[Show abstract][Hide abstract] ABSTRACT: Due to global climate change, heat and drought stress have an increasing impact on rice (Oryza sativa L.) yields and become major challenges in agriculture. Severe yield losses were observed when a single stress or a combination of stresses coincide with flowering, but the physiological responses of rice floral organs leading to spikelet sterility are not known. Metabolic and transcriptomic profiling of anthers, pistils before pollination and pollinated pistils was performed to investigate physiological responses determining fertility under heat and combined drought and heat stress using the heat- and drought-tolerant N22 and the heat-sensitive and drought-tolerant cultivar Moroberekan. Different metabolite patterns were observed between floral organs as well as between cultivars under non-stressed and stressed conditions. Several candidate metabolites were differentially accumulated in stressed anthers of N22 compared to Moroberekan under non- stress conditions. More genes were regulated under heat-drought stress than heat stress in anthers of both cultivars with N22 showing much less gene regulation under combined stress in comparison to Moroberekan. The functional class of genes related to stress was over-represented under all stress conditions in both cultivars while fewer bins were over-represented under heat stress compared to combined stress. Combining metabolite and transcriptomic analyses identified sugar metabolism in anthers as crucial for tolerance or susceptibility to stress. In the future, identification of candidate metabolites and genes will be useful for marker assisted selection procedures to breed more stress tolerant crop plants.
[Show abstract][Hide abstract] ABSTRACT: During low temperature exposure, Arabidopsis thaliana and many other plants from temperate climates increase in freezing tolerance in a process termed cold acclimation. However, the correct timing and rate of deacclimation, resulting in loss of freezing tolerance and initiation of growth is equally important for plant fitness and survival. While the molecular basis of cold acclimation has been investigated in detail, much less information is available about deacclimation. We have characterized the responses of 10 natural accessions of Arabidopsis thaliana that vary widely in their freezing tolerance, to deacclimation conditions. Sugar, proline and transcript levels declined sharply over three days in all accessions after transfer of cold acclimated plants to ambient temperatures, while freezing tolerance only declined in tolerant accessions. Correlations between freezing tolerance and the expression levels of COR genes and the content of glucose, fructose and sucrose, as well as many correlations among transcript and solute levels, that were highly significant in cold acclimated plants, were lost during deacclimation. Other correlations persisted, indicating that after three days of deacclimation, plant metabolism had not completely reverted back to the non-acclimated state. These data provide the basis for further molecular and genetic studies to unravel the regulation of deacclimation.
[Show abstract][Hide abstract] ABSTRACT: Cruciferous plants synthesize a large variety of tryptophan-derived phytoalexins in response to pathogen infection, UV irradiation, or high dosages of heavy metals. The major phytoalexins of Eutrema salsugineum (Thellungiella salsuginea), which has recently been established as an extremophile model plant, are probably derivatives of indole glucosinolates, in contrast to Arabidopsis, which synthesizes characteristic camalexin from the glucosinolate precursor indole-3-acetaldoxime.
The transcriptional response of E. salsugineum to UV irradiation and AgNO3 was monitored by RNAseq and microarray analysis. Most transcripts (respectively 70% and 78%) were significantly differentially regulated and a large overlap between the two treatments was observed (54% of total). While core genes of the biosynthesis of aliphatic glucosinolates were repressed, tryptophan and indole glucosinolate biosynthetic genes, as well as defence-related WRKY transcription factors, were consistently upregulated. The putative Eutrema WRKY33 ortholog was functionally tested and shown to complement camalexin deficiency in Atwrky33 mutant.
In E. salsugineum, UV irradiation or heavy metal application resulted in substantial transcriptional reprogramming. Consistently induced genes of indole glucosinolate biosynthesis and modification will serve as candidate genes for the biosynthesis of Eutrema-specific phytoalexins.
Full-text · Article · Jun 2015 · BMC Plant Biology
[Show abstract][Hide abstract] ABSTRACT: Heat and drought stress are projected to become major challenges to sustain rice (Oryza sativa L.) yields with global climate change. Both stresses lead to yield losses when they coincide with flowering. A significant knowledge gap exists in the mechanistic understanding of the responses of rice floral organs that determine reproductive success under stress. Our work connects the metabolomic and transcriptomic changes in anthers, pistils before pollination and pollinated pistils in a heat tolerant (N22) and a heat sensitive (Moroberekan) cultivar. Systematic analysis of the floral organs revealed contrasts in metabolic profiles across anthers and pistils. Constitutive metabolic markers were identified that can define reproductive success in rice under stress. Six out of nine candidate metabolites identified by intersection analysis of stressed anthers were differentially accumulated in N22 compared to Moroberekan under non-stress conditions. Sugar metabolism was identified to be the crucial metabolic and transcriptional component that differentiated floral organ tolerance or susceptibility to stress. While susceptible Moroberekan specifically showed high expression of the Carbon Starved Anthers (CSA) gene under combined heat and drought, tolerant N22 responded with high expression of genes encoding a sugar transporter (MST8) and a cell wall invertase (INV4) as markers of high sink strength.
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No preview · Article · Mar 2015 · Plant Cell and Environment
[Show abstract][Hide abstract] ABSTRACT: Climate models predict an increased likelihood of seasonal droughts for many areas of the world. Breeding for drought tolerance could be accelerated by marker-assisted selection. As a basis for marker identification, we studied genetic variance, predictability of field performance, and potential costs of tolerance in potato (Solanum tuberosum L.). Potato produces high calories per unit water invested, but is drought-sensitive. In 14 independent pot or field trials, 34 potato cultivars were grown under optimal and reduced water supply to determine starch yield. In an artificial data set, we tested several stress indices for their power to distinguish tolerant and sensitive genotypes independent of their yield potential. We identified DRYM (deviation of relative starch yield from its experimental median) as the most efficient index. DRYM corresponded qualitatively to the partial least square-model based metric of drought stress tolerance in a stress effect model. The DRYM identified significant tolerance variation in the European potato cultivar population to allow tolerance breeding and marker identification. Tolerance results from pot trials correlated with those from field trials, but predicted field performance worse than field growth parameters. Drought tolerance correlated negatively with yield under optimal conditions in the field. The distribution of yield data versus DRYM indicated that tolerance can be combined with average yield potentials, thus circumventing potential yield penalties in tolerance breeding.
No preview · Article · Mar 2015 · Functional Plant Biology
[Show abstract][Hide abstract] ABSTRACT: In plants from temperate climates such as Arabidopsis thaliana low, non-freezing temperatures lead to increased freezing tolerance in a process termed cold acclimation. During cold acclimation, massive changes in gene expression and in the content of primary metabolites and lipids have been observed. Here, we have analyzed the influence of cold acclimation on flavonol and anthocyanin content and on the expression of genes related to flavonoid metabolism in 54 Arabidopsis accessions covering a wide range of freezing tolerance. Most flavonols and anthocyanins accumulated upon cold exposure, but the extent of accumulation varied strongly among the accessions. This was also true for most of the investigated transcripts. Correlation analyses revealed a high degree of coordination among metabolites and among transcripts, but only little correlation between metabolites and transcripts, indicating an important role of post-transcriptional regulation in flavonoid metabolism. Similarly, levels of many flavonoid biosynthesis genes were correlated with freezing tolerance after cold acclimation, but only the pool sizes of a few flavonols and anthocyanins. Collectively, our data provide evidence for an important role of flavonoid metabolism in Arabidopsis freezing tolerance and point to the importance of post-transcriptional mechanisms in the regulation of flavonoid metabolism in response to cold.
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No preview · Article · Feb 2015 · Plant Cell and Environment
[Show abstract][Hide abstract] ABSTRACT: As multifaceted molecules, reactive oxygen species (ROS) are known to accumulate in response to various stresses. Ozone (O3) is an air pollutant with detrimental effect on plants, and O3 can also be used as a tool to study the role of ROS in signaling. Genetic variation of O3 sensitivity in different Arabidopsis accessions highlights the complex genetic architecture of plant responses to ROS. To investigate the genetic basis of O3 sensitivity, a recombinant inbred line (RIL) population between two Arabidopsis accessions with distinct O3 sensitivity, C24 (O3 tolerant) and Te (O3 sensitive), was used for quantitative trait loci (QTL) mapping. Through analysis of QTL mapping combined with transcriptome changes in response to O3, we identified three causal QTLs and several potential candidate genes regulating the response to O3. Based on gene expression data, water loss and stomatal conductance measurement, we found that a combination of relatively low stomatal conductance and constitutive activation of salicylic acid (SA) mediated defense signaling were responsible for the O3 tolerance in C24. Application of exogenous SA prior to O3 exposure can mimic the constitutive SA signaling in C24, and could attenuate O3 induced leaf damage in the sensitive Arabidopsis accessions Te and Cvi-0.
Full-text · Article · Dec 2014 · Plant Cell and Environment
[Show abstract][Hide abstract] ABSTRACT: Abstract Plants as sessile organisms are strongly challenged by environmental stresses. Many plants species are able to cold-acclimate, acquiring higher freezing tolerance upon exposure to low but non-freezing temperatures. Among a plethora of adaptational processes, this involves the accumulation of cold regulated (COR) proteins that are assumed to stabilize and protect cellular structures during freezing. However, their molecular functions are largely unknown. We recently reported a comprehensive study of two intrinsically disordered cold regulated chloroplast proteins, COR15A and COR15B from Arabidopsis thaliana. They are necessary for full cold acclimation. During freezing, they stabilize leaf cells through folding and binding to chloroplast membranes. Contrary to evidence from in-vitro experiments, they play no role in enzyme stabilization in vivo. Elucidating these major functional and structural characteristics and estimation of protein abundance allow us to propose a detailed model for the mode of action of the two COR15 proteins.
[Show abstract][Hide abstract] ABSTRACT: During cold acclimation plants increase in freezing tolerance in response to low non-freezing temperatures. This is accompanied by many physiological, biochemical and molecular changes that have been extensively investigated. In addition, plants of many species, including Arabidopsis thaliana, become more freezing tolerant during exposure to mild, non-damaging sub-zero temperatures after cold acclimation. There is hardly any information available about the molecular basis of this adaptation. Here, we have used microarrays and a qRT-PCR primer platform covering 1,880 genes encoding transcription factors (TFs) to monitor changes in gene expression in the Arabidopsis accessions Columbia-0, Rschew and Tenela during the first 3 days of sub-zero acclimation at -3 °C. The results indicate that gene expression during sub-zero acclimation follows a tighly controlled time-course. Especially AP2/EREBP and WRKY TFs may be important regulators of sub-zero acclimation, although the CBF signal transduction pathway seems to be less important during sub-zero than during cold acclimation. Globally, we estimate that approximately 5 % of all Arabidopsis genes are regulated during sub-zero acclimation. Particularly photosynthesis-related genes are down-regulated and genes belonging to the functional classes of cell wall biosynthesis, hormone metabolism and RNA regulation of transcription are up-regulated. Collectively, these data provide the first global analysis of gene expression during sub-zero acclimation and allow the identification of candidate genes for forward and reverse genetic studies into the molecular mechanisms of sub-zero acclimation.
No preview · Article · Oct 2014 · Plant Molecular Biology
[Show abstract][Hide abstract] ABSTRACT: Alkaline/neutral invertases (A/N-Invs) are now recognized as essential proteins in plant life. They catalyze the irreversible break-down of sucrose into glucose and fructose and thus supply the cells with energy as well as signaling molecules. In this study we report on a mechanism that affects the activity of the cytosolic invertase AtCINV1 (At-A/N-InvG or AT1G35580). We demonstrate that Ser547 at the extreme C-terminus of the AtCINV1 protein is a substrate of calcium-dependent kinases (CPK3 and 21) and that phosphorylation creates a high-affinity binding site for 14-3-3 proteins. The invertase as such has basal activity, but we provide evidence that interaction with 14-3-3 proteins enhances its activity. The analysis of three quadruple 14-3-3 mutants generated from six T-DNA insertion mutants of the non-epsilon family shows both specificity as well as redundancy for this function of 14-3-3 proteins. The strong reduction in hexose levels in the roots of one 14-3-3 quadruple mutant plant is in line with the activating function of 14-3-3 proteins. The physiological relevance of this mechanism that affects A/N invertase activity is underscored by the light induced activation and is another example of the central role of 14-3-3 proteins in mediating dark/light signaling. The nature of the light induced signal that travels from the shoot to root and the question whether this signal is transmitted via cytosolic Ca++ changes that activate calcium-dependent kinases, await further study.This article is protected by copyright. All rights reserved.
Full-text · Article · Sep 2014 · The Plant Journal
[Show abstract][Hide abstract] ABSTRACT: Freezing can severely damage plants, limiting geographical distribution of natural populations and leading to major agronomical losses. Plants native to cold climates acquire increased freezing tolerance during exposure to low non-freezing temperatures in a process termed cold acclimation. This involves many adaptative responses, including global changes in metabolite content and gene expression, and the accumulation of cold regulated (COR) proteins, whose functions are largely unknown. Here we report that the chloroplast proteins COR15A and COR15B are necessary for full cold acclimation in Arabidopsis thaliana. They protect cell membranes, as indicated by electrolyte leakage and chlorophyll fluorescence measurements. Recombinant COR15 proteins stabilize lactate dehydrogenase during freezing in vitro. However, a transgenic approach shows that they have no influence on the stability of selected plastidic enzymes in vivo, although cold acclimation results in increased enzyme stability. This indicates that enzymes are stabilized by other mechanisms. Recombinant COR15 proteins are disordered in water, but fold into amphipathic α-helices at high osmolyte concentrations in the presence of membranes, a condition mimicking molecular crowding induced by dehydration during freezing. X-ray scattering experiments indicate protein-membrane interactions specifically under such crowding conditions. The COR15-membrane interactions lead to liposome stabilization during freezing. Collectively, our data demonstrate the requirement for COR15 accumulation for full cold acclimation of Arabidopsis. The function of these intrinsically disordered proteins is the stabilization of chloroplast membranes during freezing through a folding and binding mechanism, but not the stabilization of chloroplastic enzymes. This indicates a high functional specificity of these disordered plant proteins.
[Show abstract][Hide abstract] ABSTRACT: This introductory chapter provides a brief overview of plant freezing tolerance and cold acclimation and describes the basic concepts and approaches that are currently followed to investigate these phenomena. We highlight the multidisciplinary nature of these investigations and the necessity to use methodologies from different branches of science, such as ecology, genetics, physiology, biochemistry, and biophysics, to come to a complete understanding of the complex adaptive mechanisms underlying plant cold acclimation.
No preview · Article · May 2014 · Methods in molecular biology (Clifton, N.J.)
[Show abstract][Hide abstract] ABSTRACT: Quantitative assessment of freezing tolerance is essential to unravel plant adaptations to cold temperatures. Not only the survival of whole plants but also impairment of detached leaves after a freeze-thaw cycle can be used to accurately quantify plant freezing tolerance in terms of LT50 values. Here we describe two methods to determine the freezing tolerance of detached leaves using different physiological parameters. Firstly, we illustrate how to assess the integrity of (predominantly) the plasma membrane during freezing using an electrolyte leakage assay. Secondly, we provide a chlorophyll fluorescence imaging protocol to determine the freezing tolerance of the photosynthetic apparatus.
No preview · Article · May 2014 · Methods in molecular biology (Clifton, N.J.)
[Show abstract][Hide abstract] ABSTRACT: Main conclusion:
Expression of eight LEA genes enhanced desiccation tolerance in yeast, including two LEA_2 genes encoding atypical, stably folded proteins. The recombinant proteins showed enzyme, but not membrane protection during drying. To screen for possible functions of late embryogenesis abundant (LEA) proteins in cellular stress tolerance, 15 candidate genes from six Arabidopsis thaliana LEA protein families were expressed in Saccharomyces cerevisiae as a genetically amenable eukaryotic model organism. Desiccation stress experiments showed that eight of the 15 LEA proteins significantly enhanced yeast survival. While none of the proteins belonging to the LEA_1, LEA_5 or AtM families provided protection to yeast cells, two of three LEA_2 proteins, all three LEA_4 proteins and three of four dehydrins were effective. However, no significantly enhanced tolerance toward freezing, salt, osmotic or oxidative stress was observed. While most LEA proteins are highly hydrophilic and intrinsically disordered, LEA_2 proteins are "atypical", since they are more hydrophobic and possess a stable folded structure in solution. Because nothing was known about the functional properties of LEA_2 proteins, we expressed the three Arabidopsis proteins LEA1, LEA26 and LEA27 in Escherichia coli. The bacteria expressed all three proteins in inclusion bodies from which they could be purified and refolded. Correct folding was ascertained by Fourier transform Infrared (FTIR) spectroscopy. None of the proteins was able to stabilize liposomes during freezing or drying, but they were all able to protect the enzyme lactate dehydrogenase (LDH) from inactivation during freezing. Significantly, only LEA1 and LEA27, which also protected yeast cells during drying, were able to stabilize LDH during desiccation and subsequent rehydration.