Tobacco Seeds Research Articles

In vitro mutated phytohemagglutinin genes expressed in tobacco seeds: role of glycans in protein targeting and stability.

Phytohemagglutinin is a glycoprotein that accumulates in the protein storage vacuoles of bean seeds. The mature glycoprotein has a high-mannose and a complex glycan. We describe here the use of site-directed mutagenesis and expression of the mutated genes in transgenic tobacco to study the role of glycans in intracellular targeting. The reading frame for phytohemagglutinin-L was mutated so that either one or both of the glycosylation signals were disrupted to specifically prevent the attachment of asparagine-linked glycans. Expression of these genes with the beta-phaseolin promoter in the seeds of transgenic tobacco plants showed that phytohemagglutinin-L with only one glycan or without glycans was correctly targeted to the protein storage vacuoles of the seeds. Furthermore, the absence of either the complex glycan or the high-mannose glycan did not alter the processing of the other glycan. On the basis of these results, we propose that the targeting signal of this vacuolar protein is contained in its polypeptide domain and not in its glycans.

Vacuole accumulation of storage protein and lectin expressed in transgenic tobacco seeds

The expression of proteins in transgenic plants offers an elegant means to examine targeting signals used for transport to intracellular sites of accumulation. We have used electron microscopic immunogold procedures to localize several different storage proteins and lectins expressed in transgenic tobacco seeds. The objective of these studies is to characterize targeting signals which permit translocation and accumulation in protein storage vacuoles (protein bodies). Vacuolar proteins such as phaseolin and phytohemagglutinin (PHA) are correctly transported to the protein storage vacuoles of transgenic tobacco seeds. Site-directed mutagenesis was used to change Asn-linked glycosylation sites of PHA. Minus glycan PHA was accumulated in the protein storage vacuoles indicating that glycans do not confer targeting information. Zein the non-vacuolar storage protein of maize accumulates in the protein storage vacuoles indicating that deposition occurs in some proteins which may lack vacuolar targeting signals.

Biological and Photometric Measurement of Light Transmission Through Soils of Various Colors

We measured light penetration photometrically and biologically through black, brown, brick-red, tan, and gray-white soils. Less than 0.005% of the incident light penetrated 4 mm of the dark-colored or 10 mm of the gray-white soils. Light-requiring (LR) and light-indifferent (LI) tobacco seeds were used for biological measurement of light through 2, 4, 6, 8, and 10 mm of each of the five soils. The LR seeds were used to measure light-penetration effects on germination, and LI seeds were used as controls to determine whether the seed had sufficient reserves for seedlings to emerge from the various depths. The germination studies were conducted in a controlled environment with daily 12-h light periods from cool-white fluorescent lamps at 20 C. About 99% of the LR seeds germinated in light and 0% when covered with 4 mm or more of the dark-colored soils. Germination for the LI seeds was ca. 98% in light or when covered with 4 mm of any color of soil.

Correct glycosylation, Golgi-processing, and targeting to protein bodies of the vacuolar protein phytohemagglutinin in transgenic tobacco

We used a heterologous system (transgenic Nicotiana tabacum L.) to investigate the processing, assembly and targeting of phytohemagglutinin (PHA), the lectin of the common bean, Phaseolus vulgaris L. In the bean, this glycoprotein accumulates in the protein bodies of the storage parenchyma cells in the cotyledons, and each polypeptide has a high-mannose glycan attached to Asn12 and a complex glycan on Asn60. The gene for PHA-L, dlec2, with 1200 basepairs (bp) 5' upstream and 1600 bp 3' downstream from the coding sequence was introduced into tobacco using Agrobacterium-mediated transformation (T. Voelker et al., 1987, EMBO J. 6, 3571-3577). Examination of thin sections of tobacco seeds by immunocytochemistry with antibodies against PHA showed that PHA-L accumulated in the amorphous matrix of the protein bodies in the embryo and endosperm. This localization was confirmed using a non-aqueous method to isolate the protein bodies from mature tobacco seeds. The biochemical analysis of tobacco PHA indicated that the signal peptide had been correctly removed, and that the polypeptides formed 6.4 S oligomers; tobacco PHA had a high-mannose glycan at Asn12 and a complex glycan at Asn60. The presence of the complex glycan shows that transport to the protein bodies was mediated by the Golgi complex. At seed maturity, a substantial portion of the PHA-L remained associated with the endoplasmic reticulum and the Golgi complex, as indicated by fractionation experiments using aqueous media and the presence of two high-mannose glycans on some of the polypeptides. Taken together, these data show that insertion of the nascent PHA into the endoplasmic reticulum, signal peptide processing, glycosylation, assembly into oligomers, glycan modification in the Golgi, and targeting of the protein occur faithfully in this heterologous system, although transport may not be as efficient as in bean cotyledons.

Endosperm-specific activity of a zein gene promoter in transgenic tobacco plants

Transgenic tobacco plants containing maize gene (Z4) encoding a 23-kd zein protein, which is normally synthesized in the endosperm of maize seeds, were obtained using a modified Ti plasmid vector. Although a polyadenylated transcript homologous to the Z4 gene was present in the seeds of some of these transgenic plants, zein protein could not be detected in any of the plants tested (35 total). To simplify the analysis of the tissue specificity of the Z4 promoter (Z4(pro)) in different organs of transformed tobacco plants, additional transgenic plants containing the chimeric genes Z4(pro)-CAT and Z4(pro)-GUS were produced. Very weak seed-specific CAT activity was observed in one out of ten Z4(pro)-CAT-transformed plants. When the more sensitive GUS assay system was used to evaluate Z4(pro) activity in tobacco, it could be shown in all 11 transgenic plants obtained that GUS activity was restricted to the endosperm tissue of transgenic tobacco seeds. To study the synthesis and stability of the zein proteins in different organs of transgenic tobacco plants, the Z4 protein-coding region and also a cDNA clone (A30) encoding a 19-kd zein protein were placed under the control of the 35S promoter (35S(pro)) of cauliflower mosaic virus. Undegraded zein proteins of both size classes were detected in roots, leaves and endosperm tissue, but not embryos, of mature seeds from 35S(pro)-zein-transformed plants. The zein proteins did not appear to be broken down during tobacco seed germination; synthesis of zeins began in green cotyledons.

Cellular localization of soybean storage protein mRNA in transformed tobacco seeds

We transformed tobacco plants with a soybean beta-conglycinin gene that encodes the 1.7-kilobase beta-subunit mRNA. We showed that the beta-conglycinin mRNA accumulates and decays during tobacco seed development and that beta-conglycinin mRNA is undetectable in the tobacco leaf. We utilized in situ hybridization to localize beta-conglycinin mRNA within the tobacco seed. beta-Conglycinin mRNA is not detectable within the endosperm but is localized within specific embryonic cell types. The highest concentration of beta-conglycinin mRNA is found in cotyledon storage parenchyma cells. We conclude that sequences required for embryo expression, temporal control, and cell specificity are linked to the beta-conglycinin gene, and that factors regulating beta-conglycinin gene expression are compartmentalized within analogous soybean and tobacco seed regions.

The sequence of a pea vicilin gene and its expression in transgenic tobacco plants

A 5.5 kb Eco RI fragment containing a vicilin gene was selected from a Pisum sativum genomic library, and the protein-coding region and adjacent 5' and 3' regions were sequenced. A DNA construction comprising this 5.5 kb fragment together with a gene for neomycin phosphotransferase II was stably introduced into tobacco using an Agrobacterium tumefaciens binary vector, and the fidelity of expression of the pea vicilin gene in its new host was studied. The seeds of eight transgenic tobacco plants showed a sixteen-fold range in the level of accumulated pea vicilin. The level of accumulation of vicilin protein and mRNA correlated with the number of integrated copies of the vicilin gene. Pea vicilin was confined to the seeds of transgenic tobacco. Using immunogold labelling, vicilin was detected in protein bodies of eight out of ten embryos (axes plus cotyledons) and, at a much lower level, in two out of eleven endosperms. Pea vicilin was synthesized early in tobacco seed development; some molecules were cleaved as is the case in pea seeds, yielding a major parental component of M r∼50000 together with a range of smaller polypeptides.

Tobacco Budworm Control in Tobacco Seed Nurseries, 1987

Abstract Several insecticides were tested for control of tobacco budworm larvae in flue-cured tobacco seed nurseries. Tobacco was transplanted on 1 Jun into a field of Helena sandy loam that had been treated on 6 May witli isopropalin (Paarlan, 1.68 kg[AI]/ha) and metalaxyl (Ridomil 2E, 0.56 kg[AI]/ha) for weed and disease control. Diphenamid (Enide 90W, 4.48 kg[AI]/ha) was applied on 26 Jun to extend weed control. Plants were placed on 56-cm centers with 1.2 m between rows. Three experiments were conducted. Plots of 3 plants each were set up in randomized complete block designs with 5 (Experiments 1 and 2) or 3 (Experiment 3) replicates. Plants were infested with 10 3- day-old tobacco budworm larvae (Experiments 1 and 2) or 10 3-day-old tobacco budworm larvae plus 20 tobacco budworm eggs (Experiment 3, except for 1 untreated check with eggs only and 1 untreated check with larvae only) from laboratory colonies on 7, 19, and 24 Aug for experiments 1-3, respectively.

Differences in expression between two seed lectin alleles obtained from normal and lectin-deficient beans are maintained in transgenic tobacco

Using Agrobacterium-mediated transformation, two genes for phytohemagglutinin-L (PHA-L), the lectin seed protein of the common bean Phaseolus vulgaris, were stably integrated into the tobacco genome. The two alleles for PHA-L, dlec2 and pdlec2, were obtained from a normal cultivar (Greensleeves) and a lectin-deficient cultivar (Pinto) respectively. In the bean embryos, the expression of dlec2 is 30 times greater than the expression of pdlec2. In the dlec2-transformed tobacco, PHA-L accumulated specifically in the seeds at the same stages as the tobacco seed storage proteins and was degraded after germination. PHA-L was found in the embryo, and at a 5-7 times lower concentration in the endosperm tissue of the mature tobacco seeds. No PHA could be detected in other parts of the plants. We conclude that the signals for temporal and spatial regulation of the dlec2 gene are present in the DNA fragment used for transformation. Transformation with the second PHA-L allele pdlec2 from the cultivar Pinto caused the accumulation of about 50 times less PHA-L in tobacco seeds when compared to dlec2. We conclude from analyzing the 5' sequences of dlec2 and Pdlec2 that the low expression phenotype of the Pdlec2 allele could be due to the absence or mutation of a cis-acting element carried by the dlec2 fragment.

Expression of the ?-subunit of ?-conglycinin in seeds of transgenic plants

Soybean (Glycine max (L.) Merr.) seeds contain the storage protein β-conglycinin, encoded by a multigene family. β-Conglycinin consists of three subunits; α', α, and β. A genomic clone for a β-subunit of β-conglycinin has been characterized by restriction-enzyme mapping and hybrid selected in-vitro translation followed by immunoprecipitation. In order to determine the developmental regulation of this β-subunit gene, its expression was studied in seeds of transgenic petunia (Petunia hybrida) and tobacco (Nicotiana tabacum L.) plants. The β-subunit expressed in seeds of petunia and tobacco was recognized by anti-β-conglycinin serum at a relative molecular mass of 53 000, equivalent to that of the native protein. Separation of the petunia-seed proteins by isoelectric focusing followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis showed that multiple isoelectric forms of the β-subunit were produced. There was approximately a twofold variation in the accumulation of the β-subunit protein in the mature seeds of transgenic petunia plants, each containing a single β-subunit gene. However, the level of protein accumulation in mature seeds and the amount of β-subunit mRNA in developing seeds was not correlated. Accumulation of the β-subunit protein in transgenic seeds was less than the α'-subunit protein that accumulated in transgenic petunia seeds containing a single α'-subunit gene and less than the amount of the β-subunit in mature soybean seeds which contain 8-13 β-subunit genes. In transgenic tobacco plants, the accumulation of the β-subunit protein in seeds was generally well correlated with the number of genes that were incorporated in the different transformants.

Synthesis and protein body deposition of maize 15-kd zein in transgenic tobacco seeds

The maize 15-Kd zein structural gene was placed under the regulation of French bean beta-phaseolin gene flanking regions. Agrobacterium tumefaciens-mediated transformation was used to insert the chimeric phaseolin-zein gene into the tobacco genome. Transgenic plants synthesized zein in a tissue-specific manner during the latter half of seed development. Transcription of the chimeric gene was initiated in phaseolin-derived sequences, and was terminated within the phaseolin gene 3' flanking region. Both zein- and phaseolin-derived polyadenylation signals were used in the processing of zein RNA in transgenic plant seeds. Zein accumulation, though subject to an 80-fold variation among 19 plants tested, could reach as much as 1.6% of the total seed protein in several plants. In developing tobacco seeds, zein was correctly processed by the removal of a 20-amino-acid signal peptide. Electron microscope immunogold localization of the zein expressed in embryo and endosperm tissue indicates that the monocot protein accumulates in the crystalloid component of vacuolar protein bodies. The density of gold label over the protein bodies is several fold greater in the embryo than the endosperm. Zein is found in roots, hypocotyls and cotyledons of germinating transgenic tobacco seeds.

Induction of Tobacco Seed Germination Synchrony through Dark Preincubation1

AbstractNicotiana tabacum cv. Speight G‐28, a popular cultivar of bright leaf tobacco, has a high percentage of seed germination but it takes a long time (nearly a week under laboratory conditions and several weeks under field conditions) to give maximum germination. This study was undertaken to develop a laboratory seed treatment to improve germination synchrony. Twenty‐three seed treatments, including the control, were developed through the use of dark‐preincubation (DPI), polyethylene glycol (PEG) or chilling alone, or in various combinations thereof. An index of synchrony (IS) was calculated from the formula, urn:x-wiley:00021962:agj2agronj198700021962007900030012x:equation:agj2agronj198700021962007900030012x-math-0001 The theoretical highest value of the IS for a given seed lot would be 1.0. In absence of an absolute synchrony, the practical objective of a given seed treatment would be to raise the IS as close to 1.0 as possible. All the 23 seed treatments tested were ranked on the basis of this consideration. Treatments giving 0.8 or greater IS are, indecreasing order: DPI at 23°C for 1 week followed by DPI for 1 week under chilling (0.95); DPI under chilling for 1 week followed by DPI at 23°C for 1 week (0.93); DPI at 23°C for 1 week followed by chilling under light for 1 week (0.90); and DPI at 23°C for 1 week (0.81). As compared to these treatments, the IS of the control was 0.52. Possible physiological mechanisms involved in DPI‐induced germination synchrony and the potential for practical application of this treatment are discussed.

Development of greenhouse solar systems for bulk tobacco curing and plant production

Among many farm crops, bright leaf tobacco is the most energy- and labor-intensive crop. The greenhouse solar system (solar bulk-curing/greenhouse system, or solar barn) was developed to provide multiple-use facilities for year-round solar energy utilization to save fossil fuels in tobacco curing and plant production and to facilitate the total mechanization of tobacco culture. Two types of full-size greenhouse solar systems, the load-supporting wall design and the shell design, both utilizing the thermal envelope concept, were designed and constructed for solar bulk-curing of tobacco, growing transplants and horticultural crops under controlled environment, and aiding automation of transplanting operations. Full-scale field tests of solar bulk curing showed that the fuel savingswere consistantly improved from 37% in 1975 to 51% in 1978 for this solar bulk-curing system as compared with a conventional bulk-curing barn as a control. The feasibility of the system to save energy by using solar energy as a first priority source was significantly demonsrated. Three-year greenhouse and field tests showed that high germination rate of 95–97% with excellent emergence frequency was obtained for tobacco seeds under the controlled environment provided by the greenhouse solar system. In general, the containerized transplants from greenhouse solar system significantly exceeded the conventional bare-root transplant in growth, leaf-quality and yield.

Soybean seed lectin gene and flanking nonseed protein genes are developmentally regulated in transformed tobacco plants.

We introduced a 17.1-kilobase soybean DNA fragment containing the lectin gene and at least four nonseed protein genes into the tobacco genome. As in soybean plants, lectin mRNA is present in tobacco seeds, accumulates and decays during tobacco seed development, and is translated into a protein that accumulates prior to dormancy. Each soybean nonseed protein mRNA is present in tobacco leaves, roots, stems, and seeds at levels similar to that found in soybean plants. We conclude that a differentially expressed soybean gene cluster is correctly regulated in transformed tobacco plants and that sequences controlling their expression are recognized by regulatory factors present in tobacco cells.

Developmentally regulated expression of the bean β-phaseolin gene in tobacco seed

Recombinant phage lambda177.4 contains a gene for beta phaseolin, a major storage glycoprotein of French bean seed. A 3.8-kilobase Bgl II-BamHI fragment containing the entire 1700-base-pair coding region, together with 863 base pairs of 5' and 1226 base pairs of 3' flanking sequence, was inserted into the A66 Ti plasmid of Agrobacterium tumefaciens and used to transform tobacco. The level of phaseolin in the seeds of plants regenerated from cloned tissue was 1000-fold higher than in other tissues. The molecular weight of the phaseolin RNA transcript in tobacco seeds was identical to that found in bean seeds. The phaseolin protein in tobacco seed was glycosylated and appeared to undergo removal of the signal peptide. However, a large proportion of the phaseolin was cleaved into discrete peptides. These same peptides were formed as phaseolin was degraded during tobacco seed germination. The phaseolin gene appeared to be inserted as a single copy, and the proportion of phaseolin per genome copy in tobacco seeds (up to 3% of the total embryo proteins) resembled that in the bean seeds (40% of total seed protein, expressed from about 14 copies per diploid genome). Furthermore, the transplanted gene was turned on during tobacco seed development, and its protein product, phaseolin, was localized in the embryonic tissues. Finally, the phaseolin gene was inherited as a Mendelian dominant trait in tobacco.

Ultracytochemical Localization of Plasma Membrane-Associated Phosphatase Activity in Developing Tobacco Seeds

Ultracytochemical Localization of Plasma Membrane-Associated Phosphatase Activity in Developing Tobacco Seeds

Evidence for Aboriginal Tobaccos in Eastern North America

SEM micrographs of seeds from four tobaccos historically cultivated by aboriginal groups in the Eastern Woodlands and adjacent Great Plains of North America are presented. The tobaccos considered are Nicotiana rustica L., N. attenuata Torr., N. bigelovii var. quadrivalvis (Pursh) East, and N. bigelovii var. multivalvis (Lindley) East. Micrographs of seeds from herbarium collections are compared with micrographs of carbonized tobacco seeds from the Extended Middle Missouri component at the Travis I site, and tobacco is reported from the Dirt Lodge Village site, both in South Dakota. The archaeological specimens compare most favorably with N. bigelovii var. quadrivalvis (Pursh) East, the tobacco historically cultivated by the Arikara, Mandan, and Hidatsa. These results strengthen the evidence for trade contacts and cultural interaction by Middle Missouri tradition populations with groups to the west and provide new data on the temporal perspective for the cultivation of N. bigelovii var. quadrivalvis (Pursh) East in the Middle Missouri subarea.

Biological changes observed on rice and biological and genetic changes observed on tabacco after space flight in the orbital station SALYUT-7 (Biobloc III experiment)

Caryopses and isolated embryos from Rice ( Oryza sativa L .) and Tobacco seeds ( Nicotiana tabacum L. variety Xanthi ) were studied in the Biobloc III container aboard the Soviet orbital space station SALYUT 7. The recovery from radiation damage under conditions of space flight was observed for rice caryopsis and embryos gamma irradiated (Co 60, 50 grays) prior to launch. There was a large decrease in the percentage of germinating seeds from the Tobacco strain tested when the seeds were exposed to heavy ions. Among the germinating plantlets there were few morphological anomalies. Furthermore, there was a significant greater amount of genetic change in those samples held in grids as compared to those in bags.

Studies on the Germiatron of Cultivated Tobacco Seeds as Affected by Sowing Density and Interspecific Interaction

Studies on the Germiatron of Cultivated Tobacco Seeds as Affected by Sowing Density and Interspecific Interaction

Myo-inositol induced growth in ethyl methanesulphonate treated tobacco

Tobacco seeds treated with ethyl methanesulphonate produces mutations as well as physiological growth debility. The addition of myo-inositol to seeds undergoing mutagenic treatment stimulated growth and increased survival of subsequent plants with negligible effect on the mutation frequency.