Genetic analysis of phytoene synthase 1 (Psy1) gene function and regulation in common wheat

Transcriptome details for three transgenic lines with the most significantly reduced YPC and non-transformed controls. (DOCX 18 kb

Highly Efficient and Heritable Targeted Mutagenesis in Wheat via the Agrobacterium tumefaciens-Mediated CRISPR/Cas9 System

The CRISPR/Cas9 system has been successfully used in hexaploid wheat. Although it has been reported that the induced mutations can be passed to the next generation, gene editing and transmission patterns in later generations still need to be studied. In this study, we demonstrated that the CRISPR/Cas9 system could achieve efficient mutagenesis in five wheat genes via Agrobacterium-mediated transformation of an sgRNA targeting the D genome, an sgRNA targeting both the A and B homologues and three tri-genome guides targeting the editing of all three homologues. High mutation rates and putative homozygous or biallelic mutations were observed in the T0 plants. The targeted mutations could be stably inherited by the next generation, and the editing efficiency of each mutant line increased significantly across generations. The editing types and inheritance of targeted mutagenesis were similar, which were not related to the targeted subgenome number. The presence of Cas9/sgRNA could cause new mutations in subsequent generations, while mutated lines without Cas9/sgRNA could retain the mutation type. Additionally, off-target mutations were not found in sequences that were highly homologous to the selected sgRNA sequences. Overall, the results suggested that CRISPR/Cas9-induced gene editing via Agrobacterium-mediated transformation plays important roles in wheat genome engineering

Comparative proteomic analysis of cold responsive proteins in two wheat cultivars with different tolerance to spring radiation frost

Spring radiation frost (SRF) is a severe environmental stress which impairs wheat yield and productivity worldwide. To better understand the mechanism of wheat (Triticum aestivum) responding to SRF, a comparative proteomic analysis was performed to analyze the changes of the key proteins in two wheat cultivars Jimai22 and Luyuan301 with high and low tolerance to SRF respectively. A total of 43 differentially expressed proteins (DEPs) which mainly involved in carbohydrate metabolism, amino acid metabolism, resistance proteins and antioxidant enzymes, photosynthesis and cellular respiration proteins, cell-wall related proteins, protein translation/processing/degradation and signal transduction were isolated and identified by two-dimensional electrophoresis and MALDI-TOF-TOF MS. The results revealed that of the 21 DEPs in Jimai22 responding to the SRF, 13 DEPs were upregulated and 8 DEPs were downregulated, and that of the 22 DEPs in Luyuan301, 9 DEPs were upregulated and 13 DEPs were downregulated. These DEPs might be responsible for the stronger cold resistance of Jimai22 compared to Luyuan301. The expression pattern and function analysis of these DEPs were very significant to understanding the mechanism of the SRF responses in wheat

Agronomic Traits and Molecular Marker Identification of Wheat–Aegilops caudata Addition Lines

Aegilops caudata is an important gene source for wheat breeding. Intensive evaluation of its utilization value is an essential first step prior to its application in breeding. In this research, the agronomical and quality traits of Triticum aestivum-Ae. caudata additions B–G (homoeologous groups not identified) were analyzed and evaluated. Disease resistance tests showed that chromosome D of Ae. caudata might possess leaf rust resistance, and chromosome E might carry stem rust and powdery mildew resistance genes. Investigations into agronomical traits suggested that the introduction of the Ae. caudata chromosome in addition line F could reduce plant height. Grain quality tests showed that the introduction of chromosomes E or F into wheat could increase its protein and wet gluten content. Therefore, wheat-Ae. caudata additions D–F are all potentially useful candidates for chromosome engineering activities to create useful wheat-alien chromosome introgressions. A total of 55 EST-based molecular markers were developed and then used to identify the chromosome homoeologous group of each of the Ae. caudata B–G chromosomes. Marker analysis indicated that the Ae. caudata chromosomes in addition lines B to G were structurally altered, therefore, a large population combined with intensive screening pressure should be taken into consideration when inducing and screening for wheat-Ae. caudata compensating translocations. Marker data also indicated that the Ae. caudata chromosomes in addition lines C–F were 5C, 6C, 7C, and 3C, respectively, while the homoeologous group of chromosomes B and G of Ae. caudata are as yet undetermined and need further research

A Novel Flower-Like Ag/AgCl/BiOCOOH Ternary Heterojunction Photocatalyst: Facile Construction and Its Superior Photocatalytic Performance for the Removal of Toxic Pollutants

Novel 3D flower-like Ag/AgCl/BiOCOOH ternary heterojunction photocatalysts were fabricated by the solvothermal and in-situ precipitation methods, followed by light reduction treatment. The Ag/AgCl nanoparticles were homogeneously distributed on 3D BiOCOOH microspheres. These obtained catalysts were characterized by XRD, SEM, TEM, diffuse reflectance spectra (DRS), and photoluminescence (PL). As expected, they exhibited extraordinary photocatalytic capabilities for the elimination of rhodamine B (RhB) and ciprofloxacin (CIP) under simulated sunlight, the results revealed that the Ag/AgCl/BiOCH-3 with 20 wt.% of Ag/AgCl possessed the maximum activity, and the rate constant for the RhB degradation reached up to 0.1353 min−1, which was about 16.5 or 12.2 times that of bare BiOCOOH or Ag/AgCl. The PL characterization further verified that Ag/AgCl/BiOCOOH heterojunctions were endowed with the effective separation of photogenerated carriers. The excellent photocatalytic ability of Ag/AgCl/BiOCOOH could be credited to the synergistic interactions between Ag/AgCl and BiOCOOH, which not only substantially widened the light absorption, but also evidently hindered the charge recombination. The trapping experiments revealed that the dominant reactive species in RhB removal were h+, •OH, and •O2− species. In addition, Ag/AgCl/BiOCOOH was quite stable and easily recyclable after multiple cycles. The above results imply that the 3D flower-like Ag/AgCl/BiOCOOH ternary heterojunction photocatalyst holds promising prospects in treating industrial wastewater

Evolution of Disease Defense Genes and Their Regulators in Plants

Biotic stresses do damage to the growth and development of plants, and yield losses for some crops. Confronted with microbial infections, plants have evolved multiple defense mechanisms, which play important roles in the never-ending molecular arms race of plant–pathogen interactions. The complicated defense systems include pathogen-associated molecular patterns (PAMP) triggered immunity (PTI), effector triggered immunity (ETI), and the exosome-mediated cross-kingdom RNA interference (CKRI) system. Furthermore, plants have evolved a classical regulation system mediated by miRNAs to regulate these defense genes. Most of the genes/small RNAs or their regulators that involve in the defense pathways can have very rapid evolutionary rates in the longitudinal and horizontal co-evolution with pathogens. According to these internal defense mechanisms, some strategies such as molecular switch for the disease resistance genes, host-induced gene silencing (HIGS), and the new generation of RNA-based fungicides, have been developed to control multiple plant diseases. These broadly applicable new strategies by transgene or spraying ds/sRNA may lead to reduced application of pesticides and improved crop yield

Targeted mutagenesis using the Agrobacterium tumefaciens-mediated CRISPR-Cas9 system in common wheat

Abstract Background Recently, the CRISPR/Cas9 system has been widely used to precisely edit plant genomes. Due to the difficulty in Agrobacterium-mediated genetic transformation of wheat, the reported applications in CRISPR/Cas9 system were all based on the biolistic transformation. Results In the present study, we efficiently applied targeted mutagenesis in common wheat (Triticum aestivum L.) protoplasts and transgenic T0 plants using the CRISPR/Cas9 system delivered via Agrobacterium tumefaciens. Seven target sites in three genes (Pinb, waxy and DA1) were selected to construct individual expression vectors. The activities of the sgRNAs were evaluated by transforming the constructed vectors into wheat protoplasts. Mutations in the targets were detected by Illumina sequencing. Genome editing, including insertions or deletions at the target sites, was found in the wheat protoplast cells. The highest mutation efficiency was 6.8% in the DA1 gene. The CRISPR/Cas9 binary vector targeting the DA1 gene was then transformed into common wheat plants by Agrobacterium tumefaciens-mediated transformation, resulting in efficient target gene editing in the T0 generation. Thirteen mutant lines were generated, and the mutation efficiency was 54.17%. Mutations were found in the A and B genomes of the transgenic plants but not in the D genome. In addition, off-target mutations were not detected in regions that were highly homologous to the sgRNA sequences. Conclusions Our results showed that our Agrobacterium-mediated CRISPR/Cas9 system can be used for targeted mutations and facilitated wheat genetic improvement

Lineage-Specific Evolved MicroRNAs Regulating NB-LRR Defense Genes in Triticeae

Disease resistance genes encoding proteins with nucleotide binding sites and Leucine-Rich Repeat (NB-LRR) domains include many members involved in the effector-triggered immunity pathway in plants. The transcript levels of these defense genes are negatively regulated by diverse microRNAs (miRNAs) in angiosperms and gymnosperms. In wheat, using small RNA expression datasets and degradome datasets, we identified five miRNA families targeting NB-LRR defense genes in monocots, some of which arose in the Triticeae species era. These miRNAs regulate different types of NB-LRR genes, most of them with coil-coiled domains, and trigger the generation of secondary small interfering RNAs (siRNA) as a phased pattern in the target site regions. In addition to acting in response to biotic stresses, they are also responsive to abiotic stresses such as heat, drought, salt, and light stress. Their copy number and expression variation in Triticeae suggest a rapid birth and death frequency. Altogether, non-conserved miRNAs as conserved transcriptional regulators in gymnosperms and angiosperms regulating the disease resistance genes displayed quick plasticity including the variations of sequences, gene copy number, functions, and expression level, which accompanied with NB-LRR genes may be tune-regulated to plants in natural environments with various biotic and abiotic stresses