The trafficking pathway of a wheat storage protein in transgenic rice endosperm

Background and Aims The trafficking of proteins in the endoplasmic reticulum (ER) of plant cells is a topic of considerable interest since this organelle serves as an entry point for proteins destined for other organelles, as well as for the ER itself. In the current work, transgenic rice was used to study the pattern and pathway of deposition of the wheat high molecular weight (HMW) glutenin sub-unit (GS) 1Dx5 within the rice endosperm using specific antibodies to determine whether it is deposited in the same or different protein bodies from the rice storage proteins, and whether it is located in the same or separate phases within these. Methods The protein distribution and the expression pattern of HMW sub-unit 1Dx5 in transgenic rice endosperm at different stages of development were determined using light and electron microscopy after labelling with antibodies. Key results The use of HMW-GS-specific antibodies showed that sub-unit 1Dx5 was expressed mainly in the sub-aleurone cells of the endosperm and that it was deposited in both types of protein body present in the rice endosperm: derived from the ER and containing prolamins, and derived from the vacuole and containing glutelins. In addition, new types of protein bodies were also formed within the endosperm cells. Conclusions The results suggest that the HMW 1Dx5 protein could be trafficked by either the ER or vacuolar pathway, possibly depending on the stage of development, and that its accumulation in the rice endosperm could compromise the structural integrity of protein bodies and their segregation into two distinct populations in the mature endosperm.

An eight-parent multiparent advanced generation inter-cross population for winter-sown wheat: creation, properties, and validation

MAGIC populations represent one of a new generation of crop genetic mapping resources combining high genetic recombination and diversity. We describe the creation and validation of an eight-parent MAGIC population consisting of 1091 F7 lines of winter-sown wheat (Triticum aestivum L.). Analyses based on genotypes from a 90,000-single nucleotide polymorphism (SNP) array find the population to be well-suited as a platform for fine-mapping quantitative trait loci (QTL) and gene isolation. Patterns of linkage disequilibrium (LD) show the population to be highly recombined; genetic marker diversity among the founders was 74% of that captured in a larger set of 64 wheat varieties, and 54% of SNPs segregating among the 64 lines also segregated among the eight founder lines. In contrast, a commonly used reference bi-parental population had only 54% of the diversity of the 64 varieties with 27% of SNPs segregating. We demonstrate the potential of this MAGIC resource by identifying a highly diagnostic marker for the morphological character "awn presence/absence" and independently validate it in an association-mapping panel. These analyses show this large, diverse, and highly recombined MAGIC population to be a powerful resource for the genetic dissection of target traits in wheat, and it is well-placed to efficiently exploit ongoing advances in phenomics and genomics. Genetic marker and trait data, together with instructions for access to seed, are available at http://www.niab.com/MAGIC/.

Antibiosis in wheat interacts with crowding stress to affect Metopolophiumdirhodum development and susceptibility to malathion

We used a laboratory study to compare the performance of rose-grain aphid, Metopolophium dirhodum(Walker)(Hemiptera:Aphididae),onthewheatcultivars‘Huntsman’(susceptible)and‘Rapier’ (partiallyresistant)inbothlowdensity(uncrowded)andhighdensity(crowded)coloniesandexamined the consequences for aphid susceptibility to malathion. Adult apterae that developed on Rapier wheat had their mean relative growth rate (MRGR) reduced by 6 and 9% under uncrowded and crowded conditions, respectively, whereas the crowding treatment reduced MRGR by 3%, but only in Rapier aphids. Rapier resistance also reduced adult dry weight by 13 and 14% under crowded and uncrowded conditions, respectively, whereas crowding reduced it by 34 and 35% in Rapier and Huntsman aphids, respectively. Development on Rapier substantially reduced the topical LC50 of malathion by 37.8 and 34.8% under crowded and uncrowded conditions, suggesting that plant antibiosis increased malathion susceptibility. By comparison, crowding only reduced the LC50 by 29.5 and 26.0% on Huntsman and Rapier. The LD50 data showed that reductions on aphid body size on Rapier and through crowding did not fully explain the differences in LC50. This was particularly in the values for crowded aphids that were actually 80% higher than for uncrowded ones. Thi sapparent tolerance of crowded aphids, however, may partly be due to loss of insecticide from small aphids at dosing. Evidence of synergy between plant resistance and insecticide susceptibility raisest he possibility of using reduced concentrations of pesticides to control aphids on resistant crop cultivars, with diminished impacts on non-target and beneficial species important in integrated pest management(IPM)program

The endophytic fungus Piriformospora indica protects wheat from Fusarium crown rot disease in simulated UK autumn conditions

The root endophytic fungus Piriformospora indica (Sebacinacea) forms mutualistic symbioses with a broad range of host plants, increasing their biomass production and resistance to fungal pathogens. We evaluated the effect of P. indica on Fusarium crown rot disease of wheat, under in vitro and glasshouse conditions. Interaction of P. indica and Fusarium isolates under axenic culture conditions indicated no direct antagonistic activity of P. indica against Fusarium isolates. Seedlings of wheat were inoculated with P. indica and pathogenic Fusarium culmorum or F. graminearum and grown in sterilised soil-free medium or in a non-sterilised mix of soil and sand. Fusarium alone reduced emergence and led to visible browning and reduced root growth. Roots of seedlings in pots inoculated with both Fusarium isolates and P. indica were free of visible symptoms; seed emergence and root biomass were equivalent to the uninoculated. DNA was quantified by real-time polymerase chain reaction (qPCR). The ratio of Fusarium DNA to wheat DNA rose rapidly in the plants inoculated with Fusarium alone; isolates and species were not significantly different. P. indica inoculation reduced the ratio of Fusarium to host DNA in the root systems. The reduction increased with time. The ratio of P. indica to wheat DNA initially rose but then declined in root systems without Fusarium. With Fusarium, the ratio rose throughout the experiment. The absolute amount of Fusarium DNA in root systems increased in the absence of P. indica but was static in plants co-inoculated with P. indica.

Endophytic bacterial community composition in wheat (Triticum aestivum) is determined by plant tissue type, developmental stage and soil nutrient availability

Aims: To understand effects of tissue type, growth stage and soil fertilisers on bacterial endophyte communities of winter wheat (Triticum aestivum cv. Hereward). Methods: Endophytes were isolated from wheat grown under six fertiliser conditions in the long term Broadbalk Experiment at Rothamsted Research, UK. Samples were taken in May and July from root and leaf tissues. Results: Root and leaf communities differed in abundance and composition of endophytes. Endophytes were most abundant in roots and the Proteobacteria were most prevalent. In contrast, Firmicutes and Actinobacteria, the Gram positive phyla, were most prevalent in the leaves. Both fertiliser treatment and sample time influenced abundance and relative proportions of each phylum and genus in the endosphere. A higher density of endophytes was found in the Nil input treatment plants. Conclusions: Robust isolation techniques and stringent controls are critical for accurate recovery of endophytes. The plant tissue type, plant growth stage, and soil fertiliser treatment all contribute to the composition of the endophytic bacterial community in wheat. These results should help facilitate targeted development of endophytes for beneficial applications in agriculture.

Effect of azole fungicide mixtures, alternations and dose on azole sensitivity in the wheat pathogen Zymoseptoria tritici

The evolution of fungicide resistance in the cereal pathogen Zymoseptoria tritici, is a serious threat to the sustainability and profitability of wheat production in Europe. Application of azole fungicides has been shown to affect fitness of Z. tritici variants differentially, so it has been hypothesised that combinations of azoles could slow the evolution of resistance. This work was initiated to assess the effects of dose, mixtures and alternations of two azoles on selection for isolates with reduced sensitivity and on disease control. Naturally infected field trials were carried out at six sites across Ireland and the sensitivity of Z. tritici isolates monitored pre- and post-treatment. The azoles epoxiconazole and metconazole were applied as solo products, in alternation with each other and as a pre-formulated mixture. Full and half label doses were tested. The two azoles were partially cross-resistant, with a common azole resistance principal component accounting for 75% of the variation between isolates. Selection for isolates with reduced azole sensitivity was correlated with disease control. Decreased doses were related to decreases in sensitivity but the effect was barely significant (P = 0.1) and control was reduced. Single applications of an active ingredient (a.i.) caused smaller decreases in sensitivity than double applications. Shifts in sensitivity to the a.i. applied to a plot were greater than to the a.i. not applied, and the decrease in sensitivity was greater to the a.i. applied at the second timing. These results confirm the need to mix a.i.s with different modes of action.

Yield and nitrogen accumulation and partitioning in winter wheat under elevated CO2: A 3-year free-air CO2 enrichment experiment

Fossil fuel combustion and deforestation have resulted in a rapid increase in atmospheric [CO2] since the 1950’s, and it will reach about 550 μmol mol−1 in 2050. Field experiments were conducted at the Free-air CO2 Enrichment facility in Beijing, China. Winter wheat was grown to maturity under elevated [CO2] (550 ± 17 μmol mol−1) and ambient [CO2] (415 ± 16 μmol mol−1), with high nitrogen (N) supply (HN, 170 kg N ha−1) and low nitrogen supply (LN, 100 kg N ha−1) for three growing seasons from 2007 to 2010. Elevated [CO2] increased wheat grain yield by 11.4% across the three years. [CO2]-induced yield enhancements were 10.8% and 11.9% under low N and high N supply, respectively. Nitrogen accumulation under elevated [CO2] was increased by 12.9% and 9.2% at the half-way anthesis and ripening stage across three years, respectively. Winter wheat had higher nitrogen demand under elevated [CO2] than ambient [CO2], and grain yield had a stronger correlation with plant N uptake after anthesis than before anthesis at high [CO2]. Our results suggest that regulating on the N application rate and time, is likely important for sustainable grain production under future CO2 climate.

Comparative in situ analyses of cell wall matrix polysaccharide dynamics in developing rice and wheat grain

Cell wall polysaccharides of wheat and rice endosperm are an important source of dietary fibre. Monoclonal antibodies specific to cell wall polysaccharides were used to determine polysaccharide dynamics during the development of both wheat and rice grain. Wheat and rice grain present near synchronous developmental processes and significantly different endosperm cell wall compositions, allowing the localisation of these polysaccharides to be related to developmental changes. Arabinoxylan (AX) and mixed-linkage glucan (MLG) have analogous cellular locations in both species, with deposition of AX and MLG coinciding with the start of grain filling. A glucuronoxylan (GUX) epitope was detected in rice, but not wheat endosperm cell walls. Callose has been reported to be associated with the formation of cell wall outgrowths during endosperm cellularisation and xyloglucan is here shown to be a component of these anticlinal extensions, occurring transiently in both species. Pectic homogalacturonan (HG) was abundant in cell walls of maternal tissues of wheat and rice grain, but only detected in endosperm cell walls of rice in an unesterified HG form. A rhamnogalacturonan-I (RG-I) backbone epitope was observed to be temporally regulated in both species, detected in endosperm cell walls from 12 DAA in rice and 20 DAA in wheat grain. Detection of the LM5 galactan epitope showed a clear distinction between wheat and rice, being detected at the earliest stages of development in rice endosperm cell walls, but not detected in wheat endosperm cell walls, only in maternal tissues. In contrast, the LM6 arabinan epitope was detected in both species around 8 DAA and was transient in wheat grain, but persisted in rice until maturity.

Influence of temperature on the composition and polymerization of gluten proteins during grain filling in spring wheat (Triticum aestivum L.)

One Norwegian and one UK spring wheat cultivar, Bjarne and Cadenza, respectively, were grown in climate chambers to investigate the effects of lower to moderate temperatures during grain filling on the gluten quality. Two experiments were carried out with weekly fertilization until anthesis, while post-anthesis fertilization was applied in a third experiment. The proportions of different gluten proteins were affected by temperature in a similar manner for both cultivars when grown without post-anthesis fertilization. However, whereas low temperature strongly decreased %UPP for Cadenza, Bjarne had high %UPP at all temperature regimes. The results indicated that the assembly of glutenin polymers in Bjarne was less sensitive to variation in temperature than in Cadenza. Thus, our results suggested that the temperature influenced the proportion of different gluten proteins in both cultivars, while its effects on the assembly of the glutenin polymers were cultivar dependent. The duration of grain filling was longer at the lower temperatures, and this was associated with increased grain weight. Temperature had little effect on the amount of protein accumulated per grain, thus the proportion of proteins was strongly decreased at lower temperatures. This was to some extent, but not fully counteracted by post-anthesis fertilization.

Comparative analysis of performance and stability among composite cross populations, variety mixtures and pure lines of winter wheat in organic and conventional cropping systems

This study investigated the effects of increased genetic diversity in winter wheat (Triticum aestivum L.), either from hybridization across genotypes or from physical mixing of lines, on grain yield, grain quality, and yield stability in different cropping environments. Sets of pure lines (no diversity), chosen for high yielding ability or high quality, were compared with line mixtures (intermediate level of diversity), and lines crossed with each other in composite cross populations (CCPn, high diversity). Additional populations containing male sterility genes (CCPms) to increase outcrossing rates were also tested. Grain yield, grain protein content, and protein yield were measured at four sites (two organically-managed and two conventionally-managed) over three years, using seed harvested locally in each preceding year. CCPn and mixtures out-yielded the mean of the parents by 2.4% and 3.6%, respectively. These yield differences were consistent across genetic backgrounds but partly inconsistent across cropping environments and years. Yield stability measured by environmental variance was higher in CCPn and CCPms than the mean of the parents. An index of yield reliability tended to be higher in CCPn, CCPms and mixtures than the mean of the parents. Lin and Binns’ superiority values of yield and protein yield were consistently and significantly lower (i.e. better) in the CCPs than in the mean of the parents, but not different between CCPs and mixtures. However, CCPs showed greater early ground cover and plant height than mixtures. When compared with the (locally non-predictable) best-yielding pure line, CCPs and mixtures exhibited lower mean yield and somewhat lower yield reliability but comparable superiority values. Thus, establishing CCPs from smaller sets of high-performing parent lines might optimize their yielding ability. On the whole, the results demonstrate that using increased within-crop genetic diversity can produce wheat crops with improved yield stability and good yield reliability across variable and unpredictable cropping environments.

The identification of QTL controlling ergot sclerotia size in hexaploid wheat implicates a role for the Rht dwarfing alleles

The fungal pathogen Claviceps purpurea infects ovaries of a broad range of temperate grasses and cereals, including hexaploid wheat, causing a disease commonly known as ergot. Sclerotia produced in place of seed carry a cocktail of harmful alkaloid compounds that result in a range of symptoms in humans and animals, causing ergotism. Following a field assessment of C. purpurea infection in winter wheat, two varieties ‘Robigus’ and ‘Solstice’ were selected which consistently produced the largest differential effect on ergot sclerotia weights. They were crossed to produce a doubled haploid mapping population, and a marker map, consisting of 714 genetic loci and a total length of 2895 cM was produced. Four ergot reducing QTL were identified using both sclerotia weight and size as phenotypic parameters; QCp.niab.2A and QCp.niab.4B being detected in the wheat variety ‘Robigus’, and QCp.niab.6A and QCp.niab.4D in the variety ‘Solstice’. The ergot resistance QTL QCp.niab.4B and QCp.niab.4D peaks mapped to the same markers as the known reduced height (Rht) loci on chromosomes 4B and 4D, Rht-B1 and Rht-D1, respectively. In both cases, the reduction in sclerotia weight and size was associated with the semi-dwarfing alleles, Rht-B1b from ‘Robigus’ and Rht-D1b from ‘Solstice’. Two-dimensional, two-QTL scans identified significant additive interactions between QTL QCp.niab.4B and QCp.niab.4D, and between QCp.niab.2A and QCp.niab.4B when looking at sclerotia size, but not between QCp.niab.2A and QCp.niab.4D. The two plant height QTL, QPh.niab.4B and QPh.niab.4D, which mapped to the same locations as QCp.niab.4B and QCp.niab.4D, also displayed significant genetic interactions.

Effects of aphid infestation on Cd and Zn concentration in wheat

The effect of infestation by the aphid Metopolophium dirhodum on the concentration and mass partitioning of Cd and Zn was studied in wheat plants. Results show that infestation did not affect the concentration of either metal in the roots or shoots of wheat, but elevated concentrations in the ears. This appeared to result from the concentration of metal in the smaller ear mass of infested plants. Infestation did not significantly affect the mass partitioning of either metal in any part of the plant, some 10% of both metals being allocated to the roots. However, the two metals contrasted in their partitioning in the aerial parts of the plant, with ca. 60% of Cd mass partitioned in the shoots and ca. 67% of Zn mass partitioned in the ears. The possible effects of infestation on the transfer of Cd and Zn from the soil to cereal aphids are discussed.

Extractability and characteristics of proteins deriving from wheat DDGS

Wheat Distillers’ Dried Grains with Solubles (DDGS) and in-process samples were used for protein extraction. Prolamins were the predominant protein components in the samples. The absence of extractable α- and γ-gliadins in DDGS indicated protein aggregation during the drum drying processing stage. Prolamin extraction was performed using 70% (v/v) ethanol or alkaline-ethanol solution in the presence of reducing agent. DDGS extracts had relatively low protein contents (14-44.9%, w/w), regardless of the condition applied. The wet solids were the most suitable raw material for protein extraction, with recovery yields of ~ 55% (w/w) and protein content of ~58% (w/w) in 70% (v/v) ethanol. Protein extracts from wet solids were significantly rich in glutamic acid and proline. Mass balance calculations demonstrated the high carbohydrate content (~ 50%, w/w) of solid residues. Overall, the feasibility of utilising in-process samples of DDGS for protein extraction with commercial potential was demonstrated.

Valorisation of side streams from wheat milling and confectionery industries for consolidated production and extraction of microbial lipids

Crude enzymes produced via solid state fermentation (SSF) using wheat milling by-products have been employed for both fermentation media production using flour-rich waste (FRW) streams and lysis of Rhodosporidium toruloides yeast cells. Filter sterilization of crude hydrolysates was more beneficial than heat sterilization regarding yeast growth and microbial oil production. The initial carbon to free amino nitrogen ratio of crude hydrolysates was optimized (80.2 g/g) in fed-batch cultures of R. toruloides leading to a total dry weight of 61.2 g/L with microbial oil content of 61.8 % (w/w). Employing a feeding strategy where the glucose concentration was maintained in the range of 12.2 – 17.6 g/L led to the highest productivity (0.32 g/L∙h). The crude enzymes produced by SSF were utilised for yeast cell treatment leading to simultaneous release of around 80% of total lipids in the broth and production of a hydrolysate suitable as yeast extract replacement.

Distribution of lipids in the grain of wheat (cv Hereward) determined by lipidomic analysis of milling and pearling fractions

Lipidomic analyses of milling and pearling fractions from wheat grain were carried out to determine differences in composition which could relate to the spatial distribution of lipids in the grain. Free fatty acids and triacylglycerols were major components in all fractions, but the relative contents of polar lipids varied, particularly lysophosphatidyl choline and digalactosyldiglyceride, which were enriched in flour fractions. By contrast, minor phospholipids were enriched in bran and offal fractions. The most abundant fatty acids in the analysed acyl lipids were C16:0 and C18:2 and their combinations, including C36:4 and C34:2. Phospholipids and galactolipids have been reported to have beneficial properties for bread making, while free fatty acids and triacylglycerols are considered detrimental. The subtle differences in the compositions of fractions determined in the present study could therefore underpin the production of flour fractions with optimised compositions for different end uses.