Two vacuole-mediated defense strategies in plants

As plants lack immune cells, each cell has to defend itself against invading pathogens. Plant cells have a large central vacuole that accumulates a variety of hydrolytic enzymes and antimicrobial compounds, raising the possibility that vacuoles play a role in plant defense. However, how plants use vacuoles to protect against invading pathogens is poorly understood. Recently, we characterized two vacuole-mediated defense strategies associated with programmed cell death (PCD). In one strategy, vacuolar processing enzyme (VPE) mediated the disruption of the vacuolar membrane, resulting in the release of vacuolar contents into the cytoplasm in response to viral infection. In the other strategy, proteasome-dependent fusion of the central vacuole with the plasma membrane caused the discharge of vacuolar antibacterial protease and cell death-promoting contents from the cell in response to bacterial infection. Intriguingly, both strategies relied on enzymes with caspase-like activities: the vacuolar membrane-collapse system required VPE, which has caspase-1-like activity and the membrane-fusion system required a proteasome that has caspase-3-like activity. Thus, plants may have evolved a cellular immune system that involves vacuolar membrane collapse to prevent the systemic spread of viral pathogens and membrane fusion to inhibit the proliferation of bacterial pathogens.Key words: plant-pathogen interaction, vacuole, hypersensitive cell death, caspase activity, vacuolar processing enzyme, proteasome     

Trypanosoma cruzi: mechanisms for entry into host cells

Infective trypomastigote stages of the obligate intracellular protozoan parasite Trypanosoma cruzi are capable of entering virtually any mammalian cell in vitro. Entry is a complex process, involving initial parasite attachment to surface moieties of the target cell, internalization of the parasite via formation of a vacuole, and finally disruption of the vacuolar membrane to permit access of the parasite to the host cell cytoplasm. Attachment requires parasite metabolic energy. At sites of parasite entry recruitment of host cell lysosomes may occur, and lysosomal membrane components contribute prominently to formation of the parasitophorous vacuole. Parasite escape from the vacuole depends upon vacuolar acidification and is mediated by the coordinated action of a parasite-derived neuramindase/trans-sialidase that is capable of desialylating host-derived vacuolar membrane constituents, and a parasite-derived trans-membrane pore-forming protein. Dissection of the entry process at both the organellar and molecular level is providing fundamental and complementary insights into microbial pathogenesis and cell biology.     

Cable properties and compartmentation in Acetabularia

The electrical cable properties of three different compartmentation types of Acetabularia cells have been investigated. These three types were: normal cells, ‘stumps’ (filled with cytoplasm, no central vacuole) and ‘tubes’ (cytoplasm depleted vacuoles). The latter two types have been obtained by centrifugation of normal cells. Qualitatively, the characteristic biphasic voltage response upon rectangular current pulses is the same in these three types. Quantitatively, however, the two conductances which can be obtained from the biphasic voltage response as well as the apparent capacity of several F · m^?2 which derives from the large time constant of the second phase, are drastically increased in stumps and decreased in tubes compared to normal cells. The resting potential is a few mV more negative in stumps, and more positive in tubes, than in normal cells.Based on the existence of the high resting potential and the apparent large capacity in the non-vacuolated stumps, it is concluded that the electrogenic Cl^? pump of Acetabularia is located in the plasmalemma membrane and that the apparent large capacity is not a result of the complicated membraneous organisation of the vacuolar system. Several possibilities are discussed, in relation to the quantitative correlation between intracellular compartmentation and electrical membrane parameters.     

K+ currents through SV-type vacuolar channels are sensitive to elevated luminal sodium levels

Non-selective slow vacuolar (SV) channels mediate uptake of K+ and Na+ into vacuolar compartment. Under salt stress plant cells accumulate Na+ in the vacuole and release vacuolar K+ into the cytoplasm. It is, however, unclear how plants mediate transport of K+ from the vacuole without concomitant efflux of toxic Na+. Here we show by patch-clamp studies on isolated Arabidopsis thaliana cell culture vacuoles that SV channels do not mediate Na+ release from the vacuole as luminal Na+ blocks this channel. Gating of the SV channel is dependent on the K+ gradient across the vacuolar membrane. Under symmetrical K+ concentrations on both sides of the vacuolar membrane, SV channels mediate potassium uptake. When cytoplasmic K+ decreases, SV channels allow K+ release from the vacuole. In contrast to potassium, Na+ can be taken up by SV channels, but not released even in the presence of a 150-fold gradient (lumen to cytoplasm). Accumulation of Na+ in the vacuole shifts the activation potential of SV channels to more positive voltages and prevents gradient-driven efflux of K+. Similar to sodium, under physiological conditions, vacuolar Ca2+ is not released from vacuoles via SV channels. We suggest that a major Arabidopsis SV channel is equipped with a positively charged intrinsic gate located at the luminal side, which prevents release of Na+ and Ca2+, but permits efflux of K+. This property of the SV channel guarantees that K+ can shuttle across the vacuolar membrane while maintaining Na+ and Ca2+ stored in this organelle.     

The ultrastructure of Blastocystis galli from chickens

The ultrastructure stages of Blastocystis galli were studied in chicken's intestine and in laboratory cultures. There were found morphological structures: surface coat (cell from chickens' intestine showed a very thick surface coat); cell membrane--there were some small electron-opaque deepening "pockets" on the membrane; inner membrane; endoplasmic reticulum with attached ribosomes, which present in the cytoplasm; all cells contained numerous of small vacuoles and large glycogen inclusions in cytoplasm; mitochondria with tubular cristae; nucleus with granules condensed chromatin; central vacuole; Golgi complex was represented by number of plates grouped in a pite; the cyst-like forms were surrounded by multilayered wall.     

Simultaneous Measurement of the Bioelectric Potential of the Cell Wall and the Vacuoles during the Oscillatory Response to the Nitella Cell

Simultaneous measurements of bioelectric potentials of the vacuole and cell wall in cells of Nitella mucronata were made by inserting glass microelectrodes into the vacuole and cell wall respeclively. During the oscillation of the bioelectric potential of the vacuole. induced by sudden changes of the external bathing solution or by the impalement of the cell with a microelectrode. the cell wall potential also exhibited fluctuations of variable intensities in phase and concomitant with spikes of the vacuolar potential oscillation. However, the polarity of the pulses of the cell wall potential was reverse to that of the spikes of the vacuolar potential. These results suggest that the same event is registered at both sides of the plasmalemma membrane across which these phenomena are occurring. The results also support the voltage clamp and tracer flux measurements on these cells which indicate that during the generation of single action potentials, induced by current, the plasma lemma transiently increases its permeability to Cl^? and K⁺ ions expelling them from the cell. The variable intensity of the transient hyperpolarizations of the cell wall potential is explained by the distance of the microelectrode in the cell wall from the plasmalemma.     

FOOD VACUOLE MEMBRANE GROWTH WITH MICROTUBULE-ASSOCIATED MEMBRANE TRANSPORT IN PARAMECIUM

Evidence from a morphological study of the oral apparatus of Paramecium caudatum using electron microscope techniques have shown the existence of an elaborate structural system which is apparently designed to recycle digestive-vacuole membrane. Disk-shaped vesicles are filtered out of the cytoplasm by a group of microtubular ribbons. The vesicles, after being transported to the cytostome-cytopharynx region in association with these ribbons, accumulate next to the cytopharynx before they become fused with the cytopharyngeal membrane. This fusion allows the nascent food vacuole to grow and increase its membrane surface area. The morphology of this cytostome-cytopharynx region is described in detail and illustrated with a three-dimensional drawing of a portion of this region and a clay sculpture of the oral apparatus of Paramecium. Evidence from the literature for the transformation of food vacuole membrane into disk-shaped vesicles both from condensing food vacuoles in the endoplasm and from egested food vacuoles at the cytoproct is presented. This transformation would complete a system of digestive vacuole membrane recycling.     

Cable properties and compartmentation in Acetabularia.

The electrical cable properties of three different compartmentation types of Acetabularia cells have been investigated. These three types were: normal cells, 'stumps' (filled with cytoplasm, no central vacuole) and 'tubes' (cytoplasm depleted vacuoles). The latter two types have been obtained by centrifugation of normal cells. Qualitatively, the characteristic biphasic voltage response upon rectangular current pulses is the same in these three types. Quantitatively, however, the two conductances which can be obtained from the biphasic voltage response as well as the apparent capacity of several F . m-2 which derives from the large time constant of the second phase, are drastically increased in stumps and decreased in tubes compared to normal cells. The resting potential is a few mV more negative in stumps, and more positive in tubes, than in normal cells. Based on the existence of the high resting potential and the apparent large capacity in the non-vacuolated stumps, it is concluded that the electrogenic Cl- pump of Acetabularia is located in the plasmalemma membrane and that the apparent large capacity is not a result of the complicated membraneous organisation of the vacuolar system. Several possibilities are discussed, in relation to the quantitative correlation between intracellular compartmentation and electrical membrane parameters.     

Osmotic effects on the electrical properties of Arabidopsis root hair vacuoles in situ

To assess the role of the vacuole in responses to hyperosmotic and hypo-osmotic stress, the electrical properties of the vacuole were measured in situ. A double-barrel micropipette was inserted into the vacuole for voltage clamping. A second double-barrel micropipette was inserted into the cytoplasm to provide a virtual ground that separated the electrical properties of the vacuole from those of the plasma membrane. Osmotic stress causes immediate electrical responses at the plasma membrane (Lew RR [1996] Plant Physiol 97: 2002-2005) and ion flux changes and turgor recovery (Shabala SN, Lew RR [2002] 129: 290-299) in Arabidopsis root cells. In situ, the vacuole also responds rapidly to changes in extracellular osmotic potential. Hyperosmotic treatment caused a very large increase in the ionic conductance of the vacuole. Hypo-osmotic treatment did not affect the vacuolar conductance. In either case, the vacuolar electrical potential was unchanged. Taken in concert with previous studies of changes at the plasma membrane, these results demonstrate a highly coordinated system in which the vacuole and plasma membrane are primed to respond immediately to hyperosmotic stress before changes in gene expression.     

ULTRASTRUCTURE OF DEVELOPING AND MATURE NONARTICULATED LATICIFERS IN THE MILKWEED ASCLEPIAS SYRIACA L. (ASCLEPIADACEAE)

The ultrastructure of developing and mature nonarticulated laticifers in Asclepias syriaca L. (the common milkweed) was studied by conventional fixation and staining techniques and by osmium impregnation techniques. The mature laticifer protoplast in A. syriaca possesses a large central vacuole with an intact vacuolar membrane. Formation of this vacuole apparently results from dilation and subsequent enlargement of endoplasmic reticulum and possibly in part by fusion of smaller vacuoles and limited cellular-lytic autophagy. Widespread digestion or autophagy of cytoplasm within vacuoles is not evident. Nuclei, mitochondria, dictyosomes, and small vesicles are the most prominent components distributed in the peripheral cytoplasm. Plastids appear to degenerate as the laticifer matures. The specialized cellular component, latex, which is the vacuolar content of the laticifer, is interpreted to be produced in the cytoplasm and subsequently incorporated into the large central vacuole. Rubber globules, the most prominent latex component, are surrounded by a membrane that does not have a trilaminate structure. Globules are associated with an electron-dense fibrillar component in the vacuole.     

Ultrastructural variation of Blastocystis hominis stocks in culture

An ultrastructural study of 10 different Blastocystis hominis stocks was undertaken. Three distinct morphological forms, vacuolar, granular and amoeboid, were distinguished. Numerous variations in the organelles and general cell structure were observed between stocks. B. hominis displayed considerable size variation in the vacuolar forms, ranging from 4 to 63 micron. Thickness and density of the surface coat varied between different stocks. Beneath the surface coat the bilaminar cell membrane displayed electron-dense pits. The nature and quantity of the vacuolar contents varied, and in the granular form four morphologically different inclusions were seen. The organelles which showed the greatest variation between stocks were the mitochondria, varying in shape, electron-density, type of cristae and presence of inclusions. There was minimal variation between stocks with regard to endoplasmic reticulum, Golgi complex and nuclei. Budding of material between the cytoplasm and central vacuole was observed in some stocks. Indications of phagocytic behaviour of B. hominis were seen in the amoeboid form and in the vacuolar form of one stock.     

Membrane trafficking in guard cells during stomatal movement: Application of an image processing technique

Pairs of guard cells form small pores called stoma in the epidermis, and the reversible swelling and shrinking of these guard cells regulate the stomatal apertures. The well-documented changes in guard cell volume have been associated with their vacuolar structures. To investigate the contribution of the guard cell vacuoles to stomatal movement, the dynamics of these vacuolar structures were recently monitored during stomatal movement in vacuolar-membrane visualized Arabidopsis plants. Calculation of the vacuolar volume and surface area after reconstruction of three-dimensional images revealed a decrease in the vacuolar volume but an increase in the vacuolar surface area upon stomatal closure. These results implied the possible acceleration of membrane trafficking to the vacuole upon stomatal closure and membrane recycling from the vacuole to the plasma membrane upon stomatal opening. To clarify and quantify membrane trafficking during stomatal movement, we describe in this addendum our development of an improved image processing system.Key words: stomata, guard cells, vacuole, membrane traffic, image processing     

Anthocyanin Vacuolar Inclusions Form by a Microautophagy Mechanism

Anthocyanins are flavonoid pigments synthesized in the cytoplasm and stored inside vacuoles. Many plant species accumulate densely packed, 3- to 10-μm diameter anthocyanin deposits called anthocyanin vacuolar inclusions (AVIs). Despite their conspicuousness and importance in organ coloration, the origin and nature of AVIs have remained controversial for decades. We analyzed AVI formation in cotyledons of different Arabidopsis thaliana genotypes grown under anthocyanin inductive conditions and in purple petals of lisianthus (Eustoma grandiorum). We found that cytoplasmic anthocyanin aggregates in close contact with the vacuolar surface are directly engulfed by the vacuolar membrane in a process reminiscent of microautophagy. The engulfed anthocyanin aggregates are surrounded by a single membrane derived from the tonoplast and eventually become free in the vacuolar lumen like an autophagic body. Neither endosomal/prevacuolar trafficking nor the autophagy ATG5 protein is involved in the formation of AVIs. In Arabidopsis, formation of AVIs is promoted by both an increase in cyanidin 3-O-glucoside derivatives and by depletion of the glutathione S-transferase TT19. We hypothesize that this novel microautophagy mechanism also mediates the transport of other flavonoid aggregates into the vacuole.     

Seed Dormancy in Red Rice : VI. Monocarboxylic Acids: A New Class of pH-Dependent Germination Stimulants

The addition of an elicitor (glucan) to Phaseolus vulgaris cell suspension cultures increased the formation of the phytoalexin phaseollin. Intracellular pH and phosphate concentrations were studied with ³¹P nuclear magnetic resonance spectroscopy on elicitor-treated cells which were aerated during the nuclear magnetic resonance measurement. The pH of the vacuole and to a lesser extent the pH of the cytoplasm were affected at 10 minutes after elicitor addition; a decrease in pH from 5.3 to 4.8 was noted in the vacuole and from 7.46 to 7.28 in the cytoplasm. The ratio between the amount of Pi in the vacuole to that in the cytoplasm also changed within 10 minutes after elicitor addition. The signal for ATP (β-ATP) was low after elicitor addition and was high again 23 hours after elicitation. Forty-eight hours after elicitor addition, vacuolar and cytoplasmic pH had almost returned to their initial values. The rapid change in vacuolar and cytoplasmic pH may cause the change of metabolism that occurs in elicitor-treated P. vulgaris cells.     

Aspartyl aminopeptidase is imported from the cytoplasm to the vacuole by selective autophagy in Saccharomyces cerevisiae

Macroautophagy is a catabolic process by which cytosolic components are sequestered by double membrane vesicles called autophagosomes and sorted to the lysosomes/vacuoles to be degraded. Saccharomyces cerevisiae has adapted this mechanism for constitutive transport of the specific vacuolar hydrolases aminopeptidase I (Ape1) and α-mannosidase (Ams1); this process is called the cytoplasm to vacuole targeting (Cvt) pathway. The precursor form of Ape1 self-assembles into an aggregate-like structure in the cytosol that is then recognized by Atg19 in a propeptide-dependent manner. The interaction between Atg19 and autophagosome-forming machineries allows selective packaging of the Ape1-Atg19 complex by the autophagosome-like Cvt vesicle. Ams1 also forms oligomers and utilizes the Ape1 transport system by interacting with Atg19. Although the mechanism of selective transport of the Cvt cargoes has been well studied, it is unclear whether proteins other than Ape1 and Ams1 are transported via the Cvt pathway. We describe here that aspartyl aminopeptidase (Yhr113w/Ape4) is the third Cvt cargo, which is similar in primary structure and subunit organization to Ape1. Ape4 has no propeptide, and it does not self-assemble into aggregates. However, it binds to Atg19 in a site distinct from the Ape1- and Ams1-binding sites, allowing it to "piggyback" on the Ape1 transport system. In growing conditions, a small portion of Ape4 localizes in the vacuole, but its vacuolar transport is accelerated by nutrient starvation, and it stably resides in the vacuole lumen. We propose that the cytosolic Ape4 is redistributed to the vacuole when yeast cells need more active vacuolar degradation.     

Membrane perforations inhibit lysosome fusion by altering pH and calcium in Listeria monocytogenes vacuoles

Listeria monocytogenes (Lm) evade microbicidal defences inside macrophages by secreting a pore-forming cytolysin listeriolysin O (LLO), which allows Lm to escape vacuoles. LLO also inhibits Lm vacuole fusion with lysosomes, which indicates LLO alters vacuole chemistry prior to release of Lm into cytoplasm. Using fluorescent probes to measure membrane permeability, calcium and pH, we identified small membrane perforations in vacuoles containing wild-type but not LLO-deficient (hly-) Lm. The small membrane perforations released small fluorescent molecules and persisted for several minutes before expanding to allow exchange of larger fluorescent molecules. Macropinosomes and hly- Lm vacuoles acidified and increased their calcium content ([Ca2+]vac) within minutes of formation; however, the small perforations made by LLO-expressing bacteria increased vacuolar pH and decreased [Ca2+]vac shortly after infection. Experimental increases in vacuolar pH inhibited Lm vacuole fusion with lysosomes. The timing of perforation indicated that LLO-dependent delays of Lm vacuole maturation result from disruption of ion gradients across vacuolar membranes.     

A novel pathway of import of alpha-mannosidase, a marker enzyme of vacuolar membrane, in Saccharomyces cerevisiae

We have investigated the vacuolar delivery of alpha-mannosidase, a marker enzyme of the vacuolar membrane in the yeast Saccharomyces cerevisiae, and found that the enzyme has several unique characteristics in its biosynthesis and vacuolar delivery. alpha-Mannosidase has no typical signal sequence (Yoshihisa, T., and Anraku, Y. (1989) Biochem. Biophys. Res. Commun. 163, 908-915) but is located on the inner surface of the vacuolar membrane. The enzyme is synthesized as a 107-kDa polypeptide and converted to a 73-kDa polypeptide. Although the conversion depends on a vacuolar processing protease, proteinase A, it is much slower (t1/2 = 10 h) than the proteinase A-dependent processing of other vacuolar proteins. None of Asn-X-Thr/Ser sites on the 107-kDa alpha-mannosidase or on two alpha-mannosidase-invertase fusion proteins that are localized inside the vacuole receives N-linked oligosaccharide, whereas those sites on a carboxypeptidase Y-alpha-mannosidase fusion protein are N-glycosylated. The newly synthesized alpha-mannosidase is normally delivered to the vacuole and converted to the 73-kDa polypeptide even when the secretory pathway is blocked by a subset of sec mutations. These characteristics are different from those of other vacuolar proteins targeted to the vacuole via the secretory pathway. We conclude that alpha-mannosidase is delivered to the vacuole in a novel pathway separate from the secretory pathway.     

A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast

We have used a lipophilic styryl dye, N-(3-triethylammoniumpropyl)-4- (p-diethylaminophenyl-hexatrienyl) pyridinium dibromide (FM 4-64), as a vital stain to follow bulk membrane-internalization and transport to the vacuole in yeast. After treatment for 60 min at 30 degrees C, FM 4- 64 stained the vacuole membrane (ring staining pattern). FM 4-64 did not appear to reach the vacuole by passive diffusion because at 0 degree C it exclusively stained the plasma membrane (PM). The PM staining decreased after warming cells to 25 degrees C and small punctate structures became apparent in the cytoplasm within 5-10 min. After an additional 20-40 min, the PM and cytoplasmic punctate staining disappeared concomitant with staining of the vacuolar membrane. Under steady state conditions, FM 4-64 staining was specific for vacuolar membranes; other membrane structures were not stained. The dye served as a sensitive reporter of vacuolar dynamics, detecting such events as segregation structure formation during mitosis, vacuole fission/fusion events, and vacuolar morphology in different classes of vacuolar protein sorting (vps) mutants. A particularly striking pattern was observed in class E mutants (e.g., vps27) where 500-700 nm organelles (presumptive prevacuolar compartments) were intensely stained with FM 4- 64 while the vacuole membrane was weakly fluorescent. Internalization of FM 4-64 at 15 degrees C delayed vacuolar labeling and trapped FM 4- 64 in cytoplasmic intermediates between the PM and the vacuole. The intermediate structures in the cytoplasm are likely to be endosomes as their staining was temperature, time, and energy dependent. Interestingly, unlike Lucifer yellow uptake, vacuolar labeling by FM 4- 64 was not blocked in sec18, sec14, end3, and end4 mutants, but was blocked in sec1 mutant cells. Finally, using permeabilized yeast spheroplasts to reconstitute FM 4-64 transport, we found that delivery of FM 4-64 from the endosome-like intermediate compartment (labeled at 15 degrees C) to the vacuole was ATP and cytosol dependent. Thus, we show that FM 4-64 is a new vital stain for the vacuolar membrane, a marker for endocytic intermediates, and a fluor for detecting endosome to vacuole membrane transport in vitro.     

ESCRT-III-Associated Protein ALIX Mediates High-Affinity Phosphate Transporter Trafficking to Maintain Phosphate Homeostasis in Arabidopsis

Prior to the release of their cargoes into the vacuolar lumen, sorting endosomes mature into multivesicular bodies (MVBs) through the action of ENDOSOMAL COMPLEX REQUIRED FOR TRANSPORT (ESCRT) protein complexes. MVB-mediated sorting of high-affinity phosphate transporters (PHT1) to the vacuole limits their plasma membrane levels under phosphate-sufficient conditions, a process that allows plants to maintain phosphate homeostasis. Here, we describe ALIX, a cytosolic protein that associates with MVB by interacting with ESCRT-III subunit SNF7 and mediates PHT1;1 trafficking to the vacuole in Arabidopsis thaliana. We show that the partial loss-of-function mutant alix-1 displays reduced vacuolar degradation of PHT1;1. ALIX derivatives containing the alix-1 mutation showed reduced interaction with SNF7, providing a simple molecular explanation for impaired cargo trafficking in alix-1 mutants. In fact, the alix-1 mutation also hampered vacuolar sorting of the brassinosteroid receptor BRI1. We also show that alix-1 displays altered vacuole morphogenesis, implying a new role for ALIX proteins in vacuolar biogenesis, likely acting as part of ESCRT-III complexes. In line with a presumed broad target spectrum, the alix-1 mutation is pleiotropic, leading to reduced plant growth and late flowering, with stronger alix mutations being lethal, indicating that ALIX participates in diverse processes in plants essential for their life.