Ozone-induced cell death occurs via two distinct mechanisms in Arabidopsis: the role of salicylic acid

Previous studies suggest that salicylic acid (SA) plays an important role in influencing plant resistance to ozone (O3). To further define the role of SA in O3-induced responses, we compared the responses of two Arabidopsis genotypes that accumulate different amounts of SA in response to O3 and a SA-deficient transgenic Col-0 line expressing salicylate hydroxylase (NahG). The differences observed in O3-induced changes in SA levels, the accumulation of active oxygen species, defense gene expression, and the kinetics and severity of lesion formation indicate that SA influences O3 tolerance via two distinct mechanisms. Detailed analyses indicated that features associated with a hypersensitive response (HR) were significantly greater in O3-exposed Cvi-0 than in Col-0, and that NahG plants failed to exhibit these HR-like responses. Furthermore, O3-induced antioxidant defenses, including the redox state of glutathione, were greatly reduced in NahG plants compared to Col-0 and Cvi-0. This suggests that O3-induced cell death in NahG plants is due to the loss of SA-mediated potentiation of antioxidant defenses, while O3-induced cell death in Cvi-0 is due to activation of a HR. This hypothesis is supported by the observation that inhibition of NADPH-oxidases reduced O3-induced H2O2 levels and the O3-induced cell death in Cvi-0, while no major changes were observed in NahG plants. We conclude that although SA is required to maintain the cellular redox state and potentiate defense responses in O3 exposed plants, high levels of SA also potentiate activation of an oxidative burst and a cell death pathway that results in apparent O3 sensitivity.     

Jasmonic acid signaling modulates ozone-induced hypersensitive cell death

Recent studies suggest that cross-talk between salicylic acid (SA)-, jasmonic acid (JA)-, and ethylene-dependent signaling pathways regulates plant responses to both abiotic and biotic stress factors. Earlier studies demonstrated that ozone (O(3)) exposure activates a hypersensitive response (HR)-like cell death pathway in the Arabidopsis ecotype Cvi-0. We now have confirmed the role of SA and JA signaling in influencing O(3)-induced cell death. Expression of salicylate hydroxylase (NahG) in Cvi-0 reduced O(3)-induced cell death. Methyl jasmonate (Me-JA) pretreatment of Cvi-0 decreased O(3)-induced H(2)O(2) content and SA concentrations and completely abolished O(3)-induced cell death. Cvi-0 synthesized as much JA as did Col-0 in response to O(3) exposure but exhibited much less sensitivity to exogenous Me-JA. Analyses of the responses to O(3) of the JA-signaling mutants jar1 and fad3/7/8 also demonstrated an antagonistic relationship between JA- and SA-signaling pathways in controlling the magnitude of O(3)-induced HR-like cell death.     

Differential ozone sensitivity among <Emphasis Type="Italic">Arabidopsis</Emphasis> accessions and its relevance to ethylene synthesis

We compared the physiological and molecular responses of two Arabidopsis accessions, Col-0 and Ws-2, to ozone (O(3)) exposure. Observation of visible injury as well as ion-leakage analysis demonstrated clear differences between the O(3)-tolerant accession Col and the O(3)-sensitive accession Ws. RNA-blot analysis showed that O(3)-induced increases in mRNA levels of several ethylene-inducible genes and a salicylic acid-inducible gene were substantially higher in Ws than in Col. The time-course of induction of various mRNA levels shows that the expression of ethylene-inducible genes was rapidly, and more strongly, induced by O(3) in Ws than in Col, suggesting that Ws exhibits higher ethylene-signaling. Both the level of mRNA for an O(3)-inducible 1-aminocyclopropane-1-carboxylate synthase and the level of ethylene generation after 3 h of O(3)-exposure were higher in Ws than in Col. O(3)-induced leaf damage was attenuated by pretreatment with ethylene biosynthesis- and signaling-inhibitors, indicating that ethylene signaling is required for O(3)-induced leaf injury in Ws. On the other hand, an ethylene-overproducing mutant of Col, eto1-1, displayed significantly increased O(3)-induced leaf injury compared to wild type plants. These results indicate that the difference in O(3) sensitivity is dependent on the difference in ethylene production rate between these two accessions. Finally, we investigated the relationship between the degree of leaf damage and the level of ethylene evolution in 20 different Arabidopsis accessions. Based on the result, the accessions were classified into four types. However, most of them showed significant correlation between the ethylene production level and the degree of leaf injury, suggesting that ethylene signaling is an important factor in the natural variety of O(3) sensitivity among Arabidopsis accessions.     

Plant growth-promoting rhizobacteria systemically protect Arabidopsis thaliana against Cucumber mosaic virus by a salicylic acid and NPR1-independent and jasmonic acid-dependent signaling pathway

Arabidopsis thaliana ecotype Columbia plants (Col-0) treated with plant growth-promoting rhizobacteria (PGPR) Serattia marcescens strain 90-166 and Bacillus pumilus strain SE34 had significantly reduced symptom severity by Cucumber mosaic virus (CMV). In some cases, CMV accumulation was also significantly reduced in systemically infected leaves. The signal transduction pathway(s) associated with induced resistance against CMV by strain 90-166 was determined using mutant strains and transgenic and mutant Arabidopsis lines. NahG plants treated with strains 90-166 and SE34 had reduced symptom severity indicating that the resistance did not require salicylic acid (SA). Strain 90-166 naturally produces SA under iron-limited conditions. Col-0 and NahG plants treated with the SA-deficient mutant, 90-166-1441, had significantly reduced CMV symptom severity with reduced virus accumulation in Col-0 plants. Another PGPR mutant, 90-166-2882, caused reduced disease severity in Col-0 and NahG plants. In a time course study, strain 90-166 reduced virus accumulation at 7 but not at 14 and 21 days post-inoculation (dpi) on the non-inoculated leaves of Col-0 plants. NahG and npr1-1 plants treated with strain 90-166 had reduced amounts of virus at 7 and 14 dpi but not at 21 dpi. In contrast, no decrease in CMV accumulation occurred in strain 90-166-treated fad3-2 fad7-2 fad8 plants. These data indicate that the protection of Arabidopsis against CMV by strain 90-166 follows a signaling pathway for virus protection that is independent of SA and NPR1, but dependent on jasmonic acid.     

Sublethal concentrations of salicylic acid decrease the formation of reactive oxygen species but maintain an increased nitric oxide production in the root apex of the ethylene-insensitive Never ripe tomato mutants

The pattern of salicylic acid (SA)-induced production of reactive oxygen species (ROS) and nitric oxide (NO) were different in the apex of adventitious roots in wild-type and in the ethylene-insensitive Never ripe (Nr) mutants of tomato (Solanum lycopersicum L. cv Ailsa Craig). ROS were upregulated, while NO remained at the control level in apical root tissues of wildtype plants exposed to sublethal concentrations of SA. In contrast, Nr plants expressing a defective ethylene receptor displayed a reduced level of ROS and a higher NO content in the apical root cells. In wild-type plants NO production seems to be ROS(H₂O₂)-dependent at cell death-inducing concentrations of SA, indicating that ROS and NO may interact to trigger oxidative cell death. In the absence of significant ROS accumulation, the increased NO production caused moderate reduction in cell viability in root apex of Nr plants exposed to 10⁻³ M SA. This suggests that a functional ethylene signaling pathway is necessary for the control of ROS and NO production induced by SA.Key words: ethylene receptor mutant, never ripe, nitric oxide, reactive oxygen species, root apex, salicylic acid, tomatoSeveral signal molecules, including salicylic acid (SA) have been implicated in the response of plants to biotic^1–3 and abiotic stressors.^4–6 SA was identified as a central regulator of local defense against (hemi)biotophic pathogens inducing a hypersensitive response (HR), which is characterized by the development of lesions that restrict pathogen spread. It has also emerged as a possible signaling component involved in the activation of certain plant defense responses in non-infected part of the plants establishing the systemic acquired resistance (SAR).⁷The SA-induced biotic and abiotic stress adaptation most likely involves reactive oxygen species (ROS) and nitric oxide (NO) in primary signaling events that activate multiple signal transduction pathways. SA-induced ROS is required for the activation of antioxidant defense mechanisms⁴ and if the generation of ROS exceeds the capacity of antioxidant systems, the cells die.⁸ NO is another important player that is required for the induction of defense mechanisms⁹ or for ROS-induced cell death.¹⁰Accumulation of SA, and two other plant hormones, ethylene (ET) and jasmonic acid (JA) are intimately associated with the initiation or spread of cell death. In HR SA and ROS have been proposed to be on a positive feedback loop that amplifies signals and leads to programmed cell death (PCD). Ethylene caused increased spreading of cell death, while lesion containment can be achieved by JA through decreasing the sensitivity of the cells to ethylene and through the suppression of SA biosynthesis and signaling.⁸Ethylene evolution is associated with diverse physiological processes such as leaf and flower senescence, abscission of organs and fruit ripening.¹¹ The biosynthesis of ethylene is stimulated by a variety of abiotic and biotic stress factors. Ethylene overproducing mutants (eto1 and eto3) of Arabidopsis were found to be more sensitive to O₃, an abiotic stressor which induces ROS-dependent cell death.¹² Cadmium-induced cell death was also accompanied by increased production of ethylene and simultaneously by H₂O₂ accumulation in tomato cell suspension, and based on the effect of specific inhibitors of ethylene biosynthesis and action the authors concluded that the cell death process required H₂O₂ production and a functional ethylene signaling pathway.¹³ Ethylene signaling is also required for the susceptible disease response of tomato plants infected with Xanthomonas campestris pv vesicatoria.¹⁴ It was found that the accumulation of SA and increased production of ethylene were important components of the disease symptoms of this pathogen in wild-type plants, while in Never ripe (Nr) mutants, which have a non-functional ethylene receptor, the infected plants failed to accumulate SA, produced less ethylene, and the leaves exhibited reduced necrotic lesions.It has been also shown that SA enhances NO synthesis in a dose-dependent manner.¹⁵ ROS, such as ^·O₂⁻ and H₂O₂ as well as NO can act together in the cell death regulation and propagation.^8,16 The compartment-specific (down)regulation of ROS can be controlled by NO, accordingly, ROS and NO homeostasis may be essential for the induction or for the avoidance of cell death.     

Salicylate accumulation inhibits growth at chilling temperature in Arabidopsis

The growth of Arabidopsis plants in chilling conditions could be related to their levels of salicylic acid (SA). Plants with the SA hydroxylase NahG transgene grew at similar rates to Col-0 wild types at 23 degrees C, and growth of both genotypes was slowed by transfer to 5 degrees C. However, at 5 degrees C, NahG plants displayed relative growth rates about one-third greater than Col-0, so that by 2 months NahG plants were typically 2.7-fold larger. This resulted primarily from greater cell expansion in NahG rosette leaves. Specific leaf areas and leaf area ratios remained similar in both genotypes. Net assimilation rates were similar in both genotypes at 23 degrees C, but higher in NahG at 5 degrees C. Chlorophyll fluorescence measurements revealed no PSII photodamage in chilled leaves of either genotype. Col-0 shoots at 5 degrees C accumulated SA, particularly in glucosylated form. SA in NahG shoots showed similar tendencies at 5 degrees C, but at greatly depleted levels. Catechol was not detected as a metabolite of the NahG transgene product. We also examined growth and SA levels in SA signaling and metabolism mutants at 5 degrees C. The partially SA-insensitive npr1 mutant displayed growth intermediate between NahG and Col-0, while the SA-deficient eds5 mutant behaved like NahG. In contrast, the cpr1 mutant at 5 degrees C accumulated very high levels of SA and its growth was much more inhibited than wild type. At both temperatures, cpr1 was the only SA-responsive genotype in which oxidative damage (measured as thiobarbituric acid-reactive substances) was significantly different from wild type.     

Loss of non-host resistance of Arabidopsis NahG to Pseudomonas syringae pv. phaseolicola is due to degradation products of salicylic acid

In plants carrying the NahG transgene, salicylate hydroxylase converts salicylic acid (SA) to catechol. Arabidopsis NahG plants are defective in non-host resistance to Pseudomonas syringae pv. phaseolicola strain 3121 (Psp), suggesting that resistance requires SA signaling. However, several mutants with defects in SA signaling, including eds1, pad4, eds5, sid2, and npr1, remain resistant to Psp, demonstrating that susceptibility of NahG plants is not due to absence of SA. SA synthesis is blocked in sid2NahG double mutants, but resistance to Psp is retained. Therefore, it must be the degradative action of NAHG on SA that causes the loss of resistance of NahG to Psp. Treatment of plants with catechol compromised Psp resistance suggesting that the effect of NahG on resistance results from catechol production. Application of catalase to NahG or catechol-treated wild-type plants partially restored resistance to Psp, suggesting that the deleterious effect of catechol results from inappropriate production of hydrogen peroxide. These results indicate that conclusions about SA requirements based solely on phenotypes of NahG plants should be re-evaluated.     

Differential induction of tobacco MAP kinases by the defense signals nitric oxide, salicylic acid, ethylene, and jasmonic acid

In tobacco, two mitogen-activated protein (MAP) kinases, designated salicylic acid (SA)-induced protein kinase (SIPK) and wounding-induced protein kinase (WIPK) are activated in a disease resistance-specific manner following pathogen infection or elicitor treatment. To investigate whether nitric oxide (NO), SA, ethylene, or jasmonic acid (JA) are involved in this phenomenon, the ability of these defense signals to activate these kinases was assessed. Both NO and SA activated SIPK; however, they did not activate WIPK. Additional analyses with transgenic NahG tobacco revealed that SA is required for the NO-mediated induction of SIPK. Neither JA nor ethylene activated SIPK or WIPK. Thus, SIPK may function downstream of SA in the NO signaling pathway for defense responses, while the signals responsible for resistance-associated WIPK activation have yet to be determined.     

Genetic evidence that expression of NahG modifies defence pathways independent of salicylic acid biosynthesis in the Arabidopsis-Pseudomonas syringae pv. tomato interaction

The salicylic acid (SA)-induction deficient (sid) mutants of Arabidopsis, eds5 and sid2 accumulate normal amounts of camalexin after inoculation with Pseudomonas syringae pv. tomato (Pst), while transgenic NahG plants expressing an SA hydroxylase that degrades SA have reduced levels of camalexin and exhibit a higher susceptibility to different pathogens compared to the sid mutants. SID2 encodes an isochorismate synthase necessary for the synthesis of SA. NahG was shown to act epistatically to the sid mutant phenotype regarding accumulation of camalexin after inoculation with Pst in eds5NahG and sid2NahG plants. The effect of the pad4 mutation on the sid mutant phenotype was furthermore tested in eds5pad4 and sid2pad4 double mutants, and it was demonstrated that PAD4 acts epistatically to EDS5 and SID2 regarding the production of camalexin after inoculation with Pst. NahG plants and pad4 mutants were also found to produce less ethylene (ET) after infection with Pst in comparison to the wild type (WT) and sid mutants. Both PAD4 and NahG acted epistatically to SID regarding the Pst-dependent production of ET that was found to be necessary for the accumulation of camalexin. Early production of jasmonic acid (JA) 12 h after inoculation with Pst/avrRpt2 was absent in all plants expressing NahG compared to the other mutants tested here. These genetic studies unravel pleiotropic changes in defence signalling of NahG plants that are unlikely to result from their low SA content. This adds unexpected difficulties in the interpretation of earlier findings based solely on NahG plants.     

Characterisation of an Arabidopsis-Leptosphaeria maculans pathosystem: resistance partially requires camalexin biosynthesis and is independent of salicylic acid, ethylene and jasmonic acid signalling

Out of 168 Arabidopsis accessions screened with isolates of Leptosphaeria maculans, one (An-1) showed clear disease symptoms. In order to identify additional components involved in containment of L. maculans in Arabidopsis, a screen for L. maculans-susceptible (lms) mutants was performed. Eleven lms mutants were isolated, which displayed differential susceptibility responses to L. maculans. lms1 was crossed with Columbia (Col-0) and Ws-0, and mapping data for both populations showed the highest linkage to a region on chromosome 2. Reduced levels of PR-1 and PDF1.2 expression were found in lms1 compared to wild-type plants 48 h after pathogen inoculation. In contrast, the lms1 mutant displayed upregulation of either marker gene upon chemical treatment, possibly as an effect of an altered ethylene (ET) response. To assess the contribution of different defence pathways, genotypes implicated in salicylic acid (SA) signalling plants expressing the bacterial salicylate hydroxylase (nahG) gene, non-expressor of PR1 (npr1)-1 and phytoalexin-deficient (pad4-1), jasmonic acid (JA) signalling (coronatine insensitive (coi)1-16, enhanced disease susceptibility (eds)8-1 and jasmonic acid resistant (jar)1-1) and ET signalling (eds4-1, ethylene insensitive (ein)2, ein3-1 and ethylene resistant (etr)1-1) were screened. All the genotypes screened were as resistant as wild-type plants, demonstrating the dispensability of the pathways in L. maculans resistance. When mutants implicated in cell death responses were assayed, responsive to antagonist 1 (ran1)-1 exhibited a weak susceptible phenotype, whereas accelerated cell death (acd)1-20 showed a rapid lesion development. Camalexin is only partially responsible for L. maculans containment in Arabidopsis, as pad3-1 and enhanced susceptibility to Alternaria (esa)1 clearly showed a susceptible response while wild-type levels of camalexin were present in An-1 and lms1. The data presented point to the existence of multiple defence mechanisms controlling the containment of L. maculans in Arabidopsis.     

Overexpression of Pto induces a salicylate-independent cell death but inhibits necrotic lesions caused by salicylate-deficiency in tomato plants

Tomato plants overexpressing the disease resistance gene Pto (35S::Pto) exhibit spontaneous cell death, accumulation of salicylic acid (SA), elevated expression of pathogenesis-related genes, and enhanced resistance to a broad range of pathogens. Because salicylate plays an important role in the cell death and defense activation in many lesion mimic mutants, we investigated the interaction of SA-mediated processes and the 35S::Pto-mediated defense pathway by introducing the nahG transgene that encodes salicylate hydroxylase. Here, we show that SA is not required for the 35S::Pto-activated microscopic cell death and plays a minor role in defense gene activation and general disease resistance in 35S::Pto plants. In contrast, temperature greatly affects the spontaneous cell death and general resistance in 35S::Pto plants, and high temperature inhibits the cell death. The NahG tomato plants develop spontaneous, unconstrained necrotic lesions on leaves. These lesions also are initiated by the inoculation of a virulent strain of Pseudomonas syringae pv. tomato. However, the NahG-dependent necrotic lesions are inhibited in the NahG/35S::Pto plants. This inhibition is most pronounced under conditions favoring the 35S::Pto-mediated spontaneous cell death development. These results indicate that the signaling pathways activated by Pto overexpression suppress the cellular damage that is caused by SA depletion. We also found that ethylene is dispensable for the 35S::Pto-mediated general defense.     

Uncoupling salicylic acid-dependent cell death and defense-related responses from disease resistance in the Arabidopsis mutant acd5

Salicylic acid (SA) is required for resistance to many diseases in higher plants. SA-dependent cell death and defense-related responses have been correlated with disease resistance. The accelerated cell death 5 mutant of Arabidopsis provides additional genetic evidence that SA regulates cell death and defense-related responses. However, in acd5, these events are uncoupled from disease resistance. acd5 plants are more susceptible to Pseudomonas syringae early in development and show spontaneous SA accumulation, cell death, and defense-related markers later in development. In acd5 plants, cell death and defense-related responses are SA dependent but they do not confer disease resistance. Double mutants with acd5 and nonexpressor of PR1, in which SA signaling is partially blocked, show greatly attenuated cell death, indicating a role for NPR1 in controlling cell death. The hormone ethylene potentiates the effects of SA and is important for disease symptom development in Arabidopsis. Double mutants of acd5 and ethylene insensitive 2, in which ethylene signaling is blocked, show decreased cell death, supporting a role for ethylene in cell death control. We propose that acd5 plants mimic P. syringae-infected wild-type plants and that both SA and ethylene are normally involved in regulating cell death during some susceptible pathogen infections.     

Epidermal cell death in rice is regulated by ethylene, gibberellin, and abscisic acid

Programmed cell death (PCD) of epidermal cells that cover adventitious root primordia in deepwater rice (Oryza sativa) is induced by submergence. Early suicide of epidermal cells may prevent injury to the growing root that emerges under flooding conditions. Induction of PCD is dependent on ethylene signaling and is further promoted by gibberellin (GA). Ethylene and GA act in a synergistic manner, indicating converging signaling pathways. Treatment of plants with GA alone did not promote PCD. Treatment with the GA biosynthesis inhibitor paclobutrazol resulted in increased PCD in response to ethylene and GA presumably due to an increased sensitivity of epidermal cells to GA. Abscisic acid (ABA) was shown to efficiently delay ethylene-induced as well as GA-promoted cell death. The results point to ethylene signaling as a target of ABA inhibition of PCD. Accumulation of ethylene and GA and a decreased ABA level in the rice internode thus favor induction of epidermal cell death and ensure that PCD is initiated as an early response that precedes adventitious root growth.     

Ethylene signaling in salt stress- and salicylic acid-induced programmed cell death in tomato suspension cells

Salt stress- and salicylic acid (SA)-induced cell death can be activated by various signaling pathways including ethylene (ET) signaling in intact tomato plants. In tomato suspension cultures, a treatment with 250 mM NaCl increased the production of reactive oxygen species (ROS), nitric oxide (NO), and ET. The 10^?3 M SA-induced cell death was also accompanied by ROS and NO production, but ET emanation, the most characteristic difference between the two cell death programs, did not change. ET synthesis was enhanced by addition of ET precursor 1-aminocyclopropane-1-carboxylic acid, which, after 2 h, increased the ROS production in the case of both stressors and accelerated cell death under salt stress. However, it did not change the viability and NO levels in SA-treated samples. The effect of ET induced by salt stress could be blocked with silver thiosulfate (STS), an inhibitor of ET action. STS reduced the death of cells which is in accordance with the decrease in ROS production of cells exposed to high salinity. Unexpectedly, application of STS together with SA resulted in increasing ROS and reduced NO accumulation which led to a faster cell death. NaCl- and SA-induced cell death was blocked by Ca²⁺ chelator EGTA and calmodulin inhibitor W-7, or with the inhibitors of ROS. The inhibitor of MAPKs, PD98059, and the cysteine protease inhibitor E-64 reduced cell death in both cases. These results show that NaCl induces cell death mainly by ET-induced ROS production, but ROS generated by SA was not controlled by ET in tomato cell suspension.     

Mi-1-Mediated aphid resistance involves salicylic acid and mitogen-activated protein kinase signaling cascades

The tomato Mi-1 gene confers resistance to root-knot nematodes (Meloidogyne spp.), potato aphids (Macrosiphum eluphorbiae), and whiteflies (Bemisia tabaci and B. tabaci biotype B). Resistance to potato aphid is developmentally regulated and is not associated with induction of a hypersensitive response. The NahG transgene that eliminates endogenous salicylic acid (SA) was used to test the role of the SA signaling pathway in the resistance mediated by Mi-1 to potato aphids. Aphids survived longer on NahG tomato plants than on wild type. However, aphid reproduction was not affected on NahG tomato. Aphid resistance in Mi-1 NahG plants was completely abolished and the phenotype was successfully rescued by application of BTH (benzo(1,2,3)-thiaiazole-7-carbothioic acid S-methyl ester), indicating that the SA signaling pathway is an important component of Mi-1-mediated aphid resistance. Using virus-induced gene silencing, one or more mitogen-activated protein kinase (MAPK) cascades required for Mi-1-mediated aphid resistance were identified. Silencing plants for MAPK kinase (LeMKK2) and MAPKs (LeMPK2 and LeMPK1, or LeMPK3) resulted in attenuation of Mi-1-mediated aphid resistance. These results further demonstrate that resistance gene-mediated signaling events against piercing-sucking insects are similar to those against other plant pathogens.     

Effects of endogenous salicylic acid on nodulation in the model legumes Lotus japonicus and Medicago truncatula

The exogenous addition of salicylic acid (SA) was previously shown to inhibit indeterminate but not determinate-type nodulation. We sought to extend these results by modulating endogenous levels of SA through the transgenic expression of salicylate hydroxylase (NahG) in both stably transformed Lotus japonicus and composite Medicago truncatula plants. NahG expression in L. japonicus resulted in a marked reduction of SA levels. This reduction correlated with an increase in the number of infections and mean nodule number when compared to controls. However, a complicating factor was that NahG-expressing plants had greater root growth. Spot inoculations of NahG-expressing L. japonicus plants confirmed increased nodulation in these plants. Consistent with the reported inhibitory effects of exogenous SA on indeterminate-type nodulation, NahG expression in M. truncatula plants led to enhanced nodulation and infection. These data point to an important role for SA-mediated plant defense pathways in controlling nodule formation on both determinate and indeterminate nodule-forming hosts.     

The role of salicylic acid in the glutathione-mediated protection against photooxidative stress in rice

Salicylic acid (SA) is known to be an essential component responsible for disease resistance in dicotyledonous plants. In rice, however, tissue contains extremely high endogenous levels of SA that do not increase after pathogen infection, suggesting that the SA has other major functions in healthy leaves. Although involvement of SA in oxidative-stress response is known in some dicotyledonous plants, antioxidative role of SA in rice is obscure. In this study, we examined the involvement of SA in the protection against oxidative stress in rice, using transgenic plants expressing the bacterial nahG gene that encodes salicylate hydroxylase, an SA-degrading enzyme. In SA-deficient NahG rice, the glutathione pool size was constitutively diminished as compared with control plants. NahG seedlings showed a delayed development phenotype, an increased susceptibility to oxidative stress and they developed light-induced lesions in their leaves without pathogen infection. Conversely, treatment with an activator of the SA-mediated defense-signaling pathway, probenazole, increased the glutathione pool size and suppressed lesion formation. These results suggest that in rice, SA has an important role in the response to high-light-induced oxidative stress, through its regulatory effects on glutathione homeostasis.     

Ethylene is one of the key elements for cell death and defense response control in the Arabidopsis lesion mimic mutant vad1

Although ethylene is involved in the complex cross talk of signaling pathways regulating plant defense responses to microbial attack, its functions remain to be elucidated. The lesion mimic mutant vad1-1 (for vascular associated death), which exhibits the light-conditional appearance of propagative hypersensitive response-like lesions along the vascular system, is a good model for studying the role of ethylene in programmed cell death and defense. Here, we demonstrate that expression of genes associated with ethylene synthesis and signaling is enhanced in vad1-1 under lesion-promoting conditions and after plant-pathogen interaction. Analyses of the progeny from crosses between vad1-1 plants and either 35SERF1 transgenic plants or ein2-1, ein3-1, ein4-1, ctr1-1, or eto2-1 mutants revealed that the vad1-1 cell death and defense phenotypes are dependent on ethylene biosynthesis and signaling. In contrast, whereas vad1-1-dependent increased resistance was abolished by ein2, ein3, and ein4 mutations, positive regulation of ethylene biosynthesis (eto2-1) or ethylene responses (35SERF1) did not exacerbate this phenotype. In addition, VAD1 expression in response to a hypersensitive response-inducing bacterial pathogen is dependent on ethylene perception and signaling. These results, together with previous data, suggest that VAD1 could act as an integrative node in hormonal signaling, with ethylene acting in concert with salicylic acid as a positive regulator of cell death propagation.