The application of quantitative telomerase activity measurement as an important indicator to monitor the cardiomyocyte differentiation process of human induced pluripotent stem cells under defined conditions.

The construction of an in vitro differentiation system for human induced pluripotent stem cells (hiPSCs) has made exciting progress, but it is still of great significance to clarify the differentiation process. The use of conventional genetic and protein-labeled microscopes to observe or detect different stages of hiPSC differentiation is not specific enough and is cumbersome and time-consuming. In this study, in addition to analyzing the expression of gene/protein-related markers, we used a previously reported simple and excellent quantitative method of cellular telomerase activity based on a quartz crystal microbalance (TREAQ) device to monitor the dynamic changes in cellular telomerase activity in hiPSCs during myocardial differentiation under chemically defined conditions. Finally, by integrating these results, we analyzed the relationship between telomerase activity and the expression of marker genes/proteins as well as the cell type at each study time point. This dynamic quantitative measurement of cellular telomerase activity should be a promising indicator for monitoring dynamic changes in a stage of hiPSC differentiation and inducing cell types. This study provided a quantitative, dynamic and simple monitoring index for the in vitro differentiation process of hiPSC-CMs, which was a certain reference value for the optimization and improvement of the induction system.

CRISPR/Cas9-targeted mutagenesis of TaDCL4, TaDCL5 and TaRDR6 induces male sterility in common wheat.

Phased, small interfering RNAs (phasiRNAs) are important for plant anther development, especially for male sterility. PhasiRNA biogenesis is dependent on genes like RNA polymerase 6 (RDR6), DICER-LIKE 4 (DCL4), or DCL5 to produce 21- or 24 nucleotide (nt) double-strand small RNAs. Here, we generated mutants of DCL4, DCL5 and RDR6 using CRISPR/Cas9 system and studied their effects on plant reproductive development and phasiRNA production in wheat. We found that RDR6 mutation caused sever consequence throughout plant development starting from seed germination and the dcl4 mutants grew weaker with thorough male sterility, while dcl5 plants developed normally but exhibited male sterility. Correspondingly, DCL4 and DCL5, respectively, specified 21- and 24-nt phasiRNA biogenesis, while RDR6 contributed to both. Also, the three key genes evolved differently in wheat, with TaDCL5-A/B becoming non-functioning and TaRDR6-A being lost after polyploidization. Furthermore, we found that PHAS genes (phasiRNA precursors) identified via phasiRNAs diverged rapidly among sub-genomes of polyploid wheat. Despite no similarity being found among phasiRNAs of grasses, their targets were enriched for similar biological functions. In light of the important roles of phasiRNA pathways in gametophyte development, genetic dissection of the function of key genes may help generate male sterile lines suitable for hybrid wheat breeding.

Intergenerational toxic effects of parental exposure to [Cn mim]NO3 (n = 2,4,6) on nervous and skeletal development in zebrafish offspring.

Ionic liquids (ILs) are thought to have negative effects on human health. Researchers have explored the effects of ILs on zebrafish development during the early stages, but the intergenerational toxicity of ILs on zebrafish development has rarely been reported. Herein, parental zebrafish were exposed to different concentrations (0, 12.5, 25, and 50 mg/L) of [Cn mim]NO3 (n = 2, 4, 6) for 1 week. Subsequently, the F1 offspring were cultured in clean water for 96 h. [Cn mim]NO3 (n = 2, 4, 6) exposure inhibited spermatogenesis and oogenesis in F0 adults, even causing obvious lacunae in the testis and atretic follicle oocytes in ovary. After parental exposure to [Cn mim]NO3 (n = 2, 4, 6), the body length and locomotor behavior were measured in F1 larvae at 96 hours post-fertilization (hpf). The results showed that the higher the concentration of [Cn mim]NO3 (n = 2, 4, 6), the shorter the body length and swimming distance, and the longer the immobility time. Besides, a longer alkyl chain length of [Cn mim]NO3 had a more negative effect on body length and locomotor behavior. RNA-seq analysis revealed several downregulated differentially expressed genes (DEGs)-grin1b, prss1, gria3a, and gria4a-enriched in neurodevelopment-related pathways, particularly the pathway for neuroactive ligand-receptor interaction. Moreover, several upregulated DEGs, namely col1a1a, col1a1b, and acta2, were mainly associated with skeletal development. Expression of DEGs was tested by RT-qPCR, and the outcomes were consistent with those obtained from RNA-Seq. We provide evidence showing the effects of parental exposure to ILs on the regulation of nervous and skeletal development in F1 offspring, demonstrating intergenerational effects.

Establishment of a venovenous extracorporeal membrane oxygenation in a rat model of acute respiratory distress syndrome.

INTRODUCTION: Venovenous extracorporeal membrane oxygenation (VV ECMO) is now considered a reasonable option to salvage acute respiratory distress syndrome (ARDS). However, we lack a rodent model for experimental studies. This study was undertaken to establish an animal model of VV ECMO in ARDS rats. METHODS: A total of 18 Sprague-Dawley (SD) rats (350 ± 50 g) were used in this study. Using a rat model of oleic acid (OA)-induced ARDS, VV ECMO was established through cavoatrial cannulation of the right jugular vein for venous drainage and venous reinfusion with a specially designed three-cavity catheter. Continuous arterial pressure monitoring was implemented by using a catheter through cannulation of the right femoral artery. The central temperature was monitored with a rectal probe. Arterial blood gas monitoring was implemented by a blood gas analyzer at three-time points: at baseline, 1-hour (after OA modeling), and 3.5-hour (after VV ECMO support). Lung tissue and bronchoalveolar lavage fluid were harvested respectively for protein concentration and pulmonary histologic evaluation to confirm the alleviation of lung injury during VV ECMO. RESULTS: Following ARDS induced by OA, ten rats were successfully established on VV ECMO without failure and survived the ECMO procedure. VV ECMO alleviated lung injury and restored adequate circulation for the return of lung function and oxygenation. VV ECMO was associated with decreased lung injury score, wet/dry weight ratio, and fluid leakage into airspaces. CONCLUSION: We have established a reliable, economical, and functioning ARDS rat model of VV ECMO.

Relationship between Proton Pump Inhibitors and Adverse Effects in Hemodialysis Patients: A Systematic Review and Meta-Analysis.

BACKGROUND: Currently, the interaction between proton pump inhibitors (PPIs) and their effects on hemodialysis (HD) patients has not been clarified. OBJECTIVES: Here, we aimed to explore the association between PPIs and adverse outcomes in HD patients. METHODS: A search was performed on the PubMed, Embase, Cochrane Library, and Web of Science databases for relevant articles published up to April 10, 2022. Studies examining the association (odds ratio [OR]) between PPIs and side effects were identified. The study followed guidelines prescribed in the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA), and was registered with PROSPERO (CRD42021291177). RESULTS: A total of 12 studies comprising 4,227,497 HD patients with PPIs were identified. Results showed that PPI use was associated with an increased risk of bone fracture and hip fracture in the HD patients (pooled OR = 1.29, 95% CI = 1.21-1.37, p < 0.00001, I2 = 0%; pooled OR = 1.37, 95% CI = 1.12-1.67, p = 0.002, I2 = 82%). Besides, HD patients who received PPIs were more likely to develop hypomagnesemia compared with those who did not receive PPIs (pooled OR = 2.79, 95% CI = 1.95-4.00, p < 0.00001, I2 = 0%). In addition, PPIs use was linked to abdominal aortic calcification and all-cause mortality (pooled OR = 2.03, 95% CI = 1.28-3.24, p = 0.003, I2 = 0%) (pooled OR = 1.44, 95% CI = 1.17-1.78, p = 0.0006, I2 = 0%). CONCLUSIONS: Taken together, the present results demonstrate that PPIs use in HD patients is independently associated with adverse reactions such as hip fracture, hypomagnesemia, abdominal aortic calcification, and all-cause mortality. Thus, the use of PPIs in HD patients should be carefully evaluated and optimized.

Risk Factors for Arteriovenous Fistula Thrombus Development: A Systematic Review and Meta-Analysis.

BACKGROUND: Risk factors like female sex, fistula location, hypertension, albumin, diabetes, arteriovenous graft (AVG), age, and other factors are related to arteriovenous fistula thrombus (AVFT), but the consistency and magnitude of their associations have not been confirmed by meta-analysis. OBJECTIVES: The purpose of this study was to provide a comprehensive and up-to-date synthesis of evidence on the association between potential risk factors and AVFT. METHODS: In this systematic review and meta-analysis, PubMed, Embase, Cochrane Library, and Web of Science databases were searched for articles published up to April 20th, 2022, and cohort, cross-sectional, and case-control studies examining the association (odds ratio [OR]) between potential risk factors and AVFT were identified. The other inclusion criteria were sufficient data for analysis and nonoverlapping datasets, excluding reviews, meta-analyses, and articles with overlapping datasets. Extracted variables included first author, publication year, study type, sample size, percentage of women, vascular access type, risk or protective factors, and measure of association (adjusted estimates of effect of all risk factors). The study protocol is registered at PROSPERO (CRD42020201884) and followed Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. RESULTS: Among the 27 identified studies, data from 24 cohort, 2 case-control, and 1 cross-sectional study were included in this review. When compared to non-AVFT, our data showed that the significant risk factors were AVG (pooled OR = 6.28, 95% CI = 1.79-22.02, p = 0.004, I2 = 87%), age (pooled OR = 1.06, 95% CI = 1.00-1.13, p = 0.05, I2 = 98%), female sex (pooled OR = 2.62, 95% CI = 2.56-2.69, p < 0.00001, I2 = 0%), C-reactive protein (pooled OR = 1.18, 95% CI = 1.08-1.30, p = 0.0005, I2 = 90%), fistula site (distal) (pooled OR = 3.64, 95% CI = 1.74-7.62, p = 0.0006, I2 = 47%), hypertension (pooled OR = 1.21, 95% CI = 1.00-1.47, p = 0.05, I2 = 46%), CD34+KDR+ cell (pooled OR = 1.85, 95% CI = 1.33-2.57, p = 0.0002, I2 = 0%), and eprex use (pooled OR = 5.36, 95% CI = 1.82-15.77, p = 0.002, I2 = 0%). CONCLUSIONS: The meta-analysis suggests that AVG, age, female sex, CRP level, fistula site (distal), hypertension, CD34+KDR+ cell, and the use of eprex are independent risk factors for AVFT. Therefore, clinical medical staff should treat these risk factors carefully, identify them early, and prevent them early to reduce the occurrence of AVFT.

Oroxin A ameliorates the oleic acid-induced A549 cell injury through the suppression of pyroptosis and degradation of alveolar surfactant.

The destruction of the pulmonary epithelial barrier in acute respiratory distress syndrome is caused by the damage of the alveolar epithelial cells. Oroxin A is an effective flavonoid component derived from the medicinal plant Oroxylum indicum (L.) Kurz. In this study, the oleic acid (OA)-induced A549 cell injury model was established in vitro to explore the protective mechanism of Oroxin A. The experiment was divided into the following groups: control, OA and OA + Oroxin A group. The OA-induced A549 cell injury was dose (time)-dependent and was detected by the CCK-8 assay. The protein and mRNA expression levels associated with pyroptosis are detected by Western blotting and RT-qPCR. After Oroxin A treatment, the levels of IL-1ß, IL-18 and LDH released were significantly lower than the OA group. In terms of pyroptosis, Oroxin A can inhibit the expression of pyroptosis-related protein and mRNA. Significantly, the surfactant protein C (SPC) level in the OA + Oroxin A group was significantly higher than that in the OA group. The treatment with Oroxin A alleviates the OA-induced injury in the A549 cells, and its mechanism may be related to the inhibition of A549 cells pyroptosis and prevention of the degradation of the SPC.

Sevoflurane alleviates lung injury and inflammatory response compared with propofol in a rat model of VV ECMO.

INTRODUCTION: Although venovenous extracorporeal membrane oxygenation (VV ECMO) is a reasonable salvage treatment for acute respiratory distress syndrome (ARDS), it requires sedating the patient. Sevoflurane and propofol have pulmonary protective and immunomodulatory properties. This study aimed to compare the effectiveness of sevoflurane and propofol on rats with induced ARDS undergoing VV ECMO. METHODS: Fifteen sprague-dawley (SD) rats were randomly divided into three groups: Con group, sevoflurane (Sevo) group and propofol (Pro) group. Arterial blood gas tests were performed at time pointsT0 (baseline), T1 (the time to ARDS), and T2 (weaning from ECMO). Oxygenation index (PaO2/FiO2) was calculated, and lung edema assessed by determining the lung wet:dry ratio. The protein concentration in bronchial alveolar lavage fluid (BALF) was determined by using bicinchoninic acid assay. Haematoxylin and eosin staining was used to evaluate the lung pathological scores in each group. IL-1ß and TNF-α were also measured in the BALF, serum and lung. RESULTS: Oxygenation index showed improvement in the Sevo group versus Pro group. The wet:dry ratio was reduced in the Sevo group compared with propofol-treated rats. Lung pathological scores were substantially lower in the Sevo group versus the Pro group. Protein concentrations in the BALF and levels of IL-1ß and TNF-α in the Sevo group were substantially lower versus Pro group. CONCLUSION: This study demonstrates that compared with propofol, sevoflurane was more efficacious in improving oxygenation and decreasing inflammatory response in rat models with ARDS subject to VV ECMO treatment.

Evolution of PHAS loci in the young spike of Allohexaploid wheat.

BACKGROUND: PhasiRNAs (phased secondary siRNAs) play important regulatory roles in the development processes and biotic or abiotic stresses in plants. Some of phasiRNAs involve in the reproductive development in grasses, which include two categories, 21-nt (nucleotide) and 24-nt phasiRNAs. They are triggered by miR2118 and miR2275 respectively, in premeiotic and meiotic anthers of rice, maize and other grass species. Wheat (Triticum aestivum) with three closely related subgenomes (subA, subB and subD), is a model of allopolyploid in plants. Knowledge about the role of phasiRNAs in the inflorescence development of wheat is absent until now, and the evolution of PHAS loci in polyploid plants is also unavailable. RESULTS: Using 261 small RNA expression datasets from various tissues, a batch of PHAS (phasiRNA precursors) loci were identified in the young spike of wheat, most of which were regulated by miR2118 and miR2275 in their target site regions. Dissection of PHAS and their trigger miRNAs among the diploid (AA and DD), tetraploid (AABB) and hexaploid (AABBDD) genomes of Triticum indicated that distribution of PHAS loci were dominant randomly in local chromosomes, while miR2118 was dominant only in the subB genome. The diversity of PHAS loci in the three subgenomes of wheat and their progenitor genomes (AA, DD and AABB) suggested that they originated or diverged at least before the occurrence of the tetraploid AABB genome. The positive correlation between the PHAS loci or the trigger miRNAs and the ploidy of genome indicated the expansion of genome was the major drive force for the increase of PHAS loci and their trigger miRNAs in Triticum. In addition, the expression profiles of the PHAS transcripts suggested they responded to abiotic stresses such as cold stress in wheat. CONCLUSIONS: Altogether, non-coding phasiRNAs are conserved transcriptional regulators that display quick plasticity in Triticum genome. They may be involved in reproductive development and abiotic stress in wheat. It could be referred to molecular research on male reproductive development in Triticum.

The genome-wide transcriptional consequences of the nullisomic-tetrasomic stocks for homoeologous group 7 in bread wheat.

BACKGROUND: Hexaploid bread wheat (Triticum aestivum L) arose by two polyploidisation events from three diploid species with homoeologous genomes. Nullisomic-tetrasomic (nulli-tetra or NT) lines are aneuploid wheat plants lacking two and adding two of six homoeologous chromosomes. These plants can grow normally, but with significantly morphological variations because the adding two chromosomes or the remaining four chromosomes compensate for those absent. Despite these interesting phenomena, detailed molecular mechanisms underlying dosage deletion and compensation in these useful genetic materials have not been determined. RESULTS: By sequencing the transcriptomes of leaves in two-week-old seedlings, we showed that the profiles of differentially expressed genes between NT stocks for homoeologous group 7 and the parent hexaploid Chinese Spring (CS) occurred throughout the whole genome with a subgenome and chromosome preference. The deletion effect of nulli-chromosomes was compensated partly by the tetra-chromosomes via the dose level of expressed genes, according to the types of homoeologous genes. The functions of differentially regulated genes primarily focused on carbon metabolic process, photosynthesis process, hormone metabolism, and responding to stimulus, and etc., which might be related to the defective phenotypes that included reductions in plant height, flag leaf length, spikelet number, and kernels per spike. CONCLUSIONS: The perturbation of the expression levels of transcriptional genes among the NT stocks for homoeologous group 7 demonstrated the gene dosage effect of the subgenome at the genome-wide level. The gene dosage deletion and compensation can be used as a model to elucidate the functions of the subgenomes in modern polyploid plants.

Genome-wide analysis and identification of the low potassium stress responsive gene SiMYB3 in foxtail millet (Setariaitalica L.).

BACKGROUND: Potassium (K) is essential to plant growth and development. Foxtail millet (Setaria italic L.) is an important fodder grain crop in arid and semi-arid regions of Asia and Africa because of its strong tolerance to drought and barren stresses. The molecular mechanisms of physiological and biochemical responses and regulations to various abiotic stresses such as low potassium conditions in foxtail millet are not fully understood, which hinders the research and exploitation of this valuable resource. RESULTS: In this research, we demonstrated that the millet variety Longgu 25 was the most insensitive variety to low potassium stress among other five varieties. The transcriptome analysis of Longgu 25 variety revealed a total of 26,192 and 26,849 genes from the K+-deficient and normal transcriptomic libraries by RNA-seq, respectively. A total of 1982 differentially expressed genes (DEGs) were identified including 866 up-regulated genes and 1116 down-regulated genes. We conducted a comparative analysis of these DEGs under low-K+ stress conditions and discovered 248 common DEGs for potassium deprivation among foxtail millet, rice and Arabidopsis. Further Gene Ontology (GO) enrichment analysis identified a series of candidate genes that may involve in K+-deficient response and in intersection of molecular functions among foxtail millet, rice and Arabidopsis. The expression profiles of randomly selected 18 candidate genes were confirmed as true DEGs with RT-qPCR. Furthermore, one of the 18 DEGs, SiMYB3, is specifically expressed only in the millet under low-K+ stress conditions. Overexpression of SiMYB3 promoted the main root elongation and improved K+ deficiency tolerance in transgenic Arabidopsis plants. The fresh weight of the transgenic plants was higher, the primary root length was longer and the root surface-area was larger than those of control plants after K+ deficiency treatments. CONCLUSIONS: This study provides a global view of transcriptomic resources relevant to the K+-deficient tolerance in foxtail millet, and shows that SiMYB3 is a valuable genetic resource for the improvement of K+ deficiency tolerance in foxtail millet.

Aegilops tauschii draft genome sequence reveals a gene repertoire for wheat adaptation.

About 8,000 years ago in the Fertile Crescent, a spontaneous hybridization of the wild diploid grass Aegilops tauschii (2n = 14; DD) with the cultivated tetraploid wheat Triticum turgidum (2n = 4x = 28; AABB) resulted in hexaploid wheat (T. aestivum; 2n = 6x = 42; AABBDD). Wheat has since become a primary staple crop worldwide as a result of its enhanced adaptability to a wide range of climates and improved grain quality for the production of baker's flour. Here we describe sequencing the Ae. tauschii genome and obtaining a roughly 90-fold depth of short reads from libraries with various insert sizes, to gain a better understanding of this genetically complex plant. The assembled scaffolds represented 83.4% of the genome, of which 65.9% comprised transposable elements. We generated comprehensive RNA-Seq data and used it to identify 43,150 protein-coding genes, of which 30,697 (71.1%) were uniquely anchored to chromosomes with an integrated high-density genetic map. Whole-genome analysis revealed gene family expansion in Ae. tauschii of agronomically relevant gene families that were associated with disease resistance, abiotic stress tolerance and grain quality. This draft genome sequence provides insight into the environmental adaptation of bread wheat and can aid in defining the large and complicated genomes of wheat species.

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.

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.

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.

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

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.

Enhancement of grain number per spike by RNA interference of cytokinin oxidase 2 gene in bread wheat.

BACKGROUND: This study aimed to validate the function of CKX gene on grain numbers in wheat. METHODS: we constructed and transformed a RNA interference expression vector of TaCKX2.4 in bread wheat line NB1. Southern blotting analysis was used to select transgenic plants with single copy. The expression of TaCKX2.4 gene was estimated by Quantitative real-time PCR (qRT-PCR) analysis. Finally, the relation between expression of TaCKX2.4 gene and grain numbers was validated. RESULTS: Totally, 20 positive independent events were obtained. Homozygous lines from 5 events with a single copy of transformed gene each were selected to evaluate the expression of TaCKX2.4 and grain numbers per spike in T3 generation. Compared with the control NB1, the average grain numbers per spike significantly increased by 12.6%, 8.3%, 6.5% and 5.8% in the T3 lines JW39-3A, JW1-2B, JW1-1A and JW5-1A, respectively. CONCLUSION: Our study indicated that the expression level of TaCKX2.4 was negatively correlated with the grain number per spike, indicating that the reduced expression of TaCKX2.4 increased grain numbers per spike in wheat.

Identification of interacting proteins of the TaFVE protein involved in spike development in bread wheat.

WD-40 repeat-containing protein MSI4 (FVE)/MSI4 plays important roles in determining flowering time in Arabidopsis. However, its function is unexplored in wheat. In the present study, coimmunoprecipitation and nanoscale liquid chromatography coupled to MS/MS were used to identify FVE in wheat (TaFVE)-interacting or associated proteins. Altogether 89 differentially expressed proteins showed the same downregulated expression trends as TaFVE in wheat line 5660M. Among them, 62 proteins were further predicted to be involved in the interaction network of TaFVE and 11 proteins have been shown to be potential TaFVE interactors based on curated databases and experimentally determined in other species by the STRING. Both yeast two-hybrid assay and bimolecular fluorescence complementation assay showed that histone deacetylase 6 and histone deacetylase 15 directly interacted with TaFVE. Multiple chromatin-remodelling proteins and polycomb group proteins were also identified and predicted to interact with TaFVE. These results showed that TaFVE directly interacted with multiple proteins to form multiple complexes to regulate spike developmental process, e.g. histone deacetylate, chromatin-remodelling and polycomb repressive complex 2 complexes. In addition, multiple flower development regulation factors (e.g. flowering locus K homology domain, flowering time control protein FPA, FY, flowering time control protein FCA, APETALA 1) involved in floral transition were also identified in the present study. Taken together, these results further elucidate the regulatory functions of TaFVE and help reveal the genetic mechanisms underlying wheat spike differentiation.

Response of microRNAs to cold treatment in the young spikes of common wheat.

BACKGROUND: MicroRNAs (miRNAs) are a class of small non-coding RNAs that play important roles in biotic and abiotic stresses by regulating their target genes. For common wheat, spring frost damage frequently occurs, especially when low temperature coincides with plants at early floral organ differentiation, which may result in significant yield loss. Up to date, the role of miRNAs in wheat response to frost stress is not well understood. RESULTS: We report here the sequencing of small RNA transcriptomes from the young spikes that were treated with cold stress and the comparative analysis with those of the control. A total of 192 conserved miRNAs from 105 families and nine novel miRNAs were identified. Among them, 34 conserved and five novel miRNAs were differentially expressed between the cold-stressed samples and the controls. The expression patterns of 18 miRNAs were further validated by quantitative real time polymerase chain reaction (qRT-PCR). Moreover, nearly half of the miRNAs were cross inducible by biotic and abiotic stresses when compared with previously published work. Target genes were predicted and validated by degradome sequencing. Gene Ontology (GO) enrichment analysis showed that the target genes of differentially expressed miRNAs were enriched for response to the stimulus, regulation of transcription, and ion transport functions. Since many targets of differentially expressed miRNAs were transcription factors that are associated with floral development such as ARF, SPB (Squamosa Promoter Binding like protein), MADS-box (MCM1, AG, DEFA and SRF), MYB, SPX (SYG1, Pho81 and XPR1), TCP (TEOSINTE BRANCHED, Cycloidea and PCF), and PPR (PentatricoPeptide Repeat) genes, cold-altered miRNA expression may cause abnormal reproductive organ development. CONCLUSION: Analysis of small RNA transcriptomes and their target genes provide new insight into miRNA regulation in developing wheat inflorescences under cold stress. MiRNAs provide another layer of gene regulation in cold stress response that can be genetically manipulated to reduce yield loss in wheat.

mRNA and Small RNA Transcriptomes Reveal Insights into Dynamic Homoeolog Regulation of Allopolyploid Heterosis in Nascent Hexaploid Wheat.

Nascent allohexaploid wheat may represent the initial genetic state of common wheat (Triticum aestivum), which arose as a hybrid between Triticum turgidum (AABB) and Aegilops tauschii (DD) and by chromosome doubling and outcompeted its parents in growth vigor and adaptability. To better understand the molecular basis for this success, we performed mRNA and small RNA transcriptome analyses in nascent allohexaploid wheat and its following generations, their progenitors, and the natural allohexaploid cultivar Chinese Spring, with the assistance of recently published A and D genome sequences. We found that nonadditively expressed protein-coding genes were rare but relevant to growth vigor. Moreover, a high proportion of protein-coding genes exhibited parental expression level dominance, with genes for which the total homoeolog expression level in the progeny was similar to that in T. turgidum potentially participating in development and those with similar expression to that in Ae. tauschii involved in adaptation. In addition, a high proportion of microRNAs showed nonadditive expression upon polyploidization, potentially leading to differential expression of important target genes. Furthermore, increased small interfering RNA density was observed for transposable element-associated D homoeologs in the allohexaploid progeny, which may account for biased repression of D homoeologs. Together, our data provide insights into small RNA-mediated dynamic homoeolog regulation mechanisms that may contribute to heterosis in nascent hexaploid wheat.