Yongfang Wan

Transcriptome analysis of grain development in hexaploid wheat

BackgroundHexaploid wheat is one of the most important cereal crops for human nutrition. Molecular understanding of the biology of the developing grain will assist the improvement of yield and quality traits for different environments. High quality transcriptomics is a powerful method to increase this understanding.ResultsThe transcriptome of developing caryopses from hexaploid wheat (Triticum aestivum, cv. Hereward) was determined using Affymetrix wheat GeneChip® oligonucleotide arrays which have probes for 55,052 transcripts. Of these, 14,550 showed significant differential regulation in the period between 6 and 42 days after anthesis (daa). Large changes in transcript abundance were observed which were categorised into distinct phases of differentiation (6–10 daa), grain fill (12–21 daa) and desiccation/maturation (28–42 daa) and were associated with specific tissues and processes. A similar experiment on developing caryopses grown with dry and/or hot environmental treatments was also analysed, using the profiles established in the first experiment to show that most environmental treatment effects on transcription were due to acceleration of development, but that a few transcripts were specifically affected. Transcript abundance profiles in both experiments for nine selected known and putative wheat transcription factors were independently confirmed by real time RT-PCR. These expression profiles confirm or extend our knowledge of the roles of the known transcription factors and suggest roles for the unknown ones.ConclusionThis transcriptome data will provide a valuable resource for molecular studies on wheat grain. It has been demonstrated how it can be used to distinguish general developmental shifts from specific effects of treatments on gene expression and to diagnose the probable tissue specificity and role of transcription factors.

Isolation and characterization of five novel high molecular weight subunit of glutenin genes from Triticum timopheevi and Aegilops cylindrica.

Abstract.Analysis by SDS-PAGE of total protein fractions from single seeds of Aegilops cylindrica (genomes C and D) and Triticum timopheevi (genomes A and G) showed the presence of three bands corresponding to high molecular weight subunits of glutenin (HMW subunits) in the former and two major bands and a minor band corresponding to HMW subunits in the latter. Three Ae. cylindrica and two T. timopheevi HMW subunit gene sequences, each comprising the entire coding region, were amplified by polymerase chain reaction (PCR) and their complete nucleotide sequences determined. A combination of N-terminal amino acid sequencing of the proteins identified by SDS-PAGE and alignments of the derived amino acid sequences of the proteins encoded by the PCR products identified the Ae. cylindrica HMW subunits as 1Cx, 1Cy and 1Dy, and the T. timopheevi HMW subunits as 1Gx, 1Ax and 1Ay. It was not clear whether or not a 1Gy HMW subunit was present in T. timopheevi. The PCR products from Ae. cyclindrica were derived from 1Cy and 1Dy genes and a silent 1Dx gene containing an in-frame internal stop codon, while those from T. timopheevi were derived from 1Ax and 1Ay genes. The 1Cx, 1Gx and 1Gy sequences were not amplified successfully. The proteins encoded by the five novel genes had similar structures to previously characterized HMW subunits of bread wheat (Triticum aestivum). Differences and similarities in sequence and structure, and in the distribution of cysteine residues (relevant to the ability of HMW subunits to form high Mr polymers) distinguished the HMW subunits of x- and y-type and of each genome rather than those of the different species. There was no evidence of a change in HMW subunit expression or structure resulting from selective breeding of bread wheat. The novel 1Ax, 1Ay, 1Cy and 1Dy HMW subunits were expressed in Escherichia coli, and the expressed proteins were shown to have very similar mobilities to the endogenous HMW subunits on SDS-PAGE. The truncated 1Dx gene from Ae. cylindrica failed to express in E. coli, and no HMW subunit-related protein of the size predicted for the truncated 1Dx subunit could be identified by immunodetection in seed extracts.

Sequence and properties of HMW subunit 1Bx20 from pasta wheat (Triticum durum) which is associated with poor end use properties.

Abstract.The gene encoding high-molecular-weight (HMW) subunit 1Bx20 was isolated from durum wheat cv. Lira. It encodes a mature protein of 774 amino acid residues with an Mr of 83,913. Comparison with the sequence of subunit 1Bx7 showed over 96% identity, the main difference being the substitution of two cysteine residues in the N-terminal domain of subunit 1Bx7 with tyrosine residues in 1Bx20. Comparison of the structures and stabilities of the two subunits purified from wheat using Fourier-transform infra-red and circular dichroism spectroscopy showed no significant differences. However, incorporation of subunit 1Bx7 into a base flour gave increased dough strength and stability measured by Mixograph analysis, while incorporation of subunit 1Bx20 resulted in small positive or negative effects on the parameters measured. It is concluded that the different effects of the two subunits could relate to the differences in their cysteine contents, thereby affecting the cross-linking and hence properties of the glutenin polymers.

Analysis of HMW glutenin subunits and their coding sequences in two diploid Aegilops species

Abstract. Considerable progress has been made in understanding the structure, function and genetic regulation of high-molecular-weight (HMW) glutenin subunits in hexaploid wheat. In contrast, less is known about these types of proteins in wheat related species. In this paper, we report the analysis of HMW glutenin subunits and their coding sequences in two diploid Aegilops species, Aegilops umbellulata (UU) and Aegilops caudata (CC). SDS-PAGE analysis demonstrated that, for each of the four Ae. umbellulata accessions, there were two HMW glutenin subunits (designated here as 1Ux and 1Uy) with electrophoretic mobilities comparable to those of the x- and y-type subunits encoded by the Glu-D1 locus, respectively. In our previous study involving multiple accessions of Ae. caudata, two HMW glutenin subunits (designated as 1Cx and 1Cy) with electrophoretic mobilities similar to those of the subunits controlled by the Glu-D1 locus were also detected. These results indicate that the U genome of Ae. umbellulata and the C genome of Ae. caudata encode HMW glutenin subunits that may be structurally similar to those specified by the D genome. The complete open reading frames (ORFs) coding for x- and y-type HMW glutenin subunits in the two diploid species were cloned and sequenced. Analysis of deduced amino acid sequences revealed that the primary structures of the x- and y-type HMW glutenin subunits of the two Aegilops species were similar to those of previously published HMW glutenin subunits. Bacterial expression of modified ORFs, in which the coding sequence for the signal peptide was removed, gave rise to proteins with electrophoretic mobilities identical to those of HMW glutenin subunits extracted from seeds, indicating that upon seed maturation the signal peptide is removed from the HMW glutenin subunit in the two species. Phylogenetic analysis showed that 1Ux and 1Cx subunits were most closely related to the 1Dx type subunit encoded by the Glu-D1 locus. The 1Uy subunit possessed a higher level of homology to the 1Dy-type subunit compared with the 1Cy subunit. In conclusion, our study suggests that the Glu-U1 locus of Ae. umbellulata and the Glu-C1 locus of Ae. caudata specify the expression of HMW glutenin subunits in a manner similar to the Glu-D1 locus. Consequently, HMW glutenin subunits from the two diploid species may have potential value in improving the processing properties of hexaploid wheat varieties.

High-molecular-weight glutenin subunits in the Cylindropyrum and Vertebrata section of the Aegilops genus and identification of subunits related to those encoded by the Dx alleles of common wheat

Abstract The high-molecular-weight (HMW) glute-nin subunit composition of seven species from the Cylindropyrum and Vertebrata sections of the Aegilops genus was studied using SDS-PAGE and Western blot analysis. Two subunits were detected in Ae. caudata and three in Ae. cylindrica. In both species, subunits showing electrophoretic mobility similar to that of 1Dx2 were present. Western blot analysis using a monoclonal antibody (IFRN 1602) specific for the 1Ax and 1Dx subunits of bread wheat showed that the 1Dx-like subunit of Ae. caudata gave only a weak reaction. This indicates that Ae. caudata expresses subunits which are more distantly related to the 1Dx subunits. Two subunits were detected in each of the 60 accessions of Ae. tauschii, including several 1Dtx subunits showing different electrophoretic mobilities from those of the 1Dx subunits commonly found in bread wheat. All of the 1Dtx subunits reacted strongly with IFRN 1602, confirming their close relationship to the 1Dx subunits of bread wheat. Three subunits were found in Ae. crassa (6 x), four in Ae. ventricosa and Ae. juvenalis and five in Ae. vavilovii. In these four species, the subunits that showed electrophoretic mobility similar, or close, to that of 1Dx2 all reacted with IFRN 1602. In addition, Ae. ventricosa contained a subunit showing electrophoretic mobility slower than that of 1Dx2.2, which also reacted with IFRN 1602. These results suggest that the D-genome component in the multiploid Aegilops species express at least one HMW glutenin subunit that is structurally related to the 1Dx subunits of bread wheat.

Comparative analysis of the D genome-encoded high-molecular weight subunits of glutenin

Four genes encoding novel 1Dx-type high-molecular weight (HMW) subunits were amplified by polymerase chain reaction, two each from Aegilops tauschii and bread wheat Triticum aestivum. The two subunits from Ae. tauschii (1Dx2.1t and 1Dx2t) were both very similar in sequence to subunit 1Dx2 from bread wheat. In contrast, the two novel bread wheat subunits (1Dx2.2 and 1Dx2.2*) differed from subunit 1Dx2 in having different internally duplicated regions (of 132 and 186 amino acid, respectively) within their repetitive domains. These duplicated sequences were located adjacent to the regions from which they had been duplicated and had complete intact repeat motifs at each end. The implications of these results for HMW subunit evolution and wheat quality improvement are discussed.

A novel family of γ-gliadin genes are highly regulated by nitrogen supply in developing wheat grain

Six wheat cultivars were grown at Rothamsted (UK) with three levels of nitrogen fertilizer (100, 200 and 350kg N/ha) in 2009 and 2010. Gene expression in developing caryopses at 21 days post-anthesis (DPA) was profiled using the Affymetrix Wheat GeneChip®. Four of 105 transcripts which were significantly upregulated by nitrogen level were annotated as γ-3 hordein and the identification of corresponding expressed sequence tags showed that they differed in sequence from previously described (typical) γ-gliadins and represented a novel form of γ-gliadin. Real-time reverse transcriptase PCR at 14, 21, 28 and 35 DPA revealed that this transcript was most abundant and most responsive to nitrogen at 21 DPA. Four novel γ-gliadin genes were isolated by PCR amplification from wheat cv. Hereward and the related species Aegilops tauschii and Triticum monococcum while three were assembled from the genomic sequence database of wheat cv. Chinese Spring (www.cerealsdb.uk.net). Comparison of the deduced amino acid sequences of the seven genes showed that they shared only 44.4–46.0% identity with the sequence of a typical γ-gliadin (accession number EF15018), but 61.8–68.3% identity with the sequence of γ-3 hordein from the wild barley species Hordeum chilense (AY338065). The novel γ-gliadin genes were localized to the group 1 chromosomes (1A, 1B, 1D).

Soil microbial respiration responses to repeated urea applications in three grasslands

Grazing animals excrete urine and create transitorily high pH, nitrogen (N)-replete soil patches. Beneath grazed pasture, we postulated the soil microbial community would be highly responsive to N application. Lesser responses were expected of soils beneath grassland without grazing animals. Soil samples were collected near Lincoln, New Zealand (43.6°S, 172.5°E), beneath pasture regularly grazed by dairy cattle, an adjacent pasture set aside from grazing 20 years ago, and a nearby grassland that has never been grazed. Soil microbial respiration responses to repeated urea (500 kg N/ha) applications were determined by laboratory incubation experiments and the soil pH and water-soluble C content were also measured. The first application induced 0.13 ± 0.04 (dairy farm), 0.15 ± 0.05 (set aside), and 0.20 ± 0.04 (ungrazed) g C/kg increases in microbial respiration over 9 days, excluding carbon dioxide production from carbonate hydrolysis. After a second application, 9-day respiration increased by 0.26 ± 0.04 (dairy farm), 0.41 ± 0.04 (set aside) and 0.20 ± 0.07 (ungrazed) g C/kg. For the dairy farm and ungrazed soils, the microbial communities responded differently to repeated urea addition. The responses included transitory changes in pH and reflected the limited amounts of readily decomposable organic matter.

A novel transcriptomic approach to identify candidate genes for grain quality traits in wheat.

A novel methodology is described in which transcriptomics is combined with the measurement of bread-making quality and other agronomic traits for wheat genotypes grown in different environments (wet and cool or hot and dry conditions) to identify transcripts associated with these traits. Seven doubled haploid lines from the Spark x Rialto mapping population were selected to be matched for development and known alleles affecting quality. These were grown in polytunnels with different environments applied 14 days post-anthesis, and the whole experiment was repeated over 2 years. Transcriptomics using the wheat Affymetrix chip was carried out on whole caryopsis samples at two stages during grain filling. Transcript abundance was correlated with the traits for approximately 400 transcripts. About 30 of these were selected as being of most interest, and markers were derived from them and mapped using the population. Expression was identified as being under cis control for 11 of these and under trans control for 18. These transcripts are candidates for involvement in the biological processes which underlie genotypic variation in these traits.

Effects of nitrogen nutrition on the synthesis and deposition of the ω-gliadins of wheat

Background and Aims The ω-gliadin storage proteins of wheat are of interest in relation to their impact on grain processing properties and their role in food allergy, particularly the ω-5 sub-group and wheat-dependent exercise-induced anaphylaxis. The ω-gliadins are also known to be responsive to nitrogen application. This study therefore compares the effects of cultivar and nitrogen availability on the synthesis and deposition of ω-gliadins in wheat grown under field conditions in the UK, including temporal and spatial analyses at the protein and transcript levels. Methods SDS–PAGE, western blotting and N-terminal amino acid sequencing were used to compare the patterns of ω-gliadin components in mature grain of six British wheat (Triticum aestivum) cultivars and their accumulation during the development of grain grown in field plots with varying nitrogen supply. Changes in gene expression during development were determined using real-time reverse transcription–PCR (RT–PCR). Spatial patterns of gene expression and protein accumulation were determined by in situ hybridization and immunofluorescence microscopy, respectively. Key Results Two patterns of ω-gliadins were identified in the six cultivars, including both monomeric ‘gliadin’ proteins and subunits present in polymeric ‘glutenin’ fractions. Increasing the level of nitrogen fertilizer in field plots resulted in increased expression of ω-gliadin transcripts and increased proportions of ω-5 gliadins. Nitrogen supply also affected the spatial patterns of ω-gliadin synthesis and deposition, which were differentially increased in the outer layers of the starchy endosperm with high levels of nitrogen. Conclusions Wheat ω-gliadins vary in amount and composition between cultivars, and in their response to nitrogen supply. Their spatial distribution is also affected by nitrogen supply, being most highly concentrated in the sub-aleurone cells of the starchy endosperm under higher nitrogen availability.