Food security: the challenge of increasing wheat yield and the importance of not compromising food safety.

Current wheat yield and consumption is considered in the context of the historical development of wheat, from early domestication through to modern plant breeding, the Green Revolution and wheat's place as one of the world's most productive and important crops in the 21st Century. The need for further improvement in the yield potential of wheat in order to meet current and impending challenges is discussed, including rising consumption and the demand for grain for fuel as well as food. Research on the complex genetics underlying wheat yield is described, including the identification of quantitative trait loci and individual genes, and the prospects of biotechnology playing a role in wheat improvement in the future are discussed. The challenge of preparing wheat to meet the problems of drought, high temperature and increasing carbon dioxide concentration that are anticipated to come about as a result of climate change is also reviewed. Wheat yield must be increased while not compromising food safety, and the emerging problem of processing contaminants is reviewed, focussing in particular on acrylamide, a contaminant that forms from free asparagine and reducing sugars during high temperature cooking and processing. Wheat breeders are strongly encouraged to consider the contaminant issue when breeding for yield.

Identification of a novel beta-turn-rich repeat motif in the D hordeins of barley.

The amino acid sequence of the C-terminal part of a barley D hordein seed protein was deduced from the nucleotide sequence of a partial cDNA. It showed high homology with the HMW glutenin subunits of wheat, both proteins consisting predominately of repeated sequences. Whereas the wheat repeats are based on tri-, hexa- and nonapeptides that are rich in glycine, proline and glutamine, the D hordein also contains eleven copies of a novel unrelated motif: Thr-Thr-Val-Ser. The repeated sequences in the wheat glutenin subunits have been demonstrated to form an unusual spiral supersecondary structure based on beta-turns. Conformational analysis of the Thr-Thr-Val-Ser motif by secondary structure prediction and by circular dichroism spectroscopy of an 18 residue synthetic peptide demonstrates that it also forms beta-turns. Thus, D hordein may also have a spiral structure like that of HMW glutenin, despite the presence of a different repeat motif. This conservation of protein conformation in D hordein and the wheat glutenin subunits may indicate a structural role, perhaps in packing of the proteins within the protein bodies of the developing grain.

Molecular cloning of the barley seed protein CMd: a variant member of the alpha-amylase/trypsin inhibitor family of cereals.

The nucleotide and deduced amino-acid sequences of a cDNA clone encoding the barley seed protein CMd are described. The sequence is homologous with those of a family of inhibitors of alpha-amylase and trypsin, except for two short insertions. The longest of these (14 residues) is at the junction between the three proposed ancestral regions that comprise this family of proteins, and has limited identity with alpha-amylases of bacterial origin.

Synthesis, expression and characterisation of peptides comprised of perfect repeat motifs based on a wheat seed storage protein.

We have developed a novel method for constructing synthetic genes that encode a series of peptides comprising perfect repeat motifs based on a high molecular weight subunit (HMW glutenin subunit), a highly repetitive storage protein from wheat seed. A series of these genes of sequentially increasing size was produced, four of which (called R3, 4, 5, 6) were expressed in Escherichia coli. Activity of the synthetic genes in E. coli was confirmed by Northern blot analysis but SDS-PAGE of crude protein extracts failed to show any expressed peptides when stained using Coomassie brilliant blue R250. However, Western blots probed with a HMW glutenin subunit-specific polyclonal antibody showed the presence of the R6 peptide (M(r) 22005) in the crude cell extracts and both this and the R3 peptide (M(r) 12005) were subsequently purified by extraction with hot aqueous ethanol followed by precipitation with acetone and separated by RP-HPLC. The R4 and R5 peptides were not purified. The purified R3 and R6 peptides absorbed Coomassie brilliant blue R250 or other protein stains only weakly and this was considered to account for their failure to be revealed by staining of separations of the crude protein extracts. Circular dichroism spectroscopy showed that both peptides had similar beta-turn rich structures similar to the repetitive sequences present in the whole HMW glutenin subunits. We conclude that expression of perfect repeat peptides in E. coli is a suitable system for the study of structure-function relationships in wheat gluten proteins and other highly repetitive proteins.

Structure and expression of a gene from Arabidopsis thaliana encoding a protein related to SNF1 protein kinase.

The AKin10 gene from Arabidopsis thaliana encoding a putative Ser/Thr protein kinase (PK) has been isolated and characterized. The AKin10-encoding gene is located on a genomic 5.4-kb BamHI fragment and contains ten introns, one being located in the 5' untranslated region. The deduced amino acid sequence of AKin10 is 65% identical over the catalytic domain to the yeast PK (SNF1). SNF1 is essential for the derepression of many glucose-repressible genes, including Suc2 which encodes invertase. Southern blot hybridization experiments suggested the presence of one copy of the gene per haploid genome of A. thaliana. Northern hybridization experiments indicated that this gene is expressed in roots, shoots and leaves. AKin10 may play an important role in a signal transduction cascade regulating gene expression and carbohydrate metabolism in higher plants.

Immunological evidence that HMG-CoA reductase kinase-A is the cauliflower homologue of the RKIN1 subfamily of plant protein kinases.

Three different antibodies against the RKIN1 and BKIN12 gene products from rye and barley recognized the 58 kDa subunit of HMG-CoA reductase kinase-A (HRK-A) from Brassica oleracea on Western blots. HRK-A was also detected by an antipeptide antibody in enzyme-linked immunoassays, and this was competed by the peptide antigen. HRK-A was not recognized by antibodies against plant, mammalian and Saccharomyces cerevisiae relatives of RKIN1, i.e. wheat PKABA1, rat AMP-activated protein kinase and S. cerevisiae Snf1p. RKIN1/HMG-CoA reductase kinase-A are now among the first protein kinases in plants to be well characterized at both the molecular and biochemical levels.

Genetic and agronomic approaches to decreasing acrylamide precursors in crop plants.

Progress in developing genetic and agronomic approaches for reducing the levels of the principal precursors of acrylamide, asparagine and sugars in crop plants is reviewed. The factors that affect asparagine and sugar accumulation, particularly in cereal seeds and potato tubers, are described. Asparagine levels appear to be the key parameter in determining acrylamide formation in processed wheat flour and agronomic strategies for reducing asparagine accumulation in wheat grain are reviewed. Sulphur availability has been shown to be particularly important, with sulphur deprivation causing a dramatic increase in grain asparagine levels and acrylamide risk. Nitrogen availability is also a factor, with increasing nitrogen availability causing grain asparagine levels and acrylamide risk to rise. In potato, attention has been focused on sugars, and there has been some success in reducing sugar accumulation in stored potatoes by genetic modification, with a resultant reduction in acrylamide formation. However, the wisdom or otherwise of this dogma is discussed. Other possible genetic targets for manipulation or development as genetic markers in breeding programmes are reviewed. Plant breeders and farmers are encouraged to exploit the varietal differences in acrylamide risk that have already been identified and to develop good agronomic practice to reduce the levels of acrylamide precursors in cereals and potato.

Genetically modified crops: methodology, benefits, regulation and public concerns.

The genetic modification of crop plants from the methodology involved in their production through to the current debate on their use in agriculture are reviewed. Techniques for plant transformation by Agrobacterium tumefaciens and particle bombardment, and for the selection of transgenic plants using marker genes are described. The benefits of currently available genetically modified (GM) crops in reducing waste and agrochemical use in agriculture, and the potential of the technology for further crop improvement in the future are discussed. The legal requirements for containment of novel GM crops and the roles of relevant regulatory bodies in ensuring that GM crops and food are safe are summarized. Some of the major concerns of the general public regarding GM crops and food: segregation of GM and non-GM crops and cross-pollination between GM crops and wild species, the use of antibiotic resistance marker genes, the prevention of new allergens being introduced in to the food chain and the relative safety of GM and non-GM foods are considered. Finally, the current debate on the use of GM crops in agriculture and the need for the government, scientists and industry to persevere with the technology in the face of widespread hostility is studied.

Cloning of DNA encoding a catalytic subunit of SNF1-related protein kinase-1 (SnRK1-alpha1), and immunological analysis of multiple forms of the kinase, in spinach leaf.

Using a PCR approach, we have cloned DNA encoding a catalytic subunit isoform (SnRK1-alpha1) of SNF1-related protein kinase-1 from spinach leaf. The predicted amino acid sequence falls into the SnRK1a sub-family, and is closely related to SnRK1a sequences expressed in cucumber, Arabidopsis thaliana, tobacco and potato. We have generated two affinity-purified antipeptide antibodies (anti-RASS and anti-AEF) based on the predicted amino acid sequence of spinach SnRK1-alpha1. They were used to analyse multiple forms of SNF1-related kinase (HRK-A, -C, -D) that were previously identified by biochemical criteria in extracts of spinach leaf (Sugden et al., Plant Physiol. 120 (1999), 257-274). Anti-AEF appears to be specific for the SnRK1-alpha1 isoform, whereas anti-RASS is a 'pan-alpha' antibody that precipitates all isoforms present in spinach leaf extracts. The activities of HRK-A and HRK-C can be entirely accounted for by the SnRK1-alpha1 catalytic subunit. By contrast, only a small proportion of HRK-D activity (ca. 20%) can be accounted for by SnRK1-alpha1, with the remainder presumably being due to other isoforms (SnRK1-alpha2?) that are currently poorly defined. A 35 kDa polypeptide recognized by an antibody against the putative Arabidopsis beta2 subunit co-precipitates with HRK-C, but not HRK-A or D.

Genetic modification and plant food allergens: risks and benefits.

Plant genetic engineering has the potential to both introduce new allergenic proteins into foods and remove established allergens. A number of allergenic plant proteins have been characterized, showing that many are related to proteins which have potentially valuable properties for use in nutritional enhancement, food processing and crop protection. It is therefore important to monitor the allergenic potential of proteins used for plant genetic engineering and major biotechnology companies have established systems for this. Current technology allows gene expression to be down-regulated using antisense or co-suppression and future developments may allow targeted gene mutation or gene replacement. However, the application of this technology may be limited at least in the short term by the presence of multiple allergens and their contribution to food processing or other properties. Furthermore, the long-term stability of these systems needs to be established as reversion could have serious consequences.

Expression of fission yeast cdc25 alters the frequency of lateral root formation in transgenic tobacco.

Lateral root formation was examined following the expression of a fission yeast mitotic regulator gene, cdc25, under the control of a tetracycline-inducible promoter in cultured roots of tobacco. Over expression of cdc25 in fission yeast results in premature cell division at a reduced cell size. Our aim was to examine whether cdc25 expression would affect cell size in the tobacco roots, and what effect this would have on lateral root morphogenesis. Transgene integration was confirmed by Southern blotting; it was inherited as a dominant Mendelian trait. Conditions for optimal expression, determined using plants transformed with gus under the control of the same promoter, were: addition of tetracycline (5 micrograms/ml) every 72 h, to cultured roots in Murashige-Skoog liquid medium in darkness at 27 degrees C. After the addition of tetracycline, cdc25 transcripts were detected using RT-PCR, initially after 48 h, and more strongly after 72 h. Appearance of cdc25 transcripts was followed by major changes in the roots. Compared with controls, lateral root primordia were initiated more frequently, were significantly smaller and comprised smaller cells at mitosis. However, cdc25 expression did not perturb normal development of the lateral roots. The data are consistent with cdc25 expression leading to a greater frequency of lateral root primordium formation and establishing a new threshold size for cell division in the primordia which was then maintained throughout subsequent development.

Tobacco plants transformed with cdc25, a mitotic inducer gene from fission yeast.

We investigated the effects of expressing a cDNA of cdc25, a mitotic inducer gene of Schizosaccharomyces pombe, on the development of transgenic tobacco plants (Nicotiana tabacum cv. Samsun). Nine independent primary transformants were regenerated containing the cdc25 sequence under the control of a cauliflower mosaic virus 35S gene promoter. Eight of the nine plants showed altered leaf morphology, the lamina being lengthened and twisted and the interveinal regions being pocketed. One of these was sacrificed for analysis of the root meristem, where the cells were found to be significantly smaller than in the wild type. The other seven were grown on and showed precocious flowering, flowers being produced earlier and in significantly greater numbers than in the wild type. They also developed abnormal flowers on short stalks developing in a position normally occupied by the most proximal axillary bud of otherwise normal flower pedicels. The presence or absence of these phenotypes in the primary transformants and in the T2 generation was associated with the presence or absence of detectable levels of cdc25 transcripts.

A beta-turn rich barley seed protein is correctly folded in Escherichia coli.

Wild-type and cysteine-containing mutant C hordeins from barley were expressed in Escherichia coli at high levels (> or = 30mg/liter). N-terminal sequence analysis, SDS-PAGE, RP-HPLC, cd spectroscopy, and small angle X-ray scattering demonstrated that their physicochemical properties were similar to those of C hordeins isolated from barley grain. This indicates that the expressed proteins were correctly folded. The cysteine-containing mutant showed evidence of polymer formation in E. coli, nonreduced preparations of the protein showing the presence of polymers that were replaced by a single protein when a reducing agent was added.

Potato SNF1-related protein kinase: molecular cloning, expression analysis and peptide kinase activity measurements.

A polymerase chain reaction product (PKIN503) was amplified from potato (Solanum tuberosum) cv. Désirée using oligonucleotide primers with sequences which are highly conserved in the plant sucrose non-fermenting 1 (SNF1)-related protein kinase gene family. Southern blot analysis showed the presence of 5-10 SNF1-related genes in the potato genome. PKIN503 was used to screen a tuber cDNA library and a genomic library, and one cDNA and five genomic clones were isolated. The nucleotide sequences of a portion of all five genomic clones were shown to be identical and only one, pgPKIN1, was analysed further. The cDNA was found to be truncated at the 5' end but the cDNA and genomic sequences contained only 15 substitutions, two of which resulted in changes in the derived amino acid sequence. PKIN1 was shown to encode an Mr 57,854 protein with 61-70% sequence similarity with other plant SNF1-related protein kinases. Northern blot analysis revealed some tissue-specific differences in PKIN1 transcript levels, the lowest being detected in leaves and the highest in stolons. However, much greater differences were found in SNF1-related activity, which was measured using a phosphorylation assay with a substrate peptide which has been shown previously to be phosphorylated by plant SNF1-related protein kinases. Activity decreased by almost 80% during development from stolons to mature tubers but it increased about seven-fold during the first seven days of storage after harvesting, before decreasing again. However, activity was highest in mini-tubers, where the levels were 37 times greater than those in mature tubers from a pot-grown plant. Transcript levels in these tissues were approximately equal, clear evidence that SNF1-related protein kinase activity in potato is regulated, in part, post-transcriptionally.

Differential expression of two barley SNF1-related protein kinase genes.

We have amplified and cloned DNA sequences derived from a gene encoding a SNF1 (sucrose-non-fermenting 1)-related protein kinase which differs from that previously reported from barley. Northern blot and polymerase chain reaction (PCR) analysis of RNA populations, using specific probes and oligonucleotide primers, indicated that the two SNF1-related genes are differentially regulated. One is expressed in all tissues, whereas the other is expressed at high levels in the seed endosperm and aleurone, but at levels undetectable by northern blot analysis in other tissues. Comparisons with other plant SNF1-related protein kinase genes suggest that the form which is expressed at greatly enhanced levels in the seed is less similar to the other plant homologues which have been reported and may be unique to cereals.

Regulation of spinach SNF1-related (SnRK1) kinases by protein kinases and phosphatases is associated with phosphorylation of the T loop and is regulated by 5'-AMP.

Members of the SNF1-related protein kinase-1 (SnRK1) subfamily of protein kinases are higher plant homologues of mammalian AMP-activated and yeast SNF1 protein kinases. Based on analogies with the mammalian system, we surmised that the SnRK1 kinases would be regulated by phosphorylation on a threonine [equivalent to Thr175 in Arabidopsis thaliana SnRK1 (AKIN10)] within the 'T loop' between the conserved DFG and APE motifs. We have raised an antibody against a phosphopeptide based on this sequence, and used it to show that inactivation of two spinach SnRK1 kinases by protein phosphatases, and reactivation by a mammalian upstream protein kinase, is associated with changes in the phosphorylation state of this threonine. We also show that dephosphorylation of this threonine by protein phosphatases, and consequent inactivation, is inhibited by low concentrations of 5'-AMP, via binding to the substrate (i.e. the kinase). This is the first report showing that the plant SnRK1 kinases are regulated by AMP in a manner similar to their mammalian counterparts. The possible physiological significance of these findings is discussed.

Two SNF1-related protein kinases from spinach leaf phosphorylate and inactivate 3-hydroxy-3-methylglutaryl-coenzyme A reductase, nitrate reductase, and sucrose phosphate synthase in vitro.

We resolved from spinach (Spinacia oleracea) leaf extracts four Ca2+-independent protein kinase activities that phosphorylate the AMARAASAAALARRR (AMARA) and HMRSAMSGLHLVKRR (SAMS) peptides, originally designed as specific substrates for mammalian AMP-activated protein kinase and its yeast homolog, SNF1. The two major activities, HRK-A and HRK-C (3-hydroxy-3-methylglutaryl-coenzyme A reductase kinase A and C) were extensively purified and shown to be members of the plant SnRK1 (SNF1-related protein kinase 1) family using the following criteria: (a) They contain 58-kD polypeptides that cross-react with an antibody against a peptide sequence characteristic of the SnRK1 family; (b) they have similar native molecular masses and specificity for peptide substrates to mammalian AMP-activated protein kinase and the cauliflower homolog; (c) they are inactivated by homogeneous protein phosphatases and can be reactivated using the mammalian upstream kinase; and (d) they phosphorylate 3-hydroxy-3-methylglutaryl-coenzyme A reductase from Arabidopsis at the inactivating site, serine (Ser)-577. We propose that HRK-A and HRK-C represent either distinct SnRK1 isoforms or the same catalytic subunit complexed with different regulatory subunits. Both kinases also rapidly phosphorylate nitrate reductase purified from spinach, which is associated with inactivation of the enzyme that is observed only in the presence of 14-3-3 protein, a characteristic of phosphorylation at Ser-543. Both kinases also inactivate spinach sucrose phosphate synthase via phosphorylation at Ser-158. The SNF1-related kinases therefore potentially regulate several major biosynthetic pathways in plants: isoprenoid synthesis, sucrose synthesis, and nitrogen assimilation for the synthesis of amino acids and nucleotides.

Molecular analyses of a barley multigene family homologous to the yeast protein kinase gene SNF1.

Genomic sequences homologous to the yeast gene SNF1 have been isolated from barley (Hordeum vulgare) cv. Sunbar. SNF1 encodes a protein serine/threonine kinase required for the derepression of a number of genes, including SUC2 (invertase) in response to glucose deprivation. Southern blotting showed the presence of a family of related genes in barley and full-length sequences have been determined for two members of the family, one of which lacks an exon and is almost certainly non-functional. A partial sequence has been obtained for a third member of the family. The transcription start site of one of the genes has been determined by S1 nuclease protection. A transcript almost identical in sequence to the exons of one of the genes has been amplified from barley endosperm mRNA using the polymerase chain reaction. One of the full-length genomic sequences contains nine introns and 10 exons and the number and position of the introns in the second full-length sequence is identical except that it lacks exon 2. However, the length and sequence of the introns vary. Northern blot analyses indicated that related transcripts are present in aleurones, coleoptiles, endosperms, internodes, leaves, ovules, roots and root tips, with highest levels of expression in the aleurones and endosperms.

Expression of antisense SnRK1 protein kinase sequence causes abnormal pollen development and male sterility in transgenic barley.

A chimaeric gene was constructed comprising a wheat high molecular weight glutenin subunit gene promoter, a 304-bp sucrose non-fermenting-1-related (SnRK1) protein kinase sequence in the antisense orientation, and the cauliflower mosaic virus 35S RNA gene terminator. Transgenic barley plants containing the antisense SnRK1 chimaeric gene were produced by particle bombardment of barley immature embryos with the aim of obtaining plants expressing the antisense SnRK1 sequence in the seeds. Despite the fact that the promoter was expected to be active only in seeds, two independent transgenic lines were found to fail to transmit the transgene to the T1 generation. These T0 plants had matured and died before this was discovered, but subsequently four other independent transgenic lines were found to be affected in the same way. Cytological analysis of the pollen grains in these lines showed that about 50% were normal but the rest had arrested at the binucleate stage of development, were small, pear-shaped, contained little or no starch and were non-functional. The presence of antisense SnRK1 transcripts was detected in the anthers of the four lines analyzed and a ubiquitin promoter/UidA (Gus) gene, one of the marker genes codelivered with the antisense gene, was found to be expressed only in the abnormal pollen. Expression analyses confirmed that SnRK1 is expressed in barley anthers and that expression of one class of SnRK1 transcripts (SnRK1b) was reduced in the abnormal lines. All of the abnormal lines showed approximately 50% seed set, and none of the transgenes were detected in the T1 generation.

Identification and molecular characterisation of hordoindolines from barley grain.

Grain texture in barley is an important quality character as soft-textured cultivars have better malting quality. In wheat, texture is considered to be determined by the puroindolines, a group of basic hydrophobic proteins present on the surface of the starch granule. Hard wheats have been proposed to lack puroindoline a or to have mutant forms of puroindoline b which do not bind to the granule surface. Analysis of six barley cultivars (three soft-textured and three hard) showed that all contained proteins homologous to wheat puroindoline b, but PCR analysis failed to show any differences in amino acid sequences similar to those which have been proposed to determine textural differences in wheat. Southern blot analysis showed two hordoindoline b genes which were isolated and shown to encode proteins with 94% sequence identity. Expression of hordoindoline b mRNA occurred in the starchy endosperm and aleurone layer of the developing seed, but not in the embryo. Analysis of seven soft- and six hard-textured barley varieties showed that all contained hordoindoline a except two hard varieties (Sundance, Hart) which were subsequently shown to both lack hordoindoline a mRNA. It was therefore concluded that there is not a clear relationship between the presence of hordoindoline a and grain texture in barley.