Freezing treatment under light conditions leads to a dramatic enhancement of freezing tolerance in cold-acclimated Arabidopsis.

Overwintering plants survive subzero temperatures by cold acclimation (CA), wherein they acquire freezing tolerance through short-term exposure to low temperatures above 0°C. The freezing tolerance of CA plants increases when they are subsequently exposed to mild subzero temperatures, a phenomenon known as second-phase cold hardening (2PH). Here, we explored the molecular mechanism and physiological conditions of 2PH. The results show that, compared with supercooling, a freezing treatment during 2PH after CA enhanced the freezing tolerance of Arabidopsis. This required CA as a pretreatment, and was designated as second-phase freezing acclimation (2PFA). Light increased the effect of 2PFA to enhance freezing tolerance after CA. C-repeat binding factor and cold-regulated genes were downregulated by light during the 2PFA treatment, a different transcription profile from that during CA. The freezing tolerance of 2PFA plants was decreased by the presence of the photosynthetic electron transfer inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea during the 2PFA treatment. Compared with wild-type plants, phototropin1,2 and phyb mutants showed lower freezing tolerance after 2PFA treatment. These results show that exposure to freezing after CA increases freezing tolerance as a secondary process, and that freezing under light conditions further increases freezing tolerance via pathways involving photoreceptors and photosynthetic electron transfer.

Plasma membrane proteomic changes of Arabidopsis DRP1E during cold acclimation in association with the enhancement of freezing tolerance.

The freezing tolerance of plants that live in cold regions increases after exposure to low temperature, a process termed cold acclimation (CA). During CA, restructuring of the plasma membrane (PM) is important to enhance freezing tolerance. We have previously shown that the function of DYNAMIN-RELATED PROTEIN 1 E (DRP1E), which regulates endocytosis by pinching vesicles from the PM, is associated with the enhancement of freezing tolerance during CA in Arabidopsis. DRP1E is predicted to play a role in reconstituting the PM composition during CA. In this study, to test the validity of this hypothesis, we studied the changes in PM proteome patterns induced by drp1e mutation. In a detailed physiological analysis, after 3 days of CA, only young leaves showed significantly less increase in freezing tolerance in the mutant than in the wild type (WT). Using nano-liquid chromatography-tandem mass spectrometry, 496 PM proteins were identified. Among these proteins, 81 or 71 proteins were specifically altered in the WT or the mutant, respectively, in response to CA. Principal component analysis showed that the proteomic pattern differed between the WT and the mutant upon cold acclimation (CA), suggesting that DRP1E contributes to reconstruction of the PM during CA. Cluster analysis revealed that proteins that were significantly increased in the mutant after CA were biased toward glycosylphosphatidylinositol-anchored proteins, such as fasciclin-like arabinogalactan proteins. Thus, a primary target of DRP1E-associated PM reconstruction during CA is considered to be glycosylphosphatidylinositol-anchored proteins, which may be removed from the PM by DRP1E in young leaves after 3 days of CA.

In Planta Monitoring of Cold-Responsive Promoter Activity Reveals a Distinctive Photoperiodic Response in Cold Acclimation.

Plant cold acclimation involves complicated pathways that integrate signals from temperature changes and light conditions. To understand plant responses to environmental signals in detail, molecular events that are regulated by temperature and light must be investigated at the whole-plant level in a nondestructive way. Using the promoter of COR15A connected to the luciferase reporter gene as a cold-responsive indicator, we developed an in planta monitoring system for gene expression under controlled temperature and photoperiod conditions. COR15A promoter activity was intensified by day-night cycles at 2�C, while its induction was abruptly suppressed in the dark at 8�C or higher, indicating a difference in responsiveness to photocycle between these two acclimation conditions. Freeze-thawing tests of whole plants proved that lower acclimation temperature resulted in higher tolerance to freezing, consistent with the temperature-dependent induction of COR15A. Inhibition of photosynthetic electron transport by 3-(3,4-dichlorophenyl)-1,1-dimethylurea eliminated the responsiveness to the day-night cycles at 2�C, indicating a possibility that the photosynthetic redox and/or the accumulation of photosynthates modulate COR15A responsiveness to photoperiod during cold acclimation, in addition to the well-known regulation by CBF (C-repeat binding factor) genes. These findings indicate that the cold-responsive promoter is regulated by distinctive mechanisms dependent on temperature and simultaneously affected by photocycle and photosynthesis.

Plasma Membrane Aquaporin Members PIPs Act in Concert to Regulate Cold Acclimation and Freezing Tolerance Responses in Arabidopsis thaliana.

Aquaporins play a major role in plant water uptake at both optimal and environmentally stressed conditions. However, the functional specificity of aquaporins under cold remains obscure. To get a better insight to the role of aquaporins in cold acclimation and freezing tolerance, we took an integrated approach of physiology, transcript profiling and cell biology in Arabidopsis thaliana. Cold acclimation resulted in specific upregulation of PIP1;4 and PIP2;5 aquaporin (plasma membrane intrinsic proteins) expression, and immunoblotting analysis confirmed the increase in amount of PIP2;5 protein and total amount of PIPs during cold acclimation, suggesting that PIP2;5 plays a major role in tackling the cold milieu. Although single mutants of pip1;4 and pip2;5 or their double mutant showed no phenotypic changes in freezing tolerance, they were more sensitive in root elongation and cell survival response under freezing stress conditions compared with the wild type. Consistently, a single mutation in either PIP1;4 or PIP2;5 altered the expression of a number of aquaporins both at the transcriptional and translational levels. Collectively, our results suggest that aquaporin members including PIP1;4 and PIP2;5 function in concert to regulate cold acclimation and freezing tolerance responses.

Season specificity in the cold-induced calcium signal and the volatile chemicals in the atmosphere.

Cold-induced Ca2+ signals in plants are widely accepted to be involved in cold acclimation. Surprisingly, despite using Arabidopsis plants grown in a growth chamber, we observed a clear seasonal change in cold-induced Ca2+ signals only in roots. Ca2+ signals were captured using Arabidopsis expressing Yellow Cameleon 3.60. In winter, two Ca2+ signal peaks were observed during a cooling treatment from 20 to 0°C, but in summer only one small peak was observed under the same cooling condition. In the spring and autumn seasons, an intermediate type of Ca2+ signal, which had a delayed first peak and smaller second peaks compared with the those of the winter type, was observed. Volatile chemicals and/or particles in the air from the outside may affect plants in the growth chamber. This idea is supported by the fact that incubation of plants with activated carbon changed the intermediate-type Ca2+ signal to the summer-type. The seasonality was also observed in the freezing tolerance of plants cold-acclimated in a low-temperature chamber. The solar radiation intensity was weakly correlated, not only with the seasonal characteristics of the Ca2+ signal but also with freezing tolerance. It has been reported that the ethylene concentration in the atmosphere seasonally changes depending on the solar radiation intensity. Ethylene gas and 1-aminocyclopropane-1-carboxylic acid treatment affected the Ca2+ signals, the shape of which became a shape close to, but not the same as, the winter type from the other types, indicating that ethylene may be one of several factors influencing the cold-induced Ca2+ signal.

A single seed treatment mediated through reactive oxygen species increases germination, growth performance, and abiotic stress tolerance in Arabidopsis and rice.

Hydroxyl radical (•OH) is considered to be the most damaging among reactive oxygen species. Although afew studies have reported on its effects on growth and stress adaptation of plants, no detailed studies have been performed using •OH in germination and early seedling growth under abiotic stresses. Here we report a single seed treatment with •OH on germination and seedling growth of Arabidopsis and rice under non-stressed (ambient) and various abiotic-stressed conditions (chilling, high temperature, heat, and salinity). The treatment resulted in faster seed germination and early seedling growth under non-stressed conditions, and, interestingly, these effects were more prominent under abiotic stresses. In addition, Arabidopsis seedlings from treated seeds showed faster root growth and developed more lateral roots. These results show apositive and potential practical use for •OH in model and crop plants for direct seeding in the field, as well as improvement of tolerance against emerging stresses. Abbreviations: AUC: area under curve; MGT: mean germination time; t50: time to reach 50% germination; U7525: time for uniform germination from 25% to 75%; ROS: reactive oxygen species; GSI: germination speed index; SI: stress index; DI: dormancy index.

Large-Scale Phosphoproteomic Study of Arabidopsis Membrane Proteins Reveals Early Signaling Events in Response to Cold.

Cold stress is one of the major factors limiting global crop production. For survival at low temperatures, plants need to sense temperature changes in the surrounding environment. How plants sense and respond to the earliest drop in temperature is still not clearly understood. The plasma membrane and its adjacent extracellular and cytoplasmic sites are the first checkpoints for sensing temperature changes and the subsequent events, such as signal generation and solute transport. To understand how plants respond to early cold exposure, we used a mass spectrometry-based phosphoproteomic method to study the temporal changes in protein phosphorylation events in Arabidopsis membranes during 5 to 60 min of cold exposure. The results revealed that brief cold exposures led to rapid phosphorylation changes in the proteins involved in cellular ion homeostasis, solute and protein transport, cytoskeleton organization, vesical trafficking, protein modification, and signal transduction processes. The phosphorylation motif and kinase-substrate network analysis also revealed that multiple protein kinases, including RLKs, MAPKs, CDPKs, and their substrates, could be involved in early cold signaling. Taken together, our results provide a first look at the cold-responsive phosphoproteome changes of Arabidopsis membrane proteins that can be a significant resource to understand how plants respond to an early temperature drop.

Calcium Signaling-Linked CBF/DREB1 Gene Expression was Induced Depending on the Temperature Fluctuation in the Field: Views from the Natural Condition of Cold Acclimation.

Environmental adaptability is essential for plant survival. Though it is well known that a simple cooling or cold shock leads to Ca2+ signals, direct evidence has not been provided that plants use Ca2+ signals as a second messenger in the cold acclimation (CA) process in the field. By developing a technique to analyze Ca2+ signals using confocal cryomicroscopy, we investigated Ca2+ signals under several temperature conditions by combining the start temperature, cooling rate and cooling time duration. In both root and leaf cells, Ca2+ signals rapidly disappeared after cooling stopped, and thereafter under a constant low temperature no Ca2+ signal was observed. Interestingly, under the cooling regime from 2�C to -2�C, non-acclimated plants grown at 23�C hardly showed Ca2+ signals, but cold-acclimated plants at 2�C were able to form Ca2+ signals in root cells. These findings suggest that plants sense temperature decreases with Ca2+ signals while adjusting the temperature sensitivity to their own temperature environment. Furthermore, if the temperature is constant, no Ca2+ signal is induced even during CA. Then, we also focused on the CA under field conditions, rich in temperature fluctuations. In CA under field conditions, the expression patterns of CBF/DREB1 genes were distinctly different from those in artificial CA. Pharmacological studies with Ca2+ channel blockers showed that the Ca2+-induced expression of CBF/DREB1 genes was closely correlated with the amplitude of temperature fluctuation, suggesting that Ca2+ signals regulate CBF/DREB1 gene expression during CA under natural conditions.

Inhibition of mRNA processing activity from ginger-, clove- and cinnamon-extract, and by two ginger constituents, 6-gingerol and 6-shogaol.

Inhibition of mRNA processing, including splicing in the nucleus, is a potential anti-cancer candidate. To obtain mRNA processing inhibitors, we have screened for active constituents from spices. Ginger, clove, and cinnamon showed an inhibitory effect on mRNA processing in the nucleus. Two components in ginger, 6-gingerol and 6-shogaol, exhibited the inhibition of mRNA processing.

Importance of the Proximity and Orientation of Ligand-Linkage to the Design of Cinnamate-GW9662 Hybrid Compounds as Covalent PPARγ Agonists.

Covalent agonists of PPARγ cause unique receptor conformational changes and behave as selective PPARγ modulators, whereas there are few covalent agonists other than endogenous unsaturated fatty acids metabolites. Previously, we established a cell-based strategy to identify new PPARγ ligands and synthesized a new-type of covalent agonist that possesses the hybrid structure of a plant-derived cinnamic acid derivative and GW9662, a covalent antagonist. Herein, we report six analogues that differ in how the two fragments are linked together. Compounds with a simplified linker showed potent agonistic activity with improved EC50 values (less than 5 nM), indicating that close proximity between the two fragments improves binding affinity. When the position of cinnamic acid moiety was placed at 4' carbon of aniline ring, PPARγ agonist activity was completely abolished. Docking studies suggested that the activation profile likely depends on interaction with the cavity around helix 3, ß-sheet, and Ω-loop region in the ligand-binding domain. Furthermore, a cell-based assay revealed that agonist-type compounds activate PPARγ transcription in a manner dependent on covalent linkage with the Cys285 residue leading to prolonged transactivation. This activation feature reflects pharmacological benefits of covalent drugs, suggesting that these hybrid compounds may serve as potential leads for a new-class of covalent PPARγ ligands.

Freezing Tolerance of Plant Cells: From the Aspect of Plasma Membrane and Microdomain.

Freezing stress is accompanied by a state change from water to ice and has multiple facets causing dehydration; consequently, hyperosmotic and mechanical stresses coupled with unfavorable chilling stress act in a parallel way. Freezing tolerance varies widely among plant species, and, for example, most temperate plants can overcome deleterious effects caused by freezing temperatures in winter. Destabilization and dysfunction of the plasma membrane are tightly linked to freezing injury of plant cells. Plant freezing tolerance increases upon exposure to nonfreezing low temperatures (cold acclimation). Recent studies have unveiled pleiotropic responses of plasma membrane lipids and proteins to cold acclimation. In addition, advanced techniques have given new insights into plasma membrane structural non-homogeneity, namely, microdomains. This chapter describes physiological implications of plasma membrane responses enhancing freezing tolerance during cold acclimation, with a focus on microdomains.

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 is accompanied by complex responses of glycosylphosphatidylinositol (GPI)-anchored proteins in Arabidopsis.

Cold acclimation results in changes of the plasma membrane (PM) composition. The PM is considered to contain specific lipid/protein-enriched microdomains which can be extracted as detergent-resistant plasma membrane (DRM). Previous studies in animal cells have demonstrated that glycosylphosphatidylinositol-anchored proteins (GPI-APs) can be targeted to microdomains and/or the apoplast. However, the functional significance of GPI-APs during cold acclimation in plants is not yet fully understood. In this study, we aimed to investigate the responsiveness of GPI-APs to cold acclimation treatment in Arabidopsis We isolated the PM, DRM, and apoplast fractions separately and, in addition, GPI-AP-enriched fractions were prepared from the PM preparation. Label-free quantitative shotgun proteomics identified a number of GPI-APs (163 proteins). Among them, some GPI-APs such as fasciclin-like arabinogalactan proteins and glycerophosphoryldiester phosphodiesterase-like proteins predominantly increased in PM- and GPI-AP-enriched fractions while the changes of GPI-APs in the DRM and apoplast fractions during cold acclimation were considerably different from those of other fractions. These proteins are thought to be associated with cell wall structure and properties. Therefore, this study demonstrated that each GPI-AP responded to cold acclimation in a different manner, suggesting that these changes during cold acclimation are involved in rearrangement of the extracellular matrix including the cell wall towards acquisition of freezing tolerance.

Cellular auxin homeostasis under high temperature is regulated through a sorting NEXIN1-dependent endosomal trafficking pathway.

High-temperature-mediated adaptation in plant architecture is linked to the increased synthesis of the phytohormone auxin, which alters cellular auxin homeostasis. The auxin gradient, modulated by cellular auxin homeostasis, plays an important role in regulating the developmental fate of plant organs. Although the signaling mechanism that integrates auxin and high temperature is relatively well understood, the cellular auxin homeostasis mechanism under high temperature is largely unknown. Using the Arabidopsis thaliana root as a model, we demonstrate that under high temperature, roots counterbalance the elevated level of intracellular auxin by promoting shootward auxin efflux in a PIN-FORMED2 (PIN2)-dependent manner. Further analyses revealed that high temperature selectively promotes the retrieval of PIN2 from late endosomes and sorts them to the plasma membrane through an endosomal trafficking pathway dependent on SORTING NEXIN1. Our results demonstrate that recycling endosomal pathway plays an important role in facilitating plants adaptation to increased temperature.

Lipid profiles of detergent resistant fractions of the plasma membrane in oat and rye in association with cold acclimation and freezing tolerance.

Cold acclimation (CA) results in alteration of the plasma membrane (PM) lipid composition in plants, which plays a crucial role in the acquisition of freezing tolerance via membrane stabilization. Recent studies have indicated that PM structure is consistent with the fluid mosaic model but is laterally non-homogenous and contains microdomains enriched in sterols, sphingolipids and specific proteins. In plant cells, the function of these microdomains in relation to CA and freezing tolerance is not yet fully understood. The present study aimed to investigate the lipid compositions of detergent resistant fractions of the PM (DRM) which are considered to represent microdomains. They were prepared from leaves of low-freezing tolerant oat and high-freezing tolerant rye. The DRMs contained higher proportions of sterols, sphingolipids and saturated phospholipids than the PM. In particular, one of the sterol lipid classes, acylated sterylglycoside, was the predominant sterol in oat DRM while rye DRM contained free sterol as the major sterol. Oat and rye showed different patterns (or changes) of sterols and 2-hydroxy fatty acids of sphingolipids of DRM lipids during CA. Taken together, these results suggest that CA-induced changes of lipid classes and molecular species in DRMs are associated with changes in the thermodynamic properties and physiological functions of microdomains during CA and hence, influence plant freezing tolerance.

A study on ice crystal formation behavior at intracellular freezing of plant cells using a high-speed camera.

Intracellular ice crystal formation (IIF) causes several problems to cryopreservation, and it is the key to developing improved cryopreservation techniques that can ensure the long-term preservation of living tissues. Therefore, the ability to capture clear intracellular freezing images is important for understanding both the occurrence and the IIF behavior. The authors developed a new cryomicroscopic system that was equipped with a high-speed camera for this study and successfully used this to capture clearer images of the IIF process in the epidermal tissues of strawberry geranium (Saxifraga stolonifera Curtis) leaves. This system was then used to examine patterns in the location and formation of intracellular ice crystals and to evaluate the degree of cell deformation because of ice crystals inside the cell and the growing rate and grain size of intracellular ice crystals at various cooling rates. The results showed that an increase in cooling rate influenced the formation pattern of intracellular ice crystals but had less of an effect on their location. Moreover, it reduced the degree of supercooling at the onset of intracellular freezing and the degree of cell deformation; the characteristic grain size of intracellular ice crystals was also reduced, but the growing rate of intracellular ice crystals was increased. Thus, the high-speed camera images could expose these changes in IIF behaviors with an increase in the cooling rate, and these are believed to have been caused by an increase in the degree of supercooling.

Effects of micro electric current load during cooling of plant tissues on intracellular ice crystal formation behavior and pH.

Cryopreservation techniques are expected to evolve further to preserve biomaterials and foods in a fresh state for extended periods of time. Long-term cryopreservation of living materials such as food and biological tissue is generally achieved by freezing; thus, intracellular freezing occurs. Intracellular freezing injures the cells and leads to cell death. Therefore, a dream cryopreservation technique would preserve the living materials without internal ice crystal formation at a temperature low enough to prevent bacterial activity. This study was performed to investigate the effect of micro electrical current loading during cooling as a new cryopreservation technique. The behavior of intracellular ice crystal formation in plant tissues with or without an electric current load was evaluated using the degree of supercooling, degree of cell deformation, and grain size and growing rate of intracellular ice crystal. Moreover, the transition of intracellular pH during plant tissue cooling with or without electric current loading was also examined using the fluorescence intensity ratio to comprehend cell activity at lower temperatures. The results indicated that micro electric current load did not only decrease the degree of cell deformation and grain size of intracellular ice crystal but also reduced the decline in intracellular pH due to temperature lowering, compared with tissues subjected to the same cooling rate without an electric current load. Thus, the effect of electric current load on cryopreservation and the potential of a new cryopreservation technique using electric current load were discussed based on these results.

Distribution of lysophosphatidylcholine in the endosperm of Oryza sativa rice.

RATIONALE: Sake is made from fermented rice and has been drunk in Japan for more than 1000 years. The rice must be polished prior to fermentation to obtain high-quality sake. It is traditionally recognized that the quality of sake is improved as the rice polishing ratio (percentage removed in the polishing process) increases. However, the underlying chemistry of the rice polishing process is incompletely understood. Herein, we analyzed the distribution of lysophosphatidylcholine (LPC) molecular species with unsaturated fatty acids in rice, as their presence is thought to exert a negative effect on the flavor of sake. METHODS: The distribution of LPC molecular species in rice was visualized via matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). RESULTS: LPC (16:0) is ubiquitously present in the endosperm of rice while LPC (18:0) is localized in the core of the endosperm. In contrast, LPC (18:2) and LPC (18:1) are present in the outer region of the endosperm. CONCLUSIONS: The enhancement of the quality of sake as the polishing ratio of the rice increases might be explained in terms of the distribution of LPC with unsaturated fatty acids in the rice.

Dietary Puerarin Translocates to Femur and Suppresses Osteoclast Differentiation in Ovariectomized Mice.

Osteoporosis is characterized by bone loss and deterioration in bone microstructure, leading to bone fragility. It is strongly correlated with menopause in women. Previously, we reported that diets supplemented with a kudzu (Pueraria lobata) vine extract suppressed bone resorption in ovariectomized (OVX) mice, a postmenopausal model. The main isoflavone in kudzu is puerarin (daidzein-8-C-glycoside). Puerarin (daidzein-8-C-glycoside), which is main isoflavone of kudzu, probably contributes to the beneficial effect. However, the underlying mechanism is unclear. Therefore, the nutrikinetics of puerarin and the comparison with the suppressive effects of kudzu isoflavones on osteoclast differentiation was examined in this study. We demonstrated that orally administered puerarin was absorbed from the gut and entered the circulation in an intact form. In addition, puerarin accumulated in RAW264.7 pre-osteoclast cells in a time-dependent manner. Tartrate-resistant acid phosphatase activity was decreased by puerarin treatment in a concentration-dependent manner in RAW264.7 cells stimulated with the receptor activator of nuclear factor kappa-B ligand. Ovariectomy-induced elevated bone resorption was suppressed, and the fragile bone strength was improved by puerarin ingestion in the diet. These findings suggested that orally administered puerarin was localized in bone tissue and suppressed bone resorption and osteoclastogenesis in ovariectomized mice.

Changes of detergent-resistant plasma membrane proteins in oat and rye during cold acclimation: association with differential freezing tolerance.

Cold acclimation (CA) results in an increase in freezing tolerance of plants, which is closely associated to functional changes of the plasma membrane (PM). Although proteomic studies have revealed compositional changes of the PM during CA, there has been no large-scale study of how the microdomains in the PM, which contains specific lipids and proteins, change during CA. Therefore, we conducted semiquantitative shotgun proteomics using microdomain-enriched detergent-resistant membrane (DRM) fractions extracted from low freezing-tolerant oat and highly freezing-tolerant rye. We identified 740 and 809 DRM proteins in oat and rye, respectively. Among the proteins identified, the abundances of a variety of proteins, such as P-type ATPase and aquaporins, were affected by CA in both oat and rye. Some CA-responsive proteins in the DRM fractions, such as heat shock protein 70, changed differently in oat and rye. In addition, changes in lipocalins and sugar transporters in the DRM fractions were different from those found in total PM fraction during CA. This is the first report to describe compositional changes in the DRM during CA. The proteomic profiles obtained in the present study hint at many possible microdomain functions associated with CA and freezing tolerance.