Abscisic acid deficiency prevents development of freezing tolerance in Arabidopsis thaliana (L.) Heynh.

Summary Abscisic acid (ABA) has been implicated as a regulatory factor in plant cold acclimation. In the present work, the cold-acclimation properties of an ABA-deficient mutant (aba) of Arabidopsis thaliana (L.) Heynh. were analyzed. The mutant had apparently lost its capability to cold acclimate: the freezing tolerance of the mutant was not increased by low temperature treatment but stayed at the level of the nonacclimated wild type. The mutational defect could be complemented by the addition of exogenous ABA to the growth medium, restoring freezing tolerance close to the wild-type level. This suggests that ABA might have a central regulatory function in the development of freezing tolerance in plants. Cold acclimation has been previously correlated to the induction of a specific set of proteins that have been suggested to have a role in freezing tolerance. However, these proteins were also induced in the aba mutant by low temperature treatment.     

Arabidopsis dynamin‐related protein 1E in sphingolipid‐enriched plasma membrane domains is associated with the development of freezing tolerance

The freezing tolerance of Arabidopsis thaliana is enhanced by cold acclimation, resulting in changes in the compositions and function of the plasma membrane. Here, we show that a dynamin‐related protein 1E (DRP1E), which is thought to function in the vesicle trafficking pathway in cells, is related to an increase in freezing tolerance during cold acclimation. DRP1E accumulated in sphingolipid and sterol‐enriched plasma membrane domains after cold acclimation. Analysis of drp1e mutants clearly showed that DRP1E is required for full development of freezing tolerance after cold acclimation. DRP1E fused with green fluorescent protein was visible as small foci that overlapped with fluorescent dye‐labelled plasma membrane, providing evidence that DRP1E localizes non‐uniformly in specific areas of the plasma membrane. These results suggest that DRP1E accumulates in sphingolipid and sterol‐enriched plasma membrane domains and plays a role in freezing tolerance development during cold acclimation.     

Cold acclimation and cold-regulated gene expression in ABA mutants of Arabidopsis thaliana

We have examined the cold-induced enhancement of freezing tolerance and expression of cold-regulated (cor) genes in Arabidopsis thaliana (L.) Heynh (Landsberg erecta) and abscisic acid (ABA)-deficient (aba) and ABA-insensitive (abi) mutants derived from it. The results indicate that the abi mutations had no apparent effect on freezing tolerance, while the aba mutations did: cold-acclimated aba mutants were markedly impaired in freezing tolerance compared to wild-type plants. In addition, it was observed that non-frozen leaves from both control and cold-treated aba mutant plants were more ion-leaky than those from corresponding wild-type plants. These data are consistent with previous observations indicating that ABA levels can affect freezing tolerance. Whether ABA has a direct role in the enhancement of freezing tolerance that occurs during cold acclimation, however, is uncertain. Several studies have suggested that ABA might mediate certain changes in gene expression that occur during cold acclimation. Our data indicate that the ABA-induced expression of three ABA-regulated Arabidopsis cor genes was unaffected in the abi2, abi3, and aba-1 mutants, but was dramatically impaired in the abi1 mutant. Cold-regulated expression of all three cor genes, however, was nearly the same in wild-type and abi1 mutant plants. These data suggest that the cold-regulated and ABA-regulated expression of the three cor genes may be mediated through independent control mechanisms.     

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.     

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.     

The genes ABI1 and ABI2 are involved in abscisic acid- and drought-inducible expression of the Daucus carota L. Dc3 promoter in guard cells of transgenic Arabidopsis thaliana (L.) Heynh.

 The ABA INSENSITIVE1 (ABI1) and ABI2 genes encode homologous type-2C protein phosphatases with redundant yet distinct functions in abscisic acid (ABA) responses. Results from Northern blot analysis showed that ABA- and mannitol-inducible expression of the COR47 and COR78/LTI78/RD29A (COR78) genes was more impaired in the abi2 mutant of Arabidopsis thaliana (L.) Heynh than in the abi1 mutant. Furthermore, ABA-plus-mannitol treatments were additive towards COR47 gene expression; however, the ABA-deficient aba1 mutant showed reduced COR expression relative to the wild type in response to mannitol and ABA-plus-mannitol treatments. These results support the notion that drought- and ABA-signalling pathways are separate yet overlapping. To facilitate quantitative analysis of the genetic control of tissue-specific ABA- and desiccation-response pathways, we analyzed ABA- and mannitol-inducible expression of a carrot (Daucus carota L.) Dc3 promoter:uidA (β-glucuronidase; GUS) chimaeric reporter (Dc3-GUS) in transgenic wild-type, ABA-deficient aba1, and ABA-insensitive abi1 and abi2 mutants. The Dc3 promoter directed ABA- and mannitol-inducible GUS expression in Arabidopsis guard cells and the two treatments were additive. The aba1, abi1, and abi2 mutant genotypes had reduced GUS expression in guard cells of cotyledons in response to mannitol, whereas abi1 and abi2 mutants were reduced in ABA-inducible GUS expression, consistent with overlapping ABA- and drought-response pathways. Quantitative fluorometric GUS assays of leaf extracts showed that abi2 mutants responded less to exogenous ABA than did abi1 mutants, and abi2 mutants responded more to mannitol than did abi1 mutants. We conclude that Dc3-GUSArabidopsis is a tractable system in which to study tissue-specific ABA and drought signalling and suggest that ABI2 functions predominantly over ABI1 in COR78 and COR47 gene expression and guard-cell Dc3-GUS expression. Received: 23 May 1999 / Accepted: 3 December 1999     

Overexpression of a <Emphasis Type="Italic">Panax ginseng</Emphasis> tonoplast aquaporin alters salt tolerance,drought tolerance and cold acclimation ability in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis> plants

Water movement across cellular membranes is regulated largely by a family of water channel proteins called aquaporins (AQPs). Since several abiotic stresses such as, drought, salinity and freezing, manifest themselves via altering water status of plant cells and are linked by the fact that they all result in cellular dehydration, we overexpressed an AQP (tonoplast intrinsic protein) from Panax ginseng, PgTIP1, in transgenic Arabidopsis thaliana plants to test its role in plant’s response to drought, salinity and cold acclimation (induced freezing tolerance). Under favorable conditions, PgTIP1 overexpression significantly increased plant growth as determined by the biomass production, and leaf and root morphology. PgTIP1 overexpression had beneficial effect on salt-stress tolerance as indicated by superior growth status and seed germination of transgenic plants under salt stress; shoots of salt-stressed transgenic plants also accumulated greater amounts of Na⁺ compared to wild-type plants. Whereas PgTIP1 overexpression diminished the water-deficit tolerance of plants grown in shallow (10 cm deep) pots, the transgenic plants were significantly more tolerant to water stress when grown in 45 cm deep pots. The rationale for this contrasting response, apparently, comes from the differences in the root morphology and leaf water channel activity (speed of dehydration/rehydration) between the transgenic and wild-type plants. Plants overexpressed with PgTIP1 exhibited lower (relative to wild-type control) cold acclimation ability; however, this response was independent of cold-regulated gene expression. Our results demonstrate a significant function of PgTIP1 in growth and development of plant cells, and suggest that the water movement across tonoplast (via AQP) represents a rate-limiting factor for plant vigor under favorable growth conditions and also significantly affect responses of plant to drought, salt and cold stresses.     

Development of freezing tolerance in different altitudinal ecotypes of <Emphasis Type="Italic">Salix paraplesia</Emphasis>

Salix paraplesia was used as an experimental model to investigate the effect of short day photoperiod (SD) and low temperature (LT) on development of freezing tolerance and on endogenous abscisic acid (ABA) contents. We characterized differences in SD and LT-induced cold acclimation in three ecotypes from different altitudes. The results demonstrated that cold acclimation could be triggered by exposing the plants to SD or LT alone, and that a combination of the different treatments had an additive effect on freezing tolerance in all ecotypes studied. However, the high altitudinal ecotype was more responsive to SD and LT than the low altitudinal ecotype. Development of freezing tolerance induced by SD and LT was accompanied by changes in ABA contents which were ecotype-dependent. Although the stem had higher initial freezing tolerance, the leaves developed freezing tolerance more quickly than the stem and thus leaves may provide an interesting experimental system for physiological and molecular studies of cold acclimation in woody plants.     

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.     

The Arabidopsis 14-3-3 Protein RARE COLD INDUCIBLE 1A Links Low-Temperature Response and Ethylene Biosynthesis to Regulate Freezing Tolerance and Cold Acclimation

In plants, the expression of 14-3-3 genes reacts to various adverse environmental conditions, including cold, high salt, and drought. Although these results suggest that 14-3-3 proteins have the potential to regulate plant responses to abiotic stresses, their role in such responses remains poorly understood. Previously, we showed that the RARE COLD INDUCIBLE 1A (RCI1A) gene encodes the 14-3-3 psi isoform. Here, we present genetic and molecular evidence implicating RCI1A in the response to low temperature. Our results demonstrate that RCI1A functions as a negative regulator of constitutive freezing tolerance and cold acclimation in Arabidopsis thaliana by controlling cold-induced gene expression. Interestingly, this control is partially performed through an ethylene (ET)-dependent pathway involving physical interaction with different ACC SYNTHASE (ACS) isoforms and a decreased ACS stability. We show that, consequently, RCI1A restrains ET biosynthesis, contributing to establish adequate levels of this hormone in Arabidopsis under both standard and low-temperature conditions. We further show that these levels are required to promote proper cold-induced gene expression and freezing tolerance before and after cold acclimation. All these data indicate that RCI1A connects the low-temperature response with ET biosynthesis to modulate constitutive freezing tolerance and cold acclimation in Arabidopsis.     

Responses of antioxidant enzymes to cold and high light are not correlated to freezing tolerance in natural accessions of Arabidopsis thaliana

Low temperatures and high light cause imbalances in primary and secondary reactions of photosynthesis, and thus can result in oxidative stress. Plants employ a range of low‐molecular weight antioxidants and antioxidant enzymes to prevent oxidative damage, and antioxidant defence is considered an important component of stress tolerance. To figure out whether oxidative stress and antioxidant defence are key factors defining the different cold acclimation capacities of natural accessions of the model plant Arabidopsis thaliana, we investigated hydrogen peroxide (H₂O₂) production, antioxidant enzyme activity and lipid peroxidation during a time course of cold treatment and exposure to high light in four differentially cold‐tolerant natural accessions of Arabidopsis (C24, Nd, Rsch, Te) that span the European distribution range of the species. All accessions except Rsch (from Russia) had elevated H₂O₂ in the cold, indicating that production of reactive oxygen species is part of the cold response in Arabidopsis. Glutathione reductase activity increased in all but Rsch, while ascorbate peroxidase and superoxide dismutase were unchanged and catalase decreased in all but Rsch. Under high light, the Scandinavian accession Te had elevated levels of H₂O₂. Te appeared most sensitive to oxidative stress, having higher malondialdehyde (MDA) levels in the cold and under high light, while only high light caused elevated MDA in the other accessions. Although the most freezing‐tolerant, Te had the highest sensitivity to oxidative stress. No correlation was found between freezing tolerance and activity of antioxidant enzymes in the four accessions investigated, arguing against a key role for antioxidant defence in the differential cold acclimation capacities of Arabidopsis accessions.     

A Subset of Cytokinin Two-component Signaling System Plays a Role in Cold Temperature Stress Response in Arabidopsis

A multistep two-component signaling system is established as a key element of cytokinin signaling in Arabidopsis. Here, we provide evidence for a function of the two-component signaling system in cold stress response in Arabidopsis. Cold significantly induced the expression of a subset of A-type ARR genes and of GUS in Pro_ARR7:GUS transgenic Arabidopsis. AHK2 and AHK3 were found to be primarily involved in mediating cold to express A-type ARRs despite cytokinin deficiency. Cold neither significantly induced AHK2 and AHK3 expression nor altered the cytokinin contents of wild type within the 4 h during which the A-type ARR genes exhibited peak expression in response to cold, indicating that cold might induce ARR expression via the AHK2 and AHK3 proteins without alterations in cytokinin levels. The ahk2 ahk3 and ahk3 ahk4 mutants exhibited enhanced freezing tolerance compared with wild type. These ahk double mutants acclimated as efficiently to cold as did wild type. The overexpression of the cold-inducible ARR7 in Arabidopsis resulted in a hypersensitivity response to freezing temperatures under cold-acclimated conditions. The expression of C-repeat/dehydration-responsive element target genes was not affected by ARR7 overexpression as well as in ahk double mutants. By contrast, the arr7 mutants showed increased freezing tolerance. The ahk2 ahk3 and arr7 mutants showed hypersensitive response to abscisic acid (ABA) for germination, whereas ARR7 overexpression lines exhibited insensitive response to ABA. These results suggest that AHK2 and AHK3 and the cold-inducible A-type ARRs play a negative regulatory role in cold stress signaling via inhibition of ABA response, occurring independently of the cold acclimation pathway.