1) Cold stress (Dr. Steven Arisz, Drs. Myrthe Praat)

Cold and freezing stress are important constraints for crop plants. When plants experience cold temperatures prior to freezing, they can become more tolerant to frost. This process, called cold acclimation, involves complex changes including, e.g. (i) the accumulation of sugars and proteins that protect macromolecules and membranes, (ii) increased fatty-acid desaturation to regulate the fluidity of membranes, and (iii) adjustments in growth and development. Many of these changes rely on the induced expression of transcription factors that occur within 15 min. However, it is still a big mystery how the acute cold response gets kick-started.

We and others have found that the signaling lipid, phosphatidic acid (PA) accumulates within minutes upon exposure of Arabidopsis seedlings to lower temperatures. Based on its rapid formation, the  proportional effect to ambient temperatures, and the emerging evidence that PA can function as a signaling lipid through interactions with protein targets, we hypothesize that PA serves as a cellular thermometer to regulate the acute response to cold.

2) Heat stress (Dr. Michael Mishkind, Dr. Essam Darwish, Drs. Max van Hooren, Drs. Nazish Annum)

Temperature is one of the key physical parameters affecting life on Earth. As a result, almost all living organisms have evolved signalling systems to sense slight temperature changes and adjust their metabolism and cell function to prevent heat-related damage. In plants, heat stress has harmful effects on most aspects of development, growth, reproduction and yield. Nonetheless, plants have an inherent ability to survive exposure to temperatures above the optimal for growth (basal thermotolerance) but also have the ability to acquire tolerance to otherwise lethal heat stress (acquired thermotolerance). At the molecular level, plants rapidly switch on a heat stress response (HSR), which is highly conserved and involves multiple pathways, regulatory networks and cellular compartments. At least four putative sensors have been proposed to trigger HSR. These include a Ca²⁺-influx channel in the plasma membrane, a histone sensor in the nucleus, and two unfolded-protein sensors at the ER and cytosol. Each of these putative sensors is thought to activate a similar set of HSR genes, but the relationship between different pathways and hierarchical order remains unclear.

We discovered that heat stress rapidly (2 min) triggers the formation of the signalling lipid, phosphatidylinositol 4,5-bisphosphate (PIP₂), and recently we identified the two PIP5Ks of Arabidopsis' 11 that are involved in this response. Using single- and double-KO mutants, as well as phosphomimetic and kinase-death mutants, we are currently addressing their functional role in basal- and aquired thermotolerance. Using FP-tagged versions and genetically encoded-PIP₂ biosensors, their subcellular localization is studied by confocal imaging, while PIP₂-affinity based MS analyses will be used to identify heat specfic-PIP₂ targets (see below). Ultimately, we hope to uncover the role of PIP₂ in the molecular mechanism by which plant cells perceive and transduce temperature signals, and exploit this knowledge to improve the yield-limiting thermotolerance in crop plants.

3) Drought stress (Drs. Max van Hooren)

A major challenge in the twenty-first century is to increase global food production to feed the continuously growing population while quality and quantity of arable land is quickly diminishing, and the climate is changing. Fundamental to this problem is the necessity to increase the yield of numerous important crop species, and to find ways to extend geographical locations suitable for agriculture. For plants, water is the most important factor to grow. Hence, drought stress is one of the biggest problems hampering global food production.

We and others have found that overexpression of phospholipase C (PLC) increases the tolerance of plants to drought in various plant species. PLC is an enzyme that hydrolyses the minor membrane lipid, PIP₂ (but also its precursor, PIP), into inositoltrisphosphate (IP₃; or IP₂ from PIP) and DAG. The inositolpolyphosphates (IPPs) are water-soluble and diffuse into the cytosol where they are phosphorylated into higher IPPs that have signalling activity. DAG is a lipid that remains in the membrane and is quickly phosphorylated by DAG kinase (DGK) to form the lipid second messenger, PA. The latter recruites and (in)activates specific PA-target proteins, which transmit - together with the downstream targets for IPPs - information of the initial signal further down the signal transduction cascade. IPPs can release Ca²⁺ from intracellular stores in guard cells to close stomata upon ABA, but have also been implicated in receptor signalling of the phytohormones, auxin and meJA, or in RNA transport during P_i signalling. IPPs and PA could also be further metabolized into compounds that would improve the plant's drought tolerance. Our research is focussed to understand the molecular mechanism by which PLC improves drought tolerance, so that we can exploit this knowledge further to improve it for crop plants.

4) Salt- and osmotic stress (Dr. Xavier Zarza, Dr. Femke de Jong, Drs. Max van Hooren; Dr. Essam Darwish)

Salt stress is another major abiotic stress that limits crop production, especially in arid and semi-arid regions. Salinity is responsible for different types of stress, including osmotic-, ionic-, and oxidative stress, which results in various hormonal and metabolic imbalances. The osmotic stress is caused by the excess of Na⁺ and Cl⁻ ions in the soil, which decrease the osmotic potential and hamper the uptake of water and nutrients. To compensate, plants accumulate compatible solutes, which are low-molecular weight compounds, such as proline, glycinebetaine, sugars, proteins, and polyols. These molecules help the plant to improve its resilience against water stress, and do not interfere with normal biochemical reactions.

We found that plant cells rapidly trigger an increase in the production of PA and PIP₂, within minutes of applying salt- or osmotic stress. PA is generated via DGK (rapidly) and PLD (slowly and at high salt concentration) while the increase of PIP₂  is caused through activation of phosphatidylinositolphosphate 5-kinase (PIP5K). The model plant Arabidopsis thaliana contains 7 DGKs, 12 PLDs and 11 PIP5Ks, and using T-DNA insertion KO mutants we are trying to identify which genes are involved. For PIP₂, we discovered that three PIP5Ks were responsible for the salt- and osmotic stress induced response. Currently, we are functionally characterizing these genes in signal transduction and in acquiring salt tolerance in Arabidopsis at the molecular level, similar to heat stress described above. Also here, we hope to uncover the molecular mechanism by which PA and PIP₂ function in salt- and osmotic stress signalling, and exploit that knowledge to improve salt- and drought tolerance of crop plants.

5) Disease resistance (Jack Dickenson)

Invading pathogens or PAMPs (Pathogen-Associated Molecular Patterns, which are molecular structures or molecules that are shared by most pathogenic bacteria and some viruses) trigger the formation of the lipid second messenger, phosphatidic acid (PA) within minutes. This is produced by activation of phospholipase D (PLD) and/or diacylglycerol kinase (DGK), for which the Arabidopsis genome encodes 12 and 7 genes, respectively. Using a reversed-genetic screen of knock-out mutants of the different PLD and DGK genes, for their sensitivity against virulent and avirulent strains of the bacterial pathogen, Pseudomonas syringae , one DGK and two PLD genes were identified to be involved in basal- and induced disease resistance, respectively. The role of the DGK is currently functionally explored with respect to its enzyme activity, localization and interacting partners, which is in close collaboration with Prof. Libo Shan's lab at Texas A&M University (TX, USA).

6) PLC signalling (Drs. Max van Hooren, Ing. Ringo van Wijk)

Phospholipase C (PLC) plays a key role in the perception and transmission of extracellular signals in all animal cells. A similar role is suggested for plants in stress signalling and development but their genomes seem to lack the primairy targets for this signalling system, i.e. the IP₃-gated Ca²⁺ channel and the DAG-activated PKC. To increase our understanding of plant-PLC signaling, we have started to address this in the model plant, Arabidopsis whose genome encodes for 9 PLC genes. Using promoter-GUS analyses, T-DNA insertion KO- and KD mutants, inducible-gene silencing and PLC-overexpression mutants, we are functionally characterizing the role of each PLC in various stress- and developmental responses. All this work is performed in close collaboration with the lab of Prof. Ana Laxalt (Mar del Plata, Argentina).

7) PIP 5-kinase (Dr. Xavier Zarza, Ing. Ringo van Wijk)

Salt- and heat stress trigger the formation of the signalling lipid, phosphatidylinositol 4,5-bisphosphate (PIP₂). In Arabidopsis seedlings, this occurs within minutes of stress application. We know that this PIP₂ is synthesised at the plasma membrane through phosphorylation of phosphatidylinositol 4-monophosphate (PI4P) by the enzyme, PIP 5-kinase (PIP5K). Arabidopsis contains 11 PIP5K genes. Using T-DNA insertion knock-out (KO) mutants, we have identified three PIP5Ks responsible for the salt stress-triggered PIP₂, and two for heat stress response, for which triple- and double-KO mutants, have been generated, respectievly, which are devoid of any salt- or heat-activated PIP₂. Currently, these mutants are being characterized and phenotyped for their development and stress responses using genetically encoded -lipid biosensor lines against DAG, PA, PI4P and PI(4,5)P₂, and various additional PIP5K mutant lines (i.e. tagged, FP-fused, phosphomimetics, kinase-death).

8) Protein targets for PIP₂ (Dr. Femke de Jong)

Within minutes, plant cells respond to heat- or osmotic stress with a dramatic increase in the production of the minor signalling lipid, phosphatidyl 4,5-bisphosphate (PIP₂). While this response has always been considered to reflect its role as precursor in phospholipase C signalling, recent data from yeast and mammalian studies indicate that the lipid itself can function as a signalling molecule. In this scenario, PIP₂ binds target proteins via specific domains with dramatic consequences for their biological activity, because binding induces changes in location and/or conformation. While several PIP₂-binding proteins have been described for yeast and animal systems, none have been characterized for plants, while many potential homologues in plant genomic databases can be observed. This project aims to identify and characterise PIP₂ targets that are involved in the early heat- and /or osmotic-stress response of the model plant system, Arabidopsis thaliana.

A PIP₂-affinity chromatography approach coupled to mass spectrometry will be applied to map the first plant PIP₂-interactome. This collection will include proteins that bind PIP₂ directly but also proteins that bind indirectly, being part of the complex that binds PIP₂. A number of them will be selected for further anaylsis to identify and biochemically characterize the first genuine plant PIP₂ targets. Selection will be based on candidates showing increased expression or affinity as a result of stress, on exhibiting known or suspected lipid-binding domains, and by data-base mining for stress-related phenomena (e.g. function). For a few candidates, cDNAs will be cloned, expressed as GST-fusions in E. coli and tested for their lipid-binding capacity. Those specific for PI(4,5)P₂ (top 5) will be characterised for their biochemical activity in vitro (GST) and subcellular localization in planta (GFP), before and after stress. To functionally characterize their role in salt-, drought- and thermo tolerance, Arabidopis over-expression and knock-out mutants will be generated (top 3). To identify potential transcriptional networks downstream, RNA-Seq analyses will be performed before and after stress. This study should identify signalling pathways and enzyme activities that play a potential role in heat- and osmotic-stress tolerance, and establish a new mode of action for lipid signalling in plants.