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1.  Changes in free polyamine levels, expression of polyamine biosynthesis genes, and performance of rice cultivars under salt stress: a comparison with responses to drought 
Soil salinity affects a large proportion of rural area and limits agricultural productivity. To investigate differential adaptation to soil salinity, we studied salt tolerance of 18 varieties of Oryza sativa using a hydroponic culture system. Based on visual inspection and photosynthetic parameters, cultivars were classified according to their tolerance level. Additionally, biomass parameters were correlated with salt tolerance. Polyamines have frequently been demonstrated to be involved in plant stress responses and therefore soluble leaf polyamines were measured. Under salinity, putrescine (Put) content was unchanged or increased in tolerant, while dropped in sensitive cultivars. Spermidine (Spd) content was unchanged at lower NaCl concentrations in all, while reduced at 100 mM NaCl in sensitive cultivars. Spermine (Spm) content was increased in all cultivars. A comparison with data from 21 cultivars under long-term, moderate drought stress revealed an increase of Spm under both stress conditions. While Spm became the most prominent polyamine under drought, levels of all three polyamines were relatively similar under salt stress. Put levels were reduced under both, drought and salt stress, while changes in Spd were different under drought (decrease) or salt (unchanged) conditions. Regulation of polyamine metabolism at the transcript level during exposure to salinity was studied for genes encoding enzymes involved in the biosynthesis of polyamines and compared to expression under drought stress. Based on expression profiles, investigated genes were divided into generally stress-induced genes (ADC2, SPD/SPM2, SPD/SPM3), one generally stress-repressed gene (ADC1), constitutively expressed genes (CPA1, CPA2, CPA4, SAMDC1, SPD/SPM1), specifically drought-induced genes (SAMDC2, AIH), one specifically drought-repressed gene (CPA3) and one specifically salt-stress repressed gene (SAMDC4), revealing both overlapping and specific stress responses under these conditions.
doi:10.3389/fpls.2014.00182
PMCID: PMC4021140  PMID: 24847340
polyamines; salt stress; drought stress; gene expression; rice; natural variety
2.  Transcriptome sequencing and microarray design for functional genomics in the extremophile Arabidopsis relative Thellungiella salsuginea (Eutrema salsugineum) 
BMC Genomics  2013;14:793.
Background
Most molecular studies of plant stress tolerance have been performed with Arabidopsis thaliana, although it is not particularly stress tolerant and may lack protective mechanisms required to survive extreme environmental conditions. Thellungiella salsuginea has attracted interest as an alternative plant model species with high tolerance of various abiotic stresses. While the T. salsuginea genome has recently been sequenced, its annotation is still incomplete and transcriptomic information is scarce. In addition, functional genomics investigations in this species are severely hampered by a lack of affordable tools for genome-wide gene expression studies.
Results
Here, we report the results of Thellungiella de novo transcriptome assembly and annotation based on 454 pyrosequencing and development and validation of a T. salsuginea microarray. ESTs were generated from a non-normalized and a normalized library synthesized from RNA pooled from samples covering different tissues and abiotic stress conditions. Both libraries yielded partially unique sequences, indicating their necessity to obtain comprehensive transcriptome coverage. More than 1 million sequence reads were assembled into 42,810 unigenes, approximately 50% of which could be functionally annotated. These unigenes were compared to all available Thellungiella genome sequence information. In addition, the groups of Late Embryogenesis Abundant (LEA) proteins, Mitogen Activated Protein (MAP) kinases and protein phosphatases were annotated in detail. We also predicted the target genes for 384 putative miRNAs. From the sequence information, we constructed a 44 k Agilent oligonucleotide microarray. Comparison of same-species and cross-species hybridization results showed superior performance of the newly designed array for T. salsuginea samples. The developed microarrays were used to investigate transcriptional responses of T. salsuginea and Arabidopsis during cold acclimation using the MapMan software.
Conclusions
This study provides the first comprehensive transcriptome information for the extremophile Arabidopsis relative T. salsuginea. The data constitute a more than three-fold increase in the number of publicly available unigene sequences and will greatly facilitate genome annotation. In addition, we have designed and validated the first genome-wide microarray for T. salsuginea, which will be commercially available. Together with the publicly available MapMan software this will become an important tool for functional genomics of plant stress tolerance.
doi:10.1186/1471-2164-14-793
PMCID: PMC3832907  PMID: 24228715
Arabidopsis thaliana; Cold acclimation; Gene annotation; LEA proteins; MAP kinases; Microarray design; microRNAs; Protein phosphatases; Thellungiella salsuginea; Transcriptome sequencing
3.  Interactions of the amphiphiles arbutin and tryptophan with phosphatidylcholine and phosphatidylethanolamine bilayers in the dry state 
BMC Biophysics  2013;6:9.
Background
Water is essential for life, but some organisms can survive complete desiccation, while many more survive partial dehydration during drying or freezing. The function of some protective molecules, such as sugars, has been extensively studied, but much less is known about the effects of amphiphiles such as flavonoids and other aromatic compounds. Amphiphiles may be largely soluble under fully hydrated conditions, but will partition into membranes upon removal of water. Little is known about the effects of amphiphiles on membrane stability and how amphiphile structure and function are related. Here, we have used two of the most intensively studied amphiphiles, tryptophan (Trp) and arbutin (Arb), along with their isolated hydrophilic moieties glycine (Gly) and glucose (Glc) to better understand structure-function relationships in amphiphile-membrane interactions in the dry state.
Results
Fourier-transform infrared (FTIR) spectroscopy was used to measure gel-to-liquid crystalline phase transition temperatures (Tm) of liposomes formed from phosphatidylcholine and phosphatidylethanolamine in the presence of the different additives. In anhydrous samples, both Glc and Arb strongly depressed Tm, independent of lipid composition, while Gly had no measurable effect. Trp, on the other hand, either depressed or increased Tm, depending on lipid composition. We found no evidence for strong interactions of any of the compounds with the lipid carbonyl or choline groups, while all additives except Gly seemed to interact with the phosphate groups. In the case of Arb and Glc, this also had a strong effect on the sugar OH vibrations in the FTIR spectra. In addition, vibrations from the hydrophobic indole and phenol moieties of Trp and Arb, respectively, provided evidence for interactions with the lipid bilayers.
Conclusions
The two amphiphiles Arb and Trp interact differently with dry bilayers. The interactions of Arb are dominated by contributions of the Glc moiety, while the indole governs the effects of Trp. In addition, only Trp-membrane interactions showed a strong influence of lipid composition. Further investigations, using the large structural diversity of plant amphiphiles will help to understand how their structure determines the interaction with membranes and how that influences their biological functions, for example under freezing or dehydration conditions.
doi:10.1186/2046-1682-6-9
PMCID: PMC3726346  PMID: 23879885
Amphiphiles; Arbutin; Desiccation; Fourier-transform infrared spectroscopy; Lipid phase transition; Model membranes; Tryptophan
4.  Identification of Drought Tolerance Markers in a Diverse Population of Rice Cultivars by Expression and Metabolite Profiling 
PLoS ONE  2013;8(5):e63637.
Rice provides about half of the calories consumed in Asian countries, but its productivity is often reduced by drought, especially when grown under rain-fed conditions. Cultivars with increased drought tolerance have been bred over centuries. Slow selection for drought tolerance on the basis of phenotypic traits may be accelerated by using molecular markers identified through expression and metabolic profiling. Previously, we identified 46 candidate genes with significant genotype × environment interaction in an expression profiling study on four cultivars with contrasting drought tolerance. These potential markers and in addition GC-MS quantified metabolites were tested in 21 cultivars from both indica and japonica background that varied in drought tolerance. Leaf blades were sampled from this population of cultivars grown under control or long-term drought condition and subjected to expression analysis by qRT-PCR and metabolite profiling. Under drought stress, metabolite levels correlated mainly negatively with performance parameters, but eight metabolites correlated positively. For 28 genes, a significant correlation between expression level and performance under drought was confirmed. Negative correlations were predominant. Among those with significant positive correlation was the gene coding for a cytosolic fructose-1,6-bisphosphatase. This enzyme catalyzes a highly regulated step in C-metabolism. The metabolic and transcript marker candidates for drought tolerance were identified in a highly diverse population of cultivars. Thus, these markers may be used to select for tolerance in a wide range of rice germplasms.
doi:10.1371/journal.pone.0063637
PMCID: PMC3661581  PMID: 23717458
5.  Dissecting Rice Polyamine Metabolism under Controlled Long-Term Drought Stress 
PLoS ONE  2013;8(4):e60325.
A selection of 21 rice cultivars (Oryza sativa L. ssp. indica and japonica) was characterized under moderate long-term drought stress by comprehensive physiological analyses and determination of the contents of polyamines and selected metabolites directly related to polyamine metabolism. To investigate the potential regulation of polyamine biosynthesis at the transcriptional level, the expression of 21 genes encoding enzymes involved in these pathways were analyzed by qRT-PCR. Analysis of the genomic loci revealed that 11 of these genes were located in drought-related QTL regions, in agreement with a proposed role of polyamine metabolism in rice drought tolerance. The cultivars differed widely in their drought tolerance and parameters such as biomass and photosynthetic quantum yield were significantly affected by drought treatment. Under optimal irrigation free putrescine was the predominant polyamine followed by free spermidine and spermine. When exposed to drought putrescine levels decreased markedly and spermine became predominant in all cultivars. There were no correlations between polyamine contents and drought tolerance. GC-MS analysis revealed drought-induced changes of the levels of ornithine/arginine (substrate), substrates of polyamine synthesis, proline, product of a competing pathway and GABA, a potential degradation product. Gene expression analysis indicated that ADC-dependent polyamine biosynthesis responded much more strongly to drought than the ODC-dependent pathway. Nevertheless the fold change in transcript abundance of ODC1 under drought stress was linearly correlated with the drought tolerance of the cultivars. Combining metabolite and gene expression data, we propose a model of the coordinate adjustment of polyamine biosynthesis for the accumulation of spermine under drought conditions.
doi:10.1371/journal.pone.0060325
PMCID: PMC3620119  PMID: 23577102
6.  Comparison of freezing tolerance, compatible solutes and polyamines in geographically diverse collections of Thellungiella sp. and Arabidopsis thaliana accessions 
BMC Plant Biology  2012;12:131.
Background
Thellungiella has been proposed as an extremophile alternative to Arabidopsis to investigate environmental stress tolerance. However, Arabidopsis accessions show large natural variation in their freezing tolerance and here the tolerance ranges of collections of accessions in the two species were compared.
Results
Leaf freezing tolerance of 16 Thellungiella accessions was assessed with an electrolyte leakage assay before and after 14 days of cold acclimation at 4°C. Soluble sugars (glucose, fructose, sucrose, raffinose) and free polyamines (putrescine, spermidine, spermine) were quantified by HPLC, proline photometrically. The ranges in nonacclimated freezing tolerance completely overlapped between Arabidopsis and Thellungiella. After cold acclimation, some Thellungiella accessions were more freezing tolerant than any Arabidopsis accessions. Acclimated freezing tolerance was correlated with sucrose levels in both species, but raffinose accumulation was lower in Thellungiella and only correlated with freezing tolerance in Arabidopsis. The reverse was true for leaf proline contents. Polyamine levels were generally similar between the species. Only spermine content was higher in nonacclimated Thellungiella plants, but decreased during acclimation and was negatively correlated with freezing tolerance.
Conclusion
Thellungiella is not an extremophile with regard to freezing tolerance, but some accessions significantly expand the range present in Arabidopsis. The metabolite data indicate different metabolic adaptation strategies between the species.
doi:10.1186/1471-2229-12-131
PMCID: PMC3464606  PMID: 22863402
Arabidopsis thaliana; Cold acclimation; Compatible solutes; Freezing tolerance; Natural variation; Polyamines; Thellungiella salsuginea
7.  Thermotropic phase behavior and headgroup interactions of the nonbilayer lipids phosphatidylethanolamine and monogalactosyldiacylglycerol in the dry state 
BMC Biophysics  2011;4:11.
Background
Although biological membranes are organized as lipid bilayers, they contain a substantial fraction of lipids that have a strong tendency to adopt a nonlamellar, most often inverted hexagonal (HII) phase. The polymorphic phase behavior of such nonbilayer lipids has been studied previously with a variety of methods in the fully hydrated state or at different degrees of dehydration. Here, we present a study of the thermotropic phase behavior of the nonbilayer lipids egg phosphatidylethanolamine (EPE) and monogalactosyldiacylglycerol (MGDG) with a focus on interactions between the lipid molecules in the interfacial and headgroup regions.
Results
Liposomes were investigated in the dry state by Fourier-transform Infrared (FTIR) spectroscopy and Differential Scanning Calorimetry (DSC). Dry EPE showed a gel to liquid-crystalline phase transition below 0°C and a liquid-crystalline to HII transition at 100°C. MGDG, on the other hand, was in the liquid-crystalline phase down to -30°C and showed a nonbilayer transition at about 85°C. Mixtures (1:1 by mass) with two different phosphatidylcholines (PC) formed bilayers with no evidence for nonbilayer transitions up to 120°C. FTIR spectroscopy revealed complex interactions between the nonbilayer lipids and PC. Strong H-bonding interactions occurred between the sugar headgroup of MGDG and the phosphate, carbonyl and choline groups of PC. Similarly, the ethanolamine moiety of EPE was H-bonded to the carbonyl and choline groups of PC and probably interacted through charge pairing with the phosphate group.
Conclusions
This study provides a comprehensive characterization of dry membranes containing the two most important nonbilayer lipids (PE and MGDG) in living cells. These data will be of particular relevance for the analysis of interactions between membranes and low molecular weight solutes or soluble proteins that are presumably involved in cellular protection during anhydrobiosis.
doi:10.1186/2046-1682-4-11
PMCID: PMC3116483  PMID: 21595868
8.  Interaction with Diurnal and Circadian Regulation Results in Dynamic Metabolic and Transcriptional Changes during Cold Acclimation in Arabidopsis 
PLoS ONE  2010;5(11):e14101.
In plants, there is a large overlap between cold and circadian regulated genes and in Arabidopsis, we have shown that cold (4°C) affects the expression of clock oscillator genes. However, a broader insight into the significance of diurnal and/or circadian regulation of cold responses, particularly for metabolic pathways, and their physiological relevance is lacking. Here, we performed an integrated analysis of transcripts and primary metabolites using microarrays and gas chromatography-mass spectrometry. As expected, expression of diurnally regulated genes was massively affected during cold acclimation. Our data indicate that disruption of clock function at the transcriptional level extends to metabolic regulation. About 80% of metabolites that showed diurnal cycles maintained these during cold treatment. In particular, maltose content showed a massive night-specific increase in the cold. However, under free-running conditions, maltose was the only metabolite that maintained any oscillations in the cold. Furthermore, although starch accumulates during cold acclimation we show it is still degraded at night, indicating significance beyond the previously demonstrated role of maltose and starch breakdown in the initial phase of cold acclimation. Levels of some conventional cold induced metabolites, such as γ-aminobutyric acid, galactinol, raffinose and putrescine, exhibited diurnal and circadian oscillations and transcripts encoding their biosynthetic enzymes often also cycled and preceded their cold-induction, in agreement with transcriptional regulation. However, the accumulation of other cold-responsive metabolites, for instance homoserine, methionine and maltose, did not have consistent transcriptional regulation, implying that metabolic reconfiguration involves complex transcriptional and post-transcriptional mechanisms. These data demonstrate the importance of understanding cold acclimation in the correct day-night context, and are further supported by our demonstration of impaired cold acclimation in a circadian mutant.
doi:10.1371/journal.pone.0014101
PMCID: PMC2990718  PMID: 21124901
9.  Predicting Arabidopsis Freezing Tolerance and Heterosis in Freezing Tolerance from Metabolite Composition 
Molecular Plant  2009;3(1):224-235.
Heterosis, or hybrid vigor, is one of the most important tools in plant breeding and has previously been demonstrated for plant freezing tolerance. Freezing tolerance is an important trait because it can limit the geographical distribution of plants and their agricultural yield. Plants from temperate climates increase in freezing tolerance during exposure to low, non-freezing temperatures in a process termed ‘cold acclimation’. Metabolite profiling has indicated a major reprogramming of plant metabolism in the cold, but it has remained unclear in previous studies which of these changes are related to freezing tolerance. In the present study, we have used metabolic profiling to discover combinations of metabolites that predict freezing tolerance and its heterosis in Arabidopsis thaliana. We identified compatible solutes and, in particular, the pathway leading to raffinose as crucial statistical predictors for freezing tolerance and its heterosis, while some TCA cycle intermediates contribute only to predicting the heterotic phenotype. This indicates coordinate links between heterosis and metabolic pathways, suggesting that a limited number of regulatory genes may determine the extent of heterosis in this complex trait. In addition, several unidentified metabolites strongly contributed to the prediction of both freezing tolerance and its heterosis and we present an exemplary analysis of one of these, identifying it as a hexose conjugate.
doi:10.1093/mp/ssp105
PMCID: PMC2807929  PMID: 20026477
Abiotic/environmental stress; cold acclimation; metabolomics; bioinformatics; biostatistics; Arabidopsis
10.  Expression profiling of rice cultivars differing in their tolerance to long-term drought stress 
Plant Molecular Biology  2008;69(1-2):133-153.
Understanding the molecular basis of plant performance under water-limiting conditions will help to breed crop plants with a lower water demand. We investigated the physiological and gene expression response of drought-tolerant (IR57311 and LC-93-4) and drought-sensitive (Nipponbare and Taipei 309) rice (Oryza sativa L.) cultivars to 18 days of drought stress in climate chamber experiments. Drought stressed plants grew significantly slower than the controls. Gene expression profiles were measured in leaf samples with the 20 K NSF oligonucleotide microarray. A linear model was fitted to the data to identify genes that were significantly regulated under drought stress. In all drought stressed cultivars, 245 genes were significantly repressed and 413 genes induced. Genes differing in their expression pattern under drought stress between tolerant and sensitive cultivars were identified by the genotype × environment (G × E) interaction term. More genes were significantly drought regulated in the sensitive than in the tolerant cultivars. Localizing all expressed genes on the rice genome map, we checked which genes with a significant G × E interaction co-localized with published quantitative trait loci regions for drought tolerance. These genes are more likely to be important for drought tolerance in an agricultural environment. To identify the metabolic processes with a significant G × E effect, we adapted the analysis software MapMan for rice. We found a drought stress induced shift toward senescence related degradation processes that was more pronounced in the sensitive than in the tolerant cultivars. In spite of higher growth rates and water use, more photosynthesis related genes were down-regulated in the tolerant than in the sensitive cultivars.
Electronic supplementary material
The online version of this article (doi:10.1007/s11103-008-9412-7) contains supplementary material, which is available to authorized users.
doi:10.1007/s11103-008-9412-7
PMCID: PMC2709230  PMID: 18931976
Abiotic stress; Expression profiling; Gene × environment interaction; QTL; Water use efficiency; Water potential
11.  Fructan and its relationship to abiotic stress tolerance in plants 
Cellular and Molecular Life Sciences   2009;66(13):2007-2023.
Numerous studies have been published that attempted to correlate fructan concentrations with freezing and drought tolerance. Studies investigating the effect of fructan on liposomes indicated that a direct interaction between membranes and fructan was possible. This new area of research began to move fructan and its association with stress beyond mere correlation by confirming that fructan has the capacity to stabilize membranes during drying by inserting at least part of the polysaccharide into the lipid headgroup region of the membrane. This helps prevent leakage when water is removed from the system either during freezing or drought. When plants were transformed with the ability to synthesize fructan, a concomitant increase in drought and/or freezing tolerance was confirmed. These experiments indicate that besides an indirect effect of supplying tissues with hexose sugars, fructan has a direct protective effect that can be demonstrated by both model systems and genetic transformation.
doi:10.1007/s00018-009-0002-x
PMCID: PMC2705711  PMID: 19290476
Inulin; Levan; Cold acclimation; Subzero acclimation; Model systems; Liposomes; Membrane stabilization; Sugar glasses; Lipid phase transitions; Plant transformation
12.  Interactions between the circadian clock and cold-response in Arabidopsis 
Plant Signaling & Behavior  2008;3(8):593-594.
Low temperature negatively affects plant growth and metabolism. Plant responses to cold involve massive transcriptional changes, and much effort has been made to identify these changes and their contribution to freezing tolerance. However, the influence of differences in environmental and developmental factors between experiments had not been investigated. We found that diurnal- and circadian-regulated genes are responsible for the majority of variation between experiments. Moreover, we demonstrated that the cyclic expression pattern of circadian clock components is affected by cold and that the cold induction of many transcription factors is dependent on the time of day. This means that genes identified so far as cold responsive are dependent on the time of day the experiment was performed and that paired diurnal controls are not sufficient to correct for this effect. Ongoing work to dissect the biological relevance of cold-diurnal regulatory interactions demonstrated that some circadian mutants have altered freezing tolerance but that time-of-day appears not to affect freezing tolerance.
PMCID: PMC2634507  PMID: 19704808
cold stress; circadian clock; transcription factors expression; gating; cold-diurnal regulation
13.  Natural variation in CBF gene sequence, gene expression and freezing tolerance in the Versailles core collection of Arabidopsis thaliana 
BMC Plant Biology  2008;8:105.
Background
Plants from temperate regions are able to withstand freezing temperatures due to a process known as cold acclimation, which is a prior exposure to low, but non-freezing temperatures. During acclimation, a large number of genes are induced, bringing about biochemical changes in the plant, thought to be responsible for the subsequent increase in freezing tolerance. Key regulatory proteins in this process are the CBF1, 2 and 3 transcription factors which control the expression of a set of target genes referred to as the "CBF regulon".
Results
To assess the role of the CBF genes in cold acclimation and freezing tolerance of Arabidopsis thaliana, the CBF genes and their promoters were sequenced in the Versailles core collection, a set of 48 accessions that maximizes the naturally-occurring genetic diversity, as well as in the commonly used accessions Col-0 and WS. Extensive polymorphism was found in all three genes. Freezing tolerance was measured in all accessions to assess the variability in acclimated freezing tolerance. The effect of sequence polymorphism was investigated by evaluating the kinetics of CBF gene expression, as well as that of a subset of the target COR genes, in a set of eight accessions with contrasting freezing tolerance. Our data indicate that CBF genes as well as the selected COR genes are cold induced in all accessions, irrespective of their freezing tolerance. Although we observed different levels of expression in different accessions, CBF or COR gene expression was not closely correlated with freezing tolerance.
Conclusion
Our results indicate that the Versailles core collection contains significant natural variation with respect to freezing tolerance, polymorphism in the CBF genes and CBF and COR gene expression. Although there tends to be more CBF and COR gene expression in tolerant accessions, there are exceptions, reinforcing the idea that a complex network of genes is involved in freezing tolerance and that the CBF genes alone cannot explain all differences in phenotype. Our study also highlights the difficulty in assessing the function of single transcription factors that are members of closely related gene families.
doi:10.1186/1471-2229-8-105
PMCID: PMC2579297  PMID: 18922165
14.  Heterosis in the freezing tolerance, and sugar and flavonoid contents of crosses between Arabidopsis thaliana accessions of widely varying freezing tolerance 
Plant, Cell & Environment  2008;31(6):813-827.
Heterosis is defined as the increased vigour of hybrids in comparison to their parents. We investigated 24 F1 hybrid lines of Arabidopsis thaliana generated by reciprocally crossing either C24 or Col with six other parental accessions (Can, Co, Cvi, Ler, Rsch, Te) that differ widely in their freezing tolerance. The crosses differed in the degree of heterosis for freezing tolerance, both in the non-acclimated state and after a 14 d cold acclimation period. Crosses with C24 showed more heterosis than crosses with Col, and heterosis was stronger in acclimated than in non-acclimated plants. Leaf content of soluble sugars and proline showed more deviation from mid-parent values in crosses involving C24 than in those involving Col, and deviations were larger in acclimated than in non-acclimated plants. There were significant correlations between the content of different sugars and leaf freezing tolerance, as well as between heterosis effects in freezing tolerance and sugar content. Flavonoid content and composition varied between accessions, and between non-acclimated and acclimated plants. In the crosses, large deviations from the mid-parent values in the contents of different flavonols occurred, and there were strikingly strong correlations between both flavonol content and freezing tolerance, and between heterosis effects in freezing tolerance and flavonol content.
doi:10.1111/j.1365-3040.2008.01800.x
PMCID: PMC2440548  PMID: 18284584
cold acclimation; compatible solutes
15.  Chlorophyll fluorescence imaging accurately quantifies freezing damage and cold acclimation responses in Arabidopsis leaves 
Plant Methods  2008;4:12.
Background
Freezing tolerance is an important factor in the geographical distribution of plants and strongly influences crop yield. Many plants increase their freezing tolerance during exposure to low, nonfreezing temperatures in a process termed cold acclimation. There is considerable natural variation in the cold acclimation capacity of Arabidopsis that has been used to study the molecular basis of this trait. Accurate methods for the quantitation of freezing damage in leaves that include spatial information about the distribution of damage and the possibility to screen large populations of plants are necessary, but currently not available. In addition, currently used standard methods such as electrolyte leakage assays are very laborious and therefore not easily applicable for large-scale screening purposes.
Results
We have performed freezing experiments with the Arabidopsis accessions C24 and Tenela, which differ strongly in their freezing tolerance, both before and after cold acclimation. Freezing tolerance of detached leaves was investigated using the well established electrolyte leakage assay as a reference. Chlorophyll fluorescence imaging was used as an alternative method that provides spatial resolution of freezing damage over the leaf area. With both methods, LT50 values (i.e. temperature where 50% damage occurred) could be derived as quantitative measures of leaf freezing tolerance. Both methods revealed the expected differences between acclimated and nonacclimated plants and between the two accessions and LT50 values were tightly correlated. However, electrolyte leakage assays consistently yielded higher LT50 values than chlorophyll fluorescence imaging. This was to a large part due to the incubation of leaves for electrolyte leakage measurements in distilled water, which apparently led to secondary damage, while this pre-incubation was not necessary for the chlorophyll fluorescence measurements.
Conclusion
Chlorophyll fluorescence imaging is an alternative method to accurately determine the freezing tolerance of leaves. It is quick and inexpensive and the system could potentially be used for large scale screening, allowing new approaches to elucidate the molecular basis of plant freezing tolerance.
doi:10.1186/1746-4811-4-12
PMCID: PMC2430023  PMID: 18505561
16.  Protection of liposomes against fusion during drying by oligosaccharides is not predicted by the calorimetric glass transition temperatures of the dry sugars 
European Biophysics Journal   2007;37(4):503-508.
Sugars play an important role in the desiccation tolerance of most anhydrobiotic organisms. It has been shown in previous studies that different structural families of oligosaccharides have different efficacies to interact with phospholipid headgroups and protect membranes from solute leakage during drying. Here, we have compared three families of linear oligosaccharides (fructans (inulins), malto-oligosaccharides, manno-oligosaccharides) for their chain-length dependent protection of egg phosphatidylcholine liposomes against membrane fusion. We found increased protection with chain length up to a degree of polymerization (DP) of 5 for malto-oligosaccharides, and a decrease for inulins and manno-oligosaccharides. Differential scanning calorimetry measurements showed that for all sugars the glass transition temperature (Tg) increased with DP, although to different degrees for the different oligosaccharide families. Higher Tg values resulted in reduced membrane fusion only for malto-oligosaccharides below DP5. Contrary to expectation, for inulins, manno-oligosaccharides and malto-oligosaccharides of a DP above five, fusion increased with increasing Tg, indicating that other physical parameters are more important in determining the ability of different sugars to protect membranes against fusion during drying. Further research will be necessary to experimentally define such parameters.
doi:10.1007/s00249-007-0245-0
PMCID: PMC2270362  PMID: 18064448
17.  LEA (Late Embryogenesis Abundant) proteins and their encoding genes in Arabidopsis thaliana 
BMC Genomics  2008;9:118.
Background
LEA (late embryogenesis abundant) proteins have first been described about 25 years ago as accumulating late in plant seed development. They were later found in vegetative plant tissues following environmental stress and also in desiccation tolerant bacteria and invertebrates. Although they are widely assumed to play crucial roles in cellular dehydration tolerance, their physiological and biochemical functions are largely unknown.
Results
We present a genome-wide analysis of LEA proteins and their encoding genes in Arabidopsis thaliana. We identified 51 LEA protein encoding genes in the Arabidopsis genome that could be classified into nine distinct groups. Expression studies were performed on all genes at different developmental stages, in different plant organs and under different stress and hormone treatments using quantitative RT-PCR. We found evidence of expression for all 51 genes. There was only little overlap between genes expressed in vegetative tissues and in seeds and expression levels were generally higher in seeds. Most genes encoding LEA proteins had abscisic acid response (ABRE) and/or low temperature response (LTRE) elements in their promoters and many genes containing the respective promoter elements were induced by abscisic acid, cold or drought. We also found that 33% of all Arabidopsis LEA protein encoding genes are arranged in tandem repeats and that 43% are part of homeologous pairs. The majority of LEA proteins were predicted to be highly hydrophilic and natively unstructured, but some were predicted to be folded.
Conclusion
The analyses indicate a wide range of sequence diversity, intracellular localizations, and expression patterns. The high fraction of retained duplicate genes and the inferred functional diversification indicate that they confer an evolutionary advantage for an organism under varying stressful environmental conditions. This comprehensive analysis will be an important starting point for future efforts to elucidate the functional role of these enigmatic proteins.
doi:10.1186/1471-2164-9-118
PMCID: PMC2292704  PMID: 18318901
18.  A Global Survey of Gene Regulation during Cold Acclimation in Arabidopsis thaliana 
PLoS Genetics  2005;1(2):e26.
Many temperate plant species such as Arabidopsis thaliana are able to increase their freezing tolerance when exposed to low, nonfreezing temperatures in a process called cold acclimation. This process is accompanied by complex changes in gene expression. Previous studies have investigated these changes but have mainly focused on individual or small groups of genes. We present a comprehensive statistical analysis of the genome-wide changes of gene expression in response to 14 d of cold acclimation in Arabidopsis, and provide a large-scale validation of these data by comparing datasets obtained for the Affymetrix ATH1 Genechip and MWG 50-mer oligonucleotide whole-genome microarrays. We combine these datasets with existing published and publicly available data investigating Arabidopsis gene expression in response to low temperature. All data are integrated into a database detailing the cold responsiveness of 22,043 genes as a function of time of exposure at low temperature. We concentrate our functional analysis on global changes marking relevant pathways or functional groups of genes. These analyses provide a statistical basis for many previously reported changes, identify so far unreported changes, and show which processes predominate during different times of cold acclimation. This approach offers the fullest characterization of global changes in gene expression in response to low temperature available to date.
Synopsis
Freezing tolerance is an important determinant of geographical distribution of plant species, and freezing damage in crop plants leads to severe losses in agriculture. Many temperate plants increase their freezing tolerance during exposure to low, but nonfreezing temperatures, a process known as cold acclimation. Freezing tolerance and cold acclimation are complex, quantitative genetic traits. The number and functional roles of the responsible genes are not known for any plant species. Using the model plant Arabidopsis thaliana, which is moderately freezing tolerant and able to cold acclimate, the global regulation of gene expression during exposure to 4 °C for 14 d was analyzed by microarray hybridization. For validation of gene expression data, triplicate biological samples were hybridized to two different oligonucleotide arrays. Results from the two platforms showed good agreement, indicating the reliability of the measurements. The authors combined their data with all publicly available data on cold-regulated gene expression in A. thaliana to compile a database detailing the cold responsiveness of 22,043 genes as a function of exposure time. In addition, thorough statistical analysis was used to identify metabolic pathways and physiological processes that are predominantly involved in the plant cold-acclimation process.
doi:10.1371/journal.pgen.0010026
PMCID: PMC1189076  PMID: 16121258
19.  Cryoprotectin: a plant lipid-transfer protein homologue that stabilizes membranes during freezing. 
Plants from temperate and cold climates are able to increase their freezing tolerance during exposure to low non-freezing temperatures. It has been shown that several genes are induced in a coordinated manner during this process of cold acclimation. The functional role of most of the corresponding cold-regulated proteins is not yet known. We summarize our knowledge of those cold-regulated proteins that are able to stabilize membranes during a freeze-thaw cycle. Special emphasis is placed on cryoprotectin, a lipid-transfer protein homologue that was isolated from cold-acclimated cabbage leaves and that protects isolated chloroplast thylakoid membranes from freeze-thaw damage.
doi:10.1098/rstb.2002.1079
PMCID: PMC1693006  PMID: 12171654

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