Jingjuan Zhang

GA-20 oxidase as a candidate for the semidwarf gene sdw1/denso in barley.

The barley sdw1/denso gene not only controls plant height but also yield and quality. The sdw1/denso gene was mapped to the long arm of chromosome 3H. Comparative genomic analysis revealed that the sdw1/denso gene was located in the syntenic region of the rice semidwarf gene sd1 on chromosome 1. The sd1 gene encodes a gibberellic acid (GA)-20 oxidase enzyme. The gene ortholog of rice sd1 was isolated from barley using polymerase chain reaction. The barley and rice genes showed a similar gene structure consisting of three exons and two introns. Both genes share 88.3% genomic sequence similarity and 89% amino acid sequence identity. A single nucleotide polymorphism was identified in intron 2 between barley varieties Baudin and AC Metcalfe with Baudin known to contain the denso semidwarf gene. The single nucleotide polymorphism (SNP) marker was mapped to chromosome 3H in a doubled haploid population of Baudin × AC Metcalfe with 178 DH lines. Quantitative trait locus analysis revealed that plant height cosegregated with the SNP. The sdw1/denso gene in barley is the most likely ortholog of the sd1 in rice. The result will facilitate understanding of the molecular mechanism controlling semidwarf phenotype and provide a diagnostic marker for selection of semidwarf gene in barley.

Water deficits in wheat: fructan exohydrolase (1‐FEH) mRNA expression and relationship to soluble carbohydrate concentrations in two varieties

Terminal drought is a risk for wheat production in many parts of the world. Robust physiological traits for resilience would enhance the preselection of breeding lines in drought-prone areas. Three pot experiments were undertaken to characterize stem water-solublecarbohydrate (WSC), fructan exohydrolase expression, grain filling and leaf gas exchange in wheat (Triticum aestivum) varieties, Kauz and Westonia, which are considered to be drought-tolerant.Water deficit accelerated the remobilization of stem WSC in Westonia but not in Kauz. The profile of WSC accumulation and loss was negatively correlated with them RNA concentration of 1-FEH, especially 1-FEH w3 (1-FEH-6B). Under water deficit, Westonia showed lower concentrations of WSC than Kauz but did not show a corresponding drop in grain yield. The results from pot experiments suggest that stem WSC concentration is not, on its own, a reliable criterion to identify potential grain yield in wheat exposed to water deficits during grain filling. The expression of 1-FEH w3 may provide a better indicator when linked to osmotic potential and green leaf retention, and this requires validation in field-grown plants.

A wheat 1‐FEH w3 variant underlies enzyme activity for stem WSC remobilization to grain under drought

In wheat stems, the levels of fructan-dominated water-soluble carbohydrates (WSC) do not always correlate well with grain yield. Field drought experiments were carried out to further explain this lack of correlation. Wheat (Triticum aestivum) varieties, Westonia, Kauz and c. 20 genetically diverse double haploid (DH) lines derived from them were investigated. Substantial genotypic differences in fructan remobilization were found and the 1-FEH w3 gene was shown to be the major contributor in the stem fructan remobilization process based on enzyme activity and gene expression results. A single nucleotide polymorphism (SNP) was detected in an auxin response element in the 1-FEH w3 promoter region, therefore we speculated that the mutated Westonia allele might affect gene expression and enzyme activity levels. A cleaved amplified polymorphic (CAP) marker was generated from the SNP. The harvested results showed that the mutated Westonia 1-FEH w3 allele was associated with a higher thousand grain weight (TGW) under drought conditions in 2011 and 2012. These results indicated that higher gene expression of 1-FEH w3 and 1-FEH w3 mediated enzyme activities that favoured stem WSC remobilization to the grains. The CAP marker residing in the 1-FEH w3 promoter region may facilitate wheat breeding by selecting lines with high stem fructan remobilization capacity under terminal drought.

The genome structure of the 1-FEH genes in wheat (Triticum aestivum L.): new markers to track stem carbohydrates and grain filling QTLs in breeding

Terminal drought tolerance of wheat is a major target in many areas in the world and is a particular focus in Western Australia. It is widely considered to relate to water soluble carbohydrate (WSC) levels such as fructan in the stem, as the head is maturing. Fructan exohydrolases are key enzymes during both fructan biosynthesis and mobilization. The wheat genome sequences of three fructan 1-exohydrolase (1-FEH) genes with seven exons and six introns were isolated by using the available 1-FEH w2 cDNA sequence. The major size differences among the three genes were located in intron 1 and intron 4. The three 1-FEH genes were mapped to Chinese Spring chromosome 6A, 6B and 6D based on polymerase chain reaction (PCR) polymorphisms and Southern hybridization. 1-FEH-6A, -6B and -6D corresponded to published cDNA sequences 1-FEH w1, w3 and w2, respectively. The overall correlation of the mRNA accumulation profile for the 1-FEH genes in stem and sheath leaf tissue in relation to the profile of soluble carbohydrate accumulation was consistent with their postulated role in stem soluble carbohydrate accumulation. The accumulation of the 1-FEH-6B (1-FEH w3) mRNA was 300 fold greater than that of 1-FEH-6A and -6D. The mRNA accumulation continued after the stem water soluble carbohydrate concentrations reached a peak, consistent with a role of 1-FEH-6B in the breakdown of soluble carbohydrate. The relationship between the 1-FEH genes and soluble carbohydrate accumulation is discussed and the 1-FEH-6B gene in particular is suggested to provide a new class of molecular marker for this trait.

Wheat genotypic variation in dynamic fluxes of WSC components in different stem segments under drought during grain filling.

In wheat, stem water soluble carbohydrates (WSC), composed mainly of fructans, are the major carbon sources for grain filling during periods of decreasing photosynthesis or under drought stress after anthesis. Here, in a field drought experiment, WSC levels and associated enzyme activities were followed in different stem segments (peduncle, penultimate internode, lower parts of stem, and sheath) during grain filling. The focus was on two double haploid (DH) lines, DH 307 and DH 338, derived from a Westonia/Kauz cross, two drought-tolerant wheat varieties that follow different drought adaptation strategies during grain filling. The results showed that in irrigated plants, in the period between 20 and 30 days after anthesis (DAA), 70–80% of WSC were fructans. Before and after this period, the fructan proportion varied from 10 to 60%, depending on the location along the stem. Under drought, the fructan proportion changed, depending on genotype, and developmental stages. After anthesis, stem fructans accumulation occurred mainly in the peduncle and penultimate internode until 14 DAA in both DH lines, with clear genotypic variation in subsequent fructan degradation under drought. In DH 307 a significant reduction of fructans with a concomitant increase in fructose levels occurred earlier in the lower parts of the stem and the sheath, as compared to DH 338 or other stem segments in both lines. This was associated with an earlier increase of grain weight and thousand grain weight in DH 307. Spatiotemporal analysis of fructan dynamics and enzymatic activities in fructan metabolism revealed that several types of FEHs are involved in fructan remobilization to the grain under drought.

Impact of mid-season sulphur deficiency on wheat nitrogen metabolism and biosynthesis of grain protein

Wheat (Triticum aestivum) quality is mainly determined by grain storage protein compositions. Sulphur availability is essential for the biosynthesis of the main wheat storage proteins. In this study, the impact of different sulphur fertilizer regimes on a range of agronomically important traits and associated gene networks was studied. High-performance liquid chromatography was used to analyse the protein compositions of grains grown under four different sulphur treatments. Results revealed that sulphur supplementation had a significant effect on grain yield, harvest index, and storage protein compositions. Consequently, two comparative sulphur fertilizer treatments (0 and 30 kg ha−1 sulphur, with 50 kg ha−1 nitrogen) at seven days post-anthesis were selected for a transcriptomics analysis to screen for differentially expressed genes (DEGs) involved in the regulation of sulphur metabolic pathways. The International Wheat Genome Sequencing Consortium chromosome survey sequence was used as reference. Higher sulphur supply led to one up-regulated DEG and sixty-three down-regulated DEGs. Gene ontology enrichment showed that four down-regulated DEGs were significantly enriched in nitrogen metabolic pathway related annotation, three of which were annotated as glutamine synthetase. The Kyoto Encyclopedia of Genes and Genomes pathway enrichment identified three significantly enriched pathways involved in nitrogen and amino acid metabolism.

Genetic characterization of cysteine-rich type-b avenin-like protein coding genes in common wheat

The wheat avenin-like proteins (ALP) are considered atypical gluten constituents and have shown positive effects on dough properties revealed using a transgenic approach. However, to date the genetic architecture of ALP genes is unclear, making it impossible to be utilized in wheat breeding. In the current study, three genes of type-b ALPs were identified and mapped to chromosomes 7AS, 4AL and 7DS. The coding gene sequence of both TaALP-7A and TaALP-7D was 855 bp long, encoding two identical homologous 284 amino acid long proteins. TaALP-4A was 858 bp long, encoding a 285 amino acid protein variant. Three alleles were identified for TaALP-7A and four for TaALP-4A. TaALP-7A alleles were of two types: type-1, which includes TaALP-7A1 andTaALP-7A2, encodes mature proteins, while type-2, represented byTaALP-7A3, contains a stop codon in the coding region and thus does not encode a mature protein. Dough quality testing of 102 wheat cultivars established a highly significant association of the type-1 TaALP-7A allele with better wheat processing quality. This allelic effects were confirmed among a range of commercial wheat cultivars. Our research makes the ALP be the first of such genetic variation source that can be readily utilized in wheat breeding.

Association Analysis of Grain-setting Rates in Apical and Basal Spikelets in Bread Wheat (Triticum aestivum L.)

The rates of grain-setting in apical and basal spikelets in wheat directly affect the kernel number per spike (KNPS). In this study, 220 wheat lines from 18 Chinese provinces and five foreign countries were used as a natural population. Phenotypic analysis showed differences in grain-setting rates between apical and basal spikelets. The broad-sense heritability of grain-setting rate in apical spikelets (18.7–21.0%) was higher than that for basal spikelets (9.4–16.4%). Significant correlations were found between KNPS and grain numbers in apical (R2 = 0.40–0.45, P < 0.01) and basal (R2 = 0.41–0.56, P < 0.01) spikelets. Seventy two of 106 SSR markers were associated with grain setting, 32 for apical spikelets, and 34 for basal spikelets. The SSR loci were located on 17 chromosomes, except 3A, 3D, 4A, and 7D, and explained 3.7–22.9% of the phenotypic variance. Four markers, Xcfa2153-1A202, Xgwm186-5A118, Xgwm156-3B319, and Xgwm537-7B210, showed the largest effects on grain numbers in apical and basal spikelets. High grain numbers in apical and basal spikelets were associated with elite alleles. Ningmai 9, Ning 0569, and Yangmai 18 with high grain-setting rates carried larger numbers of favorable alleles. Comparison of grain numbers in basal and apical spikelets of 35 Yangmai and Ningmai lines indicated that the Ningmai lines had better grain-setting rates (mean 21.4) than the Yangmai lines (16.5).

Contributions of Root WSC during Grain Filling in Wheat under Drought

As the first organ in plants to sense water-deficit in the soil, roots have important roles for improving crop adaption to water limited environments. Stem water soluble carbohydrates (WSC) are a major carbon source for grain filling under drought conditions. The contributions of root WSC during grain filling under drought has not been revealed. Wheat parental lines of Westonia, Kauz and their derived four double haploid (DH) lines, namely, DH 125, DH 139, DH 307, and DH 338 were used in a field drought experiment with four replications. Through measurements of the root and stem WSC components, and the associated enzyme activities during grain filling, we identified that the levels of root WSC and fructan were one third of the levels in stems. In particular, root glucose and 6-kestose levels were one third of the stem, while the root fructose and bifurcose level were almost half of the stem and sucrose level was two third of the stem. The accumulation and the degradation patterns of root fructan levels were similar to that in the stem, especially under drought. Correlations between root fructan levels and grain assimilation were highly significant, indicating that under terminal drought, root WSC represents a redistributed carbon source for grain filling rather than deep rooting. The significantly higher root sucrose levels under drought suggest that sucrose may act as a signal under drought stress. As compared with stem fructose levels, the earlier increased root fructose levels in DH 307, DH 139, and DH 338 provided agile response to drought stress. Our root results further confirmed that β-(2–6) linkages predominate in wheat with patterns of 6-kestose being closely correlated with overall fructan patterns. Further research will focus on the roles of 6-FEH during fructan remobilization in stems.

Wheat avenin-like protein and its significant Fusarium Head Blight resistant functions

Wheat Avenin-like proteins (TaALP) are atypical storage proteins belonging to the Prolamin superfamily. Previous studies on ALPs have focused on the proteins’ positive effects on dough strength, whilst no correlation has been made between TaALPs and the plant immune system. Here, we performed genome-wide characterization of ALP encoding genes in bread wheat. In silico analyses indicated the presence of critical peptides in TaALPs that are active in the plant immune system. Pathogenesis-related nucleotide motifs were also identified in the putative promoter regions of TaALP encoding genes. RT-PCR was performed on TaALP and previously characterised pathogenesis resistance genes in developing wheat caryopses under control and Fusarium graminearum infection conditions. The results showed that TaALP and NMT genes were upregulated upon F. graminearum inoculation. mRNA insitu hybridization showed that TaALP genes were expressed in the embryo, aleurone and sub-aleurone layer cells. Seven TaALP genes were cloned for the expression of recombinant proteins in Escherichia coli, which displayed significant inhibitory function on F. graminearum under anti-fungal tests. In addition, FHB index association analyses showed that allelic variations of two ALP genes on chromosome 7A were significantly correlated with FHB symptoms. Over-expression of an ALP gene on chromosome 7A showed an enhanced resistance to FHB. Yeast two Hybridization results revealed that ALPs have potential proteases inhibiting effect on metacaspases and beta-glucosidases. A vital infection process related pathogen protein, F. graminearum Beta-glucosidase was found to interact with ALPs. Our study is the first to report a class of wheat storage protein or gluten protein with biochemical functions. Due to its abundance in the grain and the important multi-functions, the results obtained in the current study are expected to have a significant impact on wheat research and industry.