Why is intracellular ice lethal? A microscopical study showing evidence of programmed cell death in cryo-exposed embryonic axes of recalcitrant seeds of Acer saccharinum.

BACKGROUND AND AIMS: Conservation of the genetic diversity afforded by recalcitrant seeds is achieved by cryopreservation, in which excised embryonic axes (or, where possible, embryos) are treated and stored at temperatures lower than -180 °C using liquid nitrogen. It has previously been shown that intracellular ice forms in rapidly cooled embryonic axes of Acer saccharinum (silver maple) but this is not necessarily lethal when ice crystals are small. This study seeks to understand the nature and extent of damage from intracellular ice, and the course of recovery and regrowth in surviving tissues. METHODS: Embryonic axes of A. saccharinum, not subjected to dehydration or cryoprotection treatments (water content was 1·9 g H2O g(-1) dry mass), were cooled to liquid nitrogen temperatures using two methods: plunging into nitrogen slush to achieve a cooling rate of 97 °C s(-1) or programmed cooling at 3·3 °C s(-1). Samples were thawed rapidly (177 °C s(-1)) and cell structure was examined microscopically immediately, and at intervals up to 72 h in vitro. Survival was assessed after 4 weeks in vitro. Axes were processed conventionally for optical microscopy and ultrastructural examination. KEY RESULTS: Immediately following thaw after cryogenic exposure, cells from axes did not show signs of damage at an ultrastructural level. Signs that cells had been damaged were apparent after several hours of in vitro culture and appeared as autophagic decomposition. In surviving tissues, dead cells were sloughed off and pockets of living cells were the origin of regrowth. In roots, regrowth occurred from the ground meristem and procambium, not the distal meristem, which became lethally damaged. Regrowth of shoots occurred from isolated pockets of surviving cells of peripheral and pith meristems. The size of these pockets may determine the possibility for, the extent of and the vigour of regrowth. CONCLUSIONS: Autophagic degradation and ultimately autolysis of cells following cryo-exposure and formation of small (0·2-0·4 µm) intracellular ice crystals challenges current ideas that ice causes immediate physical damage to cells. Instead, freezing stress may induce a signal for programmed cell death (PCD). Cells that form more ice crystals during cooling have faster PCD responses.

Reactive oxygen and nitrogen species in defense/stress responses activated by chitosan in sycamore cultured cells.

Chitosan (CHT) is a non-toxic and inexpensive compound obtained by deacetylation of chitin, the main component of the exoskeleton of arthropods as well as of the cell walls of many fungi. In agriculture CHT is used to control numerous diseases on various horticultural commodities but, although different mechanisms have been proposed, the exact mode of action of CHT is still unknown. In sycamore (Acer pseudoplatanus L.) cultured cells, CHT induces a set of defense/stress responses that includes production of H2O2 and nitric oxide (NO). We investigated the possible signaling role of these reactive molecules in some CHT-induced responses by means of inhibitors of production and/or scavengers. The results show that both reactive nitrogen and oxygen species are not only a mere symptom of stress conditions but are involved in the responses induced by CHT in sycamore cells. In particular, NO appears to be involved in a cell death form induced by CHT that shows apoptotic features like DNA fragmentation, increase in caspase-3-like activity and release of cytochrome c from the mitochondrion. On the contrary, reactive oxygen species (ROS) appear involved in a cell death form induced by CHT that does not show these apoptotic features but presents increase in lipid peroxidation.

Interplay of Mg2+, ADP, and ATP in the cytosol and mitochondria: unravelling the role of Mg2+ in cell respiration.

In animal and plant cells, the ATP/ADP ratio and/or energy charge are generally considered key parameters regulating metabolism and respiration. The major alternative issue of whether the cytosolic and mitochondrial concentrations of ADP and ATP directly mediate cell respiration remains unclear, however. In addition, because only free nucleotides are exchanged by the mitochondrial ADP/ATP carrier, whereas MgADP is the substrate of ATP synthase (EC 3.6.3.14), the cytosolic and mitochondrial Mg(2+) concentrations must be considered as well. Here we developed in vivo/in vitro techniques using (31)P-NMR spectroscopy to simultaneously measure these key components in subcellular compartments. We show that heterotrophic sycamore (Acer pseudoplatanus L.) cells incubated in various nutrient media contain low, stable cytosolic ADP and Mg(2+) concentrations, unlike ATP. ADP is mainly free in the cytosol, but complexed by Mg(2+) in the mitochondrial matrix, where [Mg(2+)] is tenfold higher. In contrast, owing to a much higher affinity for Mg(2+), ATP is mostly complexed by Mg(2+) in both compartments. Mg(2+) starvation used to alter cytosolic and mitochondrial [Mg(2+)] reversibly increases free nucleotide concentration in the cytosol and matrix, enhances ADP at the expense of ATP, decreases coupled respiration, and stops cell growth. We conclude that the cytosolic ADP concentration, and not ATP, ATP/ADP ratio, or energy charge, controls the respiration of plant cells. The Mg(2+) concentration, remarkably constant and low in the cytosol and tenfold higher in the matrix, mediates ADP/ATP exchange between the cytosol and matrix, [MgADP]-dependent mitochondrial ATP synthase activity, and cytosolic free ADP homeostasis.

Symplasmic, long-distance transport in xylem and cambial regions in branches of Acer pseudoplatanus (Aceraceae) and Populus tremula x P. tremuloides (Salicaceae).

PREMISE OF THE STUDY: The picture of how long-distance transport proceeds in trees is still far from being complete. Beside the apoplasmic pathway, transport undoubtedly also takes place within the system of living cells in the secondary xylem and cambial region. Because detailed, thorough studies of the symplasmic routes in woody branches, using direct localization with fluorescent tracers, had not been done, here we focused on the main routes of long-distance symplasmic transport in xylem and cambial tissues and analyzed in detail tracer distribution in the rays on the extended cambial surface in branches of Acer pseudoplatanus and Populus tremula ×P. tremuloides. METHODS: Fluorescent tracers were loaded into branches through the vascular system, then their distribution in xylem and cambial regions was analyzed. KEY RESULTS: Tracer signal was present in the symplast of axial and radial xylem parenchyma cells and in both types of cambial cells. The living cells of xylem parenchyma and of the cambium were symplasmically interconnected via xylem rays. Tracer distribution in rays was uneven on the extended cambial surface; cambial regions with intensively or sparsely dyed rays alternated along the vertical axis of analyzed branches. CONCLUSIONS: Symplasmic, long-distance transport is present between the living cells of xylem and the cambial region in woody branches. The uneven distribution of fluorescent tracers in cambial rays along the stems is surprising and suggests the presence of an intrinsic pattern caused by an unknown mechanism.

Abaxial growth and steric constraints guide leaf folding and shape in Acer pseudoplatanus.

PREMISE OF THE STUDY: How leaf shape is regulated is a long-standing question in botany. For diverse groups of dicotyledon species, lamina folding along the veins and geometry of the space available for the primordia can explain the palmate leaf morphology. Dubbed the kirigami theory, this hypothesis of fold-dependent leaf shape regulation has remained largely theoretical. Using Acer pseudoplatanus, we investigated the mechanisms behind the two key processes of kirigami leaf development. METHODS: Cytological examination and quantitative analyses were used to examine the course of the vein-dependent lamina folding. Surgical ablation and tissue culturing were employed to test the effects of physical constraints on primordia growth. The final morphology of leaves growing without steric constraints were predicted mathematically. KEY RESULTS: The cytological examination showed that the lamina's abaxial side along the veins grows substantially more than the adaxial side. The abaxial hypergrowth along the veins and the lamina extension correlated with the lamina folding. When a primordium was released from the physical constraints imposed by the other primordia, it rapidly grew into the newly available space, while maintaining the curvature inward. The morphology of such a leaf was predicted to lack symmetry in the lobe shapes. CONCLUSIONS: The enhanced growth on the abaxial side of the lamina along the veins is likely to drive lamina folding. The surgical ablation provided clear support for the space-filling nature of leaf growth; thus, steric constraints play a role in determination of the shapes of folded leaves and probably also of the final leaf morphology.

Defense/stress responses activated by chitosan in sycamore cultured cells.

Chitosan (CHT) is a natural, non-toxic, and inexpensive compound obtained by partial alkaline deacetylation of chitin, the main component of the exoskeleton of crustaceans and other arthropods. The unique physiological and biological properties of CHT make this polymer useful for a wide range of industries. In agriculture, CHT is used to control numerous pre- and postharvest diseases on various horticultural commodities. In recent years, much attention has been devoted to CHT as an elicitor of defense responses in plants, which include raising of cytosolic Ca(2+), activation of MAP kinases, callose apposition, oxidative burst, hypersensitive response, synthesis of abscisic acid, jasmonate, phytoalexins, and pathogenesis-related proteins. In this work, we investigated the effects of different CHT concentrations on some defense/stress responses of sycamore (Acer pseudoplatanus L.) cultured cells. CHT induced accumulation of dead cells, and of cells with fragmented DNA, production of H(2)O(2) and nitric oxide, release of cytochrome c from the mitochondrion, accumulation of regulative 14-3-3 proteins in the cytosol and of HSP70 molecular chaperone binding protein in the endoplasmic reticulum, accompanied by marked modifications in the architecture of this cell organelle.

Testing hypotheses that link wood anatomy to cavitation resistance and hydraulic conductivity in the genus Acer.

• Vulnerability to cavitation and conductive efficiency depend on xylem anatomy. We tested a large range of structure-function hypotheses, some for the first time, within a single genus to minimize phylogenetic 'noise' and maximize detection of functionally relevant variation. • This integrative study combined in-depth anatomical observations using light, scanning and transmission electron microscopy of seven Acer taxa, and compared these observations with empirical measures of xylem hydraulics. • Our results reveal a 2 MPa range in species' mean cavitation pressure (MCP). MCP was strongly correlated with intervessel pit structure (membrane thickness and porosity, chamber depth), weakly correlated with pit number per vessel, and not related to pit area per vessel. At the tissue level, there was a strong correlation between MCP and mechanical strength parameters, and some of the first evidence is provided for the functional significance of vessel grouping and thickenings on inner vessel walls. In addition, a strong trade-off was observed between xylem-specific conductivity and MCP. Vessel length and intervessel wall characteristics were implicated in this safety-efficiency trade-off. • Cavitation resistance and hydraulic conductivity in Acer appear to be controlled by a very complex interaction between tissue, vessel network and pit characteristics.

Ethylene is involved in stress responses induced by fusicoccin in sycamore cultured cells.

The phytohormone ethylene is involved in many physiological and developmental processes of plants, as well as in stress responses and in the development of disease resistance. Fusicoccin (FC) is a well-known phytotoxin, that in sycamore (Acer pseudoplatanus L.) cultured cells, induces a set of stress responses, including synthesis of ethylene. In this study, we investigated the possible involvement of ethylene in the FC-induced stress responses of sycamore cells by means of Co(2+), a well-known specific inhibitor of ethylene biosynthesis. Co(2+) inhibited the accumulation of dead cells in the culture, the production of nitric oxide (NO) and of the molecular chaperone Binding Protein (BiP) in the endoplasmic reticulum induced by FC. By contrast, Co(2+) was ineffective on the FC-induced accumulation of cells with fragmented DNA, production of H(2)O(2) and release of cytochrome c from the mitochondrion, and only partially reduced the accumulation of regulative 14-3-3 proteins in the cytosol. In addition, we compared the effect of FC on the above parameters with that of the ethylene-releasing compound ethephon (2-chloroethane phosphonic acid). The results suggest that ethylene is involved in several stress responses induced by FC in sycamore cells, including a form of cell death that does not show apoptotic features and possibly involves NO as a signaling molecule.

Phosphate (Pi) starvation effect on the cytosolic Pi concentration and Pi exchanges across the tonoplast in plant cells: an in vivo 31P-nuclear magnetic resonance study using methylphosphonate as a Pi analog.

In vivo (31)P-NMR analyses showed that the phosphate (Pi) concentration in the cytosol of sycamore (Acer pseudoplatanus) and Arabidopsis (Arabidopsis thaliana) cells was much lower than the cytoplasmic Pi concentrations usually considered (60-80 mum instead of >1 mm) and that it dropped very rapidly following the onset of Pi starvation. The Pi efflux from the vacuole was insufficient to compensate for the absence of external Pi supply, suggesting that the drop of cytosolic Pi might be the first endogenous signal triggering the Pi starvation rescue metabolism. Successive short sequences of Pi supply and deprivation showed that added Pi transiently accumulated in the cytosol, then in the stroma and matrix of organelles bounded by two membranes (plastids and mitochondria, respectively), and subsequently in the vacuole. The Pi analog methylphosphonate (MeP) was used to analyze Pi exchanges across the tonoplast. MeP incorporated into cells via the Pi carrier of the plasma membrane; it accumulated massively in the cytosol and prevented Pi efflux from the vacuole. This blocking of vacuolar Pi efflux was confirmed by in vitro assays with purified vacuoles. Subsequent incorporation of Pi into the cells triggered a massive transfer of MeP from the cytosol to the vacuole. Mechanisms for Pi exchanges across the tonoplast are discussed in the light of the low cytosolic Pi level, the cell response to Pi starvation, and the Pi/MeP interactive effects.

Senescence-related changes in nitrogen in fine roots: mass loss affects estimation.

The fate of nitrogen (N) in senescing fine roots has broad implications for whole-plant N economies and ecosystem N cycling. Studies to date have generally shown negligible changes in fine root N per unit root mass during senescence. However, unmeasured loss of mobile non-N constituents during senescence could lead to underestimates of fine root N loss. For N fertilized and unfertilized potted seedlings of Populus tremuloides Michx., Acer rubrum L., Acer saccharum Marsh. and Betula alleghaniensis Britton, we predicted that the fine roots would lose mass and N during senescence. We estimated mass loss as the product of changes in root mass per length and root length between live and recently dead fine roots. Changes in root N were compared among treatments on uncorrected mass, length (which is independent of changes in mass per length), calcium (Ca) and corrected mass bases and by evaluating the relationships of dead root N as a function of live root N, species and fertilization treatments. Across species, from live to dead roots, mass decreased 28-40%, N uncorrected for mass loss increased 10-35%, N per length decreased 5-16%, N per Ca declined 14-48% and N corrected for mass declined 12-28%. Given the magnitude of senescence-related root mass loss and uncertainties about Ca dynamics in senescing roots, N loss corrected for mass loss is likely the most reliable estimate of N loss. We re-evaluated the published estimates of N changes during root senescence based on our values of mass loss and found an average of 28% lower N in dead roots than in fine roots. Despite uncertainty about the contributions of resorption, leaching and microbial immobilization to the net loss of N during root senescence, live root N was a strong and proportional predictor of dead root N across species and fertilization treatments, suggesting that live root N alone could be used to predict the contributions of senescing fine roots to whole-plant N economies and N cycling.

Role of nitric oxide in actin depolymerization and programmed cell death induced by fusicoccin in sycamore (Acer pseudoplatanus) cultured cells.

Programmed cell death (PCD) plays a vital role in plant development and is involved in defence mechanisms against biotic and abiotic stresses. Different forms of PCD have been described in plants on the basis of the cell organelle first involved. In sycamore (Acer pseudoplatanus L.) cultured cells, the phytotoxin fusicoccin (FC) induces cell death. However, only a fraction of the dead cells shows the typical hallmarks of animal apoptosis, including cell shrinkage, chromatin condensation, DNA fragmentation and release of cytochrome c from the mitochondrion. In this work, we show that the scavenging of nitric oxide (NO), produced in the presence of FC, by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) and rutin inhibits cell death without affecting DNA fragmentation and cytochrome c release. In addition, we show that FC induces a massive depolymerization of actin filaments that is prevented by the NO scavengers. Finally, the addition of actin-depolymerizing drugs induces PCD in control cells and overcomes the inhibiting effect of cPTIO on FC-induced cell death. Vice versa, the addition of actin-stabilizing drugs to FC-treated cells partially inhibits the phytotoxin-induced PCD. These results suggest that besides an apoptotic-like form of PCD involving the release of cytochrome c, FC induces at least another form of cell death, likely mediated by NO and independent of cytochrome c release, and they make it tempting to speculate that changes in actin cytoskeleton are involved in this form of PCD.

Cyclosporin A inhibits programmed cell death and cytochrome c release induced by fusicoccin in sycamore cells.

Programmed cell death plays a vital role in normal plant development, response to environmental stresses, and defense against pathogen attack. Different types of programmed cell death occur in plants and the involvement of mitochondria is still under investigation. In sycamore (Acer pseudoplatanus L.) cultured cells, the phytotoxin fusicoccin induces cell death that shows apoptotic features, including chromatin condensation, DNA fragmentation, and release of cytochrome c from mitochondria. In this work, we show that cyclosporin A, an inhibitor of the permeability transition pore of animal mitochondria, inhibits the cell death, DNA fragmentation, and cytochrome c release induced by fusicoccin. In addition, we show that fusicoccin induces a change in the shape of mitochondria which is not prevented by cyclosporin A. These results suggest that the release of cytochrome c induced by fusicoccin occurs through a cyclosporin A-sensitive system that is similar to the permeability transition pore of animal mitochondria and they make it tempting to speculate that this release may be involved in the phytotoxin-induced programmed cell death of sycamore cells.

Parenchyma cell respiration and survival in secondary xylem: does metabolic activity decline with cell age?

Sapwood respiration often declines towards the sapwood/heartwood boundary, but it is not known if parenchyma metabolic activity declines with cell age. We measured sapwood respiration in five temperate species (sapwood age range of 5-64 years) and expressed respiration on a live cell basis by quantifying living parenchyma. We found no effect of parenchyma age on respiration in two conifers (Pinus strobus, Tsuga canadensis), both of which had significant amounts of dead parenchyma in the sapwood. In angiosperms (Acer rubrum, Fraxinus americana, Quercus rubra), both bulk tissue and live cell respiration were reduced by about one-half in the oldest relative to the youngest sapwood, and all sapwood parenchyma remained alive. Conifers and angiosperms had similar bulk tissue respiration despite a smaller proportion of parenchyma in conifers (5% versus 15-25% in angiosperms), such that conifer parenchyma respired at rates about three times those of angiosperms. The fact that 5-year-old parenchyma cells respired at the same rate as 25-year-old cells in conifers suggests that there is no inherent or intrinsic decline in respiration as a result of cellular ageing. In contrast, it is not known whether differences observed in cellular respiration rates of angiosperms are a function of age per se, or whether active regulation of metabolic rate or positional effects (e.g. proximity to resources and/or hormones) could be the cause of reduced respiration in older sapwood.

An important pool of sucrose linked to starch biosynthesis is taken up by endocytosis in heterotrophic cells.

We have recently shown the occurrence of endocytic sucrose uptake in heterotrophic cells. Whether this mechanism is involved in the sucrose-starch conversion process was investigated by comparing the rates of starch accumulation in sycamore cells cultured in the presence or absence of the endocytic inhibitors wortmannin and 2-(4-morpholynyl-)-8-phenyl-4H-1 benzopyran-4-1 (LY294002). These analyses revealed a two-phase process involving an initial 120 min wortmannin- and LY294002-insensitive starch accumulation period, followed by a prolonged phase that was arrested by the endocytic inhibitors. Both wortmannin and LY294002 led to a strong reduction of the intracellular levels of both sucrose and the starch precursor molecule, ADPglucose. No changes in maximum catalytic activities of enzymes closely linked to starch and sucrose metabolism occurred in cells cultured with endocytic inhibitors. In addition, starch accumulation was unaffected by endocytic inhibitors when cells were cultured with glucose. These results provide a first indication that an important pool of sucrose incorporated into the cell is taken up by endocytosis prior to its subsequent conversion into starch in heterotrophic cells. This conclusion was substantiated further by experiments showing that sucrose-starch conversion was strongly prevented by both wortmannin and LY294002 in both potato tuber discs and developing barley endosperms.

Interspecific variation in nighttime transpiration and stomatal conductance in a mixed New England deciduous forest.

Transpiration is generally assumed to be insignificant at night when stomata close in response to the lack of photosynthetically active radiation. However, there is increasing evidence that the stomata of some species remain open at night, which would allow for nighttime transpiration if there were a sufficient environmental driving force. We examined nighttime water use in co-occurring species in a mixed deciduous stand at Harvard Forest, MA, using whole-tree and leaf-level measurements. Diurnal whole-tree water use was monitored continuously with Granier-style sap flux sensors in paper birch (Betula papyrifera Marsh.), red oak (Quercus rubra L.) and red maple (Acer rubrum L.). An analysis was conducted in which nighttime water flux could be partitioned between refilling of internal water stores and transpiration. Substantial nighttime sap flux was observed in all species and much of this flux was attributed to the refilling of depleted water stores. However, in paper birch, nighttime sap flux frequently exceeded recharge estimates. Over 10% of the total daily sap flux during the growing season was due to transpiration at night in paper birch. Nighttime sap flux was over 8% of the total daily flux in red oak and 2% in red maple; however, this flux was mainly associated with recharge. On nights with elevated vapor pressure deficit, sap flux continued through the night in paper birch, whereas it reached zero during the night in red oak and red maple. Measurements of leaf-level gas exchange on a night with elevated vapor pressure deficit showed stomatal conductance dropping by only 25% in paper birch, while approaching zero in red oak and red maple. The study highlighted differences in ecophysiological controls on sap flux exerted by co-occurring species. Paper birch is a fast-growing, shade-intolerant species with an earlier successional status than red oak and red maple. Risking water loss through nighttime transpiration may provide paper birch with an ecological advantage by enabling the species to maximize photosynthesis and support rapid growth. Nighttime transpiration may also be a mechanism for delivering oxygen to respiring cells in the deep sapwood of paper birch.

Existence of two parallel mechanisms for glucose uptake in heterotrophic plant cells.

The implied existence of two mechanisms for glucose uptake into heterotrophic plant cells was investigated using the fluorescent glucose derivative 2-NBDG (2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose), two membrane impermeable fluorescent markers (3000 mol. wt. dextran-Texas Red (d-TR) and Alexa-488), hexose carrier and endocytic inhibitors (phloridzin and wortmannin-A, respectively), and fluorescent and confocal microscopy. Both phloridzin and wortmannin-A significantly reduced the uptake of 2-NBDG into sycamore cultured cells, which was confirmed by fluorescent microscopy. Phloridzin prevented 2-NBDG uptake exclusively into the cytosol, whereas the wortmannin-A effect was more general, with 2-NBDG uptake into the vacuole being the more affected. Simultaneous incubation of cells in the membrane-impermeable fluorescent probes Alexa-488 and d-TR for 24 h resulted in co-localization of the labelling in the central vacuole and other endosomal compartments. Cytoplasts, cells devoid of vacuoles, were instrumental in demonstrating the transport of 2-NBDG by separate uptake mechanisms. In cytoplasts incubated simultaneously in 2-NBDG and d-TR for 2 h, a green fluorescent cytosol was indicative of transport of hexoses across the plasmalemma, while the co-localization of 2-NBDG and d-TR in internal vesicles demonstrated transport via an endocytic system. The absence of vesicles when cytoplasts were pre-incubated in wortmannin-A authenticated the endocytic vesicular nature of the co-shared 2-NBDG and d-TR fluorescent structures. In summary, uptake of 2-NBDG occurs by two separate mechanisms: (i) a plasmalemma-bound carrier-mediated system that facilitates 2-NBDG transport into the cytosol, and (ii) an endocytic system that transports most of 2-NBDG directly into the vacuole.

Sucrose-inducible endocytosis as a mechanism for nutrient uptake in heterotrophic plant cells.

The capacity of plant heterotrophic organs to transport and accumulate incoming nutrients (mostly in the form of sucrose) directly impacts their final size, crop productivity and nutritional value. Endocytosis as a mechanism for nutrient uptake in heterotrophic cells was investigated using suspension culture cells of sycamore (Acer pseudoplatanus L.) and the endocytic inhibitors wortmannin and LY294002. Time course analysis of sucrose uptake in intact walled cells revealed a two-phase process involving an initial 90 min wortmannin- and LY294002-insensitive sucrose uptake period, followed by a prolonged phase of rapid sucrose accumulation which was greatly inhibited by the two endocytic inhibitors. Walled cells were assessed for their capacity to incorporate the fluorescent endocytosis marker lucifer yellow-CH (LY) in the presence or absence of sucrose. Rates of sucrose and LY accumulation were virtually identical, as was their response to wortmannin. In addition, LY incorporation increased as a function of external sucrose concentration. When sucrose was substituted by other sugars or amino acids, uptake of LY greatly diminished, indicating that sucrose itself is the primary signal of endocytosis. Microscopic observations revealed the formation of vesicles containing LY and its eventual accumulation on the vacuole when sucrose was present in the incubation medium. These results demonstrate the existence of a sucrose-inducible endocytic process as a viable mechanism for solute transport into the vacuole of storage cells.

Comparison between the effects of fusicoccin, Tunicamycin, and Brefeldin A on programmed cell death of cultured sycamore (Acer pseudoplatanus L.) cells.

Programmed cell death occurs in plants during several developmental processes and during the expression of resistance to pathogen attack (i.e., the hypersensitive response). An unsolved question of plant programmed cell death is whether a unique signaling pathway or different, possibly convergent pathways exist. This problem was addressed in cultured sycamore (Acer pseudoplatanus L.) cells by comparing the effects of fusicoccin, Tunicamycin and Brefeldin A, inducers of programmed cell death with well-defined molecular and cellular targets, on some of the parameters involved in the regulation of this process. In addition to cell death, the inducers are able to stimulate the production of H2O2, the leakage of cytochrome c from mitochondria, the accumulation of cytosolic 14-3-3 proteins, and changes at the endoplasmic reticulum level, such as accumulation of the molecular chaperone binding protein and modifications in the organelle architecture. Interestingly, no additive effect on any of these parameters is observed when fusicoccin is administered in combination with Tunicamycin or Brefeldin A. Thus, these inducers seem to utilize the same or largely coincident pathways to induce programmed cell death and involvement of the endoplasmic reticulum, in addition to that of mitochondria, appears to be a common step.

Fusicoccin induces in plant cells a programmed cell death showing apoptotic features.

Programmed cell death plays a pivotal role in several developmental processes of plants and it is involved in the response to environmental stresses and in the defense mechanisms against pathogen attack. It has not yet been defined which part of the death signalling mechanism and which molecules involved in programmed cell death are common to animals and plants. In this paper we show that fusicoccin, a well-known phytotoxin, induces a strong acceleration in the appearance of Evans Blue-stainable (dead) cells in sycamore (Acer pseudoplatanus L.) cultures. This fusicoccin-induced cell death shows aspects common to the form of animal programmed cell death termed apoptosis: i.e., cell shrinkage, changes in nucleus morphology, increase in DNA fragmentation detectable by a specific immunological reaction, and presence of oligonucleosomal-size fragments (laddering) in DNA gel electrophoresis. Since fusicoccin has a well-identified molecular target, the plasma membrane H(+)-ATPase, and thoroughly investigated physiological effects, this toxin appears to be a useful tool to study the transduction of death signals leading to programmed cell death in plants.

Sucrose cycling in heterotrophic plant cell metabolism: first step towards an experimental model.

Sucrose is the cornerstone of higher plant metabolism. Produced by photosynthesis, sucrose is the main substrate for respiration and biosynthesis. The emerging idea is that sucrose may act as regulator of its own metabolism, characterized in particular by a permanent process of degradation and formation. This sucrose turnover may control several important physiological functions. Of particular concern is an energy dependent cycle involving the hexokinase. This report presents an experimental approach to define quantitatively physiological states of suspension-cultured plant cells wih reference to their sucrose content and respiration rate. Sucrose depletion of normal cells incubated in a medium devoid of sugar is measured in vivo using 13C and respiration is simultaneously recorded. Results obtained with sucrose-storing cells and Arabidopsis thaliana show that respiration rate is closely linked to the available sucrose. Sucrose-depleted cells offer a stable model to study the bioenergetics of the process.