Cultured Tobacco Cells Research Articles

Multi-platform metabolomic analyses of ergosterol-induced dynamic changes in Nicotiana tabacum cells.

Metabolomics is providing new dimensions into understanding the intracellular adaptive responses in plants to external stimuli. In this study, a multi-technology-metabolomic approach was used to investigate the effect of the fungal sterol, ergosterol, on the metabolome of cultured tobacco cells. Cell suspensions were treated with different concentrations (0–1000 nM) of ergosterol and incubated for different time periods (0–24 h). Intracellular metabolites were extracted with two methods: a selective dispersive liquid-liquid micro-extraction and a general methanol extraction. Chromatographic techniques (GC-FID, GC-MS, GC×GC-TOF-MS, UHPLC-MS) and 1H NMR spectroscopy were used for quantitative and qualitative analyses. Multivariate data analyses (PCA and OPLS-DA models) were used to extract interpretable information from the multidimensional data generated from the analytical techniques. The results showed that ergosterol triggered differential changes in the metabolome of the cells, leading to variation in the biosynthesis of secondary metabolites. PCA scores plots revealed dose- and time-dependent metabolic variations, with optimal treatment conditions being found to be 300 nM ergosterol and an 18 h incubation period. The observed ergosterol-induced metabolic changes were correlated with changes in defence-related metabolites. The ‘defensome’ involved increases in terpenoid metabolites with five antimicrobial compounds (the bicyclic sesquiterpenoid phytoalexins: phytuberin, solavetivone, capsidiol, lubimin and rishitin) and other metabolites (abscisic acid and phytosterols) putatively identified. In addition, various phenylpropanoid precursors, cinnamic acid derivatives and - conjugates, coumarins and lignin monomers were annotated. These annotated metabolites revealed a dynamic reprogramming of metabolic networks that are functionally correlated, with a high complexity in their regulation.

Molecular Evolution of N-Methylputrescine Oxidase in Tobacco

Biosynthesis of nicotine in tobacco requires N-methylputrescine oxidase (MPO), which belongs to the copper-containing amine oxidase superfamily. Previous studies identified tobacco MPO1 and its close homolog NtDAO1 (formerly called MPO2), of which MPO1 has been shown preferentially to oxidize N-methylated amines. We show here that NtDAO1, as well as a homologous Arabidopsis diamine oxidase (DAO), accept non-N-methylated amines more efficiently than their corresponding N-methylated amines. MPO1 is coordinately regulated with other nicotine biosynthesis genes with regard to COI1-MYC2-dependent jasmonate induction and its dependence on nicotine-specific ERF transcription factors, whereas NtDAO1 is constitutively expressed at low basal levels in tobacco plants. Both MPO1 and NtDAO1 are targeted to peroxisomes by their C-terminal motifs, and the peroxisomal localization of MPO1 is required for it to function in nicotine biosynthesis in jasmonate-elicited cultured tobacco cells. Restricted occurrence of the MPO subfamily in Nicotiana and Solanum indicates that, during the formation of the Solanaceae, MPO has evolved from a DAO, which functions in polyamine catabolism within peroxisomes, by optimizing substrate preference and gene expression patterns to be suitable for alkaloid formation.

The longer version of Arabidopsis thaliana heat shock protein 18.2 gene terminator contributes to higher expression of stably integrated transgenes in cultured tobacco cells

In order to achieve higher expression of a transgene, it is important to optimize not only the promoter, 5′-untranslated region (UTR), and localization signal, but also the transcriptional terminator. We previously reported that the terminator derived from the Arabidopsis thaliana heat shock protein 18.2 gene (HSPT) increases gene expression in various plant species. In this study, with the goal of further increasing the expression level of transgenes, we evaluated a longer version of HSPT, corresponding to the 878 bp downstream of the stop codon, in cultured tobacco cells. The longer version of HSPT increased the expression levels of various genes including Renilla reniformis luciferase, Photinus pyralis luciferase, horseradish (Armoracia rusticana) peroxidase, and the non-toxic B subunit of Stx2e, a candidate vaccine protein for pig edema disease. This effect was not observed in a transient expression system. This element represents a useful tool for expression of transgenes integrated into the nuclear genome in plant cells.

Molecular mechanisms of herbicide-inducible gene expression of tobacco CYP71AH11 metabolizing the herbicide chlorotoluron

Tobacco cytochrome P450 (CYP) 71AH11 metabolized the herbicide chlorotoluron, and its mRNA level was increased in tobacco culture cells by the treatment of 2,4-D. In order to clarify molecular mechanisms of induced gene expression of CYP71AH11 by herbicide treatment, a 1574-bp 5′-flanking region of CYP71AH11 was cloned, ligated to the reporter β-glucuronidase (GUS) gene, and then transformed into tobacco plants. The GUS activity in the transgenic tobacco plants was highly induced by bromoxynil treatment, followed by 2,4-D. Chlorotoluron was slightly increased the GUS activity. The bromoxynil-increased GUS activity was partially repressed by the antioxidants, suggesting that reactive oxygen species may be involved in activation of the 5′-flanking region of CYP71AH11 by bromoxynil treatment. Deletion and mutation assays showed that the region CD (−1281 to −770bp from the start codon of CYP71AH11) was important, but not sufficient, for response to bromoxynil. Electrophoretic mobility shift assays and southwestern blotting revealed that the sequences AAAAAG, and GAACAAAC and GAAAATTC in the CD region were important for interaction to the nuclear proteins of <30 and ≈75kDa, respectively. Particularly, interaction between AAAAAG and <30kDa proteins was increased by bromoxynil treatment. These results gave a cue for understanding the bromoxynil-induced gene expression of CYP71AH11, which may contribute to herbicide tolerance and selectivity in crop plants.

Phosphate-induced-1 gene from Eucalyptus (EgPHI-1) enhances osmotic stress tolerance in transgenic tobacco

Environmental stresses such as drought, freezing, and high salinity induce osmotic stress in plant cells. The plant response to osmotic stress involves a number of physiological and developmental changes, which are made possible, in part, by the modulation of the expression of specific genes. Phosphate-induced-1 gene (PHI-1) was first isolated from phosphate-treated phosphate-starved tobacco cell cultures as a stress-inducible gene, which is presumably related to intracellular pH maintenance; however, the role of the PHI-1 gene product has not yet been clarified. A gene encoding a predicted protein with high similarity to tobacco PHI-1, named EgPHI-1, was previously identified in Eucalyptus by comparative transcriptome analysis of xylem cells from species of contrasting phenotypes for wood quality and growth traits. Here, we show that the overexpression of EgPHI-1 in transgenic tobacco enhances tolerance to osmotic stress. In comparison with wild-type plants, EgPHI-1 transgenic plants showed a significant increase in root length and biomass dry weight under NaCl-, polyethylene glycol, and mannitol-induced osmotic stresses. The enhanced stress tolerance of transgenic plants was correlated with increased endogenous protein levels of the molecular chaperone binding protein BiP, which in turn was correlated with the EgPHI-1 expression level in the different transgenic lines. These results provide evidence about the involvement of EgPHI-1 in osmotic stress tolerance via modulation of BiP expression, and pave the way for its future use as a candidate gene for engineering tolerance to environmental stresses in crop plants.

Sucrose transporter NtSUT1 confers aluminum tolerance on cultured cells of tobacco (Nicotiana tabacum L.)

The role of plasma membrane-localized sucrose transporter (NtSUT1) was investigated using cultured tobacco cell (Nicotiana tabacum L.) line BY-2. The wild type (WT) cells were first transformed with the NtSUT1 gene or its fragments cloned from tobacco cell line SL to form the over-expression (OX) and suppression (RNAi) cell lines, respectively. Using OX and RNAi transgenics, the role of NtSUT1 in growth capacity of actively growing cells and in aluminum (Al)-treated cells was examined. During the logarithmic phase of growth in nutrient medium containing 2,4-dichlorophenoxyacetic acid (2,4-D), both the rate of sucrose uptake measured with radio-tracer and the content of soluble sugars were higher in OX and lower in RNAi cell lines compared to WT. Overall, the content of soluble sugars negatively correlated with the time necessary for doubling mass (fresh weight). When cells were treated without (control) or with Al in a simple medium containing calcium, sucrose and 2-(N-morpholino)ethanesulfonic acid (MES; pH 5.0) for up to 18 h, the expression of NtSUT1 under its native promoter, or under the control of strong constitutive cauliflower mosaic virus (CaMV) 35S promoter, was strongly dependent on the presence of 2,4-D. Thereafter, the cells were preferentially treated in the presence of 2,4-D. During 6 h after a start of the control treatment, sucrose uptake rates were, compared to WT, slightly higher and lower in OX and RNAi lines respectively. The addition of Al reduced the sucrose uptake rates of OX and WT to the level of RNAi line, indicating that Al inhibits sucrose uptake via NtSUT1. During the post-Al culture of control and Al-treated cells in a nutrient medium, sucrose uptake rates were much higher in OX compared to WT and RNAi lines, which closely and positively correlated with the growth capacity of the cells. Judging from the growth capacity of Al-treated cells relative to that of control cells, OX cells were more tolerant to Al than WT and RNAi. In summary, we conclude that over-expression of NtSUT1 confers higher growth capacity in actively growing cells as well as in Al-treated cells.

Tobacco cell cultures transformed by the hsp101 gene exhibit an increased resistance to Clavibacter michiganensis ssp. sepedonicus

Tobacco cell cultures transformed by the hsp101 gene exhibit an increased resistance to Clavibacter michiganensis ssp. sepedonicus

Production of tENDO1 in stably transformed tobacco cell cultures for mismatch detection

Scanning DNA sequences for polymorphisms and mutations often involve the mismatch specific cleavage by endonucleases at the mismatch sites and subsequent analysis of the digested product for mutation discovery. One of the limitations of using enzymatic mutation detection methods are the cost and availability of a mismatch specific endonuclease. We report the establishment of Nicotiana tabacum L. Cv. Bright Yellow 2 cells stably expressing the truncated ENDO1 (tENDO1) mismatch specific endonuclease. The 5′-Untranslated region of N. tabacum alcohol dehydrogenase gene (NtADH 5′-UTR) under the control of cauliflower mosaic virus (CaMV 35S) promoter was employed to improve the tENDO1 protein yield. To ease the purification process, tENDO1 was secreted into the culture medium and isolated using nickel affinity chromatography. The tENDO1 was estimated to be stably produced in an average of 0.7–0.9 % total soluble protein. Functional test on tENDO1 for mismatch detection demonstrated that tENDO1 retained mismatch specific endonuclease activity resembles its native protein. Further biochemical analysis showed that tENDO1 exhibited mismatch detection specificity and efficiency comparable to other commonly used endonucleases.

Cation selectivity of the plasma membrane of tobacco protoplasts in the electroporated state

Cation selectivity of the cellular membrane of tobacco culture cells (cell line ‘bright yellow-2’) exposed to pulsed electric fields in the millisecond range was investigated. The whole cell configuration of the patch clamp technique was established on protoplasts prepared from these cells. Ion selectivity of the electroporated membrane was investigated by measuring the reversal potential of currents passing through field-induced pores. To this end the membrane was hyper- or depolarized for 10ms (prepulse); subsequently the voltage was driven to opposite polarity at a constant rate (+40 or −40mV/ms, respectively). The experiment was started by polarizing the membrane to moderately negative or positive voltages (prepulse potential ±150mV) that would not induce pore formation. Subsequently, an extended voltage range was scanned in the porated state of the membrane (prepulse potential ±600mV). IV curves in the porated and the non-porated state (obtained at the same prepulse polarity) were superimposed to determine the voltage at which both curves intersected (‘Intersection potential’). Using a modified version of the Goldmann–Hodgkin–Katz equation relative permeabilities to Ca2+ and various monovalent alkali and organic cations were calculated. Pores were found to be fairly cation selective, with a selectivity sequence determined to be Ca2+>Li+>Rb+≈K+≈Na+>TEA+≈TBA+>Cl−. Relative permeability to monovalent cations was inversely related to the ionic diameter. By fitting a formalism suggested by Dwyer at al. (J. Gen. Physiol. 75 (1980), 469–492) the effective average diameter of field induced pores was estimated to be about 1.8nm. Implications of these results for biotechnology and electroporation theory are discussed.

Histone H3 is absent from organelle nucleoids in BY‐2 cultured tobacco cells

The core histone proteins (H2A, H2B, H3 and H4) are nuclear-localised proteins that play a central role in the formation of nucleosome structure. They have long been considered to be absent from extra-nuclear, DNA-containing organelles; that is plastids and mitochondria. Recently, however, the targeting of core histone H3 to mitochondria, and the presence of nucleosome-like structures in mitochondrial nucleoids, were proposed in cauliflower and tobacco respectively. Thus, we examined whether histone H3 was present in plant organelles and participated in the organisation of nucleoid structure, using highly purified organelles and organelle nucleoids isolated from BY-2 cultured tobacco cells. Immunofluorescence microscopic observations and Western blotting analyses demonstrated that histone H3 was absent from organelles and organelle nucleoids, consistent with the historical hypothesis. Thus, the organisation of organelle nucleoids, including putative nucleosome-like repetitive structures, should be constructed and maintained without participation of histone H3.

Hyaluronan synthesis in cultured tobacco cells (BY‐2) expressing a chlorovirus enzyme: Cytological studies

Extraction of hyaluronan from animals or microbial fermentation has risks including contamination with pathogens and microbial toxins. In this work, tobacco cultured-cells (BY-2) were successfully transformed with a chloroviral hyaluronan synthase (cvHAS) gene to produce hyaluronan. Cytological studies revealed accumulation of HA on the cells, and also in subcellular fractions (protoplasts, miniplasts, vacuoplasts, and vacuoles). Transgenic BY-2 cells harboring a vSPO-cvHAS construct containing the vacuolar targeting signal of sporamin connected to the N-terminus of cvHAS accumulated significant amounts of HA in vacuoles. These results suggested that cvHAS successfully functions on the vacuolar membrane and synthesizes/transports HA into vacuoles. Efficient synthesis of HA using this system provides a new method for practical production of HA.

Enantioselective Demethylation of Nicotine as a Mechanism for Variable Nornicotine Composition in Tobacco Leaf

Nicotine and its N-demethylation product nornicotine are two important alkaloids in Nicotiana tabacum L. (tobacco). Both nicotine and nornicotine have two stereoisomers that differ from each other at 2'-C position on the pyrrolidine ring. (S)-Nicotine is the predominant form in the tobacco leaf, whereas the (R)-enantiomer only accounts for ∼0.2% of the total nicotine pool. Despite considerable past efforts, a comprehensive understanding of the factors responsible for generating an elevated and variable enantiomer fraction of nornicotine (EF(nnic) of 0.04 to 0.75) from the consistently low EF observed for nicotine has been lacking. The objective of this study was to determine potential roles of enantioselective demethylation in the formation of the nornicotine EF. Recombinant CYP82E4, CYP82E5v2, and CYP82E10, three known tobacco nicotine demethylases, were expressed in yeast and assayed for their enantioselectivities in vitro. Recombinant CYP82E4, CYP82E5v2, and CYP82E10 demethylated (R)-nicotine 3-, 10-, and 10-fold faster than (S)-nicotine, respectively. The combined enantioselective properties of the three nicotine demethylases can reasonably account for the nornicotine composition observed in tobacco leaves, which was confirmed in planta. Collectively, our studies suggest that an enantioselective mechanism facilitates the maintenance of a reduced (R)-nicotine pool and, depending on the relative abundances of the three nicotine demethylase enzymes, can confer a high (R)-enantiomer percentage within the nornicotine fraction of the leaf.

Isolation of mitochondrial and plastid ftsZ genes and analysis of the organelle targeting sequence in the diatom Chaetoceros neogracile (Diatoms, Bacillariophyceae)

SUMMARYMitochondria and plastids multiply by division in eukaryotic cells. Recently, the eukaryotic homolog of the bacterial cell division protein FtsZ was identified and shown to play an important role in the organelle division process inside the inner membrane. To explore the evolution of FtsZ proteins, and to accumulate data on the protein import system in mitochondria and plastids of the red algal lineage, one mitochondrial and three plastid ftsZ genes were isolated from the diatom Chaetoceros neogracile, whose plastids were acquired by secondary endosymbiotic uptake of a red alga. Protein import into organelles depends on the N‐terminal organelle targeting sequences. N‐terminal bipartite presequences consisting of an endoplasmic reticulum signal peptide and a plastid transit peptide are required for protein import into diatom plastids. To characterize the organelle targeting peptides of C. neogracile, we observed the localization of each green fluorescent protein‐tagged predicted organelle targeting peptide in cultured tobacco cells and diatom cells. Our data suggested that each targeting sequences functioned both in tobacco cultured cells and diatom cells.

Expression of a recombinant human erythropoietin in suspension cell cultures of Arabidopsis, tobacco and Medicago

In the last two decades, the use of plants to produce recombinant proteins and particularly biopharmaceuticals has become an attractive alternative to established systems. This is due to advantages in scalability, economy and safety. In addition the expression of recombinant proteins in plants can also be achieved utilizing in vitro cell suspensions with all the advantages such systems confer, such as product consistency, production “on demand” and the ability to perform the entire process according to good manufacturing practices. In this study we have produced the glycosylated human hormone Erythropoietin (EPO), in Medicago truncatula and Arabidopsis thaliana plants and also in cultured cell lines of tobacco, Medicago and Arabidopsis. We have also tested two different versions of the protein, one with a KDEL tag for targeted expression in the Endoplasmic Reticulum, and an untagged version expected to be secreted to the apoplast. The recombinant protein was detected in the plant leaf extracts and in the cultured cell lines. In the latter, the rEPO was detected in the cell extracts and in the spent culture medium. It was possible to recover the KDEL version of rEPO from crude cell extracts by nickel affinity chromatography, however the secreted form did not bind to the Ni- agarose beads which may indicate a possible internalization of the his-tag in the folded protein. Although the yield of rEPO obtained in cell suspensions was not as high as expected, the protein was successfully produced and secreted into the culture medium, reinforcing that plant cell suspension cultures are a promising system for production of human biopharmaceuticals.

Carbon Nanotubes Induce Growth Enhancement of Tobacco Cells

Carbon nanotubes have shown promise as regulators of seed germination and plant growth. Here, we demonstrate that multiwalled carbon nanotubes (MWCNTs) have the ability to enhance the growth of tobacco cell culture (55-64% increase over control) in a wide range of concentrations (5-500 μg/mL). Activated carbon (AC) stimulated cell growth (16% increase) only at low concentrations (5 μg/mL) while dramatically inhibited the cellular growth at higher concentrations (100-500 μg/mL). We found a correlation between the activation of cells growth exposed to MWCNTs and the upregulation of genes involved in cell division/cell wall formation and water transport. The expression of the tobacco aquaporin (NtPIP1) gene, as well as production of the NtPIP1 protein, significantly increased in cells exposed to MWCNTs compared to control cells or those exposed to AC. The expression of marker genes for cell division (CycB) and cell wall extension (NtLRX1) was also up-regulated in cells exposed to MWCNTs compared to control cells or those exposed to activated carbon only.

Isolation of polyphenol oxidase genes from Portulaca oleracea and evaluation of their ability to metabolize endocrine-disrupting chemicals

Portulaca (Portulaca oleracea cv.), a garden plant, e ciently removes endocrine-disrupting chemicals (EDCs) including bisphenol A (BPA) from hydroponic solution. We hypothesized that polyphenol oxidase (PPO) was involved in the initial steps of detoxifying EDCs in portulaca roots. In order to elucidate the molecular basis of portulaca’s ability to metabolize EDCs, we rst isolated ve PPO genes (PoPPO1–5) that were expressed mainly in portulaca roots. Among these genes, PoPPO2, PoPPO4 and PoPPO5 were introduced into cultured tobacco cells and expressed as active forms. We found that crude extracts from the cells expressing PoPPO2, PoPPO5, and to a lesser extent PoPPO4, could metabolize BPA. In addition, we found that the BPA metabolites from crude extracts of cells expressing PoPPO2, PoPPO4 and PoPPO5 were identical to those of portulaca. Moreover, PoPPO2 and PoPPO5 also caused hydroxylation of octylphenol, nonylphenol and 17β-estradiol. erefore, these results strongly suggest that PoPPOs signi cantly contribute to the superior ability of portulaca to metabolize EDCs.

DNA-binding and transcriptional activation properties of tobacco &lt;i&gt;NIC2&lt;/i&gt;-locus ERF189 and related transcription factors

In tobacco (Nicotiana tabacum), several transcription factors belonging to the IXa group of APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) cluster at the regulatory NIC2 locus for the leaf nicotine level, and directly activate structural genes required for nicotine biosynthesis and transport. Solanaceous Atropa and Hyoscyamus plants synthesize medicinal tropane alkaloids by utilizing enzymes that partially overlap with the nicotine pathway. We tested whether the tobacco NIC2-locus ERF189 binds to potential ERF binding sites in the promoters of various structural genes involved in biosynthesis of nicotine and tropane alkaloids by an electrophoresis mobility shift assay. ERF189 bound four new GCC-box-like sequences in three nicotine structural genes, but did not recognize other candidate binding sites. This binding preference was used to optimize the consensus binding sequence of ERF189 as 5′-(A/C)GC(A/C)NNCC(A/T)-3′. Five group IXa ERFs (tobacco ERF189, tobacco ERF163, Catharanthus ORCA3, Arabidopsis AtERF13, and Arabidopsis AtERF1) were examined whether they bind to the ERF189-recognition site in the promoter of the tobacco putrescine N-methyltransferase gene in vitro, and transactivate the promoter in a transient expression assay using cultured tobacco cells. The more the protein sequences were similar to ERF189, the more effective these ERFs were in these functional assays. The transient expression assay also identified transactivation domains in an N-terminal acidic region of ERF189, and in a C-terminal Ser-rich region of AtERF13.

Expression of the major mugwort pollen allergen Art v 1 in tobacco plants and cell cultures: problems and perspectives for allergen production in plants

An economic and cheap production of large amounts of recombinant allergenic proteins might become a prerequisite for the common use of microarray-based diagnostic allergy assays which allow a component-specific diagnosis. A molecular pharming strategy was applied to express the major allergen of Artemisia vulgaris pollen, Art v 1, in tobacco plants and tobacco cell cultures. The original Art v 1 with its endogenous signal peptide which directs Art v 1 to the secretory pathway, was expressed in transiently transformed tobacco leaves but was lost in stable transformed tobacco plants during the alternation of generations. Using a light-regulated promoter and “hiding” the recombinant Art v 1 in the ER succeeded in expression of Art v 1 over three generations of tobacco plants and in cell cultures generated from stable transformed plants. However, the amounts of the recombinant allergen were sufficient for analysis but not high enough to allow an economic production. Although molecular pharming has been shown to work well for the production of non-plant therapeutic proteins, it might be less efficient for closely related plant proteins.Electronic supplementary materialThe online version of this article (doi:10.1007/s00299-011-1199-3) contains supplementary material, which is available to authorised users.

Differential patterns of dehydroabietic acid biotransformation by Nicotiana tabacum and Catharanthus roseus cells

The aim of this study was to use whole cell catalysts as tools for modification of selected resin acids in order to obtain value-added functional derivatives. The enzymatic bioconversion capacities of two plant species were tested towards dehydroabietic acid. Dehydroabietic acid (DHA) is an abundant resin acid in conifers, representing a natural wood protectant. It is also one of the constituents found in by-products of the kraft chemical pulping industry. DHA was fed to tobacco (Nicotiana tabacum) and Madagascar periwinkle (Catharanthus roseus) plant cell and tissue cultures and bioconversion product formation was monitored using NMR analysis. Both plant species took up DHA from culture medium, and various types of typical detoxification processes occurred in both cultures. In addition, diverse responses to DHA treatment were observed, including differences in uptake kinetics, chemical modification of added substrate and changes in overall metabolism of the cells. Interestingly, Catharanthus roseus, a host species for pharmaceutically valuable terpenoid indole alkaloids, exhibited a very different bioconversion pattern for exogenously applied DHA than tobacco, which does not possess a terpenoid indole pathway. In tobacco, DHA is readily glycosylated in the carbonyl group, whereas in periwinkle it is proposed that a cytochrome P450-catalyzed enzymatic detoxification reaction takes place before the formation of glycosylated product.

Autophagy in Tobacco BY-2 Cells Cultured under Sucrose Starvation Conditions: Isolation of the Autolysosome and its Characterization

Tobacco culture cells carry out a large-scale degradation of intracellular proteins in order to survive under sucrose starvation conditions. We have previously suggested that this bulk degradation of cellular proteins is performed by autophagy, where autolysosomes formed de novo act as the major lytic compartments. The digestion process in autolysosomes can be retarded by addition of the cysteine protease inhibitor E-64c to the culture medium, resulting in the accumulation of autolysosomes. In the present study, we have investigated several properties of autolysosomes in tobacco cells. Electron microscopy showed that the autolysosomes contain osmiophilic particles, some of which resemble partially degraded mitochondria. It also revealed the presence of two kinds of autolysosome precursor structures; one resembled the isolation membrane and the other the autophagosome of mammalian cells. Immunofluorescence microscopy showed that autolysosomes contain acid phosphatase, in accordance with cytochemical enzyme analyses by light and electron microscopy in a previous study. Autolysosomes isolated by cell fractionation on Percoll gradients showed the localization of acid phosphatase, vacuolar H(+)-ATPase and cysteine protease. These results show that starvation-induced autophagy in tobacco cells follows a macroautophagic-type response similar to that described for other eukaryotes. However, our results indicate that, although the plant vacuole is often described as being equivalent to the lysosome of the animal cell, a new low pH lytic compartment-the autolysosome-also contributes to proteolytic degradation when tobacco cells are subjected to sucrose deprivation.