Genome‐wide association mapping of time‐dependent growth responses to moderate drought stress in Arabidopsis

Large areas of arable land are often confronted with irregular rainfall resulting in limited water availability for part(s) of the growing seasons, which demands research for drought tolerance of plants. Natural variation was observed for biomass accumulation upon controlled moderate drought stress in 324 natural accessions of Arabidopsis. Improved performance under drought stress was correlated with early flowering and lack of vernalization requirement, indicating overlap in the regulatory networks of flowering time and drought response or correlated responses of these traits to natural selection. In addition, plant size was negatively correlated with relative water content (RWC) independent of the absolute water content (WC), indicating a prominent role for soluble compounds. Growth in control and drought conditions was determined over time and was modelled by an exponential function. Genome‐wide association (GWA) mapping of temporal plant size data and of model parameters resulted in the detection of six time‐dependent quantitative trait loci (QTLs) strongly associated with drought. Most QTLs would not have been identified if plant size was determined at a single time point. Analysis of earlier reported gene expression changes upon drought enabled us to identify for each QTL the most likely candidates.     

pH‐dependent conformational dynamics of beta‐secretase 1: A molecular dynamics study

Beta‐secretase 1 (BACE‐1) is an aspartyl protease implicated in the overproduction of β‐amyloid fibrils responsible for Alzheimer disease. The process of β‐amyloid genesis is known to be pH dependent, with an activity peak between solution pH of 3.5 and 5.5. We have studied the pH‐dependent dynamics of BACE‐1 to better understand the pH dependent mechanism. We have implemented support for graphics processor unit (GPU) accelerated constant pH molecular dynamics within the AMBER molecular dynamics software package and employed this to determine the relative population of different aspartyl dyad protonation states in the pH range of greatest β‐amyloid production, followed by conventional molecular dynamics to explore the differences among the various aspartyl dyad protonation states. We observed a difference in dynamics between double‐protonated, mono‐protonated, and double‐deprotonated states over the known pH range of higher activity. These differences include Tyr 71‐aspartyl dyad proximity and active water lifetime. This work indicates that Tyr 71 stabilizes catalytic water in the aspartyl dyad active site, enabling BACE‐1 activity.     

Trehalose phosphate synthase 5‐dependent trehalose metabolism modulates basal defense responses in Arabidopsis thaliana

Despite the recent discovery that trehalose synthesis is important for plant development and abiotic stress tolerance, the effects of trehalose on biotic stress responses remain relatively unknown. In this study, we demonstrate that TREHALOSE PHOSPHATE SYNTHASE 5 (TPS5)-dependent trehalose metabolism regulates Arabidopsis thaliana defenses against pathogens (necrotrophic Botrytis cinerea and biotrophic Pseudomonas syringae). Pathogen infection increased trehalose levels and upregulated TPS5 expression. Application of exogenous trehalose significantly improved plant defenses against B. cinerea, but increased the susceptibility of plants to P. syringae. We demonstrate that elevated trehalose biosynthesis, in transgenic plants over-expressing TPS5, also increased the susceptibility to P. syringae, but decreased the disease symptoms caused by B. cinerea. The knockout of TPS5 prevented the accumulation of trehalose and enhanced defense responses against P. syringae. Additionally, we observed that a TPS5-interacting protein (multiprotein bridging factor 1c) was required for induced expression of TPS5 during pathogen infections. Furthermore, we show that trehalose promotes P. syringae growth and disease development, via a mechanism involving suppression of the plant defense gene, Pathogenesis-Related Protein 1. These findings provide insight into the function of TPS5-dependent trehalose metabolism in plant basal defense responses.     

Wounding of Arabidopsis leaves induces indole‐3‐carbinol‐dependent autophagy in roots of Arabidopsis thaliana

In cruciferous plants insect attack or physical damage induce the synthesis of the glucosinolate breakdown product indole‐3‐carbinol, which plays a key role in the defense against attackers. Indole‐3‐carbinol also affects plant growth and development, acting as an auxin antagonist by binding to the TIR1 auxin receptor. Other potential functions of indole‐3‐carbinol and the underlying mechanisms in plant biology are unknown. Here we show that an indole‐3‐carbinol‐dependent signal induces specific autophagy in root cells. Leaf treatment with exogenous indole‐3‐carbinol or leaf‐wounding induced autophagy and inhibited auxin response in the root. This induction is lost in glucosinolate‐defective mutants, indicating that the effect of indole‐3‐carbinol is transported in the plants. Thus, indole‐3‐carbinol is not only a defensive metabolite that repels insects, but is also involved in long‐distance communication regulating growth and development in plants.     

Phototropin‐ and photosynthesis‐dependent mitochondrial positioning in Arabidopsis thaliana mesophyll cells

Mitochondria are frequently observed in the vicinity of chloroplasts in photosynthesizing cells, and this association is considered necessary for their metabolic interactions. We previously reported that, in leaf palisade cells of Arabidopsis thaliana, mitochondria exhibit blue‐light‐dependent redistribution together with chloroplasts, which conduct accumulation and avoidance responses under the control of blue‐light receptor phototropins. In this study, precise motility analyses by fluorescent microscopy revealed that the individual mitochondria in palisade cells, labeled with green fluorescent protein, exhibit typical stop‐and‐go movement. When exposed to blue light, the velocity of moving mitochondria increased in 30 min, whereas after 4 h, the frequency of stoppage of mitochondrial movement markedly increased. Using different mutant plants, we concluded that the presence of both phototropin1 and phototropin2 is necessary for the early acceleration of mitochondrial movement. On the contrary, the late enhancement of stoppage of mitochondrial movement occurs only in the presence of phototropin2 and only when intact photosynthesis takes place. A plasma‐membrane ghost assay suggested that the stopped mitochondria are firmly adhered to chloroplasts. These results indicate that the physical interaction between mitochondria and chloroplasts is cooperatively mediated by phototropin2‐ and photosynthesis‐dependent signals. The present study might add novel regulatory mechanism for light‐dependent plant organelle interactions.     

Internal transport of CO2 from the root‐zone to plant shoot is pH dependent

We investigated the fate of carbon dioxide (CO₂) absorbed by roots or internally produced by respiration using gas exchange and stable isotopic labeling. CO₂ efflux from detached leaves supplied with bicarbonate/CO₂ solutions was followed over six cycles. CO₂ effluxes were detected when bicarbonate solution at high pH was used, corresponding to 71–85% of the expected efflux. No CO₂ efflux was detected when CO₂ solutions at low pH were used but CO₂ efflux was subsequently detected as soon as bicarbonate solutions at high pH were supplied. By sealing the leaf and petiole in a plastic bag to reduce diffusion to the atmosphere, a small CO₂ efflux signal (14–30% of the expected efflux) was detected suggesting that CO₂ in the xylem stream can readily escape to the atmosphere before reaching the leaf. When the root‐zones of intact plants were exposed to CO₂ solutions, a significant efflux from leaf surface was observed (13% of the expected efflux). However, no signal was detected when roots were exposed to a high pH bicarbonate solution. Isotopic tracer experiments confirmed that CO₂ supplied to the root‐zone was transported through the plant and was readily lost to the atmosphere. However, little ¹³C moved to the shoot when roots were exposed to bicarbonate solutions at pH 8, suggesting that bicarbonate does not pass into the xylem.     

Cytosolic NADP‐dependent isocitrate dehydrogenase contributes to redox homeostasis and the regulation of pathogen responses in Arabidopsis leaves

Cytosolic NADP‐dependent isocitrate dehydrogenase (cICDH) produces 2‐oxoglutarate (2‐OG) and NADPH, and is encoded by a single gene in Arabidopsis thaliana. Three allelic lines carrying T‐DNA insertions in this gene showed less than 10% extractable leaf ICDH activity, but only relatively small decreases in growth compared to wild‐type Col0. Metabolite profiling by gas chromatography–time of flight–mass spectrometry (GC–TOF–MS) and high‐performance liquid chromatography (HPLC) revealed that loss of cICDH function produced only small effects on leaf compounds involved in carbon and nitrogen assimilation. To analyse whether cICDH contributes to NADPH production under conditions of oxidative stress, the icdh mutation was introduced into the cat2 background, in which increased availability of H₂O₂ causes perturbed redox homeostasis and induction of stress‐related genes. Accumulation of oxidized glutathione and pathogen‐related responses were enhanced in double cat2 icdh mutants compared to cat2. Single icdh mutants presented constitutive induction of PR genes, and enhanced resistance to bacteria in icdh, cat2 and cat2 icdh was quantitatively correlated with PR gene expression. However, the effect of icdh in both Col0 and cat2 backgrounds was not associated with enhanced accumulation of salicylic acid (SA). The results suggest that cICDH, previously considered mainly as an enzyme involved in amino acid synthesis, plays a role in redox signalling linked to pathogen responses.     

Substrate‐dependent dynamics of UDP‐galactopyranose mutase: Implications for drug design

Trypanosoma cruzi is the causative agent of Chagas disease, a neglected tropical disease that represents one of the major health challenges of the Latin American countries. Successful efforts were made during the last few decades to control the transmission of this disease, but there is still no treatment for the 10 million adults in the chronic phase of the disease. In T. cruzi, as well as in other pathogens, the flavoenzyme UDP‐galactopyranose mutase (UGM) catalyzes the conversion of UDP‐galactopyranose to UDP‐galactofuranose, a precursor of the cell surface β‐galactofuranose that is involved in the virulence of the pathogen. The fact that UGM is not present in humans makes inhibition of this enzyme a good approach in the design of new Chagas therapeutics. By performing a series of computer simulations of T. cruzi UGM in the presence or absence of an active site ligand, we address the molecular details of the mechanism that controls the uptake and retention of the substrate. The simulations suggest a modular mechanism in which each moiety of the substrate controls the flexibility of a different protein loop. Furthermore, the calculations indicate that interactions with the substrate diphosphate moiety are especially important for stabilizing the closed active site. This hypothesis is supported with kinetics measurements of site‐directed mutants of T. cruzi UGM. Our results extend our knowledge of UGM dynamics and offer new alternatives for the prospective design of drugs.     

Physiological responses of Plantago algarbiensis and P. almogravensis shoots and plantlets to low pH and aluminum stress

We investigated the impact of low pH and aluminum (Al) stress on the growth, nutrients concentration, chlorophyll a fluorescence, photosynthetic pigment contents, proline and carbohydrate accumulation in shoots and plantlets (leaves and roots) of Plantago almogravensis and P. algarbiensis. Both species accumulated considerable and similar amounts of Al in their tissues, mainly in the roots. The presence of Al caused a significant reduction on root elongation in P. algarbiensis. Low pH and Al induced significant changes on nutrient accumulation, but no significant alterations on the maximum efficiency of PSII (F _v/F _m), quantum yield of PSII photochemistry (?_PSII), quantum yield of regulated energy dissipation (?_NPQ) and quantum yield of non-regulated energy dissipation (?_NO) were detected in both species in response to these stresses. However, Al increased significantly the non-photochemical quenching and the chlorophyll b content and decreased the PSII excitation pressure (1 ? q _p) in P. almogravensis leaves. Both stress treatments induced carbohydrate accumulation in the shoots and roots of this species, but not in leaves. In P. algarbiensis, low pH and Al decreased the photosynthetic pigment contents in the shoots, whereas Al stimulated the carbohydrate accumulation in the leaves. Although our data showed that both species are tolerant to Al³⁺ and H⁺, P. almogravensis appeared to be more adapted to maintain cellular physiology and growth under those conditions.     

Growth and functional responses of different cultivars of Lotus corniculatus to aluminum and low pH stress

Aluminum toxicity is an important stress factor in acid soils. Growth, respiration and permeability properties of root cells were studied in five cultivars of Lotus corniculatus subjected to aluminum (Al) or low pH stress. The cultivars showed significant differences in root elongation under stress conditions, which correlated with changes in membrane potential (E_M) of root cortical cells. A pH drop from 5.5 to 4.0 resulted in significant membrane depolarization and root growth inhibition. The strongest inhibition was observed in cv. São Gabriel (33.6%) and least in cv. UFRGS (25.8%). Application of an extremely high Al concentration (2 mM) stopped the root growth in cv. INIA Draco, while inhibition in cv. UFRGS reached only 75%.The E_M values of cortical cells of Lotus roots varied between −115 and −144 mV. Treatment with 250 μM of AlCl₃ (pH 4) resulted in rapid membrane depolarization. The extent of the membrane depolarization ranged between 51 mV (cv. UFGRS) and 16 mV (cv. INIA Draco). The membrane depolarization was followed by a loss of K⁺ from Al-treated roots (2 mM Al) and resulted in a decrease of the diffusion potential (E_D). The total amount of K⁺ in Al-treated roots dropped from 31.4 to 16.8 μmol g⁻¹ FW in sensitive cv. INIA Draco, or from 26.1 to 22.7 μmol g⁻¹ FW in tolerant cv. UFGRS. The rate of root respiration under control conditions as well as under Al treatment was higher in cv. INIA Draco than in cv. UFRGS. Al-induced inhibition of root respiration was 21–34% of the control.     

Miro proteins coordinate microtubule‐ and actin‐dependent mitochondrial transport and distribution

In the current model of mitochondrial trafficking, Miro1 and Miro2 Rho‐GTPases regulate mitochondrial transport along microtubules by linking mitochondria to kinesin and dynein motors. By generating Miro1/2 double‐knockout mouse embryos and single‐ and double‐knockout embryonic fibroblasts, we demonstrate the essential and non‐redundant roles of Miro proteins for embryonic development and subcellular mitochondrial distribution. Unexpectedly, the TRAK1 and TRAK2 motor protein adaptors can still localise to the outer mitochondrial membrane to drive anterograde mitochondrial motility in Miro1/2 double‐knockout cells. In contrast, we show that TRAK2‐mediated retrograde mitochondrial transport is Miro1‐dependent. Interestingly, we find that Miro is critical for recruiting and stabilising the mitochondrial myosin Myo19 on the mitochondria for coupling mitochondria to the actin cytoskeleton. Moreover, Miro depletion during PINK1/Parkin‐dependent mitophagy can also drive a loss of mitochondrial Myo19 upon mitochondrial damage. Finally, aberrant positioning of mitochondria in Miro1/2 double‐knockout cells leads to disruption of correct mitochondrial segregation during mitosis. Thus, Miro proteins can fine‐tune actin‐ and tubulin‐dependent mitochondrial motility and positioning, to regulate key cellular functions such as cell proliferation.     

State‐dependent dynamics of cycles in red grouse abundance

Fluctuating populations are frequently demonstrated to co‐vary in abundance over space, but the dynamics of coupling between populations that gives rise to this synchrony are poorly understood. Synchrony may arise through coupling that is weak and continuous, but in populations that cycle with a characteristic period, synchrony can be maintained through stronger coupling that acts only intermittently. Here, we apply a discrete Markov model that describes the state of a population trajectory to be in one of four possible states. The Markov model reveals the nature of the coupling that gives rise to the weakly synchronous cycles of red grouse abundance. Using time‐series data from 287 populations across the species range in the UK, we show that grouse populations appear mostly uncoupled through time, but that approximately one year in six, “collective forcing events” occur, where populations in a region are forced into synchrony to a significantly greater degree than would be expected if their dynamics proceeded independently. In the absence of these events, synchrony between populations dissipates within ~3 yr. Smaller, low abundance populations tend to make the less probable phase shifts required to synchronize with nearby high abundance populations, suggesting that these low abundance populations are more susceptible to the perturbations responsible for phase shifts than larger populations.     

Plant dual‐specificity tyrosine phosphorylation‐regulated kinase optimizes light‐regulated growth and development in Arabidopsis

Light controls vegetative and reproductive development of plants. For a plant, sensing the light input properly ensures coordination with the ever‐changing environment. Previously, we found that LIGHT‐REGULATED WD1 (LWD1) and LWD2 regulate the circadian clock and photoperiodic flowering. Here, we identified Arabidopsis YET ANOTHER KINASE1 (AtYAK1), an evolutionarily conserved protein and a member of dual‐specificity tyrosine phosphorylation‐regulated kinases (DYRKs), as an interacting protein of LWDs. Our study revealed that AtYAK1 is an important regulator for various light responses, including the circadian clock, photomorphogenesis and reproductive development. AtYAK1 could antagonize the function of LWDs in regulating the circadian clock and photoperiodic flowering. By examining phenotypes of atyak1, we found that AtYAK1 regulated light‐induced period‐length shortening and photomorphogenic development. Moreover, AtYAK1 mediated plant fertility especially under inferior light conditions including low light and short‐day length. This study discloses a new regulator connecting environmental light to plant growth.     

Stimulus‐dependent dynamics of p53 in single cells

Many biological networks respond to various inputs through a common signaling molecule that triggers distinct cellular outcomes. One potential mechanism for achieving specific input–output relationships is to trigger distinct dynamical patterns in response to different stimuli. Here we focused on the dynamics of p53, a tumor suppressor activated in response to cellular stress. We quantified the dynamics of p53 in individual cells in response to UV and observed a single pulse that increases in amplitude and duration in proportion to the UV dose. This graded response contrasts with the previously described series of fixed pulses in response to γ‐radiation. We further found that while γ‐triggered p53 pulses are excitable, the p53 response to UV is not excitable and depends on continuous signaling from the input‐sensing kinases. Using mathematical modeling and experiments, we identified feedback loops that contribute to specific features of the stimulus‐dependent dynamics of p53, including excitability and input‐duration dependency. Our study shows that different stresses elicit different temporal profiles of p53, suggesting that modulation of p53 dynamics might be used to achieve specificity in this network.     

Concentration dependent ion selectivity in VDAC: a molecular dynamics simulation study

The voltage-dependent anion channel (VDAC) forms the major pore in the outer mitochondrial membrane. Its high conducting open state features a moderate anion selectivity. There is some evidence indicating that the electrophysiological properties of VDAC vary with the salt concentration. Using a theoretical approach the molecular basis for this concentration dependence was investigated. Molecular dynamics simulations and continuum electrostatic calculations performed on the mouse VDAC1 isoform clearly demonstrate that the distribution of fixed charges in the channel creates an electric field, which determines the anion preference of VDAC at low salt concentration. Increasing the salt concentration in the bulk results in a higher concentration of ions in the VDAC wide pore. This event induces a large electrostatic screening of the charged residues promoting a less anion selective channel. Residues that are responsible for the electrostatic pattern of the channel were identified using the molecular dynamics trajectories. Some of these residues are found to be conserved suggesting that ion permeation between different VDAC species occurs through a common mechanism. This inference is buttressed by electrophysiological experiments performed on bean VDAC32 protein akin to mouse VDAC.     

Microtubule‐dependent retrograde transport of bovine immunodeficiency virus

Microtubules are essential components of the cytoskeleton that participate in a variety of cellular processes such as cell division and migration. In addition, there is a growing body of evidence implicating a role for microtubules in intracellular viral transport. In this study, we found that pharmacological disruption of microtubules remarkably blocked bovine immunodeficiency virus (BIV) movement from the cell periphery to the perinuclear region, a process known as retrograde transport. A similar effect was observed by inhibiting function of the microtubule‐associated motor protein dynein. By yeast two‐hybrid assay, we found that the capsid protein (CA) of BIV interacted with the dynein light‐chain component LC8. Immunoprecipitation and GST‐pulldown assays further demonstrated an interaction between CA and LC8 in mammalian cells. In addition, our data revealed LC8 as a linker between BIV particles and microtubules. Retrograde transport of BIV was significantly inhibited by knockdown of LC8 expression. Our findings present the first evidence that incoming BIV particles employ host microtubule/dynein machinery for transport towards the perinuclear region. In addition, our data indicate that the LC8–CA interaction is a potential target for the design of antiviral strategies.