Two native pools of phytochrome A in monocots: Evidence from fluorescence investigations of phytochrome mutants of rice

Fluorescence investigations of phytochrome (phy) in rice (Oryza sativa L. cv. Nipponbare) mutants deficient in phyA, phyB and phyA plus phyB were performed. Total content of the pigment (P(tot)) and its spectroscopic and photochemical characteristics were determined in different parts of the dark-grown and far-red light (FR)-grown coleoptiles. Spectroscopically, phyA in the phyB mutant was identical to phyA in the wild-type (WT) and the extent of the conversion from Pr to lumi-R at 85 K was the same for phyA in both lines and varied similarly, depending on the part of the coleoptile used. The latter finding proved that phyA in rice is heterogeneous and comprises two phyA populations, phyA' and phyA". Functional properties of phyA were also determined. In the dark the phyB mutant had a higher content of phyA, inactive protochlorophyllide (Pchlide633) and active protochlorophyllide (Pchlide655) than WT and its coleoptile was longer, indicating that phyB may affect the development of WT seedlings in the dark. Constant FR drastically reduced the content of phyA, Pchlide633 and Pchlide655 and brought about coleoptile shortening and appearance of the first leaf, whereas pulsed FR of equal fluence was less effective. This suggested that the reactions were primarily of the high irradiance responses type, which are likely to be mediated by phyA'. The effects on protochlorophyllide biosynthesis and growth responses type were more pronounced in the phyB mutant than in the WT seedlings, which can be connected with the higher phyA' content in the phyB mutant and/or phyB interference with its action in WT seedlings. In the phyA mutant induction of Pchlide633 and Pchlide655 biosynthesis was observed under constant FR, indicating that phyC may be responsible for this effect.     

Fluorescence and photochemical characterization of phytochromes A and B in transgenic potato expressing Arabidopsis phytochrome B

Phytochrome in etiolated sprouts of wild type (WT) potato and its transgenic strains (DARA5 and DARA12) expressing Arabidopsis thaliana phytochrome B (phyB) was investigated using low-temperature (85 K) fluorescence spectroscopy and photochemistry. Phytochrome content, [Ptot], position of the Pr emission and excitation spectra, lambda(max), and extent of the Pr-->lumi-R, gamma1, and Pr-->Pfr, gamma2, phototransformations (at 85 and 273 K, respectively) were shown to vary in the transgenic lines and WT depending on tissue used (upper vs. lower parts of etiolated sprouts) and light-induced phytochrome depletion. Differences in the parameters between the transgenic lines and WT were detected which were interpreted in terms of the two phenomenological Pr types: a labile Pr' with gamma1 approximately 0.5 consisting of a major phytochrome A (phyA) fraction (phyA') and a relatively conserved Pr" with gamma1 = 0 comprising a minor phyA fraction (phyA") and phyB. Both DARA lines had higher [Pr"] as compared with WT in the lower parts of etiolated stems, especially after light-induced phytochrome depletion (residual phytochrome in DARA5 and DARA12 made up to one-third of its initial level vs. <5% in WT). These differences were associated with the expression of Arabidopsis phyB in the DARA lines and its higher light stability than that of phyA. Arabidopsis phyB expressed in potato was characterised by lambda(max) = 683/669 nm in the emission/excitation (absorption) spectra and gamma1 = 0. PhyB also revealed a relatively low gamma2 (approx. 0.5) and its early red drop as compared with the gamma2 wavelength dependence for phyA. This is believed to contribute to the lower signalling ability of phyB and to confine the region (red) of its physiological activity.     

Recombinant phytochrome of the moss Ceratodon purpureus (CP2): fluorescence spectroscopy and photochemistry

The recombinant phytochrome of the moss Ceratodon purpureus (CP2) expressed in Saccharomyces cerevisiae and reconstituted with phycocyanobilin (PCB) was investigated using fluorescence spectroscopy. The pigment had an emission maximum at 670 nm at low temperature (85 K) and at 667 nm at room temperature (RT) and an excitation maximum at 650-652 nm at 85 K (excitation spectra could not be measured at RT). Both spectra had a half-band width of approx. 30-35 nm at 85 K. The fluorescence intensity revealed a steep temperature dependence with an activation energy of fluorescence decay (Ea) of 5.9-6.4 and 12.6-14.7 kJ mol(-1) in the interval from 85 to 210 K and from 210 to 275 K, respectively. The photochemical properties of CP2/PCB were characterised by the extent of the red-induced (lambda(a) = 639 nm) Pr conversion into the first photoproduct lumi-R at 85 K (gamma1) of approximately 0.07 and into Pfr at RT (gamma2) of approximately 0.7. From these characteristics, CP2/PCB can be attributed to the Pr" photochemical type with gamma1 < or = 0.05, which comprises the minor phyA fraction (phyA"), phyB, Adiantum phy1 and Synechocystis Cph1 in contrast to the major phyA' fraction (Pr' type with gamma1 = 0.5). Within the Pr" type, it is closer to phyA" than to phyB and Cph1.     

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.

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 possibility Arabidopsis and rice (Oryza) phyA were expressed in yeast and the pigments were assembled in vivo with phycocyanobilin (PCB) and phytochromobilin (P phi 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 temperature dependence of Pr fluorescence intensity and activation energy of fluorescence decay; and (3) the extent of photoconversion of Pr into photoproduct lumi-R (gamma 1) or far-red-light absorbing form (Pfr) (gamma 2). Both Arabidopsis phyA/PCB and Oryza phyA/P phi B had low gamma 1 of ca 0.05, allowing their attribution to the Pr" phenomenological type of phytochrome comprising phyA", phyB and cryptogam phytochromes. The spectroscopic properties of Oryza phyA/P phi B were also very close to phyA". However, both investigated holoproteins differed from phyA", both with respect to the character of temperature dependence of the fluorescence yield and activation energy. Thus, recombinant Oryza phyA/P phi 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.     

Effects of Moderately Low Temperature (20°C) on Phytochrome Responses During Preirradiation: Anthocyanin Synthesis in Sorghum bicolor at High- and Low-Pfr/Ptot Ratios

Abstract— Effects on phytochrome-mediated anthocyanin synthesis of moderately low temperature (MLT) given during the preirradiation culture period were studied with seedlings of broom sorghum ( Sorghum bicolor Moench, cvs. Acme Broomcorn and Sekishokuzairai-Fukuyama). Seedlings were grown in the dark at 20°C (MLT) and 24°C (control). The MLT treatment strikingly enhanced the action induced by a red light (R) pulse above ca 200 μmol m⁻² and suppressed the action induced by an R pulse below ca 30 μmol m⁻² and by a far-red light (FR) pulse alone. We refer to these MLT-affected distinct responses as "high-Pfr/Ptot response" and "low-Pfr/Ptot response," as they have features different from the high-irradiance and very-low-fluence responses, respectively. The destruction rate of spectroscopically detectable phytochrome (phyA) and the time course of escape of anthocyanin synthesis from FR reversibility did not match, and hence the possibility of phyA being involved in high-Pfr/Ptot response was rejected, although it might be involved in low-Pfr/Ptot response. Possible mechanisms for the two distinct phytochrome responses are discussed.     

Two photobiological pathways of phytochrome A activity, only one of which shows dominant negative suppression by phytochrome B

The plant receptor phytochrome A (phyA) mediates responses like hypocotyl growth inhibition and cotyledon unfolding that require continuous far-red (FR) light for maximum expression (high-irradiance responses, HIR), and responses like seed germination that can be induced by a single pulse of FR (very-low-fluence responses, VLFR). It is not known whether this duality results from either phyA interaction with different end-point processes or from the intrinsic properties of phyA activity. Etiolated seedlings of Arabidopsis thaliana were exposed to pulses of FR (3 min) separated by dark intervals of different duration. Hypocotyl-growth inhibition and cotyledon unfolding showed two phases. The first phase (VLFR) between 0.17 and 0.5 pulses.h-1, a plateau between 0.5 and 2 pulses.h-1 and a second phase (HIR) at higher frequencies. Reciprocity between fluence rate and duration of FR was observed within phases, not between phases. The fluence rate for half the maximum effect was 0.1 and 3 mumol.m-2.s-1 for hourly pulses of FR (VLFR) and continuous FR (HIR), respectively. Overexpression of phytochrome B caused dominant negative suppression under continuous but not under hourly FR. We conclude that phyA is intrinsically able to initiate two discrete photoresponses even when a single end-point process is considered.     

Phytochromes, cryptochromes, phototropin: photoreceptor interactions in plants

In higher plants, natural radiation simultaneously activates more than one photoreceptor. Five phytochromes (phyA through phyD), two cryptochromes (cry1, cry2) and phototropin have been identified in the model species Arabidopsis thaliana. There is light-dependent epistasis among certain photoreceptor genes because the action of one pigment can be affected by the activity of others. Under red light, phyA and phyB are antagonistic, but under far-red light, followed by brief red light, phyA and phyB are synergistic in the control of seedling morphology and the expression of some genes during de-etiolation. Under short photoperiods of red and blue light, cry1 and phyB are synergistic, but under continuous exposure to the same light field the actions of phyB and cry1 become independent and additive. Phototropic bending of the shoot toward unilateral blue light is mediated by phototropin, but cry1, cry2, phyA and phyB positively regulate the response. Finally, cry2 and phyB are antagonistic in the induction of flowering. At least some of these interactions are likely to result from cross talk of the photoreceptor signaling pathways and uncover new avenues to approach signal transduction. Experiments under natural radiation are beginning to show that the interactions create a phototransduction network with emergent properties. This provides a more robust system for light perception in plants.     

Fluorescence and Photochemistry of Recombinant Phytochrome from the Cyanobacterium Synechocystis

Fluorescence and photochemical properties of phytochrome from the cyanobacterium Synechocystis were investigated in the temperature interval from 293 to 85 K. The apoprotein was obtained by overexpression in Escherichia coli and assembled to a holophytochrome with phycocyanobilin (PCB) and phytochromobilin (PφB), Syn(PCB)phy and Syn(PφB)phy, respectively. Its red-absorbing form, Pr, is characterized at 85 K by the emission and excitation maxima at 682 and 666 nm in Syn(PCB)phy and at 690 and 674 nm in Syn(PφB)phy. At room temperature, the spectra are blue shifted by 5–10 nm. The fluorescence intensity dropped down by ?15–20-fold upon warming from 85 to 293 K and activation energy of the fluorescence decay was estimated to be ca 5.4 and 4.9 kJ mol^?1 in Syn(PCB)phy and Syn(PφB)phy, respectively. Phototransformation of Pr upon red illumination was observed at temperatures above 160–170 K in Syn(PCB)phy and above 140–150 K in Syn(PφB)phy with a 2–3 nm shift of the emission spectrum to the blue and increase of the intensity of its shorter wavelength part. This was interpreted as a possible formation of the photoproduct of the meta-Ra type of the plant phytochrome. At ambient temperatures, the extent of the Pr phototransformation to the far-red-absorbing form, Pfr, was ca 0.7–0.75 and 0.85–0.9 for Syn(PCB)phy and Syn(PφB)phy, respectively. Fluorescence of Pfr and of the photoproduct similar to lumi-R was not observed. With respect to the photochemical parameters, Syn(PCB)phy and Syn(PφB)phy are similar to each other and also to a small fraction of phyA (phyA″) and to phyB. The latter were shown to have low photochemical activity at low temperatures in contrast to the major phyA pool (phyA″), which is distinguished by the high extent (ca 50%) of Pr photoransformation at 85 K. These photochemical features are interpreted in terms of different activation barriers for the photoreaction in the Pr excited state.     

Two modes of the light-induced phytochrome A decline--with and without changes in the proportion of its isoforms (phyA' and phyA''): evidence from fluorescence investigations of mutant phyA-3D pea

Different modes of the phytochrome function are connected with its polymorphism, the major isoforms being phytochromes A and B (phyA and phyB). In its turn, phyA comprises two native species, phyA' and phyA', whose precise nature and functions remain obscure. With the use of in situ fluorescence spectroscopy, we investigated their properties in a mutant of pea, phyA-3D, characterized by exaggerated photoresponses and impaired photodestruction of phyA. The mutation is a substitution of alanine by valine at the position 194 in phyA. The phyA-3DphyB and phyB mutants were also investigated. In dark-grown plants, all the lines had the content and properties of the two phyA species very similar to the wild type. However, a considerably more intense reduction in [phyA] without changes in the phyA'/phyA' equilibrium was found in far-red grown mutant plants suggesting a hypersensitivity of phyA-3D with regard to its autoregulation. On the contrary, under red illumination, a higher stability of phyA-3D was observed confirming our earlier findings. This allows a conclusion that the A194V substitution in phyA-3D not only impairs its destruction but also enhances its signaling ability, suggesting a role of this locus in modulation of its activity.     

Fluorescence Analysis of Oat phyA Deletion Mutants Expressed in Tobacco Suggests that the N-Terminal Domain Determines the Photochemical and Spectroscopic Distinctions between phyA' and phyA"†

Abstract— In vivo low-temperature (85 K) fluorescence spectroscopy has defined two phytochrome A (phyA) subpopulations, designated phyA' and phyA", in etiolated seedlings (V. A. Sineshchekov, J. Photochem. Photobiol. 28, 53–55, 1995). Phytochrome A' is the more abundant but light-labile species characterized by longer wavelength emission/absorption maxima (687/673 nm) and by a higher extent of the photoconversion of its red-absorbing form (Pr) into photoproduct (lumi-R) at 85 K (γ₁≈ 0.5). Phytochrome A" is the minor but relatively light-stable species, characterized by shorter wavelength maxima (682/668 nm) and by a lower γ₁ (<0.05). To help define domains within phyA responsible for these differences, the low-temperature spectral properties of transgenic tobacco expressing full-length (FL) oat phyA and C-and N-terminally truncated versions (CD [Δ786–1129] and NA [Δ7–69], respectively) were compared. Oat phytochrome expression was more pronounced than that of tobacco in the basal section of etiolated seedlings following 2 h irradiation with white light. Seedlings expressing FL and CD phyA had spectral properties for phyA' and phyA" that were indistinguishable from that of wild-type tobacco. Conversely, expression of NA phyA generated an abundant phy species that behaved like phyA". From this we conclude that the N-terminal domain of phyA is involved in determining the photochemical and spectroscopic distinctions between the native phyA' and phyA" species.     

Phytochrome Control of Anthocyanin Biosynthesis in Tomato Seedlings: Analysis Using Photomorphogenic Mutants

Anthocyanin biosynthesis has been studied in hypocotyls and whole seedlings of tomato (Lycoperskon esculentum Mill.) wild types (WTs) and photomorphogenic mutants. In white light (WL)/dark (D) cycles the fri¹ mutant, deficient in phytochrome A (phyA), shows an enhancement of anthocyanin accumulation, whereas the tri¹ mutant, deficient in phytochrome Bl (phyBl) has a WT level of anthocyanin. Under pulses of red light (R) or R followed by far-red light (FR) given every 4 h, phyA is responsible for the non-R/FR reversible response, whereas phyBl is partially responsible for the R/FR reversible response. From R and blue light (B) pretreatment studies, B is most effective in increasing phytochrome responsiveness, whereas under R itself it appears to be dependent on the presence of phyBl. Anthocyanin biosynthesis during a 24 h period of monochromatic irradiation at different flu-ence rates of 4 day-old D-grown seedlings has been studied. At 660 nm the fluence rate-response relationships for induction of anthocyanin in the WT are similar, yet complex, showing a low fluence rate response (LFRR) and a fluence rate-dependent high irradiance response (HIR). The high-pigment-1 (hp-1) mutant exhibits a strong amplification of both the LFRR and HIR. The fri¹ mutant lacks the LFRR while retaining a normal HIR. In contrast, a transgenic tomato line overexpressing the oat PHYA3 gene shows a dramatic amplification of the LFRR. The tri¹ mutant, retains the LFRR but lacks the HIR, whereas the fri¹, tri¹ double mutant lacks both components. Only an LFRR is seen at 729 nm in WT; however, an appreciable HIR is observed at 704 nm, which is retained in the tri¹ mutant and is absent in the fri¹ mutant, indicating the labile phyA pool regulates this response component.     

Light quality influences indigo precursors production and seed germination in Isatis tinctoria L. and Isatis indigotica Fort

Isatis tinctoria L. and Isatis indigotica Fort. are biennial herbaceous plants belonging to the family of Cruciferae that are used as a source of natural indigo and show several morphological and genetic differences. Production of indigo (indigotin) precursors, indican (indoxyl beta-D glucoside) and isatan B (indoxyl ketogluconate), together with seed germination ability were compared in Isatis tinctoria and Isatis indigotica grown under six different light conditions (darkness, white, red, far red, blue, yellow light) at 25 degrees C. Light quality influenced both germination and production of indigo precursors in the two Isatis species. Different responsiveness to far red and blue light was observed. Indeed, a detrimental effect on germination by blue and far red light was found in I. tinctoria only. Different amounts of isatan B were produced under red and far red light in the two Isatis species. In I. tinctoria, the level of main indigo precursor isatan B was maximal under red light and minimal under far red light. Whereas in I. indigotica far red light promoted a large accumulation of isatan B. The photon fluence rate dependency for white and yellow light responses showed that the accumulation of indigo precursors was differently influenced in the two Isatis species. In particular, both white and yellow light enhanced above 40 micromol m(-2) s(-1) the production of isatan B in I. indigotica while only white light showed a photon fluence dependency in I. tinctoria. These results suggest a different role played by the labile and stable phytochrome species (phyA and phyB) in the isatan B production in I. tinctoria and I. indigotica. I. indigotica, whose germination percentage was not influenced by light quality, demonstrated higher germination capability compared with I. tinctoria. In fact, I. tinctoria showed high frequency of germination in darkness and under light sources that establish high phytochrome photoequilibrium (red, white and yellow light). Germination in I. tinctoria was negatively affected by far red and blue light. I. indigotica seeds appear to be indifferent to canopy-like light (far red). Our results provide further insights on the distinct behaviour of I. tinctoria and I. indigotica that belong to two different genetic clusters and different original environments.     

Up-regulation by phytochrome A of the active protochlorophyllide, Pchlide655, biosynthesis in dicots under far-red light

It is well-documented that phytochrome A (phyA) down-regulates the synthesis of NADPH:protochlorophyllide (Pchlide) oxidoreductase and active Pchlide(655) under far-red light (FR). In this work, we demonstrate that phyA can up-regulate the synthesis of Pchlide(655) under FR as well and that its sign and extent depend on plant species and tissue. With the use of fluorescence spectroscopy, it was found that [Pchlide(655)] in the upper stems of FR-grown seedlings of pea and tobacco increased > or =10-fold and much lower in cotyledons or leaves as compared with the dark-grown. In the upper stems of Arabidopsis and tomato, the positive effect of FR was low, 1.2- to 1.5-fold, and the negative effect of FR was seen in cotyledons. In stems of wild-type (WT) tobacco and its line overexpressing full-length oat phyA (FL), we observed gross stimulating effect of FR while in its line overexpressing N-terminally truncated (Delta7-69) oat phyA (NA) it was low. Because WT and FL comprise both native phyA forms, phyA' and phyA", while NA, only phyA", the regulation under FR can be associated with phyA', while phyA" inhibits the action of phyA'. In etiolated seedlings of the NA line, [Pchlide(655)] was much higher than in those of WT and FL suggesting that phyA" may have relation to this enhancement. The regulation of Pchlide(633) in contrast to Pchlide(655) was positive independent of the plant species and tissue.     

A SIMPLE AND IMPROVED METHOD OF ISOLATION AND PURIFICATION FOR NATIVE OAT PHYTOCHROME

Abstract— A simple procedure for the isolation and purification of 124 kDa phytochrome (phyA) from etiolated Avena seedlings has been developed employing ammonium sulfate back-extraction. After solubilization of the ammonium sulfate precipitate (250 g/L) an additional ammonium sulfate fractionation with 17 g per 100 mL rather than column chromatography was performed. After several steps of the "washing-out" procedure with 100 mM phosphate buffer, phytochrome was solubilized in 10 m M phosphate buffer. The resulting phytochrome had a specific absorbance ratio (SAR = A₆₆₆/A₂₈o) ranging from 0.60 to 0.85. These values are equivalent to those of phytochrome preparations after hydroxyla-patite chromatography-ammonium sulfate back-extraction. The total isolation-purification time was 8 h and yield of the chromoprotein was 50% higher than the yield using conventional techniques. The phytochrome preparation, after application to a Toyopearl HW-65S gel filtration column, produced very pure 124 kDa phyA with a specific absorbance ratio greater than 1.00. The spectral characteristics are identical to those described for the best of the highly purified native chromoprotein preparations.     

PHYTOCHROME CONVERSION AS AN IN SITU ASSAY FOR EFFECTIVE LIGHT GRADIENTS IN ETIOLATED SEEDLINGS OF Zea mays

Abstract— In situ photochemical conversion and measurement of phytochrome can be used as a non-intrusive assay for axial light conduction along etiolated plant tissues of Zea maysL. A computerized single beam spectrophotometer was used to measure phytochrome conversion just below the coleoptilar node following irradiation of the plant shoot with 632 nm light at various distances from the measurement point. Photochemical conversion decreased log-linearly with increasing distance between the irradiation and measurement points. The distance over which the light was attenuated by 50% was 1.80 mm for mesocotyl, 1.60 mm for coleoptile plus primary leaf, and 1.15 mm for primary leaf alone (coleoptile removed). These results verify the photochemical significance of axially conducted light and thereby provide supporting evidence that such light conduction is important for photomorphogenesis.     

OPTICAL PROPERTIES OF PHYTOCHROME IN THE BLUE- SPECTRAL REGION AS MEASURED IN VIVO AND IN VITRO

In the blue spectral region, the phototransformation difference spectrum of oat phytochrome extracted as P_fr differs from that of phytochrome extracted as P_r. The difference absorbance maximum for phytochrome extracted as P_fr is at 420 nm, while that extracted as P_r is at 412 nm. The phototransformation difference spectrum measured in the blue in oat coleoptile tips without inner leaves, corresponds very well with that of phytochrome as extracted in its P_fr form. There is, however, a slight apparent attenuation of the blue difference band relative to those in the red-far-red. In coleoptile tissue containing inner leaves, the blue difference band is relatively even more highly attenuated. A similar attenuation is observed in the blue, in the protochlorophyllide to chlorophyllide phototransformation difference spectrum. In the spectrum measured with excised coleoptile without inner leaves, there is a small attenuation, while in coleptile tissue with inner leaves the attentuation is nearly 9-fold. These data suggest that the observed attenuation is probably artifactual. Neither instrumental non-linearity nor fluorescence induced by the measuring beam could explain the observed attenuation. It is suggested that the observed attenuation is probably mainly the result of wavelength dependent scatter amplification, the amplification in the blue being attenuated by the high background absorption of other pigments in this region.     

Extreme dehydration of plant tissues irreversibly converts the major and variable phyA' into the minor and conserved phyA'

Effect of dehydration of plant tissues on the two native phenomenological phytochrome A (phyA) pools - major, variable and soluble phyA' and minor, relatively conserved and presumably membrane(protein)-associated phyA' - was investigated on etiolated seedlings of barley and maize. With the use of in situ low-temperature fluorescence spectroscopy and photochemistry, it was found that even a considerable loss of water (up to 75-85% of the initial fresh weight) by coleoptiles does not bring about noticeable alterations of the spectroscopic and photochemical parameters of phytochrome pointing to a relative stability of the phyA'/phyA' system in this regard. However, extreme dehydration (loss of weight 90%) of plant tissues including freeze-drying caused dramatic changes of the phytochrome properties - blue shift of the emission maximum and its widening and reduction in the extent of the Pr photoconversion into lumi-R at 85 K and into Pfr at 273 K. Rehydration of the dried tissues did not reverse the spectroscopic changes and did not recover the Pr-->lumi-R phototransformation at 85 K but restored the ability of Pr to photoconvert into Pfr at ambient temperatures. At the same time, the total phytochrome content was not affected by these treatments. These effects were interpreted as an irreversible transformation of phyA' into phyA' upon extreme loss of water by plant tissues suggesting that water may play a role in stabilizing the conformation of the major and soluble phyA' species. The data also imply that phyA in dry and imbibing seeds is likely represented primarily by its phyA' isoform.     

Phytochromes with noncovalently bound chromophores: the ability of apophytochromes to direct tetrapyrrole photoisomerization

Chromophore-apoprotein interactions were studied with recombinant apoproteins, oat phytochrome (phyA) and CphB of the cyanobacterium Calothrix PCC7601, which were both incubated with the bilin compounds biliverdin (BV) IXalpha, phycocyanobilin (PCB) and the 3'-methoxy derivative of PCB. Previously it was shown that CphB and its homolog in Calothrix, CphA, show strong sequence similarities with each other and with the phytochromes of higher and lower plants, despite the fact that CphB carries a leucine instead of a cysteine at the chromophore attachment position and thus holds the chromophore only noncovalently. CphA binds tetrapyrrole chromophores in a covalent, phytochrome-like manner. For both eyanobacterial phytochromes, red and far-red light-induced photochemistry has been reported. Thus, the role of the binding site of CphB in directing the photochemistry of noncovalently bound tetrapyrroles was analyzed in comparison with the apoprotein from phyA phytochrome. Both the aforementioned compounds, which were used as chromophores, are not able to form covalent bonds with a phytochrome-type apoprotein because of their chemical structure (vinyl group at position 3 or methoxy group at position 3'). The BV adducts of both apoproteins showed phytochrome-like photochemistry (formation of red and far-red-absorbing forms of phytochrome [P(r) and P(fr) forms]). However, incubation of the oat apophytochrome with BV primarily yields a 700 nm form from which the P(r)-P(fr) photochemistry can be initiated and to which the system relaxes in the dark after illumination. The results for CphB were compared with a CphB mutant where the chromophore-binding cysteine had been introduced, which, upon incubation with PCB, shows spectral properties nearly identical with its (covalently binding) CphA homolog. A comparison of the spectral properties (P(r) and P(fr) forms) of all the PCB- and BV-containing chromoproteins reveals that the binding site of the cyanobacterial apoprotein is better suited than the plant (oat) phytochrome to noncovalently incorporate the chromophore and to regulate its photochemistry. Our findings support the proposal that the recently identified phytochrome-like prokaryotic photoreceptors, which do not contain a covalently bound chromophore, may trigger a light-induced physiological response.     

The Heterotrimeric G-protein Complex Modulates Light Sensitivity in Arabidopsis thaliana Seed Germination

Release of dormancy and induction of seed germination are complex traits finely regulated by hormonal signals and environmental cues such as temperature and light. The Red (R):Far-Red (FR) phytochrome photoreceptors mediate light regulation of seed germination. We investigated the possible involvement of heterotrimeric G-protein complex in the phytochrome signaling pathways of Arabidopsis thaliana seed germination. Germination rates of null mutants of the alpha (Gα) and beta (Gβ) subunits of the G-protein (At gpa1-4 and agb1-2 , respectively) and the double mutant ( agb1-2/gpa1-4 ) are lower than the wildtype (WT) under continuous or pulsed light. The Gα and Gβ subunits play a role in seed germination under hourly pulses of R lower than 0.1 μmol m⁻² s⁻¹ whereas the Gβ subunit plays a role in higher R fluences. The germination of double mutants of G-protein subunits with phyA-211 and phyB-9 suggests that AtGPA1 seems to act as a positive regulator of phyA and probably phyB signaling pathways, while the role of AGB1 is ambiguous. The imbibition of seeds at 4°C and 35°C alters the R and FR light responsiveness of WT and G-protein mutants to a similar magnitude. Thus, Gα and Gβ subunits of the heterotrimeric G-protein complex modulate light induction of seed germination by phytochromes and are dispensable for the control of dormancy by low and high temperatures prior to irradiation. We discuss the possible indirect role of the G-protein complex on the phytochrome-regulated germination through hormonal signaling pathways.     

EXPRESSION OF OAT phyA cDNA IN THE MOSS Ceratodon purpureus

The possibility of transforming Ceratodon purpureus protoplasts by PEG-mediated direct DNA uptake was tested. Transformation with a plasmid carrying a kanamycin-resistance gene resulted in kanamycin-resistant colonies of C. purpureus protonemata. A full-length cDNA clone coding for oat phyA phytochrome was isolated. The clone HM4.1 which is 3.7-kb long exhibits about 99% nucleotide sequence identity to the known phytochrome clone AP3. The expression of HM4.1 in C. purpureus protonemata was tested. A construct with the 35S-promotor and the structural gene of HM4.1 was cotransformed with the plasmid containing the kanamycin-resistance. Kanamycin-resistant colonies were tested for the presence of HM4.1 sequences in a genomic Southern experiment. Two out of 19 kanamycin-resistant colonies reacted positively with a HM4.1 specific probe. The expression of phyA in the positive colonies was examined with monoclonal antibodies specific for oat phytochrome. The Western blot experiment with protein extracts of the two positive colonies grown in the dark revealed clear signals at 124-kDa which were not detected in control plants. These data demonstrate the possibility of expressing oat phyA-apoprotein in C. purpureus protonemata. The transgenic moss protonemata did not show phenotypical alterations in response to the foreign phytochrome polypeptide; it is not known at the moment if the tetrapyrole chromophore is attached to the oat polypeptide in the protonemata or not.