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Faba bean (Vicia faba L.) flour, starch concentrate (60% starch), protein concentrate (~60% protein) and protein isolate (~85% protein) were added to replace one-quarter of durum wheat semolina to enrich the nutritional quality and physiological functions of durum wheat (Triticum turgidum L.) pasta. The raw pasta samples prepared with protein concentrate or isolate had higher (p ≤ 0.001) protein and lower (p ≤ 0.001) total starch concentrations, along with increased total dietary fiber and slowly digestible starch (p ≤ 0.001) than durum wheat semolina control or those with added whole faba-bean flour or isolated starch. The faba bean fortified pasta had altered starch with increased proportion of medium B-type glucan chains and long C-type glucan chains, reduced starch digestibility and were associated with glycaemia related effects in the human diet. The faba bean fortified pasta had increased protein and dietary fiber that influenced food intake and satiety. The results suggest differential contributions of food ingredients in human health outcomes., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Fusarium head blight (FHB) in wheat (Triticum aestivum L.), predominantly caused by Fusarium graminearum, has been categorized into three chemotypes depending on the major mycotoxin produced. The three mycotoxins, namely, 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON) and nivalenol (NIV) also determine their aggressiveness and response to fungicides. Furthermore, prevalence of these chemotypes changes over time and dynamic changes in chemotypes population in the field have been observed. The objective of this study was to identify spike culture derived variants (SCDV) exhibiting resistance to multiple chemotypes of F. graminearum. First, the optimal volume of inoculum for point inoculation of the spikelets was determined using the susceptible AC Nanda wheat genotype. Fifteen μL of 105 macroconidia/mL was deemed optimal based on FHB disease severity assessment with four chemotypes. Following optimal inoculum volume determination, five chemotypes (Carman-NIV, Carman-705-2-3-ADON, M9-07-1-3-ADON, M1-07-2-15-ADON and China-Fg809-15-ADON) were used to point inoculate AC Nanda spikelets to confirm the mycotoxin produced and FHB severity during infection. Upon confirmation of the mycotoxins produced by the chemotypes, 55 SCDV were utilized to evaluate FHB severity and mycotoxin concentrations. Of the 55 SCDV, five (213.4, 244.1, 245.6, 250.2 and 252.3) resistant lines were identified with resistance to multiple chemotypes and are currently being utilized in a breeding program to develop wheat varieties with improved FHB resistance., Competing Interests: The authors have declared that no competing interests exist.

Wheat is the primary grain fed to poultry in western Canada, but its nutritional quality, including the nature of its starch digestibility, may be affected by wheat market class. The objectives of this study were to determine the rate and extent of starch digestibility of wheat market classes in broiler chickens, and to determine the relationship between starch digestibility and wheat apparent metabolizable energy (AME). In vitro starch digestion was assessed using gastric and small intestinal phases mimicking the chicken digestive tract, while in vivo evaluation used 468 male broiler chickens randomly assigned to dietary treatments from 0 to 21 d of age. The study evaluated 2 wheat cultivars from each of 6 western Canadian wheat classes: Canadian Prairie Spring (CPS), Canadian Western Amber Durum (CWAD), CW General Purpose (CWGP), CW Hard White Spring (CWHWS), CW Red Spring (CWRS), and CW Soft White Spring (CWSWS). All samples were analyzed for relevant grain characteristics. Data were analyzed as a randomized complete block design and cultivars were nested within market class. Pearson correlation was used to determine relationships between measured characteristics. Significance level was P ≤ 0.05. The starch digestibility range and wheat class rankings were: proximal jejunum - 23.7 to 50.6% (CWHWSc, CPSbc, CWSWSbc, CWRSab, CWGPa, CWADa); distal jejunum - 63.5 to 76.4% (CWHWSc, CPSbc, CWSWSbc, CWRSab, CWGPa, CWADa); proximal ileum - 88.7 to 96.9% (CWSWSc, CPSbc, CWHWSbc, CWRSb, CWGPb, CWADa); distal ileum - 94.4 to 98.5% (CWSWSb, CWHWSb, CPSb, CWRSab, CWGPab, CWADa); excreta - 98.4 to 99.3% (CPSb, CWRSb, CWHWSb, CWSWSab, CWGPab, CWADa). Wheat class affected wheat AMEn with levels ranging from 3,203 to 3,411 kcal/kg at 90% DM (CWRSc, CWSWSc, CPSb, CWGPb, CWADa, CWHWSa). Significant and moderately strong positive correlations were observed between in vitro and in vivo starch digestibility, but no correlations were found between AME and starch digestibility. In conclusion, rate and extent of starch digestibility and AME were affected by western Canadian wheat class, but starch digestibility did not predict AME.

An in vitro spike culture method was optimized to evaluate Fusarium head blight (FHB) resistance in wheat (Triticum aestivum) and used to screen a population of ethyl methane sulfonate treated spike culture-derived variants (SCDV). Of the 134 SCDV evaluated, the disease severity score of 47 of the variants was ≤30%. Single nucleotide polymorphisms (SNP) in the UDP-glucosyltransferase (UGT) genes, TaUGT-2B, TaUGT-3B, and TaUGT-EST, differed between AC Nanda (an FHB-susceptible wheat variety) and Sumai-3 (an FHB-resistant wheat cultivar). SNP at 450 and 1,558 bp from the translation initiation site in TaUGT-2B and TaUGT-3B, respectively were negatively correlated with FHB severity in the SCDV population, whereas the SNP in TaUGT-EST was not associated with FHB severity. Fusarium graminearum strain M7-07-1 induced early expression of TaUGT-2B and TaUGT-3B in FHB-resistant SCDV lines, which were associated with deoxynivalenol accumulation and reduced FHB disease progression. At 8 days after inoculation, deoxynivalenol concentration varied from 767 ppm in FHB-resistant variants to 2,576 ppm in FHB-susceptible variants. The FHB-resistant SCDV identified can be used as new sources of FHB resistance in wheat improvement programs.

Wheat grain development is a complex process and is characterized by changes in physicochemical and structural properties of starch. The present study deals with endosperm starch physicochemical properties and structure during development in different granule-bound starch synthase I (GBSSI) null also known as waxy (Wx) genotypes. The study was conducted with pure starch isolated from wheat grains at 3-30 days post anthesis (DPA), at 3-day intervals. Amylose concentration increased throughout grain development in non-waxy (7.2-30.5%) and partial waxy genotypes (6.0-26.8%). Completely waxy genotype showed 7.0% amylose at 3 and 6 DPA, which declined during development and reached non-detectable quantities by 30 DPA. Amylopectin structure had a higher content of short chains at 3 DPA, which decreased continuously until 12 DPA, after which there were only minor changes in amylopectin chain length distribution. Similarly, the average degree of polymerization (DP) increased from 3 DPA (12.3) to 12 DPA (15.0), and then did not differ significantly up to 30 DPA (15.0). This suggests the formation of basic amylopectin architecture in wheat by 12 DPA. Wx-B and Wx-D affected amylopectin short chains mostly of DP 6-8 at 3 and 6 DPA. Wx-A affected the same fraction of chains at 9 and 12 DPA, and Wx-D affected DP 18-25 chains from 18 to 30 DPA, suggesting differential effect of waxy isoproteins on amylopectin structure formation., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Near-isogenic wheat ( Triticum aestivum L.) lines differing at the Waxy locus were studied for the influence of genome-specific granule-bound starch synthase I (GBSSI/Waxy; Wx-A, Wx-B, Wx-D) on starch composition, structure, and in vitro starch enzymatic hydrolysis. Grain composition, amylose concentration, amylopectin unit-chain length distribution, and starch granule size distribution varied with the loss of functional GBSSI. Amylose concentration was more severely affected in genotypes with GBSSI missing from two genomes (double nulls) than from one genome (single nulls). Unit glucan chains (DP 6-8) of amylopectin were reduced with the complete loss of GBSSI as compared to wheat starch with a full complement of GBSSI. Wx-A and Wx-B had an additive effect toward short-chain phenotype of waxy amylopectin. Loss of Wx-D isoprotein alone significantly (p < 0.05) reduced the C-type starch granules. However, the absence of Wx-D in combination with Wx-A or Wx-B increased the B-type and C-type starch granules but decreased the volume of A-type starch granules. The rate of in vitro starch enzymatic hydrolysis was highest in completely waxy grain meal and purified starch. However, the presence of Wx-D reduced wheat starch hydrolysis as it increased the large A-type starch granule content (volume %) and reduced short chains (DP 6-8) in amylopectin. Factors such as small C-type starch granules, amylose concentration, and long chains of amylopectin (DP 23-45) also influenced wheat starch hydrolysis.

Background: An immature wheat spike culture system was used to monitor cadmium (Cd) accumulation in grains, hulls and awns of bread wheat and durum wheat. Immature spikes were cultured prior to anthesis in a medium containing 50 g L(-1) sucrose and 0.4 g L(-1) L-glutamine, supplemented with 0, 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20 or 25 mg L(-1) cadmium chloride (CdCl(2)). Grains were collected at maturity and their Cd accumulation was determined using inductively coupled plasma mass spectrometry (ICP-MS)., Results: Cd accumulation at CdCl(2) concentrations of 3 mg L(-1) and above was higher in grains of durum wheat compared with bread wheat. In hulls a similar trend was observed at CdCl(2) concentrations above 15 mg L(-1) . Starch concentration in grains increased slightly at 3 and 4 mg L(-1) CdCl(2). Cd accumulation negatively affected grain protein concentration. Expression patterns of Cd-related genes glutathione reductase (TaGR), metallothionein (MT) and phytochelatin synthase (PCS) in spikes cultured in media containing 0, 5, 10, 15 and 25 mg L(-1) CdCl(2) at 5 days post-anthesis showed that TaGR and PCS expression in bread wheat was up-regulated at 5 mg L(-1) CdCl(2) but down-regulated at other CdCl(2) concentrations. However, in durum wheat, expression of all three genes was down-regulated or remained unchanged., Conclusion: This study demonstrates that immature spike culture can be used to study Cd accumulation in grains and can delineate hyper-accumulating durum wheat from bread wheat at CdCl(2) concentrations of 2 mg L(-1) and above., (Copyright © 2011 Society of Chemical Industry.)

Low-temperature (LT) tolerance in winter wheat (Triticum aestivum L.) is an economically important but complex trait. Four selected wheat genotypes, a winter hardy cultivar, Norstar, a tender spring cultivar, Manitou and two near-isogenic lines with Vrn-A1 (spring Norstar) and vrn-A1 (winter Manitou) alleles of Manitou and Norstar were cold-acclimated at 6°C and crown and leaf tissues were collected at 0, 2, 14, 21, 35, 42, 56 and 70 days of cold acclimation. cDNA-AFLP profiling was used to determine temporal expression profiles of transcripts during cold-acclimation in crown and leaf tissues, separately to determine if LT regulatory circuitries in crown and leaf tissues could be delineated using this approach. Screening 64 primer combinations identified 4,074 and 2,757 differentially expressed transcript-derived fragments (TDFs) out of which 38 and 16% were up-regulated as compared to 3 and 6% that were down-regulated in crown and leaf tissues, respectively. DNA sequencing of TDFs revealed sequences common to both tissues including genes coding for DEAD-box RNA helicase, choline-phosphate cytidylyltransferase and delta-1-pyrroline carboxylate synthetase. TDF specific to crown tissues included genes coding for phospahtidylinositol kinase, auxin response factor protein and brassinosteroid insensitive 1-associated receptor kinase. In leaf, genes such as methylene tetrahydrofolate reductase, NADH-cytochrome b5 reductase and malate dehydrogenase were identified. However, 30 and 14% of the DNA sequences from the crown and leaf tissues, respectively, were hypothetical or unknown proteins. Cluster analysis of up-, down-regulated and unique TDFs, DNA sequence and real-time PCR validation, infer that mechanisms operating in crown and leaf tissue in response to LT are differently regulated and warrant further studies.

An in vitro culture system is useful to study grain development under defined conditions to minimize confounding effects associated with whole plant studies and metabolite movement into the developing grains. The objective of this study was to monitor the expression patterns of carbohydrate metabolism genes during grain development in an in vitro wheat spike culture system. Immature spikes were cultured prior to anthesis, and grains were collected at various days postanthesis (DPA). Grains from cultured spikes showed maximum expression of starch metabolic genes by 10 DPA, with a rapid decline thereafter. The rapid increase and decrease in expression rate in the in vitro system was thought to be due to fructan exohydrolase (1-FEH and 6-FEH) or sucrose transporter 1 (SUT1) and sucrose synthase (SuSy) genes being highly expressed. SUT1 reached peak expression at 8 DPA, two days earlier than the other genes, and may account for the rapid early stage trigger in expression of the other genes. However, expression of 1-FEH and 6-FEH genes in in vitro-cultured spikes peaked at 12 DPA, two days later than the other genes, and could indicate that fructan catabolism was not a factor in the rapid accumulation of starch in the in vitro-cultured spikes. Accumulation of GBSSI polypeptides generally showed similar patterns in both systems, with the maximum amount in the in vitro system observed four days later than in the in planta spikes, reflecting different turnover controls of GBSSI transcripts. The in vitro system offers opportunities for further refinement and detailed grain development studies.

Autumn-seeded winter cereals acquire tolerance to freezing temperatures and become vernalized by exposure to low temperature (LT). The level of accumulated LT tolerance depends on the cold acclimation rate and factors controlling timing of floral transition at the shoot apical meristem. In this study, genomic loci controlling the floral transition time were mapped in a winter wheat (T. aestivum L.) doubled haploid (DH) mapping population segregating for LT tolerance and rate of phenological development. The final leaf number (FLN), days to FLN, and days to anthesis were determined for 142 DH lines grown with and without vernalization in controlled environments. Analysis of trait data by composite interval mapping (CIM) identified 11 genomic regions that carried quantitative trait loci (QTLs) for the developmental traits studied. CIM analysis showed that the time for floral transition in both vernalized and non-vernalized plants was controlled by common QTL regions on chromosomes 1B, 2A, 2B, 6A and 7A. A QTL identified on chromosome 4A influenced floral transition time only in vernalized plants. Alleles of the LT-tolerant parent, Norstar, delayed floral transition at all QTLs except at the 2A locus. Some of the QTL alleles delaying floral transition also increased the length of vegetative growth and delayed flowering time. The genes underlying the QTLs identified in this study encode factors involved in regional adaptation of cold hardy winter wheat.

Unacceptably dark bran color has prevented the white-kernelled variety Argent from meeting grain color marketing standards for hard white wheats (Triticum aestivum L.). The objective of this research was to identify phenolic compounds that negatively affect bran color in white wheat using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) and vanillin-HCl and NaOH staining methods. In mature bran, FT-ICR-MS detected derivatives of the flavonol quercetin in varieties Argent and RL4137 (red-kernelled wheat) but not in W98616, a white wheat variety with acceptable grain color. Derivatives of the isoflavone formononetin were more abundant in W98616 relative to RL4137 and Argent. Vanillin-HCl staining indicated that RL4137 sequestered high levels of proanthocyanidin (PA) throughout its entire seed coat, whereas white wheats sequestered PAs as discrete speckles. Argent possessed abundant speckles over its entire seed coat, whereas speckles were almost undetectable in W98616. In mature kernels, flavonoids throughout the seed coat of RL4137 reacted with NaOH, but only the speckles appeared to react in white wheats. W98616 consistently had lighter grain than Argent before and after NaOH treatment. Free and bound phenolic differences in bran samples confirmed that the darker seed coat color of Argent, relative to W98616, was likely due to higher total phenolic acid content. Although isoflavones accumulated in Argent and RL4137, it appears that the majority of the flux through the flavonoid pathway ultimately accumulates quercetin derivatives and PAs. In W98616, PAs accumulate, but it appears that flavonoid biosynthesis ultimately accumulates isoflavones. Argent, compared to W98616, generally accumulated higher levels of total phenolics (flavonols, stilbenes, and PAs) within its darker pigmented bran.

A number of COR genes (COld-Regulated genes) have been implicated in the acquisition of low temperature (LT) tolerance in wheat (Triticum aestivum L.). This study compared the relative expression patterns of selected COR genes in leaf and crown tissues of wheat near-isogenic lines to increase understanding of the molecular mechanisms underlying LT acclimation. Reciprocal near-isogenic lines were generated such that the dominant Vrn-A1 and recessive vrn-A1 loci were interchanged in a spring cv. Manitou and a winter cv. Norstar. Phenological development, acquisition of LT tolerance, and WCS120 polypeptide accumulation in these genotypes proceeded at rates similar to those previously reported for 6 degrees C acclimation from 0 to 98 d. However, a differential accumulation of WCS120 polypeptide and expression of the COR genes Wcs120, Wcor410, and Wcor14 was observed in the leaf and crown tissues. COR gene transcript levels peaked at 2 d of the acclimation period in both tissues and differences among genotypes were most evident at this time. COR gene expression was highest for the LT-tolerant and lowest for the tender genotypes. However, expression rates were divergent enough in genotypes with intermediate hardiness that comparisons among tissues and/or times during acclimation often resulted in variable interpretations of the relative expression of the COR genes in the determination of LT tolerance. These observations emphasize the need to pay close attention to experimental conditions, sampling times, and genotype and tissue selection in experiments designed to identify the critical genetic components that interact to determine LT acclimation.

Low-temperature (LT) tolerance is an important economic trait in winter wheat (Triticum aestivum L.) that determines the plants' ability to cope with below freezing temperatures. Essential elements of the LT tolerance mechanism are associated with the winter growth habit controlled by the vernalization loci (Vrn-1) on the group 5 chromosomes. To identify genomic regions, which in addition to vrn-1 determine the level of LT tolerance in hexaploid wheat, two doubled haploid (DH) mapping populations were produced using parents with winter growth habit (vrn-A1, vrn-B1, and vrn-D1) but showing different LT tolerance levels. A total of 107 DH lines were analyzed by genetic mapping to produce a consensus map of 2,873 cM. The LT tolerance levels for the Norstar (LT(50)=-20.7 degrees C) x Winter Manitou (LT(50)=-14.3 degrees C) mapping population ranged from -12.0 to -22.0 degrees C. Single marker analysis and interval mapping of phenotyped lines revealed a major quantitative trait locus (QTL) on chromosome 5A and a weaker QTL on chromosome 1D. The 5A QTL located 46 cM proximal to the vrn-A1 locus explained 40% of the LT tolerance variance. Two C-repeat Binding Factor (CBF) genes expressed during cold acclimation in Norstar were located at the peak of the 5A QTL.

D hordein, a prolamin storage protein of barley endosperms, is highly homologous to the high molecular weight (HWM) glutenin subunits, which are the major determinants of bread-making quality in wheat flour. In hexaploid wheat (AABBDD), each genome contains two paralogous copies of HMW-glutenin genes that encode the x- and y-type HMW-glutenin subunits. Previously, we reported the sequence analysis of a 102-kb genomic region that contains the HMW-glutenin locus of the D genome from Aegilops tauschii, the donor of the D genome of hexaploid wheat. Here, we present the sequence analysis of a 120-kb D-hordein region of the barley genome, a more distantly related member of the Triticeae grass tribe. Comparative sequence analysis revealed that gene content and order are generally conserved. Genes included in both of these orthologous regions are arranged in the following order: a Xa21-like receptor kinase, an endosperm globulin, an HMW prolamin, and a serine (threonine) protein kinase. However, in the wheat D genome, a region containing both the globulin and HMW-glutenin gene was duplicated, indicating that this duplication event occurred after the separation of the wheat and barley genomes. The intergenic regions are divergent with regard to the sequence and structural organization. It was found that different types of retroelements are responsible for the intergenic structure divergence in the wheat and barley genomes. In the barley region, we identified 16 long terminal repeat (LTR) retrotransposons in three distinct nested clusters. These retroelements account for 63% of the contig sequence. In addition, barley D hordein was compared with wheat HMW glutenins in terms of cysteine residue conservation and repeat domain organization.

We characterized three near-full-length putative homoeologous cDNA (Ss2a-1, Ss2a-2, and Ss2a-3) in wheat endosperm most similar to the maize zSSIIa. Polypeptide sequences deduced from three Ss2a cDNA clones share a 95% overall sequence similarity, and may thus have similar biochemical properties and may make identical contributions to starch biosynthesis in wheat endosperm. The accumulation of RNA transcripts corresponding to three Ss2a genes in developing endosperm varies among three cultivars studied, but usually peaks in young endosperm at about 10 days post anthesis (DPA). The polyclonal antibody for the SSIIa-1 recombinant protein strongly reacted to three previously identified granule-bound starch synthases of 100 to 115 kDa. The polyclonal antibody for the granule-bound starch synthases strongly reacted to the SSIIa-1 recombinant protein. Sequences of the N-terminal and an internal peptide of these three granule-bound starch synthases match well with those of three predicted mature SSIIa polypeptides. These granule-bound starch synthases may therefore be SSIIa polypeptides. The antibodies also recognized a group of three polypeptides with the same gel mobility as the three granule-bound starch synthases, a polypeptide of 90 kDa, and a group of three polypeptides of about 80 to 82 kDa. Thus, the wheat SSIIa may exist in several functional forms in the stroma of amyloplasts.

Two starch granule-bound proteins (SGP), SGP-140 and SGP-145, were preferentially associated with A-type starch granules (>10 microm) in developing and mature wheat (Triticum aestivum) kernels. Immunoblotting and N-terminal sequencing suggested that the two proteins were different variants of SBEIc, a 152-kD isoform of wheat starch-branching enzyme. Both SGP-140 and SGP-145 were localized to the endosperm starch granules but were not found in the endosperm soluble fraction or pericarp starch granules younger than 15 d post anthesis (DPA). Small-size starch granules (<10 microm) initiated before 15 DPA incorporated SGP-140 and SGP-145 throughout endosperm development and grew into full-size A-type starch granules (>10 microm). In contrast, small-size starch granules harvested after 15 DPA contained only low amounts of SGP-140 and SGP-145 and developed mainly into B-type starch granules (<10 microm). Polypeptides of similar mass and immunologically related to SGP-140 and/or SGP-145 were also preferentially incorporated into A-type starch granules of barley (Hordeum vulgare), rye (Secale cereale), and triticale (x Triticosecale Wittmack) endosperm, which like wheat endosperm have a bimodal starch granule size distribution.

Screening of a wheat (Triticum aestivum) cDNA library for starch-branching enzyme I (SBEI) genes combined with 5'-rapid amplification of cDNA ends resulted in isolation of a 4,563-bp composite cDNA, Sbe1c. Based on sequence alignment to characterized SBEI cDNA clones isolated from plants, the SBEIc predicted from the cDNA sequence was produced with a transit peptide directing the polypeptide into plastids. Furthermore, the predicted mature form of SBEIc was much larger (152 kD) than previously characterized plant SBEI (80-100 kD) and contained a partial duplication of SBEI sequences. The first SBEI domain showed high amino acid similarity to a 74-kD wheat SBEI-like protein that is inactive as a branching enzyme when expressed in Escherichia coli. The second SBEI domain on SBEIc was identical in sequence to a functional 87-kD SBEI produced in the wheat endosperm. Immunoblot analysis of proteins produced in developing wheat kernels demonstrated that the 152-kD SBEIc was, in contrast to the 87- to 88-kD SBEI, preferentially associated with the starch granules. Proteins similar in size and recognized by wheat SBEI antibodies were also present in Triticum monococcum, Triticum tauschii, and Triticum turgidum subsp. durum.

The genetic relationships among the five groups of hexaploid wheat: common, spelta, macha, vavilovii, and semi-wild wheat (SWW) are not clear. Random amplified polymorphic DNA (RAPD) analysis was used to assess phylogenetic relationships among these five morphological groups of hexaploid wheat. RAPD data were analyzed using the NTSYS-PC computer program to generate Jaccard genetic similarity coefficients. A dendrogram based on RAPD analysis grouped 15 accessions into five distinct clusters. These results are in agreement with those based on morphological classification, suggesting that common wheat is most closely related to SWW, followed by spelta, vavilovii, and macha.

A wheat gene, denoted Sbe1, encoding a type I starch-branching enzyme (SBEI) was isolated from a genomic library and shown to comprise 14 exons distributed over a 5.7 kb DNA region. Analyses of kernel RNA by 5' rapid amplification of cDNA ends (5'-RACE) and reverse transcription-polymerase chain reaction (RT-PCR) demonstrated a considerable sequence variation at the 5' ends of SBEI gene transcripts. DNA sequence alignments between the 5'-RACE products and the Sbe1 genomic DNA indicated that the first two exons and first intron were differentially processed to generate three classes of the mature transcript. One form of the SBEI gene transcript in 12-day old kernels contained the exon I+II+III combination at the 5' end, whereas other forms differed by inclusion of intron 1 or exclusion of exon II sequences. RT-PCR analysis of Sbe1-uidA::nptII chimeric mRNA produced in transgenic wheat cultured cells confirmed that the isolated Sbe1 was able to produce all three forms of SBEI gene transcripts by alternative splicing of the primary mRNA. The variants of processed Sbe1 mRNA were potentially translated into N-terminal variants of the SBEI precursor with different transit peptide sequences.

A PCR-based screening approach was used to isolate genomic clones from wheat encoding peroxidase isozymes. Three complete genes (pox1, pox2 and pox4) and one truncated gene (pox3) were characterized. The nucleotide sequences predicted mature proteins of 31 kDa, in which all the highly conserved motifs of secreted plant peroxidases were preserved. The coding regions showed 73-83% DNA sequence identity, with the highest level of similarity noted for the tandemly oriented pox2 and pox3. Expression of respective pox genes in various tissues of wheat was assessed by the RT-PCR technique, which showed that all four genes are active. The primary pox1 mRNA was spliced to remove three introns, whereas processing of the other pox transcripts involved only two intervening sequences. Splicing occurred at consensus GU/AG splice sites except for the first introns of pox1, pox2 and pox4 transcripts, where processing took place at unusual GC donor sites. The RNA analysis suggested that the pox1, pox2 and pox4 genes are predominantly expressed in roots. Lower levels of expression were found for pox4 and pox3 in leaves. Infection of wheat by the powdery mildew fungus selectively induced expression of pox2 in leaves.