Interaction of cryptochrome 1, phytochrome, and ion fluxes in blue-light-induced shrinking of Arabidopsis hypocotyl protoplasts

Protoplasts isolated from red-light-adapted Arabidopsis hypocotyls and incubated under red light exhibited rapid and transient shrinking within a period of 20 min in response to a blue-light pulse and following the onset of continuous blue light. Long-persisting shrinkage was also observed during continuous stimulation. Protoplasts from a hy4 mutant and the phytochrome-deficient phyA/phyB double mutant of Arabidopsis showed little response, whereas those from phyA and phyB mutants showed a partial response. It is concluded that the shrinking response itself is mediated by the HY4 gene product, cryptochrome 1, whereas the blue-light responsiveness is strictly controlled by phytochromes A and B, with a greater contribution by phytochrome B. It is shown further that the far-red-absorbing form of phytochrome (Pfr) was not required during or after, but was required before blue-light perception. Furthermore, a component that directly determines the blue-light responsiveness was generated by Pfr after a lag of 15 min over a 15-min period and decayed with similar kinetics after removal of Pfr by far-red light. The anion-channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid prevented the shrinking response. This result, together with those in the literature and the kinetic features of shrinking, suggests that anion channels are activated first, and outward-rectifying cation channels are subsequently activated, resulting in continued net effluxes of Cl- and K+. The postshrinking volume recovery is achieved by K+ and Cl- influxes, with contribution by the proton motive force. External Ca2+ has no role in shrinking and the recovery. The gradual swelling of protoplasts that prevails under background red light is shown to be a phytochrome-mediated response in which phytochrome A contributes more than phytochrome B.     

Missense mutations define a restricted segment in the C-terminal domain of phytochrome A critical to its regulatory activity

The phytochrome family of photoreceptors has dual molecular functions: photosensory, involving light signal perception, and regulatory, involving signal transfer to downstream transduction components. To define residues necessary specifically for the regulatory activity of phytochrome A (phyA), we undertook a genetic screen to identify Arabidopsis mutants producing wild-type levels of biologically defective but photochemically active and dimeric phyA molecules. Of eight such mutants identified, six contain missense mutations (including three in the same residue, glycine 727) clustered within a restricted segment in the C-terminal domain of the polypeptide. Quantitative photobiological analysis revealed retention of varying degrees of partial activity among the different alleles--a result consistent with the extent of conservation at the position mutated. Together with additional data, these results indicate that the photoreceptor subdomain identified here is critical to the regulatory activity of both phyA and phyB.     

Different phototransduction kinetics of phytochrome A and phytochrome B in Arabidopsis thaliana

The kinetics of phototransduction of phytochrome A (phyA) and phytochrome B (phyB) were compared in etiolated Arabidopsis thaliana seedlings. The responses of hypocotyl growth, cotyledon unfolding, and expression of a light-harvesting chlorophyll a/b-binding protein of the photosystem II gene promoter fused to the coding region of beta-glucuronidase (used as a reporter enzyme) were mediated by phyA under continuous far-red light (FR) and by phyB under continuous red light (R). The seedlings were exposed hourly either to n min of FR followed by 60 minus n min in darkness or to n min of R, 3 min of FR (to back-convert phyB to its inactive form), and 57 minus n min of darkness. For the three processes investigated here, the kinetics of phototransduction of phyB were faster than that of phyA. For instance, 15 min R h-1 (terminated with a FR pulse) were almost as effective as continuous R, whereas 15 min of FR h-1 caused less than 30% of the effect of continuous FR. This difference is interpreted in terms of divergence of signal transduction pathways downstream from phyA and phyB.     

Multiple sclerosis: in situ evidence for antibody- and complement-mediated demyelination

Blue light responses in higher plants can be mediated not only by specific blue light receptors, but also by the red/far-red photoreversible phytochrome system. The question of interdependence between these photoreceptors has been debated over many years. The availability of Arabidopsis mutants for the blue light receptor CRY1 and for the two major phytochromes phyA and phyB allows a reinvestigation of this question. The analysis of photocontrol of seed germination, inhibition of hypocotyl growth and anthocyanin accumulation clearly demonstrates that (i) phyA shows a strong control in blue light responses especially at low fluence rates; (ii) phyB mediated induction reactions can be reversed by subsequent blue light irradiations; and (iii) CRY1 mediates blue light controlled inhibition of hypocotyl growth only at fluence rates higher than 5 mumol m-2s-1 and independently of phytochrome A and B.     

Chromophore-bearing NH2-terminal domains of phytochromes A and B determine their photosensory specificity and differential light lability

In early seedling development, far-red-light-induced deetiolation is mediated primarily by phytochrome A (phyA), whereas red-light-induced deetiolation is mediated primarily by phytochrome B (phyB). To map the molecular determinants responsible for this photosensory specificity, we tested the activities of two reciprocal phyA/phyB chimeras in diagnostic light regimes using overexpression in transgenic Arabidopsis. Although previous data have shown that the NH2-terminal halves of phyA and phyB each separately lack normal activity, fusion of the NH2-terminal half of phyA to the COOH-terminal half of phyB (phyAB) and the reciprocal fusion (phyBA) resulted in biologically active phytochromes. The behavior of these two chimeras in red and far-red light indicates: (i) that the NH2-terminal halves of phyA and phyB determine their respective photosensory specificities; (ii) that the COOH-terminal halves of the two photoreceptors are necessary for regulatory activity but are reciprocally inter-changeable and thus carry functionally equivalent determinants; and (iii) that the NH2-terminal halves of phyA and phyB carry determinants that direct the differential light lability of the two molecules. The present findings suggest that the contrasting photosensory information gathered by phyA and phyB through their NH2-terminal halves may be transduced to downstream signaling components through a common biochemical mechanism involving the regulatory activity of the COOH-terminal domains of the photoreceptors.     

An Arabidopsis mutant hypersensitive to red and far-red light signals

A new mutant called psi2 (for phytochrome signaling) was isolated by screening for elevated activity of a chlorophyll a/b binding protein-luciferase (CAB2-LUC) transgene in Arabidopsis. This mutant exhibited hypersensitive induction of CAB1, CAB2, and the small subunit of ribulose-1,5-bisphosphate carboxylase (RBCS) promoters in the very low fluence range of red light and a hypersensitive response in hypocotyl growth in continuous red light of higher fluences. In addition, at high- but not low-light fluence rates, the mutant showed light-dependent superinduction of the pathogen-related protein gene PR-1a and developed spontaneous necrotic lesions in the absence of any pathogen. Expression of genes responding to various hormone and environmental stress pathways in the mutant was not significantly different from that of the wild type. Analysis of double mutants demonstrated that the effects of the psi2 mutation are dependent on both phytochromes phyA and phyB. The mutation is recessive and maps to the bottom of chromosome 5. Together, our results suggest that PSI2 specifically and negatively regulates both phyA and phyB phototransduction pathways. The induction of cell death by deregulated signaling pathways observed in psi2 is reminiscent of retinal degenerative diseases in animals and humans.     

Overexpression of rice phytochrome A partially complements phytochrome B deficiency in Arabidopsis

The red/far-red reversible phytochromes play a central role in regulating the development of plants in relation to their light environment. Studies on the roles of different members of the phytochrome family have mainly focused on light-labile, phytochrome A and light-stable, phytochrome B. Although these two phytochromes often regulate identical responses, they appear to have discrete photosensory functions. Thus, phytochrome A predominantly mediates responses to prolonged far-red light, as well as acting in a non-red/far-red-reversible manner in controlling responses to light pulses. In contrast, phytochrome B mediates responses to prolonged red light and acts photoreversibly under light-pulse conditions. However, it has been reported that rice (Oryza sativa L.) phytochrome A operates in a classical red/far-red reversible fashion following its expression in transgenic tobacco plants. Thus, it was of interest to determine whether transgenic rice phytochrome A could substitute for loss of phytochrome B in phyB mutants of Arabidopsis thaliana (L.) Heynh. We have observed that ectopic expression of rice phytochrome A can correct the reduced sensitivity of phyB hypocotyls to red light and restore their response to end-of-day far-red treatments. The latter is widely regarded as a hallmark of phytochrome B action. However, although transgenic rice phytochrome A can correct other aspects of elongation growth in the phyB mutant it does not restore other responses to end-of-day far-red treatments nor does it restore responses to low red:far-red ratio. Furthermore, transgenic rice phytochrome A does not correct the early-flowering phenotype of phyB 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.     

Phytochromes and cryptochromes in the entrainment of the Arabidopsis circadian clock

Circadian clocks are synchronized by environmental cues such as light. Photoreceptor-deficient Arabidopsis thaliana mutants were used to measure the effect of light fluence rate on circadian period in plants. Phytochrome B is the primary high-intensity red light photoreceptor for circadian control, and phytochrome A acts under low-intensity red light. Cryptochrome 1 and phytochrome A both act to transmit low-fluence blue light to the clock. Cryptochrome 1 mediates high-intensity blue light signals for period length control. The presence of cryptochromes in both plants and animals suggests that circadian input pathways have been conserved throughout evolution.     

New Arabidopsis cue mutants suggest a close connection between plastid- and phytochrome regulation of nuclear gene expression

We searched for new components that are involved in the positive regulation of nuclear gene expression by light by extending a screen for Arabidopsis cue (chlorophyll a/b-binding [CAB] protein-underexpressed) mutants (H.-M. Li, K. Culligan, R.A. Dixon, J. Chory [1995] Plant Cell 7: 1599-1610). cue mutants display reduced expression of the CAB3 gene, which encodes light-harvesting chlorophyll protein, the main chloroplast antenna. The new mutants can be divided into (a) phytochrome-deficient mutants (hy1 and phyB), (b) virescent or delayed-greening mutants (cue3, cue6, and cue8), and (c) uniformly pale mutants (cue4 and cue9). For each of the mutants, the reduction in CAB expression correlates with the visible phenotype, defective chloroplast development, and reduced abundance of the light-harvesting chlorophyll protein. Levels of protochlorophyllide oxidoreductase (POR) were reduced to varying degrees in etiolated mutant seedlings. In the dark, whereas the virescent mutants displayed reduced CAB expression and the lowest levels of POR protein, the other mutants expressed CAB and accumulated POR at near wild-type levels. All of the mutants, with the exception of cue6, were compromised in their ability to derepress CAB expression in response to phytochrome activation. Based on these results, we propose that the previously postulated plastid-derived signal is closely involved in the pathway through which phytochrome regulates the expression of nuclear genes encoding plastid proteins.     

Dominant negative suppression of arabidopsis photoresponses by mutant phytochrome A sequences identifies spatially discrete regulatory domains in the photoreceptor

We used the exaggerated short hypocotyl phenotype induced by oat phytochrome A overexpression in transgenic Arabidopsis to monitor the biological activity of mutant phytochrome A derivatives. Three different mutations, which were generated by removing 52 amino acids from the N terminus (delta N52), the entire C-terminal domain (delta C617), or amino acids 617-686 (delta 617-686) of the oat molecule, each caused striking dominant negative interference with the ability of endogenous Arabidopsis phytochrome A to inhibit hypocotyl growth in continuous far-red light ("far-red high irradiance response" conditions). By contrast, in continuous white or red light, delta N52 was as active as the unmutagenized oat phytochrome A protein in suppressing hypocotyl elongation, while delta C617 and delta 617-686 continued to exhibit dominant negative behavior under these conditions. These data suggest that at least three spatially discrete molecular domains coordinate the photoregulatory activities of phytochrome A in Arabidopsis seedlings. The first is the chromophore-bearing N-terminal domain between residues 53 and 616 that is apparently sufficient for the light-induced initiation but not the completion of productive interactions with transduction chain components. The second is the C-terminal domain between residues 617 and 1129 that is apparently necessary for completion of productive interactions under all irradiation conditions. The third is the N-terminal 52 amino acids that are apparently necessary for completion of productive interactions only under far-red high irradiance conditions and are completely dispensable under white and red light regimes.     

Suppressors of an Arabidopsis thaliana phyB mutation identify genes that control light signaling and hypocotyl elongation

Ambient light controls the development and physiology of plants. The Arabidopsis thaliana photoreceptor phytochrome B (PHYB) regulates developmental light responses at both seedling and adult stages. To identify genes that mediate control of development by light, we screened for suppressors of the long hypocotyl phenotype caused by a phyB mutation. Genetic analyses show that the shy (short hypocotyl) mutations we have isolated fall in several loci. Phenotypes of the mutants suggest that some of the genes identified have functions in control of light responses. Other loci specifically affect cell elongation or expansion.     

Red light-induced appearance of phosphotyrosine-like epitopes on nuclear proteins from pea (Pisum sativum L.) plumules

As assayed by western blot analysis, red light induces the appearance of epitopes recognized by anti-phosphotyrosine antibodies in several pea nuclear proteins. The immunostaining is blocked by preadsorbing the antibodies with phosphotyrosine but not by preadsorbing them with phosphoserine or phosphothreonine. This light response is observed whether the red light irradiation is given to pea plumules or nuclei isolated from the plumules. The red-light-induced response seen in plumules is reversible by a subsequent far-red-light irradiation, indicating that the likely photoreceptor for this response may be phytochrome. By immunoblot analysis pea phytochrome A, but not phytochrome B, can be detected in proteins extracted from pea nuclear chromatin-matrix preparations. Phytochrome A and the protein bands immunostained by anti-phosphotyrosine antibodies can be solubilized from unirradiated pea chromatin by 0.3 M NaCl, but irradiating this preparation with red light does not induce the appearance of phosphotyrosine-like epitopes in any nuclear proteins. These results suggest that the association of phytochrome with purified pea nuclei is such that its conversion to the far-red light-absorbing form can induce a post-translational epitope change in nuclear proteins in vivo.     

In vivo characterization of chimeric phytochromes in yeast

Phytochromes are plant photoreceptors that play a major role in photomorphogenesis. Two members of the phytochrome family have been characterized in some detail. Phytochrome A, which controls very low fluence and high irradiance responses, is rapidly degraded in the light, forms sequestered areas of phytochrome (SAPs), and does not exhibit dark reversion in monocotyledonous seedlings. Phytochrome B mediates red/far-red reversible responses, is stable in the light, and does not form SAPs. We report on the behavior in yeast of the phytochrome apoproteins of rice PHYA, tobacco PHYB, and chimeric PHYAB and PHYBA and on the behavior of the respective holoprotein adducts after assembly with phycocyanobilin chromophore (PHY*). SAP-like formation in yeast was not observed for PHYB, but was detectable for PHYA, PHYAB, and PHYBA. Rice PHYA* did not undergo dark reversion in yeast. Surprisingly, all other tested phytochrome constructs did exhibit dark reversion, including chimeric phytochromes with a short N-terminal part of tobacco PHYB or parsley PHYA fused to rice PHYA. Furthermore, the proportion of phytochrome undergoing dark reversion and the rate of reversion were increased for both the N terminus-swapped constructs and PHYBA*. These results are discussed with respect to structure/function analysis of phytochromes A and B.     

Mutational analysis of the pea phytochrome A chromophore pocket: chromophore assembly with apophytochrome A and photoreversibility

Ten site-specific mutants of pea apophytochrome A were expressed in Saccharomyces cerevisiae and analyzed for chromophore assembly with apoprotein and photoreversible absorbance changes. The mutants constitute two specific changes for each of five conserved amino acid residues located in the microenvironment of the chromophore attachment residue, which is Cys-323 in pea phytochrome A. All mutant apophytochromes were autocatalytically able to covalently attach phycocyanobilin, indicating that there were no major structural perturbations in the apoproteins. However, the rate of chromophore ligation varied significantly among the mutants. Spectrally, the mutant holophytochromes are of three types: mutant phytochromes that are indistinguishable from the wild-type adduct, mutants with blue-shifted Pr and Pfr absorption maxima compared to the wild-type adduct, and mutants that are not photoreversible. From an analysis of the results, we concluded that the residues Asp-309, Arg-318, His-321, and Gln-326 are probably not catalytically involved in the chromophore ligation reaction, but some residues may play significant structural and stereochemical roles. Arg-318 might anchor the chromophore, as has been suggested [Partis, M. D., & Grimm, R. (1990) Z. Naturforsch, 45c, 987-998; Parker, W., et al. (1993) Bioconjugate Chem. (in press)]. The conserved Gln-326, three residues downstream from the chromophore attachment site, is not electrostatically critical for the spectral integrity and photoreversibility of phytochrome, but this residue is sterically important to the lyase activity. It appears that the role of the five amino acid residues in the N- and C-terminal vicinities of the chromophore binding Cys-323 is structural rather than catalytic for the ligation reaction.     

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.     

Role of a raf proto-oncogene during Caenorhabditis elegans vulval development

During Caenorhabditis elegans vulval induction, multipotent precursors respond to an inductive signal by generating vulval cells as opposed to non-specialized epidermal cells. Both classical and 'reverse' genetic approaches have revealed that a cascade of nematode homologues of mammalian proto-oncogenes is necessary for induction of the vulva. The inductive signal is a growth factor encoded by the lin-3 gene and its candidate receptor is a tyrosine kinase encoded by the let-23 gene. let-23 acts via a Ras protein encoded by the let-60 gene. A nematode homologue of mammalian raf family protein serine/threonine kinases has been cloned and found to be encoded by the lin-45 gene. Dominant negative lin-45 raf mutants prevent vulval induction. A recessive lin-45 raf mutation prevents the excessive vulval differentiation caused by activated ras, indicating that raf might act downstream of ras during vulval induction.