Anna Nadal

Natural variation explains most transcriptomic changes among maize plants of MON810 and comparable non-GM varieties subjected to two N-fertilization farming practices

The introduction of genetically modified organisms (GMO) in many countries follows strict regulations to ensure that only safety-tested products are marketed. Over the last few years, targeted approaches have been complemented by profiling methods to assess possible unintended effects of transformation. Here we used a commercial (Affymertix) microarray platform (i.e. allowing assessing the expression of ~1/3 of the genes of maize) to evaluate transcriptional differences between commercial MON810 GM maize and non-transgenic crops in real agricultural conditions, in a region where about 70% of the maize grown was MON810. To consider natural variation in gene expression in relation to biotech plants we took two common MON810/non-GM variety pairs as examples, and two farming practices (conventional and low-nitrogen fertilization). MON810 and comparable non-GM varieties grown in the field have very low numbers of sequences with differential expression, and their identity differs among varieties. Furthermore, we show that the differences between a given MON810 variety and the non-GM counterpart do not appear to depend to any major extent on the assayed cultural conditions, even though these differences may slightly vary between the conditions. In our study, natural variation explained most of the variability in gene expression among the samples. Up to 37.4% was dependent upon the variety (obtained by conventional breeding) and 31.9% a result of the fertilization treatment. In contrast, the MON810 GM character had a very minor effect (9.7%) on gene expression in the analyzed varieties and conditions, even though similar cryIA(b) expression levels were detected in the two MON810 varieties and nitrogen treatments. This indicates that transcriptional differences of conventionally-bred varieties and under different environmental conditions should be taken into account in safety assessment studies of GM plants.

Lack of repeatable differential expression patterns between MON810 and comparable commercial varieties of maize

The introduction of genetically modified organisms (GMO) in many countries follows strict regulations to assure that only products that have been safety tested in relation to human health and the environment are marketed. Thus, GMOs must be authorized before use. By complementing more targeted approaches, profiling methods can assess possible unintended effects of transformation. We used microarrays to compare the transcriptome profiles of widely commercialized maize MON810 varieties and their non-GM near-isogenic counterparts. The expression profiles of MON810 seedlings are more similar to those of their corresponding near-isogenic varieties than are the profiles of other lines produced by conventional breeding. However, differential expression of ∼1.7 and ∼0.1% of transcripts was identified in two variety pairs (AristisBt/Aristis and PR33P67/PR33P66) that had similar cryIA(b) mRNA levels, demonstrating that commercial varieties of the same event have different similarity levels to their near-isogenic counterparts without the transgene (note that these two pairs also show phenotypic differences). In the tissues, developmental stage and varieties analyzed, we could not identify any gene differentially expressed in all variety-pairs. However, a small set of sequences were differentially expressed in various pairs. Their relation to the transgenesis was not proven, although this is likely to be modulated by the genetic background of each variety.

Effect of volunteers on maize gene flow

Regulatory approvals for deliberate release of GM maize events into the environment have lead to real situations of coexistence between GM and non-GM, with some fields being cultivated with GM and conventional varieties in successive seasons. Given the common presence of volunteer plants in maize fields in temperate areas, we investigated the real impact of GM volunteers on the yield of 12 non-GM agricultural fields. Volunteer density varied from residual to around 10% of plants in the field and was largely reduced using certain cultural practices. Plant vigour was low, they rarely had cobs and produced pollen that cross-fertilized neighbour plants only at low—but variable—levels. In the worst-case scenario, the estimated content of GMO was 0.16%. The influence of GM volunteers was not enough to reach the 0.9% adventitious GM threshold but it could potentially contribute to adventitious GM levels, especially at high initial densities (i.e. above 1,000 volunteers/ha).

Stress proteins co-expressed in suberized and lignified cells and in apical meristems

Abstract We report the cloning of a small heat shock protein, Qs HSP17, and an osmotin like protein, Qs_OLP, from cork oak phellem tissue (cork cells). Both genes are expressed in suberizing cells and in other cells subject to endogenous stress associated with free radicals. We provide evidence that smHSPs and OLPs accumulate in overwintering buds and speculate that their role is similar to that in seed dormancy. We also show that both stress proteins are mainly located in the region of the quiescent center in root apex and in central meristem in the shoot apex. We emphasize that smHSPs and OLPs are expressed in cells growing under endogenous stress or facing long life-span. We discuss a possible role of these stress proteins against oxidative stress.

Proteomic analysis of MON810 and comparable non-GM maize varieties grown in agricultural fields

Worldwide maize is the second major agricultural commodity and around one-fourth is currently biotech, with significant application of the insect resistant event MON810 particularly in the European Union. Grains are the major commercialized part of the plant, and can be harvested after maturity (for food and feed purposes) or at late milky-starchy stage (for forage uses, with the whole plant). We assessed possible proteomic unintended effects of the MON810 transgene using two-dimensional gel electrophoresis coupled to mass spectrometry. To keep in a realistic scenario we used plants grown in agricultural fields in a region where ~50% of maize was MON810, and analyzed grains at milky-starchy stage. In maize, differential transcripts and metabolites between GM and comparable non-GM varieties tend to be variety specific. Thus, we analyzed two variety pairs, DKC6575/Tietar and PR33P67/PR33P66 which are considered representative of Food and Agriculture Organization 700 and 600 varieties commercially grown in the region. MON810 and non-GM milky-starchy grains had virtually identical proteomic patterns, with a very small number of spots showing fold-variations in the 1-1.8 range. They were all variety specific and had divergent identities and functions. Although 2DE allows the analysis of a limited dataset our results support substantial equivalence between MON810 and comparable non-GM varieties.

Gene expression profiles of MON810 and comparable non-GM maize varieties cultured in the field are more similar than are those of conventional lines

Maize is a major food crop and genetically modified (GM) varieties represented 24% of the global production in 2007. Authorized GM organisms have been tested for human and environmental safety. We previously used microarrays to compare the transcriptome profiles of widely used commercial MON810 versus near-isogenic varieties and reported differential expression of a small set of sequences in leaves of in vitro cultured plants of AristisBt/Aristis and PR33P67/PR33P66 (Coll et al. 2008). Here we further assessed the significance of these differential expression patterns in plants grown in a real context, i.e. in the field. Most sequences that were differentially expressed in plants cultured in vitro had the same expression values in MON810 and comparable varieties when grown in the field; and no sequence was found to be differentially regulated in the two variety pairs grown in the field. The differential expression patterns observed between in vitro and field culture were similar between MON810 and comparable varieties, with higher divergence between the two conventional varieties. This further indicates that MON810 and comparable non-GM varieties are equivalent except for the introduced character.

Only half the transcriptomic differences between resistant genetically modified and conventional rice are associated with the transgene

Besides the intended effects that give a genetically modified (GM) plant the desired trait, unintended differences between GM and non-GM comparable plants may also occur. Profiling technologies allow their identification, and a number of examples demonstrating that unintended effects are limited and diverse have recently been reported. Both from the food safety aspect and for research purposes, it is important to discern unintended changes produced by the transgene and its expression from those that may be attributed to other factors. Here, we show differential expression of around 0.40% transcriptome between conventional rice var. Senia and Senia-afp constitutively expressing the AFP antifungal protein. Analysis of one-fifth of the regulated sequences showed that around 35% of the unintended effects could be attributed to the process used to produce GM plants, based on in vitro tissue culture techniques. A further ∼15% were event specific, and their regulation was attributed to host gene disruption and genome rearrangements at the insertion site, and effects on proximal sequences. Thus, only around half the transcriptional unintended effects could be associated to the transgene itself. A significant number of changes in Senia-afp and Senia are part of the plant response to stress conditions, and around half the sequences for which up-regulation was attributed to the transgene were induced in conventional (but not transgenic) plants after wounding. Unintended effects might, as such, putatively result in widening the self-resistance characteristics because of the transgene in GM plants.

Constitutive expression of transgenes encoding derivatives of the synthetic antimicrobial peptide BP100: impact on rice host plant fitness

BackgroundThe Biopeptide BP100 is a synthetic and strongly cationic α-helical undecapeptide with high, specific antibacterial activity against economically important plant-pathogenic bacteria, and very low toxicity. It was selected from a library of synthetic peptides, along with other peptides with activities against relevant bacterial and fungal species. Expression of the BP100 series of peptides in plants is of major interest to establish disease-resistant plants and facilitate molecular farming. Specific challenges were the small length, peptide degradation by plant proteases and toxicity to the host plant. Here we approached the expression of the BP100 peptide series in plants using BP100 as a proof-of-concept.ResultsOur design considered up to three tandemly arranged BP100 units and peptide accumulation in the endoplasmic reticulum (ER), analyzing five BP100 derivatives. The ER retention sequence did not reduce the antimicrobial activity of chemically synthesized BP100 derivatives, making this strategy possible. Transformation with sequences encoding BP100 derivatives (bp100der) was over ten-fold less efficient than that of the hygromycin phosphotransferase (hptII) transgene. The BP100 direct tandems did not show higher antimicrobial activity than BP100, and genetically modified (GM) plants constitutively expressing them were not viable. In contrast, inverted repeats of BP100, whether or not elongated with a portion of a natural antimicrobial peptide (AMP), had higher antimicrobial activity, and fertile GM rice lines constitutively expressing bp100der were produced. These GM lines had increased resistance to the pathogens Dickeya chrysanthemi and Fusarium verticillioides, and tolerance to oxidative stress, with agronomic performance comparable to untransformed lines.ConclusionsConstitutive expression of transgenes encoding short cationic α-helical synthetic peptides can have a strong negative impact on rice fitness. However, GM plants expressing, for example, BP100 based on inverted repeats, have adequate agronomic performance and resistant phenotypes as a result of a complex equilibrium between bp100der toxicity to plant cells, antimicrobial activity and transgene-derived plant stress response. It is likely that these results can be extended to other peptides with similar characteristics.

Sensitive and label-free biosensing of RNA with predicted secondary structures by a triplex affinity capture method

A novel biosensing approach for the label-free detection of nucleic acid sequences of short and large lengths has been implemented, with special emphasis on targeting RNA sequences with secondary structures. The approach is based on selecting 8-aminoadenine-modified parallel-stranded DNA tail-clamps as affinity bioreceptors. These receptors have the ability of creating a stable triplex-stranded helix at neutral pH upon hybridization with the nucleic acid target. A surface plasmon resonance biosensor has been used for the detection. With this strategy, we have detected short DNA sequences (32-mer) and purified RNA (103-mer) at the femtomol level in a few minutes in an easy and level-free way. This approach is particularly suitable for the detection of RNA molecules with predicted secondary structures, reaching a limit of detection of 50 fmol without any label or amplification steps. Our methodology has shown a marked enhancement for the detection (18% for short DNA and 54% for RNA), when compared with the conventional duplex approach, highlighting the large difficulty of the duplex approach to detect nucleic acid sequences, especially those exhibiting stable secondary structures. We believe that our strategy could be of great interest to the RNA field.

The production of recombinant cationic α-helical antimicrobial peptides in plant cells induces the formation of protein bodies derived from the endoplasmic reticulum.

Synthetic linear antimicrobial peptides with cationic α-helical structures, such as BP100, are valuable as novel therapeutics and preservatives. However, they tend to be toxic when expressed at high levels as recombinant peptides in plants, and they can be difficult to detect and isolate from complex plant tissues because they are strongly cationic and display low extinction coefficient and extremely limited immunogenicity. We therefore expressed BP100 with a C-terminal tag which preserved its antimicrobial activity and demonstrated significant accumulation in plant cells. We used a fluorescent tag to trace BP100 following transiently expression in Nicotiana benthamiana leaves and showed that it accumulated in large vesicles derived from the endoplasmic reticulum (ER) along with typical ER luminal proteins. Interestingly, the formation of these vesicles was induced by BP100. Similar vesicles formed in stably transformed Arabidopsis thaliana seedlings, but the recombinant peptide was toxic to the host during latter developmental stages. This was avoided by selecting active BP100 derivatives based on their low haemolytic activity even though the selected peptides remained toxic to plant cells when applied exogenously at high doses. Using this strategy, we generated transgenic rice lines producing active BP100 derivatives with a yield of up to 0.5% total soluble protein.