Water-soluble chlorophyll-binding proteins from Arabidopsis thaliana and Raphanus sativus target the endoplasmic reticulum body.

BACKGROUND: Non-photosynthetic chlorophyll (Chl) proteins called water-soluble Chl-binding proteins are distributed in Brassicaceae plants. Brassica oleracea WSCP (BoWSCP) and Lepidium virginicum WSCP (LvWSCP) are highly expressed in leaves and stems, while Arabidopsis thaliana WSCP (AtWSCP) and Raphanus sativus WSCP (RshWSCP) are highly transcribed in floral organs. BoWSCP and LvWSCP exist in the endoplasmic reticulum (ER) body. However, the subcellular localization of AtWSCP and RshWSCP is still unclear. To determine the subcellular localization of these WSCPs, we constructed transgenic plants expressing Venus-fused AtWSCP or RshWSCP. RESULTS: Open reading frames corresponding to full-length AtWSCP and RshWSCP were cloned and ligated between the cauliflower mosaic virus 35S promoter and Venus, a gene encoding a yellow fluorescent protein. We introduced the constructs into A. thaliana by the floral dip method. We succeeded in constructing a number of transformants expressing Venus-fused chimeric AtWSCP (AtWSCP::Venus) or RshWSCP (RshWSCP::Venus). We detected fluorescence derived from the chimeric proteins using a fluorescence microscope system. In cotyledons, fluorescence derived from AtWSCP::Venus and RshWSCP::Venus was detected in spindle structures. The spindle structures altered their shape to a globular form under blue light excitation. In true leaves, the number of spindle structures was drastically reduced. These observations indicate that the spindle structure was the ER body. CONCLUSIONS: AtWSCP and RshWSCP have the potential for ER body targeting like BoWSCP and LvWSCP.

Accumulation of alkaline earth metals by the green macroalga Bryopsis maxima.

Twenty-five days after the disaster at the Fukushima Daiichi nuclear power plant in 2011, we collected samples of the green macroalga Bryopsis maxima from the Pacific coast of Japan. Bryopsis maxima is a unicellular, multinuclear, siphonous green macroalga. Radiation analysis revealed that B. maxima emitted remarkably high gamma radiation of (131)I, (134)Cs, (137)Cs, and (140)Ba as fission products of (235)U. Interestingly, B. maxima contained naturally occurring radionuclides derived from (226)Ra and (228)Ra. Analysis of element content revealed that B. maxima accumulates many ocean elements, especially high quantities of the alkaline earth metals Sr (15.9 g per dry-kg) and Ba (3.79 g per dry-kg), whereas Ca content (12.5 g per dry-kg) was lower than that of Sr and only 61 % of the mean content of 70 Japanese seaweed species. Time-course analysis determined the rate of radioactive (85)Sr incorporation into thalli to be approximately 0.13 g Sr per dry-kg of thallus per day. Subcellular fractionation of B. maxima cells showed that most of the (85)Sr was localized in the soluble fraction, predominantly in the vacuole or cytosol. Given that (85)Sr radioactivity was permeable through a dialysis membrane, the (85)Sr was considered to be a form of inorganic ion and/or bound with a small molecule. Precipitation analysis with sodium sulfate showed that more than 70% of the Sr did not precipitate as SrSO4, indicating that a proportion of the Sr may bind with small molecules in B. maxima.

Effect of near-infrared irradiation on photoconversion of the water-soluble chlorophyll-binding protein of Chenopodium album.

We investigated the effects of near-infrared irradiation on the photoconversion of Chenopodium album water-soluble chlorophyll-binding protein (CaWSCP) in the presence of sodium hydrosulfite and found a further photoconversion from CP742 to CP763, a novel form of CaWSCP. Interestingly, one-third of the absorption peak at 668 nm was recovered in CP763, but re-irradiation under oxidative conditions eliminated the photo convertibility of CaWSCP.

Identification of genes encoding photoconvertible (Class I) water-soluble chlorophyll-binding proteins from Chenopodium ficifolium.

Photoconvertible water-soluble chlorophyll-binding proteins, called Class I WSCPs, have been detected in Chenopodiaceae, Amaranthaceae and Polygonaceae plant species. To date, Chenopodium album WSCP (CaWSCP) is the only cloned gene encoding a Class I WSCP. In this study, we identified two cDNAs encoding Chenopodium ficifolium Class I WSCPs, CfWSCP1, and CfWSCP2. Sequence analyses revealed that the open reading frames of CfWSCP1 and CfWSCP2 were 585 and 588 bp, respectively. Furthermore, both CfWSCPs contain cystein2 and cystein30, which are essential for the chlorophyll-binding ability of CaWSCP. Recombinant CfWSCP1 and CfWSCP2, expressed in Escherichia coli as hexa-histidine fusion proteins (CfWSCP1-His and CfWSCP2-His), formed inclusion bodies; however, we were able to solubilize these using a buffer containing 8 M urea and then refold them by dialysis. The refolded CfWSCP1-His and CfWSCP2-His could bind chlorophylls and exhibited photoconvertibility, confirming that the cloned CfWSCPs are further examples of Class I WSCPs.

Cysteine-2 and Cys30 are essential for chlorophyll-binding activity of the water-soluble chlorophyll-binding protein (WSCP) of Chenopodium album.

Chenopodium album has a non-photosynthetic chlorophyll protein known as the water-soluble chlorophyll (Chl)-binding protein (WSCP). The C. album WSCP (CaWSCP) is able to photoconvert the chlorin skeleton of Chl a into a bacteriochlorin-like skeleton. Reducing reagents such as ß-mercaptoethanol or dithiothreitol inhibit photoconversion, indicating that S-S bridge(s) in CaWSCP are quite important for it. Recently, we found that the mature region of CaWSCP contains five cysteine residues; Cys2, Cys30, Cys48, Cys63, and Cys144. To identify which cysteine residues are involved in the photoconversion, we generated five mutants (C2S, C30S, C48S, C63S, and C144S) by site-directed mutagenesis. Interestingly, C48S, C63S, and C144S mutants showed the same Chl-binding activity and photoconvertibility as those of the recombinant wild-type CaWSCP-His, while the C2S and C30S mutants completely lost Chl-binding activity. Our findings indicated that the S-S bridge between Cys2 and Cys30 in each CaWSCP subunit is essential for Chl-binding activity.

Three-step photoconversion of only three subunits of the water-soluble chlorophyll-binding protein tetramer from Chenopodium album.

Water-soluble chlorophyll (Chl)-binding proteins (WSCPs) have been found in various plants. WSCPs are categorized into two classes based on their photoconvertibility: Class I (photoconvertible) and Class II (non-photoconvertible). Based on their absorption peaks, which occur in the red wavelengths, the pre- and post-photoconverted forms of Chenopodium album WSCP (CaWSCP) are called CP668 and CP742, respectively. Although various biochemical and biophysical properties of CaWSCP have already been characterized, questions remain regarding the structural dynamics of the photoconversion from CP668 to CP742, and the relationship between the photoconversion activity and incident light wavelength. To address how the wavelength of incident light affects the photoconversion, we performed time-course analyses of CaWSCP photoconversion by using light-emitting diodes that emit either white light, or at the discrete wavelengths 670, 645, 525, 470, or 430 nm. The most efficient photoconversion was observed under irradiation at 430 nm. Less efficient photoconversion was observed under irradiation with 670, 645, 470, or 525 nm light, in that order. The relationship between photoconversion activity and wavelength corresponded with the absorption peak intensities of Chls in the CaWSCP complex. The observed time dependence of the A(742)/A(668) ratio during photoconversion of the CaWSCP complex indicated that the photoconversion from CP668 to CP742 occurs in a three-step reaction, and that only three subunits in the complex could be photoconverted.

The C-terminal extension peptide of non-photoconvertible water-soluble chlorophyll-binding proteins (Class II WSCPs) affects their solubility and stability: comparative analyses of the biochemical and chlorophyll-binding properties of recombinant Brassica, Raphanus and Lepidium WSCPs with or without their C-terminal extension peptides.

Numerous members of the Brassicaceae possess non-photoconvertible water-soluble chlorophyll (Chl)-binding proteins (Class II WSCPs), which function as Chl scavengers during cell disruption caused by wounding, pest/pathogen attacks, and/or environmental stress. Class II WSCPs have two extension peptides, one at the N-terminus and one at the C-terminus. The N-terminal peptide acts as a signal peptide, targeting the protein to the endoplasmic reticulum body, a unique defensive organelle found only in the Brassicaceae. However, the physiological and biochemical functions of the C-terminal extension peptide had not been characterized previously. To investigate the function of the C-terminal extension peptide, we produced expression constructs of recombinant WSCPs with or without the C-terminal extension peptide. The WSCPs used were of Brussels sprouts (Brassica oleracea), Japanese wild radish (Raphanus sativus) and Virginia pepperweed (Lepidium virginicum). The solubility of all of the WSCPs with the C-terminal extension peptide was drastically lower than that of the recombinant WSCPs without the C-terminal extension peptide. In addition, the stability of the reconstituted WSCPs complexes with the C-terminal extension peptide was altered compared with that of the proteins without the C-terminal extension peptide. These finding indicate that the C-terminal extension peptide affects not only the solubility, but also the stability of Class II WSCP. Furthermore, we characterized the Chl-binding properties of the recombinant WSCP from Japanese wild radish (RshWSCP-His) in a 40 % methanol solution. An electrophoretic mobility shift assay revealed that RshWSCP-His required a half-molar ratio of Chls to form a tetramer.

Molecular cloning, characterization and analysis of the intracellular localization of a water-soluble chlorophyll-binding protein (WSCP) from Virginia pepperweed (Lepidium virginicum), a unique WSCP that preferentially binds chlorophyll b in vitro.

Various plants possess non-photosynthetic, hydrophilic chlorophyll (Chl) proteins called water-soluble Chl-binding proteins (WSCPs). WSCPs are categorized into two classes; Class I (photoconvertible type) and Class II (non-photoconvertible type). Among Class II WSCPs, only Lepidium virginicum WSCP (LvWSCP) exhibits a low Chl a/b ratio compared with that found in the leaf. Although the physicochemical properties of LvWSCP have been characterized, its molecular properties have not yet been documented. Here, we report the characteristics of the LvWSCP gene, the biochemical properties of a recombinant LvWSCP, and the intracellular localization of LvWSCP. The cloned LvWSCP gene possesses a 669-bp open reading frame. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis revealed that the precursor of LvWSCP contains both N- and C-terminal extension peptides. RT-PCR analysis revealed that LvWSCP was transcribed in various tissues, with the levels being higher in developing tissues. A recombinant LvWSCP and hexa-histidine fusion protein (LvWSCP-His) could remove Chls from the thylakoid in aqueous solution and showed an absorption spectrum identical to that of native LvWSCP. Although LvWSCP-His could bind both Chl a and Chl b, it bound almost exclusively to Chl b when reconstituted in 40 % methanol. To clarify the intracellular targeting functions of the N- and C-terminal extension peptides, we constructed transgenic Arabidopsis thaliana lines expressing the Venus protein fused with the LvWSCP N- and/or C-terminal peptides, as well as Venus fused at the C-terminus of LvWSCP. The results showed that the N-terminal peptide functioned in ER body targeting, while the C-terminal sequence did not act as a trailer peptide.

The photoconvertible water-soluble chlorophyll-binding protein of Chenopodium album is a member of DUF538, a superfamily that distributes in Embryophyta.

Various plants possess hydrophilic chlorophyll (Chl) proteins known as water-soluble Chl-binding proteins (WSCPs). WSCPs exist in two forms: Class I and Class II, of which Class I alone exhibits unique photoconvertibility. Although numerous genes encoding Class II WSCPs have been identified and the molecular properties of their recombinant proteins have been well characterized, no Class I WSCP gene has been identified to date. In this study, we cloned the cDNA and a gene encoding the Class I WSCP of Chenopodium album (CaWSCP). Sequence analyses revealed that CaWSCP comprises a single exon corresponding to 585bp of an open reading frame encoding 195 amino acid residues. The CaWSCP protein sequence possesses a signature of DUF538, a protein superfamily of unknown function found almost exclusively in Embryophyta. The recombinant CaWSCP was expressed in Escherichia coli as a hexa-histidine fusion protein (CaWSCP-His) that removes Chls from the thylakoid. Under visible light illumination, the reconstituted CaWSCP-His was successfully photoconverted into a different pigment with an absorption spectrum identical to that of native CaWSCP. Interestingly, while CaWSCP-His could bind both Chl a and Chl b, photoconversion occurred only in CaWSCP-His reconstituted with Chl a.

The promoter of the carotenoid cleavage dioxygenase 4a-5 gene of Chrysanthemum morifolium (CmCCD4a-5) drives petal-specific transcription of a conjugated gene in the developing flower.

Carotenoids comprise one of the major groups of pigments in flowers. Because carotenoids are physiologically indispensable pigments for all photosynthetic plants, their catabolism must be discretely regulated in photosynthetic organs and non-photosynthetic organs such as petals or fruits. In the chrysanthemum, carotenoid cleavage dioxygenase 4a (CmCCD4a), which is dominantly expressed in petals, cleaves carotenoid, leading to a white flower. CmCCD4a-5 was recently identified as a new member of the CmCCD4a family, but its detailed expression profile in plant tissues has not yet been established. In this study, we sequenced a 1094-bp region upstream of CmCCD4a-5 and assessed its petal-specific promoter activity. To evaluate the activity of this gene, we constructed two types of transgenic Arabidopsis thaliana that possessed, respectively, a fusion gene of a 1090-bp or 505-bp segment of the upstream region plus the ß-d-glucuronidase (GUS) gene (1090bUR::GUS and 505bUR::GUS). GUS activity in the 505bUR::GUS strain was observed mainly in the anthers/pollen in flower buds, whereas GUS activity of the 1090bUR::GUS strain was observed in immature petals of the flower buds. Among the cis-acting elements located between positions -505 and -1090, no elements that have previously been reported to enhance the expression in petals or to suppress it in anthers/pollen were detected by PLACE analysis, indicating the existence of unknown cis-element(s). A semiquantitative reverse transcription-polymerase chain reaction analysis revealed that CmCCD4a-5 transcription was prominent in petals but was undetectable in roots, stems and leaves.

Molecular cloning and functional expression of a water-soluble chlorophyll-binding protein from Japanese wild radish.

Hydrophilic chlorophyll (Chl)-binding proteins have been isolated from various Brassicaceae plants and are categorized into Class II water-soluble Chl-binding proteins (WSCPs). Although the molecular properties of class II WSCPs including Brassica-type (e.g., cauliflower WSCP, Brussels sprouts WSCP and BnD22, a drought- and salinity-stress-induced 22 kDa protein of rapeseed), a Lepidium-type, and an Arabidopsis-type WSCPs have been well characterized, those of Raphanus-type WSCPs are poorly understood. To gain insight into the molecular diversity of Class II WSCPs, we cloned a novel cDNA encoding a Raphanus sativus var. raphanistroides (Japanese wild radish called 'Hamadaikon') WSCP (RshWSCP). Sequence analysis revealed that the open reading frame of the RshWSCP gene consisted of 666 bp encoding 222 aa residues, including 23 residues of a deduced signal peptide. Functional recombinant RshWSCP was expressed in Escherichia coli as a hexa-histidine fusion protein (RshWSCP-His). Although the RshWSCP-His was expressed as a soluble protein in E. coli, the apo-protein was highly unstable and tended to aggregate during a series of purification steps. When the soluble fraction of RshWSCP-His-expressing E. coli was mixed immediately with homogenate of spinach leaves containing thylakoid, RshWSCP-His was able to remove Chl molecules from the thylakoid and formed a stable Chl-WSCP complex with high hydrophilicity. UV-visible absorption spectra of the reconstituted RshWSCP-His revealed that RshWSCP-His is one of the Class IIA WSCP with the highest Chl a/b ratio analyzed thus far. A semi-quantitative reverse transcription-polymerase chain reaction analysis revealed that RshWSCP was transcribed in buds and flowers but not in roots, stems and various leaves.

Molecular cloning, characterization and analysis of the intracellular localization of a water-soluble Chl-binding protein from Brussels sprouts (Brassica oleracea var. gemmifera).

A water-soluble Chl-binding protein from Brussels sprouts (Brassica oleracea var. gemmifera), hereafter termed BoWSCP, is categorized into the Class II WSCPs (non-photoconvertible WSCPs). Previous studies on BoWSCP have focused mainly on its biochemical characterization. In this study, we cloned the cDNA encoding BoWSCP. Sequence analysis revealed that the BoWSCP gene was composed of a single exon corresponding to 654 bp of an open reading frame encoding 218 amino acid residues, including 19 residues of a deduced signal peptide targeted to the endoplasmic reticulum (ER). Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of native BoWSCP revealed that the molecular mass of the subunit was 19,008.523 Da, corresponding to a mature protein of 178 amino acids, indicating the removal of 21 residues in the C-terminal region. Functional BoWSCP was expressed in Escherichia coli as a hexa-histidine fusion protein (BoWSCP-His). When BoWSCP-His was mixed with thylakoid membranes in aqueous solution, BoWSCP-His was able to remove Chls from the thylakoid membranes. The absorption spectrum of the reconstituted BoWSCP-His was identical to that of the native BoWSCP. Chl binding analyses of BoWSCP-His revealed that the BoWSCP-His bound both Chl a and Chl b with almost the same affinity in 40% methanol solution, although the native BoWSCP had a higher content of Chl a. To reveal the intracellular localization of BoWSCP, we constructed a transgenic plant expressing the fluorescent protein fused with the N-terminal deduced signal peptide of BoWSCP. The fluorescence emitted from the chimeric protein was detected in the ER body, an ER-derived compartment observed only in Brassicaceae plants.