Grain Softness Protein Research Articles

Characterising avenin-like proteins (ALPs) from albumin/globulin fraction of wheat grains by RP-HPLC, SDS-PAGE, and MS/MS peptides sequencing

BackgroundWheat grain avenin-like proteins (ALPs) belong to a recently discovered class of wheat grain storage protein. ALPs in wheat grains not only have beneficial effects on dough quality but also display antifungal activities, which is a novel observation for wheat storage proteins. Previous studies have shown that ALPs are likely present in the albumin/globulin fractions of total protein extract from wheat flour. However, the accumulation characteristics of these ALPs in the mature wheat grain remains unknown.ResultsIn the present study, a total of 13 ALPs homologs were isolated and characterized in the albumin/globulin fractions of the wheat protein extract. A combination of multiple techniques including RP-HPLC, SDS-PAGE, MALDI-TOF and peptide sequencing were used for accurate separation and identification of individual ALP homolog. The C-terminal TaALP-by-4AL/7DS, TaALP-by-4AL/7AS/7DS, TaALP-bx/4AL/7AS/7DS, TaALP-ay-7DS, TaALP-ay-4AL, TaALP-ax-4AL, TaALP-ax-7AS, and TaALP-ax-7DS, were separated as individual protein bands from wheat flour for the first time. These unique ALPs peptides were mapped to the latest wheat genome assembly in the IWGSC database. The characteristic defence related proteins present in albumin and globulin fractions, such as protein disulfide-isomerase (PDI), grain softness protein (GSP), alpha-amylase inhibitors (AAIs) and endogenous alpha-amylase/subtilisin inhibitor were also found to co-segregate with these identified ALPs, avenin-3 and α-gliadins. The molecular weight range and the electrophoresis segregation properties of ALPs were characterised in comparison with the proteins containing the tryp_alpha_amyl domain (PF00234) and the gliadin domain (PF13016), which play a role in plant immunity and grain quality. We examined the phylogenetic relationships of the AAIs, GSP, avenin-3, α-gliadins and ALPs, based on the alignment of their functional domains. MALDI-TOF profiling indicated the occurrence of certain post-translations modifications (PTMs) in some ALP subunits.ConclusionsWe reported for the first time the complete profiling of ALPs present in the albumin/globulin fractions of wheat grain protein extracts. We concluded that majority of the ALPs homologs are expressed in wheat grains. We found clear evidence of PTMs in several ALPs peptides. The identification of both gliadin domain (PF13016) and Tryp_alpha_amyl domain (PF00234) in the mature forms of ALPs highlighted the multiple functional properties of ALPs in grain quality and disease resistance.

Identification and sequence analysis of grain softness protein in selected wheat, rye and triticale

Grain softness protein (GSP) is an important protein for overcoming milling and grain defenses in the innate immunity systems of cereals. The objective of this study was to evaluate and understand GSP sequences in selected wheat, rye and triticale. Using sequences for this gene from a sequence database, we performed clustering analysis to compare the sequences obtained from 3 germplasms with other studied sequences for GSP. The maximum difference between the Hirmand GSP genotype in wheat and the database sequences was 23% in EF109396 and EF109399. Most amino acid variation between the GSP sequences involved the same amino acids. The Nikita rye GSP gene showed 64% identity with DQ269918 and AY667063. The isoelectric point in the GSP of wheat and Lasko triticale was significantly higher than that of rye GSP. In addition, parameters such as optical density, grand average of hydrophobicity, percentage of hydrophobicity and hydrophilic amino acids, and number of alpha helices and beta sheets in GSP were similar in wheat and triticale but not in wheat and rye.

Development of a PCR Assay and Marker‐Assisted Transfer of Leaf Rust and Stripe Rust Resistance Genes Lr57 and Yr40 into Hard Red Winter Wheats

ABSTRACTThe wheat (Triticum aestivum L.)‐Aegilops geniculata introgression T5DL·5DS‐5MgS(0.95), with stripe rust resistance gene Yr40 and leaf rust resistance gene Lr57, is an effective source of resistance against most isolates of the rust pathogen in Kansas and India. Rust resistance genes Lr57 and Yr40 were transferred to the hard red winter wheat (HRWW) cultivars Jagger and Overley by standard backcrossing and marker‐assisted selection (MAS). PCR‐based cleaved amplified polymorphic sequence (CAPS) markers were developed and used to select backcross F1 plants with rust resistance. Diagnostic polymorphism detected by CAPS marker XLr57/Yr40‐MAS‐CAPS16 was consistent between the resistant source and a set of wheat cultivars selected by the U.S. Wheat Coordinated Agricultural Project. Homozygous BC3F2 plants with rust resistance genes were selected using the Restriction Fragment Length Polymorphisms (RFLP)‐based diagnostic DNA marker polymorphism detected by the wheat grain softness protein (GSP). Deletion of Puroindoline a (Pina‐Mg1) and Puroindoline b (Pinb‐Mg1) gene sequences in the chromosome segment of 5MgS substituting for 5DS of wheat suggests that the backcross derivatives with Lr57/Yr40 can be used to develop hard textured wheats. The HRWW germplasm with Lr57 and Yr40 will provide breeders in the Southern Great Plains with adapted lines having an additional source of resistance that can be used to achieve durability of rust resistance.

Correlation between the kernel structure and the quality parameters on some Hungarian winter wheat varieties

Introduction The kernel hardness have great effect on the baking properties of the resulting flour. Flour, which is made from hard wheat generally have a medium to high protein content and stronger gluten than flour, which is made from soft wheat. The Hardness-locus on chromosome 5D is the main determinant of grain texture in bread wheat. Puroindoline-a (pin-a), puroindoline-b (pin-b) and Grain Softness Protein (GSP) genes are tightly linked at this locus and their products are the predominant components of friabilin, a 15 kDa endosperm protein complex. The friabilin protein complex determines the kernel hardness. Generally, when the amount of the friabilin is high, the kernel hardness is soft and when the amount of the friabilin is low the kernel hardness is hard (Acsne, 2001). We can sort in these two groups the kernel hardness. Hardness in wheat is largely controlled by genetic factors but it can be affected by the environment. (Gyimes, 2004). The transgenic expression of wild type Pina sequence in the Pina null genotype gave soft grain with the characteristics of soft wheat including increased starch bound friabilin. The results of Martin et al (2006) support the hypothesis that both wild type Pin genes need to be present for friabilin formation and soft grain. Vitreousness is also have impact on Middle-European. A recent French study allowed distinguishing consequences of hardness and vitreousness. Hardness is suggested to influence the adhesion forces between starch granules and protein matrix whereas vitreousness would rather be related to the endosperm microstructure (Grefeuille et al, 2006). The high gluten, hard wheat flour generally contains about 12,0-13,0 % (or more) crude protein under Mid-European conditions. The relationship between wheat protein content and kernel texture is usually positive and kernel texture influences the eg during milling. Hard textured wheat grains require more eg than soft textured grains. The aim of our research was to determine the relationships between kernel hardness and other technologically important traits in wheat varieties widely used in the Hungarian agriculture (Veha, 2005.)

Novel puroindoline and grain softness protein alleles in Aegilops species with the C, D, S, M and U genomes

DNA from six hexaploid, tetraploid and diploid species of Aegilops with the C, D, S, M and U genomes was amplified with specific PCR primers to identify sequences encoding puroindolines (Pins) a and b and grain softness protein (GSP), all of which are encoded by genes at the Ha (hardness) locus, with Ae. tauschii (DD) and bread wheat (T. aestivum) (AABBDD) cv Hiline being studied as controls. Seven new allelic forms of Pin a and Pin b were identified, including forms with mutations within or close to the tryptophan motif. In addition, five new forms of GSP were detected. In all species both genomic DNA from leaves and cDNA from developing grain were analysed. This revealed the presence of both silent genes (with premature stop codons) and multiple genes, with the latter being confirmed by Southern blot analysis. Freeze fracture analysis demonstrated that all except one accession (Ae. sharonensis) were soft textured. However, this difference cannot be accounted for by the sequences of the Pin alleles present in this line.

Isolation of cereal arabinogalactan-peptides and structural comparison of their carbohydrate and peptide moieties

Abstract Arabinogalactan (AG)-peptides were isolated and purified from wheat and durum wheat and for the first time from spelt, triticale, rye and barley using three dedicated isolation procedures. The AG-peptide molecules have molecular weights of approximately 23,500 with the exception of triticale (27,500) and rye (33,000). The fine structure of the carbohydrate parts revealed close resemblances among the purified cereal AG-peptide samples. They consist of a (1→6)-β- d -galactopyranosyl backbone substituted in the C(O)3-position with a single α- l -arabinofuranosyl or a single β- d -galactopyranosyl residue. The latter can also be substituted in its C(O)3-position with a single α- l -arabinofuranosyl residue. The AG-peptide peptide cores typically exist of 15 amino acids including three highly conserved hydroxyprolines (Hyp), each linked to a carbohydrate chain. The peptide amino acid sequence of spelt and durum wheat AG-peptides showed high similarity with the wheat AG-peptide peptide sequence while triticale, rye and barley AG-peptide peptide cores displayed less similarity. Homology with the N-terminal part of cereal grain softness protein (GSP) precursors indicates that the cereal AG-peptide peptides are a processing product of GSP synthesis. An overall structural model is proposed.

Isolation and characterisation of friabilin genes in rye

The Hardness locus on chromosome 5D is the main determinant of grain texture in hexaploid wheat. Puroindoline-a (pin-a), puroindoline-b (pin-b) and Grain Softness Protein (GSP) genes are tightly linked at this locus and their products are the predominant components of friabilin, a 15 kDa endosperm protein complex. Differences in grain texture are mainly caused by specific puroindoline genes mutations and are known to play a large impact on the end-product quality of wheat, contributing to the distinction of well-suited market classes for specific end-uses. We investigated friabilin genes in rye (Secale cereale L.), aiming at an increase information on the friabilin molecular system in cereals and to further investigate their potential use for the genetic improvement of this crop. Using a PCR approach, puroindoline-b and GSP-like sequences were identified for the first time in several rye and triticale cultivars, sequenced and located on specific chromosomal arms. The primary structures of deduced proteins were determined and compared with those from other cereal species. In contrast with data from previous studies, secaloindoline-a was not found. Our results introduce new evidence for a discrete allelic variation at the secaloindoline loci in rye, indicating that future larger screenings may facilitate preliminary selection of germplasm for rye breeding purposes and triticale production.

Mapping and sequence analysis of barley hordoindolines

Barley (Hordeum vulgare L.) cultivars vary in traits such as grain hardness and malt quality. However, little is known about the genetic basis of these grain quality traits in barley, while more is known about the basis of grain hardness in wheat (Triticum aestivum L.). Puroindolines are endosperm-specific proteins found in wheat and barley, as well as other members of the Triticeae. In wheat, variation of puroindoline sequence is associated with most of the variability in wheat grain texture. However, no information exists on sequence variation of the barley homologs of puroindolines, the hordoindolines. We have therefore chosen to isolate and characterize the hordoindoline (hin) sequences of eight North American barley cultivars. The barley sequences contain numerous non-conservative amino-acid substitutions relative to their wheat counterparts. However, no significant rearrangements were found in either hinA or hinB of barley. Three hinA and two hinB sequence types were found among the eight barley cultivars examined, indicating substantial allelic variation at this locus. The hinB sequence variability was used to map hinB to the short arm of chromosome 5H in a Steptoe/Morex mapping population, which is coincident with the previously mapped location of hinA and Gsp (grain-softness protein). This chromosomal location also coincides with a small barley malt-extract QTL, suggesting that hordoindoline sequence variation may play a small role in barley grain quality. Efforts to correlate barley seed textural differences and malting quality with hordoindoline sequence type are ongoing.

Cloning of a wheat 15‐kDa grain softness protein (GSP)

The wheat starch 15-kDa protein (called grain softness protein or GSP) consists of a major polypeptide and several minor polypeptides. An antiserum raised against GSP was used to screen a wheat cDNA library. A cDNA family encoding approximately 15-kDa proteins that included a heptapeptide sequence previously isolated from protease digests of GSP was identified. A partial cDNA was used in a prokaryotic expression system to produce a fusion protein which reacted strongly against the original anti-GSP serum. A new antiserum raised against the fusion protein produced a weak reaction against a 15-kDa polypeptide extracted from wheat seeds. The results suggest that the proteins encoded by the cDNA family form a minor component of the mixture of 15-kDa polypeptides defined as GSP. RNA complementary to the cDNAs could be extracted from both soft and hard wheat grains from about half-way through grain filling. The encoded proteins are novel members of the 2S superfamily of seed proteins, a diverse family of proteins which maintain a characteristic framework of cysteine residues. The deduced proteins show the highest similarity to the oat 16-kDa avenin and to wheat puroindoline (a lipid-binding 15-kDa protein from wheat). Review of previously published data shows that puroindoline is also closely related to the major polypeptide of GSP, suggesting that the lipid-binding properties of GSP polypeptides may influence grain softness.

Characterisation of the wheat Mr 15000 ?grain-softness protein? and analysis of the relationship between its accumulation in the whole seed and grain softness

The Mr 15000 protein associated with water-washed wheat starch granules from soft wheats was shown to be heterogeneous: it could be divided into a fraction containing one or moreα-amylase inhibitor subunits and a fraction largely composed of a previously uncharacterised polypeptide(s) referred to as the "grainsoftness protein" (GSP). The major N-terminal sequence and sequences of peptides derived from protease digests of GSP are reported. An antiserum specific for GSP was used to show that GSP accumulated in both hard and soft wheat grains, but the GSP in soft grains associated more strongly with starch granules than the GSP in hard grains. A positive correlation between grain softness and accumulation of GSP in the seed was demonstrated for a range of cultivars. This differs from the qualitative relationship, based on the isolated starch fraction, between GSP and grain softness that has already been reported. Analysis of wholemeal extracts with the antiserum demonstrated that the accumulation of GSP in the seed was dependent on the short arm of chromosome 5D, which also encodes theHa locus. In addition, examination of near-isogenic lines differing in hardness indicated that the gene(s) controlling GSP was (were) linked with theHa locus. The findings indicate that GSP may be the product of theHa locus and thus be the major factor that determines the milling characteristics of bread wheats.