Publications by authors named "Ken Motohashi"

39 Publications

Evaluation of CBSX Proteins as Regulators of the Chloroplast Thioredoxin System.

Front Plant Sci 2021 16;12:530376. Epub 2021 Feb 16.

Department of Frontier Life Sciences, Faculty of Life Sciences, Kyoto Sangyo University, Kyoto, Japan.

The chloroplast-localized cystathionine β-synthase X (CBSX) proteins CBSX1 and CBSX2 have been proposed as modulators of thioredoxins (Trxs). In this study, the contribution of CBSX proteins to the redox regulation of thiol enzymes in the chloroplast Trx system was evaluated both and . The biochemical studies evaluated whether CBSX proteins alter the specificities of classical chloroplastic Trx and Trx for their target proteins. However, addition of CBSX proteins did not alter the specificities of Trx and Trx for disulfide bond reduction of the photosynthesis-related major thiol enzymes, FBPase, SBPase, and NADP-MDH. analysis showed that CBSX-deficient mutants grew similarly to wild type plants under continuous normal light conditions and that CBSX deficiency did not affect photo-reduction of photosynthesis-related thiol enzymes by Trx system at several light intensities. Although CBSX proteins have been suggested as modulators in the chloroplast Trx system, our results did not support this model, at least in the cases of FBPase, SBPase, and NADP-MDH in leaves. However, fresh weights of the mutants were decreased under short day. Since Trxs regulate many proteins participating in various metabolic reactions in the chloroplast, CBSX proteins may function to regulate other chloroplast Trx target proteins, or serve as modulators in non-photosynthetic plastids of flowers. As a next stage, further investigations are required to understand the modulation of Trx-dependent redox regulation by plastidal CBSX proteins.
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http://dx.doi.org/10.3389/fpls.2021.530376DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7921703PMC
February 2021

The evolutionary conserved iron-sulfur protein TCR controls P700 oxidation in photosystem I.

iScience 2021 Feb 13;24(2):102059. Epub 2021 Jan 13.

Department of Life Science and Technology, Tokyo Institute of Technology, Yokohama 226-8501, Japan.

In natural habitats, plants have developed sophisticated regulatory mechanisms to optimize the photosynthetic electron transfer rate at the maximum efficiency and cope with the changing environments. Maintaining proper P700 oxidation at photosystem I (PSI) is the common denominator for most regulatory processes of photosynthetic electron transfers. However, the molecular complexes and cofactors involved in these processes and their function(s) have not been fully clarified. Here, we identified a redox-active chloroplast protein, the triplet-cysteine repeat protein (TCR). TCR shared similar expression profiles with known photosynthetic regulators and contained two triplet-cysteine motifs (CxxxCxxxC). Biochemical analysis indicated that TCR localizes in chloroplasts and has a [3Fe-4S] cluster. Loss of TCR limited the electron sink downstream of PSI during dark-to-light transition. double mutant reduced growth significantly and showed unusual oxidation and reduction of plastoquinone pool. These results indicated that TCR is involved in electron flow(s) downstream of PSI, contributing to P700 oxidation.
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http://dx.doi.org/10.1016/j.isci.2021.102059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7848650PMC
February 2021

Molecular and Biochemical Differences in Leaf Explants and the Implication for Regeneration Ability in (Brassicaceae).

Plants (Basel) 2020 Oct 15;9(10). Epub 2020 Oct 15.

Faculty of Life Sciences, Kyoto Sangyo University, Kyoto 603-8555, Japan.

Plants have a high regeneration capacity and some plant species can regenerate clone plants, called plantlets, from detached vegetative organs. We previously outlined the molecular mechanisms underlying plantlet regeneration from (Brassicaceae) leaf explants. However, the fundamental difference between the plant species that can and cannot regenerate plantlets from vegetative organs remains unclear. Here, we hypothesized that the viability of leaf explants is a key factor affecting the regeneration capacity of . To test this hypothesis, the viability of and leaf explants were compared, with respect to the maintenance of photosynthetic activity, senescence, and immune response. Time-course analyses of photosynthetic activity revealed that leaf explants can survive longer than those of . Endogenous abscisic acid (ABA) and jasmonic acid (JA) were found at low levels in leaf explant of . Time-course transcriptome analysis of and leaf explants suggested that senescence was suppressed at the transcriptional level in . Application of exogenous ABA reduced the efficiency of plantlet regeneration. Overall, our results propose that in nature, plant species that can regenerate in nature can survive for a long time.
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http://dx.doi.org/10.3390/plants9101372DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7602576PMC
October 2020

M-Type Thioredoxins Regulate the PGR5/PGRL1-Dependent Pathway by Forming a Disulfide-Linked Complex with PGRL1.

Plant Cell 2020 12 9;32(12):3866-3883. Epub 2020 Oct 9.

Department of Frontier Life Sciences, Faculty of Life Sciences, Kyoto Sangyo University, Kamigamo Motoyama, Kita-Ku, Kyoto 603-8555, Japan

In addition to linear electron transport, photosystem I cyclic electron transport (PSI-CET) contributes to photosynthesis and photoprotection. In Arabidopsis (), PSI-CET consists of two partially redundant pathways, one of which is the PROTON GRADIENT REGULATION5 (PGR5)/PGR5-LIKE PHOTOSYNTHETIC PHENOTYPE1 (PGRL1)-dependent pathway. Although the physiological significance of PSI-CET is widely recognized, the regulatory mechanism behind these pathways remains largely unknown. Here, we report on the regulation of the PGR5/PGRL1-dependent pathway by the -type thioredoxins (Trx ). Genetic and phenotypic characterizations of multiple mutants indicated the physiological interaction between Trx and the PGR5/PGRL1-dependent pathway in vivo. Using purified Trx proteins and ruptured chloroplasts, in vitro, we showed that the reduced form of Trx specifically decreased the PGR5/PGRL1-dependent plastoquinone reduction. In planta, Trx 4 directly interacted with PGRL1 via disulfide complex formation. Analysis of the transgenic plants expressing PGRL1 Cys variants demonstrated that Cys-123 of PGRL1 is required for Trx 4-PGRL1 complex formation. Furthermore, the Trx 4-PGRL1 complex was transiently dissociated during the induction of photosynthesis. We propose that Trx directly regulates the PGR5/PGRL1-dependent pathway by complex formation with PGRL1.
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http://dx.doi.org/10.1105/tpc.20.00304DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7721319PMC
December 2020

Cyclic Electron Transport around PSI Contributes to Photosynthetic Induction with Thioredoxin .

Plant Physiol 2020 11 11;184(3):1291-1302. Epub 2020 Sep 11.

Department of Frontier Life Sciences, Faculty of Life Sciences, Kyoto Sangyo University, Kamigamo Motoyama, Kita-ku, Kyoto 603-8555, Japan.

In response to light, plants efficiently induce photosynthesis. Light activation of thiol enzymes by the thioredoxin (Trx) systems and cyclic electron transport by the PROTON GRADIENT REGULATION5 (PGR5)-dependent pathway contribute substantially to regulation of photosynthesis. Arabidopsis () mutants lacking -type Trxs () show delayed activation of carbon assimilation due to impaired photoreduction of Calvin-Benson cycle enzymes. To further study regulatory mechanisms that contribute to efficiency during the induction of photosynthesis, we analyzed the contributions of PSI donor- and acceptor-side regulation in the mutant background. The cytochrome complex is involved in PSI donor-side regulation, whereas PGR5-dependent PSI cyclic electron transport is required for both donor and acceptor functions. Introduction of the mutation, which is conditionally defective in cytochrome complex activity, into the mutant background did not further affect the induction of photosynthesis, but the combined deficiency of Trx and PGR5 severely impaired photosynthesis and suppressed plant growth under long-day conditions. In the mutant, the acceptor-side of PSI was almost completely reduced, and quantum yields of PSII and PSI hardly increased during the induction of photosynthesis. We also compared the photoreduction of thiol enzymes between the and mutants. The mutation did not result in further impaired photoreduction of Calvin-Benson cycle enzymes or ATP synthase in the mutant background. These results indicated that acceptor-side limitations in the mutant suppress photosynthesis initiation, suggesting that PGR5 is required for efficient photosynthesis induction.
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http://dx.doi.org/10.1104/pp.20.00741DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7608166PMC
November 2020

Chloroplast ATP synthase is reduced by both f-type and m-type thioredoxins.

Biochim Biophys Acta Bioenerg 2020 11 11;1861(11):148261. Epub 2020 Jul 11.

Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology, Nagatsuta-cho 4259-R1-8, Midori-Ku, Yokohama 226-8503, Japan; School of Life Science and Technology, Tokyo Institute of Technology, Nagatsuta-cho 4259-R1-8, Midori-ku, Yokohama 226-8503, Japan. Electronic address:

The activity of the molecular motor enzyme, chloroplast ATP synthase, is regulated in a redox-dependent manner. The γ subunit, CF-γ, is the central shaft of this enzyme complex and possesses the redox-active cysteine pair, which is reduced by thioredoxin (Trx). In light conditions, Trx transfers the reducing equivalent obtained from the photosynthetic electron transfer system to the CF-γ. Previous studies showed that the light-dependent reduction of CF-γ is more rapid than those of other Trx target proteins in the stroma. Although there are multiple Trx isoforms in chloroplasts, it is not well understood as to which chloroplast Trx isoform primarily contributes to the reduction of CF-γ, especially under physiological conditions. We therefore performed direct assessment of the CF-γ reduction capacity of each of the Trx isoforms. The kinetic analysis of the reduction process showed no significant difference in the reduction efficiency between two major chloroplast Trxs, namely Trx-f and Trx-m. Based on the thorough analyses of the CF-γ redox dynamics in Arabidopsis thaliana Trx mutant plants, we found that lack of Trx-f or Trx-m had no significant impact on the in vivo light-dependent reduction of CF-γ. The results showed that CF-γ can accept the reducing power from both Trx-f and Trx-m in chloroplasts.
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http://dx.doi.org/10.1016/j.bbabio.2020.148261DOI Listing
November 2020

A Simple and Fast Manual Centrifuge to Spin Solutions in 96-Well PCR Plates.

Authors:
Ken Motohashi

Methods Protoc 2020 May 25;3(2). Epub 2020 May 25.

Department of Frontier Life Sciences, Faculty of Life Sciences, Kyoto Sangyo University, Kamigamo Motoyama, Kita-ku, Kyoto 603-8047, Japan.

A simple and fast manual centrifuge was developed to spin down solutions in 96-well polymerase chain reaction (PCR) plates. A commercially available salad spinner was utilized for this purpose. Acceleration and deceleration of the centrifuge were faster than those of a conventional electric centrifuge using 96-well PCR plates. Solutions in a 96-well PCR plate settled quickly after centrifuging for only 3 s. This lightweight centrifuge can be stored under a laboratory bench or on a shelf and can be put on the bench only when required, whereas the electric centrifuge is immobile due to its weight and the requirement of electric cables. This simple centrifuge is inexpensive, requires minimal effort for making, and can be used anywhere.
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http://dx.doi.org/10.3390/mps3020041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7359706PMC
May 2020

Development of highly sensitive and low-cost DNA agarose gel electrophoresis detection systems, and evaluation of non-mutagenic and loading dye-type DNA-staining reagents.

Authors:
Ken Motohashi

PLoS One 2019 9;14(9):e0222209. Epub 2019 Sep 9.

Department of Frontier Life Sciences, Faculty of Life Sciences, Kyoto Sangyo University, Kamigamo Motoyama, Kita-ku, Kyoto, Japan.

Highly sensitive and low-cost DNA agarose gel detection systems were developed using non-mutagenic and loading dye-type DNA-staining reagents. The DNA detection system that used Midori Green Direct and Safelook Load-Green, both with an optimum excitation wavelength at ~490 nm, could detect DNA-fragments at the same sensitivity to that of the UV (312 nm)-transilluminator system combined with ethidium bromide, after it was excited by a combination of cyan LED light and a shortpass filter (510 nm). The cyan LED system can be also applied to SYBR Safe that is widely used as a non-toxic dye for post-DNA-staining. Another DNA-detection system excited by black light was also developed. Black light used in this system had a peak emission at 360 nm and caused less damage to DNA due to lower energy of UV rays with longer wavelength when compared to those of short UV rays. Moreover, hardware costs of the black light system were ~$100, less than 1/10 of the commercially available UV (365 nm) transilluminator (>$1,000). EZ-Vision and Safelook Load-White can be used as non-mutagenic and loading dye-type DNA-staining reagents in this system. The black light system had a greater detection sensitivity for DNA fragments stained by EZ-Vision and Safelook Load-White compared with the commercially available imaging system using UV (365 nm) transilluminator.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0222209PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6733488PMC
March 2020

A novel series of high-efficiency vectors for TA cloning and blunt-end cloning of PCR products.

Authors:
Ken Motohashi

Sci Rep 2019 04 23;9(1):6417. Epub 2019 Apr 23.

Department of Frontier Life Sciences, Faculty of Life Sciences, Kyoto Sangyo University, Kamigamo Motoyama, Kita-ku, Kyoto, 603-8555, Japan.

An efficient PCR cloning method is indispensable in modern molecular biology, as it can greatly improve the efficiency of DNA cloning processes. Here, I describe the development of three vectors for TA cloning and blunt-end cloning. Specifically, pCRT and pCRZeroT were designed to improve the efficiency of TA cloning. pCRZeroT can also be used with pCRZero to facilitate blunt-end cloning using the ccdB gene. Using pCRZero and pCRZeroT and applying the Golden Gate reaction, I developed a direct PCR cloning protocol with non-digested circular vectors and PCR products. This direct PCR cloning protocol yielded colony-formation rates and cloning efficiencies that are comparable with those obtained by conventional PCR cloning with pre-digested vectors and PCR products. The three plasmids I designed are available from Addgene ( https://www.addgene.org/ ).
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http://dx.doi.org/10.1038/s41598-019-42868-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6478821PMC
April 2019

Comparative transcriptomics with self-organizing map reveals cryptic photosynthetic differences between two accessions of North American Lake cress.

Sci Rep 2018 02 19;8(1):3302. Epub 2018 Feb 19.

Department of Bioresource and Environmental Sciences, Kyoto Sangyo University, Kamigamo-Motoyama, Kita-Ku, Kyoto, 603-8555, Japan.

Because natural variation in wild species is likely the result of local adaptation, it provides a valuable resource for understanding plant-environmental interactions. Rorippa aquatica (Brassicaceae) is a semi-aquatic North American plant with morphological differences between several accessions, but little information available on any physiological differences. Here, we surveyed the transcriptomes of two R. aquatica accessions and identified cryptic physiological differences between them. We first reconstructed a Rorippa phylogeny to confirm relationships between the accessions. We performed large-scale RNA-seq and de novo assembly; the resulting 87,754 unigenes were then annotated via comparisons to different databases. Between-accession physiological variation was identified with transcriptomes from both accessions. Transcriptome data were analyzed with principal component analysis and self-organizing map. Results of analyses suggested that photosynthetic capability differs between the accessions. Indeed, physiological experiments revealed between-accession variation in electron transport rate and the redox state of the plastoquinone pool. These results indicated that one accession may have adapted to differences in temperature or length of the growing season.
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http://dx.doi.org/10.1038/s41598-018-21646-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5818620PMC
February 2018

Evaluation of the efficiency and utility of recombinant enzyme-free seamless DNA cloning methods.

Authors:
Ken Motohashi

Biochem Biophys Rep 2017 Mar 26;9:310-315. Epub 2017 Jan 26.

Department of Bioresource and Environmental Sciences, Faculty of Life Sciences, Kyoto Sangyo University, Kamigamo Motoyama, Kita-ku, Kyoto 603-8555, Japan.

Simple and low-cost recombinant enzyme-free seamless DNA cloning methods have recently become available. cloning (iVEC) can directly transform a mixture of insert and vector DNA fragments into , which are ligated by endogenous homologous recombination activity in the cells. Seamless ligation cloning extract (SLiCE) cloning uses the endogenous recombination activity of cellular extracts to ligate insert and vector DNA fragments. An evaluation of the efficiency and utility of these methods is important in deciding the adoption of a seamless cloning method as a useful tool. In this study, both seamless cloning methods incorporated inserting DNA fragments into linearized DNA vectors through short (15-39 bp) end homology regions. However, colony formation was 30-60-fold higher with SLiCE cloning in end homology regions between 15 and 29 bp than with the iVEC method using DH5α competent cells. AQ3625 strains, which harbor a A gene mutation that activates the RecE homologous recombination pathway, can be used to efficiently ligate insert and vector DNA fragments with short-end homology regions . Using AQ3625 competent cells in the iVEC method improved the rate of colony formation, but the efficiency and accuracy of SLiCE cloning were still higher. In addition, the efficiency of seamless cloning methods depends on the intrinsic competency of cells. The competency of chemically competent AQ3625 cells was lower than that of competent DH5α cells, in all cases of chemically competent cell preparations using the three different methods. Moreover, SLiCE cloning permits the use of both homemade and commercially available competent cells because it can use general A strains such as DH5α as host cells for transformation. Therefore, between the two methods, SLiCE cloning provides both higher efficiency and better utility than the iVEC method for seamless DNA plasmid engineering.
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http://dx.doi.org/10.1016/j.bbrep.2017.01.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5614619PMC
March 2017

Seamless Ligation Cloning Extract (SLiCE) Method Using Cell Lysates from Laboratory Escherichia coli Strains and its Application to SLiP Site-Directed Mutagenesis.

Authors:
Ken Motohashi

Methods Mol Biol 2017 ;1498:349-357

Department of Bioresource and Environmental Sciences, Faculty of Life Sciences, Kyoto Sangyo University, Kamigamo Motoyama, Kita-ku, Kyoto, 603-8555, Japan.

Cell lysates from laboratory Escherichia coli strains endogenously exhibit homologous recombination activity, which can be utilized for seamless DNA cloning in vitro. This method, termed Seamless Ligation Cloning Extract (SLiCE) cloning, enables high cloning efficiency with simultaneous integration of two unpurified DNA fragments into a vector. In addition, the SLiCE method is highly cost-effective, as several laboratory E. coli strains may be utilized as sources of SLiCE. Previously, the SLiCE technique has been applied to site-directed mutagenesis to develop a novel technique termed SLiCE-mediated polymerase chain reaction (PCR)-based site-directed mutagenesis (SLiP site-directed mutagenesis). Two DNA fragments containing a mutation site can be simultaneously integrated into a vector while avoiding the introduction of undesirable mutations in the vector. Therefore, SLiP site-directed mutagenesis simplifies multiple procedures involved in PCR-based site-directed mutagenesis such as overlap extension method PCR or the Megaprimer method.
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http://dx.doi.org/10.1007/978-1-4939-6472-7_23DOI Listing
January 2018

Expression of spinach ferredoxin-thioredoxin reductase using tandem T7 promoters and application of the purified protein for in vitro light-dependent thioredoxin-reduction system.

Protein Expr Purif 2016 May 8;121:46-51. Epub 2016 Jan 8.

Department of Bioresource and Environmental Sciences, Faculty of Life Sciences, Kyoto Sangyo University, Kamigamo Motoyama, Kita-ku, Kyoto 603-8555, Japan. Electronic address:

Thioredoxins (Trxs) regulate the activity of target proteins in the chloroplast redox regulatory system. In vivo, a disulfide bond within Trxs is reduced by photochemically generated electrons via ferredoxin (Fd) and ferredoxin-thioredoxin reductase (FTR: EC 1.8.7.2). FTR is an αβ-heterodimer, and the β-subunit has a 4Fe-4S cluster that is indispensable for the electron transfer from Fd to Trxs. Reconstitution of the light-dependent Fd/Trx system, including FTR, is required for the biochemical characterization of the Trx-dependent reduction pathway in the chloroplasts. In this study, we generated functional FTR by simultaneously expressing FTR-α and -β subunits under the control of tandem T7 promoters in Escherichia coli, and purifying the resulting FTR complex protein. The purified FTR complex exhibited spectroscopic absorption at 410 nm, indicating that it contained the Fe-S cluster. Modification of the expression system and simplification of the purification steps resulted in improved FTR complex yields compared to those obtained in previous studies. Furthermore, the light-dependent Trx-reduction system was reconstituted by using Fd, the purified FTR, and intact thylakoids.
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http://dx.doi.org/10.1016/j.pep.2016.01.005DOI Listing
May 2016

Application of preparative disk gel electrophoresis for antigen purification from inclusion bodies.

Protein Expr Purif 2016 Feb 19;118:77-82. Epub 2015 Oct 19.

Department of Bioresource and Environmental Sciences, Faculty of Life Sciences, Kyoto Sangyo University, Kamigamo Motoyama, Kita-ku, Kyoto 603-8555, Japan. Electronic address:

Specific antibodies are a reliable tool to examine protein expression patterns and to determine the protein localizations within cells. Generally, recombinant proteins are used as antigens for specific antibody production. However, recombinant proteins from mammals and plants are often overexpressed as insoluble inclusion bodies in Escherichia coli. Solubilization of these inclusion bodies is desirable because soluble antigens are more suitable for injection into animals to be immunized. Furthermore, highly purified proteins are also required for specific antibody production. Plastidic acetyl-CoA carboxylase (ACCase: EC 6.4.1.2) from Arabidopsis thaliana, which catalyzes the formation of malonyl-CoA from acetyl-CoA in chloroplasts, formed inclusion bodies when the recombinant protein was overexpressed in E. coli. To obtain the purified protein to use as an antigen, we applied preparative disk gel electrophoresis for protein purification from inclusion bodies. This method is suitable for antigen preparation from inclusion bodies because the purified protein is recovered as a soluble fraction in electrode running buffer containing 0.1% sodium dodecyl sulfate that can be directly injected into immune animals, and it can be used for large-scale antigen preparation (several tens of milligrams).
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http://dx.doi.org/10.1016/j.pep.2015.10.008DOI Listing
February 2016

Chloroplastic thioredoxin m functions as a major regulator of Calvin cycle enzymes during photosynthesis in vivo.

Plant J 2015 Dec;84(5):900-13

Department of Bioresource and Environmental Sciences, Faculty of Life Sciences, Kyoto Sangyo University, Kamigamo Motoyama, Kita-ku, Kyoto, 603-8555, Japan.

Thioredoxins (Trxs) regulate the activity of various chloroplastic proteins in a light-dependent manner. Five types of Trxs function in different physiological processes in the chloroplast of Arabidopsis thaliana. Previous in vitro experiments have suggested that the f-type Trx (Trx f) is the main redox regulator of chloroplast enzymes, including Calvin cycle enzymes. To investigate the in vivo contribution of each Trx isoform to the redox regulatory system, we first quantified the protein concentration of each Trx isoform in the chloroplast stroma. The m-type Trx (Trx m), which consists of four isoforms, was the most abundant type. Next, we analyzed several Arabidopsis Trx-m-deficient mutants to elucidate the physiological role of Trx m in vivo. Deficiency of Trx m impaired plant growth and decreased the CO2 assimilation rate. We also determined the redox state of Trx target enzymes to examine their photo-reduction, which is essential for enzyme activation. In the Trx-m-deficient mutants, the reduction level of fructose-1,6-bisphosphatase and sedoheptulose-1,7-bisphosphatase was lower than that in the wild type. Inconsistently with the historical view, our in vivo study suggested that Trx m plays a more important role than Trx f in the activation of Calvin cycle enzymes.
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http://dx.doi.org/10.1111/tpj.13049DOI Listing
December 2015

A simple and ultra-low cost homemade seamless ligation cloning extract (SLiCE) as an alternative to a commercially available seamless DNA cloning kit.

Biochem Biophys Rep 2015 Dec 11;4:148-151. Epub 2015 Sep 11.

Department of Bioresource and Environmental Sciences, Faculty of Life Sciences, Kyoto Sangyo University, Kamigamo Motoyama, Kita-ku, Kyoto 603-8555, Japan.

The seamless ligation cloning extract (SLiCE) method is a novel seamless DNA cloning tool that utilizes homologous recombination activities in cell lysates to assemble DNA fragments into a vector. Several laboratory strains can be used as a source for the SLiCE extract; therefore, the SLiCE-method is highly cost-effective.The SLiCE has sufficient cloning ability to support conventional DNA cloning, and can simultaneously incorporate two unpurified DNA fragments into vector. Recently, many seamless DNA cloning kits have become commercially available; these are generally very convenient, but expensive. In this study, we evaluated the cloning efficiencies between a simple and highly cost-effective SLiCE-method and a commercial kit under various molar ratios of insert DNA fragments to vector DNA. This assessment identified that the SLiCE from a laboratory strain yielded 30-85% of the colony formation rate of a commercially available seamless DNA cloning kit. The cloning efficiencies of both methods were highly effective, exhibiting over 80% success rate under all conditions examined. These results suggest that SLiCE from a laboratory strain can efficiently function as an effective alternative to commercially available seamless DNA cloning kits.
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http://dx.doi.org/10.1016/j.bbrep.2015.09.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5668909PMC
December 2015

Evaluation of seamless ligation cloning extract preparation methods from an Escherichia coli laboratory strain.

Anal Biochem 2015 Oct 29;486:51-3. Epub 2015 Jun 29.

Department of Bioresource and Environmental Sciences, Faculty of Life Sciences, Kyoto Sangyo University, Kita-ku, Kyoto 603-8555, Japan. Electronic address:

Seamless ligation cloning extract (SLiCE) is a simple and efficient method for DNA cloning without the use of restriction enzymes. Instead, SLiCE uses homologous recombination activities from Escherichia coli cell lysates. To date, SLiCE preparation has been performed using an expensive commercially available lytic reagent. To expand the utility of the SLiCE method, we evaluated different methods for SLiCE preparation that avoid using this reagent. Consequently, cell extracts prepared with buffers containing Triton X-100, which is a common and low-cost nonionic detergent, exhibited sufficient cloning activity for seamless gene incorporation into a vector.
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http://dx.doi.org/10.1016/j.ab.2015.06.031DOI Listing
October 2015

A simple and efficient seamless DNA cloning method using SLiCE from Escherichia coli laboratory strains and its application to SLiP site-directed mutagenesis.

Authors:
Ken Motohashi

BMC Biotechnol 2015 Jun 3;15:47. Epub 2015 Jun 3.

Department of Bioresource and Environmental Sciences, Faculty of Life Sciences, Kyoto Sangyo University, Kamigamo Motoyama, Kita-ku, Kyoto, 603-8555, Japan.

Background: Seamless ligation cloning extract (SLiCE) is a simple and efficient method for DNA assembly that uses cell extracts from the Escherichia coli PPY strain, which expresses the components of the λ prophage Red/ET recombination system. This method facilitates restriction endonuclease cleavage site-free DNA cloning by performing recombination between short stretches of homologous DNA (≥ 15 base pairs).

Results: To extend the versatility of this system, I examined whether, in addition to bacterial extracts from the PPY strain, other E. coli laboratory strains were suitable for the SLiCE protocol. Indeed, carefully prepared cell extracts from several strains exhibited sufficient cloning activity for seamless gene incorporation into vectors with short homology lengths (approximately 15-20 bp). Furthermore, SLiCE was applied to the polymerase chain reaction (PCR)-based site-directed mutagenesis method, in a process termed "SLiCE-mediated PCR-based site-directed mutagenesis (SLiP site-directed mutagenesis)". SLiP site-directed mutagenesis simplifies the steps of PCR-based site-directed mutagenesis, as it exploits the capability of the SLiCE method to insert multiple fragments.

Conclusions: SLiCE can be performed in the laboratory with no requirement for a special E. coli strain, and the technique is easily established. This method increases the cloning efficiency, shortens the time for DNA manipulation, and greatly reduces the cost of seamless DNA cloning.
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http://dx.doi.org/10.1186/s12896-015-0162-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4453199PMC
June 2015

Method for enhancement of plant redox-related protein expression and its application for in vitro reduction of chloroplastic thioredoxins.

Protein Expr Purif 2014 Sep 10;101:152-6. Epub 2014 Jul 10.

Department of Bioresources and Environmental Sciences, Faculty of Life Sciences, Kyoto Sangyo University, Kamigamo Motoyama, Kita-ku, Kyoto 603-8555, Japan.

Plant redox-related proteins were overexpressed using a genetic codon substitution downstream of the translation initiation codon. This method significantly improved recombinant protein expression levels of Arabidopsis chloroplastic thioredoxins and cytosolic nicotinamide adenine dinucleotide phosphate (NADPH)-dependent thioredoxin reductase (E.C. 1.8.1.9) in Escherichia coli. Using these proteins, the in vitro chloroplastic thioredoxins-reduction system was reconstituted in an NADPH-dependent manner. This system could convert the five classes of chloroplastic Arabidopsis thioredoxins and two chloroplastic Spinach thioredoxins to their reduced forms, independent of dithiothreitol and the photosynthetic electron transport system.
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http://dx.doi.org/10.1016/j.pep.2014.07.001DOI Listing
September 2014

Systematic exploration of thioredoxin target proteins in plant mitochondria.

Plant Cell Physiol 2013 Jun 26;54(6):875-92. Epub 2013 Feb 26.

Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuta 4259-R1-8, Midori-ku, Yokohama 226-8503, Japan.

The thioredoxin (Trx) system is known to play a pivotal role in cellular redox regulation, but its target proteins in plant mitochondria remain largely uncharacterized. In this study, we systemically screened Trx target candidates in plant mitochondria. Mitochondrial protein extracts were prepared from Arabidopsis shoots, spinach leaves and potato tubers, and then subfractionated into soluble matrix and insoluble membrane fractions. Protein extracts were loaded onto an affinity column immobilizing Arabidopsis mitochondria-localized o-type Trx mutant protein, in which one of two internal cysteines at the active site was substituted by serine. Proteins forming mixed-disulfide intermediates with the mutated Trx were identified by proteomic approaches. This procedure allowed the determination of 101 Trx target candidate proteins involved in a broad spectrum of mitochondrial processes. Furthermore, biochemical assay revealed that one of the potential Trx target proteins, alternative oxidase, is actually redox regulated by Trx. This study provides insights into the regulatory mechanism of diverse functions in mitochondrial biology that are mediated through the Trx system.
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http://dx.doi.org/10.1093/pcp/pct037DOI Listing
June 2013

CcdA is a thylakoid membrane protein required for the transfer of reducing equivalents from stroma to thylakoid lumen in the higher plant chloroplast.

Antioxid Redox Signal 2010 Oct;13(8):1169-76

Department of Bioresource and Environmental Sciences, Faculty of Life Sciences, Kyoto Sangyo University, Kamigamo Motoyama, Kyoto, Japan.

In order to transfer reducing equivalents into the thylakoid lumen, a specific thylakoid membrane transfer system is suggested that mediates the disulfide bond reduction of proteins in the thylakoid lumen of higher plant chloroplasts. In this system, although stromal thioredoxin can supply the reducing equivalents to a thioredoxin-like protein HCF164 in the thylakoid lumen, a mediator protein for electron transfer in the thylakoid membranes is proposed to be required to link the two suborganellar compartments. CcdA is a candidate protein as a component for this transfer system since CcdA- and HCF164-deficient mutants in Arabidopsis thaliana show the same phenotype. We now show that CcdA is localized in the thylakoid membrane and that its redox state, as well as that of HCF164, is modulated in thylakoids by stromal m-type thioredoxin. Our results strongly suggest that CcdA may act as a mediator in thylakoid membranes by transferring reducing equivalents from the stromal to the lumenal side of the thylakoid membrane in chloroplasts.
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http://dx.doi.org/10.1089/ars.2010.3138DOI Listing
October 2010

Regulation of translation by the redox state of elongation factor G in the cyanobacterium Synechocystis sp. PCC 6803.

J Biol Chem 2009 Jul 15;284(28):18685-91. Epub 2009 May 15.

Department of Biochemistry and Molecular Biology, Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570, Japan.

Elongation factor G (EF-G), a key protein in translational elongation, was identified as a primary target of inactivation by reactive oxygen species within the translational machinery of the cyanobacterium Synechocystis sp. PCC 6803 (Kojima, K., Oshita, M., Nanjo, Y., Kasai, K., Tozawa, Y., Hayashi, H., and Nishiyama, Y. (2007) Mol. Microbiol. 65, 936-947). In the present study, we found that inactivation of EF-G (Slr1463) by H(2)O(2) was attributable to the oxidation of two specific cysteine residues and formation of a disulfide bond. Substitution of these cysteine residues by serine residues protected EF-G from inactivation by H(2)O(2) and allowed the EF-G to mediate translation in a translation system in vitro that had been prepared from Synechocystis. The disulfide bond in oxidized EF-G was reduced by thioredoxin, and the resultant reduced form of EF-G regained the activity to mediate translation in vitro. Western blotting analysis showed that levels of the oxidized form of EF-G increased under strong light in a mutant that lacked NADPH-thioredoxin reductase, indicating that EF-G is reduced by thioredoxin in vivo. These observations suggest that the translational machinery is regulated by the redox state of EF-G, which is oxidized by reactive oxygen species and reduced by thioredoxin, a transmitter of reducing signals generated by the photosynthetic transport of electrons.
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http://dx.doi.org/10.1074/jbc.M109.015131DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2707220PMC
July 2009

Identification of thioredoxin targeted proteins using thioredoxin single cysteine mutant-immobilized resin.

Methods Mol Biol 2009 ;479:117-31

Chemical Resources Laboratory, Tokyo Institute of Technology, Yokohama, Japan.

Thioredoxins (Trx) are a ubiquitous family of proteins that modulate the enzymatic activity of their substrate proteins by redox regulation. This is achieved by reduction of a disulfide bond within their target proteins. A conserved pair of cysteine residues in Trx is required for catalysis of the dithiol-disulfide exchange with their target proteins. A single-cysteine mutant capable of forming a stable mixed disulfide bond with target proteins was immobilized on resin and used to capture potential target proteins. By using this method, a number of previously unidentified Trx-target protein candidates were captured from various organisms and organelles. Following the development of this technique, more than one hundred proteins have been reported as potential Trx targets, allowing significant progress to be made in our knowledge and understanding of Trx-target proteins.
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http://dx.doi.org/10.1007/978-1-59745-289-2_8DOI Listing
March 2009

Functional analysis of Arabidopsis thaliana isoforms of the Mg-chelatase CHLI subunit.

Photochem Photobiol Sci 2008 Oct 9;7(10):1188-95. Epub 2008 Jun 9.

Department of General Systems Studies, Graduate School of Arts and Sciences, The University of Tokyo, Komaba 3-8-1, Tokyo, 153-8902, Japan.

The first step of chlorophyll biosynthesis is catalyzed by a Mg-chelatase composed of the subunits CHLI, CHLD and CHLH. Mg-chelatase requires ATP hydrolysis that can be attributed to CHLI. Arabidopsis has two CHLI isoforms, CHLI1 and CHLI2, that have similar expression profiles, but it has been suggested that CHLI2 has limited function in the Mg-chelatase complex. Recently, we showed that Arabidopsis CHLI1 is an ATPase and a target of chloroplast thioredoxin. Here, we demonstrate that CHLI2 also has ATPase activity but with a lower Vmax and higher Km ATP than CHLI1. We confirmed the thioredoxin-dependent reduction of a disulfide bond in CHLI2 and thiol-modulation of its ATPase activity. We then examined the physiological contribution of CHLI2 using a chli2 T-DNA knockout line. Although visible phenotype of homozygous chli2 mutants was almost comparable to wild type, the mutant accumulated significantly less chlorophyll. Furthermore, cs/cs; chli2/chli2 double mutants were almost albino. There were three phenotypes among progenies segregated from the cs/cs; CHLI2/chli2 parent: cs-like pale green, yellow, and almost albino were obtained in the approximate ratio of 1:2:0.7. PCR analysis confirmed that the chli2 mutation is semidominant on a homozygous cs background. These results reveal that although CHLI2 plays a limited role in chlorophyll biosynthesis, this subunit certainly contributes to the assembly of the Mg-chelatase complex.
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http://dx.doi.org/10.1039/b802604cDOI Listing
October 2008

Binary reducing equivalent pathways using NADPH-thioredoxin reductase and ferredoxin-thioredoxin reductase in the cyanobacterium Synechocystis sp. strain PCC 6803.

Plant Cell Physiol 2008 Jan 14;49(1):11-8. Epub 2007 Nov 14.

Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuta 4259-R1-8, Midori-Ku, Yokohama, 226-8503 Japan.

Thioredoxin (Trx) is a small ubiquitous protein involved in the disulfide-dithiol exchange reaction occurring in cells and organelles. In vivo, Trx is reduced by Trx reductase using NADPH or photosynthetically produced reducing equivalents, and the reduced form Trx takes on the physiological functions. In the cyanobacterium Synechocystis sp. PCC6803, two Trx reductases, ferredoxin-Trx reductase (FTR) and NADPH-Trx reductase (NTR), and four typical Trx isoforms have been identified by genomic analysis. Based on analysis of the physiological features of the Trx reductase disruptants, we found that the NTR-Trx pathway is important for the antioxidant system, whereas the FTR-Trx pathway may play a more important role in the control of cell growth rate. In addition, by quantification of Trx abundance in the wild-type and the disruptant Synechocystis cells, we found that the gene product of slr0623, the homolog of m-type Trx in higher plants, is the most abundant Trx, and that accumulation of Trx isoforms occurs dependent on the expression of the other redox-related proteins. A study of the binary reducing equivalent pathways in cyanobacterial cells is reported here.
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http://dx.doi.org/10.1093/pcp/pcm158DOI Listing
January 2008

The significance of type II and PrxQ peroxiredoxins for antioxidative stress response in the purple bacterium Rhodobacter sphaeroides.

J Biol Chem 2007 Sep 20;282(38):27792-801. Epub 2007 Jul 20.

Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama 226-8501, Japan.

Two peroxiredoxins, classified as Type II and PrxQ, were characterized in the purple non-sulfur photosynthetic bacterium Rhodobacter sphaeroides. Both recombinant proteins showed remarkable thioredoxin-dependent peroxidase activity with broad substrate specificity in vitro. Nevertheless, PrxQ of R. sphaeroides, unlike typical PrxQs studied to date, does not contain one of the two conserved catalytic Cys residues. We found that R. sphaeroides PrxQ and other PrxQ-like proteins from several organisms conserve a different second Cys residue, indicating that these proteins should be categorized into a novel PrxQ subfamily. Disruption of either the Type II or PrxQ gene in R. sphaeroides had a dramatic effect on cell viability when the cells were grown under aerobic light or oxidative stress conditions created by exogenous addition of reactive oxygen species to the medium. Growth rates of the mutants were significantly decreased compared with that of wild type under aerobic but not anaerobic conditions. These results indicate that the peroxiredoxins are crucial for antioxidative stress response in this bacterium. The gene disruptants also demonstrated reduced levels of photopigment synthesis, suggesting that the peroxiredoxins are directly or indirectly involved in regulated synthesis of the photosynthetic apparatus.
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http://dx.doi.org/10.1074/jbc.M702855200DOI Listing
September 2007

The CHLI1 subunit of Arabidopsis thaliana magnesium chelatase is a target protein of the chloroplast thioredoxin.

J Biol Chem 2007 Jul 1;282(27):19282-91. Epub 2007 May 1.

Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta 4259, Yokohama 226-8501, Japan.

Insertion of magnesium into protoporphyrin IX by magnesium chelatase is a key step in the chlorophyll biosynthetic pathway, which takes place in plant chloroplasts. ATP hydrolysis by the CHLI subunit of magnesium chelatase is an essential component of this reaction, and the activity of this enzyme is a primary determinant of the rate of magnesium insertion into the chlorophyll molecule (tetrapyrrole ring). Higher plant CHLI contains highly conserved cysteine residues and was recently identified as a candidate protein in a proteomic screen of thioredoxin target proteins (Balmer, Y., Koller, A., del Val, G., Manieri, W., Schurmann, P., and Buchanan, B. B. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 370-375). To study the thioredoxin-dependent regulation of magnesium chelatase, we first investigated the effect of thioredoxin on the ATPase activity of CHLI1, a major isoform of CHLI in Arabidopsis thaliana. The ATPase activity of recombinant CHLI1 was found to be fully inactivated by oxidation and easily recovered by thioredoxin-assisted reduction, suggesting that CHLI1 is a target protein of thioredoxin. Moreover, we identified one crucial disulfide bond located in the C-terminal helical domain of CHLI1 protein, which may regulate the binding of the nucleotide to the N-terminal catalytic domain. The redox state of CHLI was also found to alter in a light-dependent manner in vivo. Moreover, we successfully observed stimulation of the magnesium chelatase activity in isolated chloroplasts by reduction. Our findings strongly suggest that chlorophyll biosynthesis is subject to chloroplast biogenesis regulation networks to coordinate them with the photosynthetic pathways in chloroplasts.
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http://dx.doi.org/10.1074/jbc.M703324200DOI Listing
July 2007

HCF164 receives reducing equivalents from stromal thioredoxin across the thylakoid membrane and mediates reduction of target proteins in the thylakoid lumen.

J Biol Chem 2006 Nov 22;281(46):35039-47. Epub 2006 Sep 22.

The ATP System Project, ERATO, JST, Nagatsuta 5800-3, Midori-ku, Yokohama, Japan.

HCF164 is a membrane-anchored thioredoxin-like protein known to be indispensable for assembly of cytochrome b6 f in the thylakoid membranes. In this study, we report the finding that chloroplast stroma m-type thioredoxin is the source of reducing equivalents for reduction of HCF164 in the thylakoid lumen, providing strong evidence that higher plant chloroplasts possess a trans-membrane reducing equivalent transfer system similar to that found in bacteria. To probe the function of HCF164 in the lumen, a screen to identify the reducing equivalent acceptor proteins of HCF164 was carried out by using a resin-immobilized HCF164 single cysteine mutant, leading to the isolation of putative target thylakoid proteins. Among the newly identified target proteins, the reduction of the PSI-N subunit of photosystem I by HCF164 was confirmed both in vitro and in isolated thylakoids. Two components of the cytochrome b6 f complex, the cytochrome f and Rieske FeS proteins, were also identified as novel potential target proteins. The data presented here suggest that HCF164 serves as an important transducer of reducing equivalents to proteins in the thylakoid lumen.
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http://dx.doi.org/10.1074/jbc.M605938200DOI Listing
November 2006

Thioredoxin-h1 reduces and reactivates the oxidized cytosolic malate dehydrogenase dimer in higher plants.

J Biol Chem 2006 Oct 31;281(43):32065-71. Epub 2006 Aug 31.

Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuta 4259-R1-8, Midori-Ku, Yokohama 226-8503, Japan.

Cytosolic malate dehydrogenase (cytMDH) was captured by thioredoxin affinity chromatography as a possible target protein of cytosolic thioredoxin (Yamazaki, D., Motohashi, K., Kasama, T., Hara, Y., and Hisabori, T. (2004) Plant Cell Physiol. 45, 18-27). To further dissect this interaction, we aimed to determine whether cytMDH can interact with the cytosolic thioredoxin and whether its activity is redox-regulated. We obtained the active recombinant cytMDH that could be oxidized and rendered inactive. Inactivation was reversed by incubation with low concentrations of dithiothreitol in the presence of recombinant Arabidopsis thaliana thioredoxin-h1. Inactivation of cytMDH was found to result from formation of a homodimer. By cysteine mutant analysis and peptide mapping analysis, we were able to determine that the cytMDH homodimer occurs by formation of a disulfide bond via the Cys(330) residue. Moreover, we found this bond to be efficiently reduced by the reduced form of thioredoxin-h1. These results demonstrate that the oxidized form cytMDH dimer is a preferable target protein of the reduced form thioredoxin-h1 as suggested by thioredoxin affinity chromatography.
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http://dx.doi.org/10.1074/jbc.M605784200DOI Listing
October 2006

In vitro reconstitution of monogalactosyldiacylglycerol (MGDG) synthase regulation by thioredoxin.

FEBS Lett 2006 Jul 30;580(17):4086-90. Epub 2006 Jun 30.

Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259-B-14 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8501, Japan.

Monogalactosyldiacylglycerol (MGDG), a major membrane lipid of chloroplasts, is synthesized by MGDG synthase (MGD) localized in chloroplast envelope membranes. We investigated whether MGD activity is regulated in a redox-dependent manner using recombinant cucumber MGD overexpressed in Escherichia coli. We found that MGD activity is reversibly regulated by reduction and oxidation in vitro and that an intramolecular disulfide bond(s) is involved in MGD activation. Because thioredoxin efficiently reduced disulfide bonds to enhance MGD activity in vitro, MGD is potentially an envelope-bound thioredoxin target protein in higher plants.
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http://dx.doi.org/10.1016/j.febslet.2006.06.050DOI Listing
July 2006
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