Comprehensive analysis of polygalacturonase gene family highlights candidate genes related to pollen development and male fertility in wheat (Triticum aestivum L.).

MAIN CONCLUSION: Four polygalacturonase gene family members were highlighted that contribute to elucidate the roles of polygalacturonase during the fertility conversion process in male-sterile wheat. Polygalacturonase (PG) belongs to a large family of hydrolases with important functions in cell separation during plant growth and development via the degradation of pectin. Specific expressed PGs in anthers may be significant for male sterility research and hybrid wheat breeding, but they have not been characterized in wheat (Triticum aestivum L.). In this study, we systematically studied the PG gene family using the latest published wheat reference genomic information. In total, 113 wheat PG genes were identified, which could be classified into six categories A-F according to their structure characteristics and phylogenetic comparisons with Arabidopsis and rice. Polyploidy and segmental duplications in wheat were proved to be mainly responsible for the expansion of the wheat PG gene family. RNA-seq showed that TaPGs have specific temporal and spatial expression characteristics, in which 12 TaPGs with spike-specific expression patterns were detected by qRT-PCR in different fertility anthers of KTM3315A, a thermo-sensitive cytoplasmic male-sterile wheat. Four of them specific upregulated (TaPG09, TaPG95, and TaPG93) or downregulated (TaPG87) at trinucleate stage of fertile anthers, and further aligning with the homologous in Arabidopsis revealed that they may undertake functions such as anther dehiscence, separation of pollen, pollen development, and pollen tube elongation, thereby inducing male fertility conversion in KTM3315A. These findings facilitate function investigations of the wheat PG gene family and provide new insights into the fertility conversion mechanism in male-sterile wheat.

Phytoene synthase 1 (Psy-1) and lipoxygenase 1 (Lpx-1) Genes Influence on Semolina Yellowness in Wheat Mediterranean Germplasm.

Phytoene synthase 1 (Psy1) and lipoxygenase 1 (Lpx-1) are key genes involved in the synthesis and catalysis of carotenoid pigments in durum wheat, regulating the increase and decrease in these compounds, respectively, resulting in the distinct yellow color of semolina and pasta. Here, we reported new haplotype variants and/or allele combinations of these two genes significantly affecting yellow pigment content in grain and semolina through their effect on carotenoid pigments. To reach the purpose of this work, three complementary approaches were undertaken: the identification of QTLs associated to carotenoid content on a recombinant inbred line (RIL) population, the characterization of a Mediterranean panel of accessions for Psy1 and Lpx-1 genes, and monitoring the expression of Psy1 and Lpx-1 genes during grain filling on two genotypes with contrasting yellow pigments. Our data suggest that Psy1 plays a major role during grain development, contributing to semolina yellowness, and Lpx-1 appears to be more predominant at post-harvest stages and during pasta making.

Toxic levels of manganese in an acidic Cambisol alters antioxidant enzymes activity, element uptake and subcellular distribution in Triticum aestivum.

In the Montado system, in Portuguese Alentejo region, some Eutric Cambisols are known to promote manganese (Mn) toxicity in wheat. Variation on bioavailable Mn concentration depends on soil acidity, which can be increased by natural events (e.g. waterlogging) or human activity (e.g. excess use of chemical fertilizers). The effect of increasing soil Mn on crop element uptake, element distribution and oxidative stress was evaluated on winter wheat (Triticum aestivum). Plants were grown for 3 weeks in an acidic Cambisol spiked with increasing Mn concentrations (0, 45.2 and 90.4 mg MnCl2/Kg soil). Calcium (Ca), phosphorus (P), magnesium (Mg) and Mn were quantified in the soil solution, root and shoot tissues and respective subcellular fractions. The activity of the antioxidant enzymes ascorbate peroxidase (APX), catalase (CAT), glutathione reductase (GR), guaiacol peroxidase (GPX) and superoxide dismutase (SOD) were determined in extracts of wheat shoots and roots. Overall, increase in soil bioavailable Mn inhibited the uptake of other elements, increased the Ca proportion in the root apoplast, promoted the translocation of Mn and P to shoot tissues and increased their proportion in the shoot vacuoles. Wheat roots showed greater antioxidant enzymes activities than shoots. These activities decreased at the highest soil Mn concentration in both plant parts. Wheat roots appear to be more sensitive to oxidative stress derived from excess soil Mn and promote Mn translocation and storage in shoot vacuoles, probably in Mn and P complexes, as a detoxification strategy. Improvement in wheat production, in acidic soils, may rely on the enhancement of its Mn detoxification strategies.

Policosanol profiles and adenosine 5'-monophosphate-activated protein kinase (AMPK) activation potential of Korean wheat seedling extracts according to cultivar and growth time.

Policosanols is a health promoting aliphatic alcohol known as lipid-lowing agent. To enable maximising the functional properties of wheat, this research investigates the policosanol profiles and adenosine 5'-monophosphate-activated protein kinase (AMPK) activation potential of Korean wheat seedlings according to cultivars and growth times. GC-MS revealed six policosanols that differed markedly in content between 17 cultivars, especially, octacosanol (8) showed the most predominant component (49-83%), varying significantly in average concentrations with growth times as 361.4 (3 days) â†’ 613.0 (6 days) â†’ 203.1 (9 days) â†’ 196.5 (12 days) â†’ 50.9 mg/100 g (19 days). The highest average policosanol (738.7 mg/100 g) exhibited after 6 days, while the lowest was 104.4 mg/100 g on 19 days. Moreover, the wheat cultivars including Shinmichal 1, Anbaek, Namhae, and Joah at 6 days may be recommended as potential sources because of high policosanols (921.7-990.6 mg/100 g). Western blot analysis revealed markedly higher AMPK activation in cells treated with the hexane extracts (150-370% at 100 µg/ml) and octacosanol (8) possessed potent AMPK activator (control; 100 â†’ 280% at 200 µg/ml). It is confirmed that the AMPK activation by wheat seedlings are positively related to the highest policosanol content at the 6 days of growth time, independent of the cultivar. Our results may be contributed to enhance the wheat value regarding development of new cultivars and functional foods.

Application of Rice Grain Husk Derived Biochar in Ameliorating Toxicity Impacts of Cu and Zn on Growth, Physiology and Enzymatic Functioning of Wheat Seedlings.

Livestock and poultry manure containing high levels of copper and zinc have led to contamination of farmland and products which could have an impact on human health. Biochar is an inexpensive and efficient heavy metal absorbent. In the present study, we have used biochar to mitigate the effects of heavy metals on the growth of wheat seedlings. The study showed that the effects of heavy metals on wheat seedlings growth were mitigated by increasing exposure to biochar. Compared to the control group, the germination potential, germination rate, germination index and vigor index of wheat seedlings with supplemented biochar increased significantly. Moreover, the specific activity of catalase, peroxidase, superoxide dismutase decreased and chlorophyll contents increased, which promote wheat growth and suggests that the addition of biochar could reduce the effects of heavy metals on wheat seedlings.

Effect of ZnO, TiO2, Al2O3 and ZrO2 nanoparticles on wheat callus cells.

Effect of metal oxide nanoparticles on calli of two wheat varieties: Parabola (stress tolerant) and Raweta (sensitive) was studied. ZnO induced 10% larger membrane damage in Raweta calli. TiO2, Al2O3, and ZrO2 caused nearly 30% greater lactate dehydrogenase leakage for Raweta compared to Parabola. UV-irradiation of samples containing ZnO particles intensified this effect. Membrane lipid peroxidation in ZnO treated Raweta calli was twice as high as in Parabola and further increased after UV-irradiation. TiO2, Al2O3, and ZrO2 nanoparticles caused a 4-fold increase in malondialdehyde concentration in Raweta calli in comparison to Parabola calli. The nanoparticles studied damaged the cellular defense system by inactivating the antioxidative enzymes.

Molybdenum-Induced Effects on Nitrogen Metabolism Enzymes and Elemental Profile of Winter Wheat (Triticum aestivum L.) Under Different Nitrogen Sources.

Different nitrogen (N) sources have been reported to significantly affect the activities and expressions of N metabolism enzymes and mineral elements concentrations in crop plants. However, molybdenum-induced effects in winter wheat cultivars have still not been investigated under different N sources. Here, a hydroponic study was carried out to investigate these effects on two winter wheat cultivars ('97003' and '97014') as Mo-efficient and Mo-inefficient, respectively, under different N sources (NO3-, NH4NO3, and NH4+). The results revealed that the activities of nitrate reductase (NR) and nitrite reductase (NiR) followed the order of NH4NO3 > NO3- > NH4+ sources, while glutamine synthetase (GS) and glutamate synthase (GOGAT) followed the order of NH4+ > NH4NO3 > NO3- in both the wheat cultivars. However, Mo-induced effects in the activities and expressions of N metabolism enzymes under different N sources followed the order of NH4NO3 > NO3- > NH4+ sources, indicating that Mo has more complementary effects towards nitrate nutrition than the sole ammonium source in winter wheat. Interestingly, under -Mo-deprived conditions, cultivar '97003' recorded more pronounced alterations in Mo-dependent parameters than '97014' cultivar. Moreover, Mo application increased the proteins, amino acids, ammonium, and nitrite contents while concomitantly decreasing the nitrate contents in the same order of NH4NO3 > NO3- > NH4+ sources that coincides with the Mo-induced N enzymes activities and expressions. The findings of the present study indicated that Mo plays a key role in regulating the N metabolism enzymes and assimilatory products under all the three N sources; however, the extent of complementation exists in the order of NH4NO3 > NO3- > NH4+ sources in winter wheat. In addition, it was revealed that mineral elements profiles were mainly affected by different N sources, while Mo application generally had no significant effects on the mineral elements contents in the winter wheat leaves under different N sources.

Graphene oxide effects in early ontogenetic stages of Triticum aestivum L. seedlings.

Nanomaterials are being used increasingly in various areas such as electronic devices manufacture, medicine, mechanical devices production, and even food industry. Therefore, the evaluation of their toxicity is mandatory. Graphene oxide (GO) has been shown to have both positive as well as negative impact on different crop plants, depending on species, dose, and duration of exposure. The current study evaluated the impact of GO sheets at different concentrations (500, 1000 and 2000 mg/L) on physiological, biochemical and genetic levels to determine the possible toxic action. Wheat caryopses were treated with GO for 48 h and 7 days. The germination rate and roots elongation decreased in a dose-response manner, except the sample treated with GO at a concentration of 1000 mg/L. Mitotic index has ascendant trend; its increase may be due to the accumulation of prophases GO induced significant accumulation of the cells with aberrations, their presence suggests a clastogenic/aneugenic effect of these carbon nanomaterials. Regarding enzymatic and non-enzymatic antioxidant system defence, the activity varied depending on the dose of GO. Thus, chlorophyll a pigments content decreased significantly at high dose (2000 mg/L), while the carotenoid pigments had lower content at 500 mg/L of GO, and no statistical difference encountered in case of chlorophyll b amount. The antioxidant enzyme activity (CAT, POD, and SOD) was higher at low dose of GO, indicating the presence of oxidative stress generated as a response to the GO treatment. Also, the free radical scavenging activity of the polyphenolic compounds was enhanced upon GO exposure. The GO accumulation has been identified by transmission electron microscopy only at plumules level, near the intercellular space.

Roles of reactive oxygen species and antioxidant enzymes on formaldehyde removal from air by plants.

The roles of enzymatic reactions and redox reactions caused by reactive oxygen species (ROS) in formaldehyde metabolism in tomatoes and wheat seedlings and the changes in peroxidase (POD) and catalase (CAT) activities in plants were investigated. Differences in the breakdown of added formaldehyde between fresh and boiled plant extracts were determined to calculate the contributions of different removal mechanisms. Two plant seedlings efficiently removed formaldehyde from air when its level varied from 0.65 to 1.91 mg m-3; meanwhile, the maximum rate at which tomato seedlings transported formaldehyde from air to the rhizosphere solution reached 182.26 µg h-1 kg-1 FW (fresh weight). Metabolism in plants was mainly responsible for the formaldehyde dissipation. The enzymatic contribution to formaldehyde dissipation decreased with increasing shoot exposure time or air formaldehyde level, while the redox contribution increased in importance because of an increasing level of ROS. The different enzymatic antioxidant activities of plants resulted in different levels of ROS and hence different tolerance and removal efficiencies toward formaldehyde. The self-enhancing ability of plants to remove formaldehyde via redox reactions suggested that the formaldehyde removal efficiency could be enhanced by plant adaptation to environmental stress.

Homeologue Specific Gene Expression Analysis of Two Vital Carbon Metabolizing Enzymes-Citrate Synthase and NADP-Isocitrate Dehydrogenase-from Wheat (Triticum aestivum L.) Under Nitrogen Stress : Homeologue Specific gene expression of CS and NADP-ICDH.

Citrate synthase (CS) and NADP-dependent isocitrate dehydrogenase (NADP-ICDH) have been considered as candidate enzymes to provide carbon skeletons for nitrogen assimilation, i.e., production of 2-oxoglutarate required by the glutamine synthetase/glutamate synthase cycle. The CS and NADP-ICDH cDNAs were encoded for polypeptides of 402 and 480 amino acids with an estimated molecular weight of 53.01 and 45 kDa and an isoelectric point of 9.08 and 5.98, respectively. Phylogenetic analysis of these proteins in wheat across kingdoms confirmed the close relationship with Aegilops tauschii and Hordeum vulgare. Further, their amino acid sequences were demonstrated to have some conserved motifs such as Mg2+ or Mn2 binding site, catalytic sites, NADP binding sites, and active sites. In-silico-identified genomic sequences for the three homeologues A, B, and Dof CS and NADP-ICDH were found to be located on long arm of chromosomes 5 and 3, and sequence analysis also revealed that the three homeologues consisted of 13 and 15 exons, respectively. The total expression analysis indicated that both genes are ubiquitously expressed in shoot and root tissues under chronic as well as transient nitrogen stress. However, they are differentially and contrastingly expressed but almost in a coordinated manner in both the tissues. Under chronic as well as transient stress, both the genes in shoot tissue showed downregulation, lowest at 6 h of transient stress. However, in the root tissue, trend was found opposite except with exceptions. Moreover, all the three homeologues of both the genes were transcribed differentially, and the ratio of the individual homeologues transcripts to total homeologues transcripts also varied with the tissue, i.e., shoots or roots, as well as with nitrogen stress treatments. Thus, cDNA as well as genomic sequence information, apparent expression at different time point of nitrogen stress, and coordination between these enzymes would be ultimately linked to nitrate assimilation and nitrogen use efficiency in wheat.

Development of wheat genotypes expressing a glutamine-specific endoprotease from barley and a prolyl endopeptidase from Flavobacterium meningosepticum or Pyrococcus furiosus as a potential remedy to celiac disease.

Ubiquitous nature of prolamin proteins dubbed gluten from wheat and allied cereals imposes a major challenge in the treatment of celiac disease, an autoimmune disorder with no known treatment other than abstinence diet. Administration of hydrolytic glutenases as food supplement is an alternative to deliver the therapeutic agents directly to the small intestine, where sensitization of immune system and downstream reactions take place. The aim of the present research was to evaluate the capacity of wheat grain to express and store hydrolytic enzymes capable of gluten detoxification. For this purpose, wheat scutellar calli were biolistically transformed to generate plants expressing a combination of glutenase genes for prolamin detoxification. Digestion of prolamins with barley endoprotease B2 (EP-HvB2) combined with Flavobacterium meningosepticum prolyl endopeptidase (PE-FmPep) or Pyrococcus furiosus prolyl endopeptidase (PE-PfuPep) significantly reduced (up to 67%) the amount of the indigestible gluten peptides of all prolamin families tested. Seven of the 168 generated lines showed inheritance of transgene to the T2 generation. Reversed phase high-performance liquid chromatography of gluten extracts under simulated gastrointestinal conditions allowed the identification of five T2 lines that contained significantly reduced amounts of immunogenic, celiac disease-provoking gliadin peptides. These findings were complemented by the R5 ELISA test results where up to 72% reduction was observed in the content of immunogenic peptides. The developed wheat genotypes open new horizons for treating celiac disease by an intraluminal enzyme therapy without compromising their agronomical performance.

A cupin domain is involved in α-amylase inhibitory activity.

Proteinaceous α-amylase inhibitors have specialized activities that make some strong inhibition of α-amylases. New α-amylase inhibitors continue to be discovered so far. A proteinaceous α-amylase inhibitor CL-AI was isolated and identified from chickpea seeds. CL-AI, encoded by Q9SMJ4, was a storage legumin precursor containing one α-chain and one ß-chain, and each chain possessed a same conserved cupin domain. Amino acid mutation and deficiency of cupin domain would lead to loss of α-amylase inhibitory activity, indicating that it was essential for inhibitory activity. CL-AI(α + ß) in its single stranded state in vivo had inhibitory activity. After it was processed into one α-chain and one ß-chain, the two chains were connected to each other via disulfide bond, which would cover the cupin domains and lead to the loss of inhibitory activity. The CL-AI(α + ß), α-chain and ß-chain could inhibit various α-amylases and delay the seed germination of wheat, rice and maize as well as the growth and development of potato beetle larva. Two cupin proteins, Glycinin G1 in soybean and Glutelinin in rice were also found to have inhibitory activity. Our results indicated that the cupin domain is involved in α-amylase inhibitory activity and the proteins with a cupin domain may be a new kind of proteinaceous α-amylase inhibitor.

Identification of new abscisic acid receptor agonists using a wheat cell-free based drug screening system.

Abscisic acid (ABA) is the main phytohormone involved in abiotic stress response and its adaptation, and is a candidate agrichemical. Consequently, several agonists of ABA have been developed using the yeast two-hybrid system. Here, we describe a novel cell-free-based drug screening approach for the development and validation of ABA receptor agonists. Biochemical validation of this approach between 14 ABA receptors (PYR/PYL/RCARs) and 7 type 2C-A protein phosphatases (PP2CAs) revealed the same interactions as those of previous proteome data, except for nine new interactions. By chemical screening using this approach, we identified two novel ABA receptor agonists, JFA1 (julolidine and fluorine containing ABA receptor activator 1) and JFA2 as its analog. The results of biochemical validation for this approach and biological analysis suggested that JFA1 and JFA2 inhibit seed germination and cotyledon greening of seedlings by activating PYR1 and PYL1, and that JFA2 enhanced drought tolerance without inhibiting root growth by activating not only PYR1 and PYL1 but also PYL5. Thus, our approach was useful for the development of ABA receptor agonists and their validation.

Extracellular Secretion of Phytase from Transgenic Wheat Roots Allows Utilization of Phytate for Enhanced Phosphorus Uptake.

A significant portion of organic phosphorus comprises of phytates which are not available to wheat for uptake. Hence for enabling wheat to utilize organic phosphorus in form of phytate, transgenic wheat expressing phytase from Aspergillus japonicus under barley root-specific promoter was developed. Transgenic events were initially screened via selection media containing BASTA, followed by PCR and BASTA leaf paint assay after hardening. Out of 138 successfully regenerated To events, only 12 had complete constructs and thus further analyzed. Positive T1 transgenic plants, grown in sand, exhibited 0.08-1.77, 0.02-0.67 and 0.44-2.14 fold increase in phytase activity in root extracts, intact roots and external root solution, respectively, after 4 weeks of phosphorus stress. Based on these results, T2 generation of four best transgenic events was further analyzed which showed up to 1.32, 56.89, and 15.40 fold increase in phytase activity in root extracts, intact roots and external root solution, respectively, while in case of real-time PCR, maximum fold increase of 19.8 in gene expression was observed. Transgenic lines showed 0.01-1.18 fold increase in phosphorus efficiency along with higher phosphorus content when supplied phytate or inorganic phosphorus than control plants. Thus, this transgenic wheat may aid in reducing fertilizer utilization and enhancing wheat yield.

P and Ca digestibility is increased in broiler diets supplemented with the high-phytase HIGHPHY wheat.

Around 70% of total seed phosphorus is represented by phytate which must be hydrolysed to be bioavailable in non-ruminant diets. The limited endogenous phytase activity in non-ruminant animals make it common practice to add an exogenous phytase source to most poultry and pig feeds. The mature grain phytase activity (MGPA) of cereal seeds provides a route for the seeds themselves to contribute to phytate digestion, but MGPA varies considerably between species and most varieties in current use make negligible contributions. Currently, all phytases used for feed supplementation and transgenic improvement of MGPA are derived from microbial enzymes belonging to the group of histidine acid phosphatases (HAP). Cereals contain HAP phytases, but the bulk of MGPA can be attributed to phytases belonging to a completely different group of phosphatases, the purple acid phosphatases (PAPhy). In recent years, increased MGPAs were achieved in cisgenic barley holding extra copies of barley PAPhy and in the wheat HIGHPHY mutant, where MGPA was increased to ~6200 FTU/kg. In the present study, the effect of replacing 33%, 66% and 100% of a standard wheat with HIGHPHY wheat was compared with a control diet with and without 500 FTU of supplemental phytase. Diets were compared by evaluating broiler performance, ileal Ca and P digestibility and tibia development, using nine replicate pens of four birds per diet over 3 weeks from hatch. There were no differences between treatments in any tibia or bird performance parameters, indicating the control diet did not contain sufficiently low levels of phosphorus to distinguish effect of phytase addition. However, in a comparison of the two wheats, the ileal Ca and P digestibility coefficients for the 100% HIGHPHY wheat diets are 22.9% and 35.6% higher, respectively, than for the control diet, indicating the wheat PAPhy is functional in the broiler digestive tract. Furthermore, 33% HIGHPHY replacement of conventional wheat, significantly improved Ca and P digestibility over the diet-supplemented exogenous phytase, probably due to the higher phytase activity in the HIGHPHY diet (1804 v. 1150 FTU). Full replacement by HIGHPHY gave 14.6% and 22.8% higher ileal digestibility coefficients for Ca and P, respectively, than for feed supplemented with exogenous HAP phytase at 500 FTU. This indicates that in planta wheat PAPhys has promising potential for improving P and mineral digestibility in animal feed.

Lutein ester profile in wheat and tritordeum can be modulated by temperature: Evidences for regioselectivity and fatty acid preferential of enzymes encoded by genes on chromosomes 7D and 7Hch.

The increase of lutein retention through the food chain is desirable for wheat breeding. Lutein esters are more stable than free lutein during post-harvest storage and two loci on chromosomes 7D and 7Hch are important for esterification. We investigated the effect of temperature during grain filling on carotenoid accumulation and lutein ester profile including fatty acid selectivity (palmitic vs. linoleic) and regioselectivity (esterification at positions 3 vs. 3'). Three different temperature regimes were assayed (controlled, semi-controlled and non-controlled). Lutein esters were more stable than free carotenoids in vivo and the enzymes encoded by chromosomes 7Hch and 7D are complementary. Indeed, they show differential preferences for the fatty acid (palmitic and linoleic, respectively) and regioselectivity (3 and 3', respectively). Besides, H. chilense has additional genes for esterification. Finally, the increase of temperature favoured the accumulation of lutein esters with linoleic acid and the synthesis of regioisomers at position 3'.

Inactivation of Lipase and Lipoxygenase of Wheat Germ with Temperature-Controlled Short Wave Infrared Radiation and Its Effect on Storage Stability and Quality of Wheat Germ Oil.

Wheat germ (WG) is quite susceptible to deterioration due to the presence of lipase (LA) and lipoxygenase (LOX). Therefore it is indispensable to adopt a stabilization step to decrease the activity of LA and LOX while retaining a maximum level of nutrients. But over-drying can make foodstuffs more susceptible to autoxidation. So a stabilization protocol for inactivating LA and LOX of WG with a temperature- controlled short wave infrared (SIR) radiation system was adopted to retard its rancidity and retain a maximum level of fat-soluble nutrients. Meanwhile, the critical storage water activity (Aw) of WG for inhibiting both hydrolytic and oxidative rancidity was appraised. Results indicate that WG irradiated at 90°C for 20 min acquired the optimal stabilization effect, and its residual LA and LOX activity were 18.02% and 19.21%, respectively. At this condition, the free fatty acids (FFA) content and peroxide value (PV) increment of WG oil at 40°C remained below 5% and 2.24 meq O2/kg for 60 days, respectively. The residual Aw of this WG sample was 0.13, and it is near the Aw corresponding to its monolayer. No significant decrease of fatty acids was observed during SIR processing, while about 96.42% of its original tocopherols still retained in WG treated at 90°C for 20 min.

Distinct expression and function of carotenoid metabolic genes and homoeologs in developing wheat grains.

BACKGROUND: ß-carotene, the most active provitamin A molecule produced by plants, plays important roles in human nutrition and health. ß-carotene does not usually accumulate in the endosperm (i.e. flour) of mature wheat grains, which is a major food source of calories for humans. Therefore, enriching ß-carotene accumulation in wheat grain endosperm will enable a sustainable dietary supplementation of provitamin A. Several metabolic genes affecting ß-carotene accumulation have already been isolated from wheat, including phytoene synthase 1 (PSY1), lycopene ε-cyclase (LCYe) and carotenoid ß-ring hydroxylase1/2 (HYD1/2). RESULTS: In this work, we cloned and biochemically characterized two carotenoid cleavage dioxygenases (CCDs), CCD1 and CCD4, from wheat. While CCD1 homoeologs cleaved ß-apo-8'-carotenal, ß-carotene, lutein and zeaxanthin into apocarotenoid products, CCD4 homoeologs were inactive towards these substrates in in vitro assays. When analyzed by real-time qPCR, PSY1, LCYe, HYD1/2 and CCD1/4 homoeologs showed distinct expression patterns in vegetative tissues and sections of developing tetraploid and hexaploid wheat grains, suggesting that carotenoid metabolic genes and homoeologs are differentially regulated at the transcriptional level in wheat. CONCLUSIONS: The CCD1/4 enzyme activity and the spatial-temporal gene expression data provide critical insights into the specific carotenoid metabolic gene homoeologs that control ß-carotene accumulation in wheat grain endosperm, thus establishing the knowledge base for generation of wheat varieties with enhanced ß-carotene in the endosperm through breeding and genome editing approaches.

The wheat calcium-dependent protein kinase TaCPK7-D positively regulates host resistance to sharp eyespot disease.

Sharp eyespot, caused mainly by the necrotrophic fungus Rhizoctonia cerealis, limits wheat production worldwide. Here, TaCPK7-D, encoding a subgroup III member of the calcium-dependent protein kinase (CPK) family, was identified from the sharp eyespot-resistant wheat line CI12633 through comparative transcriptomic analysis. Subsequently, the defence role of TaCPK7-D against R. cerealis infection was studied by the generation and characterization of TaCPK7-D-silenced and TaCPK7-D-overexpressing wheat plants. Rhizoctonia cerealis inoculation induced a higher transcriptional level of TaCPK7-D in the resistant wheat line CI12633 than in the susceptible cultivar Wenmai 6. The expression of TaCPK7-D was significantly induced after exogenous application of 1-aminocyclopropane-1-carboxylic acid (an ethylene biosynthesis precursor). The green fluorescent protein signal distribution assays indicated that TaCPK7-D localizes to the plasma membrane in both onion epidermal cells and wheat protoplasts. Following R. cerealis inoculation, TaCPK7-D-silenced wheat CI12633 plants displayed more severe sharp eyespot symptoms than control CI12633 plants. Four defence-associated genes (ß-1,3-glucanase, chitinase 1, defensin and TaPIE1) and an ethylene biosynthesis key gene, ACO2, were significantly suppressed in the TaCPK7-D-silenced wheat plants compared with control plants. Conversely, TaCPK7-D-overexpressing wheat lines showed increased resistance to sharp eyespot compared with untransformed recipient wheat Yangmai 16. Furthermore, the transcriptional levels of these four defence-related genes and ACO2 gene were significantly elevated in TaCPK7-D-overexpressing plants compared with untransformed recipient wheat plants. These results suggest that TaCPK7-D positively regulates the wheat resistance response to R. cerealis infection through the modulation of the expression of these defence-associated genes, and that TaCPK7-D is a candidate to improve sharp eyespot resistance in wheat.

Decreasing methylation of pectin caused by nitric oxide leads to higher aluminium binding in cell walls and greater aluminium sensitivity of wheat roots.

Nitric oxide (NO) is an important bioactive molecule involved in cell wall metabolism, which has been recognized as a major target of aluminium (Al) toxicity. We have investigated the effects of Al-induced NO production on cell wall composition and the subsequent Al-binding capacity in roots of an Al-sensitive cultivar of wheat (Triticum aestivum L. cv. Yang-5). We found that Al exposure induced NO accumulation in the root tips. Eliminating NO production with an NO scavenger (cPTIO) significantly alleviated the Al-induced inhibition of root growth and thus reduced Al accumulation. Elimination of NO, however, did not significantly affect malate efflux or rhizosphere pH changes under Al exposure. Levels of cell wall polysaccharides (pectin, hemicelluloses 1, and hemicelluloses 2) and pectin methylesterase activity, as well as pectin demethylation in the root apex, significantly increased under Al treatment. Exogenous cPTIO application significantly decreased pectin methylesterase activity and increased the degree of methylation of pectin in the root cell wall, thus decreasing the Al-binding capacity of pectin. These results suggest that the Al-induced enhanced production of NO decreases cell wall pectin methylation, thus increasing the Al-binding capacity of pectin and negatively regulating Al tolerance in wheat.