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.

Carotenogenesis during tuber development and storage in potato.

Germplasm of Solanum tuberosum and Solanum phureja exhibit a wide (over 20-fold) variation in tuber carotenoid content. The levels of carotenoids during tuber development and storage were compared in a high carotenoid-accumulating S. phureja accession (DB375\1) with two S. tuberosum cultivars (Pentland Javelin and Desiree) that accumulate lower levels of tuber carotenoid. On a dry weight basis, total carotenoid levels were at a maximum early in tuber development. However, in the S. phureja accession, carotenoid levels remained at a high level throughout tuber development, whereas in the S. tuberosum accessions, carotenoid content decreased as dry weight increased. The carotenoid profiles of tissues during tuber development were analysed in greater detail by reverse phase HPLC. In S. phureja tubers at maturity the major carotenoids were zeaxanthin, antheraxanthin, and violaxanthin. Following 9 months storage at 4 degrees C the levels of zeaxanthin and antheraxanthin decreased, whereas the level of lutein increased; overall, however, there was only a small decrease in total carotenoid content. In order to explore the reasons for the wide variation in tuber carotenoid content, the expression patterns of the major genes encoding the enzymes of the carotenoid biosynthetic pathway were compared. Significant differences in the profiles were detected, suggesting that transcriptional control or mRNA stability gives rise to the large differences in tuber carotenoid content. In particular, there was an inverse trend between the level of zeaxanthin epoxidase transcript level and tuber carotenoid content in a range of potato germplasm, giving rise to an hypothesis for the regulation of carotenogenesis in potato tubers.

Copy-DNA cloning and characterisation of a potato alpha-glucosidase: expression in Escherichia coli and effects of down-regulation in transgenic potato.

Polymerase chain reaction-based methodology was used to obtain a cDNA clone (MAL2) from potato (Solanum tuberosum L.) with the sequence characteristics of an alpha-glucosidase. Phylogenetic analysis of the deduced polypeptide encoded by this cDNA demonstrated that the most similar sequences were alpha-glucosidases and alpha-xylosidases of plant origin. The MAL2 cDNA was expressed in Escherichia coli and the recombinant MAL2 protein was affinity-purified. MAL2 catalysed the hydrolysis of a range of maltooligomers and p-nitrophenyl-alpha-D-glucopyranoside with a pH optimum of 5.5-5.7. The substrate with the lowest Km value was maltotetraose (3.7 mM). The MAL2 expression product did not catalyse the hydrolysis of xyloglucan oligosaccharides, p-nitrophenyl-alpha-D-xylopyranoside or gelatinised potato starch. MAL2 was down-regulated in transgenic potato plants using an antisense approach. In several independent transgenic antisense lines, MAL2 expression was severely down-regulated. Despite this, no decrease in total extractable alpha-glucosidase and alpha-xylosidase activity could be detected in tissues from the transgenic plants. In glasshouse trials, no visible phenotype, change in tuber yield or carbohydrate content was associated with MAL2 down-regulation. The implications of these results are discussed.

Functional characterisation of urease accessory protein G (ureG) from potato.

The activation of the nickel metalloenzyme urease is a complex process. In bacteria, several urease accessory proteins are essential for incorporation of nickel into the active centre of urease. Comparatively little is known about the activation process and the proteins involved in plants. We cloned five different cDNAs encoding isoforms of urease accessory protein G (ureG) in potato. The 5'-coding region of these cDNAs is highly polymorphic within Solanum tuberosum ssp. tuberosum, containing mainly a simple sequence repeat encoding histidine and aspartate. Mapping on an ultrahigh-density map of the potato genome and Southern blot analysis showed that the isoforms arise from allelic differences of a single-copy gene which was located on chromosome 2. Expression analysis at the mRNA and protein levels indicated the presence of ureG in almost all tissues examined, consistent with the ubiquitous expression of urease. An attempt to correlate urease activity with ureG expression levels in different tissues was made. Allelic copies of ureG were expressed in a tissue-specific manner. UreG from potato and the Klebsiella aerogenes urease operon defective in bacterial ureG were co-expressed in Escherichia coli. The plant gene complements the K. aerogenes ureG mutation, demonstrating that it encodes a urease accessory protein and indicating a structural conservation between the plant and the bacterial urease activation complexes.

Ripening-related changes in raspberry cell wall composition and structure.

Cell walls were prepared from the fruit of two cultivars of raspberry at three stages of ripening; green, white and red (ripe). The cultivars. Glen Clova and Glen Prosen, are subjectively classified, at harvest by growers, as soft and firm fruit, respectively. The cell walls were analysed for neutral sugar composition, uronic acid content, degree of methyl esterification, lignin and ferulic acid-derived dehydrodimers. Solid-state 31C NMR and diffuse reflectance infrared (DRIFT) spectra were acquired for the cell wall residues. For both cultivars the progression from green to white produced minimal changes, save for a reduction in pectin. NMR analyses indicated that the solubilized pectin was acetylated. Progression to the red (ripe) stage, in both cultivars, was accompanied by a reduction in the ordered cellulose and a dramatic reduction in pectin content and the degree of methyl-esterification. Significantly, the softer fruit (Glen Clova) exhibited greater reductions in both parameters, implicating increased pectin hydrolysis, as one of the main factors contributing to the difference in firmness between the cultivars. A relative increase in cell wall-associated protein was seen at the red stage. The nature and function of the protein(s) are, as yet unknown.

A potato alpha-glucosidase gene encodes a glycoprotein-processing alpha-glucosidase II-like activity. Demonstration of enzyme activity and effects of down-regulation in transgenic plants.

In order to elucidate more fully the function of a potato gene (MAL1) encoding alpha-glucosidase activity, transgenic plants in which MAL1 expression was down-regulated were generated using antisense technology. In transgenic lines severely down-regulated in the expression of MAL1, total alpha-glucosidase activity was not decreased in leaves and tubers, and the contents of starch, glucose, fructose and sucrose remained unchanged in tubers. Phylogenetic analysis indicated that the MAL1 gene product was more similar to the glycoprotein-processing alpha-glucosidase II of mammalian and yeast origin than to other plant alpha-glucosidases. Using [14C-Glc]-labelled Glc2Man9GlcNAc2 as a substrate, it was demonstrated that glucosidase II activity was markedly down-regulated in microsomes isolated from tubers of four independent antisense lines studied in detail, strongly suggesting that MAL1 encodes glucosidase II activity. In field trials (but not in the glasshouse), MAL1 down-regulation produced an extremely stunted phenotype - the leaves were curled and tuber yield was decreased by 90% compared to control values. Microscopic analysis of leaves revealed significant differences between the antisense and control samples. Plants with down-regulated glucosidase II activity showed a greater degree of plasmolysis, and an increase in the size of mesophyll intracellular spaces. Analysis of cell walls also indicated changes in structure as a result of MAL1 down-regulation. In leaves from four antisense lines, the steady-state transcript level corresponding to the endoplasmic reticulum chaperone, BiP, was enhanced. This is diagnostic of stress in the endoplasmic reticulum.

Profiling of changes in gene expression during raspberry (Rubus idaeus) fruit ripening by application of RNA fingerprinting techniques.

Processes that contribute to the overall phenomenon of fruit ripening are not well understood for many soft fruit species, including raspberry (Rubus idaeus L.) Recent biochemical data implicate ethylene and a range of cell wall hydrolases in ripening processes. However, the genes encoding these activities, and others related to ripening, remain to be characterised. With the advent of high-throughput RNA-fingerprinting techniques it is possible to characterise rapidly the changes in gene expression during developmental processes. This paper describes the application of two RNA-fingerprinting techniques (cDNA amplified fragment length polymorphism and differential display reverse transcription-polymerase chain reaction) to the ripening fruit of Rubus idaeus. Copy-DNA tags were isolated representing 34 genes, up-regulated during fruit ripening. The expression profiles of these genes were determined by RNA-blot analysis and their sequences were compared with those in public databases. Potential roles for some of these gene products are considered, providing valuable insights into the processes that underpin fruit ripening. Many of the cDNAs isolated in this study provide tools for the biotechnological improvement of fruit quality.

Two cDNAs representing alleles of the nitrate reductase gene of potato (Solanum tuberosum L. cv. Desirée): sequence analysis, genomic organization and expression.

Two, different, full-length cDNAs, StNR2 and StNR3, of 3049 and 3066 nucleotides, respectively, were isolated from a Solanum tuberosum L. cv. Desirée leaf cDNA library by an RT-PCR approach. Conceptual translation of the longest open reading frame of each cDNA showed that they could encode a protein of 911 amino acids in each case with an M(r) of 102.6 and 102.5 kDa, respectively, and with 99.7% identity with each other. The cDNAs have a high degree of sequence similarity with all higher plant nitrate reductases (NRs) and possess structural characteristics expected of NADH-NR proteins, consistent with enzyme activity data. Southern analysis of genomic DNA suggested the presence of a single NR gene in the potato genome whilst studies using the mapping population F1840, and the full-length StNR2 cDNA as hybridization probe, identified a single NR locus within the potato genome that is located on chromosome XI. The two cDNAs identified here are probably derived from two transcribing alleles of this single gene. Distribution of total NR transcript and of NADH-NR activity, in different organs of compost-grown plants, depended on the level of nitrate supplied: at low nitrate level transcript and activity were detected only in leaf and stem tissue whilst at high nitrate level they could also be detected in root and stolon. An RT-PCR approach was used to differentiate between the transcripts derived from the two identified alleles and to show that the pattern of transcription of the two identified alleles of the potato nia gene, in the organs studied, is the same.

Purification and characterisation of a novel starch synthase selective for uridine 5'-diphosphate glucose from the red alga Gracilaria tenuistipitata.

Red algae (Rhodophyceae) are photosynthetic eukaryotes that accumulate starch granules in the cytosol. Starch synthase activity in crude extracts of Gracilaria tenuistipitata Chang et Xia was almost 9-fold higher with UDP[U-14C]glucose than with ADP[U-14C]glucose. The activity with UDP[U-14C]glucose was sensitive to proteolytic and oxidative inhibition during extraction whilst the activity with ADP[U-14C]glucose appeared unaffected. This indicates the presence of separate starch synthases with different substrate specificities in G. tenuistipitata. The UDPglucose: starch synthase was purified and characterised. The enzyme appears to be a homotetramer with a native M(r) of 580 kDa and displays kinetic properties similar to other alpha-glucan synthases such as stimulation by citrate, product (UDP) inhibition and broad primer specificity. We propose that this enzyme is involved in cytosolic starch synthesis in red algae and thus is the first starch synthase described that utilises UDPglucose in vivo. The biochemical implications of the different compartmentalisation of starch synthesis in red algae and green algae/plants are also discussed.

Manipulation of S-adenosylmethionine decarboxylase activity in potato tubers. An increase in activity leads to an increase in tuber number and a change in tuber size distribution.

S-Adenosylmethionine decarboxylase (SAMDC; EC 4.1.1.50) is a key enzyme in the biosynthesis of the polyamines spermidine and spermine from putrescine and its activity is rate limiting in this pathway. Transgenic potato (Solanum tuberosum L. cv. Desiree) plants containing both sense and antisense SAMDC constructs driven by the tuber-specific patatin promoter have been generated and analysed. In sense transformants, developing tubers expressed higher steady-state levels of the SAMDC-specific transcript, had higher levels of SAMDC activity and contained significantly higher levels of spermidine than vector-transformed controls. Additionally, there was a significant shift in tuber size distribution with larger numbers of smaller tubers but no overall change in tuber yield. In developing tubers from the antisense transformed lines, there was a decrease in SAMDC transcript level, SAMDC activity and total polyamine levels. However, no obvious phenotypic effect was detected in the tuberisation physiology of the antisense lines.

The isolation of genomic DNA from blackcurrant (Ribes nigrum L.).

A method is described for isolating DNA of high molecular mass (M(r)) from blackcurrant and other softfruit species. Following a hexacethylytimethyl ammonium bromide (CTAB)-based extraction procedure, samples are treated with a glycosidic hydrolase mixture and RNase, and then purified. The suitability of this DNA for Southern analysis and genomic-library construction is demonstrated.

cDNA cloning and characterisation of an alpha-glucosidase gene from potato (Solanum tuberosum L.).

Using an Arabidopsis thaliana expressed sequence tag with sequence similarity to human lysosomal alpha-glucosidase as a probe, a potato cDNA was isolated. The cDNA encodes a polypeptide with an Mr value of 105,400 and the most significant matches of the deduced amino acid sequence are with members of family 31 of glucosyl transferase. The potato cDNA was expressed in a strain of Saccharomyces cerevisiae that is deficient in maltase activity and unable to grow using maltose as a carbon source (ABYSMAL81). Expression of the potato cDNA in the mutant yeast strain restores its ability to use maltose as a carbon source for growth. Additionally, alpha-glucosidase activity could be measured in extracts of the yeast cells following complementation. A range of maltodextrins were substrates for this activity. The steady-state expression level of the potato alpha-glucosidase gene was low in most tissues examined, the highest levels occurring in sprouting tubers and source leaves.

Isolation of RNA from blackcurrant (Ribes nigrum L.) fruit.

Extraction of high-quality RNA from blackcurrant fruit has hitherto proved difficult, probably owing to high levels of phenolic and polysaccharide components in the berries. The procedure described here is a modification of one described for grape berries, and yields RNA suitable for in vitro translations, RNA blot analysis, and cDNA library construction.

The isolation of RNA from raspberry (Rubus idaeus) fruit.

Previous attempts to extract high-quality, total RNA from raspberry (Rubus idaeus) fruits using published protocols have proven to be unsuccessful. Even the use of protocols developed for the extraction of RNA from other fruit tissue has resulted in low yields (1) or the isolation of degraded RNA (2). Here, we report on the development of a quick and simple method of extracting total RNA from raspberry fruit. Using this method, high yields of good quality, undegraded RNA were obtained from fruit at all stages of ripening. The RNA is of sufficient quality for northern analysis and cDNA library construction.

Sucrolytic Enzyme Activities in Cotyledons of the Faba Bean (Developmental Changes and Purification of Alkaline Invertase).

In terms of maximum extractable catalytic activity, sucrose synthase is the predominant sucrolytic enzyme in developing cotyledons of faba bean (Vicia faba L.). Although acid invertase activity is extremely low, there is significant activity of alkaline invertase, the majority of which is extractable only with high concentrations of NaCl. Calculations of potential activity in vivo indicate that alkaline invertase is the predominant sucrolytic enzyme from 50 days after anthesis onward. However, at almost all stages of cotyledon development analyzed, the maximum extractable catalytic activities of both enzymes is in excess of the actual rate of starch deposition. Two forms of alkaline invertase were identified in developing cotyledons. The major form has been purified to homogeneity, and antibodies have been raised against it. The native protein has a molecular mass of about 238 [plus or minus] 4.5 kD. It is apparently a homotetramer (subunit molecular mass 53.4 [plus or minus] 0.9 kD). The enzyme has a pH optimum of 7.4, an isoelectric point of 5.2, and a Km[sucrose] of 10 mM and is inhibited by Tris (50% inhibition at 5 mM) and fructose (30% inhibition at 10 mM). Bean alkaline invertase is a [beta]-fructofuranosidase with no significant activity against raffinose, stachyose, trehalose, maltose, or lactose.

Characterisation of the cDNA clones of two beta-tubulin genes and their expression in the potato plant (Solanum tuberosum L.).

The cDNA clones of two potato beta-tubulin genes were isolated from a tuberising stolon tip library. Analysis of 20 positive clones showed that they represented one or another of two different but very similar beta-tubulin genes, designated TUBST1 and TUBST2. The expression pattern of beta-tubulin genes in the potato plant was investigated by RNA blot analysis and by RT-PCR. Southern analysis of potato genomic DNA with coding and non-coding beta-tubulin probes revealed that there are multiple beta-tubulin genes in the potato genome and that there is likely to be considerable divergence in the 3' non-coding sequences. Phylogenetic analysis of plant beta-tubulin genes is described.

Characterisation of the S-adenosylmethionine decarboxylase (SAMDC) gene of potato.

S-adenosylmethionine decarboxylase (SAMDC) is involved in the biosynthesis of the polyamines, spermidine and spermine. Recently, we reported the isolation of a putative cDNA clone of the SAMDC clone of potato (Plant Mol Biol 20; 641-651). In order to confirm that the potato genes does encode SAMDC, a complementation experiment with a yeast strain that possesses a null mutation in the SAMDC gene was performed. The yeast strain contains a deletion-insertion mutation in the SAMDC gene and has an absolute requirement for the addition of exogenous spermidine for growth. When the full-length potato cDNA was expressed in the mutant yeast strain there was no longer a requirement for exogenous spermidine. Immunoblotting experiments suggest that the potato SAMDC gene product has an apparent molecular mass of 39 kDa. Expression of the SAMDC gene was high in the young and actively dividing tissues and low in the mature and non-dividing tissues of both vegetative and reproductive organs. Additionally, isolation and characterisation of the corresponding genomic clone is reported. The gene has one intron in its 5'-untranslated sequence but otherwise the transcribed portion is identical to the cDNA clone.

Characterisation of a complementary DNA encoding a novel plant enzyme with sucrolytic activity.

The cloning of a 1332 bp cDNA from a potato (Solanum tuberosum L.) cv. Cara leaf cDNA expression library, using an antibody raised against a purified tuber protein preparation with sucrolytic activity, is described. The corresponding gene in potato is of low copy number, is expressed in a variety of tissues, and encodes a protein which includes several domains with similarity to database sequences, including ferredoxin from Clostridium pasteurianum. Expression of the cDNA in E. coli yields a fusion protein with sucrolytic activity.

Potato (Solanum tuberosum) invertase-encoding cDNAs and their differential expression.

A full-length cDNA clone encoding a potato invertase (Inv) has been isolated. It is highly related (77% nucleotide identity) to a previously characterised potato cDNA clone encoding a putative extracellular Inv. These Inv genes encode a subfamily of apoplastic enzymes which are shown to be distinct, on the basis of sequence similarity, from the related subfamily of vacuolar enzymes. In order to differentiate between the expression of the two potato genes encoding apoplastic Inv, a single-stranded conformational polymorphism (SSCP) assay was developed for products generated by reverse transcription-polymerase chain reaction (RT-PCR) utilising primers designed to amplify both potato sequences. Using this approach, we have shown that these two identified Inv from potato are expressed in a tissue-specific and developmentally regulated manner.