An antioxidant redox system in the nucleus of wheat seed cells suffering oxidative stress

Cereal seed cells contain different mechanisms for protection against the oxidative stress that occurs during maturation and germination. One such mechanism is based on the antioxidant activity of a 1-Cys peroxiredoxin (1-Cys Prx) localized in the nuclei of aleurone and scutellum cells. However, nothing is known about the mechanism of activation of this enzyme. Here, we describe the pattern of localization of NADPH thioredoxin reductase (NTR) in developing and germinating wheat seeds using an immunocytochemical analysis. The presence of NTR in transfer cells, vascular tissue, developing embryo and root meristematic cells, agrees with the localization of thioredoxin h (Trx h ), and supports the important function of the NTR/Trx system in cell proliferation and communication. Interestingly, NTR is found in the nuclei of seed cells suffering oxidative stress, thus showing co-localization with Trx h and 1-Cys Prx. To test whether the NTR/Trx system serves as a reductant of the 1-Cys Prx, we cloned a full-length cDNA encoding 1-Cys Prx from wheat, and expressed the recombinant protein in Escherichia coli . Using the purified components, we show NTR-dependent activity of the 1-Cys Prx. Mutants of the 1-Cys Prx allowed us to demonstrate that the peroxidatic residue of the wheat enzyme is Cys46, which is overoxidized in vitro under oxidant conditions. Analysis of extracts from developing and germinating seeds confirmed 1-Cys Prx overoxidation in vivo . Based on these results, we propose that NADPH is the source of the reducing power to regenerate 1-Cys Prx in the nuclei of seed cells suffering oxidative stress, in a process that is catalyzed by NTR.     

Metabolic Flux Analysis of Plastidic Isoprenoid Biosynthesis in Poplar Leaves Emitting and Nonemitting Isoprene

The plastidic 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway is one of the most important pathways in plants and produces a large variety of essential isoprenoids. Its regulation, however, is still not well understood. Using the stable isotope ¹³C-labeling technique, we analyzed the carbon fluxes through the MEP pathway and into the major plastidic isoprenoid products in isoprene-emitting and transgenic isoprene-nonemitting (NE) gray poplar (Populus × canescens). We assessed the dependence on temperature, light intensity, and atmospheric [CO₂]. Isoprene biosynthesis was by far (99%) the main carbon sink of MEP pathway intermediates in mature gray poplar leaves, and its production required severalfold higher carbon fluxes compared with NE leaves with almost zero isoprene emission. To compensate for the much lower demand for carbon, NE leaves drastically reduced the overall carbon flux within the MEP pathway. Feedback inhibition of 1-deoxy-d-xylulose-5-phosphate synthase activity by accumulated plastidic dimethylallyl diphosphate almost completely explained this reduction in carbon flux. Our data demonstrate that short-term biochemical feedback regulation of 1-deoxy-d-xylulose-5-phosphate synthase activity by plastidic dimethylallyl diphosphate is an important regulatory mechanism of the MEP pathway. Despite being relieved from the large carbon demand of isoprene biosynthesis, NE plants redirected only approximately 0.5% of this saved carbon toward essential nonvolatile isoprenoids, i.e. β-carotene and lutein, most probably to compensate for the absence of isoprene and its antioxidant properties.Isoprenoids represent the largest and most diverse group (over 50,000) of natural compounds and are essential in all living organisms (Gershenzon and Dudareva, 2007; Thulasiram et al., 2007). They are economically important for humans as flavor and fragrance, cosmetics, drugs, polymers for rubber, and precursors for the chemical industry (Chang and Keasling, 2006). The broad variety of isoprenoid products is formed from two building blocks, dimethylallyl diphosphate (DMADP) and isopentenyl diphosphate (IDP). In plants, the plastidic 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway (Zeidler et al., 1997) produces physiologically and ecologically important volatile organic compounds (VOCs), the carotenoids (tetraterpenes; Giuliano et al., 2008; Cazzonelli and Pogson, 2010), diterpenes, the prenyl side-chains of chlorophylls (Chls) and plastoquinones, isoprenylated proteins, the phytohormones gibberellins, and side-chain of cytokinins (for review, see Dudareva et al., 2013; Moses et al., 2013). Industrially important prokaryotes (e.g. Escherichia coli) also use the MEP pathway for the biosynthesis of isoprenoids (Vranová et al., 2012), and there is an increasing interest in manipulating the MEP pathway of engineered microbes to increase production of economically relevant isoprenoids (Chang and Keasling, 2006). To achieve this, a mechanistic understanding of the regulation of the MEP pathway is needed (Vranová et al., 2012).Some plants, including poplars (Populus spp.), produce large amounts of the hemiterpene VOC isoprene. Worldwide isoprene emissions from plants are estimated to be 600 teragrams per year and to account for one-third of all hydrocarbons emitted to the atmosphere (Arneth et al., 2008; Guenther, 2013). Isoprene has strong effects on air chemistry and climate by participating in ozone formation reactions (Fuentes et al., 2000), by prolonging the lifespan of methane, a greenhouse gas (Poisson et al., 2000; Archibald et al., 2011), and by taking part in the formation of secondary organic aerosols (Kiendler-Scharr et al., 2012).Poplar leaves invest a significant amount of recently fixed carbon in isoprene biosynthesis (Delwiche and Sharkey, 1993; Schnitzler et al., 2010; Ghirardo et al., 2011) to cope with abiotic stresses (Sharkey, 1995; Velikova and Loreto, 2005; Behnke et al., 2007, 2010b, 2013; Vickers et al., 2009; Loreto and Schnitzler, 2010; Sun et al., 2013b), although there are indications that other protective mechanisms can partially compensate the lack of isoprene emission in genetically transformed poplars (Behnke et al., 2012; Way et al., 2013). It has been suggested that in isoprene-emitting (IE) species, most of the carbon that passes through the MEP pathway is used for isoprene biosynthesis (Sharkey and Yeh, 2001). However, a recent study using pulse-chase labeling with ¹⁴C has shown continuous synthesis and degradation of carotenes and Chl a in mature leaves of Arabidopsis (Arabidopsis thaliana; Beisel et al., 2010), and the amount of flux diverted to carotenoid and Chl synthesis compared with isoprene biosynthesis in poplar leaves is not known.Isoprene emission is temperature, light, and CO₂ dependent (Schnitzler et al., 2005; Rasulov et al., 2010; Way et al., 2011; Monson et al., 2012; Li and Sharkey, 2013a). It has been demonstrated that isoprene biosynthesis depends on the activities of IDP isomerase (EC 5.3.3.2), isoprene synthase (ISPS; EC 4.2.3.27), and the amount of ISPS substrate, DMADP (Brüggemann and Schnitzler, 2002a, 2002b; Schnitzler et al., 2005; Rasulov et al., 2009b). In turn, DMADP concentration has been hypothesized to act as a feedback regulator of the MEP pathway by inhibiting 1-deoxy-d-xylulose-5-phosphate synthase (DXS; EC 2.2.1.7), the first enzyme of the MEP pathway (Banerjee et al., 2013). Understanding the controlling mechanism of isoprene biosynthesis is not only of fundamental relevance, but also necessary for engineering the MEP pathway in various organisms and for accurate simulation of isoprene emissions by plants in predicting atmospheric reactivity (Niinemets and Monson, 2013).There is ample evidence that silencing the ISPS in poplar has a broad effect on the leaf metabolome (Behnke et al., 2009, 2010a, 2013; Way et al., 2011; Kaling et al., 2014). While some of those changes (e.g. ascorbate and α-tocopherol) are compensatory mechanisms to cope with abiotic stresses, others (e.g. shikimate pathway and phenolic compounds) might be related to the alteration of the MEP pathway (Way et al., 2013; Kaling et al., 2014). The perturbation of these metabolic pathways can be attributed to the removal of a major carbon sink of the MEP pathway and the resulting change in the energy balance within the plant cell (Niinemets et al., 1999; Ghirardo et al., 2011). In this work, we analyzed the carbon fluxes through the MEP pathway into the main plastidic isoprenoid products.We used the ¹³C-labeling technique as a tool to measure the carbon fluxes through the MEP pathway at different temperatures, light intensities, and CO₂ concentrations in mature leaves of IE and transgenic, isoprene-nonemitting (NE) gray poplar (Populus × canescens). Isoprene emission was drastically reduced in the transgenic trees through knockdown of PcISPS gene expression by RNA interference, resulting in plants with only 1% to 5% of isoprene emission potential compared with wild-type plants (Behnke et al., 2007).We measured the appearance of ¹³C in the isoprenoid precursors 2-C-methyl-d-erythritol-2,4-cyclodiphosphate (MEcDP) and DMADP as well as isoprene and the major downstream products of the MEP pathway, i.e. carotenoids and Chls. To reliably detect de novo synthesis of the pigments, which occur at very low rates (Beisel et al., 2010), we used isotope ratio mass spectrometry (IRMS).Here, (1) we quantify the effect of isoprene biosynthesis on the MEP pathway in poplar, and (2) we show that suppression of isoprene biosynthesis negatively affects the carbon flux through the MEP pathway by accumulating plastidic DMADP, which feeds back to inhibit PcDXS, leading to (3) a slight increase of carbon flux toward production of greater chain-length isoprenoids and (4) a strong decrease in the overall isoprenoid carbon fluxes to compensate for the much lower MEP pathway demand for carbon. This study strongly supports the hypothesis that an important regulatory mechanism of the MEP pathway is the feedback regulation of plastidic DMADP on DXS. The large carbon flux through the MEP pathway of IE poplar plastids demonstrates the potential of transgenically altered IE plant species to produce economically valuable isoprenoids at high rates in, for instance, industrial applications.     

In situ O2 dynamics in submerged Isoetes australis: varied leaf gas permeability influences underwater photosynthesis and internal O2

A unique type of vernal pool are those formed on granite outcrops, as the substrate prevents percolation so that water accumulates in depressions when precipitation exceeds evaporation. The O(2) dynamics of small, shallow vernal pools with dense populations of Isoetes australis were studied in situ, and the potential importance of the achlorophyllous leaf bases to underwater net photosynthesis (P(N)) and radial O(2) loss to sediments is highlighted. O(2) microelectrodes were used in situ to monitor pO(2) in leaves, shallow sediments, and water in four vernal pools. The role of the achlorophyllous leaf bases in gas exchange was evaluated in laboratory studies of underwater P(N), loss of tissue water, radial O(2) loss, and light microscopy. Tissue and sediment pO(2) showed large diurnal amplitudes and internal O(2) was more similar to sediment pO(2) than water pO(2). In early afternoon, sediment pO(2) was often higher than tissue pO(2) and although sediment O(2) declined substantially during the night, it did not become anoxic. The achlorophyllous leaf bases were 34% of the surface area of the shoots, and enhanced by 2.5-fold rates of underwater P(N) by the green portions, presumably by increasing the surface area for CO(2) entry. In addition, these leaf bases would contribute to loss of O(2) to the surrounding sediments. Numerous species of isoetids, seagrasses, and rosette-forming wetland plants have a large proportion of the leaf buried in sediments and this study indicates that the white achlorophyllous leaf bases may act as an important area of entry for CO(2), or exit for O(2), with the surrounding sediment.     

Phosphorylation of the kinase interaction motif in mitogen-activated protein (MAP) kinase phosphatase-4 mediates cross-talk between protein kinase A and MAP kinase signaling pathways

MAP kinase phosphatase 4 (DUSP9/MKP-4) plays an essential role during placental development and is one of a subfamily of three closely related cytoplasmic dual-specificity MAPK phosphatases, which includes the ERK-specific enzymes DUSP6/MKP-3 and DUSP7/MKP-X. However, unlike DUSP6/MKP-3, DUSP9/MKP-4 also inactivates the p38α MAP kinase both in vitro and in vivo. Here we demonstrate that inactivation of both ERK1/2 and p38α by DUSP9/MKP-4 is mediated by a conserved arginine-rich kinase interaction motif located within the amino-terminal non-catalytic domain of the protein. Furthermore, DUSP9/MKP-4 is unique among these cytoplasmic MKPs in containing a conserved PKA consensus phosphorylation site (55)RRXSer-58 immediately adjacent to the kinase interaction motif. DUSP9/MKP-4 is phosphorylated on Ser-58 by PKA in vitro, and phosphorylation abrogates the binding of DUSP9/MKP-4 to both ERK2 and p38α MAP kinases. In addition, although mutation of Ser-58 to either alanine or glutamic acid does not affect the intrinsic catalytic activity of DUSP9/MKP-4, phospho-mimetic (Ser-58 to Glu) substitution inhibits both the interaction of DUSP9/MKP-4 with ERK2 and p38α in vivo and its ability to dephosphorylate and inactivate these MAP kinases. Finally, the use of a phospho-specific antibody demonstrates that endogenous DUSP9/MKP-4 is phosphorylated on Ser-58 in response to the PKA agonist forskolin and is also modified in placental tissue. We conclude that DUSP9/MKP-4 is a bona fide target of PKA signaling and that attenuation of DUSP9/MKP-4 function can mediate cross-talk between the PKA pathway and MAPK signaling through both ERK1/2 and p38α in vivo.     

Developmental refinement of hair cell synapses tightens the coupling of Ca2+ influx to exocytosis

Cochlear inner hair cells (IHCs) develop from pre‐sensory pacemaker to sound transducer. Here, we report that this involves changes in structure and function of the ribbon synapses between IHCs and spiral ganglion neurons (SGNs) around hearing onset in mice. As synapses matured they changed from holding several small presynaptic active zones (AZs) and apposed postsynaptic densities (PSDs) to one large AZ/PSD complex per SGN bouton. After the onset of hearing (i) IHCs had fewer and larger ribbons; (ii) Ca_V1.3 channels formed stripe‐like clusters rather than the smaller and round clusters at immature AZs; (iii) extrasynaptic Ca_V1.3‐channels were selectively reduced, (iv) the intrinsic Ca²⁺ dependence of fast exocytosis probed by Ca²⁺ uncaging remained unchanged but (v) the apparent Ca²⁺ dependence of exocytosis linearized, when assessed by progressive dihydropyridine block of Ca²⁺ influx. Biophysical modeling of exocytosis at mature and immature AZ topographies suggests that Ca²⁺ influx through an individual channel dominates the [Ca²⁺] driving exocytosis at each mature release site. We conclude that IHC synapses undergo major developmental refinements, resulting in tighter spatial coupling between Ca²⁺ influx and exocytosis.     

The impact of CO2 emission scenarios and nutrient enrichment on a common coral reef macroalga is modified by temporal effects

Future coral reefs are expected to be subject to higher pCO₂ and temperature due to anthropogenic greenhouse gas emissions. Such global stressors are often paired with local stressors thereby potentially modifying the response of organisms. Benthic macroalgae are strong competitors to corals and are assumed to do well under future conditions. The present study aimed to assess the impact of past and future CO₂ emission scenarios as well as nutrient enrichment on the growth, productivity, pigment, and tissue nutrient content of the common tropical brown alga Chnoospora implexa. Two experiments were conducted to assess the differential impacts of the manipulated conditions in winter and spring. Chnoospora implexa's growth rate averaged over winter and spring declined with increasing pCO₂ and temperature. Furthermore, nutrient enrichment did not affect growth. Highest growth was observed under spring pre‐industrial (PI) conditions, while slightly reduced growth was observed under winter A1FI (“business‐as‐usual”) scenarios. Productivity was not a good proxy for growth, as net O₂ flux increased under A1FI conditions. Nutrient enrichment, whilst not affecting growth, led to luxury nutrient uptake that was greater in winter than in spring. The findings suggest that in contrast with previous work, C. implexa is not likely to show enhanced growth under future conditions in isolation or in conjunction with nutrient enrichment. Instead, the results suggest that greatest growth rates for this species appear to be a feature of the PI past, with A1FI winter conditions leading to potential decreases in the abundance of this species from present day levels.     

Foraging behaviour of Blue Tits Parus caeruleus in a patchy environment under contrasting levels of natural food supply

We analysed the foraging behaviour of free-ranging Blue Tits Parus caeruleus in open holm oak Quercus ilex woodlands of western Spain during winter. Such woodlands are patchy for foraging tits because of the scattered distribution of trees and the patterns of abundance of canopy arthropods within and among trees. Results were compared with those obtained in spring of the same year, when we found that the foraging behaviour and spatial distribution of Blue Tits were largely unaffected by food availability (Pulido and Díaz 1997). Patch (tree) residence time was highly variable both within and among individual birds, and it was uncorrelated with either previous travel time or patch quality. Contrary to a priori expectations, the behaviour of tits did not conform to short-term energy maximizing rules in winter, in spite of a 2.5-fold decrease in food supply from spring to winter and a likely 2-fold increase in bird requirements. Instead, birds tended to fly towards patches that were further away than locally available. Overall, we conclude that energy intake rate was not the fitness-related currency that birds were trying to maximize while foraging.     

Epiphyte Orchid Establishment on Termite Carton Trails1

In the coastal zone of central Veracruz, Mexico, we recorded the abundance of orchid plants growing on termite carton trails or directly on the peeling bark of Bursera fagaroides. Although only 19 percent of the surveyed trees contained termite carton trails, trees with termites hosted 92 percent of all orchid individuals. A disproportionate number of orchid seedlings occurred on termite cartons given the relative availability of carton and bark substrate. Our results show for the first time that orchids can establish on termite trails, and we discuss the potential for termite facilitation of orchid populations.