Different Red Light Requirements for Phytochrome-Induced Accumulation of cab RNA and rbcS RNA.

For several species of plants the abundance of those transcripts encoding the chlorophyll a/b binding protein (cab RNA) and the small subunit of ribulose-1,5-biphosphate carboxylase-oxygenase (rbcS RNA) has been established as being under the control of phytochrome. However, this conclusion does not take into account the various types of phytochrome control based on both the fluence of red light necessary to induce the response and the ability of far red light either to induce or to reverse the response. The fluence of red light necessary to induce the accumulation of rbcS RNA was found to be 10,000 times greater than that necessary to induce the accumulation of cab RNA. Furthermore, far red light alone was capable of inducing the accumulation of cab RNA. It is possible, therefore, that developing pea buds accumulate cab RNA before rbcS and that cab RNA is not subject to the normal end-of-day signals affecting many phytochrome responses.

Arabidopsis NPH1: a protein kinase with a putative redox-sensing domain.

The NPH1 (nonphototropic hypocotyl 1) gene encodes an essential component acting very early in the signal-transduction chain for phototropism. Arabidopsis NPH1 contains a serine-threonine kinase domain and LOV1 and LOV2 repeats that share similarity (36 to 56 percent) with Halobacterium salinarium Bat, Azotobacter vinelandii NIFL, Neurospora crassa White Collar-1, Escherichia coli Aer, and the Eag family of potassium-channel proteins from Drosophila and mammals. Sequence similarity with a known (NIFL) and a suspected (Aer) flavoprotein suggests that NPH1 LOV1 and LOV2 may be flavin-binding domains that regulate kinase activity in response to blue light-induced redox changes.

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.

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.

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.

Labeling of membranes from erythrocytes and corn with fluorescamine.

Fluorescamine was used as a fluorescent label for intact human erythrocytes and slices of corn coleoptile tissue. This reagent has a greater affinity for membranous than for soluble proteins, and also labels membrane lipids which contain primary amine groups. In addition, some membrane fractions from labeled coleoptiles have a higher affinity for fluorescamine than do others. The relative labeling of the various fractions can be altered by changing the pH of the external labeling medium. Because the pH of the medium determines the rate of hydrolysis of fluorescamine to an unreactive form, this result suggests that the specificity of this reagent towards different cellular structures is determined by the lifetime of the active reagent. Fluorescamine was not found to be a specific reagent for the cell surface.

A Pea Plasma Membrane Protein Exhibiting Blue Light-Induced Phosphorylation Retains Photosensitivity following Triton Solubilization.

Phosphorylation of a polypeptide of approximately 120 kD in pea (Pisum sativum L.) plasma membranes in response to blue light has been shown to be involved in phototropic curvature, but the relationship of this protein to the kinase and photoreceptor acting upon it is uncertain. Using two-phase aqueous partitioning to isolate right-side-out plasma membrane vesicles, we have obtained evidence suggesting that the photoreceptor, kinase, and substrate are localized to the plasma membrane fraction. Latent phosphorylation accessible through Triton X-100 or freeze/thaw treatments of purified plasma membrane vesicles indicates that at least the kinase moiety is present on the internal face of the plasma membrane. Effects of solubilization of vesicles on fluence-response characteristics and on phosphorylation levels provide evidence that the receptor, kinase, and protein substrate are present together in individual mixed detergent micelles, either as a stable complex or as domains of a single polypeptide. In vivo blue-light irradiation results in a small but significant decrease in mobility of the 120-kD phosphorylated protein on sodium dodecylsulfate gel electrophoresis. This mobility shift is evident on Coomassie-stained gels and on western blots probed with polyclonal antibodies raised against the 120-kD protein. Among the plasma membrane proteins bound to the reactive nucleotide analog fluorosulfonylbenzoyladenine (FSBA), a distinct protein band at 120 kD can be detected on blots probed with anti-FSBA antibodies. This band exhibits an in vivo light-dependent mobility shift identical to that observed for the protein band and antibodies specific for the 120-kD protein, implying that the 120-kD protein has an integral nucleotide binding site and consistent with the possibility that the substrate protein is also a kinase.

Blue Light-Induced Phosphorylation of a Plasma Membrane-Associated Protein in Zea mays L.

Blue light induces a variety of photomorphogenic responses in higher plants, among them phototropic curvature, the bending of seedlings toward a unidirectional light source. In dark-grown coleoptiles of maize (Zea mays L.) seedlings, blue light induces rapid phosphorylation of a 114-kD protein at fluence levels that are sufficient to stimulate phototropic curvature. Phosphorylation in response to blue light can be detected in vivo in coleoptile tips preincubated in 32Pi or in vitro in isolated membranes supplemented with [[gamma]-32P]ATP. Phosphorylation reaches a maximum level in vitro within 2 min following an inductive light pulse, but substantial labeling occurs within the first 15 s. Isolated membranes remain activated for several minutes following an in vitro blue light stimulus, even in the absence of exogenous ATP. Phosphoamino acid analysis of the 114-kD protein detected phosphoserine and a trace of phosphothreonine. The kinase involved in phosphorylating the protein in vitro is not dependent on calcium. The 114-kD protein itself has an apparent binding site for ATP, detected by incubating with the nonhydrolyzable analog, 5[prime]-p-fluorosulfonyl-benzoyladenosine. This result suggests that the 114-kD protein, which becomes phosphorylated in response to blue light, may also be capable of kinase activity.

Correlation of Blue Light-Induced Phosphorylation to Phototropism in Zea mays L.

The physiology of light-induced phototropic curvature has been studied extensively in coleoptiles of grasses, particularly in Avena and Zea mays L. In Z. mays L., we have found that, in addition to curvature, blue light also induces rapid phosphorylation of a 114-kD protein in the tips of coleoptiles, and, in a previous report, we reported several characteristics of the phosphorylated substrate protein and kinase (J.M. Palmer, T.W. Short, S. Gallagher, W.R. Briggs [1993] Plant Physiol 102: 1211-1218). Here, we compare the phosphorylation response to several known aspects of phototropism physiology. Blue light-induced phosphorylation occurs only in the upper portion of the coleoptile and is absent from the node and mesocotyl. The specific activity of phosphorylation is highest in the extreme apical portion of the tip, which is also the site of maximal sensitivity to phototropic stimuli (A. W. Galston [1959] In Physiology of Movements, Encyclopedia of Plant Physiology, Springer, Berlin). Fluence-response determinations indicate that light dosage levels that stimulate curvature also stimulate phosphorylation. However, the threshold for inducing detectable phosphorylation in maize cannot be matched to the threshold for curvature induction. The recovery of sensitivity to phototropic stimuli after exposure to high fluences of light occurs with kinetics that are very similar to those for recovery of the phosphorylation response after a previous high-fluence light exposure. In addition, wavelengths of light in the blue and near-ultraviolet regions of the spectrum that maximally stimulate phototropic curvature also maximally stimulate in vitro phosphorylation in maize. The pattern of stimulation matches the absorption spectra of flavoproteins, which have been proposed as candidates for blue light photoreceptors.

Individual Members of the Cab Gene Family Differ Widely in Fluence Response.

Chlorophyll a/b-binding protein genes (Cab genes) can be extremely sensitive to light. Transcript accumulation following a red light pulse increases with fluence over 8 orders of magnitude (L.S. Kaufman, W.F. Thompson, W.R. Briggs [1984] Science 226: 1447-1449). We have constructed fluence-response curves for individual Cab genes. At least two Cab genes (Cab-8 and AB96) show a very low fluence response to a single red light pulse. In contrast, two other Cab genes (AB80 and AB66) fail to produce detectable transcript following a single pulse of either red or blue light but are expressed in continuous red light. Thus, very low fluence responses and high irradiance responses occur in the same gene family.

Blue Light Induces Phosphorylation at Seryl Residues on a Pea (Pisum sativum L.) Plasma Membrane Protein.

We have partially characterized the blue-light-stimulated in vitro phosphorylation of a membrane protein from etiolated Pisum sativum L. stems. Properties of the response have implicated its involvement in signal transduction of phototropic stimuli (T.W. Short, W.R. Briggs [1990] Plant Physiol 92: 179-185; P. Reymond, T.W. Short, W.R. Briggs [1992] Proc Natl Acad Sci USA 89: 4718- 4721). Analysis of proteolysis products and phosphoamino acidanalysis indicate that the substrate protein is phosphorylated on multiple seryl residues. Kinetics of the in vitro reaction show phosphorylation to be complete within 2 to 5 min at 30[deg]C in either light-exposed or dark-control plasma membrane preparations, regardless of whether the membranes were first solubilized in Triton X-100. Nucleotide competition assays show the kinase to be ATP specific. The pH optimum covers a broad range with a maximum near 7.5. A wide array of salts inhibits the phosphorylation at high concentrations, but millimolar concentrations of Mg2+ are required to form Mg.ATP complexes for maximal activity, whereas excess free Mg2+ or Ca2+ are not required for the reaction.

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.

Rhythmic chloroplast migration in the green alga Ulva: dissection of movement mechanism by differential inhibitor effects.

The chloroplasts of the green alga, Ulva lactuca L., migrate rhythmically between the outer (periclinal) cell walls in the daytime ("face" position) and the anticlinal cell walls at night ("profile" position). Both NaN3 and colchicine inhibit chloroplast movement mainly in the direction from profile towards face position. Differential drug sensitivity is suggestive of different mechanisms for the two directions of chloroplast migration. UV light reverses the inhibition by colchicine, presumably through the formation of lumicolchicine, the non-tubulin-binding isomer of colchicine. This result is indicative of microtubule involvement. Interpretation of the effects of azide is complicated by changes in the biological clock (phase delay and lengthening of the period). Cytochalasin B has no effect on chloroplast movement when added alone, but when added with colchicine it prevents colchicine inhibition of movement. We hypothesize that chloroplast position is controlled by a balance between two opposing movement systems with differential drug sensitivity.

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.

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.