Wheat Leaf Tissue Research Articles

Reduction of Fusarium head blight and trichothecene contamination in transgenic wheat expressing Fusarium graminearum trichothecene 3-O-acetyltransferase.

Fusarium graminearum, the causal agent of Fusarium head blight (FHB), produces various mycotoxins that contaminate wheat grains and cause profound health problems in humans and animals. Deoxynivalenol (DON) is the most common trichothecene found in contaminated grains. Our previous study showed that Arabidopsis-expressing F. graminearum trichothecene 3-O-acetyltransferase (FgTRI101) converted DON to 3-acetyldeoxynivalenol (3-ADON) and excreted it outside of Arabidopsis cells. To determine if wheat can convert and excrete 3-ADON and reduce FHB and DON contamination, FgTRI101 was cloned and introduced into wheat cv Bobwhite. Four independent transgenic lines containing FgTRI101 were identified. Gene expression studies showed that FgTRI101 was highly expressed in wheat leaf and spike tissues in the transgenic line FgTri101-1606. The seedlings of two FgTri101 transgenic wheat lines (FgTri101-1606 and 1651) grew significantly longer roots than the controls on media containing 5 µg/mL DON; however, the 3-ADON conversion and excretion was detected inconsistently in the seedlings of FgTri101-1606. Further analyses did not detect 3-ADON or other possible DON-related products in FgTri101-1606 seedlings after adding deuterium-labeled DON into the growth media. FgTri101-transgenic wheat plants showed significantly enhanced FHB resistance and lower DON content after they were infected with F. graminearum, but 3-ADON was not detected. Our study suggests that it is promising to utilize FgTRI101, a gene that the fungus uses for self-protection, for managing FHB and mycotoxin in wheat production.

Genomic and biological characteristics of a novel leafhopper-transmitted marafivirus infecting Triticum aestivum.

Here, we report a novel wheat-infecting marafivirus, tentatively named "Triticum aestivum marafivirus" (TaMRV). The full-length genome sequence of TaMRV comprises 6,437 nucleotides, excluding the poly(A) tail. Pairwise sequence comparisons and phylogenetic analysis revealed that TaMRV may represent a novel species within the genus Marafivirus in the family Tymoviridae. We also observed a mass of isometric particles with a diameter of about 30 nm in ultrathin sections of infected wheat leaf tissue. In addition, the leafhopper Psammotettix alienus was identified as a vector for this virus. This is the first report of the occurrence of a wheat-infecting marafivirus.

Hymenobacter endophyticus sp. nov., isolated from wheat leaf tissue.

A bacterium, designated strain ZK17L-C2T, was isolated from the leaf tissues of wheat (Triticum aestivum) collected in Chengdu, Sichuan Province, PR China. It is aerobic, non-motile, Gram-negative, rod-shaped and red-to-pink in colour. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain ZK17L-C2T belonged to the genus Hymenobacter and was most closely related to Hymenobacter rigui KCTC 12533T (98.68 %) and Hymenobacter metallilatus 9PBR-2T (98.19 %). Digital DNA-DNA hybridization (dDDH) values between strain ZK17L-C2T and these two type strains were 26.6 and 26.5 %, and average nucleotide identity (ANI) values were 84.9 and 84.8 %, respectively; these values are lower than the proposed and generally accepted species boundaries for dDDH and ANI. The genomic DNA G+C content of strain ZK17L-C2T was 59.4 mol%. It can grow at pH 5.5-7.5 and 15-30 °C, which is different from the closely related type strains. The major fatty acids of strain ZK17L-C2T were iso-C15 : 0, C16 : 0 and C18 : 0. Overall, the results from biochemical, chemical taxonomy and phylogenetic analyses indicate that strain ZK17L-C2T (=CGMCC 1.19373T=KCTC 92184 T) represents a new species of the genus Hymenobacter, for which the name Hymenobacter endophyticus sp. nov. is proposed.

Unraveling the regulatory role of MYC2 on ASMT gene expression in wheat: Implications for melatonin biosynthesis and drought tolerance.

Recognized for its multifaceted functions, melatonin is a hormone found in both animals and plants. In the plant kingdom, it plays diverse roles, regulating growth, development, and stress responses. Notably, melatonin demonstrates its significance by mitigating the effects of abiotic stresses like drought. However, understanding the precise regulatory mechanisms controlling melatonin biosynthesis genes, especially during monocots' response to stresses, requires further exploration. Seeking to understand the molecular basis of drought stress tolerance in wheat, we analyzed RNA-Seq libraries of wheat exposed to drought stress using bioinformatics methods. In light of our findings, we identified that the Myelocytomatosis oncogenes 2 (MYC2) transcription factor is a hub gene upstream of a main melatonin biosynthesis gene, N-acetylserotonin methyltransferase (ASMT), in the wheat drought response-gene network. Promoter analysis of the ASMT gene suggested that it might be a target gene of MYC2. We conducted a set of molecular and physiochemical assays along with robust machine learning approaches to elevate those findings further. MYC2 and ASMT were co-regulated under Jasmonate, drought, and a combination of them in the leaf tissues of wheat was detected. A meaningful correlation was observed among gene expression profiles, melatonin contents, photosynthetic activities, antioxidant enzyme activities, H2 O2 levels, and plasma membrane damage. The results indicated an evident relationship between jasmonic acid and the melatonin biosynthesis pathway. Moreover, it seems that the MYC2-ASMT module might contribute to wheat drought tolerance by regulating melatonin contents.

BZIP transcription factors repress the expression of wheat (Triticum aestivum L.) high molecular weight glutenin subunit genes in vegetative tissues

High molecular weight glutenin subunits (HMW GS) represent an important fraction of endosperm-specific seed-storage proteins that provide elasticity to bread dough. Previously, the second cis-regulatory module (CRM2) was found to be one of the most conserved part of HMW GS promoters, which indicated its pre-eminent role in their gene regulation. Here, we observed that deletion of CRM2 from the promoters of the Bx7 and By8 HMW GS genes increased the leakage of their transient expression in wheat leaf tissue. The effect of a VP1, an Myb and an antisense bZIP transcription factor (TF)-binding site, potentially involved in endosperm-specific regulation within CRM2, was then studied. The activity of a Bx7 gene promoter containing a mutant CRM2 with altered VP1 and Myb TF-binding sites, but an intact bZIP TF-binding site, was similarly low to that of the wild type in leaves. Transactivation analysis and EMSA indicated the binding of TFs TabZIP34 and TabZIP115 to the Skn-1 motif GTCAT in CRM2 and the repression of Bx7 and By8 HMW GS gene promoter activity in leaves. TabZIP34 and TabZIP115 may be involved in the downregulation of HMW GS gene expression in vegetative tissues and early-stage endosperm as well its modulation during seed maturation.

Complete Genome Sequence Resource for Xanthomonas translucens pv. undulosa MAI5034, a Wheat Pathogen from Uruguay.

Complete Genome Sequence Resource for Xanthomonas translucens pv. undulosa MAI5034, a Wheat Pathogen from Uruguay.

Abscisic acid signaling reduced transpiration flow, regulated Na+ ion homeostasis and antioxidant enzyme activities to induce salinity tolerance in wheat (Triticum aestivum L.) seedlings

Salinity, one of the catastrophic abiotic stresses that uces wheat production around the globe. Abscisic acid (ABA) is a stress phytohormone as a signaling molecule that led us to investigate its potential to improve morpho-physiological characteristics, antioxidant metabolism, and ion homeostasis in wheat ( Triticum aestivum L.) seedlings grown under salinity stress (0, 50, and 100 mM NaCl). The findings suggested that salt-induced toxicity significantly ( P < 0.05) damaged root morphological characteristics, plant growth, photosynthetic pigments, and water contents, while trigger the oxidative injury, Na + ion accumulation and uptake in wheat leaf and root tissues with the increasing NaCl concentration in the nutrient media. However, root-zone supply of ABA (0, 5, and 10 μ M) prominently alleviated salt induced phytotoxicity. The 10 μ M concentration of ABA promoted shoot (81.7%) and root (102.1%) dry weight, root length (38.2%), Chl. a (65.3%), Chl. b (149.0%), carotenoids (95.7%) and membrane damage (36.7%) when NaCl was added at 100 mM, relative to the corresponding treatment without ABA. Moreover, ABA (10 μ M) supply decreased Na + ion uptake (root to leaf) due to reduced transpiration rate (81.1%), and thereby ameliorated oxidative injury by ucing leaf malondialdehyde (MDA) and H 2 O 2 contents by 36.8% and 29.9%, respectively, at 100 mM NaCl stress, relative to the similar treatment without ABA. In addition, the activities of catalase (CAT), superoxide dismutase (SOD), and ascorbate peroxidase (APX) were upregulated by 143.9%, 20.2%, and 19.5% in leaves and by 144.9%, 23.4% and 41.1% in roots respectively, with 10 μ M ABA application under 100 mM salinity stress, compa to the 100 mM NaCl treatment without ABA. Conclusively, this study proposed that root-zone ABA application promoted salinity tolerance in wheat seedlings and could be a practical approach for wheat production in salt-affected regions to ensure food security. • Na + ion uptake was increased by wheat tissues under salinity stress. • Abscisic acid (ABA) alleviated salt injury by promoting growth and physiology. • ABA reduced Na+ uptake and accumulation by ucing transpirational flow. • ABA regulated antioxidant metabolism in wheat tissues exposed to salinity stress.

Genome-wide identification and characterization of the BES/BZR gene family in wheat and foxtail millet

BackgroundBES/BZR family genes have vital roles in plant growth, development, and adaptation to environmental stimuli. However, they have not yet been characterized and systematically analyzed in wheat and foxtail millet.ResultsIn the current study, five common and two unique BES/BZR genes were identified by genome-wide analysis in wheat and foxtail millet, respectively. These genes were unevenly distributed on 14 and five chromosomes of wheat and foxtail millet, respectively, and clustered in two subgroups in a phylogenetic analysis. The BES/BZR gene family members in each subgroup contained similar conserved motifs. Investigation of cis-acting elements and expression profile analysis revealed that the BES/BZR genes were predominantly expressed in leaf tissues of wheat and foxtail millet seedlings and responded to brassinosteroid, abscisic acid, and NaCl treatments.ConclusionsOur results provide a basis for future studies on the function and molecular mechanisms of the BES/BZR gene family in wheat, foxtail millet, and other plants.

Molecular Analysis of Bacterial Isolates From Necrotic Wheat Leaf Lesions Caused by Xanthomonas translucens, and Description of Three Putative Novel Species, Sphingomonas albertensis sp. nov., Pseudomonas triticumensis sp. nov. and Pseudomonas foliumensis sp. nov.

Xanthomonas translucens is the etiological agent of the wheat bacterial leaf streak (BLS) disease. The isolation of this pathogen is usually based on the Wilbrink’s-boric acid–cephalexin semi-selective medium which eliminates 90% of other bacteria, some of which might be novel species. In our study, a general purpose nutrient agar was used to isolate 49 bacterial strains including X. translucens from necrotic wheat leaf tissues. Maximum likelihood cluster analysis of 16S rRNA sequences grouped the strains into 10 distinct genera. Pseudomonas (32.7%) and Pantoea (28.6%) were the dominant genera while Xanthomonas, Clavibacter and Curtobacterium had 8.2%, each. Erwinia and Sphingomonas had two strains, each. BLAST and phylogenetic analyses of multilocus sequence analysis (MLSA) of specific housekeeping genes taxonomically assigned all the strains to validly described bacterial species, except three strains (10L4B, 12L4D and 32L3A) of Pseudomonas and two (23L3C and 15L3B) of Sphingomonas. Strains 10L4B and12L4D had Pseudomonas caspiana as their closest known type strain while strain 32L3A was closest to Pseudomonas asturiensis. Sphingomonas sp. strains 23L3C and 15L3B were closest to S. faeni based on MLSA analysis. Our data on MLSA, whole genome-based cluster analysis, DNA-DNA hybridization and average nucleotide identity, matrix-assisted laser desorption/ionization-time-of-flight, chemotaxonomy and phenotype affirmed that these 5 strains constitute three novel lineages and are taxonomically described in this study. We propose the names, Sphingomonas albertensis sp. nov. (type strain 23L3CT = DOAB 1063T = CECT 30248T = LMG 32139T), Pseudomonas triticumensis sp. nov. (type strain 32L3AT = DOAB 1067T = CECT 30249T = LMG 32140T) and Pseudomonas foliumensis sp. nov. (type strain 10L4BT = DOAB 1069T = CECT 30250T = LMG 32142T). Comparative genomics of these novel species, relative to their closest type strains, revealed unique repertoires of core secretion systems and secondary metabolites/antibiotics. Also, the detection of CRISPR-Cas systems in the genomes of these novel species suggests an acquired mechanism for resistance against foreign mobile genetic elements. The results presented here revealed a cohabitation, within the BLS lesions, of diverse bacterial species, including novel lineages.

Elucidating the biochemical basis of trans-16:1 fatty acid change in leaves during cold acclimation in wheat.

In plant cells, phosphatidylglycerol (PG) in the chloroplast has a characteristic trans-∆3-hexadecenoic acid (t16:1) at the sn-2 position. The t16:1 content in wheat leaf tissues decreases during cold treatment, but the significance of this fatty acid compositional change and the underlying biochemical mechanism remains poorly understood. Using a large collection of wheat cultivars displaying a varying capacity of freezing tolerance, we show for the first time under field conditions that this low temperature induced t16:1 change is associated with winter hardiness. To explore the metabolic mechanism responsible for the reduction of t16:1, we performed detailed lipid analysis and comparative transcriptome study with four selected wheat lines under cold acclimation. Our results show that wheat leaf tissues experience a gradual decrease in chloroplast lipid pathway activity during cold acclimation and that the decline in chloroplast lipid synthesis manifests itself in the decrease of t16:1 in PG. Comparative RNA-seq analyses with leaf tissues further reveal concerted transcriptome shifts indicating a rebalancing of chloroplast and cytosolic lipid synthesis during cold acclimation. Our study, thus, provides mechanistic understanding on chloroplast lipid adjustments as a "molecular ideotype" and the t16:1 change as a specific metabolite marker for screening freezing tolerance in wheat.

Silicon soil amendment as a complement to manage tan spot and fusarium head blight in wheat

Tan spot, caused by Pyrenophora tritici-repentis, and fusarium head blight (FHB), caused by the Fusarium graminearum species complex, are among the main wheat diseases worldwide. This 3-year field study evaluated the effect of soil incorporation of calcium silicate, a source of silicon (Si), to manage tan spot and FHB and improve grain yield and quality. The effect of Si was compared on two cultivars contrasting in disease resistance and associated with one or two fungicide sprays. Calcium silicate fertilization increased the Si concentration in the soil and wheat leaf and spike tissues. The increase of Si concentration in wheat tissues was associated with reduction in the severity of both tan spot and FHB, consequently increased the quality and grain yield of wheat. The reduction of disease severity conferred by Si was greater for tan spot than FHB. The greatest control of tan spot and FHB was obtained with the moderately resistant cultivar grown in soil amended with calcium silicate (+Si) and treated with two fungicide sprayings. On the other hand, the highest grain yield, under high disease pressure, was obtained in +Si plants, regardless of the cultivar, treated with two fungicide sprays. The results of this study show for the first time that the incorporation of Si in the soil complemented the effect of genetic resistance and fungicide treatments in controlling both tan spot and FHB. Furthermore, results indicate that calcium silicate fertilization is useful as part of integrated management of these wheat diseases.

A Streamlined Protocol for Wheat (Triticum aestivum) Protoplast Isolation and Transformation With CRISPR-Cas Ribonucleoprotein Complexes.

The genetic engineering method CRISPR has been touted as an efficient, inexpensive, easily used, and targeted genetic modification technology that is widely suggested as having the potential to solve many of the problems facing agriculture now and in the future. Like all new technologies, however, it is not without challenges. One of the most difficult challenges to anticipate and detect is gene targets that are inaccessible due to the chromatin state at their specific location. There is currently no way to predict this during the process of designing a sgRNA target, and the only way to detect this issue before spending time and resources on full transformations is to test the cleavage ability of the sgRNA in vivo. In wheat, this is possible using protoplast isolation and PEG transformation with Cas9 ribonucleoprotein complexes. Therefore, we have developed a streamlined protocol for testing the accessibility of sgRNA targets in wheat. The first steps involve digesting wheat leaf tissue in an enzymatic solution and then isolating viable protoplasts using filters and a sucrose gradient. The protoplasts are then transformed using Cas9 ribonucleoprotein complexes via PEG-mediated transformation. DNA is isolated from the CRISPR-Cas-edited protoplasts and PCR is performed to amplify the gene target region. The PCR product is then used to assess the editing efficiency of the chosen sgRNA using Sanger sequencing. This simplified protocol for the isolation and transformation of wheat protoplast cells using Cas9 ribonucleoprotein complexes streamlines CRISPR transformation projects by allowing for a fast and easy test of sgRNA accessibility in vivo.

Transcriptome analysis reveals rapid defence responses in wheat induced by phytotoxic aphid Schizaphis graminum feeding

BackgroundSchizaphis graminum is one of the most important and devastating cereal aphids worldwide, and its feeding can cause chlorosis and necrosis in wheat. However, little information is available on the wheat defence responses triggered by S. graminum feeding at the molecular level.ResultsHere, we collected and analysed transcriptome sequencing data from leaf tissues of wheat infested with S. graminum at 2, 6, 12, 24 and 48 hpi (hours post infestation). A total of 44,835 genes were either up- or downregulated and differed significantly in response to aphid feeding. The expression levels of a number of genes (9761 genes) were significantly altered within 2 hpi and continued to change during the entire 48 h experiment. Gene Ontology analysis showed that the downregulated DEGs were mainly enriched in photosynthesis and light harvesting, and the total chlorophyll content in wheat leaves was also significantly reduced after S. graminum infestation at 24 and 48 hpi. However, a number of related genes of the salicylic acid (SA)-mediated defence signalling pathway and MAPK-WRKY pathway were significantly upregulated at early feeding time points (2 and 6 hpi). In addition, the gene expression and activity of antioxidant enzymes, such as peroxidase and superoxide dismutase, were rapidly increased at 2, 6 and 12 hpi. DAB staining results showed that S. graminum feeding induced hydrogen peroxide (H2O2) accumulation at the feeding sites at 2 hpi, and increased H2O2 production was detected with the increases in aphid feeding time. Pretreatment with diphenylene iodonium, an NADPH oxidase inhibitor, repressed the H2O2 accumulation and expression levels of SA-associated defence genes in wheat.ConclusionsOur transcriptomic analysis revealed that defence-related pathways and oxidative stress in wheat were rapidly induced within hours after the initiation of aphid feeding. Additionally, NADPH oxidase plays an important role in aphid-induced defence responses and H2O2 accumulation in wheat. These results provide valuable insight into the dynamic transcriptomic responses of wheat leaves to phytotoxic aphid feeding and the molecular mechanisms of aphid-plant interactions.

Pedobacter chitinilyticus sp. nov., a chitin-degrading bacterium isolated from wheat leaf tissue.

A bacterium, designated strain CM134L-2T, was isolated from a chitin-enriched wheat leaf microbiome in Chengdu, Sichuan province, China. It was Gram-stain-negative, strictly aerobic, non-spore-forming, motile, rod-shaped, and bright yellow in colour. Strain CM134L-2T grew at 4-35 °C, at pH 6.0-9.0 and could use chitin as the only carbon resource. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain CM134L-2T was most closely related to Pedobacter nanyangensis Q-4T (97.7 %) and Pedobacter zeaxanthinifaciens TDMA-5T (97.4 %). Digital DNA-DNA hybridization (dDDH) values between strain CM134L-2T with these two type strains were 26.8 and 20.8 %, respectively, and average nucleotide identity (ANI) values were 83.2 and 76.2 %; these values are lower than the proposed and generally accepted species boundaries of 70 % for dDDH and 95-96 % for ANI, which suggests strain CM134L-2T represents a novel species. The genomic DNA G+C content of strain CM134L-2T was 39.3 mol%, menaquinone-7 was the major respiratory quinone, phosphatidylethanolamine was the major polar lipid and the major components of the cellular fatty acids were iso-C15 : 0, and C16 : 1ω7c/C16 : 1ω6c (summed feature 3); these features supported the affiliation of strain CM134L-2T to the genus Pedobacter. Overall, strain CM134L-2T belongs to the genus Pedobacter, but can be classified as a novel species, for which the name Pedobacter chitinilyticus sp. nov. is proposed. The type strain is CM134L-2T (=CGMCC 1.16520T=KCTC 62643T).

Effect of Pseudomonas Bacteria on Peroxidase Activity in Wheat Plants when Infected with Bipolaris sorokiniana

We investigated the effect of treating soft wheat seeds (Triticum aestivum L.) with two Pseudomonas bacteria strains, isolated from earthworm coprolites, showing a significant antifungal and growth-promoting action in preliminary screening on the activity of guaiacol-dependant peroxidase under phytopathogenic load in the presence of Bipolaris sorokiniana (Sacc.) Shoemaker as a mechanism for inducing plant resistance to the pathogen. We established a statistically significant decrease (P < 0.05) in root rot disease incidence and severity during bacterization, which is indicative both of antifungal activity of the used bacterial isolates and of their successful colonizing the rhizosphere of wheat plants. We noted a response of free and weakly bound peroxidase of wheat plants to infection with B. sorokiniana: the enzyme activity increased during pathogenesis. Bacterization also increased peroxidase activity in plant leaves and roots, the greatest differences from non-bacterized plants being observed in wheat roots in the presence of the pathogen. We detected a direct link between peroxidase activity in wheat roots and leaf tissues in the absence of the pathogen and the feedback between peroxidase activity and plant infestation by the root rot pathogen. In the presence of the phytopathogen, there is a lack of correlation between peroxidase activity in wheat roots and leaves, and there is a shift of activity towards its increase in roots, which plays an important role in the development of systemic resistance against the root rot pathogen that penetrates into plants through the roots and root collar.

Optimization and Comparative Analysis of Plant Organellar DNA Enrichment Methods Suitable for Next-generation Sequencing.

Plant organellar genomes contain large, repetitive elements that may undergo pairing or recombination to form complex structures and/or sub-genomic fragments. Organellar genomes also exist in admixtures within a given cell or tissue type (heteroplasmy), and an abundance of subtypes may change throughout development or when under stress (sub-stoichiometric shifting). Next-generation sequencing (NGS) technologies are required to obtain deeper understanding of organellar genome structure and function. Traditional sequencing studies use several methods to obtain organellar DNA: (1) If a large amount of starting tissue is used, it is homogenized and subjected to differential centrifugation and/or gradient purification. (2) If a smaller amount of tissue is used (i.e., if seeds, material, or space is limited), the same process is performed as in (1), followed by whole-genome amplification to obtain sufficient DNA. (3) Bioinformatics analysis can be used to sequence the total genomic DNA and to parse out organellar reads. All these methods have inherent challenges and tradeoffs. In (1), it may be difficult to obtain such a large amount of starting tissue; in (2), whole-genome amplification could introduce a sequencing bias; and in (3), homology between nuclear and organellar genomes could interfere with assembly and analysis. In plants with large nuclear genomes, it is advantageous to enrich for organellar DNA to reduce sequencing costs and sequence complexity for bioinformatics analyses. Here, we compare a traditional differential centrifugation method with a fourth method, an adapted CpG-methyl pulldown approach, to separate the total genomic DNA into nuclear and organellar fractions. Both methods yield sufficient DNA for NGS, DNA that is highly enriched for organellar sequences, albeit at different ratios in mitochondria and chloroplasts. We present the optimization of these methods for wheat leaf tissue and discuss major advantages and disadvantages of each approach in the context of sample input, protocol ease, and downstream application.

Optimization and Comparative Analysis of Plant Organellar DNA Enrichment Methods Suitable for Next-generation Sequencing

Plant organellar genomes contain large, repetitive elements that may undergo pairing or recombination to form complex structures and/or sub-genomic fragments. Organellar genomes also exist in admixtures within a given cell or tissue type (heteroplasmy), and an abundance of subtypes may change throughout development or when under stress (sub-stoichiometric shifting). Next-generation sequencing (NGS) technologies are required to obtain deeper understanding of organellar genome structure and function. Traditional sequencing studies use several methods to obtain organellar DNA: (1) If a large amount of starting tissue is used, it is homogenized and subjected to differential centrifugation and/or gradient purification. (2) If a smaller amount of tissue is used (i.e., if seeds, material, or space is limited), the same process is performed as in (1), followed by whole-genome amplification to obtain sufficient DNA. (3) Bioinformatics analysis can be used to sequence the total genomic DNA and to parse out organellar reads. All these methods have inherent challenges and tradeoffs. In (1), it may be difficult to obtain such a large amount of starting tissue; in (2), whole-genome amplification could introduce a sequencing bias; and in (3), homology between nuclear and organellar genomes could interfere with assembly and analysis. In plants with large nuclear genomes, it is advantageous to enrich for organellar DNA to reduce sequencing costs and sequence complexity for bioinformatics analyses. Here, we compare a traditional differential centrifugation method with a fourth method, an adapted CpG-methyl pulldown approach, to separate the total genomic DNA into nuclear and organellar fractions. Both methods yield sufficient DNA for NGS, DNA that is highly enriched for organellar sequences, albeit at different ratios in mitochondria and chloroplasts. We present the optimization of these methods for wheat leaf tissue and discuss major advantages and disadvantages of each approach in the context of sample input, protocol ease, and downstream application.

Three MYB genes co-regulate the phloem-based defence against English grain aphid in wheat.

Plant phloem-based defence (PBD) against phloem-feeding insects is characteristic of the sieve occlusion by phloem lectins and β-1,3-glucan callose, both of which are produced under regulation by ethylene and MYB transcription factors. Wheat PBD requires β-1,3-glucan synthase-like proteins GSL2, GSL10, and GSL12, and may also require insect-resistant mannose-binding lectins Hfr-1 and Wci-1, which can accumulate in the phloem upon aphid feeding. This study elucidates whether any of the 73 MYB genes identified previously in the common wheat Triticum aestivum genome plays a role in wheat PBD activation with regard to the GSLs and lectins. Wheat MYB genes TaMYB19, TaMYB29, and TaMYB44 are highly activated in response to infestation of English grain aphid, and their silencing facilitates aphid feeding on wheat phloem and represses wheat PBD responses. Repressed PBD is shown to decrease aphid-induced callose deposition in wheat leaf epidermis and decrease aphid-induced expression of genes GSL2, GSL10, GSL12, Hfr-1, and Wci-1 in wheat leaf tissues. Based on single gene silencing effects, TaMYB19, TaMYB29, and TaMYB44 contribute 55-82% of PBD responses. However, the contributions of TaMYB genes to PBD are eliminated by ethylene signalling inhibitors, while simultaneous silencing of the three TaMYB genes cancels the tested PBD responses. Therefore, TaMYB19, TaMYB29, and TaMYB44 are co-regulators of wheat PBD and execute this function through crosstalk with the ethylene signalling pathway.

An improved method for RNA extraction from urediniospores of and wheat leaves infected by an obligate fungal pathogen, Puccinia striiformis f. sp. tritici

Stripe rust, caused by Puccinia striiformis f. sp. tritici, is an important wheat disease in China, seriously threatening wheat production. Understanding the winter survival of the fungus is a key for predicting the spring epidemics of the disease, which determines the crop loss. Estimation of P. striiformis f. sp. tritici winter survival requires processing a large number of samples for sensitive detection of the pathogen in wheat leaf tissue using real-time quantitative reverse transcription PCR (qRT-PCR). A bottleneck for the analysis is the acquisition of a good yield of high quality RNA suitable for qRT-PCR to distinguish dead and alive fungal hyphae inside leaves. Although several methods have been described in the literatures and commercial kits are available for RNA extraction, these methods are mostly too complicated, expensive and inefficient. Thus, we modified three previously reported RNA extraction methods with common and low-cost reagents (LiCl, SDS and NaCl) to solve the problems and selected the best to obtain high quality and quantity RNA for use in qRT-PCR. In the three improved methods, the NaCl method was proven to be the best for extracting RNA from urediniospores of and wheat leaves infected by P. striiformis f. sp. tritici, although the modified LiCl and SDS methods also increased yield of RNA compared to the previous methods. The improved NaCl method has the following advantages: 1) Complete transfer of urediniospores of P. striiformis f. sp. tritici from the mortar and pestle can ensure the initial amount of RNA for the qRT-PCR analysis; 2) the use of low-cost NaCl to replace more expensive Trizol can reduce the cost; 3) the yield and quality of RNA can be increased; 4) the improved method is more suitable for a large number and high quantity of samples from fields. Using the improved NaCl method, the amount of RNA was increased three times from urediniospores of P. striiformis f. sp. tritici compared from the extraction kit. Approximately, 10.11 μg total RNA of high quality was obtained from 100 mg of infected leaves, which was 8.8, 6.5, 3.4 and 2.1 folds of the amounts obtained from the previous LiCl, SDS, NaCl and traditional Trizol methods, respectively. The method could be used to study the overwintering rates of P. striiformis f. sp. tritici over a large region of wheat production for predicting epidemic levels by determining pathogen survival levels after winter. The method can also be used in any studies which need a large number of high quality RNA samples.

Diurnal variation of apoplastic ascorbate in winter wheat leaves in relation to ozone detoxification

Besides stomatal closure, biological detoxification is an important protection mechanism for plants against ozone (O3). This study investigated the diurnal changes of ascorbate (a major detoxification agent) in the apoplast and leaf tissues of winter wheat grown under ambient air field conditions. Results showed the reduced ascorbate in the apoplast (ASCapo) exhibited a peak in late morning or midday, mismatching with either the maximum external O3 concentrations in mid-afternoon or the maximum stomatal O3 uptake between late morning and mid-afternoon. In contrast, the ASC in leaf tissues remained stable throughout the day. The investigations conducted in a Free-Air Concentration Elevation of O3 system confirmed that the diurnal variations of the ASCapo were induced more by the daily variations of O3 concentrations rather than the cumulative O3 effects. In conclusion, the O3-stress detoxification should be a dynamic variable rather than a fixed threshold as assumed in the stomatal flux-based O3 dose metrics.