Topology independent protein structural alignment

Background  

Identifying structurally similar proteins with different chain topologies can aid studies in homology modeling, protein folding, protein design, and protein evolution. These include circular permuted protein structures, and the more general cases of non-cyclic permutations between similar structures, which are related by non-topological rearrangement beyond circular permutation. We present a method based on an approximation algorithm that finds sequence-order independent structural alignments that are close to optimal. We formulate the structural alignment problem as a special case of the maximum-weight independent set problem, and solve this computationally intensive problem approximately by iteratively solving relaxations of a corresponding integer programming problem. The resulting structural alignment is sequence order independent. Our method is also insensitive to insertions, deletions, and gaps.     

长江经济带湖南区域外来植物入侵现状及防控对策

[目的]了解长江经济带湖南区域外来入侵植物的种类构成、区系组成和区域分布,推进长江经济带湖南区域的生物多样性保护。[方法]采用实地踏查法开展长江经济带湖南区域外来入侵植物调查,以文献访查法对调查区域的入侵植物物种进行补充分析。[结果]长江经济带湖南区域有外来入侵植物41科102属146种,其中,菊科植物最多(35种),占入侵植物总种数的23.97%;外来入侵植物原产于美洲的最多(99种),占入侵植物总种数的67.81%;从生活型组成看,草本植物是长江经济带湖南区域外来入侵植物的主要组成部分,占比82.88%;从科的分布区系特征看,世界广布类型是入侵植物的主要区系类型(19科),占总科数的46.34%;从属的分布区系特征看,泛热带分布类型最多(32属),占总属数的31.38%。[结论]长江经济带湖南区域外来植物入侵呈现种类多、增速快、危害程度加重等特点,应加强对菊科入侵植物和来源于美洲入侵植物的监管。     

Amino acids regulate salinity-induced potassium efflux in barley root epidermis

The amino acid content increases substantially in salt-stressed plants. The physiological relevance of this phenomenon remains largely unknown. Using the MIFE ion flux measuring technique, we studied the effects of physiologically relevant concentrations of 26 amino acids on NaCl-induced K⁺ flux from barley root epidermis. We show that 21 (of 26) amino acids caused a significant mitigation of the NaCl-induced K⁺ efflux, while valine and ornithine substantially enhanced the detrimental effects of salinity on K⁺ homeostasis. Our results suggest that physiologically relevant concentrations of free amino acids might contribute to plant adaptive responses to salinity by regulating K⁺ transport across the plasma membrane, thus enabling maintenance of an optimal K⁺/Na⁺ ratio as opposed to being merely a symptom of plant damage by stress. Investigating the specific mechanisms of such amelioration remains a key issue for future studies.     

Effect of secondary metabolites associated with anaerobic soil conditions on ion fluxes and electrophysiology in barley roots

The effects of secondary metabolites produced by waterlogged soils on net K(+), H(+), and Ca(2+) fluxes were studied in the mature zone of roots of two barley (Hordeum vulgare) cultivars contrasting in their waterlogging (WL) tolerance using the noninvasive microelectrode ion flux measuring technique. In WL-sensitive variety 'Naso Nijo', all three lower monocarboxylic acids (formic, acetic, and propionic acids) and three phenolic acids (benzoic, 2-hydroxybenzoic, 4-hydroxybenzoic acids) caused a substantial shift toward steady K(+) efflux, accompanied by an immediate net influx of H(+). Detrimental effects of secondary metabolites on K(+) homeostasis in root cells were absent in WL-tolerant 'TX' variety. Root treatment with Mn(2+) caused only a temporary K(+) loss that returned to the initial level 10 min after treatment. Phenolic acids slightly increased Ca(2+) influx immediately after treatment, while other metabolites tested resulted in transient Ca(2+) efflux from the root. In the long-term (24 h) treatment, all metabolites tested significantly reduced K(+) uptake and the adverse effects of phenolic acids were smaller than for monocarboxylic acids and Mn(2+). Treatment with monocarboxylic acids for 24 h shifted H(+) from net efflux to net influx, while all three phenolic acids did not cause significant effects compared with the control. Based on results of pharmacological experiments and membrane potential measurements, a model explaining the effects of secondary metabolites on membrane transport activity is proposed. We also suggest that plant tolerance to these secondary metabolites could be considered a useful trait in breeding programs.     

Expression of animal CED-9 anti-apoptotic gene in tobacco modifies plasma membrane ion fluxes in response to salinity and oxidative stress

Apoptosis, one form of programmed cell death (PCD), plays an important role in mediating plant adaptive responses to the environment. Recent studies suggest that expression of animal anti-apoptotic genes in transgenic plants may significantly improve a plant’s ability to tolerate a variety of biotic and abiotic stresses. The underlying cellular mechanisms of this process remain unexplored. In this study, we investigated specific ion flux “signatures” in Nicotiana benthamiana plants transiently expressing CED-9 anti-apoptotic gene and undergoing salt- and oxidative stresses. Using a range of electrophysiological techniques, we show that expression of CED-9 increased plant salt and oxidative stress tolerance by altering K⁺ and H⁺ flux patterns across the plasma membrane. Our data shows that PVX/CED-9 plants are capable of preventing stress-induced K⁺ efflux from mesophyll cells, so maintaining intracellular K⁺ homeostasis. We attribute these effects to the ability of CED-9 to control at least two types of K⁺-permeable channels; outward-rectifying depolarization-activating K⁺ channels (KOR) and non-selective cation channels (NSCC). A possible scenario linking CED-9 expression and ionic relations in plant cell is suggested. To the best of our knowledge, this study is the first to link “ion flux signatures” and mechanisms involved in regulation of PCD in plants.     

Endomembrane Ca2+-ATPases play a significant role in virus-induced adaptation to oxidative stress

Although the role of Ca²⁺ influx channels in oxidative stress signaling and cross-tolerance in plants is well established, little is known about the role of active Ca²⁺ efflux systems in this process. In our recent paper,¹⁷ we reported Potato Virus X (PVX)-induced acquired resistance to oxidative stress in Nicotiana benthamiana and showed the critical role of plasma membrane Ca²⁺/H⁺ exchangers in this process. The current study continues this research. Using biochemical and electrophysiological approaches, we reveal that both endomembrane P_2A and P_2B Ca²⁺-ATPases play significant roles in adaptive responses to oxidative stress by removing excessive Ca²⁺ from the cytosol, and that their functional expression is significantly altered in PVX-inoculated plants. These findings highlight the crucial role of Ca²⁺ efflux systems in acquired tolerance to oxidative stress and open up prospects for practical applications in agriculture, after in-depth comprehension of the fundamental mechanisms involved in common responses to environmental factors at the genomic, cellular and organismal levels.Key words: cytosolic calcium, reactive oxygen species, cross-tolerance, calcium pumpThe phenomenon of cross-tolerance to a variety of biotic and abiotic stresses is well-known.^1,2 Some of the demonstrated examples include the correlation between oxidative stress tolerance and pathogen resistance.^3–5 At the mechanistic level, changes in cytosolic Ca²⁺ levels [Ca²⁺]_cyt, have long been implicated as a quintessential component of this process.⁶ The rise in [Ca²⁺]_cyt is proven to be essential for the development of the oxidative burst required for triggering the activation of several plant defense reactions.^7,8 The observed elevation in H₂O₂ level is believed to result from Ca²⁺-dependent activation of the NADPH oxidase,⁸ which then causes a further increase in [Ca²⁺]_cyt via a positive feedback mechanism. This process is further accomplished by defense gene activation, phytoalexin synthesis and eventual cell death.⁹ Downstream from the stimulus-induced [Ca²⁺]_cyt elevation, cells possess an array of proteins that can respond to a message. Such proteins include calmodulin (CaM),¹⁰ Ca²⁺-dependent protein kinases¹¹ and CaM binding proteins.¹² Of note is that when Ca²⁺ channels are blocked, biosynthesis of ROS is prevented.¹³While the role of Ca²⁺ influx channels in oxidative stress signaling and cross-tolerance in plants is well established, little is known about the involvement of active Ca²⁺ efflux systems in this process. In contrast, in animal systems the essential role of re-establishing [Ca²⁺]_cyt to resting levels is widely reported. A sustained increase in [Ca²⁺]_cyt in the alveolar macrophage is thought to be the consequence of membrane Ca²⁺-ATPase dysfunction.¹⁴ In endothelial cells, inhibition of the Ca²⁺/Na⁺ electroneutral exchanger of the mitochondria was named as one of the reasons for [Ca²⁺]_cyt increases.¹⁵ A significant loss of the plasma membrane Ca²⁺-ATPase (PMCA) activity was reported in brain synapses in response to oxidative stress,¹⁶ suggesting that PMCA may be a downstream target of oxidative stress.In our recently published paper¹⁷ we reported the phenomenon of Potato Virus X (PVX)-induced acquired resistance to oxidative stress in Nicotiana benthamiana plants and showed the critical role of plasma membrane Ca²⁺/H⁺ exchangers in this process. Nonetheless, questions remain, is this transporter the only active Ca²⁺ efflux system involved in this process?In addition to Ca²⁺/H⁺ exchangers, active Ca²⁺ extrusion could also be achieved by Ca²⁺-ATPases. Two major types of Ca²⁺-ATPases that differ substantially in their pharmacology and sensitivity to CaM are known.¹⁸ Type P_2A pumps (also called ER-type or ECA^19,20) are predominantly ER-localized,¹⁹ although they are also present at other endomembranes (e.g., tonoplast and Golgi). Four members of this group have been identified in the Arabidopsis genome (named AtECAs 1 to 4).^18,21 These pumps lack an N-terminal autoregulatory domain, are insensitive to CaM and suppressed by cyclopropiazonic acid (CPA).¹⁹ P_2B (or ACA) pumps contain an autoinhibitory N-terminal domain that possesses a binding site for Ca²⁺-CaM.¹⁸ Ten members are known in Arabidopsis (termed AtACA1, 2, 4 and 7 to 13).²¹ Plant P_2B pumps are located at the plasma membrane²⁰ as well as in inner membranes such as tonoplast (e.g., ACA4), ER (e.g., ACA2) and plastids.^18,19 These pumps probably constitute the basis for precise cytosolic Ca²⁺ regulation; as the Ca²⁺ concentration increases, CaM is activated and binds to the autoinhibitory domain of the Ca²⁺ pump. This results in the activation of the pump.In our recent study,¹⁷ we found no significant difference between the purified plasma membranes fractions isolated from control and UV-treated tobacco plants (with or without PVX inoculation) either in the Ca²⁺-ATPase activity or in the Ca²⁺-ATPase expression level and its ability to bind CaM. This suggests that the plasma membrane P_2B type pumps (the only pump type known to be expressed at the plasma membrane) play no major role in removing excess Ca²⁺ from the cytosol under oxidative stress conditions. This led to an obvious question: what about endomembrane Ca²⁺-ATPases?To address this issue, microsomal membrane fractions were isolated from tobacco leaves in a manner previously described for plasma membrane fractions¹⁷ (Fig. 1A). Western blot and CaM overlay assays were then made to investigate the role of endomembrane P_2B Ca²⁺-ATPases in our reported phenomena of acquired resistance. The results show that the expression of the P_2B Ca²⁺ pumps in PVX-inoculated plants is significantly higher than in control plants (Fig. 1B), correlating well with the CaM overlay assay (Fig. 1C). As no difference was observed for the P_2B Ca²⁺-ATPase expression levels in the plasma membranes,¹⁷ the observed difference in the microsomal fractions of PVX-infected plants must be due to an increased expression of endomembrane P_2B Ca²⁺-ATPases. Given the fact that Ca²⁺ pumps have a high affinity for calcium, the observed increase in endomembrane P_2B-type Ca²⁺-ATPases expression in PVX-inoculated plants may be advantageous for more efficient Ca²⁺ removal from the cytosol into internal organelles.Open in a separate windowFigure 1Expression of P_2B Ca²⁺ in purified microsomal fractions from tobacco leaves. Measurements were undertaken C = mock controls; C-UV = mock controls treated with UV-light; PVX = PVX infected plants; PVX-UV = PVX inoculated plants treated with UV-light. (A) Coomassie Brilliant Blue-stained gel; (B) Protein blot immunostained with a non isoform-specific polyclonal antibody for P_2B Ca²⁺-ATPases; (C) CaM overlay assay.To decipher the possible role of P_2A Ca²⁺-ATPases in acquired resistance, a series of electrophysiological experiments were conducted using inhibitors of P_2A-type Ca²⁺-ATPases, such as thapsigargin (TG)²² and cyclopiazonic acid (CPA).²³ Ion-selective Ca²⁺ microelectrodes were prepared as described elsewhere in reference ²⁴ and ²⁵, and net Ca²⁺ fluxes were measured from tobacco mesophyll tissue following previously described protocols.¹⁷ Leaf pre-treatment for 2 h in either of these inhibitors dramatically suppressed the net Ca²⁺ efflux measured from tobacco mesophyll cells 2 h after UV light exposure (Fig. 2). Given the specificity of TG and CPA inhibitors for P_2A-type Ca²⁺-ATPases, these results strongly support a hypothesis that both endomembrane P_2A and P_2B Ca²⁺-ATPases play significant roles in plant adaptive responses to oxidative stress. This is achieved by removing excess Ca²⁺ from the cytosol.Open in a separate windowFigure 2Effect of known Ca²⁺-ATPase blockers on light-induced Ca²⁺ flux kinetics after 20 min of UV-C treatment. Leaf mesophyll segments were pre-treated in either 5 µM TG (thapsigargin) or 50 µM CPA (cyclopiazonic acid) for 1–1.5 h prior to exposure to UV-C light. Net Ca²⁺ fluxes were measured 2 h after the end of UV treatment. These were compared with two controls: (1) no pre-treatment/no UV exposure (closed circles) and (2) no pre-treatment/20 min UV exposure (open squares). Mean ± SE (n = 4 to 7).Combining these results with our previously reported observations in reference ¹⁷, the following model is proposed (Fig. 3). Oxidative stress (such as UV) causes increased ROS production in leaf chloroplasts, leading to the elevated [Ca²⁺]_cyt. Several Ca²⁺ efflux systems are involved in restoring basal cytosolic Ca²⁺ levels. Two of these, the plasma membrane Ca²⁺/H⁺ exchanger¹⁷ and endomembrane P_2A and P_2B Ca²⁺-ATPases (as reported in this study) are upregulated in PVX inoculated plants and contribute to the improved tolerance to oxidative stress. Overall, these findings highlight the potential role of Ca²⁺ efflux systems in virus-induced tolerance to oxidative stress in plants. This is consistent with our previous reports on the important role of Ca²⁺ efflux systems in biotic stress tolerance²⁶ and brings forth possibilities for genetic engineering of more tolerant plants by targeting expression and regulation of active Ca²⁺ efflux systems at either the plasma or endomembranes.Open in a separate windowFigure 3The proposed model of oxidative stress signaling and the role of Ca²⁺-efflux systems in acquired resistance and plant adaptation to oxidative stress.Overall, a better adaptation of virus-infected plants to a short wave UV irradiation as compared to uninfected controls may suggest that infection triggers common defense mechanisms that could be efficient against secondary unrelated stresses. This observation may lead to the development of novel strategies to protect plants against complex environmental stress conditions.     

Proteomic profiling of oil bodies isolated from the unicellular green microalga Chlamydomonas reinhardtii: with focus on proteins involved in lipid metabolism

Oil bodies are sites of energy and carbon storage in many organisms including microalgae. As a step toward deciphering oil accumulation mechanisms in algae, we used proteomics to analyze purified oil bodies from the model microalga Chlamydomonas reinhardtii grown under nitrogen deprivation. Among the 248 proteins (≥ 2 peptides) identified by LC-MS/MS, 33 were putatively involved in the metabolism of lipids (mostly acyl-lipids and sterols). Compared with a recently reported Chlamydomonas oil body proteome, 19 new proteins of lipid metabolism were identified, spanning the key steps of the triacylglycerol synthesis pathway and including a glycerol-3-phosphate acyltransferase (GPAT), a lysophosphatidic acid acyltransferase (LPAT) and a putative phospholipid:diacylglycerol acyltransferase (PDAT). In addition, proteins putatively involved in deacylation/reacylation, sterol synthesis, lipid signaling and lipid trafficking were found to be associated with the oil body fraction. This data set thus provides evidence that Chlamydomonas oil bodies are not only storage compartments but also are dynamic structures likely to be involved in processes such as oil synthesis, degradation and lipid homeostasis. The proteins identified here should provide useful targets for genetic studies aiming at increasing our understanding of triacyglycerol synthesis and the role of oil bodies in microalgal cell functions.