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.     

Differential responses of Arabidopsis ecotypes to cold, chilling and freezing temperatures

Arabidopsis plants show an increase in freezing tolerance in response to exposure to low nonfreezing temperatures, a phenomenon known as cold acclimation. In the present study, we evaluated the physiological and morphological responses of various Arabidopsis ecotypes to continuous growth under chilling (14°C) and cold (6°C) temperatures and evaluated their basal freezing tolerance levels. Seedlings of Arabidopsis plants were extremely sensitive to low growth temperatures: the hypocotyls and petioles were much longer and the angles of the second pair of true leaves were much greater in plants grown at 14°C than in those grown at 22°C, whereas just intermediate responses were observed under the cold temperature of 6°C. Flowering time was also markedly delayed at low growth temperatures and, interestingly, lower growth temperatures were accompanied by longer inflorescences. Other marked responses to low temperatures were changes in pigmentation, which appeared to be both ecotype specific and temperature dependent and resulted in various visual phenotypes such as chlorosis, necrosis or enhanced accumulation of anthocyanins. The observed decreases in chlorophyll contents and accumulation of anthocyanins were much more prominent in plants grown at 6°C than in those grown at 14°C. Among the various ecotypes tested, Mt‐0 plants markedly accumulated the highest levels of anthocyanins upon growth at 6°C. Freezing tolerance examination revealed that among 10 ecotypes tested, only C24 plants were significantly more sensitive to subzero temperatures. In conclusion, Arabidopsis ecotypes responded differentially to cold (6°C), chilling (14°C) and freezing temperatures, with specific ecotypes being more sensitive in particular traits to each low temperature.     

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.     

Differential remodeling of the lipidome during cold acclimation in natural accessions of Arabidopsis thaliana

Freezing injury is a major factor limiting the geographical distribution of plant species and the growth and yield of crop plants. Plants from temperate climates are able to increase their freezing tolerance during exposure to low but non‐freezing temperatures in a process termed cold acclimation. Damage to cellular membranes is the major cause of freezing injury in plants, and membrane lipid composition is strongly modified during cold acclimation. Forward and reverse genetic approaches have been used to probe the role of specific lipid‐modifying enzymes in the freezing tolerance of plants. In the present paper we describe an alternative ecological genomics approach that relies on the natural genetic variation within a species. Arabidopsis thaliana has a wide geographical range throughout the Northern Hemisphere with significant natural variation in freezing tolerance that was used for a comparative analysis of the lipidomes of 15 Arabidopsis accessions using ultra‐performance liquid chromatography coupled to Fourier‐transform mass spectrometry, allowing the detection of 180 lipid species. After 14 days of cold acclimation at 4°C the plants from most accessions had accumulated massive amounts of storage lipids, with most of the changes in long‐chain unsaturated triacylglycerides, while the total amount of membrane lipids was only slightly changed. Nevertheless, major changes in the relative amounts of different membrane lipids were also evident. The relative abundance of several lipid species was highly correlated with the freezing tolerance of the accessions, allowing the identification of possible marker lipids for plant freezing tolerance.     

Responses of the bed bug,Cimex lectularius,to temperature extremes and dehydration: levels of tolerance,rapid cold hardening and expression of heat shock proteins

This study of the bed bug, Cimex lectularius, examines tolerance of adult females to extremes in temperature and loss of body water. Although the supercooling point (SCP) of the bed bugs was approximately −20°C, all were killed by a direct 1 h exposure to −16°C. Thus, this species cannot tolerate freezing and is killed at temperatures well above its SCP. Neither cold acclimation at 4°C for 2 weeks nor dehydration (15% loss of water content) enhanced cold tolerance. However, bed bugs have the capacity for rapid cold hardening, i.e. a 1‐h exposure to 0°C improved their subsequent tolerance of −14 and −16°C. In response to heat stress, fewer than 20% of the bugs survived a 1‐h exposure to 46°C, and nearly all were killed at 48°C. Dehydration, heat acclimation at 30°C for 2 weeks and rapid heat hardening at 37°C for 1 h all failed to improve heat tolerance. Expression of the mRNAs encoding two heat shock proteins (Hsps), Hsp70 and Hsp90, was elevated in response to heat stress, cold stress and during dehydration and rehydration. The response of Hsp90 was more pronounced than that of Hsp70 during dehydration and rehydration. Our results define the tolerance limits for bed bugs to these commonly encountered stresses of temperature and low humidity and indicate a role for Hsps in responding to these stresses.     

Winter cold tolerance and the geographic range separation of Bromus tectorum and Bromus rubens,two severe invasive species in North America

The invasive grasses Bromus rubens and Bromus tectorum are responsible for widespread damage to semiarid biomes of western North America. Bromus. tectorum dominates higher and more northern landscapes than its sister species B. rubens, which is a severe invader in the Mojave desert region of the American Southwest. To assess climate thresholds controlling their distinct geographic ranges, we evaluated the winter cold tolerance of B. tectorum and B. rubens. Freezing tolerance thresholds were determined using electrolyte leakage and whole‐plant mortality. The responses of the two species to winter cold and artificial freezing treatments were similar in 2007–2008 and 2009–2010. When grown at minimum temperatures of 10 °C, plants of both species had cold tolerance thresholds near ?10 °C, while plants acclimated to a daily minimum of ?10 to ?30 °C survived temperatures down to ?31 °C. In the winter of 2010–2011, a sudden severe cold event on December 9, 2010 killed all B. rubens populations, while B. tectorum was not harmed; all tested plants were 7–8 weeks old. Controlled acclimation experiments demonstrated that 8‐week‐old plants of B. rubens had a slower acclimation rate to subzero temperatures than B. tectorum and could not survive a rapid temperature drop from 1 to ?14 °C. Four‐month‐old B. rubens populations were as cold tolerant as B. tectorum. Our results show that severe and sudden freeze events in late autumn can kill young plants of B. rubens but not B. tectorum. Such events could exclude B. rubens from the relatively cold, Intermountain steppe biome of western North America where B. tectorum predominates.     

Heterosis in the freezing tolerance, and sugar and flavonoid contents of crosses between Arabidopsis thaliana accessions of widely varying freezing tolerance

Heterosis is defined as the increased vigour of hybrids in comparison to their parents. We investigated 24 F(1) 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.     

Cold acclimation in warmer extended autumns impairs freezing tolerance of perennial ryegrass (Lolium perenne) and timothy (Phleum pratense)

The effect of variable autumn temperatures in combination with decreasing irradiance and daylength on photosynthesis, growth cessation and freezing tolerance was investigated in northern‐ and southern‐adapted populations of perennial ryegrass (Lolium perenne) and timothy (Phleum pratense) intended for use in regions at northern high latitudes. Plants were subjected to three different acclimation temperatures; 12, 6 and 9/3°C (day/night) for 4 weeks, followed by 1 week of cold acclimation at 2°C under natural light conditions. This experimental setup was repeated at three different periods during autumn with decreasing sums of irradiance and daylengths. Photoacclimation, leaf elongation and freezing tolerance were studied. The results showed that plants cold acclimated during the period with lowest irradiance and shortest day had lowest freezing tolerance, lowest photosynthetic activity, longest leaves and least biomass production. Higher acclimation temperature (12°C) resulted in lower freezing tolerance, lower photosynthetic activity, faster leaf elongation rate and higher biomass compared with the other temperatures. Photochemical mechanisms were predominant in photoacclimation. The northern‐adapted populations had a better freezing tolerance than the southern‐adapted except when grown during the late autumn period and at the highest temperature; then there were no differences between the populations. Our results indicate that the projected climate change in the north may reduce freezing tolerance in grasses as acclimation will take place at higher temperatures and shorter daylengths with lower irradiance.     

Freezing tolerance, cold acclimation and oxidative stress in potato. Paraquat tolerance is related to acclimation but is a poor indicator of freezing tolerance

Potato is a species commonly cultivated in temperate areas where the growing season may be interrupted by frosts, resulting in loss of yield. Cultivated potato, Solanum tuberosum, is freezing sensitive, but it has several freezing-tolerant wild potato relatives, one of which is S. commersonii. Our study was aimed to resolve the relationship between enhanced freezing tolerance, acclimation capacity and capacity to tolerate active oxygen species. To be able to characterize freezing tolerant ideotypes, a potato population (S1), which segregates in freezing tolerance, acclimation capacity and capacity to tolerate superoxide radicals, was produced by selfing a somatic hybrid between a freezing-tolerant Solanum commersonii (LT₅₀=-4.6°C) and -sensitive S. tuberosum (LT₅₀=-3.0°C). The distribution of non-acclimated freezing tolerance (NA-freezing tolerance) of the S1 population varied between the parental lines and we were able to identify genotypes, having significantly high or low NA-freezing tolerance. When a population of 25 genotypes was tested both for NA-freezing and paraquat (PQ) tolerance, no correlation was found between these two traits (R = 0.02). However, the most NA-freezing tolerant genotypes were also among the most PQ tolerant plants. Simultaneously, one of the NA-freezing sensitive genotypes (2022) (LT₅₀=-3.0°C) was observed to be PQ tolerant. These conflicting results may reflect a significant, but not obligatory, role of superoxide scavenging mechanisms in the NA-freezing tolerance of S. commersonii. The freezing tolerance after cold acclimation (CA-freezing tolerance) and the acclimation capacity (AC) was measured after acclimation for 7 days at 4/2°C. Lack of correlation between NA-freezing tolerance and AC (R =-0.05) in the S1 population points to independent genetic control of NA-freezing tolerance and AC in Solanum sp. Increased freezing tolerance after cold acclimation was clearly related to PQ tolerance of all S1 genotypes, especially those having good acclimation capacity. The rapid loss of improved PQ tolerance under deacclimation conditions confirmed the close relationship between the process of cold acclimation and enhanced PQ tolerance. Here, we report an increased PQ tolerance in cold-acclimated plants compared to non-acclimated controls. However, we concluded that high PQ tolerance is not a good indicator of actual freezing tolerance and should not be used as a selectable marker for the identification of a freezing-tolerant genotype.     

The role of the photosynthetic apparatus in cold acclimation of <Emphasis Type="Italic">Lolium multiflorum</Emphasis>. Characteristics of novel genotypes low-sensitive to PSII over-reduction

During cold acclimation by higher plants, temperature perception via changes in redox state of Photosystem II (PSII) and subsequent acclimation of the photosynthetic apparatus to cold is very important for achieving freezing tolerance. These properties were studied in two groups (A and B) of the same backcross 3 (BC₃) progeny derived from a triploid hybrid of Festuca pratensis (2×) × Lolium multiflorum (4×) backcrossed three times onto diploid L. multiflorum cultivars. Leaves of Group A plants formed at 20°C at medium-low light were unable to acclimate their photosynthetic apparatus to cold. Compared to Group B, the Group A plants were also more frost sensitive. This acclimation ability correlated with the freezing tolerance of the plants. However, leaves of the same Group A plants developed at 20°C, but under higher-light conditions had increased ability to acclimate their photosynthetic apparatus to cold. It was concluded that Group A plants may have impaired PSII temperature perception, and this then resulted in their poor capability to cold acclimate.     

Modification of the intracellular sugar content alters the incidence of freeze-induced membrane lesions of protoplasts isolated from Arabidopsis thaliana leaves

Sugar content and freezing tolerance of protoplasts of Arabidopsis thaliana leaves were manipulated by incubating seedlings in a sucrose solution before protoplast isolation. Incubation in a 400 mM sucrose solution at 2 °C in the dark increased their freezing tolerance equivalent to that achieved after a conventional cold acclimation at 2 °C. The increased freezing tolerance was due to a decrease in the incidence of freeze‐induced lesions: expansion‐induced lysis (EIL) between ?2 and ?4 °C and loss of osmotic responsiveness (LOR) between ?5 and ?12 °C. The concentration of sucrose in the incubation medium required to minimize the incidence of the lesions was substantially different: 10–35 mM for EIL and 30–400 mM for LOR. Incubation in the sucrose solution at 23 °C decreased LOR only at ?5 and ?6 °C but less than that incubated at 2 °C, and there was no effect on EIL. Incubation in sorbitol solutions at 2 °C also decreased LOR at ?5 and ?6 °C but much less than in the sucrose solution. These results suggest that low concentrations of sucrose act as a metabolic substrate for the low‐temperature‐induced alterations required for the amelioration of EIL and, at higher concentrations, sucrose has a direct cryoprotective effect to minimize LOR.     

Accumulation of glycinebetaine during cold acclimation and freezing tolerance in leaves of winter and spring barley plants

A study was performed to examine whether or not betaine (glycinebetaine), a compatible solute, is accumulated in response to cold stress and is involved in mechanisms that protect plants from freezing injury. For this purpose, we used near-isogenic lines of barley, with each line differing only in a single gene for the spring type of growth habit; the various lines were produced by back-crosses to a recurrent cultivar of the winter type. The winter type of growth habit requires a low temperature for triggering of flower development (vernalization), whereas the spring type does not. Betaine was accumulated to five times the basal level over the course of 3 weeks at low temperature (5 °C) in the winter-type cultivar and in a spring-sh line having the sh gene for the spring-type growth habit, but the level was only doubled in the spring-Sh3 line, which carried the Sh₃ gene for the spring-type growth habit. Among near-isogenic lines of the same cultivar, the levels of betaine accumulated in leaves at low temperature were well correlated with the percentages (on a dry weight basis) of green leaves that survived freezing injury (-5 °C). This observation indicates the possibility, separate from the recognized role of betaine in the response to salinity and/or drought, that betaine accumulates in response to cold stress and that the accumulation of betaine during cold acclimation is associated to some extent with freezing tolerance in leaves of barley plants.     

RAPID COLD HARDENING PROVIDING HIGHER COLD TOLERANCE THAN COLD ACCLIMATION IN THE PINE NEEDLE GALL MIDGE THECODIPLOSIS JAPO‐NENSIS LARVAE*

Abstract The responses of overwintering larvae of the pine needle gall midge Thecodiplosis japonensis Uchida et Inouye to rapid cold hardening and cold acclimation were studied. A rapid cold hardening response is found in the 3rd instar larvae of T. japonensis. When overwintering larvae are transferred directly from 27°C to ‐ 15°C for 3 h, there is only 17.9% survival, whereas exposure to 4°C for 2 h prior to transfer to ‐ 15°C increases survival to 40.0%. The acquired cold tolerance is transient and is rapidly lost (after 15 min at 27°C). Rapid cold hardening is more effective in maintaining larval survival than cold acclimation. Different mechanisms are suggested to regulate the insect's cold hardiness under rapid cold hardening and cold acclimation.     

Plant Cold Acclimation: The Role of Abscisic Acid

The freezing tolerance or cold acclimation of plants is enhanced over a period of time by temperatures below 10°C and by a short photoperiod in certain species of trees and grasses. During this process, freezing tolerance increases 2–8°C in spring annuals, 10–30°C in winter annuals, and 20–200°C in tree species. Gene upregulation and downregulation have been demonstrated to be involved in response to environmental cues such as low temperature. Evidence suggests ABA can substitute for the low temperature stimulus, provided there is also an adequate supply of sugars. Evidence also suggests there may be ABA-dependent and ABA-independent pathways involved in the acclimation process. This review summarizes the role of ABA in cold acclimation from both a historical and recent perspective. It is concluded that it is highly unlikely that ABA regulates all the genes associated with cold acclimation; however, it definitely regulates many of the genes associated with an increase in freezing tolerance.     

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.     

Effects of acclimation on the thermal tolerance of the brown planthopper Nilaparvata lugens (Stål)

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The oatmeal nematode <Emphasis Type="Italic">Panagrellus redivivus</Emphasis> survives moderately low temperatures by freezing tolerance and cryoprotective dehydration

The cold tolerance abilities of only a few nematode species have been determined. This study shows that the oatmeal nematode, Panagrellus redivivus, has modest cold tolerance with a 50% survival temperature (S ₅₀) of −2.5°C after cooling at 0.5°C min⁻¹ and freezing for 1 h. It can survive low temperatures by freezing tolerance and cryoprotective dehydration; although freezing tolerance appears to be the dominant strategy. Freezing survival is enhanced by low temperature acclimation (7 days at 5°C), with the S ₅₀ being lowered by a small but significant amount (0.42°C). There is no cold shock or rapid cold hardening response under the conditions tested. Cryoprotective dehydration enhances the ability to survive freezing (the S ₅₀ is lowered by 0.55°C, compared to the control, after 4 h freezing at −1°C) and this effect is in addition to that produced by acclimation. Breeding from survivors of a freezing stress did not enhance the ability to survive freezing. The cold tolerance abilities of this nematode are modest, but sufficient to enable it to survive in the cold temperate environments it inhabits.     

Magnetic resonance imaging (MRI) of water during cold acclimation and freezing in winter wheat

Nuclear magnetic resonance (NMR) spectroscopy and magnetic resonance imaging (MRI) were used to analyse changes in the physical state of water in wheat crowns during cold acclimation and during the freezing/thawing cycle. Spectroscopically measured average spin-spin relaxation times (T₂) decreased during cold acclimation and increased when plants were grown at normal temperature. Spin-spin relaxation images whose contrast is proportional to T₂, times were calculated allowing association of water relaxation with regions of tissue in spin-echo images during acclimation and freezing. Images taken during freezing revealed nonuniform freezing of tissue in crowns and roots. Acclimated and non-acclimated wheat crowns were imaged during freezing and after thawing. Spin-echo image signal intensity and T₂ times decreased dramatically between -4°C and -8°C as a result of a decrease in water mobility during freezing. Images collected during thawing were diffuse with less structure and relaxation times were longer, consistent with water redistribution in tissue after membrane damage.