Plants have ubiquitous response to abiotic stresses causing severe damage to structural organization and physiological mechanism of plant cell. The capacity of surviving under those adverse condition makes the plant Physcomitrella patens as a model for functional genomics of adaptive environments. In higher plants, exogenous ABA application and artificial cold acclimation were found to be the tools of enhancing stress. But the mechanism is still unclear in lower plants like P patens. Analysis of ABA-insensitive lines would be important tools for identification of gene expression pathway involved in ABA perception of signal transduction. Present study was undertaken to clarify the mechanism underlying physiological processes leading to the development of freezing, desiccation, salt and osmotic stress tolerance in ABA insensitive lines of P. patens. In this study we used the transgenic D2-1 line expressing the catalytic domain of protein phosphatase 2C negatively regulating ABA signaling, and the ABA-insensitive AR7 mutant isolated by ultraviolet mutagenesis and wild type of P.patens. Both mutant and transgenic line of P. patens demonstrated as a negative regulator of ABA signaling and were sensitive to freezing, desiccation and osmotic stresses. In wild type, ABA enhanced tolerance to all type of abiotic stresses. Accumulated sugar and proteins in mutant, transgenic and wild type lines explained their role in stress tolerance under ABA treatment. Changes in cold-acclimated protonema cells explained the influence of freezing stresses in P. patens. Protonema cells of the wild Physcomitrella patens acquired freezing tolerance in response to cold and abscisic acid treatment. Effects of low temperature treatment on freezing tolerance indicated that the low temperature treatment for seven days of wild type plants increased the freezing tolerance significantly but the treatment had little effect on that of ABA-insensitive lines. In contrast, non-acclimated protonemata from both wild type and ABA-insensitive plants were of less survival rate. These data were consistent with ABA-induced stress tolerance in P.patens, indicating that ABA treatment increased freezing tolerance in wild P.patens compared to ABA-insensitive plants. Protein and sugar analyses indicated that cold treatment induces accumulation of specific LEA-like proteins and soluble sugars in wild type, whereas the treatment increased only soluble sugars but not LEA-like proteins in the ABA-insensitive lines. As the cold acclimation could not increase freezing tolerance in ABA insensitive lines, it can be concluded that Physcomitrella patens had an ABA dependent pathway of cold induced gene expression.
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Environmental stresses like cold, salt and desiccation have enormous impact on crop yield and distribution around the world. They adversely affect crop productivity through the regulation of spatial distribution and genetic expression of plants . The alteration of genetic expression under cold stress is mainly due to the changes in metabolic reactions, though indirect effect like osmotic (chilling induced inhibition of water uptake and freezing induced cellular dehydration), oxidative and other stresses are associated with cold stress. Desiccation and salinity cause osmotic stress to living terrestrial plants and lead to water deficit in plants, which consequently affects plant growth and development . The response of plants cells to stresses also depends upon the plant species and type of induction. Bryophytes are considered as an important source of abiotic stress-tolerant genes due to their high degree of adaptation under extreme environments. They are widely distributed from tropical rain forest to dessert and frigid zones. 'Poikilohydry', a primitive trait of land plants, had been lost in the evolutionary process of vascular plants; which is still retained in the tissues of bryophytes. Most tropical plants are chilling sensitive and cannot even tolerate exposure to non-freezing low temperature, but some bryophyte species readily tolerate -20ËšC or lower temperatures. When desiccated, bryophytes lose most of water retained in the tissues but when watered they can equilibrate rapidly with surrounding water potential and fully hydrated without significant damage. However, some members of bryophytes are also important for their tolerance to other stresses like salt stress.
The moss Physcomitrella patens, a representative of bryophytes, has newly emerged as a stress-tolerant model for functional genomics approaches . Its simple body plan and the small number of different cell types makes this plant suitable for elucidation of developmental processes . The sequenced genome of the moss Physcomitrella patens provides a powerful tool for comparative analysis of land plant genomes . Investigation of the molecular mechanisms involved in the abiotic stress response of plants has made substantial progress in recent years . Recently, Minami et al. investigated the freezing tolerance of Physcomitrella protonemata, which was markedly enhanced upon pre-treatment with abscisic acid (ABA). Benito and Rodriguez-Navarro first analysed the salt tolerance in P. patens and indicated that the plants were able to tolerate NaCl concentrations up to 600 mM when the plants had been slowly adapted to increasing salt concentrations. Kroemer et al. have characterized the stress-responsive expression pattern of two Physcomitrella genes homologous to the Arabidopsis RCI2A and RCI2B genes. The regulation of these genes upon different stress treatments indicates that stress-related signaling pathways might have been altered during the evolutionary development of land plants. Recent investigation on functional analysis of ABI1-related protein phosphatise type 2C revealed the evolutionary conserved regulation of abscisic acid signalling between Arabiodopsis and the moss P. patens . But the physiological data and information about the molecular events underlying the abiotic stress response in Physcomitrella are still limited.
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Cold acclimation is the process by which plants enhances freezing tolerance in response to seasonal changes from autumn to winter. Most of the terrestrial higher land plants growing in the temperate zone developing tolerance level to freezing in winter by a process of seasonal cold acclimation. The process of cold acclimation is associated with combination of biochemical and physiological changes within the plant cells (Levitt, 1980; Sakai and Larcher, 1987) including the expression of numbers of genes, acquirement of LEA-like proteins, accumulate compatible solutes as soluble sugars and activation of metabolic enzymes (Pearce 1999; Thomashow 1999; Xin and Browse 2000; Seki et al. 2001). During cold acclimation, increases in levels of endogenous abscisic acid (ABA), which include the majority of the response to water stress, is common among higher plants such as tomato (Daie and Campbell 1981), potato (Chen et al. 1983) , winter wheat (Lalk and Dorffling 1985), cacti (Loik and Nobel 1993), maize (Anderson et al. 1994), barley (Bravo et al. 1998), Arabidopsis (Lang et al. 1994), poplar (Jouve et al. 2000), and silver birch (Li et al. 2002). The expression of stress-responsive genes also found to be regulated by ABA rendering the enhancement of freezing tolerance in higher plants (Zeevart and Creelman 1988). Scientific evidences suggested the existence of both ABA independent and independent pathways with cold acclimation in plants . Investigation on Arabidopsis thaliana that the ABA-insensitive mutant abi1-1 of cannot acquire freezing tolerance by ABA treatment though it retains capacity to increase freezing tolerance by cold acclimation . But, little is known how ABA is involved in cold acclimation in lower plants like bryophytes. However, ABA is also involved in other abiotic stress adaptation of P. patens, since exogenously applied ABA increases freezing tolerance and increased endogenous ABA levels were detected upon osmotic stress treatment .
Extensive analyses were made on molecular and cellular responses to stresses through the study of accumulation of various kind of proteins and smaller molecules, including sugars, proline, and glycine betaine . We previously reported that the protonema cells of the moss Physcomitrella patens acquire freezing tolerance when they were treated with 10 μM ABA for one day. At the cellular level, ABA treatment resulted in reduction in sizes of chloroplasts and vacuoles, and also thickening of cell wall. These morphological changes were associated with reduction in the amount of starch and accumulation of low molecular weight soluble sugars . We found that the major soluble sugars accumulated in association with ABA-induced freezing tolerance are disaccharide sucrose and trisaccharide theanderose . Though it is suggested that these low-molecular-weight sugars serve mainly as (1) osmolytes against hydraulic pressure across cellular membranes generated by extracellular ice crystals and (2) protectants for cellular membranes and enzymes from irreversible damage caused by freezing. Detail mechanism of involvement of sugar in freezing tolerance is yet to be elucidated.
Present study therefore, was aimed to clarify mechanisms underlying physiological processes leading to development of abiotic stress tolerance in moss protonema cells. Here we particularly emphasized on the exploration of the role of ABA and soluble sugars in the pathway of cold acclimated freezing tolerance using ABA insensitive AR7 mutant and ABA insensitive D2-1 transgenic strain.
Materials and Methods
Plant materials and growth condition
Three different lines (D2-1, AR7 and wild type) of Physcomitrella patens were used as the plant materials for this study. D2-1 is a transgenic strain of P. patens, expressing catalytic domain of protein phosphatase 2C and showing an ABA-insensitive phenotype . AR7 is a ABA insensitive mutant isolated by Minami et al . Protonema tissue of Physcomitrella patens were grown on cellophane-overlaid 0.8% agar plates of modified BCD medium supplemented with 0.5% glucose. P. patens transplanted medium was cultured in a controlled environment growth chamber at 25°C under continuous illumination (35 µmol photons m-2s-1) for 5 days. The protonema tissue was then transferred into ABA containing BCD medium and cultured for 1 day.
Cold-Acclimation and freezing stress
Cold treatment was carried out by transferring colonies of protonemata onto fresh agar plates of BCD medium and incubating them at 0°C (on ice) under continuous light for different times (3, 7 and 10 days). Freezing tolerance was estimated by equilibrium freezing of protonema tissue, followed by measurement of electrolyte leakage by electro-conductivity meter . Freezing treatment used in this study were -3°C, -6°C and -9°C, and 4°C as control.
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Wild type, AR7 and D2-1 protonema cells were subjected to treatment with various concentrations (0, 0.4, 0.8, 1, and 1.2 M) of mannitol and NaCl ((0, 0.2, 0.4, 0.6, 0.8, 1 and 1.2 M) for 10 minutes with or without ABA treatment, and then cultured on normal BCD medium to determine effects of the treatment on colony growth. Protonemata were grown on cellophane-overlaid 0.8% agar plates of modified BCD medium for 7 days. ABA treatment was carried out by transferring colonies of protonemata onto fresh agar plates of BCD medium containing 1 μM ABA and incubated at 4-c under dim light for 1 day. After that colonies were then planted on normal BCD medium to determine effects of the treatment on colony growth. Survival rate was measured by staining death cells of protonema.
Measurement of plant growth
Fresh weight of one week old protonemal tissues was measured after aseptic removal of excess water. A five g of fresh tissue was homogenized in 2 ml of 0.1% (w/v) agar solution, and spotted on BCD agar medium (5µl each spot). After 1 weeks of culture, colony areas were digitally measured using LIA for Win32 image analysis.
Moss colonies were grown for only one day on BCD medium with cellophane supplemented with 1 μM ABA, after which the tissues were transferred to sterile filter papers in plant tissue culture petri-dishes. Slow drying was performed according to the method of Tetteroo et al that was developed for drying of somatic embryos. Drying was perfomed in the dark condition. The moss tissue was exposed to descending relative humidities (RH) in desiccators with different salt solution at 22 °C. Silica gel (11% RH), Ca(NO3)2 (55% RH), NaCl (75% RH), KCl (85% RH), KNO3 (95% RH), K2S04(97% RH), and H20(100% RH). Moss tissue remained at each relative humidity for 1 day. Prior to rehydration, the dried samples were pre-humidified in a chamber with moisture saturated air for 1 h. Sterile water was then added on the pre-humidified samples. Subsequently, the rehydrated samples were transferred to fresh agar plates and incubated in dim-light for 1 h before they were placed in normal light conditions. Rate of survival was noted following 24 hrs desiccation.
Staining of protonemata caused by freezing, salt and desiccation stress
.Evans blue was used as a stain to proronema cells after stress treatment. Non-viable cells appear blue, whereas vigorous cells do not absorb the blue color dye. For stress experiments, protonemata were stained for 1 hr on 0.5% evans blue, after which the healthy cells was counted to determine survival rate.
Soluble sugar extraction and analysis
Protonema cells were weighed and crushed using a mortar and a pestle for soluble sugar extraction. Samples were suspended in 80% (v/v) ethanol and insoluble materials were removed by centrifugation at 14,000g at 4-C. The supernatant was dried and suspended in H2O. After removal of water-insoluble material by centrifugation, the supernatants were quantified by the anthron-sulfuric acid assay using glucose as a standard . For thin-layer chromatography (TLC), sugar samples extracted in water were subjected to ascendant chromatography on a thin-layer silica gel plate. The samples were developed by acetonitrile (75%). The mobilized sugar spots were visualized by spraying 50% H2SO4 , followed by baking at 110-C for 10 min .
Preparation of Protein and electrophoresis
Proteins were extracted from the protonema cells by the procedure as described by . SDS-Polyacrylamide gel electrophoresis technique was used for protein analysis. Protonema cells were homogenized and extracted with a solution containing 50mM Tris (pH 7.5), 100mM NaCl, 1 mM EDTA, 1 mM DTT and 1mM PMSF on ice. After centrifugation at 14,000 g for 10 min at 4-C, supernatants were used as crude fractions.The proteins in the crude fractions, adjusted to equal concentrations, were boiled for 10 min, and insoluble materials were removed by centrifugation at 14,000 g for 10 min at 4-C. These soluble fractions were used as boiling soluble proteins. The boiling soluble proteins corresponding to 20 μg of crude proteins were used for gel electrophoresis. The crude proteins and boiling soluble proteins were electrophorsed on 12% (w/v) SDS-polyacrylamide gels.
RNA extraction and Northern blot analysis were performed as described by Minami et al .
AR-7 mutant and D2-1 transgenic lines were ABA insensitive
We characterised the phenotypic expression of all three lines (AR7, D2-1 and WT) for ABA sensitivity. Growth of normal protonema cells is strongly inhibited by 10µM ABA, and by incubation with ABA for several days, the cells undergo morphological changes that subsequently lead to formation of round-shaped cells known as brood cells . The growth of protonema cells were thus examined in BCD medium containing 10 µM ABA. Protonemal growth of AR7 and D2-1 were not inhibited by ABA treatments, though strong inhibition of wild type protonema cells was observed at 10µM ABA (incubated for five days) through the formation of round-shaped brood cells (Fig. 1). This demonstrates that AR7 and D2-1 functions as a negative regulator of ABA signaling in P. patens.
AR7 mutant and D2-1 transgenic lines were sensitive to freezing, desiccation, and salt stresses with or without ABA treatment
Wild type strain of P. patens is naturally sensitive to freezing stresses. We, therefore, tested if the ABA insensitive mutant AR7 and transgenic line D2-1 have altered sensitivity to freezing tolerance under ABA treatment. All three lines (AR7, D2-1 and WT) showed high sensitivity to freezing at different temperature when their protonema cells had been cultured on medium without ABA . Measurement of electrolyte leakage after freeze-thawing revealed ABA-induced freezing tolerance in wild type of P. patens (Fig 2), as previously observed . ABA treatment for one day remarkably increased the freezing tolerance of wild type protonema cells in a dose-dependent manner. Whereas, AR7 and D2-1 did not response significantly even at h ABA treatment. The transgenic plant D2-1 and AR7 mutant significantly lower freezing tolerance, even at 1µM ABA, compared to the wild type. However, the effects of the ABA application resulted in the same level of freezing tolerance as low temperature induction in AR7 and D2-1.
Effect of salt stress on growth of P. patens was examined by treating protonema cells with different concentration of NaCl. ABA induced salt and osmotic stress adaptation was monitored in WT, AR7 and D2-1. After treating with Mannitol and NaCl, protonema cells were further transferred to normal BCD and cultured for 5 day to assess the degree of salt and osmotic stress tolerance. Hyper-osmotic stress treatment by mannitol severely inhibited the growth of protonema cells of three lines in ABA free medium (Fig 3). Thus, all three lines showed similar level of mannitol induced osmotic stress response, manifested in the impaired growth phenotypes of the plants under stress conditions. However, incubation with 1 µM ABA, wild type cells acquired tolerance to hyper-osmotic stress even at (1.2 M) of mannitol. In contrast, AR7 mutant and D2-1 transgene could survive only at very low concentration of mannitol (0.4 M) in ABA free medium and ABA induction did not change their levels of tolerance to mannitol induced osmotic stress. Phenotypic characterization made on three lines of P. patens under different concentration of NaCl showed similar type of response to salt stress. Without ABA treatment, three lines were severely damaged by treatment with 0.4 m or greater concentration of NaCl (Fig 3a). In contrast, tolerance levels of AR7 mutant and D2-1 transgenic cells were unchanged by one-day incubation on medium containing 1 µM ABA.
Therefore analyzed their tolerance to osmotic stress (Fig. 3c, d, e and f). Protonemata were cultured on media containing various concentration of mannitol and NaCl, which causes changes in media osmolality. As the mannitol and NaCl concentration increased, the growth of the wild type protonemat was inhibited. Growth inhibition caused by salt-induce osmotic stress was more distinct in ABA-insensitive lines. Both of these results demonstrate that reduce tolerance of AR7 and D2-1 protonemata to osmotic stress.
Chloronemal tissue that was grown in the presence or absence of ABA was subjected to different relative humidity (100%, 97% ,95%, 86%,75%, 55% and 11%). Both ABA treated and untreated moss tissues were dehydrated for 24 hrs followed by grown with RH 100% (H2O) for re-hydration. Untreated wild-type protonema tissues of P. patens could survive at 97% RH, but severely damaged even at low desiccation treatment (Fig 4) and cannot recover from desiccation. When WT was incubated with 1µM ABA, protonema tissue survived at all desiccation treatments, though few damage cells appeared at 11% RH. Whereas AR7 mutant and D2-1 transgene showed insensitivity to ABA even at low desiccation treatment (97%RH). It was thus clear that WT of P. patens can survive in presence of ABA (1µM) when they are subjected to a drying process. In contrast, ABA insensitive AR7 mutant and D2-1 transgene showed high sensitivity to desiccation. Khandelwal et al also observed similar result and suggested that both ABA and ABAI are required for P. patens vegetative tissue to survive desiccation.
Cold acclimation increases freezing tolerance in wild type but not in ABA-insensitive mutant and transgenic lines
We examined the effects of low temperature treatment on freezing tolerance of the ABA-insensitive mutant and transgenic lines of P. patens protonemata. The protonemata grown at 25°C for 7 days were transferred to fresh BCD medium and incubated at 0°C for 3, 7 and 10 days for low temperature treatment. These incubation periods had differential effects on freezing tolerance of the protonema cells (Fig. 5a). By freezing to -2°C, only 25% of non-treated protonemata survived in wild type. On the other hand, protonemata subjected to low temperature had increased freezing tolerance. During cold treatment at 0°C, the survival rate of cells subjected to freezing to -2°C gradually increased and reached 72% after 7 days and 60% of the cells survived freezing to -3°C after 7 days cold acclimated protonemata. The degree of freezing tolerance of protonemata increased progressively with increasing duration of cold acclimation, and after 10 days of cold treatment. Furthermore, staining analysis using evans blue raised against the blue cell like death cell stained by dye, which had been counted as one of alive cell upon cold treatment revealed that the cold-induced increased the survival after freezing in WT but not in AR7 and D2-1 plants (Fig 5d).
Effect of Cold acclimation on proteins accumulation
Accumulation of proteins from cold-acclimated and Non treated protonemata was analyzed by SDS-PAGE. When total soluble proteins were analyzed, electrophoretic profiles were not significantly changed by cold acclimation . While LEA like proteins are highly hydrophilic, total soluble proteins were boiled for 3 min and analyzed the accumulation of boiling soluble proteins (Fig. 6). The results indicated that the profiles of Lea like/boiling soluble proteins changed remarkably during cold acclimation in wild type, but the levels of these proteins in tissues during cold treatment for 3, 7 and 10 days did not increase in the ABA insensitive lines of AR7 and D2-1 plant.
Accumulation of soluble sugars during cold acclimation
We examined the effects of low-temperature on the profiles of soluble sugar composition in AR7, D2-1 and P.patens protonemata. To determine the roles of soluble sugars in cold-induced freezing tolerance of P. patens, we investigated sugar contents in protonemata. The amount of sugar determined by anthron assay, (Fig 7a) shows sugar levels in tissues during cold treatment at 0°C for 3, 7 and 10 days was analyzed by thin layer chromatography (TLC) (Fig. 7b). When sugars from a wild type extract were analyzed, sugar content profiles were not significantly changed by cold acclimation. Since most abundantly expressed sucrose upon cold treatment revealed that the cold-induced accumulation of the sucrose was decreased in AR7 and D2-1 plants than wild type.
Expression level of cold stress related genes
We are on the way of preparation of protonema cells for the study of mRNA expression. We hope to finish the work within a month. So, we didn't get the result in hand.
Abiotic stresses have an adverse effect on crop productivity through their negative influence on genetic expression and physiological mechanisms in the cell. It is postulated that the cellular responses to abiotic stress is regulated by the plant hormone Abscisic acid (ABA) . In higher plants both exogenous and endogenous application of ABA increased freezing and other abiotic stress tolerance. But its role in bryophyte like Physcomitrella patens is still unclear, though previous studies on wild P. patens suggested ABA induced freezing tolerant gene expression . Here we demonstrated the role of ABA on the pathway of cold induced gene expression in P patens using ABA insensitive mutant and transgenic lines.
In this study, we demonstrated that ABA induction clearly enhanced stress tolerance in wild type P. patens protonema cells, but AR7 mutant and D2-1 transgenic lines are certainly ABA insensitive. Many land plants including bryophytes have been shown to accumulate ABA, and bryophytes also respond to exogenous ABA, suggesting that ABA evolved to protect the land plants from such stresses. However, the role of endogenous ABA or the signaling pathways in response to environmental stresses has yet to be elucidated. P.patens displays a high degree of tolerance against osmotic stress (Frank et al. 2005), and ABA levels in protonemata increase upon 0.5 M mannitol treatment (Minami et al. 2005). Our results demonstrated that growth of growth of ABA-insensitive lines reduced the ABA sensitivity and tolerance against osmotic stress of protonemata, strongly indicating that endogenous ABA signaling is involved in the abiotic stress response of bryophytes. Though all three lines were sensitive to stresses without ABA, but only wild type showed increased regrowth and survival after ABA treatment. (Fig. 2, 3and 4). Results thus indicated that AR7 and D2-1 function as a negative regulator of ABA signaling in P.patens and both ABA and ABAI gene are important for stress resistance.
It has been reported that sucrose is a soluble sugar that commonly accumulates in various moss species during seasonal cold acclimation, and increase in sucrose amounts are thought to be associated with freezing tolerance . However, our results in the present study showing increased sucrose accumulation in the freezing and osmotic stress sensitive AR7 mutant and D2-1 transgenic line suggest that sucrose plays a limited or no role in tolerance to these stresses. Results of our previous report indicated that treatment of the protonema cells with cycloheximide and okadaic acid, which inhibits ABA-induced freezing tolerance, did not affect sucrose accumulation in wild type P. patens . it is therefore suggested that the accumulation of sucrose might be required but not sufficient for acquisition of freezing, desiccation, salt and osmotic stress tolerance.
In addition with soluble sugars (Ristic and Ashworth, 1993; Wanner and junttila, 1999), reports also stated the accumulation of amino acids (Lalk and Dorfflung, 1985; Wanner and Junttila,1999) and glycine betaine (Kishitani et al., 1994) after cold acclimation. We previously reported that accumulation of LEA-like proteins with boiling soluble traits are induced by ABA and cold treatment in protonema cells of wild type P. patens, indicating that these proteins, as well as soluble sugars, are involved in stress tolerance . Analysis by SDS-polyacrylamide gel electrophoresis showed that accumulation of boiling-soluble proteins was remarkably increased by ABA treatment in protonema cells of wild type but the increase of those protein in the AR7 mutant and D2-1 transgenic due to ABA treatment was less or none. These results thus indicated that ABA induced highly hydrophilic proteins such as LEA proteins play important role in enhancement of stress tolerance in P. patens, as reported in other plants . Protonemata cells treated at low temperature exhibited not only an increase in freezing tolerance but also increase levels of accumulation of LEA-like proteins and soluble sugars (Fig. 6) in wild type, whereas the treatment increased only soluble sugars but not LEA like proteins in the ABA-insensitive lines. These results suggest that low temperature affects ABA signaling in P.patens, leading accumulation of LEA-like proteins, which are required for freezing tolerance, It is well known that higher plants sense low temperature and transduce signals to change biochemical processes leading to the expression of cold-induced genes, mediated by cold regulated second messengers and transcription factors (Xiong et al. 2002). Previous reports showed that P.patens (Minami et al. 2005) possess mechanisms of low temperature sensing and signal transduction required for stress responses, such as expression of LEA-like genes, similar to higher plants (Kobayasi et al. 2004). Protein and sugar analysis indicated that cold acclimation induces accumulation of specific LEA-like proteins and soluble sugars in wild type, but there is the treatment increased only soluble sugars but not LEA-like proteins in the ABA-insensitive lines. These facts suggest that low temperature affects ABA signaling in P.patens, leading accumulation of LEA-like proteins, which are required for freezing tolerance. As the cold acclimation could not increase freezing tolerance in ABA insensitive lines, it can be concluded that Physcomitrella patens had an ABA dependent pathway of cold induced gene expression where soluble sugar had limited role on stress tolerance.
Fig. Cold acclimation processes of A. thaliana (Angiosperm) and P. patens