2S storage protein gene of Douglas-fir: characterization and activity of promoter in transgenic tobacco seeds.

To date a few sequences regulating expression of conifer seed-specific genes have been reported. To characterize Douglas-fir (Pseudotsuga menziesii [Mirb] Franco) 2S albumin storage protein genes, a genomic DNA sequence containing upstream promoter sequences was isolated by screening a Douglas-fir genomic library. Sequence analysis of the Douglas-fir gPm2S1 promoter revealed the presence of RY-repeated elements (GCATGC), and multiple E-box motifs (CANNTG) and ACGT-core elements, features characteristic of 2S storage protein genes in angiosperms. When fused to the GUS reporter gene, the 1.16 kb Douglas-fir 2S promoter sequence was sufficient to direct transient expression in both developing Douglas-fir embryos and maternally derived haploid megagametophytes. Analysis of this promoter construct in transgenic tobacco showed that expression was restricted to embryo and endosperm in developing seeds and was not detected in vegetative tissues of two-week-old seedlings. These results strongly suggest that both structural and regulatory elements as well as upstream signaling components controlling the expression of 2S albumin genes are highly conserved during evolution.     

Soluble methionine enhances accumulation of a 15 kDa zein, a methionine-rich storage protein, in transgenic alfalfa but not in transgenic tobacco plants

With the general aim of elevating the content of the essential amino acid methionine in vegetative tissues of plants, alfalfa (Medicago sativa L.) and tobacco plants, as well as BY2 tobacco suspension cells, were transformed with a beta-zein::3HA gene under the 35S promoter of cauliflower mosaic virus encoding a rumen-stable methionine-rich storage protein of 15 kDa zein. To examine whether soluble methionine content limited the accumulation of the 15 kDa zein::3HA, methionine was first added to the growth medium of the different transgenic plants and the level of the alien protein was determined. Results demonstrated that the added methionine enhanced the accumulation of the 15 kDa zein::3HA in transgenic alfalfa and tobacco BY2 cells, but not in whole transgenic tobacco plants. Next, the endogenous levels of methionine were elevated in the transgenic tobacco and alfalfa plants by crossing them with plants expressing the Arabidopsis cystathionine gamma-synthase (AtCGS) having significantly higher levels of soluble methionine in their leaves. Compared with plants expressing only the 15 kDa zein::3HA, transgenic alfalfa co-expressing both alien genes showed significantly enhanced levels of this protein concurrently with a reduction in the soluble methionine content, thus implying that soluble methionine was incorporated into the 15 kDa zein::3HA. Similar phenomena also occurred in tobacco, but were considerably less pronounced. The results demonstrate that the accumulation of the 15 kDa zein::3HA is regulated in a species-specific manner and that soluble methionine plays a major role in the accumulation of the 15 kDa zein in some plant species but less so in others.     

Properties of beta-propeller phytase expressed in transgenic tobacco

Phytases are enzymes that liberate inorganic phosphates from phytate. In a previous study, a beta-propeller phytase (168phyA) from Bacillus subtilis was introduced into transgenic tobacco, which resulted in certain phenotypic changes. In the study described herein, the recombinant phytase (t168phyA) was purified from transgenic tobacco to near homogeneity by a three-step purification scheme. The biochemical properties and kinetic parameters of t168phyA were compared with those of its counterpart from B. subtilis. t168phyA was glycosylated, and it showed a 4 kDa increase in molecular size in SDS-PAGE (44 kDa vs. 40 kDa). Although its thermostability remained unchanged, its temperature optimum shifted from 60 degrees C to 45-50 degrees C and its pH optimum shifted from pH 5.5 to 6.0. Kinetic data showed that the t168phyA had a lower Kcat, but a higher Km than the native enzyme. Despite these changes, t168phyA remained catalytically active and has a specific activity of 2.3 U/mg protein. These results verify the activity of recombinant Bacillus phytase that is expressed in plants.     

Limits to sulfur accumulation in transgenic lupin seeds expressing a foreign sulfur-rich protein

The low sulfur amino acid content of legume seeds restricts their nutritive value for animals. We have investigated the limitations to the accumulation of sulfur amino acids in the storage proteins of narrow leaf lupin (Lupinus angustifolius) seeds. Variation in sulfur supply to lupin plants affected the sulfur amino acid accumulation in the mature seed. However, when sulfur was in abundant supply, it accumulated to a large extent in oxidized form, rather than reduced form, in the seeds. At all but severely limiting sulfur supply, addition of a transgenic (Tg) sink for organic sulfur resulted in an increase in seed sulfur amino acid content. We hypothesize that demand, or sink strength for organic sulfur, which is itself responsive to environmental sulfur supply, was the first limit to the methionine (Met) and cysteine (Cys) content of wild-type lupin seed protein under most growing conditions. In Tg, soil-grown seeds expressing a foreign Met- and Cys-rich protein, decreased pools of free Met, free Cys, and glutathione indicated that the rate of synthesis of sulfur amino acids in the cotyledon had become limiting. Homeostatic mechanisms similar to those mediating the responses of plants to environmental sulfur stress resulted in an adjustment of endogenous protein composition in Tg seeds, even when grown at adequate sulfur supply. Uptake of sulfur by lupin cotyledons, as indicated by total seed sulfur at maturity, responded positively to increased sulfur supply, but not to increased demand in the Tg seeds.     

Analysis of kafirin promoter activity in transgenic tobacco seeds

Sequences corresponding to 855 bp of 5 promoter region and the transit peptide from GK.1, a genomic clone encoding a 22 kDa -kafirin seed protein from sorghum, were translationally fused to a cloned -glucuronidase (GUS) coding sequence from uidA and transferred to tobacco via Agrobacterium tumefaciens-mediated transformation. No GUS expression was detectable at any stage of growth in stems or leaves of these plants. However, GUS expression was detected in both embryo and endosperm tissues of resulting tobacco seeds 10–15 days after flowering. Dissected tissues indicate endosperm expression was localized within the bulk endosperm and not within the parenchyma cell layer underlying the integument. These studies also demonstrate that within dissected tobacco embryos, expression from the kafirin promoter was restricted to the mesocotyl region.     

Normal and lysine-containing zeins are unstable in transgenic tobacco seeds

Chimeric genes composed of the -phaseolin promoter, an -zein coding sequence and its modified versions containing lysine codons, and a -zein polyadenylation signal were inserted into the genome of tobacco by Agrobacterium-mediated transformation. -Zein mRNA levels in the transgenic tobacco seeds 20 days after self-pollination varied between 1.0% and 2.5% of the total mRNA population. At 25 days after pollination the 19 kDa -zein was immunologically detected with a polyclonal antiserum in protein extracts from the seeds of transgenic plants. The transgenic plant with the highest level of zein gene expression had an -zein content that was approximately 0.003% of the total seed protein. The amount of -zein in other transgenic plants varied between 1 × 10^–4% and 1 × 10^–5% of the total seed protein. The differences in the amounts of mRNA and protein did not correlate with the lysine substitutions introduced into the -zein protein. Polysomes translating -zein mRNA isolated from tobacco seeds contained fewer ribosomes than those from maize endosperm, but this did not appear to be the cause of the inefficient protein synthesis. In vivo labelling and immunoprecipitation indicated that newly synthesized -zein was degraded in tobacco seeds with a half-life of less than 1 hour.     

Correct targeting of the bean storage protein phaseolin in the seeds of transformed tobacco

The storage protein phaseolin accumulates during seed development in protein bodies in cotyledons of the common bean Phaseolus vulgaris. Hall et al. (In L Van Vloten-Doting, TC Hall, eds, Molecular Form and Function of the Plant Genome, 1985 Plenum Press, In press) recently reported the expression of a gene coding for phaseolin and the accumulation of phaseolin protein in developing seeds of tobacco plants regenerated from transformed callus cells. The protein did not accumulate in other organs of the plants. Mature seeds from normal and transformed tobacco plants were obtained and the subcellular distribution of phaseolin in the seeds was examined using both light and electron microscopic immunocytochemical methods. Phaseolin was found in six of seven transformed tobacco embryos examined, but was present in only one endosperm of five. When present, phaseolin was located exclusively in the protein bodies of the embryonic and endospermic cells. Furthermore, phaseolin was restricted solely to the amorphous matrix of the protein bodies and was excluded from the globoid and proteinaceous crystalloid components of these organelles. The subcellular location of phaseolin in seeds from transformed tobacco plants is similar to that seen in mature seeds of the common bean indicating that in the transformed cells the protein is targeted to the right subcellular compartment.     

Mannityl opine accumulation and exudation by transgenic tobacco

Three genes from the T_R region of pTi15955 were introduced into tobacco (Nicotiana tabacum L.) to direct the synthesis of the mannityl opines from hexose sugars and glutamine or glutamate. Opines were present in all tissue types tested and accumulated to levels of 100 to 150 micrograms per milligram dry weight in root, stem, and leaf tissues. Opine-producing plants appeared normal with respect to morphology and development. Transgenic plants grown for 60 days under sterile autotrophic conditions produced up to 540 micrograms of the mannityl opines per milligrams dry weight of tissue as root exudates. Opines were also detected in leaf and seed washes from soil-grown plants.     

Synthesis and accumulation of pea plastocyanin in transgenic tobacco plants

The pea plastocyanin gene in a 3.5 kbp Eco RI fragment of pea nuclear DNA was introduced into tobacco by Agrobacterium-mediated transformation. Regenerated plants contained pea plastocyanin located within the chloroplast thylakoid membrane system. Analysis of seedlings from a self-pollinated transgenic plant containing a single copy of the pea plastocyanin gene indicated that seedlings homozygous for the pea gene contained almost twice as much pea plastocyanin as seedlings hemizygous for the pea gene. Homozygous seedlings contained approximately equal amounts of pea and tobacco plastocyanins. The amount of tobacco plastocyanin in leaves of transgenic plants was unaffected by the expression of the pea plastocyanin gene. The mRNA from the pea gene in tobacco was indistinguishable by northern blotting and S1 nuclease protection from the mRNA found in pea. In both pea and transgenic tobacco, expression of the pea plastocyanin gene was induced by light in leaves but was suppressed in roots. Pea plastocyanin free of contaminating tobacco plastocyanin was purified from transgenic tobacco plants and shown to be indistinguishable from natural pea plastocyanin by N-terminal protein sequencing and ¹H NMR spectroscopy.     

Differential accumulation of four phaseolin glycoforms in transgenic tobacco

An intron-less phaseolin gene [15] was used to express phaseolin polypeptides in transgenic tobacco plants. The corresponding amounts of phaseolin immunoreactive polypeptides and mRNA were similar to those found in plants transformed with a bean genomic DNA sequence that encodes an identical -phaseolin subunit. These results justified the use of the intron-less gene for engineering of the phaseolin protein by oligonucleotide-directed mutagenesis. Each and both of the two Asn residues that serve as glycan acceptors in wild-type phaseolin were modified to prevent N-linked glycosylation. Wild-type (wt^i–) and mutant phaseolin glycoforms (dgly ₁, dgly ₂ and dgly _1,2) were localized to the protein body matrix by immunogold microscopy. Although quantitative slot-blot hybridization analysis showed similar levels of phaseolin mRNA in transgenic seed derived from all constructs, seed from the dgly ₁ and dgly ₂ mutations contained only 41% and 73% of that expressed from the wild-type control; even less (23%) was present in seed of plants transformed with the phaseolin dgly _1,2 gene. Additionally, the profile of 25–29 kDa processed peptides was different for each of the glycoforms, indicating that processing of the full-length phaseolin polypeptides was modified. Thus, although targeting of phaseolin to the protein body was not eliminated by removal of the glycan side-chains, decreased accumulation and stability of the full-length phaseolin protein in transgenic tobacco seed were evident.Abbreviations bp base pair(s) - DAF days after flowering - GUS -glucuronidase - kb kilobase - kDa kilodalton     

Type II fish antifreeze protein accumulation in transgenic tobacco does not confer frost resistance

Type II fish antifreeze protein (AFP) is active in both freezing point depression and the inhibition of ice recrystallization. This extensively disulfidebonded 14 kDa protein was targeted for accumulation in its pro and mature forms in the cytosol and apoplast of transgenic tobacco plants. Type II AFP gene constructs under control of a duplicate cauliflower mosaic virus 35S promoter, both with and without a native plant transit peptide sequence, were introduced into tobacco by Agrobacterium tumefaciensmediated transformation. AFP did not accumulate in the cytosol of transgenic plants, but active AFP was present as 2% of the total protein present in the apoplast. Plantproduced AFP was the same size as mature Type II AFP isolated from fish, and was comparable to wildtype AFP in thermal hysteresis activity and its effect on ice crystal morphology. Field trials conducted in late summer on R1 generation transgenic plants showed similar AFP accumulation in plants under field conditions at levels suitable for largescale production: but no difference in frost resistance was observed between transgenic and wildtype plants during the onset of early fall frosts.     

Colocalization of barley lectin and sporamin in vacuoles of transgenic tobacco plants.

Various targeting motifs have been identified for plant proteins delivered to the vacuole. For barley (Hordeum vulgare) lectin, a typical Gramineae lectin and defense-related protein, the vacuolar information is contained in a carboxyl-terminal propeptide. In contrast, the vacuolar targeting information of sporamin, a storage protein from the tuberous roots of the sweet potato (Ipomoea batatas), is encoded in an amino-terminal propeptide. Both proteins were expressed simultaneously in transgenic tobacco plants to enable analysis of their posttranslational processing and subcellular localization by pulse-chase labeling and electron-microscopic immunocytochemical methods. The pulse-chase experiments demonstrated that processing and delivery to the vacuole are not impaired by the simultaneous expression of barley lectin and sporamin. Both proteins were targeted quantitatively to the vacuole, indicating that the carboxyl-terminal and amino-terminal propeptides are equally recognized by the vacuolar protein-sorting machinery. Double-labeling experiments showed that barley lectin and sporamin accumulate in the same vacuole of transgenic tobacco (Nicotiana tabacum) leaf and root cells.     

N-Glycosylation of a mouse IgG expressed in transgenic tobacco plants

Since plants are emerging as an important system for the expression of recombinant glycoproteins, especially those intended for therapeutic purposes, it is important to scrutinize to what extent glycans harbored by mammalian glycoproteins produced in transgenic plants differ from their natural counterpart. We report here the first detailed analysis of the glycosylation of a functional mammalian glycoprotein expressed in a transgenic plant. The structures of the N-linked glycans attached to the heavy chains of the monoclonal antibody Guy's 13 produced in transgenic tobacco plants (plantibody Guy's 13) were identified and compared to those found in the corresponding IgG1 of murine origin. Both N-glycosylation sites located on the heavy chain of the plantibody Guy's 13 are N-glycosylated as in mouse. However, the number of Guy's 13 glycoforms is higher in the plant than in the mammalian expression system. Despite the high structural diversity of the plantibody N-glycans, glycosylation appears to be sufficient for the production of a soluble and biologically active IgG in the plant system. In addition to high-mannose-type N-glycans, 60% of the oligosaccharides N-linked to the plantibody have beta(1, 2)-xylose and alpha(1, 3)-fucose residues linked to the core Man3GlcNAc2. These plant-specific oligosaccharide structures are not a limitation to the use of plantibody Guy's 13 for topical immunotherapy. However, their immunogenicity may raise concerns for systemic applications of plantibodies in human.     

Characterisation of pea cytosolic glutathione reductase expressed in transgenic tobacco

 Expression in transgenic tobacco (Nicotiana tabacum L.) of a pea (Pisum sativum L.) GOR2 cDNA, encoding an isoform of glutathione reductase (GOR2), resulted in a 3- to 7-fold elevation of total foliar glutathione reductase (GR) activity. The enzyme encoded by GOR2 was confirmed to be extraplastidial in organelle fractionation studies and was considered most likely to be localised in the cytosol. A partial purification of GOR2 was achieved but a standard affinity chromatography step, using adenosine-2′,5′-diphosphate-Sepharose and often employed in the purification of GR from diverse sources, was unsuccessful with this isoform. Preparative isoelectric focussing was employed as part of the purification procedure of GOR2 and a complete separation from plastidial/mitochondrial glutathione reductase (GOR1) was achieved. The isoform GOR2 was shown to have a slower migration on non-denaturing polyacrylamide gels compared with GOR1 and properties typical of GR enzymes from plant sources. Received: 9 November 1999 / Accepted: 28 February 2000     

Immunogenicity of the capsid protein VP2 from porcine parvovirus expressed in low alkaloid transgenic tobacco

Porcine parvovirus is a widespread infectious viral disease with serious consequences to the reproductive health of swine. We have expressed the VP2 capsid protein of porcine parvovirus in the leaves of low alkaloid transgenic tobacco at approximately 0.3% of total soluble protein. Self-assembled virus-like particles were observed in planta by electron microscopy. Total soluble protein was extracted from the plant tissue and administered to mice by subcutaneous injection. An immune response was detected in these mice. The ability of serum antibodies to neutralize the infectivity of porcine parvovirus was further examined by a serum neutralization assay and was determined to be 1:2700–1:3900, a clear indication of the potential of VP2 expressed in plant material as a subunit vaccine against porcine parvovirus.     

A modified storage protein is synthesized,processed, and degraded in the seeds of transgenic plants

In vitro mutagenesis was used to supplement the sulfur amino acid codon content of a gene encoding -phaseolin, a Phaseolus vulgaris storage protein. The number of methionine codons in the phaseolin gene was increased from three to nine by insertion of a 45 base pair (bp) synthetic duplex. Either modified or normal phaseolin genes were integrated into the genome of tobacco plants through Agrobacterium tumefaciens-mediated transformation. Although similar levels of phaseolin RNA are detected in seeds of plants transformed with either the normal or modified (himet) gene, the quantity of himet protein is consistently much lower than normal -phaseolin. Himet phaseolin is expressed in a temporal- and organ-specific fashion, and is N-glycosylated and assembled into trimers in the manner of normal phaseolin. After germination, both types of phaseolin are hydrolyzed, but the himet protein is more quickly degraded. Electron microscopic immunocytochemical observations of developing seeds indicate that the himet protein is primarily localized in the endoplasmic reticulum (ER) and in Golgi apparatus secretion vesicles. Himet phaseolin is absent from protein storage vacuoles, termed protein bodies, where normal phaseolin is deposited in transgenic tobacco. We interpret the immunocytochemical data to indicate that himet phasolin is transported through the ER and Golgi apparatus and is then degraded in Golgi secretion vesicles or the protein bodies.Mention of trademark, proprietary product, or vendor does not constitute a guarantee of warranty of the product by the United States Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable. Publication No. 70 from Agrigenetics Advanced Science Company.     

The maize gamma-zein sequesters alpha-zein and stabilizes its accumulation in protein bodies of transgenic tobacco endosperm.

Zeins are seed storage proteins that form accretions called protein bodies in the rough endoplasmic reticulum of maize endosperm cells. Four types of zeins, alpha, beta, gamma, and delta, aggregate in a distinctive spatial pattern within the protein body. We created transgenic tobacco plants expressing alpha-zein, gamma-zein, or both to examine the interactions between these proteins leading to the formation of protein bodies in the endosperm. Whereas gamma-zein accumulated in seeds of these plants, stable accumulation of alpha-zein required simultaneous synthesis of gamma-zein. The zein proteins formed accretions in the endoplasmic reticulum similar to those in maize endosperm. Protein bodies were also found in protein storage vacuoles. The accumulation of both types of zeins peaked early in development and declined during maturation. Even in the presence of gamma-zein, there was a turnover of alpha-zein, suggesting that the interaction between the two proteins might be transitory. We suggest that gamma-zein plays an important role in protein body formation and demonstrate the utility of tobacco for studying interactions between different zeins.     

Amyloid-like protein inclusions in tobacco transgenic plants

The formation of insoluble protein deposits in human tissues is linked to the onset of more than 40 different disorders, ranging from dementia to diabetes. In these diseases, the proteins usually self-assemble into ordered β-sheet enriched aggregates known as amyloid fibrils. Here we study the structure of the inclusions formed by maize transglutaminase (TGZ) in the chloroplasts of tobacco transplastomic plants and demonstrate that they have an amyloid-like nature. Together with the evidence of amyloid structures in bacteria and fungi our data argue that amyloid formation is likely a ubiquitous process occurring across the different kingdoms of life. The discovery of amyloid conformations inside inclusions of genetically modified plants might have implications regarding their use for human applications.