Convergence and divergence of stress-induced mitogen-activated protein kinase signaling pathways at the level of two distinct mitogen-activated protein kinase kinases

Plants respond to biotic and abiotic stresses by inducing overlapping sets of mitogen-activated protein kinases (MAPKs) and response genes. To define the mechanisms of how different signals can activate a common signaling pathway, upstream activators of SIMK, a salt stress- and pathogen-induced alfalfa MAPK, were identified. Here, we compare the properties of SIMKK, a MAPK kinase (MAPKK) that mediates the activation of SIMK by salt stress, with those of PRKK, a distantly related novel MAPKK. Although both SIMKK and PRKK show strongest interaction with SIMK, SIMKK can activate SIMK without stimulation by upstream factors. In contrast, PRKK requires activation by an upstream activated MAPKK kinase. SIMKK mediates pathogen elicitor signaling and salt stress, but PRKK transmits only elicitor-induced MAPK activation. Of four tested MAPKs, PRKK activates three of them (SIMK, MMK3, and SAMK) upon elicitor treatment of cells. However, PRKK is unable to activate any MAPK upon salt stress. In contrast, SIMKK activates SIMK and MMK3 in response to elicitor, but it activates only SIMK upon salt stress. These data show that (1) MAPKKs function as convergence points for stress signals, (2) MAPKKs activate multiple MAPKs, and (3) signaling specificity is obtained not only through the inherent affinities of MAPKK-MAPK combinations but also through stress signal-dependent intracellular mechanisms.     

A MAPK pathway mediates ethylene signaling in plants

Ethylene signal transduction involves ETR1, a two-component histidine protein kinase receptor. ETR1 functions upstream of the negative regulator CTR1. The similarity of CTR1 to members of the Raf family of mitogen-activated protein kinase kinase kinases (MAPKKKs) suggested that ethylene signaling in plants involves a MAPK pathway, but no direct evidence for this has been provided. Here we show that distinct MAPKs are activated by the ethylene precursor aminocyclopropane-1-carboxylic acid (ACC) in Medicago and ARABIDOPSIS: In Medicago, the ACC-activated MAPKs were SIMK and MMK3, while in Arabidopsis MPK6 and another MAPK were identified. Medicago SIMKK specifically mediated ACC-induced activation of SIMK and MMK3. Transgenic Arabidopsis plants overexpressing SIMKK have constitutive MPK6 activation and ethylene-induced target gene expression. SIMKK overexpressor lines resemble ctr1 mutants in showing a triple response phenotype in the absence of ACC. Whereas MPK6 was not activated by ACC in etr1 mutants, ein2 and ein3 mutants showed normal activation profiles. In contrast, ctr1 mutants showed constitutive activation of MPK6. These data indicate that a MAPK cascade is part of the ethylene signal transduction pathway in plants.     

Differential activation of four specific MAPK pathways by distinct elicitors

Plant cells respond to elicitors by inducing a variety of defense responses. Some of these reactions are dependent on the activity of protein kinases. Recently, mitogen-activated protein kinases (MAPKs) have been identified to be activated by fungal and bacterial elicitors as well as by pathogen infection. In gel kinase assays of alfalfa cells treated with yeast cell wall-derived elicitor (YE) revealed that 44- and 46-kDa MAPKs are rapidly and transiently activated. Immunokinase assays with specific MAPK antibodies revealed that YE mainly activated the 46-kDa SIMK and the 44-kDa MMK3 and to a lesser extent the 44-kDa MMK2 and SAMK. When cells were treated with chemically defined elicitors potentially contained in the YE (chitin and N-acetylglucosamine oligomers, beta-glucan, and ergosterol), the four MAPKs were found to be activated to different levels and with different kinetics. Whereas SIMK and SAMK have been found to be activated by a number of diverse stimuli, MMK3 is activated during mitosis and was therefore assumed to participate in cell division (). No physiological process could be associated with MMK2 activity so far. This is the first report that MMK2 and MMK3 can be activated by external stimuli. Overall, our findings indicate that plant cells can sense different cues of a given microorganism through the activation of multiple MAPKs.     

Stress-induced protein phosphatase 2C is a negative regulator of a mitogen-activated protein kinase

Protein phosphatases of type 2C (PP2Cs) play important roles in eukaryotic signal transduction. In contrast to other eukaryotes, plants such as Arabidopsis have an unusually large group of 69 different PP2C genes. At present, little is known about the functions and substrates of plant PP2Cs. We have previously shown that MP2C, a wound-induced alfalfa PP2C, is a negative regulator of mitogen-activated protein kinase (MAPK) pathways in yeast and plants. In this report, we provide evidence that alfalfa salt stress-inducible MAPK (SIMK) and stress-activated MAPK (SAMK) are activated by wounding and that MP2C is a MAPK phosphatase that directly inactivates SIMK but not the wound-activated MAPK, SAMK. SIMK is inactivated through threonine dephosphorylation of the pTEpY motif, which is essential for MAPK activity. Mutant analysis indicated that inactivation of SIMK depends on the catalytic activity of MP2C. A comparison of MP2C with two other PP2Cs, ABI2 and AtP2CHA, revealed that although all three phosphatases have similar activities toward casein as a substrate, only MP2C is able to dephosphorylate and inactivate SIMK. In agreement with the notion that MP2C interacts directly with SIMK, the MAPK was identified as an interacting partner of MP2C in a yeast two-hybrid screen. MP2C can be immunoprecipitated with SIMK in a complex in vivo and shows direct binding to SIMK in vitro in protein interaction assays. Wound-induced MP2C expression correlates with the time window when SIMK is inactivated, corroborating the notion that MP2C is involved in resetting the SIMK signaling pathway.     

Differential oxidation of thioredoxin-1, thioredoxin-2, and glutathione by metal ions

Metal toxicity often includes the generation of reactive oxygen species (ROS) and subsequent oxidative stress, but whether metals have different effects on the major thiol antioxidant systems is unknown. Here, we examine the effects of arsenic, cadmium, cesium, copper, iron, mercury, nickel, and zinc on glutathione (GSH), cytoplasmic thioredoxin-1 (Trx1), and mitochondrial thioredoxin-2 (Trx2) redox states. GSH/GSSG redox states were determined by HPLC, and Trx1 and Trx2 redox states were determined by Redox Western blot methods. Copper, iron, and nickel showed significant oxidation of GSH but relatively little oxidation of either Trx1 or Trx2. Arsenic, cadmium, and mercury showed little oxidation of GSH but significantly oxidized both Trx1 and Trx2. The magnitude of effects of arsenic, cadmium, and mercury was greater for the mitochondrial Trx2 (>60 mV) compared to the cytoplasmic Trx1 (20 to 40 mV). Apoptosis signal-regulating kinase 1 (ASK1) may be activated by two different pathways, one dependent upon GSH and glutaredoxin and the other independent of GSH and dependent upon thioredoxin. ASK1 activation and cell death were observed with metals that oxidized thioredoxins but not with metals that oxidized GSH. These findings show that metals have differential oxidative effects on the major thiol antioxidant systems and that activation of apoptosis may be associated with metal ions that oxidize thioredoxin and activate ASK1. The differential oxidation of the major thiol antioxidant systems by metal ions suggest that the distinct thiol/disulfide redox couples represented by GSH/GSSG and the thioredoxins may convey different levels of control in apoptotic and toxic signaling pathways.     

植物重金属转运蛋白研究进展

土壤中的有毒重金属不仅对植物有害,也可通过食物链危害人类和动物的健康.重金属转运蛋白在植物吸收、抵抗重金属的复杂机制中起着关键作用.植物重金属转运蛋白分为吸收蛋白和排出蛋白,其中,吸收蛋白转运必需重金属进入细胞,同时也会因为必需重金属的缺乏或离子之间的竞争而运载有毒重金属;排出蛋白是一类解毒蛋白,可将过量的或有毒的重金属逆向转运出细胞,或区室化于液泡中.目前,细胞内多种重金属转运蛋白基因的转录水平与重金属离子积累之间的联系已被揭示,并分离克隆出诸多相关蛋白家族成员.本文综述了近年来发现并鉴定的主要重金属转运蛋白的金属亲和性、器官表达特异性及细胞内定位等的研究进展.     

Halophytes--an emerging trend in phytoremediation

Halophytic plants are of special interest because these plants are naturally present in environments characterized by an excess of toxic ions, mainly sodium and chloride. Several studies have revealed that these plants may also tolerate other stresses including heavy metals based on the findings that tolerance to salt and to heavy metals may, at least partly, rely on common physiological mechanisms. In addition, it has been shown that salt-tolerant plants may also be able to accumulate metals. Therefore, halophytes have been suggested to be naturally better adapted to cope with environmental stresses, including heavy metals compared to salt-sensitive crop plants commonly chosen for phytoextraction purposes. Thus, potentially halophytes are ideal candidates for phytoextraction orphytostabilization of heavy metal polluted soils and moreover of heavy metal polluted soils affected by salinity. Some halophytes use excretion processes in order to remove the excess of salt ions from their sensitive tissues and in some cases these glandular structures are not always specific to Na+ and Cl- and other toxic elements such as cadmium, zinc, lead, or copper are accumulated and excreted by salt glands or trichomes on the surface of the leaves--a novel phytoremediation process called "phytoexcretion". Finally, the use of halophytes has also been proposed for soil desalination through salt accumulation in the plant tissue or dissolution of soil calcite in the rhizosphere to provide Ca2+ that can be exchanged with Na+ at cation exchange sites.     

Opposite changes in membrane fluidity mimic cold and heat stress activation of distinct plant MAP kinase pathways

Mitogen-activated protein kinases (MAPKs) appear to be ubiquitously involved in signal transduction during eukaryotic responses to extracellular stimuli. In plants, no heat shock-activated MAPK has so far been reported. Also, whereas cold activates specific plant MAPKs such as alfalfa SAMK, mechanisms of such activation are unknown. Here, we report a heat shock-activated MAPK (HAMK) immunologically related to ERK (Extracellular signal-Regulated Kinase) superfamily of protein kinases. Molecular mechanisms of heat-activation of HAMK and cold-activation of SAMK were investigated. We show that cold-activation of SAMK requires membrane rigidification, whereas heat-activation of HAMK occurs through membrane fluidization. The temperature stress- and membrane structure-dependent activation of both SAMK and HAMK is mimicked at 25 degrees C by destabilizers of microfilaments and microtubules, latrunculin B and oryzalin, respectively; but is blocked by jasplakinolide, a stabilizer of actin microfilaments. Activation of SAMK or HAMK by temperature, chemically modulated membrane fluidity, or by cytoskeleton destabilizers is inhibited by blocking the influx of extracellular calcium. Activation of SAMK or HAMK is also prevented by an antagonist of calcium-dependent protein kinases (CDPKs). In summary, our data indicate that cold and heat are sensed by structural changes in the plasma membrane that translates the signal via cytoskeleton, Ca2+ fluxes and CDPKs into the activation of distinct MAPK cascades.     

Heavy metal resistance of biofilm and planktonic Pseudomonas aeruginosa

A study was undertaken to examine the effects of the heavy metals copper, lead, and zinc on biofilm and planktonic Pseudomonas aeruginosa. A rotating-disk biofilm reactor was used to generate biofilm and free-swimming cultures to test their relative levels of resistance to heavy metals. It was determined that biofilms were anywhere from 2 to 600 times more resistant to heavy metal stress than free-swimming cells. When planktonic cells at different stages of growth were examined, it was found that logarithmically growing cells were more resistant to copper and lead stress than stationary-phase cells. However, biofilms were observed to be more resistant to heavy metals than either stationary-phase or logarithmically growing planktonic cells. Microscopy was used to evaluate the effect of copper stress on a mature P. aeruginosa biofilm. The exterior of the biofilm was preferentially killed after exposure to elevated concentrations of copper, and the majority of living cells were near the substratum. A potential explanation for this is that the extracellular polymeric substances that encase a biofilm may be responsible for protecting cells from heavy metal stress by binding the heavy metals and retarding their diffusion within the biofilm.     

A proteome analysis of the cadmium and mercury response in Corynebacterium glutamicum

Cadmium and mercury are well-known toxic heavy metals, but the basis of their toxicity is not well understood. In this study, we analyzed the cellular response of Corynebacterium glutamicum to sublethal concentrations of cadmium and mercury ions using 2-DE and MS. Mercury induced the over-expression of 13 C. glutamicum proteins, whereas 35 proteins were induced, and 8 proteins were repressed, respectively, under cadmium stress. The principal response to these metals was protection against oxidative stress, as demonstrated by upregulation of, e.g., Mn/Zn superoxide dismutase. Thioredoxin and oxidoreductase responded most strongly to cadmium and mercury. The increased level of heat-shock proteins, enzymes involved in energy metabolism, as well as in lipoic acid and terpenoid biosynthesis after the treatment of cells with cadmium was also registered. Identification of these proteins and their mapping into specific cellular processes enable a global understanding of the way in which C. glutamicum adapts to heavy-metal stress and may help to gain deeper insight into the toxic mechanism of these metals.     

Metallothionein accumulation may account for intracellular copper retention in Menkes' disease

Cultured lymphoblasts derived from infants with Menkes' disease exhibit the same increased avidity for copper as do fibroblasts and most extrahepatic tissues from these patients. The Menkes' cells preferentially take up not only copper but also, on exposure to elevated metal concentrations, the other metallothionein-binding metals, zinc and cadmium. Menkes' lymphoblasts contain larger amounts of metallothionein than normal cells following exposure to each of these metals; the amount bound to this protein quantitatively accounted for the total cellular increment in metal in Menkes' cells. Induction of metallothionein synthesis caused both normal and Menkes' cells to subsequently take up increased amounts of 67Cu. These observations suggest that an enhanced capacity of Menkes' cells to accumulate metallothionein may be responsible for their increased uptake and retention of copper.     

Heavy Metal Resistance of Biofilm and Planktonic Pseudomonas aeruginosa

A study was undertaken to examine the effects of the heavy metals copper, lead, and zinc on biofilm and planktonic Pseudomonas aeruginosa. A rotating-disk biofilm reactor was used to generate biofilm and free-swimming cultures to test their relative levels of resistance to heavy metals. It was determined that biofilms were anywhere from 2 to 600 times more resistant to heavy metal stress than free-swimming cells. When planktonic cells at different stages of growth were examined, it was found that logarithmically growing cells were more resistant to copper and lead stress than stationary-phase cells. However, biofilms were observed to be more resistant to heavy metals than either stationary-phase or logarithmically growing planktonic cells. Microscopy was used to evaluate the effect of copper stress on a mature P. aeruginosa biofilm. The exterior of the biofilm was preferentially killed after exposure to elevated concentrations of copper, and the majority of living cells were near the substratum. A potential explanation for this is that the extracellular polymeric substances that encase a biofilm may be responsible for protecting cells from heavy metal stress by binding the heavy metals and retarding their diffusion within the biofilm.     

Disease-toxicant screen reveals a neuroprotective interaction between Huntington's disease and manganese exposure

Recognizing the similarities between Huntington's disease (HD) pathophysiology and the neurotoxicology of various metals, we hypothesized that they may exhibit disease-toxicant interactions revealing cellular pathways underlying neurodegeneration. Here, we utilize metals and the ST Hdh mouse striatal cell line model of HD to perform a gene–environment interaction screen. We report that striatal cells expressing mutant Huntingtin exhibit elevated sensitivity to cadmium toxicity and resistance to manganese toxicity. This neuroprotective gene–environment interaction with manganese is highly specific, as it does not occur with iron, copper, zinc, cobalt, cadmium, lead, or nickel ions. Analysis of the Akt cell stress signaling pathway showed diminished activation with manganese exposure and elevated activation after cadmium exposure in the mutant cells. Direct examination of intracellular manganese levels found that mutant cells have a significant impairment in manganese accumulation. Furthermore, YAC128Q mice, a HD model, showed decreased total striatal manganese levels following manganese exposure relative to wild-type mice. Thus, this disease-toxicant interaction screen has revealed that expression of mutant Huntingtin results in heightened sensitivity to cadmium neurotoxicity and a selective impairment of manganese accumulation.     

Defense responses of soybean roots during exposure to cadmium, excess of nitrogen supply and combinations of these stressors

Heavy metal pollution is a serious environmental problem in agricultural soils since the uptake of heavy metals by plants represents an entry point into the food chain and is influenced by the form and amount of nitrogen (N) fertilization. Here we studied the defense responses in soybean roots exposed to ions of cadmium (applied as 50?mg?l^?1 Cd²⁺) when combined with an excessive dose of N in form of NH₄NO₃. Our data indicate that despite of stunted root growth, several stress symptoms typically observed upon cadmium treatment, e.g. peroxidation of lipid membranes or activation of chitinase isoforms, become suppressed at highly excessive N. At the same time, other defense mechanisms such as catalases and proline accumulation were elevated. Most importantly, the interplay of ongoing responses resulted in a decreased uptake of the metal into the root tissue. This report points to the complexity of plant defense responses under conditions of heavy metal pollution combined with intensive fertilization in agriculture.     

Mass balance of heavy metal uptake by encapsulated cultures ofKlebsiella aerogenes

Dialysis was employed as a method of speciating heavy metals in cultures of an extracellular polymer forming strain ofKlebsiella aerogenes. A noncapsulated strain of the same bacterium was used as a control, and a mass balance of copper, cadmium, cobalt, nickel, and manganese in batch culture at pH 4.5 and pH 6.8 and in continuous culture at pH 6.8 was constructed. Copper and cadmium were accumulated by the cell during rapid proliferation whereas all 5 metals were bound nonspecifically by extracellular polymer produced during stationary phase and at low dilution rates. The presence of extracellular polymer appeared to inhibit cellular uptake of nickel. At the lower pH, metal uptake was considerably reduced. The results are discussed in the context of metal removal in the activated sludge process of waste water treatment.