Presence of small resistant peptides from new in vitro digestion assays detected by liquid chromatography tandem mass spectrometry: An implication of allergenicity prediction of novel proteins?

The susceptibility of newly expressed proteins to digestion by gastrointestinal proteases (e.g., pepsin) has long been regarded as one of the important endpoints in the weight-of-evidence (WOE) approach to assess the allergenic risk of genetically modified (GM) crops. The European Food Safety Authority (EFSA) has suggested that current digestion study protocols used for this assessment should be modified to more accurately reflect the diverse physiological conditions encountered in human populations and that the post-digestion analysis should include analytical methods to detect small peptide digestion products.The susceptibility of two allergens (beta-lactoglobin (ß-Lg) and alpha-lactalbumin (α-La)) and two non-allergens (hemoglobin (Hb) and phosphofructokinase (PFK)) to proteolytic degradation was investigated under two pepsin digestion conditions (optimal pepsin digestion condition: pH 1.2, 10 U pepsin/µg test protein; sub-optimal pepsin digestion condition: pH 5.0, 1 U pepsin/10 mg test protein), followed by 34.5 U trypsin/mg test protein and 0.4 U chymotrypsin/mg test protein digestion in the absence or presence of bile salts. All samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in conjunction with Coomassie Blue staining and, in parallel, liquid chromatography tandem mass spectrometry (LC-MS) detection. The results provide following insights: 1) LC-MS methodology does provide the detection of small peptides; 2) Peptides are detected in both allergens and non-allergens from all digestion conditions; 3) No clear differences among the peptides detected from allergen and non-allergens; 4) The differences observed in SDS-PAGE between the optimal and sub-optimal pepsin digestion conditions are expected and align with kinetics and properties of the specific enzymes; 5) The new methodology with new digestion conditions and LC-MS detection does not provide any differentiating information for prediction whether a protein is an allergen. The classic pepsin resistance assay remains the most useful assessment of the potential exposure of an intact newly expressed protein as part of product safety assessment within a WOE approach.

Engineered selective plant male sterility through pollen-specific expression of the EcoRI restriction endonuclease.

Unintended gene flow from transgenic plants via pollen, seed and vegetative propagation is a regulatory concern because of potential admixture in food and crop systems, as well as hybridization and introgression to wild and weedy relatives. Bioconfinement of transgenic pollen would help address some of these concerns and enable transgenic plant production for several crops where gene flow is an issue. Here, we demonstrate the expression of the restriction endonuclease EcoRI under the control of the tomato pollen-specific LAT52 promoter is an effective method for generating selective male sterility in Nicotiana tabacum (tobacco). Of nine transgenic events recovered, four events had very high bioconfinement with tightly controlled EcoRI expression in pollen and negligible-to-no expression other plant tissues. Transgenic plants had normal morphology wherein vegetative growth and reproductivity were similar to nontransgenic controls. In glasshouse experiments, transgenic lines were hand-crossed to both male-sterile and emasculated nontransgenic tobacco varieties. Progeny analysis of 16 000-40 000 seeds per transgenic line demonstrated five lines approached (>99.7%) or attained 100% bioconfinement for one or more generations. Bioconfinement was again demonstrated at or near 100% under field conditions where four transgenic lines were grown in close proximity to male-sterile tobacco, and 900-2100 seeds per male-sterile line were analysed for transgenes. Based upon these results, we conclude EcoRI-driven selective male sterility holds practical potential as a safe and reliable transgene bioconfinement strategy. Given the mechanism of male sterility, this method could be applicable to any plant species.

Phytohormonal networks promote differentiation of fiber initials on pre-anthesis cotton ovules grown in vitro and in planta.

The number of cotton (Gossypium sp.) ovule epidermal cells differentiating into fiber initials is an important factor affecting cotton yield and fiber quality. Despite extensive efforts in determining the molecular mechanisms regulating fiber initial differentiation, only a few genes responsible for fiber initial differentiation have been discovered. To identify putative genes directly involved in the fiber initiation process, we used a cotton ovule culture technique that controls the timing of fiber initial differentiation by exogenous phytohormone application in combination with comparative expression analyses between wild type and three fiberless mutants. The addition of exogenous auxin and gibberellins to pre-anthesis wild type ovules that did not have visible fiber initials increased the expression of genes affecting auxin, ethylene, ABA and jasmonic acid signaling pathways within 1 h after treatment. Most transcripts expressed differentially by the phytohormone treatment in vitro were also differentially expressed in the ovules of wild type and fiberless mutants that were grown in planta. In addition to MYB25-like, a gene that was previously shown to be associated with the differentiation of fiber initials, several other differentially expressed genes, including auxin/indole-3-acetic acid (AUX/IAA) involved in auxin signaling, ACC oxidase involved in ethylene biosynthesis, and abscisic acid (ABA) 8'-hydroxylase an enzyme that controls the rate of ABA catabolism, were co-regulated in the pre-anthesis ovules of both wild type and fiberless mutants. These results support the hypothesis that phytohormonal signaling networks regulate the temporal expression of genes responsible for differentiation of cotton fiber initials in vitro and in planta.

Functional analyses of cotton (Gossypium hirsutum L.) immature fiber (im) mutant infer that fiber cell wall development is associated with stress responses.

BACKGROUND: Cotton fiber maturity is an important factor for determining the commercial value of cotton. How fiber cell wall development affects fiber maturity is not well understood. A comparison of fiber cross-sections showed that an immature fiber (im) mutant had lower fiber maturity than its near isogenic wild type, Texas marker-1 (TM-1). The availability of the im mutant and TM-1 provides a unique way to determine molecular mechanisms regulating cotton fiber maturity. RESULTS: Transcriptome analysis showed that the differentially expressed genes (DEGs) in the im mutant fibers grown under normal stress conditions were similar to those in wild type cotton fibers grown under severe stress conditions. The majority of these DEGs in the im mutant were related to stress responses and cellular respiration. Stress is known to reduce the activity of a classical respiration pathway responsible for energy production and reactive oxygen species (ROS) accumulation. Both energy productions and ROS levels in the im mutant fibers are expected to be reduced if the im mutant is associated with stress responses. In accord with the prediction, the transcriptome profiles of the im mutant showed the same alteration of transcriptional regulation that happened in energy deprived plants in which expressions of genes associated with cell growth processes were reduced whereas expressions of genes associated with recycling and transporting processes were elevated. We confirmed that ROS production in developing fibers from the im mutant was lower than that from the wild type. The lower production of ROS in the im mutant fibers might result from the elevated levels of alternative respiration induced by stress. CONCLUSION: The low degree of fiber cell wall thickness of the im mutant fibers is associated with deregulation of the genes involved in stress responses and cellular respiration. The reduction of ROS levels and up-regulation of the genes involved in alternative respirations suggest that energy deprivation may occur in the im mutant fibers.

Molecular markers associated with the immature fiber (im) gene affecting the degree of fiber cell wall thickening in cotton (Gossypium hirsutum L.).

Cotton fiber fineness and maturity measured indirectly as micronaire (MIC) are important properties of determining fiber grades in the textile market. To understand the genetic control and molecular mechanisms of fiber fineness and maturity, we studied two near isogenic lines, Gossypium hirsutum, Texas Marker-1 wild type (TM-1) and immature fiber (im) mutant showing a significant difference in MIC values. The fibers from im mutant plants were finer and less mature with lower MIC values than those from the recurrent parent, TM-1. A comprehensive fiber property analysis of TM-1 and im mutant showed that the lower MIC of fibers in im mutant was due to the lower degree of fiber cell wall thickening as compared to the TM-1 fibers. Using an F(2) population comprising 366 progenies derived from a cross between TM-1 and im mutant, we confirmed that the immature fiber phenotype present in a mutant plant was controlled by one single recessive gene im. Furthermore, we identified 13 simple sequence repeat markers that were closely linked to the im gene located on chromosome 3. Molecular markers associated with the im gene will lay the foundation to further investigate genetic information required for improving cotton fiber fineness and maturity.

A SABATH Methyltransferase from the moss Physcomitrella patens catalyzes S-methylation of thiols and has a role in detoxification.

Known SABATH methyltransferases, all of which were identified from seed plants, catalyze methylation of either the carboxyl group of a variety of low molecular weight metabolites or the nitrogen moiety of precursors of caffeine. In this study, the SABATH family from the bryophyte Physcomitrella patens was identified and characterized. Four SABATH-like sequences (PpSABATH1, PpSABATH2, PpSABATH3, and PpSABATH4) were identified from the P. patens genome. Only PpSABATH1 and PpSABATH2 showed expression in the leafy gametophyte of P. patens. Full-length cDNAs of PpSABATH1 and PpSABATH2 were cloned and expressed in soluble form in Escherichia coli. Recombinant PpSABATH1 and PpSABATH2 were tested for methyltransferase activity with a total of 75 compounds. While showing no activity with carboxylic acids or nitrogen-containing compounds, PpSABATH1 displayed methyltransferase activity with a number of thiols. PpSABATH2 did not show activity with any of the compounds tested. Among the thiols analyzed, PpSABATH1 showed the highest level of activity with thiobenzoic acid with an apparent Km value of 95.5µM, which is comparable to those of known SABATHs. Using thiobenzoic acid as substrate, GC-MS analysis indicated that the methylation catalyzed by PpSABATH1 is on the sulfur atom. The mechanism for S-methylation of thiols catalyzed by PpSABATH1 was partially revealed by homology-based structural modeling. The expression of PpSABATH1 was induced by the treatment of thiobenzoic acid. Further transgenic studies showed that tobacco plants overexpressing PpSABATH1 exhibited enhanced tolerance to thiobenzoic acid, suggesting that PpSABATH1 have a role in the detoxification of xenobiotic thiols.

Transgene excision in pollen using a codon optimized serine resolvase CinH-RS2 site-specific recombination system.

Transgene escape, a major environmental and regulatory concern in transgenic crop cultivation, could be alleviated by removing transgenes from pollen, the most frequent vector for transgene flow. A transgene excision vector containing a codon optimized serine resolvase CinH recombinase (CinH) and its recognition sites RS2 were constructed and transformed into tobacco (Nicotiana tabacum cv. Xanthi). CinH recombinase recognized 119 bp of nucleic acid sequences, RS2, in pollen and excised the transgene flanked by the RS2 sites. In this system, the pollen-specific LAT52 promoter from tomato was employed to control the expression of CinH recombinase. Loss of expression of a green fluorescent protein (GFP) gene under the control of the LAT59 promoter from tomato was used as an indicator of transgene excision. Efficiency of transgene excision from pollen was determined by flow cytometry (FCM)-based pollen screening. While a transgenic event in the absence of CinH recombinase contained about 70% of GFP-synthesizing pollen, three single-copy transgene events contained less than 1% of GFP-synthesizing pollen based on 30,000 pollen grains analyzed per event. This suggests that CinH-RS2 recombination system could be effectively utilized for transgene biocontainment.

Transgene introgression in crop relatives: molecular evidence and mitigation strategies.

Incorporation of crop genes into wild and weedy relative populations (i.e. introgression) has long been of interest to ecologists and weed scientists. Potential negative outcomes that result from crop transgene introgression (e.g. extinction of native wild relative populations; invasive spread by wild or weedy hosts) have not been documented, and few examples of transgene introgression exist. However, molecular evidence of introgression from non-transgenic crops to their relatives continues to emerge, even for crops deemed low-risk candidates for transgene introgression. We posit that transgene introgression monitoring and mitigation strategies are warranted in cases in which transgenes are predicted to confer selective advantages and disadvantages to recipient hosts. The utility and consequences of such strategies are examined, and future directions provided.

An efficient and rapid transgenic pollen screening and detection method using flow cytometry.

Assaying for transgenic pollen, a major vector of transgene flow, provides valuable information and essential data for the study of gene flow and assessing the effectiveness of transgene containment. Most studies have employed microscopic screening methods or progeny analyses to estimate the frequency of transgenic pollen. However, these methods are time-consuming and laborious when large numbers of pollen grains must be analyzed to look for rare transgenic pollen grains. Thus, there is an urgent need for the development of a simple, rapid, and high throughput analysis method for transgenic pollen analysis. In this study, our objective was to determine the accuracy of using flow cytometry technology for transgenic pollen quantification in practical application where transgenic pollen is not frequent. A suspension of non-transgenic tobacco pollen was spiked with a known amount of verified transgenic tobacco pollen synthesizing low or high amounts of green fluorescent protein (GFP). The flow cytometric method detected approximately 75% and 100% of pollen grains synthesizing low and high amounts of GFP, respectively. The method is rapid, as it is able to count 5000 pollen grains per minute-long run. Our data indicate that this flow cytometric method is useful to study gene flow and assessment of transgene containment.

Sustainable use of biotechnology for bioenergy feedstocks.

Done correctly, cellulosic bioenergy should be both environmentally and economically beneficial. Carbon sequestration and decreased fossil fuel use are both worthy goals in developing next-generation biofuels. We believe that biotechnology will be needed to significantly improve yield and digestibility of dedicated perennial herbaceous biomass feedstocks, such as switchgrass and Miscanthus, which are native to the US and China, respectively. This Forum discusses the sustainability of herbaceous feedstocks relative to the regulation of biotechnology with regards to likely genetically engineered traits. The Forum focuses on two prominent countries wishing to develop their bioeconomies: the US and China. These two countries also share a political desire and regulatory frameworks to enable the commercialization and wide release of transgenic feedstocks with appropriate and safe new genetics. In recent years, regulators in both countries perform regular inspections of transgenic field releases and seriously consider compliance issues, even though the US framework is considered to be more mature and stringent. Transgene flow continues to be a pertinent environmental and regulatory issue with regards to transgenic plants. This concern is largely driven by consumer issues and ecological uncertainties. Regulators are concerned about large-scale releases of transgenic crops that have sexually compatible crops or wild relatives that can stably harbor transgenes via hybridization and introgression. Therefore, prior to the commercialization or extensive field testing of transgenic bioenergy feedstocks, we recommend that mechanisms that ensure biocontainment of transgenes be instituted, especially for perennial grasses. A cautionary case study will be presented in which a plant's biology and ecology conspired against regulatory constraints in a non-biomass crop perennial grass (creeping bentgrass, Agrostis stolonifera), in which biocontainment was not attained. Appropriate technologies that could be applied to perennial grass feedstocks for biocontainment are discussed.

FLP/FRT recombination from yeast: application of a two gene cassette scheme as an inducible system in plants.

Phytosensors are plants that are genetically engineered for sensing and reporting the presence of a specific contaminant, including agriculturally important biological agents. Phytosensors are constructed by transforming plants to contain specific biotic- or abiotic-inducible promoters fused to a reporter gene. When such transgenic plants encounter the target biotic or abiotic agent, the specific inducible promoter is triggered and subsequently drives the expression of the reporter gene, which produces a signal for detection. However, several systems lack robustness, rapid induction and promoter strength. Here, we tested the FLP/FRT recombination system in a construct containing a two gene cassette organization and examined its potential in transgenic Arabidopsis and tobacco plants using a ß-glucuronidase (GUS) reporter. In this model system, a heat-shock inducible promoter was employed to control the expression of the FLP recombinase gene. Upon heat induction and subsequent active FLP-mediated excision event, the GUS gene was placed in close proximity to the 35S promoter resulting in an active GUS reporter expression. Our results demonstrate that the two gene cassette scheme of inducible FLP/FRT recombination system is functional in tobacco and Arabidopsis, providing additional insights into its possible application in phytosensing such as creating strong readout capabilities.

Keeping the genie in the bottle: transgene biocontainment by excision in pollen.

Gene flow from transgenic plants is an environmental and regulatory concern. While biocontainment might be achieved using male sterility or transgenic mitigation tools, we believe that perhaps the optimal solution might be simply to remove transgenes from pollen. Male sterility might not be ideal for many pollinators, and might not be implementable using standardized genes. Transgenic mitigation might not be useful to control conspecific gene flow (e.g. crop to crop), and relies on competition and not biocontainment per se. Site-specific recombination systems could allow highly efficient excision of transgenes in pollen to eliminate, or at least minimize, unwanted transgene movement via pollen dispersal. There are other potential biotechnologies, such as zinc finger nucleases, that could be also used for transgene excision.

Genetic load and transgenic mitigating genes in transgenic Brassica rapa (field mustard) x Brassica napus (oilseed rape) hybrid populations.

BACKGROUND: One theoretical explanation for the relatively poor performance of Brassica rapa (weed) x Brassica napus (crop) transgenic hybrids suggests that hybridization imparts a negative genetic load. Consequently, in hybrids genetic load could overshadow any benefits of fitness enhancing transgenes and become the limiting factor in transgenic hybrid persistence. Two types of genetic load were analyzed in this study: random/linkage-derived genetic load, and directly incorporated genetic load using a transgenic mitigation (TM) strategy. In order to measure the effects of random genetic load, hybrid productivity (seed yield and biomass) was correlated with crop- and weed-specific AFLP genomic markers. This portion of the study was designed to answer whether or not weed x transgenic crop hybrids possessing more crop genes were less competitive than hybrids containing fewer crop genes. The effects of directly incorporated genetic load (TM) were analyzed through transgene persistence data. TM strategies are proposed to decrease transgene persistence if gene flow and subsequent transgene introgression to a wild host were to occur. RESULTS: In the absence of interspecific competition, transgenic weed x crop hybrids benefited from having more crop-specific alleles. There was a positive correlation between performance and number of B. napus crop-specific AFLP markers [seed yield vs. marker number (r = 0.54, P = 0.0003) and vegetative dry biomass vs. marker number (r = 0.44, P = 0.005)]. However under interspecific competition with wheat or more weed-like conditions (i.e. representing a situation where hybrid plants emerge as volunteer weeds in subsequent cropping systems), there was a positive correlation between the number of B. rapa weed-specific AFLP markers and seed yield (r = 0.70, P = 0.0001), although no such correlation was detected for vegetative biomass. When genetic load was directly incorporated into the hybrid genome, by inserting a fitness-mitigating dwarfing gene that that is beneficial for crops but deleterious for weeds (a transgene mitigation measure), there was a dramatic decrease in the number of transgenic hybrid progeny persisting in the population. CONCLUSION: The effects of genetic load of crop and in some situations, weed alleles might be beneficial under certain environmental conditions. However, when genetic load was directly incorporated into transgenic events, e.g., using a TM construct, the number of transgenic hybrids and persistence in weedy genomic backgrounds was significantly decreased.

Characterization of directly transformed weedy Brassica rapa and introgressed B. rapa with Bt cry1Ac and gfp genes.

Crop to weed transgene flow, which could result in more competitive weed populations, is an agricultural biosafety concern. Crop Brassica napus to weedy Brassica rapa hybridization has been extensively characterized to better understand the transgene flow and its consequences. In this study, weedy accessions of B. rapa were transformed with Bacillus thuringiensis (Bt) cry1Ac- and green fluorescence protein (gfp)-coding transgenes using Agrobacterium to assess ecological performance of the wild biotype relative to introgressed hybrids in which the transgenic parent was the crop. Regenerated transgenic B. rapa events were characterized by progeny analysis, Bt protein enzyme-linked immunosorbent assay (ELISA), Southern blot analysis, and GFP expression assay. GFP expression level and Bt protein concentration were significantly different between independent transgenic B. rapa events. Similar reproductive productivity was observed in comparison between transgenic B. rapa events and B. rapa x B. napus introgressed hybrids in greenhouse and field experiments. In the greenhouse, Bt transgenic plants experienced significantly less herbivory damage from the diamondback moth (Plutella xylostella). No differences were found in the field experiment under ambient, low, herbivore pressure. Directly transformed transgenic B. rapa plants should be a helpful experimental control to better understand crop genetic load in introgressed transgenic weeds.

Expression of green fluorescent protein in pollen of oilseed rape (Brassica napus L.) and its utility for assessing pollen movement in the field.

Transgene movement via pollen is an important component of gene flow from transgenic plants. Here, we present proof-of-concept studies that demonstrate the monitoring of short distant movement of pollen expressing a genetically encoded fluorescent tag in oilseed rape (Brassica napus L. cv. Westar). Transgenic oilseed rape plants were produced using Agrobacterium-mediated transformation method with the pBINDC1 construct containing a green fluorescent protein (GFP) variant, mGFP5-ER, under the control of the pollen-specific LAT59 promoter from tomato. Transgenic pollen was differentiated from non-transgenic pollen in vivo by a unique spectral signature, and was shown to be an effective tool to monitor pollen movement in the greenhouse and field. GFP-tagged pollen also served as a practical marker to determine the zygosity of plants. In a greenhouse pollen flow study, more pollen was captured at closer distances from the source plant plot with consistent wind generated by a fan. Under field conditions, GFP transgenic pollen grains were detected up to a distance of 15 m, the farthest distance from source plants assayed. GFP-tagged pollen was easily distinguishable from non-transgenic pollen using an epifluorescence microscope.