Crop biotechnology. Where now?

Nature Biotechnology organized a conference in London on Agobiotech 99: Biotechnology and World Agriculture (November 14-16, 1999). The conference focused entirely on crop biotechnology and covered both societal and scientific aspects. Below is an account of the more important issues raised by the speakers and the audience.

Primary structure and differential expression of glutamine synthetase genes in nodules, roots and leaves of Phaseolus vulgaris.

In plants, glutamine synthetase (GS) is the enzyme primarily responsible for the assimilation of ammonia into organic nitrogen. In Phaseolus vulgaris a number of isoenzymic forms of GS are found, each of which consists of eight subunits of mol. wt 41 000-45 000. The GS subunits of P. vulgaris have previously been shown to be encoded by a small multigene family and a partial cDNA clone for a nodule-specific GS subunit has been obtained. We report here the isolation and nucleotide sequencing of two essentially full-length GS cDNA clones (pR-1 and pR-2) from a root cDNA library and the deduced amino acid sequences of the corresponding GS subunits (355 amino acid residues each). The coding sequences of pR-1 and pR-2 are closely related (80% nucleotide homology, 88% amino acid homology), but their 5'- and 3'-untranslated regions have diverged almost completely. Both pR-1 and pR-2 are related to, but distinct from, the nodule GS clone, pcPvNGS-01 (or pN-1). Hybridization to genomic Southern blots showed that the three GS mRNAs are encoded by three seperate genes and indicated the existence of a fourth class of GS gene. An S1 nuclease protection assay demonstrated the presence of R-1 and R-2 mRNA in both roots and leaves and confirmed that expression of the N-1 gene is nodule-specific. Expression of the R-1 and R-2 genes in the roots did not change significantly during nodulation. However, only the R-1 gene is expressed in the nodules themselves, indicating that the R-2 gene is specifically repressed during nodule development.

The nucleotide sequence of a HMW glutenin subunit gene located on chromosome 1A of wheat (Triticum aestivum L.).

A cloned 8.2 kb EcoRI fragment has been isolated from a genomic library of DNA derived from Triticum aestivum L. cv. Cheyenne. This fragment contains sequences related to the high molecular weight (HMW) subunits of glutenin, proteins considered to be important in determining the elastic properties of gluten. The cloned HMW subunit gene appears to be derived from chromosome 1A. The nucleotide sequence of this gene has provided new information on the structure and evolution of the HMW subunits. However, hybrid-selection translation experiments suggest that this gene is silent.

Molecular evolution of the seed storage proteins of barley, rye and wheat.

The major storage proteins (prolamins) of barley, rye and wheat are characterized by the presence of two or more unrelated structural domains, one of which contains repeated sequences. Because of this repetitive structure and their restricted distribution (only in grasses), it has been suggested that the prolamins are of recent origin. Contrary to this hypothesis, we show that parts of the non-repetitive domain of one group of prolamins are homologous with sequences present in a large group of seed proteins from monocotyledonous and dicotyledonous plants; including Bowman-Birk protease inhibitors, cereal inhibitors of alpha-amylase and trypsin, and 2 S globulin storage proteins of castor bean and oil seed rape. This implies an ancient origin for these non-repetitive domains. The origins of the repetitive domains are not known but may lie within the grasses.

Short tandem repeats shared by B- and C-hordein cDNAs suggest a common evolutionary origin for two groups of cereal storage protein genes.

We have identified cDNA clones coding for the major sulphur-rich and sulphur-poor groups of barley storage proteins (the B- and C-hordeins, respectively). Hybridization studies have revealed unexpected homologies between B- and C-hordein mRNAs. Using a deletion mutant (Risø 56), we have mapped some C-hordein-related sequences within, or closely associated with, B-hordein genes at the Hor 2 locus. Nucleotide sequencing has shown that the primary structure of B-hordein polypeptides can be divided into at least two domains: domain 1 (repetitive, proline-rich, sulphur-poor), which is homologous to C-hordein sequences, and domain 2 (non-repetitive, proline-poor, sulphur rich), which makes up two-thirds of the polypeptide and is partially homologous to a 2S globulin storage protein found in dicotyledons. The coding sequences that are homologous in B- and C-hordein mRNAs have an asymmetric base composition (>80% C-A) and are largely composed of a degenerate tandem repeat based on a 24 nucleotide consensus that encodes Pro-Gln-Gln-Pro-Phe-Pro-Gln-Gln. We discuss the evolutionary implications of the domain structure of the B-hordeins and the unusual relationship between the two groups of barley storage proteins.

Specificity of an antibody to a subunit of high-molecular-weight storage protein from wheat seed and its reaction with other cereal storage proteins (prolamins).

An antiserum to subunit 2 from the high-molecular-weight (HMW) subunits of the glutenin fraction of Triticum aestivum cv. Highbury was shown to react with related subunits from other cultivars of wheat. The reaction was measured quantitatively by laser nephelometry in polyethylene glycol phosphate-buffered saline after dissolving the HMW fraction in 0.1 M acetic acid; urea used to dissolve the HMW prolamins inhibited the reaction, in some cases at the low concentration of 0.06 M. A study of the comparative reactions of other cereal prolamins was made. 'D' hordein, the homologous HMW protein of barley, showed less reaction, which was more inhibited by urea than the wheat subunits. Some ω-gliadins from the wheat cultivars Chinese Spring and Cheyenne reacted more strongly than the injected fraction and there was less inhibition by urea. A-, ß- and γ3 of wheat also reacted with the antiserum while a secalin of rye of Mr 40000 gave a weak reaction.

Hordein-gene expression during development of the barley (Hordeum vulgare) endosperm.

A previous study [Rahman, Shewry & Miflin (1982) J. Exp. Bot. 33, 717-728] showed differential accumulation of the major storage proteins (called B and C hordeins) in developing endosperms of barley (Hordeum vulgare). To determine how this accumulation is regulated, we have studied mRNA fractions prepared from similar endosperms. Hordein-related mRNA species were detected some days before the deposition of hordeins in vivo. The translation products in vivo directed by polyribosomes, polysomal RNA and total cellular RNA showed similar changes in the proportions of the hordein products to those observed in the accumulations of the proteins in vivo. There was a relative increase in one of the subfamilies of B hordeins (called B1 hordein) and a decrease in the second subfamily of B hordeins (B3 hordein) and in C hordeins. The populations of RNA species related to these three groups of hordeins were studied by 'dot hybridization', with specific complementary-DNA probes for B1-, B3- and C-hordein-related sequences. This showed a 10-15-fold increase in sequences related to the B1 hordein during endosperm development, but only a 4-fold increase in sequences related to B3 and C hordeins. These results indicate that the rates of synthesis of hordeins are related to the abundance of their respective mRNA species. The different results observed for the two subfamilies of B hordeins are of interest, since they indicate differential expression of two subfamilies of genes present at a single multigenic locus.

Molecular analysis of the effects of the lys 3a gene on the expression of Hor loci in developing endosperms of barley (Hordeum vulgare L.).

The lys 3a gene present in the barley mutant Ris phi 1508 results in an increased content of lysine in the grain. Previous studies have shown that this increase results from a decreased accumulation of hordein and an increase in other more lysine-rich proteins and in free amino acids. We report here a detailed examination of the effects of this gene on the different groups of hordein polypeptides and the mRNAs encoding them. The amounts of the two major groups of hordein polypeptides (B and C hordein ) were reduced to about 20 and 7%, respectively, of those present in the parental variety ( Bomi ), with a greater effect on one of the two subfamilies of B polypeptides. In contrast, the amounts of D hordein polypeptides were increased fourfold. In vitro translations of polysomal and total cellular RNA fractions showed similar effects on the relative amounts of hordein products synthesized. More detailed analyses of the populations of hordein mRNAs were made using specific cDNA clones and hybrid-selection translation, Northern hybridization, and "hybrid-dot" analysis. Only traces of mRNAs for "C" hordein were detected, while the abundances of mRNAs for the two subfamilies of B hordeins were reduced to 40 and 5% of those in Bomi . The amount of mRNA for D hordein was increased twofold. A cDNA clone related to B hordein was used to analyze genomic DNA fractions by Southern hybridization. The lys 3a gene had no effect on either the number (about 10) or the organization of the B hordein genes. These studies clearly demonstrate that the effects of the lys 3a gene on the amounts of the hordein polypeptides are closely related to changes in the amounts of the mRNAs encoding them. Although the exact effect of the gene remains unknown, it is most likely to be either at transcription or on the early processing of the mRNA.

Glutamine synthetase of Phaseolus vulgaris L.: organ-specific expression of a multigene family.

A recombinant plasmid (pcPvNGS-01) containing sequences related to glutamine synthetase (GS) has been identified from a cDNA library constructed from poly (A)+ RNA isolated from root nodules of Phaseolus vulgaris L. The identification of this recombinant relied on the observations that: (a) the clone hybridized strongly to purified GS mRNA; (b) in hybrid-select translation experiments, the clone selected mRNA that produced a polypeptide identical in molecular weight to purified GS subunits which was immunoprecipitated with anti-GS-antiserum; and (c) the translated nucleotide sequence of the cloned cDNA was homologous to a partial amino acid sequence of higher plant GS. The cloned cDNA hybridized to poly (A)+ RNA of different mobilities from leaves, roots, and nodules of P. vulgaris. In RNA "dot" blots washed at different stringencies, differences were observed both in the relative amounts of GS mRNA in different tissues and in the strength of their hybridization to the cDNA probe. The cloned probe hybridized to several fragments of restricted P. vulgaris DNA but not to DNA from Rhizobium phaseoli. These results suggest that GS is coded for by a small multigene family showing organ-specific expression.

Immunochemical studies on barley seed storage proteins : The specificity of an antibody to 'C' hordein and its reaction with prolamins from other cereals.

The antigenic relationships between the prolamins of barley, rye and wheat have been studied by examining the specificity of an antibody to 'C' hordein in a quantitative study using a laser nephelometer. The antibody reacts weakly with 'B' hordein and strongly with 75-kdalton and 40-kdalton γ-secalins from rye and γ3 some ω-gliadins from wheat. Absorption experiments and immunodiffusion tests indicate that there are shared antigenic determinants for most of the prolamins. All the species with reacting prolamins belong to the sub-family Festucoideae of the Gramineae. The prolamins of maize, pearl millet and sorghum, species of the sub-family Panicoideae, do not react. The results confirm the known lack of homology between the prolamins of the two sub-families and also indicate the presence of relationships not yet established between 'C' hordein, the 75-kdalton and 40-kdalton γ-secalins and also γ3 gliadin.

Molecular analysis of a mutation conferring the high-lysine phenotype on the grain of barley (Hordeum vulgare).

We have analyzed the molecular nature of the Riso 56 mutation that occurs in barley. This mutation results in a depression of hordein accumulation in the grain and consequently in a higher overall lysine content. In particular, the amount of B hordein, which is encoded by the complex locus Hor-2, is decreased by about 75% because of the absence of the major components. The synthesis of certain minor polypeptides, with properties similar to the major B hordeins, remains unaffected. Analysis of endosperm RNA, by in vitro translation and hybridization to various cloned cDNAs derived from hordein mRNA, shows that mRNA for the major B hordeins is not present in the endosperm. Hybridization of a B hordein cDNA clone to gel-fractionated restriction digests of mutant and wild-type DNA indicates that at least 85 kb of DNA has been deleted from the Hor-2 locus in the high-lysine mutant.

Intracellular localization of aspartate kinase and the enzymes of threonine and methionine biosynthesis in green leaves.

The intracellular localization of several aspartate pathway enzymes has been studied in pea (Pisum sativum cv Feltham First) and barley (Hordeum vulgare cv Julia) leaves. Protoplast lysates were fractionated by differential or sucrose density gradient centrifugation, in media optimized for each enzyme. The results show that aspartate kinase, homoserine kinase, threonine synthase, and cystathionine gamma-synthase are confined to the chloroplast. Cystathionine beta-lyase appears to be present in several fractions, though more than 50% of the total activity is associated with the chloroplasts. In contrast, neither methionine synthase nor methionine adenosyl-transferase were significantly associated with chloroplasts, and only a small proportion of the methionine synthase was associated with the mitochondrial fraction. Methionine adenosyltransferase, and hence S-adenosylmethionine synthesis, is not found in any organelle fraction. The conclusion is that whereas threonine, like lysine, is synthesized only in the chloroplast, the last step in methionine biosynthesis occurs largely in the cytoplasm.

Nutritional control of storage-protein synthesis in developing grain of barley (Hordeum vulgare L.).

Sulphur starvation of barley results in decreased accumulation of the "sulphur-rich" B-hordein polypeptides, with little or no effect on the 'sulphur-poor' C hordeins. The populations of mRNAs for C hordeins and the two major subfamilies of B-hordein polypeptides have been assessed by in-vitro translations in a wheat-germ extract and by 'dot' hybridisations to (32)P-labelled B- and C-hordein complementary-DNA clones. The results show that the relative changes in the rates of accumulation of the three groups of polypeptides are correlated with similar changes in the abundances of their respective mRNAs. In addition, the deficiency of sulphur also appears to increase the efficiency of translation of C-hordein mRNA and to decrease the efficiency of translation of the B-hordein mRNAs. Thus the results indicate that there may be two components to the effect of sulphur deficiency on hordein accumulation, one acting at the level of transcription, or mRNA degradation, and one at the level of translation.

The synthesis and deposition of the prolamin storage proteins (secalins) of rye.

The synthesis and deposition of the endosperm storage proteins of rye, usually termed secalins, has been studied. The rate of accumulation of secalin in developing rye grain was at a maximum between 3 and 5 weeks after anthesis. Some changes in the proportions of the four major groups of secalin polypeptides were observed during maturation, notably an increase in γ-secalins of Mr 75k and a decrease in ω-secalins. In-vitro translation of mRNA fractions prepared from 4-week-old endosperms showed that secalin polypeptides were synthesised on membrane-bound polysomes. The secalin products were identified by their mobilities on SDS-PAGE and their relative incorporation of radioactive lysine, glycine, proline, leucine and methionine. Protein bodies prepared by sucrose density ultracentrifugation contained reduced amounts of γ-secalins of Mr 40k and ω-secalins compared with the total secalin fraction, but these components were present in the expected amounts when 1.0 M NaCl was added to the buffers. Treatment of the protein bodies with proteinase-k resulted in the digestion of their contents regardless of the presence of NaCl, indicating that the surrounding membrane was incomplete. It was concluded that the NaCl reduced the loss of secalins from the protein bodies by decreasing secalin solubility rather than by affecting the integrity of the protein body membrane. The results reported for the synthesis and deposition of secalins are consistent with the results of previous studies on the prolamins of wheat and barley.

The isolation and characterisation of a catalase-deficient mutant of barley (Hordeum vulgare L.).

A mutant line of barley, R(othamsted)-Pr 79/4, has been isolated which grows poorly in natural air, but normally in air enriched to 0.2% CO2. Analysis of the products of (14)CO2 fixation showed that there was no major block in photosynthetic or photorespiratory carbon metabolism in the mutant and that rates of CO2 fixation were only slightly lower than those measured in the wild type (c.v. Maris Mink). Leaves of the mutant line contained only 10% of the catalase (EC 1.11.1.6) activity found in the wild type; and the two major bands of catalase activity detected after starch-gel electrophoresis of extracts of normal leaves were missing from similar extracts of RPr 79/4. Peroxisomes isolated from mutant leaves contained negligible catalase activity, but normal levels of other enzymes involved in photorespiration. Genetic analysis has shown that the mutation is recessive and that both air-sensitivity and catalase-deficiency segregate together in F2 plants derived from a cross between the mutant and the cultivar Golden Promise. [1-(14)C]Glycollate was not converted to (14)CO2 faster in the mutant leaves than in the normal leaves. Thus there was no evidence that photorespiratory CO2 may be obtained by the chemical action of H2O2 on glyoxylate or hydroxypyruvate.

Feedback-insensitive aspartate kinase isoenzymes in barley mutants resistant to lysine plus threonine.

The regulatory properties of aspartate kinase (EC 2.7.2.4) and homoserine dehydrogenase (EC 1.1.1.3) in two barley (Hordeum vulgare L.) mutants resistant to growth inhibition by lysine plus threonine, Rothamsted (R) 3004 and R3202, were compared with those in the normal, sensitive parent line cv. Bomi. Three forms of aspartate kinase (AKI, AKII, AKIII) were chromatographically separated and were considered to represent at least three independently regulated isoenzymes. Aspartate kinase I was inhibited by threonine; AKII and AKIII by lysine or lysine plus S-adenosylmethionine. The characteristics of AKI were unchanged in the mutants. Aspartate kinase II and AKIII from Bomi were both inhibited by lysine and by lysine plus S-adenosylmethionine. Aspartate kinase II from mutant R3202 was altered in its properties such that it was insensitive to lysine or lysine plus S-adenosylmethionine; AKII from mutant R3004 did not differ in its properties from AKII of Bomi. The concentration of lysine required to give half maximal inhibition of AKIII from R3004 was ten times that required for AKIII of Bomi; AKIII from R3202 did not differ from that of Bomi in this regard. There was no change in the properties of homoserine dehydrogenase of the mutants as compared with that of Bomi. We conclude that the lt1 and lt2 loci code for structural genes for lysine- and lysine plus S-adenosylmethionine-sensitive aspartate kinase isoenzymes. The mutant genes Lt1b and Lt2 in R3202 and R3004 respectively code for feedback-desensitized isoenzymes. The presence of one of these is sufficient to allow the synthesis of methionine to overcome the growth inhibition by lysine plus threonine.