Tobacco Bright Yellow-2 Cells Research Articles

Why does Anatabine, But not Nicotine, Accumulate in Jasmonate-Elicited Cultured Tobacco BY-2 Cells?

Suspension-cultured cells of Nicotiana tabacum cv. Bright Yellow-2 (BY-2) grow rapidly in a highly homogenous population and still exhibit the general behavior of plant cells, and thus are often used as model systems in several areas of plant molecular and cellular biology, including secondary metabolism. While the parental tobacco variety synthesizes nicotine as a major alkaloid, the cultured tobacco cells mainly produce a related alkaloid anatabine, instead of nicotine, when elicited with jasmonates. We report here that cultured BY-2 cells scarcely express N-methylputrescine oxidase (MPO) genes even after jasmonate elicitation. MPO is the second enzyme in the biosynthetic pathway that supplies the pyrrolidine moiety of nicotine and nornicotine, but is predicted to be dispensable for the biosynthesis of anatabine, anabasine and anatalline, which do not contain the pyrrolidine moiety. When MPO was overexpressed in tobacco BY-2 cells, nicotine synthesis was dramatically enhanced while anatabine formation was effectively suppressed. As a complementary approach, we suppressed MPO expression by RNA interference in tobacco hairy roots that normally accumulate nicotine. In the MPO-suppressed roots, the contents of anatabine, anabasine and anatalline, as well as N-methylputrescine and putrescine, markedly increased to compensate for suppressed formation of nicotine and nornicotine. These results identify the transcriptional regulation of MPO as a critical rate-limiting step that restricts nicotine formation in cultured tobacco BY-2 cells.

SCAMPs Highlight the Developing Cell Plate during Cytokinesis in Tobacco BY-2 Cells

We previously demonstrated that rice (Oryza sativa) SECRETORY CARRIER MEMBRANE PROTEIN1 (OsSCAMP1)-yellow fluorescent protein in transgenic tobacco (Nicotiana tabacum) Bright Yellow-2 cells locates to the plasma membrane and to motile punctate structures, which represent the trans-Golgi network/early endosome and are tubular-vesicular in nature. Here, we now show that SCAMPs are diverted to the cell plate during cytokinesis dividing Bright Yellow-2 cells. As cells progress from metaphase to cytokinesis, punctate OsSCAMP1-labeled structures begin to collect in the future division plane. Together with the internalized endosomal marker FM4-64, they then become incorporated into the cell plate as it forms and expands. This was confirmed by immunogold electron microscopy. We also monitored for the Golgi apparatus and the prevacuolar compartment (PVC)/multivesicular body. Golgi stacks tend to accumulate in the vicinity of the division plane, but the signals are clearly separate to the cell plate. The situation with the PVC (labeled by green fluorescent protein-BP-80) is not so clear. Punctate BP-80 signals are seen at the advancing periphery of the cell plate, which was confirmed by immunogold electron microscopy. Specific but weak labeling was observed in the cell plate, but no evidence for a fusion of the PVC/multivesicular body with the cell plate could be obtained. Our data, therefore, support the notion that cell plate formation is mainly a secretory process involving mass incorporation of domains of the trans-Golgi network/early endosome membrane. We regard the involvement of multivesicular late endosomes in this process to be equivocal.

The Plant Defense Elicitor Cryptogein Stimulates Clathrin-Mediated Endocytosis Correlated with Reactive Oxygen Species Production in Bright Yellow-2 Tobacco Cells

The plant defense elicitor cryptogein triggers well-known biochemical events of early signal transduction at the plasma membrane of tobacco (Nicotiana tabacum) cells, but microscopic observations of cell responses related to these early events were lacking. We determined that internalization of the lipophilic dye FM4-64, which is a marker of endocytosis, is stimulated a few minutes after addition of cryptogein to tobacco Bright Yellow-2 (BY-2) cells. This stimulation is specific to the signal transduction pathway elicited by cryptogein because a lipid transfer protein, which binds to the same receptor as cryptogein but without triggering signaling, does not increase endocytosis. To define the nature of the stimulated endocytosis, we quantified clathrin-coated pits (CCPs) forming on the plasma membrane of BY-2 cells. A transitory stimulation of this morphological event by cryptogein occurs within the first 15 min. In the presence of cryptogein, increases in both FM4-64 internalization and clathrin-mediated endocytosis are specifically blocked upon treatment with 5 microm tyrphostin A23, a receptor-mediated endocytosis inhibitor. The kinetics of the transient increase in CCPs at the plasma membrane coincides with that of transitory reactive oxygen species (ROS) production occurring within the first 15 min after elicitation. Moreover, in BY-2 cells expressing NtrbohD antisense cDNA, which are unable to produce ROS when treated with cryptogein, the CCP stimulation is inhibited. These results indicate that the very early endocytic process induced by cryptogein in tobacco is due, at least partly, to clathrin-mediated endocytosis and is dependent on ROS production by the NADPH oxidase NtrbohD.

Proline and glycinebetaine enhance antioxidant defense and methylglyoxal detoxification systems and reduce NaCl-induced damage in cultured tobacco cells

Salt stress impairs reactive oxygen species (ROS) and methylglyoxal (MG) detoxification systems, and causes oxidative damage to plants. Up-regulation of the antioxidant and glyoxalase systems provides protection against NaCl-induced oxidative damage in plants. Thiol-disulfide contents, glutathione content and its associated enzyme activities involved in the antioxidant defense and glyoxalase systems, and protein carbonylation in tobacco Bright Yellow-2 cells grown in suspension culture were investigated to assess the protection offered by proline and glycinebetaine against salt stress. Salt stress increased protein carbonylation, contents of thiol, disulfide, reduced (GSH) and oxidized (GSSG) forms of glutathione, and the activity of glutathione-S-transferase and glyoxalase II enzymes, but decreased redox state of both thiol-disulfide and glutathione, and the activity of glutathione peroxidase and glyoxalase I enzymes involved in the ROS and MG detoxification systems. Exogenous application of proline or glycinebetaine resulted in a reduction of protein carbonylation, and in an increase in glutathione redox state and activity of glutathione peroxidase, glutathione-S-transferase and glyoxalase I under salt stress. Neither proline nor glycinebetaine, however, had any direct protective effect on NaCl-induced GSH-associated enzyme activities. The present study, therefore, suggests that both proline and glycinebetaine provide a protective action against NaCl-induced oxidative damage by reducing protein carbonylation, and enhancing antioxidant defense and MG detoxification systems.

Visualization of phosphatidylinositol 4,5‐bisphosphate in the plasma membrane of suspension‐cultured tobacco BY‐2 cells and whole Arabidopsis seedlings

Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] is an important signalling lipid in mammalian cells, where it functions as a second-messenger precursor in response to agonist-dependent activation of phospholipase C (PLC) but also operates as a signalling molecule on its own. Much of the recent knowledge about it has come from a new technique to visualize PtdIns(4,5)P(2)in vivo, by expressing a green or yellow fluorescent protein (GFP or YFP) fused to the pleckstrin homology (PH) domain of human PLCdelta1 that specifically binds PtdIns(4,5)P(2). In this way, YFP-PH(PLCdelta1) has been shown to predominantly label the plasma membrane and to transiently translocate into the cytoplasm upon PLC activation in a variety of mammalian cell systems. In plants, biochemical studies have shown that PtdIns(4,5)P(2) is present in very small quantities, but knowledge of its localization and function is still very limited. In this study, we have used YFP-PH(PLCdelta1) to try monitoring PtdIns(4,5)P(2)/PLC signalling in stably-transformed tobacco Bright Yellow-2 (BY-2) cells and Arabidopsis seedlings. In both plant systems, no detrimental effects were observed, indicating that overexpression of the biosensor did not interfere with the function of PtdIns(4,5)P(2). Confocal imaging revealed that most of the YFP-PH(PLCdelta1) fluorescence was present in the cytoplasm, and not in the plasma membrane as in mammalian cells. Nonetheless, four conditions were found in which YFP-PH(PLCdelta1) was concentrated at the plasma membrane: (i) upon treatment with the PLC inhibitor U73122; (ii) in response to salt stress; (iii) as a gradient at the tip of growing root hairs; (iv) during the final stage of a BY-2 cell division. We conclude that PtdIns(4,5)P(2), as in animals, is present in the plasma membrane of plants, but that its concentration in most cells is too low to be detected by YFP-PH(PLCdelta1). Hence, the reporter remains unbound in the cytosol, making it unsuitable to monitor PLC signalling. Nonetheless, YFP-PH(PLCdelta1) is a valuable plant PtdIns(4,5)P(2) reporter, for it highlights specific cells and conditions where this lipid becomes abnormally concentrated in membranes, raising the question of what it is doing there. New roles for PtdIns(4,5)P(2) in plant cell signalling are discussed.

Metabolic fate of nicotinamide in higher plants

Metabolism of [carbonyl-14C]nicotinamide was surveyed in various plant materials including the model plants, Arabidopsis thaliana, Oryza sativa and Lotus japonicus. In all plants studied, nicotinamide was used for the pyridine (nicotinamide adenine) nucleotide synthesis, probably after conversion to nicotinic acid. Radioactivity from [carbonyl-14C]nicotinamide was incorporated into trigonelline (1-N-methylnicotinic acid) and/or into nicotinic acid 1N-glucoside (Na-Glc). Trigonelline is formed mainly in leaves and cell cultures of O. sativa and L. japonicus and in seedlings of Trifolium incarnatum, Medicago sativa and Raphanus sativus. Trigonelline synthesis from nicotinamide is generally greater in leaves than in roots. Na-Glc was formed as the major nicotinic acid conjugate in A. thaliana and in tobacco Bright Yellow-2 cells. In seedlings of Chrysanthemum coronarium and Theobroma cacao, both trigonelline and Na-Glc were synthesized from [carbonyl-14C]nicotinamide. Trigonelline is accumulated in some seeds, mainly Leguminosae species. The pattern of formation of the nicotinic acid conjugates differs between species and organs.

Diverse Subcellular Locations of Cryptogein-Induced Reactive Oxygen Species Production in Tobacco Bright Yellow-2 Cells

Reactive oxygen species (ROS) play a crucial role in many cellular responses and signaling pathways, including the oxidative burst defense response to pathogens. We have examined very early events in cryptogein-induced ROS production in tobacco (Nicotiana tabacum) Bright Yellow-2 suspension cells. Using Amplex Red and Amplex Ultra Red reagents, which report real-time H2O2 accumulation in cell populations, we show that the internal signal for H2O2 develops more rapidly than the external apoplastic signal. Subcellular accumulation of H2O2 was also followed in individual cells using the 2',7'-dichlorofluorescein diacetate fluorescent probe. Major accumulation was detected in endomembrane, cytoplasmic, and nuclear compartments. When cryptogein was added, the signal developed first in the nuclear region and, after a short delay, in the cell periphery. Interestingly, isolated nuclei were capable of producing H2O2 in a calcium-dependent manner, implying that nuclei can serve as a potential active source of ROS production. These results show complex spatial compartmentalization for ROS accumulation and an unexpected temporal sequence of events that occurs after cryptogein application, suggesting novel intricacy in ROS-signaling cascades.

Expression of B2-Type Cyclin-Dependent Kinase is Controlled by Protein Degradation in Arabidopsis thaliana

The eukaryotic cell cycle is controlled by cyclin-dependent kinases (CDKs). Plants possess six types of CDK, among which the B-type CDK (CDKB) is expressed specifically from the late S- to the M-phase. We demonstrate that the expression of Arabidopsis CDKB2 is under the control of the protein degradation machinery. beta-Glucuronidase fused to a putative PEST motif of CDKB2 was unstable in tobacco Bright Yellow-2 cells and Arabidopsis plants, and its degradation was arrested by the proteasome inhibitor MG132. We propose that the abundance of CDKB2 protein is regulated not only at the transcriptional level, but also through proteasome-mediated protein degradation.

Hydrogen peroxide, nitric oxide and cytosolic ascorbate peroxidase at the crossroad between defence and cell death

An increase in the production of reactive oxygen species (ROS) is a typical event occurring during different stress conditions and activating conflicting responses in plants. In order to investigate the relevance of different timing and amounts of ROS production, tobacco (Nicotiana tabacum) Bright Yellow-2 (TBY-2) cells were incubated with different amounts of glucose plus glucose oxidase, for generating H(2)O(2) during time, or directly with known amounts of H(2)O(2). Data presented here indicate that, in TBY-2 cells, a difference in H(2)O(2) level is a critical point for shifting metabolic responses towards strengthening of antioxidant defences, or their depletion with consequent cell death. Timing of ROS production is also critical because it can determine programmed cell death (PCD) or necrosis. Depending on the different kinds of activated cell death, ascorbate (ASC) and glutathione (GSH) pools are altered differently. Moreover, an H(2)O(2)-dependent activation of nitric oxide synthesis is triggered only in the conditions inducing PCD. Ascorbate peroxidase (APX) has been analysed under different conditions of H(2)O(2) generation. Under a threshold value of H(2)O(2) overproduction, a transient increase in APX occurs, whereas under conditions inducing cell necrosis, the activity of APX decreases in proportion to cell death without any evident alteration in APX gene expression. Under conditions triggering PCD, the suppression of APX involves both gene expression and alteration of the kinetic characteristics of the enzyme. The changes in ASC, GSH and APX are involved in the signalling pathway leading to PCD, probably contributing to guaranteeing the cellular redox conditions required for successful PCD.

Localization and in vitro binding studies suggest that the cytoplasmic/nuclear tobacco lectin can interact in situ with high-mannose and complex N-glycans

The possible in vivo interaction of the Nicotiana tabacum agglutinin (Nictaba) with endogenous glycoproteins was corroborated using a combination of confocal/electron microscopy of an EGFP-Nictaba fusion protein expressed in tobacco Bright Yellow-2 (BY-2) cells and biochemical analyses. In vitro binding studies demonstrated that the expressed EGFP-Nictaba possesses carbohydrate-binding activity. Microscopic analyses confirmed the previously reported cytoplasmic/nuclear location of Nictaba in jasmonate-treated tobacco leaves and provided evidence for the involvement of a nuclear localization signal-dependent transport mechanism. In addition, it became evident that the lectin is not uniformly distributed over the nucleus and the cytoplasm of BY-2 cells. Far Western blot analysis of extracts from whole BY-2 cells and purified nuclei revealed that Nictaba interacts in a glycan inhibitable way with numerous proteins including many nuclear proteins. Enzymatic deglycosylation with PNGase F indicated that the observed interaction depends on the presence of N-glycans. Glycan array screening, which showed that Nictaba exhibits a strong affinity for high-mannose and complex N-glycans, provided a reasonable explanation for this observation. The cytoplasmic/nuclear localization of a plant lectin that has a high affinity for high-mannose and complex N-glycans and specifically interacts with conspecific glycoproteins suggests that N-glycosylated proteins might be more important in the cytoplasm and nucleus than is currently believed.

Molecular Interactions of Arabinogalactan Proteins with Cortical Microtubules and F-Actin in Bright Yellow-2 Tobacco Cultured Cells

Arabinogalactan proteins (AGPs), a superfamily of plant hydroxyproline-rich glycoproteins, are present at cell surfaces. Although precise functions of AGPs remain elusive, they are widely implicated in plant growth and development. A well-characterized classical tomato (Lycopersicon esculentum) AGP containing a glycosylphosphatidylinositol plasma membrane anchor sequence was used here to elucidate functional roles of AGPs. Transgenic tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells stably expressing green fluorescent protein (GFP)-LeAGP-1 were plasmolysed and used to localize LeAGP-1 on the plasma membrane and in Hechtian strands. Cytoskeleton disruptors and beta-Yariv reagent (which binds and perturbs AGPs) were used to examine the role of LeAGP-1 as a candidate linker protein between the plasma membrane and cytoskeleton. This study used a two-pronged approach. First, BY-2 cells, either wild type or expressing GFP-microtubule (MT)-binding domain, were treated with beta-Yariv reagent, and effects on MTs and F-actin were observed. Second, BY-2 cells expressing GFP-LeAGP-1 were treated with amiprophosmethyl and cytochalasin-D to disrupt MTs and F-actin, and effects on LeAGP-1 localization were observed. beta-Yariv treatment resulted in terminal cell bulging, puncta formation, and depolymerization/disorganization of MTs, indicating a likely role for AGPs in cortical MT organization. beta-Yariv treatment also resulted in the formation of thicker actin filaments, indicating a role for AGPs in actin polymerization. Similarly, amiprophosmethyl and cytochalasin-D treatments resulted in relocalization of LeAGP-1 on Hechtian strands and indicate roles for MTs and F-actin in AGP organization at the cell surface and in Hechtian strands. Collectively, these studies indicate that glycosylphosphatidylinositol-anchored AGPs function to link the plasma membrane to the cytoskeleton.

Dynamic Response of Prevacuolar Compartments to Brefeldin A in Plant Cells

Little is known about the dynamics and molecular components of plant prevacuolar compartments (PVCs) in the secretory pathway. Using transgenic tobacco (Nicotiana tabacum) Bright-Yellow-2 (BY-2) cells expressing membrane-anchored yellow fluorescent protein (YFP) reporters marking Golgi or PVCs, we have recently demonstrated that PVCs are mobile multivesicular bodies defined by vacuolar sorting receptor proteins. Here, we demonstrate that Golgi and PVCs have different sensitivity in response to brefeldin A (BFA) treatment in living tobacco BY-2 cells. BFA at low concentrations (5-10 microg mL(-1)) induced YFP-marked Golgi stacks to form both endoplasmic reticulum-Golgi hybrid structures and BFA-induced aggregates, but had little effect on YFP-marked PVCs in transgenic BY-2 cells at both confocal and immunogold electron microscopy levels. However, BFA at high concentrations (50-100 microg mL(-1)) caused both YFP-marked Golgi stacks and PVCs to form aggregates in a dose- and time-dependent manner. Normal Golgi or PVC signals can be recovered upon removal of BFA from the culture media. Confocal immunofluorescence and immunogold electron microscopy studies with specific organelle markers further demonstrate that the PVC aggregates are distinct, but physically associated, with Golgi aggregates in BFA-treated cells and that PVCs might lose their internal vesicle structures at high BFA concentration. In addition, vacuolar sorting receptor-marked PVCs in root-tip cells of tobacco, pea (Pisum sativum), mung bean (Vigna radiata), and Arabidopsis (Arabidopsis thaliana) upon BFA treatment are also induced to form similar aggregates. Thus, we have demonstrated that the effects of BFA are not limited to endoplasmic reticulum and Golgi, but extend to PVC in the endomembrane system, which might provide a quick tool for distinguishing Golgi from PVC for its identification and characterization, as well as a possible new tool in studying PVC-mediated protein traffic in plant cells.

ENOD40 affects elongation growth in tobacco Bright Yellow-2 cells by alteration of ethylene biosynthesis kinetics

Plant developmental processes are controlled by co-ordinated action of phytohormones and plant genes encoding components of developmental response pathways. ENOD40 was identified as a candidate for such a plant factor with a regulatory role during nodulation. Although its mode of action is poorly understood, several lines of evidence suggest interaction with phytohormone response pathways. This hypothesis was investigated by analysing cytokinin-, auxin-, and ethylene-induced responses on cell growth and cell division in transgenic 35S:NtENOD40 Bright Yellow-2 (BY-2) tobacco cell suspensions. It was found that cell division frequency is controlled by the balance between cytokinin and auxin in wild-type cells and that this regulation is not affected in 35S:NtENOD40 lines. Elongation growth, on the other hand, is reduced upon overexpression of NtENOD40. Analysis of ethylene homeostasis shows that ethylene accumulation is accelerated in 35S:NtENOD40 lines. ENOD40 action can be counteracted by an ethylene perception blocker, indicating that ethylene is a negative regulator of elongation growth in 35S:NtENOD40 cells, and that the NtENOD40-induced response is mediated by alteration of ethylene biosynthesis kinetics.

A novel R2R3 MYB transcription factor NtMYBJS1 is a methyl jasmonate‐dependent regulator of phenylpropanoid‐conjugate biosynthesis in tobacco

Target metabolic and large-scale transcriptomic analyses of tobacco (Nicotiana tabacum L.) Bright Yellow-2 (BY-2) cells were employed to identify novel gene(s) involved in methyl jasmonate (MJ)-dependent function in plants. At the metabolic level, we describe the specific accumulation of several phenylpropanoid-polyamine conjugates in MJ-treated BY-2 cells. Furthermore, global gene expression analysis of MJ-treated cells using a 16K cDNA microarray containing expressed sequence tags (ESTs) from BY-2 cells revealed 828 genes that were upregulated by MJ treatment within 48 h. Using time-course expression data we identified a novel MJ-inducible R2R3 MYB-type transcription factor (NtMYBJS1) that was co-expressed in a close temporal pattern with the core phenylpropanoid genes phenylalanine ammonia-lyase (PAL) and 4-coumarate:CoA ligase (4CL). Overexpression of NtMYBJS1 in tobacco BY-2 cells caused accumulation of specific phenylpropanoid conjugates in the cells. Subsequent microarray analysis of NtMYBJS1 transgenic lines revealed that a limited number of genes, including PAL and 4CL, were specifically induced in the presence of the NtMYBJS1 transgene. These results, together with results of both antisense expression analysis and of gel mobility shift assays, strongly indicate that the NtMYBJS1 protein functions in tobacco MJ signal transduction, inducing phenylpropanoid biosynthetic genes and the accumulation of phenylpropanoid-polyamine conjugates during stress.

The role of the Arabidopsis E2FB transcription factor in regulating auxin-dependent cell division.

The molecular mechanisms by which the phytohormone auxin coordinates cell division with cell growth and differentiation are largely unknown. Here, we show that in Arabidopsis thaliana E2FB, accumulation and stability are positively regulated by auxin. Coexpression of E2FB, but not of E2FA, with its dimerization partner A, stimulated cell proliferation in the absence of auxin in tobacco (Nicotiana tabacum) Bright Yellow-2 cells. E2FB regulated the entry into both S- and M-phases, the latter corresponding to the activation of a plant-specific mitotic regulator, CDKB1;1. Increased E2FB levels led to shortened cell cycle duration, elevated cell numbers, and extremely small cell sizes. In the absence of auxin, cells elongated with concomitant increase in their ploidy level, but both were strongly inhibited by E2FB. We conclude that E2FB is one of the key targets for auxin to determine whether cells proliferate or whether they exit the cell cycle, enlarge, and endoreduplicate their DNA.

Alamethicin permeabilizes the plasma membrane and mitochondria but not the tonoplast in tobacco (Nicotiana tabacum L. cv Bright Yellow) suspension cells

The ion channel-forming peptide AlaM (alamethicin) is known to permeabilize isolated mitochondria as well as animal cells. When intact tobacco (Nicotiana tabacum L.) Bright Yellow-2 cells were treated with AlaM, the cells became permeable for low-molecular-mass molecules as shown by induced leakage of NAD(P)+. After the addition of cofactors and substrates, activities of cytosolic as well as mitochondrial respiratory enzymes could be directly determined inside the permeabilized cells. However, at an AlaM concentration at which the cytoplasmic enzymes were maximally accessible, the vacuole remained intact, as indicated by an unaffected tonoplast proton gradient. Low-flux permeabilization of plasma membranes and mitochondria at moderate AlaM concentrations was reversible and did not affect cell vigour. Higher AlaM concentrations induced cell death. After the addition of catalase that removes the H2O2 necessary for NADH oxidation by apoplastic peroxidases, mitochondrial oxygen consumption could be measured in permeabilized cells. Inhibitor-sensitive oxidation of the respiratory substrates succinate, malate and NADH was observed after the addition of the appropriate coenzymes (ATP, NAD+). The capacities of different pathways in the respiratory electron-transport chain could thus be determined directly. We conclude that AlaM permeabilization provides a very useful tool for monitoring metabolic pathways or individual enzymes in their native proteinaceous environment with controlled cofactor concentrations. Possible uses and limitations of this method for plant cell research are discussed.

Functional Analysis of Arabidopsis Ethylene-Responsive Element Binding Protein Conferring Resistance to Bax and Abiotic Stress-Induced Plant Cell Death

Arabidopsis (Arabidopsis thaliana) ethylene-responsive element binding protein (AtEBP) gene was isolated as a suppressor of Bax-induced cell death by functional screening in yeast (Saccharomyces cerevisiae). To further examine the cell death suppressive action of AtEBP in plant cells, we established transgenic tobacco (Nicotiana tabacum) plants overexpressing AtEBP as well as transgenic tobacco plants ectopically expressing mouse Bax protein under a dexamethasone-inducible promoter. We prepared the crosses of the selective lines of each transgenic plant, which were evaluated in terms of cell death suppression activity. Results indicate that AtEBP suppressed Bax-induced cell death in tobacco plants, an action also associated with a lowered level of ion leakage. Furthermore, tobacco Bright Yellow-2 cells overexpressing AtEBP conferred resistance to hydrogen peroxide (H(2)O(2)) and heat treatments. AtEBP protein localized in the nucleus and functioned as an in vivo transcription activator as confirmed in transient assays and experiments using stable transgenic system. Up-regulation of defense genes was observed in transgenic Arabidopsis plants overexpressing AtEBP. Based on the analysis of mRNA accumulation in ethylene-related mutants, the position of AtEBP in signaling pathway is presented.

Chemical regulation of abscisic acid catabolism in plants by cytochrome P450 inhibitors

Plant hormone abscisic acid (ABA) is an important factor for conferring drought stress resistance on plants. Therefore, small molecules that regulate ABA levels in plants can be useful both for investigating functions of ABA and for developing new plant growth regulators. Abscisic acid (ABA) catabolism in plants is primarily regulated by ABA 8′-hydroxylase, which is a cytochrome P450 (P450). We tested known P450 inhibitors containing a triazole group and found that uniconazole-P inhibited ABA catabolism in cultured tobacco Bright Yellow-2 cells. In a structure–activity study of uniconazole, we found a more effective ABA catabolic inhibitor (diniconazole) than uniconazole-P. Diniconazole, a fungicide, acted as a potent competitive inhibitor of recombinant Arabidopsis ABA 8′-hydroxylase, CYP707A3, in an in vitro assay. Diniconazole-treated plants retained a higher ABA content and higher transcription levels of ABA response genes during rehydration than did untreated plants and were more drought stress tolerant than untreated plants. These results strongly suggest that ABA catabolic inhibitors that target ABA 8′-hydroxylase can regulate the ABA content of plants and conferred drought stress resistance on plants. The optical resolution of diniconazole revealed that the S-form isomer, which is a weak fungicidal isomer, was more active as an ABA catabolic inhibitor than was the R-form isomer.

Proteome Analysis of Tobacco Bright Yellow-2 (BY-2) Cell Culture Plastids as a Model for Undifferentiated Heterotrophic Plastids

We analyzed the proteome of undifferentiated plastids from a tobacco BY-2 cell culture by shotgun proteomics following multidimensional protein fractionation. The fractionation strategy initiated with the serial extraction of proteins from membranes which allowed us to distinguish soluble, peripheral, and integral membrane proteins. The majority of the identified proteins have a function in the cellular metabolism and most of them are active in amino acid synthesis pathways. A significant number of the identified proteins was not identified in chloroplast proteome analyses before. This suggests BY-2 plastid specific functions that differ from the major activities of chloroplasts. We have used the BY-2 plastid proteins reported here to assess the metabolic activities of undifferentiated heterotrophic plastids and compared the functional profile with that of differentiated heterotrophic amyloplasts. Comparative shotgun proteome analyses as reported here provide information about prevalent metabolic activities of different plastid types.

Reactive oxygen species and nitric oxide affect cell wall metabolism in tobacco BY-2 cells

The effects of hydrogen peroxide (H2O2), nitric oxide (NO), and a combination of both on the metabolism of cell wall polysaccharides were studied in tobacco (Nicotiana tabacum L.) cv Bright Yellow 2 (BY-2) suspension cultured cells in the presence of d-[U-14C]glucose or d-[U-14C]galactose as radioactive tracers. We found that the radiolabelling of newly synthesised total cell wall polysaccharides (pectins, hemicelluloses and α-cellulose), buffer-soluble polysaccharides, and membrane-associated polysaccharides decreased under the influence of exogenous systems generating H2O2 and NO. However, when the total amount of newly synthesised cell wall polysaccharides was calculated as a percentage of the total cellular radioactivity (ethanol-soluble pool plus the homogenate of ethanol-insoluble material), all treatments showed negligible effects in the presence of d-[U-14C]glucose or d-[U-14C]galactose as tracers. This occurred because the treatments generating H2O2, NO and H2O2 plus NO caused a marked decrease in the concentration of the ethanol-soluble pool as well as in the total radioactivity found in the homogenate of the ethanol-insoluble material. Most of the radioactivity taken up by the cells was evolved as 14CO2 during the respiratory processes. A qualitative and quantitative characterisation of the ethanol-soluble pool showed that radioactive UDP-sugars in BY-2 suspension cultured cells were differentially reduced by all treatments. Therefore, the decrease of the newly synthesised cell wall polysaccharides seems to be strictly dependent on the reduction of the UDP-sugars pool.