NPR1-dependent salicylic acid signaling is not involved in elevated CO2-induced heat stress tolerance in Arabidopsis thaliana

Elevated CO₂ can protect plants from heat stress (HS); however, the underlying mechanisms are largely unknown. Here, we used a set of Arabidopsis mutants such as salicylic acid (SA) signaling mutants nonexpressor of pathogenesis-related gene 1 (npr1-1 and npr1-5) and heat-shock proteins (HSPs) mutants (hsp21 and hsp70-1) to understand the requirement of SA signaling and HSPs in elevated CO₂-induced HS tolerance. Under ambient CO₂ (380 µmol mol⁻¹) conditions, HS (42°C, 24 h) drastically decreased maximum photochemical efficiency of PSII (Fv/Fm) in all studied plant groups. Enrichment of CO₂ (800 µmol mol⁻¹) with HS remarkably increased the Fv/Fm value in all plant groups except hsp70-1, indicating that NPR1-dependent SA signaling is not involved in the elevated CO₂-induced HS tolerance. These results also suggest an essentiality of HSP70-1, but not HSP21 in elevated CO₂-induced HS mitigation.     

Salicylic acid biosynthesis is not from phenylalanine in Arabidopsis

The phytohormone salicylic acid(SA) regulates biotic and abiotic stress responses in plants. Two distinct biosynthetic pathways for SA have been well documented in plants: the isochorismate(IC)pathway in the chloroplast and the phenylalanine ammonia-lyase(PAL) pathway in the cytosol.However, there has been no solid evidence that the PAL pathway contributes to SA biosynthesis. Here,we report that feeding Arabidopsis thaliana with Ring-¹³C-labeled phenylalanine(¹³C₆     

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.     

Infection of Arabidopsis with a necrotrophic pathogen,Botrytis cinerea,elicits various defense responses but does not induce systemic acquired resistance (SAR)

Botrytis cinerea is a non-specific necrotrophic pathogen that attacks more than 200 plant species. In contrast to biotrophs, the necrotrophs obtain their nutrients by first killing the host cells. Many studies have shown that infection of plants by necrosis-causing pathogens induces a systemic acquired resistance (SAR), which provides protection against successive infections by a range of pathogenic organisms. We analyzed the role of SAR in B. cinerea infection of Arabidopsis. We show that although B. cinerea induced necrotic lesions and camalexin biosynthesis, it did not induce SAR-mediated protection against virulent strains of Pseudomonas syringae, or against subsequent B. cinerea infections. Induction of SAR with avirulent P. syringae or by chemical treatment with salicylic acid (SA) or benzothiadiazole also failed to inhibit B. cinerea growth, although removal of basal SA accumulation by expression of a bacterial salicylate hydroxylase (NahG) gene or by infiltration of 2-aminoindan-2-phosphonic acid, an inhibitor of phenylpropanoid pathway, increased B. cinerea disease symptoms. In addition, we show that B. cinerea induced expression of genes associated with SAR, general stress and ethylene/jasmonate-mediated defense pathways. Thus, B. cinerea does not induce SAR nor is it affected by SAR, making it a rare example of a necrogenic pathogen that does not cause SAR.     

Induction of Resistance in Melon to Didymella bryoniae and Sclerotinia sclerotiorum by Seed Treatments with Acibenzolar-S-methyl and Methyl Jasmonate but not with Salicylic Acid

The plant resistance activator acibenzolar‐S‐methyl (BTH), the signalling molecules salicylic acid (SA) and methyl jasmonate (MeJA) were tested by seed treatment for their ability to protect melon seedlings from gummy stem blight and white mould disease caused by the soil‐borne fungal pathogens Didymella bryoniae and Sclerotinia sclerotiorum, respectively. Didymella bryoniae infection on melon seedlings was completely suppressed by MeJA treatment. Necrotic lesions akin hypersensitive response occurred on all inoculated seedlings and prevented pathogen diffusion into healthy tissues. Didymella bryoniae infection was restricted following BTH seed treatment as well, although the percentage of necrotic lesions in comparison with the water soaked lesions was significantly lower than that from MeJA‐induced seedlings. BTH protected melon seedlings against S. sclerotiorum by the occurrence of a high percentage of necrotic lesions. A lower level of resistance was also achieved by MeJA seed treatment. The augmented level of resistance of tissues from BTH and MeJA‐treated seeds was associated with rapid increases in the activity of the pathogenesis‐related proteins chitinase and peroxidase. MeJA also determined a rapid and transient accumulation of lipoxygenase. Moreover, BTH and MeJA treatments determined the differential induction of particular de novo synthesized isoenzymes of these proteins. Results indicate that BTH and MeJA applied to melon seeds may activate on seedlings diverse metabolic pathways leading to the enhancement of resistance against distinct pathogens.     

Phosphoinositide-specific phospholipase C9 is involved in the thermotolerance of Arabidopsis

Intracellular calcium (Ca(2+)) increases rapidly after heat shock (HS) in the Ca(2+)/calmodulin (Ca(2+)/CaM) HS signal transduction pathway: a hypothesis proposed based on our previous findings. However, evidence for the increase in Ca(2+) after HS was obtained only through physiological and pharmacological experiments; thus, direct molecular genetic evidence is needed. The role of phosphoinositide-specific phospholipase C (PI-PLC) is poorly understood in the plant response to HS. In this work, atplc9 mutant plants displayed a serious thermosensitive phenotype compared with wild-type (WT) plants after HS. Complementation of atplc9 with AtPLC9 rescued both the basal and acquired thermotolerance phenotype of the WT plants. In addition, thermotolerance was even improved in overexpressed lines. The GUS staining of AtPLC9 promoter:GUS transgenic seedlings showed that AtPLC9 expression was ubiquitous. The fluorescence distribution of the fusion protein AtPLC9 promoter:AtPLC9:GFP revealed that the subcellular localization of AtPLC9 was restricted to the plasma membrane. The results of a PLC activity assay showed a reduction in the accumulation of inositol-1,4,5-trisphosphate (IP(3)) in atplc9 during HS and improved IP(3) generation in the overexpressed lines. Furthermore, the heat-induced increase in intracellular Ca(2+) was decreased in atplc9. Accumulation of the small HS proteins HSP18.2 and HSP25.3 was downregulated in atplc9 and upregulated in the overexpressed lines after HS. Together, these results provide molecular genetic evidence showing that AtPLC9 plays a role in thermotolerance in Arabidopsis.     

Phosphatidic acid is a major phospholipid class in reproductive organs of Arabidopsis thaliana

Phospholipids are the crucial components of biological membranes and signal transduction. Among different tissues, flower phospholipids are one of the least characterized features of plant lipidome. Here, we report that floral reproductive organs of Arabidopsis thaliana contain high levels of phosphatidic acid (PA), a known lipid second messenger. By using floral homeotic mutants enriched with specific floral organs, lipidomics study showed increased levels of PA species in ap3-3 mutant with enriched pistils. Accompanied gene expression study for 7 diacylglycerol kinases and 11 PA phosphatases revealed distinct floral organ specificity, suggesting an active phosphorylation/dephosphorylation between PA and diacylglycerol in flowers. Our results suggest that PA is a major phospholipid class in floral reproductive organs of A. thaliana.     

Flavonols are not essential for fertilization in Arabidopsis thaliana

Flavonols are plant metabolites suggested to serve a vital role in fertilization of higher plants. Petunia and maize plants mutated in their flavonol biosynthesis are not able to set seed after self-pollination. We have investigated the role of these compounds in Arabidopsis thaliana. Like in all other plant species, high levels of flavonols could be detected in pollen of wild-type A. thaliana. No flavonols were detected in reproductive organs of the A. thaliana tt4 mutant in which the chs gene is mutated. Surprisingly, this mutant did set seed after self-fertilization and no pollen tube growth aberrations were observed in vivo. The role of flavonols during fertilization of Arabidopsis is discussed.Abbreviations CHS chalcone synthase - TLC thin-layer chromatography     

Diacylglycerol pyrophosphate is a second messenger of abscisic acid signaling in Arabidopsis thaliana suspension cells

In plants, the importance of phospholipid signaling in responses to environmental stresses is becoming well documented. The involvement of phospholipids in abscisic acid (ABA) responses is also established. In a previous study, we demonstrated that the stimulation of phospholipase D (PLD) activity and plasma membrane anion currents by ABA were both required for RAB18 expression in Arabidopsis thaliana suspension cells. In this study, we show that the total lipids extracted from ABA-treated cells mimic ABA in activating plasmalemma anion currents and induction of RAB18 expression. Moreover, ABA evokes within 5 min a transient 1.7-fold increase in phosphatidic acid (PA) followed by a sevenfold increase in diacylglycerol pyrophosphate (DGPP) at 20 min. PA activated plasmalemma anion currents but was incapable of triggering RAB18 expression. By contrast, DGPP mimicked ABA on anion currents and was also able to stimulate RAB18 expression. Here we show the role of DGPP as phospholipid second messenger in ABA signaling.     

Integrity of intermediate filaments is associated with the development of acquired thermotolerance in 9L rat brain tumor cells

Withangulatin A (WA), a newly discovered withanolide isolated from an antitumor Chinese herb, has been shown to be a vimentin intermediate filament-targeting drug by using immunofluorescence microscopy. Together with cytochalasin D and colchicine, these drugs were employed to investigate the importance of vimentin intermediate filaments, actin filaments, and microtubules in the development of acquired thermotolerance in 9L rat brain tumor cells treated at 45°C for 15 min (priming heat-shock). Acquired thermotolerance was abrogated in cells incubated with WA before the priming heat-shock but it could be detected in cells treated with WA after the priming heat-shock. In contrast, cytochalasin D and colchicine do not interfere with the development of thermotolerance at all. The intracellular localizations of vimentin and the constitutive heat-shock protein70 (HSC70) in treated cells were examined by using immunofluorescence microscopy and detergent-extractability studies. In cells treated with WA before the priming heat-shock, vimentin IFs were tightly aggregated around the nucleus and unable to return to their normal organization after a recovery under normal growing conditions. In contrast, the IF network in cells treated with WA after the priming heat-shock was able to reorganize into filamentous form after a recovery period, a behavior similar to that of the cells treated with heat-shock only. HSC70 was found to be co-localized with vimentin during these changes. It is suggested that the integrity of intermediate filaments is important for the development of thermotolerance and that HSC70 may be involved in this process by stabilizing the intermediate filaments through direct or indirect binding.     

Salicylic acid is not a bacterial siderophore: a theoretical study

Using a newly available program for calculating the concentrations and speciation of various ions (Pettit, LD & Powell KJ, `SolEq' Academic Software, 1999), we have calculated that at pH 7 the amount of free Fe(III) present in an aqueous solution is 1.4×10^–9 M and not 10^–18 M as is usually quoted. In the presence of salicylic acid, included in the calculations at 10^–4 M, the solubility of Fe(III) is increased to only 9.8×10^–9 M suggesting that salicylate is unable to act as a siderophore although it is produced as an extracellular product by several bacterial genera when grown iron deficiently. In the presence of 40 mM phosphate, the soluble Fe(III) concentration is decreased by 10⁴ at pH 7 and again this is hardly affected by the presence of salicylate. Thus, for microorganisms grown either in vitro or in vivo, salicylate is unlikely to function as a iron solubilizing agent. The same conclusions may also apply to 2,3-dihydroxybenzoic acid.     

Resistance and biomass in Arabidopsis: a new model for Salicylic Acid perception

Salicylic acid (SA) is an essential hormone for plant defence and development. SA perception is usually measured by counting the number of pathogens that grow in planta upon an exogenous application of the hormone. A biological SA perception model based on plant fresh weight reduction caused by disease resistance in Arabidopsis thaliana is proposed. This effect is more noticeable when a chemical analogue of SA is used, like Benzothiadiazole (BTH). By spraying BTH several times, a substantial difference in plant biomass is observed when compared with the mock treatment. Such difference is dose‐dependent and does not require pathogen inoculation. The model is robust and allows for the comparison of different Arabidopsis ecotypes, recombinant inbreed lines, and mutants. Our results show that two mutants, non‐expresser of pathogenesis‐related genes 1 (npr1) and auxin resistant 3 (axr3), fail to lose biomass when BTH is applied to them. Further experiments show that axr3 responds to SA and BTH in terms of defence induction. NPR1‐related genotypes also confirm the pivotal role of NPR1 in SA perception, and suggest an active program of depletion of resources in the infected tissues.