Frost resistance of winter rape leaves as related to the changes in water potential and growth capability

Differential thermal analysis indicated that the frost resistance of winter rape leaves ( Brassica napus L. var. oleifera L. cv. Gòrczanski), collected from plants grown in the cold (5/2°C), relies mainly on their ability to supercool to −9 to −11°C, i.e. consists in freezing avoidance. Initiation of ice formation in the cold-acclimated leaves resulted in the death of more than 50% of the cells as determined with a conductivity method. The development of freezing tolerance appeared to be an attribute of the second stage of plant hardening and was induced by the exposure of plants to a slightly subzero temperature (−5°C) for 18 h. Such a treatment brought about a sudden and persistent water potential decrease in the leaves, despite the fact that they had reabsorbed water from the medium prior to water potential measurements. Water potential changes were associated with a higher growth capability of the leaves as checked by determinations of disk area increments. It is suggested that the increased frost tolerance of the cold-grown winter rape leaves, subjected to subfreezing temperature, is related to the decreased water potential of the tissue caused by changes in turgor and/or in osmotic pressures of the cells.     

Membrane lipids in Ceratodon purpureus protonemata grown at high and low temperatures

Ceratodon purpureus (Hedw.) Brid. was grown at two temperatures, 20 and 4°C. The protonemata grown at 4°C fixed more CO₂ at low temperatures; but their frost tolerance, tested as the recovery of photosynthesis after frost treatment, was not better than in the protonemata grown at 20°C. The effects of the growth temperature were studied on the membrane lipids of intact protonemata and on the lipid and protein contents of isolated thylakoid membranes. A large proportion, 70 to 90%, of the thylakoid membrane lipids was lost unless precautions were taken to inhibit the lipid-degrading enzyme activities. The lipid content of the thylakoid membranes of protonemata grown at 20 and 4°C was 3.9 and 4.8 mol (mol chlorophyll)⁻¹, respectively. Only minor differences were found in the lipid class composition. Monogalactosyldi-acylglycerol constituted more than 50 mol-% of the thylakoid membrane lipids at both 20 and 4°C. However, each lipid class had a higher average number of double bonds per lipid molecule in cold growth conditions. The protein content of the thylakoid membranes was low at both 20 and 4°C. These characteristics of the thylakoid membranes may be a prerequisite for the observed ability of protonemata to photosynthesize even at subzero temperatures.     

Does availability of potassium affect cold hardening of Scots pine through polyamine metabolism?

The effect of different potassium availability on the polyamines and frost resistance of Scots pine seedlings ( Pinus sylvestris L.) during cold hardening was studied. Scots pine seedlings were grown applying different rates of potassium by using the relative addition rate technique followed by a 2- or 9-week hardening period with decreased light intensity, day length and temperature. After 2 weeks of treatment the seedlings were not hardened (LT₅₀, =−11°C) and showed no differences in frost resistance, although differences in the polyamine levels between the K levels were observed. After 9 weeks of hardening the seedlings at the low, medium and high K levels showed a mean frost resistance (LTs.₅₀) of −81, −63, and −47°C, respectively. A negative effect of K on the frost resistance of the needles was also found in adult trees in September. The results indicate that at the early stage of cold hardening, potassium or free polyamine levels do not affect the frost resistance of Scots pine needles. However, in hardened seedlings and adult trees potassium displays a negative and putrescine a positive correlation with frost resistance, whereas spermidine and spermine show no correlation.     

Changes in the level of MACC during cold hardening and dehardening in winter wheat

Changes in the level of 1-(malonylamino)-cyclopropane-1-carboxylic acid (MACC) were determined in 6 winter wheat cultivars during cold hardening at 4°C. The cultivars differed by one degree of frost resistance within the range of degree II to VII of the COMECON scale. The time-course of changes in MACC level showed a similar pattern in all 6 cultivars; i.e. increase till day 6, no changes for the next 10 days, and then a steady decrease till the end of the hardening period. There was little difference between the final and the initial levels. The increase of MACC level, expressed as per cent of the original level, was not directly correlated with either the degree of frost resistance of the actual percentage of survival. In some cultivars. mean errors exceeded the difference in MACC accumulation between cultivars closest on the resistance scale.
The fate of MACC during the second half of hardening and after transfer of plants to 25°C was studied in cultivars Bezostaya and San Pastore. During the second half of the hardening period the level of MACC decreased in the leaves of both cultivars, but increased significantly in the roots. Within two days of transfer of the hardened plants to 25°C, the MACC level in leaves increased again, while that in the roots decreased. This finding, together with the preliminary evidence of very low MACC metabolism, strongly suggest that MACC accumulates in roots during the hardening period and when transferred to 25°C, it moves from roots to leaves.     

Changes in parameters of the plasmalemma ATPase during cold acclimation of apple (Mains domestica) tree bark tissues

A plasmalemma fraction was isolated from homogenized apple tree (Mains domestica Borkh 'Golden Delicious') hark tissues using aqueous phase partitioning and ultra-centrifugation. Results of marker enzyme assays indicated that a membrane preparation highly enriched in plasma membranes was obtained. ATPase activity in this preparation possessed a high specificity for ATP as substrate, was inhibited by vanadate, diethylstilbcsterol and dicyclohexylcarbocHimide, and was insensitive to inhibitors of mitochondria! and tonoplasl ATPases. Specific activity of the plasma-lemma ATPase increased during cold acclimation prior to the attainment of vegetative maturity. Kinetic parameters (Km, Vln) determined from assays performed at different temperatures (10, 30°C) indicated a differential effect of cold acclimation on enzyme activity. Vm increased during cold acclimation, whereas Km increased when determined at 30°C but declined at 10°C. Acclimation treatments during April and May resulted in alteration of ATPase kinetics in the absence of any increase in bark frost hardiness. Changes in ATPase kinetics may be related more to enhanced low temperature metabolism than to frost hardiness per se.     

Frost tolerance in excised leaves of the common bugle (Ajuga reptans L.) correlates positively with the concentrations of raffinose family oligosaccharides (RFOs)

Mass increases in raffinose family oligosaccharides (RFOs, α 1,6-galactosyl extensions of sucrose) are well documented in the generative tissues of many plants upon cold acclimation, and they (i.e. mainly the two shortest RFO members, raffinose and stachyose) have been suggested as frost stress protectants. Our focus here was on the longer RFO members as they commonly occur in the frost-hardy evergreen labiate Ajuga reptans in its natural habitat, and accumulate to their highest concentrations in winter when the plant is faced with sub-zero temperatures. We examined the effects of RFO concentration and chain length on frost tolerance using excised leaves which accumulate long-chain RFOs under both cold and warm conditions, thereby uncoupling the acclimation temperature from RFO production. We demonstrated that frost tolerance in excised A. reptans leaves correlates positively with long-chain RFO accumulation under both acclimation temperatures. After 24 d post-excision in the warm, the leaves had increased their RFO concentrations (mainly long-chain RFOs) 22-fold to 78 mg g⁻¹ fresh weight, and decreased their EL₅₀ values (temperature at which 50% leakage occurred) from −10.5 to −24.5 °C, suggesting a protective role for these oligosaccharides in the natural frost tolerance of A. reptans .     

Assessment of the frost sensitivity of Trifolium species by chlorophyll fluorescence analysis

The potential of the chlorophyll fluorescence technique in screening for frost sensitivity in a range of Trifolium species from different geographical origins was assessed by measuring the decrease in variable chlorophyll fluorescence (F_var) of leaves after freezing at - 5°C for 60 min. The method was rapid and the results obtained agreed well with a visual assessment of freezing injury carried out after leaves were returned to optimal growth conditions for 72 h. Trifolium alexandrinum (Berseem clover) cv. Tabor originating from Israel was shown to be the most frost sensitive species studied and Trifolium subterraneum (subterranean clover) cv. Mt. Barker, from temperate regions of Australia, the most frost resistant. On extended periods of freezing, frost damage increased and this was associated with a further reduction in variable chlorophyll fluorescence and in quenching capacity of the thylakoid membranes. These results thus indicate that substantial thylakoid membrane dysfunction is induced at freezing temperatures. Furthermore, it was found that frost hardening of the frost sensitive species T. alexandrinum for 21 days at 5°C reduced the extent of damage sustained by the thylakoid membranes as shown by higher fluorescence quenching capacity, smaller reduction in variable fluorescence (F_var) and higher initial fluorescence (F_o) when leaves of hardened plants were frozen at -5°C and -7°C.     

Modifications of abscisic acid level in winter oilseed rape leaves during acclimation of plants to freezing temperatures

An almost twofold increase in abscisic acid (ABA) content was observed in the leaves of winter oilseed rape plants (Brassica napus L., var. oleifera L., cv. Jantar) grown in the cold (>0°C). This ABA increase took place during the first three days of cold treatment. After 6 days of plant growth in the cold, the level of ABA started to decline or remained constant, depending on the calculation basis: dry weight or disc area units, respectively. The exposure of cold-acclimated plants to night frost (–5°C for 18 h) induced a further increase (65%) in the ABA level, which begun during the first few hours after thawing. The comparison of time courses of frost resistance increments and ABA content changes showed that modifications of ABA level in the cold-treated leaves preceded those of frost resistance, whereas in the frost-pretreated tissues the ABA increase occurred later than that of frost tolerance. Possible interrelations between ABA content, frost tolerance and tissue water potential modifications in the low temperature-affected tissues are discussed.     

Drought stress-induced changes in the composition and physical state of phospholipids in wheat

Seedlings of winter wheat ( Triticum aestivum L. cv. Jubilejnaja 50) were grown under normal and dry conditions. Frost resistance (LT₅₀) of 10-day-old control seedlings was −6°C. LT₅₀ of the subsequently drought-stressed leaves shifted to −16°C. In plants of the same physiological age (28 days) but grown without stress, LT₅₀ was −12°C. Phosphatidylcholine accumulated and phosphatidylethanolamine decreased in drought-stressed leaves. Fatty acid unsaturation of these phospholipids increased with leaf age, independently of water supply. Both ageing and drought stress produced a decrease in the apparent phase separation temperature of isolated total phospholipids as determined by electron spin resonance. The possible role of dehydration-induced structural changes in the bilayer matrix in triggering adaptive alterations in membrane composition, similar to those observed during cold hardening, is discussed.     

Effects of temperature and photoperiod on frost resistance of white clover (Trifolium repens) ecotypes

Effects of photoperiod and temperature on frost resistance of seedling populations and clones of white clover (Trifolium repens L.) originating from various latitudes (58°48'–69°54'N) and altitudes (up to 1100 m above sea level) were studied in a phytotron. Low-temperature-induced cold acclimation in all populations was significantly stimulated by short photoperiod and low temperature. The highest levels of frost resistance were found in the northernmost seedling populations and in clones from northern coastal locations.     

Intracellular resistance correlates with initial stage of frost hardening in willow (Salix viminalis)

Distributed model parameters of shoots of five clones of willow ( Salix viminalis ) were examined with electrical impedance analysis at the end of the growing season and with cold acclimation. The parameters were compared with regard to frost hardiness, linoleic (18:2) and linolenic (18:3) fatty acids, unsaturation/saturation ratio of fatty acids and dry weight content. The intracellular resistance (r_i) correlated best with changes in frost hardiness. The value of r_i rose from 1–2 Ω m in non-hardened to about 12 Ω m in hardened samples. In the initial stages of frost hardening, a linear relationship was found between r_i and frost hardiness and levels of 18:2 fatty acid, and an inverse relationship between r_i and levels of 18:3 fatty acid. The unsaturation/saturation ratio of fatty acids rose fairly rapidly in the initial stage of hardening. The dry weight content increased stepwise during the experimental period, and less steadily than r_i. In addition, equivalent circuit parameters changed in the prehardening phase, and were probably connected with cell differentiation and lignification. Frost hardiness by the visual method and by extracellular resistance, determined after controlled freezing tests, correlated well in the initial stages of hardening until about − 10°C but deviated upon further hardening.     

Salt treatment induces frost hardiness in leaves and isolated thylakoids from spinach

Frost hardiness of spinach (Spinacia oleracea L.) leaves was increased by high concentrations of NaCl in the hydroponic culture medium. Freezing damage was determined by measurement of slow chlorophyll fluorescence quenching after freezing of leaves. Both the osmolality of the leaf sap and forst hardiness of the leaves were linearly correlated with the salt concentration in the hydroponic culture medium. Freezing damage occurred, irrespective of the extent of frost hardening, when dehydration of cells during extracellular ice formation decreased cellular volume to approximately 14% of the volume of unfrozen cells. The resistance of isolated, washed thylakoids against mechanical and chemical damage by freezing was investigated. Chemical damage by freezing caused by salt accumulation was measured as release of chloroplast coupling factor (CF1; EC 3.6.1.3), and mechanical damage was measured as release of the lumenal protein plastocyanin from the membranes during an in-vitro freeze-thaw cycle. Isolated thylakoids from salt-treated frost-hardy spinach and those from plants hardened under natural conditions did not exhibit improved tolerance against chemical freezing stress exerted by high salt concentrations. They were, however, more hardy than thylakoids from unhardened control leaves against mechanical damage by freezing.Abbreviation CF1 peripheral part of chloroplast coupling factor ATPase     

THE COMBINED EFFECTS OF LOW TEMPERATURE AND SO2+NO2 POLLUTION ON THE NEW SEASON'S GROWTH AND WATER RELATIONS OF PICEA SITCHENSIS

Picea sitchensis (Bong.) Carr. seedlings were exposed to SO₂, NO₂ and SO₂+ NO₂ during dormancy in controlled environments, and were taken to night temperatures of 4, 0, −5, −10 and −15 °C in a freezer. Conditions in the freezer were carefully monitored during the low–temperature treatments. In two experiments, different photoenvironments and temperature regimes were imposed prior to the cold treatments, and different effects were observed. In the first, only limited frost hardiness was achieved and night temperatures of −15 °C were lethal. Temperatures of −5 and − 10 °C led to poor survival of lateral buds, particularly in plants exposed to 45 ppb SO₂. The poor bud break in plants exposed to SO₂ and to − 5 °C resulted in a loss of the effectiveness of this temperature as a chill requirement. Pressure-volume analysis showed that the shoots of plants exposed to NO₂ had greater elasticity (lower elastic moduli, e), so that loss of turgor occurred at lower relative water contents. In contrast, a hardening period (2 weeks in night/day temperatures of 3/10 °C and 8 h days at 50 μmol m⁻² s⁻¹ PAR) gave decreased elasticity and lower solute potentials of spruce shoots. In the second experiment, exposure to 30 ppb SO₂ and SO₂+ NO₂ led to slight, but consistent, increases in frost injury to the needles of plants frozen to − 5 and − 10 °C. The results suggest that the main interaction of low temperatures and winter pollutants may be on bud survival rather than on needle damage, but that effects are subtle, only occurring with certain combinations of pollutant dose and cold treatment.     

Proteins accumulate in the apoplast of winter rye leaves during cold acclimation

During cold acclimation, winter rye ( Secale cereale L.) plants develop the ability to tolerate freezing temperatures by forming ice in intercellular spaces and xylem vessels. In this study, proteins were extracted from the apoplast of rye leaves to determine their role in controlling extracellular ice formation. Several polypeptides in the 15 to 32 kDa range accumulated in the leaf apoplast during cold acclimation at 5°C and decreased during deacclimation at 20°C. A second group of polypeptides (63, 65 and 68 kDa) appeared only when the leaves were maximally frost tolerant. Ice nucleation activity, as well as the previously reported antifreeze activity, was higher in apoplastic extracts from cold-acclimated than from nonacclimated rye leaves. These results indicate that apoplastic proteins exert a direct influence on the growth of ice. In addition, freezing injury was greater in extracted cold-acclimated leaves than in unextracted cold-acclimated leaves, which suggests that the proteins present in the apoplast are an important component of the mechanism by which winter rye leaves tolerate ice formation     

Intergenerational acclimation in aphid overwintering

Abstract.  1. When first instar nymphs and adults of the grain aphid Sitobion avenae (Fabricius) (Hemiptera: Aphidiae) were maintained in long-term cultures (>6 months) at 20 °C and 10 °C, the LT₅₀ decreased from −8 and −8.8 °C to −16.0 and −13.5 °C, respectively.
2. When aphids from the 20 °C culture were transferred to 10 °C, there was a progressive increase in cold tolerance through three successive generations. Transfer of newly moulted pre-reproductive adults reared at 10 °C for three generations back to 20 °C resulted in a rapid loss of cold hardiness in their nymphal offspring.
3. In all generations reared at 10 °C, first born nymphs were more cold hardy than those born later in the birth sequence. The LT₅₀ of nymphs produced on the first day of reproduction in the first, second and third generations maintained at 10 °C were −14.8, −17.0 and −16.6 °C, respectively. Thereafter, nymphal cold hardiness decreased over the subsequent 14 days of reproduction in each generation at 10 °C with mean LT₅₀ values of −10.3, −12.6 and −14.8 °C, respectively. By contrast, the cold tolerance of first born nymphs of aphids reared continuously at 20 °C did not differ in comparison with later born siblings. The LT₅₀ of adult aphids was also unaffected by ageing.
4. The ecological relevance of these findings is discussed in relation to the overwintering survival of aphids such as S. avenae .     

The after-effects of temperature and irradiance during early growth of winter oilseed rape (Brassica napus L. var. oleifera, cv. Górczański) seedlings on the progress of their cold acclimation

Experiments performed under controlled conditions showed that level of PPFD (photosynthetic photon flux density) during early seedlings growth (preceding cold acclimation at +2 °C) was not the key factor for the development of frost resistance. It did not modify the beneficial effects of prehardening (Rapacz 1997, in this issue) at moderately low (+12 °C) day temperature. Now I have shown that the increase of PPFD may replace to some extent prehardening in the development of frost resistance. It was particularly seen in non-prehardened plants, which had been grown under warm-day (+20 °C) conditions. Prehardening performed under controlled conditions, as well as seedlings growth under natural autumn conditions in the field, allowed to maintain a high net-photosynthesis rate at chilling temperatures. A net-photosynthesis rate during cold acclimation at +2 °C corresponded well with higher frost resistance. As a result, seedlings non subjected to prehardening and grown before cold acclimation under low PPFD acclimated better, if the cold treatment was applied only at nights (+20/2 °C day/night). Only under such conditions the photosynthetic rate was sufficiently high to allow plants to reach a higher level of frost resistance. All other plants acclimated better when they were exposed to the hardening temperature continuously during days and nights (+2/2 °C day/night).     

The effect of photoperiod and temperature on growth and frost resistance of winter rye root systems

Root growth, development and frost resistance were examined in winter rye ( Secale cereale L. cv. Puma) plants grown under 6 combinations of temperature and photoperiod (20/16°C or 5/3°C, day/night; 8, 16- or 24-h days). Overall root system growth is influenced by the interaction of temperature and photoperiod. Maximum shoot growth occurs at a 24-h photoperiod in 20°C plants and at a 16-h photoperiod in 5°C plants, and is correlated in both treatments with a high root:shoot ratio. Frost resistance of rye roots is affected by short photoperiods in 2 ways. First, short photoperiod and low temperature delay production of new adventitious roots so that newly developing roots are not exposed to freezing temperatures. Second, short photoperiod alone can induce several degrees of frost tolerance in existing roots during the lag phase of growth. Low temperature alone does not decrease the rate of dry weight accumulation in rye root systems, but cold temperature does retard developmental processes within the roots. Rye roots grown at 5°C develop first order lateral roots, differentiate metaxylem vessels and suberize endodermal cell walls more slowly than roots grown at 20°C.     

Equilibrium freezing of leaf water and extracellular ice formation in Afroalpine ‘giant rosette’ plants

The water potentials of frozen leaves of Afroalpine plants were measured psychrometrically in the field. Comparison of these potentials with the osmotic potentials of an expressed cellular sap and the water potentials of ice indicated almost ideal freezing behaviour and suggested equilibrium freezing. On the basis of the osmotic potentials of expressed cellular sap, the fractions of frozen cellular water which correspond to the measured water potentials of the frozen leaves could be determined (e.g. 74% at -3.0° C). The freezing points of leaves were found to be in the range between 0° C and -0.5° C, rendering evidence for freezing of almost pure water and thus confirming the conclusions drawn from the water-potential measurements. The leaves proved to be frost resistant down to temperatures between -5° C and -15° C, as depending on the species. They tolerated short supercooling periods which were necessary in order to start ice nucleation. Extracellular ice caps and ice crystals in the intercellular space were observed when cross sections of frozen leaves were investigated microscopically at subfreezing temperatures.Symbols T temperature - water potential Dedicated to Professor Dr. Hubert Ziegler on the occasion of his 60th birthday     

The role of ice nucleation active bacteria in supercooling of citrus tissues

The frost sensitivity of Citrus sinensis in relation to the presence of biogenic ice nuclei was studied. In commercially managed citrus groves the ice nucleation active (INA) bacterium Pseudomonas syringae reached 6 × 10⁴ colony forming units (CFU) leaf⁻¹, a population sufficiently high to catalyze ice formation. However, a transient loss of bacterial nucleation activity was noticeable at subzero field temperatures, followed by resumption as temperatures rose. This loss was apparently due to a temporary transition of INA to ice nucleation inactive (INI) bacteria. Field application of Bordeaux mixture, copper hydroxide, streptomycin, and 2-hydroxypropylmethanethiolsulfonate (HPMTS), resulted in reduction of INA bacterial populations to detectability (≤ 10² CFU leaf⁻¹) limits. However, the corresponding reduction in ice nucleation events in treated samples as compared to controls at nucleation temperature ≥−3°C was not as dramatic. It ranged from approximately 7% in samples treated with the bactericide HPMTS, to 35% in samples treated with chemicals possessing combined bactericidal - fungicidal action (coppers). Since a quantitative relationship exists between ice nucleation events on individual leaves and the INA bacterial populations harbored by these leaves, these results suggest the co-existence of a bacterial and a proteinaceous, yet non-bacterial ice nucleating source in citrus, both active at ≥−3°C.     

Cadmium and copper uptake and distribution in Mediterranean tree seedlings

Provenances of seedlings of Scots pine ( Pinus sylvestris L.) from northern Finland (65°N) and cuttings from trees of the same species about 30 to 40 years in age were studied in 3 artificial deacclimation experiments during the winter and spring of 1994. The deacclimation conditions varied in terms of temperature rise and photoperiods. Frost resistance and the pH of the cell effusate were studied in order to investigate the possibility of a connection between them. Complete deacclimation was achieved, as shown by a frost resistance of –60°C to –65°C in the seedlings and –8O°C in the cuttings at the beginning of the experiment and decreased to above –10°C by the end. The frost resistance and pH of the cell effusate responded significantly to the increase in temperature that marked the onset of the deacclimation process, whereas the response to different photoperiods was only slight. A highly significant correlation was found between frost resistance and the pH of the cell effusate. These variables were also significantly correlated with the freezing test temperature, indicating the condition of plasmalemma and tonoplast; and with sampling day, indicating the response to deacclimation. In the light of these findings, it is concluded that the pH of the cell effusate with certain reservations, can be used as a time saving, alternative viability test when determining frost resistance.