Cloning and Organelle Expression of Bamboo Mitochondrial Complex I Subunits Nad1, Nad2, Nad4, and Nad5 in the Yeast Saccharomyces cerevisiae.

Mitochondrial respiratory complex I catalyzes electron transfer from NADH to ubiquinone and pumps protons from the matrix into the intermembrane space. In particular, the complex I subunits Nad1, Nad2, Nad4, and Nad5, which are encoded by the nad1, nad2, nad4, and nad5 genes, reside at the mitochondrial inner membrane and possibly function as proton (H+) and ion translocators. To understand the individual functional roles of the Nad1, Nad2, Nad4, and Nad5 subunits in bamboo, each cDNA of these four genes was cloned into the pYES2 vector and expressed in the mitochondria of the yeast Saccharomyces cerevisiae. The mitochondrial targeting peptide mt gene (encoding MT) and the egfp marker gene (encoding enhanced green fluorescent protein, EGFP) were fused at the 5'-terminal and 3'-terminal ends, respectively. The constructed plasmids were then transformed into yeast. RNA transcripts and fusion protein expression were observed in the yeast transformants. Mitochondrial localizations of the MT-Nad1-EGFP, MT-Nad2-EGFP, MT-Nad4-EGFP, and MT-Nad5-EGFP fusion proteins were confirmed by fluorescence microscopy. The ectopically expressed bamboo subunits Nad1, Nad2, Nad4, and Nad5 may function in ion translocation, which was confirmed by growth phenotype assays with the addition of different concentrations of K+, Na+, or H+.

Optimal timing of blastocyst vitrification after trophectoderm biopsy for preimplantation genetic screening.

Is the timing of vitrification after trophectoderm (TE) biopsy associated with successful implantation and pregnancy after the embryo transfer of blastocysts subjected to preimplantation genetic screening (PGS)? In this retrospective cohort study, 1329 blastocysts from 223 patients were subjected to TE biopsy for performing array comparative genomic hybridization (CGH) tests. The PGS and frozen blastocyst transfer (FET) cycles were performed from December 2012 to May 2015. Only the good quality and expanded blastocysts on day 5 or 6 were selected for biopsy. After TE biopsy, the re-expansion grades relative to the original blastocoel were (1) collapsed blastocysts (CB), (2) 3/4 re-expansion but not full expansion (RE), and (3) full re-expansion or hatching (FE). All biopsied blastocysts were subjected to vitrification within 0.5-6 h after biopsy; the time intervals between TE biopsy and vitrification and the expansion grades at the time of vitrification were recorded. By combining two factors, namely the expansion grades and culture intervals between biopsy and vitrification, the patients were further divided into four groups, namely CB with a < 3 h culture interval (n = 34 cycles, Group I), RE and FE blastocysts with a < 3 h culture interval (n = 10 cycles, Group II); CB blastocysts with a ≥ 3 h culture interval (n = 6 cycles, Group III); and RE or FE blastocysts with a ≥ 3 h culture interval (n = 173 cycles, Group IV). The implantation (63.7%, 179/281) and clinical pregnancy (74.0%, 128/173) rates in Group IV were significantly higher than those in Group I (45.3%, 24/53; 50.0%, 17/34; P = 0.012 and 0.005, respectively). According to our findings, optimal vitrification timing > 3 hours to enable blastocysts to reach RE or FE provides improved implantation and pregnancy rates after FET. TRIAL REGISTRATION: ClinicalTrials.gov NCT03065114.

Leaf structure affects a plant's appearance: combined multiple-mechanisms intensify remarkable foliar variegation.

The presence of foliar variegation challenges perceptions of leaf form and functioning. But variegation is often incorrectly identified and misinterpreted. The striking variegation found in juvenile Blastus cochinchinensis (Melastomataceae) provides an instructive case study of mechanisms and their ecophysiological implications. Variegated (white and green areas, vw and vg) and non-variegated leaves (normal green leaves, ng) of seedlings of Blastus were compared structurally with microtechniques, and characterized for chlorophyll content and fluorescence. More limited study of Sonerila heterostemon (Melastomataceae) and Kaempferia pulchra (Zingiberaceae) tested the generality of the findings. Variegation in Blastus combines five mechanisms: epidermal, air space, upper mesophyll, chloroplast and crystal, the latter two being new mechanisms. All mesophyll cells (vw, vg, ng) have functional chloroplasts with dense thylakoids. The vw areas are distinguished by flatter adaxial epidermal cells and central trichomes containing crystals, the presence of air spaces between the adaxial epidermis and a colorless spongy-like upper mesophyll containing smaller and fewer chloroplasts. The vw area is further distinguished by having the largest spongy-tissue chloroplasts and fewer stomata. Both leaf types have similar total chlorophyll content and similar  F v/F m (maximum quantum yield of PSII), but vg has significantly higher F v/F m than ng. Variegation in Sonerila and Kaempferia is also caused by combined mechanisms, including the crystal type in Kaempferia. This finding of combined mechanisms in three different species suggests that combined mechanisms may occur more commonly in nature than current understanding. The combined mechanisms in Blastus variegated leaves represent intricate structural modifications that may compensate for and minimize photosynthetic loss, and reflect changing plant needs.

Knockdown of PsbO leads to induction of HydA and production of photobiological H2 in the green alga Chlorella sp. DT.

Green algae are able to convert solar energy to H2 via the photosynthetic electron transport pathway under certain conditions. Algal hydrogenase (HydA, encoded by HYDA) is in charge of catalyzing the reaction: 2H(+)+2e(-)↔H2 but usually inhibited by O2, a byproduct of photosynthesis. The aim of this study was to knockdown PsbO (encoded by psbO), a subunit concerned with O2 evolution, so that it would lead to HydA induction. The alga, Chlorella sp. DT, was then transformed with short interference RNA antisense-psbO (siRNA-psbO) fragments. The algal mutants were selected by checking for the existence of siRNA-psbO fragments in their genomes and the low amount of PsbO proteins. The HYDA transcription and the HydA expression were observed in the PsbO-knockdown mutants. Under semi-aerobic condition, PsbO-knockdown mutants could photobiologically produce H2 which increased by as much as 10-fold in comparison to the wild type.

Natural foliar variegation without costs? The case of Begonia.

BACKGROUND AND AIMS: Foliar variegation is recognized as arising from two major mechanisms: leaf structure and pigment-related variegation. Begonia has species with a variety of natural foliar variegation patterns, providing diverse examples of this phenomenon. The aims of this work are to elucidate the mechanisms underlying different foliar variegation patterns in Begonia and to determine their physiological consequences. METHODS: Six species and one cultivar of Begonia were investigated. Light and electron microscopy revealed the leaf structure and ultrastructure of chloroplasts in green and light areas of variegated leaves. Maximum quantum yields of photosystem II were measured by chlorophyll fluorescence. Comparison with a cultivar of Ficus revealed key features distinguishing variegation mechanisms. KEY RESULTS: Intercellular space above the chlorenchyma is the mechanism of variegation in these Begonia. This intercellular space can be located (a) below the adaxial epidermis or (b) below the adaxial water storage tissue (the first report for any taxa), creating light areas on a leaf. In addition, chlorenchyma cell shape and chloroplast distribution within chlorenchyma cells differ between light and green areas. Chloroplasts from both areas showed dense stacking of grana and stroma thylakoid membranes. The maximum quantum yield did not differ significantly between these areas, suggesting minimal loss of function with variegation. However, the absence of chloroplasts in light areas of leaves in the Ficus cultivar led to an extremely low quantum yield. CONCLUSIONS: Variegation in these Begonia is structural, where light areas are created by internal reflection between air spaces and cells in a leaf. Two forms of air space structural variegation occur, distinguished by the location of the air spaces. Both forms may have a common origin in development where dermal tissue becomes loosely connected to mesophyll. Photosynthetic functioning is retained in light areas, and these areas do not include primary veins, potentially limiting the costs of variegation.

Mitochondrial energy metabolism in young bamboo rhizomes from Bambusa oldhamii and Phyllostachys edulis during shooting stage.

The energy metabolism of mitochondria in young rhizomes of the bamboo species Bambusa oldhamii, which favors shooting during the summer, and Phyllostachys edulis, which favors shooting during the winter, was characterized. The mitochondrial energy-converting system was clarified in terms of respiratory activity and structural organization. The respiration rates were measured at 15, 28, and 42 °C by NADH, succinate, and malate oxidation. NADH was shown to act as an efficient substrate regardless of the temperature. The structural organization of functional mitochondrial respiratory supercomplexes was studied using blue native PAGE and in-gel activity staining. In both species, almost 90% of the total complex I was assembled into supercomplexes, and P. edulis contained a greater amount of complex-I-comprising supercomplexes than B. oldhamii. Approximately 50% of complex III and 75% of complex V were included in supercomplexes, whereas P. edulis mitochondria possessed a greater amount of complex-V-comprising supercomplexes. The alternative oxidase (AOX), plant mitochondrial uncoupling protein (PUCP), plant mitochondrial potassium channel (PmitoK(ATP)), rotenone-insensitive external/internal NADH:ubiquinone oxidoreductase [NDH(e/i)], and superoxide dismutase (SOD) activities of the energy-dissipating systems were investigated. P. edulis mitochondria had higher levels of the PUCP1 and AOX1 proteins than B. oldhamii mitochondria. The activity of PmitoK(ATP) in P. edulis was higher than that in B. oldhamii. However, P. edulis mitochondria possessed lower NDH(e/i) and SOD activities than B. oldhamii mitochondria. The results suggest that the adaptation of P. edulis to a cooler environment may correlate with its greater abundance of functional mitochondrial supercomplexes and the higher energy-dissipating capacity of its AOX, PUCP and PmitoK(ATP) relative to B. oldhamii.

A new approach to image copy detection based on extended feature sets.

Conventional image copy detection research concentrates on finding features that are robust enough to resist various kinds of image attacks. However, finding a globally effective fealure is difficult and, in many cases, domain dependent. Instead of imply extracting features from copyrighted images directly, we propose a new framework called the extended feature set for detecting copies of images. In our approach, virtual prior attacks are applied to copyrighted images to generate novel features, which serve as training data. The copy-detection problem can be solved by learning classifiers from the training data, thus, generated. Our approach can be integrated into existing copy detectors to further improve their performance. Experiment results demonstrate that the proposed approach can substantially enhance the accuracy of copy detection.

Functional roles of arginine residues in mung bean vacuolar H+-pyrophosphatase.

Plant vacuolar H+-translocating inorganic pyrophosphatase (V-PPase EC 3.6.1.1) utilizes inorganic pyrophosphate (PPi) as an energy source to generate a H+ gradient potential for the secondary transport of ions and metabolites across the vacuole membrane. In this study, functional roles of arginine residues in mung bean V-PPase were determined by site-directed mutagenesis. Alignment of amino-acid sequence of K+-dependent V-PPases from several organisms showed that 11 of all 15 arginine residues were highly conserved. Arginine residues were individually substituted by alanine residues to produce R-->A-substituted V-PPases, which were then heterologously expressed in yeast. The characteristics of mutant variants were subsequently scrutinized. As a result, most R-->A-substituted V-PPases exhibited similar enzymatic activities to the wild-type with exception that R242A, R523A, and R609A mutants markedly lost their abilities of PPi hydrolysis and associated H+-translocation. Moreover, mutation on these three arginines altered the optimal pH and significantly reduced K+-stimulation for enzymatic activities, implying a conformational change or a modification in enzymatic reaction upon substitution. In particular, R242A performed striking resistance to specific arginine-modifiers, 2,3-butanedione and phenylglyoxal, revealing that Arg242 is most likely the primary target residue for these two reagents. The mutation at Arg242 also removed F- inhibition that is presumably derived from the interfering in the formation of substrate complex Mg2+-PPi. Our results suggest accordingly that active pocket of V-PPase probably contains the essential Arg242 which is embedded in a more hydrophobic environment.

Expression of mercuric reductase from Bacillus megaterium MB1 in eukaryotic microalga Chlorella sp. DT: an approach for mercury phytoremediation.

A eukaryotic microalga, Chlorella sp. DT, was transformed with the Bacillus megaterium strain MB1 merA gene, encoding mercuric reductase (MerA), which mediates the reduction of Hg2+ to volatile elemental Hg0. The transformed Chlorella cells were selected first by hygromycin B and then by HgCl2. The existence of merA gene in the genomic DNA of transgenic strains was shown by polymerase chain reaction amplification, while the stable integration of merA into genomic DNA of transgenic strains was confirmed by Southern blot analysis. The ability to remove Hg2+ in merA transgenic strains was higher than that in the wild type. The merA transgenic strains showed higher growth rate and photosynthetic activity than the wild type did in the presence of a toxic concentration of Hg2+. Cultured with Hg2+, the expression level of superoxide dismutase in transgenic strains was lower than that in the wild type, suggesting that the transgenic strains faced a lower level of oxidative stress. All the results indicated that merA gene was successfully integrated into the genome of transgenic strains and functionally expressed to promote the removal of Hg2+.

Proton pumping inorganic pyrophosphatase of endoplasmic reticulum-enriched vesicles from etiolated mung bean seedlings.

Endoplasmic reticulum (ER)-enriched vesicles from etiolated hypocotyls of mung bean seedlings (Vigna radiata) were successfully isolated using Ficoll gradient and two-phase (polyethylene glycol-dextran) partition. The ER-enriched vesicles contained inorganic pyrophosphate (PPi) hydrolysis and its associated proton translocating activities. Antiserum prepared against vacuolar H+-pyrophosphatase (V-PPase, EC 3.6.1.1) did not inhibit this novel pyrophosphatase-dependent proton translocation, excluding the possible contamination of tonoplast vesicles in the ER-enriched membrane preparation. The optimal ratios of Mg2+/PPi (inorganic pyrophosphate) for enzymatic activity and PPi-dependent proton translocation of ER-enriched vesicles were higher than those of vacuolar membranes. The PPi-dependent proton translocation of ER-enriched vesicles absolutely required the presence of monovalent cations with preference for K+, but could be inhibited by a common PPase inhibitor, F-. Furthermore, ER H+-pyrophosphatase exhibited some similarities and differences to vacuolar H+-PPases in cofactor/substrate ratios, pH profile, and concentration dependence of F-, imidodiphosphate (a PPi analogue), and various chemical modifiers. These results suggest that ER-enriched vesicles contain a novel type of proton-translocating PPase distinct from that of tonoplast from higher plants.