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.

https://science.sciencemag.org/content/sci/282/5394/1698.full.pdf

Arabidopsis NPH1: A Flavoprotein with the Properties of a Photoreceptor for Phototropism

The phototropic response is an important component of seedling establishment in higher plants because it orients the young seedlings for maximal photosynthetic light capture. Despite their obvious importance, little is known about the mechanisms underlying the perception and transduction of the light signals that induce phototropic curvatures. Here, we report the isolation of eight mutants of Arabidopsis that lack or have severely impaired phototropic responses. These nph (for nonphototropic hypocotyl) mutants comprise four genetic loci: nph1, nph2, nph3, and nph4. Physiological and biochemical characterization of the nph1 allele series indicated that the NPH1 locus may encode the apoprotein for a dual-chromophoric or multichromophoric holoprotein photoreceptor capable of absorbing UV-A, blue, and green light and that this photoreceptor regulates all the phototropic responses of Arabidopsis. It appears that the NPH1 protein is most likely a 120-kD plasma membrane-associated phosphoprotein because all of the nph1 mutations negatively affected the abundance of this protein. In addition, the putative NPH1 photoreceptor protein is genetically and biochemically distinct from the HY4 protein, which most likely acts as a photoreceptor for blue light-mediated hypocotyl growth inhibition. Furthermore, the NPH1 and HY4 proteins are not functionally redundant because mutations in either gene alone affect only one physiological response but not the other, thus providing strong support for the hypothesis that more than one blue light photoreceptor is required for the normal growth and development of a seedling.

Mutations in the NPH1 Locus of Arabidopsis Disrupt the Perception of Phototropic Stimuli

Phototropins (phot1 and phot2) are autophosphorylating serine/threonine kinases that function as photoreceptors for phototropism, light-induced chloroplast movement, and stomatal opening in Arabidopsis. The N-terminal region of phot1 and phot2 contains two specialized PAS domains, designated LOV1 and LOV2, which function as binding sites for the chromophore flavin mononucleotide (FMN). Both LOV1 and LOV2 undergo a self-contained photocycle, which involves the formation of a covalent adduct between the FMN chromophore and a conserved active-site cysteine residue (Cys39). Replacement of Cys39 with alanine abolishes the light-induced photochemical reaction of LOV1 and LOV2. Here we have used the Cys39Ala mutation to investigate the role of LOV1 and LOV2 in regulating phototropin function. Photochemical analysis of a bacterially expressed LOV1 + LOV2 fusion protein indicates that LOV2 functions as the predominant light-sensing domain for phot1. LOV2 also plays a major role in mediating light-dependent autophosphorylation of full-length phot1 expressed in insect cells and transgenic Arabidopsis. Moreover, photochemically active LOV2 alone in full-length phot1 is sufficient to elicit hypocotyl phototropism in transgenic Arabidopsis, whereas photochemically active LOV1 alone is not. Further photochemical and biochemical analyses also indicate that the LOV1 and LOV2 domains of phot2 exhibit distinct roles. The significance for the different roles of the phototropin LOV domains is discussed.

https://www.onlinelibrary.wiley.com/doi/pdf/10.1046/j.1365-313X.2002.01415.x

Phototropin LOV domains exhibit distinct roles in regulating photoreceptor function

UV and blue light control the expression of flavonoid biosynthesis genes in a range of higher plants. To investigate the signal transduction processes involved in the induction of chalcone synthase (CHS) gene expression by UV-B and UV-A/blue light, we examined the effects of specific agonists and inhibitors of known signaling components in mammalian systems in a photomixotrophic Arabidopsis cell suspension culture. CHS expression is induced specifically by these wavelengths in the cell culture, in a manner similar to that in mature Arabidopsis leaf tissue. Both the UV-B and UV-A/blue phototransduction processes involve calcium, although the elevation of cytosolic calcium is insufficient on its own to stimulate CHS expression. The UV-A/blue light induction of CHS expression does not appear to involve calmodulin, whereas the UV-B response does; this difference indicates that the signal transduction pathways are, at least in part, distinct. We provide evidence that both pathways involve reversible protein phosphorylation and require protein synthesis. The UV-B and UV-A/blue light signaling pathways are therefore different from the phytochrome signal transduction pathway regulating CHS expression in other species.

Distinct UV-B and UV-A/blue light signal transduction pathways induce chalcone synthase gene expression in Arabidopsis cells.

CONTENTS INTRODUCTION ....... . , 144 LIGHT-INDUCED PHOSPHORYLATION OF A PLASMA MEMBRANE PROTEIN .... . . . 145 Preliminary Characterization and Membrane Localization 145 Correlation of Light-induced Phosphorylation with Phototropism 145 Biochemical Properties of the Reaction . . . ... ...... . . . . . . ... .... . . . . . . . ... ... . . .... . . . . . . . .. 146 LIGHT-INDUCED REDUCTION OF A b-TYPE CYTOCHROME-THE LIGHTINDUCED ABSORBANCE CHANGE (LIAC) ... . . 149 GTP-BINDING PROTEINS . . . . . . . . . 1 5 1 G-Proteins Associated with the Plasma Membrane o f Pea Apical Buds 151 PHOTO PERCEPTION : GENERAL CONSIDERATIONS ... . 151 Physiological Clues to the Nature of B Photo receptors lSI B REGULATION OF GENE EXPRESSION ......... .... . . . . 154 Measurements of Transcription and Transcript Abundance 154 Cisand TranS-Acting Elements in B-Mediated Gene Expression 158 Genetic Mutants In H Signal Transduction ....... 159

The Transduction of Blue Light Signals in Higher Plants

— We have continued to characterize the blue light-regulated phosphorylation of a 120 kDa pea plasma membrane protein thought to be involved in sensory transduction for phototropism (Short and Briggs, 1990, Plant Physiol.92, 179–185). By incubating pea stem sections in 32P-phosphate, we show that the 120 kDa protein is phosphorylated in vivo only after blue light irradiation and that the photosensitive fluence range matches that for phototropism. Blue light induces phosphorylation of the protein in vitro as well, but the fluences required to elicit the response are at least 30-fold higher. Triton solubilization of the plasma membrane vesicles does not further alter the fluence-response relationship. Very little turnover was detected over 20 min phosphorylation time courses or by pulse-chase experiments on unirradiated, blue light pulse-irradiated, or continuously irradiated membranes. Experiments with a dark period intervening between irradiation and addition of adenosine triphosphate show the light-induced change to persist for several minutes at 30°c. Agents that disrupt the normal photochemistry of flavins preferentially inhibit the light-induced enhancement of phosphorylation, suggesting a flavin chromophore. However, exogenous free flavins do not affect the sensitivity of the response. Staphylococcus aureus V-8 proteolysis of the phosphorylated protein from membranes subjected to a range of fluences before phosphorylation shows that the radiolabel on each of three peptides increases in proportion to the phosphorylation level of the undigested polypeptide. These studies may be valuable for assessing the nature of the photoreceptor and for unravelling the early sensory transduction steps in phototropism.

Winslow R. Brigg

Papers

A PHOTORECEPTOR SYSTEM REGULATING in vivo AND in vitro PHOSPHORYLATION OF A PEA PLASMA MEMBRANE PROTEIN