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Cellobiohydrolase II (CBH II) is an exo-glucanase that is part of a fungal mixture of enzymes from a wood-rot fungus, Trichoderma reesei. It is therefore difficult to purify and to establish a specific activity assay. The gene for this enzyme, driven by the rice Os glutelin promoter, was transformed into High II tissue culture competent corn, and the enzyme accumulated in the endosperm of the seed. The transgenic line recovered from tissue culture was bred into male and female elite Stine inbred corn lines, stiff stalk 16083-025 (female) and Lancaster MSO411 (male), for future production in their hybrid. The enzyme increases its accumulation throughout its 6 generations of back crosses, 27-266-fold between T1 and T2, and 2-10-fold between T2 and T3 generations with lesser increases in T4-T6. The germplasm of the inbred lines replaces the tissue culture corn variety germplasm with each generation, with the ultimate goal of producing a high-yielding hybrid with the transgene. The CBH II enzyme was purified from T5 inbred male grain 10-fold to homogeneity with 47.5% recovery. The specific activity was determined to be 1.544 units per µg protein. The corn-derived CBH II works in biopolishing of cotton by removing surface fibers to improve dyeability and increasing glucose from corn flour for increasing ethanol yield from starch-based first-generation processes., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Background: Enzymes are biocatalysts that play a vital role in the production of biomolecules. Plants can be a valuable and cost-effective source for producing well-structured recombinant enzymes. Glucose is one of the most important biological molecules, providing energy to most living systems. An electrochemical method for immobilization of enzyme is promising because it is economic, generates less component waste, improves the signal-to-noise ratio, leads to a lower limit of detection, and stabilizes and protects the enzyme structure., Results: A glucose biosensor was constructed using polyaniline (PANI) and a recombinant enzyme from corn, plant-produced manganese peroxidase (PPMP), with polymerization of aniline as a monomer in the presence of gold nanoparticles (AuNPs)-glucose oxidase (GOx), and bovine serum albumin. Using linear sweep voltammetry and cyclic voltammetry techniques, PANI-AuNPs-GOx-PPMP/Au electrode exhibited a superior sensing property with a wider linear range of 0.005-16.0 mm, and a lower detection limit of 0.001 mm compared to PANI-GOx-PPMP/Au electrode and PANI-GOx-PPMP/AuNPs/Au electrode. The biosensor selectivity was assessed by determining glucose concentrations in the presence of ascorbic acid, dopamine, aspartame, and caffeine., Conclusion: We conclude that a plant-produced Mn peroxidase enzyme combined with conductive polymers and AuNPs results in a promising nanocomposite biosensor for detecting glucose. The use of such devices for quality control in the food industry can have a significant economic impact. © 2022 Society of Chemical Industry., (© 2022 Society of Chemical Industry.)

Expression of recombinant proteins in plants is a technology for producing vaccines, pharmaceuticals and industrial enzymes. For the past several years, we have produced recombinant proteins in maize kernels using only the embryo, primarily driving expression of foreign genes with the maize globulin-1 promoter. Although strong expression is obtained, these lines use only 10-12% of the seed tissue. If strong embryo expression could be combined with strong endosperm expression, much more recombinant protein could be recovered from a set amount of seed biomass. In this study, we tested three endosperm promoters for expression of a cellulase gene. Promoters tested were rice globulin and glutelin promoters and a maize 19 kDa α-zein promoter. The rice promoters were used in two tandem expression constructs as well. Although the rice promoters were active in producing stable amounts of cellulase, the α-zein promoter was by far the most effective: as much as 9% of total soluble protein was recovered from seed of several independent events and plants. One or two inserts were detected by Southern blot in several lines, indicating that copy number did not appear to be responsible for the differences in protein accumulation. Tissue print analysis indicated that expression was primarily in the endosperm.

To be commercialized and grown in the US, genetically engineered (GE) crops typically go through an extensive food, feed, and environmental safety assessment process which, in certain instances, requires complex consultations with three different US regulatory agencies. Many small market, niche, and specialty crops have been genetically engineered using the modern tools of recombinant DNA but few have been commercialized due to real or perceived regulatory constraints. This workshop discussed the practical aspects of developing dossiers on GE specialty, niche, or small-market crops/products for submission to US regulatory agencies. This workshop focused on actual case studies, and provided an opportunity for public or private sector scientists and crop developers to spend time with regulatory officials to learn the specifics of compiling a dossier for regulatory approval. The objective of the workshop was to explain and demystify data requirements and regulatory dossier compilation by small companies, academics, and other developers.

Loblolly pine residues have enormous potential to be the raw material for advanced biofuel production due to extensive sources and high cellulose content. Hot water (HW) pretreatment, while being a relatively economical and clean technology for the deconstruction of lignocellulosic biomass, could also inhibit the ensuing enzymatic hydrolysis process because of the production of inhibitors. In this study, we investigated the effect of oligosaccharide fractions purified from HW pre-hydrolyzate of pinewood using centrifugal partition chromatography (CPC) on three recombinant cellulolytic enzymes (E1, CBHI and CBHII), which were expressed in the transgenic corn grain system. The efficiency of recombinant enzymes was measured using either a 4-methylumbelliferyl-β-D-cellobioside (MUC) or a cellulose-dinitrosalicylic acid (DNS) assay system. The results showed that HW pre-hydrolyzate CPC fractions contain phenolics, furans, and monomeric and oligomeric sugars. Among CPC fractions, oligomers composed of xylan, galactan, and mannan were inhibitory to the three recombinant enzymes and to the commercial cellulase cocktail, reducing the enzymatic efficiency to as low as 10%.

Plant cell wall degradation into fermentable sugars by cellulases is one of the greatest barriers to biofuel production. Expansin protein loosens the plant cell wall by opening up the complex of cellulose microfibrils and polysaccharide matrix components thereby increasing its accessibility to cellulases. We over-expressed cucumber expansin in maize kernels to produce enough protein to assess its potential to serve as an industrial enzyme for applications particularly in biomass conversion. We used the globulin-1 embryo-preferred promoter to express the cucumber expansin gene in maize seed. Expansin protein was targeted to one of three sub-cellular locations: the cell wall, the vacuole, or the endoplasmic reticulum (ER). To assess the level of expansin accumulation in seeds of transgenic kernels, a high throughput expansin assay was developed. The highest expressing plants were chosen and enriched crude expansin extract from those plants was tested for synergistic effects with cellulase on several lignocellulosic substrates. Activity of recombinant cucumber expansin from transgenic kernels was confirmed on these pretreated substrates. The best transgenic lines (ER-targeted) can now be used for breeding to increase expansin expression for use in the biomass conversion industry. Results of these experiments show the success of expansin over-expression and accumulation in transgenic maize seed without negative impact on growth and development and confirm its synergistic effect with cellulase on deconstruction of complex cell wall substrates.

This review is a short synopsis of some of the latest breakthroughs in the areas of lignocellulosic conversion to fuels and utilization of oils for biodiesel. Although four lignocellulosic ethanol factories have opened in the USA and hundreds of biodiesel installations are active worldwide, technological improvements are being discovered that will rapidly evolve the biofuels industry into a new paradigm. These discoveries involve the feedstocks as well as the technologies to process them.

Transgenic plants in the US and abroad generated using genetic engineering technology are regulated with respect to release into the environment and inclusion into diets of humans and animals. For crops incorporating pharmaceuticals or industrial enzymes regulations are even more stringent. Notifications are not allowed for movement and release, therefore a permit is required. However, growing under permit is cumbersome and more expensive than open, non- regulated growth. Thus, when the genetically engineered pharmaceutical or industrial crop is ready for scale-up, achieving non-regulated status is critical. Regulatory compliance in the US comprises petitioning the appropriate agencies for permission for environmental release and feeding trials. For release without yearly permits, a petition for allowing non-regulated status can be filed with the United States Department of Agriculture with consultations that include the Food and Drug Administration and possibly the Environmental Protection Agency, the latter if the plant includes an incorporated pesticide. The data package should ensure that the plants are substantially equivalent in every parameter except for the engineered trait. We undertook a preliminary study on transgenic maize field-grown hybrids that express one of two cellulase genes, an exo-cellulase or an endo-cellulase. We performed field observations of whole plants and numerous in vitro analyses of grain. Although some minor differences were observed when comparing genetically engineered hybrid plants to control wild type hybrids, no significant differences were seen.

The corn grain biofactory was used to produce Cel7A, an exo-cellulase (cellobiohydrolase I) from Hypocrea jecorina. The enzymatic activity on small molecule substrates was equivalent to its fungal counterpart. The corn grain-derived enzyme is glycosylated and 6 kDa smaller than the native fungal protein, likely due to more sugars added in the glycosylation of the fungal enzyme. Our data suggest that corn seed-derived cellobiohydrolase (CBH) I performs as well as or better than its fungal counterpart in releasing sugars from complex substrates such as pre-treated corn stover or wood. This recombinant protein product can enter and expand current reagent enzyme markets as well as create new markets in textile or pulp processing. The purified protein is now available commercially.

The technology of converting lignocellulose to biofuels has advanced swiftly over the past few years, and enzymes are a significant constituent of this technology. In this regard, cost effective production of cellulases has been the focus of research for many years. One approach to reach cost targets of these enzymes involves the use of plants as bio-factories. The application of this technology to plant biomass conversion for biofuels and biobased products has the potential for significantly lowering the cost of these products due to lower enzyme production costs. Cel6A, one of the two cellobiohydrolases (CBH II) produced by Hypocrea jecorina, is an exoglucanase that cleaves primarily cellobiose units from the non-reducing end of cellulose microfibrils. In this work we describe the expression of Cel6A in maize endosperm as part of the process to lower the cost of this dominant enzyme for the bioconversion process. The enzyme is active on microcrystalline cellulose as exponential microbial growth was observed in the mixture of cellulose, cellulases, yeast and Cel6A, Cel7A (endoglucanase), and Cel5A (cellobiohydrolase I) expressed in maize seeds. We quantify the amount accumulated and the activity of the enzyme. Cel6A expressed in maize endosperm was purified to homogeneity and verified using peptide mass finger printing.

Background: It is important for industries to find green chemistries for manufacturing their products that have utility, are cost-effective and that protect the environment. The paper industry is no exception. Renewable resources derived from plant components could be an excellent substitute for the chemicals that are currently used as paper binders. Air laid pressed paper products that are typically used in wet wipes must be bound together so they can resist mechanical tearing during storage and use. The binders must be strong but cost-effective. Although chemical binders are approved by the Environmental Protection Agency, the public is demanding products with lower carbon footprints and that are derived from renewable sources., Results: In this project, carbohydrates, proteins and phenolic compounds were applied to air laid, pressed paper products in order to identify potential renewable green binders that are as strong as the current commercial binders, while being organic and renewable. Each potential green binder was applied to several filter paper strips and tested for strength in the direction perpendicular to the cellulose fibril orientation. Out of the twenty binders surveyed, soy protein, gelatin, zein protein, pectin and Salix lignin provided comparable strength results to a currently employed chemical binder., Conclusions: These organic and renewable binders can be purchased in large quantities at low cost, require minimal reaction time and do not form viscous solutions that would clog sprayers, characteristics that make them attractive to the non-woven paper industry. As with any new process, a large-scale trial must be conducted along with an economic analysis of the procedure. However, because multiple examples of "green" binders were found that showed strong cross-linking activity, a candidate for commercial application will likely be found.

Background: Maize is one of the most important crops in the world. With the exponentially increasing population and the need for ever increased food and feed production, an increased yield of maize grain (as well as rice, wheat and other grains) will be critical. Maize grain development is understood from the perspective of morphology, hormone responses, and storage reserve accumulation. This includes various studies on gene expression during embryo development and maturation but a global study of gene expression of the embryo has not been possible until recently. Transcriptome analysis is a powerful new tool that can be used to understand the genetic basis of embryo maturation., Results: We undertook a transcriptomic analysis of normal maturing embryos at 15, 21 and 27 days after pollination (DAP), of one elite maize germplasm line that was utilized in crosses to transgenic plants. More than 19,000 genes were analyzed by this method and the challenge was to select subsets of genes that are vitally important to embryo development and maturation for the initial analysis. We describe the changes in expression for genes relating to primary metabolic pathways, DNA synthesis, late embryogenesis proteins and embryo storage proteins, shown through transcriptome analysis and confirmed levels of transcription for some genes in the transcriptome using qRT-PCR., Conclusions: Numerous genes involved in embryo maturation have been identified, many of which show changes in expression level during the progression from 15 to 27 DAP. An expected array of genes involved in primary metabolism was identified. Moreover, more than 30% of transcripts represented un-annotated genes, leaving many functions to be discovered. Of particular interest are the storage protein genes, globulin-1, globulin-2 and an unidentified cupin family gene. When expressing foreign proteins in maize, the globulin-1 promoter is most often used, but this cupin family gene has much higher expression and may be a better candidate for foreign gene expression in maize embryos. Results such as these allow identification of candidate genes and promoters that may not otherwise be available for use. mRNA seq data archived in NCBI SRA; Accession number: ACC=SRA060791 subid=108584.

In this article, the general principles of genetically modified (GM) plant risk assessment and the regulatory framework for contained use and open field production of plant-made pharmaceuticals/plant-made industrials (PMP/PMI) are described. While significant progress has been made for the containment grown (plant cell culture) production of PMPs, with the first regulatory approval made by the FDA in 2012, the commercialization of medicinal or industrial products produced in the field has yet to emerge in either Europe or the US. In the current paper, we discribe the regulatory environment in Europe and the US surrounding GM crops, and provide case studies for experimental field releases of PMP and PMI producing plants in both regions. Suggestions for reducing the regulatory burden for GM plants will be discussed, also in light of the emerging new technologies to modify the genetics of plants. Since regulations surrounding the commercialization of GM crops are very costly and not appropriate for most of the PMP/PMI applications in Europe, we propose that amendments to the EU Directive 2001/18/EC are necessary to allow for the commercialization of products from GM plants without the need of an 'authorization'. To fully acknowledge the overall outcome of adopting plants to produce PMP/PMI, the conclusion is that broader and more balanced legislative oversight is needed in Europe; while specific legislation is not needed in the US.

Using plants as biofactories for industrial enzymes is a developing technology. The application of this technology to plant biomass conversion for biofuels and biobased products has potential for significantly lowering the cost of these products because of lower enzyme production costs. However, the concentration of the enzymes in plant tissue must be high to realize this goal. We describe the enhancement of the accumulation of cellulases in transgenic maize seed as a part of the process to lower the cost of these dominant enzymes for the bioconversion process. We have used breeding to move these genes into elite and high oil germplasm to enhance protein accumulation in grain. We have also explored processing of the grain to isolate the germ, which preferentially contains the enzymes, to further enhance recovery of enzyme on a dry weight basis of raw materials. The enzymes are active on microcrystalline cellulose to release glucose and cellobiose., (© 2011 The Authors. Plant Biotechnology Journal © 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.)

In a recent presentation at the 2010 International Association for Plant Biotechnology meeting, Dr. Richard Flavell (Ceres, Malibu, CA, USA) motivated the plant community to act quickly and with purpose to move a multitude of traits into crop plants to improve their productivity. Current progress toward understanding of plants is too slow and will not achieve our communal goal of doubling agricultural productivity by 2050. Major breakthroughs are necessary! Thus, high-throughput methods that couple gene identification and phenotype observations are required to put potential products into the hands of plant breeders to make varieties with good agronomic characteristics that will be approved by the regulatory agencies. These first improved crops must be on the market in the next 10 years, according to Flavell, in order to begin to meet our doubled productivity goals in 30 years. Because it takes approximately 10 years to produce a characterized variety from an identified gene and move it through product development and regulatory approval, we must begin now. Presumably, by employing the techniques in the following -chapters, we can do that.

Objective: To determine the utility of re-testing pregnant patients0.05 meaning that having a negative initial screen was not predictive of also having a negative result upon re-testing at 34-36 weeks., Conclusion: First trimester Chlamydia trachomatis test results are not predictive of Chlamydia trachomatis status during the third trimester.

Ethanol from lignocellulosic biomass is being pursued as an alternative to petroleum-based transportation fuels. To succeed in this endeavour, efficient digestion of cellulose into monomeric sugar streams is a key step. Current production systems for cellulase enzymes, i.e. fungi and bacteria, cannot meet the cost and huge volume requirements of this commodity-based industry. Transgenic maize (Zea mays L.) seed containing cellulase protein in embryo tissue, with protein localized to the endoplasmic reticulum, cell wall or vacuole, allows the recovery of commercial amounts of enzyme. E1 cellulase, an endo-beta-1,4-glucanase from Acidothermus cellulolyticus, was recovered at levels greater than 16% total soluble protein (TSP) in single seed. More significantly, cellobiohydrolase I (CBH I), an exocellulase from Trichoderma reesei, also accumulated to levels greater than 16% TSP in single seed, nearly 1000-fold higher than the expression in any other plant reported in the literature. The catalytic domain was the dominant form of E1 that was detected in the endoplasmic reticulum and vacuole, whereas CBH I holoenzyme was present in the cell wall. With one exception, individual transgenic events contained single inserts. Recovery of high levels of enzyme in T2 ears demonstrated that expression is likely to be stable over multiple generations. The enzymes were active in cleaving soluble substrate.

Manganese peroxidase (MnP) has been implicated in lignin degradation and thus has potential applications in pulp and paper bleaching, enzymatic remediation and the textile industry. Transgenic plants are an emerging protein expression platform that offer many advantages over traditional systems, in particular their potential for large-scale industrial enzyme production. Several plant expression vectors were created to evaluate the accumulation of MnP from the wood-rot fungus Phanerochaete chrysosporium in maize seed. We showed that cell wall targeting yielded full-length MnP, whereas cytoplasmic localization resulted in multiple truncated peroxidase polypeptides as detected by immunoblot analysis. In addition, the use of a seed-preferred promoter dramatically increased the expression levels and reduced the negative effects on plant health. Multiple independent transgenic lines were backcrossed with elite inbred corn lines for several generations with the maintenance of high-level expression, indicating genetic stability of the transgene.

The availability of foods low in sugar content yet high in flavour is critically important to millions of individuals conscious of carbohydrate intake for diabetic or dietetic purposes. Brazzein is a sweet protein occurring naturally in a tropical plant that is impractical to produce economically on a large scale, thus limiting its availability for food products. We report here the use of a maize expression system for the production of this naturally sweet protein. High expression of brazzein was obtained, with accumulation of up to 4% total soluble protein in maize seed. Purified corn brazzein possessed a sweetness intensity of up to 1200 times that of sucrose on a per weight basis. In addition, application tests demonstrated that brazzein-containing maize germ flour could be used directly in food applications, providing product sweetness. These results demonstrate that high-intensity sweet protein engineered into food products can give sweetener attributes useful in the food industry.

The maize polyubiquitin-1 (Ubi-1) promoter is one of a few select promoters used to express foreign genes in monocots, such that recombinant proteins can be produced at commercially viable levels. Modifying the activity, specificity and responsiveness of such promoters provides a means to achieve desired levels and patterns of expression of genes encoding target products. Ubi-1 is constitutively expressed but is further induced by heat shock. The promoter contains two overlapping sequences with similarity to defined heat shock elements and we show that these sequences are also present upstream of the Ubi-1 homologue isolated from teosinte. Both the maize and teosinte promoters can mediate a heat shock response in transgenic maize. We have dissected the overlapping maize Ubi-1 promoter heat shock elements and demonstrate that the 3' element is required to mediate a heat shock response. The Ubi-1 promoter is particularly active in tissues consisting of rapidly dividing cells, and within the seed it is strongly biased towards driving expression in the embryo. However, replacement of the heat shock elements with a trimer of a basic domain/leucine zipper factor binding site of a pea lectin promoter shifts the balance in seed expression towards the endosperm. The Ubi-1 variants described here differ in their overall activity in the seed, but they all show potential for driving high levels of heterologous gene expression in maize.

A recent publication by R. Chikwamba and colleagues highlights interesting issues in recombinant protein expression in transgenic plants. In the study they expressed a bacterial antigen in maize seed and obtained aberrant localization data. This work is of great importance to the biotechnology industry and raises fascinating questions in plant cell biology that require creative thinking.

Lignolytic enzymes such as laccase have been difficult to over-express in an active form. This paper describes the expression, characterization, and application of a fungal laccase in maize seed. The transgenic seed contains immobilized and extractable laccase. Fifty ppm dry weight of aqueously extractable laccase was obtained, and the remaining solids contained a significant amount of immobilized laccase that was active. Although a portion of the extractable laccase was produced as inactive apoenzyme, laccase activity was recovered by treatment with copper and chloride. In addition to allowing the apoenzyme to regain activity, treatment with copper also provided a partial purification step by precipitating other endogenous corn proteins while leaving >90% of the laccase in solution. The data also demonstrate the application of maize-produced laccase as a polymerization agent. The apparent concentration of laccase in ground, defatted corn germ is approximately 0.20% of dry weight.

Bovine trypsin (EC 3.4.21.4) is an enzyme that is widely used for commercial purposes to digest or process other proteins, including some therapeutic proteins. The biopharmaceutical industry is trying to eliminate animal-derived proteins from manufacturing processes due to the possible contamination of these products by human pathogens. Recombinant trypsin has been produced in a number of systems, including cell culture, bacteria and yeast. To date, these expression systems have not produced trypsin on a scale sufficient to fulfill the need of biopharmaceutical manufacturers where kilogram quantities are often required. The present paper describes commercial-level production of trypsin in transgenic maize (Zea mays) and its physical and functional characterization. This protease, the first enzyme to be produced on a large-scale using transgenic plant technology, is functionally equivalent to native bovine pancreatic trypsin. The availability of this reagent should allow for the replacement of animal-derived trypsin in the processing of pharmaceutical proteins.

Generation of transgenic plants exhibiting traits of interest requires the marriage of several technologies including gene transfer, selection and plant regeneration. Variety is the driver for these technologies because of the breadth of plant species requiring modification. A new selectable marker gene, pflp, has been applied to the recovery of orchid plants that exhibit resistance to a major bacterial disease that plagues the orchid industry. pflp as a selection system might be adaptable to many crops.

Expression of industrial enzymes in transgenic plants offers an alternative system to fungal fermentation for large-scale production. Very high levels of expression are required to make the enzymes cost-effective. We tested several parameters to determine the best method for achieving high levels of expression for a fungal laccase gene. Transgenic maize plants were generated using an Agrobacterium-mediated system. The molecular parameters that induced the highest expression were the maize embryo-preferred globulin 1 promoter and targeting of the protein to the cell wall. Two independent transgenic events that yielded multiple clonal plants were characterized in detail. Independent transgenic events 01 and 03 contained two or one copies of T-DNA, respectively. Plants derived from a single transgenic event varied in expression level, and the variation in expression levels was heritable. Within the seed, expression in these plants was primarily within the embryo, and was associated with seed browning and limited germination. High oil germplasm was used to increase germination, as well as to assist in increasing expression 20-fold in five generations through breeding and selection.

The synthesis of selected antigens in plants and their oral delivery has great potential for reducing the costs of vaccine production and administration. The application of this technology requires antigen concentrations in final plant material to be uniform to ensure consistent dosing. In addition, antigen levels should be such as to allow the volume of each dose, containing a set amount of antigen, to be practical for oral delivery. Here, we demonstrate that the Lt-B protein of enterotoxigenic E. coli is evenly distributed in defatted corn germ prepared from transgenic grain. Furthermore, the choice of sub-cellular location for Lt-B affects accumulation of the protein in excess of four orders of magnitude.

The use of recombinant gene technologies by the vaccine industry has revolutionized the way antigens are generated, and has provided safer, more effective means of protecting animals and humans against bacterial and viral pathogens. Viral and bacterial antigens for recombinant subunit vaccines have been produced in a variety of organisms. Transgenic plants are now recognized as legitimate sources for these proteins, especially in the developing area of oral vaccines, because antigens have been shown to be correctly processed in plants into forms that elicit immune responses when fed to animals or humans. Antigens expressed in maize (Zea mays) are particularly attractive since they can be deposited in the natural storage vessel, the corn seed, and can be conveniently delivered to any organism that consumes grain. We have previously demonstrated high level expression of the B-subunit of Escherichia coli heat-labile enterotoxin and the spike protein of swine transmissible gastroenteritis in corn, and have demonstrated that these antigens delivered in the seed elicit protective immune responses. Here we provide additional data to support the potency, efficacy, and stability of recombinant subunit vaccines delivered in maize seed.

The number and types of antibodies expressed in plants has increased steadily since the first reports of this accomplishment in the 1980s, illustrating the versatility of plants as a production system for antibodies. Many recent reviews have detailed the antibody forms that have been derived from plant expression systems. This contribution focuses on the remaining challenges to develop plant-derived therapeutic antibodies into products, and some of the progress that is being made in addressing these challenges.

The telomerase reverse transcriptase can recognize broken chromosome ends and add new telomeres de novo in a reaction termed "chromosome healing". Here we investigate new telomere formation in vitro by telomerases from a variety of flowering plant species. Comparing the electrophoretic mobilities and nucleotide sequences of the products, we uncovered three different modes of new telomere formation. The soybean telomerase, designated a Class I enzyme, only elongated DNA primers ending in telomeric nucleotides. Arabidopsis and maize telomerases, designated Class II enzymes, efficiently extended completely non-telomeric sequences by positioning the 3' terminus at a preferred site on the RNA template. Silene latifolia and sorghum telomerases constituted class III enzymes that elongated non-telomeric DNA primers by annealing them at alternative sites on the RNA template. For all enzymes, errors were prevalent during synthesis of the first two repeats, likely reflecting lateral instability of the primer 3' terminus on the template during the initial rounds of elongation. Class III telomerases, however, were five- to 13-fold more error prone than class II, generating more mistakes in distal repeats added to the primers. This remarkable variability in enzyme-DNA interactions among plant telomerases does not reflect phylogenetic relationships, and therefore implies that the telomerase active site can evolve rapidly.

Numerous studies have shown that viral epitopes and subunits of bacterial toxins can be expressed and correctly processed in transgenic plants. The recombinant proteins induce immune responses and have several benefits over current vaccine technologies, including increased safety, economy, stability, versatility and efficacy. Antigens expressed in corn are particularly advantageous since the seed can be produced in vast quantities and shipped over long distances at ambient temperature, potentially allowing global vaccination. We have expressed the B-subunit of Escherichia coli heat-labile enterotoxin and the spike protein of swine transmissible gastroenteritis virus at high levels in corn, and demonstrate that these antigens delivered in the seed elicit protective immune responses.

Foreign protein production in transgenic plants has been successful, from the generation of transgenic plant lines to the marketing of purified proteins. Antigenic proteins from disease organisms, monoclonal antibodies raised against antigens of disease organisms, and proteins with industrial process applications have been produced and tested. For vaccines, clinical trials in humans and feeding trials in animals are in progress to demonstrate their efficacy. For industrial proteins, high expression and downstream processing efficiency are key concerns, with application and test market trials in progress.

Recombinant egg white avidin and bacterial B-glucuronidase (GUS) from transgenic maize have been commercially produced. High levels of expression were obtained in seed by employing the ubiquitin promoter from maize. The recombinant proteins had activities that were indistinguishable from their native counterparts. We have illustrated that down-stream activities in the production of these recombinant proteins, such as stabilizing the germplasm and processing for purification, were accomplished without any major obstacles. Avidin (A8706) and GUS (G2035) are currently marketed by Sigma Chemical Co.

The tools of plant biotechnology that have been developed to improve agronomic traits are now being applied to generate recombinant protein products for the food, feed, and pharmaceutical industry. This study addresses several processing and protein recovery issues that are relevant to utilizing transgenic corn as a protein production system. The gus gene coding for beta-glucuronidase (rGUS) was stably integrated and expressed over four generations. The accumulation level of rGUS reached 0.4% of total extractable protein. Within the kernel, rGUS was preferentially accumulated in the germ even though a constitutive ubiquitin promoter was used to direct gus expression. Fourth-generation transgenic seed was used to investigate the effect of seed processing on the activity and the recovery of rGUS. Transgenic seed containing rGUS could be stored at an ambient temperature for up to two weeks and for at least three months at 10 degrees C without a significant loss of enzyme activity. rGUS exposed to dry heat was more stable in ground than in whole kernels. The enzyme stability was correlated with the moisture loss of the samples during the heating. Transgenic seed was dry-milled, fractionated, and hexane extracted to produce full-fat and defatted germ fractions. The results of the aqueous extraction of rGUS from ground kernels, full-fat germ, and defatted-germ samples revealed that approximately 10 times more rGUS per gram of solids could be extracted from the ground full-fat germ and defatted-germ than from the kernel samples. The extraction of corn oil from ground germ with hot hexane (60 degrees C) did not affect the extractable rGUS activity. rGUS was purified from ground kernels and full-fat germ extracts by ion exchange, hydrophobic interaction, and size exclusion chromatography. Similar purity and yield of rGUS were obtained from both extracts. Biochemical properties of rGUS purified from transgenic corn seed were similar to those of E. coli GUS., (Copyright 1998 John Wiley & Sons, Inc.)

This study reports the production, purification, and characterization of recombinant Escherichia coli beta-glucuronidase (GUS) and chicken egg-white avidin from transgenic corn seed. The avidin and gus genes were stably integrated in the genome and expressed over seven generations. The accumulation levels of avidin and GUS in corn kernel were 5.7% and 0.7% of extractable protein, respectively. Within the kernel, avidin and GUS accumulation was mainly localized to the germ, indicating possible tissue preference of the ubiquitin promoter. The storage-stability studies demonstrated that processed transgenic seed containing GUS or avidin can be stored at 10 degrees C for at least 3 months and at 25 degrees C for up to 2 weeks without a significant loss of activity. The heat-stability experiments indicated that GUS and avidin in the whole kernels were stable at 50 degrees C for up to 1 week. The buffer composition also had an affect on the aqueous extraction of avidin and GUS from ground kernels. Avidin was purified in one step by using 2-iminobiotin agarose, whereas GUS was purified in four steps consisting of adsorption, ion-exchange, hydrophobic interaction, and size-exclusion chromatography. Biochemical properties of purified avidin and GUS were similar to those of the respective native proteins.

Amaranthus includes approximately 60 species, of which three are cultivated as a grain source. Many wild Amaranthus species possess agriculturally desirable traits such as drought and salt tolerance, and pathogen resistance. We examined relationships among wild and cultivated Amaranthus species based upon restriction-site variation in two chloroplast DNA regions and in a nuclear DNA region. The chloroplast regions consisted of (1) an intergenic spacer in transfer RNA genes and (2) the ribulose-1,5-bisphosphate carboxylase gene with a flanking open reading frame. The nuclear region was the internal transcribed spacers ITS-1 and ITS-2 flanking the 5.8S gene in the ribosomal DNA. These regions were amplified by the polymerase chain reaction and digested with a total of 38 restriction endonucleases. We detected 11 potentially informative restriction-site mutations and seven length-polymorphisms among the 28 Amaranthus species. Parsimony analysis was used to find the shortest tree for each separate data set (chloroplast, nuclear, and length) and for two combined matrices (chloroplast/nuclear and all data sets). Overall, there was a low level of variation which generated poorly resolved trees among the 28 species. Congruence analyses revealed that the chloroplast and nuclear data sets were congruent with each other but not to the length data set. The congruence of the chloroplast and nuclear data sets suggested that cytoplasmic gene flow may not be a confounding factor in our analyses. The phylogeny also suggested that drought tolerance evolved independently several times. The molecular phylogeny provides a basis for selection of species pairs for crop development.

This study concerned the developmental regulation of wall-localized, hydroxyproline-containing proteins in maize tissues and organs. Silk and pericarp cell walls contained more peptidyl hydroxyproline than did walls of any vegetative tissue, although all tissues and organs accumulated these proteins as they matured. In many tissues, hydroxyproline-rich proteins are first associated with the wall in a soluble form before being insolubilized through covalent attachment to the matrix. Because hydroxyproline was more soluble earlier than later in development, it appears that insolubilization was occurring in maize tissues and organs as well. Tissue prints reacted with an anti-extensin antibody gave positive results, indicating the presence of a soluble form of this common hydroxyproline-rich glycoprotein (HRGP). Silk and pericarp cells actively synthesized this extensin from abundant transcripts. In vegetative tissues, extensin transcripts were somewhat more abundant in seedlings than in pre-anthesis or mature plants, but levels were much lower than in silk and pericarp. Southern blots of maize genomic DNA indicated that these extensin transcripts are encoded by a small multigene family. Potential roles for extensin in reproductive/protective tissues versus the embryo or vegetative tissues are suggested.

This study concerned the molecular basis for the protein size heterogeneity of extensin from two maize (Zea mays L.) varieties. We studied the physical properties of extensin, a hydroxyproline-rich glycoprotein (HRGP), from the silk and pericarp of Golden X Bantam (GXB) sweet corn and Japanese Hulless (JHL) popcorn. Extensin from GXB has a molecular mass of 66 kDa whereas extensins from JHL have molecular masses of 76 and 66 kDa. Treatment with anhydrous hydrogen fluoride to deglycosylate proteins reduced the size of all extensins by 5 kDa. Probing with a 500 bp fragment from a genomic clone of maize extensin identified two transcripts (1.9 and 1.5 kb) on northern blots. JHL contained both transcripts and GXB contained only the 1.5 kb transcript. The probe also hybridized to two larger transcripts (6.2 and 4.5 kb) that were found in both varieties. We immunoprecipitated two proteins (66 and 56 kDa) from translated RNA isolated from JHL and one protein (56 kDa) from GXB. These results demonstrate that these extensins differ in the size of their peptide moiety and not in their extent of glycosylation.

In an effort to understand the role of hydroxyproline-rich glycoproteins (HRGPs) in plant cell wall structure, we studied the distribution and physical properties of PC-1-like proteins (PC-1 being the major pericarp HRGP) throughout sporophytic tissues of two maize (Zea mays L.) varieties. We determined total amounts of hydroxyproline, an indicator of HRGPs, and did tissue print and Western blot analysis. We found hydroxyproline in cell walls of stems, leaves, roots, tassels, and silks. We also observed reactivity of anti-PC-1 monoclonal antibodies with anatomical prints of these tissues on nitrocellulose paper. Stem nodes and silks contained the most hydroxyproline and exhibited the strongest reaction with the antibody. PC-1 was localized in vascular bundles and the epidermis of stem tissue. However, localization to a specific cell type in the silk could not be determined at the resolution of the tissue print. The stem node protein had the same electrophoretic mobility as the pericarp protein as determined on Western blots prepared from cationic neutral gels. Protein extracts from silk tissues of both varieties studied contained one protein of the same size/charge as that found in pericarp, as well as some minor variant bands. The data presented here document that cell wall proteins are present in many tissues of the maize plant, although they are primarily in cell types contributing to support.

The purpose of this study was to investigate the genetic variation of susceptibility to Agrobacterium within a Picea abies population. Tumor formation was studied in 16 open-pollinated families belonging to a central Swedish population of Picea abies. Strain A281 of Agrobacterium tumefaciens was used to infect 3-monthold seedlings in a five-block greenhouse experiment. The analysis of variance showed strong significance for the between-family variation of tumor-formation percentages, varying from 28% to 73%. The most susceptible material will be suitable for experiments on the production of transgenic plants in vitro using disarmed Agrobacterium strains.

The hypervirulent Agrobacterium tumefaciens strain A281 formed frequent tumors (31%) on Picea abies (Norway spruce), an economically important tree species in Swedish forests. Three-month-old seedlings were inoculated and tumors were established that grew hormone-independently in culture. Tumors contained agropine and mannopine/mannopinic acid as determined by acid pH paper electrophoresis. In addition, DNA hybridization studies showed that the DNA from these tumor lines contained sequences homologous to Ti plasmid T-DNA, whereas wild-type spruce seedling DNA did not. These results suggest that Agrobacterium vectors can be used for gene transfer into this important forest species.

We have studied the accumulation of peptidyl hydroxyproline in the pericarp of developing maize (Zea mays L., Golden cross Bantam sweet corn) kernels. Although this hydroxyproline accumulates throughout development, it is most soluble and its content per milligram dry weight greatest at midmaturation stages of development. Salt-soluble proteins containing this hydroxyproline from isolated cell walls of developing kernels were fractionated on a CsCl density gradient and on a Chromatofocusing column, resulting in the purification of an hydroxyproline-rich glycoprotein, PC-1. PC-1 is a basic protein of approximately 65 to 70 kilodaltons in molecular weight with an isoelectric point of at least 10.2 and a density of 1.38 to 1.39 in CsCl. Amino acid composition data indicate that it is rich in hydroxyproline, threonine, proline, lysine, and glycine. Its relation to dicot extensin is discussed.

Low levels of adenosine 3',5'-monophosphate (cyclic AMP) were detected in the cyanobacterium Anabaena variabilis using a protein binding assay and two radioisotopic labelling methods. The basal concentration of intracellular cyclic AMP ranged from 0.27 pmol/mg protein in A. variabilis Kutz grown under heterotrophic conditions to 1.0--2.7 pmol/mg protein in A. variabilis strain 377 grown autotrophically. Extracellular cyclic AMP was found to comprise as much as 90% of the total cyclic AMP in rapidly growing cultures. When A. variabilis strain 377 was starved of nitrogen, a 3--4-fold increase in intracellular cyclic AMP was observed during the 24 h period coincident with early heterocyst development.

This study addresses the basis of host range on legumes of Agrobacterium tumefaciens strain A281, an l,l-succinamopine strain. We tested virulence of T-DNA and vir region constructs from this tumor-inducing (Ti) plasmid with complementary Ti plasmid regions from heterologous nopaline and octopine strains.

We report here the molecular characterization of transferred DNA (T-DNA) in leguminous tumors incited by Agrobacterium tumefaciens A281 harboring the tumor-inducing plasmid pTiBo542. The T-DNA is composed of two regions named TL (left portion)-DNA and TR (right portion)-DNA, in accordance with the nomenclature for the octopine strains. TL-DNA is defined by several internal HindIII restriction fragments totaling 10.8 kilobase pairs (kbp) in uncloned soybean and alfalfa tumors. Alfalfa tumor DNA may contain one more HindIII fragment at the left end of TL-DNA than does soybean tumor DNA. TR-DNA has a 5.8-kbp BamHI-EcoRI internal fragment. All borders other than the left border of TL-DNA appear to be the same within the detection limits of Southern blot hybridization experiments. The two T-DNA regions are separated by 16 to 19 kbp of DNA not stably maintained in tumors. The distance from the left border of TL-DNA to the right border of TR-DNA is approximately 40 kbp. Loci for the mannityl opines are situated in TR-DNA, based on genetic and biochemical criteria.

We used a binary-vector strategy to study the hypervirulence of Agrobacterium tumefaciens A281, an L,L-succinamopine strain. Strain A281 is hypervirulent on several solanaceous plants. We constructed plasmids (pCS65 and pCS277) carrying either the transferred DNA (T-DNA) or the remainder of the tumor-inducing (Ti) plasmid (pEHA101) from this strain and tested each of these constructs in trans with complementary regions from heterologous Ti plasmids. Hypervirulence on tobacco could be reconstructed in a bipartite strain with the L,L-succinamopine T-DNA and the vir region on separate plasmids. pEHA101 was able to complement octopine T-DNA to hypervirulence on tobacco and tomato plants. Nopaline T-DNA was complemented better on tomato plants by pEHA101 than it was by its own nopaline vir region, but not to hypervirulence. L,L-Succinamopine T-DNA could not be complemented to hypervirulence on tobacco and tomato plants with either heterologous vir region. From these results we suggest that the hypervirulence of strain A281 is due to non-T-DNA sequences on the Ti plasmid.