Identification of traits underpinning good breadmaking performance of wheat grown with reduced nitrogen fertilisation.

BACKGROUND: Nitrogen fertiliser is the major input and cost for wheat production, being required to support the development of the canopy to maximise yield and for the synthesis of the gluten proteins that are necessary for breadmaking. Consequently, current high-yielding cultivars require the use of nitrogen fertilisation levels above the yield optimum to achieve the grain protein content needed for breadmaking. This study aimed to reduce this requirement by identifying traits that allow the use of lower levels of nitrogen fertiliser to produce wheat for breadmaking. RESULTS: A range of commercial wheat genotypes (cultivars) were grown in multiple field trials (six sites over 3 years) in the UK with optimal (200 kg Ha-1 ) and suboptimal (150 kg Ha-1 ) application of nitrogen. Bulked grain samples from four sites per year were milled and white flours were baked using three types of breadmaking process. This identified five cultivars that consistently exhibited good breadmaking quality when grown with the lower nitrogen application. Chemical and biochemical analyses showed that the five cultivars were characterised by exhibiting grain protein deviation (GPD) and high dough elasticity. CONCLUSIONS: It is possible to develop novel types of wheat that exhibit good breadmaking quality by selecting for GPD and high dough strength. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Subcellular dynamics studies of iron reveal how tissue-specific distribution patterns are established in developing wheat grains.

Understanding the mechanisms of iron trafficking in plants is key to enhancing the nutritional quality of crops. Because it is difficult to image iron in transit, we currently have an incomplete picture of the route(s) of iron translocation in developing seeds and how the tissue-specific distribution is established. We have used a novel approach, combining iron-57 (57 Fe) isotope labelling and nanoscale secondary ion mass spectrometry (NanoSIMS), to visualize iron translocation between tissues and within cells in immature wheat grain, Triticum aestivum. This enabled us to track the main route of iron transport from maternal tissues to the embryo through the different cell types. Further evidence for this route was provided by genetically diverting iron into storage vacuoles, with confirmation provided by histological staining and transmission electron microscopy energy dispersive X-ray spectroscopy (TEM-EDS). Almost all iron in both control and transgenic grains was found in intracellular bodies, indicating symplastic rather than apoplastic transport. Furthermore, a new type of iron body, highly enriched in 57 Fe, was observed in aleurone cells and may represent iron being delivered to phytate globoids. Correlation of the 57 Fe enrichment profiles obtained by NanoSIMS with tissue-specific gene expression provides an updated model of iron homeostasis in cereal grains with relevance for future biofortification strategies.

Loss of TaIRX9b gene function in wheat decreases chain length and amount of arabinoxylan in grain but increases cross-linking.

Wheat contains abundant xylan in cell walls of all tissues, but in endosperm, there is an unusual form of xylan substituted only by arabinose (arabinoxylan; AX) that has long chains and low levels of feruloylation, a fraction of which is extractable in water (WE-AX). WE-AX acts as soluble dietary fibre but also gives rise to viscous extracts from grain, a detrimental trait for some non-food uses of wheat. Here, we show that a glycosyl transferase family 43 wheat gene abundantly expressed in endosperm complements the Arabidopsis irx9 mutant and so name the three homoeologous genes TaIRX9b. We generated wheat lines with a constitutive knockout of TaIRX9b by stacking loss-of-function alleles for these homeologues from a mutagenized hexaploid wheat population resulting in decreases in grain extract viscosity of 50%-80%. The amount and chain length of WE-AX molecules from grain of these triple-stack lines was decreased accounting for the changes in extract viscosity. Imaging of immature wheat grain sections of triple-stacks showed abolition of immunolabelling in endosperm with LM11 antibody that recognizes epitopes in AX, but also showed apparently normal cell size and shape in all cell types, including endosperm. We identified differentially expressed genes from endosperm of triple-stacks suggesting that compensatory changes occur to maintain this endosperm cell wall integrity. Consistent with this, we observed increased ferulate dimerization and increased cross-linking of WE-AX molecules in triple-stacks. These novel wheat lines lacking functional TaIRX9b therefore provide insight into control of wheat endosperm cell walls.

Genetic variation and heritability of grain protein deviation in European wheat genotypes.

There is a well-established negative relationship between the yield and the concentration of protein in the mature wheat grain. However, some wheat genotypes consistently deviate from this relationship, a phenomenon known as Grain Protein Deviation (GPD). Positive GPD is therefore of considerable interest in relation to reducing the requirement for nitrogen fertilization for producing wheat for breadmaking. We have carried out two sets of field experiments on multiple sites in South East England. The first set comprised 11 field trials of 6 cultivars grown over three years (2008-2011) and the second comprised 9 field trials of 40 genotypes grown over two years (2015-2017) and 5 field trials of 30 genotypes grown in a single year (2017-2018). All trials comprised three replicate randomized plots of each genotype and nutrient regime. These studies showed strong genetic variation in GPD, which also differed in stability between genotypes, with cultivars bred in the UK generally having higher GPD and higher stability than those bred in other European countries. The heritability of GPD was estimated as 0.44, based on data from the field trials of 30 and 40 genotypes. The largest component contributing to the genetic variance was genotype (0.30), with a smaller contribution of the interaction between genotype and year/site (0.11) and a small (but statistically significant) contribution of nitrogen level. These studies suggest that selection for GPD is a viable target for breeders.

Evaluation of the prebiotic potential of arabinoxylans extracted from wheat distillers' dried grains with solubles (DDGS) and in-process samples.

Distillers' dried grains with solubles (DDGS) is a low-value agro-industrial by-product, rich in arabinoxylans (AX), which is produced by commercial distillery and bioethanol plants. In a first approach, we investigated the prebiotic potential of four fractions comprising arabinoxylan oligosaccharides (AXOS) and xylooligosaccharides (XOS) obtained by enzymatic hydrolysis of AX fractions derived from DDGS and wet solids (in-process sample of DDGS production process). Anaerobic batch cultures in controlled pH conditions were used to test the prebiotic activity of the samples. Results did not show significant differences between the enzymatic treatments used, and all AXOS/XOS were extensively fermented after 24 h. In addition, significant increases (P < 0.05) in Bifidobacterium and total short-chain fatty acids (SCFAs) were observed after 24 h of fermentation. Finally, DDGS-derived hydrolysates were separated on an anionic semi-preparative column to prepare AXOS/XOS fractions with degree of polymerisation (DP) greater than 3. Bifidogenic activity and an increase of SCFAs were again observed after 24 h of fermentation, although this time, the selectivity was higher and the fermentation slower, suggesting that the fermentation of this substrate could take place (at least partially) in the distal part of the colon with highly desirable beneficial effects.

Gradients in compositions in the starchy endosperm of wheat have implications for milling and processing.

BACKGROUND: Wheat is the major food grain consumed in temperate countries. Most wheat is consumed after milling to produce white flour, which corresponds to the endosperm storage tissue of the grain. Because the starchy endosperm accounts for about 80% of the grain dry weight, the miller aims to achieve flour yields approaching this value. SCOPE AND APPROACH: Bioimaging can be combined with biochemical analysis of fractions produced by sequential pearling of whole grains to determine the distributions of components within the endosperm tissue. KEY FINDINGS AND CONCLUSIONS: This reveals that endosperm is not homogeneous, but exhibits gradients in composition from the outer to the inner part. These include gradients in both amount and composition. For example, the content of gluten proteins decreases but the proportion of glutenin polymers increases from the outside to the centre of the tissue. However, the content of starch increases with changes in the granule size distribution, the proportions of amylose and amylopectin, and their thermal properties. Hence these parts of the endosperm differ in the functional properties for food processing. Gradients also exist in minor components which may affect health and processing, such as dietary fibre and lipids. The gradients in grain composition are reflected in differences in the compositions of the mill streams which are combined to give white flour (which may number over 20). These differences could therefore be exploited by millers and food processors to develop flours with compositions and properties for specific end uses.

Intrinsic wheat lipid composition effects the interfacial and foaming properties of dough liquor.

Doughs were prepared from a single variety breadmaking flour (cv. Hereward), from three successive harvests (years; 2011, 2012 and 2013). A preparation of the aqueous phase from dough, known as dough liquor (DL), was prepared by ultracentrifugation and its physico-chemical properties were investigated. Surface tension and interfacial rheology, showed that the interface of DL was lipid-dominated and that 2013 DL had a different type of interface to 2011 and 2012 DL. This data was consistent with the improved foam stability observed for 2013 DL and with the types of lipids identified. All foams collapsed quickly, but the most stable foam was from 2013 DL with 89.2% loss in foam, followed by 2011 DL with 91.7% loss and 2012 had the least stable foam with a loss of 92.5% of the foam structure. Glycolipids (DGDG and MGDG) were enriched in 2013 DL, and were also present in DL foam, contributing towards improved stability. Neutral lipids, such as FFAs, were enriched in DL foams contributing towards instability and rapid foam collapse. Baking trials using 2012 and 2013 flour, showed increased loaf volumes and gas bubble diameter in 2013 bread compared to 2012 bread, highlighting the potential impact that surface active polar lipids, enriched in the aqueous phase of dough, could have on improving breadmaking quality.

Feruloylation and structure of arabinoxylan in wheat endosperm cell walls from RNAi lines with suppression of genes responsible for backbone synthesis and decoration.

Arabinoxylan (AX) is the major component of the cell walls of wheat grain (70% in starchy endosperm), is an important determinant of end-use qualities affecting food processing, use for animal feed and distilling and is a major source of dietary fibre in the human diet. AX is a heterogeneous polysaccharide composed of fractions which can be sequentially extracted by water (WE-AX), then xylanase action (XE-AX) leaving an unextractable (XU-AX) fraction. We determined arabinosylation and feruloylation of AX in these fractions in both wild-type wheat and RNAi lines with decreased AX content (TaGT43_2 RNAi, TaGT47_2 RNAi) or decreased arabinose 3-linked to mono-substituted xylose (TaXAT1 RNAi). We show that these fractions are characterized by the degree of feruloylation of AX, <5, 5-7 and 13-19 mg bound ferulate (g-1 AX), and their content of diferulates (diFA), <0.3, 1-1.7 and 4-5 mg (g-1 AX), for the WE, XE and XU fractions, respectively, in all RNAi lines and their control lines. The amount of AX and its degree of arabinosylation and feruloylation were less affected by RNAi transgenes in the XE-AX fraction than in the WE-AX fraction and largely unaffected in the XU-AX fraction. As the majority of diFA is associated with the XU-AX fraction, there was only a small effect (TaGT43_2 RNAi, TaGT47_2 RNAi) or no effect (TaXAT1 RNAi) on total diFA content. Our results are compatible with a model where, to maintain cell wall function, diFA is maintained at stable levels when other AX properties are altered.

Differences in gluten protein composition between old and modern durum wheat genotypes in relation to 20th century breeding in Italy.

The impact of breeding on grain yields of wheat varieties released during the 20th century has been extensively studied, whereas less information is available on the changes in gluten quality associated with effects on the amount and composition of glutenins and gliadins. In order to explore the effects of breeding during the 20th century on gluten quality of durum wheat for processing and health we have compared a set of old and modern Italian genotypes grown under Mediterranean conditions. The better technological performance observed for the modern varieties was found to be due not only to the introgression of superior alleles of high (HMW-GS) and low molecular weight (LMW-GS) glutenin subunits encoded at Glu-B1 and Glu-B3 loci, but also to differential expression of specific storage proteins. In particular, the higher gluten index observed in modern genotypes was correlated with an increased glutenin/gliadin ratio and the expression of B-type LMW-GS which was, on average, two times higher in the modern than in the old group of durum wheat genotypes. By contrast, no significant differences were found between old and modern durum wheat genotypes in relation to the expression of α-type and γ-type gliadins which are major fractions that trigger coeliac disease (CD) in susceptible individuals. Furthermore, a drastic decrease was observed in the expression of ω-type gliadins in the modern genotypes, mainly ω-5 gliadin (also known as Tri a 19) which is a major allergen in wheat dependent exercise induced anaphylaxis (WDEIA). Immunological and 2DE SDS-PAGE analyses indicated that these differences could be related either to a general down-regulation or to differences in numbers of isoforms. Lower rainfall during grain filling period was related to overall higher expression of HMW-GS and ω-gliadins. In conclusion, breeding activity carried out in Italy during the 20th century appears to have improved durum wheat gluten quality, both in relation to technological performance and allergenic potential.

The dynamics of protein body formation in developing wheat grain.

Wheat is a major source of protein in the diets of humans and livestock but we know little about the mechanisms that determine the patterns of protein synthesis in the developing endosperm. We have used a combination of enrichment with (15) N glutamine and NanoSIMS imaging to establish that the substrate required for protein synthesis is transported radially from its point of entrance in the endosperm cavity across the starchy endosperm tissues, before becoming concentrated in the cells immediately below the aleurone layer. This transport occurs continuously during grain development but may be slower in the later stages. Although older starchy endosperm cells tend to contain larger protein deposits formed by the fusion of small protein bodies, small highly enriched protein bodies may also be present in the same cells. This shows a continuous process of protein body initiation, in both older and younger starchy endosperm cells and in all regions of the tissue. Immunolabeling with specific antibodies shows that the patterns of enrichment are not related to the contents of gluten proteins in the protein bodies. In addition to providing new information on the dynamics of protein deposition, the study demonstrates the wider utility of NanoSIMS and isotope labelling for studying complex developmental processes in plant tissues.

(1)H-NMR screening for the high-throughput determination of genotype and environmental effects on the content of asparagine in wheat grain.

Free asparagine in cereals is known to be the precursor of acrylamide, a neurotoxic and carcinogenic product formed during cooking processes. Thus, the development of crops with lower asparagine is of considerable interest to growers and the food industry. In this study, we describe the development and application of a rapid (1)H-NMR-based analysis of cereal flour, that is, suitable for quantifying asparagine levels, and hence acrylamide-forming potential, across large numbers of samples. The screen was applied to flour samples from 150 bread wheats grown at a single site in 2005, providing the largest sample set to date. Additionally, screening of 26 selected cultivars grown for two further years in the same location and in three additional European locations in the third year (2007) provided six widely different environments to allow estimation of the environmental (E) and G x E effects on asparagine levels. Asparagine concentrations in the 150 genotypes ranged from 0.32 to 1.56 mg/g dry matter in wholemeal wheat flours. Asparagine levels were correlated with plant height and therefore, due to recent breeding activities to produce semi-dwarf varieties, a negative relationship with the year of registration of the cultivar was also observed. The multisite study indicated that only 13% of the observed variation in asparagine levels was heritable, whilst the environmental contribution was 36% and the GxE component was 43%. Thus, compared to some other phenotypic traits, breeding for low asparagine wheats presents a difficult challenge.

Influence of temperature during grain filling on gluten viscoelastic properties and gluten protein composition.

BACKGROUND: The aim of this study was to investigate the effects of low to moderate temperatures on gluten functionality and gluten protein composition. Four spring wheat cultivars were grown in climate chambers with three temperature regimes (day/night temperatures of 13/10, 18/15 and 23/20 °C) during grain filling. RESULTS: The temperature strongly influenced grain weight and protein content. Gluten quality measured by maximum resistance to extension (Rmax ) was highest in three cultivars grown at 13 °C. Rmax was positively correlated with the proportion of sodium dodecyl sulfate-unextractable polymeric proteins (%UPP). The proportions of ω-gliadins and D-type low-molecular-weight glutenin subunits (LMW-GS) increased and the proportions of α- and γ-gliadins and B-type LMW-GS decreased with higher temperature, while the proportion of high-molecular-weight glutenin subunits (HMW-GS) was constant between temperatures. The cultivar Berserk had strong and constant Rmax between the different temperatures. CONCLUSION: Constant low temperature, even as low as 13 °C, had no negative effects on gluten quality. The observed variation in Rmax related to temperature could be explained more by %UPP than by changes in the proportions of HMW-GS or other gluten proteins. The four cultivars responded differently to temperature, as gluten from Berserk was stronger and more stable over a wide range of temperatures.

Comparative in situ analyses of cell wall matrix polysaccharide dynamics in developing rice and wheat grain.

Cell wall polysaccharides of wheat and rice endosperm are an important source of dietary fibre. Monoclonal antibodies specific to cell wall polysaccharides were used to determine polysaccharide dynamics during the development of both wheat and rice grain. Wheat and rice grain present near synchronous developmental processes and significantly different endosperm cell wall compositions, allowing the localisation of these polysaccharides to be related to developmental changes. Arabinoxylan (AX) and mixed-linkage glucan (MLG) have analogous cellular locations in both species, with deposition of AX and MLG coinciding with the start of grain filling. A glucuronoxylan (GUX) epitope was detected in rice, but not wheat endosperm cell walls. Callose has been reported to be associated with the formation of cell wall outgrowths during endosperm cellularisation and xyloglucan is here shown to be a component of these anticlinal extensions, occurring transiently in both species. Pectic homogalacturonan (HG) was abundant in cell walls of maternal tissues of wheat and rice grain, but only detected in endosperm cell walls of rice in an unesterified HG form. A rhamnogalacturonan-I (RG-I) backbone epitope was observed to be temporally regulated in both species, detected in endosperm cell walls from 12 DAA in rice and 20 DAA in wheat grain. Detection of the LM5 galactan epitope showed a clear distinction between wheat and rice, being detected at the earliest stages of development in rice endosperm cell walls, but not detected in wheat endosperm cell walls, only in maternal tissues. In contrast, the LM6 arabinan epitope was detected in both species around 8 DAA and was transient in wheat grain, but persisted in rice until maturity.

A novel approach to identify genes that determine grain protein deviation in cereals.

Grain yield and protein content were determined for six wheat cultivars grown over 3 years at multiple sites and at multiple nitrogen (N) fertilizer inputs. Although grain protein content was negatively correlated with yield, some grain samples had higher protein contents than expected based on their yields, a trait referred to as grain protein deviation (GPD). We used novel statistical approaches to identify gene transcripts significantly related to GPD across environments. The yield and protein content were initially adjusted for nitrogen fertilizer inputs and then adjusted for yield (to remove the negative correlation with protein content), resulting in a parameter termed corrected GPD. Significant genetic variation in corrected GPD was observed for six cultivars grown over a range of environmental conditions (a total of 584 samples). Gene transcript profiles were determined in a subset of 161 samples of developing grain to identify transcripts contributing to GPD. Principal component analysis (PCA), analysis of variance (ANOVA) and means of scores regression (MSR) were used to identify individual principal components (PCs) correlating with GPD alone. Scores of the selected PCs, which were significantly related to GPD and protein content but not to the yield and significantly affected by cultivar, were identified as reflecting a multivariate pattern of gene expression related to genetic variation in GPD. Transcripts with consistent variation along the selected PCs were identified by an approach hereby called one-block means of scores regression (one-block MSR).

G-fibre cell wall development in willow stems during tension wood induction.

Willows (Salix spp.) are important as a potential feedstock for bioenergy and biofuels. Previous work suggested that reaction wood (RW) formation could be a desirable trait for biofuel production in willows as it is associated with increased glucose yields, but willow RW has not been characterized for cell wall components. Fasciclin-like arabinogalactan (FLA) proteins are highly up-regulated in RW of poplars and are considered to be involved in cell adhesion and cellulose biosynthesis. COBRA genes are involved in anisotropic cell expansion by modulating the orientation of cellulose microfibril deposition. This study determined the temporal and spatial deposition of non-cellulosic polysaccharides in cell walls of the tension wood (TW) component of willow RW and compared it with opposite wood (OW) and normal wood (NW) using specific antibodies and confocal laser scanning microscopy and transmission electron microscopy. In addition, the expression patterns of an FLA gene (SxFLA12) and a COBRA-like gene (SxCOBL4) were compared using RNA in situ hybridization. Deposition of the non-cellulosic polysaccharides (1-4)-ß-D-galactan, mannan and de-esterified homogalacturonan was found to be highly associated with TW, often with the G-layer itself. Of particular interest was that the G-layer itself can be highly enriched in (1-4)-ß-D-galactan, especially in G-fibres where the G-layer is still thickening, which contrasts with previous studies in poplar. Only xylan showed a similar distribution in TW, OW, and NW, being restricted to the secondary cell wall layers. SxFLA12 and SxCOBL4 transcripts were specifically expressed in developing TW, confirming their importance. A model of polysaccharides distribution in developing willow G-fibre cells is presented.

Differentially penalized regression to predict agronomic traits from metabolites and markers in wheat.

BACKGROUND: Genomic prediction of agronomic traits as targets for selection in plant breeding programmes is increasingly common. The methods employed can also be applied to predict traits from other sources of covariates, such as metabolomics. However, prediction combining sets of covariates can be less accurate than using the best of the individual sets. RESULTS: We describe a method, termed Differentially Penalized Regression (DiPR), which uses standard ridge regression software to combine sets of covariates while applying independent penalties to each. In a dataset of wheat varieties, field traits are better predicted, on average, by seed metabolites than by genetic markers, but DiPR using both sets of predictors is best. CONCLUSION: DiPR is a simple and accessible method of using existing software to combine multiple sets of covariates in trait prediction when there are more predictors than observations and the contribution to accuracy from each set differs.

Postprandial plasma betaine and other methyl donor-related responses after consumption of minimally processed wheat bran or wheat aleurone, or wheat aleurone incorporated into bread.

The bran and particularly the aleurone fraction of wheat are high in betaine and other physiological methyl donors, which may exert beneficial physiological effects. We conducted two randomised, controlled, cross-over postprandial studies to assess and compare plasma betaine and other methyl donor-related responses following the consumption of minimally processed bran and aleurone fractions (study A) and aleurone bread (study B). For both studies, standard pharmacokinetic parameters were derived for betaine, choline, folate, dimethylglycine (DMG), total homocysteine and methionine from plasma samples taken at 0, 0·5, 1, 2 and 3 h. In study A (n 14), plasma betaine concentrations were significantly and substantially elevated from 0·5 to 3 h following the consumption of both bran and aleurone compared with the control; however, aleurone gave significantly higher responses than bran. Small, but significant, increases were also observed in DMG measures; however, no significant responses were observed in other analytes. In study B (n 13), plasma betaine concentrations were significantly and substantially higher following consumption of the aleurone bread compared with the control bread; small, but significant, increases were also observed in DMG and folate measures in response to consumption of the aleurone bread; however, no significant responses were observed in other analytes. Peak plasma betaine concentrations, which were 1·7-1·8 times the baseline levels, were attained earlier following the consumption of minimally processed aleurone compared with the aleurone bread (time taken to reach peak concentration 1·2 v. 2·1 h). These results showed that the consumption of minimally processed wheat bran, and particularly the aleurone fraction, yielded substantial postprandial increases in plasma betaine concentrations. Furthermore, these effects appear to be maintained when aleurone was incorporated into bread.

Glycosyl transferases in family 61 mediate arabinofuranosyl transfer onto xylan in grasses.

Xylan, a hemicellulosic component of the plant cell wall, is one of the most abundant polysaccharides in nature. In contrast to dicots, xylan in grasses is extensively modified by α-(1,2)- and α-(1,3)-linked arabinofuranose. Despite the importance of grass arabinoxylan in human and animal nutrition and for bioenergy, the enzymes adding the arabinosyl substitutions are unknown. Here we demonstrate that knocking-down glycosyltransferase (GT) 61 expression in wheat endosperm strongly decreases α-(1,3)-linked arabinosyl substitution of xylan. Moreover, heterologous expression of wheat and rice GT61s in Arabidopsis leads to arabinosylation of the xylan, and therefore provides gain-of-function evidence for α-(1,3)-arabinosyltransferase activity. Thus, GT61 proteins play a key role in arabinoxylan biosynthesis and therefore in the evolutionary divergence of grass cell walls.

Secondary cell wall composition and candidate gene expression in developing willow (Salix purpurea) stems.

The properties of the secondary cell wall (SCW) in willow largely determine the suitability of willow biomass feedstock for potential bioenergy and biofuel applications. SCW development has been little studied in willow and it is not known how willow compares with model species, particularly the closely related genus Populus. To address this and relate SCW synthesis to candidate genes in willow, a tractable bud culture-derived system was developed in Salix purpurea, and cell wall composition and RNA-Seq transcriptome were followed in stems during early development. A large increase in SCW deposition in the period 0-2 weeks after transfer to soil was characterised by a big increase in xylan content, but no change in the frequency of substitution of xylan with glucuronic acid, and increased abundance of putative transcripts for synthesis of SCW cellulose, xylan and lignin. Histochemical staining and immunolabeling revealed that increased deposition of lignin and xylan was associated with xylem, xylem fibre cells and phloem fibre cells. Transcripts orthologous to those encoding xylan synthase components IRX9 and IRX10 and xylan glucuronyl transferase GUX1 in Arabidopsis were co-expressed, and showed the same spatial pattern of expression revealed by in situ hybridisation at four developmental stages, with abundant expression in proto-xylem, xylem fibre and ray parenchyma cells and some expression in phloem fibre cells. The results show a close similarity with SCW development in Populus species, but also give novel information on the relationship between spatial and temporal variation in xylan-related transcripts and xylan composition.

Safety assessment of genetically modified plants with deliberately altered composition.

The development and marketing of 'novel' genetically modified (GM) crops in which composition has been deliberately altered poses a challenge to the European Union (EU)'s risk assessment processes, which are based on the concept of substantial equivalence with a non-GM comparator. This article gives some examples of these novel GM crops and summarizes the conclusions of a report that was commissioned by the European Food Safety Authority on how the EU's risk assessment processes could be adapted to enable their safety to be assessed.