Contiguous hydroxyproline residues direct hydroxyproline arabinosylation in Nicotiana tabacum

Hydroxyproline (Hyp) O-glycosylation characterizes the hydroxyproline-rich glycoprotein (HRGP) superfamily of the plant extracellular matrix. Hyp glycosylation occurs in two modes: Arabinosylation adds short oligoarabinosides (Hyp-arabinosides) while galactosylation leads to the addition of larger arabinogalactan polysaccharides (Hyp-polysaccharides). We hypothesize that sequence-dependent glycosylation of small peptide motifs results in glycomodules. These small functional units in combination with other repetitive peptide modules define the properties of HRGPs. The Hyp contiguity hypothesis predicts arabinosylation of contiguous Hyp residues and galactosylation of clustered noncontiguous Hyp residues. To determine the minimum level of Hyp contiguity that directs arabinosylation, we designed a series of synthetic genes encoding repetitive (Ser-Pro(2))(n), (Ser-Pro(3))(n), and (Ser-Pro(4))(n). A signal sequence targeted these endogenous substrates to the endoplasmic reticulum/Golgi for post-translational proline hydroxylation and glycosylation in transformed Nicotiana tabacum cells. The fusion glycoproteins also contained green fluorescence protein, facilitating their detection and isolation. The (Ser-Pro(2))(n) and (Ser-Hyp(4))(n) fusion glycoproteins yielded Hyp-arabinosides but no Hyp-polysaccharide. The motif (Ser-Pro(3))(n) was incompletely hydroxylated, yielding mixed contiguous/noncontiguous Hyp and a corresponding mixture of Hyp-arabinosides and Hyp-polysaccharides. These results plus circular dichroic spectra of the glycosylated and deglycosylated (Ser-Pro(2))(n), (Ser-Pro(3))(n), and (Ser-Pro(4))(n) modules corroborate the Hyp contiguity hypothesis and indicate that Hyp O-glycosylation is indeed sequence-driven.     

Expression of a Self-Incompatibility Glycoprotein (S2-Ribonuclease) from Nicotiana alata in Transgenic Nicotiana tabacum

In Nicotiana alata, self-incompatibility is controlled by a single locus, designated the S-locus, with multiple alleles. Stylar products of these alleles are ribonucleases that are secreted mainly in the transmitting tract tissues. N. tabacum plants were transformed with constructs containing the S2-cDNA and genomic S2-sequences from N. alata that were linked to the cauliflower mosaic virus 35S promoter. Unlike other genes controlled by this promoter, the genes were expressed most highly in mature floral organs. This pattern of expression was observed at both the protein and RNA levels. The S2-glycoprotein was detected in the stylar transmitting tract tissues of the transgenic plants. The transgene product was secreted, had ribonuclease activity, and was glycosylated with the correct number of glycan chains. However, the maximum level of S2-glycoprotein in styles of the transgenic plants was approximately 100-fold lower than that found in N. alata styles carrying the S2-allele. Perhaps because of this lower protein level, the plants showed no changes in the incompatibility phenotype.     

Synthesis and self-assembly of a functional monoclonal antibody in transgenic Nicotiana tabacum

Immunoglobulin light and heavy chains are synthesized in mammalian cells as precursors containing a signal peptide. Processing and assembling result in formation of active antibodies. Chimeric genes have been made containing the coding sequence of the barley -amylase signal peptide which has been fused to cDNAs coding for either the mature light or the mature heavy chain of a monoclonal antibody. A plasmid was constructed linking both chimeric genes under the control of plant active promoters in an expression cassette. This DNA fragment was stably integrated into the genome of Nicotiana tabacum by Agrobacterium tumefaciens mediated gene transfer. Synthesis of light and heavy chains and assembly to antibodies was detected in transgenic tobacco tissue using specific secondary antibodies. By electron microscopic immunogold labeling, the presence of assembled antibody could be detected within the endoplasmic reticulum. Affinity chromatography indicated biological activity of the assembled immunoglobulin produced in plant cells. Unexpectedly, a significant amount of assembled antibodies was found within chloroplasts.     

Pollen Heteromorphism in Nicotiana tabacum (Solanaceae)

Pollen heteromorphism is defined as the production by a single plant of different fertile pollen types in all its anthers, and thus all flowers, throughout its life cycle. Eight cultivars of Nicotiana tabacum, as well as its ancestors (N. tabacum is an amphiploid hybrid 4 × from a cross between N. sylvestris and N. tomentosiformis) and recent hybrids were analyzed. Most cultivars and the hybrids are heteromorphic (producing 3- and 4-aperturate pollen grains), whereas both parent species are homomorphic (3-aperturate). Heteromorphism is a common consequence of polyploidization and these results agree with this interpretation. There is a significant variation in the proportions of the two pollen types among cultivars (genetic component), but also (with a much lower component of variance) within each cultivar, between plants (genets), flowers of a plant, and even anthers of a flower. This is interpreted as a release of the selective pressure: the cultivars of N. tabacum were obtained after several generations of selfing and are themselves selfers. Selfing, by removing pollen mixtures on a stigma, removes pollen competition, which is the drive for heteromorphism, and allows for a large variation of the proportions of the different pollen types.     

Large-scale phenotyping of transgenic tobacco plants (Nicotiana tabacum) to identify essential leaf functions

Two of the major challenges in functional genomics are to identify genes that play a key role in biological processes, and to elucidate the biological role of the large numbers of genes whose function is poorly characterized or still completely unknown. In this study, a combination of large-scale expressed sequence tag sequencing, high-throughput gene silencing and visual phenotyping was used to identify genes in which partial inhibition of expression leads to marked phenotypic changes, mostly on leaves. Three normalized tobacco (Nicotiana tabacum) cDNA libraries were prepared directly in a binary vector using different tissues of tobacco as an RNA source, randomly sequenced and clustered. The Agrobacterium-tobacco leaf disc transformation system was used to generate sets of antisense or co-suppression transgenic tobacco plants for over 20 000 randomly chosen clones, each representing an independent cluster. After transfer to the glasshouse, transgenic plants were scored visually after 10-14 days for changes in growth, leaf form and chlorosis or necrosis. Putative hits were validated by repeating the transformation. This procedure is more stringent than the analysis of knockout mutants, because it requires that even a partial decrease in expression generates a phenotype. This procedure identified 88 validated gene/phenotype relations. These included several previously characterized gene/phenotype relationships, demonstrating the validity of the approach. For about one-third, a function could be inferred, but a loss-of-function phenotype had not been described previously. Strikingly, almost one-half of the validated genes were poorly annotated, or had no known function. For 77 of these tobacco sequences, a single or small number of potential orthologues were identified in Arabidopsis. The genes for which orthologues were identified in Arabidopsis included about one-half of the genes whose function was completely unknown. Comparison with published gene/phenotype relations for Arabidopsis knockout mutants revealed surprisingly little overlap with the present study. Our results indicate that partial gene silencing identifies novel gene/phenotype relationships, which are distinct from those uncovered by knockout screens. They also show that it is possible to perform these analyses in a crop species in which full genome sequence information is lacking, and subsequently to transfer the information to a reference species in which functional studies can be performed more effectively.     

An S-RNase promoter from Nicotiana alata functions in transgenic N. alata plants but not Nicotiana tabacum

Nicotiana tabacum and Nicotiana alata plants were transformed with genomic clones of two S-RNase alleles from N. alata. Neither the S ₂ clone, with 1.6 kb of 5 sequence, nor the S ₆ clone, with 2.8 kb of 5 sequence, were expressed at detectable levels in transgenic N. tabacum plants. In N. alata, expression of the S ₂ clone was not detected, however the S ₆ clone was expressed (at low levels) in three out of four transgenic plants. An S ₆-promoter-GUS fusion gene was also expressed in transgenic N. alata but not N. tabacum. Although endogenous S-RNase genes are expressed exclusively in floral pistils, the GUS fusion was expressed in both styles and leaves.     

Molecular characterization of profilin isoforms from tobacco (Nicotiana tabacum) pollen

Profilins are actin-binding proteins in eukaryotes which participate in the phosphoinositide pathway via binding to PIP₂. Using polyclonal rabbit sera raised against plant profilins, the occurrence of several profilin isoforms is demonstrated in two-dimensionally analyzed tobacco pollen extracts. The cDNAs coding for two novel tobacco profilin isoforms (ntPro2, ntPro3) were isolated from a pollen cDNA library by antibody screening. When the cDNA and deduced amino acid sequences of the two isoforms were compared with a previously isolated tobacco pollen profilin cl)NA (ntPro1), significant differences were noted in the non-coding regions, whereas the coding sequences, in particular the functional domains, showed little variation. The cDNAs coding for the three tobacco profilin isoforms were expressed inEscherichia coli and shown to bind comparably to different anti-profilin antisera. The high degree of similarity among the different tobacco pollen profilin isoforms points to functional equivalence. Assuming that the presence of profilin is indispensable to the control of the large amounts of actin present in pollen, the occurrence of different profilin isoforms in pollen is interpreted to represent a protective mechanism against loss of profilin functions.     

Cell-wall-bound oxidases from tobacco (Nicotiana tabacum) xylem participate in lignin formation

A band of cells closest to the cambium in the xylem of tobacco (Nicotiana tabacum L. cv. Samsun) stems oxidized 2,2-azinobis-(3-ethylbenzo-thiazoline-6-sulphonate) (ABTS), o-dianisidine and syringaldazine in the absence of exogenously added hydrogen peroxide. The oxidation was not prevented by catalase which suggests that the oxidation is not dependent on the production and utilisation of endogenous hydrogen peroxide by cell-wall peroxidases. Cell walls, isolated from tobacco xylem, also oxidized these substrates in the absence of added hydrogen peroxide. The cell walls consumed molecular oxygen whilst oxidizing a range of compounds including coniferyl alcohol. The substrate preference and sensitivity to inhibitors suggest the presence of laccasetype polyphenol oxidases (p-diphenol:O₂ oxidoreductase EC 1.14.18.1) which are covalently bound to the wall. The oxidation of coniferyl alcohol by the xylem cell walls was confirmed by assays based on the disappearance of coniferyl alcohol and was not affected by the presence of 500 units·mi^-1 catalase or Superoxide dismutase. Prolonged incubation of cell walls with coniferyl alcohol led to the production of a yellow-orange water-insoluble material that precipitated with the cell walls. Although a proportion of this material was soluble in methanol, the majority was tightly associated with the cell walls. These coloured cell walls had elevated lignin contents when assayed by the acetyl-bromide method. Fourier transforminfrared spectroscopic analysis of the coloured cell walls indicated that the increased lignin content is due to the deposition of guaiacyl-type lignin. Digestion of the xylem cell walls with Driselase, a mixture of fungal glycases, produced a wall residue that had a dramatically reduced ability to oxidize ABTS in the absence of added H₂O₂. However, oxidase activity could not be detected in the Driselase-solubilized extract, although small amounts of oxidase activity could be recovered from the Driselaseresistant wall residue by extraction in 3 M CaCl₂.Abbreviations ABTS 2,2-azinobis-(3-ethylbenzo-thiazoline-6-sulphonate) - dl-DOPA 3-(3,4-dihydroxyphenyl)-alanine - FTIR Fourier transform infra-red - o-D o-dianisidine - o-pD o-phenylenediamine - SYR syringaldazine The authors acknowledge funding from the Scottish Office Agriculture and Food Department. They would like to thank Professor J.R. Hillman for his support, Dr. G.D. Lyon for his help and advice with the oxygen electrode and Mrs F. Carr for lignin determinations.     

Transmission of paternal chloroplasts in tobacco (Nicotiana tabacum)

Medgyesy et al. (1986, Mol. Gen. Genet. 204, 195–198) have described in Nicotiana plumbaginifolia and in an interspecific cross involving N. plumbaginifolia and N. tabacum a procedure for selecting cell lines derived from seedlings carrying paternal chloroplasts by taking advantage of a plastid-encoded mutation which confers resistance to streptomycin. We have extended their demonstration of occasional transmission of chloroplasts through pollen to the case of an intraspecific cross in N. tabacum. The line used as maternal parent, ITB19(sua), displayed a cytoplasmic male sterility due to the presence of a cytoplasm originating from N. suaveolens. The line used as paternal parent, SR1, was fertile and possessed mutant chloroplasts conferring resistance to streptomycin. From cell lines derived from 204 seedlings, three were regenerated into streptomycin-resistant buds. The plants derived from these three clones were male-sterile. Their progeny, after crossing with a wild type tobacco line, XHFD8, was resistant to streptomycin. Tests of resistance of the seedlings to tentoxin and restriction analyses of the chloroplast DNA indicated that two clones still had the maternal chloroplasts and were thus probably new streptomycin-resistant mutants, whereas the third one had acquired the chloroplasts of the paternal parent, but had retained the mitochondria of the maternal parent.Abbreviations cp-DNA chloroplast DNA - mt-DNA mitochondrial DNA - Np Nicotiana plumbaginifolia - Nt Nicotiana tabacum     

Mismatch-specific DNA breakdown in nuclear extract from tobacco (Nicotiana tabacum) callus

Mismatch-specific enzymatic activity was sought for in nuclei from normal and transformed plant cells originating from tobacco (Nicotiana tabacum) callus and crown gall tumor induced by Agrobacterium tumefaciens. The specific enzymatic activity was assayed with substrates derived from synthetic oligonucleotides (19-mer sequences corresponding to the human K-ras gene). Single-base changes in the middle of the sequence were the basis for creating heteroduplexes with all eight mismatches. Homo- and heteroduplexes were ligated in a size ladder and used as substrates. We detected mismatch-specific DNA breakdown and determined basic requirements for the reations. Kinetic analysis indicates the following reactivity order of preference: C : A=C : C=C : T>G : TA : AG : AG : GT : T>>G : C. It can be said now that specific mismatch recognition and repair activities have been detected in all kingdoms of living species.     

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.     

Ethylene Production by Tobacco (Nicotiana tabacum) Callus

Tobacco callus cultures grown on defined agar-solidified media produced ethylene in differing amounts, which were related to cultural treatment and age of the callus. There was a close correlation between the rate of ethylene production and growth. In darkness, maximal rates occurred in the third week of growth with ethylene production in the range of 750 nl (callus piece)^?1 d^?1 (fr. wt. = 1.5 g), and in the light, maximal rates occurred in the first week of growth, 200 nl (callus piece)^?1 d^?1 (fr. wt. = 200 mg). Growth was also correlated with ethylene production when the latter was altered by exposure of the callus to inhibitors of ethylene synthesis, L-canaline, benzyl isothiocyanate, and 3,5-diiodo-4-hydroxy-benzoic acid. No correlation was found following treatment with AgNO₃, a presumptive inhibitor of ethylene action. The inhibition of growth and ethylene production by L-canaline was partially reversed by gassing the cultures with ethylene (1 μl/1). A mercuric perchlorate sink had no significant effect on growth. A possible relationship between ethylene evolution and growth is discussed.