and PHYB. Whereas PHYB* showed dark reversion and no sequestering, the PHYA performed sequestering but not dark reversion.

3) PHYA* and PHYB* expressed in yeast possess biological activity. Since the yeast system was not suitable for studies concerning the functionality (differential gene expression) of phytochrome, we decided to perform microinjection experiments.^2 The injection experiments with the aurea mutant of tomato revealed that both proteins induce a variety of physiological reactions like, for example, the formation of chlorophyll.

These points clearly show that yeast-derived phytochrome is in a native conformation. We have therefore come to the conclusion that the observed differences between the rice phytochrome A and the tobacco phytochrome B reflect molecular properties of different types of phytochrome.


FOOTNOTES

*
This work was supported by the Deutsche Forschungsgemeinschaft SFB 388 and an International Human Frontier Science Organization (HSFPO) grant (to E. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Tel.: 49-761-2032683; Fax: 49-761-203-2629.

(^1)
The abbreviations used are: Pr, red light absorbing form of phytochrome; Pfr, far-red light absorbing form of phytochrome; PHYA*, rice phytochrome A-phycocyanobilin adduct; PHYB*, tobacco phytochrome B-phycocyanobilin adduct; SAP sequestered areas of phytochrome.

(^2)
T. Kunkel, G. Neuhaus, A. Batschauer, N.-H. Chua, and E. Schäfer, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Professor Dr. M. Furuya and Dr. D. H. Wolf for fruitful discussion and help with the yeast system and Professor Dr. N.-H. Chua for providing the phytochrome genes. We also would like to thank Professor Dr. R. Hertel, Professor Dr. M. Blatt and J. Blatt for critical reading of the manuscript, and E. Mummert for providing purified oat phytochrome A.


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