Identification of a metabolic bottleneck for cold acclimation in Arabidopsis thaliana

Central carbohydrate metabolism of Arabidopsis thaliana is known to play a crucial role during cold acclimation and the acquisition of freezing tolerance. During cold exposure, many carbohydrates accumulate and a new metabolic homeostasis evolves. In the present study, we analyse the diurnal dynamics of carbohydrate homeostasis before and after cold exposure in three natural accessions showing distinct cold acclimation capacity. Diurnal dynamics of soluble carbohydrates were found to be significantly different in cold-sensitive and cold-tolerant accessions. Although experimentally determined maximum turnover rates for sucrose phosphate synthase in cold-acclimated leaves were higher for cold-tolerant accessions, model simulations of diurnal carbohydrate dynamics revealed similar fluxes. This implied a significantly higher capacity for sucrose synthesis in cold-tolerant than cold-sensitive accessions. Based on this implication resulting from mathematical model simulation, a critical temperature for sucrose synthesis was calculated using the Arrhenius equation and experimentally validated in the cold-sensitive accession C24. At the critical temperature suggested by model simulation, an imbalance in photosynthetic carbon fixation ultimately resulting in oxidative stress was observed. It is therefore concluded that metabolic capacities at least in part determine the ability of accessions of Arabidopsis thaliana to cope with changes in environmental conditions.     

A global reorganization of the metabolome in Arabidopsis during cold acclimation is revealed by metabolic fingerprinting

Many plants, including Arabidopsis , increase their freezing tolerance in response to low, non-freezing temperatures. This process is known as cold acclimation and involves many complex biochemical changes at the level of the metabolome. Our goal was to examine the effects of cold acclimation on the metabolome using a non-targeted metabolic fingerprinting approach. Multivariate data analyses indicate that, in Arabidopsis, a global reprogramming of metabolism occurs as a result of cold acclimation. By measuring an entire spectrum of putative metabolites based on mass-to-charge ( m / z ) ratios, vs. an individual or group of metabolite(s), a comprehensive, unbiased assessment of metabolic processes relative to cold acclimation was determined. Whereas leaves shifted to low temperature present metabolic profiles that are constantly changing, leaves developed at low temperature demonstrate a stable complement of components. Although it appears that some metabolic networks are modulated by the environment, others require development under low-temperature conditions for adjustment. Understanding how metabolism as a whole is regulated allows the integration of cellular, physiological and ecological attributes in a biological system, a necessity if complex traits, such as freezing tolerance, are to be modified by breeding or genetic manipulation.     

Altering flux through the sucrose biosynthesis pathway in transgenic Arabidopsis thaliana modifies photosynthetic acclimation at low temperatures and the development of freezing tolerance

To test the hypothesis that the up‐regulation of sucrose biosynthesis during cold acclimation is essential for the development of freezing tolerance, the acclimation responses of wild‐type (WT) Arabidopsis thaliana (Heynh.) were compared with transgenic plants over‐expressing sucrose phosphate synthase (over‐sps) or with antisense repression of either cytosolic fructose‐1,6‐bisphosphatase (antifbp) or sucrose phosphate synthase (antisps). Plants were grown at 23 °C and then shifted to 5 °C. The leaves shifted to 5 °C for 10 d and the new leaves that developed at 5 °C were compared with control leaves on plants at 23 °C. Plants over‐expressing sucrose phosphate synthase showed improved photosynthesis and increased flux of fixed carbon into sucrose when shifted to 5 °C, whereas both antisense lines showed reduced flux into soluble sugars relative to WT. The improved photosynthetic performance by the over‐sps plants shifted to 5 °C was associated with an increase in freezing tolerance relative to WT (?9.1 and ?7.2 °C, respectively). In contrast, both antisense lines showed impaired development of freezing tolerance (? 5.2 and ?5.8 °C for antifbp and antisps, respectively) when shifted to 5 °C. In the new leaves developed at 5 °C the recovery of photosynthesis as typically seen in WT was strongly inhibited in both antisense lines and this inhibition was associated with a further failure of both antisense lines to cold acclimate. Thus, functional sucrose biosynthesis at low temperature in the over‐sps plants reduced the inhibition of photosynthesis, maintained the mobilization of carbohydrates from source leaves to sinks and increased the rate at which freezing tolerance developed. Modification of sucrose metabolism therefore represents an additional approach that will have benefits both for the development of freezing tolerance and over‐wintering, and for the supply of exportable carbohydrate to support growth at low temperatures.     

Sucrose metabolism of perennial ryegrass in relation to cold acclimation

Sugar metabolism is one of the important factors involved in winter hardiness and since the discovery of sucrose biosynthesis, considerable advances have been made in understanding its regulation and crucial role. This investigation examined the changes in activities of sucrose metabolizing enzymes and sugar content during cold hardening of perennial ryegrass (Lolium perenne L.). Changes in acid invertase (AI), sucrose synthase (SS) and sucrose phosphate synthase (SPS) along with all the three soluble sugars glucose, fructose and sucrose were measured in leaves and stem base tissue during cold acclimation. Although fructans were the predominant carbohydrate the changes in glucose, fructose and sucrose were significant. All the three soluble sugars in both leaf and stem tissues started to decrease from the first day and continued up to day 7 and thereafter started to increase until day 28. AI in the soluble fraction showed a higher activity than that in the cell wall bound fraction. In both the leaf and stem bases soluble AI activity increased during the first week and after that it started to decrease gradually. On the other hand both the SS and SPS increased gradually throughout the acclimation period. Sucrose content was negatively correlated with AI and positively correlated with SS and SPS accounting well for the relation between the substrate and enzyme activity. These results suggest that AI, SS and SPS in ryegrass are regulated by cold acclimation and play an important role in sugar accumulation and acquisition of 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.     

植物抗寒及其基因表达研究进展

植物经过逐渐降低的温度从而提高抗寒能力 ,这个过程被人们称为低温驯化。植物低温驯化过程是一个复杂的生理、生化和能量代谢变化过程 ,这些变化主要包括膜系统的稳定性、可溶性蛋白的积累和小分子渗透物质 ,比如脯氨酸、糖等 ,这些变化中的一些是植物抗寒必需的 ,而另外一些变化不是必需的。主要对冷害和低温生理生化变化、低温诱导表达基因的功能和作用、低温驯化的调节机制及其信号转导方面进行了综述。通过差别筛选 c DNA文库的方法已经鉴定了许多低温诱导表达、进而提高植物抗寒能力的基因 ,其中有脱水素、COR基因和 CBF1转录因子等。低温信号的感受、转导和调节表达是低温驯化的关键环节 ,低温信号的转导过程与干旱胁迫之间具有一定的交叉 ,这为利用 ABA等来提高植物抗寒能力成为可能 ,相信不久的将来人们可以通过提高植物抗寒能力从而增加经济产量成为现实。     

Klebsormidium flaccidum, a charophycean green alga, exhibits cold acclimation that is closely associated with compatible solute accumulation and ultrastructural changes

To elucidate the fundamental mechanisms and subsequent evolutionary aspects of plant cold acclimation, we examined the effect of cold acclimation on freezing tolerance in Klebsormidium flaccidum, a green alga belonging to Charophyceae, a sister group of land plants. Freezing tolerance of K. flaccidum was significantly enhanced by cold treatment: survival increased from 15% at -10 degrees C when grown at 18 degrees C to 55 and 85% after exposure at 2 degrees C for 2 and 7 d, respectively. Accompanying the development of freezing tolerance, soluble sugars (glucose and sucrose), a putative glycoside and amino acids, including gamma-aminobutyric acid (GABA), accumulated to high levels in the alga, suggesting that these solutes play a crucial role in the cold acclimation of K. flaccidum. Interestingly, the application of abscisic acid (ABA) did not change the freezing tolerance of the alga. We also observed changes in cell structure, including increased numbers and sizes of starch grains in chloroplasts, chloroplast enlargement, vacuole size reduction and cytoplasmic volume increase. These results suggest that K. flaccidum responds well to cold treatment and develops freezing tolerance in a process comparable to that of land plants.     

磷脂酶Dδ参与植物的低温驯化过程

对经低温驯化和未经低温驯化的磷脂酶Dδ（PLDδ）基因敲除突变体与野生型植株进行冻害胁迫处理后,比较2种基因型植株的抗冻性。结果发现,经低温驯化的PLDδ敲除突变体的抗冻性明显低于野生型,而未经低温驯化的PLD礅除突变体与野生型的抗冻性没有显著差异,表明PLDδ参与植物的低温驯化过程。对PLDδ的作用途径进行分析,发现PLDδ在低温驯化过程中不参与抗氧化酶活性的调节,对脯氨酸和可溶性糖的积累起负调节作用,但是参与低温信号转导物质ABA诱导抗冻性的过程。     

磷脂酶Dδ参与植物的低温驯化过程

对经低温驯化和未经低温驯化的磷脂酶Dδ (PLDδ)基因敲除突变体与野生型植株进行冻害胁迫处理后, 比较2种基因型植株的抗冻性。结果发现, 经低温驯化的PLDδ敲除突变体的抗冻性明显低于野生型, 而未经低温驯化的PLDδ敲除突变体与野生型的抗冻性没有显著差异, 表明PLDδ参与植物的低温驯化过程。对PLDδ的作用途径进行分析, 发现PLDδ在低温驯化过程中不参与抗氧化酶活性的调节, 对脯氨酸和可溶性糖的积累起负调节作用, 但是参与低温信号转导物质ABA诱导抗冻性的过程。     

Changes in carbohydrates, ABA and bark proteins during seasonal cold acclimation and deacclimation in Hydrangea species differing in cold hardiness

Cold injury is frequently seen in the commercially important shrub Hydrangea macrophylla but not in Hydrangea paniculata. Cold acclimation and deacclimation and associated physiological adaptations were investigated from late September 2006 to early May 2007 in stems of field-grown H. macrophylla ssp. macrophylla (Thunb.) Ser. cv. Blaumeise and H. paniculata Sieb. cv. Kyushu. Acclimation and deacclimation appeared approximately synchronized in the two species, but they differed significantly in levels of mid-winter cold hardiness, rates of acclimation and deacclimation and physiological traits conferring tolerance to freezing conditions. Accumulation patterns of sucrose and raffinose in stems paralleled fluctuations in cold hardiness in both species, but H. macrophylla additionally accumulated glucose and fructose during winter, indicating species-specific differences in carbohydrate metabolism. Protein profiles differed between H. macrophylla and H. paniculata, but distinct seasonal patterns associated with winter acclimation were observed in both species. In H. paniculata concurrent increases in xylem sap abscisic acid (ABA) concentrations ([ABA](xylem)) and freezing tolerance suggests an involvement of ABA in cold acclimation. In contrast, ABA from the root system was seemingly not involved in cold acclimation in H. macrophylla, suggesting that species-specific differences in cold hardiness may be related to differences in [ABA](xylem). In both species a significant increase in stem freezing tolerance appeared long after growth ceased, suggesting that cold acclimation is more regulated by temperature than by photoperiod.     

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.     

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.     

Environmental regulation and physiological basis of freezing tolerance in woody plants

Cold acclimation of plants is a complex process involving a number of biochemical and physiological changes. The ability to cold acclimate is under genetic control. The development of freezing tolerance in woody plants is generally triggered by non-freezing low temperatures but can also be induced by mild drought or exogenous abscisic acid, as well as by short photoperiod. In nature, the extreme freezing tolerance of woody plants is achieved during sequential stages of cold acclimation the first of which is initiated by short photoperiods and non-freezing low temperatures, and the second by freezing temperatures. Although recent breakthroughs have increased our knowledge on the physiological molecular basis of freezing tolerance in herbaceous species, which acclimate primarily in response to non-freezing low temperatures, very little is known about cold acclimation of woody plants. This article attempts to review our current understanding of the physiological aspects that underline cold acclimation in woody plants.