ABA and gibberellin-like substances during prehardening,cold acclimation,de- and reacclimation of oilseed rape

Previously published results showed that high relative reduction state of PSII (PSII excitation pressure) during both early seedling growth (prehardening) as well as cold deacclimation caused significant changes in growth pattern. The differences in elongation growth rate were related to the cold acclimation of photosynthetic apparatus and to frost resistance. To study changes in the hormonal balance connected with alterations in elongation growth rate observed during prehardening and deacclimation under different PSII excitation pressure (modulated by day-temperatures), endogenous concentration of ABA, GA₃ and GA-like substances (GAs) were analysed. Analyses were also performed during cold acclimation and reacclimation of plants characterized by different elongation growth rate triggered by prehardening or deacclimation under different day-temperatures. Growth under high PSII excitation pressure (prehardening) resulted in a significant increase in ABA and a considerable decrease in GAs contents. On the other hand, different ABA content played almost no role in controlling growth rate during cold deacclimation and subsequent reacclimation, when the induction of elongation growth was connected with the changes in concentration of GAs including GA₃. The possible role of ABA and GAs in controlling prehardening, cold acclimation and deacclimation is discussed.     

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.     

Low temperature effects on growth and actin cytoskeleton organisation in suspension cells of winter oilseed rape

Rhodamine-phalloidin staining of winter oilseed rape suspension cells revealed that the structure of actin cytoskeleton changes with the phase of cell growth. In small, 4-day-old cells, entering the exponential phase of growth, a dense and uniformly distributed cortical microfilament networks was seen. In six-day-old vacuolated cells, which reached the stationary phase of growth, the actin cytoskeleton was composed of thicker microfilament cables in irregular arrangements. In cells acclimated in cold for 7 days a dense, uniformly distributed and cortical microfilament network was still seen. The fine microfilament network was sensitive to extracellular freezing since the structures underwent depolymerization at −3 °C (in the presence of extracellular ice), both in non-acclimated and cold-acclimated cells. The thicker transvacuolar cables in cells of the stationary growth phase resisted freezing to −7 °C. Acclimation of suspensions at 2 °C resulted in slowing down growth of cells and in the increased freezing tolerance of cells as indicated by a decrease of LT₅₀ from −11 °C to −17.5^o or to −25 °C when determined 7 or 20 days after the beginning of the cold treatment, respectively. Freezing tolerance of non-acclimated cells decreased from −11 °C to −8 °C during subculture, showing a transient increase to −17 °C on the day 6. Results indicate that the arrangement of actin microfilaments and their sensitivity to freezing-induced depolymerization depends on the phase of cell growth rather than on cell acclimation status. Possible mechanisms involved in the freezing-induced depolymerization of actin microfilaments are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

The effects of low temperature acclimation and photoinhibitory treatments on Photosystem 2 studied by thermoluminescence and fluorescence decay kinetics

The effects of low temperature acclimation and photoinhibitory treatment on Photosystem 2 (PS 2) have been studied by thermoluminescence and chlorophyll fluorescence decay kinetics after a single turnover saturating flash. A comparison of unhardened and hardened leaves showed that, in the hardened case, a decrease in overall and B-band thermoluminescence emissions occurred, indicating the presence of fewer active PS 2 reaction centers. A modification in the form of the B-band emission was also observed and is attributed to a decrease in the apparent activation energy of recombination in the hardened leaves. The acclimated leaves also produced slower Q_A ^– reoxidation kinetics as judged from the chlorophyll fluorescence decay kinetics. This change was mainly seen in an increased lifetime of the slow reoxidation component with only a small increase in its amplitude. Similar changes in both thermoluminescence and fluorescence decay kinetics were observed when unhardened leaves were given a high light photoinhibitory treatment at 4°C, whereas the hardened leaves were affected to a much lesser extent by a similar treatment. These results suggest that the acclimated plants undergo photoinhibition at 4°C even at low light intensities and that a subsequent high light treatment produces only a small additive photoinhibitory effect. Furthermore, it can be seen that photoinhibition eventually gives rise to PS 2 reaction centers which are no longer functional and which do not produce thermoluminescence or variable chlorophyll fluorescence.Abbreviations D1 The 32 kDa protein of Photosystem 2 reaction center - F_m maximum chlorophyll fluorescence yield - F₀ minimal chlorophyll fluorescence yield obtained when all PS 2 centers are open - F_i intermediate fluorescence level corresponding to PS 2 centers which are loosely or not connected to plastoquinone (non-B centers) - F_v maximum variable chlorophyll fluorescence yield (F_v=F_m–F₀) - PS 2 Photosystem 2 - Q_A and Q_B respectively, primary and secondary quinonic acceptors of PS 2 - S1, S2 and S3 respectively, the one, two and three positively charged states of the oxygen evolving system - Z secondary donor of PS 2     

Reaction of winter oilseed rape callus to different concentrations of elicitors: pectinase or chitosan

The aim of the presented methodical experiments was 1) the evaluation if callus of winter oilseed rape (Brassica napus var. oleifera L.) initiates a defence reaction to fungal elicitors: pectinase (polygalacturonase) or chitosan, and 2) the choice of the elicitor doses, which evoke the strongest tissue reaction. The results obtained will be used in the next experiments relating the studies of pathogenesis mechanisms proceeding in rape plants infected by necrotrophic fungi. The defence response was estimated on the basis of changes in electrolyte leakage from cells, metabolic efficiency, phenolic content and catalase activity. In the experiment pectinase was used at concentration of 3, 8, 16, 133 and 166 μl per 1 cm³ of culture medium while chitosan at: 25, 50, 75 and 100 μg·cm⁻³. Both elicitors increased cell membrane permeability: pectinase at the doses equal or greater to 16 μl·cm⁻³ while chitosan of 25 μg·cm⁻³. The greatest metabolic inefficiency was observed in calli elicited with 16 μl·cm⁻³ pectinase and with chitosan of 100 μg·cm⁻³. The decrease in phenolic content was noted under influence of most doses of both elicitors. The highest catalase activity was evoked by pectinase of 8 μl·cm⁻³ and chitosan of 75 and 100 μg·cm⁻³. The results indicated that 8–16 μl·cm⁻³ of pectinase and 100 μg·cm⁻³ of chitosan caused the strongest defence reaction of oilseed rape tissue.     

The Pattern of Changes in Photosynthetic Apparatus in Response to Cold Acclimation and De-acclimation in Two Contrasting Cultivars of Oilseed Rape

In spring and winter cultivars of oilseed rape (Brassica napus var. oleifera), acclimation of photosynthetic apparatus to cold was connected with the increase in activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO) and sucrose-phosphate synthase (SPS). Conversely, cold de-acclimation entailed the decline of RuBPCO and SPS activities. The rate of this photosynthetic de-acclimation might depend on day temperature. On the other hand, temperature rise during de-acclimation (identical during the day and night) resulted in the improvement of photosynthetic activity measured by means of chlorophyll fluorescence. An increase in SPS activity (and even transitory increase in RuBPCO activity) was observed when the elongation growth rate (EGR) accelerated during de-acclimation. Throughout re-acclimation, plants with high EGR were unable to maintain or recover higher photosynthetic capacity, despite the fact that SPS activity remained high or even increased during re-acclimation.     

Does the lack of vernalization requirement interfere with winter survival of oilseed rape plants?

Recent studies (Rapacz 1999) have shown that cultivars of spring-type oilseed rape are able to cold-acclimate to the level comparable with winter cultivars, but only after prehardening which results both in the increase of photosynthetic activity and in growth cessation. It is commonly known that under field conditions spring-type cultivars could not survive winter. Present studies were undertaken to explain the reasons for low winter hardiness of spring type rape plants. Six cultivars of spring and two of winter rape were sown in the open-air vegetation room at the end of August. The obtained results indicate that the degree of frost damage in spring-type plants increased in the course of winter and this increase was parallel to elongation of generative shoots observed after periods of warming. Each spring cultivar was completely killed by frost just after its generative shoot reached 15–20 cm, irrespective of its frost resistance level, determined previously under laboratory conditions. In the case of winter cultivars survival rate was consistent with laboratory-estimated frost resistance. It is suggested that spring rape could not survive winter because of its limited ability to prevent shoot elongation during winter at temperatures slightly above 0 °C. It was also found that less efficient photosynthetic electron transport in autumn was observed in these spring cultivars in which the elongation of generative shoots was observed already during the first warm break in winter.     

Comparison of responses of bean, pea and rape plants to UV-B radiation in darkness and in light

Effect of UV-B radiation on leaves of bean, pea and rape plants was studied. UV-B radiation (11.2 kJ·m⁻²) induced more distinct reduction of the primary photosynthesis activity when applied in darkness than the same UV-B dose, extended in time, and applied with photosynthetic active radiation (PAR). The pea plants were more susceptible to UV-B in darkness, but in the presence of PAR their tolerance was higher. The CO₂ fixation in the bean and rape plants, exposed to UV-B was decreased, but for the pea plants it remained unchanged. The UV-B irradiation caused an increase in the content of ultraviolet-absorbing pigments. Additionally, the bean plants grown at UV-B increased the thickness of leaves, described as SLW.     

Abscisic acid concentrations are correlated with leaf area reductions in two salt-stressed rapid-cycling Brassica species

The greater sensitivity of B. carinata to salinity in comparison to B. napus has been linked to a greater reduction in net assimilation rate. Apparently this is not due to ion toxicity; the cause is unknown. In this report, we test the hypothesis that increases in abscisic acid (ABA) are involved in the reduction of growth by salinity. Salinity (8 dS m^–1) caused an increase of ABA concentrations in the shoot, root and callus of both species. ABA concentrations were lower in the salt-tolerant species, B. napus, than the salt-sensitive species, B. carinata, both in the whole plant and callus. Leaf expansion for both species was equally sensitive to ABA; salt stress did not significantly alter sensitivity to applied ABA. The growth inhibition increased in a hyperbolic manner with an increase in endogenous ABA concentration. These results indicate that ABA in salt-stressed plants may play a role in the inhibition of growth. The photosynthesis of salt-sensitive species, B. carinata, was also decreased by salinity, corresponding to the reduction in growth. The decreased photosynthesis does not appear to be the cause of the growth reduction, because photosynthesis was not inhibited by short-term exposure to salinity and photosynthesis was poorly correlated with endogenous ABA concentrations.     

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.     

Relationship between quantum efficiency of PSII and cold-induced plant resistance to fungal pathogens

The aim of the presented work was to study whether the efficiency of photosynthesis may influence resistance of hardened plants to disease. Seedlings of spring barley, meadow fescue and winter oilseed rape were chilled at 5 °C for 2, 4 or 6 weeks and at these deadlines the changes in cell membrane permeability (expressed as electrolyte leakage), chlorophyll fluorescence (initial fluorescence - F₀, maximal fluorescence - F_m, quantum yield of PSII - F_v/F_m) and net photosynthesis rate (F_N) were measured. Also, the influence of cold on the degree of plant resistance to economically important pathogens -Bipolaris sorokiniana or Phoma lingam was estimated. Two, four or six week-hardened plants were artificially infected: barley and fescue by B. sorokiniana, and oilseed rape by P. lingam. Hardening at 5 °C stimulated resistance of barley, fecue and rape to their specific pathogens. Six-week long acclimation was the most effective for plant resistance. Cold significantly changed cell membrane permeability and decreased chlorophyll fluorescence (F₀, F_m and F_v/F_m) of all studied plant species, while net photosynthesis rate was found to decrease only in barley. The results indicate that cold-induced resistance of plants to pathogens was correlated with a decrease in cell membrane permeability. In the case of fescue and barley a significant connection between the quantum yield of PSII and their resistance to B. sorokiniana was shown. Additionally, the resistance of barley to fungus was depended on net photosynthesis rate. In general this research shows that the efficiency of photosynthesis may be used as an indicator of plant resistance to disease.     

Influence of the triazole growth retardant BAS 111..W on phytohormone levels in senescing intact pods of oilseed rape

Foliar treatment of oilseed rape plants (Brassica napus L.ssp. napus cv. Linetta) with the growth retardant BAS 111..W at the 5th leaf stage delayed pod senescence during early maturation. Changes of immunoreactive cytokinin- and abscisic acid (ABA)- like substances and of the ethylene precursor 1-aminocyclo-propane-1-carboxylic acid (ACC) and its malonyl-conjugate (MACC) were determined in intact whole pods. When compared with control plants, higher levels of total chlorophyll correlated with four-fold and three-fold increases of trans-zeatin riboside- and dihydrozeatin riboside-type cytokinins, respectively, in the pods of plants treated with 0.25 mg BAS 111..W per plant. Isopentenyladenosine-type cytokinins and ACC and MACC contents remained virtually unchanged, whereas ABA levels dropped considerably below those of controls (60% reduction). However, when analysed at late pod maturity, BAS 111..W treatment no longer affected the total chlorophyll content, or the levels of cytokinins, ABA, ACC and MACC. We hypothesize that the retardant-induced changes in the hormonal status of the pods, favouring the senescence-delaying cytokinins as opposed to abscisic acid, could contribute to the developmental delay.     

Photosynthesis in leaves and siliques of winter oilseed rape (Brassica napus L.)

The rate of photosynthesis and its relation to tissue nitrogen content was studied in leaves and siliques of winter oilseed rape (Brassica napus L.) growing under field conditions including three rates of nitrogen application (0, 100 or 200 kg N ha^-1) and two levels of irrigation (rainfed or irrigated at a deficit of 20 mm). The predominant effect of increasing N application under conditions without water deficiency was enhanced expansion of photosynthetically active leaf and silique surfaces, while the rate of photosynthesis per unit leaf or silique surface area was similar in the different N treatments. Thus, oilseed rape did not increase N investment in leaf area expansion before a decline in photosynthetic rate per unit leaf area due to N deficiency could be avoided. Much less photosynthetically active radiation penetrated into high-N canopies than into low-N canopies. The specific leaf area increased markedly in low light conditions, causing leaves in shade to be less dense than leaves exposed to ample light. In both leaves and siliques the photosynthetic rate per unit surface area responded linearly to increasing N content up to about 2 g m^-2, thus showing a constant rate of net CO₂ assimilation per unit increment in N (constant photosynthetic N use efficiency). At higher tissue N contents, photosynthetic rate responded less to changes in N status. Expressed per unit N, light saturated photosynthetic rate was three times higher in leaves than in silique valves, indicating a more efficient photosynthetic N utilization in leaves than in siliques. Nevertheless, from about two weeks after completion of flowering and onwards total net CO₂ fixation in silique valves exceeded that in leaves because siliques received much higher radiation intensities than leaves and because the leaf area declined rapidly during the reproductive phase of growth. Water deficiency in late vegetative and early reproductive growth stages reduced the photosynthetic rate in leaves and, in particular, siliques of medium- and high-N plants, but not of low-N plants.     

Effects of Ozone Fumigation on Photosynthesis and Membrane Permeability in Leaves of Spring Barley, Meadow Fescue, and Winter Rape

Seedlings of spring barley, meadow fescue, and winter rape were fumigated with 180 μg kg⁻¹ of ozone for 12 d, and effect of O₃ on photosynthesis and cell membrane permeability of fumigated plants was determined. Electrolyte leakage and chlorophyll fluorescence were measured after 6, 9, and 12 d of fumigation, while net photosynthetic rate (P _N) and stomatal conductance (g _s) were measured 9 d after the start of ozone exposure. O₃ treatment did not change membrane permeability in fescue and barley leaves, while in rape a significant decrease in ion leakage was noted within the whole experiment. O₃ did not change the photochemical efficiency of photosystem 2 (PS2), i.e., F_v/F_m, and the initial fluorescence (F₀). The values of half-rise time (t_1/2) from F₀ to maximal fluorescence (F_m) decreased in fescue and barley after 6 and 9 d of fumigation. P _N decreased significantly in ozonated plants, in the three species. The greatest decrease in P _N was observed in ozonated barley plants (17 % of the control). The ozone-induced decrease in P _N was due to the closure of stomata. Rape was more resistant to ozone than fescue or barley. Apparently, the rape plants show a large adaptation to ozone and prevent loss of membrane integrity leading to ion leakage. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effects of temperature on parameters of root growth relevant to nutrient uptake: Measurements on oilseed rape and barley grown in flowing nutrient solution

Summary Effects of root temperature on the growth and morphology of roots were measured in oilseed rape (Brassica napus L.) and barley (Hordeum vulgare L.). Plants were grown in flowing solution culture and acclimatized over several weeks to a root temperature of 5°C prior to treatment at a range of root temperatures between 3 and 25°C, with common shoot temperature. Root temperature affected root extension, mean radius, root surface area, numbers and lengths of root hairs. Total root length of rape plants increased with temperature over the range 3–9°C, but was constant at higher temperatures. Root length of barley increased with temperature in the range 3–25°C, by a factor of 27 after 20 days. Root radii had a lognormal distribution and their means decreased with increasing temperature from 0.14 mm at 3°C to 0.08 mm at 25°C. The density of root hairs on the root surface increased by a factor of 4 in rape between 3 and 25°C, but in barley the highest density was at 9°C. The contribution of root hairs to total root surface area was relatively greater in rape than in barley. The changes in root system morphology may be interpreted as adaptive responses to temperature stress on nutrient uptake, providing greater surface area for absorption per unit root weight or length.     

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.     

Gibberellin and abscisic acid effects on the activity of hydrolytic enzymes in de-embryonated barley seeds

The production of hydrolytic enzymes by embryo-less barley seeds in response to various gibberellins and abscisic acid was investigated. The data support the hypothesis that plant growth substances may affect the mechanisms of hydrolase production and secretion in cereal seeds in different ways and at different point.