Oat Phytochrome Research Articles

A light-regulated pool of phytochrome and rudimentary high-irradiance responses under far-red light in Pinus elliottii and Pseudotsuga menziesii

The presence of a phytochrome pool down-regulated by light and the occurrence of high-irradiance responses to far-red light are well documented in angiosperms but not in gymnosperms. A pool of phytochrome was identified in Pinus elliottii and Pseudotsuga menziesii seedlings grown in darkness with a monoclonal antibody developed against oat phytochrome A. This pool was barely detectable in light-grown tissues. Dark-grown conifer seedlings transferred to continuous red light showed a gradual decrease of the levels of immunodetectable phytochrome. This decrease was significantly slower in gymnosperms than in angiosperms. Dark-grown seedlings of P. elliottii and P. menziesii showed enhanced growth of the cotyledonary whorl and increased anthocyanin pigmentation of the hypocotyl, but no hypocotyl-growth inhibition in response to continuous far-red light. Hourly pulses were significantly less effective than continuous far-red light. The response to far-red light was not observed in seedlings pretreated with red light to reduce the levels of immunodetectable phytochrome. Rudiments of phytochrome A-like function and kinetics are present in P. elliottii and P. menziesii.

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.

Mass spectrometric characterization of oat phytochrome A: isoforms and posttranslational modifications.

At least four mRNAs for oat phytochrome A (phyA) are present in etiolated oat tissue. The complete amino acid sequences of two phyA isoforms (A3 and A4) and the N-terminal amino acid sequence of a third isoform (A5) were deduced from cDNA sequencing (Hershey et al., 1985). In the present study, heterogeneity of phyA on a protein level was studied by tryptic mapping using electrospray ionization mass-spectrometry (ESIMS). The total tryptic digest of iodoacetamide-modified phyA was fractionated by gel filtration chromatography followed by reversed-phase high-performance liquid chromatography. ESIMS was used to identify peptides. Amino acid sequences of the peptides were confirmed or determined by collision-induced dissociation mass spectrometry (CID MS), MS/MS, or by subdigestion of the tryptic peptides followed by ESIMS analysis. More than 97% of the phyA3 sequence (1,128 amino acid residues) was determined in the present study. Mass-spectrometric analysis of peptides unique to each form showed that phyA purified from etiolated oat seedling is represented by three isoforms A5, A3, and A4, with ratio 3.4:2.3:1.0. Possible light-induced changes in phytochrome in vivo phosphorylation site at Ser7 (Lapko VN et al., 1997, Biochemistry 36:10595-10599) as well at Ser17 and Ser598 (known as in vitro phosphorylation sites) were also analyzed. The extent of phosphorylation at Ser7 appears to be the same for phyA isolated from dark-grown and red-light illuminated seedlings. In addition to Ser7, Ser598 was identified as an in vivo phosphorylation site in oat phyA. Ser598 phosphorylation was found only in phyA from the red light-treated seedlings, suggesting that the protein phosphorylation plays a functional role in the phytochrome A-mediated light-signal transduction.

Recombinant phytochrome of the moss Ceratodon purpureus: heterologous expression and kinetic analysis of Pr-->Pfr conversion.

The phytochrome-encoding gene Cerpu;PHY;2 (CP2) of the moss Ceratodon purpureus was heterologously expressed in Saccharomyces cerevisiae as a polyhistidine-tagged apoprotein and assembled with phytochromobilin (P phi B) and phycocyanobilin (PCB). Nickel-affinity chromatography yielded a protein fraction containing approximately 80% phytochrome. The holoproteins showed photoreversibility with both chromophores. Difference spectra gave maxima at 644/716 nm (red-absorbing phytochrome [Pr]/far-red-absorbing phytochrome [Pfr]) for the PCB adduct, and 659/724 nm for the P phi B-adduct, the latter in close agreement with values for phytochrome extracted from Ceratodon itself, implying that P phi B is the native chromophore in this moss species. Immunoblots stained with the antiphytochrome antibody APC1 showed that the recombinant phytochrome had the same molecular size as phytochrome from Ceratodon extracts. Further, the mobility of recombinant CP2 holophytochrome on native size-exclusion chromatography was similar to that of native oat phytochrome, implying that CP2 forms a dimer. Kinetics of absorbance changes during the Pr-->Pfr photoconversion of the PCB adduct, monitored between 620 and 740 nm in the microsecond range, revealed the rapid formation of a red-shifted intermediate (I700), decaying with a time constant of approximately 110 microseconds. This is similar to the behavior of phytochromes from higher plants when assembled with the same chromophore. When following the formation of the Pfr state, two major processes were identified (with time constants of 3 and 18 ms) that are followed by slow reactions in the range of 166 ms and 8 s, respectively, albeit with very small amplitudes.

Isolation, partial purification and differential DNA-binding properties of putative high-mobility-group proteins from rice

Putative high-mobility-group (HMG) proteins were isolated from 10–11-day-old rice ( Oryza sativa L. cv. IR36) shoots and roots, and from ungerminated rice grains. Nine proteins below 30 kDa which fulfilled the criteria for HMG proteins were identified from rice shoots and subjected to reversed-phase perfusion chromatography as an initial purification step. Southwestern hybridisation analysis established novel selective and differential binding of individual pea, wheat and rice HMG proteins to A/T- and G/C-rich DNA probes. Gel retardation assays using sequences derived from the pea plastocyanin and oat phytochrome A3 gene promoters have suggested that rice shoot HMG protein extracts contain a DNA-binding protein factor resembling PCF1 from pea in its binding characteristics, and that this factor is closely related to the HMG-I/Y-like PF1 protein from rice. An analysis of fractionated rice shoot HMG proteins suggests that specific promoter-binding activity resides in the 26 kDa HMG α protein.

Eukaryotic phytochromes: light-regulated serine/threonine protein kinases with histidine kinase ancestry.

The discovery of cyanobacterial phytochrome histidine kinases, together with the evidence that phytochromes from higher plants display protein kinase activity, bind ATP analogs, and possess C-terminal domains similar to bacterial histidine kinases, has fueled the controversial hypothesis that the eukaryotic phytochrome family of photoreceptors are light-regulated enzymes. Here we demonstrate that purified recombinant phytochromes from a higher plant and a green alga exhibit serine/threonine kinase activity similar to that of phytochrome isolated from dark grown seedlings. Phosphorylation of recombinant oat phytochrome is a light- and chromophore-regulated intramolecular process. Based on comparative protein sequence alignments and biochemical cross-talk experiments with the response regulator substrate of the cyanobacterial phytochrome Cph1, we propose that eukaryotic phytochromes are histidine kinase paralogs with serine/threonine specificity whose enzymatic activity diverged from that of a prokaryotic ancestor after duplication of the transmitter module.

The Complexity of the Pr to Pfr Phototransformation Kinetics Is an Intrinsic Property of Native Phytochrome*

AbstractSeveral possible origins of the complex phytochrome red to far‐red light‐absorbing phytochrome (Pr Pfr) phototransformation kinetics in the nanosecond‐to‐second time range have been examined. Heterogeneity based on protein sequence is ruled out as an origin of the multi‐component kinetics because recombinant 124 kDa oat phytochrome A apoprotein reconstituted with phytochro‐mobilin and the native protein are very similar in this regard throughout this time range. The Pr forms of native 124 kDa oat phytochrome A and of a homogeneous recombinant 65 kDa chromoprotein fragment exhibit thermochromic properties interpreted as arising in each case from the presence of two Pr species in thermal equilibrium. They exhibit identical photochemical properties. The complex kinetics therefore cannot result from Pr heterogeneity either. Thus, the presence of two Pr forms in equilibrium (Pr,675 and Pr,655) and the complex multiex‐ponential PrPfr phototransformation kinetics observed in all time ranges are intrinsic properties of the homogeneous holoprotein of oat phytochrome A.

Spatial distribution and temporal regulation of phytochromes A and B levels in maize seedlings

Phytochrome distribution and putative level in dark- and light-grown maize ( Zea mays L.) seedlings were analyzed using antibodies against oat phytochrome A (PHYA) and tobacco phytochrome B (PHYB) by immunoblotting. During the course of leaf elongation (4–8 d), the putative level of both PHYA and PHYB declined with age. Exposure of etiolated seedlings to light induced severe down-regulation of PHYA level, and transfer of light-grown seedlings to darkness induced reaccumulation of PHYA. For PHYB, no large effect of dark to light, or light to dark, transitions could be observed. Immunoblotting and spectroscopy of phytochromes along the length of 7-d old leaves showed that the level of both PHYA and PHYB was maximal at the leaf base and in the upper half of the leaf, but their level was low in the lower half of the leaf. In light-grown leaves, PHYB distribution was similar to the dark-grown leaves, though its overall level was lower. In light-grown leaves, PHYA could not be detected by immunoblotting, but when the leaves were transferred to darkness, PHYA reappeared with a distribution pattern more or less similar to that of dark-grown leaves. These results indicate that the temporal regulation and spatial distribution of PHYA and PHYB levels in maize appear to be governed by a common regulatory element during the development of seedlings.

Surface topography of phytochrome A deduced from specific chemical modification with iodoacetamide.

Phytochromes are a photoreversible photochromic light switch for photomorphogenesis in plants. The molecular structure and functional mechanism of phytochromes are not fully understood. On the basis of complete mapping of total tryptic digest of the iodoacetamide-modified oat phytochrome A (phyA), the molecular surface topography of phyA was probed by specific chemical modification of cysteine residues with [14C]iodoacetamide. Under native conditions, only two cysteines (Cys-158 and Cys-311) of eleven half-cystines of the N-terminal chromophore binding domain were modified to a significant extent. In the C-terminal domain, six cysteine residues (Cys-715, Cys-774, Cys-809, Cys-869, Cys-961, Cys-995) were readily accessible to iodoacetamide. Among the reactive cysteine residues, only cysteine-311 displayed reactivity that was dependent on the photochromic form (Pr left arrow over right arrow Pfr) of the photoreceptor. Surprisingly, the modification of Cys-311 in the vicinity of the chromophore attachment site (Cys-321) did not have any detectable effect on spectral properties of phyA. Most of the cysteines of the N-terminal domain (Cys-83, Cys-175, Cys-291, Cys-370, Cys-386, Cys-445, Cys-506) are deeply buried in the core of the chromophore binding domain, as they can be modified only after denaturation of the chromoprotein. In the C-terminal domain, modification of only one cysteine residue (Cys-939) required protein denaturation. Since all 22 half-cystines can be modified with iodoacetamide without reduction of the chromoprotein, it follows that oat phyA does not have any disulfide bonds. We found that Cys-311, Cys-774, Cys-961, and Cys-995 could be easily partially oxidized under the conditions used for phytochrome isolation. The surface topography/conformation of oat phyA and its role in protein-protein recognition in phytochrome-mediated signal transduction are discussed in terms of the relative reactivity of cysteine residues.

Chromophore incorporation, Pr to Pfr kinetics, and Pfr thermal reversion of recombinant N-terminal fragments of phytochrome A and B chromoproteins.

N-Terminal apoprotein fragments of oat phytochrome A (phyA) of 65 kDa (amino acids 1-595) and potato phyB of 66 kDa (1-596) were heterologously expressed in Escherichia coli and in the yeasts Saccharomyces cerevisiae and Pichia pastoris, and assembled with phytochromobilin (PthetaB; native chromophore) and phycocyanobilin (PCB). The phyA65 apoprotein from yeast showed a monoexponential assembly kinetics after an initial steep rise, whereas the corresponding apoprotein from E. coli showed only a slow monoexponential assembly. The phyB66 apoprotein incorporated either chromophore more slowly than the phyA65s, with biexponential kinetics. With all apoproteins, PthetaB was incorporated faster than PCB. The thermal stabilities of the Pfr forms of the N-terminal halves are similar to those known for the full-length recombinant phytochromes: oat phyA65 Pfr is highly stable, whereas potato phyB66 Pfr is rapidly converted into Pr. Thus, neither the C-terminal domain nor homodimer formation regulates this property. Rather, it is a characteristic of the phytochrome indicating its origin from mono- or dicots. The Pr to Pfr kinetics of the N-terminal phyA65 and phyB66 are different. The primary photoproduct I700 of phyA65-PCB decayed monoexponentially and the PthetaB analogue biexponentially, whereas the phyB66 I700 decayed monoexponentially irrespective of the chromophore incorporated. The formation of Pfr from Pr is faster with the N-terminal halves than with the full-length phytochromes, indicating an involvement of the C-terminal domain in the relatively slow protein conformational changes.

Light-Enhanced Target Gene Expression in Tobacco BY-2 by the Combination of Overexpressed Phytochrome and rbcS3A Promoter

Enhanced expression of the rbcS3A promoter was achieved by overproducing phytochrome in BY-2 tobacco cells. The transformed cells contained the oat phytochrome A gene fused the 35S promoter of Cauliflower Mosaic Virus (CaMV) and the gus gene to the rbcS3A promoter. The transformed cells exhibited 1.5- to -2.8 fold enhanced the β-glucuronidase (GUS) activity when subjected to light.

Ectopic expression of oat phytochrome A in hybrid aspen changes critical daylength for growth and prevents cold acclimatization

Survival of temperate‐zone tree species under the normal summer‐winter cycle is dependent on proper timing of apical growth cessation and cold acclimatization. This timing is primarily based on the perception of daylength, and through evolution many tree species have developed photoperiodic ecotypes which are closely adapted to the local light conditions. The longest photoperiod inducing growth cessation, the critical photoperiod, is inherited as a quantitative character. The phytochrome pigment family is the probable receptor of daylength, but the exact role of phytochrome and the physiological basis for the different responses between photoperiodic ecotypes are not known. This report shows for the first time that over‐expression of the oat phytochrome A gene (PHYA) in a tree significantly changes the critical daylength and effectively prevents cold acclimatization. While the critical daylength for elongation growth in the wild‐type of hybrid aspen (Populus tremula × tremuloides) was approximately 15 h, transgenic lines with a strong expression of the oat PHYA gene did not stop growing even under a photoperiod of 6 h. Quantitative analysis of gibberellins (GA) as well as indole‐3‐acetic acid (IAA) revealed that levels of these were not down‐regulated under short days in the transgenic plants expressing high levels of oat PHYA, as in the wild‐type. These results indicate that photoperiodic responses in trees might be regulated by the amount of PHYA gene expressed in the plants, and that the amount of phytochrome A (phyA) affects the metabolism of GAs and IAA.

Expression of heterologous phytochromes A, B or C in transgenic tobacco plants alters vegetative development and flowering time.

In this study, oat phytochrome A (phyA), Arabidopsis phytochrome B (phyB) or Arabidopsis phytochrome C (phyC) were expressed in both day-neutral and photo-period-sensitive (short-day) tobacco (Nicotiana tabacum cv. Hicks). Introgression of the Maryland Mammoth (MM) gene into cv Hicks was used to confer short-day photo-periodic sensitivity. Expression of oat phyA led to characteristic hypersensitivity of hypocotyls to red light (R) and far-red light (FR) and an overall dwarfing of the mature plant. Expression of Arabidopsis phyB enhanced the sensitivity of hypocotyls to R and caused even more marked dwarfing of the mature plant. In contrast, the expression of Arabidopsis phyC had no detectable consequences for the photocontrol of hypocotyl elongation. However, phyC expression did lead to a R-dependent increase in cotyledon expansion in de-etiolating seedlings and to a significant increase in leaf area in mature plants. This provides the first experimental evidence that phyC is biologically active. The flowering time of cv Hicks plants grown under 8 h photoperiods was virtually unaffected by a 30 min white light (W) night break given 8 h into the dark period. In contrast, cv Hicks MM plants responded to a night break with a delay in flowering. Expression of phyA or phyB led to a night break-dependent delay in flowering in cv Hicks plants. For cv Hicks MM plants, the expression of any of phyA, phyB or phyC caused a marked enhancement of the flower-delaying effect of a night break. These observations indicate that transgenic phyA, phyB or phyC can interact with the endogenous mechanisms controlling flowering time in tobacco.

Light-induced transcriptional repression of the pea AS1 gene: identification of cis-elements and transfactors.

Here, we examine the cis-elements and trans-factors affecting the expression of asparagine synthetase (AS) genes whose transcription is negatively regulated by light. The promoters for the AS1 and AS2 genes of pea were isolated, sequenced, and functionally dissected for their ability to confer regulated expression to the GUS reporter gene in transgenic tobacco. Histochemical analysis of transgenic plants demonstrated that the AS1 and AS2 promoters show identical patterns of cell-specific expression. The more highly active AS1 promoter was further demonstrated to confer negative light-regulation to the GUS gene in transgenic tobacco. Deletion analysis and gain-of-function experiments showed that 124 bp of the AS1 promoter was sufficient to confer light-activated repression to a heterologous promoter. Potential conserved transcription regulatory elements, Box B, Box C, and Box C' within this region were shown to bind to nuclear proteins by gel shift analysis. A light-specific DNA:protein interaction was detected with Box B. The nuclear factors that bind to Box C and C' elements of AS1 are competed by a putative repressor element 'RE1' defined previously in the oat phytochrome gene whose transcription is also repressed by light. The Box B and C/C'-Box/RE1-binding factors were found in nuclear extracts of tobacco, pea, and Arabidopsis and may therefore be universal factors involved in light-activated transcriptional repression.

Effect of chromophore exchange on the resonance Raman spectra of recombinant phytochromes

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

Phytochrome photoconversion

ABSTRACTThe spectral properties of native and modified phytochromes and the molecular events during phytochrome photoconversion, , are reviewed. Steady‐state and time‐resolved absorption spectra of native phytochrome A, as well as recombinant phytochromes (oat and potato phytochrome A and potato phytochrome B) reconstituted with phycocyanobilin and phytochromobilin as chromophores, are analysed. The vinyl double bond, present at position 18 in phytochromobilin and substituted by an ethyl group in phycocyanobilin, has a considerable influence on the photo‐transformation kinetics of phytochromes A and B, evidently due to a strong interaction of this region of the chromophore with the protein surrounding. The kinetics of the phototransformation of potato phytochrome B differs from that of oat phytochrome A (wild‐type and recombinant), indicating that the chromophore‐protein interaction in phytochrome B is different from that in phytochrome A. It remains to be seen whether this difference is due to the di‐ versus monocotyledon origin of the phytochromes. Optoacoustic spectroscopy, applied to native oat phytochrome A, afforded thermo‐dynamic, structural and kinetic parameters of the Pr→I700 and the I700→Pr phototransformations. Raman and infrared spectroscopic data for wild‐type phytochrome A suggest that the protonated chromophore in Pr undergoes torsions around two single bonds in addition to the Z→E isomerization of the 15,16 double bond, and that all transients, possibly with the exception of IbI, are protonated at the central pyrrole ring.

Purification and characterization of recombinant affinity peptide-tagged oat phytochrome A.

Full-length Avena sativa (oat) phytochrome A (ASPHYA) was expressed in the yeast Saccharomyces cerevisiae and purified to apparent homogeneity. Expression of an ASPHYA cDNA that encoded the full-length photoreceptor with a 15 amino acid 'strep-tag' peptide at its C-terminus produced a single polypeptide with a molecular mass of 124 kDa. This strep-tagged polypeptide (ASPHYA-ST) bound tightly to streptavidin agarose and was selectively eluted using diaminobiotin, with a chromatographic efficiency of 45%. Incubation of ASPHYA-ST with phytochromobilin (P phi B) and the unnatural chromophore precursors, phycocyanobilin (PCB) and phycoerythrobilin (PEB), produced covalent adducts that were similarly affinity purified. Both P phi B and PCB adducts of ASPHYA-ST were photoactive--the P phi B adduct displaying spectrophotometric properties nearly indistinguishable from those of the native photoreceptor, and the PCB adduct exhibiting blue-shifted absorption maxima. Although the PEB adduct of ASPHYA-ST was photochemically inactive, it was intensely fluorescent with an excitation maximum at 576 nm and emission maxima at 586 nm. The superimposability of its absorption and fluorescence excitation spectra established that a single biliprotein species was responsible for fluorescence from the adduct produced when ASPHYA-ST was incubated with PEB. Steric exclusion HPLC also confirmed that ASPHYA-ST and its three bilin adducts were homodimers, as has been established for phytochrome A isolated from natural sources. The ability to express and purify recombinant phytochromes with biochemical properties very similar to those of the native molecule should facilitate detailed structural analysis of this important class of photoreceptors.

Photocontrol of anthocyanin biosynthesis in tomato.

Juvenile anthocyanin biosynthesis has been studied in dark-grown seedlings of tomato (Lycopersicon esculentum Mill.) wild types (WTs) and photomorphogenic mutants. During a subsequent 24-hr period of monochromatic irradiation at different fluence rates of red light (R) the fluence-rate response relationships for induction of anthocyanin in all the WTs are similar, yet complex, showing a response at low fluence rates (LFRR) followed by a fluence rate-dependent high irradiance response (HIR). In the hypocotyl this response is restricted to the sub-epidermal layer of cells. The high-pigment-1 (hp-1) mutant exhibits a strong amplification of both response components. Theatroviolacea (atv) mutant shows strongest amplification of the HIR component. In contrast, a transgenic line overexpressing an oat phytochrome A gene (PHYA3 (+)) shows a most dramatic amplification of the LFRR component. The far-red light (FR)-insensitive (fri) mutant, deficient in phytochrome A (phyA), lacks the LFRR component whilst retaining a normal HIR. The temporarily R-insensitive (tri) mutant, deficient in phytochrome B1 (phyB1) retains the LFRR, but lacks the HIR. Thehp-1,fri andhp-1,tri double mutant, exhibit amplified, yet qualitatively similar responses to the monogenicfri andtri mutants. Thefri,tri double mutant lacks both response components in R, but a residual response to blue light (B) remains. Similarly, theaurea (au) mutant deficient in phytochrome chromophore biosynthesis and presumably all phytochromes, lacks both response components in the R and FR regions of the spectrum. Experiments at other wavelengths demonstrate that while there is only a small response in the FR spectral region (729 nm) in tomato, there is an appreciable HIR response in the near FR at 704 nm, which is retained in thetri mutant. This suggests that the labile phyA pool participates in the HIR at this wavelength. The intense pigmentation (Ip) mutant appears to be specifically deficient in the B1 induced anthocyanin biosynthesis. Adult plants, grown under fluorescent light/dark cycles, show a reduction of anthocyanin content of young developing leaves upon application of supplemtary or end-of-day FR. The involvement of different phytochrome species in anthocyanin biosynthesis based on micro-injection studies into theau mutant and studies using type specific phytochrome mutants is discussed.

High-irradiance responses induced by far-red light in grass seedlings of the wild type or overexpressing phytochrome A

The occurrence of phytochrome-mediated highirradiance responses (HIR), previously characterised largely in dicotyledonous plants, was investigated in Triticum aestivum L., Zea mays L., Lolium multiflorum Lam. and in both wild-type Oryza sativa L. and in transgenic plants overexpressing oat phytochrome A under the control of a 35S promoter. Coleoptile growth was promoted (maize, ryegrass) or inhibited (wild-type rice) by continuous far-red light (FRc). However, at equal fluences, hourly pulses of far-red light (FRp) were equally effective, indicating that the growth responses to FRc were not true HIR. In contrast, in maize and rice, FRc increased anthocyanin content in the coleoptile in a fluence-rate dependent manner. This response was a true HIR as FRp had reduced effects. In maize, anthocyanin levels were significantly higher under FRc than under continuous red light. In rice, overexpression of phytochrome A increased the inhibition of coleoptile growth and the levels of anthocyanin under FRc but not under FRp or under continuous red light. The effect of FRc was fluence-rate dependent. In light-grown rice, overexpression of phytochrome A reduced leaf-sheath length, impaired the response to supplementary far-red light, but did not affect the response to canopy shade-light. In grasses, typical HIR, i.e. fluence-rate dependent responses showing reciprocity failure, can be induced by FRc. Under FRc, overexpressed phytochrome A operates through this action mode in transgenic rice.

Novel members of a family of AT hook-containing DNA-binding proteins from rice are identified through theirin vitro interaction with consensus target sites of plant and animal homeodomain proteins

The AT hook is an AT-rich DNA-binding domain that occurs three times in mammalian high-mobility-group I/Y chromosomal proteins and has recently also been identified in DNA-binding proteins from plants. We unexpectedly isolated three rice cDNA clones encoding AT hook-containing proteins in an attempt to isolate homeobox cDNA clones by south-western screening of an expression library with known binding sites for Arabidopsis and animal homeodomain proteins. One of these clones (Os-PF1) has previously been identified due to the binding of its encoded protein to PE1, a cis-acting element from the oat phytochrome promoter. The other two clones represent newly described cDNA clones, designated Os-AT1 and Os-AT2. The Os-AT1 and Os-AT2 proteins were found to have the same specificities as Os-PF1 with respect to in vitro binding of wild-type and mutant PE1 versions. However, all three proteins appeared to bind much stronger in south-western assays to two of the rather AT-rich sequences used in our screening than to the PE1 element. In none of the AT hook proteins clear homologies to transcriptional activation domains could be identified, but the N-terminal regions of Os-AT1 and Os-PF1 were found to show similarity to histone H1 chromosomal proteins. Given their structural characteristics it is conceivable that the rice AT hook proteins bind to gene promoter regions as accessory proteins that may alter the accessibility of chromatin to other nuclear factors. Their predominant expression in young and meristematic tissues suggests that the presence of the AT hook proteins may affect the expression of genes that determine the differentiation status of cells.