Development and Characterization of a New TILLING Population of Common Bread Wheat (Triticum aestivum L.)

Mutagenesis is an important tool in crop improvement. However, the hexaploid genome of wheat (Triticum aestivum L.) presents problems in identifying desirable genetic changes based on phenotypic screening due to gene redundancy. TILLING (Targeting Induced Local Lesions IN Genomes), a powerful reverse genetic strategy that allows the detection of induced point mutations in individuals of the mutagenized populations, can address the major challenge of linking sequence information to the biological function of genes and can also identify novel variation for crop breeding. Wheat is especially well-suited for TILLING due to the high mutation densities tolerated by polyploids. However, only a few wheat TILLING populations are currently available in the world, which is far from satisfying the requirement of researchers and breeders in different growing environments. In addition, current TILLING screening protocols require costly fluorescence detection systems, limiting their use, especially in developing countries. We developed a new TILLING resource comprising 2610 M(2) mutants in a common wheat cultivar 'Jinmai 47'. Numerous phenotypes with altered morphological and agronomic traits were observed from the M(2) and M(3) lines in the field. To simplify the procedure and decrease costs, we use unlabeled primers and either non-denaturing polyacrylamide gels or agarose gels for mutation detection. The value of this new resource was tested using PCR with RAPD and Intron-spliced junction (ISJ) primers, and also TILLING in three selected candidate genes, in 300 and 512 mutant lines, revealing high mutation densities of 1/34 kb by RAPD/ISJ analysis and 1/47 kb by TILLING. In total, 31 novel alleles were identified in the 3 targeted genes and confirmed by sequencing. The results indicate that this mutant population represents a useful resource for the wheat research community. We hope that the use of this reverse genetics resource will provide novel allelic diversity for wheat improvement and functional genomics.     

Genome-Wide Identification and Characterization of Germin and Germin-Like Proteins (GLPs) and Their Response Under Powdery Mildew Stress in Wheat (Triticum aestivum L.)

Plant Molecular Biology Reporter - Germin and germin-like proteins (GLPs) play multifaceted roles in plants and participate in signaling processes associated with host–pathogen interactions...     

Thiourea-mediated Nitric Oxide Production Enhances Tolerance to Boron Toxicity by Reducing Oxidative Stress in Bread Wheat (Triticum aestivum L.) and Durum Wheat (Triticum durum Desf.) Plants

Two independent experiments were performed to assess the role of thiourea (TU)-mediated nitric oxide (NO) in mitigating boron toxicity (BT) in bread wheat (Triticum aestivum L. cv. Pandas) and durum wheat (Triticum durum cv. Altıntoprak 98) plants. In the first experiment, plants of the two wheat species were grown under control (0.05 mM B) and BT (0.2 mM B) supplied to nutrient solution for 4 weeks after germination. These two treatments were also combined with TU spray at 200 or 400 mg L⁻¹ once a week during the period of stress. Boron toxicity reduced dry weights of shoot and root, leaf total chlorophyll, efficiency of photosystem II (F_v/F_m) and leaf relative water content, whereas it increased endogenous nitric oxide (NO), nitric oxide synthase (NOS), electrolyte leakage (EL), hydrogen peroxide (H₂O₂), malondialdehyde (MDA) and leaf B content. Reductions in total dry matter were 33% and 61% of control in cvs. Pandas and Altintoprak, respectively. Exogenous application of TU improved the plant growth attributes and led to further increases in NO in the leaves. An additional experiment was set up to further understand whether or not TU mediated NO production played a significant role in mitigation of BT using 0.1 mM scavenger of NO, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (cPTIO) combined with the TU treatments by spraying once a week for 4 weeks. TU-induced BT tolerance was totally eliminated by cPTIO by reversing endogenous NO levels. BT enhanced the activities of superoxide dismutase (SOD; EC 1.15.1.1), catalase (CAT; EC. 1.11.1.6), peroxidase (POD; EC. 1.11.1.7) and lipoxygenase (LOX; EC. 1.12.11.12) as well as the contents of soluble sugars (SS), soluble proteins and phenols, but decreased NR. TU treatments enhanced enzyme activities, but reduced contents of soluble sugars (SS), soluble protein and phenols. The present results clearly indicate that TU mediated endogenous NO significantly improved BT tolerance of wheat plants. This evidence was also supported by the increase in hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) as well as plant growth inhibition with the application of TU combined with cPTIO.

     

Nitrogen and Photosynthesis in the Flag Leaf of Wheat (Triticum aestivum L.)

Wheat (Triticum aestivum L. cv Yecora 70) plants were grown with various concentrations of nitrate nitrogen available to the roots. Sampling of flag leaves began after they had reached full expansion and continued throughout senescence. Rates of gas exchange, ribulose-1,5-bisphosphate (RuP₂) carboxylase activity, and the amounts of chlorophyll, soluble protein, nitrogen, and phosphorus were determined for each flag leaf. Rate of CO₂ assimilation was uniquely related to total leaf nitrogen irrespective of nutrient treatment, season, and leaf age. Assimilation rate increased with leaf nitrogen, but the slope of the relationship declined markedly when leaf nitrogen exceeded 125 millimoles nitrogen per square meter. Chlorophyll content and RuP₂ carboxylase activity were approximately proportional to leaf nitrogen content. As leaves aged, RuP₂ carboxylase activity and calculated Hill activity declined in parallel. With normal ambient partial pressure of CO₂, the intercellular partial pressure of CO₂ was always such that rate of assimilation appeared colimited by RuP₂ carboxylation and RuP₂ regeneration capacity.

The initial slope of rate of CO₂ assimilation against intercellular partial pressure of CO₂ varied nonlinearly with carboxylase activity. It is suggested that this was due to a finite conductance to CO₂ diffusion in the wall and liquid phase which causes a drop in CO₂ partial pressure between the intercellular spaces and the site of carboxylation. A double reciprocal plot was used to obtain an estimate of the transfer conductance.

     

Purification and Characterization of Lipase from Wheat (Triticum aestivum L.) Germ

A method of isolation and purification of lipase (EC 3.1.1.3) from the germ of wheat (Triticum aestivumL.) is described. An electrophoretically homogeneous preparation of the enzyme (specific activity, 622.5 × 10^–3 mol/min per mg protein) was obtained after 61-fold purification. The molecular weight of the enzyme, determined by gel chromatography, was 143 ± 2 kDa. The optimal conditions for the enzyme were 37°C and pH 8.0. The homogeneous preparation of the lipase exhibited high thermal stability: over 20% of the original activity was retained after incubation of the preparation at high temperatures (60–90°C) for 1 h at pH 8.0.     

Comparative Analysis of GS2 and Fd-GOGAT Genes in Cultivated Wheat and Their Progenitors Under N Stress

Plant Molecular Biology Reporter - Polyploidization plays an important role in the genesis of cultivated wheat (hexaploid and tetraploid) from its diploid progenitors. Thus, evolution during...     

Molecular Characterization and Global Expression Analysis of Lectin Receptor Kinases in Bread Wheat (Triticum aestivum)

Lectin receptor kinases (LRKs) play a critical role in plants during development and stress conditions, but a comprehensive analysis at genome level is still not carried out in Triticum aestivum. Herein, we performed the genome wide identification, characterization and expression analysis of these genes in T. aestivum (TaLRK). In-total 263 TaLRK genes were identified, which were further classified into three groups based on the nature of lectin domain. We identified, two TaLRKs consisted of calcium-dependent lectin (C-LRK), while 84 legume-lectin (L-LRK) and 177 bulb-lectin (B-LRK) domains. The L-LRK and B-LRK genes were distributed throughout the genome of T. aestivum. Most of the TaLRKs were clustered as homologs, which were distributed either in proximity on same chromosome or on homoeologous chromosomes of A, B and D sub-genomes. A total of 9 and 58 duplication events were also predicted in L-LRK and B-LRK, respectively. Phylogenetic analysis indicated conserved evolutionary relationship of homologous and orthologous genes from multiple plant species. Gene ontology analysis indicated TaLRKs role in binding, signaling and receptor activities. Most of the TaLRKs consisted of a trans-membrane domain and predicted to be localized in the plasma-membrane. A diverse expression pattern of TaLRK genes was found in various developmental stages and stress conditions. Some TaLRKs were found to be highly affected during a particular stress, which indicated a specialized role of each LRK gene in a specific stress condition. These results described various characteristic feature and expression pattern of TaLRK genes, which will pave the way for functional characterization in wheat.     

Response of Spring Wheat (Triticum aestivum L.) Quality Traits and Yield to Sowing Date

The unpredictability and large fluctuation of the climatic conditions in rainfed regions do affect spring wheat yield and grain quality. These variations offer the opportunity for the production of better quality wheat. The effect of variable years, locations and sowing managements on wheat grain yield and quality was studied through field experiments using three genotypes, three locations for two years under rainfed conditions. The two studied years as contrasting years at three locations and sowing dates depicted variability in temperature and water stress during grain filling which resulted considerable change in grain yield and quality. Delayed sowing, years (2009–10) and location (Talagang) with high temperature and water stress resulted increased proline, and grain quality traits i.e. grain protein (GP) and grain ash (GA) than optimum conditions (during 2008–09, at Islamabad and early sowing). However, opposite trend was observed for dry gluten (DG), sodium dodecyl sulphate (SDS), SPAD content and grain yield irrespective of genotypes. The influence of variable climatic conditions was dominant in determining the quality traits and inverse relationship was observed among some quality traits and grain yield. It may be concluded that by selecting suitable locations and different sowing managements for subjecting the crop to desirable environmental conditions (temperature and water) quality traits of wheat crop could be modified.     

QTL Mapping of Diffuse Reflectance Indices of Leaves in Hexaploid Bread Wheat (Triticum aestivum L.)

Russian Journal of Plant Physiology - Quantitative trait loci (QTL) mapping of diffuse reflectance indices of laminas in bread wheat (Triticum aestivum L.) has been first performed under controlled...     

Free Polyamines in Senescing Wheat(Triticum aestivum L.)

The free polyamine content of flag leaves, peduncles, rachis,glumes, and grains of wheat (Triticum aestivum L., cv. Castell)plants, ripening under field conditions, has been investigatedduring three consecutive growing seasons. Putrescine was quantitativelythe most important of all polyamines detected in these organs.Concentrations were highest in the grains, glumes and flag leaves.No correlation was found between polyamine content and the onsetof senescence of flag leaves and other organs. Excised primaryleaves, however, showed a decrease in polyamine content in thedark and also in light/dark cycles, but in the latter case onlyafter an initial increase. Sink removal of otherwise intactwheat plants caused an accumulation of putrescine in flag leavesat the later stages of senescence, whereas removal of all otherleaves was without any significant effect. Putrescine was alsorecovered in phloem-exudate samples collected throughout theperiod of grain development. In both grains and glumes, peakconcentrations of polyamines were found early during seed development. Key words: Triticum aestivum, polyamines, ripening, senescence     

The Morphology and Development of Cross Veins in the Leaves of Bread Wheat (Triticum aestivum L.)

In the leaves of bread wheat Triticum aestivum L. the longitudinalvascular bundles are linked by small transverse bundles Pairsof similar small vascular bundles also link the upper ends ofminor longitudinal bundles to their neighbours in a Y-shapedarrangement The cross-vein procambial strands arise from unexpanded cellsof one layer of the mesophyll tissue. Lines of these cells connectone longitudinal procambial strand to the next The procambialcells subsequently undergo two tangential divisions to producecells which differentiate to form the conducting and parenchymatouselements of the mature cross veins. Anomalous cross veins are sometimes found. possible modes oforigin of these anomalous cross veins are considered.     

Genetic Diversity and Population Structure Analysis of European Hexaploid Bread Wheat (Triticum aestivum L.) Varieties

Progress in plant breeding is facilitated by accurate information about genetic structure and diversity. Here, Diversity Array Technology (DArT) was used to characterize a population of 94 bread wheat (Triticum aestivum L.) varieties of mainly European origin. In total, 1,849 of 7,000 tested markers were polymorphic and could be used for population structure analysis. Two major subgroups of wheat varieties, GrI and GrII, were identified using the program STRUCTURE, and confirmed by principal component analysis (PCA). These subgroups were largely separated according to origin; GrI comprised varieties from Southern and Eastern Europe, whereas GrII contained mostly modern varieties from Western and Northern Europe. A large proportion of the markers contributing most to the genetic separation of the subgroups were located on chromosome 2D near the Reduced height 8 (Rht8) locus, and PCR-based genotyping suggested that breeding for the Rht8 allele had a major impact on subgroup separation. Consistently, analysis of linkage disequilibrium (LD) suggested that different selective pressures had acted on chromosome 2D in the two subgroups. Our data provides an overview of the allele composition of bread wheat varieties anchored to DArT markers, which will facilitate targeted combination of alleles following DArT-based QTL studies. In addition, the genetic diversity and distance data combined with specific Rht8 genotypes can now be used by breeders to guide selection of crossing parents.     

Nitrogen Metabolism in Senescent Flag Leaves of Wheat (Triticum aestivum L.) in the Light

Nitrogen metabolism was examined in senescent flag leaves of 90- to 93-day-old wheat (Triticum aestivum L. cv Yecora 70) plants. CO₂ assimilation and the levels of protein, chlorophyll, and nitrogen in the leaves decreased with age. Glutamine synthetase activity decreased to one-eighth of the level in young flag leaves. Detached leaves were incubated (with the cut base) in ¹⁵N-labeled NH₃, glutamate, or glycine in the light (1.8 millieinstein per square meter per second) at 25°C in an open gas exchange system under normal atmospheric conditions for up to 135 minutes. The ¹⁵N-enrichment of various amino acids derived from these ¹⁵N-substrates were examined. The amido-N of glutamine was the first ¹⁵N-labeled product in leaves incubated with ¹⁵NH₄Cl whereas serine, closely followed by the amido- and amino-N of glutamine, were the most highly ¹⁵N-labeled products during incubation with [¹⁵N]glycine. In contrast, aspartate and alanine were the first ¹⁵N-labeled products when [¹⁵N] glutamate was used. These results indicate that NH₃ was assimilated via glutamine synthetase and glutamate synthase activities and the photorespiratory nitrogen cycle remained functional in these senescent wheat flag leaves. In contrast, an involvement of glutamate dehydrogenase in the assimilation of ammonia could not be detected in these tissues.     

Overexpression of Avenin-Like b Proteins in Bread Wheat (Triticum aestivum L.) Improves Dough Mixing Properties by Their Incorporation into Glutenin Polymers

Avenin-like b proteins are a small family of wheat storage proteins, each containing 18 or 19 cysteine residues. The role of these proteins, with high numbers of cysteine residues, in determining the functional properties of wheat flour is unclear. In the present study, two transgenic lines of the bread wheat overexpressing avenin-like b gene were generated to investigate the effects of Avenin-like b proteins on dough mixing properties. Sodium dodecyl sulfate sedimentation (SDSS) test and Mixograph analysis of these lines demonstrated that overexpression of Avenin-like b proteins in both transgenic wheat lines significantly increased SDSS volume and improved dough elasticity, mixing tolerance and resistance to extension. These changes were associated with the increased proportion of polymeric proteins due to the incorporation of overexpressed Avenin-like b proteins into the glutenin polymers. The results of this study were critical to confirm the hypothesis that Avenin-like b proteins could be integrated into glutenin polymers by inter-chain disulphide bonds, which could help understand the mechanism behind strengthening wheat dough strength.     

Floret Initiation and Development in Spring Wheat (Triticum aestivum L.)

The number and developmental stages of florets were determinedin each spikelet of the spike in the main shoots of spring wheat.Samples were taken frequently from plants grown in a phytotronand in a nitrogen application field-test. Ten stages of development,from floret initiation until anthesis, were recognized and described. Inter-spikelet variation in the total number of initiated floretswas rather small. However, the number of florets at advancedstages of development, as well as the number of grains, washighest in the central spikelets in which florets initiatedfirst. Floret initiation did not proceed beyond spike emergence,whereafter the distal florets and the spikelet apex degenerated.Grain-set was restricted to florets which had developed at leastto the stage of visible anther lobes at spike emergence. Thenumber of these florets was increased significantly by nitrogenapplication. Wheat, Triticum aestivum L., spikelet, floret, grain set, nitrogen     

A Study of Floret Development in Wheat (Triticum aestivum L.)

Plants of wheat (Triticum aestlvum L.) cv. Aotea were grownat high or low nitrogen levels and in a natural photoperiodor continuous light. Starting 17–21 days from the double-ridgestage, eight plants from each treatment were sampled every 3days until anthesis, and the two basal, the sixth, and the terminalspikelets were sectioned longitudinally. A developmental scorewas assigned to each floret and rates of development calculated.Continuous light hastened development but reduced the numberof spikelets per ear, while high nitrogen delayed developmentbut increased spikelet numbers. The number of florets initiatedin each spikelet varied within narrow limits, but grain settingdepended strongly on spikelet position and on treatment. Althoughflorets were initiated in acropetal succession, the rate ofdevelopment tended to increase up to floret 4 but then declinedmarkedly. As a result grain setting was confined to basal floretpositions, although the two basal spikelets developed so slowlythat they contributed relatively little to grain yield. Distalflorets degenerated almost simultaneously at or before ear emergence,but those in intermediate positions continued to develop untilafter fertilization in the lower florets. It is argued thatthe spikelet is an integrated system in which correlative mechanismsplay a part throughout the development of the florets.