Direct evidence of active and rapid nuclear degradation triggered by vacuole rupture during programmed cell death in Zinnia

Differentiation into a tracheary element (TE) is a typical example of programmed cell death (PCD) in the developmental processes of vascular plants. In the PCD process the TE degrades its cellular contents and becomes a hollow corpse that serves as a water conduct. Using a zinnia (Zinnia elegans) cell culture we obtained serial observations of single living cells undergoing TE PCD by confocal laser scanning microscopy. Vital staining was performed and the relative fluorescence intensity was measured, revealing that the tonoplast of the swollen vacuole in TEs loses selective permeability of fluorescein just before its physical rupture. After the vacuole ruptured the nucleus was degraded rapidly within 10 to 20 min. No prominent chromatin condensation or nuclear fragmentation occurred in this process. Nucleoids in chloroplasts were also degraded in a similar time course to that of the nucleus. Degradations did not occur in non-TEs forced to rupture the vacuole by probenecid treatment. These results demonstrate that TE differentiation involves a unique type of PCD in which active and rapid nuclear degradation is triggered by vacuole rupture.     

Vacuolar Lucifer Yellow Uptake in Plants: Endocytosis or Anion Transport; A Critical Opinion

Because of its membrane-impermeant-properties Lucifer Yellow-CH (LY) is regarded by animal cell biologists as an ideal tracer for fluid-phase endocytosis. When presented to plant cells or protoplasts this fluoroprobe accumulates in the vacuole. On the other hand there are many cases where LY does not enter the vacuole when loaded into the plant cytosol. These, superficially divergent, results have previously been explained in terms of endocytosis whereby access to the vacuole is considered to occur through vesicle transport. This interpretation has now been challenged in three recent papers where the benzoic acid derivative, probenecid, has been shown to prevent vacuolar LY accumulation in plants. Since probenecid is a well-known inhibitor of organic anion transport in animal cells it has been argued that anion carriers capable of transporting LY might also exist at the plasma membrane and tonoplast of plant cells. Unfortunately probenecid has rarely, if ever, been used in plant transport studies. The fact that it is a weak acid, whose inhibitory effects are observed at concentrations of around 1 mM suggests that caution should prevail when interpreting results obtained with probenecid. The purpose of this article is therefore to highlight the current controversy surrounding LY uptake by plants and to critically evaluate the recent probenecid data.     

Cysteine proteases XCP1 and XCP2 aid micro-autolysis within the intact central vacuole during xylogenesis in Arabidopsis roots

Establishing the mechanisms regulating the autolysis of xylem tracheary elements (TEs) is important for understanding this programmed cell death process. These data demonstrate that two paralogous Arabidopsis thaliana proteases, XYLEM CYSTEINE PROTEASE1 (XCP1) and XCP2, participated in micro-autolysis within the intact central vacuole before mega-autolysis was initiated by tonoplast implosion. The data acquisition was aided by the predictable pattern of seedling root xylogenesis, the availability of single and double total knock-out T-DNA lines, anti-sera that recognized XCP1 and XCP2, and the microwave-assisted processing of whole seedlings prior to immunolabeling and observation in the transmission electron microscope. During secondary wall thickening, XCP1 and XCP2 (in wild type), XCP1 (in xcp2 seedlings) or XCP2 (in xcp1 seedlings) were imported into the TE central vacuole. Both XCP1 and XCP2 heavily labeled dense aggregates of material within the vacuole. However, because of XCP1 deficiency in xcp1 and xcp1 xcp2 TEs, non-degraded cellular remnants first accumulated in the vacuole and then persisted in the TE lumen (longer than in the wild type) after the final mega-autolysis was otherwise complete. This delayed TE clearing phenotype in xcp1 was rescued by complementation with wild-type XCP1. Although TEs in the xcp2 single knock-out cleared comparably with wild type, the non-degraded remnants in xcp1 xcp2 TEs were more densely packed than in xcp1 TEs. Therefore, XCP2 has a minor but distinct role in micro-autolysis. After tonoplast implosion, XCP1 and XCP2 remained associated with disintegrating cellular material as mega-autolysis, aided by additional lytic enzymes, destroyed the bulk of the cellular contents.     

Functions of the Vacuole in Higher Plant Cells

Current views of the activities inherent in the vacuole as a multifunctional compartment in higher plant cells are outlined. The available data indicate that the vacuole is involved in ion homeostasis of the cytosol, storing products of the primary and secondary metabolism, osmoregulation, generation of defense responses of plant cells under biotic and abiotic stress, and programmed cell death. Transport of diverse molecules and ions across the vacuolar membrane, i.e., the tonoplast, plays the major role in all these functional activities of the vacuole. Much progress toward the identification of the transport systems located in this membrane and the elucidation of the mechanisms underlying their functioning has been achieved in the last 10–15 years and has given new insights into the role of the vacuole in the integration and regulation of plant cell metabolism.     

Programmed cell death of tracheary elements as a paradigm in plants

Plant development involves various programmed cell death (PCD) processes. Among them, cell death occurring during differentiation of procambium into tracheary elements (TEs), which are a major component of vessels or tracheids, has been studied extensively. Recent studies of PCD during TE differentiation mainly using an in vitro differentiation system of Zinnia have revealed that PCD of TEs is a plant-specific one in which the vacuole plays a central role. Furthermore, there are recent findings of several factors that may initiate PCD of TEs and that act at autonomous degradation of cell contents. Herein I summarize the present knowledge about cell death program during TE differentiation as an excellent example of PCD in plants.     

Uptake and compartmentalisation of fluorescent probes byPisolithus tinctorius hyphae: evidence for an anion transport mechanism at the tonoplast but not for fluid-phase endocytosis

Summary Membrane-impermeant fluorescent probes, such as Lucifer Yellow carbohydrazide, 6-carboxyfluorescein, and high-molecular-mass fluorescent dextrans (10 and 70 kDa) are not internalised by actively-growing hyphal tip-cells ofPisolithus tinctorius even after prolonged exposure to the probe. These findings suggest that fluid-phase endocytosis may not occur in these fully turgid tip-growing hyphae. In contrast, a number of membrane-permeant fluorescent probes, including 6-carboxfluorescein diacetate, the novel fluorescein-substitute Oregon Green 488 carboxylic acid diacetate, and the thiol-reactive Cell Tracker reagents 7-amino-4-chloro-methylcoumarin and 5-chloromethylfluorescein diacetate, are taken up by these hyphae and their fluorescent products accumulate in the vacuole system. Accumulation of the fluorescent products of both 6-carboxyfluorescein diacetate and Oregon Green 488 carboxylic acid diacetate in the vacuole system is inhibited by the anion transport inhibitor probenecid and instead these fluorochromes remain in the cytoplasm. These results suggest that the membrane-permeant esters 6-carboxyfluorescein diacetate and Oregon Green 488 carboxylic acid diacetate are first hydrolysed in the cytoplasm and that their fluorescent products are subsequently sequestered across the tonoplast via an anion transport mechanism. Such an anion transport mechanism has been hitherto unrecognised in fungi and may serve to detoxify the fungal cytoplasm by the removal of naturally-occurring unwanted anions. Probenecid-inhibitable organic anion transporters are also located at the limiting membrane of the animal endosomal/lysosomal system and at the tonoplast of higher plants. Our results further support the idea that the tubular vacuole system inP. tinctorius is similar to animal endosomal/lysosomal and plant vacuole systems.     

Programmed cell death of plant tracheary elements differentiating in vitro

Summary We used various microscopic and labeling techniques to examine events occurring during the programmed cell death (PCD) of plant tracheary elements (TEs) developing in vitro. TEs differentiating in vitro synthesize a secondary cell wall which is complex in composition and pattern at approximately 72 h after hormone manipulation. The timing of PCD events was established relative to this developmental marker. Cytoplasmic streaming continues throughout secondary wall synthesis, which takes 6 h to complete in a typical cell. Vital dye staining and ultrastructural analysis show that the vacuole and plasma membrane are intact during secondary cell wall synthesis, but the cytoplasm becomes less dense in appearance, most likely through the action of confined hydrolysis by small vacuoles which are seen throughout the cell at this time. The final, preeminent step of TE PCD is a rapid collapse of the vacuole occurring after completion of secondary cell wall synthesis. Vacuole collapse is an irreversible commitment to death which results in the immediate cessation of cytoplasmic streaming and leads to the complete degradation of cellular contents, which is probably accomplished by release of hydrolytic enzymes sequestered in the vacuole. This event represents a novel form of PCD. The degradation of nuclear DNA is detectable by TUNEL, an in situ labeling method, and appears to occur near or after vacuole collapse. Our observations indicate that the process of cellular degradation that produces the hollow TE cell corpse is an active and cell-autonomous process which is distinguishable morphologically and kinetically from necrosis. Although TE PCD does not resemble apoptosis morphologically, we describe the production of spherical protoplast fragments by cultured cells that resemble apoptotic bodies but which are not involved in TE PCD. We also present evidence that, unlike the hypersensitive response (HR), TE PCD does not involve an oxidative burst. While this evidence does not exclude a role for reactive oxygen intermediates in TE PCD, it does suggest TE PCD is mechanistically distinct from cell death during the HR.Abbreviations BA 6-benzylamino-purine - DAPI 4,6-diamidino-2-phenylindole diacetate - DCF 2,7-dichlorofluorescein diacetate - DPI diphenyleneiodonium - FDA fluorescein diacetate - HR hypersensitive response - NAA -naphthalene-acetic acid - PCD programmed cell death - ROI reactive oxygen intermediate - TE tracheary element - TUNEL TdT-mediated dUTP nick end labeling     

Movement of Abscisic Acid Across the Plasmalemma and the Tonoplast of Guard Cells of Valerianella locusta

Uptake experiments and efflux compartmental analyses of abscisic acid (ABA) with acid treated epidermal peels of Valerianella locusta were performed to elucidate the mechanisms of transport of ABA across the plasmalemma and tonoplast of guard cells. ABA uptake across the plasmalemma is linearly correlated with external ABA concentration in the incubation medium. Under alkaline conditions ABA-uptake was not significantly above background, indicating that ABA uptake occurs mainly by diffusion of undissociated ABAH as the most permeable species, which is trapped afterwards in the alkaline cytosol as impermeable ABA^?. Efflux analysis of ABA revealed a saturable component of ABA transfer across the tonoplast. A Woolf-Augustinsson-Hofstee analysis suggested the existence of two transport systems for ABA at the tonoplast. The high affinity transport system had a K_M of 0.21 mol m^?3 and a V_max 85.8 amol ABA cell^?1 h^?1. Using the data of the uptake and efflux experiments we calculated the permeability coefficients of ABA for the plasmalemma and the tonoplast of guard cells, which are 2.46 10^?7 m s–¹ and 1.26 10^?8m s^?1, respectively. The distribution of the pH-probe (¹⁴C)-DMO between medium, cytosol and vacuole was investigated and used to calculate cytosolic and vacuolar pH. The vacuolar pH is too low to explain the high vacuolar ABA concentration by trapping of ABA^?, whereas the cytosol is sufficiently alkaline to act as an efficient anion trap. Therefore we conclude that ABA transport across the guard cell tonoplast is catalyzed by a saturable uptake component.     

Cytoplasmic chloride regulates cation channels in the vacuolar membrane of plant cells

Summary This study is concerned with the characterization of the ionic currents in the vacuolar membrane (tonoplast) of plant cells. Voltage patch-clamp experiments at the whole vacuole and single channel levels were employed to study the effects of cytoplasmic chloride on the tonoplast inward rectifying currents of sugar beet cultured cells. Whole vacuole experiments showed that removal of cytoplasmic chloride induced a decrease in the level of the inward currents, an effect that was reversed upon returning to control levels of cytoplasmic chloride. Substitution of cytoplasmic chloride by any other anion (organic or inorganic) resulted in a reduction in the level of the inward currents. At a given negative tonoplast potential, the inward currents showed a linear relationship with the concentration of cytoplasmic chloride between 10 and 100 mM, with the slope of these relationships increasing as the potential was made more negative. Single channel experiments showed that reduction of cytoplasmic chloride changed the gating mechanism of the channels without affecting the single channel conductance. Reduction of cytoplasmic chloride caused a decrease in the open probability of the tonoplast cation channels by reducing their mean open time and by inducing the appearance of an additional closed state.This work was supported by the National Science and Engineering Research Council of Canada.     

Progress of lignification mediated by intercellular transportation of monolignols during tracheary element differentiation of isolated Zinnia mesophyll cells

Tracheary element (TE) differentiation is a typical example of programmed cell death (PCD) in higher plants, and maturation of TEs is completed by degradation of all cell contents. However, lignification of TEs progresses even after PCD. We investigated how and whence monolignols are supplied to TEs which have undergone PCD during differentiation of isolated Zinnia mesophyll cells into TEs. Higher densities of cell culture induced greater lignification of TEs. Whereas the continuous exchanging of culture medium suppressed lignification of TEs, further addition of coniferyl alcohol into the exchanging medium reduced the suppression of lignification. Analysis of the culture medium by HPLC and GC-MS showed that coniferyl alcohol, coniferaldehyde, and sinapyl alcohol accumulated in TE inductive culture. The concentration of coniferyl alcohol peaked at the beginning of secondary wall thickening, decreased rapidly during secondary wall thickening, then increased again. These results indicated that lignification on TEs progresses by supply of monolignols from not only TEs themselves but also surrounding xylem parenchyma-like cells through medium in vitro.     

A Protease Activity Displaying Some Thrombin-like Characteristics in Conditioned Medium of Zinnia Mesophyll Cells Undergoing Tracheary Element Differentiation

The normal development of tracheary elements (TE) requires a selective degradation of the cytoplasm without loss of the extracellular wall that remains behind as the water-conducting units of xylem. Using zinnia-(Zinnia elegans L. cv. Green Envy) cultured mesophyll cells that synchronously transdifferentiate into TEs, extracellular and intracellular proteases, respectively, have been shown to both trigger death and to execute autolysis as the final component of a programmed cell death (PCD). We report here the appearance in the medium of an unusual proteolytic activity correlated with the PCD process just prior to the autolysis. The activity has a pH optimum of 5.5–6.0 and displays some thrombin characteristics. This protease activity has 1) a 10-fold higher affinity towards a thrombin-specific chromogenic substrate than toward a trypsin-specific chromogenic substrate; 2) a 1000-fold lower sensitivity to soybean trypsin inhibitor (STI) compared to trypsin; and 3) limited ability to cleave the protease-activated receptor-1, the native thrombin substrate. However, the addition of partially purified fraction containing the thrombin-like protease activity to the medium of PCD-competent cells does not prematurely trigger PCD, and the thrombin-specific peptide inhibitor phenylalanine-proline-aspartic acid-chloromethylketone fails to inhibit PCD or tracheary element (TE) formation. This suggests that this protease activity may play a role within the cells in execution of the autolysis or in the collapse of the tonoplast rather than as an extracellular proteolytic activity participating in the chain of events leading to cell death. Online publication: 7 April 2005     

ZEN1 is a key enzyme in the degradation of nuclear DNA during programmed cell death of tracheary elements

Tracheary elements (TEs) have a unique cell death program in which the rapid collapse of the vacuole triggers the beginning of nuclear degradation. Although various nucleases are known to function in nuclear DNA degradation in animal apoptosis, it is unclear what hydrolase is involved in nuclear degradation in plants. In this study, we demonstrated that an S1-type nuclease, Zinnia endonuclease 1 (ZEN1), functions directly in nuclear DNA degradation during programmed cell death (PCD) of TEs. In-gel DNase assay demonstrated the presence of a 24-kD Ca(2+)/Mg(2+)-dependent nuclease and a 40-kD Zn(2+)-dependent nuclease as well as ZEN1 in 60-h-cultured cells that included differentiating TEs. Such cell extracts possessed the ability to degrade the nuclear DNA isolated from Zinnia elegans cells in the presence of Zn(2+), and its activity was suppressed by an anti-ZEN1 antibody, indicating that ZEN1 is a central DNase responsible for nuclear DNA degradation. The introduction of the antisense ZEN1 gene into Zinnia cells cultured for 40 h specifically suppressed the degradation of nuclear DNA in TEs undergoing PCD but did not affect vacuole collapse. Based on these results, a common mechanism between animal and plant PCD is discussed.     

Caspase inhibitors affect the kinetics and dimensions of tracheary elements in xylogenic Zinnia (<Emphasis Type="Italic">Zinnia elegans</Emphasis>) cell cultures

Background  

The xylem vascular system is composed of fused dead, hollow cells called tracheary elements (TEs) that originate through trans-differentiation of root and shoot cambium cells. TEs undergo autolysis as they differentiate and mature. The final stage of the formation of TEs in plants is the death of the involved cells, a process showing some similarities to programmed cell death (PCD) in animal systems. Plant proteases with functional similarity to proteases involved in mammalian apoptotic cell death (caspases) are suggested as an integral part of the core mechanism of most PCD responses in plants, but participation of plant caspase-like proteases in TE PCD has not yet been documented.     

The Rab GTPase RabG3b functions in autophagy and contributes to tracheary element differentiation in Arabidopsis

The tracheary elements (TEs) of the xylem serve as the water‐conducting vessels of the plant vascular system. To achieve this, TEs undergo secondary cell wall thickening and cell death, during which the cell contents are completely removed. Cell death of TEs is a typical example of developmental programmed cell death that has been suggested to be autophagic. However, little evidence of autophagy in TE differentiation has been provided. The present study demonstrates that the small GTP binding protein RabG3b plays a role in TE differentiation through its function in autophagy. Differentiating wild type TE cells were found to undergo autophagy in an Arabidopsis culture system. Both autophagy and TE formation were significantly stimulated by overexpression of a constitutively active mutant (RabG3bCA), and were inhibited in transgenic plants overexpressing a dominant negative mutant (RabG3bDN) or RabG3b RNAi (RabG3bRNAi), a brassinosteroid insensitive mutant bri1‐301, and an autophagy mutant atg5‐1. Taken together, our results suggest that autophagy occurs during TE differentiation, and that RabG3b, as a component of autophagy, regulates TE differentiation.     

Voltage sensitivity of H+/Ca2+ antiport in higher plant tonoplast suggests a role in vacuolar calcium accumulation

The electrogenicity of H+/Ca2+ exchange in vacuolar membrane (tonoplast) vesicles from Beta was studied to elucidate the role of this transport system in vacuolar Ca2+ accumulation. To overcome the inherently high proton permeability of tonoplast vesicles, the pH difference established by the primary H(+)-ATPase was titrated to a uniform value by variation of the concentration either of ATP or of a permanent anion (Cl-). This enabled manipulation of membrane potential independently of the transmembrane pH difference, with a higher inside-positive membrane potential produced at lower Cl- concentrations. The rate and the extent of uncoupler-sensitive Ca2+ uptake are both stimulated about 2-fold in conditions of more positive membrane potential, suggesting that the transport system translocates positive charge outward during Ca2+ uptake. A minimum integral H+:Ca2+ stoichiometry of 3 results in a driving force for Ca2+ accumulation in the vacuole amounting to -140 mV in typical physiological conditions. It is concluded that the antiporter is thermodynamically competent to account for Ca2+ accumulation in plant vacuoles and that its reversal in vivo is unlikely.     

Vacuolar chloride regulation of an anion-selective tonoplast channel

Fluctuations in intravacuolar chloride concentrations affected the tonoplast inward (anion flux into the vacuole) currents of sugar beets (Beta vulgaris). Rising vacuolar chloride concentrations induced increases in the levels of nitrate, acetate and phosphate inward currents. These currents, evoked at physiological vacuolar potentials, showed a linear relationship with the concentration of vacuolar chloride between 6 and 100 mm. Single channel currents revealed that rises in vacuolar chloride increased the frequency and probability of channel openings at a given tonoplast potential by reducing the mean closed time of the anion channel. In addition, there was an increase in the gating charge for the channel and a decrease in the free-energy favoring the transition of the channel from the closed to the open state. Vacuolar chloride had a very different effect on malate currents. Increasing chloride concentrations resulted in decreased frequency and open probability of the channel openings, a decrease in the gating charge and an increase in the mean closed time of the channel. Our results support the role for vacuolar chloride concentrations regulating the influx of anions into the vacuole, in addition to osmoregulation. The activation of channel activity by chloride will provide a pathway for the storage of nutrients, such as nitrate and phosphate into the vacuole, while the reduction of the malate currents will allow the use of malate for mitochondrial oxidation and cytoplasmic pH control.This work was supported by the National Science and Engineering Research Council of Canada.     

Regulation of Water Permeability of Vacuolar Symplast

Effect of exogenous ABA and an inhibitor of energy metabolism NaN₃ on water permeability of the desmotubules and tonoplast as the structural elements of vacuolar symplast ensuring water permeability of this transport system was investigated. The methodological approach based on the use of NMR with magnetic field pulse gradient is described in detail. It was shown that ABA affects water permeability of the vacuolar symplast in the root cells of maize (Zea mays L.) seedlings by temporary increase in water permeability of its membrane (tonoplast) and does not modify water permeability of desmotubules. At the same time, the effect of sodium azide is related to the disturbance of water permeability in the latter, and this evidence is corroborated by the additivity in the effects of the two above-mentioned agents on diffusion decay of spin echo produced by vacuolar symplast water molecules. ABA effect was detected only at high exogenous concentrations (10^?4 M). The effect of ABA on water permeability of the tonoplast did not depend on or was weakly related to intracellular concentration of ATP, whereas the open state of desmotubules was ATP-dependent. Observations were made on the role of aquaporins in the ABA influence on tonoplast water permeability and the physiological role of high ABA concentrations.     

Inhibition of proteasome activity by the TED4 protein in extracellular space: a novel mechanism for protection of living cells from injury caused by dying cells

In maturation process of tracheary element (TE) differentiation, many hydrolases are activated to execute programmed cell death of TEs. Such hydrolases are released from maturing TEs into extracellular space. The release of hydrolases should be harmful to surrounding cells. The TED4 protein, a tentative plant non-specific lipid transfer protein that is expressed preferentially in TE-induced culture of zinnia (Zinnia elegans L.), is secreted into the apoplastic space prior to and associated with morphological changes of TEs. Our studies on the interrelationship between the TED4 protein and proteolytic activities using an in vitro TE differentiation system of zinnia revealed the following facts. (1) Active proteasome is released into medium at maturation stage of TE differentiation. (2) The TED4 protein forms a complex with proteasome in culture medium. (3) The TED4 protein inhibits proteasome activity in the medium and crude extracts of zinnia cells. (4) The depletion of the TED4 protein from culture medium results in an increase in mortality of other living cells. These results strongly suggest that the secreted TED4 protein acts as an inhibitor of proteasome to protect other cells from undesirable injury due to proteolytic activities exudated from dying TEs.     

Characterization of vacuolar transport of the endogenous alkaloid berberine in Coptis japonica

Alkaloids comprise one of the largest groups of plant secondary metabolites. Many of them exhibit strong biological activities, and, in most cases, they are accumulated in the central vacuole of alkaloid-producing plants after synthesis. However, the mechanisms involved in alkaloid transport across the tonoplast are only poorly understood. In this study, we analyzed the vacuolar transport mechanism of an isoquinoline alkaloid, berberine, which is produced and accumulated in the vacuole of cultured cells of Coptis japonica. The characterization of berberine transport using intact vacuoles and a tonoplast vesicle system showed that berberine uptake was stimulated by Mg/ATP, as well as GTP, CTP, UTP, and Mg/pyrophosphate. Berberine uptake was strongly inhibited by NH4(+) and bafilomycin A1, while vanadate, which is commonly used to inhibit ATP-binding cassette transporters, had only a slight effect, which suggests the presence of a typical secondary transport mechanism. This is contrary to the situation in the plasma membrane of this plant cell, where the ATP-binding cassette transporter is involved in berberine transport. Model experiments with liposomes demonstrated that an ion-trap mechanism was hardly implicated in berberine transport. Further studies suggested that berberine was transported across the tonoplast via an H+/berberine antiporter, which has a Km value of 43.7 microM for berberine. Competition experiments using various berberine analogs, as well as other classes of alkaloids, revealed that this transporter is fairly specific, but not exclusive, for berberine.