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 cloning and analysis of cDNA sequences derived from poly A+ RNA from barley endosperm: identification of B hordein related clones.

A collection of over 130 cDNA clones has been constructed in the bacterial plasmids pPH207 and pBR322 using as template the poly A+ RNA from membrane-bound polysomes of barley endosperm (cv. Sundance). Fifty four B hordein cDNA clones have been identified by cross-hybridization analysis and in vitro translation of plasmid-selected mRNAs. Hybridization of 11 of the B hordein cDNA clones to Northern blots of size-fractionated RNA indicated that the B hordein mRNA is ca. 1300 nucleotides long. One cDNA clone, pHvE-c16, has been partially sequenced and shown by comparison with C-terminal and other peptide sequences to be related to B1 hordein polypeptides. The results obtained from the analysis of the B hordein cDNA clones support the idea that the Hor 2 locus, which specifies the B hordeins, is complex and codes for a family of related mRNA species.     

Quantification of Hordeins by ELISA: The Correct Standard Makes a Magnitude of Difference

Background

Coeliacs require a life-long gluten-free diet supported by accurate measurement of gluten (hordein) in gluten-free food. The gluten-free food industry, with a value in excess of $6 billion in 2011, currently depends on two ELISA protocols calibrated against standards that may not be representative of the sample being assayed.

Aim

The factors affecting the accuracy of ELISA analysis of hordeins in beer were examined.

Results

A simple alcohol-dithiothreitol extraction protocol successfully extracts the majority of hordeins from barley flour and malt. Primary hordein standards were purified by FPLC. ELISA detected different classes of purified hordeins with vastly different sensitivity. The dissociation constant (Kd) for a given ELISA reaction with different hordeins varied by three orders of magnitude. The Kd of the same hordein determined by ELISA using different antibodies varied by up to two orders of magnitude. The choice of either ELISA kit or hordein standard may bias the results and confound interpretation.

Conclusions

Accurate determination of hordein requires that the hordein standard used to calibrate the ELISA reaction be identical in composition to the hordeins present in the test substance. In practice it is not feasible to isolate a representative hordein standard from each test food. We suggest that mass spectrometry is more reliable than ELISA, as ELISA enumerates only the concentration of particular amino-acid epitopes which may vary between different hordeins and may not be related to the absolute hordein concentration. MS quantification is undertaken using peptides that are specific and unique enabling the quantification of individual hordein isoforms.     

A role for γ3 hordein in the transport and targeting of prolamin polypeptides to the vacuole of developing barley endosperm

Hordein synthesis, transport and deposition was analysed by immunocytochemistry in developing endosperm cells of wild-type (Carlsberg II) and mutant varieties deficient in B hordein ( hor2ca ), γ1 hordein (Donetsky), γ2 hordein and minor B hordein polypeptides (Haisa), or γ3 hordein (Nevsky). In all varieties, hordein polypeptides were detected both in the cytoplasm as globules, ranging in diameter from 50 nm to 1.24 µm, and in the vacuole as protein bodies. In the cytoplasmic globules B and C hordein polypeptides are assembled as a core and are surrounded by an outer layer of γ1 and γ2 hordein. The globules apparently fuse several times in the cytoplasm before entering the vacuole. Absence of γ3 hordein in the mutant Nevsky leads to a dramatic change in hordein polypeptide targeting, the hordein storage proteins being largely deposited in the lumen of the rough endoplasmic reticulum. γ3 Hordein is unique among the sulphur-rich hordein polypeptides, being monomeric and forming only intramolecular disulphide bridges, while the other B and γ hordein polypeptides are aggregated by intermolecular disulphide bridges. Retention of hordein in the rough endoplasmatic reticulum in the absence of γ3 hordein suggests that γ3 hordein may maintain the prolamin storage polypeptides in a transport competent state. The sequence of the mature γ3 hordein polypeptide was deduced from a cDNA clone, and compared with γ2 hordein. The epitope recognized by the γ1 + γ2 hordein-specific BX monoclonal antibody used for immunocytochemistry was mapped to include E¹⁹⁰ and K¹⁹³, by synthesizing overlapping oligopeptides.     

Analysis of genetic diversity of hordein in wild close relatives of barley from Tibet

We analyzed genetic diversity in the storage protein hordein encoded at Hor-1, Hor-2 and Hor-3 loci in seeds from 211 accessions of wild close relatives of barley, Hordeum vulgare ssp. agriocrithon and H. vulgare ssp. spontaneum. Altogether 32, 27 and 13 different phenotypes were found for Hor-1, Hor-2 and Hor-3, respectively. A comparison of our results with those of previous studies indicates that Tibetan samples reflect the highest diverse level of hordein phenotypes when compared to samples from Israel and Jordan. This high degree of polymorphism supports the hypothesis that Tibet is one of the original centers of H. vulgare L.Communicated by H.F. Linskens     

Genetic change in the waxy trait of barley under the influence of wild type DNA. Analysis of the composition of starch and the electrophoretic spectrum of caryopsis hordein from altered plants and the retention of these changes to the fourth generation

Injection of DNA isolated from the wild type of barley into grains of recipient mutant plants (waxy mutants) at the milk stage of maturity leads to a change in starch synthesis; type of spikes and hordein composition. In the first generation of injected plants the wild type starch synthesis was observed in some separate plants (these observations were made at a haploid level in pollen cells). In the second generation of transformed plants along with the change in starch and hordein synthesis a modification of the type of spikes was also revealed. Recipient plants had six-rowded (hexastichous) spikes, and donor plants--two rowded (distichous) spikes. Disc-electrophoresis of hordeins of the wild type barley (Yuzhny var.), hordeins of the waxy mutant (defected in synthesis of normal starch) and barley plants transformed under the action of wild type exogenous DNA reveals differences in the protein spectrum between donor, recipient and transformants. In the second generation in many of the transformed plants starch synthesis reverted to the recipient mutant type. Simultaneously a reversion of hordein composition to the initial mutant type was observed, and the distichous pikes became hexastichous. Analysis of the components of starch revealed that donor plant that have amilose and amilopectin in starch, and the recipient plants that lack amilose, can be distinguished by the spectra of light absorption of starch. For characterizing these differences the plot of absoprtions at 490 versus that at 590 nm was used. The tangens of angles of these curves for the waxy mutant were equal to 1.05 +/- 0.07 and 1.81 +/- 0.04 for the wild type barley. All transformants have a 1.78 ratio and for revertants this value was 1.02.     

Recombination of alleles conferring specific resistance to powdery mildew at the Mla locus in barley.

In barley (Hordeum vulgare L.), the Mla locus conditions reaction to the powdery mildew fungus Erysiphe graminis f.sp. hordei. Enrichment for genetic recombinants in the Mla region is possible by screening for recombination events between the flanking endosperm storage proteins hordeins C and B. Reciprocal crosses were made between the Franger (C.I. 16151) and Rupee (C.I. 16155) lines carrying the (Mla6 + Mla14) and Mla13 alleles, respectively. Recombinants were identified from F2 segregants by analyzing the extracted hordein polypeptides by sodium dodecyl sulphate - polyacrylamide gel electrophoresis. Two hundred and seventy-six recombinant gametes were identified from the 1800 seeds that were screened. Recombination of Mla alleles was analyzed by inoculating F4 recombinant lines with three isolates of E. graminis (A27, 5874, and CR3), which recognize specific Mla alleles. The linkage order established is Hor1-Mla6-Mla13-Mla14-Hor2. The genetic distances between Hor1-Mla6, Mla6-Mla13, and Mla13-Hor2, obtained using Mapmaker 3.0b F3 intercross analysis, are 3.9, 0.2, and 5.2 cM, respectively.     

Fine Structure and Instability of the Ml-a Locus in Barley

There are many naturally occurring variants at the Ml-a locus in barley that confer resistance to the powdery mildew fungus Erysiphe graminis f. sp. hordei. Since the Ml-a locus is bracketed by Hor-1 and Hor-2, genes that encode storage proteins in the endosperm, the Ml-a locus is amenable to fine structure analysis. Rare susceptible recombinants, as judged by exchange of flanking markers, were recovered in F3 families from the Ml-a10 X Ml-a1, Ml-a1 X Ml-a15 and Ml-a6 X Ml-a13 crosses. Some susceptible recombinants were recovered from the Ml-a6 X Ml-a13 cross that did not fit the expected F3 family segregation ratios. The Ml-a6/Ml-a13 recombinants often reverted to resistance in subsequent generations. No recombinants were recovered in the reciprocal cross, Ml-a13 X Ml-a6. The possibility of a transposable element and a possible linear order of six "alleles" at the Ml-a locus is discussed.     

Polymorphism of the cultivated barley (Hordeum vulgare L.) of South Arabia at hordein-coding loci

Electrophoresis in starch gel was used to study the polymorphism of hordeins controlled by loci Hrd A, Hrd B, and Hrd F in 89 samples of the local barleys from South Arabia (Yemen). Overall, 36 alleles were detected for locus Hrd A; 48 alleles, for Hrd B; and 5 alleles, for Hrd F. The existence of the blocks of hordein components controlled by loci Hrd A and Hrd B was demonstrated. Calculation of genetic distances allows us to conclude that the barley populations from Yemen and Ethiopia are more similar compared with the populations from Egypt. This confirms the hypothesis of Bakhteev on the origin of Ethiopian barleys.     

Mapping of the Hor-3 locus encoding D hordein in Barley

Summary The hordein storage proteins of barley (Hordeum vulgare L.) are of intense interest due to their genetic diversity and prominence and impact on the industrial and agricultural uses of the seed. Two major hordein loci have been previously mapped on chromosome 5 (Hor-1 and Hor-2 encoding the C and B hordeins, respectively). A third major locus, Hor-3, which codes for D hordein, has been located in the centromeric region of chromosome 5, probably on the long arm. Two allelic variants with apparent molecular weights of 83,000 and 91,000 and similar isoelectric points of 8.0 comprise the products of this locus in the barley varieties Advance and Triple Awned Lemma. The D hordein examined is similar in molecular weight and isoelectric point to the high molecular weight (HMW) glutenin proteins encoded by the 1B chromosome of wheat (Triticum aestivum L.)Scientific Paper No. 6229. College of Agriculture Research Center, Washington State University, Pullman, Washington, Project Number 1006. This investigation supported in part by funds provided to Washington State University through the NIH Biomedical Research Support Grant     

Heritable somaclonal variation in wild barley (Hordeum spontaneum)

Summary Progenies of H. spontaneum plants regenerated from immature embryo derived calli were analysed for somaclonal variation on the following traits: (1) organization of the intergenic spacer of the rRNA genes; (2) B and C hordein pattern on SDS-PAGE; (3) genomic organization of the B and C hordein coding sequences; (4) mitochondrial DNA organization assayed by hybridization of Southern blots of total DNA with mitochondrial coding genes; (5) cytology. One out of twelve progeny plants was characterized as variant for two traits: (a) a loss of 1.8 and 2.5 kb Taq I intergenic rDNA spacer fragments and (b) a variant pattern of hordeins on 1-D SDS-PAGE. No numerical or structural chromosome variation was detected among the control plants therefore it is assumed that the variation was caused by the in vitro culture and transmitted, through sexual reproduction, to the analysed progeny.     

Differential Protein Accumulation during Barley Grain Development

Barley grains were analysed during development for the presenceof salt-soluble proteins, hordeins and glutelins. Characteristictemporal differences between the fractions were observed withhordeins being produced relatively late during maturation. Analysisof this fraction by gel electrophoresis revealed differentialaccumulation of its component polypeptides. The C hordeins madeup a relatively higher percentage of total hordein at the earlystages, decreasing from 20% of the total at 33% final dry weightto 15% at maturity. The relative amount of the lowest molecularweight B hordein band (Bl) increased throughout developmentfrom 30% of the total at 33% final dry weight to about 45% atmaturity. Electrophoresis of the salt-soluble fraction showedthat a group of low molecular weight polypeptides appeared atthe same stage of development as did the hordeins. There wereonly relatively minor changes in the polypeptide compositionof the glutelin fraction.