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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Uptake of calcium in wheat and cucumber roots

Uptake of Ca²⁺(⁴⁵Ca) was investigated in plants of wheat ( Triticum aestivum L. var. Svenno) and cucumber ( Cucumis sativus L. var. Cilla) cultivated in a nutrient solution with various Ca²⁺ concentrations. The adsorption of Ca²⁺ was higher in cucumber roots than in wheat roots especially at lower Ca²⁺ levels in the external medium. The intracellular fraction of Ca²⁺ was less than 20% of the total Ca²⁺ in wheat roots and less than 10% of the total Ca²⁺ in cucumber roots. The uptake of Ca²⁺ in cucumber was about 40 times higher than in wheat. Transport of Ca²⁺ in the roots towards the endodermis is suggested to take place mainly in the apoplastic pathway regulated by the availability of negatively charged binding sites along the cell wall continuum. Further transport of Ca²⁺ towards the stele may involve diffusion of Ca²⁺ into the symplasm in the vicinity of the endodermis. An active extrusion of Ca²⁺ towards the stele or towards the external medium is suggested to play a role in the regulation of Ca²⁺ uptake.     

Influence of low temperature on regulation of Rb+ and Ca2+ influx in roots of winter wheat

Influx of Rb⁺(⁸⁶Rb⁺) and Ca²⁺(⁴⁵Ca²⁺) was determined in roots of winter wheat (Triticum aestivum L. cv. Weibulls Starke II) after 14 days at 16°C/16 h light, after 1 and 8 weeks of cold acclimation (2°C/8 h light) and at intervals after deacclimation (16°C/16 h light) for up to 14 days. The plants were cultivated at 3 ionic strengths: 100, 10 and 1% of a full strength nutrient solution, containing 3.0 mM K⁺ and 1.0 mM Ca²⁺. K⁺ concentrations in roots and shoots increased during cold treatment, while Ca²⁺ in the roots decreased. In the shoots Ca²⁺ concentrations remained the same. Influx of Rb⁺ as a function of average K⁺ concentration in the roots of 14-day-old, non-cold-treated plants was high at a certain K⁺ level in the root and decreased at higher root K⁺ levels (negative feedback). The pattern for Ca²⁺ influx versus average concentration of Ca²⁺ in the root was the reverse. Independent of duration of treatment (1–8 weeks), cold acclimation partly changed the regulation of Rb⁺ influx, so that it became less dependent upon negative feedback and more dependent on the ionic strength of the cultivation solution. After exposure to 2°C, Ca²⁺ influx increased at high Ca²⁺ concentrations in the root as compared with influx in roots of 14-day-old non-cold-treated plants. Under deacclimation, Ca²⁺ influx gradually decreased again, and reached the level observed before cold treatment within 7–14 days at 16°C; the number of days depending on the exposure time at 2°C. It is suggested that Rb⁺(K⁺) influx became adjusted to low temperature and that abscisic acid (ABA) may be involved in this mechanism. It is also suggested that extrusion of Ca²⁺ was impaired and/or Ca²⁺ channels were activated at 2°C in roots of plants grown in the full-strength solution and that extrusion was gradually restored and/or Ca²⁺ channels were closed under deacclimation conditions.     

Interactions among factors regulating phenological development and acclimation rate determine low-temperature tolerance in wheat

BACKGROUND AND AIMS: Exposure to low temperatures (LT) produces innumerable changes in morphological, biochemical and physiological characteristics of plants, with the result that it has been difficult to separate cause and effect adjustments to LT. Phenotypic studies have shown that the LT-induced protective mechanisms in cereals are developmentally regulated and involve an acclimation process that can be stopped, reversed and restarted. The present study was initiated to separate the developmental factors determining duration from those responsible for rate of acclimation, to provide the opportunity for a more in depth analysis of the critical mechanisms that regulate LT tolerance in wheat (Triticum aestivum). METHODS: The non-hardy spring wheat cultivar 'Manitou' and the very cold-hardy winter wheat cultivar 'Norstar' were used to produce reciprocal near-isogenic lines (NILs) in which the vrn-A1 (winter) alleles of 'Norstar' were inserted into the non-hardy 'Manitou' genetic background and the Vrn-A1 (spring) alleles of 'Manitou' were inserted in the hardy 'Norstar' genetic background so that the effects of duration and rate of LT acclimation could be quantified. KEY RESULTS: Comparison of the acclimation curves of the NILs and their parents grown at 2, 6 and 10 degrees C established that the full expression of LT-induced genetic systems was revealed only under genotypically dependent optimum combinations of time and temperature. Both duration and rate of acclimation were found to contribute significantly to the 13.8 degrees C difference in lowest survival temperature between 'Norstar' and 'Manitou'. CONCLUSIONS: Duration of LT acclimation was dependent upon the rate of phenological development, which, in turn, was determined by acclimation temperatures and vernalization requirements. Rate of acclimation was faster for genotypes with the 'Norstar' genetic background but the ability to sustain a high rate of acclimation was dependent upon the length of the vegetative stage. Complex time/temperature relationships and unexplained genetic interactions indicated that detailed functional genomic or phenomic analyses of natural allelic variation will be required to identify the critical genetic components of a highly integrated system, which is regulated by environmentally responsive, complex pathways.     

The coleoptile sheath of wheat impedes oxygen transport to seminal roots

Abstract Wheat, Triticum aestivum cv. Gamenya, seedlings were grown for 4d in acrated 0.5 mol m^?3 CaSO₄ solution. A cylindrical platinum-electrode and vernier microscope were used to examine the effects of the colcoptile sheath on O₂ transport from shoots to seminal roots via the internal gas-space system, and on the clongation of the root tip. By removing the colcoptile sheath, O₂ concentrations at the root tip in an O₂-free medium at 5°C increased from 0.017 to 0.17 mol m^?3 O₂ when 100 kPa O₂ was around the shoots. When the shoots were in air, the separation of the colcoptile sheath from the primary leaf caused the rate of root clongation in the anoxic medium to increase by two fold.     

Effects of donor plant environmental conditions on immature embryo cultures derived from worldwide origin wheat genotypes

The influence of donor plant growth conditions in years 2003, 2004, and 2005 on tissue culture response (TCR) traits of 96 wheat genotypes was evaluated. Immature embryos, collected 12–15 days after anthesis from field-grown plants, were cultured on Murashige and Skoog medium containing 2 mg/l 2,4-D followed by their transfer to a growth regulator-free medium. Donor plants growth in a season 2003 characterized by drought and heat stress, as compared to favorable growth seasons 2004 and 2005, resulted in a increased variability and a decreased percentage of callus formation and the number of regenerating calli and plants regenerated per embryo in all genotypes, except genotype Florida that exhibited a significantly increased number of regenerating calli in 2003. The reduction rate of regenerating callus formation depended on genotype, and it was the highest in Magnif 41, NS66/92, and Mexico 3. The results suggest that the impact of donor plant growth conditions on TCR traits can be as large as the effect of the genotype, which should be taken into account when planning transformation work with wheat. Published in Russian in Fiziologiya Rastenii, 2009, vol. 56, No. 4, pp. 596–602. This text was submitted by the authors in English.