Plant Cold Acclimation Research Articles

Potassium Application Boosts Photosynthesis and Sorbitol Biosynthesis and Accelerates Cold Acclimation of Common Plantain (Plantago major L.).

Potassium (K) is essential for the processes critical for plant performance, including photosynthesis, carbon assimilation, and response to stress. K also influences translocation of sugars in the phloem and regulates sucrose metabolism. Several plant species synthesize polyols and transport these sugar alcohols from source to sink tissues. Limited knowledge exists about the involvement of K in the above processes in polyol-translocating plants. We, therefore, studied K effects in Plantago major, a species that accumulates the polyol sorbitol to high concentrations. We grew P. major plants on soil substrate adjusted to low-, medium-, or high-potassium conditions. We found that biomass, seed yield, and leaf tissue K contents increased in a soil K-dependent manner. K gradually increased the photosynthetic efficiency and decreased the non-photochemical quenching. Concomitantly, sorbitol levels and sorbitol to sucrose ratio in leaves and phloem sap increased in a K-dependent manner. K supply also fostered plant cold acclimation. High soil K levels mitigated loss of water from leaves in the cold and supported cold-dependent sugar and sorbitol accumulation. We hypothesize that with increased K nutrition, P. major preferentially channels photosynthesis-derived electrons into sorbitol biosynthesis and that this increased sorbitol is supportive for sink development and as a protective solute, during abiotic stress.

Растворимые углеводы и холодовая акклимация растений

Растворимые углеводы и холодовая акклимация растений

The cold response regulator CBF1 promotes Arabidopsis hypocotyl growth at ambient temperatures.

Light and temperature are two core environmental factors that coordinately regulate plant growth and survival throughout their entire life cycle. However, the mechanisms integrating light and temperature signaling pathways in plants remain poorly understood. Here, we report that CBF1, an AP2/ERF-family transcription factor essential for plant cold acclimation, promotes hypocotyl growth under ambient temperatures in Arabidopsis. We show that CBF1 increases the protein abundance of PIF4 and PIF5, two phytochrome-interacting bHLH-family transcription factors that play pivotal roles in modulating plant growth and development, by directly binding to their promoters to induce their gene expression, and by inhibiting their interaction with phyB in the light. Moreover, our data demonstrate that CBF1 promotes PIF4/PIF5 protein accumulation and hypocotyl growth at both 22°C and 17°C, but not at 4°C, with a more prominent role at 17°C than at 22°C. Together, our study reveals that CBF1 integrates light and temperature control of hypocotyl growth by promoting PIF4 and PIF5 protein abundance in the light, thus providing insights into the integration mechanisms of light and temperature signaling pathways in plants.

Causes of leaf reddening in subterranean clover cultivars

ABSTRACT Subterranean clover (Trifolium subterraneum L.) is an important legume in dryland hill pastures in New Zealand. In this environment plants adapt to low temperatures (<10°C) through a series of physiological mechanisms, such as the foliar biosynthesis of pigments, to protect vulnerable tissues. The biosynthesis of anthocyanins is one of the most important physiological responses for cold acclimation in plants. In this study we investigated anthocyanin concentration by accessing leaf reddening in 14 cultivars from three subspecies (yanninicum, brachycalycinum or subterraneum) during winter growth. The effect of anthocyanin production on spring herbage productivity was further quantified in the field and in the glasshouse. Leaf concentration of anthocyanins increased 10-fold as a consequence of cold exposure (mean daily air temperature was below ∼7°C) in red leaves compared with green leaves. There were large cultivar differences with yanninicum ssp. cultivars having the highest leaf reddening at >60%. Lowest values were observed in ‘Antas’ (brachycalycinum ssp.) and ‘Denmark’ (subterraneum spp.) at <20% of leaf reddening. Australian subterraneum ssp. cultivars had 13% higher leaf reddening values than Italian cultivars. Leaf reddening was a temporary, cold induced, anthocyanin response that did not affect subsequent subterranean clover plant growth.

Proteomic Approaches to Identify Cold-Regulated Plasma Membrane Proteins.

Plasma membrane is the primary determinant of freezing tolerance in plants because of its central role in freeze-thaw cycle. Changes in plasma membrane protein composition have been one of the major research areas in plant cold acclimation. To obtain comprehensive profiles of the plasma membrane proteomes and their changes during the cold acclimation process, a plasma membrane purification method using a dextran-polyethylene glycol two polymer system and a mass spectrometry-based shotgun proteomics method using nano-LC-MS/MS for the plasma membrane proteins are described. The proteomic results obtained are further applied to label-free protein semiquantification.

Introduction: Plant Cold Acclimation and Winter Survival.

This introductory chapter provides a brief overview of plant freezing tolerance, cold acclimation, including subzero acclimation, and the subsequent deacclimation when plants return to warm conditions favoring growth and development. We describe the basic concepts and approaches that are currently followed to investigate these phenomena. We highlight the multidisciplinary nature of these investigations and the necessity to use methodologies from different branches of science, such as ecology, genetics, physiology, cell biology, biochemistry, and biophysics to gain a complete understanding of the complex adaptive mechanisms ultimately underlying plant winter survival.

Analysis of Changes in Plant Cell Wall Composition and Structure During Cold Acclimation.

The cell wall has a crucial influence on the mechanical properties of plant cells. It therefore has a strong impact on the freezing behavior and very likely also the freezing tolerance of plants. However, not many studies have addressed the question how cell wall composition and structure impact plant freezing tolerance and cold acclimation. In this chapter, we describe a comprehensive workflow to extract total cell wall material from leaves of Arabidopsis thaliana and to separate this material into fractions enriched in crystalline cellulose, pectins, and hemicelluloses by sequential fractionation. We further describe methods for the analysis of chemical structure, monosaccharide composition, and cellulose and uronic acid contents in the total cell wall material and the fractions in response to cold acclimation. Structural properties of cell wall material are analyzed by attenuated total reflectance-Fourier-transform infrared spectrometry (ATR-FTIR) and monosaccharide composition by gas chromatography-mass spectrometry (GC-MS) after isolation of alditol acetate derivatives of the sugars.

Mapping of Quantitative Trait Loci (QTL) Associated with Plant Freezing Tolerance and Cold Acclimation.

Most agronomic traits are determined by quantitative trait loci (QTL) and exhibit continuous distribution in natural or especially built segregating populations. The genetic architecture and the hereditary characteristics of these traits are much more complicated than those of oligogenic traits and need adapted strategies for deciphering. The model plant Arabidopsis thaliana is widely studied for quantitative traits, especially via the utilization of genetic natural diversity. Here we describe a QTL-mapping protocol for analyzing freezing tolerance after cold acclimation in this species, based on its specific genetic tools. Nevertheless, this approach can be applied for the elucidation of complex traits in others species.

Biochemical and molecular responses during overwintering of red clover populations recurrently selected for improved freezing tolerance

Low freezing tolerance reduces the persistence of red clover under northern climate. The incidence of winter damages in perennial crops could increase in the future due to the adverse effects of the predicted warmer fall temperature on plant cold acclimation. To accelerate breeding progress, two cultivars of red clover Christie (C-TF0) and Endure (E-TF0) were exposed to a recurrent selection protocol for freezing tolerance performed indoor. New populations were obtained after five (C-TF5 and E-TF5), six (C-TF6 and E-TF6), and seven (C-TF7 and E-TF7) cycles of recurrent selection. These populations were overwintered under natural conditions and monitored for freezing tolerance and cold-induced molecular traits. Freezing tolerance was improved by up to 6 °C in recurrently selected populations when compared to initial cultivars confirming that further progress are achieved with advanced cycles of selection. Monthly analysis of biochemical changes shows that higher starch concentrations at the onset of the fall hardening period are contributing to the acquisition of superior freezing tolerance through its impact on sucrose accumulation. They also contribute to the vigor of spring regrowth by sustaining more pinitol and proline synthesis. Larger concentrations of these metabolites in populations with higher levels of freezing tolerance (TF7) hint at their involvement in winter survival of red clover. Among genes differentially expressed in response to both cold acclimation and recurrent selection, a concomitant cold induction of APPR9 and cold repression of 1-aminocyclopropane-carboxylate synthase suggests a link between the repression of a pathway regulated by ethylene and the improvement of freezing tolerance in red clover.

Dynamics of Plant Metabolism during Cold Acclimation.

Plants have evolved strategies to tightly regulate metabolism during acclimation to a changing environment. Low temperature significantly constrains distribution, growth and yield of many temperate plant species. Exposing plants to low but non-freezing temperature induces a multigenic processes termed cold acclimation, which eventually results in an increased freezing tolerance. Cold acclimation comprises reprogramming of the transcriptome, proteome and metabolome and affects communication and signaling between subcellular organelles. Carbohydrates play a central role in this metabolic reprogramming. This review summarizes current knowledge about the role of carbohydrate metabolism in plant cold acclimation with a focus on subcellular metabolic reprogramming, its thermodynamic constraints under low temperature and mathematical modelling of metabolism.

PUB25 and PUB26 Promote Plant Freezing Tolerance by Degrading the Cold Signaling Negative Regulator MYB15.

Cold stress adversely affects plant growth, development, and crop productivity and quality. Plants employ cold acclimation strategy to protect them from cold damage. The transcription-factor-CBF-dependent cold signaling pathway plays a key role in plant cold acclimation. However, how this signaling pathway is dynamically and precisely regulated remains unknown. Here, we report that two U-box type E3 ubiquitin ligases, PUB25 and PUB26, positively regulate freezing tolerance in Arabidopsis thaliana. Both PUB25 and PUB26 poly-ubiquitinate MYB15, a transcriptional repressor of the CBF-dependent cold signaling pathway, leading to MYB15 degradation and thus enhanced CBF expression under cold stress. Furthermore, cold-activated OST1 specifically phosphorylates PUB25 and PUB26 at conserved threonine residues, enhancing their E3 activity and facilitating the cold-induced degradation of MYB15. Our results thus unravel the regulatory role of the OST1-PUB25/26 module in regulating the duration and amplitude of the cold response by controlling the homeostasis of the negative regulator MYB15.

The involvements of calcium-dependent protein kinases and catechins in tea plant [Camellia sinensis (L.) O. Kuntze] cold responses

Temperature is one of the most important environmental factors limiting tea plant growth and tea production. Previously we reported that both Ca2+ and ROS signals play important roles in tea plant cold acclimation. Here, we identified 26 CsCPK transcripts, analyzed their phylogenetic and sequence characters, and detected their transcriptions to monitor Ca2+ signaling status. Tissue-specific expression profiles indicated that most CsCPK genes were constitutively expressed in tested tissues, suggesting their possible roles in development. Cold along with calcium inhibitor assays suggested that CsCPKs are important cold regulators and CsCPK30/5/4/9 maybe the key members. Moreover, LaCl3 or EGTA pre-treatment could result in impaired Ca2+ signaling and compromised cold-responding network, but higher catechins accumulation revealed their potential positive roles in cold responses. Those findings indicated that catechins and other secondary metabolites in tea plant may form an alternative cold-responding network that closely correlated with Ca2+ signaling status.

Influence of acclimation conditions on winter hardiness of wheat lines with introgressions from Aurotica amphidiploid

Aim. Genome substituted amphidiploid Aurotica and developed common wheat introgressive lines have higher winter hardiness. Gene TaAGL21, which encodes MADS-box TF controlling auxin transport in the root, plays some role in providing winter hardiness. The aim of this work was to determine the influence of this TF on the root architecture in different cold acclimation conditions. Methods. Quantitative assessment of the root system parameters, genotyping of plants using PCR, the statistical method for comparison of normalized deviations of genotypes in two groups with different alleles of TaAGL21 gene. Results. The root system characteristics such as the maximum root length, the root number, the root quantity, and the root system volume have been determined for Aurora, Aurotica, and introgressive lines grown without natural cold acclimation. Genotypes were grouped for TaAGL21 alleles: allele 2 inherent to Aurotica with better winter hardiness than Aurora, and allele 1 inherent to Aurora. A comparative analysis of the arithmetic mean values for the studied traits with the arithmetic mean values of the same traits assessed in 2015 on plants grown in conditions of natural cold acclimation were conducted. For the plants grown in the natural cold acclimation conditions genotypes with allele 2 of TaAGL gene differed from genotypes with allele 1 of the same gene; whereas, in the absence of cold acclimation no difference was observed for this trait between two groups of plants. Сontrasting results were obtained for the plants grown under the conditions of the natural cold acclimation for their normalized deviations. Conclusions. Allele specific influence of TaAGL21 gene on the root system architecture of common wheat plants and amphidiploid Aurotica depends on the conditions of cold acclimation of wheat plants on the developmental stage before branching of the stem.

Transcriptional read-through of the long non-coding RNA SVALKA governs plant cold acclimation

Most DNA in the genomes of higher organisms does not encode proteins, yet much is transcribed by RNA polymerase II (RNAPII) into long non-coding RNAs (lncRNAs). The biological significance of most lncRNAs is largely unclear. Here, we identify a lncRNA (SVALKA) in a cold-sensitive region of the Arabidopsis genome. Mutations in SVALKA affect CBF1 expression and freezing tolerance. RNAPII read-through transcription of SVALKA results in a cryptic lncRNA overlapping CBF1 on the antisense strand, termed asCBF1. Our molecular dissection reveals that CBF1 is suppressed by RNAPII collision stemming from the SVALKA-asCBF1 lncRNA cascade. The SVALKA-asCBF1 cascade provides a mechanism to tightly control CBF1 expression and timing that could be exploited to maximize freezing tolerance with mitigated fitness costs. Our results provide a compelling example of local gene regulation by lncRNA transcription having a profound impact on the ability of plants to appropriately acclimate to challenging environmental conditions.

The Arabidopsis THADA homologue modulates TOR activity and cold acclimation.

Low temperature is one of the most important environmental factors that affect global survival of humans and animals and equally importantly the distribution of plants and crop productivity. Survival of metazoan cells under cold stress requires regulation of the sensor-kinase Target Of Rapamycin (TOR). TOR controls growth of eukaryotic cells by adjusting anabolic and catabolic metabolism. Previous studies identified the Thyroid Adenoma Associated (THADA) gene as the major effect locus by positive selection in the evolution of modern human adapted to cold. Here we investigate the role of THADA in TOR signaling and cold acclimation of plants. We applied BLAST searches and homology modeling to identify the AtTHADA (AT3G55160) in Arabidopsis thaliana as the highly probable orthologue protein. Reverse genetics approaches were combined with immunological detection of TOR activity and metabolite profiling to address the role of the TOR and THADA for growth regulation and cold acclimation. Depletion of the AtTHADA gene caused complete or partial loss of full-length mRNA, respectively, and significant retardation of growth under non-stressed conditions. Furthermore, depletion of AtTHADA caused hypersensitivity towards low-temperatures. Atthada displayed a lowered energy charge. This went along with decreased TOR activity, which offers a molecular explanation for the slow growth phenotype of Atthada. Finally, we used TOR RNAi lines to identify the de-regulation of TOR activity as one determinant for sensitivity towards low-temperatures. Taken together our results provide evidence for a conserved function of THADA in cold acclimation of eukaryotes and suggest that cold acclimation in plants requires regulation of TOR.

12. 植物の凍結耐性増大過程における細胞内適合溶質の役割(平成13年度第47回低温生物工学会研究報告)

12. 植物の凍結耐性増大過程における細胞内適合溶質の役割(平成13年度第47回低温生物工学会研究報告)

Knockdown of Ice-Binding Proteins in Brachypodium distachyon Demonstrates Their Role in Freeze Protection.

Sub-zero temperatures pose a major threat to the survival of cold-climate perennials. Some of these freeze-tolerant plants produce ice-binding proteins (IBPs) that offer frost protection by restricting ice crystal growth and preventing expansion-induced lysis of the plasma membranes. Despite the extensive in vitro characterization of such proteins, the importance of IBPs in the freezing stress response has not been investigated. Using the freeze-tolerant grass and model crop, Brachypodium distachyon, we characterized putative IBPs (BdIRIs) and generated the first ‘IBP-knockdowns’. Seven IBP sequences were identified and expressed in Escherichia coli, with all of the recombinant proteins demonstrating moderate to high levels of ice-recrystallization inhibition (IRI) activity, low levels of thermal hysteresis (TH) activity (0.03−0.09°C at 1 mg/mL) and apparent adsorption to ice primary prism planes. Following plant cold acclimation, IBPs purified from wild-type B. distachyon cell lysates similarly showed high levels of IRI activity, hexagonal ice-shaping, and low levels of TH activity (0.15°C at 0.5 mg/mL total protein). The transfer of a microRNA construct to wild-type plants resulted in the attenuation of IBP activity. The resulting knockdown mutant plants had reduced ability to restrict ice-crystal growth and a 63% reduction in TH activity. Additionally, all transgenic lines were significantly more vulnerable to electrolyte leakage after freezing to −10°C, showing a 13−22% increase in released ions compared to wild-type. IBP-knockdown lines also demonstrated a significant decrease in viability following freezing to −8°C, with some lines showing only two-thirds the survival seen in control lines. These results underscore the vital role IBPs play in the development of a freeze-tolerant phenotype and suggests that expression of these proteins in frost-susceptible plants could be valuable for the production of more winter-hardy crops.

Insights from the Cold Transcriptome and Metabolome of Dendrobium officinale: Global Reprogramming of Metabolic and Gene Regulation Networks during Cold Acclimation.

Plant cold acclimation (CA) is a genetically complex phenomenon involving gene regulation and expression. Little is known about the cascading pattern of gene regulatroy network and the link between genes and metabolites during CA. Dendrobium officinale (DOKM) is an important medicinal and ornamental plant and hypersensitive to low temperature. Here, we used the large scale metabolomic and transcriptomic technologies to reveal the response to CA in DOKM seedlings based on the physiological profile analyses. Lowering temperature from 4 to –2°C resulted in significant increase (P < 0.01) in antioxidant activities and electrolyte leakage (EL) during 24 h. The fitness CA piont of 0°C and control (20°C) during 20 h were firstly obtained according to physiological analyses. Subsequently, massive transcriptome and metabolome reprogramming occurred during CA. The gene to metabolite network demonstrated that the CA associated processes are highly energy demanding through activating hydrolysis of sugars, amino acids catabolism and citrate cycle. The expression levels of 2,767 genes were significantly affected by CA, including 153-fold upregulation of CBF transcription factor, 56-fold upregulation of MAPKKK16 protein kinase. Moreover, the gene interaction and regulation network analysis revealed that the CA as an active process, was regulated at the transcriptional, post-transcriptional, translational and post-translational levels. Our findings highligted a comprehensive regulatory mechanism including cold signal transduction, transcriptional regulation, and gene expression, which contributes a deeper understanding of the highly complex regulatory program during CA in DOKM. Some marker genes identified in DOKM seedlings will allow us to understand the role of each individual during CA by further functional analyses.

Rice mutants deficient in ω-3 fatty acid desaturase (FAD8) fail to acclimate to cold temperatures

To investigate the role of ω-3 fatty acid (FA) desaturase (FAD8) during cold acclimation in higher plants, we characterized three independent T-DNA insertional knock-out mutants of OsFAD8 from rice (Oryza sativa L.). At room temperature (28 °C), osfad8 plants exhibited significant alterations in fatty acid (FA) unsaturation for all four investigated plastidic lipid classes. During a 5-d acclimation period at 4 °C, further changes in FA unsaturation in both wild-type (WT) and mutant plants varied according to the type of lipid. We also monitored the fluidity of the thylakoid membrane using a threshold temperature to represent the change in fluorescence. The values were altered significantly by both FAD8 mutation and cold acclimation, suggesting that factors other than FAD8 are involved in C18 FA unsaturation and fluctuations in membrane fluidity. Similarly, significant changes were noted for both the mutant and WT samples in terms of their FA compositions as well as activities related to photosystem (PS) I, PSII, and photoprotection. This included the development of non-photochemical quenching and increased zeaxanthin accumulation. Despite the relatively small changes in FA composition during cold acclimation, cold-inducible FAD8 knock-out mutants displayed strong differences in photoprotective activities and a further drop in membrane fluidity. The mutants were more sensitive than WT to short-term low-temperature stress that resulted in increased production of reactive oxygen species after 5 d of chilling. Taken together, our findings suggest that FA unsaturation by OsFAD8 is crucial for the acclimation of higher plants to low-temperature stress.

Flavonoids are determinants of freezing tolerance and cold acclimation in Arabidopsis thaliana.

In plants from temperate climates such as Arabidopsis thaliana low, non-freezing temperatures lead to increased freezing tolerance in a process termed cold acclimation. This process is accompanied by massive changes in gene expression and in the content of primary metabolites and lipids. In addition, most flavonols and anthocyanins accumulate upon cold exposure, along with most transcripts encoding transcription factors and enzymes of the flavonoid biosynthetic pathway. However, no evidence for a functional role of flavonoids in plant freezing tolerance has been shown. Here, we present a comprehensive analysis using qRT-PCR for transcript, LC-MS for flavonoid and GC-MS for primary metabolite measurements, and an electrolyte leakage assay to determine freezing tolerance of 20 mutant lines in two Arabidopsis accessions that are affected in different steps of the flavonoid biosynthetic pathway. This analysis provides evidence for a functional role of flavonoids in plant cold acclimation. The accumulation of flavonoids in the activation tagging mutant line pap1-D improved, while reduced flavonoid content in different knock-out mutants impaired leaf freezing tolerance. Analysis of the different knock-out mutants suggests redundancy of flavonoid structures, as the lack of flavonols or anthocyanins could be compensated by other compound classes.