Masamitsu Wada

phot1 and phot2 mediate blue light regulation of stomatal opening

The stomatal pores of higher plants allow for gaseous exchange into and out of leaves. Situated in the epidermis, they are surrounded by a pair of guard cells which control their opening in response to many environmental stimuli, including blue light. Opening of the pores is mediated by K+ accumulation in guard cells through a K+ channel and driven by an inside-negative electrical potential. Blue light causes phosphorylation and activation of the plasma membrane H+-ATPase that creates this potential. Thus far, no blue light receptor mediating stomatal opening has been identified, although the carotenoid, zeaxanthin, has been proposed. Arabidopsis mutants deficient in specific blue-light-mediated responses have identified four blue light receptors, cryptochrome 1 (cry1), cryptochrome 2 (cry2), phot1 and phot2. Here we show that in a double mutant of phot1 and phot2 stomata do not respond to blue light although single mutants are phenotypically normal. These results demonstrate that phot1 and phot2 act redundantly as blue light receptors mediating stomatal opening.

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 nph1npl1 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 nph1npl1 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.

Chloroplast avoidance movement reduces photodamage in plants

When plants are exposed to light levels higher than those required for photosynthesis, reactive oxygen species are generated in the chloroplasts and cause photodamage. This can occur even under natural growth conditions. To mitigate photodamage, plants have developed several protective mechanisms. One is chloroplast avoidance movement, in which chloroplasts move from the cell surface to the side walls of cells under high light conditions, although experimental support is still awaited. Here, using different classes of mutant defective in chloroplast avoidance movement, we show that these mutants are more susceptible to damage in high light than wild-type plants. Damage of the photosynthetic apparatus and subsequent bleaching of leaf colour and necrosis occur faster under high light conditions in the mutants than in wild-type plants. We conclude that chloroplast avoidance movement actually decreases the amount of light absorption by chloroplasts, and might therefore be important to the survival of plants under natural growth conditions.

The Phototropin Family of Photoreceptors

The past decade has seen dramatic advances in our knowledge of plant photoreceptors and in our understanding of the signal transduction pathways that they activate ([Briggs and Olney, 2001][1]). A major part of these advances has been the identification and characterization of photoreceptors that

The plant MITE mPing is mobilized in anther culture

Transposable elements constitute a large portion of eukaryotic genomes and contribute to their evolution and diversification. Miniature inverted-repeat transposable elements (MITEs) constitute one of the main groups of transposable elements and are distributed ubiquitously in the genomes of plants and animals such as maize, rice, Arabidopsis, human, insect and nematode. Because active MITEs have not been identified, the transposition mechanism of MITEs and their accumulation in eukaryotic genomes remain poorly understood. Here we describe a new class of MITE, called miniature Ping (mPing), in the genome of Oryza sativa (rice). mPing elements are activated in cells derived from anther culture, where they are excised efficiently from original sites and reinserted into new loci. An mPing-associated Ping element, which has a putative PIF family transposase, is implicated in the recent proliferation of this MITE family in a subspecies of rice.

Chloroplast unusual positioning1 is essential for proper chloroplast positioning.

The intracellular distribution of organelles is a crucial aspect of effective cell function. Chloroplasts change their intracellular positions to optimize photosynthetic activity in response to ambient light conditions. Through screening of mutants of Arabidopsis defective in chloroplast photorelocation movement, we isolated six mutant clones in which chloroplasts gathered at the bottom of the cells and did not distribute throughout cells. These mutants, termed chloroplast unusual positioning (chup), were shown to belong to a single genetic locus by complementation tests. Observation of the positioning of other organelles, such as mitochondria, peroxisomes, and nuclei, revealed that chloroplast positioning and movement are impaired specifically in this mutant, although peroxisomes are distributed along with chloroplasts. The CHUP1 gene encodes a novel protein containing multiple domains, including a coiled-coil domain, an actin binding domain, a Pro-rich region, and two Leu zipper domains. The N-terminal hydrophobic segment of CHUP1 was expressed transiently in leaf cells of Arabidopsis as a fusion protein with the green fluorescent protein. The fusion protein was targeted to envelope membranes of chloroplasts in mesophyll cells, suggesting that CHUP1 may localize in chloroplasts. A glutathione S-transferase fusion protein containing the actin binding domain of CHUP1 was found to bind F-actin in vitro. CHUP1 is a unique gene identified that encodes a protein required for organellar positioning and movement in plant cells.

Responses of ferns to red light are mediated by an unconventional photoreceptor

Efficient photosynthesis is essential for plant survival. To optimize photosynthesis, plants have developed several photoresponses. Stems bend towards a light source (phototropism), chloroplasts move to a place of appropriate light intensity (chloroplast photorelocation) and stomata open to absorb carbon dioxide. These responses are mediated by the blue-light receptors phototropin 1 (phot1) and phototropin 2 (phot2) in Arabidopsis (refs 1–5). In some ferns, phototropism and chloroplast photorelocation are controlled by red light as well as blue light. However, until now, the photoreceptor mediating these red-light responses has not been identified. The fern Adiantum capillus-veneris has an unconventional photoreceptor, phytochrome 3 (phy3), which is a chimaera of the red/far-red light receptor phytochrome and phototropin. We identify here a function of phy3 for red-light-induced phototropism and for red-light-induced chloroplast photorelocation, by using mutational analysis and complementation. Because phy3 greatly enhances the sensitivity to white light in orienting leaves and chloroplasts, and PHY3 homologues exist among various fern species, this chimaeric photoreceptor may have had a central role in the divergence and proliferation of fern species under low-light canopy conditions.

AUREOCHROME, a photoreceptor required for photomorphogenesis in stramenopiles

A blue light (BL) receptor was discovered in stramenopile algae Vaucheria frigida (Xanthophyceae) and Fucus distichus (Phaeophyceae). Two homologs were identified in Vaucheria; each has one basic region/leucine zipper (bZIP) domain and one light–oxygen–voltage (LOV)-sensing domain. We named these chromoproteins AUREOCHROMEs (AUREO1 and AUREO2). AUREO1 binds flavin mononucleotide via its LOV domain and forms a 390-nm-absorbing form, indicative of formation of a cysteinyl adduct to the C(4a) carbon of the flavin mononucleotide upon BL irradiation. The adduct decays to the ground state in ≈5 min. Its bZIP domain binds the target sequence TGACGT. The AUREO1 target binding was strongly enhanced by BL treatment, implying that AUREO1 functions as a BL-regulated transcription factor. The function of AUREO1 as photoreceptor for BL-induced branching is elucidated through RNAi experiments. RNAi of AUREO2 unexpectedly induces sex organ primordia instead of branches, implicating AUREO2 as a subswitch to initiate development of a branch, but not a sex organ. AUREO sequences are also found in the genome of the marine diatom Thalassiosira pseudonana (Bacillariophyceae), but are not present in green plants. AUREOCHROME therefore represents a BL receptor in photosynthetic stramenopiles.

Short actin-based mechanism for light-directed chloroplast movement in Arabidopsis

Organelle movement is essential for proper function of living cells. In plants, these movements generally depend on actin filaments, but the underlying mechanism is unknown. Here, in Arabidopsis, we identify associations of short actin filaments along the chloroplast periphery on the plasma membrane side associated with chloroplast photorelocation and anchoring to the plasma membrane. We have termed these chloroplast-actin filaments (cp-actin filaments). Cp-actin filaments emerge from the chloroplast edge and exhibit rapid turnover. The presence of cp-actin filaments depends on an actin-binding protein, chloroplast unusual positioning1 (CHUP1), localized on the chloroplast envelope. chup1 mutant lacked cp-actin filaments but showed normal cytoplasmic actin filaments. When irradiated with blue light to induce chloroplast movement, cp-actin filaments relocalize to the leading edge of chloroplasts before and during photorelocation and are regulated by 2 phototropins, phot1 and phot2. Our findings suggest that plants evolved a unique actin-based mechanism for organelle movement.

Blue light activates calcium-permeable channels in Arabidopsis mesophyll cells via the phototropin signaling pathway

Light is a central regulator of plant growth and development. Among the processes triggered by blue and UV-A light, phototropism, stomatal movement, and chloroplast orientation rely on the activation of blue-light receptors known as phototropins. So far, these photoreceptors constitute a class of light receptor kinases unique to the plant kingdom. In Arabidopsis thaliana, the two members phot1 and phot2 have been shown to display partially overlapping functions. Up to now little is known about the signaling cascade, which links these phototropins to the physiological responses downstream of blue-light perception. Here, we show that on illumination with blue light, but not red light, voltage-dependent and calcium-permeable channels activate in the plasma membrane of mesophyll cells. Blue-light stimulation in the presence of the photosynthetic electron transport inhibitor, 3-(3,4-dichlorophenyl)-1,1-dimethylurea, indicates that blue-light receptors rather than photosynthesis control channel activity. Sensitivity toward the protein kinase inhibitor K252a further pointed to the possible involvement of light receptor kinases. In support of this hypothesis, in the photoreceptor mutant phot1-5, blue-light induction of calcium currents was dramatically reduced and was eliminated in the double mutant phot1-5 phot2-1. By contrast, in cry1-304 cry2-1, an Arabidopsis mutant lacking another class of plant blue-light receptors, the channel remained sensitive to blue light. We thus conclude that blue light triggers calcium fluxes via the phototropin-activated calcium-permeable channel.