Plasma Treatment Modifies Element Distribution in Seed Coating and Affects Further Germination and Plant Growth through Interaction with Soil Microbiome.

This study investigates the impact of plasma-seed interaction on germination and early plant development, focusing on Arabidopsis thaliana and Brassica napus. The investigation delves into changes in chemical composition, water absorption, and surface morphology induced by plasma filaments generated in synthetic air. These analyses were conducted using scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). Although plasma treatment enhanced water absorption and modified surface chemistry, its impact on germination demonstrated species- and context-dependent variations. Notably, the accelerated germination and morphogenesis of seedlings in microbiome-enriched (MB+) soil could be achieved also in microbiome-deprived (MB-) soil by short-term plasma treatment of seeds. Remarkably, the positive effects of plasma treatment on early developmental events (germination, morphogenesis) and later events (formation of inflorescences) were more pronounced in the context of MB- soil but were accompanied by a slight decrease in disease resistance, which was not detected in MB+ soil. The results underscore the intricate dynamics of plasma-plant interactions and stress the significance of accounting for the soil microbiome while designing experiments with potential field application.

DIACYLGLYCEROL KINASE 5 participates in flagellin-induced signaling in Arabidopsis.

Flagellin perception is a keystone of pattern-triggered immunity in plants. The recognition of this protein by a plasma membrane (PM) receptor complex is the beginning of a signaling cascade that includes protein phosphorylation and the production of reactive oxygen species (ROS). In both Arabidopsis (Arabidopsis thaliana) seedlings and suspension cells, we found that treatment with flg22, a peptide corresponding to the most conserved domain of bacterial flagellin, caused a rapid and transient decrease in the level of phosphatidylinositol (PI) 4,5-bisphosphate along with a parallel increase in phosphatidic acid (PA). In suspension cells, inhibitors of either phosphoinositide-dependent phospholipases C (PLC) or diacylglycerol kinases (DGKs) inhibited flg22-triggered PA production and the oxidative burst. In response to flg22, receptor-like kinase-deficient fls2, bak1, and bik1 mutants (FLAGELLIN SENSITIVE 2, BRASSINOSTEROID INSENSITIVE 1-associated kinase 1, and BOTRYTIS-INDUCED KINASE 1, respectively) produced less PA than wild-type (WT) plants, whereas this response did not differ in NADPH oxidase-deficient rbohD (RESPIRATORY BURST OXIDASE HOMOLOG D) plants. Among the DGK-deficient lines tested, the dgk5.1 mutant produced less PA and less ROS after flg22 treatment compared with WT seedlings. In response to flg22, dgk5.1 plants showed lower callose accumulation and impaired resistance to Pseudomonas syringae pv. tomato DC3000 hrcC-. Transcriptomics revealed that the basal expression of defense-related genes was altered in dgk5.1 seedlings compared with the WT. A GFP-DGK5 fusion protein localized to the PM, where RBOHD and PLC2 (proteins involved in plant immunity) are also located. The role of DGK5 and its enzymatic activity in flagellin signaling and fine-tuning of early immune responses in plant-microbe interactions is discussed.

Isolation and Characterization of Two Lytic Phages Efficient Against Phytopathogenic Bacteria From Pseudomonas and Xanthomonas Genera.

Pseudomonas syringae is a bacterial pathogen that causes yield losses in various economically important plant species. At the same time, P. syringae pv. tomato (Pst) is one of the best-studied bacterial phytopathogens and a popular model organism. In this study, we report on the isolation of two phages from the market-bought pepper fruit showing symptoms of bacterial speck. These Pseudomonas phages were named Eir4 and Eisa9 and characterized using traditional microbiological methods and whole-genome sequencing followed by various bioinformatics approaches. Both of the isolated phages were capable only of the lytic life cycle and were efficient against several pathovars from Pseudomonas and Xanthomonas genera. With the combination of transmission electron microscopy (TEM) virion morphology inspection and comparative genomics analyses, both of the phages were classified as members of the Autographiviridae family with different degrees of novelty within the known phage diversity. Eir4, but not Eisa9, phage application significantly decreased the propagation of Pst in the leaf tissues of Arabidopsis thaliana plants. The biological properties of Eir4 phage allow us to propose it as a potential biocontrol agent for use in the prevention of Pst-associated bacterioses and also as a model organism for the future research of mechanisms of phage-host interactions in different plant systems.

An Arabidopsis mutant deficient in phosphatidylinositol-4-phosphate kinases ß1 and ß2 displays altered auxin-related responses in roots.

Phosphatidylinositol 4-kinases (PI4Ks) are the first enzymes that commit phosphatidylinositol into the phosphoinositide pathway. Here, we show that Arabidopsis thaliana seedlings deficient in PI4Kß1 and ß2 have several developmental defects including shorter roots and unfinished cytokinesis. The pi4kß1ß2 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.

Specialist Aphid Feeding Causes Local Activation of Salicylic and Jasmonic Acid Signaling in Arabidopsis Veins.

Aphids, the phloem sap feeders, probe into leaf tissues and activate a complex network of plant defense responses. Phytohormonal signaling plays a major role in this network; however, the dynamics of the signal spreading is yet to be clarified. Despite the growing knowledge about transcriptomic changes upon infestation, results often differ due to sampling, varying strongly between the tissues collected at the single feeding site, individual leaves, pooled infested leaves, or whole plant rosettes. This study focuses on activation of salicylic acid (SA) and jasmonic acid (JA) signals in Arabidopsis leaves during infestation by cabbage aphid (Brevicoryne brassicae) in high spatio-temporal resolution. We used genetically encoded fluorescent biosensors, histochemistry, and quantitative reverse transcription-PCR to precisely map activation of distinct branches of phytohormonal signaling. We found a rapid induction of SA and JA signaling markers in cells surrounding stylet puncture, colocalizing with callose deposition. For both PR1 and JAZ10, we detected activation at 24 h postinfestation (hpi), increasing and spreading along the veins until 72 hpi and, to a lesser extent, within the epidermal pavement cells. The SA signaling wave appeared in parallel with JA-associated signaling and continued to increase in time. Our results first show a local activation of SA- and JA-related responses after stylet penetration of Arabidopsis leaves and bring a detailed insight into the spatio-temporal complexity of plant defense activation during specialist aphid attack.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

BODIPY Conjugate of Epibrassinolide as a Novel Biologically Active Probe for In Vivo Imaging.

Brassinosteroids (BRs) are plant hormones of steroid nature, regulating various developmental and adaptive processes. The perception, transport, and signaling of BRs are actively studied nowadays via a wide range of biochemical and genetic tools. However, most of the knowledge about BRs intracellular localization and turnover relies on the visualization of the receptors or cellular compartments using dyes or fluorescent protein fusions. We have previously synthesized a conjugate of epibrassinolide with green fluorescent dye BODIPY (eBL-BODIPY). Here we present a detailed assessment of the compound bioactivity and its suitability as probe for in vivo visualization of BRs. We show that eBL-BODIPY rapidly penetrates epidermal cells of Arabidopsis thaliana roots and after long exposure causes physiological and transcriptomic responses similar to the natural hormone.

Regulation of the microsomal proteome by salicylic acid and deficiency of phosphatidylinositol-4-kinases ß1 and ß2 in Arabidopsis thaliana.

Phosphatidylinositol-4-kinases ß1 and ß2 (PI4Kß1/PI4Kß2), which are responsible for phosphorylation of phosphatidylinositol to phosphatidylinositol-4-phosphate, have important roles in plant vesicular trafficking. Moreover, PI4Kß1/PI4Kß2 negatively regulates biosynthesis of phytohormone salicylic acid (SA), a key player in plant immune responses. The study focused on the effect of PI4Kß1/PI4Kß2 deficiency and SA level on the proteome of microsomal fraction. For that purpose we used four Arabidopsis thaliana genotypes: wild type; double mutant with impaired function of PI4Kß1/PI4Kß2 (pi4kß1/pi4kß2) exhibiting high SA level; sid2 mutant with impaired SA biosynthesis depending on the isochorismate synthase 1 and triple mutant sid2/pi4kß1/pi4kß2. We identified 1797 proteins whose levels were changed between genotypes. We showed that increased SA concentration affected the levels of 473 proteins. This includes typical SA pathway markers but also points to connections between SA pathway and clathrin-independent endocytosis (flotillins) and exocytosis/protein secretion (syntaxins, tetraspanin) to be investigated in future. In contrast to SA, the absence of PI4Kß1/PI4Kß2 itself affected only 27 proteins. Among them we identified CERK1, a receptor for chitin. Although PI4Kß1/PI4Kß2 deficiency itself did not have a substantial impact on the proteome of the microsomal fraction, our data clearly show that it enhances proteome changes when SA pathway is modulated in parallel.

Brassinosteroids application induces phosphatidic acid production and modify antioxidant enzymes activity in tobacco in calcium-dependent manner.

Brassinosteroids (BRs) are steroid hormones regulating various aspects of plant metabolism, including growth, development and stress responses. However, little is known about the mechanism of their impact on antioxidant systems and phospholipid turnover. Using tobacco plants overexpressing H+/Ca2+vacuolar Arabidopsis antiporter CAX1, we showed the role of Ca2+ ion balance in the reactive oxygen species production and rapid phosphatidic acid accumulation induced by exogenous BR. Combination of our experimental results with public transcriptomic and proteomic data revealed a particular role of Ca2+-dependent phospholipid metabolizing enzymes in BR signaling. Here we provide novel insights into the role of calcium balance and lipid-derived second messengers in plant responses to exogenous BRs and propose a complex model integrating BR-mediated metabolic changes with phospholipid turnover.

Disrupted actin: a novel player in pathogen attack sensing?

The actin cytoskeleton is widely involved in plant immune responses. The majority of studies show that chemical disruption of the actin cytoskeleton increases plant susceptibility to pathogen infection. Similarly, several pathogens have adopted this as a virulence strategy and produce effectors that affect cytoskeleton integrity. Such effectors either exhibit actin-depolymerizing activity themselves or prevent actin polymerization. Is it thus possible for plants to recognize the actin's status and launch a counterattack? Recently we showed that chemical depolymerization of actin filaments can trigger resistance to further infection via the specific activation of salicylic acid (SA) signalling. This is accompanied by several defence-related, but SA-independent, effects (e.g. callose deposition, gene expression), relying on vesicular trafficking and phospholipid metabolism. These data suggest that the role of actin in plant-pathogen interactions is more complex than previously believed. It raises the question of whether plants have evolved a mechanism of sensing pathological actin disruption that eventually triggers defence responses. If so, what is the molecular basis of it? Otherwise, why does actin depolymerization specifically influence SA content but not any other phytohormone? Here we propose an updated model of actin's role in plant-microbe interactions and suggest some future directions of research to be conducted in this area.

Identification of salicylic acid-independent responses in an Arabidopsis phosphatidylinositol 4-kinase beta double mutant.

BACKGROUND AND AIMS: We have recently shown that an Arabidopsis thaliana double mutant of type III phosphatidylinositol-4-kinases (PI4Ks), pi4kß1ß2, constitutively accumulated a high level of salicylic acid (SA). By crossing this pi4kß1ß2 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 PI4Kß1 and PI4Kß2. METHODS: To achieve this we used the sid2pi4kß1ß2 triple mutant. High-throughput analyses of phytohormones were performed on this mutant together with pi4kß1ß2 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 pi4kß1ß2 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 pi4kß1ß2 after flagellin treatment was independent of SA. Concerning the response to Blumeria graminis, both callose accumulation and fungal penetration were enhanced in the pi4kß1ß2 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 sid2pi4kß1ß2 triple mutant revealed the role of PI4Kß1/ß2 per se, thus showing the importance of these enzymes in plant defence responses.

"Salicylic Acid Mutant Collection" as a Tool to Explore the Role of Salicylic Acid in Regulation of Plant Growth under a Changing Environment.

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 pi4kß1ß2) 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.

Actin depolymerization is able to increase plant resistance against pathogens via activation of salicylic acid signalling pathway.

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.

Diacylglycerol kinases activate tobacco NADPH oxidase-dependent oxidative burst in response to cryptogein.

Cryptogein is a 10 kDa protein secreted by the oomycete Phytophthora cryptogea that activates defence mechanisms in tobacco plants. Among early signalling events triggered by this microbial-associated molecular pattern is a transient apoplastic oxidative burst which is dependent on the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity of the RESPIRATORY BURST OXIDASE HOMOLOG isoform D (RBOHD). Using radioactive [33 P]-orthophosphate labelling of tobacco Bright Yellow-2 suspension cells, we here provide in vivo evidence for a rapid accumulation of phosphatidic acid (PA) in response to cryptogein because of the coordinated onset of phosphoinositide-dependent phospholipase C and diacylglycerol kinase (DGK) activities. Both enzyme specific inhibitors and silencing of the phylogenetic cluster III of the tobacco DGK family were found to reduce PA production upon elicitation and to strongly decrease the RBOHD-mediated oxidative burst. Therefore, it appears that PA originating from DGK controls NADPH-oxidase activity. Amongst cluster III DGKs, the expression of DGK5-like was up-regulated in response to cryptogein. Besides DGK5-like is likely to be the main cluster III DGK isoform silenced in one of our mutant lines, making it a strong candidate for the observed response to cryptogein. The relevance of these results is discussed with regard to early signalling lipid-mediated events in plant immunity.

Importance of phosphoinositide-dependent signaling pathways in the control of gene expression in resting cells and in response to phytohormones.

"Phosphoinositide" refers to phosphorylated forms of phosphatidylinositol, including phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate. Both of these molecules could be in vivo substrates of plant phospholipase C. These phosphoinositides can also be biologically active "per se," by directly binding to proteins and thus altering their location and/or activity. The use of pharmacological agents in Arabidopsis suspension cells allowed us to identify genes whose expression was positively or negatively controlled, in the basal state, by products of phosphoinositide-dependent phospholipase C. In this basal state, it seems that no genes exhibit a phosphoinositide-dependent expression "per se." However, many genes whose expression is altered in the presence of phospholipase C inhibitors appeared to be responsive to salicylic acid. This allowed us to show that salicylic acid acts both by increasing the phosphoinositide pool and by inhibiting the phospholipase C. In response to salicylic acid it is possible to identify genes whose expression is controlled by products of PI-PLC, but also genes whose expression is controlled by phosphoinositides "per se." Our data highlight the importance of phosphoinositide-dependent pathways in gene expression in resting cells and in response to phytohormones.

Involvement of phospholipase D and NADPH-oxidase in salicylic acid signaling cascade.

Salicylic acid is associated with the primary defense responses to biotic stress and formation of systemic acquired resistance. However, molecular mechanisms of early cell reactions to phytohormone application are currently undisclosed. The present study investigates the participation of phospholipase D and NADPH-oxidase in salicylic acid signal transduction cascade. The activation of lipid signaling enzymes within 15 min of salicylic acid application was shown in Arabidopsis thaliana plants by measuring the phosphatidic acid accumulation. Adding of primary alcohol (1-butanol) to the incubation medium led to phosphatidylbutanol accumulation as a result of phospholipase D (PLD) action in wild-type and NADPH-oxidase RbohD deficient plants. Salicylic acid induced rapid increase in NADPH-oxidase activity in histochemical assay with nitroblue tetrazolium but the reaction was not observed in presence of 1-butanol and NADPH-oxidase inhibitor diphenylene iodide (DPI). The further physiological effect of salicylic acid and inhibitory analysis of the signaling cascade were made in the guard cell model. Stomatal closure induced by salicylic acid was inhibited by 1-butanol and DPI treatment. rbohD transgenic plants showed impaired stomatal reaction upon phytohormone effect, while the reaction to H2O2 did not differ from that of wild-type plants. Thus a key role of NADPH-oxidase D-isoform in the process of stomatal closure in response to salicylic acid has been postulated. It has enabled to predict a cascade implication of PLD and NADPH oxidase to salicylic acid signaling pathway.