Tobacco Cell Suspension Research Articles

Acidothermus cellulolyticus E1 endoglucanase expressed in planta undergoes extensive hydroxyproline-O-glycosylation and exhibits enhanced impact on biomass digestibility.

E1 holoenzyme was extensively Hyp-O-glycosylated at the proline rich linker region in plants, which substantially increased the molecular size and improved the enzymatic digestibility of the biomass of transgenic plants. Thermophilic E1 endo-1,4-β-glucanase derived from Acidothermus cellulolyticus has been frequently expressed in planta to reconstruct the plant cell wall to overcome biomass recalcitrance. However, the expressed holoenzyme exhibited a larger molecular size (~ 100kDa) than the theoretical one (57kDa), possibly due to posttranslational modifications in the recombinant enzyme within plant cells. This study investigates the glycosylation of the E1 holoenzyme expressed in tobacco plants and determines its impact on enzyme activity and biomass digestibility. The E1 holoenzyme, E1 catalytic domain (E1cd) and E1 linker (E1Lk) were each expressed in tobacco plants and suspension cells. The accumulation of holoenzyme was 2.0- to 2.3- times higher than that of E1cd. The proline-rich E1Lk region was extensively hydroxyproline-O-glycosylated with arabinogalactan polysaccharides. Compared with E1cd, the holoenzyme displayed a broader optimal temperature range (70 to 85 ºC). When grown in greenhouse, the expression of E1 holoenzyme induced notable phenotypic changes in plants, including delayed flowering and leaf variegation post-flowering. However, the final yield of plant biomass was not significantly affected. Finally, plant biomass engineering with E1 holoenzyme showed 1.7- to 1.8-fold higher saccharification efficiency than the E1cd lines and 2.4- to 2.7-fold higher than the wild-type lines, which was ascribed to the synergetic action of the E1Lk and cellulose binding module in reducing cell wall recalcitrance.

Development and characterization of tobacco suspension cell chassis NBS-1

Plant synthetic biology has significant theoretical advantages in exploration and production of plant natural products. However, its contribution to the field of biosynthesis is currently limited due to the lack of efficient chassis systems and related enabling technologies. Synthetic biologists often avoid tobacco as a chassis system because of its long operation cycle, difficulties in genetic and metabolic modification, complex metabolism and purification background, nicotine toxicity, and challenges in accurately controlling for agricultural production. Nevertheless, the tobacco suspension cell chassis system offers a viable solution to these challenges. The objective of this research was to develop a tobacco suspension cell chassis with high scientific and industrial potential. This chassis should exhibit rapid growth, high biomass, excellent dispersion, high transformation efficiency, and minimal nicotine content. Nicotiana benthamiana, which has high applicability in molecular technology, was used to induce suspension cells. The induced suspension cells, named NBS-1, exhibited rapid growth, excellent dispersion, and high biomass, reaching a maximum biomass of 476.39 g/L (fresh weight), which was significantly higher than that of BY-2. The transformation efficiency of the widely utilized pEAQ-HT transient expression system in NBS-1 reached 81%, which was substantially elevated compared to BY-2. The metabolic characteristics and bias of BY-2 and NBS-1 were analyzed using transcriptome data. It was found that the gene expression of pathways related to biosynthesis of flavonoids and their derivatives in NBS-1 was significantly higher, while the pathways related to alkaloid biosynthesis were significantly lower compared to BY-2. These findings were further validated by the total content of flavonoid and alkaloid. In summary, our research demonstrates NBS-1 possesses minimal nicotine content and provides valuable guidance for selecting appropriate chassis for specific products. In conclusion, this study developed NBS-1, a tobacco suspension cell chassis with excellent growth and transformation, high flavonoid content and minimal nicotine content, which has important guiding significance for the development of tobacco suspension cell chassis.

Fusion gene 4CL-CCR promotes lignification in tobacco suspension cells

The fusion gene 4CL-CCR promotes lignification and activates lignin-related MYB expression in tobacco but inhibits auxin-related gene expression and hinders the auxin absorption of cells. Given the importance of lignin polymers in plant growth and their industrial value, it is necessary to investigate how plants synthesize monolignols and regulate the level of lignin in cell walls. In our previous study, expression of the Populus tomentosa fusion gene 4CL-CCR significantly promoted the production of 4-hydroxycinnamyl alcohols. However, the function of 4CL-CCR in organisms remains poorly understood. In this study, the fusion gene 4CL-CCR was heterologously expressed in tobacco suspension cells. We found that the transgenic suspension cells exhibited lignification earlier. Furthermore, 4CL-CCR significantly reduced the content of phenolic acids and increased the content of aldehydes in the medium, which led to an increase in lignin deposition. Moreover, transcriptome results showed that the genes related to lignin synthesis, such as PAL, 4CL, CCoAOMT and CAD, were significantly upregulated in the 4CL-CCR group. The expression of genes related to auxin, such as ARF3, ARF5 and ARF6, was significantly downregulated. The downregulation of auxin affected the expression of transcription factor MYBs. We hypothesize that the upregulated genes MYB306 and MYB315 are involved in the regulation of cell morphogenesis and lignin biosynthesis and eventually enhance lignification in tobacco suspension cells. Our findings provide insight into the function of 4CL-CCR in lignification and how secondary cell walls are formed in plants.

Plant-Based Cell-Free Transcription and Translation of Recombinant Proteins.

Plant cell-free lysates contain all the cellular components of the protein biosynthesis machinery, providing an alternative to intact plant cells, tissues, and whole plants for the production of recombinant proteins. Cell-free lysates achieve rapid protein production (within hours or days) and allow the synthesis of proteins that are cytotoxic or unstable in living cells. The open nature of cell-free lysates and their homogeneous and reproducible performance is ideal for protein production, especially for screening applications, allowing the direct addition of nucleic acid templates encoding proteins of interest, as well as other components such as enzyme substrates, chaperones, artificial amino acids, or labeling molecules. Here we describe procedures for the production of recombinant proteins in the ALiCE (Almost Living Cell-free Expression) system, a lysate derived from tobacco cell suspension cultures that can be used to manufacture protein products for molecular and biochemical analysis as well as applications in the pharmaceutical industry.

Melatonin Protects Tobacco Suspension Cells against Pb-Induced Mitochondrial Dysfunction.

Recent studies have shown that melatonin is an important molecule in plant physiology. It seems that the most important is that melatonin effectively eliminates oxidative stress (direct and indirect antioxidant) and switches on different defence strategies (preventive and interventive actions) during environmental stresses. In the presented report, exogenous melatonin potential to protect Nicotiana tabacum L. line Bright Yellow 2 (BY-2) exposed to lead against death was examined. Analyses of cell proliferation and viability, the level of intracellular calcium, changes in mitochondrial membrane potential (ΔΨm) as well as possible translocation of cytochrome c from mitochondria to cytosol and subsequent caspase-like proteolytic activity were conducted. Our results indicate that pretreatment BY-2 with melatonin protected tobacco cells against mitochondrial dysfunction and caspase-like activation caused by lead. The findings suggest the possible role of this indoleamine in the molecular mechanism of mitochondria, safeguarding against potential collapse and cytochrome c release. Thus, it seems that applied melatonin acted as an effective factor, promoting survival and increasing plant tolerance to lead.

Purification, immunological, and functional characterization of MxFIT in Malus xiaojinensis

MxFIT is a FER-like iron deficiency induced transcriptional factor in Malus xiaojinensis. Here, we described the heterologous expression of MxFIT in Escherichia coli BL21 (DE3) host cells. The E. coli harboring the recombinant construct pET-MxFIT was efficiently induced to express the MxFIT protein at a high level and the optimal profile for MxFIT expression was investigated. By inoculating a New Zealand rabbit with purified MxFIT-His fusion protein, a high specific anti-MxFIT antiserum was achieved. Western blot analysis showed MxFIT protein expression was induced in roots when iron supply was limiting and was inhibited when iron supply was excessive. In leaves, there was almost no expression irrespective of iron supply. The localization of MxFIT on transverse section of root indicated that MxFIT participated in iron deficiency response. Over-expression of MxFIT in transgenic tobacco suspension cells showed that MxFIT increased iron absorption under insufficient iron supply. This study provides a basis for further investigating the underlying mechanism of high iron absorption efficiency in M. xiaojinensis.

Application of tetrahedral -deoxyribonucleic acid electrochemistry platform coupling aptazymes and hybridized hairpin reactions for the measurement of extracellular adenosine triphosphate in plants

Extracellular ATP (eATP) is an important biological signal transduction molecule. Although a variety of detection methods have been extensively used in ATP sensing and analysis, accurate detection of eATP remains difficult due to its extremely low concentration and spatiotemporal distribution. Here, an eATP measurement strategy based on tetrahedral DNA (T-DNA)-modified electrode sensing platform and hybridization chain reaction (HCR) combined with G-quadruplex/Hemin (G4/Hemin) DNAzyme dual signal amplification is proposed. In this strategy, ATP aptamer and RNA-cleaving DNAzyme were combined to form a split aptazyme. In the presence of ATP, this aptazyme hydrolyzes the cleaving substrate strand with high selectivity, releasing cleaved ssDNA, which are captured by the T-DNA assembled on the electrode surface, triggering an HCR on the electrode surface to form numerous linker sequences of the HCR dsDNA product. When G-quadruplex@AuNPs (G4) spherical nucleic acid enzymes (SNAzymes) with other linkers are used as nanocatalyst tags, they are captured by HCR dsDNA through sticky linkers present on the electrode surface. An amplified electrochemical redox current signal is generated through SNAzyme-mediated catalysis of H2O2, enabling easy detection of picomole amounts of ATP. Using this strategy, eATP levels released by tobacco suspension cells were accurately measured and the distribution and concentration of eATP released on the surface of an Arabidopsis leaf was determined.

In vitro inhibition of shikimate hydroxycinnamoyltransferase by acibenzolar acid, the first metabolite of the plant defence inducer acibenzolar-S-methyl

Acibenzolar acid, the first metabolite formed in planta from the defence inducer acibenzolar-S-methyl (ASM), has been shown to be an inhibitor of the enzyme shikimate hydroxycinnamoyltransferase (HST), extracted from grapevine or tobacco cell suspension cultures. Using a purified recombinant Arabidopsis thaliana HST, the inhibition was found to be competitive, acibenzolar acid binding reversibly to the shikimate binding site of the HST:p-coumaroyl-CoA complex, with a Ki value of 250 μM. The other hydroxycinnamoyltransferases tested in the course of this study, using either hydroxypalmitic acid, putrescine, tyramine, or quinic acid as acyl acceptors were not, or only slightly, inhibited by acibenzolar acid. To understand the specificity of the interaction of acibenzolar acid with HST, we analyzed the structure-activity relationship of a series of benzoic or acibenzolar acid analogues, tested either as AtHST substrates or as inhibitors. This analysis confirmed previously published data on the substrate flexibility of HST and demonstrated that both the carboxyl group and the thiadiazole moiety of acibenzolar acid are playing an important role in the interaction with the shikimate binding site. Acibenzolar acid, which cannot form an ester bond with p-coumaric acid, was however a less potent inhibitor than protocatechuic or 3-hydroxybenzoic acids, which are used as acyl acceptors by HST. Our results show that the interaction of acibenzolar acid with HST, which is probably directly linked to the substrate promiscuity of HST, is unlikely to play a direct role in the defence-inducing properties of ASM in plants.

H2O2 signaling modulates Glycoprotein-1 induced programmed cell death in tobacco suspension cells

Glycoprotein (GP)-1 is a glycoprotein elicitor with antiviral activity found in Streptomyces kanasensis zx01. GP-1 can induce programmed cell death (PCD) in vitro; however, the underlying mechanism is unclear. In the present study, we demonstrated that GP-1 induced PCD in tobacco suspension cells, which was modulated by hydrogen peroxide (H2O2). GP-1 participated in and modulated biologically relevant signaling in plant cells. GP-1 induced tobacco cell death in a dose- and time-dependent manner; affected the expression of BRI1-associated receptor kinase 1 (BAK1) and the accumulation of salicylic acid (SA), which are related to PCD; and enzymatic activities and mitochondrial functions. In conclusion, GP-1-induced PCD in tobacco may be mediated by H2O2 which alters BAK1 and SA levels, as well as mitochondrial and gene function. This cell signal cascade played an important role in the process of GP-1 induced plant disease resistance.

From Survival to Productivity Mode: Cytokinins Allow Avoiding the Avoidance Strategy Under Stress Conditions.

Growth retardation and stress-induced premature plant senescence are accompanied by a severe yield reduction and raise a major agro-economic concern. To improve biomass and yield in agricultural crops under mild stress conditions, the survival must be changed to productivity mode. Our previous successful attempts to delay premature senescence and growth inhibition under abiotic stress conditions by autoregulation of cytokinins (CKs) levels constitute a generic technology toward the development of highly productive plants. Since this technology is based on the induction of CKs synthesis during the age-dependent senescence phase by a senescence-specific promoter (SARK), which is not necessarily regulated by abiotic stress conditions, we developed autoregulating transgenic plants expressing the IPT gene specifically under abiotic stress conditions. The Arabidopsis promoter of the stress-induced metallothionein gene (AtMT) was isolated, fused to the IPT gene and transformed into tobacco plants. The MT:IPT transgenic tobacco plants displayed comparable elevated biomass productivity and maintained growth under drought conditions. To decipher the role and the molecular mechanisms of CKs in reverting the survival transcriptional program to a sustainable plant growth program, we performed gene expression analysis of candidate stress-related genes and found unexpectedly clear downregulation in the CK-overproducing plants. We also investigated kinase activity after applying exogenous CKs to tobacco cell suspensions that were grown in salinity stress. In-gel kinase activity analysis demonstrated CK-dependent deactivation of several stress-related kinases including two of the MAPK components, SIPK and WIPK and the NtOSAK, a member of SnRK2 kinase family, a key component of the ABA signaling cascade. A comprehensive phosphoproteomics analysis of tobacco cells, treated with exogenous CKs under salinity-stress conditions indicated that >50% of the identified phosphoproteins involved in stress responses were dephosphorylated by CKs. We hypothesize that upregulation of CK levels under stress conditions desensitize stress signaling cues through deactivation of kinases that are normally activated under stress conditions. CK-dependent desensitization of environmental stimuli is suggested to attenuate various pathways of the avoidance syndrome including the characteristic growth arrest and the premature senescence while allowing normal growth and metabolic maintenance.

Comparison of two systems of tonoplast purification from tobacco cells of suspension culture BY-2

The tonoplast is an intracellular membrane, important for numerous functions of the vacuoles in plant cells. Transport, signaling, enzymatic activity and other processes of the tonoplast are the focus of a wide spectrum of studies. Multiple advanced analyses demand highly purified vacuoles or vesicles of the tonoplast membrane fraction. Since 1960 several approaches have been developed for such purification, but new goals and new model objects require adjustment of already existing techniques. The presented investigation aimed to compare two methods of tonoplast membrane fraction purification from tobacco suspension cell culture BY-2 (Nicotiana tabacum L., cv Bright Yellow) based on sucrose and sucrose/sorbitol gradients. The functional activity of obtained purified tonoplast fractions was measured by the hydrolytic activity of vacuolar H+-ATPase and H+-PPase.

Gene expression variability between randomly and targeted transgene integration events in tobacco suspension cell lines

Genome editing tools such as zinc-finger nucleases provide novel strategies for genetic manipulation in plants. Unlike agrobacterium-mediated or direct gene transfer, which introduce genes randomly into the genome and thereby potentially resulting in high variation of gene expression, the targeted gene addition provides predictable integration of DNA sequences into a specified location of the plant genome. We investigated whether various independent cell lines that all contain a transgene placed in the same genomic locus by zinc-finger nuclease-mediated homologous recombination (HR) would yield a more reproducible and homogeneous level of expression compared to integration events generated via agrobacterium-mediated transformation at random sites. The variance of gene expression of targeted HR events and random integration events was analyzed in Nicotiana tabacum L cv. Bright Yellow 2 (BY-2) suspension cells by measuring protein amount produced from the transgene by flow cytometry, thus providing the first report on positional effects of marker gene expression in a quickly proliferating plant suspension cell line. Marker protein levels of targeted HR and single-copy random events covered a similar range; however, the uniformity of protein expression in a given cell line was significantly higher in targeted events than in lines with randomly inserted transgene; the same is true for the overall viability of protoplasts from HR lines. In conclusion, using targeted insertion into a qualified locus of a well-characterized line leads to more reliable results than random insertion into the genome.

Extracellular ATP affects cell viability, respiratory O2 uptake, and intracellular ATP production of tobacco cell suspension culture in response to hydrogen peroxide-induced oxidative stress

Extracellular adenosine 5′-triphosphate (ATP) is a well-known signalling molecule in plants and plays important roles during plant stress response. In the present work, the treatment of tobacco cell suspension culture with exogenous hydrogen peroxide (H2O2) caused the decreases of respiratory O2 uptake, intracellular ATP production, extracellular ATP level, and reduction of cell viability. Combining this observation with the finding that the oxidative phosphorylation uncoupler or the respiratory inhibitor not only decreased intracellular ATP production but also decreased the extracellular ATP level and cell viability, it is suggested that the decrease of extracellular ATP level and cell viability under H2O2 stress could be a result of the suppressed production of intracellular ATP. Treatment with ATP-degrading enzyme, apyrase, also caused the decreases of cell viability, respiratory O2 uptake, and intracellular ATP production. More importantly, addition of exogenous ATP alleviated the H2O2-induced decreases of cell viability, respiratory O2 uptake, and production of intracellular ATP. These results indicate that extracellular ATP could be an important effector in regulating the cell viability, respiratory O2 uptake, and intracellular ATP production of tobacco cell suspension culture under H2O2 stress.

MAMP-triggered Medium Alkalinization of Plant Cell Cultures.

Plants recognize a wide variety of microbial molecules to detect and respond to potential invaders. Recognition of Microbe-Associated Molecular Patterns (MAMPs) by cell surface receptors initiate a cascade of biochemical responses that include, among others, ion fluxes across the plasma membrane. A consequence of such event is a decrease in the concentration of extracellular H+ ions, which can be experimentally detected in plant cell suspensions as a shift in the pH of the medium. Thus, similarly to reactive oxygen species (ROS) accumulation, phosphorylation of MAP kinases and induction of defense-related genes, MAMP-induced medium alkalinization can be used as a proxy for the activation of plant immune responses. Here, we describe a detailed protocol for the measurement of medium alkalinization of tobacco BY-2 cell suspensions upon treatment with two different MAMPs: chitohexamers derived from fungal cell walls (NAG6; N-acetylglucosamine) and the flagellin epitope flg22, found in the bacterial flagellum. This method provides a reliable and fast platform to access MAMP-Triggered Immunity (MTI) in tobacco cell suspensions and can be easily adapted to other plant species as well as to other MAMPs.

Optimizing the particle bombardment conditions in cell suspension cultures of Nicotiana tabacum and expression of the recombinant antihypertensive amarantin

The first successful attempt to produce transgenic tobacco cell suspensions expressing a modified version of amarantin, through direct gene transfer using particle gun is described. The physical parameters for transformation were optimized using the gene encoding β-glucuronidase (gusA) as the reporter gene. Statistical analysis revealed that helium pressure, target distance, and osmotic pretreatment had significant influence on transformation efficiency. Of the different variables evaluated, a combination of sorbitol 0.5 M pretreatment, shooting distance of 7 cm and firing pressure of 900 psi were found ideal. Selection of putative transgenic calli was done on Hygromicine-selection medium. Presence of the transgene was confirmed using histological glucuronidase assay and PCR analysis. Accumulation and stable expression of the recombinant amarantin was detected using Western blot analysis. Transformation efficiency in terms of biochemical GUS assay was of 60%. The present results demonstrate the possibility of incorporating and expressing transgene encoding recombinant proteins of interest, in tobacco cell suspension through biolistic delivery system.

Redox- and bio-activity of apocynin (acetovanillone) in tobacco, a plant phenolic that alleviates symptoms of autoimmune diseases in animals

Apocynin (APO) was shown to be the major constituent of early herbal medicines used to reduce painful suffering from many ailments. Because of its proven utility in rat models, it is now an active candidate for treating humans for several major autoimmune diseases such as Alzheimer's, arthritis, and cardiovascular diseases. In plant research, APO is referred to as acetovanillone, a secondary metabolite that is induced in the leaf apoplast during bacterial invasion. Since its role in plants is unknown, we examined whether any of the attributes found in medical studies could found in plants. In medical studies, APO is considered to be an NADPH oxidase (NOX) inhibitor, however when tested in tobacco suspension cells, APO failed to inhibit NOX activity. In addition, when APO was used to pretreat plants against virulent or avirulent pathovars of Pseudomonas syringae, there was no reduction of symptoms. When pathovars were incubated with 100 μM APO for 90 min, there was only a slight decrease in the bacterial population, suggesting that it was not toxic. In animals, a key step for the inhibition of NOX by APO appears to be its oxidation by myeloperoxidase, yielding dimers and trimers. In vitro studies using pH 6, which is more comparable the plant environment, APO oxidation by horseradish peroxidase yielded similar products. We monitored the redox potential during some of these experiments, trying to begin looking at this parameter, which like pH, can have a more global effect on closed systems such as the apoplast. Phenolics such as APO that are induced upon bacterial ingress, seem likely to play a role in the plant bacterial interaction, but it will require additional and creative research to find and fully understand this role.

Stronger cellulose microfibril network structure through the expression of cellulose-binding modules in plant primary cell walls

Cellulose-binding modules (CBMs) are non-catalytic domains typically occurring in glycoside hydrolases. Their specific interaction with diverse polysaccharides assists hydrolysis by the catalytic subunits. In this work, we have exploited the interactions between a CBM from family 3 (CBM3) and cell wall polysaccharides to alter the structure and mechanical properties of cellulose microfibrils from BY-2 tobacco cell suspension cultures. A CBM3 from Clostridium thermocellum was overexpressed in the cells using Agrobacterium-mediated transformation. Water suspensions of cellulose microfibrils were prepared by the removal of the non-cellulosic components of the primary cell walls, followed by mild disintegration using sonication. The morphology of the microfibrils was characterized by transmission electron microscopy and atomic force microscopy. These cellulose microfibrils were further hydrolyzed with 64 wt% sulfuric acid to produce cellulose nanocrystals (CNCs). The average length of CNCs prepared from the CBM3-transformed cells was 201 nm, higher than that from the wild-type cells (122 nm). In addition, the mechanical properties and deformation mechanism of nanopapers prepared from suspensions of cellulose microfibrils were investigated. The nanopapers obtained from the CBM3-transformed cells exhibited enhanced tensile strength and work of fracture, 40% and 128% higher than those prepared from wild-type tobacco cells, respectively.Graphical abstract

Why are the anionic porphyrins so efficient to induce plant cell death? A structure-activity relationship study to solve the puzzle

Previous work showed that compared to cationic porphyrins under light irradiation, the anionic ones were the strongest tobacco bright yellow cell death inducers. As this result was unexpected in light to literature, it was of importance to try to understand why anionic porphyrins were able to efficiently induce cell detah in tobacco cell suspension. Thus we decided to investigate structure-activity relationships of a serie of anionic porphyrins toward Tobacco Bright Yellow - 2 (TBY-2) cells, namely 5,10,15,20-tetrakis(carboxyphenyl) porphyrin TPPC, its zinc analogue Zn-TPPC, 5,10,15,20-tetrakis(4-sulfonatophenyl) porphyrin TPPS, and its zinc analogue Zn-TPPS, 5,10,15,20-tetrakis(4-phosphonatophenyl) porphyrin TPPP. In particular, their charge states as a function of the pH medium, their abilities to generate reactive oxygen species as well as their spectroscopic and photophysical properties have been investigated and correlated to their biological effect.

Characterization and abolishment of the cyclopiazonic acids produced by Aspergillus oryzae HMP-F28.

Extracellular alkalinization and H2O2 production are important early events during induced resistance establishment in plants. In a screen for metabolites as plant resistance activators from 98 fungal isolates associated with marine sponge Hymeniacidon perleve, the cyclopiazonic acids (CPAs) produced by Aspergillus oryzae HMP-F28 induced significant extracellular alkalinization coupled with augmented H2O2 production in tobacco cell suspensions. Bioassay-guided fractionation led to the isolation and structural elucidation of a new CPA congener (4, 3-hydroxysperadine A) and three known ones (1-3). To construct a mutasynthetic strain to generate unnatural CPA analogues, a hybrid pks-nrps gene (cpaS) was disrupted to abolish the production of the critical precursor of cyclo-acetoacetyl-L-tryptophan (cAATrp) and all the downstream CPA products. Elimination of cAATrp will allow cAATrp mimics being processed by the CPA biosynthetic machinery to produce CPA derivatives with designed structural features.

Extracellular ATP released by copper stress could act as diffusible signal in alleviating the copper stress-induced cell death.

In the present work, by using tobacco cell suspension and wheat seedlings, we studied that eATP (extracellular ATP) released by copper (Cu) stress could act as diffusible signal in alleviating the Cu stress-induced cell death. A semipermeable membrane was fixed in the middle of a plastic box to divide the box into two equal compartments (A and B, respectively). This semipermeable membrane can prevent direct cell-to-cell (or seedling-to-seedling) contact and the diffusion of the macromolecules [such as ATPase (adenosine 5'-triphosphatase)] between these two compartments. The cell suspension directly stressed with CuCl2 was placed in compartment A and was incubated with the untreated cell suspension in compartment B. Such treatment significantly increased the levels of cell death and eATP content of the cell suspension in these two compartments. In contrast, addition of ATPase into the cell suspension directly stressed with CuCl2 decreased the eATP level in these two compartments but further increased the level of cell death in compartment B, compared to no addition of ATPase. Similar results were obtained when tobacco cell suspension was replaced by wheat seedlings. These observations indicate that when Cu stress from compartment A induced the plant cell death in compartment B, ATP transferred from compartment A could play a role in alleviating this cell death. Thus, it is suggested that eATP released by copper stress could act as diffusible signal in alleviating the Cu stress-induced cell death.