The starch granule associated proteomes of commercially purified starch reference materials from rice and maize

Commercially available reference materials are integral components of many experimental protocols, as it is critical to compare one's results to those derived from well-characterized standards. Most reference materials are well defined, with all their components being cataloged. However, certain reference materials, such as commercially prepared starch samples, can have undefined components, potentially limiting their usefulness as standards. The proteome of commercially prepared starch has not been documented, and to that end, we initiated a mass spectrometry-based survey of the proteins associated with starch granules in commercially prepared rice and maize starch samples. We performed direct trypsin treatments of starch samples and sequenced both the water-soluble peptides liberated into the aqueous supernatant and the peptides released from the starch granule surface by isopropanol solvent washing. We discovered that the majority of proteins, in both rice and maize samples, were involved in either carbohydrate metabolism or storage. We also documented proteins that are markers for seed maturity and for starch mobilization.     

Expression of nitrous oxide reductase from Pseudomonas stutzeri in transgenic tobacco roots using the root-specific rolD promoter from Agrobacterium rhizogenes

The nitrous oxide (N(2)O) reduction pathway from a soil bacterium, Pseudomonas stutzeri, was engineered in plants to reduce N(2)O emissions. As a proof of principle, transgenic plants expressing nitrous oxide reductase (N(2)OR) from P. stutzeri, encoded by the nosZ gene, and other transgenic plants expressing N(2)OR along with the more complete operon from P. stutzeri, encoded by nosFLZDY, were generated. Gene constructs were engineered under the control of a root-specific promoter and with a secretion signal peptide. Expression and rhizosecretion of the transgene protein were achieved, and N(2)OR from transgenic Nicotiana tabacum proved functional using the methyl viologen assay. Transgenic plant line 1.10 showed the highest specific activity of 16.7 μmol N(2)O reduced min(-1) g(-1) root protein. Another event, plant line 1.9, also demonstrated high specific activity of N(2)OR, 13.2 μmol N(2)O reduced min(-1) g(-1) root protein. The availability now of these transgenic seed stocks may enable canopy studies in field test plots to monitor whole rhizosphere N flux. By incorporating one bacterial gene into genetically modified organism (GMO) crops (e.g., cotton, corn, and soybean) in this way, it may be possible to reduce the atmospheric concentration of N(2)O that has continued to increase linearly (about 0.26% year(-1)) over the past half-century.     

Expression of the nos operon proteins from Pseudomonas stutzeri in transgenic plants to assemble nitrous oxide reductase

Nitrous oxide (N(2)O) is a stable greenhouse gas that plays a significant role in the destruction of the ozone layer. Soils are a significant source of atmospheric N(2)O. It is important to explore some innovative and effective biology-based strategies for N(2)O mitigation. The enzyme nitrous oxide reductase (N(2)OR), naturally found in soil bacteria, is responsible for catalysing the final step of the denitrification pathway, conversion of N(2)O to dintrogen gas (N(2)). To transfer this catalytic pathway from soil into plants and amplify the abundance of this essential mechanism (to reduce global warming), a mega-cassette of five coding sequences was assembled to produce transgenic plants heterologously expressing the bacterial nos operon in plant leaves. Both the single-gene transformants (nosZ) and the multi-gene transformants (nosFLZDY) produced active recombinant N(2)OR. Enzymatic activity was detected using the methyl viologen-linked enzyme assay, showing that extracts from both types of transgenic plants exhibited N(2)O-reducing activity. Remarkably, the single-gene strategy produced higher reductase capability than the whole-operon approach. The data indicate that bacterial N(2)OR expressed in plants could convert N(2)O into inert N(2) without involvement of other Nos proteins. Silencing by homologous signal sequences, or cryptic intracellular targeting are possible explanations for the low activities obtained. Expressing N(2)OR from Pseudomonas stutzeri in single-gene transgenic plants indicated that such ag-biotech solutions to climate change have the potential to be easily incorporated into existing genetically modified organism seed germplasm.     

Expression of Cry1Ac in Transgenic Tobacco Plants Under the Control of a Wound-Inducible Promoter (AoPR1) Isolated from Asparagus officinalis to Control Heliothis virescens and Manduca sexta

Expression of cry1Ac gene from Bacillus thuringiensis (Bt) was evaluated under the control of a wound-inducible AoPR1 promoter from Asparagus officinalis in transgenic tobacco plants. The leaves of transgenic plants were mechanically wounded to evaluate the activity of the AoPR1 promoter in driving the expression of Cry1Ac protein at the wound site. Our results indicate that mechanical wounding of transgenic plants was effective in inducing the expression of Cry1Ac protein. As a result of this induction, the accumulated levels of Cry1Ac protein increased during 6–72 h post-wounding period. The leaves of transgenic tobacco plants were evaluated for resistance against Heliothis virescens and Manduca sexta in insect bioassays in two different ways. The detached tobacco leaves were either fed directly to the insect larvae or they were first mechanically wounded followed by a 72 h post-wounding feeding period. Complete protection of mechanically wounded leaves of transgenic plants was observed within 24 h of the bioassay. The leaves of transgenic plants fed directly (without pre-wounding) to the larvae achieved the same level of protection between 24 and 72 h of the bioassay.     

Screening and cloning of antimicrobial DNA sequences using a vital staining method

A novel method for cloning of genes coding for cytotoxic molecules based on a cell viability assay is described. The working hypothesis is that expression of DNA sequences coding for cytotoxic molecules in bacterial cells will lead to cell death or impairment, and the isolation of the impaired or dead cells could lead to identification of DNA sequences responsible for debilitating the host cells. We verified this concept by isolating the well known antimicrobial Puroindoline b gene in Escherichia coli cells. We further demonstrated the feasibility to use this approach for isolating DNA encoding for antimicrobials from cDNA expression libraries. Sequence analysis and bioassay indicated that the isolated clones encoded previously characterized antimicrobial proteins (AMPs), proteins not previously characterized as AMPs, as well as novel antimicrobial peptides. In addition, clones harboring ribosomal protein encoding cDNA were also identified. Therefore, this method could also be used to identify host genes important in maintaining bacterial cell viability.     

Characterization of developing oat seed mRNA: evidence for many globulin mRNAs

Polyadenylated mRNA from developing oat (Avena sativa L.) seeds was isolated and analyzed. Prominent mRNA species of 18S, 15S and 12S were observed; the 18S mRNA was judged to be esentially free of ribosomal RNA by hybridization analysis. Size fractionation andin vitro translation of this mRNA was performed. SDS, IEF-SDS gel electrophoresis and immunoprecipitation were used to analyze the translation products. It is shown that globulin mRNA (18S) accounts for roughly 30% of the total mRNA in developing seeds, the 12S and 15S mRNAs accounting for the remainder. The 18S mRNA directs the synthesis of a series of distinct but related polypeptides, suggesting that some of the heterogeneity seen in the oat globulins is at the amino acid sequence level.     

EFFECTS OF BT RICE ON THE FOOD CONSUMPTION,GROWTH AND SURVIVAL OF CHILO SUPPRESSAUS LARVAE UNDER DIFFERENT TEMPERATURES*

Abstract The Bt transgenic rice line, i.e. KMD1 containing a synthetic cry 1 Ab gene from Bacillus thuringiensis Berliner was tested in the laboratory for evaluating its effects on larval food consumption, growth and survival of striped stem borer (SSB), Chilo suppressalis (Walker) under different temperatures. The food consumption and body weight growth as well as the survival of both 3rd and 5th instar larvae fed on Bt rice were significantly reduced, as compared with those on the control. The temperature showed no marked effect on both food consumption and body weight growth of larvae fed on Bt rice stems since the onset of 3rd instar, conversely had significant effects as since the initial stage of 5th instar. In contrast, the temperature did not distinctly influence the survival of both 3rd and 5th instar larvae. There was a linearly positive relationship between the corrected mortality of larvae and the accumulative amount of Bt rice tissues ingested by larvae.     

Transgenic rice plants with a synthetic cry1Ab gene from Bacillus thuringiensis were highly resistant to eight lepidopteran rice pest species

To fully explore the resistance potential of transgenic rice produced by Agrobacterium-mediated transformation, an elite line KMD1 was assessed for its resistance to eight lepidopteran rice pest species. KMD1 contained a synthetic cry1Ab gene from Bacillus thuringiensis under the control of a maize ubiquitin promoter. It was derived from a commercial japonica Chinese rice variety Xiushui 11, and bred true for both agronomic traits and a cry1Ab gene when the bioassays were done in 1998 in the R₅ generation. The eight lepidopteran pest species were: four Pyralidae species: Chilo suppressalis (striped stem borer, SSB), Scirpophaga incertulas (yellow stem borer, YSB), Cnaphalocrocis medinalis (leaf folder), Herpitogramma licarisalis; two Noctuidae: Sesamia inferens (pink stem borer, PSB) and Naranga anescens; one Stayridae: Mycalesis gotama; and one Hesperiidae, Parnara guttata. In laboratory bioassays, 100% mortality was observed in all insect species when their newly hatched or third-instar larvae were fed KMD1 leaf tissues, whereas only 9.65% of the neonates and none of the third-instar larvae died when fed the leaf tissues of non-transgenic control. Moreover, the leaf area of control tissues consumed in four days by stem borers was 20 to 40 times higher than that of KMD1 tissues, and the area of control tissues eaten by leaf-feeding species was 120 to 180 times greater than that of the transgenic tissues. Under natural infestation, no KMD1 plant was visibly damaged by the SSB, YSB and leaf folder in field evaluation. On the other hand, 80, 9.3 and 88.7% of control plants were injured by SSB, YSB, and leaf folder, respectively. These data disclosed that the transgenic line was highly resistant to a broad spectrum of lepidopteran insect species and could be useful in insect resistance breeding programs.     

Human CD14 Expressed in Seeds of Transgenic Tobacco Displays Similar Proteolytic Resistance and Bioactivity with its Mammalian-produced Counterpart

Human CD14 plays an important role in innate immunity by being the key receptor of lipopolysaccharide found on Gram-negative bacteria. The recently discovered widespread localization of CD14 in secretions and mucosal surfaces reveals its extensive anti-microbial properties and numerous potential medical applications. To produce active recombinant human CD14 (rhCD14) for massive distribution, transgenic tobacco plants were successfully generated to express rhCD14 in the seed endosperm under the control of two versions (1.8 kb and 5.1 kb) of the rice glutelin Gt-1 promoter. Plant-made rhCD14 proteins reached a concentration of 16 μg/g of seeds and showed stability, proteolytic resistance to pepsin digestion and ability to induce the release of pro-inflammatory IL-6 and IL-8 cytokines in presence of LPS. The expression of plant rhCD14 in tobacco seeds constitutes a promising low-cost and abundant supply of this immune protein to further investigate its roles in, impacts on and potential medical applications for the innate immune system.     

Increased yield of heterologous viral glycoprotein in the seeds of homozygous transgenic tobacco plants cultivated underground.

The use of transgenic plants in the production of recombinant proteins for human therapy, including subunit vaccines, is being investigated to evaluate the efficacy and safety of these emerging biopharmaceutical products. We have previously shown that synthesis of recombinant glycoprotein B (gB) of human cytomegalovirus can be targeted to seeds of transgenic tobacco when directed by the rice glutelin 3 promoter, with gB retaining critical features of immunological reactivity (E.S. Tackaberry et al. 1999. Vaccine, 17: 3020-3029). Here, we report development of second generation transgenic plant lines (T1) homozygous for the transgene. Twenty progeny plants from two lines (A23T(1)-2 and A24T(1)-3) were grown underground in an environmentally contained mine shaft. Based on yields of gB in their seeds, the A23T(1)-2 line was then selected for scale-up in the same facility. Analyses of mature seeds by ELISA showedthat gB specific activity in A23T(1)-2 seeds was over 30-fold greater than the best T0 plants from the same transformation series, representing 1.07% total seed protein. These data demonstrate stable inheritance, an absence of transgene inactivation, and enhanced levels of gB expression in a homozygous second generation plant line. They also provide evidence for the suitability of using this environmentally secure facility to grow transgenic plants producing therapeutic biopharmaceuticals.