Overexpression of epsps transgene in weedy rice: insufficient evidence to support speculations about biosafety

Forum Letters Overexpression of epsps transgene in weedy rice: insufficient evidence to support speculations about biosafety The recent paper published in New Phytologist, Wang et al. (2014; this issue pp. 679–683), purports in its title that ‘A novel 5enolpyruvoylshikimate-3-phosphate (EPSP) synthase transgene for glyphosate resistance stimulates growth and fecundity in weedy rice (Oryza sativa) without herbicide’ and in the paper claims that weedy rice expressing the transgene is more competitively fit than the wild type in the absence of glyphosate treatment. While this may be so, there is a lack of evidence in the paper that the transgene confers glyphosate resistance, or that the transgenic weedy rice is more competitively fit than its wild type. As this paper has generated extensive media coverage facilitated by the authors, we feel it imperative to analyze the paper in depth to ascertain whether the claims made in title, summary, and text have been met, whether criteria necessary for publication have been achieved, as well as whether the media reportage was justified in the form released by the authors. In many respects, the findings are not as novel as the authors imply. This is not the first instance when genes from a crop have increased the rate of growth and/or fecundity of a related weed with which the crop can cross-pollinate. Many such cases have been discussed before (Ellstrand, 2003). Whenever yield-enhancing traits are introduced into crops they can quickly introgress in related infesting weeds. For example, weedy Aegilops spp. growing in or near wheat fields typically introgress wheat genetic material (Weissmann et al., 2005), have larger flag leaves, ears, and grain than the same Aegilops spp. in wild habitats (Rehman et al., 2006; Arrigo et al., 2011). Indeed after disease resistance was crossed into a US wheat from a nonindigenous Aegilops spp., it quickly introgressed from wheat into indigenous Aegilops cylindrica, a major weed in parts of the United States (Perez-Jones et al., 2006). Most crop-related weeds, and especially weedy rice, have co-domesticated with the respective crops. There is considerable evidence that most weedy rice strains derive from de-domestication of cultivated rice and are not derived directly from the wild progenitors (Gross et al., 2010; Lawton-Rauh & Burgos, 2010; Thurber et al., 2013, and references cited therein). Thus, weedy rice fecundity has increased through the ages because cultivated rice yield has increased. None of this very relevant information was intimated or cited, and the authors made it appear as if it is novel that a trait introduced in a crop enhanced and raised fecundity when it introgressed to a related weed. 360 New Phytologist (2014) 202: 360–362 www.newphytologist.com A basic tenet of published science is that other scientists should be able to reproduce the experimental findings, based on the Materials and Methods section of peer-reviewed papers. This was not the case here. Particularly crucial, no sequence data were provided or deposited at EMBL/GenBank for the transgene construct used. The authors say they used a modified rice epsps, but there was no information provided on the sequence modifications, or its protein products Km or Ki for glyphosate, yet they claim that the modified gene confers resistance to glyphosate. No data are provided that demonstrate resistance to glyphosate or its extent, nor is a reference given to any peer-reviewed study where such data are presented, despite implying that such a critical and influential connection is supported by this study. They do show a doubled level of the EPSPS protein. In other cases where there was a doubled level of EPSPS expression (Boerboom et al., 1990; Baerson et al., 2002) there was only a c. seven-fold increase in the I50 resistance level to glyphosate, which is usually not enough for commercial use in the field. They say that the gene was described in a published paper (Xu et al., 2002) (correct citation given here and not in the manuscript in question). This paper (Xu et al., 2002) only presents data on cloning the rice epsps gene and presents nothing about its modification. Is this the gene the authors used to produce the transgenic rice in question, which was then further crossed to weedy rice? They cite no paper on the original transgenic cultivated rice containing this gene nor on the variety they used. Additionally, there is a surprising paucity of molecular data about the transgenic rice plants, for example, there is an absence of Southern blot data, resequencing hybrids to confirm identity, gene insert number, number of transgenic events recovered, the variation of gene expression and phenotypes among the transgenic events, or even enough gene expression analysis of the glasshouse-grown plants to be certain of increased transgene expression. However, we can assume here that transgenic plants had higher expression. In some places in the paper, the authors state that the overexpressed epsps gene in the transgenics is the ‘native rice epsps gene’ (e.g. the Summary), and in other places they confusingly refer to it as a modified gene. In the Materials and Methods section, they state that the transgene is different from the native gene in size (700 and 1000 bp), but there is no sequence information on how it is different, or even if it includes the entire coding sequence. Since the entirety of the findings of Wang et al. rests on the material composition of the transgenic plants, we do not understand how the findings could be validated or replicated by others in rice or another species of plant and thus how this paper can be seen as a valid contribution deserving publication. No data are given on the fecundity of the transgenic rice vs the parent variety. Did all cultivated rice transformants give such an increase or just one – that is, is this a positional effect that has nothing to do with the gene inserted? There is no sequencing and mapping of flanking regions that would supply insights into this Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust New Phytologist possibility. The absence of molecular data on the transgenics and the absence of data on the magnitude of yield increase conferred to the cultivated rice by the transgenic trait are needed as a baseline to compare with the yield increment it confers on weedy rice. This would place the data on the increase in fecundity in weedy rice in the perspective of whether this is just analogous to other cases where yield increases in crops become introgressed into their related weeds. The paper would be quite novel if the transgene insertion resulted in a significantly higher increase in fecundity in the weedy rice than the cultivated wild type, but this is not supported by the data presented. The authors competitive fitness experiment – a key to the paper – is flawed. They used transplants spaced at 25 m 2 to measure competition between the transgenic and nontransgenic strains, without a phase of self-thinning, one of the most critical stages of intra-specific competition. It is the balance between inter-/intraspecific competition that defines the relative competitive ability of a species growing in mixture with another (Boddy et al., 2012). Thus, their experimental procedure is highly artificial and probably not relevant to field conditions considering that under typical field conditions the density of weedy rice at the early seedling stage could be c. 1000 plants m 2. After self-thinning, a heavily weed-infested field reaches maturity with c. 50 plants m 2 of cultivated rice and 250 plants m 2 of weedy rice, that is, 75% of the weedy rice succumbed to intra-specific self-thinning competition at very close spacing. Relative competitiveness is just one component of the overall ecological fitness of individuals. Wang et al. only measured fitness of the transgenic hybrid weedy rice in competition with the nontransgenic hybrid at 20 cm spacing that is, 25 plants m 2, which is not close enough to show a competitive fitness difference. This is borne out in published studies, for example, Boddy et al. (2012), and many studies cited therein (but not cited by Wang et al., 2014). Wang et al. used an 11 : 1 ratio, which is unlikely to occur in the field because much of the weedy rice in a field experiment should be wild type, due to the very low outcrossing of rice as well as emerging dormant weedy rice from previous seasons. Studies involving different densities and ratios (replacement series) of wild type and transgenic, or having shorter distances between plants, which enhances competition that are typical of such ecological experiments (Boddy et al., 2012) were not performed. The redundant analyses of variance and the figures referred to actually say nothing about enhanced fitness of the hybrids. Thus, despite the claim in the title, it is doubtful that there are any significant data in the paper on the enhanced competitive fitness of the transgenic weedy rice. We can only conclude that the transgenic material has enhanced fecundity. The data on tryptophan overproduction seem solid – but the missing data would help in understanding the significance of their results. There are many ways to obtain tryptophan overproduction nontransgenically – for example, selection for mutants resistant to methyl tryptophan. Would the authors then say that the movement of such nontransgenic mutations would ‘have broad implications for biotech risk assessment’? Is only the movement of transgenes a worry, as the authors strongly imply, or should one worry about all novel traits as the Canadian regulatory authorities do with their Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust Letters Forum 361 product-oriented (not process-oriented) regulation (Ammann, 2014)? The paper does mention that there are ways to ameliorate transgene flow, should it pose a risk – and cites papers to that effect. They repeatedly emphasize the gene flow problem throughout the paper but the ‘minor’ solution does not appear in the Summary which is meant to abstract the whole paper, including the concluding take-home message. The possibility that such transgene flow could be controlled was not mentioned in any of the authors’ press releases (e.g. http://researchnews.osu.edu/archive/geneflow. htm) nor in their interviews (Qiu, 2013). This is probably because such transgene flow control was sufficiently well hidden such that the journalists who may have read the actual paper might not discover this or overlook the significance. Thus, besides the title and the summary not supplying data on many of the contentions, the summary and the conclusions are at odds with each other. There is no statement that the experimental material used to support the paper’s conclusions is available to any scientists wishing to further validate the conclusions based on the data presented, as required by many journals. Of even greater importance, as we discuss earlier, the paucity of molecular data renders the experiments impossible to reproduce. This lack of critical information means that there is little opportunity for independent validation of the presented results and no possibility for follow-up directly addressing the significant issues discussed regarding transgene movement in the future. This is especially important given that the media immediately interpreted and summarized the results making strong assertions. Clearly this was either based only on the misleading title and overstated summary, and/or on a press release by an author (http://researchnews.osu.edu/archive/geneflow.htm). There was non-critical acceptance of their conclusions in the paper, even in Nature News (Qiu, 2013) and reproduced verbatim in Scientific American. The journalist assumed that the rice and weedy rice were herbicide resistant and the weeds with the transgene were more reproductively fit. The press release from a co-author (Qiu, 2013) echoes these contentions. Such reportage with the resulting hype, dealing with agricultural biotechnology, often influence public perception and policy far beyond the scope of the published study and thus directly impact on the potential benefits of specific transgenic crops. In this case, there is not only insufficient evidence provided in the study to justify the conclusions, but there is insufficient description of the materials used in the study to allow the scientific community to try to replicate, interpret, or even understand, the study. In summary, to directly quote a recent Nature Editorial on the potential misuse of inappropriately presented and described work on genetic studies of human intelligence and behaviour: ‘Scientists have a responsibility to do what they can to prevent abuses of their work’. To do so: scientists must ‘First: be patient. Do not speculate about the possibility of finding certain results, or about the implications of those results.’ ‘Second: be accurate. Researchers should design studies on the basis of sound scientific reasoning.’ ‘Third: be sensitive. Even if scientists have truly honourable intentions, they must realize how easy it can be for studies … to seem self-serving’ (Anonymous, 2013).’ We do not believe that any of these simple criteria have been met in the present instance. New Phytologist (2014) 202: 360–362 www.newphytologist.com 362 Forum New Phytologist Letters Indeed, the authors’ interviews and press releases appear to promulgate their speculative over-interpretation of very limited data. Acknowledgements Useful insights and information for this letter to the Editor were supplied by Drs Micheal Owen, Bernal Valverde, Aldo Ferrero and Francesco Vidotto. Jonathan Gressel1*, C. Neal Stewart Jr2, L. Val Giddings3, Albert J. Fischer4, Jens Carl Streibig5, 6 Nilda R. Burgos , Anthony Trewavas7, Aldo Merotto Jr8, Christopher John Leaver9, Klaus Ammann10, Vivian Moses11 and Amy Lawton-Rauh12 1 Plant Sciences Department, Weizmann Institute of Science, Rehovot 76100, Israel; 2 Plant Sciences, University of Tennessee, Knoxville, TN 37996, USA; 3 Information Technology & Innovation Foundation, 1101 K Street NW Suite 610, Washington, DC 20005, USA; 4 Department of Plant Sciences, University of California, Davis, CA 95616, USA; 5 Department of Plant and Environmental Sciences, University of Copenhagen, DK-2630 Taastrup Denmark; 6 Department of Crop, Soil, and Environmental Sciences, University of Arkansas, 1366 W. Altheimer Drive, Fayetteville, AR, 72704, USA; 7 Institute of Molecular Plant Science, The University of Edinburgh, Mayfield Road, Edinburgh EH9 3JH, UK; 8 Crop Science Department, Federal University of Rio Grande do Sul, 7712 Bento Goncalves Ave, Porto Alegre RS 91501-970, Brazil; 9 Department of Plant Sciences, University of Oxford, Oxford OX1 3RB, UK; 10 University of Bern, Monruz 20, 2000 Neuch^atel, Switzerland 11 King’s College, University of London, London SE1 9NH, UK; 12 Department Genetics and Biochemistry, Clemson University, Clemson, SC 29634-0318, USA (*Author for correspondence: tel +972 8 934 3481; email jonathan.gressel@weizmann.ac.il) References Ammann K. 2014. Genomic misconception. A fresh look at the biosafety regulation of transgenic and conventional crops: a plea for a process of agnostic regulation. New Biotechnology 31: 1–17. Anonymous. 2013. Editorial, Dangerous work. Behavioural geneticists must tread carefully to prevent their research being misinterpreted. Nature 502: 5–6. Arrigo N, Guadagnuolo R, Lappe S, Pasche S, Parisod C, Felber F. 2011. Gene flow between wheat and wild relatives: empirical evidence from Aegilops geniculata, Ae. neglecta and Ae. triuncialis. Evolutionary Applications 4: 685–695. Baerson SR, Rodriguez DJ, Biest NA, Tran M, You JS, Kreuger RW, Dill GM, Pratley JE, Gruys KJ. 2002. Investigating the mechanism of glyphosate resistance in rigid ryegrass (Lolium ridigum). Weed Science 50: 721–730. Boddy LG, Streibig JC, Yamasue Y, Fischer AJ. 2012. Biomass, fecundity, and interference ability of multiple herbicide-resistant and -susceptible late watergrass (Echinochloa phyllopogon). Weed Science 60: 401–410. Boerboom CM, Wyse DL, Somers DA. 1990. Mechanism of glyphosate tolerance in birdsfoot trefoil (Lotus corniculatus). Weed Science 38: 463–467. Ellstrand NC. 2003. Dangerous liaisons – when cultivated plants mate with their wild relatives. Baltimore, MD, USA: Johns Hopkins University Press. Gross BL, Reagon M, Hsu SC, Caicedo AL, Jia YL, Olsen KM. 2010. Seeing red: the origin of grain pigmentation in US weedy rice. Molecular Ecology 19: 3380–3393. Lawton-Rauh A, Burgos N. 2010. Cultivated and weedy rice interactions and the domestication process. Molecular Ecology 19: 3243–3245. Perez-Jones A, Mallory-Smith CA, Riera-Lizarazu O, Watson CJW, Wang Z, Rehman M, Zemetra RS. 2006. Introgression of a strawbreaker foot rot resistance gene from winter wheat into jointed goatgrass. Crop Science 46: 2155–2160. Qiu J. 2013. Genetically modified crops pass benefits to weeds: herbicide resistance and other genetic modifications could confer an advantage on plants in the wild. Nature News. doi: 10.1038/nature.2013.13517. Rehman M, Hansen JL, Brown J, Price W, Zemetra RS, Mallory-Smith CA. 2006. Effect of wheat genotype on the phenotype of wheat x jointed goatgrass (Aegilops cylindrica) hybrids. Weed Science 54: 690–694. Thurber CS, Jia MH, Jia YL, Caicedo AL. 2013. Similar traits, different genes? Examining convergent evolution in related weedy rice populations. Molecular Ecology 22: 685–698. Wang W, Xia H, Yang X, Xu T, Si HJ, Cai XX, Wang F, Su J, Snow AA, Lu B-R. 2014. A novel 5-enolpyruvoylshikimate-3-phosphate (EPSP) synthase transgene for glyphosate resistance stimulates growth and fecundity in weedy rice (Oryza sativa) without herbicide. New Phytologist 202: 679–683. Weissmann S, Feldman M, Gressel J. 2005. Sporadic inter–generic DNA introgression from wheat into wild Aegilops spp. Molecular Biology and Evolution 22: 2055–2062. Xu J, Feng D, Li X, Chang T, Zhu Z. 2002. Cloning of genomic DNA of rice 5-enolpyruvylshikimate 3-phosphate synthase gene and chromosomal localization of the gene. Science in China (Series C) 45: 251–259. Key words: critique, EPSP synthase transgene, glyphosate resistance, Oryza sativa, Wang et al. (2014). Editorial Note: the Letter from Gressel et al. states that sequence data were not supplied in Wang et al. (2014), despite this being a requirement of the Journal. In light of this, New Phytologist has taken steps to ensure that authors must, if their paper includes new sequence data, provide the relevant accession numbers. In addition, we have made our policy on data deposition explicit by amending our Author Guidelines. These now state that New Phytologist requires authors to make their data available to readers and interested parties upon reasonable request. New Phytologist (2014) 202: 360–362 www.newphytologist.com Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust