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.     

The effects of freezing on membrane electric potential in winter oilseed rape leaves

Extracellular ice formation in winter oilseed rape leaf discs (Brassica napus L. var. oleifera L. cv. Jantar) at different temperatures resulted in a transient membrane depolarization, which was followed by a decrease in membrane electric potential. In discs which underwent supercooling (no extracellular ice was formed), no membrane depolarization was observed. The inhibitors of calcium ion channels, gadolinium and lanthanum, decreased to some extend the amplitude of the frost-induced (−6 °C) depolarization and completely eliminated the decrease in membrane potential. Changes in membrane potential were associated with the increased electrolyte efflux, measured after thawing of the discs. No efflux from supercooled discs was observed. Application of calcium channel blockers decreased the level of the efflux induced by freezing at −6°C. It is suggested that membrane depolarization is one of the primary events induced by ice formation at a leaf surface. The possible reasons for changes in the membrane electric potential and their physiological consequences are discussed.     

Effects of cold on NAD+ kinase activity in winter rape plants

Low temperature (2°C) caused an increase in the activity of NAD⁺ kinase (EC 2.7.1.23) in leaves of winter rape plants ( Brassica napus L. var. oleifera L. cv. Górezański). The enzyme activity markedly increased between day 4 and 11 of plant exposure to cold, then tended to decrease. Changes in activity of NAD⁺ kinase coincided with the previously observed changes in the levels of pyridine nucleotides, NADP(H) (U. Maciejewska and A. Kacperska, Physiol. Plant. 69: 687–691, 1987). As a result of cold treatment, Ca²⁺–calmodulin–dependent and Ca²⁺–calmodulin–independent NAD⁺ kinase activities increased to almost the same extent. It seems therefore, that the cold–induced activation of NAD⁺ kinase does not depend on the Ca²⁺–calmodulin complex.     

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.     

Low temperature-induced modifications in cell ultrastructure and localization of phenolics in winter oilseed rape (Brassica napus L. var. oleifera L.) leaves

Acclimation of winter oilseed plants in the cold (i.e. at temperatures >0 degrees C) followed by short exposure to sub-lethal freezing temperatures resulted in pronounced ultrastructural changes of leaf epidermal and mesophyll cells. The following major changes were observed upon acclimation at 2 degrees C: increased thickness of cell walls; numerous invaginations of plasma membranes; the appearance of many large vesicles localized in the cytoplasm in close proximity to the central vacuole; the occurrence of abundant populations of microvesicles associated with the endoplasmic reticulum (ER) cisternae or located in the vicinity of dictyosomes; and the occurrence of paramural bodies and myelin-like structures. In addition, large phenolic deposits were observed in the vicinity of the plasma membrane and membrane-bound organelles such as chloroplasts, large vesicles or cytoplasm/tonoplast interfaces. Transient freezing (-5 degrees C for 18 h) of the cold-acclimated leaves led to reversible disorganization of the cytoplasm and to pronounced structural changes of the cellular organelles. Chloroplasts were swollen, with the stroma occupying one half of their volume and the thylakoid system being displaced to the other half. Large phenolic aggregates disappeared but distinct layers of phenolic deposits were associated with mitochondrial membranes and with chloroplast envelopes. In frost-thawed cells recovered at 2 degrees C for 24 h, dictyosomes and dictyosome- or ER-derived small vesicles reappeared in the ribosome-rich cytoplasm. Aberrations in the structure of chloroplasts and mitochondria were less pronounced. Few phenolic deposits were seen as small grains associated with chloroplast envelopes and vesicle membranes. These observations demonstrate that plants undergo different changes in cell ultrastructure depending on whether they are subjected to chilling or freezing temperatures. Results are discussed in relation to membrane recycling and the possible role of phenolics during the first and second stages of plant acclimation at low temperature.     

Frost resistance and water status of winter rape leaves as affected by differential shoot/root temperature

Twenty day-old winter rape ( Brassica napus L. var. oleifera L. cv. Jantar) seedlings, grown in nutrient solution, were exposed to different shoot/root temperature (i. e. 20/20, 20/3, 3/20 and 3/3°C) for 2 or 4 weeks. Chilling treatments modified markedly the pattern of plant growth as indicated by changes in dry matter accumulation in individual plant parts (leaves, hypocotyls, roots) and decreased leaf specific area. Growth of roots was less sensitive to low temperature than that of shoots. This was reflected by a decrease in shoot/root biomass ratio. Chilling treatments increased freezing resistance, decreased water content and water potential and modified reducing sugar, soluble protein and phospholipid contents in the leaves. A biphasic character of tissue responses to chilling temperature was observed, the most remarkable changes being registered during the first 7 or 14 days of the treatment. Effects of root or shoot exposure to chilling temperature on ice nucleation temperature, LT₅₀, water potential, accumulation of sugars and phospholipids in leaves were additive. All the observations point to the important role of the root system in plant acclimation to cold. Its impact on water status of leaves is emphasized and some mechanisms of root involvement in acclimation processes are proposed.     

Participation of the cyanide-resistant pathway in respiration of winter rape leaves as affected by plant cold acclimation

Leaf slices sampled from winter rape plants ( Brassica napus L., var. oleifera L., cv. GórczaánAski), grown in cold (5°C), showed an increase in the dark respiration rate (measured at 25°C) as compared to slices cut from control plants (grown at 20/15°C). The effect of low temperature was most pronounced after 4 days of plant growth in the cold. Oxygen uptake by control slices was 60% inhibited by 1 m M KCN and was insensitive to 2.5 m M salicylhydroxamic acid (SHAM). On the contrary, respiration of leaf slices from cold-pretreated plants was more resistant to cyanide (35% inhibition after 4 days of cold treatment) and was 30% inhibited by SHAM. The patterns of cold-induced changes in total respiratory activity and in the estimated activity of alternative pathway were similar. It seems that in leaf slices from plants grown in the cold, the cyanide-resistant, alternative pathway participates in oxygen uptake. Cold treatment of plants also brought about a 4-fold increase in the level of soluble sugars, which reached a maximum on day 4 of exposure to cold. Addition of sucrose to the incubation medium resulted in an immediate increase in oxygen uptake by slices with low endogenous sugar level. The respiration stimulated by sucrose addition was more resistant to cyanide than the basal respiration and it was inhibited by SHAM. It is concluded that the operation of the alternative pathway is responsible for the increased oxygen uptake by the cold-grown winter rape leaves and it may be induced by an increased sugar supply for respiratory processes.     

The role of calcium ions in freezing injuries of winter oilseed rape leaves. Effects of calcium channel blockers and mimesis by calcium ionophore

Calcium ionophore A23187 allowing for a calcium ion influx from an apoplast to a cytoplasm, mimicked symptoms of the frost-induced injuries in winter oilseed rape leaves, as estimated by the conductivity method. Both calcium ionophore and freezing treatment induced degradation of phosphatidylcholine. On the other hand lanthanum and gadolinum ions as well as verapamil, the inhibitors of calcium ion channels, decreased the degree of the frost-induced injuries. Lanthanum ions prevented the frost-induced degradation of PC. It is proposed that freezing alters the functioning of calcium ion channels which results in calcium ion influx into a cytosol. This in turn may lead to a degradation of cell membranes.     

Cold-deacclimation of oilseed rape (Brassica napus var. oleifera) in response to fluctuating temperatures and photoperiod

The aim of this work was to establish the role of factors that may trigger elongation growth in the dehardening response, namely temperature during daylight, photoperiod and vernalization. Fully cold-acclimated seedlings of winter (with incomplete vernalization) and spring oilseed rape were subjected to deacclimation under temperatures of 2/12, 12/2, 12/12, 12/20, 20/12 and 20/20 degrees C (day/night) and a 12 h photoperiod. Plants were also deacclimated under photoperiods of 8 and 16 h at constant temperatures of 12 and 20 degrees C. After deacclimation, plants were subjected to reacclimation. Results suggest that the level of growth activity induced during deacclimation affects both the deacclimation rate and the capacity for reacclimation. Deacclimation is fully reversible if it is not accompanied by induction of elongation growth. In such cases the rate of the decrease in freezing tolerance depends on the mean temperature of deacclimation. Deacclimation becomes partially or completely irreversible when it is connected with promotion of elongation growth. The stimuli triggering elongation growth during deacclimation may be the growth-promoting temperature (20 degrees C) during the day and the lack of vernalization blockage of elongation growth. When elongation growth was stimulated by other factors such as long-day treatments, rehardening was also disturbed.     

Uniconazole-induced alleviation of freezing injury in relation to changes in hormonal balance, enzyme activities and lipid peroxidation in winter rape

Winter rape (Brassica napus L. cv. 601) seedlings were treated with 50 mg.l-1 of foliar-applied uniconazole and then exposed to freezing stress with a light/dark temperature regime of 2 °C/–3 °C for 5 days at the seedling stage. Stressed plants contained lower endogenous GA3 and IAA contents than the controls, while zeatin and ABA contents and ethylene levels were significantly increased. Uniconazole-treated plants had lower endogenous GA3 and IAA contents, and higher zeatin and ABA contents and ethylene levels. Leaf chlorophyll content and respiratory capacity of roots were reduced significantly after plants were subjected to freezing stress, and foliar sprays of uniconazole retarded the degradation of chlorophyll and increased respiratory capacity of roots. Uniconazole-induced freezing tolerance was accompanied by increased activities of various antioxidant enzymes, including superoxide dismutase, catalase and peroxidase. Foliar applications of uniconazole reduced electrolyte leakage and malondialdehyde accumulation caused by freezing stress, suggesting that uniconazole may have decreased freezing-induced lipid peroxidation and membrane damage.     

Regulation of elongase activity by abscisic acid and temperature in microspore‐derived embryos of oilseed rape (Brassica napus)

Growth temperature and ABA both affect the level of erucic acid (22:1) in microspore‐derived embryos (MDEs) of oilseed rape. We have previously shown that these stimuli act independently. In the present study we investigated the effects of growth temperature (15 vs 25°C) and ABA (0 vs 5 µ M ) on the characteristics and activity of the elongase complex, the enzymes synthesising 22:1. Due to inhibition by the substrate oleoyl‐CoA at low concentrations (< 10 µ M ) it was not possible to determine values for K_m and V_max. Elongase activities from preparations extracted from MDEs grown under different conditions showed an optimum temperature higher than 30°C, with a Q₁₀ value of about 3. We found considerable effects of temperature and exogenous ABA on total elongase activity in MDEs. Our results suggest that the accumulation of 22:1 is regulated by the amount of elongase enzyme and not by changes in the intrinsic characteristics of the enzyme. Elongase activity correlated closely with the absolute amount of 22:1 (µmol), whereas the correlation between elongase activity and the fraction of 22:1 (% of fatty acids) in oil was poorer. Including the total activity of acyltransferases did not improve the correlation. Acyltransferase activity itself correlated poorly with the total amount of oil formed.     

Effects of water stress on growth, osmotic potential and abscisic acid content of maize roots

Under water stress conditions, induced by mannitol solutions (0 to 0.66 M ) applied to the apical 12 mm of intact roots of Zea mays L. (cv. LG 11), a growth inhibition, a decrease in the osmotic potential of the cell sap and a significant accumulation of abscisic acid (ABA) were observed. When the roots were placed in a humid atmosphere after the stress, the growth rate increased again, even if elongation had been totally inhibited. Under a stress corresponding to an osmotic potential of -1.09 MPa in the solution, growth was totally inhibited, which means that the root cell turgor pressure was reduced to the yield threshold. These conditions led to the largest accumulation of ABA. The effect of water stress on the level of ABA was studied for three parts of the root. The greatest increase in ABA (about 10 fold) was obtained in the growth zone and this increase was apparently independent of the hydrolysis of the conjugated form. With a mannitol treatment of 1 h equivalent to a stress level of -1.39 MPa, a 4-fold increase in ABA efflux into the medium was obtained. These results suggest that there are interactions between water stress, root growth, osmotic potential and the ABA level. The growth under conditions of stress and the role of endogenous ABA in the control of plant metabolism, specially in the growth zone, are discussed.     

Morphological and physiological modifications of Cistus salvifolius L. winter leaves in response to the rise in winter temperatures

The global climate is predicted to change in the next century; for the Mediterranean Basin, an increase in air temperature more than 4°C and a higher frequency of extreme climatic events such as drought and heat waves are expected. In this work, the response of Cistus salvifolius L. to the rise in winter temperature has been studied. Plants acclimated to winter conditions [outdoor (OUT)] were moved into a greenhouse [indoor (IND)] at higher temperature and eco-physiological behaviour was analysed on leaves after 15 days from plant transferring (IND_15d) and on leaves developed IND. IND leaves were characterized by reduced thickness, higher specific leaf area, higher CO₂ mesophyll conductance and photosynthetic rate, and lower respiratory rate than leaves grown OUT upon current winter conditions. In IND_15d leaves, no improvement of photochemical activity was found. When IND leaves were subjected to a rapid increase in air temperature, the CO₂ fixation was not limited indicating a high thermotolerance of photosynthetic machinery. The results for IND leaves indicate the occurrence of a strategy that merging changes in leaf structure as well as in photosynthetic regulation allow C. salvifolius to maintain an elevated carbon gain in response to temperature increase.     

Distant olfactory response of the cabbage seed weevil, Ceutorhynchus assimilis, to oilseed rape odour in the field

Abstract. The olfactory orientation of adult cabbage seed weevils (Ceutorhynchus assimilis Payk.) from a distance of 20 m to extracts of oilseed rape (Brassica napus L.) was studied in the field using mark–release–recapture experiments. Male and female weevils dispersed upwind from the centre of a circular array of traps baited with Oilseed rape extracts. In the absence of host-plant odour, female weevils dispersed randomly, and male weevils downwind. Percentage recapture of weevils was significantly greater in traps baited with rape flower than rape leaf extracts. Significantly fewer weevils were recaptured in unbaited traps compared to traps baited with rape flower or leaf extracts. We suggest that seed weevils can use odour-mediated upwind anemotaxis to locate their host-plants from distances of at least 20 m.     

Thermodynamic analysis of the physical state of water during freezing in plant tissue, based on the temperature dependence of proton spin-spin relaxation

Multi-proton spin-echo images were collected from cold-acclimated winter wheat crowns (Triticum aestivum L.) cv. Cappelle Desprez at 400 MHz between 4 and ?4 °C. Water proton relaxation by the spin-spin (T2) mechanism from individual voxels in image slices was found to be mono-exponential. The temperature dependence of these relaxation rates was found to obey Arrhenius or absolute rate theory expressions relating temperature, activation energies and relaxation rates, Images whose contrast is proportional to the Arrhenius activation energy (E_a), Gibb's free energy of activation (ΔG^?), and the entropy of activation (ΔS^?) for water relaxation on a voxel basis were constructed by post-image processing. These new images exhibit contrast based on activation energies rather than rules of proton relaxation. The temperature dependence of water proton T₂ relaxation rates permits prediction of changes in the physical state of water in this tissue over modest temperature ranges. A simple model is proposed to predict the freezing temperature kof various tissue in wheat crowns. The average E_a and ΔH^? for water proton T₂ relaxation over the above temperature range in winter wheat tissue were ?6.4 ± 14.8 and ?8.6 ± 14.8kj mol^?1, respectively. This barrier is considerably lower than the E_a for proton translation in ice at 0°C, which is reported to be between 46.0 and 56.5 kj mol^?1     

Commercially available plant growth regulators and promoters modify bulk tissue abscisic acid concentrations in spring barley, but not root growth and yield response to drought

Field and lysimeter experiments were conducted in 2002 to investigate the effects of an antigibberellin growth regulator (Moddus, active ingredient trinexapac‐ethyl, Syngenta Crop Protection UK Ltd, Whittlesford, Cambridge, UK) and an auxin‐stimulating (Route, active ingredient zinc ammonium acetate, De Sangosse Ltd, Swaffham Bulbeck, Cambridge, UK) growth promoter on root growth, soil water extraction and the drought response of spring barley. The effects on root growth and distribution were investigated in the field. The effects on the drought response were studied in 1.2‐m‐deep lysimeters packed with a loamy sand subsoil and sandy loam topsoil. Lysimeters were located under a fixed rain shelter, and drought was imposed by withholding irrigation. In both field and lysimeter experiments, growth regulator/promoters were applied to cv. Optic at early tillering according to the manufacturers’ recommendations. After withholding irrigation from lysimeters at Zadoks growth stage (GS) 21 (37 days after sowing), 50% of the profile available water had been depleted by flag leaf emergence (GS 37/39; 62 days after sowing). Drought significantly reduced stem biomass at ear emergence (GS 59; 78 days after sowing) but not leaf or ear dry weight; this was before there was any significant reduction in leaf water potential or stomatal conductance to water vapour. The reduction in stem biomass may reflect a change in partitioning between shoot and root in response to soil drying. When averaged over growth regulator/promoter treatments, drought reduced grain yield by approximately 1 t ha^?1. This was associated with a reduction in both ears per m² and grains per ear. The mean grain weight was not reduced by drought, in spite of significant reductions in stomatal conductance and canopy lifespan post‐anthesis. Route, and to a lesser extent Moddus, significantly increased abscisic acid accumulation in the stem base of droughted plants, and there was some indication of a possible delay in stomatal closure in Route‐treated plants as the soil moisture deficit developed. However, there was no significant effect on the amount of soil water extracted or grain yield under drought. Similarly, in field experiments, neither Route nor Moddus significantly altered total root length, biomass or distribution. There is little evidence from these experiments or in the literature to support the use of antigibberellin or auxin‐simulating growth regulator/promoters to modify root growth and drought avoidance of spring barley.