Aluminum tolerance in Arabidopsis thaliana as affected by endogenous salicylic acid

Endogenous salicylic acid (SA) functions in plant response to an aluminum stress were assessed. We used different Arabidopsis thaliana genotypes including snc1 with a constitutively high content of SA, sid2 and nahG (transgenic lines) both with a low content of SA, SA insensitive mutant npr1-1, and snc1/nahG (i.e., the nahG expression in the snc1 background) with a similar SA content as in wild type (WT) plants. Results show that the snc1 plants displayed obvious growth retardation of roots and shoots under the Al³⁺ stress, whereas the sid2, nahG, and npr1-1 plants exhibited alleviated symptoms in comparison with the WT plants. The Al³⁺ content increased in all the tested genotypes with the increasing AlCl₃ concentration applied, but no significant variations were detected among the tested genotypes. The snc1 had much higher superoxide dismutase and peroxidase activities, and a lower catalase activity and the ratio of reduced to oxidized glutathione accompanied by higher accumulations of H₂O₂ and malondialdehyde compared with the WT plants. These changes were largely reversed by the introduction of nahG; the sid2, nahG, and npr1-1 plants were less affected than WT plants in all the above-mentioned parameters. The Al³⁺ stress significantly enhanced malate exudation in all the tested genotypes, but no significant correlation was observed between the SA-involved response to the Al³⁺ stress and the malate exudation. Based on these data, it was concluded that the SA-related functions in Arabidopsis response to the Al³⁺ stress were associated with the control of oxidative stress, but not of malate exudation.     

Endogenous Salicylic Acid Levels and Signaling Positively Regulate <Emphasis Type="Italic">Arabidopsis</Emphasis> Response to Polyethylene Glycol-Simulated Drought Stress

Salicylic acid (SA) functions in the plant response to drought stress were assessed using SA-altering Arabidopsis mutants, including snc1 (with constitutively high levels of SA) and its nahG-transformed plants (named as snc1/nahG, with a comparable SA level to the wild type), sid2 and transgenic line nahG (both with SA deficiency), and npr1-1 (with SA signaling blockage). The drought stress was simulated by polyethylene glycol (PEG)-6000 treatment. Compared with wild-type (wt) plants, the snc1 plants displayed obvious easing of PEG-induced growth inhibition, leaf water loss, and photosynthesis-related impairment, whereas in nahG, sid2, and npr1-1 mutants the effect was more severe. PEG stress reduced stomatal conductance, to a higher extent in the snc1 line, whereas it was lower in nahG, sid2, and npr1-1 lines as compared with the wt. The snc1 plants accumulated higher levels of H₂O₂ than the other genotypes tested. PEG stress increased activities of superoxide dismutase and peroxidase, but decreased activities of catalase in all lines tested, to a greater extent in snc1 and less in sid2, nahG, and npr1-1 relative to wt. Proline was significantly increased, especially in snc1 line at 6 % and higher PEG stress. Noticeably, the performance of snc1 under PEG stress was dependent on SA levels, as the expression of nahG in snc1 plants did not only significantly reduce SA levels, but largely reversed the above-mentioned parameters, as well as eliminated the drought tolerance. Based on these data, it was concluded that endogenous SA levels and signaling provided a protective role in the Arabidopsis response to PEG-simulated drought.     

Salicylic acid-altering Arabidopsis mutants response to salt stress

Aims

The role of salicylic acid (SA) in plant responses to salinity is still a matter of controversy. To address the effect of endogenous SA variation in level and signaling on plant responses to salinity, biochemical and physiological analyses were performed on SA-altering Arabidopsis mutants including snc1 with high level of SA, transgenic line nahG with low SA, npr1-1 with SA signaling blockage, snc1/nahG plants (expression of nahG in the snc1 background), as well as wild type plants.

Methods

Plants were cultured in 1?×?Hoagland solution under controlled conditions. For salt exposure, NaCl at final concentrations of 100?mM, 200?mM, and 300?mM, respectively, was added to the culture solution after 25?d of seed germination. Except where mentioned, plant leaves were harvested after 14?d of salt stress, and used for physiological and chemical analyses.

Results

Salt stress caused all plants growth retardation with a dose-effect relationship relative to control. However, compared to wild type plants, a greater growth inhibition occurred in snc1, while a less inhibition was observed in nahG and npr1-1 plants, and a comparable extent was detected in snc1/nahG plants in which the SA level was near to that in wild type plants. The snc1 plants had lower net photosynthetic rate, variable to maximum fluorescence ratio, quantum efficiency of photosystem 2, reduced glutathione/oxidized glutathione ratio, proline levels, and higher malondiadehyde levels and electrolyte leakage rates as compared to wild type plants under salt stress. These values were effectively reversed by the expression of nahG gene in snc1 plants. The nahG and npr1-1 plants always exhibited more tolerance to salinity in above-mentioned indices than wild type plants. However, higher activities of superoxide dismutase and peroxidase in snc1 plants did not contribute to salt tolerance.

Conclusions

These data showed that SA deficit or signaling blockage in Arabidopsis plants was favorable to salt adaptation, while a high accumulation of SA potentiated salt-induced damage to Arabidopsis plants.     

Reducing basal salicylic acid enhances Arabidopsis tolerance to lead or cadmium

Aims

Phytoremediation is an emerging strategy for the removal of heavy metal contaminants. However, one of the prerequisite is to understand adequately plant resistant mechanisms. The present study was performed to assess the role of endogenous SA in plant response to Pb or Cd using wild-type (wt) Arabidopsis and its SA-accumulating mutant snc1, SA-reducing transgenic line nahG, SA signal-blocking npr1-1, and snc1/nahG (i.e. expression of nahG in snc1 plant) with a comparable level of SA to the wt.

Methods

Plants were grown hydroponically in controlled conditions. For heavy metal exposure, Pb²⁺ or Cd²⁺ at final concentrations of 50 μM, 100 μM, and 150 μM, respectively, was added to the culture solution. Unless otherwise indicated, samples were harvested after 7 d of exposure, and used for analyses.

Results

Compared to the wt level, the high endogenous SA significantly potentiated Pb- and Cd-induced plant growth retardation, whereas SA deficiency decreased the growth inhibition, and SA signaling blockage also had some protective effect. The expression of nahG in snc1 plant mitigated effectively the growth inhibition. The SA-related mechanism was involved in redox homeostasis, photosynthetic process, and soluble matter accumulation.

Conclusions

These results suggest that Pb- or Cd-induced phytotoxicity in Arabidopsis was intensified by elevated endogenous SA, whereas ameliorated by reduced SA.     

Combined effect of ethylene- and salicylic acid-signaling insensitive mutation on Arabidopsis response to low temperature

The roles of ethylene (ET) or salicylic acid (SA) in plant response to low temperature (LT, 5 °C) have been implicated. However, the combined effect of ET- and SA-signaling on plant growth and metabolism under LT remains to be evaluated. In this study, we comparatively analyzed the response of Arabidopsis ethylene insensitive (ein) 2-1 (an ET insensitive mutant), nonexprressor of pathogenesis relative (npr)1-1 (an SA insensitive mutant) and double mutant ein2-1/npr1-1 plants to LT. The results show that a LT of 5 °C induced plant growth retardation to a less degree in ein2-1, an intermediate degree in npr1-1, but a much larger in ein2-1/npr1-1 compared to the wild-type (WT) plants. The LT susceptibility of the ein2-1/npr1-1 plants was correlated to a lower net photosynthetic rate and proline content, and a higher content of H₂O₂ and malondialdehyde and electrolyte leakage relative to the WT plants. Lower activities of superoxide dismutase, peroxidase, and catalase, as well as a lower glutathione content and a ratio of its reduced form to its oxidized form were also observed in the double mutant plants as compared with the WT plants. However, at normal conditions (23 °C), all the tested physiological and biochemical parameters were comparable between the ein2-1/npr1-1 and WT plants, and plant growth was even better in the double mutant than in the WT plants. On the contrary, most of the above-mentioned parameters were advantageous in the ein2-1 and npr1-1 plants over the WT plants under the LT conditions. These data suggest that a parallel function or physiological redundancy of nonexpressor of pathogenesis relative 1 and ethylene insensitive 2 existed in the Arabidopsis plant response to the LT. On the other hand, an interaction between ET- and SA-signaling occurred during this process.     

Activation of an EDS1-mediated R-gene pathway in the snc1 mutant leads to constitutive, NPR1-independent pathogen resistance

The Arabidopsis NPR1 protein is an essential regulatory component of systemic acquired resistance (SAR). Mutations in the NPR1 gene completely block the induction of SAR by signals such as salicylic acid (SA). An Arabidopsis mutant, snc1 (suppressor of npr1-1, constitutive 1), was isolated in a screen for suppressors of npr1-1. In the npr1-1 background, the snc1 mutation resulted in constitutive resistance to Pseudomonas syringae maculicola ES4326 and Peronospora parasitica Noco2. High levels of SA were detected in the mutant and shown to be required for manifestation of the snc1 phenotype. The snc1 mutation was mapped to the RPP5 resistance (R) gene cluster and the eds1 mutation that blocks RPP5-mediated resistance suppressed snc1. These data suggest that a RPP5-related resistance pathway is activated constitutively in snc1. This pathway does not employ NPR1 but requires the signal molecule SA and the function of EDS1. Moreover, in snc1, constitutive resistance is conferred in the absence of cell death, which is often associated with R-gene mediated resistance.     

NPR1-dependent salicylic acid signaling is not involved in elevated CO2-induced heat stress tolerance in Arabidopsis thaliana

Elevated CO₂ can protect plants from heat stress (HS); however, the underlying mechanisms are largely unknown. Here, we used a set of Arabidopsis mutants such as salicylic acid (SA) signaling mutants nonexpressor of pathogenesis-related gene 1 (npr1-1 and npr1-5) and heat-shock proteins (HSPs) mutants (hsp21 and hsp70-1) to understand the requirement of SA signaling and HSPs in elevated CO₂-induced HS tolerance. Under ambient CO₂ (380 µmol mol⁻¹) conditions, HS (42°C, 24 h) drastically decreased maximum photochemical efficiency of PSII (Fv/Fm) in all studied plant groups. Enrichment of CO₂ (800 µmol mol⁻¹) with HS remarkably increased the Fv/Fm value in all plant groups except hsp70-1, indicating that NPR1-dependent SA signaling is not involved in the elevated CO₂-induced HS tolerance. These results also suggest an essentiality of HSP70-1, but not HSP21 in elevated CO₂-induced HS mitigation.     

Jasmonic acid and salicylic acid play minor roles in stomatal regulation by CO2, abscisic acid,darkness, vapor pressure deficit and ozone

Jasmonic acid (JA) and salicylic acid (SA) regulate stomatal closure, preventing pathogen invasion into plants. However, to what extent abscisic acid (ABA), SA and JA interact, and what the roles of SA and JA are in stomatal responses to environmental cues, remains unclear. Here, by using intact plant gas-exchange measurements in JA and SA single and double mutants, we show that stomatal responsiveness to CO₂, light intensity, ABA, high vapor pressure deficit and ozone either did not or, for some stimuli only, very slightly depended upon JA and SA biosynthesis and signaling mutants, including dde2, sid2, coi1, jai1, myc2 and npr1 alleles. Although the stomata in the mutants studied clearly responded to ABA, CO₂, light and ozone, ABA-triggered stomatal closure in npr1-1 was slightly accelerated compared with the wild type. Stomatal reopening after ozone pulses was quicker in the coi1-16 mutant than in the wild type. In intact Arabidopsis plants, spraying with methyl-JA led to only a modest reduction in stomatal conductance 80 min after treatment, whereas ABA and CO₂ induced pronounced stomatal closure within minutes. We could not document a reduction of stomatal conductance after spraying with SA. Coronatine-induced stomatal opening was initiated slowly after 1.5–2.0 h, and reached a maximum by 3 h after spraying intact plants. Our results suggest that ABA, CO₂ and light are major regulators of rapid guard cell signaling, whereas JA and SA could play only minor roles in the whole-plant stomatal response to environmental cues in Arabidopsis and Solanum lycopersicum (tomato).     

Salicylic Acid and a Chitin Elicitor Both Control Expression of the CAD1 Gene Involved in the Plant Immunity of Arabidopsis

The Arabidopsis mutant cad1 (constitutively activated cell death 1) shows a phenotype that mimics hypersensitive response (HR)-like cell death. The CAD1 gene, which encodes a protein containing a domain with significant homology to the MACPF (membrane attach complex and perforin) domain of complement components and perforin, is likely to control plant immunity negatively and has a W-box cis-element in its promoter region. We found that expression of the CAD1 gene and other W-box containing genes, such as NPR1 and PR2, was promoted by salicylic acid (SA) and benzothiadiazole (BTH) as a SA agonist. The CAD1 gene was also stimulated by a purified chitin oligosaccharide elicitor (degree of polymerization = 8). This latter control was not under SA, because CAD1 expression was not suppressed in 35SnahG transgenic plants, which are unable to accumulate SA. These expression profiles were confirmed by promoter analysis using pCAD1::GUS transgenic plants. The CAD1 expression promoted by BTH and the chitin elicitor was not suppressed in the npr1 mutant, which is insensitive to SA signaling. These results indicate that the CAD1 gene is regulated by two distinct pathways involving SA and a chitin elicitor: viz., SA signaling mediated through an NPR1-independent pathway, and chitin elicitor signaling, through an SA-independent pathway. Three CAD1 homologs that have multiple W-box elements in their promoters were also found to be under the control of SA.     

Accumulation of Four Metals in Tissues of Corchorus olitorius and Possible Mechanisms of Their Tolerance

Corchorus olitorius plants treated by 5 μg cm⁻³ of Cd, Pb, Al or Cu in hydroponic culture accumulated in leaves 190, 150, 350 and 325 μg g⁻¹(d.m.) of these metals, respectively, after 6 d of exposure. Exposure of Corchorus plants to tested metals resulted in a sharp rise in content of amino acids in leaf tissues, however the magnitude of accumulation was different from one metal to another. Presence of sulphur in the growth medium significantly increased uptake of Cd and Pb and cysteine (cyst) was more effective than K₂SO₄. Similarly, addition of salicylic acid (SA) in the growth medium significantly enhanced the ability of Corchorus plants to accumulate all these metals. Growth of Corchorus plants was significantly reduced by treatment with any of the four metals except Cu and added cyst, K₂SO₄ or SA alleviated the growth retarding effect of metals. This revised version was published online in July 2006 with corrections to the Cover Date.     

Chitosan signaling in guard cells requires endogenous salicylic acid

An elicitor chitosan (CHT) induces stomatal closure but the mechanism remains to be clarified. A phytohormone salicylic acid (SA) is crucial for elicitor-induced defense signaling in plants. Here we investigated whether endogenous SA is required for CHT signaling in guard cells. In the SA-deficient nahG mutant, treatment of CHT did not induce either apoplastic reactive oxygen species (ROS) production or stomatal closure but co-treatment of CHT and SA induced both apoplastic ROS production and stomatal closure, indicating the involvement of endogenous SA in CHT-induced apoplastic ROS production and CHT-induced stomatal closure. Furthermore, CHT induced transient cytosolic free calcium concentration increments in the nahG mutant in the presence of exogenous SA but not in the absence of exogenous SA. These results provide evidence that endogenous SA is a crucial element in CHT-induced stomatal closure.     

Mechanistic Elucidation of Salicylic Acid and Sulphur-Induced Defence Systems,Nitrogen Metabolism,Photosynthetic, and Growth Potential of Mungbean (Vigna radiata) Under Salt Stress

The potential of plant nutrients (such as sulphur, S) and phytohormones (such as salicylic acid, SA) has been explored in isolated studies by researchers in controlling the impact of abiotic stresses such as salinity in plants. However, information is scanty on the major mechanisms underlying the role of S and/or SA in modulation of enzymes involved in nitrogen (N) assimilation, GOGAT cycle, and antioxidant defence system; the cellular status of N-containing osmolyte proline, glucose, S-containing compounds; and their cumulative role in photosynthesis functions and growth in crop plants. The present study aimed to assess the role of cumulative effect of SA and S (SO₄²⁻) mediated induction of N assimilatory enzymes, GOGAT cycle, N-osmolyte proline and its metabolizing enzymes, glyoxylase enzymes, and antioxidant capacity in mungbean (Vigna radiata L.) exposed to NaCl with or without SO₄²⁻ and SA. Salt-exposed V. radiate showed differential elevations in damage (O^.₂⁻, H₂O₂, lipid peroxidation; glucose) and defence (ascorbate peroxidase, APX; glutathione reductase, GR; superoxide dismutase, SOD; reduced GSH; proline) and inhibitions in the activities of NR and NiR; N content, photosynthesis, photosynthetic N-use-efficiency (NUE), and growth. The separate supplementation of SA and SO₄²⁻ to 50 mM NaCl almost equally strengthened the antioxidant machinery and diminished NaCl-accrued damages. However, combined supply of SA and SO₄²⁻ to NaCl-exposed cultivars led to significant improvements in NR and NiR activities, the accumulation of N, GSH, proline, enhanced activity of APX, GR, and reduced activity of SOD, and also decreases in O^.₂⁻, H₂O₂, lipid peroxidation and glucose. These observations were corroborated with SA, SO₄²⁻ and NaCl-mediated changes in the traits of photosynthesis and growth, stomatal behaviour, and the polypeptide patterns of Rubisco in V. radiata. Overall, in V. radiata, SA-mediated higher enhancements in the activity of N assimilatory enzymes (NR, NiR, and GS), increase in the N and proline, and GSH; and decreases in the contents of Na⁺ and Cl⁻ ions, and glucose (a photosynthesis repressor); maintenance of a fine tuning among SOD, APX, and GR enzymes; and higher minimization of ROS (O^.₂⁻, H₂O₂) and lipid peroxidation finally led to a higher promotion in photosynthesis and growth.

     

Effects of buffer capacity on growth,photosynthesis, and solute accumulation of a glycophyte (wheat) and a halophyte (<Emphasis Type="Italic">Chloris virgata</Emphasis>)

Two species with different resistances to alkaline pH, the glycophylic Triticum aestivum (wheat) and the halophilic Chloris virgata, were chosen as test organisms. The salt-alkaline (SA) mixed stress conditions with different buffer capacities (BC) but with the same salt molarities and pH were established by mixing neutral (NaCl, Na₂SO₄), and alkaline salts (NaHCO₃ and Na₂CO₃) in various proportions. Growth, photosynthetic characteristics, and solute accumulation of the seedlings were monitored to test the validity of BC as a decisive index of alkali-stress (AS) intensity in SA mixed stress. At the same salinities and pHs, the relative growth rate, the content of photosynthetic pigments, and net photosynthetic rates of wheat and C. virgata decreased, while Na⁺ content and Na⁺/K⁺ ratios in shoots increased with increasing BC. Hence BC was a true measure of AS intensity at mixed SA stress and the alkali-resistance mechanism of plants was easy to interpret. BC of soil solution is an important parameter for estimating the alkalization degree of salt-alkalized soil.     

Arabidopsis snc2-1D Activates Receptor-Like Protein-Mediated Immunity Transduced through WRKY70

Plant immune receptors belonging to the receptor-like protein (RLP) family contain extracellular leucine-rich repeats (LRRs) and a short cytoplasmic tail linked by a single transmembrane motif. Here, we report the identification of snc2-1D (for suppressor of npr1-1, constitutive 2), a semidominant Arabidopsis thaliana mutant with constitutively activated defense responses. Map-based cloning of snc2-1D showed that it encodes an RLP. The point mutation in snc2-1D leads to substitution of the second Gly for Arg in the conserved GXXXG motif of the transmembrane helix, suggesting that this residue is important for negative regulation of the protein. Epistasis analysis revealed that the snc2-1D mutant phenotype is not affected by mutations in genes known to be required for the nucleotide binding (NB)-LRR Resistance (R) protein signaling. A suppressor screen of snc2-1D was performed, and map-based cloning of one suppressor revealed that mutations in WRKY70 suppress the constitutive defense responses in snc2-1D, suggesting that WRKY70 functions downstream of snc2-1D. The identification of snc2-1D provides us with a unique system for genetic analysis of resistance pathways downstream of RLPs, which may be distinct from those downstream of NB-LRR type R proteins.     

Response of xanthophyll cycle and chloroplastic antioxidant enzymes to chilling stress in tomato over-expressing glycerol-3-phosphate acyltransferase gene

Over-expression of chloroplastic glycerol-3-phosphate acyltransferase gene (LeGPAT) increased unsaturated fatty acid contents in phosphatidylglycerol (PG) of thylakoid membrane in tomato. The effect of this increase on the xanthophyll cycle and chloroplast antioxidant enzymes was examined by comparing wild type (WT) tomato with the transgenic (TG) lines at chilling temperature (4 °C) under low irradiance (100 μmol m⁻² s⁻¹). Net photosynthetic rate and the maximal photochemical efficiency of photosystem (PS) 2 (F_v/F_m) in TG plants decreased more slowly during chilling stress and F_v/F_m recovered faster than that in WT plants under optimal conditions. The oxidizable P700 in both WT and TG plants decreased during chilling stress under low irradiance, but recovered faster in TG plants than in the WT ones. During chilling stress, non-photochemical quenching (NPQ) and the de-epoxidized ratio of xanthophyll cycle in WT plants were lower than those of TG tomatoes. The higher activities of superoxide dismutase (SOD) and ascorbate peroxidase (APX) in TG plants resulted in the reduction of O₂ ^−· and H₂O₂ contents during chilling stress. Hence the increase in content of unsaturated fatty acids in PG by the over-expression of LeGPAT could alleviate photoinhibition of PS2 and PS1 by improving the de-epoxidized ratio of xanthophyll cycle and activities of SOD and APX in chloroplast.     

A gain-of-function mutation in a plant disease resistance gene leads to constitutive activation of downstream signal transduction pathways in suppressor of npr1-1, constitutive 1

Plants have evolved sophisticated defense mechanisms against pathogen infections, during which resistance (R) genes play central roles in recognizing pathogens and initiating defense cascades. Most of the cloned R genes share two common domains: the central domain, which encodes a nucleotide binding adaptor shared by APAF-1, certain R proteins, and CED-4 (NB-ARC), plus a C-terminal region that encodes Leu-rich repeats (LRR). In Arabidopsis, a dominant mutant, suppressor of npr1-1, constitutive 1 (snc1), was identified previously that constitutively expresses pathogenesis-related (PR) genes and resistance against both Pseudomonas syringae pv maculicola ES4326 and Peronospora parasitica Noco2. The snc1 mutation was mapped to the RPP4 cluster. In snc1, one of the TIR-NB-LRR-type R genes contains a point mutation that results in a single amino acid change from Glu to Lys in the region between NB-ARC and LRR. Deletions of this R gene in snc1 reverted the plants to wild-type morphology and completely abolished constitutive PR gene expression and disease resistance. The constitutive activation of the defense responses was not the result of the overexpression of the R gene, because its expression level was not altered in snc1. Our data suggest that the point mutation in snc1 renders the R gene constitutively active without interaction with pathogens. To analyze signal transduction pathways downstream of snc1, epistasis analyses between snc1 and pad4-1 or eds5-3 were performed. Although the resistance signaling in snc1 was fully dependent on PAD4, it was only partially affected by blocking salicylic acid (SA) synthesis, suggesting that snc1 activates both SA-dependent and SA-independent resistance pathways.