A Spectroscopy-based Methodology for Rapid Screening and Characterization of Phytochrome Photochemistry in Search of Pfr-favored Variants

Phytochromes are photosensitive proteins with a covalently bound open-chain chromophore that can switch between two principal states: red light absorbing Pr and far-red light absorbing Pfr. Our group has previously shown that the bacteriophytochrome from Xanthomonas campestris pv. campestris (XccBphP) is a bathy-like phytochrome that uses biliverdin IXα as a co-factor and is involved in bacterial virulence. To date, the XccBphP crystal structure could only be solved in the Pr state, while the structure of its Pfr state remains elusive. The aims of this work were to develop an efficient screening methodology for the rapid characterization and to identify XccBphP variants that favor the Pfr form. The screening approach developed here consists in analyzing the UV-Vis absorption behavior of clarified crude extracts containing recombinant phytochromes. This strategy has allowed us to quickly explore over a hundred XccBphP variants, characterize multiple variants and identify Pfr-favored candidates. The high-quality data obtained enabled not only a qualitative, but also a quantitative characterization of their photochemistry. This method could be easily adapted to other phytochromes or other photoreceptor families.     

Response of brassinosteroid-treated oilseed rape cotyledons to infection with the wild type and HR-mutant of <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> or with <Emphasis Type="Italic">P. fluorescence</Emphasis>

The aim of the study has been to determine the protection effect of brassinosteroid (BR₂₇) in oilseed rape cotyledons against infection by an incompatible wild type of, a hypersensitive response mutant of and saprophytic Pseudomonas bacteria. In this paper, membrane permeability, PSII efficiency and metabolic activity were analysed. The following strains of Pseudomonans were used: P. syringae pv. syringae (Ps), P. syringae pv. syringae hrcC mutant (Pm) and P. fluorescence (Pf). The study was carried out using two cultivars of spring oilseed rape (Brassica napus L.): ‘Licosmos’ and ‘Huzar’. Pre-treatment of cotyledons with BR₂₇ caused about 50–70% increase in ion leakage for both cultivars. However, BR₂₇ significantly decreased ion leakage from cotyledons inoculated with Ps in both cultivars. Infection with Ps and Pf caused disturbances of energy flow in PSII by lowering its efficiency in rape cotyledons. We noted insignificant impact of 24-epibrassinolide on PSII efficiency if compared to absolute control, but generally it had a positive effect in plants infected with bacteria. The values of heat flow in all treatments, except for cotyledons infected with Ps, decreased during 20 h after inoculation. However, the curves of heat flow for Ps-infected cotyledons showed a completely different pattern with at least two peaks. BR₂₇ pre-treated cotyledons infected with Ps had higher heat flow in comparison to Ps infected ones. BR₂₇ treatment did not change specific enthalpy of cotyledon growth (Δgh) for both cultivars if compared with absolute control. However, infection with Ps markedly increased Δgh values by about 200% for both cultivars. We suggested protective action of BR₂₇ in oilseed rape cotyledons after bacterial infection with Pseudomonas.     

THE MODE OF INTERACTION BETWEEN BLUE (UV) LIGHT PHOTORECEPTOR AND PHYTOCHROME IN ANTHOCYANIN FORMATION OF THE SORGHUM SEEDLING

Two non-photosynthetic photoreceptors (phytochrome and a blue light photoreceptor) are involved in light-mediated anthocyanin synthesis in the mesocotyl of Sorghum seedlings. The present study was undertaken to investigate the kind of interaction between phytochrome and the blue light photoreceptor. The data show that phytochrome (P_fr) can only act once a blue light effect has occurred. On the other hand, the blue light effect cannot express itself without P_fr. It is concluded that there is an obligatory dependency (or sequential interaction) between the blue light effect and the light effect occurring through phytochrome, although the blue light photoreaction per se is not affected by the presence or absence of phytochrome. The latter statement is based on the results of dichromatic experiments, i.e. simultaneous, high fluence rate irradiation with two kinds of light. Blue light can be replaced by UV light. It is not clarified yet whether the effect of blue and UV light is due to the same photoreceptor.     

Common Structural Elements in the Chromophore Binding Pocket of the Pfr State of Bathy Phytochromes

Phytochromes are bimodal photoreceptors which, upon light absorption by the tetrapyrrole chromophore, can be converted between a red‐absorbing state (Pr) and far‐red‐absorbing state (Pfr). In bacterial phytochromes, either Pr or Pfr are the thermally stable states, thereby constituting the classes of prototypical and bathy phytochromes, respectively. In this work, we have employed vibrational spectroscopies to elucidate the origin of the thermal stability of the Pfr states in bathy phytochromes. Here, we present the first detailed spectroscopic analysis of RpBphP6 (Rhodopseudomas palustris), which together with results obtained for Agp2 (Agrobacterium tumefaciens) and PaBphP (Pseudomonas aeruginosa) allows identifying common structural properties of the Pfr state of bathy phytochromes, which are (1) a homogenous chromophore structure, (2) the protonated ring C propionic side chain of the chromophore and (3) a retarded H/D exchange at the ring D nitrogen. These properties are related to the unique strength of the hydrogen bonding interactions between the ring D N‐H group with the side chain of the conserved Asp194 (PaBphP numbering). As revealed by a comparative analysis of homology models and available crystal structures of Pfr states, these interactions are strengthened by an Arg residue (Arg453) only in bathy but not in prototypical phytochromes.     

PHOTORECEPTOR INTERACTION IN PLANT PHOTOMORPHOGENESIS: THE LIMITS OF EXPERIMENTAL TECHNIQUES AND THEIR INTERPRETATIONS

Abstract— Problems concerning the interpretation of interactions of higher plant photomorphogenetic receptors are discussed. The theory that action of a blue light photoreceptor serves only to maintain responsiveness to phytochrome (Responsiveness Theory) is demonstrated to be unable to be properly tested with present techniques. This theory is also unable to explain experimental results any better than an alternative theory that a blue light photoreceptor may require the presence of the active form of phytochrome to express its activity (Presence Theory). This tatter theory is also incapable of being fully tested. There does not appear to be an adequate current theory to explain photoreceptor interactions. Other issues discussed include the use of displacement transducers in growth studies, the induction of phytochrome-type responses by blue light, and the relative importance of the photoreceptors. New data are introduced on the effect of blue light in the end-of-day growth response to phytochrome of the light-grown Cucumis sativus L. hypocotyl, and on the light equivalence principle in the same species.     

IDENTITY OF THE PHOTORECEPTORS FOR THE PERCEPTION OF IRRADIANCE IN PHOTOSYNTHETIC LIGHT ACCLIMATION IN TOMATO*

The photoreceptors involved in the photosynthetic acclimation of tomato (Lycopersicon esculentum Mill.) to increased irradiance were investigated. Plants were transferred from 100 p.mol m^?2 s^?1 cool white fluorescent light to higher irradiances of white light or white light supplemented with blue, red, green or yellow light. In these experiements light of all wavelengths tested was capable of causing acclimation as measured by the rate of light-saturated photosynthesis. It was concluded that the photosynthetic system rather than the blue-absorbing photoreceptor or phytochrome system acts as the photoreceptor for increased irradiance. No acclimation was observed in response to increased CO₂ levels, but increasing light integral at a constant irradiance was effective in bringing about acclimation. We conclude that acclimation is a response to increased photosynthetic light capture rather than increased photosynthetic carbon fixation, and involves a photon counting mechanism.     

PHOTOMORPHOGENIC RESPONSES TO UV RADIATION III: A COMPARATIVE STUDY OF UVB EFFECTS ON ANTHOCYANIN and FLAVONOID ACCUMULATION IN WILD-TYPE and aurea MUTANT OF TOMATO (Lycopersicon esculentum MILL.)

The UV-mediated induction of anthocyanin and UV-absorbing compounds was characterized in etiolated hypocotyls of wild-type and aurea (au) mutant tomato seedlings. Ultraviolet radiation induced significant increases of anthocyanin and UV-absorbing compounds in hypocotyls of die au mutant and of its isogenic wild-type, but the differences in the time courses of UV-induced pigment accumulation indicate mat different photoregulatory mechanisms are involved for each of these two groups of pigments. It appears mat prolonged presence of adequate levels of UVB (290–320nm) energy and consequently the action of a specific UVB photoreceptor are indispensable for the photoinduction of anthocyanin accumulation in UV-irradiated hypocotyl of the au mutant that is missing the labile phytochrome pool. The large difference found between the wild-type and the au mutant strongly indicate the involvement of labile phytochrome as the primary functional photoreceptor for the photoinduction of anthocyanin accumulation in wild-type tomato hypocotyls. The UVB photoreceptor could at least partly replace the action of labile phytochrome (as far as anthocyanin accumulation is concerned) when the functional phytochrome pool is missing as in the au mutant. The general picture of UV-mediated induction of total UV-absorbing compounds shows only a macroscopic difference between wild-type and die au mutant of tomato: the higher initial level (in darkness) of these compounds in die wild-type in contrast to the au mutant. Although there is UV-induced accumulation of UV-absorbing compounds in bom genotypes, the levels in the au mutant never reach mat of the wild-type under the same UV exposure. A UVB photosensor may play a more important role in the photoinduction of UV-absorbing compounds. Indeed, in the absence of labile phytochrome, i.e. in the au mutant, a UVB-absorbing photoreceptor alone is able to establish high responsiveness for the UV-induced flavonoid accumulation.     

PHOTOMORPHOGENIC RESPONSES TO UV RADIATION. II: A COMPARATIVE STUDY OF UV EFFECTS ON HYPOCOTYL ELONGATION IN A WILD-TYPE AND AN aurea MUTANT OF TOMATO (Lycopersicon esculentum Mill.)

Hypocotyl growth in etiolated seedlings of wild-type and an aurea mutant of tomato (Lycopersicon esculenturn Mill.), that appears to be deficient in labile phytochrome, is strongly inhibited by UV radiation in the region of 300–400 nm. The role of phytochrome in the UV-mediated inhibition of hypocotyl growth was studied using different experimental approaches: (1) by comparing the effectiveness of treatments of increasing duration of exposure to 692 nm and UV radiation; (2) by modifying the UV spectral range with specific cut-off filters. The experimental results suggest that the UV-induced inhibition of growth in wild-type tomato is mediated to a large extent by the longer wavelengths of the UV-A region and is mediated mainly by phytochrome. In contrast, at wavelengths < 305 nm a strong UV-B effect was found in the aurea mutant, suggesting a preeminent action of a specific UV-B absorbing photoreceptor that displays less action in the wild-type.     

In vivo PHYTOCHROME-MEDIATED PERCEPTION OF REFLECTED LIGHT SIGNALS

The spectrophotometric assay of phytochrome in vivo in etiolated plant material was used to determine the effects of changes in reflected light on the state of the photoreceptor in etiolated seedlings exposed simultaneously to direct and reflected light. Changes in reflected light that were small in terms of the total (direct + reflected) radiation incident on the seedlings produced detectable changes in the state of phytochrome in vivo. The contribution of reflected light to the state of phytochrome in vertical organs was greater than expected from its low contribution to total incident light. These data from laboratory studies complement and are consistent with results of field studies on the effects of light reflected from neighboring vegetation on plant growth under natural radiation conditions.     

RELATIVE IMPORTANCE OF BLUE LIGHT AND LIGHT ABSORBED BY PHYTOCHROME IN GROWTH OF MUSTARD (Sinapis alba L.) SEEDLINGS*

Abstract— Hypocotyl straight growth in mustard (Sinapis alba L.) responds very strongly and in precisely the same way to low fluence rate red (RL) and white light (WL). The effect of weak light can be attributed fully to light absorption by phytochrome. Only with increasing fluence rate an effect of blue light (BL) comes into play which cannot be explained by the action of phytochrome. However, this specific action of BL can be demonstrated in hypocotyl growth of mustard seedlings only up to 5 days after sowing (25°C). With older seedlings control of hypocotyl growth seems to be exerted exclusively via phytochrome. Regarding the far-red light dependent “high irradiance reaction” (FR-HIR) it was found that it plays a dominant role in growth of mustard only during a relatively short period. It tends to disappear in favor of a RL-HIR between 3 and 4 days after sowing. It is concluded that the seedling exhibits a largely endogenous temporal pattern of responsiveness to light. Phototropism of the mustard seedling can be elicited by low fluence rates (< 1 mW m^?2) of unilateral BL. This same light has no effect on straight growth. It is concluded that BL-dependent phototropic growth response of a hypocotyl and the effect of BL on longitudinal growth of the hypocotyl are unrelated phenomena.     

MULTIPLICATIVE ACTION OF A UV-B PHOTORECEPTOR and PHYTOCHROME IN ANTHOCYANIN SYNTHESIS

Using 290-nm light, which excites only a UV-B photoreceptor, and 385- and 660-nm light, which activate only phytochrome, the fluence rate-response curves of monochromatic irradiations for anthocyanin synthesis in the first internodes of broom sorghum (Sorghum bicolor Moench, cv. Acme Broomcorn) were analyzed. Although the two photoreceptors absorbed light independently, they multiplicatively increased the action of each other. Accordingly, when the fluence rates of both wavelengths were changed together, the resulting slopes of the fluence rate-response curves of double-log plots were steep compared with the slopes obtained with the respective monochromatic irradiations. The slopes of fluence rate-response curves for monochromatic irradiations at 325 to 345 nm were steeper than those at other wavelengths. This difference was shown to be due to the multiplicative actions of both photoreceptors.     

PHYSIOLOGICAL CHARACTERIZATION OF A HIGH-PIGMENT MUTANT OF TOMATO

Abstract— A high-pigment (hp) mutant, which shows exaggerated phytochrome responses and three other genotypes of Lycopersicon esculenrum Mill. cv. Ailsa Craig: the aurea (au) mutant deficient in the bulk light-labile phytochrome (PI) pool, the au, hp double mutant, and their isogenic wild type, were used in this study. Measurements of phytochrome destruction in red light (R) revealed that the exaggerated responses of the hp mutant are not caused by a higher absolute phytochrome level or a reduced rate of phytochrome destruction. Fluence-response relationships for anthocyanin synthesis after a blue-light pretreatment were studied to test if the hp mutant conveys hypersensitivity to the far-red light (FR)-absorbing form of phytochrome (Pfr), i.e. the threshold of Pfr required to initiate the response is lower. The response range for the hp mutant and wild type was identical, although the former exhibited a 6-fold larger response. Moreover, the kinetics of anthocyanin accumulation in continuous R were similar in the wild-type and hp-mutant seedlings, despite the latter accumulating 9-fold more anthocyanin. Since the properties of phytochrome are the same, the hp mutation appears to affect the state of responsiveness amplification, i.e. the same amount of Pfr leads to a higher response in the hp mutant. We therefore propose that the hp mutation is associated with an amplification step in the phytochrome transduction chain. Escape experiments showed that the anthocyanin synthesis after different light pretreatments terminated with a R pulse was still 50% FR reversible after 4–6 h darkness, indicating that the Pfr pool regulating this response must be relatively stable. However, fluence-rate response relationships for anthocyanin synthesis and hypocotyl growth induced by a 24-h irradiation with 451, 539, 649, 693, 704 and 729 nm light showed no or a severely reduced response in the au and au, hp mutants, suggesting the importance of PI in these responses. We therefore propose that the capacity for anthocyanin synthesis (state of responsiveness amplification) could be established by PI, while the anthocyanin synthesis is actually photoregulated via a stable Pfr pool. The Hp gene product is proposed to be an inhibitor of the state of responsiveness amplification for responses controlled by this relatively stable Pfr species.     

Phytochrome,the Visual Pigment of Plants

Plants require light for photosynthesis. In order to adapt to the light conditions in their particular habitat, they have developed various photoreceptor systems. Of these, phytochrome allows even two-color vision in the red/far-red region. The photoreceptor phytochrome is of interest not only to botanists, but also to natural product chemists, photochemists, biochemists, photobiologists, and recently molecular biologists. Despite numerous studies, there are still considerable gaps in our knowledge of this photoreceptor. This article first describes the basic structural studies of the tetrapyrrole chromophore and its photochemical cis–trans isomerization, which is the source of the chromoprotein's photochromism. In the section on the protein moiety, beside other topics, the domain structure of phytochrome and the conformational changes during phototransformation are discussed. Finally, the known phytochrome genes are used to derive phylogenetic relationships, and possible structure–function relationships are discussed.     

PHOTOREGULATION OF ROOT: SHOOT RATIO IN SOYBEAN SEEDLINGS*

Abstract— The partitioning of plant growth between shoot and root has the potential to affect diverse physiological processes including water and nutrient uptake, nitrogen fixation, light interception, and interactions between plant and soil microorganisms. Root: shoot ratio is determined both by genetics and developmental status as well as by availability of water, nutrients and light. It is shown here that relative root growth was modulated by photomorphogenetic treatments designed to affect phytochrome (supplemental far-red radiation given either as an end-of-day treatment or continuously during the photoperiod) or blue light photoreceptors (blue light-deficient low pressure sodium lamps ± low irradiances of supplemental blue [i.e. 5% of total photon flux: 25 μ.mol m^?2 s^?1]). Photomorphogenetic control of root: shoot ratio was apparent within1–2 days when light treatments were initiated at emergence, and did not necessarily involve changes in net seedling growth. On the other hand, shortened daylength inhibited early seedling growth but had little effect on partitioning. Changes in relative root dry matter induced by supplemental far red radiation or blue light deficiency were similar to those caused by low irradiances, suggesting that phytochrome or blue light photoreceptors may be involved in regulating the partitioning of growth between shoot and root as a part of adaptation to vegetation shade. The influence of spectral quality on root: shoot ratio should be considered when comparing plants grown under different types of lighting or with different spacing.     

All‐Trans Retinal Mediates Light‐Induced Oxidation in Single Living Rod Photoreceptors†

All‐trans retinal is a potent photosensitizer that is released in photoreceptor outer segments by the photoactivated visual pigment following the detection of light. Photoreceptor outer segments also contain high concentrations of polyunsaturated fatty acids, and are thus particularly susceptible to oxidative damage such as that initiated by light via a photosensitizer. Upon its release, all‐trans retinal is reduced within the outer segment to all‐trans retinol, through a reaction requiring metabolic input in the form of NADPH. The phototoxic potential of physiologically generated all‐trans retinal was examined in single living rod photoreceptors obtained from frog (Rana pipiens) retinas. Light‐induced oxidation was measured with fluorescence imaging using an oxidation‐sensitive indicator dye from the shift in fluorescence between the intact and oxidized forms. Light‐induced oxidation was highest in metabolically compromised rod outer segments following photoactivation of the visual pigment rhodopsin, and after a time interval, sufficiently long to ensure the release of all‐trans retinal. Furthermore, light‐induced oxidation increased with the concentration of exogenously added all‐trans retinal. The results show that the all‐trans retinal generated during the detection of light can mediate light‐induced oxidation. Its removal through reduction to all‐trans retinol protects photoreceptor outer segments against light‐induced oxidative damage.     

The Evolution and Functional Role of Flavin‐based Prokaryotic Photoreceptors

Flavin‐based photoreceptor proteins of the LOV (light, oxygen and voltage) superfamily are ubiquitous and appear to be essential blue‐light sensing systems not only in plants, algae and fungi, but also in prokaryotes, where they are represented in more than 10% of known species. Despite their broad occurrence, only in few cases LOV proteins have been correlated with important phenomena such as bacterial infectivity, selective growth patterns or/and stress responses; nevertheless these few known roles are helping us understand the multiple ways by which prokaryotes can exploit these soluble blue‐light photoreceptors. Given the large number of sequences now deposited in databases, it becomes meaningful to define a signature for bona fide LOV domains, a procedure that facilitates identification of proteins with new properties and phylogenetic analysis. The latter clearly evidences that a class of LOV proteins from alpha‐proteobacteria is the closest prokaryotic relative of eukaryotic LOV domains, whereas cyanobacterial sequences cluster with the archaeal and the other bacterial LOV domains. Distance trees built for LOV domains suggest complex evolutionary patterns, possibly involving multiple horizontal gene transfer events. Based on available data, the in vivo relevance and evolution of prokaryotic LOV is discussed.     

Time‐Resolved Energetics of Photoprocesses in Prokaryotic Phytochrome‐Related Photoreceptors

Time‐resolved photoacoustics (PA) is uniquely able to explore the energy landscape of photoactive proteins and concomitantly detects light‐induced volumetric changes (ΔV) accompanying the formation and decay of transient species in a time window between ca. 20 ns and 5 μs. Here, we report PA measurements on diverse photochromic bilin‐binding photoreceptors of prokaryotic origin: (1) the chromophore‐binding GAF3 domain of the red (R)/green (G) switching cyanobacteriochrome 1393 (Slr1393g3) from Synechocystis; (2) the red/far red (R/FR) Synechocystis Cph1 phytochrome; (3) full‐length and truncated constructs of Xanthomonas campestris bacteriophytochrome (XccBphP), absorbing up to the NIR spectral region. In almost all cases, photoisomerization results in a large fraction of energy dissipated as heat (up to 90%) on the sub‐ns scale, reflecting the low photoisomerization quantum yield (<0.2). This “prompt” step is accompanied by a positive ΔV₁ = 5–12.5 mL mol^?1. Formation of the first intermediate is the sole process accessible to PA, with the notable exception of Slr1393g3‐G for which ΔV₁ = +4.5 mL mol^?1 is followed by a time‐resolved, energy‐conserving contraction ΔV₂ = ?11.4 mL mol^?1, τ₂ = 180 ns at 2.4°C. This peculiarity is possibly due to a larger solvent occupancy of the chromophore cavity for Slr1393g3‐G.     

Influence of Heterogeneity on the Ultrafast Photoisomerization Dynamics of Pfr in Cph1 Phytochrome

Photoisomerization of a protein‐bound chromophore is the basis of light sensing and signaling in many photoreceptors. Phytochrome photoreceptors can be photoconverted reversibly between the Pr and Pfr states through photoisomerization of the methine bridge between rings C and D. Ground‐state heterogeneity of the chromophore has been reported for both Pr and Pfr. Here, we report ultrafast visible (Vis) pump–probe and femtosecond polarization‐resolved Vis pump–infrared (IR) probe studies of the Pfr photoreaction in native and ¹³C/¹⁵N‐labeled Cph1 phytochrome with unlabeled PCB chromophore, demonstrating different S₀ substates, Pfr‐I and Pfr‐II, with distinct IR absorptions, orientations and dynamics of the carbonyl vibration of ring D. We derived time constants of 0.24 ps, 0.7 ps and 6 ps, describing the complete initial photoreaction. We identified an isomerizing pathway with 0.7 ps for Pfr‐I, and silent dynamics with 6 ps for Pfr‐II. We discuss different origins of the Pfr substates, and favor different facial orientations of ring D. The model provides a quantum yield for Pfr‐I of 38%, in line with ~35% ring D rotation in the electronic excited state. We tentatively assign the silent form Pfr‐II to a dark‐adapted state that can convert to Pfr‐I upon light absorption.