Phosphatidylinositol 4-kinases (PI4Ks) are the first enzymes that commit phosphatidylinositol into the phosphoinositide pathway. Here, we show that Arabidopsis thaliana seedlings deficient in PI4Kβ1 and β2 have several developmental defects including shorter roots and unfinished cytokinesis. The pi4kβ1β2 double mutant was insensitive to exogenous auxin concerning inhibition of root length and cell elongation; it also responded more slowly to gravistimulation. The pi4kß1ß2 root transcriptome displayed some similarities to a wild type plant response to auxin. Yet, not all the genes displayed such a constitutive auxin-like response. Besides, most assessed genes did not respond to exogenous auxin. This is consistent with data with the transcriptional reporter DR5-GUS. The content of bioactive auxin in the pi4kß1ß2 roots was similar to that in wild-type ones. Yet, an enhanced auxin-conjugating activity was detected and the auxin level reporter DII-VENUS did not respond to exogenous auxin in pi4kß1ß2 mutant. The mutant exhibited altered subcellular trafficking behavior including the trapping of PIN-FORMED 2 protein in rapidly moving vesicles. Bigger and less fragmented vacuoles were observed in pi4kß1ß2 roots when compared to the wild type. Furthermore, the actin filament web of the pi4kß1ß2 double mutant was less dense than in wild-type seedling roots, and less prone to rebuilding after treatment with latrunculin B. A mechanistic model is proposed in which an altered PI4K activity leads to actin filament disorganization, changes in vesicle trafficking, and altered auxin homeostasis and response resulting in a pleiotropic root phenotypes.

• MeSH
• Arabidopsis * metabolism   MeSH
• Phosphatidylinositol Phosphates MeSH
• Phosphatidylinositols metabolism   MeSH
• Plant Roots genetics   metabolism   MeSH
• Indoleacetic Acids metabolism   MeSH
• Arabidopsis Proteins * genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Phosphatidylinositol Phosphates MeSH
• Phosphatidylinositols MeSH
• Indoleacetic Acids MeSH
• phosphatidylinositol 4-phosphate MeSH Browser
• Arabidopsis Proteins * MeSH

Phosphoinositides are low-abundant lipids that participate in the acquisition of membrane identity through their spatiotemporal enrichment in specific compartments. Phosphatidylinositol 4-phosphate (PI4P) accumulates at the plant plasma membrane driving its high electrostatic potential, and thereby facilitating interactions with polybasic regions of proteins. PI4Kα1 has been suggested to produce PI4P at the plasma membrane, but how it is recruited to this compartment is unknown. Here, we pin-point the mechanism that tethers Arabidopsis thaliana phosphatidylinositol 4-kinase alpha1 (PI4Kα1) to the plasma membrane via a nanodomain-anchored scaffolding complex. We established that PI4Kα1 is part of a complex composed of proteins from the NO-POLLEN-GERMINATION, EFR3-OF-PLANTS, and HYCCIN-CONTAINING families. Comprehensive knockout and knockdown strategies revealed that subunits of the PI4Kα1 complex are essential for pollen, embryonic, and post-embryonic development. We further found that the PI4Kα1 complex is immobilized in plasma membrane nanodomains. Using synthetic mis-targeting strategies, we demonstrate that a combination of lipid anchoring and scaffolding localizes PI4Kα1 to the plasma membrane, which is essential for its function. Together, this work opens perspectives on the mechanisms and function of plasma membrane nanopatterning by lipid kinases.

• MeSH
• Arabidopsis enzymology   genetics   MeSH
• Phosphotransferases (Alcohol Group Acceptor) genetics   metabolism   MeSH
• Matrix Attachment Regions * MeSH
• Arabidopsis Proteins genetics   metabolism   MeSH
• Minor Histocompatibility Antigens genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Phosphotransferases (Alcohol Group Acceptor) MeSH
• phosphatidylinositol phosphate 4-kinase MeSH Browser
• Arabidopsis Proteins MeSH
• Minor Histocompatibility Antigens MeSH

BACKGROUND AND AIMS: We have recently shown that an Arabidopsis thaliana double mutant of type III phosphatidylinositol-4-kinases (PI4Ks), pi4kβ1β2, constitutively accumulated a high level of salicylic acid (SA). By crossing this pi4kβ1β2 double mutant with mutants impaired in SA synthesis (such as sid2 impaired in isochorismate synthase) or transduction, we demonstrated that the high SA level was responsible for the dwarfism phenotype of the double mutant. Here we aimed to distinguish between the SA-dependent and SA-independent effects triggered by the deficiency in PI4Kβ1 and PI4Kβ2. METHODS: To achieve this we used the sid2pi4kβ1β2 triple mutant. High-throughput analyses of phytohormones were performed on this mutant together with pi4kβ1β2 and sid2 mutants and wild-type plants. Responses to pathogens, namely Hyaloperonospora arabidopsidis, Pseudomonas syringae and Botrytis cinerea, and also to the non-host fungus Blumeria graminis, were also determined. Callose accumulation was monitored in response to flagellin. KEY RESULTS: We show here the prominent role of high SA levels in influencing the concentration of many other tested phytohormones, including abscisic acid and its derivatives, the aspartate-conjugated form of indole-3-acetic acid and some cytokinins such as cis-zeatin. We show that the increased resistance of pi4kβ1β2 plants to the host pathogens H. arabidopsidis, P. syringae pv. tomato DC3000 and Bothrytis cinerea is dependent on accumulation of high SA levels. In contrast, accumulation of callose in pi4kβ1β2 after flagellin treatment was independent of SA. Concerning the response to Blumeria graminis, both callose accumulation and fungal penetration were enhanced in the pi4kβ1β2 double mutant compared to wild-type plants. Both of these processes occurred in an SA-independent manner. CONCLUSIONS: Our data extensively illustrate the influence of SA on other phytohormone levels. The sid2pi4kβ1β2 triple mutant revealed the role of PI4Kβ1/β2 per se, thus showing the importance of these enzymes in plant defence responses.

• Keywords
• Arabidopsis thaliana, biotic stress, callose, isochorismate synthase 1, pathogens, phytohormones, pi4kβ1β2/PI4Ks, salicylic acid,
• MeSH
• 1-Phosphatidylinositol 4-Kinase * MeSH
• Arabidopsis * MeSH
• Salicylic Acid MeSH
• Mutation MeSH
• Plant Diseases MeSH
• Arabidopsis Proteins genetics   MeSH
• Pseudomonas syringae MeSH
• Gene Expression Regulation, Plant MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 1-Phosphatidylinositol 4-Kinase * MeSH
• AT5G09350 protein, Arabidopsis MeSH Browser
• AT5G64070 protein, Arabidopsis MeSH Browser
• Salicylic Acid MeSH
• Arabidopsis Proteins MeSH

The phytohormone salicylic acid (SA) has a crucial role in plant physiology. Its role is best described in the context of plant response to pathogen attack. During infection, SA is rapidly accumulated throughout the green tissues and is important for both local and systemic defences. However, some genetic/metabolic variations can also result in SA overaccumulation in plants, even in basal conditions. To date, more than forty Arabidopsis thaliana mutants have been described as having enhanced endogenous SA levels or constitutively activated SA signalling pathways. In this study, we established a collection of mutants containing different SA levels due to diverse genetic modifications and distinct gene functions. We chose prototypic SA-overaccumulators (SA-OAs), such as bon1-1, but also "non-typical" ones such as exo70b1-1; the selection of OA is accompanied by their crosses with SA-deficient lines. Here, we extensively studied the plant development and SA level/signalling under various growth conditions in soil and in vitro, and showed a strong negative correlation between rosette size, SA content and PR1/ICS1 transcript signature. SA-OAs (namely cpr5, acd6, bon1-1, fah1/fah2 and pi4kβ1β2) had bigger rosettes under high light conditions, whereas WT plants did not. Our data provide new insights clarifying a link between SA and plant behaviour under environmental stresses. The presented SA mutant collection is thus a suitable tool to shed light on the mechanisms underlying trade-offs between growth and defence in plants.

• Keywords
• Arabidopsis mutants, Salicylic acid, gene transcription, growth, light,
• MeSH
• Arabidopsis genetics   metabolism   microbiology   MeSH
• Host-Pathogen Interactions MeSH
• Salicylic Acid metabolism   MeSH
• Mutation * MeSH
• Plant Diseases genetics   microbiology   MeSH
• Arabidopsis Proteins genetics   metabolism   MeSH
• Gene Expression Regulation, Plant * MeSH
• Plant Growth Regulators metabolism   MeSH
• Signal Transduction genetics   MeSH
• Plant Development genetics   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Salicylic Acid MeSH
• Arabidopsis Proteins MeSH
• Plant Growth Regulators MeSH

The integrity of the actin cytoskeleton is essential for plant immune signalling. Consequently, it is generally assumed that actin disruption reduces plant resistance to pathogen attack. Here, we demonstrate that actin depolymerization induced a dramatic increase in salicylic acid (SA) levels in Arabidopsis thaliana. Transcriptomic analysis showed that the SA pathway was activated due to the action of isochorismate synthase (ICS). The effect was also confirmed in Brassica napus. This raises the question of whether actin depolymerization could, under particular conditions, lead to increased resistance to pathogens. Thus, we explored the effect of pretreatment with actin-depolymerizing drugs on the resistance of Arabidopsis thaliana to the bacterial pathogen Pseudomonas syringae, and on the resistance of an important crop Brassica napus to its natural fungal pathogen Leptosphaeria maculans. In both pathosystems, actin depolymerization activated the SA pathway, leading to increased plant resistance. To our best knowledge, we herein provide the first direct evidence that disruption of the actin cytoskeleton can actually lead to increased plant resistance to pathogens, and that SA is crucial to this process.

• MeSH
• Actins metabolism   MeSH
• Arabidopsis metabolism   microbiology   MeSH
• Ascomycota pathogenicity   MeSH
• Brassica napus metabolism   microbiology   MeSH
• Intramolecular Transferases metabolism   MeSH
• Salicylic Acid metabolism   MeSH
• Plant Diseases microbiology   MeSH
• Arabidopsis Proteins metabolism   MeSH
• Pseudomonas syringae pathogenicity   MeSH
• Gene Expression Regulation, Plant physiology   MeSH
• Signal Transduction physiology   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Actins MeSH
• Intramolecular Transferases MeSH
• isochorismate synthase MeSH Browser
• Salicylic Acid MeSH
• Arabidopsis Proteins MeSH

BACKGROUND AND AIMS: The non-specific phospholipase C (NPC) is a new member of the plant phospholipase family that reacts to abiotic environmental stresses, such as phosphate deficiency, high salinity, heat and aluminium toxicity, and is involved in root development, silicon distribution and brassinolide signalling. Six NPC genes (NPC1-NPC6) are found in the Arabidopsis genome. The NPC2 isoform has not been experimentally characterized so far. METHODS: The Arabidopsis NPC2 isoform was cloned and heterologously expressed in Escherichia coli. NPC2 enzyme activity was determined using fluorescent phosphatidylcholine as a substrate. Tissue expression and subcellular localization were analysed using GUS- and GFP-tagged NPC2. The expression patterns of NPC2 were analysed via quantitative real-time PCR. Independent homozygous transgenic plant lines overexpressing NPC2 under the control of a 35S promoter were generated, and reactive oxygen species were measured using a luminol-based assay. KEY RESULTS: The heterologously expressed protein possessed phospholipase C activity, being able to hydrolyse phosphatidylcholine to diacylglycerol. NPC2 tagged with GFP was predominantly localized to the Golgi apparatus in Arabidopsis roots. The level of NPC2 transcript is rapidly altered during plant immune responses and correlates with the activation of multiple layers of the plant defence system. Transcription of NPC2 decreased substantially after plant infiltration with Pseudomonas syringae, flagellin peptide flg22 and salicylic acid treatments and expression of the effector molecule AvrRpm1. The decrease in NPC2 transcript levels correlated with a decrease in NPC2 enzyme activity. NPC2-overexpressing mutants showed higher reactive oxygen species production triggered by flg22. CONCLUSIONS: This first experimental characterization of NPC2 provides new insights into the role of the non-specific phospholipase C protein family. The results suggest that NPC2 is involved in the response of Arabidopsis to P. syringae attack.

• Keywords
• Arabidopsis thaliana, MAMP-triggered immunity, Pseudomonas syringae, effector-triggered immunity, flagellin, non-specific phospholipase C, phosphatidylcholine-specific phospholipase C, reactive oxygen species,
• MeSH
• Arabidopsis enzymology   immunology   microbiology   MeSH
• Phosphatidylcholines metabolism   MeSH
• Type C Phospholipases genetics   physiology   MeSH
• Golgi Apparatus enzymology   MeSH
• Plant Immunity physiology   MeSH
• Cloning, Molecular MeSH
• Microscopy, Confocal MeSH
• Real-Time Polymerase Chain Reaction MeSH
• Plant Diseases immunology   microbiology   MeSH
• Arabidopsis Proteins genetics   physiology   MeSH
• Protoplasts enzymology   MeSH
• Pseudomonas syringae * MeSH
• Reactive Oxygen Species MeSH
• Gene Expression Regulation, Plant MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Phosphatidylcholines MeSH
• Type C Phospholipases MeSH
• NPC2 protein, Arabidopsis MeSH Browser
• Arabidopsis Proteins MeSH
• Reactive Oxygen Species MeSH

To achieve host colonization, successful pathogens need to overcome plant basal defences. For this, (hemi)biotrophic pathogens secrete effectors that interfere with a range of physiological processes of the host plant. AvrLm4-7 is one of the cloned effectors from the hemibiotrophic fungus Leptosphaeria maculans 'brassicaceae' infecting mainly oilseed rape (Brassica napus). Although its mode of action is still unknown, AvrLm4-7 is strongly involved in L. maculans virulence. Here, we investigated the effect of AvrLm4-7 on plant defence responses in a susceptible cultivar of B. napus. Using two isogenic L. maculans isolates differing in the presence of a functional AvrLm4-7 allele [absence ('a4a7') and presence ('A4A7') of the allele], the plant hormone concentrations, defence-related gene transcription and reactive oxygen species (ROS) accumulation were analysed in infected B. napus cotyledons. Various components of the plant immune system were affected. Infection with the 'A4A7' isolate caused suppression of salicylic acid- and ethylene-dependent signalling, the pathways regulating an effective defence against L. maculans infection. Furthermore, ROS accumulation was decreased in cotyledons infected with the 'A4A7' isolate. Treatment with an antioxidant agent, ascorbic acid, increased the aggressiveness of the 'a4a7' L. maculans isolate, but not that of the 'A4A7' isolate. Together, our results suggest that the increased aggressiveness of the 'A4A7' L. maculans isolate could be caused by defects in ROS-dependent defence and/or linked to suppressed SA and ET signalling. This is the first study to provide insights into the manipulation of B. napus defence responses by an effector of L. maculans.

• Keywords
• AvrLm4-7, Brassica napus, Leptosphaeria, ROS, effector, ethylene, salicylic acid,
• MeSH
• Alleles MeSH
• Antioxidants pharmacology   MeSH
• Ascomycota drug effects   isolation & purification   metabolism   MeSH
• Brassica napus drug effects   growth & development   metabolism   microbiology   MeSH
• Chromatography, Liquid MeSH
• Cyclopentanes metabolism   MeSH
• Ethylenes metabolism   MeSH
• Fungal Proteins metabolism   MeSH
• Mass Spectrometry MeSH
• Host-Pathogen Interactions drug effects   MeSH
• Cotyledon drug effects   metabolism   microbiology   MeSH
• Abscisic Acid metabolism   MeSH
• Ascorbic Acid pharmacology   MeSH
• Salicylic Acid metabolism   MeSH
• Oxylipins metabolism   MeSH
• Hydrogen Peroxide metabolism   MeSH
• Reverse Transcriptase Polymerase Chain Reaction MeSH
• Plant Growth Regulators metabolism   MeSH
• Signal Transduction * drug effects   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antioxidants MeSH
• Cyclopentanes MeSH
• Ethylenes MeSH
• Fungal Proteins MeSH
• jasmonic acid MeSH Browser
• Abscisic Acid MeSH
• Ascorbic Acid MeSH
• Salicylic Acid MeSH
• Oxylipins MeSH
• Hydrogen Peroxide MeSH
• Plant Growth Regulators MeSH

Even though resistance (R) genes are among the most studied components of the plant immunity, there remain still a lot of aspects to be explained about the regulation of their function. Many gain-of-function mutants of R genes and loss-of-function of their regulators often demonstrate up-regulated defense responses in combination with dwarf stature and/or spontaneous leaf lesions formation. For most of these mutants, phenotypes are a consequence of an ectopic activation of R genes. Based on the compilation and comparison of published results in this field, we have concluded that the constitutively activated defense phenotypes recurrently arise by disruption of tight, constitutive and multilevel negative control of some of R proteins that might involve also their targeting to the autophagy pathway. This mode of R protein regulation is supported also by protein-protein interactions listed in available databases, as well as in silico search for autophagy machinery interacting motifs. The suggested model could resolve some explanatory discrepancies found in the studies of the immunity responses of autophagy mutants.

• Keywords
• Avr, ETI, R, autophagy, dwarf, exocyst, lesions, resistance,
• Publication type
• Journal Article MeSH

BACKGROUND: The use of light emitting diodes (LEDs) brings several key advantages over existing illumination technologies for indoor plant cultivation. Among these are that LEDs have predicted lifetimes from 50-100.000 hours without significant drops in efficiency and energy consumption is much lower compared to traditional fluorescent tubes. Recent advances allow LEDs to be used with customized wavelengths for plant growth. However, most of these LED growth systems use mixtures of chips emitting in several narrow wavelengths and frequently they are not compatible with existing infrastructures. This study tested the growth of five different plant species under phosphor coated LED-chips fitted into a tube with a standard G13 base that provide continuous visible light illumination with enhanced blue and red light. RESULTS: The LED system was characterized and compared with standard fluorescence tubes in the same cultivation room. Significant differences in heat generation between LEDs and fluorescent tubes were clearly demonstrated. Also, LED lights allowed for better control and stability of preset conditions. Physiological properties such as growth characteristics, biomass, and chlorophyll content were measured and the responses to pathogen assessed for five plant species (both the model plants Arabidopsis thaliana, Nicotiana bentamiana and crop species potato, oilseed rape and soybean) under the different illumination sources. CONCLUSIONS: We showed that polychromatic LEDs provide light of sufficient quality and intensity for plant growth using less than 40% of the electricity required by the standard fluorescent lighting under test. The tested type of LED installation provides a simple upgrade pathway for existing infrastructure for indoor plant growth. Interestingly, individual plant species responded differently to the LED lights so it would be reasonable to test their utility to any particular application.

• Keywords
• Arabidopsis thaliana, Fluorescent tubes, LED, Light, Nicotiana bentamiana, Oilseed rape, Plant physiology, Potato, Soybean,
• Publication type
• Journal Article MeSH

Phytohormone salicylic acid (SA) is a crucial component of plant-induced defense against biotrophic pathogens. Although the key players of the SA pathway are known, there are still gaps in the understanding of the molecular mechanism and the regulation of particular steps. In our previous research, we showed in Arabidopsis suspension cells that n-butanol, which specifically modulates phospholipase D activity, significantly suppresses the transcription of the pathogenesis related (PR-1) gene, which is generally accepted as the SA pathway marker. In the presented study, we have investigated the site of n-butanol action in the SA pathway. We were able to show in Arabidopsis plants treated with SA that n-butanol inhibits the transcription of defense genes (PR-1, WRKY38). Fluorescence microscopy of Arabidopsis thaliana mutants expressing 35S::NPR1-GFP (nonexpressor pathogenesis related 1) revealed significantly decreased nuclear localization of NPR1 in the presence of n-butanol. On the other hand, n-butanol did not decrease the nuclear localization of NPR1 in 35S::npr1C82A-GFP and 35S::npr1C216A-GFP mutants constitutively expressing NPR1 monomers. Mass spectrometric analysis of plant extracts showed that n-butanol significantly changes the metabolic fingerprinting while t-butanol had no effect. We found groups of the plant metabolites, influenced differently by SA and n-butanol treatment. Thus, we proposed several metabolites as markers for n-butanol action.

• Keywords
• NPR1, PR-1, metabolome, n-butanol, phospholipase D, salicylic acid, signaling,
• Publication type
• Journal Article MeSH