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.

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.

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.

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.

Mapping of Plasma Membrane Proteins Interacting With Arabidopsis thaliana Flotillin 2.

Arabidopsis flotillin 2 (At5g25260) belongs to the group of plant flotillins, which are not well characterized. In contrast, metazoan flotillins are well known as plasma membrane proteins associated with membrane microdomains that act as a signaling hub. The similarity of plant and metazoan flotillins, whose functions most likely consist of affecting other proteins via protein-protein interactions, determines the necessity of detecting their interacting partners in plants. Nevertheless, identifying the proteins that form complexes on the plasma membrane is a challenging task due to their low abundance and hydrophobic character. Here we present an approach for mapping Arabidopsis thaliana flotillin 2 plasma membrane interactors, based on the immunoaffinity purification of crosslinked and enriched plasma membrane proteins with mass spectrometry detection. Using this approach, 61 proteins were enriched in the AtFlot-GFP plasma membrane fraction, and 19 of them were proposed to be flotillin 2 interaction partners. Among our proposed partners of Flot2, proteins playing a role in the plant response to various biotic and abiotic stresses were detected. Additionally, the use of the split-ubiquitin yeast system helped us to confirm that plasma-membrane ATPase 1, early-responsive to dehydration stress protein 4, syntaxin-71, harpin-induced protein-like 3, hypersensitive-induced response protein 2 and two aquaporin isoforms interact with flotillin 2 directly. Based on the results of our study and the reported properties of Flot2 interactors, we propose that Flot2 complexes may be involved in plant-pathogen interactions, water transport and intracellular trafficking.

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.

Arabidopsis non-specific phospholipase C1: characterization and its involvement in response to heat stress.

The Arabidopsis non-specific phospholipase C (NPC) protein family is encoded by the genes NPC1 - NPC6. It has been shown that NPC4 and NPC5 possess phospholipase C activity; NPC3 has lysophosphatidic acid phosphatase activity. NPC3, 4 and 5 play roles in the responses to hormones and abiotic stresses. NPC1, 2 and 6 has not been studied functionally yet. We found that Arabidopsis NPC1 expressed in Escherichia coli possesses phospholipase C activity in vitro. This protein was able to hydrolyse phosphatidylcholine to diacylglycerol. NPC1-green fluorescent protein was localized to secretory pathway compartments in Arabidopsis roots. In the knock out T-DNA insertion line NPC1 (npc1) basal thermotolerance was impaired compared with wild-type (WT); npc1 exhibited significant decreases in survival rate and chlorophyll content at the seventh day after heat stress (HS). Conversely, plants overexpressing NPC1 (NPC1-OE) were more resistant to HS compared with WT. These findings suggest that NPC1 is involved in the plant response to heat.

Changes in actin dynamics are involved in salicylic acid signaling pathway.

Changes in actin cytoskeleton dynamics are one of the crucial players in many physiological as well as non-physiological processes in plant cells. Positioning of actin filament arrays is necessary for successful establishment of primary lines of defense toward pathogen attack, depolymerization leads very often to the enhanced susceptibility to the invading pathogen. On the other hand it was also shown that the disruption of actin cytoskeleton leads to the induction of defense response leading to the expression of PATHOGENESIS RELATED proteins (PR). In this study we show that pharmacological actin depolymerization leads to the specific induction of genes in salicylic acid pathway but not that involved in jasmonic acid signaling. Life imaging of leafs of Arabidopsis thaliana with GFP-tagged fimbrin (GFP-fABD2) treated with 1 mM salicylic acid revealed rapid disruption of actin filaments resembling the pattern viewed after treatment with 200 nM latrunculin B. The effect of salicylic acid on actin filament fragmentation was prevented by exogenous addition of phosphatidic acid, which binds to the capping protein and thus promotes actin polymerization. The quantitative evaluation of actin filament dynamics is also presented.

The plant non-specific phospholipase C gene family. Novel competitors in lipid signalling.

Non-specific phospholipases C (NPCs) were discovered as a novel type of plant phospholipid-cleaving enzyme homologous to bacterial phosphatidylcholine-specific phospholipases C and responsible for lipid conversion during phosphate-limiting conditions. The six-gene family was established in Arabidopsis, and growing evidence suggests the involvement of two articles NPCs in biotic and abiotic stress responses as well as phytohormone actions. In addition, the diacylglycerol produced via NPCs is postulated to participate in membrane remodelling, general lipid metabolism and cross-talk with other phospholipid signalling systems in plants. This review summarises information concerning this new plant protein family and focusses on its sequence analysis, biochemical properties, cellular and tissue distribution and physiological functions. Possible modes of action are also discussed.

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.

The role for the exocyst complex subunits Exo70B2 and Exo70H1 in the plant-pathogen interaction.

Recently, the octameric vesicle-tethering complex exocyst was found in plants and its importance for Arabidopsis morphogenesis was demonstrated. Exo70 exocyst subunits in plants, unlike in yeasts and mammals, are represented by a multigene family, comprising 23 members in Arabidopsis. For Exo70B2 and Exo70H1 paralogues, transcriptional up-regulation was confirmed on treatment with an elicitor peptide, elf18, derived from the bacterial elongation factor. Their ability to participate in the exocyst complex formation was inferred by the interaction of both the Exo70s with several other exocyst subunits using the yeast two-hybrid system. Arabidopsis plants mutated in these two genes were used to analyse their local reaction upon inoculation with Pseudomonas syringae pv. maculicola and the fungal pathogen Blumeria graminis f. sp. hordei. The Pseudomonas sensitivity test revealed enhanced susceptibility for the two exo70B2 and one H1 mutant lines. After Blumeria inoculation, an increase in the proportion of abnormal papilla formation, with an unusual wide halo made of vesicle-like structures, was found in exo70B2 mutants. Intracellular localization of both Exo70 proteins was studied following a GFP fusion assay and Agrobacterium-mediated transient expression of the constructs in Nicotiana benthamiana leaf epidermis. GFP-Exo70H1 localizes in the vesicle-like structures, while GFP-Exo70B2 is localized mainly in the cytoplasm. It is concluded that both Exo70B2 and Exo70H1 are involved in the response to pathogens, with Exo70B2 having a more important role in cell wall apposition formation related to plant defence.

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.