A systems biology approach for the analysis of carbohydrate dynamics during acclimation to low temperature in Arabidopsis thaliana

Low temperature is an important environmental factor affecting the performance and distribution of plants. During the so-called process of cold acclimation, many plants are able to develop low-temperature tolerance, associated with the reprogramming of a large part of their metabolism. In this study, we present a systems biology approach based on mathematical modelling to determine interactions between the reprogramming of central carbohydrate metabolism and the development of freezing tolerance in two accessions of Arabidopsis thaliana. Different regulation strategies were observed for (a) photosynthesis, (b) soluble carbohydrate metabolism and (c) enzyme activities of central metabolite interconversions. Metabolism of the storage compound starch was found to be independent of accession-specific reprogramming of soluble sugar metabolism in the cold. Mathematical modelling and simulation of cold-induced metabolic reprogramming indicated major differences in the rates of interconversion between the pools of hexoses and sucrose, as well as the rate of assimilate export to sink organs. A comprehensive overview of interconversion rates is presented, from which accession-specific regulation strategies during exposure to low temperature can be derived. We propose this concept as a tool for predicting metabolic engineering strategies to optimize plant freezing tolerance. We confirm that a significant improvement in freezing tolerance in plants involves multiple regulatory instances in sucrose metabolism, and provide evidence for a pivotal role of sucrose-hexose interconversion in increasing the cold acclimation output.     

Isolation and characterization of cold-regulated transcriptional activator <Emphasis Type="Italic">LpCBF3</Emphasis> gene from perennial ryegrass (<Emphasis Type="Italic">Lolium perenne</Emphasis> L.)

In plants, low temperatures can activate the CBF cold response pathway playing a prominent role in cold acclimation by triggering a set of cold-related gene expressions. CBF homologous gene, designated as LpCBF3, from a cold-tolerant perennial ryegrass (Lolium perenne L.) accession was identified. It carries the sequences for nuclear localization signal (NLS), AP2 DNA-binding domains and an acidic activation present in most of the plant CBF proteins. Southern analysis indicated the presence of three homologs of LpCBF3 gene in perennial ryegrass genome, and only one amino acid variation in LpCBF3 protein between cold-tolerant and -sensitive perennial ryegrass accessions. In their putative promoter regions, some differential regions were found. Northern blotting and RT-PCR analysis found that LpCBF3 reached the highest expression after 1.5 h of cold treatment (4 degrees C). The COR homologous gene, a downstream gene of CBF, can be expressed in the plant stem of cold-tolerant perennial ryegrass accessions without cold treatment. Without cold treatment, the COR gene cannot be activated in cold-sensitive perennial ryegrass accessions. Cold treatment can prompt expression levels of COR homologous genes in both perennial ryegrass accessions. In transgenic Arabidopsis, the overexpression of LpCBF3 with the 35S promoter resulted in dwarf-like plants, later flowering and greater freezing tolerance.     

Impact of soluble sugar concentrations on the acquisition of freezing tolerance in accessions of Arabidopsis thaliana with contrasting cold adaptation – evidence for a role of raffinose in cold acclimation

In the present study the cold acclimation potential of two accessions of Arabidopsis thaliana was investigated. Significant variation was found for basic tolerance as well as the capacity to acclimate to freezing temperatures. During cold acclimation, levels of soluble sugars increased in both genotypes, but raffinose accumulation discriminated the more tolerant accession Col‐0 from C24. Concentrations of other compatible solutes such as proline and glutamine were also higher in cold‐acclimated Col‐0 than C24 plants. Changes of invertase activity during cold exposure corresponded to changes in sucrose and fructose, but not glucose concentrations and were consistent with an initial chilling response and a later decline in hexose metabolization. When vacuolar invertase was suppressed by siRNA expression, reduced sucrolytic activity resulted in elevated leaf sucrose concentration, whereas the fructose content was strongly reduced. This led to elevated freezing tolerance in the cold‐tolerant genotype Col‐0, but not in C24. The most pronounced metabolic changes in invertase‐inhibited Col‐0 plants occurred for proline and glutamine concentrations, indicating indirect metabolic effects of altered sugar concentrations.     

Subcellular reprogramming of metabolism during cold acclimation in Arabidopsis thaliana

Metabolite changes in plant leaves during exposure to low temperatures involve re‐allocation of a large number of metabolites between sub‐cellular compartments. Therefore, metabolite determination at the whole cell level may be insufficient for interpretation of the functional significance of cellular compounds. To investigate the cold‐induced metabolite dynamics at the level of individual sub‐cellular compartments, an integrative platform was developed that combines quantitative metabolite profiling by gas chromatography coupled to mass spectrometry (GC‐MS) with the non‐aqueous fractionation technique allowing separation of cytosol, vacuole and the plastidial compartment. Two mutants of Arabidopsis thaliana representing antipodes in the diversion of carbohydrate metabolism between sucrose and starch were compared to Col‐0 wildtype before and after cold acclimation to investigate interactions of cold acclimation with subcellular re‐programming of metabolism. A multivariate analysis of the data set revealed dominant effects of compartmentation on metabolite concentrations that were modulated by environmental condition and genetic determinants. While for both, the starchless mutant of plastidial phospho‐gluco mutase (pgm) and a mutant defective in sucrose‐phosphate synthase A1, metabolic constraints, especially at low temperature, could be uncovered based on subcellularly resolved metabolite profiles, only pgm had lowered freezing tolerance. Metabolic profiles of pgm point to redox imbalance as a possible reason for reduced cold acclimation capacity.     

Interaction of temperature and irradiance effects on photosynthetic acclimation in two accessions of Arabidopsis thaliana

The effect of temperature and irradiance during growth on photosynthetic traits of two accessions of Arabidopsis thaliana was investigated. Plants were grown at 10 and 22?°C, and at 50 and 300?μmol photons?m(-2)?s(-1) in a factorial design. As known from other cold-tolerant herbaceous species, growth of Arabidopsis at low temperature resulted in increases in photosynthetic capacity per unit leaf area and chlorophyll. Growth at high irradiance had a similar effect. However, the growth temperature and irradiance showed interacting effects for several capacity-related variables. Temperature effects on the ratio between electron transport capacity and carboxylation capacity were also different in low compared to high irradiance grown Arabidopsis. The carboxylation capacity per unit Rubisco, a measure for the in vivo Rubisco activity, was low in low irradiance grown plants but there was no clear growth temperature effect. The limitation of photosynthesis by the utilization of triose-phosphate in high temperature grown plants was less when grown at low compared to high irradiance. Several of these traits contribute to reduced efficiency of the utilization of resources for photosynthesis of Arabidopsis at low irradiance. The two accessions from contrasting climates showed remarkably similar capabilities of developmental acclimation to the two environmental factors. Hence, no evidence was found for photosynthetic adaptation of the photosynthetic apparatus to specific climatic conditions.     

Changes in carbohydrates and freezing tolerance during cold acclimation of red raspberry cultivars grown in vitro and in vivo

Changes in LT50 and carbohydrate levels in response to cold acclimation were monitored in vitro and in vivo in red raspberry ( Rubus idaeus L.) cultivars with different levels of cold hardiness. Entire micropropagated plantlets or shoot tips from 3 cultivars were harvested before, during and after cold acclimation. Cane samples from container-grown plants of 4 cultivars were harvested before and during cold acclimation and deacclimation. Samples were evaluated for cold hardiness (LT50) by controlled freezing, then analyzed for carbohydrates, including starch, sucrose, glucose, fructose and raffinose. Hardiness of cold-acclimated 'Muskoka' and 'Festival' was superior to that of 'Titan' or 'Willamette'. In vitro plantlets had higher levels of soluble carbohydrates on a dry weight basis and higher ratios of sucrose:(glucose+fructose) than the container-grown plants. Total soluble carbohydrates, primarily sucrose, accumulated during cold acclimation in both plantlets (33–56% relative increase) and plants (143–191% relative increase). Sucrose increased 124–165% in plantlets and 253–582% in container-grown plants during acclimation and declined rapidly to the level of control plants during deacclimation. Glucose and fructose also accumulated, but to a lesser extent than sucrose. Raffinose concentrations were very low, but increased significantly during cold acclimation. In vitro, genotype hardiness was related to the high concentrations of total soluble carbohydrates, sucrose and raffinose. In vivo, hardier genotypes had lower concentrations of starch than the less hardy genotypes. These results demonstrated the importance of soluble carbohydrates, especially sucrose, in cold hardening of red raspberry and that the in vitro conditions or controlled acclimation conditions do not necessarily reflect the phenomena observed in vivo.     

Clinal variation in the non-acclimated and cold-acclimated freezing tolerance of Arabidopsis thaliana accessions

Arabidopsis thaliana is a geographically widely spread species consisting of local accessions differing both genetically and phenotypically. These differences may constitute environmental adaptations and a latitudinal cline in freezing tolerance has been shown previously. Many plants, including Arabidopsis, exhibit increased freezing tolerance after cold exposure (cold acclimation). Here we present evidence for geographical clines (both latitudinal and longitudinal) in acclimated (ACC) and non-acclimated (NA) freezing tolerance, estimated from electrolyte leakage measurements on 54 accessions. Leaf Pro contents were not correlated with freezing tolerance, while sugar contents (Glc, Fru, Suc, Raf) were in the ACC, but not the NA state. Expression levels of 14 cold-induced genes were investigated before and after 2 weeks of cold acclimation by quantitative RT-PCR. Expression of the CBF1, 2 and 3 genes was not correlated with freezing tolerance. The expression of some CBF-regulated (COR) genes, however, was correlated specifically with ACC freezing tolerance. A tight correlation between CBF and COR gene expression was only observed under non-acclimating conditions, where CBF and COR expression were also correlated with the expression of PRR5, a component of the circadian clock. Collectively, this study sheds new light on the molecular determinants of plant-freezing tolerance and cold acclimation and their geographical dependence.     

Chill-tolerant Gryllus crickets maintain ion balance at low temperatures

Insect cold tolerance is both phenotypically-plastic and evolutionarily labile, but the mechanisms underlying this variation are uncertain. Chill-susceptible insects lose ion and water homeostasis in the cold, which contributes to the development of injuries and eventually death. We thus hypothesized that more cold-tolerant insects will better maintain ion and water balance at low temperatures. We used rapid cold-hardening (RCH) and cold acclimation to improve cold tolerance of male Gryllus pennsylvanicus, and also compared this species to its cold-tolerant relative (Gryllus veletis). Cold acclimation and RCH decreased the critical thermal minimum (CT_min) and chill coma recovery time (CCR) in G. pennsylvanicus, but while cold acclimation improved survival of 0 °C, RCH did not; G. veletis was consistently more cold-tolerant (and had lower CCR and CT_min) than G. pennsylvanicus. During cold exposure, hemolymph water and Na⁺ migrated to the gut of warm-acclimated G. pennsylvanicus, which increased hemolymph [K⁺] and decreased muscle K⁺ equilibrium potentials. By contrast, cold-acclimated G. pennsylvanicus suffered a smaller loss of ion and water homeostasis during cold exposure, and this redistribution did not occur at all in cold-exposed G. veletis. The loss of ion and water balance was similar between RCH and warm-acclimated G. pennsylvanicus, suggesting that different mechanisms underlie decreased CCR and CT_min compared to increased survival at 0 °C. We conclude that increased tolerance of chilling is associated with improved maintenance of ion and water homeostasis in the cold, and that this is consistent for both phenotypic plasticity and evolved cold tolerance.     

Cucumber carbohydrate metabolism and translocation under chilling night temperature

A cold-tolerant line (NY-1) and a cold-sensitive cultivar (Jinyan 4) of cucumber (Cucumis sativus) were treated with temperatures of 28 degrees C/22 degrees C or 28 degrees C/12 degrees C (day/night) in a 10-h photoperiod. Carbohydrates and related enzymes were assayed from 0 to 4 h after the start of the dark period. Compared to the normal night temperature (22 degrees C, control), sucrose, stachyose and galactinol increased in mature leaves under cold-night treatment (12 degrees C) while sucrose, glucose and fructose in fruits remained unchanged. In peduncles, where stachyose is catabolized to sucrose after long-distance transport, cold nights simultaneously induced a significant increase of stachyose (substrate) and a decrease of sucrose (product), indicating that the metabolic step from stachyose to sucrose in peduncles is crucial to translocation inhibition in cold nights. This decrease was more pronounced in the cold-sensitive cultivar. Similar growth rates of fruits on one-fruit and two-fruit plants under cold-night treatment further confirmed that it is sink activity rather than source supply that is limiting the source-sink translocation. No significant genotypic differences in enzyme activities involved in the stachyose-sucrose conversion, including alkaline alpha-galactosidase, acid alpha-galactosidase, galactokinase, uridine diphosphate (UDP)-galactose pyrophosphorylase, UDP-glucose-4'-epimerase and sucrose synthase, were observed when assayed in an adenosine triphosphate (ATP)-rich in vitro environment. However, the ATP concentration was much higher in peduncles of the cold-tolerant line, indicating that a limiting ATP supply may be partially responsible for the stronger inhibition of the stachyose-sucrose pathway observed in the cold-sensitive cultivar (Jinyan 4).     

Fitness benefits and costs of cold acclimation in Arabidopsis thaliana

Abstract When resources are limited, there is a trade-off between growth/reproduction and stress defense in plants. Most temperate plant species, including Arabidopsis thaliana, can enhance freezing tolerance through cold acclimation at low but nonfreezing temperatures. Induction of the cold acclimation pathway should be beneficial in environments where plants frequently encounter freezing stress, but it might represent a cost in environments where freezing events are rare. In A. thaliana, induction of the cold acclimation pathway critically involves a small subfamily of genes known as the CBFs. Here we test for a cost of cold acclimation by utilizing (1) natural accessions of A. thaliana that originate from different regions of the species' native range and that have experienced different patterns of historical selection on their CBF genes and (2) transgenic CBF overexpression and T-DNA insertion (knockdown/knockout) lines. While benefits of cold acclimation in the presence of freezing stress were confirmed, no cost of cold acclimation was detected in the absence of freezing stress. These findings suggest that cold acclimation is unlikely to be selected against in warmer environments and that naturally occurring mutations disrupting CBF function in the southern part of the species range are likely to be selectively neutral. An unanticipated finding was that cold acclimation in the absence of a subsequent freezing stress resulted in increased fruit production, that is, fitness.     

Cold responses of Arabidopsis mutants impaired in freezing tolerance

Mutants of Arabidopsis thaliana L. (Heynh), characterized asdeficient in their freezing tolerance after cold acclimation,were surveyed for some of the normal responses to cold exposure.In foliar tissue, the coldinducibility of three proteins, thelevels of sucrose and glucose, the fatty acyl composition oflipids, and the accumulation of anthocyanin was examined. Fourmutations (sfr3, sfr4, sfr6, and sfr7) reduced or eliminatedthe accumulation of anthocyanin during cold acclimation. Onemutation (sfr4) prevented the normally cold-induced elevationof sucrose and glucose levels; both sfr4 and another mutation(sfr7) affected fatty acid composition after (and only after)cold acclimation. On the other hand mutations sfr1, sfr2 andsfr5 did not differ significantly from the wild type in anyof the parameters tested, suggesting that they have other, perhapshighly specific, effects on lowtemperature responses. Key words: Arabidopsis thaliana, cold acclimation, freezing tolerance, mutation     

Distinction between cold-sensitive and -tolerant jute by DNA polymorphisms

Jute is the principal coarse fiber for commercial production and use in Bangladesh. Therefore, the development of a high-yielding and environmental-stress tolerant jute variety would be beneficial for the agro economy of Bangladesh. Two molecular fingerprinting techniques, random-amplified polymorphic DNA (RAPD) and amplified-fragment length polymorphism (AFLP) were applied on six jute samples. Two of them were cold-sensitive varieties and the remaining four were cold-tolerant accessions. RAPD and AFLP fingerprints were employed to generate polymorphism between the cold-sensitive varieties and cold-tolerant accessions because of their simplicity, and also because there is no available sequence information on jute. RAPD data were obtained by using 30 arbitrary oligonucleotide primers. Five primers were found to give polymorphism between the varieties that were tested. AFLP fingerprints were generated using 25 combinations of selective-amplification primers. Eight primer combinations gave the best results with 93 polymorphic fragments, and they were able to discriminate the two cold-sensitive and four cold-tolerant jute populations. A cluster analysis, based on the RAPD and AFLP fingerprint data, showed the population-specific grouping of individuals. This information could be useful later in marker-aided selection between the cold-sensitive varieties and cold-tolerant jute accessions.     

The role of raffinose in the cold acclimation response of Arabidopsis thaliana

In many plants raffinose family oligosaccharides are accumulated during cold acclimation. The contribution of raffinose accumulation to freezing tolerance is not clear. Here, we investigated whether synthesis of raffinose is an essential component for acquiring frost tolerance. We created transgenic lines of Arabidopsis thaliana accessions Columbia-0 and Cape Verde Islands constitutively overexpressing a galactinol synthase (GS) gene from cucumber. GS overexpressing lines contained up to 20 times as much raffinose as the respective wild-type under non-acclimated conditions and up to 2.3 times more after 14 days of cold acclimation at 4 degrees C. Furthermore, we used a mutant carrying a knockout of the endogenous raffinose synthase (RS) gene. Raffinose was completely absent in this mutant. However, neither the freezing tolerance of non-acclimated leaves, nor their ability to cold acclimate were influenced in the RS mutant or in the GS overexpressing lines. We conclude that raffinose is not essential for basic freezing tolerance or for cold acclimation of A. thaliana.     

Carbon partitioning and export in transgenic Arabidopsis thaliana with altered capacity for sucrose synthesis grown at low temperature: a role for metabolite transporters

We investigated the role of metabolite transporters in cold acclimation by comparing the responses of wild-type (WT) Arabidopsis thaliana (Heynh.) with that of transgenic plants over-expressing sucrose-phosphate synthase (SPSox) or with that of antisense repression of cytosolic fructose-1,6-bisphosphatase (FBPas). Plants were grown at 23 degrees C and then shifted to 5 degrees C. We compared the leaves shifted to 5 degrees C for 3 and 10 d with new leaves that developed at 5 degrees C with control leaves on plants at 23 degrees C. At 23 degrees C, ectopic expression of SPS resulted in 30% more carbon being fixed per day and an increase in sucrose export from source leaves. This increase in fixation and export was supported by increased expression of the plastidic triose-phosphate transporter AtTPT and, to a lesser extent, the high-affinity Suc transporter AtSUC1. The improved photosynthetic performance of the SPSox plants was maintained after they were shifted to 5 degrees C and this was associated with further increases in AtSUC1 expression but with a strong repression of AtTPT mRNA abundance. Similar responses were shown by WT plants during acclimation to low temperature and this response was attenuated in the low sucrose producing FBPas plants. These data suggest that a key element in recovering flux through carbohydrate metabolism in the cold is to control the partitioning of metabolites between the chloroplast and the cytosol, and Arabidopsis modulates the expression of AtTPT to maintain balanced carbon flow. Arabidopsis also up-regulates the expression of AtSUC1, and to lesser extent AtSUC2, as down-stream components facilitate sucrose transport in leaves that develop at low temperatures.     

Energy response and fatty acid metabolism in Onychostoma macrolepis exposed to low-temperature stress

Temperature is a key environmental factor, and understanding how its fluctuations affect physiological and metabolic processes is critical for fish. The present study characterizes the energy response and fatty acid metabolism in Onychostoma macrolepis exposed to low temperature (10 °C). The results demonstrated that cold stress remarkably disrupted the energy homeostasis of O. macrolepis, then the AMP-activated protein kinase (AMPK) could strategically mobilize carbohydrates and lipids. In particular, when the O. macrolepis were faced with cold stress, the lipolysis was stimulated along with the enhanced fatty acid β-oxidation for energy, while the fatty acid synthesis was supressed in the early stage. Additionally, the fatty acid composition analysis suggested that saturated fatty acid (SFA) might accumulate while monounsaturated fatty acid (MUFA) and polyunsaturated fatty acid (PUFA) in storage lipids (mainly containing non-polar lipid, NPL) could be utilized to supply energy during cold acclimation. Altogether, this study may provide some meritorious for understanding the cold-tolerant mechanism of fish in the viewpoint of energy balance combined with fatty acid metabolism, and thus to contribute to this species rearing in fish farms in the future.     

Heterosis in the freezing tolerance of crosses between two Arabidopsis thaliana accessions (Columbia-0 and C24) that show differences in non-acclimated and acclimated freezing tolerance

Heterosis is broadly defined as the increased vigour of hybrids in comparison to their parents. In the model plant Arabidopsis thaliana, a significant heterosis effect on leaf-freezing tolerance was observed in the F(1) generation of a cross between the accessions Columbia-0 (Col) and C24. Parental Col plants were significantly more freezing-tolerant than C24 plants in both the acclimated and non-acclimated (NA) states. Mid-parent heterosis was observed in the F(1) plants, both in the basic tolerance of non-adapted plants and in freezing tolerance after cold acclimation. Best-parent heterosis, on the other hand, was only found after cold acclimation. The heterosis effect was reduced in the F(2) populations such that only mid-parent heterosis was evident. The leaf content of soluble sugars (fructose (Fru), glucose (Glc), sucrose (Suc) and raffinose (Raf)) increased dramatically in the F(1) plants after cold acclimation as compared to the parental lines. The content of proline (Pro), however, was only moderately increased in the F(1) plants under the same conditions. Correlation analyses showed that only Raf content was consistently related to leaf-freezing tolerance in both the acclimated and NA states. A quantification of mRNA levels in leaves of parental and F(1) lines using quantitative real-time RT-PCR showed no clear indication for an involvement of the investigated genes (CBF (C-repeat binding factor)1, CBF2, (cold-regulated protein (COR) 6.6, COR15a, COR15b, COR47 and COR78) in the heterosis effect.