Phytochrome-regulated Gene Expression Research Articles

Geomagnetic field impacts on cryptochrome and phytochrome signaling

The geomagnetic field (GMF) is an environmental element whose instability affects plant growth and development. Despite known plant responses to GMF direction and intensity, the mechanism of magnetoreception in plants is still not known. Magnetic field variations affect many light-dependent plant processes, suggesting that the magnetoreception could require light. The objective of this work was to comprehensively investigate the influence of GMF on Arabidopsis thaliana (Col-0) photoreceptor signaling. Wild-type Arabidopsis seedlings and photoreceptor-deficient mutants (cry1cry2, phot1, phyA and phyAphyB) were exposed to near null magnetic field (NNMF, ≤40 nT) and GMF (~43 μT) under darkness and different light wavelengths. The GMF did not alter skotomorphogenic or photomorphogenic seedling development but had a significant impact on gene expression pathways downstream of cryptochrome and phytochrome photoactivation. GMF-induced changes in gene expression observed under blue light were partially associated with an alteration of cryptochrome activation. GMF impacts on phytochrome-regulated gene expression could be attributed to alterations in phytochrome protein abundance that were also dependent on the presence of cry1, cry2 and phot1. Moreover, the GMF was found to impact photomorphogenic-promoting gene expression in etiolated seedlings, indicating the existence of a light-independent magnetoreception mechanism. In conclusion, our data shows that magnetoreception alters photoreceptor signaling in Arabidopsis, but it does not necessarily depend on light.

Arabidopsis PSEUDO-RESPONSE REGULATOR7 is a signaling intermediate in phytochrome-regulated seedling deetiolation and phasing of the circadian clock.

To identify new components in the phytochrome (phy) signaling network in Arabidopsis, we used a sensitized genetic screen for deetiolation-defective seedlings. Two allelic mutants were isolated that exhibited reduced sensitivity to both continuous red and far-red light, suggesting involvement in both phyA and phyB signaling. The molecular lesions responsible for the phenotype were shown to be mutations in the Arabidopsis PSEUDO-RESPONSE REGULATOR7 (PRR7) gene. PRR7 is a member of a small gene family in Arabidopsis previously suggested to be involved in circadian rhythms. A PRR7-beta-glucuronidase fusion protein localized to the nucleus, implying a possible function in the regulation of photoresponsive gene expression. Consistent with this suggestion, prr7 seedlings were partially defective in the regulation of the rapidly light-induced genes CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY), observable as a premature increase in expression level during the second peak of the biphasic induction profile that is elicited upon initial exposure of dark-grown seedlings to light. A similar 3- to 6-h coordinated advance in peak free-running expression of CCA1, LHY, and TIMING-OF-CAB1, which are considered to encode the molecular components of the circadian oscillator in Arabidopsis, was observed in entrained fully green prr7 seedlings compared with wild-type seedlings. Collectively, these data suggest that PRR7 functions as a signaling intermediate in the phytochrome-regulated gene expression responsible for both seedling deetiolation and phasing of the circadian clock in response to light.

Nuclear and cytosolic events of light-induced, phytochrome-regulated signaling in higher plants.

Plants as sessile organisms cannot walk away but have to adapt to changes in their natural environments. Light is obviously the most important external factor, being the energy source. To regulate their fitness, the photosynthetic capacity of plants must be adapted to changes in the ambient light environment. Therefore, plants have evolved a large set of photoreceptors to monitor light quality and quantity, ranging from the UV to the infrared part of the spectrum. At least three different photoreceptor classes have been identified and analyzed: (i) UV‐B receptors, primarily characterized by action spectroscopy (Wellmann, 1983); (ii) the blue UV‐A photoreceptors, cry1 and cry2 and phototropine (Ahmad and Cashmore, 1993; Christie et al ., 1998; Lin et al ., 1998); and (iii) the red/far‐red reversible phytochromes. These photoreceptors control different aspects of plant growth and development, and in this review we focus on the physiological and molecular events regulated by the most characterized plant photoreceptors, the phytochromes. In higher plants, phytochromes are encoded by a small multigene family; five genes ( PHYA–PHYE , Sharrock and Quail, 1989; Clack et al ., 1994) have been identified in Arabidopsis . Phytochromes exist as dimers composed of two 125 kDa polypeptides, each carrying a covalently linked tetrapyrrol chromophore in the N‐terminal domain and dimerization domains in the C‐terminal domain. The photosensory function of the molecule is based on its capacity for reversible interconversion between the red light‐absorbing Pr form and the far‐red light absorbing Pfr form following sequential absorption of red and far‐red light. Photosignal perception by the receptor is followed by conformational changes, and activates, through an as yet poorly understood mechanism, signaling pathways leading to changes in the expression of genes that underlie developmental responses to light (Quail et al ., 1995). In order to finely tune their …

Phytochrome signal-transduction: characterization of pathways and isolation of mutants.

The study of phytochrome signalling has yielded a wealth of data describing both the perception of light by the receptor, and the terminal steps in phytochrome-regulated gene expression by a number of transcription factors. We are now focusing on establishing the intervening steps linking phytochrome photoactivation to gene expression, and the regulation and interactions of these signalling pathways. Recent work has utilized both a pharmacological approach in phototrophic soybean suspension cultures and microinjection techniques in tomato to establish three distinct phytochrome signal-transduction pathways: (i) a calcium-dependent pathway that regulates the expression of genes encoding the chlorophyll a/b binding protein (CAB) and other components of photosystem II; (ii) a cGMP-dependent pathway that regulates the expression of the gene encoding chalcone synthase (CHS) and the production of anthocyanin pigments; and (iii) a pathway dependent upon both calcium and cGMP that regulates the expression of genes encoding components of photosystem I and is necessary for the production of mature chloroplasts. To study the components and the regulation of phytochrome signal-transduction pathways, mutants with altered photomorphogenic responses have been isolated by a number of laboratories. However, with several possible exceptions, little real progress has been made towards the isolation of mutants in positive regulatory elements of the phytochrome signal-transduction pathway. We have characterized a novel phytochrome A (PhyA)-mediated far-red light (FR) response in Arabidopsis seedlings which we are currently using to screen for specific phyA signal-transduction mutants.

Phytochrome regulated gene expression

Light is used by plants as a signal for many physiological and developmental processes. Phytochrome is the most extensively studies family of photoreceptors that plants use to perceive the presence and quality of light in their environment. While the initial action of the phytochrome molecule is not yet known, one important kind of response, changes in the expression of specific nuclear genes, has been intensively investigated. Although phytochrome-regulated promoters are complex and can also respond to other signals, specific DNA elements that are involved in conferring phytochrome responsiveness have been identified. Potential signal transduction pathway components include G proteins, cyclic GMP and Ca 2+/calmodulin. In addition, the study of transcription factors involved in phytochrome-regulated gene expression has yielded insights into some of the final steps of transcriptional regulation by phytochrome.

Guanine nucleotide binding protein involvement in early steps of phytochrome-regulated gene expression.

The transmission process of light signals from plant photoreceptors to target cellular events is largely unknown. In the present work, we show that treatment of dark-adapted soybean cells (SB-P) with cholera toxin or pertussis toxin uncouples phytochrome-dependent gene expression. Addition of as little as 10 ng of toxin per ml is sufficient to activate expression of genes encoding the major chlorophyll a/b-binding protein (cab) in the dark. Significant levels of cab transcript accumulation are detected within 0.5 h after addition of the toxins and expression of these genes is desensitized to further light treatments. Treatment of SB-P cells with the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphtha-lenesulfonamide hydrochloride (W-7) prevents induction of the photoregulated gene by phytochrome or bacterial toxins. These results indicate the involvement of guanine nucleotide binding protein(s) in phytochrome-mediated cab gene activation. A likely site of action for this step is between the photoreceptor and a downstream W-7-sensitive effector.

Characterization of phytochrome-regulated gene expression in a photoautotrophic cell suspension: possible role for calmodulin.

A photoautotrophic suspension culture of soybeans was found to exhibit light-dependent expression of the genes encoding the major chlorophyll a- and b-binding protein (CAB). The expression was mediated by phytochrome, since it was induced by red light and reversed by far-red light. The maximal level as well as the kinetics of the induction were comparable between the suspension culture and soybean seedlings. Using this cell culture, we addressed the question of whether a calcium- and/or calmodulin-mediated step is involved in the signal transduction process between phytochrome and CAB expression. We found that W-7, a potent calmodulin antagonist, severely attenuated the induction of CAB mRNA by light, whereas W-5, a weak calmodulin antagonist, had little effect. Control experiments demonstrated that W-7 treatment did not block the induction of hsp-75 by heat shock. The addition of ionomycin, a calcium ionophore, induced a low level of CAB mRNA accumulation in the dark which could be further enhanced by light treatment. We propose that calmodulin activation by light is necessary but not sufficient to induce maximal CAB expression.

Characterization of phytochrome-regulated gene expression in a photoautotrophic cell suspension: possible role for calmodulin.

A photoautotrophic suspension culture of soybeans was found to exhibit light-dependent expression of the genes encoding the major chlorophyll a- and b-binding protein (CAB). The expression was mediated by phytochrome, since it was induced by red light and reversed by far-red light. The maximal level as well as the kinetics of the induction were comparable between the suspension culture and soybean seedlings. Using this cell culture, we addressed the question of whether a calcium- and/or calmodulin-mediated step is involved in the signal transduction process between phytochrome and CAB expression. We found that W-7, a potent calmodulin antagonist, severely attenuated the induction of CAB mRNA by light, whereas W-5, a weak calmodulin antagonist, had little effect. Control experiments demonstrated that W-7 treatment did not block the induction of hsp-75 by heat shock. The addition of ionomycin, a calcium ionophore, induced a low level of CAB mRNA accumulation in the dark which could be further enhanced by light treatment. We propose that calmodulin activation by light is necessary but not sufficient to induce maximal CAB expression.

Expression of Light-Harvesting Chlorophyll a/b-Protein Genes Is Phytochrome-Regulated in Etiolated Arabidopsis thaliana Seedlings

Phytochrome action results in a large and rapid increase in the light-harvesting chlorophyll a/b-protein (LHCP) mRNA level in etiolated seedlings of Arabidopsis thaliana: the RNA increase is detectable within 1 hour after 1 minute red illumination, reaches a maximum 30-fold higher than the dark level at ca. 2 hours, and decays back to dark levels by about 8 hours after the brief red illumination. S1 nuclease analysis distinguishes two kinds of mRNAs transcribed from the three members of the LHCP gene family previously characterized for Arabidopsis (LS Leutwiler, EM Meyerowitz, EM Tobin, 1986 Nucleic Acids Res 14: 4051-4064). One of these arises from the AB140 gene, while the other represents the product(s) of the AB165 and/or AB180 gene(s) (AB165/AB180 mRNA). In mature, white light-grown plants, the two kinds of mRNAs are present in nearly equal amounts. In contrast, in etiolated seedlings, 1 minute red light causes a sixfold greater increase in the level of AB140 mRNA than in the level of AB165/AB180 mRNA, although both levels are regulated by phytochrome action. The kinetics of the responses to 1 minute red light are similar for both kinds of transcripts. Additional evidence suggests that this differential expression is developmentally regulated. Because the AB140 gene offers an attractive target for further analysis of phytochrome-regulated gene expression in Arabidopsis, we have further characterized this gene by mapping its 5' and 3' transcript termini.

Phytochrome: molecular properties and biogenesis

Native Avena phytochrome, recently shown to have a monomeric molecular mass of 124 kDa, has molecular properties that differ significantly from those of the extensively characterized ‘ 120’ kDa or ‘large’ phytochrome preparations now known to contain a mixture of proteolytically degraded 118 and 114 kDa polypeptides. For example, 124 kDa phytochrome has a blocked N-terminus, a P fr λ max of 730 nm, a higher photostationary state in red light (86% P fr ), exhibits no dark reversion and shows no differential reactivity of P r and P fr toward a chemical probe of hydrophobic domains. The data indicate that the proteolytically removed 6-10 kDa polypeptide segment (s) is critical to the spectral and structural integrity of the photoreceptor; that at least part of the cleaved domain is located at the N-terminus of the molecule; that this domain influences the chemical reactivity of the chromophore with the external medium; and that a current hypothesis that P r —P fr photoconversion results in the exposure of a hydrophobic domain on the molecule is inconsistent with the properties of native phytochrome. Phytochrome has been found to exert rapid negative feedback control over the level of its own translatable mRNA. P fr formation in etiolated tissue causes a decline in translatable phytochrome mRNA that is detectable within 15—30 min and that results in more than a 95 % reduction within 2 h. Less than 1 % P fr is sufficient to induce 60 % of the maximum response, which is saturated at 20 % P fr or less. The rapidity of this autoregulatory control makes phytochrome itself an attractive system for investigating phytochrome-regulated gene expression. A project to clone phytochrome complementary DNA (cDNA) has been initiated. A major obstacle in this work has been the unexpectedly low abundance of phytochrome mRNA, less than 0.005 % of the poly (A) RNA in etiolated tissue. cDNA made from poly (A) RNA enriched ca. 200-fold in phytochrome mRNA has been cloned and bacterial colonies have been screened with a synthetic oligodeoxynucleotide hybridization probe. The sequence of this probe was derived from a known partial amino acid sequence of the phytochrome protein. Difficulties encountered with this approach are discussed.

Western blot analysis of a lytic process in vitro specific for the red light absorbing form of phytochrome

Phytochrome, which has been isolated from several species as a soluble protein [1-4], is regarded as the photoreceptor of all photoresponses induced by red light and reversed by far-red light, as well as of photoresponses with the characteristics of the so-called high intensity reaction (review [5]). Several examples of phytochrome-regulated gene expression have been recognized [6-8]. The photochemical interconversion between the 2 spectrally distinct forms of phytochrome, Pr and Pfr, is the basis of photoreception in vivo and occurs also in cell-flee preparations. However, reactions that might be related to signal transmission have never been observed in vitro. Perhaps loss of the native protein structure soon after extraction is the reason of these failures. Undoubtedly, this applies to the 60 000 Mr species purified after extensive proteolysis [1]. But also preparations termed 'undegraded phytochrome' seem to have undergone limited degradation, for after discontinuous electrophoresis, they showed a heterogeneous band pattern with main bands at 114000 and 118000 Mr [2,4,9]. These bands, however, were neither observed after extraction of seedlings with denaturing buffer [10] nor after cell-flee translation of the phytochrome message [11]. In both