Jan Martinec

Brassinosteroids (BRs) are essential regulators of plant development and stress responses, but the distinct contributions of BR biosynthesis and signaling to hormonal crosstalk remain poorly defined. Here, we investigated the effects of the BR biosynthesis inhibitor brassinazole (BRZ) and the BR-insensitive mutant bri1-6 on endogenous phytohormone profiles in Arabidopsis thaliana. Using multivariate analysis and targeted hormone quantification, we show that BRZ treatment and BRI1 disruption alter hormone balance through partially overlapping but mechanistically distinct pathways. Principal component analysis (PCA) and hierarchical clustering revealed that BRZ and the bri1-6 mutation do not phenocopy each other and that BRZ still alters hormone profiles even in the bri1-6 mutant, suggesting potential BRI1-independent effects. Both BRZ treatment and the bri1-6 mutation tend to influence cytokinins and auxin conjugates divergently. On the contrary, their effects on stress-related hormones converge: BRZ decreases salicylic acid (SA), jasmonic acid (JA), and abscisic acid (ABA) in the WT leaves; similarly, bri1-6 mutants show reduced SA, JA, and ABA. These results indicate that BR biosynthesis and BRI1-mediated perception may contribute independently to hormonal reprogramming, with BRZ eliciting additional effects, possibly via metabolic feedback, compensatory signaling, or off-target action. Hormone correlation analyses revealed conserved co-regulation clusters that reflect underlying regulatory modules. Altogether, our findings provide evidence for a partial uncoupling of BR levels and BR signaling and illustrate how BR pathways intersect with broader hormone networks to coordinate growth and stress responses.

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.

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.

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.

Magnesium 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-Mg ²⁺ -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 Mg ²⁺ . A knockout mutant of PLDα1 ( plda1-1 ), exhibits premature leaf senescence under high-Mg ²⁺ conditions. In pldα1-1 plants, higher accumulation of abscisic and jasmonic acid and impaired magnesium, potassium and phosphate homeostasis were observed under high-Mg ²⁺ conditions. High Mg ²⁺ also led to an increase of starch and proline content in Arabidopsis plants. While the starch content was higher in plda1-1 plants, proline content was significantly lower in plda1-1 compared with WT. Our results show that PLDα1 is essential for Arabidopsis plants to cope with the pleiotropic effects of high-Mg ²⁺ stress and delay the leaf senescence.

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.

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.

Summary: 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.

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.