The ocs element in the soybean GH2/4 promoter is activated by both active and inactive auxin and salicylic acid analogues

The octopine synthase (ocs or ocs-like) element has been previously reported to be responsive to the plant hormones, auxin, salicylic acid, and methyl jasmonate. Using transient assays with carrot protoplasts, we have demonstrated that an ocs element from the soybean auxin-inducible GH2/4 promoter is not only activated by strong auxins (i.e, 2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, -naphthalene acetic acid) and salicylic acid, but also by weak auxin analogues (-naphthalene acetic acid), inactive auxin analogs (i.e., 2,3-dichlorophenoxyacetic acid, 2,4,6-trichlorophenoxyacetic acid), and inactive salicylic acid analogs (3-hydroxybenzoic acid and 4-hydroxybenzoic acid). Our results indicate that the ocs element in the GH2/4 promoter is not selectively induced by plant hormones and might function similarly to tandem AP-1 sites in some animal glutathione S-transferase (GST) genes. The ocs element, like the AP-1 sites in animal GST promoters, may be induced not only by certain hormones but also by some non-hormonal stress-inducing or electrophilic agents.Abbreviations GST glutathione S-transferase - MUG 4-methyl-umbelliferyl-glucuronide - GUS -glucuronidase - 2,4-D 2,4-dichlorophenoxyacetic acid - 2,3-D 2,3-dichlorophenoxyacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - 2,4,6-T 2,4,6-trichlorophenoxyacetic acid - NAA naphthalene acetic acid - SA salicylic acid - SARE putative salicylic acid-responsive element - BA benzoic acid - UTR untranslated region - nos nopaline synthase - ocs octopine synthase - mas mannopine synthase - ocs element-(–)46 CaMV 35S promoter-GUS reporter gene: the ocs element fused to a minimal –46 cauliflower mosaic virus 35S promoter fused to a GUS reporter gene with a 3 nos untranslated region     

CLE9 peptide‐induced stomatal closure is mediated by abscisic acid,hydrogen peroxide,and nitric oxide in Arabidopsis thaliana

CLE peptides have been implicated in various developmental processes of plants and mediate their responses to environmental stimuli. However, the biological relevance of most CLE genes remains to be functionally characterized. Here, we report that CLE9, which is expressed in stomata, acts as an essential regulator in the induction of stomatal closure. Exogenous application of CLE9 peptides or overexpression of CLE9 effectively led to stomatal closure and enhanced drought tolerance, whereas CLE9 loss‐of‐function mutants were sensitivity to drought stress. CLE9‐induced stomatal closure was impaired in abscisic acid (ABA)‐deficient mutants, indicating that ABA is required for CLE9‐medaited guard cell signalling. We further deciphered that two guard cell ABA‐signalling components, OST1 and SLAC1, were responsible for CLE9‐induced stomatal closure. MPK3 and MPK6 were activated by the CLE9 peptide, and CLE9 peptides failed to close stomata in mpk3 and mpk6 mutants. In addition, CLE9 peptides stimulated the induction of hydrogen peroxide (H₂O₂) and nitric oxide (NO) synthesis associated with stomatal closure, which was abolished in the NADPH oxidase‐deficient mutants or nitric reductase mutants, respectively. Collectively, our results reveal a novel ABA‐dependent function of CLE9 in the regulation of stomatal apertures, thereby suggesting a potential role of CLE9 in the stress acclimatization of plants.     

Cell death mediated by MAPK is associated with hydrogen peroxide production in Arabidopsis.

Rapid and localized programmed cell death, known as the hypersensitive response (HR) is frequently associated with plant disease resistance. In contrast to our knowledge about the regulation and execution of apoptosis in animal system, information about plant HR is limited. Recent studies implicated the mitogen-activated protein kinase (MAPK) cascade in regulating plant HR cell death as well as several other defense responses during incompatible interactions between plants and pathogens. Here, we report the generation of transgenic Arabidopsis plants that express the active mutants of AtMEK4 and AtMEK5, two closely related MAPK kinases under the control of a steroid-inducible promoter. Induction of the transgene expression by the application of dexamethasone, a steroid, leads to HR-like cell death, which is preceded by the activation of endogenous MAPKs and the generation of hydrogen peroxide. Both prolonged MAPK activation and reactive oxygen species generation have been implicated in the regulation of HR cell death induced by incompatible pathogens. As a result, we speculate that the prolonged activation of the MAPK pathway in cells could disrupt the redox balance, which leads to the generation of reactive oxygen species and eventually HR cell death.     

Enhanced lipoxygenase activity is involved in barley root tip swelling induced by cadmium,auxin or hydrogen peroxide

Lipoxygenases (EC 1.13.11.12) catalyse the formation of hydroperoxy derivates by oxygenation of polyunsaturated fatty acids. They act as signal molecules, triggering several developmental processes and defence responses under stress conditions. Incubation of Cd-, IAA- or H₂O₂-short-term treated seedlings in the presence of LOX inhibitors efficiently inhibited both Cd-, IAA- or H₂O₂-induced LOX activity and root swelling in a concentration dependent manner, suggesting a key role of LOX or LOX signalling pathway in radial expansion of root cells. Application of antioxidants (ascorbate or N-acetyl cysteine) to the treated seedlings at low 2 mM concentration did not affect the Cd-, IAA- or H₂O₂-induced LOX activity and root swelling. At higher, 10 mM concentration antioxidants markedly inhibited root growth, significantly increased the activity of LOX and evoked the radial expansion of root cells leading to root swelling with well developed root hairs already in control roots. By contrast, the lipophilic antioxidant trolox, a scavenger of hydroperoxides, severely inhibited the development of Cd-, IAA- or H₂O₂-induced root swelling, indicating that not directly LOX, but probably oxylipins, products of LOX pathway, are involved in the induction of root swelling in barley root tip. The results of this study suggest a strong connection between abiotic stress-induced alteration in redox and hormone status caused root growth inhibition and LOX pathway mediated radial expansion of root tip cells.     

OsAGAP, an ARF-GAP from rice, regulates root development mediated by auxin in Arabidopsis

Arf (ADP-ribosylation factor) proteins, which mediate vesicular transport, have little or no intrinsic GTPase activity. They rely on the action of GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs) for their function. In the present study the OsAGAP gene in rice, which encoded a protein with predicted structure similar to ArfGAP, was identified. The purified OsAGAP-GST fusion protein was able to stimulate the GTPase activity of rice Arf. Furthermore, OsAGAP can rescue the defect of vesicular transport in the yeast gcs1 delta glo3 delta double-mutant cells. Transgenic Arabidopsis with OsAGAP constitutively expression showed reduced apical dominance, shorter primary roots, increasing number of longer adventitious roots. Many of the phenotypes can be phenocopied by treatment of exogenous indoleacetic acid level (IAA) in wild-type plants. Determination of whole-plant IAA level showed that there is a sharp increase of free IAA in OsAGAP transgenic Arabidopsis seedlings. In addition, removal of the 4-day-old shoot apex could inhibit the adventitious root formation in the transgenic seedlings. These results suggest OsAGAP, an ARF-GAP of rice, maybe involved in the mediation of plant root development by regulating auxin level.     

ACD6, a novel ankyrin protein, is a regulator and an effector of salicylic acid signaling in the Arabidopsis defense response

The previously reported Arabidopsis dominant gain-of-function mutant accelerated cell death6-1 (acd6-1) shows spontaneous cell death and increased disease resistance. acd6-1 also confers increased responsiveness to the major defense signal salicylic acid (SA). To further explore the role of ACD6 in the defense response, we cloned and characterized the gene. ACD6 encodes a novel protein with putative ankyrin and transmembrane regions. It is a member of one of the largest uncharacterized gene families in higher plants. Steady state basal expression of ACD6 mRNA required light, SA, and an intact SA signaling pathway. Additionally, ACD6 mRNA levels were increased in the systemic, uninfected tissue of Pseudomonas syringae-infected plants as well as in plants treated with the SA agonist benzothiazole (BTH). A newly isolated ACD6 loss-of-function mutant was less responsive to BTH and upon P. syringae infection had reduced SA levels and increased susceptibility. Conversely, plants overexpressing ACD6 showed modestly increased SA levels, increased resistance to P. syringae, and BTH-inducible and/or a low level of spontaneous cell death. Thus, ACD6 is a necessary and dose-dependent activator of the defense response against virulent bacteria and can activate SA-dependent cell death.     

The rib1 mutant is resistant to indole-3-butyric acid, an endogenous auxin in Arabidopsis

The presence of indole-3-butyric acid (IBA) as an endogenous auxin in Arabidopsis has been recently demonstrated. However, the in vivo role of IBA remains to be elucidated. We present the characterization of a semi-dominant mutant that is affected in its response to IBA, but shows a wild-type response to indole-3-acetic acid (IAA), the predominant and most studied form of auxin. We have named this mutant rib1 for resistant to IBA. Root elongation assays show that rib1 is specifically resistant to IBA, to the synthetic auxin 2,4-dichlorophenoxyacetic acid, and to auxin transport inhibitors. rib1 does not display increased resistance to IAA, to the synthetic auxin naphthalene acetic acid, or to other classes of plant hormones. rib1 individuals also have other root specific phenotypes including a shortened primary root, an increased number of lateral roots, and a more variable response than wild type to a change in gravitational vector. Adult rib1 plants are morphologically indistinguishable from wild-type plants. These phenotypes suggest that rib1 alters IBA activity in the root, thereby affecting root development and response to environmental stimuli. We propose models in which RIB1 has a function in either IBA transport or response. Our experiments also suggest that IBA does not use the same mechanism to exit cells as does IAA and we propose a model for IBA transport.     

Enhanced chilling tolerance in Zoysia matrella by pre-treatment with salicylic acid, calcium chloride, hydrogen peroxide or 6-benzylaminopurine

Following leaf application of salicylic acid (SA), calcium chloride, hydrogen peroxide and 6-benzylaminopurine (BA), Manila grass (Zoysia matrella) plants were exposed to day/night temperature of 7/2 °C for 120 h in a growth chamber. The lower content of malondialdehyde (MDA) and H₂O₂ and higher activities of ascorbate peroxidase (APX), guaiacol peroxidase (POD) and catalase (CAT) during exposure to low temperature in pre-treated plants in comparison with control plants demonstrated that these compounds improved the chilling tolerance of Manila grass.     

Stimulation by auxin of phospholipase A in membrane vesicles from an auxin-sensitive tissue is mediated by an auxin receptor

Microsomal vesicles were prepared from zucchini (Cucurbita pepo L.) hypocotyls containing radioactive phosphatidylethanolamine or phosphatidylcholine, and these lipids were used as substrates by phospholipase A which is activated by auxins. Phospholipase D and phospholipase C hydrolysed the same substrates but were not influenced by auxin. Phospholipase A was activated by the auxins indolyl-3-acetic acid, 2,4-dichlorophenoxyacetic acid and, to a lesser extent, by -naphthaleneacetic acid whereas the weak auxins 2,3-dichlorophenoxyacetic acid and -naphthaleneacetic acid were almost inactive. This hormone specificity was also found in growth tests with etiolated zucchini hypocotyls. Phospholipase A activation by auxin was blocked by a polyclonal antibody against the maize auxin-binding protein. We propose that phospholipase A activation is a primary reaction in the signal transduction leading from hormone-binding to the growth response.Abbreviations IAA indolyl-3-acetic acid - 2,3-D, 2,4-D 2,3- and 2,4-dichlorophenoxyacetic acid - -NAA; -NAA - and -naphthaleneacetic acid This work was supported by the Deutsche Forchungsgemeinschaft. We thank D. Klämbt (Botanical Institute, University of Bonn, FRG) for a generous gift of polyclonal antibody (IgG fraction) against auxin-binding protein and U. Kutschera (Botanical Institute, University of Bonn, FRG) for advice with the growth tests.     

The Arabidopsis extensin gene is developmentally regulated, is induced by wounding, methyl jasmonate, abscisic and salicylic acid, and codes for a protein with unusual motifs

A single-copy extensin gene (atExt1) has been isolated from Arabidopsis thaliana (L.) Heynh. The deduced amino acid sequence consists of 374 amino acids which are organised into highly ordered repeating blocks in which Ser(Pro)₄ and Ser(Pro)₃ motifs alternate. Two copies of the Tyr-X-Tyr-Lys motif and 13 copies of the Val-Tyr-Lys motif are present, showing that this extensin may be highly cross-linked, possessing the capacity for both intra and inter-molecular bond formation. The gene atExt1 is normally expressed in the root and is silent in the leaf; wounding reverses this pattern, turning on the gene in the leaf and repressing it in the root. The promoter contains motifs which have been found to activate plant defence genes in response to salicylic acid, abscisic acid and methyl jasmonate; when these compounds are applied to the roots, the atExt1 gene is activated in the leaf. Received: 11 September 1998 / Accepted: 20 December 1998     

Induction of a salicylic acid glucosyltransferase, AtSGT1, is an early disease response in Arabidopsis thaliana

Endogenous salicylic acid (SA) and its predominant conjugates, SA 2-O-beta-D-glucoside (SAG) and the glucose ester of SA (SGE), increase dramatically during plant defense responses. Here I report the isolation and characterization of an Arabidopsis thaliana UDP-glucose:SA glucosyltransferase1 (AtSGT1) gene using a tobacco SGT gene previously reported, whose product catalyzes the formation of both SAG and SGE. The recombinant AtSGT1 protein had significant activities with SA and benzoic acid, and synthesized SAG and SGE. Northern blot analysis showed that AtSGT1 was rapidly induced both by exogenous SA and infection with the bacterial pathogen Pseudomonas syringae, indicating that pathogen-inducible AtSGT1 expression is an early disease response and may be involved in the accumulation of glucosyl SA during pathogenesis.     

Induction of defence gene expression by oligogalacturonic acid requires increases in both cytosolic calcium and hydrogen peroxide in Arabidopsis thaliana

Responses to oligogalacturonic acid (OGA) were determined in transgenic Arabidopsis thaliana seedlings express-ing the calcium reporter protein aequorin. OGA stimulated a rapid, substantial and transient increase in the concentration of cytosolic calcium ([Ca^2 ]cyt) that peaked after ca. 15 s. This increase was dose-dependent, saturating at ca. 50 μg Gal equiv/ml of OGA. OGA also stimulated a rapid generation of H202. A small, rapid increase in H2O2 content was followed by a much larger oxidative burst, with H2O2 content peaking after ca. 60 min and declining thereafter. Induction of the oxidative burst by OGA was also dose-dependent, with a maximum response again being achieved at ca. 50 μg Gal equiv/mL. Inhibitors of calcium fluxes inhibited both increases in [Ca^2 ]cyt and [H2O2], whereas inhibitors of NADPH oxidase blocked only the oxidative burst. OGA increased strongly the expression of the defence-related genes CHS,GST, PAL and PR-1. This induction was suppressed by inhibitors of calcium flux or NADPH oxidase, indicating that increases in both cytosolic calcium and H2O2 are required for OGA-induced gene expression.     

Ethylene antagonizes the inhibition of germination in Arabidopsis induced by salinity by modulating the concentration of hydrogen peroxide

The capacity of plants to achieve successful germination and early seedling establishment under high salinity is crucial for tolerance of plants to salt. The gaseous hormone ethylene has been implicated in modulating salt tolerance, but the detailed role of how ethylene modulates the response of early seedling establishment to salt is unclear. To better understand the role of the ethylene signal transduction pathway during germination and seedling establishment, an ethylene insensitive mutation (ein2-5) and an ethylene sensitive mutation (ctr1-1) of Arabidopsis were analyzed under saline conditions and compared with the wild type plant (Col-0) as control. High salinity (>100?mM NaCl) inhibited and delayed germination. These effects were more severe in the ethylene insensitive mutants (ein2-5) and less severe in the constitutive ethylene sensitive plants (ctr1-1) compared with Col-0 plants. Addition of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) or inhibitors of ethylene action implied that ethylene was essential for early seedling establishment under normal and saline conditions. Salt stress increased the endogenous concentration of hydrogen peroxide (H₂O₂) in germinating seeds and ACC reduced its concentration. Our results suggest that ethylene promotes germination under salinity by modulating the endogenous concentration of H₂O₂ in germinating seeds. These findings demonstrate that ethylene is involved in regulating germination as an initiator of the process rather than consequence, and that ethylene promotes germination by modulating the endogenous concentration of H₂O₂ in germinating seeds under salinity.     

Thermotolerance and antioxidant systems in Agrostis stolonifera: involvement of salicylic acid, abscisic acid, calcium, hydrogen peroxide, and ethylene

This study investigated whether pre-treating plants with specific putative signaling components and heat acclimation would induce tolerance of a cool-season grass, creeping bentgrass (Agrostis stolonifera var. palustris), to subsequent heat stress and whether thermotolerance induction of those pretreatments was associated with the regulation of antioxidant regenerating enzymes. The treatments included foliar application of salicylic acid (SA), abscisic acid (ABA), calcium chloride (CaCl2), hydrogen peroxide (H2O2), 1-aminocyclopropane-1-carboxylic acid (ACC, a precursor of ethylene prior to the exposure of plants to heat stress (35 degrees C) in a growth chamber. Physiological measurements including turf quality, leaf photosynthetic rate, and levels of oxidative damage demonstrated that all treatments increased heat tolerance. The better heat tolerance for pre-treated plants as compared to controls was related to the protection of oxidative damage under heat stress. APX activity increased over the first 2 days and 5 days of heating for ACC and CaCl2 respectively, but for only 12 h for H2O2. SA and ABA pre-treatments had no effects on APX activity earlier, but maintained APX activity at a significantly higher level than in controls after 24 h of heating. SA and ABA pre-treatments had no effects on POX activity. ACC treatment significantly increased POX activity. Pre-treatment with CaCl2, H2O2, and HA reduced POX activity, particularly during the later phase of heating. Plants treated with SA, CaCl2, H2O2 and HA had lower CAT activity than their control plants prior to heating and within 48 h of heat stress. ABA and ACC pre-treatments maintained higher CAT activity than the controls after 48 h of heating. ACC, CaCl2, or HA pre-treatments increased SOD activity only before 5 days of heat stress. SA and ABA pre-treatments had less effect on APX activity earlier under heat stress. These results suggest that specific groups of potential signaling molecules may induce tolerance of creeping bentgrass to heat stress by reducing oxidative damage.