Cold Acclimation Phase Research Articles

Evaluation of Fall and Winter Trinexapac-ethyl Applications on Ultradwarf Bermudagrass Putting Green Color, Quality, and Green Cover

Ultradwarf bermudagrass (UDB) putting greens grown in subtropical and temperate climates can face elevated risk of winter injury from cold temperatures. Trinexapac-ethyl (TE) inhibits UDB growth potentially reducing spring green-up and overexertion of carbohydrate reserves for UDB during the cold de-acclimation period. A field study was conducted to determine the effect of fall and winter TE applications on the visual quality and color of UDB putting greens in Virginia from the cold acclimation phase through the cold de-acclimation phase. A second controlled-environment study was conducted to determine how TE applications to UDB during cold acclimation affected UDB cold tolerance. In the first study, plots were treated with 0.026 kg⋅ha−1 a.i. every 14 days, 0.013 kg⋅ha−1 a.i. every 14 days, or 0.013 kg⋅ha−1 a.i. every 7 days either in the fall only or in the fall and winter. A nontreated control was included for comparison. For the second study, cup-cutter plugs (10.8-cm diameter) of UDB were treated with 0.026 kg⋅ha−1 a.i. every 14 days from the time growth resumed after green-up through cold acclimation or not treated with TE. Plugs were then exposed to −9.4 °C for 4, 6, 8, or 10 hours and placed into a greenhouse to green up. The GC50 values (exposure time to reduce bermudagrass green cover by 50% 6 days after exposure to −9.4 °C) for the treatments were then calculated based on exposure time and percent green-up. In the first study, TE applications improved UDB quality >3.8%. However, TE applications reduced UDB color, and trends exhibited this reduction in color particularly during the late cold acclimation, winter dormancy, and early cold de-acclimation phases. In the second study, TE applications reduced GC50 values by >10.9% compared with nontreated plugs, suggesting TE reduces UDB cold tolerance during the cold acclimation phase.

Temperature-phase transcriptomics reveals that hormones and sugars in the phloem of grape participate in tolerance during cold acclimation.

Most of the upregulated genes contributed to the accumulation of soluble sugars and ABA in the phloem of 'Vitis amurensis' compared to 'Merlot' during cold acclimation. Extreme cold is one of the dominant abiotic factors affecting grape yield and quality. However, the changes in sugars, phytohormones, and gene expression in the branch phloem of different tolerant grape varieties during cold acclimation remain elusive. The data supported that with decreasing temperature, the contents of fructose, sucrose, and ABA in the phloem of Vitis amurensis (cold-tolerant, T) and 'Merlot' (cold-sensitive, S) increased during cold acclimation, and these indicators were higher in T than in S. Furthermore, the activities of sucrose synthetase, sucrose phosphate synthetase, and acid invertase peaked in the early phase of cold acclimation (approximately 5°C) compared to other phases (approximately 28°C, 0°C, - 5°C and - 10°C). Moreover, the RNA sequencing results helped identify a total of 11,343 differentially expressed genes in the phloem of T and S, among which 4912 were upregulated and 6431 were downregulated. In the abscisic acid pathway, CRTISO, PSPY1-1, CYCP707A4-2, PYL4-1, PYL4-2, P2C08, SAPK2, TARAB1, and DBF3 were more highly expressed in T than in S. In the starch and sucrose metabolism pathway, HXK1, PGMP, GLGL1, SUS6, VCINV, BGL11, SSY1, GPS, BAM1 and BAM3 were also more highly expressed in T than in S. Moreover, the genes related to oxidative phosphorylation, such as NDHF, ND4, ND1, NAD7, NAD2, ATPB, YMF19, ATP9, PMA1 and AHA8, were upregulated in T. These results will be beneficial for understanding the potential differences in tolerance across two different cold-tolerant grapes with respect to sugar metabolism and gene expression.

Disturbances in the Biosynthesis or Signalling of Brassinosteroids That Are Caused by Mutations in the HvDWARF, HvCPD and HvBRI1 Genes Increase the Tolerance of Barley to the Deacclimation Process

Tolerance to deacclimation is an important physiological feature in plants in the face of global warming, which is resulting in incidents of increases in winter temperatures. The aim of the work was to explore how disturbances in the signalling and synthesis of brassinosteroids (BR) influence the deacclimation tolerance of barley. One group of mutants and their reference cultivars (Bowman and Delisa) was cold-acclimated, deacclimated and then tested for frost tolerance at − 12 °C. After cold acclimation, the second group of plants was additionally exposed to frost (− 6 °C) and then, deacclimated and tested for frost tolerance at − 12 °C. The deacclimated brassinosteroid mutants were characterised by an increased tolerance to frost, and consequently, had a higher tolerance to deacclimation than their wild-type cultivars. The mechanism of this phenomenon may be partly explained by analysing the hormonal homeostasis in the crowns. For all of the tested plants, a characteristic feature of the response to the deacclimation phase was an increase in the growth-promoting hormones and abscisic acid compared to the cold acclimation phase. The increase was greater in the BR-deficient (BW084) and BR-insensitive (BW312) mutants compared to the Bowman reference cultivar. Mutant 522DK was characterised by a lower accumulation of total cytokinins and gibberellins as well as an enhanced auxin deactivation compared to the Delisa. In the second group, when the plants were exposed to a temperature of − 6 °C before deacclimation, the hormonal homeostasis was further altered in both the mutants and reference cultivars, but all of the mutants had a higher frost tolerance than the wild types.

De novo Synthesis and Assembly of rRNA into Ribosomal Subunits during Cold Acclimation in Escherichia coli

During the cold adaptation that follows a cold stress, bacterial cells undergo many physiological changes and extensive reprogramming of their gene expression pattern. Bulk gene expression is drastically reduced, while a set of cold shock genes is selectively and transiently expressed. The initial stage of cold acclimation is characterized by the establishment of a stoichiometric imbalance of the translation initiation factors (IFs)/ribosomes ratio that contributes to the preferential translation of cold shock transcripts. Whereas de novo synthesis of the IFs following cold stress has been documented, nothing was known concerning the activity of the rrn operons during the cold acclimation period. In this work, we focus on the expression of the rrn operons and the fate of rRNA after temperature downshift. We demonstrate that in Escherichia coli, rRNA synthesis does not stop during the cold acclimation phase, but continues with greater contribution of the P2 compared to the P1 promoter and all seven rrn operons are active, although their expression levels change with respect to pre-stress conditions. Eight hours after the 37°→10°C temperature downshift, the newly transcribed rRNA represents up to 20% of total rRNA and is preferentially found in the polysomes. However, with respect to the de novo synthesis of the IFs, both rRNA transcription and maturation are slowed down drastically by cold stress, thereby accounting in part for the stoichiometric imbalance of the IFs/ribosomes. Overall, our data indicate that new ribosomes, which are possibly suitable to function at low temperature, are slowly assembled during cold acclimation.

Interscapular brown adipose tissue metabolic reprogramming during cold acclimation: Interplay of HIF-1α and AMPKα

Background Brown adipose tissue thermogenic program includes complex molecular and structural changes. However, energetic aspects of this process are poorly depicted. Methods We investigated time-dependent reprogramming of interscapular brown adipose tissue (IBAT) energy metabolism during cold-acclimation, as well as the effects of nitric oxide ( NO) on those changes. Rats were exposed to cold (4 ± 1 °C) for periods of 1, 3, 7, 12, 21, and 45 days, and divided into three groups: control, treated with L-arginine, and treated with N ω-nitro-L-arginine methyl ester (L-NAME). Results In the early phase of cold-acclimation (up to 7 days), the protein levels of all metabolic parameters and oxidative phosphorylation components were below the control. However, metabolic parameters and respiratory chain components entered a new homeostatic level in the late phase of cold-acclimation. These changes were accompanied with increased protein levels of phospho-AMP-dependent protein kinase-α (phospho-AMPKα) on the first day of cold-acclimation, and hypoxia-inducible factor-1α (HIF-1α) throughout early cold-acclimation. L-arginine positively affected protein expression of enzymes involved in glucose metabolism and β-oxidation of fatty acids in the early phase of cold-acclimation, and oxidative phosphorylation components throughout cold-acclimation. In contrast, L-NAME had the opposite effects. Conclusion Results suggest that IBAT structural remodeling is followed by energy metabolism reprogramming, which control might be orchestrated by the action of AMPKα and HIF-1α. Data also indicated the involvement of L-arginine- NO in the regulation of IBAT metabolism. General significance Results obtained in this study might be of great importance for elucidating regulatory pathways governing energy metabolism in both physiological and pathophysiological states.

Interaction with Diurnal and Circadian Regulation Results in Dynamic Metabolic and Transcriptional Changes during Cold Acclimation in Arabidopsis

In plants, there is a large overlap between cold and circadian regulated genes and in Arabidopsis, we have shown that cold (4°C) affects the expression of clock oscillator genes. However, a broader insight into the significance of diurnal and/or circadian regulation of cold responses, particularly for metabolic pathways, and their physiological relevance is lacking. Here, we performed an integrated analysis of transcripts and primary metabolites using microarrays and gas chromatography-mass spectrometry. As expected, expression of diurnally regulated genes was massively affected during cold acclimation. Our data indicate that disruption of clock function at the transcriptional level extends to metabolic regulation. About 80% of metabolites that showed diurnal cycles maintained these during cold treatment. In particular, maltose content showed a massive night-specific increase in the cold. However, under free-running conditions, maltose was the only metabolite that maintained any oscillations in the cold. Furthermore, although starch accumulates during cold acclimation we show it is still degraded at night, indicating significance beyond the previously demonstrated role of maltose and starch breakdown in the initial phase of cold acclimation. Levels of some conventional cold induced metabolites, such as γ-aminobutyric acid, galactinol, raffinose and putrescine, exhibited diurnal and circadian oscillations and transcripts encoding their biosynthetic enzymes often also cycled and preceded their cold-induction, in agreement with transcriptional regulation. However, the accumulation of other cold-responsive metabolites, for instance homoserine, methionine and maltose, did not have consistent transcriptional regulation, implying that metabolic reconfiguration involves complex transcriptional and post-transcriptional mechanisms. These data demonstrate the importance of understanding cold acclimation in the correct day-night context, and are further supported by our demonstration of impaired cold acclimation in a circadian mutant.

Autogenous regulation of Escherichia coli polynucleotide phosphorylase during cold acclimation by transcription termination and antitermination

Adaptation of Escherichia coli at low temperature implicates a drastic reprogramming of gene expression patterns. Mechanisms operating downstream of transcription initiation, such as control of transcription termination, mRNA stability and translatability, play a major role in controlling gene expression in the cold acclimation phase. It was previously shown that Rho-dependent transcription termination within pnp, the gene encoding polynucleotide phosphorylase (PNPase), was suppressed in pnp nonsense mutants, whereas it was restored by complementation with wild type allele. Using a tRNA gene as a reporter and the strong Rho-dependent transcription terminator t ( imm ) of bacteriophage P4 as a tester, here we show that specific sites in the 5'-untranslated region of pnp mRNA are required for PNPase-sensitive cold-induced suppression of Rho-dependent transcription termination. We suggest that suppression of Rho-dependent transcription termination within pnp and its restoration by PNPase is an autogenous regulatory circuit that modulates pnp expression during cold acclimation.

Genetic analysis of polynucleotide phosphorylase structure and functions

Polynucleotide phosphorylase (PNPase) is a phosphate-dependent 3′ to 5′ exonuclease widely diffused among bacteria and eukaryotes. The enzyme, a homotrimer, can also be found associated with the endonuclease RNase E and other proteins in a heteromultimeric complex, the RNA degradosome. PNPase negatively controls its own gene ( pnp) expression by destabilizing pnp mRNA. A current model of autoregulation maintains that PNPase and a short duplex at the 5′-end of pnp mRNA are the only determinants of mRNA stability. During the cold acclimation phase autoregulation is transiently relieved and cellular pnp mRNA abundance increases significantly. Although PNPase has been extensively studied and widely employed in molecular biology for about 50 years, several aspects of structure–function relationships of such a complex protein are still elusive. In this work, we performed a systematic PCR mutagenesis of discrete pnp regions and screened the mutants for diverse phenotypic traits affected by PNPase. Overall our results support previous proposals that both first and second core domains are involved in the catalysis of the phosphorolytic reaction, and that both phosphorolytic activity and RNA binding are required for autogenous regulation and growth in the cold, and give new insights on PNPase structure–function relationships by implicating the α-helical domain in PNPase enzymatic activity.

Hematological Changes in Cold-Acclimated Xenopus laevis.

While hematopoiesis in individuals is strictly regulated for maintenance of homeostasis, it has been reported that the numbers of peripheral blood cells are modulated in response to environmental temperature in vertebrates including hamsters, rats, squirrels, dogs, and bullfrogs. To date, the physiological and molecular mechanisms have not been elucidated yet. Amphibians are poikilothermic vertebrates exposed to various fluctuations of environmental conditions; therefore they need to exert their capacities to acclimate to such changes. Additionally numerous studies have demonstrated a measurable metabolic reduction in the metabolic rate of cold-acclimated frogs. We examined hematological changes in response to environmental temperature in an aquatic amphibian, Xenopus laevis. Frogs initially maintained at 25°C were acclimated to 10°C, and hematological changes were observed. The numbers of erythrocytes, leukocytes and thrombocytes gradually reduced as the transient phase by 4 weeks, and subsequently reached the steady-state that sustained for more than 4 months. Whereas the reduction in the numbers of white blood cells and thrombocytes were moderate, the number of erythrocytes and the level of hemoglobin at nadir were remarkably low (approximately 40% of the initial values). It is known that oxygen levels may reduce in ice-cold water, and cold-acclimated animals therefore tolerate prolonged severe hypoxia; nevertheless cold-acclimated Xenopus exhibited severe erythrocytopenia. In addition, morphological change of peripheral blood cells and hematopoietic tissues (liver, spleen, kidney, and bone marrow) were examined. There were no remarkable cellular changes in cellular size and shape. However, increased numbers of mature erythrocytes were observed in the bone marrow of the steady state cold-acclimated Xenopus, while mature erythrocytes were not found in the bone marrow in Xenopus at 25 °C. This cold-temperature-induced pancytopenia was reversible when the temperature was put back to 25°C, as all of blood cell counts returned to the normal levels within 4 weeks in a reverse fashion as the transient phase of cold-acclimation. During the recovery phase, immature erythrocytes that were scarcely existed in the normal peripheral blood appeared in the circulation, suggesting that erythrocytes were newly produced at 25 °C after prolonged exposure to cold temperature. The possible explanations for the reduction in the numbers of circulating peripheral blood cells might be due to a number of various reasons such as reduced productions of hamatopoietic progenitors and/or related cytokines, alternation in the storage capacities and/or the life span of blood cells, and systemic suspension of normal activities. To compare the lifespan of erythrocytes between normal and cold-acclimated Xenopus, erythrocytes were covalently labeled with biotin. The surviving biotinylated erythrocytes in the circulation were quantitatively detected as avidin-biotin complex by microscopy and flowcytometry. Furthermore expression levels of several genes responsible for the hematopoietic regulation were comparatively examined. The cold-acclimated Xenopus model developed here may allow for a valuable approach aiming at exploring undiscovered systems in hematopoietic regulation.

Starch-Related α-Glucan/Water Dikinase Is Involved in the Cold-Induced Development of Freezing Tolerance in Arabidopsis

Cold-induced soluble sugar accumulation enhances the degree of freezing tolerance in various cold-hardy plants including Arabidopsis (Arabidopsis thaliana), where soluble sugars accumulate in only a few hours at 2 degrees C. Hence, along with photosynthesis, starch degradation might play a significant role in cold-induced sugar accumulation and enhanced freezing tolerance. Starch-related alpha-glucan/water dikinase (EC 2.7.9.4), encoded by Arabidopsis STARCH EXCESS 1 (SEX1), is hypothesized to regulate starch degradation in plastids by phosphorylating starch, thereby ensuring better accessibility by starch-degrading enzymes. Here, we show that Arabidopsis sex1 mutants, when incubated at 2 degrees C for 1 d, were unable to accumulate maltooligosaccharides or normal glucose and fructose levels. In addition, they displayed impaired freezing tolerance. After 7 d at 2 degrees C, sex1 mutants did not show any of the above abnormal phenotypes but displayed slightly higher leaf starch contents. The impaired freezing tolerance of sex1 mutants was restored by overexpression of wild-type SEX1 cDNA using the cauliflower mosaic virus 35S promoter. The results demonstrate a genetic link between the SEX1 locus and plant freezing tolerance, and show that starch degradation is important for enhanced freezing tolerance during an early phase of cold acclimation. However, induction of starch degradation was not accompanied by significant changes in alpha-glucan/water dikinase activity in leaf extracts and preceded cold-induced augmentation of SEX1 transcripts. Therefore, we conclude that augmentation of SEX1 transcripts might be a homeostatic response to low temperature, and that starch degradation during an early phase of cold acclimation could be regulated by a component(s) of a starch degradation pathway(s) downstream of SEX1.

Transcriptional and post-transcriptional control of polynucleotide phosphorylase during cold acclimation in Escherichia coli.

Polynucleotide phosphorylase (PNPase, polyribonucleotide nucleotidyltransferase, EC 2.7.7.8) is one of the cold shock-induced proteins in Escherichia coli and pnp, the gene encoding it, is essential for growth at low temperatures. We have analysed the expression of pnp upon cold shock and found a dramatic transient variation of pnp transcription profile: within the first hour after temperature downshift the amount of pnp transcripts detectable by Northern blotting increased more than 10-fold and new mRNA species that cover pnp and the downstream region, including the cold shock gene deaD, appeared; 2 h after temperature downshift the transcription profile reverted to a preshift-like pattern in a PNPase-independent manner. The higher amount of pnp transcripts appeared to be mainly due to an increased stability of the RNAs. The abundance of pnp transcripts was not paralleled by comparable variation of the protein: PNPase steadily increased about twofold during the first 3 h at low temperature, as determined both by Western blotting and enzymatic activity assay, suggesting that PNPase, unlike other known cold shock proteins, is not efficiently translated in the acclimation phase. In experiments aimed at assessing the role of PNPase in autogenous control during cold shock, we detected a Rho-dependent termination site within pnp. In the cold acclimation phase, termination at this site depended upon the presence of PNPase, suggesting that during cold shock pnp is autogenously regulated at the level of transcription elongation.

The role of ABA in freezing tolerance and cold acclimation in barley

The role of ABA in freezing resistance in nonacclimated and cold‐acclimated barley (Hordeum vulgare L.) was studied. Eleven nonacclimated cultivars differed in their LT50, ranging from −10.8 to −4.8°C. Sugars, free proline, soluble proteins and ABA were analyzed in nonacclimated cultivars and during cold acclimation of one cultivar. There was an inverse correlation between LT50 and both ABA and sucrose contents. Exogenous ABA caused a decrease in the freezing point of leaf tissue in the cultivar with the lowest level of endogenous ABA, but not in the cultivar with the highest level, suggesting that ABA in the latter may be near the optimum endogenous level to induce freezing tolerance. Plants of cv. Aramir treated with ABA or allowed to acclimate to cold temperature increased their soluble sugar content to a similar level. The LT50 of leaves of cold‐acclimated cv. Aramir decreased from −5.8 to −11.4°C, with biphasic kinetics, accumulating proline and soluble sugars with similar kinetics. The biphasic profile observed during cold acclimation could be a direct consequence of cryoprotectant accumulation kinetics. ABA and soluble protein accumulation showed a single step profile, associated mainly with the second phase of the LT50 decrease. Thus, a significant increase in endogenous ABA is part of the response of barley to low temperature and may be required as a signal for the second phase of cold acclimation. Endogenous ABA contents in the nonacclimated state may determine constitutive freezing tolerance.

Water Permeability and Cold Hardiness of Cortex Cells in Cornus stolonifera Michx.—A Preliminary Report

The relationship of freezing resistance to water permeability of cortex cells was studied in stems of red osier dogwood (Cornus stolonifera Michx.). Permeability was estimated by determining the diffusion flux of tritiated water from cortex slices previously equilibrated in tritiated water. Energy of activation and diffusion time comparisons of tritiated water flux from living cortex slices and slices killed by immersion in liquid N(2) verified that intact membranes of uninjured cortex cells limited water flux.Water permeability of living phloem and cortical parenchyma cells increased during the initial (photoperiodically induced) phase of cold acclimation. This accompanied an increase in hardiness from -3 to -12 C. Little if any further increase in permeability was noted during subsequent acclimation to below -65 C.Permeability measurements on nonhardy cortex samples yielded consistent results, but measurements on samples from hardy twigs were often difficult to reproduce. This unexplained variability precludes specific conclusions, but the tritiated water diffusion flux technique may provide an alternative to traditional plasmolytic techniques in studying water permeability in woody plant tissues.

Effects of Red and Far Red Light on the Initiation of Cold Acclimation in Cornus stolonifera Michx.

Red and far red light distinctly influence the initial phytochrome-mediated phase of cold acclimation in red-osier dogwood (Cornus stolonifera). Under controlled conditions, short days and end-of-day far red light exposure after long days promote growth cessation, cold acclimation, and subsequent cold hardening of dogwood stems in response to low temperature. Nuclear magnetic resonance absorption spectra of the water in internode stem sections imply that the short day-induced phase of cold acclimation involves a change in tissue hydration, at least in part, due to a substantial reduction in bulk phase water as a result of senescence and loss of water from the pith. Seasonal responses to light and an attempt to induce early acclimation under natural conditions with end-of-day far red light are discussed.