A cytogenetic study of the blue-grain line of the common wheat cultivar Saratovskaya 29

The chromosome composition of the blue-grain line i:S29Ba of the cultivar Saratovskaya 29 was identified by cytological, GISH, and microsatellite analyses and C-banding. It was found that common wheat chromosome 4B of the cultivar Saratovskaya 29 was substituted with the Agropyron elongatum Host. chromosome carrying the gene for blue grain (s:S294Ag(4B)) during the construction of this nearly isogenic line. The blue-grain line was tested for productivity. The substitution of total chromosome 4B of the cultivar Saratovskaya 29 by Ag. elongatum chromosome 4 did not significantly affect the spike productivity parameters and grain quality with the exception of spike length (plus effect), spike density, and vitreousness (minus effects). The blue-grain line with s:S294Ag(4B) can be used in further studies associated with chromosome engineering in cereals and wheat breeding.     

Analysis of spike productivity traits in near-isogenic lines of the common wheat cultivar Saratovskaya 29 carrying alien marker genes

Six near-isogenic lines of the wheat cultivar Saratovskaya 29 carrying five marker genes from different species (Triticum compactum L., T. polonicum L., T. petropavlovskyi Udacz. et Migusch., Aegilops elongatum Host. and Secale cereale L.) were studied. It was shown that the introduced marker genes of taxonomic significance, C and P, have strong pleiotropic effects on quantitative traits of the spike productivity.     

Construction of immune lines with complex resistance to leaf rust and powdery mildew in common spring wheat cultivar Saratovskaya 29

Immune lines resistant both to leaf rust and to powdery mildew were constructed on the basis of common wheat cultivar Saratovskaya 29. Synthetic wheat Triticum timopheevii/Aegilops squarrosa (AAGGDD, 2n = 42) of Savov (Bulgaria) was used as a source of resistance genes. Using cytological analysis of BC2, we selected resistant plants (21") free from meiosis 1 (M1) defects. With these plants and continuous selection, BC8-BC9 immune lines were obtained. The lines were shown to carry new resistance genes differing from the known ones, and were proposed as donors of immunity to the diseases.     

The role of chromosomes from homeologous group 5 in regulation of grain hardness and protein content in substitution lines of common wheat cultivar Saratovskaya 29

Genetic regulation of grain hardness and protein content in intervarietal substitution lines for chromosomes of homeologous group 5 was examined. Common wheat cultivar Saratovskaya 29 with high bread-backing properties served as the recipient. Donors of chromosomes 5A and 5D were 18 cultivars with variable traits examined, including high-protein cultivars (Atlas 66 and Diamant 2), and soft-grain cultivars (Ul’yanovka and Chinese Spring). Analysis of substitution lines pointed to a substantial effect of chromosome 5D on the regulation of both traits. It was demonstrated that as a result of intervarietal substitution for chromosome 5D from donor cultivars Ul’yanovka and Chinese Spring, the endosperm softness was increased compared to the recipient cultivar Saratovskaya 29. Substitution lines Saratovskaya 29/Atlas 66 5D and Saratovskaya 29/Diamant 2 5D were characterized by high grain protein content, as well as by high endosperm hardness. In addition, the line Saratovskaya 29/Novosibirskaya 67 5D, characterized by grain hardness higher than in Saratovskaya 29, was isolated. In the lines with intervarietal substitution of chromosome 5A, grain protein content was found to be lower than in recipient cultivar Saratovskaya 29.     

Linkage mapping of genes controlling endosperm storage proteins in wheat

Summary A translocation mapping procedure was used to map gene-centromere distances for the genes controlling endosperm proteins on the short arm of each of the chromosomes 1A, 1B and 1D in wheat. The genes controlling triplet proteins (tentatively designated Tri-1) were found to be closely linked to the centromere on chromosome arms 1AS and 1DS and loosely linked to the gliadin genes (Gli-1) on the same arms. The Gli-1 genes segregated independently or were very loosely linked to their respective centromeres. The Gli-B1-centromere map distance on 1BS was also estimated using conventional telocentric mapping and the result was similar to that obtained with the translocation mapping. A simple two-step one-dimensional electrophoretic procedure is described which allows the low-molecular-weight (LMW) glutenin subunits to be separated from the gliadin bands, thus facilitating the genetic analysis of these LMW subunits. No recombination was observed between the genes (designated Glu-3) controlling some major LMW glutenin subunits and those controlling gliadins on chromosome arms 1AS and 1DS. However, in a separate experiment, the genes controlling LMW glutenin subunits on 1BS (Glu-B3) showed a low frequency of recombination with the gliadin genes.Portion of the Ph.D. thesis submitted by the senior author     

Isolation and identification of mRNA for the high-molecular-weight storage proteins of wheat endosperm

The prolamin storage proteins of the wheat endosperm contain a sub-class of high-molecular-weight (HMW) polypeptides which have been implicated in determining breadmaking quality. Membrane-bound polysomes isolated from developing wheat endosperms contain mRNA for these HMW components. Although unfractionated polyadenylated RNA derived from the polysomes did not direct the synthesis of these components in an in-vitro wheat-germ system, it did when incubated with a rabbit reticulocyte lysate system. Identification of the translation products as HMW prolamins was based on their large incorporation of [³H]leucine and [³H]glycine relative to [³H]lysine, their mobility on polyacrylamide-gel electrophoresis and the observation that the changes of mobility in response to change in wheat genotype were the same as those observed for the authentic protein. The mRNA was fractionated by electrophoresis and density-gradient centrifugation. The mRNA for the HMW prolamins was found to have a relative molecular mass of about 1.6·10⁶.Abbreviations HMW high molecular weight - PAGE polyacrylamide-gel electrophoresis - poly(A)⁺RNA polyadenylated RNA - SDS sodium dodecyl sulphate     

Two forms of fructose 1,6-bisphosphatase from immature wheat endosperm

Fructose 1,6-bisphosphatase (α-D-fructose 1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11; FBPase) from immature wheat endosperm has been resolved into two forms, FBPase-I and FBPase-II. Their specific activities over crude homogenate increased 47- and 77-fold, respectively, by using ammonium sulfate fractionation, DEAE-cellulose chromatography and gel filtration through Sephadex G-200. The pH optimum was 7.6 for FBPase-I and 8.4 for FBPase-II. The two forms were highly specific for the substrate FBP with K_m values of 0.17 and 0.08 m M , respectively, for FBPase-I and FBPase-II at their respective pH optimum and saturating Mg²⁺ concentration. pH had no effect on the K_m value for FBPase-I, but that for FBPase-II increased below optimum pH. Neither of the forms had an absolute requirement for Mg²⁺, although it was essential for maximum activity. Mg²⁺ could not be replaced by Cu²⁺, Ca²⁺, Ba²⁺, Co²⁺ or Ni²⁺. Sulfhydryl reagents inactivated both FBPase-I and FBPase-II. Of the metabolites, only 6-phosphogluconate was inhibitory with 50% inhibition at 2 and 4 m M for FBPase-I and FBPase-II, respectively.     

Composition of root exometabolites of the symbiotically effective pea cultivar triumph and its parental forms

The qualitative and quantitative composition of low-molecular exometabolites in roots of pea (Pisum sativum L.) was studied with a cultivar Triumph and its parental forms (a symbiotically effective variety k-8274 and a modern highly productive cv. Classic). A relationship between root exudation and the ability of cultivars to establish symbiosis was analyzed. In the early stages of plant growth, the roots of cv. Triumph exhibited low exudation of organic acids, sugars, and amino acids. The quantitative composition of organic acids in the root exudates of cv. Triumph was close to that of cv. k-8274, whereas the composition of sugars and amino acids was similar to that of cv. Classic. In the field experiment, the effect of inoculation with a mixture of rhizobium strains and mycorrhizal fungus on plant growth was more evident in cv. Triumph than in cvs. Classic and k-8274. The results suggest that the high symbiotic potential of cv. Triumph is related to exudation of pyruvic and succinic acids that were the major components of root exometabolites both in Triumph and k-8274 cultivars.     

Efficient recovery of recombinant proteins from cereal endosperm is affected by interaction with endogenous storage proteins

Cereal seeds are versatile platforms for the production of recombinant proteins because they provide a stable environment for protein accumulation. Endogenous seed storage proteins, however, include several prolamin-type polypeptides that aggregate and crosslink via intermolecular disulfide bridges, which could potentially interact with multimeric recombinant proteins such as antibodies, which assemble in the same manner. We investigated this possibility by sequentially extracting a human antibody expressed in maize endosperm, followed by precipitation in vitro with zein. We provide evidence that a significant proportion of the antibody pool interacts with zein and therefore cannot be extracted using non-reducing buffers. Immunolocalization experiments demonstrated that antibodies targeted for secretion were instead retained within zein bodies because of such covalent interactions. Our findings suggest that the production of soluble recombinant antibodies in maize could be enhanced by eliminating or minimizing interactions with endogenous storage proteins.     

The trafficking pathway of a wheat storage protein in transgenic rice endosperm

Background and Aims

The trafficking of proteins in the endoplasmic reticulum (ER) of plant cells is a topic of considerable interest since this organelle serves as an entry point for proteins destined for other organelles, as well as for the ER itself. In the current work, transgenic rice was used to study the pattern and pathway of deposition of the wheat high molecular weight (HMW) glutenin sub-unit (GS) 1Dx5 within the rice endosperm using specific antibodies to determine whether it is deposited in the same or different protein bodies from the rice storage proteins, and whether it is located in the same or separate phases within these.

Methods

The protein distribution and the expression pattern of HMW sub-unit 1Dx5 in transgenic rice endosperm at different stages of development were determined using light and electron microscopy after labelling with antibodies.

Key results

The use of HMW-GS-specific antibodies showed that sub-unit 1Dx5 was expressed mainly in the sub-aleurone cells of the endosperm and that it was deposited in both types of protein body present in the rice endosperm: derived from the ER and containing prolamins, and derived from the vacuole and containing glutelins. In addition, new types of protein bodies were also formed within the endosperm cells.

Conclusions

The results suggest that the HMW 1Dx5 protein could be trafficked by either the ER or vacuolar pathway, possibly depending on the stage of development, and that its accumulation in the rice endosperm could compromise the structural integrity of protein bodies and their segregation into two distinct populations in the mature endosperm.     

Effects of an Agropyron chromosome on endosperm proteins in common wheat (Triticum aestivum L.)

An Agropyron chromosome having a gene conferring blue color on the aleurone layer of the kernel endosperm causes a 15% increase in total grain protein content when it is added to the common wheat (2n=42) complement. In contrast, there is no effect of this chromosome on total protein content if it replaced part of a wheat chromosome. Endosperm protein components of isolines having blue aleurone due to the Agropyron chromosome being added (2n=44) or translocated (2n=42) were compared to normal nonblue isoline counterparts. Gliadin proteins separated by aluminum lactate (pH 3.2) polyacrylamide gel electrophoresis (PAGE) in one or two dimensions showed greater staining intensity for the blue addition isolines (2n=44) than nonblue (2n=42) isolines. However, the 42-chromosome blue isoline did not show increased protein staining over the nonblue isoline, but at least five protein differences were detected between the lines. SDS-PAGE showed that blue and nonblue differences were expressed primarily in the gliadins, but also in the glutenin, globulin, and albumin proteins.This research was supported by a D. F. Jones Postdoctoral Fellowship to K. M. Soliman and by Western Regional Project W-132, Genotype-environment interactions related to end-product uses in small grains.     

Chromosomal location by F1 monosomic analysis of endosperm proteins in bread wheat

Summary A new methodology to determine the chromosomal location and allelic differences of endosperm proteins in common wheat cultivars by analysis of monosomic intervarietal hybrids is reported. Endosperm proteins from the common wheats Chinese Spring, Capelle Desprez, Holdfast and Pane 247 are studied using monosomic F1 for the chromosomes of homoeologous groups 1 and 6. The proteins were fractionated by two electrophoretical techniques, SDS- and A-PAGE. The use of monosomic offers a remarkable advantage over the utilization of intervarietal substitution lines, because it permits a quicker characterization and earlier evaluation of basic material to be used in breeding programmes.     

Chromosomal location by F1 monosomic analysis of endosperm proteins in bread wheat

Summary The gliadin components from four bread wheat cultivars: Chinese Spring, Capelle Desprez, Holdfast and Pane-247 and their monosomic F1s for the chromosomes of homoeologous groups 1 and 6 have been analyzed by two-dimensional (2-pH) polyacrylamide gel electrophoresis. Chromosomal location of gliadin genes and the allelic differences were well established by analyzing the different F1 monosomic hybrids, electrophoretical patterns and differences in relative staining intensity. A new gliadin encoded by a gene located on chromosome 6B in Chinese Spring is described. The two-dimensional patterns of gliadin in the other three varieties and the chromosomal location of their genes are reported for the first time. Relationships between gliadins in the two-dimensional patterns and the traditional system for their nomenclature are discussed.     

Specific reduction of wheat storage proteins by thioredoxin h

Gliadins and glutenins, the major storage proteins of wheat endosperm (Triticum durum, Desf. cv Monroe), were reduced in vitro by the NADP/thioredoxin system (NADPH, NADP-thioredoxin reductase and thioredoxin; in plants, the h type) from either the same source or the bacterium Escherichia coli. A more limited reduction of certain members of these protein groups was achieved with the reduced form of glutathione or glutaredoxin, a protein known to replace thioredoxin in certain bacterial and mammalian enzyme systems but not known to occur in higher plants. Endosperm extracts contained the enzymes necessary to reduce NADP by the oxidative pentose phosphate pathway (hexokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase). The gliadins and glutenins were also reduced in vivo during germination--an event that accompanied their proteolytic breakdown. The results suggest that thioredoxin, reduced by NADPH generated via the oxidative pentose phosphate pathway, functions as a signal in germination to enhance metabolic processes such as the mobilization of storage proteins and, as found earlier, the activation of enzymes.