Arabidopsis PROTODERMAL FACTOR2 binds lysophosphatidylcholines and transcriptionally regulates phospholipid metabolism.

Plant homeodomain leucine zipper IV (HD-Zip IV) transcription factors (TFs) contain an evolutionarily conserved steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain. While the START domain is required for TF activity, its presumed role as a lipid sensor is not clear. Here we used tandem affinity purification from Arabidopsis cell cultures to demonstrate that PROTODERMAL FACTOR2 (PDF2), a representative member that controls epidermal differentiation, recruits lysophosphatidylcholines (LysoPCs) in a START-dependent manner. Microscale thermophoresis assays confirmed that a missense mutation in a predicted ligand contact site reduces lysophospholipid binding. We additionally found that PDF2 acts as a transcriptional regulator of phospholipid- and phosphate (Pi) starvation-related genes and binds to a palindromic octamer with consensus to a Pi response element. Phospholipid homeostasis and elongation growth were altered in pdf2 mutants according to Pi availability. Cycloheximide chase experiments revealed a role for START in maintaining protein levels, and Pi starvation resulted in enhanced protein destabilization, suggesting a mechanism by which lipid binding controls TF activity. We propose that the START domain serves as a molecular sensor for membrane phospholipid status in the epidermis. Our data provide insights toward understanding how the lipid metabolome integrates Pi availability with gene expression.

Natural Variation among Arabidopsis Accessions in the Regulation of Flavonoid Metabolism and Stress Gene Expression by Combined UV Radiation and Cold.

Plants are constantly exposed to stressful environmental conditions. Plant stress reactions were mainly investigated for single stress factors. However, under natural conditions plants may be simultaneously exposed to different stresses. Responses to combined stresses cannot be predicted from the reactions to the single stresses. Flavonoids accumulate in Arabidopsis thaliana during exposure to UV-A, UV-B or cold, but the interactions of these factors on flavonoid biosynthesis were unknown. We therefore investigated the interaction of UV radiation and cold in regulating the expression of well-characterized stress-regulated genes, and on transcripts and metabolites of the flavonoid biosynthetic pathway in 52 natural Arabidopsis accessions that differ widely in their freezing tolerance. The data revealed interactions of cold and UV on the regulation of stress-related and flavonoid biosynthesis genes, and on flavonoid composition. In many cases, plant reactions to a combination of cold and UV were unique under combined stress and not predictable from the responses to the single stresses. Strikingly, all correlations between expression levels of flavonoid biosynthesis genes and flavonol levels were abolished by UV-B exposure. Similarly, correlations between transcript levels of flavonoid biosynthesis genes or flavonoid contents, and freezing tolerance were lost in the presence of UV radiation, while correlations with the expression levels of cold-regulated genes largely persisted. This may indicate different molecular cold acclimation responses in the presence or absence of UV radiation.

Cell wall modification by the xyloglucan endotransglucosylase/hydrolase XTH19 influences freezing tolerance after cold and sub-zero acclimation.

Freezing triggers extracellular ice formation leading to cell dehydration and deformation during a freeze-thaw cycle. Many plant species increase their freezing tolerance during exposure to low, non-freezing temperatures, a process termed cold acclimation. In addition, exposure to mild freezing temperatures after cold acclimation evokes a further increase in freezing tolerance (sub-zero acclimation). Previous transcriptome and proteome analyses indicate that cell wall remodelling may be particularly important for sub-zero acclimation. In the present study, we used a combination of immunohistochemical, chemical and spectroscopic analyses to characterize the cell walls of Arabidopsis thaliana and characterized a mutant in the XTH19 gene, encoding a xyloglucan endotransglucosylase/hydrolase (XTH). The mutant showed reduced freezing tolerance after both cold and sub-zero acclimation, compared to the Col-0 wild type, which was associated with differences in cell wall composition and structure. Most strikingly, immunohistochemistry in combination with 3D reconstruction of centres of rosette indicated that epitopes of the xyloglucan-specific antibody LM25 were highly abundant in the vasculature of Col-0 plants after sub-zero acclimation but absent in the XTH19 mutant. Taken together, our data shed new light on the potential roles of cell wall remodelling for the increased freezing tolerance observed after low temperature acclimation.

Target of Rapamycin Inhibition in Chlamydomonas reinhardtii Triggers de Novo Amino Acid Synthesis by Enhancing Nitrogen Assimilation.

The Target of Rapamycin (TOR) kinase is a central regulator of growth and metabolism in all eukaryotic organisms, including animals, fungi, and plants. Even though the inputs and outputs of TOR signaling are well characterized for animals and fungi, our understanding of the upstream regulators of TOR and its downstream targets is still fragmentary in photosynthetic organisms. In this study, we employed the rapamycin-sensitive green alga Chlamydomonas reinhardtii to elucidate the molecular cause of the amino acid accumulation that occurs after rapamycin-induced inhibition of TOR. Using different growth conditions and stable 13C- and 15N-isotope labeling, we show that this phenotype is accompanied by increased nitrogen (N) uptake, which is induced within minutes of TOR inhibition. Interestingly, this increased N influx is accompanied by increased activities of glutamine synthetase and glutamine oxoglutarate aminotransferase, the main N-assimilating enzymes, which are responsible for the rise in levels of several amino acids, which occurs within a few minutes. Accordingly, we conclude that even though translation initiation and autophagy have been reported to be the main downstream targets of TOR, the upregulation of de novo amino acid synthesis seems to be one of the earliest responses induced after the inhibition of TOR in Chlamydomonas.

Characterization of the Heat-Stable Proteome during Seed Germination in Arabidopsis with Special Focus on LEA Proteins.

During seed germination, desiccation tolerance is lost in the radicle with progressing radicle protrusion and seedling establishment. This process is accompanied by comprehensive changes in the metabolome and proteome. Germination of Arabidopsis seeds was investigated over 72 h with special focus on the heat-stable proteome including late embryogenesis abundant (LEA) proteins together with changes in primary metabolites. Six metabolites in dry seeds known to be important for seed longevity decreased during germination and seedling establishment, while all other metabolites increased simultaneously with activation of growth and development. Thermo-stable proteins were associated with a multitude of biological processes. In the heat-stable proteome, a relatively similar proportion of fully ordered and fully intrinsically disordered proteins (IDP) was discovered. Highly disordered proteins were found to be associated with functional categories development, protein, RNA and stress. As expected, the majority of LEA proteins decreased during germination and seedling establishment. However, four germination-specific dehydrins were identified, not present in dry seeds. A network analysis of proteins, metabolites and amino acids generated during the course of germination revealed a highly connected LEA protein network.

Characterisation of the ERF102 to ERF105 genes of Arabidopsis thaliana and their role in the response to cold stress.

KEY MESSAGE: The four phylogenetically closely related ERF102 to ERF105 transcription factors of Arabidopsis thaliana are regulated by different stresses and are involved in the response to cold stress. The ETHYLENE RESPONSE FACTOR (ERF) genes of Arabidopsis thaliana form a large family encoding plant-specific transcription factors. Here, we characterise the four phylogenetically closely related ERF102/ERF5, ERF103/ERF6, ERF104 and ERF105 genes. Expression analyses revealed that these four genes are similarly regulated by different hormones and abiotic stresses. Analyses of tissue-specific expression using promoter:GUS reporter lines revealed their predominant expression in root tissues including the root meristem (ERF103), the quiescent center (ERF104) and the root vasculature (all). All GFP-ERF fusion proteins were nuclear-localised. The analysis of insertional mutants, amiRNA lines and 35S:ERF overexpressing transgenic lines indicated that ERF102 to ERF105 have only a limited impact on regulating shoot and root growth. Previous work had shown a role for ERF105 in the cold stress response. Here, measurement of electrolyte leakage to determine leaf freezing tolerance and expression analyses of cold-responsive genes revealed that the combined activity of ERF102 and ERF103 is also required for a full cold acclimation response likely involving the CBF regulon. These results suggest a common function of these ERF genes in the response to cold stress.

Repair of sub-lethal freezing damage in leaves of Arabidopsis thaliana.

BACKGROUND: The detrimental effects of global climate change direct more attention to the survival and productivity of plants during periods of highly fluctuating temperatures. In particular in temperate climates in spring, temperatures can vary between above-zero and freezing temperatures, even during a single day. Freeze-thaw cycles cause cell membrane lesions that can lead to tissue damage and plant death. Whereas the processes of cold acclimation and freeze-thaw injury are well documented, not much is known about the recovery of plants after a freezing event. We therefore addressed the following questions: i. how does the severity of freezing damage influence repair; ii. how are respiration and content of selected metabolites influenced during the repair process; and iii. how do transcript levels of selected genes respond during repair? RESULTS: We have investigated the recovery from freezing to sub-lethal temperatures in leaves of non-acclimated and cold acclimated Arabidopsis thaliana plants over a period of 6 days. Fast membrane repair and recovery of photosynthesis were observed 1 day after recovery (1D-REC) and continued until 6D-REC. A substantial increase in respiration accompanied the repair process. In parallel, concentrations of sugars and proline, acting as compatible solutes during freezing, remained unchanged or declined, implicating these compounds as carbon and nitrogen sources during recovery. Similarly, cold-responsive genes were mainly down regulated during recovery of cold acclimated leaves. In contrast, genes involved in cell wall remodeling and ROS scavenging were induced during recovery. Interestingly, also the expression of genes encoding regulatory proteins, such as 14-3-3 proteins, was increased suggesting their role as regulators of repair processes. CONCLUSIONS: Recovery from sub-lethal freezing comprised membrane repair, restored photosynthesis and increased respiration rates. The process was accompanied by transcriptional changes including genes encoding regulatory proteins redirecting the previous cold response to repair processes, e.g. to cell wall remodeling, maintenance of the cellular proteome and to ROS scavenging. Understanding of processes involved in repair of freeze-thaw injury increases our knowledge on plant survival in changing climates with highly fluctuating temperatures.

Evaluation of Seven Different RNA-Seq Alignment Tools Based on Experimental Data from the Model Plant Arabidopsis thaliana.

Quantification of gene expression is crucial to connect genome sequences with phenotypic and physiological data. RNA-Sequencing (RNA-Seq) has taken a prominent role in the study of transcriptomic reactions of plants to various environmental and genetic perturbations. However, comparative tests of different tools for RNA-Seq read mapping and quantification have been mainly performed on data from animals or humans, which necessarily neglect, for example, the large genetic variability among natural accessions within plant species. Here, we compared seven computational tools for their ability to map and quantify Illumina single-end reads from the Arabidopsis thaliana accessions Columbia-0 (Col-0) and N14. Between 92.4% and 99.5% of all reads were mapped to the reference genome or transcriptome and the raw count distributions obtained from the different mappers were highly correlated. Using the software DESeq2 to determine differential gene expression (DGE) between plants exposed to 20 °C or 4 °C from these read counts showed a large pairwise overlap between the mappers. Interestingly, when the commercial CLC software was used with its own DGE module instead of DESeq2, strongly diverging results were obtained. All tested mappers provided highly similar results for mapping Illumina reads of two polymorphic Arabidopsis accessions to the reference genome or transcriptome and for the determination of DGE when the same software was used for processing.

Similar Yet Different-Structural and Functional Diversity among Arabidopsis thaliana LEA_4 Proteins.

The importance of intrinsically disordered late embryogenesis abundant (LEA) proteins in the tolerance to abiotic stresses involving cellular dehydration is undisputed. While structural transitions of LEA proteins in response to changes in water availability are commonly observed and several molecular functions have been suggested, a systematic, comprehensive and comparative study of possible underlying sequence-structure-function relationships is still lacking. We performed molecular dynamics (MD) simulations as well as spectroscopic and light scattering experiments to characterize six members of two distinct, lowly homologous clades of LEA_4 family proteins from Arabidopsis thaliana. We compared structural and functional characteristics to elucidate to what degree structure and function are encoded in LEA protein sequences and complemented these findings with physicochemical properties identified in a systematic bioinformatics study of the entire Arabidopsis thaliana LEA_4 family. Our results demonstrate that although the six experimentally characterized LEA_4 proteins have similar structural and functional characteristics, differences concerning their folding propensity and membrane stabilization capacity during a freeze/thaw cycle are obvious. These differences cannot be easily attributed to sequence conservation, simple physicochemical characteristics or the abundance of sequence motifs. Moreover, the folding propensity does not appear to be correlated with membrane stabilization capacity. Therefore, the refinement of LEA_4 structural and functional properties is likely encoded in specific patterns of their physicochemical characteristics.

Utilizing PacBio Iso-Seq for Novel Transcript and Gene Discovery of Abiotic Stress Responses in Oryza sativa L.

The wide natural variation present in rice is an important source of genes to facilitate stress tolerance breeding. However, identification of candidate genes from RNA-Seq studies is hampered by the lack of high-quality genome assemblies for the most stress tolerant cultivars. A more targeted solution is the reconstruction of transcriptomes to provide templates to map RNA-seq reads. Here, we sequenced transcriptomes of ten rice cultivars of three subspecies on the PacBio Sequel platform. RNA was isolated from different organs of plants grown under control and abiotic stress conditions in different environments. Reconstructed de novo reference transcriptomes resulted in 37,500 to 54,600 plant-specific high-quality isoforms per cultivar. Isoforms were collapsed to reduce sequence redundancy and evaluated, e.g., for protein completeness (BUSCO). About 40% of all identified transcripts were novel isoforms compared to the Nipponbare reference transcriptome. For the drought/heat tolerant aus cultivar N22, 56 differentially expressed genes in developing seeds were identified at combined heat and drought in the field. The newly generated rice transcriptomes are useful to identify candidate genes for stress tolerance breeding not present in the reference transcriptomes/genomes. In addition, our approach provides a cost-effective alternative to genome sequencing for identification of candidate genes in highly stress tolerant genotypes.

Season Affects Yield and Metabolic Profiles of Rice (Oryza sativa) under High Night Temperature Stress in the Field.

Rice (Oryza sativa) is the main food source for more than 3.5 billion people in the world. Global climate change is having a strong negative effect on rice production. One of the climatic factors impacting rice yield is asymmetric warming, i.e., the stronger increase in nighttime as compared to daytime temperatures. Little is known of the metabolic responses of rice to high night temperature (HNT) in the field. Eight rice cultivars with contrasting HNT sensitivity were grown in the field during the wet (WS) and dry season (DS) in the Philippines. Plant height, 1000-grain weight and harvest index were influenced by HNT in both seasons, while total grain yield was only consistently reduced in the WS. Metabolite composition was analysed by gas chromatography-mass spectrometry (GC-MS). HNT effects were more pronounced in panicles than in flag leaves. A decreased abundance of sugar phosphates and sucrose, and a higher abundance of monosaccharides in panicles indicated impaired glycolysis and higher respiration-driven carbon losses in response to HNT in the WS. Higher amounts of alanine and cyano-alanine in panicles grown in the DS compared to in those grown in the WS point to an improved N-assimilation and more effective detoxification of cyanide, contributing to the smaller impact of HNT on grain yield in the DS.

Plant Temperature Acclimation and Growth Rely on Cytosolic Ribosome Biogenesis Factor Homologs.

Arabidopsis (Arabidopsis thaliana) REI1-LIKE (REIL) proteins, REIL1 and REIL2, are homologs of a yeast ribosome biogenesis factor that participates in late cytoplasmic 60S ribosomal subunit maturation. Here, we report that the inhibited growth of the reil1-1 reil2-1 mutant at 10°C can be rescued by the expression of amino-terminal FLUORESCENT PROTEIN (FP)-REIL fusions driven by the UBIQUITIN10 promoter, allowing the analysis of REIL function in planta. Arabidopsis REIL1 appears to be functionally conserved, based on the cytosolic localization of FP-REIL1 and the interaction of native REIL1 with the 60S subunit in wild-type plants. In contrast to its yeast homologs, REIL1 also was present in translating ribosome fractions. Systems analysis revealed that wild-type Arabidopsis remodels the cytosolic translation machinery when grown at 10°C by accumulating cytosolic ribosome subunits and inducing the expression of cytosolic ribosomal RNA, ribosomal genes, ribosome biogenesis factors, and translation initiation or elongation factors. In the reil1-1 reil2-1 mutant, all processes associated with inhibited growth were delayed, although the plants maintained cellular integrity or acquired freezing tolerance. REIL proteins also were implicated in plant-specific processes: nonacclimated reil1-1 reil2-1 exhibited cold-acclimation responses, including activation of the DREB/CBF regulon. In addition, acclimated reil1-1 reil2-1 plants failed to activate FLOWERING LOCUS T expression in mature leaves. Therefore, in the wild type, REIL function may contribute to temperature perception by suppressing premature cold responses during growth at nonstressful temperatures. In conclusion, we suggest that Arabidopsis REIL proteins influence cold-induced plant ribosome remodeling and enhance the accumulation of cytosolic ribosome subunits after cold shift either by de novo synthesis or by recycling them from the translating ribosome fraction.

Molecular signatures associated with increased freezing tolerance due to low temperature memory in Arabidopsis.

Alternating temperatures require fast and coordinated adaptation responses of plants. Cold acclimation has been extensively investigated and results in increased freezing tolerance in Arabidopsis thaliana. Here, we show that the two Arabidopsis accessions, Col-0 and N14, which differ in their freezing tolerance, showed memory of cold acclimation, that is, cold priming. Freezing tolerance was higher in plants exposed to cold priming at 4°C, a lag phase at 20°C, and a second triggering cold stress (4°C) than in plants that were only cold primed. To our knowledge, this is the first report on cold memory improving plant freezing tolerance. The triggering response was distinguishable from the priming response at the levels of gene expression (RNA-Seq), lipid (ultraperformance liquid chromatography-mass spectrometry), and metabolite composition (gas chromatography-mass spectrometry). Transcriptomic responses pointed to induced lipid, secondary metabolism, and stress in Col-0 and growth-related functions in N14. Specific accumulation of lipids included arabidopsides with possible functions as signalling molecules or precursors of jasmonic acid. Whereas cold-induced metabolites such as raffinose and its precursors were maintained in N14 during the lag phase, they were strongly accumulated in Col-0 after the cold trigger. This indicates genetic differences in the transcriptomic and metabolic patterns during cold memory.

Deacclimation after cold acclimation-a crucial, but widely neglected part of plant winter survival.

Temperate and boreal plants show natural low temperature acclimation during autumn. This cold acclimation process results in increased freezing tolerance. Global climate change is leading to increasing spring and autumn temperatures that can trigger deacclimation and loss of freezing tolerance, making plants susceptible to both late-autumn and late-spring freezing events. In particular, spring frosts can have devastating effects on whole ecosystems and can significantly reduce the yield of crop plants. Although the timing and speed of deacclimation are clearly of crucial importance for plant winter survival, the molecular basis of this process is still largely unknown. The regulation of deacclimation is, however, not only related to freezing tolerance, but also to the termination of dormancy, and the initiation of growth and development. In this paper, we provide an overview of what is known about deacclimation in both woody and herbaceous plants. We use publicly available transcriptome data to identify a core set of deacclimation-related genes in Arabidopsis thaliana that highlight physiological determinants of deacclimation, and suggest important directions for future research in this area.

Conformational selection of the intrinsically disordered plant stress protein COR15A in response to solution osmolarity - an X-ray and light scattering study.

The plant stress protein COR15A stabilizes chloroplast membranes during freezing. COR15A is an intrinsically disordered protein (IDP) in aqueous solution, but acquires an α-helical structure during dehydration or the increase of solution osmolarity. We have used small- and wide-angle X-ray scattering (SAXS/WAXS) combined with static and dynamic light scattering (SLS/DLS) to investigate the structural and hydrodynamic properties of COR15A in response to increasing solution osmolarity. Coarse-grained ensemble modelling allowed a structure-based interpretation of the SAXS data. Our results demonstrate that COR15A behaves as a biomacromolecule with polymer-like properties which strongly depend on solution osmolarity. Biomacromolecular self-assembly occurring at high solvent osmolarity is initiated by the occurrence of two specific structural subpopulations of the COR15A monomer. The osmolarity dependent structural selection mechanism is an elegant way for conformational regulation and assembly of COR15A. It highlights the importance of the polymer-like properties of IDPs for their associated biological function.

Folding and Lipid Composition Determine Membrane Interaction of the Disordered Protein COR15A.

Plants from temperate climates, such as the model plant Arabidopsis thaliana, are challenged with seasonal low temperatures that lead to increased freezing tolerance in fall in a process termed cold acclimation. Among other adaptations, this involves the accumulation of cold-regulated (COR) proteins, such as the intrinsically disordered chloroplast-localized protein COR15A. Together with its close homolog COR15B, it stabilizes chloroplast membranes during freezing. COR15A folds into amphipathic α-helices in the presence of high concentrations of low-molecular-mass crowders or upon dehydration. Under these conditions, the (partially) folded protein binds peripherally to membranes. In our study, we have used coarse-grained molecular dynamics simulations to elucidate the details of COR15A-membrane binding and its effects on membrane structure and dynamics. Simulation results indicate that at least partial folding of COR15A and the presence of highly unsaturated galactolipids in the membranes are necessary for efficient membrane binding. The bound protein is stabilized on the membrane by interactions of charged and polar amino acids with galactolipid headgroups and by interactions of hydrophobic amino acids with the upper part of the fatty acyl chains. Experimentally, the presence of liposomes made from a mixture of lipids mimicking chloroplast membranes induces additional folding in COR15A under conditions of partial dehydration, in agreement with the simulation results.

Metabolite and transcript markers for the prediction of potato drought tolerance.

Potato (Solanum tuberosum L.) is one of the most important food crops worldwide. Current potato varieties are highly susceptible to drought stress. In view of global climate change, selection of cultivars with improved drought tolerance and high yield potential is of paramount importance. Drought tolerance breeding of potato is currently based on direct selection according to yield and phenotypic traits and requires multiple trials under drought conditions. Marker-assisted selection (MAS) is cheaper, faster and reduces classification errors caused by noncontrolled environmental effects. We analysed 31 potato cultivars grown under optimal and reduced water supply in six independent field trials. Drought tolerance was determined as tuber starch yield. Leaf samples from young plants were screened for preselected transcript and nontargeted metabolite abundance using qRT-PCR and GC-MS profiling, respectively. Transcript marker candidates were selected from a published RNA-Seq data set. A Random Forest machine learning approach extracted metabolite and transcript markers for drought tolerance prediction with low error rates of 6% and 9%, respectively. Moreover, by combining transcript and metabolite markers, the prediction error was reduced to 4.3%. Feature selection from Random Forest models allowed model minimization, yielding a minimal combination of only 20 metabolite and transcript markers that were successfully tested for their reproducibility in 16 independent agronomic field trials. We demonstrate that a minimum combination of transcript and metabolite markers sampled at early cultivation stages predicts potato yield stability under drought largely independent of seasonal and regional agronomic conditions.

Natural Variation in Freezing Tolerance and Cold Acclimation Response in Arabidopsis thaliana and Related Species.

During low-temperature exposure, temperate plant species increase their freezing tolerance in a process termed cold acclimation. The molecular mechanisms involved in cold acclimation have been mostly investigated in Arabidopsis thaliana. In addition, other Brassicaceae species related to A. thaliana have been employed in recent years to study plant stress responses on a phylogenetically broader basis and in some cases with extremophile species with a much higher stress tolerance. In this paper, we briefly summarize cold acclimation responses in A. thaliana and current knowledge about cold acclimation in A. thaliana relatives with special emphasis on Eutrema salsugineum and two closely related Thellungiella species. We then present a transcriptomic and metabolomic analysis of cold acclimation in five A. thaliana and two E. salsugineum accessions that differ widely in their freezing tolerance. Differences in the cold responses of the two species are discussed.

Intrinsically Disordered Stress Protein COR15A Resides at the Membrane Surface during Dehydration.

Plants from temperate climate zones are able to increase their freezing tolerance during exposure to low, above-zero temperatures in a process termed cold acclimation. During this process, several cold-regulated (COR) proteins are accumulated in the cells. One of them is COR15A, a small, intrinsically disordered protein that contributes to leaf freezing tolerance by stabilizing cellular membranes. The isolated protein folds into amphipathic α-helices in response to increased crowding conditions, such as high concentrations of glycerol. Although there is evidence for direct COR15A-membrane interactions, the orientation and depth of protein insertion were unknown. In addition, although folding due to high osmolyte concentrations had been established, the folding response of the protein under conditions of gradual dehydration had not been investigated. Here we show, using Fourier transform infrared spectroscopy, that COR15A starts to fold into α-helices already under mild dehydration conditions (97% relative humidity (RH), corresponding to freezing at -3°C) and that folding gradually increases with decreasing RH. Neutron diffraction experiments at 97 and 75% RH established that the presence of COR15A had no significant influence on the structure of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes. However, using deuterated POPC we could clearly establish that COR15A interacts with the membranes and penetrates below the headgroup region into the upper part of the fatty acyl chain region. This localization is in agreement with our hypothesis that COR15A-membrane interaction is at least, in part, driven by a hydrophobic interaction between the lipids and the hydrophobic face of the amphipathic protein α-helix.

Effects of flavonol glycosides on liposome stability during freezing and drying.

Flavonoids are a large and diverse group of plant secondary metabolites that are mainly present as glycosides. They are often accumulated in response to abiotic stresses such as UV radiation, drought, cold and freezing. The most extensively studied function of flavonoids is their antioxidant activity although their importance as antioxidants in plants has been questioned. We therefore aim to study effects of flavonols on cellular stress tolerance that are independent of their antioxidant function. Here we investigate the effects of the glycosylated flavonols kaempferol-3-O-glucoside, kaempferol-7-O-glucoside, quercetin-3-O-glucoside and quercetin-3-O-rhamnoside on liposome stability after freezing and drying. Insertion of flavonols in lipid bilayers destabilized egg phosphatidylcholine (EPC) liposomes and to a lesser extent vesicles made from equal proportions of EPC and egg phosphatidylethanolamine (EPE) during a freeze-thaw cycle, while liposomes containing the unsaturated non-bilayer lipid 18:2 PE were either unaffected or slightly stabilized. In general, the kaempferol derivatives were more destabilizing for liposomes during freezing than the quercetin derivatives. Fourier-transform infrared spectroscopy revealed that all flavonols were localized in the interfacial region of the lipid bilayers, forming H-bonds with the lipid phosphate and carbonyl groups. The phase transition temperature of dry 16:0/18:1 PC (POPC) and POPC/EPE liposomes was decreased by 75°C and 55°C, respectively. Changes in the vibration bands attributed to the phenolic ring structures of the flavonols in the presence of liposomes provided further evidence of interactions of these molecules in particular with the interfacial region of the bilayers.