Exploring freeze-injury mechanism through ion-specific analysis of leachate from reversibly versus irreversibly injured spinach (Spinacia oleracea L.) leaves.

The present study analyzed four cations (K+, Ca2+, Mg2+, Fe2+) in leachate from freeze-injured spinach (Spinacia oleracea L. 'Reflect') leaves exposed for four freezing-durations (FDs) (0.5, 3.0, 5.5, 10.5 h) at -4.8 °C. Comparison of electrolyte leakage from right-after-thaw with that after 6-d recovery revealed that injury at 0.5 or 3 h FDs was recoverable but irreversible at 5.5 or 10.5 h FDs. Data suggests leakage of K+, the most abundant cation in leachate, can serve as a proxy for total electrolyte-leakage in determining plant freezing-tolerance and an ionic marker discerning moderate vs. severe injury. Quantitative correspondence between Ca2+- and K+-leakage supports earlier proposition that leaked K+ induces loss of membrane-Ca2+, which, in turn, promotes further K+-leakage due to weakened membrane. Reduced/undetectable Fe2+ in leachate at longer FDs suggests activation of Fenton reaction converting soluble Fe2+ into insoluble Fe3+. Enhanced Mg2+-leakage at greater freeze-injury suggests structural/functional impairment of chlorophyll/chloroplast complex.

Infrared thermography of in situ natural freezing and mechanism of winter-thermonasty in Rhododendron maximum.

Evergreen leaves of Rhododendron species inhabiting temperate/montane climates are typically exposed to both high radiation and freezing temperatures during winter when photosynthetic biochemistry is severely inhibited. Cold-induced "thermonasty," that is, lamina rolling and petiole curling, can reduce the amount of leaf area exposed to solar radiation and has been associated with photoprotection in overwintering rhododendrons. The present study was conducted on natural, mature plantings of a cold-hardy and large-leaved thermonastic North American species (Rhododendron maximum) during winter freezes. Infrared thermography was used to determine initial sites of ice formation, patterns of ice propagation, and dynamics of the freezing process in leaves to understand the temporal and mechanistic relationship between freezing and thermonasty. Results indicated that ice formation in whole plants is initiated in the stem, predominantly in the upper portions, and propagates in both directions from the original site. Ice formation in leaves initially occurred in the vascular tissue of the midrib and then propagated into other portions of the vascular system/venation. Ice was never observed to initiate or propagate into palisade, spongy mesophyll, or epidermal tissues. These observations, together with the leaf- and petiole-histology, and a simulation of the rolling effect of dehydrated leaves using a cellulose-based, paper-bilayer system, suggest that thermonasty occurs due to anisotropic contraction of cell wall cellulose fibers of adaxial versus abaxial surface as the cells lose water to ice present in vascular tissues.

Pre-stress salicylic-acid treatment as an intervention strategy for freeze-protection in spinach: Foliar versus sub-irrigation application and duration of efficacy.

Exogenous application of salicylic acid (SA) to plant tissues has been shown to confer tolerance against various abiotic stresses. Recently, SA application through sub-irrigation was shown to improve plant freezing tolerance (FT). For SA treatment to be employable as an effective intervention strategy for frost protection under field conditions, it is important to study its effect on FT when applied as a foliar spray to whole plants. It is also important to determine for how long the FT-improvement by SA lasts. Present study was conducted to compare SA-induced FT of spinach (Spinacia oleracea L. 'Reflect') seedlings following SA-application by foliar spray vs. sub-irrigation. Durability of FT-promotive effect of SA was evaluated using three freeze-tests over a 4-d period, i.e., at 10-d, 12-d, and 14-d after the SA application. Freezing stress was applied using a temperature-controlled freeze-thaw protocol, and FT was assessed by visual observations (leaf flaccidness vs. turgidity) as well as ion-leakage assay. Data indicated that both foliar spray and sub-irrigation methods improved FT of the seedlings against a relatively moderate (-5.5 °C) as well as severe stress (-6.5 °C). Moreover, improved FT against moderate stress was sustained over a 4-d period, whereas such benefit waned somewhat against the severe stress. SA-treated leaves' growth performance was similar to the non-treated control based on dry weight, fresh weight, leaf area, and dry weight/leaf area parameters. Our results suggest that SA application as a foliar spray can potentially be used to protect field-grown transplants against episodic frosts.

Factors affecting freezing tolerance: a comparative transcriptomics study between field and artificial cold acclimations in overwintering evergreens.

Cold acclimation (CA) is a well-known strategy employed by plants to enhance freezing tolerance (FT) in winter. Global warming could disturb CA and increase the potential for winter freeze-injury. Thus, developing robust FT through complete CA is essential. To explore the molecular mechanisms of CA in woody perennials, we compared field and artificial CAs. Transcriptomic data showed that photosynthesis/photoprotection and fatty acid metabolism pathways were specifically enriched in field CA; carbohydrate metabolism, secondary metabolism and circadian rhythm pathways were commonly enriched in both field and artificial CAs. When compared with plants in vegetative growth in the chamber, we found that the light signals with warm air temperatures in the fall might induce the accumulation of leaf abscisic acid (ABA) and jasmonic acid (JA) concentrations, and activate Ca2+ , ABA and JA signaling transductions in plants. With the gradual cooling occurrence in winter, more accumulation of anthocyanin, chlorophyll degradation, closure/degradation of photosystem II reaction centers, and substantial accumulation of glucose and fructose contributed to obtaining robust FT during field CA. Moreover, we observed that in Rhododendron 'Elsie Lee', ABA and JA decreased in winter, which may be due to the strong requirement of zeaxanthin for rapid thermal dissipation and unsaturated fatty acids for membrane fluidity. Taken together, our results indicate that artificial CA has limitations to understand the field CA and field light signals (like short photoperiod, light intensity and/or light quality) before the low temperature in fall might be essential for complete CA.

The relationship of cold acclimation and extracellular ice formation to winter thermonasty in two Rhododendron species and their F1 hybrid.

PREMISE: Thermonastic leaf movements in evergreen Rhododendron species have been used to study plant strategies for winter photoprotection. To add to the current fundamental understanding of this behavior, we addressed the following questions: (1) Is the cold-acclimated (CA) state necessary for thermonasty, and do cold-induced leaf movements also occur in non-acclimated (NA) plants? (2) Which of the two movements, leaf rolling versus curling, is more responsive to freezing, if any, in a non-thermonastic species? (3) What is the temporal relationship between extracellular freezing and thermonasty? (4) What genetic inferences can be drawn from leaf movement in an F1 hybrid relative to its parents? METHODS: A temperature-controlled, gradual cooling regime was used to quantify freeze-induced leaf movements. Infrared thermography was used to confirm extracellular ice-formation in leaves. RESULTS: Both NA and CA plants of thermonastic species exhibited thermonasty, but leaf rolling/curling increased significantly in CA plants. In the cold-acclimated condition, a non-thermonastic species showed almost no rolling during freezing, while the thermonastic species and F1 hybrid did, the latter exhibiting a response intermediate to the parents. Freezing-induced leaf curling in the non-thermonastic species and the F1 hybrid was equivalent and significantly less than the degree of curling in the thermonastic species. CONCLUSIONS: Milder thermonasty in NA than CA leaves could be associated with differential anisotropy in the rolling forces and/or response of aquaporins to freezing. Leaf movements in the hybrid suggest that leaf rolling and curling are additive and dominant genetic traits, respectively. Infrared thermography confirms that ice formation in tissues precedes cold-induced thermonasty in R. catawbiense.

Repair of sub-lethal freezing damage in leaves of Arabidopsis thaliana.

BACKGROUND: The detrimental effects of global climate change direct more attention to the survival and productivity of plants during periods of highly fluctuating temperatures. In particular in temperate climates in spring, temperatures can vary between above-zero and freezing temperatures, even during a single day. Freeze-thaw cycles cause cell membrane lesions that can lead to tissue damage and plant death. Whereas the processes of cold acclimation and freeze-thaw injury are well documented, not much is known about the recovery of plants after a freezing event. We therefore addressed the following questions: i. how does the severity of freezing damage influence repair; ii. how are respiration and content of selected metabolites influenced during the repair process; and iii. how do transcript levels of selected genes respond during repair? RESULTS: We have investigated the recovery from freezing to sub-lethal temperatures in leaves of non-acclimated and cold acclimated Arabidopsis thaliana plants over a period of 6 days. Fast membrane repair and recovery of photosynthesis were observed 1 day after recovery (1D-REC) and continued until 6D-REC. A substantial increase in respiration accompanied the repair process. In parallel, concentrations of sugars and proline, acting as compatible solutes during freezing, remained unchanged or declined, implicating these compounds as carbon and nitrogen sources during recovery. Similarly, cold-responsive genes were mainly down regulated during recovery of cold acclimated leaves. In contrast, genes involved in cell wall remodeling and ROS scavenging were induced during recovery. Interestingly, also the expression of genes encoding regulatory proteins, such as 14-3-3 proteins, was increased suggesting their role as regulators of repair processes. CONCLUSIONS: Recovery from sub-lethal freezing comprised membrane repair, restored photosynthesis and increased respiration rates. The process was accompanied by transcriptional changes including genes encoding regulatory proteins redirecting the previous cold response to repair processes, e.g. to cell wall remodeling, maintenance of the cellular proteome and to ROS scavenging. Understanding of processes involved in repair of freeze-thaw injury increases our knowledge on plant survival in changing climates with highly fluctuating temperatures.

Short versus prolonged freezing differentially impacts freeze - thaw injury in spinach leaves: mechanistic insights through metabolite profiling.

Plant tissues subjected to short or prolonged freezing to a fixed sub-freezing temperature are expected to undergo similar freeze-desiccation but the former causes substantially less injury than the latter. To gain metabolic insight into this differential response, metabolome changes in spinach (Spinacia oleracea L.) leaves were determined following short-term (0.5 and 3.0 h) vs. prolonged freezing (5.5 and 10.5 h) at -4.5°C resulting in reversible or irreversible injury, respectively. LD50 , the freezing duration causing 50% injury, was estimated to be ∼3.1 h and defined as the threshold beyond which tissues were irreversibly injured. From 39 identified metabolites, 19 were selected and clustered into 3 groups: (1) signaling-related (salicylic acid, aliphatic and aromatic amino acids), (2) injury-related (GABA, lactic acid, maltose, fatty acids, policosanols, TCA intermediates) and (3) recovery-related (ascorbic acid, α-tocopherol). Initial accumulation of salicylic acid during short-term freezing followed by a decline may be involved in triggering tolerance mechanisms in moderately injured tissues, while its resurgence during prolonged freezing may signal programmed cell death. GABA accumulated with increasing freezing duration, possibly to serve as a 'pH-stat' against cytoplasmic acidification resulting from lactic acid accumulation. Mitochondria seem to be more sensitive to prolonged freezing than chloroplasts since TCA intermediates decreased after LD50 while salicylic acid and maltose, produced in chloroplasts, accumulate even at 10.5-h freezing. Fatty acids and policosanols accumulation with increasing freezing duration indicates greater injury to membrane lipids and epicuticular waxes. Ascorbic acid and α-tocopherol accumulated after short-term freezing, supposedly facilitating recovery while their levels decreased in irreversibly injured tissues.

Exogenous salicylic acid improves freezing tolerance of spinach (Spinacia oleracea L.) leaves.

Salicylic acid (SA)-treatment has been reported to improve plant tolerance to various abiotic stresses. However, its effect on freezing tolerance has not been well investigated. We investigated the effect of exogenous SA on freezing tolerance of spinach (Spinacia oleracea L.) leaves. We also explored if nitric oxide (NO) and/or hydrogen peroxide (H2O2)-mediation was involved in this response, since these are known as primary signaling molecules involved in many physiological processes. A micro-centrifuge tube-based system used to apply SA to petiolate spinach leaves (0.5 mM over 4-d) was effective, as evident by SA content of leaf tissues. SA-treatment did not hamper leaf growth (fresh and dry weight; equatorial and longitudinal length) and was also not significantly different from 25% Hoagland controls vis-à-vis growth. SA application significantly improved freezing tolerance as evidenced by reduced ion-leakage and alleviated oxidative stress (lower accumulation of O2·- and H2O2) following freeze-thaw stress treatments (-6.5, -7.5, and -8.5 °C). Improved freezing tolerance of SA-treated leaves was paralleled by increased proline and ascorbic acid (AsA) accumulation. A 9-d cold acclimation (CA) treatment also improved leaf freezing tolerance (compared to non-acclimated control) and was accompanied by accumulation of SA and proline. Our results indicate that increased freezing tolerance may be associated with accumulation of compatible solutes (proline) and antioxidants (AsA). Notably, the beneficial effect of SA on freezing tolerance was abolished when either H2O2- or NO-scavenger (1 µM N-acetylneuraminic acid, NANA or 100 µM hemoglobin, HB, respectively) was added to SA as pretreatment. Our data suggest that SA-induced freezing tolerance in spinach may be mediated by NO and H2O2 signaling.

Understanding the cellular mechanism of recovery from freeze-thaw injury in spinach: possible role of aquaporins, heat shock proteins, dehydrin and antioxidant system.

Recovery from reversible freeze-thaw injury in plants is a critical component of ultimate frost survival. However, little is known about this aspect at the cellular level. To explore possible cellular mechanism(s) for post-thaw recovery (REC), we used Spinacia oleracea L. cv. Bloomsdale leaves to first determine the reversible freeze-thaw injury point. Freeze (-4.5°C)-thaw-injured tissues (32% injury vs <3% in unfrozen control) fully recovered during post-thaw, as assessed by an ion leakage-based method. Our data indicate that photosystem II efficiency (Fv/Fm) was compromised in injured tissues but recovered during post-thaw. Similarly, the reactive oxygen species (O2 (•-) and H2 O2 ) accumulated in injured tissues but dissipated during recovery, paralleled by the repression and restoration, respectively, of activities of antioxidant enzymes, superoxide dismutase (SOD) (EC. 1.14.1.1), and catalase (CAT) (EC.1.11.1.6) and ascorbate peroxidase (APX) (EC.1.11.1.11). Restoration of CAT and APX activities during recovery was slower than SOD, concomitant with a slower depletion of H2 O2 compared to O2 (•-) . A hypothesis was also tested that the REC is accompanied by changes in the expression of water channels [aquaporines (AQPs)] likely needed for re-absorption of thawed extracellular water. Indeed, the expression of two spinach AQPs, SoPIP2;1 and SoδTIP, was downregulated in injured tissues and restored during recovery. Additionally, a notion that molecular chaperones [heat shock protein of 70 kDa (HSP70s)] and putative membrane stabilizers [dehydrins (DHNs)] are recruited during recovery to restore cellular homeostasis was also tested. We noted that, after an initial repression in injured tissues, the expression of three HSP70s (cytosolic, endoplasmic reticulum and mitochondrial) and a spinach DHN (CAP85) was significantly restored during the REC.

Redox regulation of the Calvin-Benson-Bassham cycle during cold acclimation.

NADPH-dependent thioredoxin reductase C (NTRC) redox interaction with protein CP12 plays a role in cold acclimation. A recent study by Teh et al. describes the underlying molecular mechanisms that leads to dissociation of the autoinhibitory PRK/CP12/GAPDH (phosphoribulokinase/CP12/glyceraldehyde-3-phosphate dehydrogenase) supracomplex. We propose that chloroplast-to-nucleus retrograde signaling precedes the described mechanism.

Proteomic changes associated with freeze-thaw injury and post-thaw recovery in onion (Allium cepa L.) scales.

The ability of plants to recover from freeze-thaw injury is a critical component of freeze-thaw stress tolerance. To investigate the molecular basis of freeze-thaw recovery, here we compared the proteomes of onion scales from unfrozen control (UFC), freeze-thaw injured (INJ), and post-thaw recovered (REC) treatments. Injury-related proteins (IRPs) and recovery-related proteins (RRPs) were differentiated according to their accumulation patterns. Many IRPs decreased right after thaw without any significant re-accumulation during post-thaw recovery, while others were exclusively induced in INJ tissues. Most IRPs are antioxidants, stress proteins, molecular chaperones, those induced by physical injury or proteins involved in energy metabolism. Taken together, these observations suggest that while freeze-thaw compromises the constitutive stress protection and energy supply in onion scales, it might also recruit 'first-responders' (IRPs that were induced) to mitigate such injury. RRPs, on the other hand, are involved in the injury-repair program during post-thaw environment conducive for recovery. Some RRPs were restored in REC tissues after their first reduction right after thaw, while others exhibit higher abundance than their 'constitutive' levels. RRPs might facilitate new cellular homeostasis, potentially by re-establishing ion homeostasis and proteostasis, cell-wall remodelling, reactive oxygen species (ROS) scavenging, defence against possible post-thaw infection, and regulating the energy budget to sustain these processes.

Winter survival and deacclimation of perennials under warming climate: physiological perspectives.

Appropriate timing and rate of cold deacclimation and the ability to reacclimate are important components of winter survival of perennials in temperate and boreal zones. In association with the progressive increase in atmospheric CO2, temperate and boreal winters are becoming progressively milder, and temperature patterns are becoming irregular with increasing risk of unseasonable warm spells during the colder periods of plants' annual cycle. Because deacclimation is mainly driven by temperature, these changes pose a risk for untimely/premature deacclimation, thereby rendering plant tissue vulnerable to freeze-injury by a subsequent frost. Research also indicates that elevated CO2 may directly impact deacclimation. Hence, understanding the underlying cellular mechanisms of how deacclimation and reacclimation capacity are affected by changes in environmental conditions is important to ensure winter survival and the sustainability of plant sources under changing climate. Relative to cold acclimation, deacclimation is a little studied process, but the limited evidence points to specific changes occurring in the transcriptome and proteome during deacclimation. Loss of freezing tolerance is additionally associated with substantial changes in cell/tissue-water relations and carbohydrate metabolism; the latter also impacted by temperature-driven, altered respiratory metabolism. This review summarizes recent progress in understanding the physiological mechanisms of deacclimation and how they may be impacted by climate change.

Post-translational activation of CBF for inducing freezing tolerance.

Plants can acquire increased freezing tolerance through cold-acclimation involving the ICE1-CBF-COR pathway. Recently, Lee et al. investigated a potential link between the functional activation of CBF and cellular redox state. We propose that redox-mediated CBF activation could be a hub of low temperature as well as light signaling in the cold-acclimation process.

Ice recrystallization inhibition proteins of perennial ryegrass enhance freezing tolerance.

Ice recrystallization inhibition (IRI) proteins are thought to play an important role in conferring freezing tolerance in plants. Two genes encoding IRI proteins, LpIRI-a and LpIRI-b, were isolated from a relatively cold-tolerant perennial ryegrass cv. Caddyshack. Amino acid alignments among the IRI proteins revealed the presence of conserved repetitive IRI-domain motifs (NxVxxG/NxVxG) in both proteins. Quantitative reverse transcriptase PCR (qRT-PCR) analysis indicated that LpIRI-a was up-regulated approximately 40-fold while LpIRI-b was up-regulated sevenfold after just 1 h of cold acclimation, and by 7 days of cold acclimation the transcripts had increased 8,000-fold for LpIRI-a and 1,000-fold for LpIRI-b. Overexpression of either LpIRI-a or LpIRI-b gene in Arabidopsis increased survival rates of the seedlings following a freezing test under both cold-acclimated and nonacclimated conditions. For example, without cold acclimation a -4 degrees C treatment reduced the wild type's survival rate to an average of 73%, but resulted in survival rates of 85-100% for four transgenic lines. With cold acclimation, a -12 degrees C treatment reduced the wild type's survival rate to an average of 38.7%, while it resulted in a survival rate of 51-78.5% for transgenic lines. After cold acclimation, transgenic Arabidopsis plants overexpressing either LpIRI-a or LpIRI-b gene exhibited a consistent reduction in freezing-induced ion leakage at -8, -9, and -10 degrees C. Furthermore, the induced expression of the LpIRI-a and LpIRI-b proteins in transgenic E. coli enhanced the freezing tolerance in host cells. Our results suggest that IRI proteins play an important role in freezing tolerance in plants.

A metabolomics study of ascorbic acid-induced in situ freezing tolerance in spinach (Spinacia oleracea L.).

Freeze-thaw stress is one of the major environmental constraints that limit plant growth and reduce productivity and quality. Plants exhibit a variety of cellular dysfunctions following freeze-thaw stress, including accumulation of reactive oxygen species (ROS). This means that enhancement of antioxidant capacity by exogenous application of antioxidants could potentially be one of the strategies for improving freezing tolerance (FT) of plants. Exogenous application of ascorbic acid (AsA), as an antioxidant, has been shown to improve plant tolerance against abiotic stresses but its effect on FT has not been investigated. We evaluated the effect of AsA-feeding on FT of spinach (Spinacia oleracea L.) at whole plant and excised-leaf level, and conducted metabolite profiling of leaves before and after AsA treatment to explore metabolic explanation for change in FT. AsA application did not impede leaf growth, instead slightly promoted it. Temperature-controlled freeze-thaw tests revealed AsA-fed plants were more freezing tolerant as indicated by: (a) less visual damage/mortality; (b) lower ion leakage; and (c) less oxidative injury, lower abundance of free radicals ( O 2 · - and H2O2). Comparative leaf metabolite profiling revealed clear separation of metabolic phenotypes for control versus AsA-fed leaves. Specifically, AsA-fed leaves had greater abundance of antioxidants (AsA, glutathione, alpha- & gamma-tocopherol) and compatible solutes (proline, galactinol, and myo-inositol). AsA-fed leaves also had higher activity of antioxidant enzymes (superoxide dismutase, ascorbate peroxidase, and catalase). These changes, together, may improve FT via alleviating freeze-induced oxidative stress as well as protecting membranes from freeze desiccation. Additionally, improved FT by AsA-feeding may potentially include enhanced cell wall/lignin augmentation and bolstered secondary metabolism as indicated by diminished level of phenylalanine and increased abundance of branched amino acids, respectively.

Identification and Characterization of Five Cold Stress-Related Rhododendron Dehydrin Genes: Spotlight on a FSK-Type Dehydrin With Multiple F-Segments.

Dehydrins are a family of plant proteins that accumulate in response to dehydration stresses, such as low temperature, drought, high salinity, or during seed maturation. We have previously constructed cDNA libraries from Rhododendron catawbiense leaves of naturally non-acclimated (NA; leaf LT50, temperature that results in 50% injury of maximum, approximately -7°C) and cold-acclimated (CA; leaf LT50 approximately -50°C) plants and analyzed expressed sequence tags (ESTs). Five ESTs were identified as dehydrin genes. Their full-length cDNA sequences were obtained and designated as RcDhn 1-5. To explore their functionality vis-à-vis winter hardiness, their seasonal expression kinetics was studied at two levels. Firstly, in leaves of R. catawbiense collected from the NA, CA, and de-acclimated (DA) plants corresponding to summer, winter and spring, respectively. Secondly, in leaves collected monthly from August through February, which progressively increased freezing tolerance from summer through mid-winter. The expression pattern data indicated that RcDhn 1-5 had 6- to 15-fold up-regulation during the cold acclimation process, followed by substantial down-regulation during deacclimation (even back to NA levels for some). Interestingly, our data shows RcDhn 5 contains a histidine-rich motif near N-terminus, a characteristic of metal-binding dehydrins. Equally important, RcDhn 2 contains a consensus 18 amino acid sequence (i.e., ETKDRGLFDFLGKKEEEE) near the N-terminus, with two additional copies upstream, and it is the most acidic (pI of 4.8) among the five RcDhns found. The core of this consensus 18 amino acid sequence is a 11-residue amino acid sequence (DRGLFDFLGKK), recently designated in the literature as the F-segment (based on the pair of hydrophobic F residues it contains). Furthermore, the 208 orthologs of F-segment-containing RcDhn 2 were identified across a broad range of species in GenBank database. This study expands our knowledge about the types of F-segment from the literature-reported single F-segment dehydrins (FSKn) to two or three F-segment dehydrins: Camelina sativa dehydrin ERD14 as F2S2Kn type; and RcDhn 2 as F3SKn type identified here. Our results also indicate some consensus amino acid sequences flanking the core F-segment in dehydrins. Implications for these cold-responsive RcDhn genes in future genetic engineering efforts to improve plant cold hardiness are discussed.

Rhododendron catawbiense plasma membrane intrinsic proteins are aquaporins, and their over-expression compromises constitutive freezing tolerance and cold acclimation ability of transgenic Arabidopsis plants.

Extracellular freezing results in cellular dehydration caused by water efflux, which is likely regulated by aquaporins (AQPs). In a seasonal cold acclimation (CA) study of Rhododendron catawbiense, two AQP cDNAs, RcPIP2;1 and RcPIP2;2, were down-regulated as the leaf freezing tolerance (FT) increased from -7 to approximately -50 degrees C. We hypothesized this down-regulation to be an adaptive component of CA process allowing cells to resist freeze-induced dehydration. Here, we characterize full-length cDNAs of the two Rhododendron PIPs, and demonstrate that RcPIP2s have water channel activity. Moreover, RcPIP2s were over-expressed in Arabidopsis, and FT of transgenic plants was compared with that of wild-type (WT) controls. Data indicated a significantly lower constitutive FT and CA ability of RcPIP2-OXP plants (compared with WT) due, presumably, to their lower ability to resist freeze desiccation. A relatively higher dehydration rate of RcPIP2-OXP leaves (than WT) supports this notion. Phenotypic and microscopic observations revealed bigger leaf size and mesophyll cells of RcPIP2-OXP plants than WT. It is proposed that lower FT of transgenic plants may be associated with their leaf cells' propensity to greater mechanical stress, that is, volume strain per unit surface, during freeze-thaw-induced contraction or expansion. Additionally, greater freeze injury in RcPIP2-OXP plants could also be attributed to their susceptibility to potentially faster rehydration (than WT) during a thaw.

Functional dissection of hydrophilins during in vitro freeze protection.

In plants, Late Embryogenesis Abundant (LEA) proteins typically accumulate in response to low water availability conditions imposed during development or by the environment. Analogous proteins in other organisms are induced when exposed to stress conditions. Most of this diverse set of proteins can be grouped according to properties such as high hydrophilicity and high content of glycine or other small amino acids in what we have termed hydrophilins. Previously, we showed that hydrophilins protect enzyme activities in vitro from low water availability effects. Here, we demonstrate that hydrophilins can also protect enzyme activities from the adverse effects induced by freeze-thaw cycles in vitro. We monitored conformational changes induced by freeze-thaw on the enzyme lactate dehydrogenase (LDH) using the fluorophore 1-anilinonaphthalene-8-sulfonate (ANS). Hydrophilin addition prevents enzyme inactivation and this effect is reflected in changes in the ANS-fluorescence levels determined for LDH. We further show that for selected plant hydrophilins, removal of certain conserved domains affects their protecting capabilities. Thus, we propose that hydrophilins, and in particular specific protein domains, have a role in protecting cell components from the adverse effects caused by low water availability such as those present during freezing conditions by preventing deleterious changes in protein secondary and tertiary structure.

Phylogenetic analysis and seasonal cold acclimation-associated expression of early light-induced protein genes of Rhododendron catawbiense.

The early light-induced proteins (ELIPs) are nuclear-encoded, light stress-induced proteins located in thylakoid membranes and related to light-harvesting Chl a/b-binding proteins. Recent evidence from physiological and genetic (mutant) studies supports a photoprotective function for ELIPs, particularly when green tissues are exposed to high light intensities at suboptimal temperatures. Broad-leaved evergreens belonging to genus Rhododendron are often exposed to a combination of low temperatures and high light in their natural habitat as the understory plants in deciduous forests and, therefore, are expected to employ photoprotective strategies during overwintering phase. Here we report analysis and characterization of previously identified ELIP expressed sequence tags (ESTs) from winter-collected Rhododendron catawbiense leaves. 5' or 3' rapid amplification of complementary DNA ends (RACEs) coupled with bioinformatic analyses were used to identify seven unique ELIPs from the 40 ESTs and were designated as RcELIP1-RcELIP7. Phylogenetic analysis revealed separate clustering of ELIP homologs from lower plants, monocots and eudicots (including RcELIPs) and further indicated an evolutionary divergence of ELIPs among angiosperms and gymnosperms. To gain insights into the cold acclimation (CA) physiology of rhododendrons, relative and absolute quantitative expression of RcELIPs was examined during seasonal CA of R. catawbiense leaves using real time reverse transcriptase-polymerase chain reaction. All seven RcELIPs were distinctly upregulated during the CA. It is postulated that RcELIPs expression constitutes an adaptive response to cold and high light in winter-adapted rhododendron leaves and perhaps plays a key role in the protection of photosynthetic apparatus from these stresses.

Changes in carbohydrates, ABA and bark proteins during seasonal cold acclimation and deacclimation in Hydrangea species differing in cold hardiness.

Cold injury is frequently seen in the commercially important shrub Hydrangea macrophylla but not in Hydrangea paniculata. Cold acclimation and deacclimation and associated physiological adaptations were investigated from late September 2006 to early May 2007 in stems of field-grown H. macrophylla ssp. macrophylla (Thunb.) Ser. cv. Blaumeise and H. paniculata Sieb. cv. Kyushu. Acclimation and deacclimation appeared approximately synchronized in the two species, but they differed significantly in levels of mid-winter cold hardiness, rates of acclimation and deacclimation and physiological traits conferring tolerance to freezing conditions. Accumulation patterns of sucrose and raffinose in stems paralleled fluctuations in cold hardiness in both species, but H. macrophylla additionally accumulated glucose and fructose during winter, indicating species-specific differences in carbohydrate metabolism. Protein profiles differed between H. macrophylla and H. paniculata, but distinct seasonal patterns associated with winter acclimation were observed in both species. In H. paniculata concurrent increases in xylem sap abscisic acid (ABA) concentrations ([ABA](xylem)) and freezing tolerance suggests an involvement of ABA in cold acclimation. In contrast, ABA from the root system was seemingly not involved in cold acclimation in H. macrophylla, suggesting that species-specific differences in cold hardiness may be related to differences in [ABA](xylem). In both species a significant increase in stem freezing tolerance appeared long after growth ceased, suggesting that cold acclimation is more regulated by temperature than by photoperiod.