Unintended effects and their detection in genetically modified crops.

The commercialisation of GM crops in Europe is practically non-existent at the present time. The European Commission has instigated changes to the regulatory process to address the concerns of consumers and member states and to pave the way for removing the current moratorium. With regard to the safety of GM crops and products, the current risk assessment process pays particular attention to potential adverse effects on human and animal health and the environment. This document deals with the concept of unintended effects in GM crops and products, i.e. effects that go beyond that of the original modification and that might impact primarily on health. The document first deals with the potential for unintended effects caused by the processes of transgene insertion (DNA rearrangements) and makes comparisons with genetic recombination events and DNA rearrangements in traditional breeding. The document then focuses on the potential value of evolving "profiling" or "omics" technologies as non-targeted, unbiased approaches, to detect unintended effects. These technologies include metabolomics (parallel analysis of a range of primary and secondary metabolites), proteomics (analysis of polypeptide complement) and transcriptomics (parallel analysis of gene expression). The technologies are described, together with their current limitations. Importantly, the significance of unintended effects on consumer health are discussed and conclusions and recommendations presented on the various approaches outlined.

Field tolerance to fungal pathogens of Brassica napus constitutively expressing a chimeric chitinase gene.

Constitutive overexpression of a protein involved in plant defense mechanisms to disease is one of the strategies proposed to increase plant tolerance to fungal pathogens. A hybrid endochitinase gene under a constitutive promoter was introduced by Agrobacterium-mediated transformation into a winter-type oilseed rape (Brassica napus var. oleifera) inbred line. Progeny from transformed plants was challenged using three different fungal pathogens (Cylindrosporium concentricum, Phoma lingam, Sclerotinia sclerotiorum) in field trials at two different geographical locations. These plants exhibited an increased tolerance to disease as compared with the nontransgenic parental plants.

Regulation of the maize HRGP gene expression by ethylene and wounding. mRNA accumulation and qualitative expression analysis of the promoter by microprojectile bombardment.

The expression of the maize gene coding for a hydroxyproline-rich glycoprotein (HRGP) has been studied by measuring the mRNA accumulation after wounding or ethylene treatment. RNA blot and in situ hybridization techniques have been used. The temporal and tissue-specific expression has been observed: the cells related to the vascular system show the more intense HRGP mRNA accumulation. Transcriptional constructions of the maize HRGP promoter have been tested on different maize tissues by microbombarding. A 582 bp promoter is able to direct the expression of the gus gene on calli and young leaves. Constructions having shorter promoter sequences lose this ability. The 582 bp construction retains the general specificity of expression observed for the HRGP gene.

Common responses of cultured soybean cells to 2,4-D starvation and fungal elicitor treatment.

The patterns of in vivo protein synthesis in soybean cell suspensions were compared by polyacrylamide gel electrophoresis after the cells had been submitted to different stress conditions : treatment with Phytophthora megasperma (Pmg) cell wall elicitors, 2,4-D starvation and heat shock (HS) temperatures. Changes in protein synthesis patterns induced after elicitation of cell suspensions or after infection of soybean hypocotyls by Pmg were found to be similar to changes brought about by auxin starvation of the cells. Changes common to both stress situations involve a prominent 17 kDa peptide family and 27, 29, 35 and about 45 kDa peptides. Moreover, "defense" reactions, i.e. glyceollin accumulation and synthesis of chalcone synthase (CHS) were also strongly stimulated in auxin-starved cells. On the contrary, although characteristic sets of low molecular weight heat shock (HS) proteins were synthesized by cells grown at 37°C, no clear similarity was observed with peptides characteristic of auxin-starved cells.

Patterns of protein synthesis in dividing and auxin-starved soybean cell suspensions. Differential expression of a major group of Mr-17000 peptides.

Auxin starvation of soybean cell suspensions results in the arrest of cell growth after about 4 days. The addition of 4 µM 2,4-D enables cells to divide again after a lag phase of 1 day. By comparing the patterns of in vivo and in vitro protein synthesis, we have identified two sets of polypeptides whose synthesis is positively or negatively regulated by auxin. Several major peptide bands (17, 26, 31, 35, 38 kD) are characteristic of auxin-starved cells. The 17 kD peptide group, containing one major (75%) component, accounts for 6% and 25% of the radiolabeling in vivo of proteins from respectively dividing and auxin-starved cells. Our results suggest the 17 kD major component to be a direct translation product whose synthesis is regulated by the abundance or the activity of the relevant mRNA. The soybean suspension culture system here described provides a model to study auxin-mediated control of gene expression.

Auxin Conjugation by Tobacco Mesophyll Protoplasts : Correlations between Auxin Cytotoxicity under Low Density Growth Conditions and Induction of Conjugation Processes at High Density.

Auxin induction of the proliferation of Nicotiana tabacum (cv Xanthi) mesophyll protoplasts and of protoplast-derived cells was studied. The growth-promoting properties and cytotoxicities at high concentrations of IAA and naphthaleneacetic acid were strongly affected by cell density. The induction of growth by 2,4-dichlorophenoxyacetic acid and picloram was not affected by cell density. The comparison of catabolism of these [(14)C]-labeled auxins by protoplasts showed that IAA and naphthalene-acetic acid were rapidly accumulated and conjugated unlike 2,4-dichlorophenoxyacetic acid and picloram. The major catabolite derived from naphthaleneacetic acid was identified as naphthaleneacetyl-l-aspartate. The biosynthesis of this conjugate in protoplasts was inducible by naphthaleneacetic acid concentrations found to be cytotoxic under low density growth conditions. However, although it was taken up by cells, the conjugate was not cytotoxic at concentrations as high as 0.2 mm under low density growth conditions. The relationship between conjugation processes and auxin cytotoxicity is discussed.

A P Nuclear Magnetic Resonance Study of Intracellular pH of Plant Cells Cultivated in Liquid Medium.

(31)P nuclear magnetic resonance has been used to study the vacuolar and cytoplasmic pH of Acer pseudoplatanus, Catharanthus roseus, and Glycine max cells grown as cell suspensions. The adaptation of this technique to plant cells grown in liquid medium is described with emphasis on the removal of Mn(2+) and phosphate from the extracellular medium and on providing the O(2) supply of the cells in the nuclear magnetic resonance tube and the various problems of calibration. Aerobic and anaerobic cells show large differences in their glucose-6-phosphate, their cytoplasmic inorganic phosphate pools, and their cytoplasmic pH. Differences in the relative sizes of the cytoplasmic and vacuolar inorganic phosphate pools have been observed for the three cell strains studied.

Influence of fusicoccin on the control of cell division by auxins.

An indirect stimulation of cell division by fusicoccin is demonstrated. The distribution of 2,4-dichlorophenoxyacetic acid molecules between Acer pseudoplatanus cells and their culture medium is strongly modified by a fusicoccin treatment, through the extracellular acidification induced by the toxin. As a consequence, a stimulation of cell division is observed when the intracellular auxin concentration is sufficiently increased to reach the threshold control level (Leguay JJ, J Guern 1977 Plant Physiol 60: 265-270).Independently of this indirect action on cell division, the number of cells per cluster is decreased and the volume of the cells increased in fusicoccintreated cells which show a typical enlargement response.

Measurement of intracellular pH and aspects of its control in higher plant cells cultivated in liquid medium.

The 5,5-dimethyloxazolidine-2,4-dione (DMO) method for the determination of intracellular pH has been adapted to Acer pseudoplatanus cells cultivated in liquid medium. DMO is metabolized by these cells and this parameter has to be taken into account in the measurement of the intracellular concentration of free DMO. As the cells contain a large acidic vacuolar compartment, the intracellular pH estimated from the DMO technique is an overall pHi', the dependence of which on various cytoplasmic and vacuolar characteristics has been discussed. Important modifications of these overall pHi values under the influence of CO2 or NH4+ have been measured. These pHi modifications strongly affect the regulation of cell division through the control of the intracellular concentration of hormones of the auxin type.

Quantitative Effects of 2,4-Dichlorophenoxyacetic Acid on Growth of Suspension-cultured Acer pseudoplatanus Cells: II. Influence of 2,4-D Metabolism and Intracellular pH on the Control of Cell Division by Intracellular 2,4-D Concentration.

The utilization of 2,4-dichlorophenoxyacetic acid (2,4-D) molecules by Acer pseudoplatanus cells is governed mainly by a glucosylation process. Evidence that 2,4-D glucoside molecules are biologically inactive is presented. 2,3,5-Triiodobenzoic acid (TIBA), by inhibiting 2,4-D glucosylation, has a sparing effect on 2,4-D molecules; thus TIBA treatments increase growth yield (expressed as the ratio of the maximum number of cells produced to the initial concentration of 2,4-D in the culture medium).Significant amounts of intact 2,4-D molecules remain outside and inside the cells when cell division stops at the onset of the stationary phase. This result and the previous demonstration that, at the onset of the stationary phase, 2,4-D is the specific limiting factor of cell division (Leguay JJ, J Guern 1975 Plant Physiol 56: 356-359) suggest that a threshold concentration of auxin is needed for cell division to proceed.The distribution of 2,4-D molecules between the cells and the culture medium is dependent on the population density at the stationary phase. The extracellular 2,4-D concentration at that time is a linear function of the population density whereas intracellular amounts of 2,4-D and 2,4-D metabolites are constant. By using a modified 2-(14)C,-5,5-dimethyloxazolidine-2,4-dione technique, it has been shown that the intracellular pH is markedly lowered as the population density at the plateau is increased. This intracellular pH modification is likely to be responsible for a large modification of the ratio between intracellular and extracellular auxin concentrations.The intracellular auxin concentration reaches a constant value (about 3 x 10(-7)m), independent of population density when cell division stops at the onset of the stationary phase suggesting that it represents the threshold value of the control for cell division.

The 5,5-dimethyloxazolidine-2[14C],4-dione distribution technique and the measurement of intracellular pH in Acer pseudoplatanus cells.

The intracellular pH of suspension-cultured Acer pseudoplatanus cells, was estimated from the distribution of 5,5-dimethyloxazolidine-2[14C],4-dione (DMO) between the culture medium and the cells. The metabolization of DMO in this biological system introduces an error in the calculated intracellular pH value. Three methods are given to overcome this difficulty and to estimate the equilibrium between intracellular and extracellular DMO molecules. A preliminary study has shown that the intracellular pH remains constant about 6.5 when the extracellular pH increases from 5.6 to 7.3.

Quantitative Effects of 2,4-Dichlorophenoxyacetic Acid on Growth of Suspension-cultured Acer pseudoplatanus Cells.

Using suspension-cultured Acer pseudoplatanus cells requiring 2,4-dichlorophenoxyacetic acid for growth, the dependence of the population doubling time and the maximum increase in cell population density on the auxin concentration was studied. It appears that in the range of 2,4-dichlorophenoxyacetic acid concentration from 4 x 10(-8) to 4 x 10(-6) M, the rate of cell division during the logarithmic growth phase is independent of the auxin concentration, while the maximum number of cell generations obtained is limited by the initial auxin concentration. The significance of these two aspects of auxin action are discussed.