Photomorphogenesis of the root system in developing sunflower seedlings: a role for sucrose.

The domestic sunflower (Helianthus annuus L. cv. 'Giganteus') has been used since the 19th century as a model plant for the study of seedling development in darkness and white light (WL) (scoto- versus photomorphogenesis). However, most pertinent studies have focused on the developmental patterns of the hypocotyl and cotyledons, whereas the root system has been largely ignored. In this study, we analysed entire sunflower seedlings (root and shoot) and quantified organ development in the above- and belowground parts of the organism under natural (non-sterile) conditions. We document that seedlings, raised in moist vermiculite, are covered with methylobacteria, microbes that are known to promote root development in Arabidopsis. Quantitative data revealed that during photomorphogenesis in WL, the root system expands by 90%, whereas stem elongation is inhibited, and hook opening/cotyledon expansion occurs. Root morphogenesis may be mediated via imported sucrose provided by the green, photosynthetically active cotyledons. This hypothesis is supported by the documented effect of sucrose on the induction of lateral root initials in sunflower cuttings. Under these experimental conditions, phytohormones (auxin, cytokinin, brassinolide) exerted little effect on root and cotyledon expansion, and no hormone-induced initiation of lateral roots was observed. It is concluded that sucrose not only acts as an energy source to fuel cell metabolism but is also a shoot-derived signalling molecule that triggers root morphogenesis.

The blue light receptor Phototropin 1 suppresses lateral root growth by controlling cell elongation.

We investigated the relationship between the blue light receptor phototropin 1 (phot1) and lateral root growth in Arabidopsis thaliana seedlings. Fluorescence and confocal microscopy images, as well as PHOT1 mRNA expression studies provide evidence that it is highly expressed in the elongation zone of lateral roots where auxin is accumulating. However, treatment with the auxin transport inhibitor N-1-naphthylphthalamic acid significantly reduced PHOT1 expression in this zone. In addition, PHOT1 expression was higher in darkness than in light. The total number of lateral roots was higher in the phot1 mutant than in wild-type Arabidopsis. Cells in the elongation zone of lateral roots of the phot1 mutant were longer than those of wild-type lateral roots. These findings suggest that PHOT1 plays a role(s) in elongation of lateral roots through the control of an auxin-related signalling pathway.

Seedling development in buckwheat and the discovery of the photomorphogenic shade-avoidance response.

Numerous botanists of the early 19th century investigated the effect of sunlight on plant development, but no clear picture developed. One hundred and fifty years ago, Julius Sachs (1863) systematically analysed the light-plant relationships, using developing garden nasturtium (Tropaeolum majus) and seedlings of buckwheat (Fagopyron esculentum) as experimental material. From these studies, Sachs elucidated the phenomenon of photomorphogenesis (plant development under the influence of daylight) and the associated 'shade-avoidance response'. We have reproduced the classical buckwheat experiments of Sachs (1863) and document the original shade-avoidance syndrome with reference to hypocotyl elongation and cotyledon development in darkness (skotomorphogenesis), white light and shade induced by a canopy of green leaves. In subsequent publications, Sachs elaborated his concepts of 1863 and postulated the occurrence of 'flower-inducing substances'. In addition, he argued that the shade-avoidance response in cereals, such as wheat and maize, is responsible for lodging in crowded plant communities. We discuss these processes with respect to the red- to far-red light/phytochrome B relationships. Finally, we summarise the phytochrome B-phytohormone (auxin, brassinosteroids) connection within the cells of shaded Arabidopsis plants, and present a simple model to illustrate the shade-avoidance syndrome. In addition, we address the relationship between plant density and health of the corresponding population, a topic that was raised for the first time by Sachs (1863) in his seminal paper and elaborated in his textbooks.

From Charles Darwin's botanical country-house studies to modern plant biology.

As a student of theology at Cambridge University, Charles Darwin (1809-1882) attended the lectures of the botanist John S. Henslow (1796-1861). This instruction provided the basis for his life-long interest in plants as well as the species question. This was a major reason why in his book On the Origin of Species, which was published 150 years ago, Darwin explained his metaphorical phrase 'struggle for life' with respect to animals and plants. In this article, we review Darwin's botanical work with reference to the following topics: the struggle for existence in the vegetable kingdom with respect to the phytochrome-mediated shade avoidance response; the biology of flowers and Darwin's plant-insect co-evolution hypothesis; climbing plants and the discovery of action potentials; the power of movement in plants and Darwin's conflict with the German plant physiologist Julius Sachs; and light perception by growing grass coleoptiles with reference to the phototropins. Finally, we describe the establishment of the scientific discipline of Plant Biology that took place in the USA 80 years ago, and define this area of research with respect to Darwin's work on botany and the physiology of higher plants.

A carnation anthocyanin mutant is complemented by the glutathione S-transferases encoded by maize Bz2 and petunia An9.

Particle bombardment was used to elucidate the function of Flavonoid3, a late-acting anthocyanin gene of the ornamental plant, carnation ( Dianthus caryophyllus L.). The fl3 mutation conditions dilute anthocyanin coloration that closely resembles phenotypes produced by the anthocyanin mutants bz2 of maize and an9 of petunia. Bz2 and An9 encode glutathione S-transferases (GSTs) involved in vacuolar sequestration of anthocyanins. Constructs containing either of these or another late-function maize gene, Bronze1 (UDPglucose:flavonol 3- O-glucosyltransferase), were introduced via microprojectile bombardment into fl3 petals. Complementation resulted only from Bz2 and An9, indicating that Fl3 encodes a GST involved in the transport of anthocyanins to the vacuole. The observed result in carnation, an angiosperm phylogenetically distant from maize and petunia, indicates that GST activity might be a universal step in the anthocyanin pathway. Microprojectile bombardment was used to identify late-pathway anthocyanin mutations, which may be responsible for the pale anthocyanin coloration of important cultivars in many species but which can be difficult to characterize by other means.

The photocycle of a flavin-binding domain of the blue light photoreceptor phototropin.

The plant blue light receptor, phot1, a member of the phototropin family, is a plasma membrane-associated flavoprotein that contains two ( approximately 110 amino acids) flavin-binding domains, LOV1 and LOV2, within its N terminus and a typical serine-threonine protein kinase domain at its C terminus. The LOV (light, oxygen, and voltage) domains belong to the PAS domain superfamily of sensor proteins. In response to blue light, phototropins undergo autophosphorylation. E. coli-expressed LOV domains bind riboflavin-5'-monophosphate, are photochemically active, and have major absorption peaks at 360 and 450 nm, with the 450 nm peak having vibronic structure at 425 and 475 nm. These spectral features correspond to the action spectrum for phototropism in higher plants. Blue light excitation of the LOV2 domain generates, in less than 30 ns, a transient approximately 660 nm-absorbing species that spectroscopically resembles a flavin triplet state. This putative triplet state subsequently decays with a 4-micros time constant into a 390 nm-absorbing metastable form. The LOV2 domain (450 nm) recovers spontaneously with half-times of approximately 50 s. It has been shown that the metastable species is likely a flavin-cysteine (Cys(39) thiol) adduct at the flavin C(4a) position. A LOV2C39A mutant generates the early photoproduct but not the adduct. Titrations of LOV2 using chromophore fluorescence as an indicator suggest that Cys(39) exists as a thiolate.

Arabidopsis nph1 and npl1: blue light receptors that mediate both phototropism and chloroplast relocation.

UV-A/blue light acts to regulate a number of physiological processes in higher plants. These include light-driven chloroplast movement and phototropism. The NPH1 gene of Arabidopsis encodes an autophosphorylating protein kinase that functions as a photoreceptor for phototropism in response to low-intensity blue light. However, nph1 mutants have been reported to exhibit normal phototropic curvature under high-intensity blue light, indicating the presence of an additional phototropic receptor. A likely candidate is the nph1 homologue, npl1, which has recently been shown to mediate the avoidance response of chloroplasts to high-intensity blue light in Arabidopsis. Here we demonstrate that npl1, like nph1, noncovalently binds the chromophore flavin mononucleotide (FMN) within two specialized PAS domains, termed LOV domains. Furthermore, when expressed in insect cells, npl1, like nph1, undergoes light-dependent autophosphorylation, indicating that npl1 also functions as a light receptor kinase. Consistent with this conclusion, we show that a nph1 npl1 double mutant exhibits an impaired phototropic response under both low- and high-intensity blue light. Hence, npl1 functions as a second phototropic receptor under high fluence rate conditions and is, in part, functionally redundant to nph1. We also demonstrate that both chloroplast accumulation in response to low-intensity light and chloroplast avoidance movement in response to high-intensity light are lacking in the nph1 npl1 double mutant. Our findings therefore indicate that nph1 and npl1 show partially overlapping functions in two different responses, phototropism and chloroplast relocation, in a fluence rate-dependent manner.

Phototropins: a new family of flavin-binding blue light receptors in plants.

Phototropin is the designation originally assigned to a recently characterized chromoprotein that serves as a photoreceptor for phototropism. Phototropin is a light-activated autophosphorylating serine/threonine kinase that binds two flavin mononucleotide (FMN) molecules that function as blue light-absorbing chromophores. Each FMN molecule is bound in a rigid binding pocket within specialized PAS (PER-ARNT-SIM superfamily) domains, known as LOV (light, oxygen, or voltage) domains. This article reviews the detailed photobiological and biochemical characterization of the light-activated phosphorylation reaction of phototropin and follows the sequence of events leading to the cloning, sequencing, and characterization of the gene and the subsequent biochemical characterization of its encoded protein. It then considers recent biochemical and photochemical evidence that light activation of phototropin involves the formation of a cysteinyl adduct at the C(4a) position of the FMN chromophores. Adduct formation causes a major conformational change in the chromophores and a possible conformational change in the protein moiety as well. The review concludes with a brief discussion of the evidence for a second phototropin-like protein in Arabidopsis and rice. Possible roles for this photoreceptor are discussed.

Photochemical and mutational analysis of the FMN-binding domains of the plant blue light receptor, phototropin.

The plant photoreceptor phototropin is an autophosphorylating serine-threonine protein kinase activated by UV-A/blue light. Two domains, LOV1 and LOV2, members of the PAS domain superfamily, mediate light sensing by phototropin. Heterologous expression studies have shown that both domains function as FMN-binding sites. Although three plant blue light photoreceptors, cry1, cry2, and phototropin, have been identified to date, the photochemical reactions underlying photoactivation of these light sensors have not been described so far. Herein, we demonstrate that the LOV domains of Avena sativa phototropin undergo a self-contained photocycle characterized by a loss of blue light absorbance in response to light and a spontaneous recovery of the blue light-absorbing form in the dark. Rate constants and quantum efficiencies for the photoreactions indicate that LOV1 exhibits a lower photosensitivity than LOV2. The spectral properties of the photoproduct produced for both LOV domains are unrelated to those found for photoreduced flavins and flavoproteins, but are consistent with those of a flavin-cysteinyl adduct. Flavin-thiol adducts are generally short-lifetime reaction intermediates formed during the flavoprotein-catalyzed reduction of protein disulfides. By site-directed mutagenesis, we have identified several amino acid residues within the putative chromophore binding site of LOV1 and LOV2 that appear to be important for FMN binding and/or the photochemical reactivity. Among those is Cys39, which plays an important role in the photochemical reaction of the LOV domains. Replacement of Cys39 with Ala abolished the photochemical reactions of both LOV domains. We therefore propose that light sensing by the phototropin LOV domains occurs via the formation of a stable adduct between the FMN chromophore and Cys39.

Safety and immunogenicity of a three-component blood-stage malaria vaccine in adults living in an endemic area of Papua New Guinea.

A Phase I safety and immunogenicity study with a three-component blood-stage malaria vaccine was conducted in adult male subjects living in an endemic area of Papua New Guinea. The preparations were recombinant proteins which corresponded to parts of the two merozoite surface proteins of Plasmodium falciparum (MSP1 and 2), and of the ring-infected erythrocyte surface antigen (RESA). The three proteins were emulsified with the adjuvant Montanide ISA720. Ten subjects were injected twice (four weeks apart) with the vaccine formulation and two with the adjuvant alone. Mild pain at the site of injection was reported by about half of the subjects but no systemic reaction related to the formulation occurred. There was a sharp rise in geometric mean stimulation index after the second dose compared to baseline for MSP1 and RESA, while the rise was small for MSP2. Geometric mean antibody titres increased for MSP1 during the study, whereas they hardly changed for MSP2 and RESA. The vaccine formulation was safe when used in an already immune population. The vaccine induced good cellular responses, especially for MSP1 and RESA. Boosting of humoral responses was weak, probably because of high baseline antibody levels.

Blue-light photoreceptors in higher plants.

In the past few years great progress has been made in identifying and characterizing plant photoreceptors active in the blue/UV-A regions of the spectrum. These photoreceptors include cryptochrome 1 and cryptochrome 2, which are similar in structure and chromophore composition to the prokaryotic DNA photolyases. However, they have a C-terminal extension that is not present in photolyases and lack photolyase activity. They are involved in regulation of cell elongation and in many other processes, including interfacing with circadian rhythms and activating gene transcription. Animal cryptochromes that play a photoreceptor role in circadian rhythms have also been characterized. Phototropin, the protein product of the NPH1 gene in Arabidopsis, likely serves as the photoreceptor for phototropism and appears to have no other role. A plasma membrane protein, it serves as photoreceptor, kinase, and substrate for light-activated phosphorylation. The carotenoid zeaxanthin may serve as the chromophore for a photoreceptor involved in blue-light-activated stomatal opening. The properties of these photoreceptors and some of the downstream events they are known to activate are discussed.

Human phase I vaccine trials of 3 recombinant asexual stage malaria antigens with Montanide ISA720 adjuvant.

Two phase I vaccine trials were conducted to test the immunogenicity and safety of a vaccine containing three recombinant malaria antigens from the asexual stage of Plasmodium falciparum. The three antigens are a fragment of MSP1 (190LCS.T3); MSP2 and a portion of RESA and were formulated in Montanide ISA720 adjuvant. These trials investigated the dose response of each antigen for eliciting both antibody and T-cell responses and the immunogenicity of a mixture of the antigens compared with the antigens injected separately. All three antigens elicited both antibody and T-cell responses. Strong T-cell responses were observed with 190LCS.T3 and RESA with stimulation indices exceeding 100 for peripheral blood leucocytes in some individuals. The antibody responses were generally weak. The human antibody responses observed with MSP2 in Montanide ISA720 were not significantly different from those obtained in an earlier trial which used MSP2 with alum as the adjuvant. No antigenic competition was observed: volunteers receiving a mixture of antigens had similar responses to those receiving the three antigens at separate sites. Tenderness and pain at the injection site were common over the first few days following immunization. In some volunteers, especially those receiving the highest doses tested, there was a delayed reaction at the injection site with pain and swelling occurring approximately 10 days after injection.

LOV (light, oxygen, or voltage) domains of the blue-light photoreceptor phototropin (nph1): binding sites for the chromophore flavin mononucleotide.

Phototropism, the bending response of plant organs to or away from a directional light source, is one of the best studied blue light responses in plants. Although phototropism has been studied for more than a century, recent advances have improved our understanding of the underlying signaling mechanisms involved. The NPH1 gene of Arabidopsis thaliana encodes a blue light-dependent autophosphorylating protein kinase with the properties of a photoreceptor for phototropism. NPH1 apoprotein noncovalently binds FMN to form the holoprotein nph1. The N-terminal region of the protein contains two LOV (light, oxygen, or voltage) domains that share homology with sensor proteins from a diverse group of organisms. These include the bacterial proteins NIFL and AER, both of which bind FAD, and the phy3 photoreceptor from Adiantium capillus-veneris. The LOV domain has therefore been proposed to reflect a flavin-binding site, regulating nph1 kinase activity in response to blue light-induced redox changes. Herein we demonstrate that the LOV domains of two nph1 proteins and phy3 bind stoichiometric amounts of FMN when expressed in Escherichia coli. The spectral properties of the chromopeptides are similar to the action spectrum for phototropism, implying that the LOV domain binds FMN to function as a light sensor. Thus, our findings support the earlier model that nph1 is a dual-chromophoric flavoprotein photoreceptor regulating phototropic responses in higher plants. We therefore propose the name phototropin to designate the nph1 holoprotein.

Arabidopsis contains at least four independent blue-light-activated signal transduction pathways.

We have investigated the stomatal and phototropic responses to blue light of a number of single and double mutants at various loci that encode proteins involved in blue-light responses in Arabidopsis. The stomatal responses of light-grown mutant plants (cry1, cry2, nph1, nph3, nph4, cry1cry2, and nph1cry1) did not differ significantly from those of their wild-type counterparts. Second positive phototropic responses of etiolated mutant seedlings, cry1, cry2, cry1cry2, and npq1-2, were also similar to those of their wild-type counterparts. Although npq1 and single and double cry1cry2 mutants showed somewhat reduced amplitude for first positive phototropism, threshold, peak, and saturation fluence values for first positive phototropic responses of etiolated seedlings did not differ from those of wild-type seedlings. Similar to the cry1cry2 double mutants and to npq1-2, a phyAphyB mutant showed reduced curvature but no change in the position or shape of the fluence-response curve. By contrast, the phototropism mutant nph1-5 failed to show phototropic curvature under any of the irradiation conditions used in the present study. We conclude that the chromoproteins cry1, cry2, nph1, and the blue-light photoreceptor for the stomatal response are genetically separable. Moreover, these photoreceptors appear to activate separate signal transduction pathways.

Shade-avoidance responses in two common coastal redwood forest species, Sequoia sempervirens (Taxodiaceae) and Satureja douglasii (Lamiaceae), occurring in various light quality environments.

Shade-avoidance responses were examined for two species common to the coastal redwood forest, Sequoia sempervirens and Satureja douglasii. Sequoia seedlings demonstrated a shade-avoidance response when given end-of-day far-red light by increased hypocotyl, epicotyl, and first-node extension, and greater total number of needles and reduced anthocyanin concentration. Thus, Sequoia seedlings respond as sun-adapted plants. Satureja has several leaf monoterpene chemotypes that occur in different light environments including the redwood forest, and the types responded differently to the light treatments. The pulegone type responded to end-of-day far-red light as a sun-adapted plant with significant extension growth, increased leaf area and chlorophyll, and reduced anthocyanin. The isomenthone type responded as a shade-tolerant plant and did not exhibit extension growth nor a change in other parameters with end-of-day far-red light. However, the carvone and bicyclic types had variable responses depending on the parameter studied, which indicated genetic variation for these traits.

Arabidopsis NPH1: a flavoprotein with the properties of a photoreceptor for phototropism.

The NPH1 gene of Arabidopsis thaliana encodes a 120-kilodalton serine-threonine protein kinase hypothesized to function as a photoreceptor for phototropism. When expressed in insect cells, the NPH1 protein is phosphorylated in response to blue light irradiation. The biochemical and photochemical properties of the photosensitive protein reflect those of the native protein in microsomal membranes. Recombinant NPH1 noncovalently binds flavin mononucleotide, a likely chromophore for light-dependent autophosphorylation. The fluorescence excitation spectrum of the recombinant protein is similar to the action spectrum for phototropism, consistent with the conclusion that NPH1 is an autophosphorylating flavoprotein photoreceptor mediating phototropic responses in higher plants.