Alain Zachowski

Cold signalling and cold acclimation in plants

Exposure to low temperatures is one of the most important plant abiotic stress factors. In this review we describe the damages that chilling and/or freezing temperatures can cause to plant cells. Confronted to these damages, some plants are able to adapt through mechanisms based on protein synthesis, membrane composition changes, and activation of active oxygen scavenging systems. These adaptive mechanisms rely in part on gene induction. The best understood genetic pathway leading to gene induction upon a temperature downshift is based on C-repeat-binding factors (CBF) activating promoters through the C-repeat (CRT) cis-element. Such activation of transcription factors suggests that cold, as a signal, has been transduced into the cells. Calcium entry is a major signalling event occurring immediately after a temperature downshift. The increase in cytosolic calcium will activate many enzymes, such as phospholipases and calcium dependent-protein kinases. A MAP-kinase module has been shown to be involved in the cold response. Ultimately, the activation of those signalling pathways leads to changes to the transcriptome. In this review we have focused on the genetic and signalling pathways activated early after cold exposure. Much of the data cited is from the model plant Arabidopsis but when possible evidence from other plants is presented.

Desaturase mutants reveal that membrane rigidification acts as a cold perception mechanism upstream of the diacylglycerol kinase pathway in Arabidopsis cells

Membrane rigidification could be the first step of cold perception in poikilotherms. We have investigated its implication in diacylglycerol kinase (DAGK) activation by cold stress in suspension cells from Arabidopsis mutants altered in desaturase activities. By lateral diffusion assay, we showed that plasma membrane rigidification with temperature decrease was steeper in cells deficient in oleate desaturase than in wild type cells and in cells overexpressing linoleate desaturase. The threshold for the activation of the DAGK pathway in each type of cells correlated with this order of rigidification rate, suggesting that cold induced‐membrane rigidification is upstream of DAGK pathway activation.

Regulation of phosphatidylcholine biosynthesis under salt stress involves choline kinases in Arabidopsis thaliana

Increasing evidence suggests a major role for phosphatidylcholine (PC) in plant stress adaptation. The present work investigated the regulation of choline, PC and interconnected phosphatidylethanolamine biosynthesis in Arabidopsis thaliana L. as a function of cold‐ and salt‐ or mannitol‐mediated hyperosmotic stresses. While PC synthesis is accelerated in both salt‐ and cold‐treated plants, the choline kinase (CK) and phosphocholine cytidylyltransferase genes are oppositely regulated with respect to these abiotic treatments. Salt stress also stimulates CK activity in vitro. A possible regulatory role of CK in stimulating PC biosynthesis rate in salt‐stressed plants is discussed.

Signal transduction pathways involving phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate: Convergences and divergences among eukaryotic kingdoms

Phosphoinositides are minor constituents of eukaryotic membranes but participate in a wide range of cellular processes. The most abundant and best characterized phosphoinositide species are phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) and its main precursor, phosphatidylinositol 4-phosphate (PI4P). PI4P and PI(4,5)P₂ regulate various structural and developmental functions but are also centrally involved in a plethora of signal transduction pathways in all eukaryotic models. They are not only precursors of second messengers but also directly interact with many protein effectors, thus regulating their localisation and/or activity. Furthermore, the discovery of independent PI(4,5)P₂ signalling functions in the nucleus of mammalian cells have open a new perspective in the field. Striking similarities between mammalian, yeast and higher plant phosphoinositide signalling are noticeable, revealing early appearance and evolutionary conservation of this intracellular language. However, major differences have also been highlighted over the years, suggesting that organisms may have evolved different PI4P and PI(4,5)P₂ functions over the course of eukaryotic diversification. Comparative studies of the different eukaryotic models is thus crucial for a comprehensive view of this fascinating signalling system. The present review aims to emphasize convergences and divergences between eukaryotic kingdoms in the mechanisms underlying PI4P and PI(4,5)P₂ roles in signal transduction, in response to extracellular stimuli.

Lipid-transfer proteins from plants: Structure and binding properties

Plant cells contain lipid-transfer proteins (LTPs) able to transfer phospholipids between membranes in vitro. Plant LTPs share in common structural and functional features. Recent structural studies carried out by NMR and X-ray crystallography on an LTP isolated from maize seeds have showed that this protein involves four helices packed against a C-terminal region and stabilized by four disulfide bridges. A most striking feature of this structure is the existence of an internal hydrophobic cavity running through the whole molecule and able to accomodate acyl chains. It was thus of interest to study the ability of maize LTP to bind hydrophobic ligands such as acyl chains or lysophosphatidylcholine and to determine the effect of this binding on phospholipid transfer. The binding abilities of maize LTP, presented in this paper, are discussed and compared to those of lipid-binding proteins from animal tissues.

A New Method for Accurately Measuring Δ 1 -Pyrroline-5-Carboxylate Synthetase Activity

Proline is a key factor in plant adaptation to environmental stresses. The Delta(1)-pyrroline-5-carboxylate synthetase catalyzes the first committed step and the rate-limiting step for proline biosynthesis in both plants and mammals. This enzyme catalyzes the reduction of glutamate to pyrroline-5-carboxylate in two sequential steps including the phosphorylation and the reduction of its precursor. Several methods were established to assay P5CS activity but however none of them are fully reliable. Therefore, we developed a new simple and reliable assay which is based on the quantification of Pi. This assay allowed us to determine the optimal pH, the apparent K(m) and V(m) of P5CS with regard to ATP and glutamate.

Over-expression of the Anti-apoptotic Protein Bcl-2 Affects Membrane Lipid Composition in HL-60 Cells

We studied modifications induced at the membrane lipid level by over-expression of the anti-apoptotic protein Bcl-2. When total cell phospholipids were analyzed, the transformation led to a moderate decrease in poly-unsaturated fatty acids, compensated by an increase in mono-unsaturated species. At the mitochondrial membrane level, the changes were more important and occurred in saturated and dimethyl acetal fatty acids, which became more abundant, while unsaturated fatty acid content diminished. This indicates a decline in oxidation-sensitive fatty acids (unsaturated species) together with a gain in oxidation-insensitive saturated fatty acids and in plasmalogen (as detected by dimethyl acetal fatty acids) considered as oxygen species scavengers. Theses changes, combined with the protective role of Bcl-2 against oxidation due to its effect on the redox potential, should protect cells from apoptosis starting in mitochondria.

Purification and characterization of phosphate carrier from potato mitochondria

Abstract The phosphate carrier of the inner mitochondrial membrane has been purified from potato tuber in a nearly homogenous form and characterized after reconstitution into liposomes. The mitochondrial membrane was solubilised by Triton X-114 in the presence of cardiolipin and the soluble fraction was chromatographied on hydroxylapatite. The flow-through fractions were collected and analyzed by SDS-gel electrophoresis. In some fractions, a single protein band of Mr=35 kDa was found. This protein was reconstituted into phosphatidylcholine vesicles and its capability to transport phosphate was measured by phosphate–phosphate exchange. A maximum velocity of 28.1 μmol min−1. (mg of protein)−1 was detected. Phosphate transport was sensitive to mersalyl and N-ethylmaleimide. On the other hand, no proton transport was apparent in these proteoliposomes, unlike to what was seen with similar hydroxylapatite-purified fractions obtained after solubilization of the membrane by decyl-pentaoxyethylene ether, which contained several polypeptides. Thus the phosphate carrier is distinct from the uncoupling protein (UCP) family and its behavior shows that not all anion carriers are able to act as UCPs.

Genetically Modified Arabidopsis thaliana Cells Reveal the Involvement of the Mitochondrial Fatty Acid Composition in Membrane Basal and Uncoupling Protein-Mediated Proton Leaks

We investigated the role of membrane fatty acids in basal proton leaks and uncoupling protein (UCP)-dependent proton conductance in Arabidopsis mitochondria. Using wild-type cells, cold-sensitive fad2 mutant cells, deficient in omega-6-oleate desaturase, and cold-tolerant FAD3(+) transformant cells, overexpressing omega-3-linoleate desaturase, we showed that basal proton leak in the non-phosphorylating state was dependent on lipid composition. The extent of membrane proton leak was drastically reduced in the fad2 mutant, containing low amounts of polyunsaturated fatty acids. Conversely, this proton leak was higher in FAD3(+) mitochondria that exhibit a higher polyunsaturated fatty acid content and high protein to lipid ratio. The dependency of membrane leaks upon membrane potential was higher in FAD3(+) and lower in fad2. UCP content was higher in both the fad2 mutant and FAD3(+) transgenic lines compared with wild-type cells and so was the UCP activity, assayed by the reduction of phosphorylation yield (ADP/O) triggered by palmitate as UCP activator. This UCP assay was validated by measurements of UCP-proton leak in the non-phosphorylating state (flux-force relationships between proton flux and membrane potential). The potential uncoupling capacity of the UCP was high enough to allow the loss of respiratory control in the three genotypes. Taken together, the data reported here suggest that the cold tolerance of FAD3(+) cells and the cold sensitivity of fad2 cells are associated with changes in their mitochondrial membrane basal proton leaks, whereas differences in functional expression of UCP are not simply related to cold adaptation in Arabidopsis cells.

Changes in electron transport pathways in endoplasmic reticulum of rapeseed in response to cold

We studied changes induced by cold on electron transfer pathways (linked to NADH or NADPH oxidation) in endoplasmic reticulum of rapeseed hypocotyls (Brassica napus L.) from a freezing-sensitive variety (ISL) and freezing-tolerant variety (Tradition). Plantlets were grown at 22°C then submitted to a cold shock of 13 or 35xa0days at 4°C. We measured the content in NADH, NADPH, NAD and NADP of the hypocotyls and the redox power was estimated by the reduced versus oxidized nucleotide ratio. The contents in cytochromes b5 and P-450, electron acceptors of NADH and NADPH respectively, were determined by differential spectrophotometry. Finally, activity of both NADH–cytochrome b5 reductase (E.C.1.6.2.2) and NADPH cytochrome P-450 reductase (E.C.1.6.2.4) was determined by reduction of exogenous cytochrome c. Results show that during cold shock, along with an increase of linolenic acid content, there was a general activation of the NADPH pathway which was observed more quickly in Tradition plantlets than in ISL ones. Due to transfer of electrons that can occur between NADPH reductase and cytochrome b5, this could favor fatty acid desaturation in Tradition, explaining why linolenic acid accumulation was more pronounced in this variety. Besides, more cytochrome P-450 accumulated in ISL that could compete for electrons needed by the FAD3 desaturase, resulting in a relative slower enrichment in 18:3 fatty acid in these plantlets.