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.     

Morphological and physiological changes in roots of various wheat genotypes as related to cytoskeleton disruption

A depolymerizing effect of anti-microtubule drug oryzalin on the roots of three winter wheat (Triticum aestivum L.) cultivars contrasting in their frost-resistance was studied. The influence of plant cold acclimation (3°C, 7 days) and ABA treatment (30 μM) on oryzalin action was evaluated. Plant growing in the presence of 10 μM oryzalin under optimum temperature of 23°C resulted in the root-length decrease by 19–24% and root-apex swelling. All cells, especially in the root cortex, changed their radial dimensions. The cells acquired a rounded or irregular shape and increased in size. This indicates the loss of correct cell growth polarity. Most pronounced changes in the root apex diameter and most severe linear growth suppression were observed in the cultivar of moderate frost-resistance. The roots of this cultivar contained the highest amounts of actin and tubulins, as was evident from the immunoblot analysis. The effect of oryzalin on root growth and apex swelling was correlated with the content of actin in the roots of different wheat cultivars. Cold acclimation and exogenous ABA reduced (or prevented) oryzalin action on roots in a cultivar-specific manner. The conclusion was made that the bulk of the cytoskeletal net determined the efficiency of the cytoskeletal control of plant growth and morphogenesis. During autumn and winter periods, this is important for a better adaptation to temperature fluctuations of moderately frost-resistant plants, which are characterized by a high ecological plasticity.     

Lectin and mitotic activities in root meristems of winter wheat under oryzalin effect

The effect of oryzalin, a microtubule polymerization inhibitor (10 MM), on lectin and mitotic activities (mitotic index and duration of mitotic phases) was studied in unhardened (23 degrees C) and hardened (7 days, 2-3 degrees C) winter wheat seedlings. Three wheat cultivars differing in their frost tolerance were compared. Oryzalin treatment (3 h) decreased activity of soluble lectins, increased activity of cell wall lectin mitotic index. Under these conditions, prolongation of anaphases and disappearance of telophases were detected. Plant hardening reduced the sensitivity of cell wall lectins and mitotic activity to the cytoskeleton inhibitor due, presumably, to the appearance of cold-stable microtubules. Plant growing and hardening with oryzalin stopped mitoses and caused the appearance of polyploid cells and cells with micronuclei. These abnormalities were preserved after hardening. The results obtained demonstrate an important role of microtubules in adaptation of plants to low temperature.     

Effect of cartolin on oryzalin-induced changes in lectin activity during low-temperature plant hardening

The effect of cartolin (0.33 μM), an antistress regulator of cytokinin type, on the cytoskeleton-dependent changes in lectin activity in the roots of unhardened (23°C) and cold-hardened (3°C, 7 days) 7-day-old plants of three cultivars of winter wheat (Triticum aestivum L.) was studied. In unhardened plants, cartolin increased activity of soluble and cell wall-bound lectins in a cultivar-specific mode. This is evidently important for subsequent enhancement of adaptation processes in the cell. The inhibitor of microtubule polymerization, oryzalin, reduced the activity of soluble lectins and increased that of cell wall-bound lectins. A reduced sensitivity of lectin activity to oryzalin after cartolin treatment could result from its stabilizing action on the cytoskeletal structures and on the interaction between cell-wall lectins and microtubules. The most efficient cartolin action, the suppression of oryzalin effect on lectin activity in hardened plants, was observed in the frost-sensitive wheat cultivar. It is likely that cartolin treatment is more efficient in the activation of adaptation processes occurring with the involvement of cytoskeletal structures in the cultivars of lower tolerance.     

The effect of low temperature on the microtubules in root meristem cells of spring and winter cultivars of wheat <Emphasis Type="Italic">Triticum aestivum L.</Emphasis>

We have studied the response of interphase and mitotic microtubule arrays in root meristem cells of spring and winter cultivars of wheat Triticum aestivum L. (Moskovskaya 35 and Moskovskaya 39) to cold stress (1 h at 0°C) and acclimation to cold (3–48 h at 0°C). We show that, in general, interphase microtubules are more resistant to cold then mitotic arrays in both cultivars. During cold stress, no changes are detected in the microtubule system of interphase cells of spring wheat, whereas the density of endoplasmic microtubules increases in interphase cells of winter wheat. During mitosis, the density of the kinetochore fibers of the spindle decreases in the cells of both cultivars, but it is prevailing in the cells of spring cultivar of wheat. During acclimation to cold, the disorganization of the cortical microtubule bundles and the enhanced growth of the endoplasmic microtubule network, which is comprised of microtubule converging centers, are observed in cells of both cultivars. However, the mitotic microtubule systems of winter and spring cultivars respond differently to cold acclimation. During prophase, a diffuse tubulin “halo,”followed by the assembly of microtubule converging centers, accumulate at the perinuclear area in the cells of winter wheat. In cells of spring cultivar, the prophase spindle is only detected during initial stages of cold acclimation. During metaphase, aberrant mitotic spindles, abnormal metaphase plates, and the excessive appearance of microtubule converging centers are observed in cells of both cultivars. Acclimation induces the disorganization of the phragmoplast and the formation of multiple microtubule converging centers during telophase in the cells of both cultivars. Microtubule converging centers are detected at the perinuclear area of daughter cells in winter wheat and in the cortical cytoplasm in spring wheat. The excessive formation of microtubule converging centers suggests the activation of microtubule assembly during prolonged exposure to low temperature. Our data also demonstrates common pathways of microtubule response to cold treatment (0°C).     

The relationship between cell injury and osmotic volume reduction: III. Freezing injury and frost resistance in winter wheat

In order to distinguish between several possible mechanisms of frost hardening in winter wheat (Triticum aestivum L.) cells from two hardy and two tender cultivars were plasmolyzed in CaCl₂ solution at room temperature and cell volumes estimated by microscopic examination. Analyses of Boyle-van't Hoff plots of these data reveal that all cells from cultivars progressively increase their intracellular solute concentration up to 20 days hardening. This increase, which we had predicted from published calorimetric data to be the sole mechanism of hardening explained less than half of the increase in hardening seen in the most hardy cultivar, Kharkov. Hardening also increased the osmotically inactive volume.At CaCl₂ concentrations greater than 5%, plasmolyzed protoplasts departed further from the Boyle-van't Hoff prediction, remaining larger than expected until some higher concentration of CaCl₂, where protoplast volume again sharply decreased. In all cultivars except hardened Kharkov, the concentration of CaCl₂ producing this abrupt volume decrease had a freezing point corresponding to the killing temperature. If this concentration was exceeded during plasmolysis, then the protoplasts burst during deplasmolysis at some volume less than their original volume.We interpret these data to mean that, in addition to the often described hardening mechanism of increased cell solute and water binding, winter wheat shows a third mechanism, a mechanical resistance to protoplast shrinkage which produces volumes larger than those predicted during osmotic stress. The resisting element appears to be the plasma membrane itself. Shrinkage brings the membrane under compressive stress, developing tangential pressure within it. Cell injury occurs when the cell membrane area has been reduced to the point at which irreversible loss of membrane material is inevitable. Cell death occurs during deplasmolysis when the protoplast bursts because its membrane contains insufficient material to subtend the area of the cell wall.Of the cultivars tested, hardened Kharkov was unique in avoiding injury. Hardened Kharkov was injured only after the volume inflection had been greatly exceeded. Refractile droplets of lipid appeared in the cytoplasm of hardened Kharkov protoplasts during plasmolysis but disappeared during deplasmolysis suggesting that hardy Kharkov was able reversibly to store membrane lipids in cytoplasmic vesicles and return them to the membrane on deplasmolysis.     

微管骨架在轮藻节间细胞伸长生长中的作用

利用免疫荧光定位及激光共聚焦扫描显微镜,结合细胞生长曲线的定量测定,对不同生长阶段的轮藻节间细胞微管骨架进行了观察研究,结果如下：轮藻顶端生长活跃的新生细胞中,与细胞长轴垂直的周质微管(cortical microtubules)占绝对优势,随着生长速率的减慢,周质微管由垂直于细胞长轴逐渐转为平行排列;基部生长基本停止的节间细胞中,胞内微管则以平行细胞长轴为主;不同生长阶段节间细胞的微管骨架,对微管特异解聚剂黄草消(oryzalin)处理的敏感性表现不相同。顶端生长活跃的节间细胞经oryzalin处理40min后,绝大多数周质微管发生解聚;而基部生长基本停止的老细胞中,即使延长处理时间,仍残留一些尚未完全解聚的微管片段;10μmol／L微管解聚剂oryzalin处理轮藻顶端新生细胞,在高精度的细胞伸长生长测定装置监测下,发现oryzalin对细胞的伸长生长速率有明显的抑制作用,去掉药剂后,伸长生长又有一定的恢复。并且发现,经oryzalin处理后,微管的解聚(40min左右)与顶端节间细胞伸长生长的停止(100min左右)两者间存在着时间上的差异,即微管解聚在先,细胞伸长停止在后。以上结果均说明微管骨架在轮藻节间细胞生长中具有重要作用。     

Cytoskeleton-induced alterations of the lectin activity in winter wheat under cold hardening and abscisic acid (ABA)

The roots and leaves of 7-day seedlings of three winter wheat cultivars differing in frost resistant were used to study changes in lectin activity under cytoskeleton modifiers (DMSO-7%; colchicine-1 m m; oryzalin-15 microm; cytochalasin B-15 microm) of non-hardened (23 degrees C) and hardened (2-3 degrees C, 3-7 day) plants. Plants were grown with ABA (30 microm) or without ABA. Pretreatment with colchicine, oryzalin [inhibitors of microtubules (MT) polymerization], cytochalasin B [inhibitor of microfilament (MF) polymerization] increased the activity of cell wall lectins, although pretreatment with DMSO (stabilizer of microtubules) decreased the activity. Both hardening and ABA decreased the effect of the cytoskeletal modifiers. These results could be explained by the appearance of tolerant MTs with less affinity. It is probable that increase in the activity of cell wall lectins may be the compensatory mechanism which stabilizes the cytoskeleton structure in conditions tending to disrupt it. The genotype with low resistance had higher sensitivity of lectin activity to cytoskeleton modifiers than the frost resistant genotype. The results suggest that leaves have more stable MTs and MFs and stronger MT-MF binding than roots.     

Reorganization of the tubulin and actin cytoskeleton under acclimation and abscisic acid treatment of Triticum aestivum L. plants

Only scanty and contradictory data are available concerning effects of low temperatures and ABA on the structural organization of microtubules (MTs) and microfilaments (MFs), and no information exists on the interaction of these parameters at cold acclimation of plants. Therefore, in cold acclimate and ABA-treated winter wheat plants, a comparative study was made of the state (localization, orientation, structure) and stability of actin and tubulin cytoskeleton in root cells taken from different zones, using indirect immunofluorescent microscope. The plant cold acclimation caused MT aggregation, the rise of MT and MF fluorescence, and the increase of their stability (a decrease of oryzalin effect) mainly in the root differentiation zone, that may testify to the strengthening of contacts between MTs and MFs. Like the cold acclimation, ABA induced the formation of MT bunches only in meristem and elongation zone cells. However in the zone of differentiation, the hormone stimulated the increase of tubulin structure stability, well correlating with a decrease in MT content, aggregation degree, and immunofluorescence, and, in addition with a complete depolymerization of MFs. Low temperatures removed the hormone effect on the structural organization of tubulin and actin cytoskeleton in the zone of differentiation. It is suggested that MT destruction, the decrease of instable MT populations, and the increase of stable MT populations may slow down growth processes in ABA-treated plants, similarly as in seedlings being on the initial stages of cold acclimation. By the end of this process, the induction of plant growth is determined evidently by the increase in the number of instable, highly labile MT populations, and in the status of MF polymerization.     

The effect of low temperature on the microtubules in root meristem cells of spring and winter cultivars of wheat Triticum aestivum L

We have studied the response of the interphase and mitotis microtubule arrays in root meristem cells of spring and winter cultivars of wheat Triticum aestivum L. (Moskovskaya 35 and Moskovskaya 39) during cold stress (1 h at 0 degrees C) and acclimation to cold (3-48 h at 0 degrees C). Our data show that interphase microtubules are more resistant to cold than mitotic arrays in both cultivars. During cold stress the density of endoplasmic microtubules increases in interphase cells of winter plants, yet no changes are detected in cells of spring plants. In mitotic cells of both wheat cultivars the density of microtubules within the kinetochore fibers decreases, yet this effect is more evident in the cells of spring plants. During acclimation to cold of both cultivars, we have observed the disorganization of the interphase cortical arrays and the enhanced growth of endoplasmic microtubule arrays, composed of microtubule converging centers. However, the reaction of mitotic microtubule arrays differs in the cells of winter and spring plants. In winter plants, during prophase diffuse tubulin "halo" accumulates first at perinuclear area, followed by the appearance of the microtubule converging centers. In spring plants, we have observed the formation of the prophase spindle, yet later the prophase spindle is not detected. Metaphase cells of both cultivars show similar aberrations of the mitotic spindle, accumulation of abnormal metaphases and the excessive formation of microtubule converging centers. In telophase cells of both cultivars, acclimation induces similar reaction, resulting in the disorganization of the phragmoplast and the formation of multiple microtubule converging centers. The latter are detected in the perinuclear areas of the daughter cells in winter plants and in the cortical cytoplasm of cells in spring plants. Our data point to the common pathways of microtubule response to cold treatment (0 degrees C). The excessive formation of the microtubule converging centers indicates the activation of microtubule assembly during prolonged cold treatment.     

Impact of taxol-mediated stabilization of microtubules on nuclear morphology,ploidy levels and cell growth in maize roots

Direct contact of the radiating perinuclear microtubules (MTs) with the nuclear envelope was visualized with an immunogold technique using specific monoclonal tubulin antibody. The possibility that these perinuclear MT arrays are involved in establishing and maintaining nuclear organization during the interphase of cycling cells in maize root meristems was tested using taxol, a MT-stabilizing agent. Taxol not only stabilized all MTs against the action of the MT-disrupters colchicine and oryzalin but also prevented these agents from their usual induction of nuclear enlargement and decondensation of nuclear chromatin. On the contrary, nuclear size decreased and the chromatin became more compact in mitotically cycling cells of the taxol-treated root apices. Moreover, taxol prevented the stimulation, by colchicine and oryzalin, of the onset of the S phase in cells of the quiescent centre and proximal root meristem. Exposure of maize roots to taxol strongly decreased final cell volumes, suggesting that the more condensed nuclear chromatin is less efficient in genome expression and that this accounts for the restriction of cellular growth. All these findings support the hypothesis that MT arrays, radiating from the nuclear surface, are an essential part of an integrated plant ‘cell body’ consisting of nucleus and the MT cytoskeleton, and that they regulate, perhaps via their impact on chromatin condensation and activity, progress through the plant cell cycle.     

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

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

Mitochondrial energy-dissipating systems (alternative oxidase,uncoupling proteins,and external NADH dehydrogenase) are involved in development of frost-resistance of winter wheat seedlings

Gene expression, protein synthesis, and activities of alternative oxidase (AOX), uncoupling proteins (UCP), adenine nucleotide translocator (ANT), and non-coupled NAD(P)H dehydrogenases (NDex, NDPex, and NDin) were studied in shoots of etiolated winter wheat (Triticum aestivum L.) seedlings after exposure to hardening low positive (2°C for 7 days) and freezing (?2°C for 2 days) temperatures. The cold hardening efficiently increased frost-resistance of the seedlings and decreased the generation of reactive oxygen species (ROS) during further cold shock. Functioning of mitochondrial energy-dissipating systems can represent a mechanism responsible for the decrease in ROS under these conditions. These systems are different in their response to the action of the hardening low positive and freezing temperatures. The functioning of the first system causes induction of AOX and UCP synthesis associated with an increase in electron transfer via AOX in the mitochondrial respiratory chain and also with an increase in the sensitivity of mitochondrial non-phosphorylating respiration to linoleic and palmitic acids. The increase in electron transfer via AOX upon exposure of seedlings to hardening freezing temperature is associated with retention of a high activity of NDex. It seems that NDex but not the NDPex and NDin can play an important role in maintaining the functional state of mitochondria in heterotrophic tissues of plants under the influence of freezing temperatures. The involvement of the mitochondrial energy-dissipating systems and their possible physiological role in the adaptation of winter crops to cold and frost are discussed.     

Role of cytoskeleton in cell shaping of developing mesophyll of wheat (Triticum aestivum L.).

The effects of oryzalin and cytochalasin B (CB) on microtubule and actin microfilament arrays and on cell shaping were investigated in developing wheat mesophyll. Excised immature leaf sections capable of differentiating were incubated with the drugs. The behavior of the cytoskeleton was monitored by fluorescence microscopy after labeling with fluorescent dyes. Brief incubation with oryzalin (40 min, 10 microM) caused disassembly of microtubules. Recovery of microtubule arrays was comparatively slow after removal of the drug. Cells failed to establish transverse cortical bands of microtubules and transverse hoops of wall reinforcement. They expanded isodiametrically rather than longitudinally without forming lobes typical of wheat mesophyll cells. Brief treatment with CB (60 min, 20 micrograms ml-1) appeared to disrupt the microfilament arrays. Filaments recovered rapidly after removal of CB, and cells were able to shape in an apparently normal fashion. Continuous incubation at comparatively low concentration of CB (4 micrograms ml-1) appeared to cause selective loss of the fine transverse cortical microfilament arrays. Cortical transverse microtubule arrays persisted, but failed to form distinct bands in the majority of the cells. Cells were able to elongate in an almost normal fashion, but no lobes were formed.     

Changes in the activity of proteases and trypsin inhibitors in wheat leaves in the initial period of cold hardening and subsequent dehardening

The dynamics of amidase, cysteine protease, and trypsin inhibitor activities were studied in the leaves of wheat (Triticum aestivum L.) seedlings grown under controlled conditions (25°C, illuminance 10 kLx, 14-h photoperiod) and subjected to cold hardening (5°C, 10 kLx, 14-h photoperiod). Changes in the activity of amidases and cysteine proteases proved to precede an increase in cold resistance during cold hardening and a decrease in cold resistance after the end of cold hardening. The activity of trypsin inhibitors changed only during cold hardening. It is suggested that amidases, cysteine proteases, and trypsin inhibitors are involved in the cold adaptation of plants.     

Nod factors alter the microtubule cytoskeleton in Medicago truncatula root hairs to allow root hair reorientation

The microtubule (MT) cytoskeleton is an important part of the tip-growth machinery in legume root hairs. Here we report the effect of Nod factor (NF) on MTs in root hairs of Medicago truncatula. In tip-growing hairs, the ones that typically curl around rhizobia, NF caused a subtle shortening of the endoplasmic MT array, which recovered within 10 min, whereas cortical MTs were not visibly affected. In growth-arresting root hairs, endoplasmic MTs disappeared shortly after NF application, but reformed within 20 min, whereas cortical MTs remained present in a high density. After NF treatment, growth-arresting hairs were swelling at their tips, after which a new outgrowth formed that deviated with a certain angle from the former growth axis. MT depolymerization with oryzalin caused a growth deviation similar to the NF; whereas, combined with NF, oryzalin increased and the MT-stabilizing drug taxol suppressed NF-induced growth deviation. The NF-induced disappearance of the endoplasmic MTs correlated with a loss of polar cytoarchitecture and straight growth directionality, whereas the reappearance of endoplasmic MTs correlated with the new set up of polar cytoarchitecture. Drug studies showed that MTs are involved in determining root hair elongation in a new direction after NF treatment.     

Cytomorphogenesis in cenocytic green algae. V. Segregative cell division and cortical microtubules in Dictyosphaeria cavernosa (Siphonocladales,Chlorophyceae)*

Disassembly and reassembly of cortical microtubules (MT) during and after segregative cell division (SCO) in Dictyosphaeria cavernosa (Forssk.) Børgesen were observed using fluorescence microscopy. Parallel cortical MT in a mother cell were intact just after the initiation of SCD, but soon circular, MT-free patches appeared. Protoplasmic contraction enlarged the patches, and in these areas, the protoplasm eventually became perforated. Long and undulating cortical MT were arranged densely in the reticulate protoplasm. During further protoplasmic contraction, cortical MT appeared to be random and decreased in density. Finally, short and random cortical MT were present in the segregated protoplasts. Parallel cortical MT reassembled in the expanding daughter cells. After the daughter cells came in contact with one another, a radial system of cortical MT was constructed at the side that faced the inside of the mother cell wall. A microtubule inhibitor (amiprophos methyl, APM) had no effect on SCO. Segregative cell division was not induced directly by mechanical wounding. A comparison between SCO and wound-induced protoplasmic contraction was made.     

Cell-wall lectins during winter wheat cold hardening

The polypeptide composition and functional activity of cell-wall lectins from roots of winter wheat (Triticum aestivum L., cv. Mironovskaya 808) seedlings during cold hardening were studied. Several phases of lectin activity changes were observed, which indicates their involvement in the development of general adaptation syndrome of the cell. After 0.5-h low-temperature treatment, marked alterations occurred in the profile of protein elution: lectins with mol wts of 78 and 42.5 kD disappeared and new ones with mol wts of 72, 69, 37, and 34.5 kD appeared. It was established that 17.5-and 69-kD lectins and most lectins eluted with glucose were arabinogalactan proteins (AGP), which permitted a supposition that these lectins were involved in the interaction between the cell wall and cytoskeleton. After 7-day-long hardening, total protein content reduced and lectins with mol wts of 69 and 37 kD disappeared, which corresponded to reduced lectin activity by the end of hardening. A transient appearance of 37-and 69-kD lectins, which are AGP, might indicate their involvement in the triggering the development of plant-cell defense responses.