Freeze avoidance: a dehydrating moss gathers no ice

Using cryo‐SEM with EDX fundamental structural and mechanical properties of the moss Ceratodon purpureus (Hedw.) Brid. were studied in relation to tolerance of freezing temperatures. In contrast to more complex plants, no ice accumulated within the moss during the freezing event. External ice induced desiccation with the response being a function of cell type; water‐filled hydroid cells cavitated and were embolized at ?4 °C while parenchyma cells of the inner cortex exhibited cytorrhysis, decreasing to ～20% of their original volume at a nadir temperature of ?20 °C. Chlorophyll fluorescence showed that these winter acclimated mosses displayed no evidence of damage after thawing from ?20 °C while GCMS showed that sugar concentrations were not sufficient to confer this level of freezing tolerance. In addition, differential scanning calorimetry showed internal ice nucleation occurred in hydrated moss at ～?12 °C while desiccated moss showed no evidence of freezing with lowering of nadir temperature to ?20 °C. Therefore the rapid dehydration of the moss provides an elegantly simple solution to the problem of freezing; remove that which freezes.     

Genetic variation in photosynthetic performance and tolerance to osmotic stress (desiccation,freezing, hyposalinity) in the rocky littoral foundation species Fucus vesiculosus (Fucales,Phaeophyceae)

Genetic diversity may play an analogous role to species diversity, as it can contribute to ecosystem function and stability, and provision of ecosystem services. In the Baltic Sea, perennial algal beds are often comprised of only Fucus vesiculosus and the amount of genetic variation in fitness‐related traits (i.e., the ability of the alga to photosynthesize or withstand stress) will thus determine the alga's local persistence in a changing environment. To study genetic variation in the crucial traits behind persistence we grew replicate vegetative branches that came from the same genotype in common gardens. We quantified osmotic stress tolerance and recovery responses by exposing branches to desiccation, freezing, and hyposalinity regimens. Our results show that genetic variation among genotypes was apparent for some photosynthetic parameters (maximal electron transport rate, saturation irradiance for electron transport, nonphotochemical quenching) and growth. Algae tolerated freezing (1,440 min at ?2.5°C) and hyposalinity (1,560 min at 2.5) well, but did not recover from desiccation (70 min at 12°C, causing ~94% water loss). Furthermore, we found very little if any evidence on genetic variation in tolerance to these stressors. Our results suggest that low salinity and cold winters in the northern marginal populations selected for hyposalinity and freezing tolerant genotypes, possibly eroding genetic variation in tolerance, but that tolerance to harsh desiccation has been lost, likely due to relaxed selection. The overall availability of genetic variation in fitness related traits might be supportive for F. vesiculosus during adaptation to gradual changes of its environment.     

PROTEINS IMMUNOLOGICALLY RELATED TO DEHYDRINS IN FUCOID ALGAE

Adult fucoid algae on Atlantic shores have well-characterized, species-specific tolerances to the varying levels of desiccation that occur from the low to high intertidal zones; however, less is known about embryonic tolerances and their mechanistic basis. We investigated this by 1) exposing embryos of Fucus evanescens C. Agardh, F. spiralis L., and F. vesiculosus L. from the Maine shore to osmotic desiccation in hypersaline seawater and 2) examining whether these embryos contain species-specific dehydrins, proteins first identified in higher plants that are hypothesized to confer tolerance to dehydration. Embryonic survival when cultured in hypersaline seawater >100 practical salinity units (psu) correlated with the position of these species in the intertidal zone (F. spiralis > F. vesiculosus > F. evanescens), but all 1-day-old embryos of these species tolerated treatment with 100 psu or lower seawater. Proteins (17–105 kDa) immunologically related to dehydrins were detected on western blots with dehydrin antibodies raised against a synthetic peptide representing the conserved motif of dehydrins in higher plants. These proteins were constitutive and unstable when subjected to prolonged (>15 min) temperatures above 55° C, unlike most higher plant dehydrins, which are inducible and remain soluble at 75°–100° C. The presence of these proteins was species- and stage-specific. Sperm of F. vesiculosus had a characteristic protein of 76 kDa, whereas eggs and embryos (6 h to 3 days old) had a 92-kDa protein. By 1 week of age, expression of the 92-kDa protein decreased, and the 35-kDa protein of adults was present. Embryos of A. nodosum L. and Pelvetia compressa J. Agardh DeToni contained an 85-kDa protein rather than the 92-kDa protein of Fucus embryos (F. distichus L., F. evanescens, F. spiralis, and F. vesiculosus). The 92-kDa protein became more abundant in embryos exposed to hyperosmotic seawater at 50 psu (F. evanescens and F. vesiculosus) or 150 psu (F. spiralis); however, dehydrin-like proteins of some molecular masses decreased in abundance simultaneously. Further characterization of these proteins is required to establish whether they protect embryos against intertidal desiccation.     

Cold,freezing, and desiccation tolerance of the limpet Acmaea digitalis (Eschscholtz)

Cold, freezing, and desiccation tolerance were examined in the limpet, Acmaea digitalis (Eschscholtz). Fifty percent of the experimental population survived freezing at temperatures between ?10 ° and ?12 °C for at least 24 hr. In this temperature range, 60–80 % of the body water was frozen. The LD₅₀ for water loss by desiccation was 76.5% ± 1.3_δ. At 70–80% body water loss, solutes were concentrated between 350 and 500%. Some limpets failed to survive immersion in 450% seawater for 6 hr (100% scawater = salinity of 31‰), suggesting that much of the damage from freezing and desiccation was from an increased solute concentration. No glycerol could be detected in extracts of the foot and the visceral mass of A. digitalis during winter, confirming similar results in studies carried out on other intertidal molluscs.     

Freezing and desiccation injury resistance in the filamentous green alga Klebsormidium from the Antarctic, Arctic and Slovakia

The freezing and desiccation tolerance of 12 Klebsormidium strains, isolated from various habitats (aeroterrestrial, terrestrial, and hydro-terrestrial) from distinct geographical regions (Antarctic — South Shetlands, King George Island, Arctic — Ellesmere Island, Svalbard, Central Europe — Slovakia) were studied. Each strain was exposed to several freezing (−4°C, −40°C, −196°C) and desiccation (+4°C and + 20°C) regimes, simulating both natural and semi-natural freeze-thaw and desiccation cycles. The level of resistance (or the survival capacity) was evaluated by chlorophyll a content, viability, and chlorophyll fluorescence evaluations. No statistical differences (Kruskal-Wallis tests) between strains originating from different regions were observed. All strains tested were highly resistant to both freezing and desiccation injuries. Freezing down to −196°C was the most harmful regime for all studied strains. Freezing at −4°C did not influence the survival of studied strains. Further, freezing down to −40°C (at a speed of 4°C/min) was not fatal for most of the strains. RDA analysis showed that certain Antarctic and Arctic strains did not survive desiccation at +4°C; however, freezing at −40°C, as well as desiccation at +20°C was not fatal to them. On the other hand, other strains from the Antarctic, the Arctic, and Central Europe (Slovakia) survived desiccation at temperatures of +4°C, and freezing down to −40°C. It appears that species of Klebsormidium which occupy an environment where both seasonal and diurnal variations of water availability prevail, are well adapted to freezing and desiccation injuries. Freezing and desiccation tolerance is not species-specific nor is the resilience only found in polar strains as it is also a feature of temperate strains. Presented at the International Symposium Biology and Taxonomy of Green Algae V, Smolenice, June 26–29, 2007, Slovakia. This paper is dedicated to the memory of the late Dr. Bohuslav Fott (1908–1976), Professor of Botany at the Charles University in Prague, to mark the centenary of his birth.     

Dehydration of Onion Cells: A Comparison of Freezing vs. Desiccation and Living vs. Dead Cells

Measurements were made of the amount of liquid water present in the epidermal cells of onion at various degrees of dehydration caused by slow extracellular freezing and by desiccation. This was achieved by using a pulsed NMR spectrometer during freezing stress and by weighing the epidermal pieces during desiccation. Measurements were made on the extent of cell survival by direct microscopic observation (plasmolysis and protoplasmic streaming). Onion epidermal cells (Allium cepa L. cv. Downing Yellow Globe) were found to survive freezing temperatures as low as –20°C and an equivalent desiccation stress. This equivalence opposes the reports by others on Hordeum vulgare and on Solanum sp. of greater injury by freezing than by an equivalent dehydration due to desiccation. The discrepancy -has been explained in terms of the limitations of the conductivity method used by those authors to evaluate the injury. The freezing and desiccation curves correspond to the equation: L _t=L₀Δt_m/t+L_u where L_t and L₀ are the amounts of liquid water at temperature t and O°C respectively. Δt_m is the freezing point depression of the cell sap and L_u is the amount of liquid water which does not freeze. These results demonstrate that the dehydration of onion cells during both freezing and desiccation duplicates the dehydration of ordinary aqueous solutions. This was equally true for living and dead cells, and suggests that the negative turgor invoked by others is not significantly involved in the dehydration of living Allium cepa epidermis cells. An explanation is proposed for these contradictory results.     

Functional properties of photosynthetic apparatus in three <Emphasis Type="Italic">Fucus</Emphasis> species inhabiting the White Sea: Effect of dehydration

Three species of Fucus inhabiting different zones at the littoral and sublittoral of the White Sea (F. vesiculosus L., F. serratus L., and F. distichus L.) were compared with regard to performance of the photosynthetic apparatus (PSA) and PSA changes upon prolonged desiccation of algae. The content of chlorophyll a and the total content of carotenoids were significantly higher in F. serratus than in F. vesiculosus. The potential quantum yield (F _v/F _m) of photosystem II (PSII) was 0.69–0.74 in all species. The highest effective quantum yield of PSII (Y _II) was observed in F. vesiculosus plants inhabiting zones with the most intense insolation. The ratio of non-photochemical quenching to photochemical quenching (qN/qP) increased with the depth of algal habitats in the row from F. vesiculosus to F. serratus. The fluorescence parameters F. serratus deviated during desiccation from their normal values and did not recover upon the subsequent return to water. By contrast, the fluorescence parameters of F. vesiculosus and F. distichus recovered gradually after the return of algae from air to sea water. It is supposed that F. serratus plants, unlike other species, are characterized by comparatively low physiological plasticity.     

Thermal ecotypes of amphi-Atlantic algae. I. Algae of Arctic to cold-temperate distribution (Chaetomorpha melagonium,Devaleraea ramentacea andPhycodrys rubens)

Three species of Arctic to cold-temperate amphi-Atlantic algae, all occurring also in the North Pacific, were tested for growth and/or survival at temperatures of −20 to 30°C. When isolates from both western and eastern Atlantic shores were tested side-by-side, it was found that thermal ecotypes may occur in such Arctic algae.Chaetomorpha melagonium was the most eurythermal of the 3 species. Isolates of this alga were alike in temperature tolerance and growth rate but Icelandic plants were more sensitive to the lethal temperature of 25°C than were more southerly isolates from both east and west. With regard toDevaleraea ramentacea, one Canadian isolate grew extraordinarily well at −2 and 0°C, and all tolerated temperatures 2–3°C higher than the lethal limit (18–20°C) of isolates from Europe. ConcerningPhycodrys rubens, both eastern and western isolates died at 20°C but European plants tolerated the lethal high temperature longer, were more sensitive to freezing, and attained more rapid growth at optimal temperatures. The intertidal species,C. melagonium andD. ramentacea, both survived freezing at −5 and −20°C, at least for short time periods.C. melagonium was more susceptible thanD. ramentacea to desiccation. Patterns of thermal tolerance may provide insight into the evolutionary history of seaweed species.     

FREEZING STRESS AND OSMOTIC DEHYDRATION IN FUCUS DISTICHUS (PHAEOPHYTA): EVIDENCE FOR PHYSIOLOGICAL SIMILARITY1

The effects of osmotic dehydration and freezing on photosynthesis were studied in the brown alga Fucus distichus L. The data indicated that F. distichus exhibits similar physiological responses to both osmotic dehydration and freezing stress and that these responses resemble those in the literature for the effect of desiccation in air. Both stresses inhibited light-limited (P_subsat) and light-saturated (P_max) photosynthesis measured immediately after plants were reimmersed in seawater. The degree of initial inhibition and subsequent recovery of photosynthesis were proportional to the severity of the dehydration or freezing treatment. P_subsat and P_max recovered completely from osmotic dehydration for 3 h in 200% and 3 hr at – 10°C, but recovery was only partial following 3 h in 300%o or 3 h at – 15°C. In most cases, recovery was complete within 2 h following dehydration, with little further recovery occurring between 2 and 24 h posttreatment. No time-dependent recovery occurred following severe freezing. Observations using the vital stain fluorescein diacetate suggested that the lack of complete recovery might be due to severe damage or death of a proportion of cells in the thallus. There were no clear effects of either osmotic dehydration or freezing on dark respiration (R_d), although R_d was stimulated in all emersed treatments (frozen plants and 5° C controls) immediately following reimmersion. Measurement of chlorophyll fluorescence induction kinetics indicated that both osmotic dehydration and freezing reduced the ratio of variable to maximum florescence (F_v/F_m), indicating a decrease in the quantum efficiency of photosystem I. Based on these data, we suggest that there are common cellular and physiological components involved in the response of fucoid algae to a range of water stresses. This hypothesis was supported by experiments that showed that osmoacclimation in hyperosmotic seawater (51%o)for 2 weeks increased the ability of F. distichus to recover from freezing at – 15° C. During acclimation, mannitol content increased under hyperosmotic conditions and decreased under hypoosmotic conditions. Changes in plasma membrane integrity, determined by fresh weight: dry weight ratio, and amino acid release following freezing indicated an increasing gradient of freezing tolerance from low to high salinity. However, none of these physiological changes fully explained the marked increase in the freezing tolerance of photosynthesis observed in plants acclimated under hyperosmotic conditions.     

Comparative experiments on temperature responses of bryophytes: assimilation,respiration and freezing damage

Abstract

(1) Temperature-net assimilation and temperature-respiration curves based on manometric measurements at high carbon dioxide concentrations are presented for twenty-three mosses and five hepatics.

(2) In most of the species, the optimum temperature for net assimilation under the experimental conditions was about 25°–30°C and the temperature compensation point about 35°–40°C.

(3) Substantially lower optima and maxima were found in Orthothecium rufescens, Plagiopus oederi, Acrocladium trifarium, Fontinalis squamosa, Nardia compressa and Hookeria lucens.

(4) Several northern and montane species (e.g. Anthelia julacea, Andreaea nivalis, Rhacomitrium lanuginosum) did not differ substantially from the majority of lowland species in the response of net assimilation to temperature. Some substantial differences were found between species of differing habitats.

(5) Most of the mosses and leafy liverworts tested withstood rapid cooling to ?5°C for 6 hr. They are evidently protected from intracellular freezing at normal rates of cooling by the withdrawal of water to form extracellular ice.

(6) Conocephalum conicum, Targionia hypophylla and Pellia epiphylla were killed by rapid cooling to ?5°C.

(7) Plagiochila spinulosa and Myurium hebridarumwithstood periods of 1–2 weeks at ?5°C. Survival of bryophytes for long periods of low temperatures appears to be principally a matter of desiccation resistance.     

Efficiency of cold hardiness induction by desiccation stress in four winter cereals

A number of defined desiccation treatments without low temperature exposure were able to induce freezing tolerance in 20 cultivars of winter cereals. A maximal degree of freezing tolerance was induced in epicotyls at 24°C in 24 hours at 40% relative humidity in rye and wheat, 7 days at 54% RH in barley, and 4 days at 70% RH in oats. Freezing tolerance was not correlated to water content of the plants after desiccation treatment but was related to the genetic capacity of the cultivars to frost harden. Levels of freezing tolerance induced by desiccation were similar to those induced by cold acclimation in rye and wheat, but considerably less in barley and oats. This is associated with a more rapid desiccation injury in barley and oats, precluding the completion of the hardening process.     

FREQUENT CLONALITY IN FUCOIDS (FUCUS RADICANS AND FUCUS VESICULOSUS; FUCALES,PHAEOPHYCEAE) IN THE BALTIC SEA1

Asexual reproduction by cloning may affect the genetic structure of populations, their potential to evolve, and, among foundation species, contributions to ecosystem functions. Macroalgae of the genus Fucus are known to produce attached plants only by sexual recruitment. Recently, however, clones of attached plants recruited by asexual reproduction were observed in a few populations of Fucus radicans Bergström et L. Kautsky and F. vesiculosus L. inside the Baltic Sea. Herein we assess the distribution and prevalence of clonality in Baltic fucoids using nine polymorphic microsatellite loci and samples of F. radicans and F. vesiculosus from 13 Baltic sites. Clonality was more common in F. radicans than in F. vesiculosus, and in both species it tended to be most common in northern Baltic sites, although variation among close populations was sometimes extensive. Individual clonal lineages were mostly restricted to single or nearby locations, but one clonal lineage of F. radicans dominated five of 10 populations and was widely distributed over 550 × 100 km of coast. Populations dominated by a few clonal lineages were common in F. radicans, and these were less genetically variable than in other populations. As thalli recruited by cloning produced gametes, a possible explanation for this reduced genetic variation is that dominance of one or a few clonal lineages biases the gamete pool resulting in a decreased effective population size and thereby loss of genetic variation by genetic drift. Baltic fucoids are important habitat‐forming species, and genetic structure and presence of clonality have implications for conservation strategies.     

GENETIC AND MORPHOLOGICAL IDENTIFICATION OF FUCUS RADICANS SP. NOV. (FUCALES,PHAEOPHYCEAE) IN THE BRACKISH BALTIC SEA1

Brown seaweeds of the genus Fucus occupy a wide variety of temperate coastal habitats. The genus is evolutionary dynamic with recent radiations to form morphologically distinct taxa. In the brackish Baltic Sea, fucoids are the only perennial canopy‐forming macroalgae. The most northern populations of Fucus occur permanently submerged in extremely low salinity (3–5 psu). These are currently referred to as Fucus vesiculosus L. but are morphologically distinct with a narrow frond without bladders. We report here that a population of this unique morphotype is reproductively isolated from a truly sympatric population of common F. vesiculosus and conclude that the northern morphotype represents a previously undescribed species. We describe Fucus radicans sp. nov., which is attached and dioecious with broadly elliptic receptacles, characterized by a richly branched narrow flat frond (2–5 mm), short thallus (<26 cm), and a high capacity for vegetative recruitment of attached plants. Analysis of five highly polymorphic microsatellite DNA loci showed genetic differentiation between sympatric populations of F. radicans and F. vesiculosus, whereas allopatric populations of the same species revealed a coherent pattern of genetic variation. Sequences of the RUBISCO region in F. radicans were identical to or differing at only one to two dinucleotide positions from those of F. vesiculosus, indicating a recent common origin of the two species.     

Freezing and high temperature thresholds of photosystem 2 compared to ice nucleation, frost and heat damage in evergreen subalpine plants

Freezing and high temperature thresholds of photosystem 2 (PS2), ice formation and frost and heat damage were measured in leaves of evergreen subalpine plants under conditions of naturally low (winter) to high (summer) PS2 efficiencies (F_V/F_M). The temperature‐dependent change in basic Chl fluorescence (F₀) (T‐F₀) technique that is usually used to assess the high temperature threshold of PS2 in a new approach was applied to test freezing temperature thresholds of PS2. T‐F₀ curves (+5 °C to ?10 °C at 2 K h^?1) revealed a significant, sudden increase in F₀ on extracellular ice formation (?4.0 or ?5.5 °C). The rise in F₀ was recorded 0.3–0.6 K below ice nucleation (10–20 min later) and was produced by freeze dehydration of cells. The rise in F₀ was not caused by frost damage, as during winter LT₅₀ was lower than ?27 °C and not by formation of ice on the leaf surface. Hence, F₀ measurements during freezing are a useful tool to distinguish between surface ice and extracellular ice inside the leaf tissue which cannot be differentiated by other ice‐detecting methods. PS2 efficiency significantly affected the shape of the high temperature T‐F₀ curves (20–65 °C at 1 K min^?1). Under F_V/F_M >0.6, two F₀ maxima were recorded. The fast rise phase to the first F₀ maximum corresponded with tissue heat damage (LT₅₀: 46.9–54.3 °C). The second F₀ maximum occurred at leaf temperatures between 55 and 60 °C. Under F_V/F_M <0.2 only, the second F₀ maximum was detectable. Lack of awareness of the missing F₀ maximum would lead to an overestimation of the PS2 high temperature threshold by >10 K; hence, under low F_V/F_M, it cannot be determined by the T‐F₀ technique.     

Winter cold tolerance and the geographic range separation of Bromus tectorum and Bromus rubens,two severe invasive species in North America

The invasive grasses Bromus rubens and Bromus tectorum are responsible for widespread damage to semiarid biomes of western North America. Bromus. tectorum dominates higher and more northern landscapes than its sister species B. rubens, which is a severe invader in the Mojave desert region of the American Southwest. To assess climate thresholds controlling their distinct geographic ranges, we evaluated the winter cold tolerance of B. tectorum and B. rubens. Freezing tolerance thresholds were determined using electrolyte leakage and whole‐plant mortality. The responses of the two species to winter cold and artificial freezing treatments were similar in 2007–2008 and 2009–2010. When grown at minimum temperatures of 10 °C, plants of both species had cold tolerance thresholds near ?10 °C, while plants acclimated to a daily minimum of ?10 to ?30 °C survived temperatures down to ?31 °C. In the winter of 2010–2011, a sudden severe cold event on December 9, 2010 killed all B. rubens populations, while B. tectorum was not harmed; all tested plants were 7–8 weeks old. Controlled acclimation experiments demonstrated that 8‐week‐old plants of B. rubens had a slower acclimation rate to subzero temperatures than B. tectorum and could not survive a rapid temperature drop from 1 to ?14 °C. Four‐month‐old B. rubens populations were as cold tolerant as B. tectorum. Our results show that severe and sudden freeze events in late autumn can kill young plants of B. rubens but not B. tectorum. Such events could exclude B. rubens from the relatively cold, Intermountain steppe biome of western North America where B. tectorum predominates.     

PHOTOSYNTHESIS OF THE RED ALGA GASTROCLONIUM COULTERI (RHODOPHYTA) IN RESPONSE TO CHANGES IN TEMPERATURE,LIGHT INTENSITY,AND DESICCATION1

Previously reported transplantation experiments in the field showed that Gastroclonium coulteri (Harvey) Kylin could survive above its normal intertidal range (0.0–0.5 m above MLLW), except during periods of daytime low tides in spring. Net photosynthetic rate measurements in the laboratory were performed to determine which physical factors might determine the upper boundary for this species in the intertidal zone. Maximum net photosynthesis occurred between 15 and 20° C, but remained positive between 4 and 35° C. The air temperature extremes observed in the field were 2° C (only seen once) and 26° C. Net photosynthesis increased as expected with light intensity to the highest value obtainable in the laboratory, 1400 μEin m^?2 s^?1. Plants collected from the field under higher light intensity (up to 2000 μEin m^?2 s^?2) also showed high rates of photosynthesis. Neither the temperature nor light levels observed in the field were directly damaging to photosynthesis. Desiccation, however, resulted in a sharp decrease in both photosynthesis and respiration. G. coulteri fully recovered from successive daily treatments of about 35% desiccation, but not from successive treatments of 50% desiccation. One exposure to 70% desiccation allowed no recovery of photosynthetic capacity.     

Conservation of the germination capacity of pollen grains in three varieties of maize (Zea mays L.)

Maize pollen longevity is short under natural conditions, and the seed yield is hampered by poor synchronisation between pollen shed and ovule maturity. Hybridization during stress conditions could be improved if pollen is collected and stored for use at appropriate times. This study was initiated to determine the optimum conditions for in vitro maize pollen germination. The method of pollen conservation and its viability over time was also part of the study. The pollen from three Zea mays cultivars (Exp₁ 24 and 5057, two inbred lines, and Tuxpeño sequía which is an open pollinated cultivar) were used in this study. The conservation was evaluated using three different methods: room temperature, the refrigerator (+10°C), and deep‐freezing at ?20°C. Two base culture media (Brewbaker &; Kwack, Heslop‐Harrison) were used for in vitro pollen germination. The effects of saccharose concentrations, incubation and desiccation times, were also evaluated. Results from this study showed that, Heslop‐Harrison medium was better than Brewbaker and Kwack or and with pollen germination. Pollen stored in a refrigerator (+10°C) varying desiccation times, yielded the following findings: for a short conservation time (10 days maximum), there is no need for a prior desiccation. For a medium conservation time (between 16 days and 20 days), five to six hours desiccation time are needed to obtain maximum germination for inbred line Exp₁ 24 and for the composite variety Tuxpeño sequía respectively. It was also determined that, at room temperature, maize pollen could germination is longer than 24?hours. The use of deep‐freezer at ?20°C did not yield any consistent result. In vivo germination test in a maize field will be necessary to confirm the above in vitro germination.     

Hardening, abscisic acid, proline and freezing resistance in two winter wheat varieties

Two varieties of winter wheat ( Triticum aestivum L.) differing in freezing resistance ("Holme" from Sweden, freezing resistant, and "Amandus" from Germany, less freezing resistant) were hardened for five weeks by gradually reducing the day/night temperature from 20°C/15°C during the first week to 2° C/0° C during the fifth week and the photoperiod from 15 to 9 h. This treatment increased the freezing resistance of both varieties in comparison to unhardened control plants. Hardening caused an increase in osmolarity of cell sap and in the levels of proline and abscisic acid (ABA). Increase in osmolarity preceded the increase in ABA level, and proline levels increased later than ABA levels. Holme had higher values of osmolarity as well as higher levels of ABA and proline. but the differences between the two varieties were significant only for proline. Since the pressure potential remained constant or increased slightly during the hardening period, it is suggested that the accumulation of ABA is due to the hardening process and not to simple water stress caused by cold-induced inhibition of water uptake by the root.
Spraying hardened plants with 10⁻⁴ M ABA 24 h before a freezing test increased freezing resistance in both varieties, but did not obliterate the differences in freezing resistance between the two varieties. Spraying hardened plants with an aqueous proline solution (10%, w/v) was without effect on freezing resistance. It is concluded that the hardening procedure causes an accumulation of ABA in winter wheat leaves and that ABA is involved in the chain of events leading to freezing resistance.     

Photosynthesis in cold acclimated leaves of plants with various degrees of freezing tolerance

The objective of this study was to compare the photosynthetic changes during cold acclimation in various plant types able to acquire different degrees of freezing tolerance. Four herbaceous and six woody plants were hardened under natural or artificial conditions and – after determination of their frost resistance (LT₅₀) – the net photosynthetic rate at an ambient CO₂ of 33 Pa (P_n33), the dependencies of P_n to light and to CO₂ and the room temperature chlorophyll a fluorescence were recorded under optimal conditions. Herbaceous plants acquired freezing tolerances to temperatures between ?10 and ?15°C when hardened at temperatures around 0°C. Most leaves fully developed prior to frost hardening exhibited typical symptoms of senescence after frost hardening. In non-senescing leaves P_n33 was reduced by 15 to 50% mainly due to a reduced stomatal conductance. After hardening at temperatures around ?10°C Brassica survived down to ?24°C, but P_n33 was almost abolished as a result of disturbances in the chloroplasts. After transferring the plants to 20/15°C P_n33 recovered completely within a few days. Woody plants hardened at temperatures around 0°C tolerated – 15 to ?36°C: P_n33 was reduced by 25 to 60% and hardly recovered at 20/15°C. Hardening at ?10°C induced a tolerance of ?32 to n33 was almost totally blocked, but at 20/15°C it returned to the values of the plants hardened at 0°C within a few days. In woody plants disturbances were invariably localized in the chloroplasts. Thus, conifers, and especially Pinus cembra, can survive much lower temperatures than herbaceous plants and, at the same level of freezing tolerance, exhibit appreciably less restriction in relative P_n33.