A novel workflow for unbiased quantification of autophagosomes in 3D in Arabidopsis thaliana roots.

Macroautophagy is often quantified by live imaging of autophagosomes labeled with fluorescently tagged ATG8 protein (FP-ATG8) in Arabidopsis thaliana. The labeled particles are then counted in single focal planes. This approach may lead to inaccurate results as the actual 3D distribution of autophagosomes is not taken into account and appropriate sampling in the Z-direction is not performed. To overcome this issue, we developed a workflow consisting of immunolabeling of autophagosomes with an anti-ATG8 antibody followed by stereological image analysis using the optical disector and the Cavalieri principle. Our protocol specifically recognized autophagosomes in epidermal cells of Arabidopsis root. Since the anti-ATG8 antibody recognizes multiple AtATG8 isoforms, we were able to detect a higher number of immunolabeled autophagosomes than with the FP-AtATG8e marker, that most likely does not recognize all autophagosomes in a cell. The number of autophagosomes per tissue volume correlated with the intensity of autophagy induction. Compared to the quantification of autophagosomes in maximum intensity projections, stereological methods were able to detect the autophagosomes present in a given volume with higher accuracy. Our novel workflow provides a powerful toolkit for unbiased and reproducible quantification of autophagosomes and offers a convenient alternative to the standard of live imaging with FP-ATG8 markers.

Phosphatidic Acid in Plant Hormonal Signaling: From Target Proteins to Membrane Conformations.

Cells sense a variety of extracellular signals balancing their metabolism and physiology according to changing growth conditions. Plasma membranes are the outermost informational barriers that render cells sensitive to regulatory inputs. Membranes are composed of different types of lipids that play not only structural but also informational roles. Hormones and other regulators are sensed by specific receptors leading to the activation of lipid metabolizing enzymes. These enzymes generate lipid second messengers. Among them, phosphatidic acid (PA) is a well-known intracellular messenger that regulates various cellular processes. This lipid affects the functional properties of cell membranes and binds to specific target proteins leading to either genomic (affecting transcriptome) or non-genomic responses. The subsequent biochemical, cellular and physiological reactions regulate plant growth, development and stress tolerance. In the present review, we focus on primary (genome-independent) signaling events triggered by rapid PA accumulation in plant cells and describe the functional role of PA in mediating response to hormones and hormone-like regulators. The contributions of individual lipid signaling enzymes to the formation of PA by specific stimuli are also discussed. We provide an overview of the current state of knowledge and future perspectives needed to decipher the mode of action of PA in the regulation of cell functions.

Cell wall contributes to the stability of plasma membrane nanodomain organization of Arabidopsis thaliana FLOTILLIN2 and HYPERSENSITIVE INDUCED REACTION1 proteins.

Current models of plasma membrane (PM) postulate its organization in various nano- and micro-domains with distinct protein and lipid composition. While metazoan PM nanodomains usually display high lateral mobility, the dynamics of plant nanodomains is often highly spatially restricted. Here we have focused on the determination of the PM distribution in nanodomains for Arabidopsis thaliana flotillin (AtFLOT) and hypersensitive induced reaction proteins (AtHIR), previously shown to be involved in response to extracellular stimuli. Using in vivo laser scanning and spinning disc confocal microscopy in Arabidopsis thaliana we present here their nanodomain localization in various epidermal cell types. Fluorescence recovery after photobleaching (FRAP) and kymographic analysis revealed that PM-associated AtFLOTs contain significantly higher immobile fraction than AtHIRs. In addition, much lower immobile fractions have been found in tonoplast pool of AtHIR3. Although members of both groups of proteins were spatially restricted in their PM distribution by corrals co-aligning with microtubules (MTs), pharmacological treatments showed no or very low role of actin and microtubular cytoskeleton for clustering of AtFLOT and AtHIR into nanodomains. Finally, pharmacological alteration of cell wall (CW) synthesis and structure resulted in changes in lateral mobility of AtFLOT2 and AtHIR1. Accordingly, partial enzymatic CW removal increased the overall dynamics as well as individual nanodomain mobility of these two proteins. Such structural links to CW could play an important role in their correct positioning during PM communication with extracellular environment.

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.

Phospholipase Dα1 mediates the high-Mg2+ stress response partially through regulation of K+ homeostasis.

Intracellular levels of Mg2+ are tightly regulated, as Mg2+ deficiency or excess affects normal plant growth and development. In Arabidopsis, we determined that phospholipase Dα1 (PLDα1) is involved in the stress response to high-magnesium conditions. The T-DNA insertion mutant pldα1 is hypersensitive to increased concentrations of magnesium, exhibiting reduced primary root length and fresh weight. PLDα1 activity increases rapidly after high-Mg2+ treatment, and this increase was found to be dose dependent. Two lines harbouring mutations in the HKD motif, which is essential for PLDα1 activity, displayed the same high-Mg2+ hypersensitivity of pldα1 plants. Moreover, we show that high concentrations of Mg2+ disrupt K+ homeostasis, and that transcription of K+ homeostasis-related genes CIPK9 and HAK5 is impaired in pldα1. Additionally, we found that the akt1, hak5 double mutant is hypersensitive to high-Mg2+ . We conclude that in Arabidopsis, the enzyme activity of PLDα1 is vital in the response to high-Mg2+ conditions, and that PLDα1 mediates this response partially through regulation of K+ homeostasis.

The Arabidopsis thaliana non-specific phospholipase C2 is involved in the response to Pseudomonas syringae attack.

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.

Phosphoglycerolipids are master players in plant hormone signal transduction.

Phosphoglycerolipids are essential structural constituents of membranes and some also have important cell signalling roles. In this review, we focus on phosphoglycerolipids that are mediators in hormone signal transduction in plants. We first describe the structures of the main signalling phosphoglycerolipids and the metabolic pathways that generate them, namely the phospholipase and lipid kinase pathways. In silico analysis of Arabidopsis transcriptome data provides evidence that the genes encoding the enzymes of these pathways are transcriptionally regulated in responses to hormones, suggesting some link with hormone signal transduction. The involvement of phosphoglycerolipid signalling in the early responses to abscisic acid, salicylic acid and auxins is then detailed. One of the most important signalling lipids in plants is phosphatidic acid. It can activate or inactivate protein kinases and/or protein phosphatases involved in hormone signalling. It can also activate NADPH oxidase leading to the production of reactive oxygen species. We will interrogate the mechanisms that allow the activation/deactivation of the lipid pathways, in particular the roles of G proteins and calcium. Mediating lipids thus appear as master players of cell signalling, modulating, if not controlling, major transducing steps of hormone signals.

Influence of Exogenous 24-Epicasterone on the Hormonal Status of Soybean Plants.

Brassinosteroids (BRs) are key phytohormones involved in the regulation of major processes of cell metabolism that guide plant growth. In the past decades, new evidence has made it clear that BRs also play a key role in the orchestration of plant responses to many abiotic and biotic stresses. In the present work, we analyzed the impact of foliar treatment with 24-epicastasterone (ECS) on the endogenous content of major phytohormones (auxins, salicylic acid, jasmonic acid, and abscisic acid) and their intermediates in soybean leaves 7 days following the treatment. Changes in the endogenous content of phytohormones have been identified and quantified by LC/MS. The obtained results point to a clear role of ECS in the upregulation of auxin content (indole-3-acetic acid, IAA) and downregulation of salicylic, jasmonic, and abscisic acid levels. These data confirm that under optimal conditions, ECS in tested concentrations of 0.25 µM and 1 µM might promote growth in soybeans by inducing auxin contents. Benzoic acid (a precursor of salicylic acid (SA)), but not SA itself, has also been highly accumulated under ECS treatment, which indicates an activation of the adaptation strategies of cell metabolism to possible environmental challenges.

Noble metal nanoparticles in agriculture: impacts on plants, associated microorganisms, and biotechnological practices.

Within the past decades, nanoparticles (NPs) have become common components of electronics, batteries, cosmetics, clothing, and even dietary supplements. Despite their undisputed advantages consisting in the possibility of engineering their novel physical, thermal, optical, and biological properties, safety questions arise concerning their wide exploitation. NPs interact with living organisms, which can interfere with essential life processes. The aim of this paper is to critically review the current literature dealing with noble metals' NPs (NM-NPs) and their effects on plants and associated microorganisms. Particular attention has been given to the less studied NPs of platinum group elements, which can be considered a neglected pollutant, since they are released from vehicles' catalysts. In addition, we have provided a comprehensive overview of the biotechnology exploitation of NM-NPs in plant cultivation, where prospective nanomaterials developed as nanofertilizers and nanopesticides are introduced, and both the pros and the cons of nanomaterial plant treatments have been discussed.

Mutual regulation of plant phospholipase D and the actin cytoskeleton.

Membrane lipids and cytoskeleton dynamics are intimately inter-connected in the eukaryotic cell; however, only recently have the molecular mechanisms operating at this interface in plant cells been addressed experimentally. Phospholipase D (PLD) and its product phosphatidic acid (PA) were discovered to be important regulators in the membrane-cytoskeleton interface in eukaryotes. Here we report the mechanistic details of plant PLD-actin interactions. Inhibition of PLD by n-butanol compromises pollen tube actin, and PA rescues the detrimental effect of n-butanol on F-actin, showing clearly the importance of the PLD-PA interaction for pollen tube F-actin dynamics. From various candidate tobacco PLDs isoforms, we identified NtPLDbeta1 as a regulatory partner of actin, by both activity and in vitro interaction assays. Similarly to published data, the activity of tobacco PIP(2)-dependent PLD (PLDbeta) is specifically enhanced by F-actin and inhibited by G-actin. We then identified the NtPLDbeta1 domain responsible for actin interactions. Using sequence- and structure-based analysis, together with site-directed mutagenesis, we identified Asn323 and Thr382 of NtPLDbeta1 as the crucial amino acids in the actin-interacting fold. The effect of antisense-mediated suppression of NtPLDbeta1 or NtPLDdelta on pollen tube F-actin dynamics shows that NtPLDbeta1 is the active partner in PLD-actin interplay. The positive feedback loop created by activation of PLDbeta by F-actin and of F-actin by PA provides an important mechanism to locally increase membrane-F-actin dynamics in the cortex of plant cells.

Probing plant membranes with FM dyes: tracking, dragging or blocking?

Remarkable progress in various techniques of in vivo fluorescence microscopy has brought an urgent need for reliable markers for tracking cellular structures and processes. The goal of this manuscript is to describe unexplored effects of the FM (Fei Mao) styryl dyes, which are widely used probes that label processes of endocytosis and vesicle trafficking in eukaryotic cells. Although there are few reports on the effect of styryl dyes on membrane fluidity and the activity of mammalian receptors, FM dyes have been considered as reliable tools for tracking of plant endocytosis. Using plasma membrane-localized transporters for the plant hormone auxin in tobacco BY-2 and Arabidopsis thaliana cell suspensions, we show that routinely used concentrations of FM 4-64 and FM 5-95 trigger transient re-localization of these proteins, and FM 1-43 affects their activity. The active process of re-localization is blocked neither by inhibitors of endocytosis nor by cytoskeletal drugs. It does not occur in A. thaliana roots and depends on the degree of hydrophobicity (lipophilicity) of a particular FM dye. Our results emphasize the need for circumspection during in vivo studies of membrane proteins performed using simultaneous labelling with FM dyes.

The phosphatidylcholine-hydrolysing phospholipase C NPC4 plays a role in response of Arabidopsis roots to salt stress.

Phosphatidylcholine-hydrolysing phospholipase C, also known as non-specific phospholipase C (NPC), is a new member of the plant phospholipase family that reacts to environmental stresses such as phosphate deficiency and aluminium toxicity, and has a role in root development and brassinolide signalling. Expression of NPC4, one of the six NPC genes in Arabidopsis, was highly induced by NaCl. Maximum expression was observed from 3 h to 6 h after the salt treatment and was dependent on salt concentration. Results of histochemical analysis of P(NPC4):GUS plants showed the localization of salt-induced expression in root tips. On the biochemical level, increased NPC enzyme activity, indicated by accumulation of diacylglycerol, was observed as early as after 30 min of salt treatment of Arabidopsis seedlings. Phenotype analysis of NPC4 knockout plants showed increased sensitivity to salinity as compared with wild-type plants. Under salt stress npc4 plants had shorter roots, lower fresh weight, and reduced seed germination. Expression levels of abscisic acid-related genes ABI1, ABI2, RAB18, PP2CA, and SOT12 were substantially reduced in salt-treated npc4 plants. These observations demonstrate a role for NPC4 in the response of Arabidopsis to salt stress.

Phospholipase Dα1 Acts as a Negative Regulator of High Mg2+-Induced Leaf Senescence in Arabidopsis.

Magnesium (Mg2+) is a macronutrient involved in essential cellular processes. Its deficiency or excess is a stress factor for plants, seriously affecting their growth and development and therefore, its accurate regulation is essential. Recently, we discovered that phospholipase Dα1 (PLDα1) activity is vital in the stress response to high-magnesium conditions in Arabidopsis roots. This study shows that PLDα1 acts as a negative regulator of high-Mg2+-induced leaf senescence in Arabidopsis. The level of phosphatidic acid produced by PLDα1 and the amount of PLDα1 in the leaves increase in plants treated with high Mg2+. A knockout mutant of PLDα1 (pldα1-1), exhibits premature leaf senescence under high-Mg2+ conditions. In pldα1-1 plants, higher accumulation of abscisic and jasmonic acid (JA) and impaired magnesium, potassium and phosphate homeostasis were observed under high-Mg2+ conditions. High Mg2+ also led to an increase of starch and proline content in Arabidopsis plants. While the starch content was higher in pldα1-1 plants, proline content was significantly lower in pldα1-1 compared with wild type plants. Our results show that PLDα1 is essential for Arabidopsis plants to cope with the pleiotropic effects of high-Mg2+ stress and delay the leaf senescence.

Aluminium ions inhibit the formation of diacylglycerol generated by phosphatidylcholine-hydrolysing phospholipase C in tobacco cells.

• Aluminium ions (Al) have been recognized as a major toxic factor for crop production in acidic soils. This study aimed to assess the impact of Al on the activity of phosphatidylcholine-hydrolysing phospholipase C (PC-PLC), a new member of the plant phospholipase family. • We labelled the tobacco cell line BY-2 and pollen tubes with a fluorescent derivative of phosphatidylcholine and assayed for patterns of fluorescently labelled products. Growth of pollen tubes was analysed. • We observed a significant decrease of labelled diacylglycerol (DAG) in cells treated with AlCl(3). Investigation of possible metabolic pathways that control DAG generation and consumption during the response to Al showed that DAG originated from the reaction catalysed by PC-PLC. The growth of pollen tubes was retarded in the presence of Al and this effect was accompanied by the decrease of labelled DAG similar to the case of the BY-2 cell line. The growth of pollen tubes arrested by Al was rescued by externally added DAG. • Our observation strongly supports the role of DAG generated by PC-PLC in the response of tobacco cells to Al.

A selective autophagy cargo receptor NBR1 modulates abscisic acid signalling in Arabidopsis thaliana.

The plant selective autophagy cargo receptor neighbour of breast cancer 1 gene (NBR1) has been scarcely studied in the context of abiotic stress. We wanted to expand this knowledge by using Arabidopsis thaliana lines with constitutive ectopic overexpression of the AtNBR1 gene (OX lines) and the AtNBR1 Knock-Out (KO lines). Transcriptomic analysis of the shoots and roots of one representative OX line indicated differences in gene expression relative to the parental (WT) line. In shoots, many differentially expressed genes, either up- or down-regulated, were involved in responses to stimuli and stress. In roots the most significant difference was observed in a set of downregulated genes that is mainly related to translation and formation of ribonucleoprotein complexes. The link between AtNBR1 overexpression and abscisic acid (ABA) signalling was suggested by an interaction network analysis of these differentially expressed genes. Most hubs of this network were associated with ABA signalling. Although transcriptomic analysis suggested enhancement of ABA responses, ABA levels were unchanged in the OX shoots. Moreover, some of the phenotypes of the OX (delayed germination, increased number of closed stomata) and the KO lines (increased number of lateral root initiation sites) indicate that AtNBR1 is essential for fine-tuning of the ABA signalling pathway. The interaction of AtNBR1 with three regulatory proteins of ABA pathway (ABI3, ABI4 and ABI5) was observed in planta. It suggests that AtNBR1 might play role in maintaining the balance of ABA signalling by controlling their level and/or activity.

Temporary heat stress suppresses PAMP-triggered immunity and resistance to bacteria in Arabidopsis thaliana.

Recognition of pathogen-associated molecular patterns (PAMPs) is crucial for plant defence against pathogen attack. The best characterized PAMP is flg22, a 22 amino acid conserved peptide from flagellin protein. In Arabidopsis thaliana, flg22 is recognized by the flagellin sensing 2 (FLS2) receptor. In this study, we focused on biotic stress responses triggered by flg22 after exposure to temporary heat stress (HS). It is important to study the reactions of plants to multiple stress conditions because plants are often exposed simultaneously to a combination of both abiotic and biotic stresses. Transient early production of reactive oxygen species (ROS) is a well-characterized response to PAMP recognition. We demonstrate the strong reduction of flg22-induced ROS production in A. thaliana after HS treatment. In addition, a decrease in FLS2 transcription and a decrease of the FLS2 presence at the plasma membrane are shown after HS. In summary, our data show the strong inhibitory effect of HS on flg22-triggered events in A. thaliana. Subsequently, temporary HS strongly decreases the resistance of A. thaliana to Pseudomonas syringae. We propose that short exposure to high temperature is a crucial abiotic stress factor that suppresses PAMP-triggered immunity, which subsequently leads to the higher susceptibility of plants to pathogens.

Seed germination, respiratory processes and phosphatidic acid accumulation in Arabidopsis diacylglycerol kinase knockouts - The effect of brassinosteroid, brassinazole and salinity.

Using Arabidopsis thaliana wild type (WT) plants and diacylglycerol kinase knockouts (single mutants - dgk3, dgk1, dgk6; double mutants - dgk3dgk7, dgk5dgk6, dgk1dgk2) we observed that the inhibitor of brassinosteroid (BR) biosynthesis, brassinazole (BRZ), drastically decreased germination of dgk mutants under salt stress, while BRZ co-administration with 24-epibrassinolide (EBL) partially improved germination rates. We also observed a statistically significant decrease in alternative and cytochrome respiratory pathways in response to BRZ treatment under salinity conditions. We showed that production of the lipid second messenger phosphatidic acid (PA) is impaired in dgk mutants in response to EBL treatment and inhibitor of diacylglycerol kinase (DGK) - R59022. This study demonstrates that dgk mutants possess lower germination rates, lower total respiration rates, an alternative respiratory pathway and PA content under optimal and high salinity conditions in response to EBL treatment comparing to WT plants.

Phosphatidic acids mediate transport of Ca2+ and H+ through plant cell membranes.

Phosphatidic acids (PAs) are a key intermediate in phospholipid biosynthesis, and a central element in numerous signalling pathways. Functions of PAs are related to their fundamental role in molecular interactions within cell membranes modifying membrane bending, budding, fission and fusion. Here we tested the hypothesis that PAs are capable of direct transport of ions across bio-membranes. We have demonstrated that PAs added to the maize plasma membrane vesicles induced ionophore-like transmembrane transport of Ca2+, H+ and Mg2+. PA-induced Ca2+ fluxes increased with an increasing PAs acyl chain unsaturation. For all the PAs analysed, the effect on Ca2+ permeability increased with increasing pH (pH 8.0>pH 7.2>pH 6.0). The PA-induced Ca2+, Mg2+ and H+ permeability was also more pronounced in the endomembrane vesicles as compared with the plasma membrane vesicles. Addition of PA to protoplasts from Arabidopsis thaliana (L.) Heynh. roots constitutively expressing aequorin triggered elevation of the cytosolic Ca2+ activity, indicating that the observed PA-dependent Ca2+ transport occurs in intact plants.

The phosphatidic acid paradox: Too many actions for one molecule class? Lessons from plants.

Phosphatidic acid (PA) is a simple phospholipid observed in most organisms. PA acts as a key metabolic intermediate and a second messenger that regulates many cell activities. In plants, PA is involved in numerous cell responses induced by hormones, stress inputs and developmental processes. Interestingly, PA production can be triggered by opposite stressors, such as cold and heat, or by hormones that are considered to be antagonistic, such as abscisic acid and salicylic acid. This property questions the specificity of the responses controlled by PA. Are there generic responses to PA, meaning that cell regulation triggered by PA would be always the same, even in opposite physiological situations? Alternatively, do the responses to PA differ according to the physiological context within the cells? If so, the mechanisms that regulate the divergence of PA-controlled reactions are poorly defined. This review summarizes the latest opinions on how PA signalling is directed in plant cells and examines the intrinsic properties of PA that enable its regulatory diversity. We propose a concept whereby PA regulatory messages are perceived as complex "signatures" that take into account their production site, the availability of target proteins and the relevant cellular environments.

Round-shape gold nanoparticles: effect of particle size and concentration on Arabidopsis thaliana root growth.

Nowadays, due to a wide range of applications of nanoparticles (NPs) in many industrial areas, accumulations of those entities in environment pose a great risk. Owing to their inertness, noble metal NPs may remain in contaminated soils nearly unchanged for long time. Within this context, size-, shape-, and concentration-dependent uptake of particles by plants belongs to unexplored area. In this work, we present water solutions of biologically friendly synthesized spherical AuNPs with pretty narrow size distribution in size range from 10 to 18 nm. Their thorough characterization by atomic absorption spectroscopy, mass spectroscopy-equipped inductively coupled plasma, dynamic light scattering (DLS), and TEM methods was followed by the study of their effect on the growth of Arabidopsis thaliana (primary and lateral roots), in particle size- and concentration-dependent manner. Due to strictly round-shape form of AuNPs and absence of particle agglomeration, DLS-derived size and size distribution were in good concordance with those obtained from TEM. The length and number of A. thaliana lateral roots were significantly affected by all types of AuNPs. Smallest AuNPs at highest concentration inhibited length of primary roots and, in contrast, enhanced hair root growth.