Cellular and ultrastructural changes in mesophyll and bundle sheath cells of maize in response to water stress

Ultrastructural changes were correlated with leaf water potential, relative water content, and abscisic acid levels in the leaf. Mesophyll cells were more prone to damage than bundle sheath cells at a leaf water potential of -18.5 bars. Tonoplast breakdown and cell disruption occurred in 25% of the mesophyll cells. On rewatering, these disrupted cells did not recover. In bundle sheath cells, starch, lost at about -13.5 bars leaf water potential, reappeared within 2.5 hours of rewatering.     

Early stages of the apoptotic pathway in plant cells are reversible

Chromatin condensation and nDNA fragmentation, indicators of apoptosis in mammalian cells, occur in plant cells during senescence and following induction by chemical agents. In Nicotiana plumbaginifolia cells, camptothecin, okadaic acid, salicylic acid, hydrogen peroxide, and the calcium ionophore A23187 induced chromatin condensation and nDNA fragmentation. Exposure of cells to low concentrations or removal of the chemical agent resulted in an initial phase of chromatin condensation, followed by its reversal. A further feature of apoptosis in mammalian cells, annexin V binding, indicative of phosphotidylserine exposure, was also confirmed in relation to the other events in the apoptotic pathway. With respect to flow cytometric characteristics, apoptosis triggered by a variety of chemicals occurs in plant cells in a manner closely related to that in mammalian cells. However, the extent of chromatin condensation is substantially greater, and in the early stages is reversible.     

Low-temperature-induced transmembrane potential changes in mesophyll cells of Arabidopsis thaliana, Helianthus annuus and Vicia faba

Glass microelectrodes were inserted into mesophyll cells of intact leaves from higher plants: Arabidopsis thaliana, Helianthus annuus and Vicia faba var minor , and transient membrane potential changes were recorded in response to a sudden temperature drop. The cold-induced potential changes were unaffected by an anion channel inhibitor (anthracene-9-carboxylic acid) and potassium channel inhibitor (tetraethyl ammonium ion). Verapamil, a calcium channel inhibitor, caused significant suppression of the cold-induced potential changes. In the presence of calmoduline antagonists (trifluoperazine and N -6-aminohexyl-5-chloro-1-naphtalenesulphonamide), their amplitudes decreased and their durations were prolonged. Neomycin, which suppresses phospholipase C, also caused substantial inhibition of the amplitudes of the cold-induced potential changes. It is concluded that cold-evoked membrane potential changes are due to calcium influxes from both the apoplast and internal stores.     

VIP inhibits histamine-induced ultrastructural changes related to acid secretion by parietal cells

We have studied the in vitro effect of VIP and histamine on ultrastructure of the parietal cells in isolated guinea pig fundic glands. The morphological changes induced by histamine in the parietal cells can be compared to those observed after histamine stimulation in vivo or in vitro on gastric mucosa preparations. In contrast, VIP incubation did not produce the ultrastructural changes related to gastric acid secretion, in resting parietal cells. Pretreatment of the glands by VIP resulted in a remarkable suppression of the histamine effect, since the parietal cells assumed an almost resting state. The data (1) indicate that the parietal cells in isolated gastric glands of the guinea pig retain in vitro the capacity to undergo the ultrastructural changes that are related to acid secretion in vivo after histamine or cAMP and (2) suggest that VIP is an inhibitor of histamine-induced gastric acid secretion in the guinea pig. It is proposed that VIP could act directly on the parietal cell via cAMP-phosphodiesterase activation, or indirectly via gastric somatostatin and/or prostaglandin secretions, inhibiting the H2 receptor-cAMP system of the parietal cell.     

Utilization of metabolic energy under saline conditions: changes in properties of ATP dependent enzymes in plant cells grown under saline conditions

The effect of growth in saline medium on the activity of two ATP utilizing enzymes was studied. Hexokinase in carrot (Daucus carota L.) cells grown in suspension culture either in the absence or presence of 150 ml NaCl, and tonoplast H⁺-ATPase in tobacco (Nicotiana tabacum L. cv. Wisconsin 38) cells grown in suspension culture either in the absence of presence of 428 mM NaCl. There was no difference in the pH profiles, NaCl sensitivity and kinetic parameters towards glucose of hexokinase activities from carrot cells grown in the presence or the absence of NaCl, but the activity from cells grown in the presence of NaCl was more resistant to inhibition by N-ethylmaleimide and to inactivation by heat. Two separate apparent Km values toward ATP were delineated in the extract from cells grown in presence of NaCl while extracts from cells grown in the absence of NaCl had only one apparent Km value. The tonoplast H⁺-ATPase from NaCl grown tobacco cells showed changed kinetic compared to this activity from cells grown in the absence of NaCl. These data may indicate that growth in NaCl results in the appearance of isozymic activity that enhances the ability of plant cells to utilize metabolic energy more efficiently.     

Morphological and ultrastructural changes in vegetative cells and heterocysts of Anabaena variabilis grown with fructose.

The morphology and ultrastructure of Anabaena variabilis grown in medium with and without 40 mM fructose were compared. Vegetative cells and young heterocysts in fructose-supplemented medium were significantly larger, were filled with glycogen granules, and had fewer thylakoids. Developing heterocysts contained large numbers of glycogen granules well into mature stages, and envelope formation was precocious. As heterocysts enlarged in fructose medium, their shape became more broadly oblong compared with the more rectangular heterocysts in fructose-free medium.     

Kinetic analysis of the K(+)-selective outward rectifier in Arabidopsis mesophyll cells: a comparison with other plant species

This paper gives a kinetic analysis of the K(+)-selective outward-rectifier (IK,out) in the plasma membrane of Arabidopsis thaliana mesophyll cells in terms of the Hodgkin-Huxley formalism. We compared the kinetic characteristics of IK,out in Arabidopsis with IK,out channels in three other plant species that were subjected to a similar analysis: tobacco suspension cells, Vicia faba guard cells and Plantago media root cells. Because the activation kinetics of IK,out shows a clear voltage dependence, the time constant of half-activation (tau 1/2) and the elementary rate constant of channel opening (a) were calculated at the potential of half-activation (V1/2). The Arabidopsis IK,out activates relatively slowly and this is reflected in a tau 1/2 of approximately 1 s. The reason for this slow activation is twofold. Firstly, the value of a is 1.5 s-1 falls at the lower end of the range of values obtained for tobacco, Vicia and Plantago: 1.1 to 3.0 s-1. Secondly, IK,out in Arabidopsis has four closed states, while tobacco and Vicia have only two. As observed in other plant species, the activation kinetics of IK,out in Arabidopsis are sensitive to external K+: V1/2 shifts with EK but remains approximately 50 mV more positive than EK.     

Effects of mitochondria-selective fluorescent probes on mitochondrial movement in Arabidopsis mesophyll cells evaluated by using the quantification

Mitochondria-selective fluorescent probes such as MitoTracker are often used for mitochondria imaging in various plants. Although some of the probes are reported to induce mitochondria dysfunction in animal cells, the effect on plant cells remains to be determined. In the present study, we applied quantitative methods to analyze mitochondrial movement, speed frequency, and speed-angle changes, based on trajectory analysis of mitochondria in mesophyll protoplast cells of Arabidopsis thaliana expressing the mitochondria-localized fluorescent protein. Using the quantitative method, we assessed whether MitoTracker Red (FM and CMXRos) induce mitochondria dysfunction in A. thaliana. Although both the fluorescent probes well-stained mitochondria, the CMXRos probe, not the FM probe, gave a severe effect on mitochondrial movement at the low concentration (10 nM), indicating a MitoTracker-induced mitochondria dysfunction in A. thaliana. These results revealed that our quantitative method based on mitochondrial movement can be used to determine the appropriate concentrations of mitochondria-selective fluorescent probes in plants.     

Identification of a vacuolar sucrose transporter in barley and Arabidopsis mesophyll cells by a tonoplast proteomic approach

The vacuole is the main cellular storage pool, where sucrose (Suc) accumulates to high concentrations. While a limited number of vacuolar membrane proteins, such as V-type H(+)-ATPases and H(+)-pyrophosphatases, are well characterized, the majority of vacuolar transporters are still unidentified, among them the transporter(s) responsible for vacuolar Suc uptake and release. In search of novel tonoplast transporters, we used a proteomic approach, analyzing the tonoplast fraction of highly purified mesophyll vacuoles of the crop plant barley (Hordeum vulgare). We identified 101 proteins, including 88 vacuolar and putative vacuolar proteins. The Suc transporter (SUT) HvSUT2 was discovered among the 40 vacuolar proteins, which were previously not reported in Arabidopsis (Arabidopsis thaliana) vacuolar proteomic studies. To confirm the tonoplast localization of this Suc transporter, we constructed and expressed green fluorescent protein (GFP) fusion proteins with HvSUT2 and its closest Arabidopsis homolog, AtSUT4. Transient expression of HvSUT2-GFP and AtSUT4-GFP in Arabidopsis leaves and onion (Allium cepa) epidermal cells resulted in green fluorescence at the tonoplast, indicating that these Suc transporters are indeed located at the vacuolar membrane. Using a microcapillary, we selected mesophyll protoplasts from a leaf protoplast preparation and demonstrated unequivocally that, in contrast to the companion cell-specific AtSUC2, HvSUT2 and AtSUT4 are expressed in mesophyll protoplasts, suggesting that HvSUT2 and AtSUT4 are involved in transport and vacuolar storage of photosynthetically derived Suc.     

白粉菌侵染对小麦叶片显微及超微结构的影响

通过半薄及超薄切片,比较了正常和受白粉菌感染的小麦叶片细胞的显微及超微结构的差异。观察结果发现：（1）受感染小麦叶肉细胞的细胞壁上局部沉积大量团状电子致密颗粒;（2）叶绿体形状由原来的椭圆形转变成圆形,叶绿体膜破裂;类囊体膨大,基粒片层排列疏松,同时,叶绿体内嗜饿性颗粒数量增加;（3）线粒体膜解体,内含物分散到了细胞质中。     

Rapid responses of mesophyll conductance to changes of CO2 concentration,temperature and irradiance are affected by N supplements in rice

Photosynthesis in C₃ plants is significantly limited by mesophyll conductance (g_m), which can vary with leaf anatomical traits and nitrogen (N) supplements. Several studies have investigated the response of g_m to N supplements; however, none examined the implications of N supplements on the response of g_m to rapid environmental changes. Here we investigated the effect of N supplement on g_m and the response of g_m to change of CO₂, temperature and irradiance in rice. High N supplement (HN) increased mesophyll cell wall surface area and chloroplast surface area exposed to intercellular airspace per leaf area, and reduced cell wall thickness. These changes resulted in increased g_m. The g_m of leaves with HN was more sensitive to changes in CO₂ concentration, temperature and irradiance. The difference in leaf structural features between low N supplement and HN indicates that a rapid change in g_m is related to the regulation of diffusion through biological membranes rather than leaf structural features. These results will contribute to an understanding of the determinants of g_m response to rapid changes in environmental factors.     

Two short sequences from amaranth 11S globulin are sufficient to target green fluorescent protein and beta-glucuronidase to vacuoles in Arabidopsis cells.

Vacuolar sorting of seed storage proteins is a very complex process since several sorting pathways and interactions among proteins of different classes have been reported. In addition, although the C-terminus of several 7S proteins is important for vacuolar delivery, other signals seem also to be involved in this process. In this work, the ability of two sequences of the Amaranthus hypochondriacus 11S globulin (amaranthin) to target reporter proteins to vacuoles was studied. We show that the C-terminal pentapeptide (KISIA) and the GNIFRGF internal sequence fused at the C terminal region of genes encoding secretory versions of green fluorescent protein (GFP) and GFP-beta-glucuronidase (GFP-GUS) were sufficient to redirect these reporter proteins to the vacuole of Arabidopsis cells. According to the three-dimensional structure of 7S and 11S storage globulins, this internal vacuolar sorting sequence corresponds to the alpha helical region involved in trimer formation, and is conserved within these families. In addition, these sequences were able to interact in vitro, in a calcium dependent manner, with the sunflower vacuolar sorting receptor homolog to pea BP-80/AtVSR1/pumpkin PV72. This work shows for the first time the role of a short internal sequence conserved among 7S and 11S proteins in vacuolar sorting.     

Quantitation of ultrastructural changes in mouse pancreatic acinar cells caused by foreign serum

Quantitative changes in the pancreatic acinar cell organelles were studied in BALB/c mice injected with 1.0 ml fresh rabbit serum intraperitoneally. Groups of 5 mice were killed at 0, 1, 3, 6 and 12 h after the serum injection. Pancreatic tissue was processed for electron microscopy by glutaraldehyde and osmium tetroxide fixation and Epon embedding. The proportions of acinar cell cytoplasm (volume fractions) occupied by zymogen granules, granular endoplasmic reticulum, Golgi apparatus, mitochondria and lysosomes (including autophagosomes) were determined by the point counting method from electron micrographs. The volume fraction of lysosomes increased during the first 3 h and remained markedly elevated up to 12 h. The volume fractions of zymogen granules increased from 12 to 28% in 12 h. It was concluded that the secretory mechanism of pancreatic acinar cells was injured by the foreign serum. The injury caused accumulation of zymogen granules and increased autophagic activity in the acinar cells.     

Daily changes of neurosecretory type-II cell structure of Pieris larvae entrained by short and long days

The neurosecretory type-II cell (NS-II cell) group of each brain hemisphere consists of three kinds of cells: two small cells, six large ones, and two others having characteristic vacuolated endoplasmic reticulum (ER).Ultrastructural changes of large NS-II cells were observed through the fifth instar and diurnally when short-day and long-day larvae were compared. There were little differences between short-day and long-day larvae in cell structures on corresponding developmental days except for daily changes, but remarkable changes were observed every day through the instar. A secretory cycle through the instar was supposed being based on the ultrastructural changes in NS-II cells: reduced secretory activity on the first day, formation of organelles necessary for the synthesis of secretory materials throughout the instar on the second day, active synthesis and secretion of secretory material during the middle stage (third-fourth day), and reversion to a reduced level of cell activity after the cessation of feeding.In short-day larvae on the third to fourth day, NS-II cells contained large aggregates of secretory granules during the day except for the time of 13 hr after the onset of photophase when a decrease of secretory granules occurred. In long-day larvae, only a small amount of secretory granules was observed at 8 and 13 hr after the onset. Rough ER changed daily paralleling with the quantitative change of the secretory granules.Based on these differences of daily changes in NS-II cell activity between short-day and long-day larvae, it was concluded that photoperiodic time measurement of diapause induction depends on the daily secretory cycle entrained by the photoperiods during the larval stage.     

Characterization of plant circadian rhythms by employing Arabidopsis cultured cells with bioluminescence reporters

Recent intensive studies have begun to shed light on the molecular mechanisms underlying the plant circadian clock in Arabidopsis thaliana. During the course of these previous studies, the most powerful technique, elegantly adopted, was a real-time bioluminescence monitoring system of circadian rhythms in intact plants carrying a luciferase (LUC) fusion transgene. We previously demonstrated that Arabidopsis cultured cells also retain an ability to generate circadian rhythms, at least partly. To further improve the cultured cell system for studies on circadian rhythms, here we adopted a bioluminescence monitoring system by establishing the cell lines carrying appropriate reporter genes, namely, CCA1::LUC and APRR1::LUC, with which CCA1 (CIRCADIAN CLOCK-ASSOCIATED1) and APRR1 (or TOC1) (ARABIDOPSIS PSEUDO-RESPONSE REGULATORS1 or TIMING OF CAB EXPRESSION1) are believed to be the components of the central oscillator. We report the results that consistently supported the view that the established cell lines, equipped with such bioluminescence reporters, might provide us with an advantageous means to characterize the plant circadian clock.     

Features of ultrastructural changes of tumor cells in tissue culture under the effect of energy metabolism inhibitors

It is shown that 2,4-dinitrophenol and dicyclohexylcarbodiimide, energy metabolism inhibitors, induce regular specific changes of the HeLa culture cells ultrastructure. Peculiarities of metabolism of the tumor cells as against the normal culture cells cause weaker and slower response.     

Protein aggregates are transported to vacuoles by a macroautophagic mechanism in nutrient-starved plant cells

When a fusion protein of cytochrome b5 (Cyt b5) and the red fluorescent protein (RFP) are expressed in tobacco BY-2 cells, the expressed protein forms intracellular aggregates that emit red fluorescence. When such cells are grown to the stationary phase or incubated in nutrient limited medium, RFP fluorescence can be detected in the vacuolar lumen. We investigated this transport mechanism using a limited-nitrogen model. E-64 and 3-methyladenine, which inhibit autophagic processes, blocked the transport of the RFP signal to the vacuole. We next traced the autophagic process in tobacco cells using YFP fused with the tobacco Atg8 homologue (YFP-NtAtg8) and analyzed the contribution of autophagy to the vacuolar transport of the aggregates. Under limited-nitrogen conditions, the aggregates were degraded in preference to other organelles, and the autophagosomes colocalized with the aggregates at a higher frequency than with mitochondria. This is the first demonstration that selective macroautophagic degradation can occur in plant cells.     

Structural and functional changes of PSI-LHCI supercomplexes of Chlamydomonas reinhardtii cells grown under high salt conditions

The effect of high salt concentration (100 mM NaCl) on the organization of photosystem I-light harvesting complex I supercomplexes (PSI-LHCI) of Chlamydomonas reinhardtii was studied. The electron transfer activity was reduced by 39% in isolated PSI-LHCI supercomplexes. The visible circular dichroism (CD) spectra associated with strongly coupled chlorophyll (Chl) dimers were reduced in intensity, indicating that pigment–pigment interactions were disrupted. This data is consistent with results from fluorescence streak camera spectroscopy, which suggest that red-shifted pigments in the PSI-LHCI antenna had been lost. Denaturing gel electrophoresis and immunoblot analysis reveals that levels of the PSI reaction center proteins PsaD, PsaE and PsaF were reduced due to salt stress. PsaE is almost completely absent under high salt conditions. It is known that the membrane-extrinsic subunits PsaD and E form the ferredoxin-docking site. Our results indicate that the PSI-LHCI supercomplex is damaged by reactive oxygen species at high salt concentration, with particular impact on the ferredoxin-docking site and the PSI-LHCI interface.