Molecular details of tomato extensin and glycine-rich protein gene expression

In a recent publication (Plant Molecular Biology 16: 547–565 (1991)) Showalter et al. described the isolation and initial characterization of fifteen extensin and extensin-like tomato cDNAs. These cDNAs were determined to fall into five distinct classes; class I and II clones encoded extensins, class III and V clones encoded glycine-rich proteins (GRPs), and class IV clones encoded a portion of a GRP sequence on one DNA strand and a portion of an extensin sequence on the other DNA strand. In this publication, a more detailed analysis of the expression of these cDNA classes was performed with respect to wounding in various tomato organs, development, kinetics and systemic extent of the wound response, ethylene treatment, abscisic acid (ABA) treatment, and drought stress by using RNA gel blot hybridizations. In general, extensin gene expression was readily detected in stems and roots, but not in leaves. With both class I and II extensin cDNA probes, wound-induced accumulation of mRNA in stems was first detected between 4 and 8 h after wounding with maximal accumulation occurring after 12 h. Moreover, these extensin wound responses were detected locally at the wound site but not systemically. Expression of the class III GRP was largely limited to wounded stem tissue. Initial detection and maximal accumulation of the class III GRP mRNA was similar to the extensins mRNAs; however, this GRP wound response occurred both locally and systemically. Additionally, abscisic acid treatment and drought stress resulted in the marked accumulation of the class III GRP mRNA in tomato stems, but did not alter the expression of the other cDNA classes. In contrast, expression of the class V GRP occurred in stems and roots and to a lesser extent in leaves and decreased in response to wounding over a 24 h time period. The class V GRP wound response was further characterized by an early, transient accumulation of mRNA occurring 2–4 h after wounding in stems and by its local nature.     

A novel wound-inducible extensin gene is expressed early in newly isolated protoplasts of Nicotiana sylvestris

A cDNA clone (6PExt 1.2) encoding a novel extensin was isolated from a cDNA library made from 6 h old mesophyll protoplasts of Nicotiana sylvestris. The screening was performed with a heterologous probe from carrot. The encoded polypeptide showed features characteristic of hydroxyproline-rich glycoproteins such as Ser-(Pro)₄ repeats and a high content in Tyr and Lys residues. The presence of four Tyr-X-Tyr-Lys motifs suggests the possibility for intramolecular isodityrosine cross-links whereas three Val-Tyr-Lys motifs may participate in intermolecular cross-links. The analysis of genomic DNA gel blots using both the N. sylvestris and the carrot clones as probes showed that the 6PExt 1.2 gene belongs to a complex multigene family encoding extensin and extensin-related polypeptides in N. sylvestris as well as in related Nicotianeae including a laboratory hybrid. This was confirmed by the analysis of RNA gel blots: a set of mRNAs ranging in size from 0.3 kb to 3.5 kb was found by the carrot extensin probe. The 6PExt 1.2 probe found a 1.2 kb mRNA in protoplasts and in wounded tissues as well as a 0.9 kb mRNA which seemed to be stem-specific. The gene encoding 6PExt 1.2 was induced by wounding in protoplasts, in leaf strips and after Agrobacterium tumefaciens infection of stems.     

The regulation of enzyme activities of the nicotine pathway in tobacco

The activities of the three enzymes of the route ornithine to the N-methyl-Δ¹-pyrrolinium salt and of the eight enzymes of the route quinolinic acid to nicotinic acid (pyridine nucleotide cycle) were determined in the roots of different Nicotiana tabacum L. cultivars and for comparison in tomato ( Lysopersicon esculentum L. cv. Vollendung) roots. Enzyme activities were further followed in different plant organs, dedifferentiated tissues, root organ cultures and in the roots after decapitation of tobacco plants. The data are in accord with the concept that the two routes are predominantly regulated by the enzymes putrescine methyltransferase (EC 2.1.1.53) and quinolinic acid phosphoribosyltransferase (EC 2.4.2.19), respectively. Further regulation involves the enzymes NAD⁺-pyrophosphatase (EC 3.6.1.22) and nicotinic acid mononucleotide glycohydrolase; these findings confirm the suggestion that the transformation to nicotinic acid is performed along two routes: either via the synthesis and degradation of NAD⁺ or by a direct step through the action of a specific glycohydrolase.     

Fate of chloramphenicol in tobacco tissue culture under various light regimes

Abstract. Non-differentiated tissue cultures (calli) and differentiated tissues (shoots) of tobacco were found to differ in their sensitivity to chloramphenicol (CAP). This phenomenon is especially manifested in darkness and in an illumination regime lacking u.v. and blue light. When the latter are included, CAP's photodegradation products are shown to appear. It seems that one of the main photodegradation pathways is through the production of p -nitro-benzaldehyde ( p -NBA) which is further degraded. The possibility that either acetylation or physiological nitration of CAP is the cause for the differential tolerance was eliminated. The chromatographic and radiographic results indicate that in vivo degradation of CAP occurs both in calli and in shoot cultures. One of the in vivo degradation products is CAP-base.     

Isolation of extensin precursors by direct elution of intact tomato cell suspension cultures

Dilute salt solutions eluted peroxidase and hydroxyproline-rich glycoproteins (HRGP's) very rapidly (60 % within 10s) from the surface of intact tomato cells grown in suspension culture. Further purification of the HRGPs based on (a) their solubility in 10% trichloroacetic acid and (b) chromatography on carboxymethyl cellulose, gave two components (P1 and P2) rich in serine, tyrosine, lysine and arabinosylated hydroxyproline. The sum of the hydroxyproline arabinoside profiles of P1 and P2 approximated that of the wall. P1, unlike P2, was histidine-rich and also contained proline. Significantly, isodityrosine (IDT) was absent from P1 and P2 but present in cell wall hydrolysates where, the Hyp:IDT molar ratio was ca 15: 1. In cells 4 days after subculture, ³H-proline pulse-chase data indicated turnover of P1 and P2 presumably resulting from covalent attachment to the wall as neither P1 nor P2 appeared in the growth medium. At day four the cell mean generation time (MGT) was 4.6 days, the cell hydr oxyproline content was 0.7 % (w/w), the half lives of P1 and P2 were both ca 12 hr, and the combined CaCl₂ elutable P1 and P2 precursor pools contained ca 400 μg Hyp/g cells (dry weight). Calculated from the MGT and Hyp content, the cell demand was 44.μg Hyp/g cells (dry weight)/hr. The precursor pool size was therefore sufficient for 9 hours growth. However the pool turnover calculated from half life and pool size was 5.6 %/hr or 22.4μg Hyp/g cells (dry weight)/hr. Thus the supply of P1 and P2 precursors met > 50 % of the cell wall demand. Corroborative experiments showed that after depletion of the P1 and P2 pools by salt elution, washed cells resuspended in growth medium repleted the precursor pools at a rate corresponding to a synthesis of 43μg Hyp/g cells (dry weight)/hr, or 98 % of the demand. These data allow us to make the following suggestions: P1 and P2 represent monomeric extensin precursor subunits. Salt elution of P1 and P2 indicates their ionic binding by pectic carboxyl groups. The rapidity of elution indicates a high diffusivity of these extended rodlike macromolecules through the cell wall. This may imply a preferred orientation for P1 and P2 perpendicular rather than parallel to the plane of the wall. The lack of IDT in P1 and P2 implies that IDT forms in muro, possibly via peroxidase. We speculate that some of these IDT residues may crosslink an extensin precursor ‘tweft’ around a cellulose microfibrillar ‘twarp’. Such formation of heteromultimeric extensin interpenetrated by microfibrils would create a mechanically coupled extensin-cellulose network.     

Expression of extensin genes is dependent on the stage of the cell cycle and cell proliferation in suspension-cultured Catharanthus roseus cells

To isolate cDNAs expressed at a specific phase of the cell cycle in a higher plant, we performed differential screening of a cDNA library prepared from the S-phase cells of synchronized cultures of Catharanthus roseus. Sequence analysis shows that two of the identified cDNAs, cyc15 and cyc17, encode extensins that represent a family of cell wall hydroxyproline-rich glycoproteins. Protein sequences deduced from the two cDNAs contain the characteristic pentapeptide repeat sequence, Ser-Pro-Pro-Pro-Pro, which is commonly observed in extensins. The protein sequences also share several other extensin characteristics such as the presence of a N-terminal signal peptide and a high content of Tyr and Lys residues. When C. roseus cell suspension cultures were synchronized by phosphate starvation, the mRNAs of both cyc15 and cyc17 were transiently expressed during the S and G2 phases of the cell cycle. However, significant amounts of the mRNAs also accumulated in phosphate-starved cells arrested in the G1 phase. In asynchronous cultures, both genes were expressed during the stationary phase, when cell proliferation ceased. The observed patterns of expression suggest that the extensin genes, cyc15 and cyc17, are under two types of regulation: one that depends on the stage of the cell cycle and another that is induced during the growth arrest. Thus, the products of these genes may function both during the progression through the cell cycle and in the strengthening of the cell wall after cell division.     

Immunolocalization of extensin and potato tuber lectin in carrot, tomato and potato

Tissue-specific expression of two members of the cell wall hydroxyproline-rich glycoprotein (HRGP) family, extensin and potato tuber lectin, was examined by immunolocalization at the light microscope level in various organs (leaves, stems, roots, fruit, tuber) of carrot ( Daucus carota cv. Thumbelina), tomato ( Lycopersicon esclentum cv. Pixie Hybrid II), and potato ( Solanum tuberosum cv. Kennebec). Extensin was prominently expressed in vascular tissue, particularly xylem and also phloem, although virtually all cells displayed some degree of staining which varied as a function of the tissue, organ, and plant under study. Antibodies against potato tuber lectin (PTL) displayed a localization pattern similar to that observed for extensin; notably PTL did not stain cambium but did stain epithelial cells lining secretory cavities. These distribution patterns are consistent with a role for extensin, and possibly PTL, in providing mechanical support in tissues subjected to compression or torsional stress imparted by vascular growth, or by similar stress brought about by transport of vascular fluids.     

The overexpression of an alternative oxidase gene triggers ozone sensitivity in tobacco plants

The alternative oxidase (AOX) of plant mitochondria transfers electrons from the ubiquinione pool to oxygen without energy conservation and prevents the formation of reactive oxygen species (ROS) when the ubiquinone pool is over-reduced. Thus, AOX may be involved in plant acclimation to a number of oxidative stresses. To test this hypothesis, we exposed wild-type (WT) Xanthi tobacco plants as well as Xanthi plants transformed with the Bright Yellow tobacco AOX1a cDNA with enhanced (SN21 and SN29), and decreased (SN10) AOX capacity to an acute ozone (O3) fumigation. As a result of 5 h of O3 exposition (250 nL L(-1)), SN21 and SN29 plants surprisingly showed localized leaf damage, whereas SN10, similarly to WT plants, was undamaged. In keeping with this observation, WT and SN21 plants differed in their response to O3)for the expression profiles of catalase 1 (CAT1), catalase 2 (CAT2), glutathione peroxidase (GPX) and ascorbate peroxidase (APX) genes, and for the activity of these antioxidant enzymes, which were induced in WT. Concomitantly, although ozone induced H2O2 accumulation in WT and in all transgenic lines, only in transgenics with high AOX capacity the H2O2 level in the post-fumigation period was high. The alternative pathway of WT plants was strongly stimulated by O3, whereas in SN21 plants, the respiratory capacity was always high across the treatment. The present results show that, far from exerting a protective role, the overexpression of AOX triggers an increased O3 sensitivity in tobacco plants. We hypothesize that the AOX overexpression results in a decrease of mitochondrial ROS level that in turn alters the defensive mitochondrial to nucleus signalling pathway that activates ROS scavenging systems.     

Accumulation of aluminium in the cell wall pectin in cultured tobacco (Nicotiana tabacum L.) cells treated with a combination of aluminium and iron

In nutrient medium, aluminium (Al) accumulation in tobacco cells occurs only in the presence of ferrous ion [Fe(II)]. The localization of Al was examined to elucidate a mechanism of Al accumulation. After the digestion of Al-treated cells with cellulase and pectolyase together, the resulting spheroplasts contained as much Al as the intact cells. However, the cell walls isolated from Al-treated cells also contained as much Al as the intact cells. Comparison of sugar and Al contents in polysaccharide components extracted chemically from cell walls isolated from intact cells and spheroplasts revealed that the enzymes digested most of the cellulose and hemicellulose, but only half of the pectin, and that Al mainly existed in the pectin remaining in the spheroplasts. Gel-permeation chromatography of the pectin fraction (NH₄-oxalate extract) from the cell walls of the intact cells indicated that Al was associated with small polysaccharides of approximately 3–7 kDa. These results suggest that a minor part of pectin is a major site of Al accumulation. The content of cell wall pectin increased during Al treatment in nutrient medium. Taken together, we hypothesize that Al may bind to the pectin newly produced during Al treatment.     

Culture-induced changes in osmolality of tobacco cell suspensions using four exogenous sugars

Suspension cultures of tobacco cells were grown in B5 media supplemented with sucrose, glucose, mannitol and sorbitol as exogenous sugars to examine culture-induced changes in the osmolality of the medium. Osmolality decreases were greatest in sucrose and glucose media during the 14 days in culture, and in glucose media were essentially linear, presumably reflecting the use of this sugar as a food source. Osmolality decreases occurred during the first week of culture in mannitol- and sorbitol-supplemented media, but later stabilized. Fresh weight of cultured cells in sucrose- and glucose-supplemented media increased by <200% during 14 days in culture, whereas cultured cells in mannitol- and sorbitol-supplemented media increased by only 39 and 48%, respectively. Cells transferred to the original liquid medium (B5 medium with 3% sucrose and 3% glucose) grew vigorously if they had been cultured in sucrose- and glucose-supplemented media; however, cells grown in mannitol- and sorbitol-supplemented media needed to be subcultured several times to recover their normal growth rate. By subculturing cells into increasingly higher conditions of sugars, cells tolerant to 560 mOsmol kg-1 H2O were obtained. The high osmolality-adapted cells increased by 140% in fresh weight in 8% glucose-supplemented medium. Glucose was best suited for producing the high osmolality required because sucrose concentrations at 10% sucrose and above resulted in cell death. To limit the decrease of osmolality in these suspension cultures requires changing the medium every 3 days to maintain osmolality above the 530 mOsmol kg-1 H2O needed to co-culture these as feeder cells with gametic and zygotic cells. This revised version was published online in June 2006 with corrections to the Cover Date.     

A kinetic analysis of cadmium accumulation in a Cd hyper‐accumulator fern,Athyrium yokoscense and tobacco plants

Cadmium (Cd) accumulations in a Cd hyper‐accumulator fern, Athyrium yokoscense (Ay), and tobacco, Nicotiana tabacum (Nt), were kinetically analysed using the positron‐emitting tracer imaging system under two medium conditions (basal and no‐nutrient). In Ay, maximumly 50% and 15% of the total Cd accumulated in the distal roots and the shoots under the basal condition, respectively. Interestingly, a portion of the Cd in the distal roots returned to the medium. In comparison with Ay, a little fewer Cd accumulations in the distal roots and clearly higher Cd migration to the shoots were observed in Nt under the basal condition (maximumly 40% and 70% of the total Cd, respectively). The no‐nutrient condition down‐regulated the Cd migration in both species, although the regulation was highly stricter in Ay than in Nt (almost no migration in Ay and around 20% migration in Nt). In addition, the present work enabled to estimate physical and physiological Cd accumulation capacities in the distal roots, and demonstrated condition‐dependent changes especially in Ay. These results clearly suggested occurrences of species‐/condition‐specific regulations in each observed parts. It is probable that integration of these properties govern the specific Cd tolerance/accumulation in Ay and Nt.     

SVISS - a novel transient gene silencing system for gene function discovery and validation in tobacco plants

We developed a novel, two-component transient gene silencing system in which the satellite tobacco mosaic virus (STMV) is used as vector for the delivery of inhibitory RNA into tobacco plants and the tobacco mosaic virus strain U2 (TMV-U2) is used as helper virus for supplying replication and movement proteins in trans. The main advantage of the system is that by uncoupling virus replication components from silencing induction components, the intensity of silencing becomes more pronounced. We call this system satellite virus-induced silencing system (SVISS) and will demonstrate here its robustness, speed and effectiveness. We were able to obtain pronounced and severe knockout phenotypes for a range of targeted endogenous genes belonging to various biochemical pathways and expressed in different plant tissues, such as genes involved in leaf and flower pigmentation, genes for cell wall synthesis in leaf, stem and root tissues or a ubiquitous RNA polymerase gene. By tandem insertion of more than one target gene sequence into the vector, we were able to induce simultaneous knockouts of an endogenous gene and a transgene. SVISS is the first transient gene silencing system for Nicotiana tabacum, which is a genetically well-characterized bridging species for the Solanaceae plant family.     

Stability of salt tolerance at the cell level after regeneration of plants from a salt tolerant tobacco cell line

Plants were regenerated from both the wild type and a stable NaCI-tolerant line of tobacco cells ( Nicotiana tabacum/gossii ). The regeneration process was much more difficult in the case of the NaCI-tolerant line and was only successful in the absence of NaCI. These plants differed morphologically from those regenerated from the wild type cell line, exhibiting abnormally short internodes, small leaves and reduced growth. Cell suspension cultures derived from plants regenerated from the stable NaCI-tolerant line retained a high level of tolerance to salt. The NaCI-concentration required to reduce fresh and dry weight gain by 50% was about twice that observed in the case of the cells obtained from wild type plants.
The results presented here, together with those of Watad et al. (1985), indicate that resistance to salt is operating and stable at the cellular level before and after plant regeneration. When the regenerated plants were grown in increasing levels of salt their growth response was not clearly different from that of the plants regenerated from the wild type cell line. However, the survival of plants on high concentrations of NaCI tended to be higher in the case of plants regenerated from the NaCI-tolerant cell line.     

Short-term aluminium uptake by tobacco cells: Growth dependence and evidence for internalization in a discrete peripheral region

Short-term uptake and initial localization of aluminium (Al) were investigated in cultured cells of Nicotiana tabacum L. cv. BY-2. Graphite furnace atomic absorption spectrometry and an in vivo Al-sensitive fluorometric assay, employing morin, yielded similar results in all experiments. Aluminium uptake was critically dependent on cell growth. As opposed to negligible uptake in stationary-phase cells, Al uptake (20 μ M AlCl₃, pH 4.5, 23°C) by actively growing cells was detectable within 5 min, with an initial rate of 16 nmol Al (10⁶ cells)⁻¹ h⁻¹. Increased CaCl₂ levels (up to 20 m M ), low temperature (4°C), and pre-chelation of Al to citrate greatly reduced Al uptake (by 75–90%). A pH-associated permeabilization of cells at pH 4.5, as monitored by trypan blue, was observed in some growing cells. Although permeability to trypan blue was not a requirement for Al uptake, enhanced membrane permeability at pH 4.5, relative to pH 5.6, may contribute to Al uptake. Aluminium was observed to localize mainly in a pronounced and discrete fluorescent zone at the cell periphery (2–30 μm wide), presumably in the cortical cytosol and/or the adjoining plasma membrane section, although the possibility cannot be excluded that some Al resided in the cell wall apposing this discrete region. However, as judged by the Al-morin assay, there were no detectable Al levels in the remaining, larger portion of the cell wall. The potential of the Al-morin method in Al toxicity studies is illustrated.     

Evidence for the activation of a MAP kinase upon phosphate‐induced cell cycle re‐entry in tobacco cells

Mitogen‐activated‐protein (MAP) kinases are components of signal transduction pathways which respond to a variety of stimuli in different organisms. In quiescent mammalian cells, the reactivation of cell division induced by different mitogenic signals is mediated by the rapid phosphorylation and activation of MAP kinases. We have investigated whether a similar situation occurs in plants, arresting tobacco ( Nicotiana tabacum L.) cells in the G₁ phase of the cell cycle by phosphate starvation, and then inducing them to re‐enter the cell cycle by refeeding with phosphate. The transient activation of a kinase activity with the characteristics of a MAP kinase was observed during the first hour after refeeding, when the cells were still in G₁. Using myelin basic protein (MBP) as substrate, an increase in this phosphorylating activity, with a molecular mass of approximately 45 kDa, was detected in cell extracts between 35 and 55 min after induction, in in‐gel phosphorylation assays and after immunoprecipitation with anti‐MAP kinase antibodies. The specificity of the antibodies against recombinant tobacco MAP kinases suggested that the MAP kinase p45^ntf4 was responsible for the observed activity. These data provide experimental evidence for the activation in vivo of a plant MAP kinase, possibly mediating the reactivation of cell division in G₁‐arrested cells.     

A two-component gene (NTHK1) encoding a putative ethylene-receptor homolog is both developmentally and stress regulated in tobacco

The full-length of a two-component gene NTHK1 (Nicotiana tabacum histidine kinase-l) was isolated from tobacco (N. tabacum var. Xanthi) using a previously obtained NTHK1 cDNA fragment as a probe. Sequence analysis revealed that NTHK1 shared high homology with LeETR4 from tomato and encoded an ethylene- receptor homolog. The predicted NTHK1 protein had a putative signal peptide, three transmembrane domains, a histidine kinase domain and a receiver domain. The putative autophosphorylation site at His378 and the phosphate receiver site at Asp689 were also identified. By using the in situ hybridization technique, NTHK1 mRNA was detected during flower organ development. It is also highly expressed in the processes of pollen formation and embryo development. The expression of NTHK1 in response to wounding and other stresses was investigated using competitive RT-PCR. The results demonstrated that NTHK1 was inducible upon wounding (cutting). Floating of the cut leaf pieces in 0.5× MS, with shaking, led to a relatively rapid and strong expression. This phenomenon was confirmed by the in situ hybridization results. In addition to the up-regulation by wounding, NTHK1 expression was also induced following NaCl and PEG treatment, indicating a possible role for NTHK1 in multiple stress responses. Received: 28 June 2000 / Accepted: 1 August 2000     

The cell wall and secretory proteome of a tobacco cell line synthesising secondary wall

The utility of plant secondary cell wall biomass for industrial and biofuel purposes depends upon improving cellulose amount, availability and extractability. The possibility of engineering such biomass requires much more knowledge of the genes and proteins involved in the synthesis, modification and assembly of cellulose, lignin and xylans. Proteomic data are essential to aid gene annotation and understanding of polymer biosynthesis. Comparative proteomes were determined for secondary walls of stem xylem and transgenic xylogenic cells of tobacco and detected peroxidase, cellulase, chitinase, pectinesterase and a number of defence/cell death related proteins, but not marker proteins of primary walls such as xyloglucan endotransglycosidase and expansins. Only the corresponding detergent soluble proteome of secretory microsomes from the xylogenic cultured cells, subjected to ion‐exchange chromatography, could be determined accurately since, xylem‐specific membrane yields were of poor quality from stem tissue. Among the 109 proteins analysed, many of the protein markers of the ER such as BiP, HSP70, calreticulin and calnexin were identified, together with some of the biosynthetic enzymes and associated polypeptides involved in polymer synthesis. However 53% of these endomembrane proteins failed identification despite the use of two different MS methods, leaving considerable possibilities for future identification of novel proteins involved in secondary wall polymer synthesis once full genomic data are available.