Analysis of starch swelling power in Australian breeding lines of hexaploid wheat (Triticum aestivum L.)

Starch is the major component of wheat (Triticum aestivum L.) grain and is composed of two large glucan molecules, amylose and amylopectin. The ratio between the two polymers types influences the water absorbing properties of starch upon heating, and thus affects the end-use of grain and purified starch. In this study, we evaluated the starch swelling power (SSP) values in seven wheat populations developed from crosses involving low-SSP lines. Analysis of starch produced by the F₂ generation plants showed that the largest SSP variation (11.4–16.2) and lowest SSP mean (13.9) was obtained for a population derived from doubled haploid lines SM1028 (SSP = 14.5) and VK306 (SSP = 13.6). The population of 360 lines was advanced by single seed descent to the following generations for further studies. Starch analysis of grain produced by F₄ generation lines in two field locations during 2006 and in a greenhouse environment during 2005 showed that SSP values were relatively stably inherited. The average broad-sense heritability was 73% and significant (P < 0.001) genotype × genotype and genotype × environment interactions were seen. Starches with the highest and lowest SSP values were inversely related to amylose concentration determined by high pressure liquid chromatography (HPLC)–size exclusion chromatography (SEC) of debranched starch. Developed lines with the lowest SSP values surpassed 40% in apparent amylose concentration. The study illustrates that screening for SSP in early generations can be used to develop wheat lines with desired starch swelling characteristics.     

Identification of a GDSL-motif carboxylester hydrolase from rice bran (Oryza sativa L.)

A major esterase (designated OsEST1) showing high activity using 1-naphthyl acetate as a substrate was identified from rice bran and purified approximately 239-fold to near-homogeniety. The purified enzyme migrated as a single polypeptide band on native and SDS-polyacrylamide gels and had a molecular mass of 25 kDa under denaturing conditions. Analysis of its tryptic peptides by MALDI-TOF-MS and subsequent data mining identified a corresponding cDNA OsEST1 consisting of 714 nucleotides and encoding a 238 amino acid protein. Analysis of its primary sequence indicated that OsEST1 is a GDSL-motif carboxylester hydrolase belonging to the SGNH protein subfamily in containing the putative catalytic triad of Ser¹¹, Asp¹⁸⁷, and His¹⁹⁰. OsEST1 showed the highest catalytic activity at approximately pH 8.0–8.5 and at 45 °C with K_m and V_max values for 1-naphthyl acetate of 172 μM and 63.7 μmol/min/mg protein, respectively. However, OsEST1 showed no activity with triacylglycerol. Alignment of the primary sequence of OsEST1 and other rice GDSL-motif esterases/lipases showed that OsEST1 aligns with a specific family of plant SGNH esterases involved in response to dehydration and cuticle formation. These results suggest that OsEST1 is not a lipase but an esterase activity which has some other function in rice, especially during seed development.     

The first characterised wheat (Triticum aestivum L.) member of the nudix hydrolase family shows specificity for NAD(P)(H) and FAD

We cloned the first nudix hydrolase of wheat (Triticum aestivum L.) (TaNUDT1) and expressed it using Escherichia coli as expression host. Properties of the purified His₆-tagged protein were studied. The sequence codes for a nudix hydrolase with a molecular mass of around 43x10³ and a predicted pI of 5.68. The characteristic residues of the nudix box were highly conserved in the wheat enzyme sequence, and TaNUDT1 is most probably targeted to the peroxisomes. In the presence of dithiothreitol and MnCl₂, the enzyme hydrolyses the nicotinamide coenzymes NAD(P)(H) as could be predicted by the presence of a conserved amino acid array C-terminal to the nudix box, and the enzyme has higher affinity towards the reduced forms of the coenzymes. In light of previous reports of nudix enzymes and the physiological concentration of their substrates, we found its K_m values towards some of the coenzymes to be relatively high. As NAD(P)(H) and FAD play crucial roles in cell metabolism, extensive hydrolysis could be detrimental to cell functioning. We speculate that a decreased enzyme affinity may point to a built-in restriction on coenzyme hydrolysis in vivo. Furthermore, by hydrolysing the cofactors of several redox enzymes, this enzyme may well affect several wheat based processes.     

Characterization of seed proteome in Brachypodium distachyon

Brachypodium distachyon, an emerging model plant system for some economically important temperate grasses such as wheat, barley and switchgrass, has recently caught wide attention in modern biological research. In the current study, the glutenin, albumin and globulin components of 13 B. distachyon accessions were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) followed by peptide mass finger printing (PMF) and MS/MS protein identification. Abundant wheat low molecular weight glutenin subunit (LMW-GS) like proteins and a few high molecular weight glutenin subunits (HMW-GS) with low expression level were detected in B. distachyon. A total of 18 storage proteins and 15 albumin proteins were identified through PMF and MS/MS. The results demonstrated that the major seed storage proteins in B. distachyon are wheat LMW-GS like proteins and globulins. The identified albumins and globulins were mostly various enzymes that were classified into five groups according to their functions. The 2-DE spot distribution and MS results suggested that post-translational modifications (PTMs) such as phosphorylations and glycosylations are common phenomena in B. distachyon seed proteome.     

Amylolysis of wheat starches. II. Degradation patterns of native starch granules with varying functional properties

Scanning electron microscopy (SEM) and X-ray diffraction (XRD) were employed to investigate degradation patterns of native starch granules from wheat (Triticum aestivum L.) by different starch-degrading enzymes. The starches examined were from a waxy wheat and four varieties with slightly elevated amylose content, but with different functional properties. Differences in the digestion patterns after partial α-amylolysis of starch granules were noted between the starches. The waxy starch seemed to be degraded by endocorrosion, whereas the amylose-rich starches followed a slower mode of hydrolysis starting from the granular surface. X-ray diffractograms of the amylose-rich starches were not significantly altered by 2 h of α-amylolysis, whereas partial hydrolysis of the waxy starch decreased scattering intensity at higher 2θ angles, consistent with a different mode of attack by α-amylase in the initial digestion stages of granules of waxy and amylose-rich starches. We propose these differences are due to the combined effects of the change in packing density and partial preference for hydrolysis of amorphous material. The native starch granules were also attacked by beta-amylase, isoamylase and amyloglucosidase, which indicates that α-amylase is not the only starch-degrading enzyme that is able to initiate starch hydrolysis of native granules.     

Development of simple and co-dominant PCR markers to genotype puroindoline a and b alleles for grain hardness in bread wheat (Triticum aestivum L.)

Grain hardness is one of the most important quality characteristics of cultivated bread wheat (Triticum aestivum L.). A large deletion in the puroindoline a (Pina) gene or single nucleotide polymorphisms (SNPs) in the puroindoline b (Pinb) gene results in hard grain texture. So far, nine Pina alleles (Pina-D1a–Pina-D1b, Pina-D1k–Pina-D1q) and seventeen Pinb alleles (Pinb-D1a–Pinb-D1g, Pinb-D1p–Pinb-D1ab) have been identified in bread wheat. The major Pina and Pinb alleles identified in hard wheat cultivars are Pina-D1b, Pinb-D1b, Pinb-D1c and Pinb-D1d. In this study, a three-primer PCR system was employed to develop nine co-dominant STS markers for genotyping Pina-D1a and Pina-D1b, whereas temperature-switch (TS) PCR was used to develop six co-dominant SNP markers for genotyping the Pinb-D1a, Pinb-D1b, Pinb-D1c and Pinb-D1d alleles. These STS and TS-PCR markers were used to verify the grain hardness genotype of 100 wheat cultivars. The reliability and genotyping accuracy of TS-PCR markers were confirmed through sequencing of PCR products and a comparison with previously published results. Therefore, STS and TS-PCR markers offer a simple, cost-effective and reliable method for high-throughput genotyping Pina and Pinb alleles to select grain hardness in wheat quality breeding programs and for wheat market classification.     

PCR-based markers for identification of HMW-GS at Glu-B1x loci in common wheat

The bread wheat elasticity, which is very important for bread-making quality, is largely determined by the composition of high-molecular-weight glutenin subunits (HMW-GS). The HMW-GS encoded by Glu-B1 loci are highly polymorphic and the combinations 17+18 and 14+15 are good for bread making. Thus it is very important to identify the alleles at Glu-B1 loci for wheat quality improvement. In this study, the five common HMW-GS types encoded by Glu-B1x locus carried by 18 Chinese bread wheat cultivars (or lines) were analyzed by SDS-PAGE. Two pairs of PCR primers which could distinguish the Glu-Blx alleles of the five common HMW-GS types were designed based on the Glu-B1x gene sequences (Reddy and Appels, 1993; Genbank accession: X13927; Genbank accession:AY367771). 22 recombinant inbred lines (RILs) derived from Jing711 (contains 17 subunit on Glu-B1x) and Pm97034 (contains 14 subunit on Glu-B1x) were used to validate the accuracy of the primers, which showed that the two specific markers could be used together to distinguish alleles at Glu-B1x locus and accelerate wheat quality breeding by marker assisted selection.     

Isolation and characterization of EMS-induced Dy10 and Ax1 high molecular weight glutenin subunit deficient mutant lines of elite hexaploid wheat (Triticum aestivum L.) cv. Summit

The mixing properties of the dough are critical in the production of bread and other food products derived from wheat. The high molecular weight glutenin subunits (HMW-GS) are major determinants of wheat dough processing qualities. The different alleles of the HMW-GS genes in hexaploid wheat vary in their effect on dough quality. To determine the contribution of the individual HMW-GS alleles, lines deficient in HMW-GS proteins were generated by chemical mutagenesis in the elite bread wheat Triticum aestivum cv. Summit. In this report we describe the identification and characterization of Dy10 and Ax1 deficient lines. Examination of the effect of Dy10 and Ax1 deficiency on dough rheological properties by mixography showed shorter mixing time to reach peak resistance, and weaker and less extensible doughs relative to the wild type control. This is the first time that the role of Dy10 in vivo has been examined apart from the Dx5 + Dy10 allelic pair combination.     

Agronomic and quality characteristics of triticale (X Triticosecale Wittmack) with HMW glutenin subunits 5+10

Sets of triticale (X Triticosecale Wittmack) lines derived from the cv. Presto with HMW glutenin allele Glu-D1d (subunits 5+10) translocated from bread wheat (Triticum aestivum L.) chromosome 1D to chromosome 1R were evaluated for agronomic and grain quality characteristics in 2002–2005. Two different translocation types were used: (a) single translocation 1R.1D₅₊₁₀-2 where the long arm of 1R carries the wheat segment from 1DL with the Glu-D1d replacing a secalin locus Sec-3, (b) double translocation Valdy where the long arm of 1R has the translocation 1R.1D₅₊₁₀-2 and the short arm has a segment from 1DS carrying wheat loci Gli-D1 and Glu-D3. The presence of Glu-D1d was determined by polyacrylamide gel electrophoresis (PAGE-ISTA) and DNA markers. The tested lines of triticale were compared with the check triticale cv. Presto and with wheat cultivars of different bread making quality (E-C quality classes). Single translocation 1R.1D₅₊₁₀-2 reduced grain yield by 16% and Valdy translocation by 24% as compared with cv. Presto. The Valdy translocation had substantially shortened spike length and reduced specific weight in comparison with check cv. Presto. Wet gluten content (according to the Perten method) was 12% in both translocation types, 8% in check Presto and on average 24% in wheat. Translocations increased the Zeleny sedimentation value (Valdy — 27 ml, 1R.1D₅₊₁₀-2 – 25 ml, cv. Presto — 23 ml). Triticale had a very low Hagberg falling number (FN) of 62–70 s without significant differences, while wheat had on average 301 s. The translocations did not significantly increase loaf volume; however, they improved loaf shape (height/width ratio): Valdy — 0.61, 1R.1D₅₊₁₀-2 – 0.56, cv. Presto 0.44, wheat on average 0.70. The dough was non-sticky in Valdy, slightly sticky in 1R.1D₅₊₁₀-2 and sticky in cv. Presto. Problems with a low FN for improving bread making quality of triticale are discussed. Higher bread making quality can be influenced by appropriate combination with donors of low α-amylase activity.     

Hemicellulase production in Chrysosporium lucknowense C1

Filamentous fungi are widely used for enzyme production for the biofuel industry. The ascomycetous fungus Chrysosporium lucknowense C1 was isolated as a natural producer of neutral cellulases. It is at present an attractive alternative to well known fungi like Aspergillus sp. and Trichoderma reesei for protein production on a commercial scale. Besides many cellulases, a large number of hemicellulases (particularly xylanases and arabinofuranosidases) and esterases (acetyl xylan esterases and ferulic acid esterases) encoding genes have also been identified in the C1 genome. Many of these extracellular enzymes have been selectively expressed in C1 and then purified and characterized. Four arabinofuranosidases, two acetyl xylan esterases, two ferulic acid esterases, an α-glucuronidase and four xylanases have been purified and characterized. All these enzymes were found to be active towards arabinoxylans, demonstrating the high potential of C1 as a producer of hemicellulolytic enzymes.     

Waxy endosperm accompanies increased fat and saccharide contents in bread wheat (Triticum aestivum L.) grain

The contents of fat, starch, pentosan, fructan, β-glucan and several mono- and oligosaccharides in grain were evaluated to find out the possible effects of the Wx-D1 gene of bread wheat using two sets of near-isogenic waxy and non-waxy lines and two low-amylose mutant lines with a common genetic background of Kanto 107. These materials have two non-functional Wx-A1b and Wx-B1b alleles in common. Waxy near-isogenic lines with a non-functional Wx-D1d allele showed consistently increased contents of fat, total fructan, β-glucan, glucose, fructose, sucrose, 1-kestose, 6-kestose, neokestose, nystose and bifurcose compared with non-waxy lines with a functional Wx-D1a allele throughout three growing/harvest seasons. Starch and total pentosan contents were inconsistently influenced by the allelic status of the Wx-D1 locus, while water-soluble pentosan and raffinose contents were not affected. The compositional changes of a low-amylose mutant line with an almost non-functional Wx-D1f allele were closely similar to those of waxy near-isogenic lines, while significantly different changes were barely observed in another low-amylose mutant line with a partly functional Wx-D1g allele in two seasons. These results showed that the Wx-D1 gene has pleiotropic effects on the fat and saccharide contents of bread wheat grain.     

Effects of Fusarium culmorum and water stress on durum wheat in Tunisia

The effects of water stress on Fusarium foot and root rot in durum wheat were investigated in growth chamber, greenhouse and field tests in Tunisia. In the seedling stage, emergence of six durum wheat cultivars in the growth chamber was significantly reduced by inoculation with Fusarium culmorum and water stress (P<0.0001), with more disease under drier conditions. Additionally, the tiller number per mature plant, the 1000 grain weight and disease severity in mature stage were reduced by inoculation in greenhouse studies. In a field test, inoculation with F. culmorum significantly reduced the yield (P<0.001), by more than 17% for Om Rabiaa and 38% for Karim, the two cultivars tested. Yield was also significantly affected by precipitation and irrigation levels. The severity of the disease, estimated by the percentage of white heads, was separately affected by the cultivar (P<0.001) and inoculation (P = 0.0004). Percentage of white heads was 1.5 and 2 × higher in inoculated plants than non-inoculated for Om Rabiaa and Karim cultivars, respectively. Disease severity was highest in treatments with the greatest water stress. This is the first detailed study of water stress and F. culmorum on durum wheat in Tunisia, and indicates that cultivar resistance and irrigation management may be important in the management of Fusarium foot rot.     

Effect of the grain protein content locus Gpc-B1 on bread and pasta quality

Grain protein concentration (GPC) affects wheat nutritional value and several critical parameters for bread and pasta quality. A gene designated Gpc-B1, which is not functional in common and durum wheat cultivars, was recently identified in Triticum turgidum ssp. dicoccoides. The functional allele of Gpc-B1 improves nitrogen remobilization from the straw increasing GPC, but also shortens the grain filling period resulting in reduced grain weight in some genetic backgrounds. We developed isogenic lines for the Gpc-B1 introgression in six hexaploid and two tetraploid wheat genotypes to evaluate its effects on bread-making and pasta quality. In common wheat, the functional Gpc-B1 introgression was associated with significantly higher GPC, water absorption, mixing time and loaf volume, whereas in durum wheat, the introgression resulted in significant increases in GPC, wet gluten, mixing time, and spaghetti firmness, as well as a decrease in cooking loss. On the negative side, the functional Gpc-B1 introgression was associated in some varieties with a significant reduction in grain weight, test weight, and flour yield and significant increases in ash concentration. Significant gene × environment and gene × genotype interactions for most traits stress the need for evaluating the effect of this introgression in particular genotypes and environments.     

Effect of Acibenzolar-S-methyl (ASM) pre-treatment in inducing resistance against Pythium aphanidermatum infection in Curcuma longa

In vitro experiments were carried out to test the efficacy of plant activator Acibenzolar-S-methyl (ASM, a benzothiadiazole derivative; trade name Bion 50WG) against rhizome rot disease of turmeric (Curcuma longa L.) caused by Pythium aphanidermatum. The plant activator was applied as a liquid rhizome pre-treatment followed by inoculation with P. aphanidermatum. Cell death, activities of pathogenesis related (PR) proteins such as cysteine protease (EC 3.4.22), peroxidases (EC 1.11.1.7) both soluble and ionically bound (IB), trypsin inhibitor (EC 3.4.21.1) and chymotrypsin inhibitor (EC 3.4.21.4) were monitored. Rhizome pre-treatment was effective in controlling P. aphanidermatum infection. Anatomical observation of turmeric rhizomes indicated the presence of calcium oxalate deposits in infected tissue and an accumulation of starch grains in response to infection by P. aphanidermatum. Pathogen infection also induced new basic polypeptides corresponding to 18.0 and 41.0 kDa. Induction of protease, protease inhibitors, soluble and ionically bound peroxidase activity was observed after ASM pre-treatment and P. aphanidermatum infection. ASM treatment also enhanced activities of proteases and peroxidase in rhizomes already infected with P. aphanidermatum. Increases in enzyme activities and protease inhibitors occurred much more rapidly and were enhanced in P. aphanidermatum infected rhizomes that were previously treated with ASM suggesting that increased activities of peroxidases and protease inhibitors may play a key role in restricting the development of disease symptoms on the rhizomes infected with P. aphanidermatum as evidenced by a reduction in cell death. Hence, pretreatment with ASM suppress the P. aphanidermatum induced oxidative damage through higher accumulation of peroxidases and induced defense through activities of protease inhibitors thereby, protected turmeric rhizomes from rhizome rot disease.     

Development of an Aegilops longissima substitution line with improved bread-making quality

This study focuses on the effect of Aegilops longissima on wheat bread making quality. Chromosome 1S^l disomic addition line of Ae. longissima (DAL1S^l) had significantly higher dough strength, grain hardness, mixographic peak height, band width, and unextractable polymeric protein content compared with wheat. DAL1S^l also had additional glutenin and gliadin proteins contributed by Ae. longissima. The larger size of 1S^l coded HMW-GSs sequenced from DAL1S^l and their phylogenetic similarity to the D-genome-coded subunits were suspected to be one of the major reasons for the increased dough strength of DAL1S^l. To transfer the chromosome 1S^l genes responsible for the good bread-making quality to wheat, we generated a chromosome-specific disomic substitution line [DSL1S^l(1A)] by crossing DAL1S^l with nulli 1A tetra 1B genetic stock and further selection. Grain quality analysis revealed significantly lower grain hardness and significantly higher dough strength, farinograph development time, stability time, gluten index, bread loaf volume, and bread quality score in DSL1S^l(1A), compared with wheat. However, the increased bread loaf volume and quality were not proportional to the relatively higher increases in dough strength and gluten index, indicating importance of other traits influencing bread making quality. The presence of a minor hardness locus on chromosome 1A is speculated.     

Development of STS markers and establishment of multiplex PCR for Glu-A3 alleles in common wheat (Triticum aestivum L.)

Low-molecular-weight glutenin subunits (LMW-GS) play a key role in determining the processing quality of the end-use products of common wheat. The objectives of this study were to identify genes at Glu-A3 locus, develop the STS markers, and establish multiplex PCR with the STS markers for Glu-A3 alleles. Gene-specific PCR primers were designed to amplify six near-isogenic lines (NILs) and Glenlea with different Glu-A3 alleles (a, b, c, d, e, f and g) defined by the protein electrophoretic mobility. Three Glu-A3 genes with complete coding sequence were cloned, designated as GluA3-1, GluA3-2 and GluA3-3, respectively. Seven dominant allele-specific STS (sequence tagged sites) markers were designed based on the SNPs (single nucleotide polymorphisms) among different allelic variants for the discrimination of the Glu-A3 protein alleles a, b, c, d, e, f and g. Four multiplex PCRs were established including Glu-A3b + Glu-A3f, Glu-A3d + Glu-A3f, Glu-A3d + Glu-A3g, and Glu-A3b + Glu-A3e. These markers and multiplex-PCR systems were validated on 141 CIMMYT wheat varieties and advanced lines with different Glu-A3 alleles, confirming that they can be efficiently used in marker-assisted breeding.     

Differential response of pea (Pisum sativum) to Orobanche crenata, Orobanche foetida and Phelipanche aegyptiaca

Several broomrape species including Orobanche crenata, Orobanche foetida and Phelipanche aegyptiaca are reported to infect various grain and forage legumes in the Mediterranean and West Asia. Pea (Pisum sativum) is severely damaged by O. crenata, but there are no reports on O. foetida or P. aegyptiaca infection. We report here that pea can induce high germination of seeds of O. crenata, O. foetida and P. aegyptiaca but only O. crenata success infecting pea roots and developing further. Some differences in levels of infection by O. crenata were observed among pea accessions what can be exploited in pea resistance breeding programmes. On the contrary, all pea accessions studied were highly resistant to infection by both O. foetida and P. aegyptiaca, preventing any tubercle attachment and development. This makes pea a promising candidate as trap crop for O. foetida and P. aegyptiaca seed bank demise in infested soils.