Overexpression of MsALMT1, from the Aluminum-Sensitive Medicago sativa, Enhances Malate Exudation and Aluminum Resistance in Tobacco

Aluminum (Al) toxicity is a major limiting factor for plant growth and crop production in acidic soils. Al-induced organic acid (OA) exudation plays an important role in plant Al resistance. The exudation of OAs is mediated by membrane-localized OA transporters. In our previous study, a gene encoding the Al-induced malate transporter (MsALMT1) was identified in the roots of the Al-sensitive plant Medicago sativa L. cv. Yumu no. 1 (YM1). To further validate the function of MsALMT1, transgenic plants that overexpressed MsALMT1 under the control of the CaMV 35S (35S) promoter were generated. This transgenic tobacco showed an enhanced capacity for malate efflux and better Al resistance than wild type (WT) plants after exposure to 30 μM Al for 24 h. The Al content in the transgenic plant roots decreased to 40–52 % of that in WT plant roots. These results demonstrate that MsALMT1 is an Al-resistant gene in YM1 and encodes a malate transporter, the overexpression of which effectively enhances the Al resistance of transgenic tobacco plants.     

低磷和铝毒胁迫条件下菜豆有机酸的分泌与累积

以水培方式研究了低磷、铝毒胁迫条件下,不同菜豆基因型根系有机酸的分泌及其在植穆不同部位的累积,结果表明,低磷,铝毒胁迫诱导菜豆有机酸的分泌与累积存在显著的基因差异。低磷、铝毒胁迫诱导菜豆主要分泌柠檬酸、酒石酸和乙酸,其中,50μmol/LAl^3 诱导柠檬酸分泌量最高;低磷（小于20μmol/LH2PO4^－）胁迫诱导柠榨菜酸分泌量显著高于高磷处理,但低磷处理之间差异不明显,铝毒胁迫诱导菜豆有机酸的分泌与累积显著高于低磷胁迫处理,低磷,铝毒胁迫植株不同部位有机酸的含量为叶片大小根系,低磷,铝毒胁迫时,G842菜豆型柠檬酸有机酸分泌总量显著高于273、AFR和ZPV,其干重和磷吸收明明显于大G273,AFR和ZPV,且铝吸收量小于G273,AFR和ZPV,说明,G482菜豆基因型对低磷,铝毒的适应能力强于G273,AFR和ZPV基因型,菜豆有机酸,,尤其柠檬酸的分泌是其适应低磷、铝毒胁迫的重要生理反应。     

Overexpression of Malate Dehydrogenase in Transgenic Alfalfa Enhances Organic Acid Synthesis and Confers Tolerance to Aluminum

Al toxicity is a severe impediment to production of many crops in acid soil. Toxicity can be reduced through lime application to raise soil pH, however this amendment does not remedy subsoil acidity, and liming may not always be practical or cost-effective. Addition of organic acids to plant nutrient solutions alleviates phytotoxic Al effects, presumably by chelating Al and rendering it less toxic. In an effort to increase organic acid secretion and thereby enhance Al tolerance in alfalfa (Medicago sativa), we produced transgenic plants using nodule-enhanced forms of malate dehydrogenase and phosphoenolpyruvate carboxylase cDNAs under the control of the constitutive cauliflower mosaic virus 35S promoter. We report that a 1.6-fold increase in malate dehydrogenase enzyme specific activity in root tips of selected transgenic alfalfa led to a 4.2-fold increase in root concentration as well as a 7.1-fold increase in root exudation of citrate, oxalate, malate, succinate, and acetate compared with untransformed control alfalfa plants. Overexpression of phosphoenolpyruvate carboxylase enzyme specific activity in transgenic alfalfa did not result in increased root exudation of organic acids. The degree of Al tolerance by transformed plants in hydroponic solutions and in naturally acid soil corresponded with their patterns of organic acid exudation and supports the concept that enhancing organic acid synthesis in plants may be an effective strategy to cope with soil acidity and Al toxicity.     

Nutrient Reabsorption Mechanism Adapted to Low Phosphorus in Wild and Cultivated Soybean Varieties

Journal of Plant Growth Regulation - Phosphorus (P) plays an important role in soybean growth and reproduction. Wild soybean (Glycine soja) expressed higher tolerance to P-limited environment...     

Fatty Acid Composition and Nitrate Uptake of Soybean Roots during Acclimation to Low Temperature

Fatty acid composition of old and new roots was determined for soybeans (Glycine max [L.] Merr. cv Ransom) at root-zone temperatures of 14, 18, and 22°C during a 26-day period. New roots had a greater concentration of polyunsaturated fatty acids than old roots. The ratio of polyunsaturated to saturated fatty acid concentration in new roots exposed to 14 and 18°C peaked at 16 days and declined, while the corresponding ratio in old roots increased throughout the treatment period. Apparently the response of fatty acid composition in old and new roots to low temperature was mediated by tissue aging or differentiation. These findings were contrary to the concept that modifications in fatty acid composition remain constant at lower temperatures.

The function of root tissues exposed to lower temperature was evaluated with respect to the ability of the root systems to absorb NO₃⁻. Over the relatively long periods of exposure, the ability of whole root systems to absorb NO₃⁻ was similar at cool and warm temperatures. The effect of cool temperature on functioning of roots appeared to involve reductions in the rates of initiation and differentiation of young root tissues rather than changes in membrane permeability related to alteration of fatty acid composition.

     

Low Atmospheric Nitrogen Loads Lead to Grass Encroachment in Coastal Dunes,but Only on Acid Soils

The impact of atmospheric N-deposition on succession from open sand to dry, lichen-rich, short grassland, and tall grass vegetation dominated by Carex arenaria was surveyed in 19 coastal dune sites along the Baltic Sea. Coastal dunes with acid or slightly calcareous sand reacted differently to atmospheric wet deposition of 5–8 kg N ha⁻¹ y⁻¹. Accelerated acidification, as well as increased growth of Carex and accumulation of organic matter, was observed only at acid sites with pH_NaCl of the parent material below 6.0. At sites with slightly calcareous parent material, increased N-deposition had no effect. A trigger for grass encroachment seems to be high acidification in early successional stages to below pH_NaCl 4.0. Metals like Al or Fe become freely available and may hamper intolerant species. At acid sites, N-mineralization increases with elevated N-deposition, which may further stimulate Carex arenaria. Due to high growth plasticity, efficient resource allocation and tolerance of high metal concentrations, C. arenaria is a superior competitor under these conditions and can start to dominate the dune system. Carex-dominated vegetation is species-poor. Even at the moderate N-loads in this study, foliose lichens, forbs and grasses were reduced in short grass vegetation at acid sites. Species indicating these first effects of atmospheric deposition on dry, lichen-rich, short grasslands are identified and recommendations for restoration of grass-encroached sites given.     

Enhanced Disease Susceptibility 1 and Salicylic Acid Act Redundantly to Regulate Resistance Gene-Mediated Signaling

Resistance (R) protein–associated pathways are well known to participate in defense against a variety of microbial pathogens. Salicylic acid (SA) and its associated proteinaceous signaling components, including enhanced disease susceptibility 1 (EDS1), non–race-specific disease resistance 1 (NDR1), phytoalexin deficient 4 (PAD4), senescence associated gene 101 (SAG101), and EDS5, have been identified as components of resistance derived from many R proteins. Here, we show that EDS1 and SA fulfill redundant functions in defense signaling mediated by R proteins, which were thought to function independent of EDS1 and/or SA. Simultaneous mutations in EDS1 and the SA–synthesizing enzyme SID2 compromised hypersensitive response and/or resistance mediated by R proteins that contain coiled coil domains at their N-terminal ends. Furthermore, the expression of R genes and the associated defense signaling induced in response to a reduction in the level of oleic acid were also suppressed by compromising SA biosynthesis in the eds1 mutant background. The functional redundancy with SA was specific to EDS1. Results presented here redefine our understanding of the roles of EDS1 and SA in plant defense.     

Sensitivity of the Plant Vacuolar Malate Channel to pH, Ca2+ and Anion-Channel Blockers

The organic anion malate is accumulated in the central vacuole of most plant cells. Malate has several important roles in plant vacuoles, such as the maintenance of charge balance and pH regulation, as an osmolyte involved in the generation of cell turgor, and as a storage form of CO2. Transport of malate across the vacuolar membrane is important for the regulation of cytoplasmic pH and the control of cellular metabolism, particularly in plants showing crassulacean acid metabolism (CAM), in which large fluxes of malate occur during the day/night cycle. By applying the patch-clamp technique, in the whole-vacuole configuration, to isolated vacuoles from leaf mesophyll cells of the CAM plant Kalancho? daigremontiana, we studied the regulation of the vacuolar malate channel by pH and Ca2+, as well as its sensitivity to anion-channel blockers. Malate currents were found to be insensitive to Ca2+ on the cytoplasmic side of the membrane over a range from approximately 10(-8) M to 10(-4) M. In contrast, decreasing cytoplasmic pH below 7.5 had a significant modulatory effect on channel activity, reducing malate currents by 40%, whereas increasing cytoplasmic pH above 7.5 resulted in no change in current. Several known Cl?-channel blockers inhibited the vacuolar malate currents: niflumic acid and indanoyloxyacetic acid (IAA-94) proved to be the most effective inhibitors, exerting half-maximal effects at concentrations of approximately 20 mM, suggesting that the plant vacuolar malate channel may share certain similarities with other classes of known anion channels.     

Contribution of Malate and Amino Acid Metabolism to Cytoplasmic pH Regulation in Hypoxic Maize Root Tips Studied Using Nuclear Magnetic Resonance Spectroscopy

³¹P-, ¹³C-, and ¹⁵N-nuclear magnetic resonance spectroscopy were used to determine the roles of malate, succinate, Ala, Asp, Glu, Gln, and γ-aminobutyrate (GABA) in the energy metabolism and regulation of cytoplasmic pH in hypoxic maize (Zea mays L.) root tips. Nitrogen status was manipulated by perfusing root tips with ammonium sulfate prior to hypoxia; this pretreatment led to enhanced synthesis of Ala early in hypoxia, and of GABA at later times. We show that: (a) the ability to regulate cytoplasmic pH during hypoxia is not significantly affected by enhanced Ala synthesis. (b) Independent of nitrogen status, decarboxylation of Glu to GABA is greatest after several hours of hypoxia, as metabolism collapses. (c) Early in hypoxia, cytoplasmic malate is in part decarboxylated to pyruvate (leading to Ala, lactate, and ethanol), and in part converted to succinate. It appears that activation of malic enzyme serves to limit cytoplasmic acidosis early in hypoxia. (d) Ala synthesis in hypoxic root tips under these conditions is due to transfer of nitrogen ultimately derived from Asp and Gln, present in oxygenated tissue. We describe the relative contributions of glycolysis and malate decarboxylation in providing Ala carbons. (e) Succinate accumulation during hypoxia can be attributed to metabolism of Asp and malate; this flux to succinate is energetically negligible. There is no detectable net flux from Glc to succinate during hypoxia. The significance of the above metabolic reactions relative to ethanol and lactate production, and to flooding tolerance, is discussed. The regulation of the patterns of metabolism during hypoxia is considered with respect to cytoplasmic pH and redox state.     

Adaptation of Escherichia coli O157:H7 to pH Alters Membrane Lipid Composition, Verotoxin Secretion, and Resistance to Simulated Gastric Fluid Acid

The influence of adaptation to pH (from pH 5.0 to 9.0) on membrane lipid composition, verotoxin concentration, and resistance to acidic conditions in simulated gastric fluid (SGF) (pH 1.5, 37°C) was determined for Escherichia coli O157:H7 (HEC, ATCC 43895), an rpoS-deficient mutant of ATCC 43895 (HEC-RM, FRIK 816-3), and nonpathogenic E. coli (NPEC, ATCC 25922). Regardless of the strain, D values (in SGF) of acid-adapted cells were higher than those of non-acid-adapted cells, with HEC adapted at pH 5.0 having the greatest D value, i.e., 25.6 min. Acid adaptation increased the amounts of palmitic acid (C16:0) and decreased cis-vaccenic acid (C18:1ω7c) in the membrane lipids of all strains. The ratio of cis-vaccenic acid to palmitic acid increased at acidic pH, causing a decrease in membrane fluidity. HEC adapted to pH 8.3 and HEC-RM adapted to pH 7.3 exhibited the greatest verotoxin concentrations (2,470 and 1,460 ng/ml, respectively) at approximately 10⁸ CFU/ml. In addition, the ratio of extracellular to intracellular verotoxin concentration decreased at acidic pH, possibly due to the decrease of membrane fluidity. These results suggest that while the rpoS gene does not influence acid resistance in acid-adapted cells it does confer decreased membrane fluidity, which may increase acid resistance and decrease verotoxin secretion.     

Adaptation of Active Proton Pumping and Plasmalemma ATPase Activity of Corn Roots to Low Root Medium pH

Corn (Zea mays L.) root adaptation to pH 3.5 in comparison with pH 6.0 (control) was investigated in long-term nutrient solution experiments. When pH was gradually reduced, comparable root growth was observed irrespective of whether the pH was 3.5 or 6.0. After low-pH adaptation, H⁺ release of corn roots in vivo at pH 5.6 was about 3 times higher than that of control. Plasmalemma of corn roots was isolated for investigation in vitro. At optimum assay pH, in comparison with control, the following increases of the various parameters were caused by low-pH treatment: (a) hydrolytic ATPase activity, (b) maximum initial velocity and Michaelis constant (c) activation energy of H⁺-ATPase, (d) H⁺-pumping activity, (e) H⁺ permeability of plasmalemma, and (f) pH gradient across the membranes of plasmalemma vesicles. In addition, vanadate sensitivity remained unchanged. It is concluded that plasmalemma H⁺-ATPase contributes significantly to the adaptation of corn roots to low pH. A restricted net H⁺ release at low pH in vivo may be attributed to the steeper pH gradient and enhanced H⁺ permeability of plasmalemma but not to deactivation of H⁺-ATPase. Possible mechanisms responsible for adaptation of plasmalemma H⁺-ATPase to low solution pH during plant cultivation are discussed.     

DiGeorge Syndrome Gene tbx1 Functions through wnt11r to Regulate Heart Looping and Differentiation

DiGeorge syndrome (DGS) is the most common microdeletion syndrome, and is characterized by congenital cardiac, craniofacial and immune system abnormalities. The cardiac defects in DGS patients include conotruncal and ventricular septal defects. Although the etiology of DGS is critically regulated by TBX1 gene, the molecular pathways underpinning TBX1''s role in heart development are not fully understood. In this study, we characterized heart defects and downstream signaling in the zebrafish tbx1^−/− mutant, which has craniofacial and immune defects similar to DGS patients. We show that tbx1^−/− mutants have defective heart looping, morphology and function. Defective heart looping is accompanied by failure of cardiomyocytes to differentiate normally and failure to change shape from isotropic to anisotropic morphology in the outer curvatures of the heart. This is the first demonstration of tbx1''s role in regulating heart looping, cardiomyocyte shape and differentiation, and may explain how Tbx1 regulates conotruncal development in humans. Next we elucidated tbx1''s molecular signaling pathway guided by the cardiac phenotype of tbx1^−/− mutants. We show for the first time that wnt11r (wnt11 related), a member of the non-canonical Wnt pathway, and its downstream effector gene alcama (activated leukocyte cell adhesion molecule a) regulate heart looping and differentiation similarly to tbx1. Expression of both wnt11r and alcama are downregulated in tbx1^−/− mutants. In addition, both wnt11r ^−/− mutants and alcama morphants have heart looping and differentiation defects similar to tbx1^−/− mutants. Strikingly, heart looping and differentiation in tbx1^−/− mutants can be partially rescued by ectopic expression of wnt11r or alcama, supporting a model whereby heart looping and differentiation are regulated by tbx1 in a linear pathway through wnt11r and alcama. This is the first study linking tbx1 and non-canonical Wnt signaling and extends our understanding of DGS and heart development.     

Effects of Acid Adaptation, Product pH, and Heating on Survival of Escherichia coli O157:H7 in Pepperoni

The thermotolerance of E. coli O157:H7 cells (strain 380-94) heated in pepperoni is reported. Information on the pattern of thermal inactivation of E. coli O157:H7 in pepperoni was applied in the development of heating processes designed to reduce E. coli O157:H7 numbers therein by 5 log₁₀ units.