Ethylene Biosynthesis-Inducing Endoxylanase Is Translocated through the Xylem of Nicotiana tabacum cv Xanthi Plants

Ethylene biosynthesis-inducing xylanase (EIX) from the fungus Trichoderma viride elicits enhanced ethylene production and tissue necrosis in whole tobacco (Nicotiana tabacum cv Xanthi) plants at sites far removed from the point of EIX application when applied through a cut petiole. Symptoms develop in a specific pattern, which appears to be determined by the interconnections of the tobacco xylem. Based on results of tissue printing experiments, EIX enters the xylem of the stem from the point of application and rapidly moves up and down the stem, resulting in localized foliar symptoms on the treated side of the plant above and below the point of EIX application. The observation that a fungal protein that elicits plant defense responses can be translocated through the xylem suggests that plants respond to pathogen-derived extracellular proteins in tissues distant from the invading pathogen.     

Alterations in Nicotiana tabacum L. cv Xanthi Cell Membrane Function following Treatment with an Ethylene Biosynthesis-Inducing Endoxylanase

An ethylene biosynthesis-inducing xylanase (EIX) produced by the fungus Trichoderma viride elicited enhanced ethylene biosynthesis and leakage of potassium and other cellular components when applied to leaf disks of tobacco (Nicotiana tabacum L. cv Xanthi). Suspension-cultured cells of Xanthi tobacco responded to EIX by rapid efflux of potassium, uptake of calcium, alkalization of the medium, inhibition of ethylene biosynthesis, and increased leakage of cellular components. EIX-treated cell suspensions released 1-aminocyclopropane-1-carboxylate (ACC) into the surrounding medium, resulting in a reduction of cellular pools of ACC. The responses of both cell suspensions and leaf disks were inhibited (50-80%) by the preincubation of the tissues with the calcium channel blocker La³⁺. High concentrations of EGTA inhibited the alkalization of the medium by cell suspensions responding to EIX, but EGTA alone caused extensive loss of K⁺ and ACC and inhibited ethylene biosynthesis by tobacco cells. Alterations in membrane function appear to be important in the mode of action of EIX in Xanthi cells.     

An Ethylene Biosynthesis-Inducing Endoxylanase Elicits Electrolyte Leakage and Necrosis in Nicotiana tabacum cv Xanthi Leaves

We have previously demonstrated that a protein purified from xylan-induced culture filtrates of Trichoderma viride contains β-1,4-endoxylanase activity and induces ethylene biosynthesis in tobacco (Nicotiana tabacum cv Xanthi) leaf discs. When the ethylene biosynthesis-inducing xylanase (EIX) was applied to cut petioles of detached tobacco leaves, it induced ethylene biosynthesis within 1 hour and extensive electrolyte leakage and necrosis were observed in tobacco leaf tissue within 5 hours. Ethylene-pretreatment (120 microliters per liter ethylene for 14 hours) of tobacco leaves enhanced ethylene biosynthesis in response to EIX by more than threefold and accelerated development of cellular leakage and necrosis. In intact plants, similar symptoms could be induced in leaves that were distant from the point of the enzyme application. The evidence suggests that EIX is translocated via the vascular system and elicits plant responses similar to those observed in a hypersensitive response.     

The Presence of the N' Gene in Nicotiana tabacum L. cv. Samsun NN

The N′ gene was shown to be present in the ‘Samsun NN’ line of Nicotiana tabacum by its differential response to tobacco mosaic (TMV) and tomato mosaic (ToMV) viruses in the segregating and test-cross generations of the cross ‘Samsun NN’בSamsun nn’. The appearance of phenotypes reacting with local necrosis to ToMV and with systemic mosaic to TMV confirmed that the genes N and N′ are on distinct loci. An isogenic N′;N′ Samsun line was selected.     

In Vitro Selection of Calli Resistant to a Triazole Cytochrome-P-450-Obtusifoliol-14-Demethylase Inhibitor from Protoplasts of Nicotiana tabacum L. cv Xanthi

The selection of biochemical mutants has been undertaken in order to elucidate regulatory and functional aspects of sterol biosynthesis in plants. 2-(4-Chlorophenyl)-3-phenyl-1-(1H-1,2,4- triazol-1-yl)-2,3-oxidopropane (LAB170250F), an experimental fungicide of the triazole family, was used as a selective agent. Indeed, this compound is a strong inhibitor of the cytochrome-P-450-obtusifoliol-14-demethylase in sterol biosynthesis. The selection strategy consisted of screening large populations of microcalli derived from ultraviolet-mutagenized protoplasts of Nicotiana tabacum L. cv Xanthi for resistance to a lethal concentration of LAB170250F. The best selective conditions were first determined, i.e. strength of the selection pressure as well as the time and duration of its application in the developmental process from protoplast to whole plant. Selection experiments resulted in the recovery of 40 resistant calli. These calli were divided into three classes according to the modification of their sterol content in response to LAB170250F. Some of these calli might be impaired in sterol biosynthesis, but most have a sterol profile identical to that of the control calli. This suggests that the toxic properties of LAB170250F are due to the parallel inhibition of sterol biosynthesis and of at least one additional unidentified target in the plant cell.     

Photosynthetic Capacities and Growth Characteristics of Nicotiana tabacum (cv. Xanthi) Cell Suspension Cultures

Photoheterotrophic growth of cell suspensions of Nicotiana tabacum L. (cv. Xanthi) in organic culture medium enriched in sucrose (30 g per liter) showed a classical sigmoid growth curve. The cells developed functional chloroplast structures during the exponential growth phase, when their chlorophyll content increased steadily. A limited drop (30%) in the chlorophyll amount and structural changes of the plastids (starch accumulation) were observed during the lag phase. The measurements of photosynthetic capacities (O₂ evolution and CO₂ fixation) during the growth cycle revealed changes in the photosynthetic ratio (O₂/CO₂), which was near 1 during the lag and stationary phases and near 2 during exponential growth. During exponential growth there was also a rapid NO₃^? uptake. Analysis of label distribution among the products of ¹⁴CO₂ fixation showed that both CO₂ assimilation pathways, linked to the ribulose-biphosphate carboxylase (the autotrophic pathway) and to phosphoenolpyruvate carboxylase (the non-autotrophic pathway) were operative with an important increase of the capacity of the latter during the exponential growth phase. Maximum rate of oxygen evolution, either endogenous or with p-benzoquinone as Hill reagent, as well as the increased CO₂ Fixation capacity via the non-autotrophic pathway during the exponential phase were concomitant with a high cyanide inhibited O₂ uptake.     

Expression of recombinant antibody (single chain antibody fragment) in transgenic plant Nicotiana tabacum cv. Xanthi

Plants offer an alternative inexpensive and convenient technology for large scale production of recombinant proteins especially recombinant antibodies (plantibodies). In this paper, we describe the expression of a model single chain antibody fragment (B6scFv) in transgenic tobacco. Four different gene constructs of B6scFv with different target signals for expression in different compartments of a tobacco plant cell with and without endoplasmic reticulum (ER) retention signal were used. Agrobacterium mediated plant transformation of B6scFv gene was performed with tobacco leaf explants and the gene in regenerated plants was detected using histochemical GUS assay and PCR. The expression of B6scFv gene was detected by western blotting and the recombinant protein was purified from putative transgenic tobacco plants using metal affinity chromatography. The expression level of recombinant protein was determined by indirect enzyme-linked immunosorbent assay. The highest accumulation of protein was found up to 3.28 % of the total soluble protein (TSP) in plants expressing B6scFv 1003 targeted to the ER, and subsequently expression of 2.9 % of TSP in plants expressing B6scFv 1004 (with target to apoplast with ER retention signal). In contrast, lower expression of 0.78 and 0.58 % of TSP was found in plants expressing antibody fragment in cytosol and apoplast, without ER retention signal. The described method/system could be used in the future for diverse applications including expression of other recombinant molecules in plants for immunomodulation, obtaining pathogen resistance against plant pathogens, altering metabolic pathways and also for the expression of different antibodies of therapeutic and diagnostic uses.     

Timing of morphological and histological development in premeiotic anthers of Nicotiana tabacum cv. Xanthi (Solanaceae)

The tobacco stamen has been the object of many developmental studies, and the organ has more recently become a model for molecular genetic studies of anther differentiation. However, the spatial and temporal details of cellular differentiation of early anther development have never been thoroughly characterized. In the present study, the age of 15 tobacco flowers from plants grown under constant light and temperature was estimated using growth analysis. Prior to tissue fixation for light microscopy, moulds of stamen and anther primordia were made with a dental impression polymer so morphological and histological observations could be made on each tissue sample. Flower ages spanned an 8-d interval during which petal and stamen initiation occurred, and sporogenous cells reached the leptonema stage of meiosis. The initial development of the tetrasporangiate anther shape largely preceded periclinal division of archesporial initials. Anatomically, periclinal divisions in the hypodermal ∗∗∗(l₂) layer were observed before archesporial initials began to divide. These data indicate differences in the cellular basis of tobacco anther development compared to earlier clonal analyses of Datura. The pattern of mitotic cell division associated with microsporangial development suggested modal peaks in division over time. The ability to estimate developmental time in the tobacco anther has implications for future studies directed at understanding mechanisms of anther evolution via heterochrony.     

Developmental Pattern of Root and Shoot Organogenesis in Cultured Leaf Explants of Nicotiana tabacum cv. Xanthi nc.

Caulogenesis and rhizogenesis were studied in cultured leafexplants of Nicotiana tabacum cv. Xanthi nc. using both lightand scanning electron microscopy. The timing of organ appearancewas also recorded. The patterns of development seen were comparedto each other and to that in explants grown on growth regulator-freemedium. Shoots first appeared after 12 d in culture and rootsafter 7 d. In caulogenesis nodules appear at the explant edgeand from these the shoots arise. The nodules are mainly derivedfrom palisade mesophyll cells, along with some spongy mesophylland bundle-sheath cells. The nodules form a continuous row alongthe edge of the explant and their initiation appears to be centredon veins. Shoots are produced indirectly. Roots are produceddirectly from bundle-sheath and vein parenchyma cells. Withoutplant growth regulators bundle-sheath cells still divide, althoughonly a few divisions were seen. Key words: Nicotiana tabacum, in vitro, caulogenesis, rhizogenesis     

Stages in the Initiation of Root and Shoot Organogenesis in Cultured Leaf Explants of Nicotiana tabacum cv. Xanthi nc.

Reciprocal transfers of Nicotiana tabacum cv. Xanthi nc. leafexplants were made daily between root inducing medium (RIM)and shoot inducing medium (SIM), SIM and a basal medium containingno growth regulators (BM), and RIM and BM. It was found thatthe explants became determined for shoot production after 6d, while roots were produced after only 1 d on RIM before transferto BM. The competence of the explant to produce roots was greatlyreduced by culture on BM prior to culture on RIM. There wasfar less reduction in shoot numbers with preculture on BM. Explantswere found to be only weakly canalized for both caulogenesisand rhizogenesis for the first 2 d after determination. Thereafterthey became strongly canalized. Transfers were also made fromBM to SIM and back to BM, which revealed that the explants becamecompetent for caulogenesis in the absence of cytokinins priorto determination. The period for which SIM is required can bereduced to only 1 d. Key words: Nicotiana tabacum, in vitro, organogenesis, competence, determination     

Effects of ribavirin and adenine arabinoside on tobacco mosaic virus in Nicotiana tabacum L. var Xanthi tissue cultures

Although both ribavirin (1-β-ribofuranosyl-1,2,4-triazole-3carboxamide) and adenine arabinoside inhibited the multiplication of tobacco mosaic virus (TMV) in mechanically inoculated leaf tissues, neither chemical inhibited virus multiplication in unorganized tobacco callus after in vitro inoculation. The adenine deaminase inhibitor, pentostatin, did not increase the activity of adenine arabinoside in cultured cells. Several different developmental conditions and media did not increase the ability of either chemical to eradicate the virus from tobacco tissue cultures. However, the virus was eradicated from TMV-infected callus when grown in the presence of combinations of ribavirin and adenine arabinoside in shoot inducing medium.     

Copper-mediated oxidative burst in Nicotiana tabacum L. cv. Bright Yellow 2 cell suspension cultures

In cell suspension cultures of Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) a rapid and concentration-dependent accumulation of H(2)O(2) is induced by excess concentrations of copper (up to 100 microM). This specific and early response towards copper stress was shown to be extracellular. Addition of 300 U of catalase per ml decreased the level of H(2)O(2). Superoxide dismutase (5 U/ml) induced an increase in H(2)O(2) production by 22.2%. This indicates that at least part of the H(2)O(2) is produced by dismutation of superoxide. Pretreatment of the cell cultures with the NAD(P)H oxidase inhibitors diphenylene iodonium (2 and 10 microM) and quinacrine (1 and 5 mM) prevented the generation of H(2)O(2) under copper stress for 90%. The influence of the pH on the H(2)O(2) production revealed the possible involvement of cell-wall-dependent peroxidases in the generation of reactive oxygen species after copper stress.     

Copper-mediated oxidative burst in Nicotiana tabacum L. cv. Bright Yellow 2 cell suspension cultures

Summary.  In cell suspension cultures of Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) a rapid and concentration-dependent accumulation of H₂O₂ is induced by excess concentrations of copper (up to 100 μM). This specific and early response towards copper stress was shown to be extracellular. Addition of 300 U of catalase per ml decreased the level of H₂O₂. Superoxide dismutase (5 U/ml) induced an increase in H₂O₂ production by 22.2%. This indicates that at least part of the H₂O₂ is produced by dismutation of superoxide. Pretreatment of the cell cultures with the NAD(P)H oxidase inhibitors diphenylene iodonium (2 and 10 μM) and quinacrine (1 and 5 mM) prevented the generation of H₂O₂ under copper stress for 90%. The influence of the pH on the H₂O₂ production revealed the possible involvement of cell-wall-dependent peroxidases in the generation of reactive oxygen species after copper stress. Received May 20, 2002; accepted July 26, 2002; published online May 21, 2003 RID="*" ID="*" Correspondence and reprints: Plant Physiology, Department of Biology, University of Antwerp (RUCA), Groenenborgerlaan 171, 2020 Antwerp, Belgium.     

Effects of ribavirin and adenine arabinoside on tobacco mosaic virus in Nicotiana tabacum L. var Xanthi tissue cultures

Although both ribavirin (1--ribofuranosyl-1,2,4-triazole-3carboxamide) and adenine arabinoside inhibited the multiplication of tobacco mosaic virus (TMV) in mechanically inoculated leaf tissues, neither chemical inhibited virus multiplication in unorganized tobacco callus after in vitro inoculation. The adenine deaminase inhibitor, pentostatin, did not increase the activity of adenine arabinoside in cultured cells. Several different developmental conditions and media did not increase the ability of either chemical to eradicate the virus from tobacco tissue cultures. However, the virus was eradicated from TMV-infected callus when grown in the presence of combinations of ribavirin and adenine arabinoside in shoot inducing medium.     

Effects of the Introduction of Agrobacterium tumefaciens T-DNA ipt Gene in Nicotiana tabacum L. cv. Petit Havana SR1 Plant Cells

Transformation of tobacco leaf discs with the ‘cytokinin’ipt gene yielded several transgenic callus tissue lines, respectiveto the kind of ipt construction present in the A. tumefacienscointegrates. Those calli containing an active ipt gene wereable to grow hormone-autotrophically and showed an increasedendogenous cytokinin level in comparison with controls. Analysisof endogenous IAA level did not allow any quantitative correlationwith the cytokinin content. However, a minimal level of auxinseems to be necessary to obtain hormone-autotrophic growth.Exogenously supplied NAA significantly reduced the endogenouscytokinin content without modifying growth characteristics. The varying chlorophyll content in the different callus lineselicited the study of the ultrastructure of the plastids. Thecontrols contained small plastids, often filled with starchor accumulated vesicles that did not allow observation of theinternal membrane system. The ‘Pssu-ipt’ line, havinga higher cytokinin content, showed plastids with an internalmembrane system consisting of stroma and grana thylakoids, butthis structure was lost during subculture. Swollen thylakoidsappeared, the amount of starch was reduced and vesicles wereaccumulating. (Received November 15, 1990; Accepted March 4, 1991)