Abstract
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.
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Abbreviations
- CaMV:
-
Cauliflower mosaic virus
- Chl:
-
Chlorophyll
- C-terminal/terminus:
-
Carboxy terminal/terminus
- EMSA:
-
Electrophoretic mobility shift assay
- ER:
-
Endoplasmic reticulum
- IPTG:
-
Isopropyl β-d-1-thiogalactopyranoside
- KTI:
-
Kunitz-type trypsin inhibitor
- MALDI-TOF–MS:
-
Matrix-assisted laser desorption ionization time-of-flight mass spectrometry
- NOS:
-
Nopaline synthase
- N-terminal/terminus:
-
Amino terminal/terminus
- WSCP:
-
Water-soluble chlorophyll-binding protein
- WT:
-
Wild type
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Acknowledgments
We thank Dr. Soichiro Watanabe for his kind help in the preparation of Chls a and b.
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The nucleotide sequence reported in this paper has been submitted to DDBJ under accession number AB002589.
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Takahashi, S., Yanai, H., Oka-Takayama, Y. et al. 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 238, 1065–1080 (2013). https://doi.org/10.1007/s00425-013-1952-7
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DOI: https://doi.org/10.1007/s00425-013-1952-7