x- and y-type thioredoxins maintain redox homeostasis on photosystem I acceptor side under fluctuating light.

Plants cope with sudden increases in light intensity through various photoprotective mechanisms. Redox regulation by thioredoxin (Trx) systems also contributes to this process. Whereas the functions of f- and m-type Trxs in response to such fluctuating light conditions have been extensively investigated, those of x- and y-type Trxs are largely unknown. Here, we analyzed the trx x single, trx y1 trx y2 double, and trx x trx y1 trx y2 triple mutants in Arabidopsis (Arabidopsis thaliana). A detailed analysis of photosynthesis revealed changes in photosystem I (PSI) parameters under low light in trx x and trx x trx y1 trx y2. The electron acceptor side of PSI was more reduced in these mutants than in the wild type. This mutant phenotype was more pronounced under fluctuating light conditions. During both low- and high-light phases, the PSI acceptor side was largely limited in trx x and trx x trx y1 trx y2. After fluctuating light treatment, we observed more severe PSI photoinhibition in trx x and trx x trx y1 trx y2 than in the wild type. Furthermore, when grown under fluctuating light conditions, trx x and trx x trx y1 trx y2 plants showed impaired growth and decreased level of PSI subunits. These results suggest that Trx x and Trx y prevent redox imbalance on the PSI acceptor side, which is required to protect PSI from photoinhibition, especially under fluctuating light. We also propose that Trx x and Trx y contribute to maintaining the redox balance even under constant low-light conditions to prepare for sudden increases in light intensity.

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

In addition to linear electron transport, photosystem I cyclic electron transport (PSI-CET) contributes to photosynthesis and photoprotection. In Arabidopsis (Arabidopsis thaliana), 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 m-type thioredoxins (Trx m). Genetic and phenotypic characterizations of multiple mutants indicated the physiological interaction between Trx m 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 m specifically decreased the PGR5/PGRL1-dependent plastoquinone reduction. In planta, Trx m4 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 m4-PGRL1 complex formation. Furthermore, the Trx m4-PGRL1 complex was transiently dissociated during the induction of photosynthesis. We propose that Trx m directly regulates the PGR5/PGRL1-dependent pathway by complex formation with PGRL1.

Maintaining the Chloroplast Redox Balance through the PGR5-Dependent Pathway and the Trx System Is Required for Light-Dependent Activation of Photosynthetic Reactions.

Light-dependent activation of chloroplast enzymes is required for the rapid induction of photosynthesis after a shift from dark to light. The thioredoxin (Trx) system plays a central role in this process. In chloroplasts, the Trx system consists of two pathways: the ferredoxin (Fd)/Trx pathway and the nicotinamide adenine dinucleotide phosphate (NADPH)-Trx reductase C (NTRC) pathway. In Arabidopsis (Arabidopsis thaliana) mutants defective in either pathway, the photoreduction of thiol enzymes was impaired, resulting in decreased carbon fixation. The close relationship between the Fd/Trx pathway and proton gradient regulation 5 (PGR5)-dependent photosystem I cyclic electron transport (PSI CET) in the induction of photosynthesis was recently elucidated. However, how the PGR5-dependent pathway is involved in the NTRC pathway is unclear, although NTRC has been suggested to physically interact with PGR5. In this study, we analyzed Arabidopsis mutants lacking either the PGR5 or the chloroplast NADH dehydrogenase-like complex (NDH)-dependent PSI CET pathway in the ntrc mutant background. The ntrc pgr5 double mutant suppressed both the growth defects and the high non-photochemical quenching phenotype of the ntrc mutant when grown under long-day conditions. By contrast, the inactivation of NDH activity with the chlororespiratory reduction 2-2 mutant failed to suppress either phenotype. We discovered that the phenotypic rescue of ntrc by pgr5 is caused by the partial restoration of Trx-dependent reduction of thiol enzymes. These results suggest that electron partitioning to the PGR5-dependent pathway and the Trx system needs to be properly regulated for the activation of the Calvin-Benson-Bassham cycle enzymes during the induction of photosynthesis.

Functional division of f-type and m-type thioredoxins to regulate the Calvin cycle and cyclic electron transport around photosystem I.

Redox regulation of chloroplast proteins is necessary to adjust photosynthetic performance with changes in light. The thioredoxin (Trx) system plays a central role in this process. Chloroplast-localized classical Trx is a small redox-active protein that regulates many target proteins by reducing their disulfide bonds in a light-dependent manner. Arabidopsis thaliana mutants lacking f-type Trx (trx f1f2) or m-type Trx (trx m124-2) have been reported to show delayed reduction of Calvin cycle enzymes. As a result, the trx m124-2 mutant exhibits growth defects. Here, we characterized a quintuple mutant lacking both Trx f and Trx m to investigate the functional complementarity of Trx f and Trx m. The trx f1f2 m124-2 quintuple mutant was newly obtained by crossing, and is analyzed here for the first time. The growth defects of the trx m124-2 mutant were not enhanced by the lack of Trx f. In contrast, deficiencies of both Trxs additively suppressed the reduction of Calvin cycle enzymes, resulting in a further delay in the initiation of photosynthesis. Trx f appeared to be necessary for the rapid activation of the Calvin cycle during the early induction of photosynthesis. To perform effective photosynthesis, plants seem to use both Trxs in a coordinated manner to activate carbon fixation reactions. In contrast, the PROTON GRADIENT REGULATION 5 (PGR5)-dependent cyclic electron transport around photosystem I was regulated by Trx m, but not by Trx f. Lack of Trx f did not affect the activity and regulation of the PGR5-dependent pathway. Trx f may have a higher specificity for target proteins, whereas Trx m has a variety of target proteins to regulate overall photosynthesis and other metabolic reactions in the chloroplasts.

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

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 (Arabidopsis thaliana) mutants lacking f-type Trxs (trx f1f2) 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 trx f1f2 mutant background. The cytochrome b 6 f 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 pgr1 mutation, which is conditionally defective in cytochrome b 6 f complex activity, into the trx f1f2 mutant background did not further affect the induction of photosynthesis, but the combined deficiency of Trx f and PGR5 severely impaired photosynthesis and suppressed plant growth under long-day conditions. In the pgr5 trx f1f2 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 trx f1f2 and pgr5 trxf1f2 mutants. The pgr5 mutation did not result in further impaired photoreduction of Calvin-Benson cycle enzymes or ATP synthase in the trx f1f2 mutant background. These results indicated that acceptor-side limitations in the pgr5 trx f1f2 mutant suppress photosynthesis initiation, suggesting that PGR5 is required for efficient photosynthesis induction.

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

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.

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

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 αß-heterodimer, and the ß-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 -ß 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.

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

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).

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

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.

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

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.

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

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.

Systematic exploration of thioredoxin target proteins in plant mitochondria.

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.

Two disulfide-reducing pathways are required for the maturation of plastid c-type cytochromes in Chlamydomonas reinhardtii.

In plastids, conversion of light energy into ATP relies on cytochrome f, a key electron carrier with a heme covalently attached to a CXXCH motif. Covalent heme attachment requires reduction of the disulfide-bonded CXXCH by CCS5 and CCS4. CCS5 receives electrons from the oxidoreductase CCDA, while CCS4 is a protein of unknown function. In Chlamydomonas reinhardtii, loss of CCS4 or CCS5 yields a partial cytochrome f assembly defect. Here, we report that the ccs4ccs5 double mutant displays a synthetic photosynthetic defect characterized by a complete loss of holocytochrome f assembly. This defect is chemically corrected by reducing agents, confirming the placement of CCS4 and CCS5 in a reducing pathway. CCS4-like proteins occur in the green lineage, and we show that HCF153, a distant ortholog from Arabidopsis thaliana, can substitute for Chlamydomonas CCS4. Dominant suppressor mutations mapping to the CCS4 gene were identified in photosynthetic revertants of the ccs4ccs5 mutants. The suppressor mutations yield changes in the stroma-facing domain of CCS4 that restore holocytochrome f assembly above the residual levels detected in ccs5. Because the CCDA protein accumulation is decreased specifically in the ccs4 mutant, we hypothesize the suppressor mutations enhance the supply of reducing power through CCDA in the absence of CCS5. We discuss the operation of a CCS5-dependent and a CCS5-independent pathway controlling the redox status of the heme-binding cysteines of apocytochrome f.

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

The chloroplast-localized cystathionine ß-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 in vitro and in vivo. The in vitro biochemical studies evaluated whether CBSX proteins alter the specificities of classical chloroplastic Trx f and Trx m for their target proteins. However, addition of CBSX proteins did not alter the specificities of Trx f and Trx m for disulfide bond reduction of the photosynthesis-related major thiol enzymes, FBPase, SBPase, and NADP-MDH. In vivo 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 cbsx2 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.

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

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. Arabidopsis pgr5-tcr 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.

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

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.

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

The activity of the molecular motor enzyme, chloroplast ATP synthase, is regulated in a redox-dependent manner. The γ subunit, CF1-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 CF1-γ. Previous studies showed that the light-dependent reduction of CF1-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 CF1-γ, especially under physiological conditions. We therefore performed direct assessment of the CF1-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 CF1-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 CF1-γ. The results showed that CF1-γ can accept the reducing power from both Trx-f and Trx-m in chloroplasts.

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

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 Rorippa aquatica (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 R. aquatica. To test this hypothesis, the viability of R. aquatica and Arabidopsis thaliana leaf explants were compared, with respect to the maintenance of photosynthetic activity, senescence, and immune response. Time-course analyses of photosynthetic activity revealed that R. aquatica leaf explants can survive longer than those of A. thaliana. Endogenous abscisic acid (ABA) and jasmonic acid (JA) were found at low levels in leaf explant of R. aquatica. Time-course transcriptome analysis of R. aquatica and A. thaliana leaf explants suggested that senescence was suppressed at the transcriptional level in R. aquatica. 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.

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

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.

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.

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.