Steven A. Arisz

Rapid phosphatidic acid accumulation in response to low temperature stress in Arabidopsis is generated through diacylglycerol kinase

Phosphatidic acid (PtdOH) is emerging as an important signaling lipid in abiotic stress responses in plants. The effect of cold stress was monitored using 32P-labeled seedlings and leaf discs of Arabidopsis thaliana. Low, non-freezing temperatures were found to trigger a very rapid 32P-PtdOH increase, peaking within 2 and 5 min, respectively. In principle, PtdOH can be generated through three different pathways, i.e., (1) via de novo phospholipid biosynthesis (through acylation of lyso-PtdOH), (2) via phospholipase D hydrolysis of structural phospholipids, or (3) via phosphorylation of diacylglycerol (DAG) by DAG kinase (DGK). Using a differential 32P-labeling protocol and a PLD-transphosphatidylation assay, evidence is provided that the rapid 32P-PtdOH response was primarily generated through DGK. A simultaneous decrease in the levels of 32P-PtdInsP, correlating in time, temperature dependency, and magnitude with the increase in 32P-PtdOH, suggested that a PtdInsP-hydrolyzing PLC generated the DAG in this reaction. Testing T-DNA insertion lines available for the seven DGK genes, revealed no clear changes in 32P-PtdOH responses, suggesting functional redundancy. Similarly, known cold-stress mutants were analyzed to investigate whether the PtdOH response acted downstream of the respective gene products. The hos1, los1, and fry1 mutants were found to exhibit normal PtdOH responses. Slight changes were found for ice1, snow1, and the overexpression line Super-ICE1, however, this was not cold-specific and likely due to pleiotropic effects. A tentative model illustrating direct cold effects on phospholipid metabolism is presented.

Plant PA signaling via diacylglycerol kinase.

Accumulating evidence suggests that phosphatidic acid (PA) plays a pivotal role in the plants response to environmental signals. Besides phospholipase D (PLD) activity, PA can also be generated by diacylglycerol kinase (DGK). To establish which metabolic route is activated, a differential (32)P-radiolabelling protocol can be used. Based on this, and more recently on reverse-genetic approaches, DGK has taken center stage, next to PLD, as a generator of PA in biotic and abiotic stress responses. The DAG substrate is generally thought to be derived from PI-PLC activity. The model plant system Arabidopsis thaliana has 7 DGK isozymes, two of which, AtDGK1 and AtDGK2, resemble mammalian DGKepsilon, containing a conserved kinase domain, a transmembrane domain and two C1 domains. The other ones have a much simpler structure, lacking the C1 domains, not matched in animals. Several protein targets have now been discovered that bind PA. Whether the PA molecules engaged in these interactions come from PLD or DGK remains to be elucidated.

Identification of novel candidate phosphatidic acid binding proteins involved in the salt stress response of Arabidopsis thaliana roots

PA (phosphatidic acid) is a lipid second messenger involved in an array of processes occurring during a plants life cycle. These include development, metabolism, and both biotic and abiotic stress responses. PA levels increase in response to salt, but little is known about its function in the earliest responses to salt stress. In the present study we have combined an approach to isolate peripheral membrane proteins of Arabidopsis thaliana roots with lipid-affinity purification, to identify putative proteins that interact with PA and are recruited to the membrane in response to salt stress. Of the 42 putative PA-binding proteins identified by MS, a set of eight new candidate PA-binding proteins accumulated at the membrane fraction after 7 min of salt stress. Among these were CHC (clathrin heavy chain) isoforms, ANTH (AP180 N-terminal homology) domain clathrin-assembly proteins, a putative regulator of potassium transport, two ribosomal proteins, GAPDH (glyceraldehyde 3-phosphate dehydrogenase) and a PI (phosphatidylinositol) 4-kinase. PA binding and salt-induced membrane recruitment of GAPDH and CHC were confirmed by Western blot analysis of the cellular fractions. In conclusion, the approach of the present study is an effective way to isolate biologically relevant lipid-binding proteins and provides new leads in the study of PA-mediated salt-stress responses in roots.

Polar glycerolipids of Chlamydomonas moewusii

The fatty acid and polar lipid compositions of the unicellular green alga Chlamydomonas moewusii were characterized. Since this organism is an important plant model for phospholipid-based signal transduction, interest was focused on the lipids phosphatidic acid, phosphatidylinositolphosphate and phosphatidylinositolbisphosphate. A phosphatidylinositol:phosphatidylinositolphosphate: phosphatidylinositolbisphosphate ratio of 100:1.7:1.3 was found. The polyphosphoinositides accounted for 0.8 mol% of the total phospholipids and their fatty acid compositions were similar to that of phosphatidylinositol except for the enrichment of linolenic acid in phosphatidylinositol phosphate. Phosphatidic acid accounted for 0.67 mol% of the phospholipids. Major structural glycerolipids were monogalactosyldiacylglycerol (35 mol%), digalactosyldiacylglycerol (15 mol%), sulfoquinovosyldiacylglycerol (10 mol%), diacylglyceryltrimethylhomoserine (16 mol%), phosphatidylglycerol (9 mol%), phosphatidylethanolamine (8 mol%) and phosphatidylinositol (6 mol%). Relative changes in the total fatty acid compositions found during growth on nutrient-limited medium reflected mainly alterations in the compositions of the chloroplast lipids phosphatidylglycerol and monogalactosyldiacylglycerol. [32P]Pi-incorporation studies revealed that it took 6 days before the amount of label in the major phospholipids was proportional to their abundance.

The salt stress-induced LPA response in Chlamydomonas is produced via PLA2 hydrolysis of DGK-generated phosphatidic acid

The unicellular green alga Chlamydomonas has frequently been used as a eukaryotic model system to study intracellular phospholipid signaling pathways in response to environmental stresses. Earlier, we found that hypersalinity induced a rapid increase in the putative lipid second messenger, phosphatidic acid (PA), which was suggested to be generated via activation of a phospholipase D (PLD) pathway and the combined action of a phospholipase C/diacylglycerol kinase (PLC/DGK) pathway. Lysophosphatidic acid (LPA) was also increased and was suggested to reflect a phospholipase A2 (PLA2) activity based on pharmacological evidence. The question of PAs and LPAs origin is, however, more complicated, especially as both function as precursors in the biosynthesis of phospho- and galactolipids. To address this complexity, a combination of fatty acid-molecular species analysis and in vivo 32P-radiolabeling was performed. Evidence is provided that LPA is formed from a distinct pool of PA characterized by a high α-linolenic acid (18:3n-3) content. This molecular species was highly enriched in the polyphosphoinositide fraction, which is the substrate for PLC to form diacylglycerol. Together with differential 32P-radiolabeling studies and earlier PLD-transphosphatidylation and PLA2-inhibitor assays, the data were consistent with the hypothesis that the salt-induced LPA response is primarily generated through PLA2-mediated hydrolysis of DGK-generated PA and that PLD or de novo synthesis [via endoplasmic reticulum - or plastid-localized routes] is not a major contributor.

Distinguishing Phosphatidic Acid Pools from De Novo Synthesis, PLD, and DGK

In plants, phosphatidic acid (PA) functions as a metabolic precursor in the biosynthesis of glycerolipids, but it also acts as a key signaling lipid in the response to environmental stress conditions (Testerink and Munnik, J Exp Bot 62:2349-2361, 2011). In vivo (32)P-radiolabeling assays have shown the level of PA to increase within seconds/minutes of exposure to a stimulus. This response can be due to the activity of diacylglycerol kinase (DGK) and/or phospholipase D (PLD). A method is described to investigate which of the pathways is responsible for PA accumulation under a particular stress condition. First, a differential (32)P-radiolabeling protocol is used to discriminate (32)P-PA pools that are rapidly labeled versus those requiring long prelabeling times, reflecting DGK and PLD activities, respectively. Second, to specifically monitor the contribution of PLD, a transphosphatidylation assay is applied, which makes use of the artificial lipid phosphatidylbutanol as an in vivo marker of PLD activity.

Diacylglycerol acyltransferase 1 contributes to freezing tolerance

ACYL-COA:DIACYLGLYCEROL ACYLTRANSFERASE1 confers freezing tolerance, and its expression and activity could underlie adaptive variation in freezing tolerance in Boechera stricta, a wild relative of Arabidopsis thaliana. Freezing limits plant growth and crop productivity, and plant species in temperate zones have the capacity to develop freezing tolerance through complex modulation of gene expression affecting various aspects of metabolism and physiology. While many components of freezing tolerance have been identified in model species under controlled laboratory conditions, little is known about the mechanisms that impart freezing tolerance in natural populations of wild species. Here, we performed a quantitative trait locus (QTL) study of acclimated freezing tolerance in seedlings of Boechera stricta, a highly adapted relative of Arabidopsis (Arabidopsis thaliana) native to the Rocky Mountains. A single QTL was identified that contained the gene encoding ACYL-COENZYME A:DIACYLGLYCEROL ACYLTRANSFERASE1 (BstDGAT1), whose expression is highly cold responsive. The primary metabolic enzyme DGAT1 catalyzes the final step in assembly of triacylglycerol (TAG) by acyl transfer from acyl-CoA to diacylglycerol. Freezing tolerant plants showed higher DGAT1 expression during cold acclimation than more sensitive plants, and this resulted in increased accumulation of TAG in response to subsequent freezing. Levels of oligogalactolipids that are produced by SFR2 (SENSITIVE TO FREEZING2), an indispensable element of freezing tolerance in Arabidopsis, were also higher in freezing-tolerant plants. Furthermore, overexpression of AtDGAT1 led to increased freezing tolerance. We propose that DGAT1 confers freezing tolerance in plants by supporting SFR2-mediated remodeling of chloroplast membranes.

Use of phospholipase A2 for the production of lysophospholipids.

Biological lipid extracts often contain small amounts of lysophospholipids (LPLs). Since different functions are emerging for LPLs in lipid metabolism and signalling, there is need for a reliable and cost-effective method for their identification. For this purpose, authentic LPL standards have to be synthesized from phosphoglycerides by PLA2 digestion in vitro. PLA2 specifically hydrolyzes the fatty acid ester linkage in the sn-2-position of phospholipids to liberate sn-2-linked fatty acids and the corresponding LPL. Due to this specificity, the reaction is also useful to analyze the positional distribution of fatty acids within membrane phospholipids. This chapter describes the in vitro generation of LPLs from diacyl-phosphoglycerides and their TLC analysis.