Water-soluble chlorophyll-binding proteins from Arabidopsis thaliana and Raphanus sativus target the endoplasmic reticulum body
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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.
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.
AtWSCP and RshWSCP have the potential for ER body targeting like BoWSCP and LvWSCP.
KeywordsChlorophyll ER body Water-soluble chlorophyll-binding protein WSCP
water-soluble chlorophyll-binding proteins
Chlorophyll (Chl) is a photosynthetic pigment. Most chlorophyll proteins playing a role in photosynthesis are membranous proteins, but highly hydrophilic Chl proteins called water-soluble Chl-binding proteins (WSCPs) have been isolated from various land plants of Chenopodiaceae, Amaranthaceae, Polygonaceae, and Brassicaceae . WSCPs from Chenopodiaceae, Amaranthaceae, and Polygonaceae are photoconvertible, but WSCPs from Brassicaceae do not show this ability . Generally, photoconvertible and non-photoconvertible WSCPs are called Class I and Class II WSCPs, respectively . Furthermore, Class II WSCPs are categorized into two subclasses, IIA and IIB, based on their Chl a/b ratio . To date, Lepidium virginicum WSCP (LvWSCP) is the only Class IIB WSCP . Chenopodiaceae WSCPs are members of the domain unknown function 538 superfamily and thus the biological function of these proteins remains unclear [2, 3]. On the other hand, all Class II WSCPs cloned thus far are members of the Kunitz-type trypsin inhibitor family [4, 5, 6, 7, 8]. The protease inhibitor activity of Class II WSCPs in young leaves was reported to be important for nitrogen remobilization under stressful conditions . Class II WSCPs are also able to repress reactive oxygen species generation derived from excited Chl . Additionally, Brassica oleracea WSCP (BoWSCP) and Lepidium virginicum WSCP (LvWSCP) are located in the endoplasmic reticulum (ER) body [6, 8], which is contributes to the a unique defense system in Brassicaceae [11, 12]. Thus, Class II WSCPs have the potential to act as Chl scavengers during cell disruption to protect healthy cells [6, 8]. Because the molecular structure of Class II WSCPs is quite simple and the complex is quite stable and easy to handle, Class II WSCPs are used as model proteins for characterizing the Chl–Chl and Chl–protein interactions of Chl proteins [13, 14].
BoWSCP and LvWSCP are highly accumulated in leaves and stems [15, 16], while Arabidopsis thaliana WSCP (AtWSCP) is expressed in the transmitting tract . Moreover, Raphanus sativus var. raphanistroides Makino WSCP (RshWSCP) is highly transcribed in floral organs . In contrast to BoWSCP and LvWSCP, the subcellular localization of AtWSCP and RshWSCP is still unclear. Elucidation of the subcellular localization of these proteins will provide clues for understanding the diversity and universality of Class II WSCPs.
Arabidopsis thaliana (ecotype, Col-0) was grown on 1 % agar plates containing full-strength MS medium and 1 % sucrose at 22 °C under continuous light. Three-week-old A. thaliana was transferred from the plate to Jiffy-7 for further growth.
Isolation of genomic DNA
Using a DNeasy mini kit (Qiagen, Venlo, The Netherlands), we extracted and purified genomic DNA from leaves of 2-week-old A. thaliana according to the manufacturer’s instructions.
Construction of transgenic A. thaliana
Fluorescence microscopic analysis
To obtain fluorescence images of A. thaliana expressing Venus-fused chimeric AtWSCP and RshWSCP (i.e., AtWSCP::Venus and RshWSCP::Venus, respectively), we used an Axioskop2 plus microscope (Carl Zeiss) and Axio Cam (Carl Zeiss) with the Axio Vision software (Carl Zeiss). The T2 generations of transgenic plants were used for the fluorescence microscopic analysis.
Results and discussion
Because AtWSCP has protease inhibitor activity  and accumulates in the transmitting tract in the gynoecium and silique , Becktas et al. hypothesized that the protease inhibitor activity of AtWSCP might be important for the formation of the transmitting tract. Note that we could not find any difference between the transformants and wild type A. thaliana. Further analysis of the AtWSCP null-mutant will provide clues to elucidate the biological function of AtWSCP.
To our knowledge, this is the first report describing the subcellular localization of floral organ-expressed WSCPs (i.e., AtWSCP and RshWSCP). Similar to leaf-expressed WSCPs (i.e., BoWSCP and LvWSCP), AtWSCP and RshWSCP target the ER body.
ST designed the research. ST and KA performed the experiments. ST and HS wrote the paper. NK and HS supervised the work. All authors read and approved the final manuscript.
Compliance with ethical guidelines
Competing interests The authors declare that they have no competing interests.
- 3.Takahashi S, Abe E, Nakayama K, Satoh H. Identification of genes encoding photoconvertible (Class I) water-soluble chlorophyll-binding proteins from Chenopodium ficifolium. Biosci Biotechnol Biochem. 2015 (in press). doi: 10.1080/09168451.2014.972326.
- 6.Takahashi S, Yanai H, Nakamaru Y, Uchida A, Nakayama K, Satoh H. Molecular cloning, characterization and analysis of the intracellular localization of a water-soluble chlorophyll-binding protein from Brussels sprouts (Brassica oleracea var. gemmifera). Plant Cell Physiol. 2012;53:879–91.CrossRefPubMedGoogle Scholar
- 8.Takahashi S, Yanai H, Oka-Takayama Y, Zanma-Sohtome A, Fujiyama K, Uchida A, Nakayama K, Satoh H. 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. Planta. 2013;238:1065–80.CrossRefGoogle Scholar
- 9.Desclos M, Dubousset L, Etienne P, Le Caherec F, Satoh H, Bonnefoy J, Ourry A, Avice JC. A proteomic profiling approach to reveal a novel role of Brassica napus drought 22 kD/water-soluble chlorophyll-binding protein in young leaves during nitrogen remobilization induced by stressful conditions. Plant Physiol. 2008;147:1830–44.PubMedCentralCrossRefPubMedGoogle Scholar
- 13.Renger G, Pieper J, Theiss C, Trostmann I, Paulsen H, Renger T, Eichler HJ, Schmitt FJ. Water soluble chlorophyll binding protein of higher plants: a most suitable model system for basic analyses of pigment-pigment and pigment-protein interactions in chlorophyll protein complexes. J Plant Physiol. 2011;168:1462–72.CrossRefPubMedGoogle Scholar
- 15.Murata T, Murata N. Water-soluble chlorophyll-proteins from Brassica nigra and Lepidium virginicum. Carnegie Inst Wash Yearb. 1971;70:504–7.Google Scholar
- 19.Gotté M, Ghosh R, Bernard A, Nguema-Ona E, Vicré-Gibouin M, Hara-Nishimura I, Driouich A. Methyl jasmonate affects morphology, number and activity of endplasmic reticulum bodies in Raphanus sativus root cells. Plant Cell Physiol. 2015 (in press). doi: 10.1093/pcp/pcu141.
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