Nitric oxide modulates ozone-induced cell death, hormone biosynthesis and gene expression in Arabidopsis thaliana

Nitric oxide (NO) is involved together with reactive oxygen species (ROS) in the activation of various stress responses in plants. We have used ozone (O₃) as a tool to elicit ROS-activated stress responses, and to activate cell death in plant leaves. Here, we have investigated the roles and interactions of ROS and NO in the induction and regulation of O₃-induced cell death. Treatment with O₃ induced a rapid accumulation of NO, which started from guard cells, spread to adjacent epidermal cells and eventually moved to mesophyll cells. During the later time points, NO production coincided with the formation of hypersensitive response (HR)-like lesions. The NO donor sodium nitroprusside (SNP) and O₃ individually induced a large set of defence-related genes; however, in a combined treatment SNP attenuated the O₃ induction of salicylic acid (SA) biosynthesis and other defence-related genes. Consistent with this, SNP treatment also decreased O₃-induced SA accumulation. The O₃-sensitive mutant rcd1 was found to be an NO overproducer; in contrast, Atnoa1/rif1 ( Arabidopsis nitric oxide associated 1/resistant to inhibition by FSM1 ), a mutant with decreased production of NO, was also O₃ sensitive. This, together with experiments combining O₃ and the NO donor SNP suggested that NO can modify signalling, hormone biosynthesis and gene expression in plants during O₃ exposure, and that a functional NO production is needed for a proper O₃ response. In summary, NO is an important signalling molecule in the response to O₃.     

Harpin induces mitogen-activated protein kinase activity during defence responses in Arabidopsis thaliana suspension cultures

Elicitation of Arabidopsis thaliana (L.) Heynh. suspension cultures with the bacterial protein harpin (from Pseudomonas syringae pv. syringae) induced the activation of two kinases of 39 and 44 kDa, as demonstrated by in-gel kinase assays using myelin basic protein (MBP) as a substrate. Both these kinases appeared to be tyrosine-phosphorylated upon activation, as demonstrated by treatment with tyrosine phosphatase and immunoprecipitation using an anti-phosphotyrosine monoclonal antibody. An inhibitor of mammalian mitogen-activated protein kinase (MAPK) activation, PD98059, inhibited harpin-induced MBPK activation, but did not inhibit the activity of these kinases. PD98059 also inhibited harpin-induced programmed cell death and defence gene expression, suggesting the involvement of harpin-induced MAPKs in defence responses in Arabidopsis thaliana. Received: 23 February 1999 / Accepted: 22 July 1999     

Spermine signalling in tobacco: activation of mitogen-activated protein kinases by spermine is mediated through mitochondrial dysfunction

Polyamines (PAs) play important roles in cell proliferation, growth and environmental stress responses of all living organisms. In this study, we examine whether these compounds act as signal mediators. Spermine (Spm) specifically activated protein kinases of tobacco leaves, which were identified as salicylic acid (SA)-induced protein kinase (SIPK) and wound-induced protein kinase (WIPK), using specific antibodies. Upon Spm treatment, upregulation of WIPK, but not SIPK, was observed. Spm-induced mitogen-activated protein kinases (MAPKs) activation and WIPK upregulation were prevented upon pre-treatment with antioxidants and Ca2+ channel blockers. Additionally, Spm specifically stimulated expression of the alternative oxidase (AOX) gene, which was disrupted by these antioxidants and Ca2+ channel blockers. Bongkrekic acid (BK), an inhibitor of the opening of mitochondrial permeability transition (PT) pores, suppressed MAPKs activation and accumulation of WIPK and AOX mRNA. Our data collectively suggest that Spm causes mitochondrial dysfunction via a signalling pathway in which reactive oxygen species and Ca2+ influx are involved. As a result, the phosphorylation activities of the two MAPK enzymes SIPK and WIPK are stimulated.     

The Elongator complex‐associated protein DRL1 plays a positive role in immune responses against necrotrophic fungal pathogens in Arabidopsis

DEFORMED ROOT AND LEAVES1 (DRL1) is an Arabidopsis homologue of the yeast TOXIN TARGET4 (TOT4)/KILLER TOXIN‐INSENSITIVE12 (KTI12) protein that is physically associated with the RNA polymerase II‐interacting protein complex named Elongator. Mutations in DRL1 and Elongator lead to similar morphological and molecular phenotypes, suggesting that DRL1 and Elongator may functionally overlap in Arabidopsis. We have shown previously that Elongator plays an important role in both salicylic acid (SA)‐ and jasmonic acid (JA)/ethylene (ET)‐mediated defence responses. Here, we tested whether DRL1 also plays a similar role as Elongator in plant immune responses. Our results show that, although DRL1 partially contributes to SA‐induced cytotoxicity, it does not play a significant role in SA‐mediated expression of PATHOGENESIS‐RELATED genes and resistance to the virulent bacterial pathogen Pseudomonas syringae pv. maculicola ES4326. In contrast, DRL1 is required for JA/ET‐ and necrotrophic fungal pathogen Botrytis cinerea‐induced defence gene expression and for resistance to B. cinerea and Alternaria brassicicola. Furthermore, unlike the TOT4/KTI12 gene which, when overexpressed in yeast, confers zymocin resistance, a phenotype of the tot4/kti12 mutant, overexpression of DRL1 does not change B. cinerea‐induced defence gene expression and resistance to this pathogen. Finally, DRL1 contains an N‐terminal P‐loop and a C‐terminal calmodulin (CaM)‐binding domain and is a CaM‐binding protein. We demonstrate that both the P‐loop and the CaM‐binding domain are essential for the function of DRL1 in B. cinerea‐induced expression of PDF1.2 and ORA59, and in resistance to B. cinerea, suggesting that the function of DRL1 in plant immunity may be regulated by ATP/GTP and CaM binding.     

GDSL lipase-like 1 regulates systemic resistance associated with ethylene signaling in Arabidopsis

Systemic resistance is induced by necrotizing pathogenic microbes and non-pathogenic rhizobacteria and confers protection against a broad range of pathogens. Here we show that Arabidopsis GDSL LIPASE-LIKE 1 (GLIP1) plays an important role in plant immunity, eliciting both local and systemic resistance in plants. GLIP1 functions independently of salicylic acid but requires ethylene signaling. Enhancement of GLIP1 expression in plants increases resistance to pathogens including Alternaria brassicicola , Erwinia carotovora and Pseudomonas syringae , and limits their growth at the infection site. Furthermore, local treatment with GLIP1 proteins is sufficient for the activation of systemic resistance, inducing both resistance gene expression and pathogen resistance in systemic leaves. The PDF1.2 -inducing activity accumulates in petiole exudates in a GLIP1-dependent manner and is fractionated in the size range of less than 10 kDa as determined by size exclusion chromatography. Our results demonstrate that GLIP1-elicited systemic resistance is dependent on ethylene signaling and provide evidence that GLIP1 may mediate the production of a systemic signaling molecule(s).     

Signalling and cell death in ozone-exposed plants

Experiments with Arabidopsis mutants and sensitive and tolerant pairs in several other species have elucidated the molecular basis of plant ozone sensitivity and ozone lesion development. They have indicated an important role for hormonal signalling in determining the outcome of ozone challenge at the cellular level. The reactive oxygen species (ROS) from ozone degradation can cause either direct necrotic damage or induce the process of programmed cell death. Perception of ozone or ROS from its degradation in the apoplast activates several signal transduction pathways that regulate the responses of the cells to the increased oxidative load. Plant hormones salicylic acid, jasmonic acid, ethylene and abscisic acid are involved in determining the duration and extent of ozone-induced cell death and its propagation. Salicylic acid is required for the programmed cell death, ethylene promotes endogenous ROS formation and lesion propagation, and jasmonic acid is involved in limiting the lesion spreading. Abscisic acid is most likely involved through the regulation of stomata and thus is expected to affect lesion initiation. The roles and interactions of perception of ozone, the immediate downstream responses, hormone biosynthesis and signalling during ozone lesion initiation and formation are reviewed.     

Function of a mitogen-activated protein kinase pathway in N gene-mediated resistance in tobacco

The active defense of plants against pathogens often includes rapid and localized cell death known as hypersensitive response (HR). Protein phosphorylation and dephosphorylation are implicated in this event based on studies using protein kinase and phosphatase inhibitors. Recent transient gain-of-function studies demonstrated that the activation of salicylic acid-induced protein kinase (SIPK) and wounding-induced protein kinase (WIPK), two tobacco mitogen-activated protein kinases (MAPKs) by their upstream MAPK kinase (MAPKK), NtMEK2 leads to HR-like cell death. Here, we report that the conserved kinase interaction motif (KIM) in MAPKKs is required for NtMEK2 function. Mutation of the conserved basic amino acids in this motif, or the deletion of N-terminal 64 amino acids containing this motif significantly compromised or abolished the ability of NtMEK2DD to activate SIPK/WIPK in vivo. These mutants were also defective in interacting with SIPK and WIPK, suggesting protein-protein interaction is required for the functional integrity of this MAPK cascade. To eliminate Agrobacterium that is known to activate a number of defense responses in transient transformation experiments, we generated permanent transgenic plants. Induction of NtMEK2DD expression by dexamethasone induced HR-like cell death in both T1 and T2 plants. In addition, by using PVX-induced gene silencing, we demonstrated that the suppression of all three known components in the NtMEK2-SIPK/WIPK pathway attenuated N gene-mediated TMV resistance. Together with previous report that SIPK and WIPK are activated by TMV in a gene-for-gene-dependent manner, we conclude that NtMEK2-SIPK/WIPK pathway plays a positive role in N gene-mediated resistance, possibly through regulating HR cell death.     

Salicylic acid inhibits jasmonic acid-induced resistance of Arabidopsis thaliana to Spodoptera exigua

The role of salicylic acid (SA) in plant responses to pathogens has been well documented, but its direct and indirect effects on plant responses to insects are not so well understood. We examined the effects of SA, alone and in combination with jasmonic acid (JA), on the performance of the generalist herbivore, Spodoptera exigua, in wild-type and mutant Arabidopsis thaliana genotypes that varied genetically in their ability to mount SA- and JA-mediated defence responses. In one experiment, growth of S. exigua larvae was highest on the Wassilewskija wild-type, intermediate on the Columbia wild-type and the JA-deficient fad mutant, and lowest on the nim1-1 and jar1-mutants, which are defective in the SA and JA pathways, respectively. Activity of guaiacol peroxidase, polyphenoloxidase, n-acetylglucosaminidase, and trypsin inhibitor varied by genotype but did not correlate with insect performance. SA treatment increased growth of S. exigua larvae by approximately 35% over all genotypes, but had no discernable effect on activities of the four defence proteins. In a second experiment, growth of S. exigua was highest across treatments on the cep1 mutant, a constitutive expressor of high SA levels and systemic acquired resistance, and lowest on the fad mutant, which is JA-deficient. JA treatment generally increased activity of all four defence proteins, increased total glucosinolate levels and reduced insect growth by approximately 25% over all genotypes. SA generally inhibited expression of JA-induced resistance to S. exigua when both hormones were applied simultaneously. Across genotypes and treatments, larval mass was negatively correlated with the activity of trypsin inhibitor and polyphenoloxidase and with total glucosinolate levels, and insect damage was negatively correlated with the activity of polyphenoloxidase. SA had little effect on the induction of defence protein activity by JA. However, SA attenuated the induction of glucosinolates by JA and therefore may explain better the interactive effects of SA and JA on insect performance. This study illustrates that direct and indirect cross-effects of SA on resistance to S. exigua can occur in A. thaliana. Effects of SA may be mediated through effects on plant defence chemistry or other aspects of the suitability of foliage for insect feeding and growth.     

Analysis of oxidative signalling induced by ozone in Arabidopsis thaliana

We are using acute ozone as an elicitor of endogenous reactive oxygen species (ROS) to understand oxidative signalling in Arabidopsis. Temporal patterns of ROS following a 6 h exposure to 300 nL L(-1) of ozone in ozone-sensitive Wassilewskija (Ws-0) ecotype showed a biphasic ROS burst with a smaller peak at 4 h and a larger peak at 16 h. This was accompanied by a nitric oxide (NO) burst that peaked at 9 h. An analysis of antioxidant levels showed that both ascorbate (AsA) and glutathione (GSH) were at their lowest levels, when ROS levels were high in ozone-stressed plants. Whole genome expression profiling analysis at 1, 4, 8, 12 and 24 h after initiation of ozone treatment identified 371 differentially expressed genes. Early induction of proteolysis and hormone-responsive genes indicated that an oxidative cell death pathway was triggered rapidly. Down-regulation of genes involved in carbon utilization, energy pathways and signalling suggested an inefficient defense response. Comparisons with other large-scale expression profiling studies indicated some overlap between genes induced by ethylene and ozone, and a significant overlap between genes repressed by ozone and methyl jasmonate treatment. Further, analysis of cis elements in the promoters of ozone-responsive genes also supports the view that phytohormones play a significant role in ozone-induced cell death.     

MPK9 and MPK12 function in SA-induced stomatal closure in Arabidopsis thaliana

Salicylic acid (SA) induces stomatal closure sharing several components with abscisic acid (ABA) and methyl jasmonate (MeJA) signaling. We have previously shown that two guard cell-preferential mitogen-activated protein kinases (MAPKs), MPK9 and MPK12, positively regulate ABA signaling and MeJA signaling in Arabidopsis thaliana. In this study, we examined whether these two MAPKs are involved in SA-induced stomatal closure using genetic mutants and a pharmacological, MAPKK inhibitor. Salicylic acid induced stomatal closure in mpk9 and mpk12 single mutants but not in mpk9 mpk12 double mutants. The MAPKK inhibitor PD98059 inhibited SA-induced stomatal closure in wild-type plants. Salicylic acid induced extracellular reactive oxygen species (ROS) production, intracellular ROS accumulation, and cytosolic alkalization in the mpk9, mpk12, and mpk9 mpk12 mutants. Moreover, SA-activated S-type anion channels in guard cells of wild-type plants but not in guard cells of mpk9 mpk12 double mutants. These results imply that MPK9 and MPK12 are positive regulators of SA signaling in Arabidopsis guard cells.     

Blockade of the translocation and activation of mitogen-activated protein kinase kinase 4 (MKK4) signaling attenuates neuronal damage during later ischemia-reperfusion

Mitogen-activated protein kinase kinase 4 (MKK4), as an upstream activator of c-Jun NH(2)-terminal kinase (JNK), plays a critical role in response to cellular stresses and pro-inflammatory cytokines. In this study, we investigated the subcellular localization and activation of MKK4 in response to global cerebral ischemia. Our results indicated that MKK4 had two activation peaks in both the cytosol and the nucleus, and translocated from the cytosol to the nucleus at 30 min and 6 h of reperfusion. We also detected the interaction of JNK-interacting protein 3 (JIP3) and MKK4, which reached a maximum at 6 h of reperfusion. To elucidate the mechanism of translocation and activation, we administered N-acetylcysteine, an antioxidant reagent, and a glutamate receptor 6 C-terminus-containing peptide (Tat-GluR6-9c) to rats. The data showed that N-acetylcysteine limited the translocation and activation at 30 min of reperfusion; however, the peptide perturbed the subcellular localization and activation at 6 h of reperfusion, and subsequently provided a protective role against delayed neuronal cell death. Taken together, these results demonstrate that the translocation and activation of MKK4 during early reperfusion are closely associated with reactive oxygen species, whereas, at late reperfusion, MKK4 activation may be involved in brain ischemic injury.     

Identification of genes encoding receptor-like protein kinases as possible targets of pathogen- and salicylic acid-induced WRKY DNA-binding proteins in Arabidopsis

To understand how plant host genes are regulated during the activation of plant defence responses, we are studying a group of pathogen- and salicylic acid (SA)-induced DNA-binding proteins containing the novel WRKY domain. To identify downstream target genes of these WRKY proteins, we have searched the Arabidopsis genome and identified four closely linked genes on chromosome IV that contain an unusually large number of the W-box sequences [(T)TGAC(C/T)] recognized by WRKY proteins within a few hundred base pairs upstream of their coding regions. All four genes encode proteins characteristic of receptor-like protein kinases (RLK), each consisting of an N-terminal signal sequence, an extracellular receptor domain, a single transmembrane domain and a C-terminal cytoplasmic serine/threonine protein kinase domain. All four RLK genes were induced by treatment with SA or infection by a bacterial pathogen. Studies with one of the RLK genes (RLK4) indicated that a cluster of W-box elements in its promoter region were recognized by both purified WRKY proteins and SA-induced W-box binding activities from SA-treated Arabidopsis plants. Further analysis using the RLK4 gene promoter fused to a reporter gene in transgenic Arabidopsis indicated that the consensus WRKY protein-binding sites in the RLK4 gene promoter were important for the inducible expression of the reporter gene. These results indicate that pathogen- and SA-induced W-box binding proteins regulate not only genes encoding defence proteins with direct or indirect anti-microbial activities, but also genes encoding proteins with regulatory functions.     

Concurrent activation of cell death-regulating signaling pathways by singlet oxygen in Arabidopsis thaliana

Upon a dark/light shift the conditional flu mutant of Arabidopsis starts to generate singlet oxygen ((1)O(2)), a non-radical reactive oxygen species that is restricted to the plastid compartment. Immediately after the shift, plants stop growing and develop necrotic lesions. We have established a protoplast system, which allows detection and characterization of the death response in flu induced by the release of (1)O(2). Vitamin B6 that quenches (1)O(2) in fungi was able to protect flu protoplasts from cell death. Blocking ethylene production was sufficient to partially inhibit the death reaction. Similarly, flu mutant seedlings expressing transgenic NahG were partially protected from the death provoked by the release of (1)O(2), indicating a requirement for salicylic acid (SA) in this process, whereas in cells depleted of both, ethylene and SA, the extent of cell death was reduced to the wild-type level. The flu mutant was also crossed with the jasmonic acid (JA)-depleted mutant opr3, and with the JA, OPDA and dinor OPDA (dnOPDA)-depleted dde2-2 mutant. Analysis of the resulting double mutants revealed that in contrast to the JA-induced suppression of H(2)O(2)/superoxide-dependent cell death reported earlier, JA promotes singlet oxygen-mediated cell death in flu, whereas other oxylipins such as OPDA and dnOPDA antagonize this death-inducing activity of JA.     

Arabidopsis thaliana Atvsp is homologous to soybean VspA and VspB,genes encoding vegetative storage protein acid phosphatases,and is regulated similarly by methyl jasmonate,wounding, sugars,light and phosphate

The soybean vegetative storage proteins, VSP and VSP, are acid phosphatases that accumulate to very high levels in hypocotyls, young leaves and flowers and pods. The genes encoding the soybean VSP are activated by jasmonate, wounding, sugars and light and down regulated by phosphate and auxin. In this study, expression of an Arabidopsis thaliana gene (Atvsp) encoding a protein homologous to soybean Vsp and Vsp, was examined and compared to expression of the soybean Vsp genes. Atvsp mRNA was present at high levels in flowers and buds and at low levels in roots, stems, leaves and siliques. Expression of Atvsp in leaves could be induced by wounding or by treatment of illuminated plants with methyl jasmonate and sucrose. Roots of plants with wounded leaves also accumulated Atvsp mRNA indicating that this gene can be regulated by a transmissible wound signal. Phosphate partially inhibited expression of Atvsp. Arabidopsis proteins of 29 and 30 kDa crossreacted with antibodies against soybean VSP. These proteins were very abundant in flowers and the proteins accumulated in leaves and roots of plants treated with methyl jasmonate. The level of these proteins in flowers was similar to the levels of soybean VSP in young soybean leaves. Overall, these data indicate that Arabidopsis Atvsp and soybean VspA/B genes are regulated similarly and that in both plants, the gene products can accumulate to high levels. This suggests that genes homologous to VspA/B may be of greater general significance than previously recognized.