Tobacco Seeds Research Articles

Developmental and hormonal regulation of sunflower helianthinin genes: proximal promoter sequences confer regionalized seed expression.

DNA elements involved in the regulation of two sunflower helianthinin genes were identified by analysis of beta-glucuronidase (GUS) expression in transgenic tobacco driven by sequences derived from the 5' upstream regions of these genes. A 2.4-kb upstream region of the helianthinin gene HaG3-A conferred rigorous developmental GUS expression in transgenic tobacco seeds with no significant GUS activity in nonembryonic tissues. Regions of the helianthinin upstream regulatory ensemble (URE) conferred ectopic expression in nonembryonic tissues when analyzed outside of the context of the complete helianthinin regulatory complex. A proximal promoter region was identified that conferred significant GUS expression in seeds but not in leaves of transgenic tobacco. Three sequence motifs that bind to seed nuclear proteins were identified in the proximal promoter region; mutations in these motifs significantly reduced the level of nuclear protein binding. Another important class of cis-regulatory elements was identified in the helianthinin URE that conferred abscisic acid-responsive GUS expression. In the full-length helianthinin URE, these elements only responded to abscisic acid in the developing seed, suggesting that the helianthinin gene contains additional regulatory elements, possibly in the proximal promoter region, that ensure hierarchical control in the developing seed.

Lysine synthesis and catabolism are coordinately regulated during tobacco seed development.

The regulation of synthesis and accumulation of the essential amino acid lysine was studied in seeds of transgenic tobacco plants expressing, in a seed-specific manner, two feedback-insensitive bacterial enzymes: dihydrodipicolinate synthase (EC 4.2.1.52) and aspartate kinase (EC 2.7.2.4). High-level expression of the two bacterial enzymes resulted in only a slight increase in free lysine accumulation at intermediate stages of seed development, while free lysine declined to the low level of control plants toward maturity. To test whether enhanced catabolism may have contributed to the failure of free lysine to accumulate in seeds of transgenic plants, we analyzed the activity of lysine-ketoglutarate reductase (EC 1.5.1.7), an enzyme that catabolizes lysine into saccharopine. In both the control and the transgenic plants, the timing of appearance of lysine-ketoglutarate reductase activity correlated very closely with that of dihydrodipicolinate synthase activity, suggesting that lysine synthesis and catabolism were coordinately regulated during seed development. Notably, the activity of lysine-ketoglutarate reductase was significantly higher in seeds of the transgenic plants than in the controls. Coexpression of both bacterial enzymes in the same plant resulted in a significant increase in the proportions of lysine and threonine in seed albumins. Apparently, the normal low steady-state levels of free lysine and threonine in tobacco seeds may be rate limiting for the synthesis of seed proteins, which are relatively rich in these amino acids.

Evidence for a role of β‐1,3‐glucanase in dicot seed germination

Class I β‐1,3‐glucanases are antifungal vacuolar proteins implicated in plant defense that show developmental, hormonal, and pathogenesis‐related regulation. The expression was studied in germinating tobacco seeds of a chimeric β‐glucuronidase (GUS) reporter gene fused to 1.6 kb of the 5′ flanking sequence of the tobacco class I β‐1,3‐glucanase B (GLB) promoter. Histological staining for GUS activity showed that expression of the GLB promoter is highly localized in a specific zone of the endosperm in germinating seeds. The temporal and spatial patterns of GUS and β‐1,3‐glucanase activity found, suggest a novel function for class I β‐1,3‐glucanases during seed germination in a dicotyledonous plant.

Synthesis and targeting of Brassica napus oleosin in transgenic tobacco

A repeseed oleosin gene, Bn-III, containing a 3.44-kbp promoter region and the complete coding sequence, was introduced into tobacco explants via Agrobacterium-mediated transformation. Rapessed oleosin protein was synthesised in the transgenic tobacco plants, as detected by immunoblotting, and accumulated only in the embryo and endosperm tissues of the maturing tobacco seeds. The amounts of oleosin on a per seed basis were than 5-fold higher in the embryos than in the endosperm tissue. Rapeseed oleosin accumulated significantly later than the period of maximum storage oil deposition in the tobacco seeds and was correctly targeted to the surface layer of tobacco seed oil bodies, as shown by immunogold labelling. This is the first study to demonstrate the spatial and temperal accumulation pattern and correct targeting of a heterologous oleosin protein, under the control of its own gene promoter, in an unrelated plant species. The study also shows that transgenic tobacco is a useful model system in which to perform in vivo oleosin targeting experiments.

Nutrient composition of some less-familiar oil seeds

Seven less familiar oil seeds, namely gokhru, thumba, onion, tobacco, grape, niger seeds and mango seed kernels were analysed for their proximate composition, amino acid and fatty acid composition. Gokhru, niger, tobacco and onion seeds were found to be good sources of protein and fat. Crude fibre contents of thumba and grape seeds were high. The seeds of thumba, niger, onion and mango (seed kernel) contained 4 g lysine per 16 g N. The methionine content of onion-seed protein was very high. Fatty acid profiles of fat from these oil seeds revealed a high proportion of unsaturated fatty acids with the exception of gokhru.

Functional studies on a seed-specific promoter from a Brazil nut 2S gene

The 5′ upstream regulatory region of the Brazil nut 2S albumin gene was studied in order to identify sequences involved in gene expression. Restriction fragments from the regulatory region were fused to the reporter genes chloramphenicol acetyl transferase (CAT) and β-glucuronidase (GUS). The CAT constructs were used to transform tobacco leaf discs via Agrobacterium and the GUS-promoter fusions were introduced into bean embryos by particle bombardment. With both systems all promoter deletions conferred expression of the GUS and CAT genes. In the biolistically transformed bean embryos reporter gene activity dropped at position −460 base pairs upstream from the transcription initiation site. CAT activity in stably transformed tobacco plants was less markedly affected at this site but declined sharply after the deletions reached −210 bp upstream from the transcription initiation site. The temporal expression of the CAT gene controlled by the Brazil nut 2S promoter in developing, transgenic tobacco seeds parallels that of 2S albumin expression in Brazil nut. DNA retardation assays and footprint analysis using Brazil nut 2S promoter fragments and nuclear extracts from Brazil nut seeds allowed the identification of at least two sequence motifs, ACGT and CCAC, that might be involved in promoter activity.

5'-flanking region responsible for endosperm-specific expression of rice prolamine chimeric gene in transgenic tobacco.

The 5' upstream region (-680-(+)40) containing the complete signal peptide coding sequence of the rice seed storage prolamine gene was amplified in vitro with polymerase chain reaction from the genome of Chinese super rice cultivar Zhonghua 8. Physical map and DNA sequence analysis showed strong homology with the 5'-flanking region of rice prolamine gene reported by Kim in 1988. No changes in the signal peptide coding sequence and a long leader sequence with several small ORFs were found. Chimeric gene containing 5'-flanking region of the prolamine gene has been transcriptionally fused with the beta-glucuronidase reporter gene. The fusion junction was confirmed by both physical map and DNA sequence analysis. The resultant chimeric gene was used to transform the tobacco explants by Ti binary system of Agrobacterium tumefaciens LBA4404. With dot and Southern blotting hybridization, three transgenic tobacco plants with the copies of chimeric GUS genes as many as 20 were obtained. Histochemical analysis revealed that the GUS activity in the endosperm tissues of tobacco seeds at the developmental stage was about 20 DAP. No GUS activity was found in leaves, stems, roots and flowers of the transgenic tobacco plants. Therefore, we concluded that the 5'-upstream cis-elements from -680 to -18 were enough to confer the endosperm-specific and temporal expression of rice prolamine gene.

Deletion analysis of a 2S seed storage protein promoter of Brassica napus in transgenic tobacco

The promoter and upstream region of the Brassica napus 2S storage protein napA gene were studied to identify cis-acting sequences involved in developmental seed-specific expression. Fragments generated by successive deletions of the 5' control region of the napA gene were fused to the reporter gene beta-glucuronidase (GUS). These constructs were used to transform tobacco leaf discs. Analyses of GUS activities in mature seeds from the transformed plants indicated that there were both negatively and positively acting sequences in the napin gene promoter. Deletion of sequences between -1101 and -309 resulted in increased GUS activity. In contrast, deletion of sequences between -309 and -211 decreased the expression. The minimum sequence required for seed-specific expression was a 196 bp fragment between -152 and +44. Further 5' deletion of the fragment to -126 abolished this activity. Sequence comparison showed that a G box-like sequence and two sequence motifs conserved between 2S storage protein genes are located between -148 to -120. Histochemical and fluorometric analysis of tobacco seeds showed that the spatial and developmental expression pattern was retained in the deletion fragments down to -152. However, the expression in tobacco seeds differed from the spatial and temporal expression in B. napus. In tobacco, the napA promoter directed GUS activity early in the endosperm before any visible activity could be seen in the heart-shaped embryo. Later, during the transition from heart to torpedo stages, the main expression of GUS was localized to the embryo. No significant GUS activity was found in either root or leaf.

In situ localization of faba bean and oat legumin-type proteins in transgenic tobacco seeds by a highly sensitive immunological tissue print technique.

We have used a highly sensitive immunological tissue print technique to study cell- and tissue-specific expression of heterologous genes in transgenic plants. Primary polyclonal antibodies, raised against legumin of faba bean (Vicia faba L.) and 12S globulin of oat (Avena sativa L.) were used to localize these proteins in transgenic tobacco seeds in a streptavidin-alkaline phosphatase assay in combination with biotinylated secondary antibodies producing a higher sensitivity (by several amplification steps) of the assay. Both storage protein genes were found to be expressed in a specific pattern. While legumin is preferentially accumulated in certain parts of the embryo, the oat legumin-type globulin is restricted to the endosperm. The applied technique is highly sensitive with a resolution power down to the single-cell level and allows rapid screening of large numbers of samples.

Phytase-containing Transgenic Seeds as a Novel Feed Additive for Improved Phosphorus Utilization

Phytate is the main storage form of phosphorus in many plant seeds, but bound in this form it is a poor nutrient for monogastric animals. Its availability can be significantly increased by addition of the enzyme phytase, which releases phosphate from the substrate, phytate. We have engineered a phytase from Aspergillus niger in tobacco seeds, providing a stable and convenient packaging of the enzyme that is directly applicable in animal feed. The enzyme was expressed as 1 percent of the soluble protein in mature seeds. In in vitro tests that simulated chicken crop and stomach conditions, release of phosphate from feed by addition of transgenic seeds was demonstrated. Supplementation of broiler diets with transgenic seeds resulted in an unproved growth rate, comparable to diets supplemented with fungal phytase or phosphorus. Addition of phytase-transgenic seeds to animal feed thus obviates the need for inorganic phosphorus supplementation, and is environmentally desirable because of the reduced excretion of phosphorus.

A complex ensemble of cis-regulatory elements controls the expression of a Vicia faba non-storage seed protein gene

We have identified cis-regulatory elements within the 5'-upstream region of a Vicia faba non-storage seed protein gene, called usp, by studying the expression of usp-promoter deletion fragments fused to reporter genes in transgenic tobacco seeds. 0.4 kb of usp upstream sequence contain at least six, but probably more, distinct cis-regulatory elements which are responsible for seemingly all quantitative, spatial and temporal aspects of expression. Expression-increasing and -decreasing elements are interspersed and include an AT-rich sequence, a G-box element and a CATGCATG motif. The latter acts as a negative element in contrast to what has been found for the same motif in legumin- and vicilin-type seed storage protein gene promoters. Seed specificity of expression is mainly determined by the -68/+51 region which confers, however, only very low levels of expression. The data support the combinatorial model of promoter function.

Cis-acting regulatory regions of the soybean seed storage 11S globulin gene and their interactions with seed embryo factors.

A 2.2 kb fragment containing the 5'-flanking region of the soybean glycinin A2B1a gene and its successive deletions with a shorter 5'-flanking sequence were fused, in frame, to the beta-glucuronidase (GUS) reporter gene. The resultant fusions were introduced into tobacco plants via Agrobacterium tumefaciens. Assays of the GUS activity in seeds of transgenic tobacco showed that the upstream region, -657 to -327 (relative to the transcription initiation site [+1]), of the glycinin gene is required for optimal expression of the transformed gene. Interactions between embryo nuclear factors and DNA fragments covering the downstream region of -326, in which are included the TATA box and legumin boxes, were not apparent. The embryo factors capable of binding specifically to three subregions, -653 to -527, -526 to -422, and -427 to -321, of the upstream regulatory region were detected. Such factors appeared to be organ-specific and could be found solely in developing seeds at the early middle stage of embryogenesis (around 24 days after flowering). Evidence obtained by characterizing the nature of the binding proteins and by gel mobility shift assays established that the same factor does interact with a consensus motif 5'-ATA/TATTTCN-/CTA-3' which occurs four times in the cis-acting regulatory region between -657 and -327. Moreover, this conserved motif could also be found in the 5' regulatory region of another glycinin A1aB1b gene. Thus it is likely that the observed interaction between the nuclear factor and the conserved motifs would lead to activation of transcription from the glycinin genes in maturing soybean seeds.

Chemical composition of tobacco seeds (Nicotiana tabacum L) from Argentina

AbstractThe lipid constituents, free fatty acids, triglycerides, sterol esters and free sterols of 11 cultivars of tobacco from Argentina were investigated by gas‐liquid chromatography. Palmitic, oleic and linoleic acids were the major components in all fractions studied. Sitosterol was the main component of the free sterol and sterol ester fractions. Oil and protein contents, as well as some physicochemical characteristics of the oils, are also reported.

Desiccation and the Control of Expression of β-Phaseolin in Transgenic Tobacco Seeds

Drying of seeds at certain stages prior to maturation, i.e. premature desiccation, will terminate synthetic events unique to development, for example, storage protein synthesis, and initiate processes associated with germination. In this study we have investigated the role of desiccation in the expression of a storage protein gene, β-phaseolin, to determine if such a developmentally-regulated gene remains sensitive to drying when controlled by a promoter that has no known sensitivity to this treatment. We compared, in transgenic tobacco seeds, the effects of maturation and premature drying on the expression of a full β-phaseolin gene, and β-phaseolin genes driven by a cauliflower mosaic virus 35S promoter with or without an alfalfa mosaic virus (AMV) 5' untranslated leader sequence

Effect of Protein Synthesis Inhibitors on Tobacco Seed Germination and Seedling Emergence

Summary This study is based on two earlier reports, one demonstrating that dark preincubation improves tobacco (Nicotiana tabacum L.) seed germination rate and the other demonstrating genetic variability in tobacco seed response to germination temperature. Each of these situations involves special classes of proteins: the phytochrome molecule and heat shock proteins, respectively. A better understanding of either process at the molecular level would involve investigations on the regulatory role of the protein synthesis mechanism. This study was undertaken to characterize the temporal relationship of the three major steps in protein synthesis - replication, transcription and translation - with the three major events associated with seedling morphogenesis: radicle protrusion, radicle elongation, and plumule protrusion. This was accomplished through the use of selected inhibitors of replication, transcription, and translation. Results suggest that a) radicle emergence is regulated by translation but is independent of replication and transcription; b) the translational regulation of the above event is mediated through preformed (i.e., longlived) m-RNAs; c) radicle elongation following its emergence and plumule emergence are regulated by all three components of protein synthesis; d) radicle length data obtained in laboratory investigations are a better predictor than radicle number (i.e., germination percentage) of seedling emergence potential under field conditions.

Alternaria zinniae . [Descriptions of Fungi and Bacteria

Abstract A description is provided for Alternaria zinniae . Information is included on the disease caused by the organism, its transmission, geographical distribution, and hosts. HOSTS: Ageratum, Aster, Bidens, Calendula, Callistephus, Chrysanthemum, Coreopsis, Cosmos, Dahlia, Gaillardia, Galinsoga, Gerbera, Helianthus, Notonia, Parthenium, Sphaeranthus indicus, Tagetes, Tithonia, Volutarella, Xanthium, Zinnia . Also recorded are a number of non-compositae hosts such as bean (50, 3295), rape (43, 1212b), Clarkia, Hibiscus esculentus (66. 4729), pawpaws (66. 4729) and seeds of tobacco (64, 1748) but these reports should be regarded with some doubt. Simmons (1982) also reports other hosts like Gentiana, Papaver and Reseda . DISEASE: Leaf spot of Zinnia . The fungus attacks the leaves, stems of its host and can cause damping-off of seedlings where both the root and the stems of the plant may be affected. In older plants the fungus attacks the older leaves and then spreads to the younger leaves, and when the attack is severe the spots may become confluent. GEOGRAPHICAL DISTRIBUTION: Africa: Egypt, Ethiopia, Ghana, Kenya, Libya, Malawi, Mauritius, Sierra Leone, South Africa, Sudan, Tanzania, Uganda, Zambia, Zimbabwe. Asia: Brunei, China (Liaoning). India, Indonesia (Borneo), Korea, Nepal, Pakistan. Australasia & Oceania: Australia. Europe: Cyprus. Denmark, France, Germany, Hungary, Italy, Norway, Poland, Portugal, Romania, The Netherlands. Central & North America: Bermuda, Canada, Jamaica, USA (Hawaii, Illinois, South Dakota). TRANSMISSION: On seeds by wind dispersal of airborne conidia.

Different legumin protein domains act as vacuolar targeting signals.

Legumin subunits are synthesized as precursor polypeptides and are transported into protein storage vacuoles in field bean cotyledons. We expressed a legumin subunit in yeast and found that in these cells it is also transported into the vacuoles. To elucidate vacuolar targeting information, we constructed gene fusions of different legumin propolypeptide segments with either yeast invertase or chloramphenicol acetyltransferase as reporters for analysis in yeast or plant cells, respectively. In yeast, increasing the length of the amino-terminal segment increased the portion of invertase directed to the vacuole. Only the complete legumin alpha chain (281 amino acids) directed over 90% to the vacuole. A short carboxy-terminal legumin segment (76 amino acids) fused to the carboxy terminus of invertase also efficiently targeted this fusion product to yeast vacuoles. With amino-terminal legumin-chloramphenicol acetyltransferase fusions expressed in tobacco seeds, efficient vacuolar targeting was obtained only with the complete alpha chain. We conclude that legumin contains multiple targeting information, probably formed by higher structures of relatively long peptide sequences.

Multiple nuclear factors interact with upstream sequences of differentially regulated ?-conglycinin genes

The expression of the alpha' and beta subunit genes of beta-conglycinin is differentially regulated during soybean embryo development. Although both are expressed solely in developing seeds during mid to late stages of embryo development, the alpha' subunit is expressed more highly on a per gene basis, and alpha' subunit mRNA begins to accumulate three to five days earlier than beta subunit mRNA. In cultured cotyledons, beta subunit gene(s) respond to changes in methionine or abscisic acid levels, whereas expression of the alpha' subunit gene(s) is unaffected by these changes. To investigate the mechanisms by which these genes are transcriptionally regulated, we examined the interactions of nuclear proteins with upstream sequences from the alpha' and beta subunit genes. Four distinct DNA binding factors were identified in nuclear extracts from developing soybean seeds. These factors are termed Soybean Embryo Factors (SEF) 1 through 4. SEF binding sites are distributed non-uniformly between the alpha' and beta subunit genes, and the amount of protein binding is modulated over the course of embryo development. DNA footprinting revealed the sequences recognized by three of these factors. Factors which behave in a manner similar to that of SEF3 were also identified in nuclear extracts from developing tobacco and sunflower seeds.

A novel seed protein gene from Vicia faba is developmentally regulated in transgenic tobacco and Arabidopsis plants

We have isolated a novel gene, denoted USP, from Vicia faba var. minor, which corresponds to the most abundant mRNA present in cotyledons during early seed development; however, the corresponding protein does not accumulate in cotyledons. The characterized USP gene with its two introns is 1 of about 15 members of a gene family. A fragment comprising 637 bp of 5' flanking sequence and the total 5' untranslated region was shown to be sufficient to drive the mainly seed-specific expression of two reporter genes, coding for neomycin phosphotransferase II and beta-glucuronidase, in transgenic Arabidopsis thaliana and Nicotiana tabacum plants. We showed that the USP promoter becomes active in transgenic tobacco seeds in both the embryo and the endosperm, whereas its activity in Arabidopsis is detectable only in the embryo. Moreover, we demonstrated a transient activity pattern of the USP promoter in root tips of both transgenic host species.

Expression of Bean Storage Protein Minigene in Tobacco Seeds: Introns are not Required for Seed Specific Expression

Summary A native and mini (intronless) phaseolin gene were inserted into a mutant derivative of Agrobacterium tumefaciens plasmid pTi15955, in which the oncogenic tms2 and tmr loci were deleted. This pTi vector is referred to as a rooty-shooty mutant, pTiRS 2 014. The vector pTiRS 2 014 was used for the transfer and integration of phaseolin genes into the genome of tobacco followed by the regeneration of phenotypically normal tobacco plants. The presence of phaseolin polypeptides in R 1 and R 2 seeds was confirmed by immunological methods. These results demonstrate that native and mini-phaseolin genes are integrated stably into the respective tobacco plant lines and that the absence of the phaseolin introns did not affect its developmental expression.