光敏色素分子特性及其信号转导机制

结合生物物理、分子遗传学和细胞生物学的方法已证实,光敏色素信号转导是一个空间分布的、非线形信号传递链。尤其是最近又发现了不同种类的光敏色素分子及其它们在Pr、Pfr光转换中产生的中间体,不仅说明了光敏色素信号转导链是一个多维的信号网络,而且这也暗示着光转换中产生的中间体也直接参与了早期的信号转导。在此,综述了光敏色素分子光转换及其早期信号转导的若干新进展,讨论光敏色素原初光反应及其信号转导的机制。     

Reversible phytochrome regulation influenced the severity of ozone-induced visible foliar injuries in Trifolium subterraneum L.

In Trifolium subterraneum, oxidative stress caused by ozone has been shown to result in more severe visible foliar injuries when plants were kept in dim broadband white light during the night (i.e. a long photoperiod) compared to darkness during the night (a short photoperiod). As phytochrome signalling is involved in photoperiod sensing, the effect of night-time red and far-red illumination on the ozone-induced response was studied. T. subterraneum plants were treated with ozone enriched air (70?ppb) for either 1?h for a single day or 6?h for three consecutive days. After the first ozone exposure, plants were separated into six night-time light regimes during the two subsequent nights (10?h?day, 14?h night): (1) darkness, (2) far-red light (FR), (3) a short night-break of red followed by far-red light during an otherwise dark night (R FR), (4) a short night-break of red, far-red and finally red light during an otherwise dark night (R FR R), (5) dim white light (L) and (6) red light (R). The treatments L and R resulted in significantly more severe ozone-induced visible foliar injuries relative to D and FR treatments, indicating a phytochrome-mediated response. The night-breaks resulted in a photoreversible and significantly different ozone response depending on the light quality of the last light interval (R FR or R FR R), supporting a photoreversible (between P_r and P_fr) phytochrome signalling response. Thus, in T. subterraneum, the outcome of oxidative stress due to ozone appears to depend on the photoperiod mediated by the night-time conformation of phytochrome.     

Mutants of phospholipase A (pPLA‐I) have a red light and auxin phenotype

pPLA‐I is the evolutionarily oldest patatin‐related phospholipase A (pPLA) in plants, which have previously been implicated to function in auxin and defence signalling. Molecular and physiological analysis of two allelic null mutants for pPLA‐I [ppla‐I‐1 in Wassilewskija (Ws) and ppla‐I‐3 in Columbia (Col) ] revealed pPLA‐I functions in auxin and light signalling. The enzyme is localized in the cytosol and to membranes. After auxin application expression of early auxin‐induced genes is significantly slower compared with wild type and both alleles show a slower gravitropic response of hypocotyls, indicating compromised auxin signalling. Additionally, phytochrome‐modulated responses like abrogation of gravitropism, enhancement of phototropism and growth in far red‐enriched light are decreased in both alleles. While early flowering, root coils and delayed phototropism are only observed in the Ws mutant devoid of phyD, the light‐related phenotypes observed in both alleles point to an involvement of pPLA‐I in phytochrome signalling.     

The phytochrome photoreceptor in the green alga Mesotaenium caldariorum: implication for a conserved mechanism of phytochrome action

Chloroplast movement in the unicellular green alga Mesotaenium caldariorum is one of the earliest documented photomorphogenetic responses in plants. Photobiological studies have established that this response is under the control of phytochrome, whose rigid association with the plasma membrane and/or cytoskeleton enables the algal cells to orientate the chloroplast in response to the direction and intensity of light from the environment. While many of the key components of the algal phytochrome signalling pathway have been elucidated (i.e. Ca²⁺, calmodulin, actin and myosin), the primary biochemical mechanism of algal phytochrome action is unknown. To begin to address this important question, phytochrome and its corresponding genes have been isolated and characterized in this alga. These studies reveal that Mesotaenium cells contain a single type of phytochrome which is encoded by a small family of highly related genes. On the basis of its biochemical properties, primary structure and ability to interfere with the photoregulatory activity of phytochrome in transgenic plant seedlings, it appears likely that the primary mechanism of phytochrome action has been conserved throughout its evolution.     

Evidence for type II phytochrome-induced rapid signalling leading to cab::luciferase gene expression in tobacco cotyledons

We studied the activation of cab gene expression by phytochrome-induced intercellular signalling and report insights into the mechanism of induction and outspread of a plant internal signal. By micro-beam irradiation techniques and use of a photon-imaging charge-coupled device (CCD) camera system we monitored cab::luciferase reporter gene expression in cotyledons of transgenic tobacco (Nicotiana tabacum L. cv. Xanthi) plants. We found that (i) the photoreceptor triggering intercellular signalling and reporter-gene expression is type II but not type I phytochrome, (ii) phytochrome in its far-red-absorbing (Pfr) form is necessary for the induction but not for the outspread of the signalling, (iii) red/far-red reversibility is restricted to the red-irradiated cells, and (iv) the phytochrome-induced signal spreads rapidly throughout the cotyledon and reaches its target cells within minutes. Received: 26 June 2000 / Accepted: 25 August 2000     

Phytochrome signalling modulates the SA-perceptive pathway in Arabidopsis

The interaction of phytochrome signalling with the SA signal transduction pathway has been investigated in Arabidopsis using single and multiple mutants affected in light perception (phyA and phyB deficient) and light-signal processing (psi2, phytochrome signalling). The induction of PR1 by SA and functional analogues has been found to strictly correlate with the activity of the signalling pathway controlled by both phyA and phyB photoreceptors. In darkness as well as dim light, and independently of a carbohydrate source, SA-induced PR gene expression as well as the hypersensitive response to pathogens (HR) are strongly reduced. Moreover, the initiation of HR also exhibits a strict dependence upon both the presence and the amplitude of a phytochrome-elicited signal. The growth of an incompatible strain of bacterial a pathogen (Pseudomonas syringae pv. tomato) was enhanced in phyA-phyB and decreased in psi2 mutants. While functional chloroplasts were found necessary for the development of an HR, the induction of PRs was strictly dependent on light, but independent of functional chloroplasts. Taken together, these data demonstrate that the light-induced signalling pathway interacts with the pathogen/SA-mediated signal transduction route. These results are summarized in a formalism that allows qualitative computer simulation.     

A novel protein phosphatase indirectly regulates phytochrome-interacting factor 3 via phytochrome

Light signal transduction in plants involves an intricate series of pathways which is finely regulated by interactions between specific signalling proteins, as well as by protein modifications such as phosphorylation and ubiquitination. The identification of novel phytochrome-interacting proteins and the precise signalling mechanisms that they mediate is still ongoing. In our present study, we show that the newly identified putative phytochrome-associated protein, PAPP2C (phytochrome-associated protein phosphatase type 2C), interacts in the nucleus with phyA (phytochrome A) and phyB, both in vitro and in vivo. Moreover, the phosphatase activity of PAPP2C and its association with phytochromes were found to be enhanced by red light, indicating that it plays a role in mediating phytochrome signalling. In particular, PAPP2C specifically binds to the N-terminal PHY domain of the phytochromes. We thus speculate that this interaction reflects a unique regulatory function of this phosphatase toward established phytochrome-associated proteins. We also show that PAPP2C effectively dephosphorylates phytochromes in vitro. Interestingly, PAPP2C indirectly mediates the dephosphorylation of PIF3 (phytochrome-interacting factor 3) in vitro. Taken together, we suggest that PAPP2C functions as a regulator of PIF3 by dephosphorylating phytochromes in the nucleus.     

Photomophogenesis: Phytochrome takes a partner!

How light signals are transduced by phytochromes is still poorly understood. Recent studies have provided evidence that a PAS domain protein, PIF3, physically interacts with phytochromes, plays a role in phytochrome signal transduction and might be a component of a novel signalling pathway in plants.     

The phytochrome family: dissection of functional roles and signalling pathways among family members.

There is considerable evidence that individual members of the five-membered phytochrome family of photoreceptors in Arabidopsis have differential functional roles in controlling plant photomorphogenesis. Emerging genetic evidence suggests that this differential activity may involve initially separate signalling pathway branches specific to individual family members.     

Photoreceptors and signals in the photoperiodic control of development

Many plant species are sensitive to changes in the seasons, especially with regard to their reproductive behaviour. Sexual or vegetative reproductive structures are often only formed at times of the year when days are sufficiently long, or short. Plants perceive daylength in the leaves, but reproductive changes occur in shoot apices in response to the movement of signals throughout the plant. There is good evidence that phytochrome A is an essential component of the daylength-sensing mechanism in long-day plants, and preliminary evidence suggests a potential interaction between phytochrome C and daylength sensitivity in short-day plants. The sensitivity of reproductive processes to photoperiodic control is directly altered by photoreceptor action, particularly in the case of phytochrome B, which has a major influence on whether flowering or tuberization occurs under non-inductive conditions in both long- and short-day plants, but is not involved in daylength measurement. The signals which move from leaves to the sites of reproductive development are not known, but there is good evidence that gibberellins may be important and some preliminary indication that brassinosteroids might also be involved in photoperiodic signalling.     

Light and temperature signal crosstalk in plant development

Light and temperature are two of the most important environmental stimuli regulating plant development. Recent advances have suggested considerable interaction between these signalling pathways at the molecular level. Studies of both flowering and germination have shown the phytochrome family of plant photoreceptors to display altered functional hierarchies at different growth temperatures. The existence of common signalling components in both light and temperature sensing has additionally been proposed. More recently, light quality signals have been shown to regulate plant-freezing tolerance in an ambient temperature-dependent manner. Together, these data suggest that complex crosstalk between light-signalling and temperature-signalling pathways is fundamental to the growth and development of plants in natural environments.     

Ubiquitin-phytochrome conjugates. Pool dynamics during in vivo phytochrome degradation

The plant photoreceptor chromoprotein, phytochrome, is rapidly degraded in vivo after photoconversion from a stable red light-absorbing form (Pr) to a far-red light-absorbing form (Pfr). Previously, we demonstrated that during Pfr degradation in etiolated oat seedlings, ubiquitin-phytochrome conjugates, (Ub-P), appear and disappear suggesting that phytochrome is degraded via a ubiquitin-dependent proteolytic pathway (Shanklin, J., Jabben, M., and Vierstra, R. D. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 359-363). Here, we provide additional kinetic and localization data consistent with this hypothesis by exploiting the unique ability to photoregulate phytochrome degradation in vivo. An assay for the quantitation of Ub-P was developed involving immunoprecipitation of total conjugates with anti-ubiquitin antibodies, followed by the detection of Ub-P with anti-phytochrome antibodies. Using this immunoassay, we found that Ub-P will accumulate to approximately 5% of initial phytochrome during Pfr degradation induced by a saturating red light pulse. Reducing the amount of Pfr produced initially by attenuating the red light pulse, lowered the amount of phytochrome degraded in the following dark period and concomitantly reduced the maximal accumulation of Ub-P. Continuous far-red irradiations that maintained only 4% of phytochrome as Pfr induced rapid phytochrome degradation similar to that induced by a red light pulse converting 86% of Pr to Pfr. The appearance and disappearance of Ub-P were similar for each irradiation indicating that Ub-P accumulation is independent of the level of Pfr provided rapid phytochrome degradation is maintained. Pulse-chase studies employing continuous far-red light followed by darkness showed that Ub-P are continuously synthesized during phytochrome degradation and rapidly disappear once degradation ceases. Ub-P also accumulated during "cycled Pr" degradation induced by the transformation of Pr to Pfr and back to Pr. The commitment to degrade cycled Pr and form Ub-P occurred within seconds after Pfr formation making the cause(s) underlying this phenomenon one of the fastest phytochrome reactions known. Within seconds after Pfr formation, a majority of phytochrome is also known to aggregate in vivo (previously defined as sequestered or pelletable), with aggregated phytochrome preferentially lost during phytochrome degradation. In vitro analysis of aggregated phytochrome indicated that they contain most of the Ub-P. Moreover, the appearance of Ub-P in the aggregated and soluble fractions correlated with the time that phytochrome disappeared from that fraction.(ABSTRACT TRUNCATED AT 400 WORDS)     

Real time spectral analysis during phytochrome chromophore and chromoprotein purification

The plant photoreceptor phytochrome senses light quality and quantity in the red region of the spectrum, directing adaptation and development. The functional holo-protein is a dimeric chromoprotein which is formed by an autoassembly reaction between the translation product and the open chain tetrapyrroles phytochromobilin (PPhiB) or phycocyanobilin (PCB). We are interested in structure/function relationships within the phytochrome molecule, in particular chromophore/protein interaction during the assembly and photoactivation, using IR and NMR spectroscopy. For this we use an automated F/HPLC system running in a darkroom to purify large amounts of protein and chromophore separately. To obtain highly pure PCB chromophore we developed improved extraction and purification methods in which the final step is RPC-18 HPLC. As there are many spectrally only slightly different tetrapyrroles in the extract, the triple-wavelength monitoring offered by the F/HPLC detector was inadequate for distinguishing between PCB and impurities. Furthermore, lambda(max) for the phytochrome Pfr signalling state lies between 705 and 730 nm, beyond the range of the detector. Also, as both holo-protein and chromophore are photoactive, we wished to minimize light exposure of the eluate. We therefore implemented a miniature CCD-based flow UV-vis spectrophotometer using a xenon flash and quartz fiber optics enabling us monitor the entire 250-800 nm spectrum of the eluate to an accuracy of +/-3 x 10(-3)A in real time. The instrumentation described can be added to any chromatographic system, thereby allowing the purification of any molecule to be monitored easily and efficiently.     

Loss of phytochrome photoreversibility in vitro: I. Extraction and partial purification of killer

Crude pea (Pisum sativum L. var. Alaska) phytochrome extracts contain a substance, “Killer,” which interacts with the far red-absorbing form of phytochrome causing a net loss of spectrophotometrically detectable phytochrome in vitro. Killer is absent from crude extracts of Avena phytochrome, is separable from pea phytochrome by gel filtration, and is alcohol-extractable from etiolated pea seedlings. Killer activity in alcohol extracts behaved, during partial purification, in a manner identical to that derived from pea phytochrome preparations. The mass extraction and partial purification of Killer are described.