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.     

Phytochrome-mediated inhibition of coleoptile growth in rice: age-dependency and action spectra

Phytochrome has been shown to be the major photoreceptor involved in the photo-inhibition of coleoptile growth in Japonica-type rice (Oryza sativa L.). We have characterized this typical photomorphogenetic response of rice using mutants deficient in phytochrome A (phyA) and phytochrome B (phyB) and with respect to age-dependency and action spectra. Seedlings were irradiated with a pulse of light 40 h or 80 h after germination (i.e. at an early or late developmental stage) and the final coleoptile length of these seedlings was determined. A saturating pulse of red light (R) had a stronger effect when it was given in the late stage than in the early stage. It was found that the photoinhibition is mediated by both the phyA and the phyB in the late stage but predominantly by phyB in the early stage. Consistent with many other reported responses, the photo-inhibition in the phyA mutant, which was observed in the early and late developmental stages and is thought to be mediated mainly by phyB, occurred in the low-fluence range (10(1)-10(3) micromol m(-2)) of R and was far-red-light (FR)-reversible; the photo-inhibition in the phyB mutant, which was observed in the late developmental stage and is thought to be mediated mainly by phyA, occurred in the very-low-fluence range (10(-2)-10(0) micromol m(-2)) and was FR-irreversible. The action spectra (350-800 nm at 50 nm intervals) obtained at the two developmental stages using phyA and phyB mutants indicated that both the phyB-mediated low-fluence response and the phyA-mediated very-low-fluence response have a major peak at 650 nm and a minor peak at 400 nm.     

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 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 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.     

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.     

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.     

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.     

Fluorescence lifetimes of protochlorophyllide in plants with different proportions of short-wavelength and long-wavelength protochlorophyllide spectral forms

Dark-grown leaves of maize (Zea mays), wheat (Triticum aestivum), wild-type pea (Pisum sativum) and its light-independent photomorphogenesis mutant (lip1) have different proportions of protochlorophyllide (Pchlide) forms as revealed by low-temperature fluorescence emission spectra. Four discrete spectral forms of Pchlide, with emission peaks around 633, 640, 656 and 670 nm, could be distinguished after Gaussian deconvolution. In maize and wheat the 656 nm component was the most prominent, whereas for wild-type pea and its lip1 mutant, the 633 and 640 nm components contributed mostly to the fluorescence emission spectra. For the fluorescence lifetimes measured at 77 K a double exponential model was the most adequate to describe the Pchlide fluorescence decay not only for the Pchlide(650-656) form but also for the short-wavelength Pchlide forms. A fast component in the range 0.3-0.8 ns and a slow component in the range 5.1-7.1 ns were present in all samples, but the values varied, depending on species. The long-wavelength Pchlide(650-656) form had a slow component with a lifetime between 5.1 and 6.7 ns, probably reflecting the fluorescence from aggregated Pchlide. The short-wavelength Pchlide(628-633) form had values of the slow component varying between 6.2 and 7.1 ns. This represents a monomeric but probably protein-bound Pchlide form because the free Pchlide in solution has a much longer lifetime around 10 ns at 77 K. The contribution of different Pchlide forms to the measured lifetime values is discussed.     

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.     

Spectroscopic properties of protochlorophyllide analyzed in situ in the course of etiolation and in illuminated leaves

The spectroscopic properties of photoactive (i.e. flash-transformable) and nonphotoactive protochlorophyll(ide)s (Pchl(ide)) were reinvestigated during the development of bean leaves in darkness. Two phases in the process of Pchl(ide) accumulation were apparent from quantitative measurements of pigment content: a lag phase (first week) during which photoactive Pchl(ide) accumulated faster than nonphotoactive Pchl(ide); and a fast phase (second week), showing parallel accumulation of both types of Pchl(ide). 'Flashed-minus-dark' absorbance difference spectra recorded in situ at 77 K showed that P650-655 was the predominant form of photoactive protochlorophyllide regardless of developmental stage. Quantitative analysis of energy migration processes between the Pchl(ide) forms showed the existence of energy transfer units containing a 1:8 ratio of nonphotoactive and photoactive Pchl(ide)s during development. Gaussian deconvolution of in situ 77 K fluorescence spectra indicated that the 633 nm band of nonphotoactive Pchl(ide) was made of four bands, at 625, 631, 637 and 643 nm, whose relative amplitudes only slightly changed during development. The emission band of photoactive Pchlide was also analyzed using the same method. Three components were found at 644, 652 and 657 nm. The emission band of P650-655 included the last two components, which become predominant only in fully etiolated plants. Photoactive Pchlide with an emission maximum at 653 nm was detected in the light during development of leaves of photoperiodically grown plants.     

PHOTORESPONSES OF Arabidopsis SEEDLINGS EXPRESSING AN INTRODUCED OAT phyA cDNA: PERSISTENCE OF ETIOLATED PLANT TYPE RESPONSES IN LIGHT-GROWN PLANTS

The photocontrol of hypocotyl elongation has been studied in etiolated and light-grown wild type (WT) Arabidopsis thaliana (L. Heynh) seedlings, and in two homozygous isogenic lines that have been transformed with the oat phy A gene coding sequence under the control of the cauliflower mosaic virus (CaMV) 35S promoter. For etiolated seedlings the inhibition of hypocotyl elongation by continuous broad band far-red light (FR) is saturated at much lower photon fluence rates in the transgenic seedlings compared with WT seedlings. Furthermore, whereas de-etiolation of WT seedlings leads to loss of responsiveness of the hypocotyls to prolonged FR, de-etiolated transgenic seedlings continue to show a pronounced FR-mediated inhibition of elongation. This may reflect the persistence of a FR-high irradiance response (HIR) mediated by the introduced oat phytochrome A. Although the hypocotyls of light-grown transgenic seedlings display a qualitatively normal end-of-day FR growth promotion, such seedlings display an aberrant shade-avoidance response to reduced red:far-red ratio (R:FR). These results are discussed in relation to the proposal that the constitutive expression of phytochrome A leads to the persistence of photoresponse modes normally restricted to etiolated plants.     

Chloroplast Biogenesis 81: Transient Formation of Divinyl Chlorophyll a Following a 2.5 ms Light Flash Treatment of Etiolated Cucumber Cotyledons

Abstract— The possible conversion of nascent divinyl (DV) chloro-phyllide a (Chlide a ) to DV chlorophyll a (Chi a during the early stages of greening in a dark divinyl-light divinyl-light/dark divinyl (DDV-LDV-LDDV) plant species was investigated. Etiolated cucumber cotyledons ( Cucu-mis sativus L .) were subjected to a 2.5 ms light flash followed by darkness. The DV and monovinyl (MV) components of the protochlorophyllide a (Pchlide a ), Chlide a , Pchlide a ester and Chi a pools were monitored quantitatively by high-resolution spectrofluorometry, immediately following the light treatments and after various periods in darkness. The light treatment photoconverted DV and MV Pchlide a to DV and MV Chlide a . Some photoconversion of MV Pchlide a ester to MV Chi a also appeared to take place. A sharp rise in the level of DV Chi a following the light treatment could not be accounted for by photoconversion of DV Pchlide a ester. It must have arisen by rapid esterification of nascent DV Chlide a. After illumination, the level of DV Chi a rose for 5 s and then declined. The implications of the transient rise and fall of DV Chi a content following illumination to the Chi a biosynthetic heterogeneity is discussed.     

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.     

EVIDENCE FOR INVOLVEMENT OF PHYTOCHROME REGULATION IN MALE STERILITY OF A MUTANT OF Oryza sativa L.

Abstract— A rice mutant ( Oryza sativa L. Nongken 58S) "Hubei photoperiod-sensitive genie male-sterile rice" ( ms mutant) has been found to be male sterile under long day cycles (LD) and fertile in short day cycles (SD). After formation of the secondary rachis-branch primodia the mutant plants under SD were interrupted in the middle of the long night phase (night break) for 10 days with 5 min pulses of red light (R) or far-red light (FR). Rates of normal pollen and seed setting of the mutant treated by R or R → FR → R declined significantly, while the rates after FR or R → FR treatments were similar to those under SD alone. The result of these induction reversion experiments is consistent with the operational criteria for the involvement of photochrome. Wild-type rice ( O. sativa L. Nongken 58) under the same treatment showed no change in fertility. Experiments on the effect of different dark intervals (20 s to 15 min) between R and FR on male sterility of the ms mutant showed that the longer the dark interval, the greater the escape of R induction from FR reversibility. Treatment with SD or LD after formation of pollen mother cells had no influence on fertility of the ms mutant plants treated previously with R or FR night breaks.     

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.     

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.     

Carotenoid dependence of the protochlorophyllide to chlorophyllide phototransformation in dark-grown wheat seedlings.

The influence of carotenoids on partial protochlorophyllide (Pchlide) photoreduction and the successive formation of long-wavelength chlorophyllide (Chlide) forms was studied by low-temperature fluorescence spectroscopy (77 K). Wheat leaves with a decreased content of carotenoids obtained from norflurazon-treated seedlings (10 and 100 micromol l(-1)) were compared with leaves containing normal amounts of these pigments. Partial photoreduction of Pchlide was achieved by irradiation of the leaves with one light flash in combination with a number of neutral gray and/or red Perspex filters. There were significant differences between the fluorescence emission spectra (the position and height of the peaks) of dark-grown normal and carotenoid-deficient leaves irradiated with non-saturating white light of increasing intensity. The long-wavelength Chlide forms appeared first in the leaves nearly devoid of carotenoids (treated with 100 micromol l(-1) norflurazon), then in the leaves with carotenoid deficiency (treated with 10 micromol l(-1) norflurazon), and finally in normal leaves. After irradiation with non-saturating light of the same intensity, the ratio Chlide/Pchlide(657) was always the highest in the leaves nearly deficient of carotenoids, medium in the leaves with carotenoid deficiency and lowest in the normal leaves. Similarly to white light, red light of low intensity induced faster formation of long-wavelength Chlide species in the leaves with carotenoid deficiency in comparison to the normal leaves. We propose that, in leaves with reduced carotenoid content, a greater number of Pchlide molecules transform to Chlide per light flash than in normal leaves. The results are discussed in relation to the involvement of carotenoids in competitive absorption and light screening, as well as to their influence on Pchlide-Chlide interactions.     

Early reactions of light-induced protochlorophyllide and chlorophyllide transformations analyzed in vivo at room temperature with a diode array spectrofluorometer

The steps of protochlorophyllide (Pchlide) photoreduction and subsequent chlorophyllide (Chlide) transformations which occur in the seconds to minutes time-scale were studied using a diode array spectrofluorometer in dark-grown barley leaves. The intensity of the excitation light was varied between 3 and 2,500 micromol m(-2) s(-1) and a series of fluorescence spectra were recorded at room temperature in the seconds and minutes time scales. In certain experiments, 77-K emission spectra were measured with the same equipment. The high quality of the spectra allowed us to run spectral resolution studies which proved the occurrence, at room temperature, of multiple Pchlide and Chlide forms found previously in 77-K spectra. The comparison of the 77-K and room-temperature spectra showed that the fluorescence yields of the nonphotoactive 633-nm Pchlide form and of the Chlide product emitting at 678 nm were temperature independent. The fluorescence intensity of aggregated NADPH-pigment-POR complexes (photoactive 656-nm Pchlide and 693-nm Chlide forms) were strongly increased at 77 K, while that of the NADP(+)-Chlide-POR (684-686-nm Chlide form) was much less affected by temperature. Information was obtained also about the dynamics of the transformation of pigment forms in the light at different photon densities. At low light intensities, the phototransformation of the 642-644-nm Pchlide form was faster than that of the 654-656-nm form. The relative amplitudes of Gaussian components related to different Chlide forms found after exposure to a constant amount of photons strongly depended on the light intensity used. Strong quenching of all Chlide components occurred upon prolonged exposure to high intensity light. These effects are discussed by considering the interconversion processes between different forms of the pigment-protein complexes, their relative fluorescence yields and energy migration processes.