Takeshi Kanegae

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.

Cryptochrome Nucleocytoplasmic Distribution and Gene Expression Are Regulated by Light Quality in the Fern Adiantum capillus-veneris

Numerous cellular responses are reportedly regulated by blue light in gametophytes of lower plants; however, the molecular mechanisms of these responses are not known. Here, we report the isolation of two blue light photoreceptor genes, designated cryptochrome genes 4 and 5 (CRY4 and CRY5), from the fern Adiantum capillus-veneris. Because previously we identified three cryptochrome genes, this fern cryptochrome gene family of five members is the largest identified to date in plants. The deduced amino acid sequences of the five genes show remarkable similarities with previously identified cryptochromes as well as class I photolyases. Like the other plant cryptochromes, none of the cryptochromes of this fern possesses photolyase activity. RNA gel blot analysis and competitive polymerase chain reaction analysis indicate that the expression of the newly identified CRY4 and CRY5 genes is regulated by light and is under phytochrome control. The intracellular distribution of reporter β-glucuronidase (GUS)–CRY fusion proteins indicates that GUS–CRY3 and GUS–CRY4 localize in fern gametophyte nuclei. The nuclear localization of GUS–CRY3 is regulated in a light-dependent manner. Together with our physiological knowledge, these results suggest that CRY3, CRY4, or both might be the photoreceptor that mediates inhibition of spore germination by blue light.

Chloroplast actin filaments organize meshwork on the photorelocated chloroplasts in the moss Physcomitrella patens

Cytoskeleton dynamics during phototropin-dependent chloroplast photorelocation movement was analyzed in protonemal cells of actin- and microtubule-visualized lines of Physcomitrella patens expressing GFP- or tdTomato-talin and GFP-tubulin. Using newly developed epi- and trans-microbeam irradiation systems that permit fluorescence observation of the cell under blue microbeam irradiation inducing chloroplast relocation, it was revealed that meshwork of actin filaments formed at the chloroplast-accumulating area both in the avoidance and accumulation movements. The structure disappeared soon when blue microbeam was turned off, and it was not induced under red microbeam irradiation that did not evoke chloroplast relocation movement. In contrast, no apparent change in microtubule organization was detected during the movements. The actin meshwork was composed of short actin filaments distinct from the cytoplasmic long actin cables and was present between the chloroplasts and plasma membrane. The short actin filaments emerged from around the chloroplast periphery towards the center of chloroplast. Showing highly dynamic behavior, the chloroplast actin filaments (cp-actin filaments) were rapidly organized into meshwork on the chloroplast surface facing plasma membrane. The actin filament configuration on a chloroplast led to the formation of actin meshwork area in the cell as the chloroplasts arrived at and occupied the area. After establishment of the meshwork, cp-actin filaments were still highly dynamic, showing appearance, disappearance, severing and bundling of filaments. These results indicate that the cp-actin filaments have significant roles in the chloroplast movement and positioning in the cell.

A single chromoprotein with triple chromophores acts as both a phytochrome and a phototropin

Plants sense their environmental light conditions by using three photoreceptors that absorb in the UV, blue/near UV, and red/far-red spectral ranges. These photoreceptors have specific chromophore components corresponding to their absorption spectra. Phytochrome, a red/far-red light receptor, has phytochromobilin as its chromophore, whereas the blue/near UV photoreceptors cryptochrome and phototropin have a pair of flavin derivatives. Plants use these various photoreceptors to assess the surrounding light environment. Phytochrome 3 (PHY3) is a red light receptor found in some ferns, which preferentially grow under weak light. PHY3 is composed of a phytochrome chromophore-binding domain in its N-terminal portion and an almost full-length phototropin in its C-terminal half. This unusual domain organization implies that two different light-sensing systems coexist in this single photoreceptor, although these light-sensing systems usually reside in independent photoreceptors. Here, we show that PHY3 acts as a dual-channel photoreceptor that possesses both the red light-sensing system of phytochrome and the blue light-sensing system of phototropin. Furthermore, red- and blue-light signals perceived by PHY3 are processed synergistically within this single chromoprotein. These unusual properties might confer an enhanced light sensitivity on PHY3, allowing ferns to grow under a low-light canopy.

A full lengthTy3/gypsy-type retrotransposon in the fernAdiantum

Ty3/gypsy-type LTR-retrotransposons have been found only in lily and maize but not in cryptogam. In fernAdiantum, we recently found a full-lengthTy3/gypsy-type LTR-retrotransposon (ARET-1; 8284 bp). This retrotransposon has both 5′ and 3′ LTRs (1.2 kb), a primer binding site, a polypurine tract, and an RNA binding motif and its domain arrangement in thepol region is the same as that ofTy3/gypsy-type retrotransposon. These results suggest thatTy3/gypsy-type retrotransposons are widespread among vascular plants.

A phytochrome/phototropin chimeric photoreceptor of fern functions as a blue/far-red light-dependent photoreceptor for phototropism in Arabidopsis.

In the fern Adiantum capillus-veneris, the phototropic response of the protonemal cells is induced by blue light and partially inhibited by subsequent irradiation with far-red light. This observation strongly suggests the existence of a phytochrome that mediates this blue/far-red reversible response; however, the phytochrome responsible for this response has not been identified. PHY3/NEO1, one of the three phytochrome genes identified in Adiantum, encodes a chimeric photoreceptor composed of both a phytochrome and a phototropin domain. It was demonstrated that phy3 mediates the red light-dependent phototropic response of Adiantum, and that phy3 potentially functions as a phototropin. These findings suggest that phy3 is the phytochrome that mediates the blue/far-red response in Adiantum protonemata. In the present study, we expressed Adiantum phy3 in a phot1 phot2 phototropin-deficient Arabidopsis line, and investigated the ability of phy3 to induce phototropic responses under various light conditions. Blue light irradiation clearly induced a phototropic response in the phy3-expressing transgenic seedlings, and this effect was fully inhibited by simultaneous irradiation with far-red light. In addition, experiments using amino acid-substituted phy3 indicated that FMN-cysteinyl adduct formation in the light, oxygen, voltage (LOV) domain was not necessary for the induction of blue light-dependent phototropism by phy3. We thus demonstrate that phy3 is the phytochrome that mediates the blue/far-red reversible phototropic response in Adiantum. Furthermore, our results imply that phy3 can function as a phototropin, but that it acts principally as a phytochrome that mediates both the red/far-red and blue/far-red light responses.

Phototropins of the moss Physcomitrella patens function as blue-light receptors for phototropism in Arabidopsis

ABSTRACT Four phototropin genes (PHOTA1, PHOTA2, PHOTB1, PHOTB2) have been isolated in the moss Physcomitrella patens. These genes encode phototropins that mediate blue-light–induced chloroplast movement. However, the individual functions of these phototropins, including the function of mediating blue-light–induced phototropism, remain unclear. To elucidate the individual functions of P. patens phototropins, each of these phototropin genes was expressed in a phototropin-deficient mutant of Arabidopsis (phot1-5 phot2-1). In addition, fluorescence of GFP fused to these phototropins was examined to determine the subcellular localization of each phototropin. Our results demonstrate that all four P. patens phototropins mediate blue-light–induced phototropism and are associated with the plasma membrane in Arabidopsis. Abbreviations GFP: green fluorescent protein; Pp_phot: Physcomitrella patens phototropin

Intramolecular co-action of two independent photosensory modules in the fern phytochrome 3.

Fern phytochrome3/neochrome1 (phy3/neo1) is a chimeric photoreceptor composed of a phytochrome-chromophore binding domain and an almost full-length phototropin. phy3 thus contains two different light-sensing modules; a red/far-red light receptor phytochrome and a blue light receptor phototropin. phy3 induces both red light- and blue light-dependent phototropism in phototropin-deficient Arabidopsis thaliana (phot1 phot2) seedlings. The red-light response is dependent on the phytochrome module of phy3, and the blue-light response is dependent on the phototropin module. We recently showed that both the phototropin-sensing module and the phytochrome-sensing module mediate the blue light-dependent phototropic response. Particularly under low-light conditions, these two light-sensing modules cooperate to induce the blue light-dependent phototropic response. This intramolecular co-action of two independent light-sensing modules in phy3 enhances light sensitivity, and perhaps allowed ferns to adapt to the low-light canopy conditions present in angiosperm forests.