Repression of a novel isoform of disproportionating enzyme (stDPE2) in potato leads to inhibition of starch degradation in leaves but not tubers stored at low temperature

A potato (Solanum tuberosum) cDNA encoding an isoform of disproportionating enzyme (stDPE2) was identified in a functional screen in Escherichia coli. The stDPE2 protein was demonstrated to be present in chloroplasts and to accumulate at times of active starch degradation in potato leaves and tubers. Transgenic potato plants were made in which its presence was almost completely eliminated. It could be demonstrated that starch degradation was repressed in leaves of the transgenic plants but that cold-induced sweetening was not affected in tubers stored at 4 degrees C. No evidence could be found for an effect of repression of stDPE2 on starch synthesis. The malto-oligosaccharide content of leaves from the transgenic plants was assessed. It was found that the amounts of malto-oligosaccharides increased in all plants during the dark period and that the transgenic lines accumulated up to 10-fold more than the control. Separation of these malto-oligosaccharides by high-performance anion-exchange chromatography with pulsed-amperometric detection showed that the only one that accumulated in the transgenic plants in comparison with the control was maltose. stDPE2 was purified to apparent homogeneity from potato tuber extracts and could be demonstrated to transfer glucose from maltose to oyster glycogen.     

Structural analysis and reaction mechanism of the disproportionating enzyme (D‐enzyme) from potato

Starch produced by plants is a stored form of energy and is an important dietary source of calories for humans and domestic animals. Disproportionating enzyme (D‐enzyme) catalyzes intramolecular and intermolecular transglycosylation reactions of α‐1, 4‐glucan. D‐enzyme is essential in starch metabolism in the potato. We present the crystal structures of potato D‐enzyme, including two different types of complex structures: a primary Michaelis complex (substrate binding mode) for 26‐meric cycloamylose (CA26) and a covalent intermediate for acarbose. Our study revealed that the acarbose and CA26 reactions catalyzed by potato D‐enzyme involve the formation of a covalent intermediate with the donor substrate. HPAEC of reaction substrates and products revealed the activity of the potato D‐enzyme on acarbose and CA26 as donor substrates. The structural and chromatography analyses provide insight into the mechanism of the coupling reaction of CA and glucose catalyzed by the potato D‐enzyme. The enzymatic reaction mechanism does not involve residual hydrolysis. This could be particularly useful in preventing unnecessary starch degradation leading to reduced crop productivity. Optimization of this mechanism would be important for improvements of starch storage and productivity in crops.     

Biofouling leads to reduced shell growth and flesh weight in the cultured mussel Mytilus galloprovincialis

Competitive interactions between cultured mussels and fouling organisms may result in growth and weight reductions in mussels, and compromised aquaculture productivity. Mussel ropes were inoculated with Ciona intestinalis, Ectopleura crocea or Styela clava, and growth parameters of fouled and unfouled Mytilus galloprovincialis were compared after two?months. Small mussels (≈50?mm) fouled by C. intestinalis and E. crocea were 4.0 and 3.2% shorter in shell length and had 21 and 13% reduced flesh weight, respectively, compared to the controls. Large mussels (≈68?mm) fouled by S. clava, C. intestinalis and E. crocea were 4.4, 3.9 and 2.1% shorter than control mussels, respectively, but flesh weights were not significantly reduced. A series of competitive feeding experiments indicated that S. clava and C. intestinalis did not reduce mussels’ food consumption, but that E. crocea, through interference competition, did. Fouling by these species at the densities used here reduced mussel growth and flesh weight, likely resulting in economic losses for the industry, and requires consideration when developing biofouling mitigation strategies.     

Transgenic potato overproducing L-ascorbic acid resisted an increase in methylglyoxal under salinity stress via maintaining higher reduced glutathione level and glyoxalase enzyme activity

Salt-tolerance was studied in transgenic potato. It was conferred by overexpression of ascorbate pathway enzyme (d-galacturonic acid reductase, GalUR). As genetic engineering of the GalUR gene in potato enhances its ascorbic acid content (l-AsA), and subsequently plants suffered minimal oxidative stress-induced damage, we now report on the comprehensive aptness of this engineering approach for enhanced salt tolerance in transgenic potato (Solanum tuberosum L. cv. Taedong Valley). Potatoes overexpressing GalUR grew and tuberized in continuous presence of 200 mM of NaCl. The transgenic plants maintained a higher reduced to oxidized glutathione (GSH:GSSG) ratio together with enhanced activity of glutathione dependent antioxidative and glyoxalase enzymes under salinity stress. The transgenics resisted an increase in methylglyoxal that increased radically in untransformed control plants under salinity stress. This is the first report of genetic engineering of ascorbate pathway gene in maintaining higher level of GSH homeostasis along with higher glyoxalase activity inhibiting the accumulation in methylglyoxal (a potent cytotoxic compound) under salt stress. These results suggested the engineering of ascorbate pathway enzymes as a major step towards developing salinity tolerant crop plants.     

Maize starch-branching enzyme isoforms and amylopectin structure. In the absence of starch-branching enzyme IIb, the further absence of starch-branching enzyme Ia leads to increased branching

Previous studies indicated that the deficiency of starch-branching enzyme (SBE) Ia in the single mutant sbe1a::Mu (sbe1a) has no impact on endosperm starch structure, whereas the deficiency of SBEIIb in the ae mutant is well known to reduce the branching of starch. We hypothesized that in maize (Zea mays) endosperm, the function of SBEIIb is predominant to that of SBEIa, and SBEIa would have an observable effect only on amylopectin structure in the absence of SBEIIb. To test this hypothesis, the mutant sbe1a was introgressed into lines containing either wx (lacking the granule-bound starch synthase GBSSI) or ae wx (lacking both SBEIIb and GBSSI) in the W64A background. Both western blotting and zymogram analysis confirmed the SBEIa deficiency in sbe1a wx and sbe1a ae wx, and the SBEIIb deficiency in ae wx and sbe1a ae wx. Using zymogram analysis, no pleiotropic effects of sbe1a genes on SBEIIa, starch synthase, or starch-debranching enzyme isoforms were observed. High-performance size exclusion chromatography analysis shows that the chain-length profiles of amylopectin as well as beta-limit dextrin were indistinguishable between wx and sbe1a wx, whereas significant differences for both were observed between ae wx and sbe1a ae wx, suggesting an effect of SBEIa on amylopectin biosynthesis that is observable only in the absence of SBEIIb. The amylopectin branch density and the average number of branches per cluster were both higher in endosperm starch from sbe1a ae wx than from ae wx. These results indicate possible functional interactions between SBE isoforms that may involve enzymatic inhibition. Both the cluster repeat distance and the distance between branch points on the short intracluster chains were similar for all genotypes however, suggesting a similar pattern of individual SBE isoforms in cluster initiation and the determination of branch point location.     

Lower thigh subcutaneous and higher visceral abdominal adipose tissue content both contribute to insulin resistance

It is well known that visceral adipose tissue (VAT) is associated with insulin resistance (IR). Considerable debate remains concerning the potential positive effect of thigh subcutaneous adipose tissue (TSAT). Our objective was to observe whether VAT and TSAT are opposite, synergistic or additive for both peripheral and hepatic IR. Fifty-two volunteers (21 male/31 female) between 30 and 75 years old were recruited from the general population. All subjects were sedentary overweight or obese (mean BMI 33.0 ± 3.4 kg/m(2)). Insulin sensitivity was determined by a 4-h hyperinsulinemic-euglycemic clamp with stable isotope tracer dilution. Total body fat and lean body mass were determined by dual X-ray absorptiometry. Abdominal and mid-thigh adiposity was determined by computed tomography. VAT was negatively associated with peripheral insulin sensitivity, while TSAT, in contrast, was positively associated with peripheral insulin sensitivity. Subjects with a combination of low VAT and high TSAT had the highest insulin sensitivity, subjects with a combination of high VAT and low TSAT were the most insulin resistant. These associations remained significant after adjusting for age and gender. These data confirm that visceral excess abdominal adiposity is associated with IR across a range of middle-age to older men and women, and further suggest that higher thigh subcutaneous fat is favorably associated with better insulin sensitivity. This strongly suggests that these two distinct fat distribution phenotypes should both be considered in IR as important determinants of cardiometabolic risk.     

Introduction of sense and antisense cDNA for branching enzyme in the amylose-free potato mutant leads to physico-chemical changes in the starch

One isoform of the branching enzyme (BE; EC 2.4.1.18) of potato (Solarium tuberosum L.) is known and catalyses the formation of α-1,6 bonds in a glucan chain, resulting in the branched starch component amylopectin. Constructs containing the antisense or sense-orientated distal 1.5-kb part of a cDNA for potato BE were used to transform the amylose-free (amf) mutant of potato, the starch of which stains red with iodine. The expression of the endogenous BE gene was inhibited either largely or fully as judged by the decrease or absence of the BE mRNA and protein. This resulted in a low percentage of starch granules with a small blue core and large red outer layer. There was no effect on the amylose content, degree of branching or λ_max of the iodine-stained starch. However, when the physico-chemical properties of the different starch suspensions were assessed, differences were observed, which although small indicated that starch in the transformants was different from that of theamf mutant.     

Long-term serotonin administration leads to higher bone mineral density, affects bone architecture, and leads to higher femoral bone stiffness in rats

New evidence suggests a control of bone mass by the central nervous system. We have previously shown that functional serotonin receptors are present in bone cells and that serotonin stimulates proliferation of osteoblast precursor cells in vitro. In the present study we investigated the effects of serotonin on bone tissue in vivo. Ten, 2-month-old female Sprague-Dawley rats were injected with serotonin subcutaneously (s.c.) (5 mg/kg) once daily for 3 months, controls received saline. Using microdialysis and HPLC, free circulating serotonin levels were measured. DXA scans were made after 3 months of serotonin administration. Bone architecture and mechanical properties were investigated by micro-computed tomography (microCT), histomorphometry, and mechanical testing. A long-lasting hyperserotoninemia with a >10-fold increase in serotonin appeared. Total body BMD was significantly higher (0.1976+/-0.0015 vs. 0.1913+/-0.0012 g/cm2) in rats receiving serotonin. Cortical thickness (Ct.Th) measured by microCT analysis was also higher, whereas trabecular bone volume (BV) was lower. Interestingly, the perimeter and cross-sectional moment of inertia (MOI), a proxy for geometrical bone strength, were the same in both groups. These data suggest that serotonin reduces resorption or/and increases apposition of endosteal bone. Mechanical testing showed that femoral stiffness was higher in serotonin-dosed animals. The energy absorption also seemed slightly, but not significantly higher. In conclusion, hyperserotoninemia led to a higher BMD, altered bone architecture and higher femural bone stiffness in growing rats, demonstrating that serotonin may have important effects on bone in vivo.     

Stable form of ascorbate peroxidase from the red alga Galdieria partita similar to both chloroplastic and cytosolic isoforms of higher plants

Depletion of the electron donor ascorbate causes rapid inactivation of chloroplastic ascorbate peroxidase (APX) of higher plants, while cytosolic APX is stable under such conditions. Here we report the cloning of cDNA from Galdieria partita, a unicellular red alga, encoding a novel type of APX (APX-B). The electrophoretic mobility, Km values, kcat and absorption spectra of recombinant APX-B produced in Escherichia coli were measured. Recombinant APX-B remained active for at least 180 min after depletion of ascorbate. The amino-terminal half of APX-B, which forms the distal pocket of the active site, was richer in amino acid residues conserved in chloroplastic APXs of higher plants rather than cytosolic APXs. In contrast, the sequence of the carboxyl-terminal half, which forms the proximal pocket, was similar to that of the cytosolic isoform. The stability of APX-B might be due to its cytosolic isoform-like structure of the carboxyl-terminal half.     

What leads to reduced fitness in non-photochemical quenching mutants?

Feedback de-excitation (FDE) is a process that protects photosystem II from damage during short periods of overexcitation. Arabidopsis thaliana mutants lacking this mechanism have reduced fitness in environments with variable light intensities. We have assayed the physiological consequences of mutations resulting in the lack of FDE and analysed the differences between field-grown plants and plants grown under fluctuating light in the laboratory. We show that FDE is an important mechanism in short-term responses to fluctuating light. Anthocyanin and carbohydrate levels indicated that the mutant plants were stressed to a higher degree than wild-type (WT) plants. Field-grown mutants were photo-inactivated to a greater degree than WT, whereas mutant plants in the fluctuating light environment in the laboratory seemed to downregulate the photosynthetic quantum yield, thereby avoiding photo-damage but resulting in impaired growth in the case of one mutant. Finally, we provide evidence that FDE is most important under conditions when photosynthesis limits plant growth, for example during flower and seed development.     

Warming leads to higher species turnover in a coastal ecosystem

The responses of ecological communities and ecosystems to increased rates of environmental change will be strongly influenced by variation in the diversity of community composition. Yet, our understanding of how diversity is affected by rising temperatures is inconclusive and mainly based on indirect evidence or short‐term experiments. In our study, we analyse the diversity and species turnover of benthic epilithic communities within the thermal flume of a nuclear power plant at the Swedish coast. This flume covers the range of predicted future temperature rises. Species composition was significantly different between control sites and sites with higher temperatures. We found that temperature had little effect on the number of species in three functional groups (macroinvertebrates, benthic diatoms, and macrophytes, which here comprise multicellular algae and macroscopic colonies of unicellular algae and cyanobacteria), neither at single sampling dates nor summed for the entire observation year. However, species turnover significantly increased with increasing temperature for diatoms, macrophytes and invertebrates. Different temperature regimes resulted in significantly different species composition and indicator species. Thus, increasing temperatures in the thermal flume increased temporal beta‐diversity and decreased compositional stability of communities, although observed richness did not change at any point in time. We highlight the need to investigate the consequences of such declines in compositional stability for functional stability of ecosystem processes.     

Decrease in manganese superoxide dismutase leads to reduced root growth and affects tricarboxylic acid cycle flux and mitochondrial redox homeostasis

Superoxide dismutases (SODs) are key components of the plant antioxidant defense system. While plastidic and cytosolic isoforms have been extensively studied, the importance of mitochondrial SOD at a cellular and whole-plant level has not been established. To address this, transgenic Arabidopsis (Arabidopsis thaliana) plants were generated in which expression of AtMSD1, encoding the mitochondrial manganese (Mn)SOD, was suppressed by antisense. The strongest antisense line showed retarded root growth even under control growth conditions. There was evidence for a specific disturbance of mitochondrial redox homeostasis in seedlings grown in liquid culture: a mitochondrially targeted redox-sensitive green fluorescent protein was significantly more oxidized in the MnSOD-antisense background. In contrast, there was no substantial change in oxidation of cytosolically targeted redox-sensitive green fluorescent protein, nor changes in antioxidant defense components. The consequences of altered mitochondrial redox status of seedlings were subtle with no widespread increase of mitochondrial protein carbonyls or inhibition of mitochondrial respiratory complexes. However, there were specific inhibitions of tricarboxylic acid (TCA) cycle enzymes (aconitase and isocitrate dehydrogenase) and an inhibition of TCA cycle flux in isolated mitochondria. Nevertheless, total respiratory CO2 output of seedlings was not decreased, suggesting that the inhibited TCA cycle enzymes can be bypassed. In older, soil-grown plants, redox perturbation was more pronounced with changes in the amount and/or redox poise of ascorbate and glutathione. Overall, the results demonstrate that reduced MnSOD affects mitochondrial redox balance and plant growth. The data also highlight the flexibility of plant metabolism with TCA cycle inhibition having little effect on overall respiratory rates.     

Mitochondrial dysfunction leads to reduced chronological lifespan and increased apoptosis in yeast

We previously isolated a Saccharomyces cerevisiae mutant (HsTnII), which displays 40% reduced chronological lifespan as compared to the wild type (WT). In this study, we found HsTnII cultures to be characterized by fragmented and dysfunctional mitochondria, and by increased initiation of apoptosis during chronological aging as compared to WT. Expression of genes encoding subunits of mitochondrial electron transport chain and ATP synthase is significantly downregulated in HsTnII, and as a consequence, HsTnII is not able to respire ethanol. All these data confirm the importance of functional mitochondria and respiration in determining yeast chronological lifespan and apoptosis.     

Relationships among iron deficit-induced potato callus growth inhibition,Fe distribution,chlorosis, and oxidative stress amplified by reduced antioxidative enzyme activities

Callus cultures were used to investigate and delineate responses of potato to iron (Fe) deficiency conditions over different culture durations. The morphological responses included chlorotic symptoms, reduced fresh weight and area of callus growth on Fe-deficient medium compared to calli grown under Fe sufficient conditions. Biochemically, potato calli under Fe deficit exhibited decreases in chlorophyll and carotenoid contents, reduction in activities of antioxidant enzymes (peroxidase, catalase and ascorbate peroxidase), as well as an increase in ferric chelate reductase (FCR) activity, lipid peroxidation, phenolic production and hydrogen peroxide (H₂O₂) level. Perls staining revealed sparse Fe distribution in Fe-deficient callus cells whereas Fe was widely distributed and intensely stained among numerous actively dividing cells in Fe-sufficient calli. These responses of calli to Fe deficiency were more pronounced with prolonged exposure to such stress leading to severe chlorosis and/or death of cells in chlorosis-susceptible calli but potential chlorosis-tolerant callus cells maintained their greenness and viability. Over a prolonged period in culture, significantly positive correlations were found among callus fresh weight, chlorophyll and carotenoid contents, antioxidant enzyme activities and lipid peroxidation as Fe supplies to the medium was increased. FCR activity was strongly correlated in a negative manner with Fe deficiency, chlorophyll content and peroxidase activity. The responses of calli to Fe supply can serve as reliable indicators for detecting chlorosis tolerance and/or nutrient deficiency stress.     

Suppression of nuclear Wnt signaling leads to stabilization of Rac1 isoforms

The Rac1 GTPase contains a functional nuclear localization signal (NLS) and destruction box sequence in the C-terminal polybasic region. It has been postulated that these two regulatory sequences may function together, enabling Rac1 to participate in nuclear signaling pathways that ultimately target it for degradation. We have previously shown that the NLS activity of Rac1 and the Rac1b splice variant is essential for Wnt pathway activation. In the present study, we demonstrate that suppression of nuclear Wnt signaling leads to stabilization of Rac1 protein. In addition, we show that Rac1b may be under proteasomal regulation. We propose that Rac1 and Rac1b levels are regulated by being targeted for degradation through a negative feedback loop initiated by Wnt signaling.