Influence of cytoskeletal agents on the respiratory electron transport pathways in the cells of winter wheat leaves

The effects of actin and tubulin polymerization inhibitors on the respiratory electron transport pathway activities were investigated using abscisic acid (ABA)- and cold-treated winter wheat seedling leaves. In unstressed control plants, cytochalasin B (15 microm) decreased the capacity of the cytochrome pathway, but stimulated the cyanide-resistant pathway, whereas oryzalin (15 microm) produced the opposite effects. Cold hardening (3 degrees C for 7 days) and ABA treatment 30 microm changed the respiratory pattern in a similar manner to cytochalasin B but to lesser effects. This points to cold- and ABA-induced reduction in microfilament sensitivity to these drugs and hence stabilization of actin-dependent processes. In contrast, oryzalin had only weak effects on control samples and its effects were strengthened in the presence of the cytoskeleton-modifying factors. The data suggest that the potential targets for the agent either increase and/or the degree of involvement of microtubules in the respiratory chain regulation, and therefore that the cytoskeleton can modify the functioning of the respiratory electron transport pathways in winter wheat cells.     

Metabolic responses of wheat roots to alkaline stress

Aims The aim of this study was to investigate the effects of alkaline stress on primary, secondary metabolites and metabolic pathways in the roots of wheat (Triticum aestivum). The results were used to evaluate the physiological adaptive mechanisms by which wheat tolerated alkali stress.Methods A pot experiment was carried out in the greenhouse. For each plastic pot, five wheat seeds were planted. After germination, seedlings were allowed to grow under controlled water and nutrient conditions for two months, then seedlings were exposed to alkaline stress (NaHCO₃-Na₂CO₃) for 12 days. The relative growth rate (RGR), absolute water content (AWC), metal elements, free cations and metabolites were measured.Important findings The alkaline stress caused the reduction of RGR and AWC. Alkaline stress caused a rapid increase of Na content with the concurrent decrease in K and Cl content, resulting in inhibited metal element accumulation and an ionic imbalance. In the present study, alkaline stress strongly enhanced Ca accumulation in wheat roots, suggesting that an increased Ca concentration can immediately trigger the salt overly sensitive (SOS)-Na exclusion system and reduce Na-associated injuries. Also, 70 metabolites, including organic acids, amino acids, sugars/polyols and others, behaved differently in the alkaline stress treatments according to a GC-MS analysis. The metabolic profiles of wheat were closely associated with alkaline-stress conditions. Alkaline stress caused the accumulation of organic acids, accompanied by the depletion of sugars/polyols and amino acids. Organic acids could play a central role in the regulation of intracellular pH by accumulating vacuoles to neutralize excess cations. Glycolysis and amino acid synthesis in roots were inhibited under salt stress while prolonged alkaline stress led to a progressive tricarboxylic acid (TCA) cycle. The severe negative effects of alkaline stress on sugar synthesis and storage may reflect the toxic levels of Na⁺ accumulating in plant cells in a high-pH environment, implying that the reactive oxygen species detoxification capacity was diminished by the high pH. A lack of NO₃^- in wheat roots can decrease synthase enzyme activities, limiting the synthesis of amino acids. Under salt stress, the TCA cycle and organic acid accumulation increased, but glycolysis and amino acid synthesis were inhibited in roots. Thus, energy levels and high concentrations of organic acids may be the key adaptive mechanisms by which wheat seedlings maintain their intracellular ion balance under alkaline stress.     

Cytoskeleton-Dependent Changes in the Structural Organization of Reticuloplasmins in Triticum aestivum Cells during Cold Acclimation and Treatment with Abscisic Acid

We studied the effects of the anti-microtubule drug, oryzalin, on the content and spatial organization of reticuloplasmins (Ca²⁺-binding marker proteins of the endoplasmic reticulum) in winter wheat seedlings after their cold acclimation (3°C, 7 days) and treatment with ABA (30 M). For identification and visualization of reticuloplasmins, we applied one-dimensional SDS-PAGE with subsequent Western blotting and indirect fluorescent microscopy. We used polyclonal HSP70 and CRH antibodies against BiP and calreticulin (Cal), respectively. On immunoblots, the brightest bands corresponded to polypeptides with mol wts of 58 kD (calreticulin) and 79 kD (BiP). The content of calreticulins in roots was shown to be higher than in leaves. Cold acclimation enhanced, and ABA treatment reduced, the concentration of calreticulins in root cells. Both treatments increased the BiP concentration in roots. Oryzalin (10 M, treatment for 3 h) did not affect the level of reticuloplasmins in roots of unhardened, cold acclimated, treated with ABA and with a combination of cold and ABA plants. However, both oryzalin and low-temperature treatments resulted in the accumulation of reticuloplasmins in the two spherical structures in the vicinity of the plasmalemma and nuclear envelope. After the combined action of oryzalin and low temperature, the cortical sphere of BiP proteins was shifted into the endoplasm and calreticulins appeared in the nuclear matrix. We believe that these changes in the reticuloplasmin localization are related to the rearrangement of the endoplasmic reticulum determined by the cytoskeleton modification. They result in the improved capacity of reticuloplasmins to control Ca²⁺ behavior and/or to the function as chaperones. The results obtained permit the conclusion that cytoskeletal proteins interact with reticuloplasmins, and this interaction might be involved in the transduction of the external and internal signals.     

Rearrangement of the Cytoskeleton in Triticum aestivum Cells during Cold Hardening and after Treatment with Abscisic Acid

Immunocytochemical study of the basic characteristics of the tubulin and actin cytoskeleton (total content, orientation, structure, and stability) was performed for various root zones of the seedlings of winter wheat cultivars contrasting in their freezing tolerance. Plant cold hardening (3°C, 7 days) and ABA treatment (30 M, 3 days) increased the stability of tubulin microtubules (MT), that is, reduced the depolymerizing action of oryzalin in vivo. However, the mechanisms of hardening and ABA stabilizing action on the cytoskeleton were different: low temperature enhanced spatial MT aggregation and resulted in the formation of a dense network of thick MT bundles, whereas ABA reduced the content of tubulin components and induced microfilament (MF) depolymerization. Most pronounced temperature- and ABA-induced cytoskeleton changes were observed in the differentiation zone, which indicates an important role of this root zone in plant adaptation and development of root freezing tolerance. Low temperatures reduced the hormonal effect on the structural arrangement and stability of MT and MF in wheat cultivars of high and moderate freezing tolerance but increased hormonal effects in the slightly tolerant cultivar. MF depolymerization and an increase in the proportion of stable MT are supposed to be a necessary condition for seedling growth retardation after their treatment with ABA and for seedlings at the initial phase of their adaptation to low temperature. At the final phase of cold hardening, some growth acceleration is evidently determined by the accumulation of highly labile MT and greater actin polymerization.     

Oryzalin-Induced Changes in Water Status and Cytoskeleton Proteins of Winter Wheat Seedlings upon Cold Acclimation and ABA Treatment

The effect of oryzalin (a specific inhibitor of tubulin polymerization in plant cells) on water retention by the leaves and roots of winter wheat (Triticum aestivum L.) seedlings was studied. The cultivars differing in their frost resistance were compared after their acclimation to low temperature (3°C for 3 or 7 days) and after treatment with ABA. In control untreated plants, oryzalin reduced the water-retaining capacity (WRC) of leaves and roots. Both hardening and ABA lowered the effect of the inhibitor on WRC in leaves, whereas their effects on water retention by roots were opposite, i.e., hardening weakened and ABA intensified the effect of oryzalin. Oryzalin-induced reduction of WRC decreased in the following sequence of cultivars: weakly frost resistant moderately frost resistant highly frost resistant. It was more pronounced in the leaves than in the roots, the latter being characterized by the lower WRC and lower frost resistance. After three-day-long hardening of plants, an additive effect of hypothermia and ABA on oryzalin-induced decrease in WRC of leaves and the lack of such effect in the roots were observed. The immunochemical analysis of the composition and content of cytoskeletal proteins with Western blotting showed that in the leaves the actin/tubulin ratio was higher than in the roots. The treatment of nonacclimated plants with ABA lowered the content of - and -tubulins and actin in roots but did not affect the level of actin in leaves. Hardening negated the effects of ABA on cytoskeletal proteins. Oryzalin produced the greatest inhibitory effect on WRC and an increase in frost resistance in ABA-treated plants in the experiments with leaves of the weakly frost resistant cultivar before and after hardening. Organ- and cultivar-specific and ABA-mediated dependence of WRC on cytoskeletal proteins and microtubules and microfilaments formed by them is supposed to result from their effect on the state of intracellular water and water permeability of the plasma membrane. In the course of cold acclimation of plants and upon their treatment with ABA, this dependence was more distinctly expressed in leaves than in roots, and especially in the plants of the weakly frost resistant cultivar.     

Relationships among vernalization, shoot apex development and frost tolerance in wheat

BACKGROUND AND AIMS: Frost tolerance of wheat depends primarily upon a strong vernalization requirement, delaying the transition to the reproductive phase. The aim of the present study was to learn how saturation of the vernalization requirement and apical development stage are related to frost tolerance in wheat. METHODS: 'Mironovskaya 808', a winter variety with a long vernalization requirement, and 'Leguan', a spring variety without a vernalization requirement, were acclimated at 2 degrees C at different stages of development. Plant development (morphological stage of the shoot apex), vernalization requirement (days to heading) and frost tolerance (survival of the plants exposed to freezing conditions) were evaluated. KEY RESULTS: 'Mironovskaya 808' increased its frost tolerance more rapidly; it reached a higher level of tolerance and after a longer duration of acclimation at 2 degrees C than was found in 'Leguan'. The frost tolerance of 'Mironovskaya 808' decreased and its ability to re-acclimate a high tolerance was lost after saturation of its vernalization requirement, but before its shoot apex had reached the double-ridge stage. The frost tolerance of 'Leguan' decreased after the plants had reached the floret initiation stage. CONCLUSIONS: The results support the hypothesis that genes for vernalization requirement act as a master switch regulating the duration of low temperature induced frost tolerance. In winter wheat, due to a longer vegetative phase, frost tolerance is maintained for a longer time and at a higher level than in spring wheat. After the saturation of vernalization requirement, winter wheat (as in spring wheat) established only a low level of frost tolerance.     

The absorption and translocation of diclofop-methyl and amitrole in wheat and oat roots

The absorption and translocation of diclofop-methyl (methyl 2-[4(2',4'-dichlorophenoxy)phenoxy]propanoate) was examined by using a specially designed treatment apparatus that separated excised roots or roots of seedlings into four zones. [¹⁴C]-Diclofop-methyl was absorbed along the entire root length of both wheat ( Triticum aestivum L.) and oat ( Avena sativa L.). In both species, absorption was greatest in the apical region of the root. Absorption by the apical region of wheat roots was more than three times greater than the basal portions, and more than twice as great as the apical region of oat roots. Less than 5% of the absorbed diclofop-methyl was translocated in both wheat and oat roots. Diclofop-methyl and diclofop(2-[4(2',4'-dichlorophenoxy)phenoxy]propanoic acid) were the predominant translocated forms. The absorption and translocation of amitrole (3-amino-1,2,4-triazole) were also examined. Amitrole was absorbed along the entire length of wheat roots and translocated primatily in the basipetal direction. The usefulness of the specially designed apparatus for biochemical and physiological studies is discussed.     

Effects of abscisic acid,low temperature,and plant age on cytoskeleton and phosphorylated proteins

The effects of exogenous abscisic acid (ABA), low temperature, and seedling age on the content of tubulin, actin, and phosphorylated proteins and the structural organization of microtubules (MTs) in cells of different tissues and organs of winter wheat cultivars contrasting in cold hardiness were studied by immunocytochemical methods using monoclonal (against - and -tubulin and actin) and polyclonal (phosphothreonine) antibodies. The leaves and roots of five- and nine- day-old seedlings of three cultivars were characterized by unequal proportion of actin/tubulin proteins. ABA decreased the content of the cytoskeleton and the 60-kD phosphorylated proteins, thus promoting a decrease in the number of MTs and occurrence of a less branched network of weakly fluorescent tubulin components in the cells of the root differentiating zone (which is most responsible for the development of cold hardiness in wheat). Although the cold acclimation of plants (3°C, 7 days) did not change the level of tubulin and actin proteins, it evoked the spatial aggregation of MT, leading to formation of a dense network of tubulin cytoskeleton comprised of thick bundles of intensively fluorescent MTs. In the case of a combined action of the studied factors, low temperatures abolished the hormone effect described above, evoking an increase in the content of the cytoskeletal and 60-kD phosphorylated proteins and MT structures. We suggest that the ABA-induced decrease in the levels of proteins and MTs occurs at the initial stages of plant cold acclimation (3°C, 2-3 days). It may be the signal that triggers the processes of low-temperature adaptation. As the duration of cold acclimation increased (3°C, 7 days), the role of ABA in the formation of plant tolerance decreased. Apparently, in this case other hormone-independent mechanisms of frost hardiness development are triggered, in which the role of the cytoskeleton components and cytoskeleton-associated proteins increases.     

Effect of oryzalin on root ultrastructure and respiration in various wheat cultivars subjected to cold hardening

The effect of anti-microtubular herbicide oryzalin (10 μM, 2–4 days) on the root ultrastructure and respiration in two cultivars of winter wheat (Triticum aestivum L.) contrasting in their frost-tolerance was studied during plant cold hardening (3°C, 3 days). The sensitivity of subcellular structures to oryzalin depended closely on cell metabolic activity and the extent of development of the cortical microtubule (MT) network. Most pronounced oryzalin-induced changes were related to enhanced cell vacuolation and the appearance of some signs of apoptosis (as judged from cytoplasm fragmentation) in some cells. In the root zone examined, cell heterogeneity increased, when, along with normally functioning cells, dramatically damaged and even completely destroyed cells appeared. Simultaneously, the activity of cyanide-resistant nonphosphorylating respiration pathway was activated, especially during cold hardening. In hardened cells, single cortical microtubules appeared in both wheat cultivars; this fact indicates that new cold-resistant subpopulations of MT were resistant to depolymerizing action of oryzalin.     

Magnetic resonance imaging (MRI) of water during cold acclimation and freezing in winter wheat

Nuclear magnetic resonance (NMR) spectroscopy and magnetic resonance imaging (MRI) were used to analyse changes in the physical state of water in wheat crowns during cold acclimation and during the freezing/thawing cycle. Spectroscopically measured average spin-spin relaxation times (T₂) decreased during cold acclimation and increased when plants were grown at normal temperature. Spin-spin relaxation images whose contrast is proportional to T₂, times were calculated allowing association of water relaxation with regions of tissue in spin-echo images during acclimation and freezing. Images taken during freezing revealed nonuniform freezing of tissue in crowns and roots. Acclimated and non-acclimated wheat crowns were imaged during freezing and after thawing. Spin-echo image signal intensity and T₂ times decreased dramatically between -4°C and -8°C as a result of a decrease in water mobility during freezing. Images collected during thawing were diffuse with less structure and relaxation times were longer, consistent with water redistribution in tissue after membrane damage.     

Influence of inhibitors of cytoskeleton proteins on water exchange of wheat roots under the after-action of water stress

The dynamics of water molecular state and transport in winter wheat (Triticum aestivum L.) of roots different resistance cultivars was studied by a biophysical method, Nuclear Magnetic Resonance (NMR), and a physiological method, Water-Holding Capacity (WHC). The effective coefficient of water self-diffusion (D(eff)), spin-spin relaxation times (T(2)) and WHC were measured after structural modification of cytoskeleton by colchicine and cytochalasin B after the action of water stress. New information about molecular mechanisms of water state and water transport regulation determined by the influence of dynamic cytoskeleton structure has been obtained. This is very important for the development of a fundamental theory of water exchange in plants, and for the ways of its optimization under conditions of environmental stress.     

Abscisic acid induced stress-like polyamine pattern in wheat seedlings, and its reversal by potassium ions

In 3-day-old wheat ( Triticum aestivum L. cv. Marinat) seedlings, 100 μ M ABA blocked the growth and altered the level of K⁺ in both the shoot and root. The presence of ABA increased the putrescine titer during a 24-h treatment. Increasing the endogenous level of K⁺ by the addition of 10 m M KCl to the ABA-treated seedlings, inhibited the effect of ABA on growth and putrescine level. In both tissues, ABA increased putrescine content at low concentrations (1 μ M ), reaching the maximal effect at 100 μ M . Putrescine increase induced by ABA was inhibited by both α-difluoromethylarginine (DFMA) and α-difluoromethylornithine (DFMO) in shoots while only the inhibitor of arginine decarboxylase was effective in the root. The presence of ABA modulated, in opposite ways, ornithine and arginine decarboxylase activities. These results are discussed in relation to ion balance under stress.     

The efficiency of transfer of plants cultivated in vitro to ex vitro conditions as affected by sugar supply

The greatest growth of wheat and rape plants in vitro was reached on media with 5 or 9 % sucrose, respectively. The highest efficiency for transfer of these plants to ex vitro conditions was found at the same sucrose concentrations. The content of endogenous non-structural saccharides (glucose, fructose, sucrose, starch and fructans) increased with increasing sucrose concentration in the medium up to 10 %. This revised version was published online in July 2006 with corrections to the Cover Date.     

Cultivar differences in the rate of nitrate uptake by intact wheat plants as related to growth rate

Six Argentinian wheat ( Triticum aestivum L.) cultivars grown in nutrient solutions in controlled environment were compared for their nitrate uptake rates on a root dry weight basis. Up to 3-fold differences were observed among the cultivars at 16, 20 and 24 days from germination, either when measured by depletion from the nutrient solution in short-term experiments, or by total N accumulation in the tissue during 8 days.
No differences in total N concentration in root or shoots were found among cultivars. Although the different cultivars showed significant differences in shoot/root ratio and nitrate reductase activity (EC 1.6.6.1) in the roots, none of these parameters was correlated with the nitrate uptake rate. However, nitrate uptake was found to be positively correlated (r = 0.99) with the shoot relative growth rate of the cultivars. The three cultivars with the highest nitrate uptake rates and relative growth rates showed a positive correlation between root nitrate concentration and uptake. However, this correlation was not found in the cultivars with the lowest growth and uptake rates.
Our results indicate that the difference in nitrate uptake rate among these cultivars may only be a consequence of their differences in growth rate, and it is suggested that at least two mechanisms regulate nitrate uptake, one working when plant demand is low and another when plant demand is high.     

Stomata response to changes in temperature and humidity in wheat cultivars grown under contrasting climatic conditions

Stomatal response to changes in temperature and humidity was studied in wheat (Triticum aestivum L.) cv. Iren’ cultivated under conditions of high water supply and cv. Kazakhstanskaya 10, which is relatively drought tolerant. Experiments were performed under both laboratory and field conditions. It was demonstrated that stomata of cv. Kazakhstanskaya 10 plants closed rapidly with reducing humidity (the response of the first type), whereas, in cv. Iren’, this response was less expressed and, under conditions of a high water content in soil, stomatal conductance could increase in response to reduced humidity (the response of the second type). At an increased stomatal conductance and transpiration, water content in cv. Iren’ plants was maintained due to the increase in hydraulic conductance and water inflow from the roots. A possible role of the first-type response (rapid stomata closure) for growth maintenance under drought and of the second-type response (a parallel increase in the stomatal and hydraulic conductance) for providing of rapid growth and high productivity under sufficient water supply is discussed. A possibility to use the type of stomata behavior for cultivar assessment is considered.     

ABA content in shoots and roots of pea mutants af and tl as related to their growth and morphogenesis

The comparative study of shoot and root growth was carried out, and the level of ABA therein determined in the mutant af and tl and wild-type isogenic lines of pea. The recessive af mutation transformed the leaflets into tendrils, and the tl mutation transformed the tendrils into leaflets. These mutations did not affect the length and number of internodes. In all plants, the level of ABA in the leaves was 3–10 times greater than in the roots, and in the course of vegetative growth it rose in both organs. An increase in the shoot area of tl mutant did not change the dry weight of underground and above-ground parts; therefore, the ratio shoot/root in the mutant was identical to that in the wild-type plants. The maintenance of shoot dry weight in the tl mutant at the level of wild-type plant while its area considerably increased was accounted for by a decrease in the thickness of the leaflet and stipule blades. The level of ABA in the stipules of mutant plants was greater than in the wild-type plants. A decrease in the shoot area in the af mutant brought about a decline in its dry weight; however, the ratio root/shoot was maintained at the wild-type level due to a reduced accumulation of dry weight by the root. The level of ABA in the roots of the af mutant was twice greater than in the leafy forms. ABA was assumed to participate in the control over the root growth exerted by the shoot. The absence of leaflets in the af plants was partially compensated for by expanding stipules. The level of ABA therein was three times higher than in the plants of wild type and comparable with the level in the leaflets of the tl mutant and in the wild-type plants. The role of ABA in the growth and final size of leaf blades is discussed.     

Alternation of plasma membrane lipids in aluminum-resistant and aluminum-sensitive wheat genotypes in response to aluminum stress

We have studied the effect of aluminum (A1) on lipid composition of plasma membranes from roots of an A1-resistant (PT741) and an A1-sensitive (Katepwa) cultivar of Triticum aestivum L. Several genotype–specific changes were observed in phospholipids and steryl lipids. While exposure to 20 μ M AICI₃ for 3 days had no effect on total phospholipids in either genotype, the most abundant phospholipid, phosphatidylcholine, increased significantly in the A1-sensitive Katepwa. Aluminum also decreased steryl lipids (mainly free sterols) in PT741. Such changes were not observed in Katepwa. As a result of differential changes in lipid composition, the relative abundance of one lipid class to another changed. The ratio of steryl lipids to phospholipids decreased in PT741, with no change in Katepwa. While limited information on the relationship between membrane function and lipid composition makes it difficult to relate these changes to A1 toxicity and resistance, changes observed only in the A1–resistant genotype could contribute to continued plant growth in the face of A1 stress.     

Phosphatase induction in roots of winter wheat during adaptation to phosphorus deficiency

The effect of phosphate supply during the first 4 weeks of the life cycle of wheat ( Triticum aestivum L. cv. Martonvásári-8) was investigated by following growth of seedlings, P levels in roots and shoots, changes of soluble phosphatases in roots and alteration of Ca²⁺ - and Mg²⁺-ATPase activity in the microsomal fraction. Plants were grown in complete nutrient solution supplemented with different levels of phosphate. Maximal growth rate was attained at 0.2 m M phosphate. The total P level in plants increased with increasing phosphate concentration in the growth solution, however, it decreased with age. Microsomal ATPase activity in 14-day-old plants increased with phosphorus deficiency. Using phosphocellulose column chromatography, a phosphatase (EC 3.1.3.2) induced by phosphorus deficiency was purified and partially characterized from the 30 000 g supernatant from roots of 14- to 30-day-old wheat plants. Na-pyrophosphate, p -nitrophenylphosphate, ATP, ADP, AMP, O-phosphoryl- l -serine and glucose-6-phosphate were all substrates for the enzyme. Its native molecular weight was 42 kDa as determined by Sephadex G-200 column chromatography. Readdition of phosphate to the growth solution resulted in a gradual decrease of the phosphatase activity, probably due to repression of its synthesis. We hypothesize that the extra phosphatase may participate in the adaptation mechanism under phosphorus-deficient conditions.     

Redox changes during cold acclimation affect freezing tolerance but not the vegetative/reproductive transition of the shoot apex in wheat

Cold acclimation is necessary for winter wheat (Triticum aestivum L.) to achieve its genetically determined maximum freezing tolerance, and cold also fulfils the vernalisation requirement. Chromosome 5A is a major regulator of these traits. The aim of the present study was to discover whether changes in the half‐cell redox potential of the glutathione/glutathione disulphide (GSH/GSSG) and ascorbate/dehydroascorbate (AA/DHA) couples induced by cold acclimation are related to freezing tolerance and vernalisation requirement in a specific genetic system including chromosome 5A substitution lines. The amounts of H₂O₂ and AA, and the AA/DHA ratio showed a rapid and transient increase in the crown of all genotypes during the first week of acclimation, followed by a gradual increase during the subsequent 2 weeks. The amount of GSH and its ratio compared to GSSG quickly decreased during the first day, while later these parameters showed a continuous slow increase. The H₂O₂, AA and GSH concentrations, AA/DHA and GSH/GSSG ratios and the half‐cell reduction potential of the GSH/GSSG couple were correlated with the level of freezing tolerance after 22 days at 2 °C; hence these parameters may have an important role in the acclimation process. In contrast to H₂O₂ and the non‐enzymatic antioxidants, the lipid peroxide concentration and activity of the four antioxidant enzymes exhibited a transient increase during the first week, with no significant difference between genotypes. None of the parameters studied showed any relationship with the vegetative/generative transition state monitored as apex morphology and vernalisation gene expression.