Different effects of two brassinosteroids on growth,auxin and cytokinin content in tobacco callus tissue

Two synthetic brassinosteroids, 24-epibrassinolide (24-epiBR) and 2,3, 17-trihydroxy-5-androstan-6-one (THA-BR), exhibit different effects on growth of tobacco callus tissue. When added to a culture medium containing growth-limiting amounts of auxin, 24-epiBR reduced and THA-BR increased the fresh weight yield of tissue up to 53% and 207%, respectively, after 6 weeks of cultivation. The stimulatory and inhibitory effects of the two brassinosteroids on tissue growth occurred over a broad range of concentrations without a pronounced maximum corresponding to the yes or no type of response. Different effects of 24-epiBR and THA-BR on tissue growth were inversely proportional to the content of endogenous cytokinins. Maximum contents of predominant cytokinins N⁶-(²-isopentenyl)adenine (iP) and trans-zeatin (Z) in tissues supplied with 24-epiBR in growth-inhibiting concentrations were up to 3.7 fold and 3.4 fold higher, respectively, as compared to tissues grown on media containing growth-stimulating concentrations of THA-BR. Stimulation of tissue growth by THA-BR correlated with content of endogenous IAA and an inverse correlation was found between the content of endogenous IAA and cytokinins in tissues supplied with 24-epiBR. THA-BR exhibited weak cytokinin-like activity in a bioassay based on stimulation of growth of lateral buds of pea while 24-epiBR was inactive. Results indicate that the qualitatively different effects of the two brassinosteroids on growth of tobacco tissue may reflect their different influence on content of endogenous cytokinin.Abbreviations BR(s) brassinosteroid(s) - 24-epiBR 24-epibrassinolide - THA-BR 2,33, 17-trihydroxy-5-androstan-6-one - CK(s) cytokinin(s) - iP N⁶-(²-isopentenyl)adenine - [9R]iP N⁶-(²-isopentenyl)adenosine - Z trans-zeatin - [9R]Z ribosyl-trans-zeatin - ABA abscisic acid - IAA indole-3-acetic acid - NAA naphtalene-1-acetic acid - DEAE cellulose diethylaminoethyl cellulose - HPLC high performance liquid chromatography - ELISA enzyme linked immuno-sorbent assay     

Some morphogenic effects of sodium sulfate on tobacco callus

Callus cultures of Nicotiana tabacum L cv. Wisconsin 38 were initiated and grown on shoot-forming (SF) and callus proliferation (CP) medium with or without Na₂SO₄. Two cultures were maintained on SF medium with 0, 0.75, 1 or 1.5% Na₂SO₄ for 2.5 and 3.5 years. In the older culture only callus grown on salt formed shoots throughout the maintenance period, while in the younger culture the control responded best and Na₂SO₄ was inhibitory. Callus from the older culture which had been grown on salt continued to form shoots in the absence of salt. Na₂SO₄ caused adventitious shoot formation in three cultures on CP medium. These shoots were present for 7 subcultures after removal of Na₂SO₄; but established, control callus, did not form shoots when transferred to Na₂SO₄. Callus initiated and maintained on NaCl or mannitol showed a slight increase in shoot initiation. On NaCl, Na₂SO₄ or mannitol, the tissue osmotic potential became more negative and proline concentration increased.     

Ascorbic acid enhancement of organogenesis in tobacco callus

The effect of ascorbic acid on growth and shoot formation in callus cultures of tobacco (Nicotiana tabacum L.) was investigated, using young (4–12 subcultures) and old (more than 30 subcultures) tissue. It was found that ascorbate, at levels of 4–8×10^-4M, enhanced shoot formation in both young and old callus. Treatment with ascorbate also speeded up the shoot-forming process. In addition, ascorbate completely reversed the inhibition of shoot formation by gibberellic acid in young callus, but was less effective in old callus.     

Expression of the isopentenyl transferase gene is regulated by auxin in transgenic tobacco tissues

The isopentenyl transferase gene (ipt) fromAgrobacterium tumefaciens was isolated and introduced, via a disarmed binary vector, into tobacco using theAgrobacterium tumefaciens-mediated gene transfer system. The expression of theipt gene was monitored by RNA hybridization, western blotting and cytokinin analysis. The addition of auxin to the media rapidly reduced the level of cytokinins in the transgenic tissues and this was associated with a reduction in IPT mRNA and protein levels. It is concluded that the hormone auxin can regulate expression of a gene involved in biosynthesis of the second hormone cytokinin. Although exogenous benzyladenine did not directly affectipt gene expression, it did antagonize the effect of auxin on levels of cytokinins and IPT mRNA and protein.     

Cell wall invertase activity in cultured tobacco tissues

Changes in insoluble or cell wall invertase and soluble invertase activity were examined during callus induction from tobacco pith-phloem explants and during callus proliferation on sucrose, glucose and fructose as carbon sources, or on transfer from culture on the hexoses to sucrose. In all cases there was a growth independent transitory increase in cell wall invertase early in culture. The magnitude of the increase was greatest in the presence of sucrose. Cell wall invertase was found to possess catalytic activity in situ, whether or not the tissue was grown on sucrose. It is hypothesized that the transitory increase in cell wall invertase plays a role in sucrose hydrolysis during wound respiration, which takes place early in culture.     

ABam HI family of tobacco highly repeated DNA:

ABamHI family of highly repeated DNA sequences of theNicotiana tabacum nuclear genome, denoted as a HRS60-family, was recently isolated. It comprises about 2% of the tobacco nuclear genome. Monomeric units are 182–184 bp long. Members of the HRS60-family isolated till now are closely related. DNA-DNA hybridization experiments with DNA of the two tobacco progenitors,N. tomentosiformis andN. sylvestris, revealed that the HRS60-family was present in many copies inN. sylvestris, the amount being about 1.7 times that inN. tabacum. InN. tomentosiformis as well as in some other species of the genusNicotiana, the HRS60-family is present in a small amount. Sequences related to the HRS60-family were revealed using DNA-DNA hybridization at low stringency. With respect to quantity, the HRS60-family could be considered as a species-specific DNA repeat which may be a useful genetic marker in genetic manipulations withN. tabacum.     

Effect of sodium sulfate on in vitro organogenesis of tobacco callus

Callus of tobacco (Nicotiana tabacum L. cv. Wisconsin 38) was grown on callus-proliferating (CP) and shoot-forming (SF) media with elevated sodium sulfate (Na₂SO₄) concentrations either in the light or dark for more than one year. An increase in Na₂SO₄ concentration resulted in a decrease in callus growth index, an increase in percent dry weight of callus tissues grown on both media, and a decrease in both number of calli forming shoots and number of shoots per callus in SF medium. The CP callus grown in the light spontaneously began to form shoots after the 5th monthly transfer, and spontaneous root formation occured after the 16th transfer in the presence of 0.75 and 1.0% Na₂SO₄. Both water () and osmotic (_s) potentials of the callus increased with increasing Na₂SO₄ concentration; and callus exhibited greater and _s in the light than dark for both CP and SF media.     

Abnormal growth of habituated sugarbeet callus and cell suspensions

Summary Habituated sugarbeet callus and cell suspension cultures derived therefrom, compared to hormone-dependent normal cultures, exhibit a shorter linear growth phase, although they divide actively. Microscopic observations indicate deficiencies in cell expansion and absence of cell differentiation. Cell expansion apparently is interrupted by a cell “budding” process. Some of the cells seem to be empty due to ballooning out of the protoplasm and the bursting of the cell membrane by defective cell wall development. A low amount of cellulose was confirmed by microspectrophotometric estimation. Such cultures exhibit all the characteristics of vitrified tissue.     

Nitrate assimulation in shoot-forming tobacco callus cultures

Summary The contents of nitrogen-containing compounds and the activities of nitrate assimilating enzymes were determined in tobacco callus cultured under shoot-forming and non-shoot-forming conditions. Whole tissue and tissues cut into top and bottom portions were examined. Highes levels of total-N, protein-N, nitrate and ammonium-N, as well as higher activities of nitrate and nitrite reductases were found in shoot-forming whole tissue and in the shoot-forming bottom portion of tobacco callus in comparison to the non-shoot-forming proliferating tissues throughout the culture period. These findings indicate that enhanced nitrogen assimilation occurs during de, de novo shoot organogenesis. This work was supported by NSERC of Canada Grant No. A-6467 to T.A.T.     

Protein synthesis in tobacco pollen tubes: preferential synthesis of cell-wall 69-kDa and 66-kDa glycoproteins

One- and two-dimensional electrophoresis of Nicotiana tabacum pollen and pollen tube proteins confirmed that a new protein is preferentially synthesized during pollen germination and tube growth and becomes the most abundant protein in pollen tubes. Analysis of proteins extracted with sodium dodecyl sulfate (SDS) from different pollen tube fractions showed that it is the most abundant non-covalently bound wall protein, characterized by molecular mass of 69 kDa, pI between 7.9 and 8.2, and glycosylation with glucose and/or mannose. Amino acid analysis revealed relative abundance of serine, glutamic acid and glycine, but did not show the presence of hydroxyproline. According to all these characteristics, it cannot be classified as an extensin-like protein. Another prominent wall-bound glycoprotein has a molecular mass of 66 kDa and the same pI as the 69 kDa glycoprotein. These two glycoproteins are similar also in ConA binding, rate of synthesis, and rapid incorporation into pollen tube walls. Their synthesis is strongly reduced by tunicamycin and this inhibition results in the occurrence of new polypeptides in the range of 57–61 kDa. Tunicamycin also inhibited pollen tube growth. At 10 ng ml^-1 and 50 ng ml^-1 the inhibitor reduced pollen tube mass after 24 h of culture by 30% and 85%, respectively. This indicates that tobacco pollen presents a system highly sensitive to tunicamycin and that cotranslational N-linked glycosylation on the rough endoplasmic reticulum is required for 66 and 69 kDa glycoprotein formation and for pollen tube growth. Although other proteins appear during pollen germination and tube growth, the new proteins occur at low levels and seem to originate through modifications of preexisting polypeptides. In contrast to 69 and 66 kDa proteins, most proteins detected by [¹⁴C]amino acid incorporation and fluorography of gels were not revealed by Coomassie blue staining.     

Sterol composition of normal and habituated sugarbeet callus (Beta vulgaris L.Altissima)

Summary A detailed qualitative and quantitative analysis of the sterol content of normal (auxin and cytokinin requiring) and habituated (auxin and cytokinin independent) sugarbeet callus (Beta vulgaris L.altissima) was made using mass spectrometry with gas chromatography. The total sterol content of the two lines did not differ significantly. Δ⁷-Sterols were the most important class of sterols in the two sugarbeet callus lines, as in allChenopodiaceae. Elevated levels of Δ⁸-sterols were found in the habituated callus. These sterols are considered to be badly integrated in the membrane of eucaryotic cells. A partial blocking of Δ⁸-Δ⁷-isomerase is hypothesized in the habituated cell line.     

Polyamine metabolism and ethylene biosynthesis in normal and habituated sugar beet callus

The optimal assay conditions and the trend with time in culture (28 days) of arginine decarboxylase (ADE; EC 4.1.1.19), omithine decarboxylase (ODC; EC 4.1.1.17) and diamine oxidase (DAO; EC 1.4.3.6) activities in habituated (H) and normal (N) auxin- and cytokinin-requiring sugar beet callus were compared. Although the response to variations in buffer pH and EDTA and pyridoxal phosphate (PLP) concentrations varied for ADC and ODC activities between the two callus types, pH 8.3, 50 μ M PLP and 5 m M EDTA were generally optimal or near-optimal for both H and N callus. In most cases the addition of ornithine or arginine in the ADC and ODC assays, respectively, given to block the interconversion between the two substrates, resulted in lower ¹⁴CO₂ recovery. DAO activity was very differently affected in H and N callus by the presence of polyvinylpyrrolidone in the extration buffer. However, in both cases, this activity increased with time in culure. ADC activity was always predominant in both cell lines and always higher in N callus. In the latter, ADC activity rose sharply between days 14 and 21 and then leveled off while in H callus it incresed steadily from day 14 onwards. ODC activity was also higher in N callus and peaked sharply on day 21 while in H callus it was not detectable in the second half of the culture period. In both cell lines this activity was low or nil on day 28. 3,4-[¹⁴C]-methionine incorporation into ethylene and polyamines was also compared in N and H callus. In the latter, ethylene synthesis was lower and [¹⁴C]-spermidine formation higher than in N callus. This is in accord with the significantly higher spermidine titres found in H callus.     

cDNA cloning and gene expression of ascorbate oxidase in tobacco

A cDNA clone for ascorbate oxidase (AAO) has been isolated from a cDNA library of tobacco (Nicotiana tabacum) cells. The identity of the amino acid sequence deduced from tobacco AAO cDNA to that from pumpkin AAO cDNA was 68%, which was much lower than the identity (80%) between pumpkin and cucumber AAO. AAO activity in tobacco cells was much lower than that in pumpkin cells, whereas the immunoreactive protein in tobacco cells was more abundant than that in pumpkin cells. We suppose that AAO protein in tobacco cells may be less active than that in pumpkin cells. Genomic Southern blotting suggested that AAO in tobacco was encoded by a single-copy gene. Northern blotting revealed that mRNA of AAO was highly expressed in young and growing tissues of tobacco plant.     

Mitochondrial alterations related to programmed cell death in tobacco cells under aluminium stress

The present investigation was undertaken to verify whether mitochondria play a significant role in aluminium (Al) toxicity, using the mitochondria isolated from tobacco cells (Nicotiana tabacum, non-chlorophyllic cell line SL) under Al stress. An inhibition of respiration was observed in terms of state-III, state-IV, succinate-dependent, alternative oxidase (AOX)-pathway capacity and cytochrome (CYT)-pathway capacity, respectively, in the mitochondria isolated from tobacco cells subjected to Al stress for 18 h. In accordance with the respiratory inhibition, the mitochondrial ATP content showed a significant decrease under Al treatment. An enhancement of reactive oxygen species (ROS) production under state-III respiration was observed in the mitochondria isolated from Al-treated cells, which would create an oxidative stress situation. The opening of mitochondrial permeability transition pore (MPTP) was seen more extensively in mitochondria isolated from Al-treated cells than in those isolated from control cells. This was Ca(2+) dependent and well modulated by dithioerythritol (DTE) and Pi, but insensitive to cyclosporine A (CsA). The collapse of inner mitochondrial membrane potential (DeltaPsi(m)) was also observed with a release of cytochrome c from mitochondria. A great decrease in the ATP content was also seen under Al stress. Transmission electron microscopy analysis of Al-treated cells also corroborated our biochemical data with distortion in membrane architecture in mitochondria. TUNEL-positive nuclei in Al-treated cells strongly indicated the occurrence of nuclear fragmentation. From the above study, it was concluded that Al toxicity affects severely the mitochondrial respiratory functions and alters the redox status studied in vitro and also the internal structure, which seems to cause finally cell death in tobacco cells.     

Habituation in sugarbeet callus: auxin content, auxin protectors, peroxidase pattern and inhibitors

A GC-ECD titration of IAA in normal auxin (2,4-D)-requiring and auxin-independent (habituated) sugarbeet callus revealed an equal amount in both tissues. A comparison of the content and pattern (through starch gel electrophoresis) of soluble, membrane and wall peroxidases indicated that normal tissues contained a higher level of isoperoxidases. Normal tissues also were found to contain higher levels of peroxidase inhibitors and auxin protectors. The hypothesized peroxidase-mediated higher rate of auxin destruction in normal sugarbeet callus is supposed to be counterbalanced by the 2,4-D-controlled auxin protectors.     

Expression of Agrobacterium rhizogenes auxin biosynthesis genes in transgenic tobacco plants

Plant oncogenes aux1 and aux2 carried by the TR-DNA of Agrobacterium rhizogenes strain A4 encode two enzymes involved in the auxin biosynthesis pathway in transformed plant cells. The short divergent promoter region between the two aux-coding sequences contains the main regulatory elements. This region was fused to the uidA reporter gene and introduced into Nicotiana tabacum in order to investigate the regulation and the tissue specificity of these genes. Neither wound nor hormone induction could be detected on transgenic leaf discs. However, phytohormone concentration and auxin/cytokinin balance controlled the expression of the chimaeric genes in transgenic protoplasts. The expression was localised in apical meristems, root tip meristems, lateral root primordia, in cells derived from transgenic protoplasts and in transgenic calli. Histological analysis showed that the expression was located in cells reactivated by in vitro culture. Experiments using cell-cycle inhibitors such as hydroxyurea or aphidicolin on transgenic protoplast cultures highly decreased the -glucuronidase activity of the chimaeric genes. These results as well as the histological approach suggest a correlation between expression of the aux1 and aux2 genes and cell division.     

In vitro culture of tobacco callus on medium containing peptone and phytate leads to growth improvement and higher genetic stability

Growth and genetic stability of Nicotiana tabacum L. callus were strongly improved by replacing the inorganic nitrogen and phosphorus of the Murashige and Skoog's medium by a soybean peptone and phytate, respectively. Cell proliferation after subcultivation on the modified medium was highly stimulated as evidenced by a strong biomass increase; this improvement was mainly due to the organic N source. In addition, while calluses grown under standard conditions displayed various cell sizes and DNA contents, subcultivation on the modified medium led to homogeneous cell size distribution and stable 4C–8C DNA contents through several subcultures. This improved genetic stability was due to replacement of inorganic P by phytate, provided the presence of peptone. Such new media composition could be useful for slow-growing cell suspensions or calluses.     

In-vivo measurement of cell-wall extensibility in maize coleoptiles: Effects of auxin and abscisic acid

Plastic and elastic in-vivo extensibilities (E_pl and E_el, respectively) of cell walls of growing maize (Zea mays L.) coleoptile segments were measured by stretching living tissue at constant force (creep test) in an extensiometer. The linear displacement transducer used as a measuring device permits the determination of load-induced extensions in the range of 0–1% of the segment's length, leading to a minimal disturbance of the hydraulic parameters of the tissue and allowing the measurement of unidirectional cell-wall creep at virtually unchanged turgor and metabolic activity. A rein-vestigation of the time-course of indole-3-acetic acid-promoted and abscisic acid-inhibited wall loo-sening revealed that the in-vivo creep test yields results very similar to those obtained previously with the in-vitro creep test [Kutschera and Schopfer, 1986, Planta 167, 527–535]. The hormones affect elongation rate and E_pl in a closely correlated manner both in step-up as well as step-down growth changes whereas E_el remains unaltered. It is argued that both hormones influence growth by modifying E_pl of the outer epidermis and that this effect can be quantitatively measured, in relative units, by either the in-vivo or the in-vitro creep test.Abbreviations ABA ±abscisic acid - E_el, E_pl elastic and plastic in-vivo cell-wall extensibility, respectively - E_tot E_el+E_pl - IAA indole-3-acetic acid; m, cell-wall yielding coefficient     

A lignin-specific peroxidase in tobacco whose antisense suppression leads to vascular tissue modification

A tobacco peroxidase isoenzyme (TP60) was down-regulated in tobacco using an antisense strategy, this affording transformants with lignin reductions of up to 40-50% of wild type (control) plants. Significantly, both guaiacyl and syringyl levels decreased in essentially a linear manner with the reductions in lignin amounts, as determined by both thioacidolysis and nitrobenzene oxidative analyses. These data provisionally suggest that a feedback mechanism is operative in lignifying cells, which prevents build-up of monolignols should oxidative capacity for their subsequent metabolism be reduced. Prior to this study, the only known rate-limiting processes in the monolignol/lignin pathways involved that of Phe supply and the relative activities of cinnamate-4-hydroxylase/p-coumarate-3-hydroxylase, respectively. These transformants thus provide an additional experimental means in which to further dissect and delineate the factors involved in monolignol targeting to precise regions in the cell wall, and of subsequent lignin assembly. Interestingly, the lignin down-regulated tobacco phenotypes displayed no readily observable differences in overall growth and development profiles, although the vascular apparatus was modified.     

Quantitative differences in sperm cells and organelles of tobacco (Nicotiana tabacum L.) grown under differing environmental conditions

In flowers grown at warm temperatures in environmental chambers and at cooler temperatures in the greenhouse, eight parameters of the sperm-cell organization of Nicotiana tabacum were examined during sperm cell maturation using serial ultrathin sectioning, transmission electron microscopy and quantitative cytology. Despite employing the same seed source, and similar soil and nutrient conditions, the surface area and volume of the cell, the nucleus and the chondriome were larger in flowers grown in growth chambers under warmer controlled conditions, whereas the number of plastids appeared to be the same, or slightly higher, in flowers grown under cooler greenhouse conditions. These results suggest that environmental conditions may influence the quantity of cytoplasmic organelles, including mitochondria and plastids, thus potentially influencing the likelihood of male cytoplasmic inheritance.