High content of endogenous cytokinins stimulates activity of enzymes and proteins involved in stress response in <Emphasis Type="Italic">Nicotiana tabacum</Emphasis>

Transgenic Pssu-ipt tobacco with elevated content of endogenous cytokinins grown under in vitro conditions exhibited elevated activities of antioxidant enzymes (i.e. catalase, ascorbate peroxidase, guaiacol and syringaldazine peroxidase, glutathione reductase) and some of enzymes involved in anaplerotic pathways such as glucose-6-phosphate dehydrogenase, glycolate oxidase, NADP-malic enzyme, NADP-isocitrate dehydrogenase, and glutamate dehydrogenase compared to control non-transgenic SR1 tobacco. Higher activities of peroxidases, NADP-malic enzyme, and glutamate dehydrogenase were maintained in transgenic grafts after several weeks of the growth under ex vitro conditions, while transgenic rooted plants showed only the increase in activity of glycolate oxidase compared to control non-transformed tobacco. Total activities of superoxide dismutase were lower in both types of Pssu-ipt tobacco contrary to controls under both growth conditions. The presence of PR-1 protein and proteins with elevated activities of chitinase was proved in the extracellular fluid in both transgenic types under both in vitro and ex vitro conditions.     

Boron deficiency causes accumulation of chlorogenic acid and caffeoyl polyamine conjugates in tobacco leaves

The effects of boron (B) deficiency on carbohydrate concentrations and the pattern of phenolic compounds were studied in leaves of tobacco plants (Nicotiana tabacum L.). Plants grown under B deficiency showed a notable increase in leaf carbohydrates and total phenolic compounds when compared to controls. The qualitative composition of phenolics was analyzed by HPLC-mass spectrometry. The level of caffeate conjugates (i.e., chlorogenic acid) increased in B-deficient plants. In addition, the accumulation of two caffeic acid amides (N-caffeoylputrescine and putative dicaffeoylspermidine) was observed.     

Cytokinin-induced activity of antioxidant enzymes in transgenic Pssu-ipt tobacco during plant ontogeny

Cytokinin (CK) content and activities of several antioxidant enzymes were examined during plant ontogeny with the aim to elucidate their role in delayed senescence of transgenic Pssu-ipt tobacco. Control Nicotiana tabacum L. (cv. Petit Havana SR1) and transgenic tobacco with the ipt gene under the control of the promoter of small subunit of Rubisco (Pssu-ipt) were both grown either as grafts on control rootstocks or as rooted plants. Both control plant types showed a decline in total content of CKs with proceeding plant senescence. Contrary to this both transgenic plant types exhibited at least ten times higher content of CKs than controls and a significant increase of CK contents throughout the ontogeny with maximal values in the later stages of plant development. Significantly higher portion of O-glucosides was found in both transgenic plant types compared to control ones. In transgenic plants, zeatin and zeatin riboside were predominant type of CKs. Generally, Pssu-ipt tobacco exhibited elevated activities of antioxidant enzymes compared to control tobacco particularly in the later stages of plant development. While in control tobacco activity of glutathione reductase (GR) and superoxide dismutase (SOD) showed increasing activity up to the onset of flowering and then gradually decreased, in both transgenic types GR increased and SOD activity showed only small change throughout the plant ontogeny. Ascorbate peroxidase (APOD) was stimulated in both transgenic types. The manifold enhancement of syringaldazine and guaiacol peroxidase activities was observed in transgenic grafts throughout plant ontogeny in contrast to control and transgenic rooted plants, where the increase was found only in the late stages. Electron microscopic examination showed higher number of crystallic cores in peroxisomes and abnormal interactions among organelles in transgenic tobacco in comparison with control plant. The overproduction of cytokinins resulted in the stimulation of activities of AOE throughout the plant ontogeny of transgenic Pssu-ipt tobacco.     

Response to mild water stress in transgenic Pssu-ipt tobacco

The response of antioxidant enzymes to cyclic drought was studied in control non-transformed tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) and two types of transgenic Pssu-ipt tobacco (grafted on wild rootstock and poorly rooted progeny of F1 generation) grown under different conditions of irradiation (greenhouse, referred as high light, versus growth chamber, referred as low light). Water stress cycles started with plants at two contrasting developmental stages, i.e., at the stage of vegetative growth (young) and at the onset of flowering (old). Drought reduced the growth of SR1 plants compared with transgenic ones, particularly, when treatment started in earlier stage of plant development. Relative leaf water content was significantly lower (below 70%) in all transgenic grafts and plants compared with the wild type, irrespective of age, drought, and growth conditions. The response of antioxidant enzymes was significantly dependent on plant type and plant age; nevertheless, growth conditions and water stress also affected enzyme activities. Contrary to non-transgenic tobacco, where about half of glutathione reductase activity was found in older plants, both transgenic types exhibited unchanged activities throughout plant development and stress treatment. No differences were found in catalase activity, although the growth in the greenhouse caused a moderate increase in all older plants. In contrast to non-transgenic and Pssu-ipt rooted plants, peroxidase activities (ascorbate, guaiacol, and syringaldazine peroxidase) in older Pssu-ipt grafts were up to four times higher, irrespective of growth and stress, nevertheless, the effect seemed to be age-dependent. Superoxide dismutase (SOD) activity was affected particularly by plant age but also by growth conditions. Unlike in older plants, water stress caused an increase of SOD activities in all younger plants. The differences observed in activities of enzymes of intermediary metabolism (i.e., malic enzyme and glucose-6-phosphate dehydrogenase) revealed that transgenic grafts probably compensated differently for a decrease of ATP and NADPH than control and transgenic rooted plants under stress.     

Arabidopsis LOS5/ABA3 overexpression in transgenic tobacco (Nicotiana tabacum cv. Xanthi-nc) results in enhanced drought tolerance

Drought is a major environmental stress factor that affects growth and development of plants. Abscisic acid (ABA), osmotically active compounds, and synthesis of specific proteins, such as proteins that scavenge oxygen radicals, are crucial for plants to adapt to water deficit. LOS5/ABA3 (LOS5) encodes molybdenum-cofactor sulfurase, which is a key regulator of ABA biosynthesis. We overexpressed LOS5 in tobacco using Agrobacterium-mediated transformation. Detached leaves of LOS5-overexpressing seedlings showed lower transpirational water loss than that of nontransgenic seedlings in the same period under normal conditions. When subjected to water-deficit stress, transgenic plants showed less wilting, maintained higher water content and better cellular membrane integrity, accumulated higher quantities of ABA and proline, and exhibited higher activities of antioxidant enzymes, i.e., superoxide dismutase, catalase, peroxidase and ascorbate peroxidase, as compared with control plants. Furthermore, LOS5-overexpressing plants treated with 30% polyethylene glycol showed similar performance in cellular membrane protection, ABA and proline accumulation, and activities of catalase and peroxidase to those under drought stress. Thus, overexpression of LOS5 in transgenic tobacco can enhance drought tolerance.     

甘蔗和烟草幼叶原生质体的核酸含量与核酸酶活性及其影响因素

与幼叶组织相比,酶法新鲜分离的甘蔗和烟草幼叶原生质体内的ＲＮＡ、ＤＮＡ及总核酸含量均降低。其原因可能是刚游离的原生质体内酸性和碱性ＲＮＡ酶与ＤＮＡ酶等活性提高所致。甘蔗叶原生质体内的核酸降低量和ＲＮＡ酶与ＤＮＡ酶活性的增加程度均高于烟草。随用作渗透压稳定剂的甘露醇浓度增加,甘蔗和烟草叶原生质体的ＲＮＡ酶和ＤＮＡ酶活性均相应提高。其中以甘蔗叶原生质体的核酸酶活性增加水平较明显。在细胞壁降解产物的作用下,除了甘蔗原生质体内的ＲＮＡ酶活性略被促进外,其ＤＮＡ酶和烟草叶原生质体内的核酸酶均不受影响     

Relative resistance of transgenic tomato tissues expressing high levels of tobacco anionic peroxidase to different insect species.

Different parts of genetically transformed tomato (Lycopersicon esculentum L.) plants that express the tobacco anionic peroxidase were compared for insect resistance with corresponding wild type plants. Leaf feeding by first instar Helicoverpa zea and Manduca sexta was often significantly reduced on intact transgenic plants and/or leaf disks compared to wild type plants, but the effect could depend on leaf age. Leaves of transgenic plants were generally as susceptible to feeding damage by third instar Helicoverpa zea (Boddie) and Manduca sexta (L.) as wild type plants. Green fruit was equally susceptible to third instar larvae of H. zea in both type plants, but fruit of transgenic plants were more resistant to first instar larvae as indicated by significantly greater mortality. Basal stem sections were more resistant to neonate larvae of H. zea and adults of Carpophilus lugubris Murray compared to wild type plants as indicated by significantly greater mortality and/or reduced feeding damage. Thus, tobacco anionic peroxidase activity can increase plant resistance to insects in tomato, a plant species closely related to the original source plant species, when expressed at sufficiently high levels. However, the degree of resistance is dependent on the size of insect and plant tissue involved.     

The effect of 2,4-dichlorophenol and pentachlorophenol on antioxidant system in the leaves of Phalaris arudinacea

The purpose of this work was to evaluate the effect of 2,4-dichlorophenol (2,4-DCP) and pentachlorophenol (PCP) on the activity of antioxidative system and lipid peroxidation in the leaves of reed canary grass (Phalaris arudinacea). The activity of catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPX), glutathione reductase (GR) and glutathione S-transferase (GST) as well as the content of glutathione, ascorbate and phenolic compounds were determined. An induced-increase in the APX, CAT, GPX and GR activities was stronger for PCP, while a significant increase in the GST activity was noted only for 2,4-DCP. Both compounds increased the content of phenolic compounds, oxidized and reduced glutathione as well as the content of ascorbic acid. PCP induced stronger increase in lipid peroxidation than 2,4-DCP. The observed changes revealed that chlorophenols induce oxidative stress and oxidative damage in the leaves of reed canary grass.     

Developmental changes of antioxidative systems in tobacco leaves as affected by limited sucrose export in transgenic plants expressing yeast-invertase in the apoplastic space

It is generally believed that a restricted export of carbohydrates from source leaves causes oxidative stress because of an enhanced utilisation of O₂ instead of NADP⁺ as electron acceptor in photosynthesis. To test this hypothesis, developmental changes of antioxidative systems were investigated in wild-type and transgenic tobacco (Nicotiana tabacum L.) suffering from disturbed sink-source relations by expression of yeast invertase in the apoplastic space. Young expanding leaves of the wild type contained higher activities of Superoxide dismutase (EC 1.15.1.1), ascorbate peroxidase (EC 1.11.1.11), catalase (EC 1.11.1.6), dehydroascorbate reductase (EC 1.8.5.1), glutathione reductase (EC 1.6.4.2) and a higher glutathione content than mature source leaves. The activity of monodehydroascorbate-radical reductase (EC 1.1.5.4) and the ascorbate content remained unaffected by the developmental stage in the wild type. In young expanding leaves of the transgenic plants the capacity of the antioxidative systems was similar to or higher than in corresponding leaves from the wild type. Source leaves of transgenic tobacco with an increased carbohydrate content showed a small chlorophyll loss, an increased malondialdehyde content, a selective loss of the activities of Cu/Zn-superoxide dismutase isoenzymes and a fourfold decrease in ascorbate compared with the wild type. There was no evidence that the protection from H₂O₂ was insufficient since source leaves of transgenic tobacco contained increased activities of catalase, ascorbate peroxidase, and monodehydroascorbate-radical reductase and an increased ascorbate-to-dehydroascorbate ratio compared with source leaves of the wild type. In severely chlorotic leaf sections of the transgenic plants, most components of the antioxidative system were lower than in green leaf sections, but the ascorbate-to-dehydroascorbate ratio was increased. These results suggest that carbohydrate-accumulating cells have an increased availability of reductant, which can increase the degree of reduction of the ascorbate system via glutathione-related systems or via the activity of monodehydroascorbate-radical reductase. At the same time, transgenic tobacco leaves seem to suffer from an increased oxidative stress, presumably as a result of a decreased consumption of O ₂ ^.- by Cu/Zn-superoxide dismutases in the chloroplasts. There was no evidence that carbohydrate-accumulating leaves acclimated to enhanced O ₂ ^.- production rates in the chloroplasts.     

Transgenic tobacco plants overexpressing cotton glutathione S-transferase (GST) show enhanced resistance to methyl viologen

A GST (EC 2.5.1.18) gene (Gst-cr 1) from cotton was introduced into Nicotiana tabacum by Agrobacterium tumefaciens-mediated transformation. Transgenic tobacco plants overexpressing Gst-cr1 were normal in growth and mature compared with control, but had much higher levels of GST and GPx activities and showed an enhanced resistance to oxidative stress induced by a low concentration of methyl viologen (MV). Six antioxidant enzymes, glutathione S-transferase, glutathione peroxidase (EC 1.11.1.9), superoxide dismutase (EC 1.15.1.1), peroxidase (EC 1.11.1.7), catalase (EC 1.11.1.6), and ascorbate peroxidase (EC 1.11.1.11) were monitored in transgenic lines and non-transgenic control during MV treatments. When they were treated with 0.03 mmol/L of MV, both transgenic lines and control showed a rapid increase in the activities of GST, GPx, SOD, POD, APx, while the activity of CAT seemed to be irregular. The percent of the increase in SOD and POD activities was much higher in control than in transgenic plants. When treated with 0.05 mmol/L of MV, both control and transgenic plants were severely damaged, and the activities of the six enzymes decreased sharply.     

Changes in the water binding characteristics of the cell walls from transgenic Nicotiana tabacum leaves with enhanced levels of peroxidase activity

Pore size in the cell wall matrix may affect cell wall–water relations, particularly under osmotic stress. Cross linkage of plant cell wall matrix polymers is an important step in the formation of this structure and peroxidases have been proposed to catalyse the cross-linking of phenolic constituents. Transgenic tobacco ( Nicotiana tabacum ) plants expressing a basic tomato peroxidase gene (TPX2) showed increased apoplastic ferulic acid peroxidase activity in mature leaves. This enhanced activity was not associated with a decreased leaf growth. Differential scanning calorimetry (DSC) of control dried cell walls showed a putative glass transition, after Ca²⁺ removal, that was absent in the transgenic line. This would indicate that transgenic walls were more rigid. DSC analysis of water-hydrated cell wall preparations distinguished two pools of water, freezable and non-freezable water. The amount of non-freezable water, which corresponds to strongly bound water, was higher in the transgenic line (64 versus 55%). DSC thermograms of the transgenic cell wall were displaced to lower temperatures, and this may be interpreted as the result of a stronger interaction between this freezable water and this wall. Water sorption and desorption isotherms, obtained at relative humidity ranging from 5 to 93%, demonstrated the presence of very strongly bound water in the transgenic cell walls that was absent in controls. Water sorption–desorption hysteresis of the isotherms was evident in the control wall but not in the transgenic line. These changes in cell wall–water interaction seem to be relevant at the organ level because leaf discs of transgenic plants maintained higher relative water content than control discs, at water potentials between −1.05 and−2.31 MPa.     

Effect of certain dicarboxylic acid monoesters on growth,chlorophyll content,chlorophyllase and peroxidase activities,and gas-exchange of young maize plants

The effect of some dicarboxylic acid monoesters on growth, chlorophyll content, chlorophyllase (EC 3.1.1.14), and total peroxidase (EC 1.11.1.7.) activities was examined in detached and intact leaves of maize (Zea mays) plants grown in a greenhouse. The -monomethyl ester of itaconic acid (MEIA) at 1250 ppm had no effect on growth. However, application of the monoethyl ester of succinic (MESA) and monoethyl ester of adipic (MEAdA) acids (1250 ppm) resulted in an increased leaf area, fresh and dry weight of leaves and stems. These compounds retarded chlorophyll degradation in both detached and intact leaves. Chlorophyllase activity of the control and treated leaves was measured and related to chlorophyll content. Delaying of senescence by treatment with monoesters resulted in greater chlorophyll and protein content, compared with the control. However, the chlorophyllase activity/chlorophylla ratio in the treated plants decreased. Total peroxidase activity was higher in senescent leaves, but all treatments inhibited the increase of this enzyme activity. Prolonged carbon assimilative activity and enhanced leaf water use efficiency in treated plants was noted.     

Differences in Protein Synthesis and Peroxidase isoenzymes between recalcitrant and regenerating ProtoPlasts

The reasons for the inability of recalcitrant mesophyll protoplasts to divide and re-enter the cell cycle are unknown. Changes in protein profile, indole-3-acetic acid (IAA)-oxidase and peroxidase activities, and isoenzymes were compared in protoplasts of recalcitrant grapcvine ( Vitis vinifera ) L. cv. Sultanina) and regenerating tobacco ( Nicotiana tabacum ) L. cv. Xanthi). Using [³⁵S]-methionine. SDS-PAGE and two-dimensional separation of proteins, differences in protein profile during protoplast culture were assessed. The changes in the de novo synthesized proteins were both qualitative and quantitative between the two species. The number of proteins which changed was double in tobacco compared to grapevine protoplasts. Peroxidase and IAA-oxidase activities increased significantly in tobacco protoplasts during culture whereas in grapevine they remained low. In tobacco protoplasts. 3 and 7 basic and acidic peroxidases, respectively, were induced during protoplast culture. which were not detected in the intact leaf, whereas in grapevine no new peroxidases were induced during protoplast culture.     

Differential response of antioxidative systems of maize (Zea mays L.) roots cell walls to osmotic and heavy metal stress

An analysis of peroxidase and ascorbate oxidase activity, phenolic content and antioxidant capacity of isolated maize root cell walls was performed in controls and plants stressed with polyethylene glycol (PEG) or heavy metals, zinc or copper. Peroxidase activity (oxidative and peroxidative) was more pronounced in the ionic than in the covalent cell wall fraction. PEG induced an increase and Zn²⁺ a decrease of both ionically bound peroxidase activities. In the covalent fraction, Cu²⁺ decreased oxidative and increased peroxidative activity of peroxidase. Isoelectric focusing of ionically bound proteins and activity staining for peroxidase demonstrated increased intensities and appearance of new acidic isoforms, especially in Zn²⁺ and PEG treatments. Most pronounced basic isoforms (pI ~ 7.5) in controls, decreased in intensity or completely disappeared in stressed plants. Ascorbate oxidase activity was significantly increased by PEG and decreased by Zn²⁺ treatments, and highly correlated with peroxidase activity. Antioxidant capacity and total phenolics content increased in heavy metal‐treated and decreased in PEG‐treated plants. Analysis of individual phenolic components revealed p‐coumaric and ferulic acids, as the most abundant, as well as ferulic acid dimers, trimers and tetramers in the cell walls; their quantity increased under stress conditions. Results presented demonstrate the existence of diverse mechanisms of plant response to different stresses.     

Effect of chlorsulfuron on phenylpropanoid metabolism in sunflower seedlings

The effect of the herbicide chlorsulfuron on phenylpropanoid titer and metabolism and the role of endogenous ethylene in this response was examined in light-grown sunflower (Helianthus annuus L.) seedlings. Application of chlorsulfuron to the apex resulted in large increases in both total phenolic and hydroxycinnamic acid content in hypocotyls isolated from the treated seedlings. Both of these parameters were increased within 24 h of herbicide treatment, and both reached a maximum level 3–4 days post-treatment. An increase in ethylene evolution was found to proceed in parallel with the alterations of phenolic content. The extractable activities of phenylalanine ammonia lyase,trans-cinnamic-4-hydroxylase, and soluble peroxidase were increased by chlorsulfuron treatment. Chlorsulfuron had little effect on total phenolic content when administered directly to isolated hypocotyl segments. Exogenous ethylene had no effect on the endogenous titer of phenolic compounds. Root-fed cobalt chloride (5 × 10^?4 M) inhibited chlorsulfuron-induced ethylene production by 92% and also inhibited the accumulation of phenolic materials by 56%. Exogenous ethylene was unable to reverse the inhibition caused by cobalt chloride. It was concluded that chlorsulfuron-induced increases in phenolic compounds were not mediated solely by endogenous ethylene.     

Functions of oligochitosan induced protein kinase in tobacco mosaic virus resistance and pathogenesis related proteins in tobacco

Oligochitosan (OC) can regulate plant defense responses in many aspects, but the basic signal transduction pathway is still unclear. In this study, we used transgenic (TG) tobacco (Nicotiana Tabacum var. Samsun NN) as plant material whose oligochitosan induced protein kinase (OIPK) gene was inhibited by antisense transformation, to study the role of OIPK in tobacco defense reactions. The results showed that OIPK could increase tobacco resistance against tobacco mosaic virus (TMV), in that wild-type (WT) tobacco showed longer lesion appearance time, higher lesion inhibition ratio, smaller average final lesion diameter and lower average final lesion area percent to whole leaf area. It led us to analyze some pathogenesis related (PR) enzymes' activities and mRNA level, which played roles in tobacco resistance against TMV. We found that phenylalanine ammonia-lyase (PAL) and peroxidase (POD) activities were positively related to OIPK, but not polyphenol oxidase (PPO). It was also demonstrated that OIPK mRNA could be induced by OC, wound and TMV infection. In addition, OIPK could up-regulated three PR genes, PAL, chitinase (CHI) and β-1, 3-glucanase (GLU) mRNA level to different extent. Taken together, these results implied that OIPK could function in tobacco resistance against both biotic and abiotic stress, possibly via various PR proteins.     

Effect of transgenic plants expressing high levels of a tobacco anionic peroxidase on the toxicity of Anagrapha falcifera nucleopolyhedrovirus to Helicoverpa zea (Lepidoptera: Noctuidae)

Wild type and corresponding transgenic tomato (Lycopersicon esculentum Miller) and two tobacco (Nicotiana spp.) plants that express high levels of a tobacco anionic peroxidase were used to determine what type of interactions occurred between peroxidase altered plant chemistry and the baculovirus Anagrapha falcifera nucleopolyhedrovirus (AfMNPV) for control of neonate corn earworms, Helicoverpa zea (Boddie). Transgenic plants expressed approximately five to 400 times higher peroxidase activity than corresponding tissues of wild type plants. The H. zea larvae typically fed 1.5 times less on transgenic compared with wild type leaf disks. There was only one experiment (of three with tomato leaves) where the larvae that fed on transgenic leaves were less susceptible to the virus based on nonoverlapping 95% confidence intervals for LC50 values. When the exposure dose was corrected for reduced feeding on the transgenic leaf disks, the insecticidal activity of the virus was not significantly different for larvae fed on transgenic versus wild type plants. Eight other experiments (with tomato and two species of tobacco) indicated either no significant effect or enhanced susceptibility (when corrected for feeding rates) to the virus of larvae fed on the transgenic leaves. These results indicate enhanced insect resistance in plants expressing high levels of a specific anionic peroxidase may be compatible with applications of AfMNPV. Potential reasons for this compatibility are discussed.     

Antioxidant enzymatic protection during tobacco leaf ageing is affected by cytokinin depletion

Plant ageing and senescence are associated with increased levels of reactive oxygen species. Level of cytokinins, the apparent inhibitors of plant senescence, is controlled by their irreversible degradation catalysed by cytokinin oxidase/dehydrogenase (CKX). We investigated the CKX activity, cytokinin concentration, and activities of antioxidative enzymes in tobacco (Nicotiana tabacum L. cv. Samsun NN) overexpressing the Arabidopsis gene for AtCKX2, targeted for extracellular secretion pathway. The control and AtCKX2 plants differed substantially in their phenotypes. When the lowest leaves in controls became yellow all leaves in AtCKX2 tobacco still remained green. Activities of antioxidant enzymes decreased with leaf age in both tobacco plants except for ascorbate peroxidase (APX) in the old leaves and glutathione reductase (GR) in young leaves. Enhancement of GR activity at all leaf stages, an increase of superoxide dismutase and a decline of catalase in young leaves, as well as an increase of APX in the oldest leaves were observed in AtCKX2 plant compared to control. Similar changes were detected after determination of isoenzymes on zymograms. It is evident that AtCKX2 plants had postponed onset of senescence despite the significantly lowered level of cytokinins. Enhanced antioxidant protection, especially in the oldest leaves, could subsidise this phenomenon.     

Inhibition of Ethylene Biosynthesis Enhances Vegetative Bud Formation without Affecting Growth and Development of Transgenic Tobacco Plants

The role of ethylene in vegetative bud formation was investigated using transgenic tobacco plants expressing an antisense tomato 1-aminocyclopropane-carboxylic acid synthase (ACS) gene. Northern blot hybridization showed that the accumulation of ACS mRNA was strongly reduced in the bud-forming leaf explants of the transgenic plants. Consequently, these transgenic tissues exhibited low ACS enzyme activity, 1-aminocyclopropane-carboxylic acid (ACC) content and ethylene production, and at the same time the tissue capacity to generate buds was greatly enhanced. However, it was also noted that the antisense ACS gene did not inhibit the endogenous ACS gene expression in intact transgenic tobacco plants. The growth and development of the transgenic tobacco was almost identical to control plants with respect to height, internode number, leaf morphology, and flowering time. Furthermore, mature leaves of transgenic tobacco had similar chlorophyll content, stomatal conductance, photosynthetic ability, and transpiration rates compared to control plants. These results demonstrated that ethylene plays an important role in bud formation in tobacco tissue culture.