Induction of Protection against Paraquat-induced Oxidative Damage by Abscisic Acid in Maize Leaves is Mediated through Mitogen-activated Protein Kinase

Mitogen-activated protein kinase (MAPK) cascade has been shown to be important components In stress signal trans-duction pathway. In the present study, protection of maize seedlings (Zea mays L.) against paraquat-generated oxidative toxicity by abscisic acid (ABA), its association with MAPK and ZmMPK5, a candidate for MAPK were investigated. Treatment of maize leaves with exogenous ABA led to significant decreases in the content of malondialdehyde, the percentage of ion leakage and the level of protein oxidation (in terms of carbonyl groups) under paraquat (PQ) stress. However, such decreases were blocked by the pretreatment with two MAPK kinase inhibitors PD98059 and U0126. The damage caused by PQ was further aggravated by inhibitors. Two inhibitors also suppressed the total activities of the antioxidant enzymes superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11), and glutathione reductase (GR, EC 1.6.4.2). Besides, treatment with PQ stimulated the activation of a 46 kDa MAPK, which was identified as ZmMPK5 by in-gel kinase assay with immunoprecipitation. These results reveal that ABA-induced protection against PQ-generated oxidative damage is mediated through MAPK cascade in maize leaves, in which ZmMPK5, a candidate for MAPK, is demonstrated to be involved.     

Induction of Protection against Paraquat-induced Oxidative Damage by Abscisic Acid in Maize Leaves is Mediated through Mitogen-activated Protein Kinase

Mitogen-activated protein kinase (MAPK) cascade has been shown to be important components in stress signal transduction pathway. In the present study, protection of maize seedlings ( Zea mays L.) against paraquat-generated oxidative toxicity by abscisic acid (ABA), its association with MAPK and ZmMPK5, a candidate for MAPK were investigated. Treatment of maize leaves with exogenous ABA led to significant decreases in the content of malondialdehyde, the percentage of ion leakage and the level of protein oxidation (in terms of carbonyl groups) under paraquat (PQ) stress. However, such decreases were blocked by the pretreatment with two MAPK kinase inhibitors PD98059 and U0126. The damage caused by PQ was further aggravated by inhibitors. Two inhibitors also suppressed the total activities of the antioxidant enzymes superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11), and glutathione reductase (GR, EC 1.6.4.2). Besides, treatment with PQ stimulated the activation of a 46 kDa MAPK, which was identified as ZmMPK5 by in-gel kinase assay with immunoprecipitation. These results reveal that ABA-induced protection against PQ-generated oxidative damage is mediated through MAPK cascade in maize leaves, in which ZmMPK5, a candidate for MAPK, is demonstrated to be involved.     

Hexavalent chromium (VI) stress induces mitogen-activated protein kinase activation mediated by distinct signal molecules in roots of Zea mays L.

Hexavalent chromium (VI) is a cytotoxic metal ion in plants. However, the mechanisms involved in the cellular response to the metal have not yet been well established. In plants, mitogen-activated protein kinase (MAPK) cascades play an important role in signal transduction related to biotic and abiotic stresses. In the present study, we investigated the Cr(VI)-induced MAPKs activation and the correlative mechanism of activation in maize (Zea mays L.) roots. Cr(VI) elicited a remarkable increase in a 45-kDa myelin basic protein (MBP) kinase activity with MAPK-like characteristics, which was identified as ZmMPK5 by immunokinase and immunoblot assays. Pretreatment with DMTU, a peroxide hydrogen (H₂O₂) scavenger, and DPI, a NADPH oxidase inhibitor, the Cr(VI)-induced ZmMPK5 activation was almost completely suppressed, suggesting that Cr(VI)-activated ZmMPK5 requires for H₂O₂. Application of exogenous sodium nitroprusside (SNP), a nitric oxide (NO) donor, could activate ZmMPK5. Pretreatment with cPTIO and l-NAME, a NO scavenger and a nitric oxide synthase (NOS) inhibitor, respectively, Cr(VI)-induced ZmMPK5 activation was attenuated effectively, implying that NO is involved in Cr(VI)-activated ZmMPK5. Furthermore, a calcium-dependent protein kinase (CDPK) antagonist, W7, abolished Cr(VI)-stimulated ZmMPK5 activation, indicating that CDPKs may participate in the ZmMPK5 activation. The results obtained suggest that Cr(VI)-induced activation of ZmMPK5, a candidate for MAPK signaling cascades, can be modulated by other distinct signaling pathways.     

A 45-kDa protein kinase related to mitogen-activated protein kinase is activated in tobacco cells treated with a phorbol ester

Phorbol 12-myristate 13-acetate (PMA), a potent activator of protein kinases in animals, elicits the transient activation of a 45-kDa protein kinase in tobacco cell-suspension cultures. The 45-kDa protein kinase preferentially phosphorylates myelin basic protein (MBP), a general substrate for MAPK. Studies using cycloheximide indicated that protein synthesis is not required for the activation of the kinase. Treatment of tobacco cell extracts containing the activated kinase with either serine/threonine-specific or tyrosine-specific protein phosphatase abolished the kinase activity, which consequently appears to be regulated by phosphorylation. By using an immune complex kinase assay with antibodies specific for stress-responsive MAPKs, we show that the PMA-activated kinase is immunologically related to the wound-induced protein kinase (WIPK), and not to the salicylic acid-induced protein kinase (SIPK), two representative members of the tobacco MAPK family, known to be activated by extracellular stimuli. Furthermore, the activated kinase was recognized by phospho-specific MAPK antibodies. Collectively, these results indicate that phorbol ester promotes the activation of a 45-kDa protein kinase related to WIPK in tobacco cells. Activation of WIPK in response to PMA is associated with protein phosphorylation but not with an increase in protein level.     

ZmABA2, an interacting protein of ZmMPK5, is involved in abscisic acid biosynthesis and functions

In maize (Zea mays), the mitogen‐activated protein kinase ZmMPK5 has been shown to be involved in abscisic acid (ABA)‐induced antioxidant defence and to enhance the tolerance of plants to drought, salt stress and oxidative stress. However, the underlying molecular mechanisms are poorly understood. Here, using ZmMPK5 as bait in yeast two‐hybrid screening, a protein interacting with ZmMPK5 named ZmABA2, which belongs to a member of the short‐chain dehydrogenase/reductase family, was identified. Pull‐down assay and bimolecular fluorescence complementation analysis and co‐immunoprecipitation test confirmed that ZmMPK5 interacts with ZmABA2 in vitro and in vivo. Phosphorylation of Ser173 in ZmABA2 by ZmMPK5 was shown to increase the activity of ZmABA2 and the protein stability. Various abiotic stimuli induced the expression of ZmABA2 in leaves of maize plants. Pharmacological, biochemical and molecular biology and genetic analyses showed that both ZmMPK5 and ZmABA2 coordinately regulate the content of ABA. Overexpression of ZmABA2 in tobacco plants was found to elevate the content of ABA, regulate seed germination and root growth under drought and salt stress and enhance the tolerance of tobacco plants to drought and salt stress. These results suggest that ZmABA2 is a direct target of ZmMPK5 and is involved in ABA biosynthesis and functions.     

Zinc induces mitogen-activated protein kinase activation mediated by reactive oxygen species in rice roots.

It is well known that zinc (Zn) is one of the micronutrients essential for normal growth and development of plants. However, the molecular mechanisms responsible for the regulation of plant growth by Zn are still not completely understood. The aim of this study was to investigate the signalling transduction pathways activated by Zn. We show that Zn elicited a remarkable increase in myelin basic protein (MBP) kinase activities. By immunoblot analysis, we suggest that Zn-activated 40- and 42-kDa MBP kinases are mitogen-activated protein kinases (MAPK). Pre-treatment of rice roots with reactive oxygen species (ROS) scavenger, sodium benzoate, was able to effectively prevent Zn-induced MAPK activation. However, phosphoinositide 3-kinase (PI-3K) inhibitor, LY294002, was unable to inhibit Zn-induced MAPK activation. These results suggest that the ROS may function in the Zn-triggered MAPK signalling pathway in rice roots.     

Molecular aspects of mechanical stress-induced cardiac hypertrophy

To elucidate the signal transduction pathway from external stimuli to nuclear gene expression in mechanical stress-induced cardiac hypertrophy, we examined the time course of activation of protein kinases such as Raf-1 kinase (Raf-1), mitogen-activated protein kinase kinase (MAPKK), MAP kinases (MAPKs) and 90-kDa ribosomal S6 kinase (p90^rsk) in neonatal rat cardiomyocytes. Mechanical stretch rapidly activated Raf-1 and its maximal activation was observed at 1–2 min after stretch. The activity of MAPKK was also increased by stretch, with a peak at 5 min after stretch. In addition, MAPKs and p90^rsk were maximally activated at 8 min and at 10–30 min after stretch, respectively. Next, the relationship between mechanical stress-induced hypertrophy and the cardiac renin-angiotensin system was investigated. When the stretch-conditioned culture medium was transferred to the culture dish of non-stretched cardiac myocytes, the medium activated MAPK activity slightly but significantly, and the activation was completely blocked by the type 1 angiotensin II receptor antagonist, CV-11974. However, activation of Raf-1 and MAPKs provoked by stretching cardiomyocytes was only partially suppressed by pretreatment with CV-11974. These results suggest that mechanical stress activates the protein kinase cascade of phosphorylation in cardiac myocytes in the order of Raf-1, MAPKK, MAPKs and p90^rsk, and that angiotensin II, which is secreted from stretched myocytes, activates a part of these protein kinases.Abbreviations MAPK mitogen-activated protein kinase - MAPKK MAP kinase kinase - Raf-1 - Raf- 1 kinase p90^rsk, 90 kDa ribosomal S6 kinase; AngII - angiotensin II - MAPKKK MAP kinase kinase kinase - rMAPK recombinant MAPKK fused to gluthathione S transferase - MMAKK recombinant MAPK fused to maltose binding protein - MBP myelin basic protein - ACE angiotensin-converting enzyme     

ZmMPK6, a Novel Maize MAP Kinase that Interacts with 14-3-3 Proteins

Although an increasing body of evidence indicates that plant MAP kinases are involved in a number of cellular processes, such as cell cycle regulation and cellular response to abiotic stresses, hormones and pathogen attack, very little is known about their biochemical properties and regulation mechanism. In this paper we report on the identification and characterization of a novel member of the MAP kinase family from maize, ZmMPK6. The amino acid sequence reveals a high degree of identity with group D plant MAP kinases. Recombinant ZmMPK6, expressed in Escherichia coli, is an active enzyme able to autophosphorylate. Remarkably, ZmMPK6 interacts in vitro with GF14-6, a maize 14-3-3 protein and the interaction is dependent on autophosphorylation. The interacting domain of ZmMPK6 is on the C-terminus and is comprised between amino acid 337 and amino acid 467. Our results represent the first evidence of an interaction between a plant MAP kinase and a 14-3-3 protein. Possible functional roles of this association in vivo are discussed.     

Okadaic acid mimics the action of insulin in stimulating protein kinase activity in isolated adipocytes. The role of protein phosphatase 2a in attenuation of the signal

Treatment of adipocytes with okadaic acid (a specific inhibitor of type 1 and 2a protein phosphatases) resulted in a rapid 8-10-fold stimulation of cell extract myelin basic protein (MBP) kinase activity (t1/2 = 10 min) and kinase activity toward a synthetic peptide RRLSSLRA (S6 peptide) (t1/2 = 5 min). Insulin brought about a smaller stimulation of these two activities (t1/2 = 2.5 min). MBP kinase activity from cells treated with okadaic acid or insulin was resolved by anion exchange chromatography into two well defined peaks; S6 peptide kinase activity was less well resolved. The two partially purified MBP kinases were inactivated by the protein tyrosine phosphatase CD45 or by protein phosphatase 2a (PP-2a). In contrast, partially purified S6 peptide kinase activity was inactivated only by PP-2a or protein phosphatase 1 (PP-1). Furthermore, a 38-kDa protein which co-eluted with one peak of MBP kinase and a 42-kDa protein which co-eluted with the other peak of MBP kinase were phosphorylated on tyrosine after treatment with okadaic acid. These findings illustrate several important points concerning regulation of MBP and S6 peptide kinases. First, these protein kinases are regulated by phosphorylation, and, second, in the absence of hormonal stimuli their activities are strongly suppressed by protein phosphatases. Lastly, the increased tyrosine phosphorylation accompanying the activation of MBP kinases following okadaic acid treatment suggests a role for PP-2a in events that are mediated by tyrosine phosphorylation.     

高温胁迫下葡萄叶片蛋白激酶的诱导形成与活性变化

以"京秀"葡萄(Vitis vinifera L.cv.Jingxiu)幼苗为试材,研究了高温胁迫激活的蛋白激酶的类型和活性.结果表明,高温胁迫10～60min明显地激活了一个分子量约为52 kD的蛋白激酶,该蛋白激酶能将凝胶中所嵌入的髓鞘碱性蛋白(MBP)磷酸化,在放射自显影中表现出很高的放射活性,而对凝胶中的组蛋白-Ⅲ(histone-Ⅲ)则没有这样的作用.在溶液反应体系中该蛋白激酶对MBP也表现出很高的磷酸化活性,而对histone-Ⅲ却无作用.Ca2 对其活性变化无显著影响.酪氨酸特异性蛋白磷酸酶(YOP)对该激酶的活性有显著的钝化作用.结果表明该52 kD蛋白激酶是MAPK家族中的一种.     

Biochemical identification of the OsMKK6-OsMPK3 signalling pathway for chilling stress tolerance in rice

MAPK (mitogen-activated protein kinase) pathways have been implicated in stress signalling in plants. In the present study, we performed yeast two-hybrid screening to identify partner MAPKs for OsMKK (Oryza sativa MAPK kinase) 6, a rice MAPK kinase, and revealed specific interactions of OsMKK6 with OsMPK3 and OsMPK6. OsMPK3 and OsMPK6 each co-immunoprecipitated OsMKK6, and both were directly phosphorylated by OsMKK6 in vitro. An MBP (myelin basic protein) kinase assay of the immunoprecipitation complex indicated that OsMPK3 and OsMPK6 were activated in response to a moderately low temperature (12°C), but not a severely low temperature (4°C) in rice seedlings. A constitutively active form of OsMKK6, OsMKK6DD, showed elevated phosphorylation activity against OsMPK3 and OsMPK6 in vitro. OsMPK3, but not OsMPK6, was constitutively activated in transgenic plants overexpressing OsMKK6DD, indicating that OsMPK3 is an in vivo target of OsMKK6. Enhanced chilling tolerance was observed in the transgenic plants overexpressing OsMKK6DD. Taken together, our data suggest that OsMKK6 and OsMPK3 constitute a moderately low-temperature signalling pathway and regulate cold stress tolerance in rice.     

Activation of 40-kDa Protein Kinases in Response to Hypo- and Hyperosmotic Shock in the Halotolerant Green Alga Dunaliella tertiolecta

Changes in protein kinase activities in Dunaliella tertiolectain response to hypo- and hyperosmotic shocks were assessed byassays of protein kinase activities on SDS-polyacrylamide gelsthat contained protein substrates, Hypoosmotic shock (transferfrom 0.5 to 0.2 M Nacl) transiently activated a 40-kDa proteinkinase (low osmotic pressure-activated protein kinase, LAP kinase)that phosphorylated myelin basic protein (MBP) and histone H1but not casein. By contrast, hyperosmotic shock (transfer from0.5 to 1.1 M NACl) rapidly activated another 40-kDa proteinkinase (high osmotic pressure-activated protein kinase, HAPkinase) that phosphorylated casein and histone H1 but not MBP.Inhibitors of protein kinases, namely, K-252a and staurosporine,suppressed the activation of both LAP kinase and HAP kinase.The protein kinase activities in extracts of osmotically shockedcells disappeared completely after treatment with calf intestinalalkaline phosphatase. Anti-phosphotyrosine monoclonal antibodiesdid not cross-react with either LAP kinase or HAP kinase. Theseresults indicate that at least two 40-kDa protein kinases, LAPkinase and HAP kinase, are activated by hyper- and hypoosmoticshock, respectively, and that their activities are regulatedvia phosphorylation by putative protein kinase(s) that act atan upstream position in the signaling cascade(s) that followsosmotic shock. (Received July 24, 1995; Accepted October 20, 1995)