Starch biosynthesis contributes to the maintenance of photosynthesis and leaf growth under drought stress in maize.

To understand the growth response to drought, we performed a proteomics study in the leaf growth zone of maize (Zea mays L.) seedlings and functionally characterized the role of starch biosynthesis in the regulation of growth, photosynthesis and antioxidant capacity, using the shrunken-2 mutant (sh2), defective in ADP-glucose pyrophosphorylase. Drought altered the abundance of 284 proteins overrepresented for photosynthesis, amino acid, sugar and starch metabolism, and redox-regulation. Changes in protein levels correlated with enzyme activities (increased ATP synthase, cysteine synthase, starch synthase, RuBisCo, peroxiredoxin, glutaredoxin, thioredoxin and decreased triosephosphate isomerase, ferredoxin, cellulose synthase activities, respectively) and metabolite concentrations (increased ATP, cysteine, glycine, serine, starch, proline and decreased cellulose levels). The sh2 mutant showed a reduced increase of starch levels under drought conditions, leading to soluble sugar starvation at the end of the night and correlating with an inhibition of leaf growth rates. Increased RuBisCo activity and pigment concentrations observed in WT, in response to drought, were lacking in the mutant, which suffered more oxidative damage and recovered more slowly after re-watering. These results demonstrate that starch biosynthesis contributes to maintaining leaf growth under drought stress and facilitates enhanced carbon acquisition upon recovery.

Drought Induces Distinct Growth Response, Protection, and Recovery Mechanisms in the Maize Leaf Growth Zone.

Drought is the most important crop yield-limiting factor, and detailed knowledge of its impact on plant growth regulation is crucial. The maize (Zea mays) leaf growth zone offers unique possibilities for studying the spatiotemporal regulation of developmental processes by transcriptional analyses and methods that require more material, such as metabolite and enzyme activity measurements. By means of a kinematic analysis, we show that drought inhibits maize leaf growth by inhibiting cell division in the meristem and cell expansion in the elongation zone. Through a microarray study, we observed the down-regulation of 32 of the 54 cell cycle genes, providing a basis for the inhibited cell division. We also found evidence for an up-regulation of the photosynthetic machinery and the antioxidant and redox systems. This was confirmed by increased chlorophyll content in mature cells and increased activity of antioxidant enzymes and metabolite levels across the growth zone, respectively. We demonstrate the functional significance of the identified transcriptional reprogramming by showing that increasing the antioxidant capacity in the proliferation zone, by overexpression of the Arabidopsis (Arabidopsis thaliana) iron-superoxide dismutase gene, increases leaf growth rate by stimulating cell division. We also show that the increased photosynthetic capacity leads to enhanced photosynthesis upon rewatering, facilitating the often-observed growth compensation.

The Arabidopsis lectin EULS3 is involved in stomatal closure.

Plants synthesize carbohydrate binding proteins in response to adverse environmental conditions such as drought, heat, pathogen attack, etc. The Arabidopsis EULS3 lectin (referred to as ArathEULS3, encoded by At2g39050) has recently been linked to the drought stress response. In this study, endogenous binding partners for this protein have been investigated. Tandem affinity purifications and mass spectrometry analyses allowed the identification of two putative interacting proteins, Embryo-specific protein 3A (ATS3A, At2g41475) and Embryo-specific protein 3B (ATS3B, At5g62200). Bimolecular fluorescence complementation experiments confirmed the interaction between ArathEULS3 and ATS3B in closed stomata of Nicotiana benthamiana plants. Transgenic lines with reduced ArathEULS3 expression exhibited an aberrant ABA-induced stomatal closure compared to plants overexpressing ArathEULS3 and control plants suggesting a role for ArathEULS3 in ABA-induced stomatal closure. Stomata are known as the major route for Pseudomonas syringae entry into the plant tissues. Bacterial infection of wild type Arabidopsis thaliana plants was accompanied by a 6-fold increase of transcript levels for ArathEULS3. Furthermore, infection experiments with ArathEULS3 overexpression lines resulted in a clear reduction of P. syringae disease symptoms whereas plants with reduced ArathEULS3 expression showed the highest levels of leaf damage at 3 days post infection. These data point towards the physiological importance of ArathEULS3 for stomatal movement.

Both the concentration and redox state of glutathione and ascorbate influence the sensitivity of arabidopsis to cadmium.

BACKGROUND AND AIMS: Cadmium (Cd) is a non-essential trace element that elicits oxidative stress. Plants respond to Cd toxicity via increasing their Cd-chelating and antioxidative capacities. They predominantly chelate Cd via glutathione (GSH) and phytochelatins (PCs), while antioxidative defence is mainly based on the use and recycling of both GSH and ascorbate (AsA), complemented by superoxide dismutase (SOD) and catalase (CAT). In addition, both metabolites act as a substrate for the regeneration of other essential antioxidants, which neutralize and regulate reactive oxygen species (ROS). Together, these functions influence the concentration and cellular redox state of GSH and AsA. In this study, these two parameters were examined in plants of Arabidopsis thaliana exposed to sub-lethal Cd concentrations. METHODS: Wild-type plants and mutant arabidopsis plants containing 30-45 % of wild-type levels of GSH (cad2-1) or 40-50 % of AsA (vtc1-1), together with the double-mutant (cad2-1 vtc1-1) were cultivated in a hydroponic system and exposed to sub-lethal Cd concentrations. Cadmium detoxification was investigated at different levels including gene expression and metabolite concentrations. KEY RESULTS: In comparison with wild-type plants, elevated basal thiol levels and enhanced PC synthesis upon exposure to Cd efficiently compensated AsA deficiency in vtc1-1 plants and contributed to decreased sensitivity towards Cd. Glutathione-deficient (cad2-1 and cad2-1 vtc1-1) mutants, however, showed a more oxidized GSH redox state, resulting in initial oxidative stress and a higher sensitivity to Cd. In order to cope with the Cd stress to which they were exposed, GSH-deficient mutants activated multiple alternative pathways. CONCLUSIONS: Our observations indicate that GSH and AsA deficiency differentially alter plant GSH homeostasis, resulting in opposite Cd sensitivities relative to wild-type plants. Upon Cd exposure, GSH-deficient mutants were hampered in chelation. They experienced phenotypic disturbances and even more oxidative stress, and therefore activated multiple alternative pathways such as SOD, CAT and ascorbate peroxidase, indicating a higher Cd sensitivity. Ascorbate deficiency, however, was associated with enhanced PC synthesis in comparison with wild-type plants after Cd exposure, which contributed to decreased sensitivity towards Cd.

Cell cycle-dependent O-GlcNAc modification of tobacco histones and their interaction with the tobacco lectin.

The Nicotiana tabacum agglutinin or Nictaba is a nucleocytoplasmic lectin that is expressed in tobacco after the plants have been exposed to jasmonate treatment or insect herbivory. Nictaba specifically recognizes GlcNAc residues. Recently, it was shown that Nictaba is interacting in vitro with the core histone proteins from calf thymus. Assuming that plant histones - similar to their animal counterparts - undergo O-GlcNAcylation, this interaction presumably occurs through binding of the lectin to the O-GlcNAc modification present on the histones. Hereupon, the question was raised whether this modification also occurs in plants and if it is cell cycle dependent. To this end, histones were purified from tobacco BY-2 suspension cells and the presence of O-GlcNAc modifications was checked. Concomitantly, O-GlcNAcylation of histone proteins was studied. Our data show that similar to animal histones plant histones are modified by O-GlcNAc in a cell cycle-dependent fashion. In addition, the interaction between Nictaba and tobacco histones was confirmed using lectin chromatography and far Western blot analysis. Collectively these findings suggest that Nictaba can act as a modulator of gene transcription through its interaction with core histones.

Recombinant antigens expressed in Pichia pastoris for the diagnosis of sleeping sickness caused by Trypanosoma brucei gambiense.

BACKGROUND: Screening tests for gambiense sleeping sickness, such as the CATT/T. b. gambiense and a recently developed lateral flow tests, are hitherto based on native variant surface glycoproteins (VSGs), namely LiTat 1.3 and LiTat 1.5, purified from highly virulent trypanosome strains grown in rodents. METHODOLOGY/PRINCIPAL FINDINGS: We have expressed SUMO (small ubiquitin-like modifier) fusion proteins of the immunogenic N-terminal part of these antigens in the yeast Pichia pastoris. The secreted recombinant proteins were affinity purified with yields up to 10 mg per liter cell culture. CONCLUSIONS/SIGNIFICANCE: The diagnostic potential of each separate antigen and a mixture of both antigens was confirmed in ELISA on sera from 88 HAT patients and 74 endemic non-HAT controls. Replacement of native antigens in the screening tests for sleeping sickness by recombinant proteins will eliminate both the infection risk for the laboratory staff during antigen production and the need for laboratory animals. Upscaling production of recombinant antigens, e.g. in biofermentors, is straightforward thus leading to improved standardisation of antigen production and reduced production costs, which on their turn will increase the availability and affordability of the diagnostic tests needed for the elimination of gambiense HAT.

Dynamic changes in plant secondary metabolites during UV acclimation in Arabidopsis thaliana.

Plants respond to environmental stress by synthesizing a range of secondary metabolites for defense purposes. Here we report on the effect of chronic ultraviolet (UV) radiation on the accumulation of plant secondary metabolites in Arabidopsis thaliana leaves. In the natural environment, UV is a highly dynamic environmental parameter and therefore we hypothesized that plants are continuously readjusting levels of secondary metabolites. Our data show distinct kinetic profiles for accumulation of tocopherols, polyamines and flavonoids upon UV acclimation. The lipid-soluble antioxidant α-tocopherol accumulated fast and remained elevated. Polyamines accumulated fast and transiently. This fast response implies a role for α-tocopherol and polyamines in short-term UV response. In contrast, an additional sustained accumulation of flavonols took place. The distinct accumulation patterns of these secondary metabolites confirm that the UV acclimation process is a dynamic process, and indicates that commonly used single time-point analyses do not reveal the full extent of UV acclimation. We demonstrate that UV stimulates the accumulation of specific flavonol glycosides, i.e. kaempferol and (to a lesser extent) quercetin di- and triglycosides, all specifically rhamnosylated at position seven. All metabolites were identified by Ultra Performance Liquid Chromatography (UPLC)-coupled tandem mass spectrometry. Some of these flavonol glycosides reached steady-state levels in 3-4 days, while concentrations of others are still increasing after 12 days of UV exposure. A biochemical pathway for these glycosides is postulated involving 7-O-rhamnosylation for the synthesis of all eight metabolites identified. We postulate that this 7-O-rhamnosylation has an important function in UV acclimation.

A mutant of the Arabidopsis thaliana LIPOXYGENASE1 gene shows altered signalling and oxidative stress related responses after cadmium exposure.

Lipoxygenases (LOXes, EC 1.13.11.12) are involved in growth, development and responses to stress. Earlier results suggested a role in stress generation, signalling and/or responses when Arabidopsis thaliana is exposed to cadmium (Cd), and expression of the cytosolic LOX1 was highly upregulated in the roots after Cd exposure. To investigate the involvement of LOX1 in early metal stress responses, three-week-old wild-type and lox1-1 mutant A. thaliana plants were acutely (24 h) exposed to realistic Cd concentrations (5 and 10 µM) and several oxidative stress and signalling related parameters were studied at transcriptional and biochemical levels. Transcription of several genes encoding ROS producing and scavenging enzymes failed to be induced up to wild-type levels after Cd exposure. Expression of 9-LOX enzymes was inhibited in lox1-1 mutant roots due to lack of functional LOX1 and downregulated LOX5 expression, and the lox1-1 mutation also interfered with the expression of genes involved in jasmonate biosynthesis. LOX1 and RBOHD may be involved in stress signalling from roots to shoots, as the induction of APX2 expression, which is dependent on RBOHD activity, was disrupted in lox1-1 while RBOHD failed to be upregulated. A different pattern of H(2)O(2) production and ascorbate and glutathione levels in lox1-1 mutants after Cd exposure may have indirectly influenced gene expression patterns. Although indirect effects of the lox1-1 mutation on gene expression complicate the determination of exact sensing - signalling - response pathways, the results presented here outline a more refined LOX1 functioning in Cd-induced stress responses that could be used in studies determining the exact involvement of LOX1 in these pathways.

A LiTat 1.5 variant surface glycoprotein-derived peptide with diagnostic potential for Trypanosoma brucei gambiense.

OBJECTIVE: To evaluate the accuracy of a peptide, corresponding to the variant surface glycoprotein (VSG) LiTat 1.5 amino acid (AA) sequence 268-281 and identified through alignment of monoclonal antibody selected mimotopes, for diagnosis of Trypanosoma brucei gambiense sleeping sickness. METHODS: A synthetic biotinylated peptide (peptide 1.5/268-281), native VSG LiTat 1.3 and VSG LiTat 1.5 were tested in an indirect ELISA with 102 sera from patients with HAT and 102 endemic HAT-negative controls. RESULTS: The area under the curve (AUC) of peptide 1.5/268-281 was 0.954 (95% confidence interval 0.918-0.980), indicating diagnostic potential. The areas under the curve of VSG LiTat 1.3 and LiTat 1.5 were 1.000 (0.982-1.000) and 0.997 (0.973-1.000), respectively, and significantly higher than the AUC of peptide 1.5/268-281. On a model of VSG LiTat 1.5, peptide 1.5/268-281 was mapped near the top of the VSG. CONCLUSIONS: A biotinylated peptide corresponding to AA 268-281 of VSG LiTat 1.5 may replace the native VSG in serodiagnostic tests, but the diagnostic accuracy is lower than for the full-length native VSG LiTat 1.3 and VSG LiTat 1.5.

Identification of mimotopes with diagnostic potential for Trypanosoma brucei gambiense variant surface glycoproteins using human antibody fractions.

BACKGROUND: At present, screening of the population at risk for gambiense human African trypanosomiasis (HAT) is based on detection of antibodies against native variant surface glycoproteins (VSGs) of Trypanosoma brucei (T.b.) gambiense. Drawbacks of these native VSGs include culture of infective T.b. gambiense trypanosomes in laboratory rodents, necessary for production, and the exposure of non-specific epitopes that may cause cross-reactions. We therefore aimed at identifying peptides that mimic epitopes, hence called "mimotopes," specific to T.b. gambiense VSGs and that may replace the native proteins in antibody detection tests. METHODOLOGY/PRINCIPAL FINDINGS: A Ph.D.-12 peptide phage display library was screened with polyclonal antibodies from patient sera, previously affinity purified on VSG LiTat 1.3 or LiTat 1.5. The peptide sequences were derived from the DNA sequence of the selected phages and synthesised as biotinylated peptides. Respectively, eighteen and twenty different mimotopes were identified for VSG LiTat 1.3 and LiTat 1.5, of which six and five were retained for assessment of their diagnostic performance. Based on alignment of the peptide sequences on the original protein sequence of VSG LiTat 1.3 and 1.5, three additional peptides were synthesised. We evaluated the diagnostic performance of the synthetic peptides in indirect ELISA with 102 sera from HAT patients and 102 endemic negative controls. All mimotopes had areas under the curve (AUCs) of ≥0.85, indicating their diagnostic potential. One peptide corresponding to the VSG LiTat 1.3 protein sequence also had an AUC of ≥0.85, while the peptide based on the sequence of VSG LiTat 1.5 had an AUC of only 0.79. CONCLUSIONS/SIGNIFICANCE: We delivered the proof of principle that mimotopes for T.b. gambiense VSGs, with diagnostic potential, can be selected by phage display using polyclonal human antibodies.

The phytohormone auxin is a component of the regulatory system that controls UV-mediated accumulation of flavonoids and UV-induced morphogenesis.

In plants, ultraviolet (UV)-B acclimation is a complex, dynamic process that plays an essential role in preventing UV-B damage to targets such as DNA and the photosynthetic machinery. In this study we tested the hypothesis that the phytohormone auxin is a component of the regulatory system that controls both UV-mediated accumulation of flavonoids and UV-induced morphogenesis. We found that the leaf area of Arabidopsis thaliana Col-0 plants raised under a low dose of UV radiation (0.56 kJ m(-2) daily dose) was, on average, decreased by 23% relative to plants raised in the absence of UV-B, and this was accompanied by a decrease (P = 0.063) in free auxin in young leaf tissues. Compared to Col-0, both the auxin influx mutant axr4-1 and the auxin biosynthesis mutant nit1-3 displayed significantly stronger morphogenic responses, i.e. relative decreases in leaf area were greater for these two mutants. UV exposure also induced accumulation of flavonoids. In Col-0, increases in the concentrations of specific kaempferol derivatives ranged from 2.1- to 19-fold. Thus, UV induces complex changes in flavonoid-glycosylation patterns. Compared to Col-0, three auxin mutants displayed significantly different flavonoid profiles. Thus, based on mutant analysis, it is concluded that the phytohormone auxin plays a role in UV acclimation by regulating flavonoid concentration, flavonoid-glycosylation pattern and by controlling UV-induced morphogenic responses.

Identification of peptide mimotopes of Trypanosoma brucei gambiense variant surface glycoproteins.

BACKGROUND: The current antibody detection tests for the diagnosis of gambiense human African trypanosomiasis (HAT) are based on native variant surface glycoproteins (VSGs) of Trypanosoma brucei (T.b.) gambiense. These native VSGs are difficult to produce, and contain non-specific epitopes that may cause cross-reactions. We aimed to identify mimotopic peptides for epitopes of T.b. gambiense VSGs that, when produced synthetically, can replace the native proteins in antibody detection tests. METHODOLOGY/PRINCIPAL FINDINGS: PhD.-12 and PhD.-C7C phage display peptide libraries were screened with mouse monoclonal antibodies against the predominant VSGs LiTat 1.3 and LiTat 1.5 of T.b. gambiense. Thirty seven different peptide sequences corresponding to a linear LiTat 1.5 VSG epitope and 17 sequences corresponding to a discontinuous LiTat 1.3 VSG epitope were identified. Seventeen of 22 synthetic peptides inhibited the binding of their homologous monoclonal to VSG LiTat 1.5 or LiTat 1.3. Binding of these monoclonal antibodies to respectively six and three synthetic mimotopic peptides of LiTat 1.5 and LiTat 1.3 was significantly inhibited by HAT sera (p<0.05). CONCLUSIONS/SIGNIFICANCE: We successfully identified peptides that mimic epitopes on the native trypanosomal VSGs LiTat 1.5 and LiTat 1.3. These mimotopes might have potential for the diagnosis of human African trypanosomiasis but require further evaluation and testing with a large panel of HAT positive and negative sera.

UV radiation reduces epidermal cell expansion in Arabidopsis thaliana leaves without altering cellular microtubule organization.

Upon chronic UV treatment pavement cell expansion in Arabidopsis leaves is reduced, implying alterations in symplastic and apoplastic properties of the epidermal cells. In this study, the effect of UV radiation on microtubule patterning is analysed, as microtubules are thought to serve as guiding rails for the cellulose synthase complexes depositing cellulose microfibrils. Together with hemicelluloses, these microfibrils are regarded as the load-bearing components of the cell wall. Leaves of transgenic plants with fluorescently tagged microtubules (GFP-TUA6) were as responsive to UV as wild type plants. Despite the UV-induced reduction in cell elongation, confocal microscopy revealed that cellular microtubule arrangements were seemingly not affected by the UV treatments. This indicates an unaltered deposition of cellulose microfibrils in the presence of UV radiation. Therefore, we surmise that the reduction in cell expansion in UV-treated leaves is most probably due to changes in cell wall loosening and/or turgor pressure.   

The cellular redox state as a modulator in cadmium and copper responses in Arabidopsis thaliana seedlings.

The cellular redox state is an important determinant of metal phytotoxicity. In this study we investigated the influence of cadmium (Cd) and copper (Cu) stress on the cellular redox balance in relation to oxidative signalling and damage in Arabidopsis thaliana. Both metals were easily taken up by the roots, but the translocation to the aboveground parts was restricted to Cd stress. In the roots, Cu directly induced an oxidative burst, whereas enzymatic ROS (reactive oxygen species) production via NADPH oxidases seems important in oxidative stress caused by Cd. Furthermore, in the roots, the glutathione metabolism plays a crucial role in controlling the gene regulation of the antioxidative defence mechanism under Cd stress. Metal-specific alterations were also noticed with regard to the microRNA regulation of CuZnSOD gene expression in both roots and leaves. The appearance of lipid peroxidation is dual: it can be an indication of oxidative damage as well as an indication of oxidative signalling as lipoxygenases are induced after metal exposure and are initial enzymes in oxylipin biosynthesis. In conclusion, the metal-induced cellular redox imbalance is strongly dependent on the chemical properties of the metal and the plant organ considered. The stress intensity determines its involvement in downstream responses in relation to oxidative damage or signalling.

Antioxidants in Erica andevalensis: a comparative study between wild plants and cadmium-exposed plants under controlled conditions.

Erica andevalensis is an endemic species from SW Iberian Peninsula, always growing in metal-enriched and acid soils. In the present study, a comparison was made between wild E. andevalensis plants collected from the field and cultivated ones exposed to different cadmium levels (0, 0.5, 5 and 50 µM). Wild plants contain higher levels of ascorbic acid (around 8000 nmol g(-1) FW) than lab-cultivated control plants (around 3000 nmol g(-1) FW). Glutathione levels follow an opposite trend being smaller in wild plants than lab-cultivated ones. Moreover, the total antioxidant capacity of wild plants is 90 times higher than in cultivated plants non-exposed to cadmium. Cadmium treatment of lab-cultivated plants did not affect the growth of E. andevalensis or the glutathione levels. However, the total antioxidative capacity increased in plants exposed to 50 µM of cadmium. Cadmium was added to the soil and it was transported into leaves reaching levels of 3.299 ± 0.781 µg Cd/g DW in plants exposed to 50 µM. These results underline a possible importance of antioxidants in the metal tolerance show by the high antioxidant capacity detected in both wild and lab-cultivated plants exposed to high cadmium levels.

UV radiation reduces epidermal cell expansion in leaves of Arabidopsis thaliana.

Plants have evolved a broad spectrum of mechanisms to ensure survival under changing and suboptimal environmental conditions. Alterations of plant architecture are commonly observed following exposure to abiotic stressors. The mechanisms behind these environmentally controlled morphogenic traits are, however, poorly understood. In this report, the effects of a low dose of chronic ultraviolet (UV) radiation on leaf development are detailed. Arabidopsis rosette leaves exposed for 7, 12, or 19 d to supplemental UV radiation expanded less compared with non-UV controls. The UV-mediated decrease in leaf expansion is associated with a decrease in adaxial pavement cell expansion. Elevated UV does not affect the number and shape of adaxial pavement cells, nor the stomatal index. Cell expansion in young Arabidopsis leaves is asynchronous along a top-to-base gradient whereas, later in development, cells localized at both the proximal and distal half expand synchronously. The prominent, UV-mediated inhibition of cell expansion in young leaves comprises effects on the early asynchronous growing stage. Subsequent cell expansion during the synchronous phase cannot nullify the UV impact established during the asynchronous phase. The developmental stage of the leaf at the onset of UV treatment determines whether UV alters cell expansion during the synchronous and/or asynchronous stage. The effect of UV radiation on adaxial epidermal cell size appears permanent, whereas leaf shape is transiently altered with a reduced length/width ratio in young leaves. The data show that UV-altered morphogenesis is a temporal- and spatial-dependent process, implying that common single time point or single leaf zone analyses are inadequate.

Folding properties of the hepatitis B core as a carrier protein for vaccination research.

The hepatitis B core (HBc) protein has been used successfully in numerous experiments as a carrier for heterologous peptides. Folding and capsid formation of the chimeric proteins is not always achieved easily. In silico analyses were performed to provide further comprehension of the feasibility for predicting successful capsid formation. In contrast to previous work, we show that common in silico predictions do not ensure assembly into particles. We included new considerations regarding capsid formation of HBc fusion proteins. Not only the primary sequence and the length of the inserts seem important, also the rigidity, the distance between the N and the C-terminus and the presence of cysteines, which could form disulphide bonds, could influence proper capsid formation. Furthermore, new conformational insights were formulated when linkers were added to create extra flexibility of the chimeric particles. Different hypotheses were suggested to clarify the obtained results. To this extent, the addition of glycine-rich linkers could lower high rigidity of the insert, removal of the strain of the core protein or ease interaction between the HBc and the insert. Finally, we observed specific changes in capsid formation properties when longer linkers were used. These findings have not been reported before in this and other virus-like particle carriers. In this study, we also propose a new high-yield purification protocol for fusion proteins to be used in vaccination experiments with the carrier protein or in comparative studies of particulate or non-particulate HBc fusion proteins.

Dipeptidyl peptidase 9 (DPP9) from bovine testes: identification and characterization as the short form by mass spectrometry.

The dipeptidyl peptidases (DPP) 8 and 9 belong to the DPP4 activity and/or structure homologues (DASH). Recently, a DPP9-like protein was purified from bovine testes. The aim of the present study was to prove its identity and to investigate the characteristics of this natural enzyme. We report the identification and N-terminal sequence analysis by MALDI-TOF/TOF MS, of the purified bovine enzyme as DPP9. The tryptic peptides after in-gel digestion covered 41% and 38% of the short and full-length variants of bovine DPP9, respectively. Using Asp-N digestion combined with a very recently described mass spectrometric method using DITC glass beads, the N-terminal peptide (XTGALTSERG) was isolated. It corresponds to the N-terminus of the short form of bovine DPP9. There was no evidence for glycosylation of purified bovine DPP9. The purified DPP9 was activated and stabilized by DTT. Bovine DPP9 lost its activity almost completely after alkylation with N-ethylmaleimide. Also alkylation with iodoacetamide inhibited DPP9, albeit only 70%. Other properties of bovine DPP9 are reported, including functional stability and sensitivity towards metal ions. Our results indicate that the short form of DPP9 can be isolated from bovine testes and that it behaves as a stable enzyme suitable for further functional and biochemical characterization as well as for inhibitor screening and characterization.

Energy use efficiency is characterized by an epigenetic component that can be directed through artificial selection to increase yield.

Quantitative traits, such as size and weight in animals and seed yield in plants, are distributed normally, even within a population of genetically identical individuals. For example, in plants, various factors, such as local soil quality, microclimate, and sowing depth, affect growth differences among individual plants of isogenic populations. Besides these physical factors, also epigenetic components contribute to differences in growth and yield. The network that regulates crop yield is still not well understood. Although this network is expected to have epigenetic elements, it is completely unclear whether it would be possible to shape the epigenome to increase crop yield. Here we show that energy use efficiency is an important factor in determining seed yield in canola (Brassica napus) and that it can be selected artificially through an epigenetic feature. From an isogenic canola population of which the individual plants and their self-fertilized progenies were recursively selected for respiration intensity, populations with distinct physiological and agronomical characteristics could be generated. These populations were found to be genetically identical, but epigenetically different. Furthermore, both the DNA methylation patterns as well as the agronomical and physiological characteristics of the selected lines were heritable. Hybrids derived from parent lines selected for high energy use efficiencies had a 5% yield increase on top of heterosis. Our results demonstrate that artificial selection allows the increase of the yield potential by selecting populations with particular epigenomic states.

Oxidative stress-related responses at transcriptional and enzymatic levels after exposure to Cd or Cu in a multipollution context.

The physiological effects of Cd and Cu have been highlighted in several studies over the last years. At the cellular level, oxidative stress has been reported as a common mechanism in both stress situations. Nevertheless, because of differences in their redox-related properties, the origin of the stress and regulation of these effects can be very different. Our results show a specific Cd-related induction of NADPH oxidases, whereas both metals induced lipid peroxidation via the activation of lipoxygenases. With respect to the antioxidative defense system, metal-specific patterns of superoxide dismutases (SODs) were detected, whereas gene expression levels of the H2O2-quenching enzymes were equally induced by both metals. Because monometallic exposure is very unusual in real-world situations, the metal-specific effects were compared with the mechanisms induced when the plants are exposed to both metals simultaneously. Combined exposure to Cd and Cu enhanced some of the effects that were induced when only one metal was applied to the medium. Other specific monometallically induced effects, such as a copper zinc superoxide dismutase (CSD2) downregulation due to Cd, were also sustained in a multipollution context, irrespective of the other monometallic effects. Furthermore, specific multipollution effects were unravelled, as iron superoxide dismutase 1 (FSD1) upregulation in the leaves was significant only when both Cu and Cd were applied. Additional relationships between these treatments and the common and specific stress induction mechanisms are discussed.