Search

The development of methods for long-term storage of microalgae remains an important and complex problem, the solution of which is impossible without studying the processes occurring during dehydration and rehydration of vegetative cells. The study presents the results from the investigation of changes in the dimensional characteristics of cells and trichomes of spirulina Arthrospira (Spirulina) platensis (Nordstedt) Gomont after dehydration and during reactivation (in dynamics) as well as processes occurring in them during reactivation. Methods close to natural dehydration were selected. Morphometric measurements of cells and trichomes of spirulina in their native state and during dehydration and rehydration were performed. Dehydration of A. platensis was accompanied by statistically significant changes in the morphometric characteristics of cells. For all methods of dehydration (in a thermostat at 30 and 60°C and on silica gel in a desiccator at a room temperature of 20°C) a decrease in their size, as well as elongation and flattening, were noted. The thickness and volume of trichomes after dehydration were reduced by two times. All processes occurring during reactivation were traced in dynamics, from the moment of moistening to the appearance of young threads. A relatively rapid flooding of the dried cells and a gradual restoration of their morphometric characteristics was noted. Among the reactivating trichomes, those in which the separation into viable and irreversibly areas damaged was clearly visible were found. Differential staining of trichomes with methylene blue revealed cells with strong plasmolysis inside externally intact threads. Morphometric parameters of the spirulina cell were restored within 24 h. This fact indicates that, in dehydrated cultures after rehydration, a number of complex biochemical processes occur, due to which the initial physiological state of the cells is restored.

Downy mildew is the most destructive disease of grapevines in the regions of relatively warm and humid climate causing up to 50% yield losses. Application of silicon- (Si-) based products have been extensively studied against various oomycete, fungal, bacterial, and viral plant diseases, but studies on Si application in their nanosize are limited. In this study, the field application of silica nanoparticles (SiNPs) on Thompson Seedless grapevines (H4 strain) infected with downy mildew was evaluated. In addition, molecular, physiological, ultrastructural, and toxicity investigations were also conducted. The obtained results revealed that spraying of grapevines with SiNPs at 150 ppm significantly overexpressed the transcription factor jasmonate and ethylene-responsive factor 3 recording 8.7-fold, and the defense-related genes β-1,3-glucanase (11-fold), peroxidase (10.7-fold) pathogenesis-related-protein 1 (10.6-fold), and chitinase (6.5-fold). Moreover, a reduction up to 81.5% in the disease severity was achieved in response to this treatment. Shoot length and yield per grapevine were considerably enhanced recording up to 26.3 and 23.7% increase, respectively. The berries quality was also improved. Furthermore, this treatment led to an enhancement in the photosynthetic pigments, induction of phenolic and ascorbic acid contents, an increase in the activity of peroxidase and polyphenol oxidase enzymes, and a reduction in the cellular electrolyte leakage, lipid peroxidation, and H2O2 content. Scanning electron microscopy observations showed an increase up to 86.6% in the number of closed stomata and a reduction up to 55% in the average stomatal pore area in response to this treatment. Observations of the transmission electron microscopy showed ultrastructural alterations in the cells of a grapevine leaf due to the infection with downy mildew, including plasmolysis and disruption of the cellular components, abnormal chloroplasts, and thickening of the cell wall and cell membrane. These abnormal alterations were reduced in response to SiNPs spray. In contrast, this study also showed that this treatment had considerable cytotoxic and genotoxic effects at this direct dose/concentration. So, additional investigations to determine the SiNPs residue in the produced edible plant parts are urgently needed. In addition, the pre-harvest interval, toxicity index, and risk assessment should be evaluated before any recommendation for use.

The alleviation effects of exogenous putrescine treatment on the ultrastructure of and calcium ion flow rate in lettuce (Lactuca sativa L.) leaf cells under drought stress were studied. Lettuce seedlings were treated with foliar sprays of 0.1 mM putrescine for 8 days, after which drought stress was simulated by using 10% polyethylene glycol 6000. The morphological characteristics of the seedlings and the calcium ion flow rate across stomatal guard cells were subsequently determined, and the leaf cell ultrastructure was observed via transmission electron microscopy. Under drought stress, the morphological characteristics of the seedlings decreased, and calcium ion influx was predominant in the guard cells. In addition, compared to that under control conditions, the stomatal density under drought stress conditions increased significantly, the open/closed stoma ratio was lower, and the degree of stomatal opening was smaller. Exogenous putrescine sprays effectively reduced the stomatal density, increased the degree of stomatal opening, and increased the proportion of open stomata. In addition, the chloroplasts became round in shape, the thylakoid structure became blurry in appearance, the number of starch grains decreased, many osmium granules were produced, and plasmolysis occurred in the mesophyll cells. However, the chloroplasts were elongated, the thylakoid structure was clear, the starch grains were abundant, few osmium granules were produced, and plasmolysis did not occur. The above results show that, by altering the leaf cell ultrastructure as well as the flow rate and direction of calcium ions in guard cells, exogenous putrescine effectively improves the drought tolerance of lettuce.

Grape anthracnose, caused by Elsinoe ampelina, is an important fungal disease during grape production. Little is known about the interactions of grape-E.ampelina compared with other grapevine fungal diseases. This study used potted seedlings of Vitis vinifera ‘Red Globe’ and E. ampelina to investigate their interactions under greenhouse conditions. The infection process of E. ampelina in the young leaves of grape was observed from 0 to 6 days post-inoculation (dpi) under microscope. Results showed that the first symptom on grape leaves were observed at 3 dpi. The rates of conidial germination on grape leaves were 23.4, 73.2, and 85.6% at 1, 2, and 3 dpi, respectively. Early penetration occurred in the epidermal cells with or without forming appressoria at 2 dpi. E. ampelina enlarged in intercellular spaces at 2–3 dpi and began to enter intracellular spaces from 3 dpi. New conidia were produced in acervuli on the leaf surface from 6 dpi. The ultrastructural observation revealed defense responses in infected host cells, including collapse of cell walls and chloroplasts, changes in the number of mitochondria, plasmolysis, and cell death. Overall, these results provide a detailed understanding of the grape-E.ampelina interaction and theoretical bases for infection pathway of grape anthracnose.

Heat-stable antifungal factor (HSAF), which belongs to the polycyclic tetramate macrolactam family, was isolated from Lysobacter enzymogenes fermentations and exhibited inhibitory activities against a wide range of fungal pathogens. In this study, the antifungal activity of HSAF against Fusarium graminearum in vitro and in vivo was investigated. A total of 50% of mycelial growth of F. graminearum was suppressed with 4.1 μg/ml of HSAF (EC50 value). HSAF treatment resulted in abnormal morphology of the hyphae, such as curling, apical swelling, and depolarized growth. Furthermore, HSAF adequately inhibited conidial germination and conidiation of F. graminearum with an inhibition rate of 100% when 1 and 6 μg/ml of HSAF were applied, respectively. HSAF caused ultrastructural changes of F. graminearum, including cell wall thickening and plasmolysis. Moreover, the application of HSAF significantly controlled Fusarium head blight in wheat caused by F. graminearum in the field. Overall, these results indicate that HSAF has potential for development as a fungicide against F. graminearum.

The progress of colonization of ash stems from ascospore inocula of Hymenoscyphus fraxineus was examined by light and electron microscopy. The main aim of the study was to characterize the cytology of the biotroph to necrotroph transition during lesion formation. Following direct penetration into epidermal cells, the fungus produced intracellular hyphae that invaded up to five cells before plant cells died. A lack of close attachment between the hyphal cell wall and plant cell membrane was revealed by plasmolysis of epidermal cells. Plant cells died at the centre of the infection but hyphae at the edge were typically found in living plant cells even around large lesions. During biotrophic invasion, the cytoplasm of penetrated plant cells showed very little response despite the plant cell membrane being in direct contact with the fungal cell wall. Before plant cell death, dark staining of the cytoplasm and proliferation of small vesicles was noted, but organelles retained normal ultrastructure. Dead plant cells contained dark brown, osmiophilic droplets. Penetration between epidermal or collenchyma cells was usually targeted to shared pits and involved constriction of hyphae. The transition to necrotrophy was not associated with a clear change in hyphal morphology. Biotrophic intracellular hyphae contained dense cytoplasm but hyphae in dead plant cells were more vacuolated. Remarkably little plant cell wall degradation was observed despite the fungus penetrating up to 18 cells deep into stem tissue. Features of the development of the ash dieback fungus are compared with other hemibiotrophic pathogens.

• Microtubule organization and tubulin polymerization in meristematic leaf cells of Chlorophyton comosum treated with an aqueous solution of 1 M mannitol, inducing plasmolysis, were examined with immunofluorescence and transmission electron microscopy. • Hyperosmotic treatment induced disintegration of the interphase microtubule systems. Free tubulin, either liberated from the depolymerized microtubules or pre-existing as a nonassembled pool, was incorporated into a network of paracrystals. In most of the dividing cells, mitotic and cytokinetic microtubule systems were replaced by atypical spindle-like structures displaying bipolarity and atypical phragmoplasts, respectively. These atypical mitotic and cytokinetic structures consisted of large densely packed bundles of macrotubules (32 nm diameter) or macrotubules and paracrystals. Tubulin paracrystals also occurred in ectopic positions in plasmolysed mitotic and cytokinetic cells. Dividing cells displaying paracrystals only did not form atypical mitotic and cytokinetic apparatuses. • Short hyperosmotic stress causes disintegration of all microtubule arrays in dividing cells of C. comosum. Free tubulin is incorporated into macrotubules and tubulin paracrystals. The latter exhibit definite periodicity and characteristic fine structure.

Xylem vessels, which conduct water from roots to aboveground tissues in vascular plants, are stiffened by secondary cell walls (SCWs). Protoxylem vessel cells deposit cellulose, hemicellulose, and lignin as SCW components in helical and/or annular patterns. The mechanisms underlying SCW patterning in the protoxylem vessel cells are not fully understood, although VASCULAR-RERATED NAC-DOMAIN 7 (VND7) has been identified as a master transcription factor in protoxylem vessel cell differentiation in Arabidopsis thaliana. Here, we investigated deposition patterns of SCWs throughout the tissues of Arabidopsis seedlings using an inducible transdifferentiation system that utilizes a chimeric protein in which VND7 is fused with the activation domain of VP16 and the glucocorticoid receptor (GR) (VND7-VP16-GR). In slender- and cylinder-shaped cells, such as petiole and hypocotyl cells, SCWs that were ectopically induced by the VND7-VP16-GR system were deposited linearly, resulting in helical and annular patterns similar to the endogenous patterns in protoxylem vessel cells. By contrast, concentrated linear SCW deposition was associated with unevenness on the surface of pavement cells in cotyledon leaf blades, suggesting the involvement of cell morphology in SCW patterning. When we exposed the seedlings to hypertonic conditions that induced plasmolysis, we observed aberrant deposition patterns in SCW formation. Because the turgor pressure becomes zero at the point when cells reach limiting plasmolysis, this result implies that proper turgor pressure is required for normal SCW patterning. Taken together, our results suggest that the deposition pattern of SCWs is affected by mechanical stimuli that are related to cell morphogenesis and turgor pressure.

Bryophytes are widely used to monitor air quality. Due to the lack of a cuticle, their cells can be compared to the roots of crop plants. This study aimed to test a hypothetical relation between metal tolerance and cell shape in biomonitoring mosses (Hypnum cupressiforme, Pleurozium schreberi, Pseudoscleropodium purum) and metal sensitive species (Physcomitrium patens, Plagiomnium affine). The tolerance experiments were conducted on leafy gametophytes exposed to solutions of ZnSO4, ZnCl2, and FeSO4 in graded concentrations of 1 M to 10&minus, 8 M. Plasmolysis in D-mannitol (0.8 M) was used as a viability measure. The selected species differed significantly in lamina cell shape, cell wall thickness, and metal tolerance. In those tested mosses, the lamina cell shape correlated significantly with the heavy metal tolerance, and we found differences for ZnSO4 and ZnCl2. Biomonitoring species with long and thin cells proved more tolerant than species with isodiametric cells. For the latter, &ldquo, death zones&rdquo, at intermediate metal concentrations were found upon exposure to ZnSO4. Species with a greater tolerance towards FeSO4 and ZnSO4 had thicker cell walls than less tolerant species. Hence, cell shape as a protoplast-to-wall ratio, in combination with cell wall thickness, could be a good marker for metal tolerance.

Osmotic adjustment is one of the important mechanisms to adapt to drought and it is the only one which is activated under any level of water stress in the plant cells. Grapevine pollen grains response was tested to osmotic stress in fourteen genotypes, initiated by immersion in 55% or 65% polyethylene glycol solutions without and with addition of potassium chloride, to estimate the expressions of osmotic adjustment. The pollen grain test found differences both in the measurements of projected area cytoplasm and expressions of osmotic adjustment present in the cells. Italian Riesling increased pollen grains cytoplasm in PEG solutions with added KCl much more than other genotypes and had the high values for both expressions of induced and overall osmotic adjustment. The results obtained for expression of induced osmotic adjustment underlined the high K+ accumulation capacity of ‘Italian Riesling’, ‘Burgund mare’ 86 Şt., ‘Muscat d’Adda’ 22 Şt., ‘Muscat Ottonel’ 16 Şt., ‘Pinot gris’ 14 Şt. and ‘Argessis’. The lack of correlation between expressions of induced and intrinsic osmotic adjustment indicated that induced osmotic adjustment expressed by K+ might use different mechanisms that are activated at the time of water stress with different levels of solute accumulation. Because the accumulation of K+ in the cells is important in all developmental stages and, in grape yield and quality, pollen responses to induced osmotic adjustment expressed by K+ could be used as a screening method, for establishing the level of drought sensitivity in the grape varieties under water stress.

Annexin 1 (ANN1) is the most abundant member of the evolutionary conserved multigene protein superfamily of annexins in plants. Generally, annexins participate in diverse cellular processes, such as cell growth, differentiation, vesicle trafficking, and stress responses. The expression of annexins is developmentally regulated, and it is sensitive to the external environment. ANN1 is expressed in almost all Arabidopsis tissues, while the most abundant is in the root, root hairs, and in the hypocotyl epidermal cells. Annexins were also occasionally proposed to associate with cytoskeleton and vesicles, but they were never developmentally localized at the subcellular level in diverse plant tissues and organs. Using advanced light-sheet fluorescence microscopy (LSFM), we followed the developmental and subcellular localization of GFP-tagged ANN1 in post-embryonic Arabidopsis organs. By contrast to conventional microscopy, LSFM allowed long-term imaging of ANN1-GFP in Arabidopsis plants at near-environmental conditions without affecting plant viability. We studied developmental regulation of ANN1-GFP expression and localization in growing Arabidopsis roots: strong accumulation was found in the root cap and epidermal cells (preferentially in elongating trichoblasts), but it was depleted in dividing cells localized in deeper layers of the root meristem. During root hair development, ANN1-GFP accumulated at the tips of emerging and growing root hairs, which was accompanied by decreased abundance in the trichoblasts. In aerial plant parts, ANN1-GFP was localized mainly in the cortical cytoplasm of trichomes and epidermal cells of hypocotyls, cotyledons, true leaves, and their petioles. At the subcellular level, ANN1-GFP was enriched at the plasma membrane (PM) and vesicles of non-dividing cells and in mitotic and cytokinetic microtubular arrays of dividing cells. Additionally, an independent immunolocalization method confirmed ANN1-GFP association with mitotic and cytokinetic microtubules (PPBs and phragmoplasts) in dividing cells of the lateral root cap. Lattice LSFM revealed subcellular accumulation of ANN1-GFP around the nuclear envelope of elongating trichoblasts. Massive relocation and accumulation of ANN1-GFP at the PM and in Hechtian strands and reticulum in plasmolyzed cells suggest a possible osmoprotective role of ANN1-GFP during plasmolysis/deplasmolysis cycle. This study shows complex developmental and subcellular localization patterns of ANN1 in living Arabidopsis plants.

Hechtian strands are thread-like structures in plasmolyzed plant cells that connect the cell wall to the plasma membrane. Although these strands were first observed more than 100 years ago, their physiological roles are largely unknown. Here, we used intracellular laser microdissection to examine the effects of disrupting Hechtian strands on plasmolyzed tobacco BY-2 cells. When we focused femtosecond laser pulses on Hechtian strands, targeted disruptions were induced, but no visible changes in cell morphology were detected. However, the calcofluor white signals from β-glucans was detected in plasmolyzed cells with disrupted Hechtian strands, whereas no signals were detected in untreated plasmolyzed cells. These results suggest that Hechtian strands play roles in sensing cell wall integrity.

Plasmolysis is usually introduced to cell biology students as a tool to illustrate the plasma membrane: hypertonic solutions cause the living protoplast to shrink by osmotic water loss, hence, it detaches from the surrounding cell wall. What happens, however, with the subcellular structures in the cell cortex during this process of turgor loss? Here, we investigated the cortical endoplasmic reticulum (ER) in moss protonema cells of Physcomitrella patens in a cell line carrying a transgenic ER marker (GFP-HDEL). The plasma membrane was labelled simultaneously with the fluorescent dye FM4-64 to achieve structural separation. By placing the protonemata in a hypertonic mannitol solution (0.8 M), we were able to follow the behaviour of the cortical ER and the protoplast during plasmolysis by confocal laser scanning microscopy (CLSM). The protoplast shape and structural changes of the ER were further examined after depolymerisation of actin microfilaments with latrunculin B (1 &micro, M). In its natural state, the cortical ER is a dynamic network of fine tubes and cisternae underneath the plasma membrane. Under acute and long-term plasmolysis (up to 45 min), changes in the protoplast form and the cortical ER, as well as the formation of Hechtian strands and Hechtian reticula, were observed. The processing of the high-resolution z-scans allowed the creation of 3D models and gave detailed insight into the ER of living protonema cells before, during and after plasmolysis.

Optimized cryopreservation protocols are essential for the safe, cost-effective and long-term conservation of germplasm of great economic interest, such as Guinea (Petiveria alliacea L.), a medicinal species that synthesizes a great diversity of bioactive substances, among them polysulfides with antitumor activity. Somatic embryos (SEs) produced from in vitro roots were cryopreserved using the V-cryoplate technique. Their viability was measured using the triphenyltetrazolium test and their recovery by counting the number of secondary SEs produced per cryopreserved SE 90 days after liquid nitrogen (LN) exposure. Structural alterations were evaluated qualitatively and quantitatively during the successive stages of the protocol. SEs were dehydrated in 0.5 M sucrose, attached to cryoplates using sodium alginate solution (3%) and treated with PVS2 for different periods. After immersion in LN, SEs were rewarmed in unloading solution (1.2 M sucrose) at room temperature (25 °C) for 20 min. Viability based on the triphenyltetrazolium test was 100% after 15 min of treatment with PVS2 and LN exposure, and recovery, based on multiplication rate, was 21 somatic embryos produced per cryopreserved somatic embryo after 90 days of culture. The histological analysis of cryopreserved somatic embryos showed plasmolysis in the different cell types after treatment with PVS2, with meristematic and parenchymatic cells presenting a higher plasmolysis level. However, after rewarming, the level of plasmolysis decreased over cultivation time, reaching only 2% in meristematic cells after 30 days, indicating the good ability of Petiveria alliacea SEs to develop cryotolerance under the experimental conditions tested.

Main conclusionSwelling of sweet cherry cell walls is a physical process counterbalanced by turgor. Cell turgor prevents swelling in intact cells, whereas loss of turgor allows cell walls to swell.AbstractSwelling of epidermal cell walls precedes skin failure in sweet cherry (Prunus avium) cracking. Swollen cell walls lead to diminished cell:cell adhesions. We identify the mechanism of cell wall swelling. Swelling was quantified microscopically on epidermal sections following freeze/thaw treatment or by determining swelling pressure or swelling capacity of cell wall extracts. Releasing turgor by a freeze/thaw treatment increased cell wall thickness 1.6-fold within 2 h. Pressurizing cell wall extracts at > 12 kPa prevented swelling in water, while releasing the pressure increased swelling. The effect was fully reversible. Across cultivars, cell wall thickness before and after turgor release in two subsequent seasons was significantly correlated (before release of turgor:r = 0.71**,n = 14; after release of turgor:r = 0.73**,n = 14) as was the swelling of cell walls upon turgor release (r = 0.71**,n = 14). Close relationships were also identified for cell wall thickness of fruit of the same cultivars grown in the greenhouse and the field (before release of turgor:r = 0.60,n = 10; after release of turgor:r = 0.78**,n = 10). Release of turgor by heating, plasmolysis, incubation in solvents or surfactants resulted in similar swelling (range 2.0–3.1 µm). Cell wall swelling increased from 1.4 to 3.0 µm as pH increased from pH 2.0 to 5.0 but remained nearly constant between pH 5.0 and 8.0. Increasing ethanol concentration decreased swelling. Swelling of sweet cherry cell walls is a physical process counterbalanced by turgor.

Transmission electron microscopy (TEM) combined with freeze substitution was employed to examine the ultrastructure of cells of gentian shoot tips cooled to the ultra-low temperature of slush nitrogen and liquid nitrogen. When shoot tips were cooled in ultra-low temperature without plant vitrification solution 2 (PVS2) treatment, massive ice formation was observed throughout the cells, indicating that severe injury occurred during cooling. In contrast, when shoot tips were treated with PVS2 and subsequently cooled to ultra- low temperatures, no ice crystals were observed in the cells. In addition, the cells of PVS2-treated shoot tips exhibited considerable plasmolysis and formation of small vesicles in cytoplasm. These results clearly demonstrate that the PVS2 treatment is essential for preventing damage caused by ice formation and for successful cryopreservation of plant shoot tips.

Polyamines are closely associated with drought stress in plants. However, the function which polyamines play in the maturing maize grain under drought is not still clear. Especially, whether the conjugated polyamines are involved in H+-ATPase activity and conformation of plasma membrane remains to be answered. Therefore, in the study, the contents of conjugated polyamines, H+-ATPase activity and conformation of plasma membrane were detected with maturing grain embryos of two maize (Zea mays L.) cultivars, which were different in drought stress-tolerance. Under drought stress, the cell plasmolysis of the drought-sensitive Xundan No. 22 cv. was more marked than that of the drought-tolerant Yedan No. 13 cv., while the increases in the contents of conjugated non-covalently spermidine and spermine, conjugated covalently putrescine and spermidine in plasma membrane from Yedan 13 were more obvious than those in Xundan 22. The results suggested that these conjugated polyamines might be involved in stability of plasma membrane. Furthermore, H+-ATPase activity in plasma membrane from Yedan 13 increased more significantly than that in Xundan 22, indicating that these conjugated polyamines were closely correlated with the activity of H+-ATPase. To justify further the inferences, the additional experiments with exogenous spermidine, spermine and polyamine biosynthesis inhibitors were carried out in the study. The results showed that the application of exogenous spermidine or spermine increased the levels of conjugated polyamines in plasma membrane, coupled with alleviating cell plasmolysis and increasing H+-ATPase activity. The application of inhibitors decreased the levels of conjugated polyamines, coupled with aggravating cell plasmolysis and decreasing H+-ATPase activity. From these results together, it could be concluded that non-covalently conjugated spermidine and spermine, conjugated covalently putrescine and spermidine in plasma membrane functioned in increasing H+-ATPase activity, mitigating cell plasmolysis and maintaining the plasma membrane conformation of the maturing maize grain embryos under drought stress.

Backgrounds and aims Because stomata in bryophytes occur on sporangia, they are subject to different developmental and evolutionary constraints from those on leaves of tracheophytes. No conclusive experimental evidence exists on the responses of hornwort stomata to exogenous stimulation. Methods Responses of hornwort stomata to abscisic acid (ABA), desiccation, darkness and plasmolysis were compared with those in tracheophyte leaves. Potassium ion concentrations in the guard cells and adjacent cells were analysed by X-ray microanalysis, and the ontogeny of the sporophytic intercellular spaces was compared with those of tracheophytes by cryo-scanning electron microscopy. Key results The apertures in hornwort stomata open early in development and thereafter remain open. In hornworts, the experimental treatments, based on measurements of >9000 stomata, produced only a slight reduction in aperture dimensions after desiccation and plasmolysis, and no changes following ABA treatments and darkness. In tracheophytes, all these treatments resulted in complete stomatal closure. Potassium concentrations are similar in hornwort guard cells and epidermal cells under all treatments at all times. The small changes in hornwort stomatal dimensions in response to desiccation and plasmolysis are probably mechanical and/or stress responses of all the epidermal and spongy chlorophyllose cells, affecting the guard cells. In contrast to their nascent gas-filled counterparts across tracheophytes, sporophytic intercellular spaces in hornworts are initially liquid filled. Conclusions Our experiments demonstrate a lack of physiological regulation of opening and closing of stomata in hornworts compared with tracheophytes, and support accumulating developmental and structural evidence that stomata in hornworts are primarily involved in sporophyte desiccation and spore discharge rather than the regulation of photosynthesis-related gaseous exchange. Our results run counter to the notion of the early acquisition of active control of stomatal movements in bryophytes as proposed from previous experiments on mosses.

Cryopreservation is a reliable and cost-effective method for the long-term preservation of clonally propagated species. The number of vegetatively propagated species conserved by cryopreservation is increasing through development of vitrification-based methods; droplet vitrification in particular is becoming the preferred method for many species, as it ensures fast freezing and thawing rates. This research investigated if cold, antioxidant and osmotic pre-treatments could maintain the structural integrity of cells, thence aid in developing a droplet vitrification protocol for kiwifruit using Actinidia chinensis var. chinensis 'Hort16A' as a model. Cold acclimation of donor plantlets at 4 °C for 2 weeks followed by sucrose pre-culture of shoot tips and supplementation of ascorbic acid (0.4 mM) in all media throughout the procedure registered 40% regeneration after cryopreservation. Transmission electron microscope imaging of meristematic cells confirmed sucrose and ascorbic acid pre-treatment of shoot tips from cold acclimated plantlets following treatment in vitrification solution exhibited severe plasmolysis and some disruption of membrane and vacuoles. In contrast cells without cold acclimation or sucrose and ascorbic acid pre-treatments exhibited minimal change after exposure to vitrification solution. After cryopreservation and recovery, all cells of untreated shoot tips showed rupture of the plasma membrane, loss of cytoplasmic contents and organelle distortions. By comparison, most pre-treated shoot-tip cells from cold acclimated plantlets retained their structural integrity, showing that only those cells that have been dehydrated and plasmolysed can withstand cryopreservation by vitrification.

Some studies have shown that excess molybdenum (Mo) could produce toxic effects on plants. However, little is known about the subcellular distribution of Mo and cell ultrastructure within plants under excess Mo stress. Here, we comprehensively analyzed the changes of Mo distribution in subcellular fractions, cell ultrastructure and antioxidant enzymes in leaves and roots of soybean seedlings in response to excess Mo stress. The results showed that roots exhibited higher Mo accumulation than leaves at the 100 mg L-1 Mo level, about 38.58-, 171.48- and 52.99-fold higher in cell walls, cell organelles and soluble fractions, respectively. Subcellular fractionations of Mo-containing tissues indicated that approximately 90% of Mo was accumulated in the soluble fractions and cell walls of the roots and leaves, and soluble fractions (accumulated 66.3-72.2% Mo) might serve as an effective storage site for excess Mo. Furthermore, excess Mo caused ultrastructural alterations in roots and leaves of soybean seedlings, leading to structural abnormality of chloroplast in leaf cells, plasmolysis, cellular deformity, vacuole enlargement and the swelling of cell wall and cytoplasm in root cells. Meanwhile, under excess Mo stress, the activity of POD, CAT and APX enzymes in roots was 1.43, 2.35 and 1.23 times that under standard Mo condition, while that of SOD and CAT enzymes in leaves was 1.23 and 1.94 times, respectively. This study provided novel insights into the mechanisms of excess Mo toxicity in soybean seedlings.

Rhododendron × hybridum, an ornamental plant, is usually heat sensitive. High temperatures cause an array of morphological, anatomical, physiological, biochemical changes in plants, which affect plant growth and development. Evaluating difference between two cultivars in R. may facilitate future understand the heat tolerance mechanisms. We conducted a comparative study on growing plants of R. ‘Lan Yin’ and R. ‘Liu Qiu Hong’ at 38/30 °C (day/night) and 25/17 °C (day/night) to elucidate the heat tolerance mechanism of Rhododendron in terms of morphology, physiology, anatomy and biology. After the plants were subjected to 38/30 °C (day/night) incubation for 6 d, the chlorophyll and carotenoid contents decreased, malondialdehyde (MDA) and hydrogen peroxide (H2O2) accompanying lesser increase in R. ‘Lan Yin’ revealed that the heat resistant cultivar was easier to adjust physiologically in heat disappearing. Morphologically, the xylem vessels, leaf ultrastructure and chloroplast of R. ‘Lan Yin’ is superior to R. ‘Liu Qiu Hong’, and R. ‘Lan Yin’ had a decreased stomatal opening and stomatal aperture under heat stress, confirming stomatal factors play a crucial role in heat tolerance in R. cultivars. The electrolyte leakage increased, superoxide dismutase (SOD) and peroxidase (POD) activities, osmotic adjustment solute contents (proline, total soluble protein, and sugar) decreased to scavenge reactive oxygenspecies (ROS) and maintain cell membrane stability in two cultivars, but R. ‘Lan Yin’ had a higher level relatively, which may suggests that CMT may use a reliable index for selecting heat-tolerant cultivars of R. In addition, there was evidence of plasmolysis and chloroplast damage in R. ‘Liu Qiu Hong’.

Pistachio seeds and pollen have desiccation tolerance and desiccation sensitivity ultrastructural characters at maturation, respectively. Seed cells filled with storage matter remain integrated, whereas some pollen show cell rupture. Maturation drying is a common trait of seeds and pollen. Orthodox seeds and pollen become tolerant to desiccation at maturation and remain their germinability in an inactive dry state for a period of time, whereas recalcitrant ones remain sensitive and must germinate immediately after maturation. This study investigated the moisture content, germinability and ultrastructure of pistachio (Pistacia vera L.) mature seeds and pollen in fresh and 24 h-desiccated states using microscopy methods. Seeds lost 90.5% water and remained 100% germinable demonstrating their orthodoxy. Fresh and desiccated cells of both cotyledon and ground meristem of root tip, mostly contained lipid and protein reserves. Large autophagic vacuoles in fresh cells contained autophagic bodies digesting organelles related to active metabolism and became converted to protein storage vacuoles at desiccated state. In ground meristem of root tip, most lipid bodies were tiered adjacent to plasma membrane probably with a secretory function and the cell walls were waved at desiccated state. Pollen lost 46.7% water along with 93% germinability by desiccation demonstrating its recalcitrance. After desiccation, 1.8% of pollen grains displayed intine degeneration, vegetative cell plasmolysis and detachment from intine, lobed vegetative cell nucleus, great vacuolation along with organelles destruction in both vegetative and generative cells and finally cell rupture. However, most pollen displayed normal structure indicating the recalcitrance behavior of pistachio pollen is not mainly related to cell structural damages but, lack of germinability through molecular damages that should be studied in details.

During the opening and closing of stomata, guard cells undergo rapid and reversible changes in their volume and shape, which affects the adhesion of the plasma membrane (PM) to the cell wall (CW). The dynamics of actin filaments in guard cells are involved in stomatal movement by regulating structural changes and intracellular signaling. However, it is unclear whether actin dynamics regulate the adhesion of the PM to the CW. In this study, we investigated the relationship between actin dynamics and PM-CW adhesion by the hyperosmotic-induced plasmolysis of tobacco guard cells. We found that actin filaments in guard cells were depolymerized during mannitol-induced plasmolysis. The inhibition of actin dynamics by treatment with latrunculin B or jasplakinolide and the disruption of the adhesion between the PM and the CW by treatment with RGDS peptide (Arg-Gly-Asp-Ser) enhanced guard cell plasmolysis. However, treatment with latrunculin B alleviated the RGDS peptide-induced plasmolysis and endocytosis. Our results reveal that the actin depolymerization is involved in the regulation of the PW-CW adhesion during hyperosmotic-induced plasmolysis in tobacco guard cells.

In plant cells, molecular connections link the cell wall-plasma membrane-actin cytoskeleton to form a continuum. It is hypothesized that the cell wall provides stable anchor points around which the actin cytoskeleton remodels. Here we use live cell imaging of fluorescently labelled marker proteins to quantify the organization and dynamics of the actin cytoskeleton and to determine the impact of disrupting connections within the continuum. Labelling of the actin cytoskeleton with green fluorescent protein (GFP)-fimbrin actin-binding domain 2 (FABD2) resulted in a network composed of fine filaments and thicker bundles that appeared as a highly dynamic remodelling meshwork. This differed substantially from the GFP-Lifeact-labelled network that appeared much more sparse with thick bundles that underwent 'simple movement', in which the bundles slightly change position, but in such a manner that the structure of the network was not substantially altered during the time of observation. Label-dependent differences in actin network morphology and remodelling necessitated development of two new image analysis techniques. The first of these, 'pairwise image subtraction', was applied to measurement of the more rapidly remodelling actin network labelled with GFP-FABD2, while the second, 'cumulative fluorescence intensity', was used to measure bulk remodelling of the actin cytoskeleton when labelled with GFP-Lifeact. In each case, these analysis techniques show that the actin cytoskeleton has a decreased rate of bulk remodelling when the cell wall-plasma membrane-actin continuum is disrupted either by plasmolysis or with isoxaben, a drug that specifically inhibits cellulose deposition. Changes in the rate of actin remodelling also affect its functionality, as observed by alteration in Golgi body motility.

Adventitious root cultures of Tarenaya rosea were successfully cryopreserved using the encapsulation-vitrification technique. Histological analysis revealed useful information on the successive steps of cryopreservation. Coupled with complementary histochemical approaches, these studies provided cellular and tissue descriptions of T. rosea root cultures during cryopreservation and contributed to an understanding of cellular stress responses, as well as characterization of the anatomical pattern of root regeneration. The effects of exposure duration to PVS3 solution (0–120 min), unloading treatment (direct and gradual), and recovery medium (liquid and solid) on recovery of cryopreserved roots were investigated. The highest recovery (91%) after cooling in liquid nitrogen (LN) was reached with PVS3 treatment for 90 min, gradual rehydration in unloading solution, and recovery on solid MS medium. The cryopreserved roots showed high multiplication capacity, which was maintained for up to four subcultures. The effect of cryopreservation on root structure was investigated by histological and histochemical studies. Plasmolysis intensified during exposure to loading and PVS3 solutions, but decreased after unloading treatment. The proportion of intercellular spaces increased progressively throughout the cryopreservation protocol, culminating in root cortex disruption. Histochemical analyses revealed polysaccharides, proteins, and both lipidic and pectic substances in intercellular spaces. The vascular cylinder remained intact, ensuring the formation of new roots from the pericycle, showing that proliferative capacity of cryopreserved roots had not diminished.

This article provides a study on morphological and anatomical changes during post-embryonic development of roots in plants under saline stress. The influence of salinity on the architecture of root related to the species-specificity of wheat plants is shown. The important roles of thickness of the epiblema and length of root hairs, the thickness of the endoderm and the diameter of the central cylinder under salt stress appear worthy of note. It is shown that both the water content of roots cells and its chromosomal apparatus are affected by salt stress. In addition to a very strong plasmolysis, the compression and fragmentation of the nuclei were noted, which resulted into their destruction and cell death. On the basis of all considered parameters the studied species can be arranged in the following according their resistance to salinity: T. polonicum

The effect of potassium tetraborate on germination of conidia of Colletotrichum gloeosporioides, and postharvest rot of mango were studied. An application of K2B4O7 to mango trees at flowering increased fruit set and decreased the incidence of anthracnose on harvested fruit. The effects of borate on the germination, nuclear division, endocytosis, and ultrastructure of conidia of C. gloeosporioides were studied using light, confocal, and transmission electron microscopy. The results showed that borate inhibited germination and germ tube elongation, delayed nuclear division, and impaired endocytosis of C. gloeosporioides conidia. Ultrastructural abnormalities also occurred in conidia treated with borate, and these included an increase in numbers of vacuoles, cytoplasmic disintegration, mitochondria degradation, and plasmolysis. These results suggest that borate can serve as a potential alternative to synthetic fungicides for the control of the postharvest disease of mango fruit caused by C. gloeosporioides.

Production of oospores by Peronospora effusa, the causal agent of downy mildew on spinach (Spinacia oleracea), was reported on spinach seed over three decades ago. In view of the rapid proliferation of new races of P. effusa worldwide, seedborne transmission of this pathogen has been suspected but methods to test the viability of seedborne oospores have not been available. Eighty-two seed lots of contemporary spinach cultivars were evaluated for the presence of P. effusa using a seed-wash method and the sediment was examined by microscopy. Of the analyzed seed lots, 16% were positive for oospores and an additional 6% for sporangiophores characteristic of P. effusa. Application of a P. effusa-specific quantitative polymerase chain reaction assay showed that 95% of the 59 tested seed lots were positive for P. effusa. The viability of oospores from five seed lots that were proven to carry the pathogen from the above tests was tested using two independent methods, one involving plasmolysis and the other trypan blue staining. The oospores plasmolyzed in 4 M sodium chloride and were deplasmolyzed in water, demonstrating an active and viable cell membrane. Similarly, viable oospores failed to take up the trypan blue stain. Overall, 59% of the oospores were viable in the plasmolysis test and 45% with the trypan blue test. These results indicate the presence of P. effusa oospores in contemporary spinach seed lots, and suggest that the transmission of viable oospores of P. effusa in spinach seed does occur. Elimination of the pathogen on seed, in addition to other management approaches, will be useful in reducing the extent and severity of downy mildew on spinach crops and diminishing pathogen spread through seed.

A three-domain pressure – volume relationship (PV curve) was studied in relation to leaf anatomical structure during dehydration in the grey mangrove, Avicennia marina. In domain 1, relative water content (RWC) declined 13% with 0.85 MPa decrease in leaf water potential, reflecting a decrease in extracellular water stored primarily in trichomes and petiolar cisternae. In domain 2, RWC decreased by another 12% with further reduction in leaf water potential to -5.1 MPa, the turgor loss point. Given the osmotic potential at full turgor (-4.2 MPa) and the effective modulus of elasticity (~40 MPa), domain 2 emphasized the role of cell wall elasticity in conserving cellular hydration during leaf water loss. Domain 3 was dominated by osmotic effects and characterized by plasmolysis in most tissues and cell types without cell wall collapse. Extracellular and cellular water storage could support an evaporation rate of 1 mmol m-2s-1 for up to 54 and 50 min, respectively, before turgor loss was reached. This study emphasized the importance of leaf anatomy for the interpretation of PV curves, and identified extracellular water storage sites that enable transient water use without substantive turgor loss when other factors, such as high soil salinity, constrain rates of water transport.The pressure-volume relationships in field grown leaves of the mangrove, Avicennia marina, exhibited three domains dominated successively by 1) the presence and consumption of extracellular water, 2) variable turgor and loss of intracellular water, and 3) osmotic behavior of flaccid cells and plasmolysis. Multiple sites of extracellular water storage were identified, including hollow trichomes and novel structures named “cisternae”. When fully charged, extracellular and cellular water storage could support a typical evaporation rate of 1 mmol m-2s-1 for 54 and 50 min, respectively, before turgor loss was reached. This study emphasizes the importance of leaf anatomy for the interpretation of PV curves, and identifies extracellular water storage sites that enable transient water use without substantive turgor loss when other factors, such as high soil salinity, constrain rates of water transport.

The cell wall emerged as one of the important structures in plant stress responses. To investigate the effect of cold on the cell wall properties, the content and localization of pectins and pectin methylesterase (PME) activity, were studied in two maize inbred lines characterized by different sensitivity to cold. Low temperature (14/12 °C) caused a reduction of pectin content and PME activity in leaves of chilling-sensitive maize line, especially after prolonged treatment (28 h and 7 days). Furthermore, immunocytohistological studies, using JIM5 and JIM7 antibodies, revealed a decrease of labeling of both low- and high-methylesterified pectins in this maize line. The osmotic potential, quantified by means of incipient plasmolysis was lower in several types of cells of chilling-sensitive maize line which was correlated with the accumulation of sucrose. These studies present new finding on the effect of cold stress on the cell wall properties in conjunction with changes in the osmotic potential of maize leaf cells.

The role of the root in water supply and plant viability is especially important if plants are subjected to stress at the juvenile stage. This article describes the study of morphophysiological and cytological responses, as well as elements of the anatomical structure of primary roots of three wheat species, Triticum monococcum L., Triticum dicoccum Shuebl., and Triticum aestivum L., to osmotic stress. It was shown that the degree of plasticity of root morphology in water deficit affected the growth and development of aboveground organs. It was found that in conditions of osmotic stress, the anatomical root modulations were species-specific. In control conditions the increase in absolute values of root diameter was reduced with the increase in the ploidy of wheat species. Species-specific cytological responses to water deficit of apical meristem cells were also shown. The development of plasmolysis, interpreted as a symptom of reduced viability apical meristem cells, was revealed. A significant increase in enzymatic activity of superoxide dismutase under osmotic stress was found to be one of the mechanisms that could facilitate root elongation in adverse conditions. The tetraploid species T. dicoccum Shuebl. were confirmed as a source of traits of drought tolerant primary root system for crosses with wheat cultivars.

International audience; Cryopreservation stands out as the main strategy to ensure safe and cost efficient long-term conservation of plant germplasm, especially for biotechnological materials. However, the injuries associated with the procedure may result in structural damage and low recovery rates after cooling. Histological analysis provides useful information on the effects of osmotic dehydration, LN exposure, and recovery conditions on cellular integrity and tissue organization, allowing the determination of the critical steps of the cryopreservation protocol and, thus, the use of optimized treatments. Passiflora pohlii Mast. (Passifloraceae) is a native species from Brazil with potential agronomic interest. Recent studies showed the presence of saponins in its roots, which presented antioxidant activity. The goal of this work was to develop a cryopreservation technique for root tips of in vitro-derived plants of P. pohlii using the V-Cryo-plate technique and to characterize the anatomical alterations that occurred during the successive steps of the protocol. Root tips were excised from in vitro plants and precultured before adhesion to cryo-plates and then treated for different periods with the plant vitrification solutions PVS2 or PVS3. Treatment with PVS2 for 45 min resulted in higher recovery (79%) when compared with PVS3 (43%). The greatest number of adventitious roots per cryopreserved explant was also observed after a 45-min exposure to PVS2. Plasmolysis levels were higher in cortical cells of cryopreserved explants treated with PVS2, while pericycle and central cylinder cells were not damaged after this treatment. Thirty days after rewarming, no plasmolysis could be detected, regardless of the experimental conditions.

The ability of date palm tree to survive under adverse abiotic conditions renders it as a valuable genomic resource for identifying tolerance genes. While mechanisms for salt tolerance have been heavily investigated in model as well as in some agronomic crops, no such studies have been undertaken in date palm. The aim of this study is to identify stress tolerance-related genes through transcriptomic analysis to support further functional studies. Young roots of Deglet Beida cultivar have been subjected to salt stress treatment, and used for RNA-Seq expression profiling and transmission electron microscopy (TEM) analysis. A total of 1939 genes are found to be differentially expressed between mock-treated roots and salt-stressed roots using log2FC ≥15≤−15. Many of these regulatory genes belong to DNA/RNA, protein, membrane, and signaling functional categories, suggesting that these genes play functional roles in tolerance to salt stress. Furthermore, RNA-Seq analysis has revealed activation of abscisic acid signaling pathways through SNF1-related protein kinases 2. Additionally, certain key genes involved in sodium uptake and transport are down-regulated, suggesting a potential mechanism for slowing down up-take and transport of salt solutes within plant tissues. TEM analysis has revealed that stressed roots exhibit plasmolysis in cortical cells of the distal region, while epidermal cells do not. Interestingly, root-tip regions of stressed roots do not exhibit plasmolysis, and this is likely due to higher solute contents present in these sink cells. These findings provide new information on multi-dimensional responses of date palm to salinity stress.

Previous investigations have revealed that Ectocarpus siliculosus strain Es524, isolated from a highly copper (Cu)-polluted location in Chile, displays higher activities of the antioxidant enzymes superoxide dismutase (SOD), ascorbate peroxidase (APX) and catalase (CAT) under Cu excess. This partly explains its enhanced tolerance compared with other E. siliculosus strains. However, the transcriptomic basis behind the activity of these antioxidant enzymes is still unknown. We have conducted laboratory experiments with Es524 under control conditions and exposure to 2.4 μM total Cu. Under Cu exposure, Es524 showed a decrease in cell length and cell plasmolysis, revealing a certain level of cytophysiological stress. The genes encoding for iron-SOD, APX and CAT were overexpressed under Cu excess, demonstrating that higher activities of these enzymes are transcriptionally regulated. It is now more apparent that the induction of SOD, APX and CAT are conserved molecular and biochemical mechanisms developed after ...

Discrete categories of seed physiology can be explained through a unified concept of the structural and molecular mobility responses within cells to drying. Tolerance of desiccation is typically described by a threshold or low water content limit to survival. This convention provides fairly good distinction between orthodox and recalcitrant seeds, which show thresholds of less than about 0.07 and greater than about 0.2 g H2O g DW−1, respectively. Threshold water contents, however, are not direct measures of the intensity of water stress tolerated by seeds, nor are they measures of cell response to water stress. More direct criteria, that accommodate both spatial and temporal effects of water loss, are required to explain variation of desiccation tolerance and longevity in seeds from diverse genetic backgrounds and growth conditions. This essay presents the argument that changes in cellular volume directly quantify primary responses to desiccating stress in a context that also links damage, as cellular constituents compress, and protection, as compressed molecules form stabilizing structure. During desiccation, fluid cytoplasm solidifies, and the newly formed spatial relationships among molecules determine whether and how long viability is maintained. The diversity of seed behaviors suggests complexity and opportunity to discover molecules and mechanisms that regulate survival and perception of time in cells that lack metabolic function.

The effects of 50 and 300 mg L(-1) Zn(2+) (50 Zn and 300 Zn) were investigated in Triticum aestivum (cv. Żura) grown hydroponically for 7 days. Although wheat treated with 50 Zn took up relatively high amount of the metal (8,943 and 1,503 mg kg(-1) DW in roots and shoots, respectively), none of the morphological and cytological parameters were changed. After 300 Zn, the metal concentration increased to 32,205 and 5,553 mg kg(-1) DW in roots and shoots, respectively. It was connected with the depletion of shoot and root growth, their fresh and dry weight, water content and mitotic index of root meristematic cells. Microelement contents (Cu, Mn and Fe) after 50 Zn were changed only in roots, while 300 Zn disturbed ion balance in whole plants. The most evident ultrastructural alterations of root meristematic cells caused by both tested Zn(2+) doses included increased vacuolization, accumulation of granular deposits inside vacuoles and cell wall thickening. The effect of 300 Zn on root cell ultrastructure was greater that of 50 Zn. The majority of mitochondria had condensed matrix and swollen cristae, plastids contained plastoglobuli, nucleoli were ring-shaped, thinned down cytoplasm with lipid droplets and swollen endoplasmic reticulum cisternae appeared. In mesophyll cells, 50 Zn caused slight reorganization of chloroplast thylakoids and formation of condensed mitochondria. Three hundred Zn triggered more extensive, but not degenerative, changes: plasmolysis of some cells; chloroplasts with protrusions, changed thylakoid organisation and often large starch grains; irregular, condensed mitochondria. The results indicate that T. aestivum cv. Żura is relatively tolerant to Zn stress.

Upon plasmolysis, protoplast ER becomes more persistently cisternal without changing its actin association or internal flow, while wall-associated ER is tubular, associated with altered microtubules, and has reduced flow., Plasmolysis of hypocotyl cells of transgenic Arabidopsis thaliana and Nicotiana benthamiana diminishes the dynamics of the remodeling of the endoplasmic reticulum (ER) in the central protoplast, namely that withdrawn from the cell wall, and more persistent cisternae are formed, yet little change in the actin network in the protoplast occurs. Also, protein flow within the ER network in the protoplast, as detected with fluorescence recovery after photobleaching (FRAP), is not affected by plasmolysis. After plasmolysis, another network of strictly tubular ER remains attached to the plasma membrane-wall interface and is contained within the Hechtian strands and reticulum. FRAP studies indicate that protein flow within these ER tubules diminishes. Actin is largely absent from the Hechtian reticulum and the ER becomes primarily associated with altered, branched microtubules. The smaller volume of the central protoplast is accompanied by decreased movement rates of tubules, cisternae, and spheroid organelles, but this reduced movement is not readily reversed by the increase in volume that accompanies deplasmolysis.

This study provides a simple protocol for storage of alder clones. The technique described is useful for in vitro germplasm collections, decreasing the risk of genetic changes and associated costs. The aim of this study was to develop a simple method for the medium-term storage of Alnus glutinosa (L.) Gaertn. explants obtained from trees aged 20–30 years. Several parameters were evaluated, including type of explant (shoot apex or nodal segments), pre-storage treatment (0 or 10 days after the last subculture) and duration of cold storage (3–24 months) at 2–4 °C. Explants were maintained at this temperature under dim lighting on Woody Plant Medium supplemented with 0.1 mg l−1 6-benzyladenine and 0.5 mg l−1 indole-3-acetic acid. Under these conditions, a high percentage (75–87 %) of cultures remained viable after 18 months in cold cabinets. The stored material was successfully recovered and multiplied normally in the same medium, showing good growth and developing into normal shoots that were morphologically similar to those of non-stored controls. At the histological level, the main change observed was the accumulation of starch granules in cells of the shoot apex, as well as in cells located close to the vascular bundles, after 3 months of cold storage. As the duration of cold storage increased, the number and size of the starch granules decreased but cell plasmolysis and the content of lipid droplets increased. Cold damage was generalized after 24 months at 4 °C. This study provides new insights into the changes occurring in A. glutinosa during cold storage.

Some species of the genus Passiflora (sect. Xerogona) have flowers that exhibit a mechanically-induced movement of the androgynophore that is probably involved in pollination. Despite the many reports of touch-induced movements of floral parts, the studies concerning anatomical, ultrastructural and molecular aspects of the plant movements are restricted to the vegetative parts. Rapid plant movements are highly dependent on turgor changes of a particular flexible tissue formed by specialized cells capable of losing and gaining water rapidly. Thigmotropic androgynophores of four species of Passiflora from section Xerogona were analyzed at cellular and subcellular levels. Our results show that the movement is due to a vacuolar remodelling in a group of parenchymatous cells at the base of the androgynophore. After the movement, plasmolyzed and multivacuolated cells are present at the stimulated side and turgid and univacuolated cells at the opposite side. The results suggest that the mechanisms leading to the androgynophore movement in Passiflora are, in general, analogous to those reported for the movements of legume pulvinus, motile stamen filaments and guard cells of stomata, pointing to conserved cellular mechanisms of plant movement.

Plasmolysis is a typical response of plant cells exposed to hyperosmotic stress. The loss of turgor causes the violent detachment of the living protoplast from the cell wall. The plasmolytic process is mainly driven by the vacuole. Plasmolysis is reversible (deplasmolysis) and characteristic to living plant cells. Obviously, dramatic structural changes are required to fulfill a plasmolytic cycle. In the present paper, the fate of cortical microtubules and actin microfilaments is documented throughout a plasmolytic cycle in living cells of green fluorescent protein (GFP) tagged Arabidopsis lines. While the microtubules became wavy and highly bundled during plasmolysis, cortical filamentous actin remained in close vicinity to the plasma membrane lining the sites of concave plasmolysis and adjusting readily to the diminished size of the protoplast. During deplasmolysis, cortical microtubule re-organization progressed slowly and required up to 24 h to complete the restoration of the original pre-plasmolytic pattern. Actin microfilaments, again, recovered faster and organelle movement remained intact throughout the whole process. In summary, the hydrostatic skeleton resulting from the osmotic state of the plant vacuole "overrules" the stabilization by cortical cytoskeletal elements.

Heavy metal toxicity in soils has been considered as major constraints for oilseed rape (Brassica napus L.) production. In the present study, toxic effects of chromium (Cr) were studied in 6-d-old seedlings of four different cultivars of B. napus (ZS 758, Zheda 619, ZY 50, and Zheda 622). The elevated content of Cr inhibited seedling growth, decreased the content of photosynthetic pigments, and activities of antioxidant enzymes, as well as increased the content of malondialdehyde and reactive oxygen species in all the cultivars. The Cr content in different parts of plants was higher in Zheda 622 than in other cultivars. The electron microscopic study showed changes in ultrastructure of leaf mesophyll and root tip cells at 400 μmol Cr, and these changes were more prominent in Zheda 622. An increased size and number of starch grains and number of plastoglobuli, damaged thylakoid membranes, and immature nucleoli and mitochondria were observed in leaves. In roots, enlarged vacuoles, disrupted cell walls and cell membranes, an increased number of mitochondria and a size of nucleolus, as well as plasmolysis (in Zheda 622) were observed. On the basis of these findings, it can be concluded that cv. Zheda 622 was more sensitive to Cr as compared to other three cultivars.

Phytoextraction is an established method of removal of heavy metals from contaminated soils worldwide. Phytoextraction is most efficient if local plants are used in the contaminated site. We propose that Prosopis pubescens (Screw bean mesquite) would be a successful phytoextractor of copper in our local soils. In order to determine the feasibility of using Screw bean mesquite, we utilized inductively-coupled plasma-optical emission spectroscopy (ICP-OES) and elemental analysis to observe the uptake of copper and the effects on macro and micro nutrients within laboratory-grown seedlings. We have previously shown that P. pubescens is a hyperaccumulator of copper in soil-grown seedlings. Light and transmission electron microscopy demonstrated death of root cells and ultrastructural changes due to the presence of copper from 50 mg/L - 600 mg/L. Ultrastructural changes included plasmolysis, starch accumulation, increased vacuolation and swollen chloroplasts with disarranged thylakoid membranes in cotyledons. Inductively coupled plasma-optical emission spectroscopy analyses of macro- and micro-nutrients revealed that the presence of copper sulfate in the growth medium of Petri-dish grown Prosopis pubescens seedlings resulted in dramatic decreases of magnesium, potassium and phosphorus. At 500-600 mg/L of copper sulfate, a substantial increase of sulfur was present in roots.

The control of several citrus diseases requires continuous applications of fungicides containing copper (Cu) which favor to the accumulation of this metal in the soil. Therefore, the evaluation of how nutrient availability and rootstock interact with Cu toxicity in the citrus trees is required to maintain sustainability of fruit production in Cu-contaminated soils. Valencia orange trees on Sunki mandarin (SM) or Swingle citrumelo (SC) rootstock were grown in nutrient solutions combining adequate Cu (1.0 μmol L−1), excess Cu (50.0 μmol L−1), deficient phosphorus (P) (0.01 mmol L−1) and sufficient P (0.5 mmol L−1). The excess Cu reduced root and shoot growth, chlorophyll and relative water content in the leaves of the trees compared to those under adequate Cu supply. Furthermore, excess Cu caused severe damage to the root ultra-structure, characterized by the degeneration of the middle lamella and the presence of a thin and sinuous cell wall, as well as, starch accumulation in the plastids, disruption of the mitochondrial membranes and cellular plasmolysis. The damage caused by excess Cu in the cell wall and middle lamella on the root cells of SC was less severe than SM. Sufficient P supply improved the structure of the cell wall and middle lamella of trees subjected to excess Cu in comparison to P-deficient ones. Thus, the occurrence of more preserved cell wall and middle lamella supports the idea that sufficient P availability in the rooting medium and the use of SC rootstock might contribute to increase the ability of young citrus trees to cope with Cu toxicity.

The growing pollen tube is central to plant reproduction and is a long-standing model for cellular tip growth in biology. Rapid osmotically driven growth is maintained under variable conditions, which requires osmosensing and regulation. This study explores the mechanism of water entry and the potential role of osmosensory regulation in maintaining pollen growth. The osmotic permeability of the plasmalemma of Lilium pollen tubes was measured from plasmolysis rates to be 1.32±0.31×10(-3) cm s(-1). Mercuric ions reduce this permeability by 65%. Simulations using an osmotic model of pollen tube growth predict that an osmosensor at the cell membrane controls pectin deposition at the cell tip; inhibiting the sensor is predicted to cause tip bursting due to cell wall thinning. It was found that adding mercury to growing pollen tubes caused such a bursting of the tips. The model indicates that lowering the osmotic permeability per se does not lead to bursting but rather to thickening of the tip. The time course of induced bursting showed no time lag and was independent of mercury concentration, compatible with a surface site of action. The submaximal bursting response to intermediate mercuric ion concentration was independent of the concentration of calcium ions, showing that bursting is not due to a competitive inhibition of calcium binding or entry. Bursting with the same time course was also shown by cells growing on potassium-free media, indicating that potassium channels (implicated in mechanosensing) are not involved in the bursting response. The possible involvement of mercury-sensitive water channels as osmosensors and current knowledge of these in pollen cells are discussed.

Qualitative and quantitative composition of fatty acids (FA) in the lipids of vegetative organs of the halophyte Suaeda altissima (L.) Pall. grown at different NaCl concentrations in nutrient solution was studied. Along with this, the biomass of these organs, the content of water and Na+, Cl−, and K+ ions in them, and the ultrastructure of root and leaf cells were determined. At both low (1 mM) and high (750 mM) NaCl concentrations in nutrient solution, plants could maintain growth and water content in organs, demonstrating a noticeable increase in the dry weight and a slight increase in the water content at 250 mM NaCl. At all NaCl concentrations in nutrient solution, S. altissima tissues contained a relatively high K+ amount. Under salinity, Na+ and Cl− ions contributed substantially into the increase in the cell osmotic pressure, i.e., a decrease in their water potential; in the absence of salinity, K+ fulfilled this function. In the cells of both roots and leaves, NaCl stimulated endo- and exocytosis, supposedly involved in the vesicular compound transport. 750 mM NaCl induced plasmolysis and changes in the membrane structure, which can be interpreted as degradation processes. Under optimal NaCl concentration in medium (250 mM), the content of lipids in plant aboveground organs per fresh weight was more than 2.5-fold higher than under 1 or 750 mM NaCl, whereas in the roots opposite patten was observed. When plants were grown under non-optimal conditions, substantial changes occurred in the qualitative and quantitative FA composition in lipids of both aboveground organs and roots. Observed changes are discussed in relation to processes underlying S. altissima salt tolerance and those of disintegration occurring at the high external NaCl concentration (750 mM).

Fusaric acid (FA) is a nonhost-selective toxin mainly produced by Fusarium oxysporum, the causal agent of plant wilt diseases. We demonstrate that FA can induce programmed cell death (PCD) in tobacco suspension cells and the FA-induced PCD is modulated by nitric oxide (NO) signalling. Cells undergoing cell death induced by FA treatment exhibited typical characteristics of PCD including cytoplasmic shrinkage, chromatin condensation, DNA fragmentation, membrane plasmolysis, and formation of small cytoplasmic vacuoles. In addition, caspase-3-like activity was activated upon the FA treatment. The process of FA-induced PCD was accompanied by a rapid accumulation of NO in a FA dose-dependent manner. Pre-treatment of cells with NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) or NO synthase inhibitor N G-monomethyl-arginine monoacetate (L-NMMA) significantly reduced the rate of FA-induced cell death. Furthermore, the caspase-3-like activity and the expression of PAL and Hsr203J genes were alleviated by application of cPTIO or L-NMMA to FA-treated tobacco cells. This indicates that NO is an important factor involved in the FA-induced PCD. Our results also show that pre-treatment of tobacco cells with a caspase-3-specific inhibitor, Ac-DEVD-CHO, can reduce the rate of FA-induced cell death. These results demonstrate that the FA-induced cell death is a PCD and is modulated by NO signalling through caspase-3-like activation.

a b s t r a c t The movement of sensitive stamens in flowers of the Plains Prickly Pear (Opuntia polyacantha) is described in detail along with the external and internal filament anatomy. The goals of this investigation were: (1) to provide a synthesis of floral phenology and determine whether this rather unique stamen movement is nastic or a tropism and (2) to conduct macro- and micro-morphological analyses of filaments to deter- mine if there are anatomical traits associated with this movement. To better understand the internal and external structure in sensitive filaments of O. polyacantha, we performed comparative anatomical analyses in two additional species from the Opuntioideae with stamens lacking such sensitivity. The con- sistent unidirectional movement of stamens, independent of the area stimulated, indicates a thigmonastic response. This movement serves multiple purposes, from enhancing pollen presentation to facilitating cross-pollination, protecting pollen and preventing insects from robbing pollen. Anatomically, the sen- sitive and non-sensitive filaments exhibit different tissue organization. Cuticle thickness, presence of capsular structures, two layers of curved cells, and more and larger intercellular spaces are characteristic of sensitive filaments. A thin unicellular epidermal layer is characteristic in sensitive filaments versus 2-3 epidermal layers in non-sensitive filaments. Another striking feature in sensitive filaments is the pres- ence of papillae and capsular structures. We believe that these elements are related to water mobility with subsequent contraction during the thigmonastic response. Capsular structures might have a role in fluid mobility according to the stimulus of the filaments. We hypothesize that the thigmonastic response is controlled by cells with elastic properties, as evidenced by the plasmolyzed curved and contracted cells in the filaments and the fact that the movement is activated by changes in cell turgor followed by contraction as a result of plasmolysis.

Xiao Z.D., Xin X.Y., Chen H.Y., Hu S.W. (2013): Cytological investigation of anther development in DGMS line Shaan-GMS in Brassica napus L. Czech J. Genet. Plant Breed., 49: 16– 23. The cytological mechanism of male sterility of Shaan-GMS, a natural mutant dominant genic male sterile (DGMS) line in Brassica napus L., is not well studied. Cytological observation was made on different-size buds of DGMS line 0A30A derived from Shaan-GMS line. The pollen mother cells (PMCs) of DGMS line 0A30A were degen erating at the beginning of meiosis and could not pass the anaphase I stage, with no dyads or tetrads formed, suggesting that the DNA damage checkpoint and spindle assembly checkpoint were activated in sterile anthers. During the meiosis process of sterile anthers in the sterile plants, several kinds of abnormal meiotic cells could be observed: nuclei condensed PMCs, cells with micronuclei, collapsed cells, plasmolysis cells, cells connected with nucleoplasmic bridge, and microspore analogue developed from PMCs without meiosis but enclosed by the exine wall. The results suggested Shaan-GMS to be a new type of DGMS line in B. napus .

Water status and diffusion transport were studied in the roots of yellow lupine ( Lupinus luteus L., cv. Juno) treated for 48 h with two selected concentrations of Pb(NO 3 ) 2 : 150 mg l −1 , which inhibited root growth by about 50% (medium stress intensity), as well as 350 mg l −1 , which almost entirely suppressed root elongation (severe stress intensity). Relative water content (RWC), which characterizes the degree of root water saturation, slightly increased at the lower lead concentration and remained unchanged at the higher lead dose. Ultrastructure analyses under a transmission electron microscope revealed that plasmolysis was not evoked by lead in the apical part of the meristem. Moreover, direct observation of meristem cells using Nomarsky optics indicated enhanced vacuolization in the presence of both lead concentrations. These data suggest that the water status of the roots was not affected by the metal. Due to the fact that proline is involved in the maintenance of turgor in the cells, the metabolism of this amino acid was investigated. In the roots, the activity of enzymes involved in proline synthesis, such as pyrroline-5-carboxylate synthetase (P5CS) and ornithine aminotransferase (OAT), increased at 150 mg l −1 Pb 2+ ; nevertheless, proline content was diminished at the lower lead concentration. This effect is likely the result of proline degradation by proline dehydrogenase (PDH), since the activity of this enzyme increased at the lower lead dose. On the other hand, in the presence of 350 mg l −1 Pb 2+ , a low level of proline was correlated with a decrease in the activity of P5CS and OAT, as well as unchanged PDH activity in lupine roots. These data may imply that enzymatic synthesis of proline was strongly damaged by the metal ions. The low level of proline in both experimental variants suggests that proline accumulation is inessential to maintaining the osmotic uptake of water into root cells. NMR spectroscopy showed that exposition of lupine seedlings to lead caused a deceleration in water transport in the roots due to a reduction in the water transfer rate across the membranes (transmembrane transfer) and vacuoles continuum, as well as water diffusion along the root apoplast. Fluorescence staining and immunogold labeling showed the presence of callose strands in cell walls and/or in the vicinity of them. In lead-treated lupine roots, callose was mainly localized in the parenchyma cortex placed lengthwise to the vascular cylinder. Callose deposits in the cell walls may reduce vacuolar transport, as well as increase cell wall resistance to water flow. Deceleration of diffusional water movement to the vascular system, may in turn, influence the rate of long-distance water transport to aerial parts of the plant.

The suitability of the cyanolichen Peltigera praetextata (Sommerf.) Zopf as indicator of the effects of air pollution around a cement industry was investigated. For this purpose, lichen samples taken from an unpolluted site were exposed for 1–6 months at selected sites (a cement mill, two quarries, inhabited and agricultural sites) in SW Slovakia: physiological parameters and ultrastructural features were examined prior and after exposure. The responses of P. praetextata were then compared with those of the green-algal lichens Evernia prunastri and Xanthoria parietina co-located at the same sites. The performance of the cyanobacterial photobiont of P. praetextata (as reflected by the maximum quantum yield of primary photochemistry FV/FM and by the performance index PIABS) was negatively influenced mainly by the length of exposure in the urban and agricultural sites and less by dust pollution around the cement mill and the quarries. A significant increase in membrane lipid peroxidation products reflected the effects of oxidative stresses in all sites. The alteration of the content of water soluble proteins was detected in the samples exposed around the cement mill, while dehydrogenase activity did not show any significant pattern. Similarly to green algal lichens, ultrastructural alterations featured lipid droplets increase, swelling of cellular components, thylakoid degeneration and sometimes plasmolysis, which on the whole gave the cells an aged appearance, especially in the urban and agricultural sites. On the whole, E. prunastri confirmed to be the most sensitive to dust pollution, P. praetextata was likely influenced by microclimatic conditions and habitat eutrophication and X. parietina was the most resistant. When compared with green-algal lichens, P. praetextata was not the best indicator of the effects of cement dust pollution.