Morchella Effectively Removes Microcystins Produced by Microcystis aeruginosa.

Microcystins (MCs) produced by Microcystis aeruginosa are harmful to animal and human health, and there is currently no effective method for their removal. Therefore, the development of biological approaches that inhibit cyanobacteria and remove MCs is needed. We identified strain MB1, confirmed as Morchella, using morphological and mole-cular evolution methods. To assess the impact of strain MB1 on M. aeruginosa, we conducted an experiment in which we inoculated M. aeruginosa with Morchella strain MB1. After their co-cultivation for 4| |d, the inoculation with 0.9696| |g MB1 completely inhibited and removed M. aeruginosa while concurrently removing up to 95% of the MC content. Moreover, within 3| |d of their co-cultivation, MB1 removed more than 50% of nitrogen and phosphorus from the M. aeruginosa solution. Therefore, the development of effective biological techniques for MC removal is paramount in safeguarding both the environment and human well-being. We herein successfully isolated MB1 from its natural habitat. This strain effectively inhibited and removed M. aeruginosa and also reduced the content of nitrogen and phosphorus in the M. aeruginosa solution. Most importantly, it exhibited a robust capability to eliminate MCs. The present results offer a new method and technical reference for mitigating harmful algal blooms.

Genomic and biological control of Sclerotinia sclerotiorum using an extracellular extract from Bacillus velezensis 20507.

Introduction: Sclerotinia sclerotiorum is a known pathogen that harms crops and vegetables. Unfortunately, there is a lack of effective biological control measures for this pathogen. Bacillus velezensis 20507 has a strong antagonistic effect on S. Sclerotiorum; however, the biological basis of its antifungal effect is not fully understood. Methods: In this study, the broad-spectrum antagonistic microorganisms of B. velezensis 20507 were investigated, and the active antifungal ingredients in this strain were isolated, purified, identified and thermal stability experiments were carried out to explore its antifungal mechanism. Results: The B. velezensis 20507 genome comprised one circular chromosome with a length of 4,043,341 bp, including 3,879 genes, 185 tandem repeats, 87 tRNAs, and 27 rRNAs. Comparative genomic analysis revealed that our sequenced strain had the closest genetic relationship with Bacillus velezensis (GenBank ID: NC 009725.2); however, there were significant differences in the positions of genes within the two genomes. It is predicted that B. velezensis 20507 encode 12 secondary metabolites, including difficidin, macrolactin H, fengycin, surfactin, bacillibactin, bacillothiazole A-N, butirosin a/b, and bacillaene. Results showed that B. velezensis 20507 produced various antagonistic effects on six plant pathogen strains: Exserohilum turcicum, Pyricularia oryzae, Fusarium graminearum, Sclerotinia sclerotiorum, Fusarium oxysporum, and Fusarium verticillioides. Acid precipitation followed by 80% methanol leaching is an effective method for isolating the antifungal component ME80 in B. velezensis 20507, which can damage the membranes of S. sclerotiorum hyphae and has good heat resistance. Using high-performance liquid chromatography, and Mass Spectrometry analysis, it is believed that fengycin C72H110N12O20 is the main active antifungal substance. Discussion: This study provides new resources for the biological control of S. Sclerotiorum in soybeans and a theoretical basis for further clarification of the mechanism of action of B. velezensis 20507.

Prediction Model for Postoperative Pressure Injury in Patients with Acute Type A Aortic Dissection.

OBJECTIVE: To establish a risk assessment model to predict postoperative National Pressure Injury Advisory Panel stage 2 or higher pressure injury (PI) risk in patients undergoing acute type A aortic dissection surgery. METHODS: This retrospective assessment included consecutive patients undergoing acute type A aortic dissection surgery in the authors' hospital from September 2017 to June 2021. The authors used LASSO (logistic least absolute shrinkage and selection operator) regression analysis to identify the most relevant variables associated with PI by running cyclic coordinate descent with 10-times cross-validation. The variables selected by LASSO regression analysis were subjected to multivariate logistic analysis. A calibration plot, receiver operating characteristic curve, and decision curve analysis were used to validate the model. RESULTS: There were 469 patients in the study, including 94 (27.5%) with postoperative PI. Ten variables were selected from LASSO regression: body mass index, diabetes, Marfan syndrome, stroke, preoperative skin moisture, hemoglobin, albumin, serum creatinine, platelet, and d-dimer. Four risk factors emerged after multivariate logistic regression: Marfan syndrome, preoperative skin moisture, albumin, and serum creatinine. The area under the receiver operating characteristic curve of the model was 0.765. The calibration plot and the decision curve analysis both suggested that the model was suitable for predicting postoperative PI. CONCLUSIONS: This study built an efficient predictive model that could help identify high-risk patients.

Multiplex CRISPR-Cas9 knockout of EIL3, EIL4, and EIN2L advances soybean flowering time and pod set.

BACKGROUND: Ethylene inhibitor treatment of soybean promotes flower bud differentiation and early flowering, suggested that there is a close relationship between ethylene signaling and soybean growth and development. The short-lived ETHYLENE INSENSITIVE2 (EIN2) and ETHYLENE INSENSITIVE3 (EIN3) proteins play central roles in plant development. The objective of this study was carried out gene editing of EIL family members in soybeans and to examine the effects on soybean yield and other markers of growth. METHODS AND RESULTS: By editing key-node genes in the ethylene signaling pathway using a multi-sgRNA-in-one strategy, we obtained a series of gene edited lines with variable edit combinations among 15 target genes. EIL3, EIL4, and EIN2L were editable genes favored by the T0 soybean lines. Pot experiments also show that the early flowering stage R1 of the EIL3, EIL4, and EIN2L triple mutant was 7.05 d earlier than that of the wild-type control. The yield of the triple mutant was also increased, being 1.65-fold higher than that of the control. Comparative RNA-seq revealed that sucrose synthase, AUX28, MADS3, type-III polyketide synthase A/B, ABC transporter G family member 26, tetraketide alpha-pyrone reductase, and fatty acyl-CoA reductase 2 may be involved in regulating early flowering and high-yield phenotypes in triple mutant soybean plants. CONCLUSION: Our results provide a scientific basis for genetic modification to promote the development of earlier-flowering and higher-yielding soybean cultivars.

Natural history of isolated abdominal aortic dissection: A prospective cohort study.

Objectives: Isolated abdominal aortic dissection (IAAD) is extremely rare, with its optimal treatment and intervention timing remaining poorly understood. We aimed to study the natural history of IAAD and facilitate better clinical decision. Methods: Consecutive patients admitted to our institution from January 2016 to April 2021 were enrolled and followed up prospectively. All-cause death was taken as the primary endpoint. Results: A total of 68 patients with IAAD were included. The mean age at presentation was 61.2 ± 14.8 (Range: 26.0, 93.0) years and 55 (80.9%) were male. A total of 38 (55.9%) patients were treated conservatively, 27 (39.7%) received endovascular aneurysm repair (EVAR), and 3 (4.4%) underwent open surgery. After a mean follow-up of 2.4 years (Range: 0.1, 5.5), 9 (13.2%) patients died, 8 of whom (21.0%) were treated conservatively and 1 EVAR (3.7%). Compared with EVAR/open surgery, patient treated conservatively had a much worse survival (p = 0.043). There was no significant difference between different IAAD aortic sizes regarding mortality (p = 0.220). Patients with completely thrombosed false lumen fared improved survival rate, followed by partial thrombosis and patency, respectively, although not significantly (p = 0.190). No significant difference was observed between male and female concerning survival rate (p = 0.970). Patients without symptoms had a significantly improved survival (p = 0.048). Conclusion: On the basis of patients' preference and surgeons' experience, a more aggressive treatment regimen for IAAD should be considered, with EVAR being the first choice, especially for those with persistent symptoms and patent false lumen, regardless of sex, age, or aortic size.

Risk factors for early death after surgery in patients with acute Stanford type A aortic dissection: A systematic review and meta-analysis.

BACKGROUND: There are many risk factors related to early death after surgery among patients with acute Stanford type A aortic dissection (ATAAD) that have been analyzed in previous studies, but no evidence-based study has been conducted to confirm these risk factors. AIMS: The aims of this study were to investigate risk factors for early death after surgery in patients with ATAAD via systematic review and meta-analysis and assess evidence-based strategies for preventing adverse events. METHODS: The protocol for this study was prospectively registered with PROSPERO (CRD 42022332772). The authors systematically searched PubMed, Ovid, Scopus, Web of Science and Cochrane Library following the Preferred Reporting Items for Systematic Reviews and Meta-analyses guidelines from database construction to May 2021. Studies that met the selection criteria were determined by two independent researchers, and the odds ratios (ORs) and 95% confidence intervals (95% CIs) were reported for the risk factors and were pooled using Stata 15.0. RESULTS: A total of 23 studies including 5510 patients met the inclusion criteria, and 10 risk factors were analyzed in this meta-analysis. The preoperative risk factors for early death after surgery in patients with ATAAD were age [(OR: 1.03, 95% CI (1.01, 1.06)], male sex [(OR: 1.43, 95% CI (1.06, 1.92)], shock [(OR: 1.91, 95% CI (1.06, 3.45)], malperfusion [(OR: 3.45, 95% CI (2.24, 5.31)] and cardiac tamponade [(OR: 3.89, 95% CI (1.17, 12.98)]. CONCLUSION: Patients with ATAAD who have an older age, male sex, shock, malperfusion and cardiac tamponade have a higher risk for early death after surgery. However, more highly homogenous studies are needed to demonstrate these results. Clinical staff should pay more attention to these factors and take individual actions to reduce mortality after surgery in patients with ATAAD.

Digital gene expression profiling of the transcriptional response to Sclerotinia sclerotiorum and its antagonistic bacterium Bacillus amyloliquefaciens in soybean.

Soybean Sclerotinia stem rot caused by Sclerotinia sclerotiorum is a common disease in soybean, and effective biological control is urgently needed. We have previously confirmed that Bacillus amyloliquefaciens can effectively antagonize S. sclerotiorum in a plate competition experiment and a soybean seedling inoculation experiment. In this study, the mechanisms underlying plant death caused by S. sclerotiorum and soybean resistance to S. sclerotiorum induced by B. amyloliquefaciens were evaluated. The stems of potted soybean seedlings were inoculated with S. sclerotiorum (Gm-Ss), B. amyloliquefaciens (Gm-Ba), and their combination (Gm-Ba-Ss), using scratch treatments as a control, followed by dual RNA sequencing and bioinformatics analyses. Global gene expression levels in the Gm-Ss treatment were much lower than those in the Gm-Ba, Gm-Ba-Ss, and Gm groups, suggesting that S. sclerotiorum strongly inhibited global gene expression in soybean. In a pairwise comparison of Gm-Ss vs. Gm, 19983 differentially expressed genes (DEGs) were identified. Down-regulated DEGs were involved in various KEGG pathways, including ko01110 (biosynthesis of secondary metabolites), ko01100 (metabolic pathways), ko01120 (microbial metabolism in diverse environments), ko00500 (starch and sucrose metabolism), and ko04075 (plant hormone signal transmission), suggesting that S. sclerotiorum inoculation had a serious negative effect on soybean metabolism. In Gm-Ba vs. Gm, 13091 DEGs were identified, and these DEGs were significantly enriched in ko03010 (ribosome) and ko03008 (ribosome biogenesis in eucaryotes). Our results suggest that B. amyloliquefaciens increases the expression of genes encoding the ribosomal subunit, promotes cell wall biogenesis, and induces systemic resistance. S. sclerotiorum strongly inhibited metabolism in soybean, inhibited the synthesis of the cytoskeleton, and induced the up-regulation of programmed death and senescence-related genes via an ethylene signal transduction pathway. These results improve our understanding of S. sclerotiorum-induced plant death and soybean resistance to S. sclerotiorum induced by B. amyloliquefaciens and may contribute to the improvement of strategies to avoid yield losses.

Dual RNA Sequencing Analysis of Bacillus amyloliquefaciens and Sclerotinia sclerotiorum During Infection of Soybean Seedlings by S. sclerotiorum Unveils Antagonistic Interactions.

Soybean Sclerotinia stem rot is caused by Sclerotinia sclerotiorum infection, which causes extensive and severe damage to soybean production. Here, we isolated and patented a Bacillus amyloliquefaciens strain, and used it to verify the antagonistic effect of B. amyloliquefaciens on S. sclerotiorum and to explore the possible underlying mechanism. First, we conducted a plate confrontation experiment using the two microbes. Then, inoculation of soybean (Glycine max) seedlings with S. sclerotiorum (Gm-Ss), B. amyloliquefaciens (Gm-Ba), and their combination (Gm-Ba-Ss) was performed, followed by dual RNA sequencing analysis. Plate confrontation and inoculation experiments showed that B. amyloliquefaciens significantly antagonized S. sclerotiorum growth. The average number of fragments per kilobase of transcript per million fragments mapped of S. sclerotiorum transcripts in Gm-Ss and Gm-Ba-Ss inoculation treatments were 117.82 and 50.79, respectively, indicating that B. amyloliquefaciens strongly inhibited gene expression of S. sclerotiorum. In contrast, the average number of fragments per kilobase of transcript per million fragments mapped of B. amyloliquefaciens transcripts in Gm-Ba and Gm-Ba-Ss inoculation treatments were 479.56 and 579.66, respectively, indicating that S. sclerotiorum promoted overall gene expression in B. amyloliquefaciens. For S. sclerotiorum, 507 upregulated and 4,950 downregulated genes were identified among 8,975 genes in the paired comparison Gm-Ba-Ss vs. Gm-Ss. These differentially expressed genes (DEGs) were significantly enriched in the ribosome (ko03010) KEGG pathway. Additionally, for B. amyloliquefaciens, 294 upregulated and 178 downregulated genes were identified among all 3,154 genes in the paired comparison Gm-Ba-Ss vs. Gm-Ba, and these DEGs were mainly and significantly enriched in metabolism-related KEGG pathways, including the citrate cycle (ko00020) and carbon metabolism (ko01200). We concluded that B. amyloliquefaciens inhibits the expression of genes encoding the ribosomal subunit of S. sclerotiorum, resulting in protein synthesis inhibition in S. sclerotiorum, and thus had a strong antagonistic effect on the fungus. This study provides a scientific basis for the biological control of S. sclerotiorum by B. amyloliquefaciens.

Chromosome-Level Genome Assembly and HazelOmics Database Construction Provides Insights Into Unsaturated Fatty Acid Synthesis and Cold Resistance in Hazelnut (Corylus heterophylla).

Corylus heterophylla (2n = 22) is the most widely distributed, unique, and economically important nut species in China. Chromosome-level genomes of C. avellana, C. heterophylla, and C. mandshurica have been published in 2021, but a satisfactory hazelnut genome database is absent. Northeast China is the main distribution and cultivation area of C. heterophylla, and the mechanism underlying the adaptation of C. heterophylla to extremely low temperature in this area remains unclear. Using single-molecule real-time sequencing and the chromosomal conformational capture (Hi-C) assisted genome assembly strategy, we obtained a high-quality chromosome-scale genome sequence of C. heterophylla, with a total length of 343 Mb and scaffold N50 of 32.88 Mb. A total of 94.72% of the test genes from the assembled genome could be aligned to the Embryophyta_odb9 database. In total, 22,319 protein-coding genes were predicted, and 21,056 (94.34%) were annotated in the assembled genome. A HazelOmics online database (HOD) containing the assembled genome, gene-coding sequences, protein sequences, and various types of annotation information was constructed. This database has a user-friendly and straightforward interface. In total, 439 contracted genes and 3,810 expanded genes were identified through genome evolution analysis, and 17 expanded genes were significantly enriched in the unsaturated fatty acid biosynthesis pathway (ko01040). Transcriptome analysis results showed that FAD (Cor0058010.1), SAD (Cor0141290.1), and KAT (Cor0122500.1) with high expression abundance were upregulated at the ovule maturity stage. We deduced that the expansion of these genes may promote high unsaturated fatty acid content in the kernels and improve the adaptability of C. heterophylla to the cold climate of Northeast China. The reference genome and database will be beneficial for future molecular breeding and gene function studies in this nut species, as well as for evolutionary research on species of the order Fagales.

Genome-Wide Identification of the ARF Gene Family and ARF3 Target Genes Regulating Ovary Initiation in Hazel via ChIP Sequencing.

Hazel (Corylus spp.) is an economically important nut species with a unique biological characteristic of ovary differentiation and development initiating from the ovary primordium after pollination. Auxin participates in ovary initiation and has an essential impact on hazel fruit yield and quality. The regulation of auxin in ovary development is thought to be related to auxin response factors (ARFs); however, its detailed regulatory mechanism remains unclear. The spatiotemporal expression pattern of C. heterophylla ARF3 (ChARF3) was accessed via ARF gene family member identification and expression abundance analysis as well as immunohistochemistry. ChARF3 target genes were identified via chromatin immunoprecipitation followed by next-generation sequencing (ChIP-Seq). In total, 14 ChARF members containing at least B3 and Auxin_resp domains were found to be distributed on 9 of 11 chromosomes, and the protein molecular weights were predicted to range from 70.93-139.22 kD. Among eight differentially expressed ChARFs, ChARF3 showed the most significant differences over four ovary developmental stages. Immunohistochemical analysis revealed that ChARF3 was expressed in the ovary primordium and funiculus, integument, endosperm, radicle, and cotyledon indicating its potential regulatory roles in ovary differentiation and development. In total, 3,167 ChARF3 target genes were identified through ChIP-Seq in four ovary developmental stages and were significantly enriched in the biosynthesis of secondary metabolites (ko01110), phenylpropanoid biosynthesis (ko00940), and phytohormone signal transduction (ko04075). ChARF3 was hypothesized to be involved in the regulation of auxin-induced genes and the transcription factors MADS, AP2/ERF, TCP, FT, and LFY. These results suggest that ChARF3 may regulate ovary initiation and ovule development by mediating genes related to auxin biosynthesis and transport, cell division and proliferation, and flower and fruit development. This study provides new insights into the molecular mechanism of hazel yield formation.

Elastin-like polypeptide and γ-zein fusions significantly increase recombinant protein accumulation in soybean seeds.

Soybean seeds are an ideal host for the production of recombinant proteins because of their high content of proteins, long-term stability of seed proteins under ambient conditions, and easy establishment of efficient purification protocols. In this study, a polypeptide fusion strategy was applied to explore the capacity of elastin-like polypeptide (ELP) and γ-zein fusions in increasing the accumulation of the recombinant protein in soybean seeds. Transgenic soybean plants were generated to express the γ-zein- or ELP-fused green fluorescent protein (GFP) under the control of the soybean seed-specific promoter of ß-conglycinin alpha subunit (BCSP). Significant differences were observed in the accumulation of zein-GFP and GFP-ELP from that of the unfused GFP in transgenic soybean seeds based on the total soluble protein (TSP), despite the low-copy of T-DNA insertions and similar expression at the mRNA levels in selected transgenic lines. The average levels of zein-GFP and GFP-ELP accumulated in immature seeds of these transgenic lines were 0.99% and 0.29% TSP, respectively, compared with 0.07% TSP of the unfused GFP. In mature soybean seeds, the accumulation of zein-GFP and GFP-ELP proteins was 1.8% and 0.84% TSP, an increase of 3.91- and 1.82-fold, respectively, in comparison with that of the unfused GFP (0.46% TSP). Confocal laser scanning analysis showed that both zein-GFP and GFP-ELP were abundantly deposited in many small spherical particles of transgenic seeds, while there were fewer such florescence signals in the same cellular compartments of the unfused GFP-expressing seeds. Despite increased recombinant protein accumulation, there were no significant changes in the total protein and oil content in seeds between the transgenic and non-transformed plants, suggesting the possible presence of threshold limits of total protein accumulation in transgenic soybean seeds. Overall, our results indicate that γ-zein and ELP fusions significantly increased the accumulation of the recombinant protein, but exhibited no significant influence on the total protein and oil content in soybean seeds.

Vegetative cells may perform nitrogen fixation function under nitrogen deprivation in Anabaena sp. strain PCC 7120 based on genome-wide differential expression analysis.

Nitrogen assimilation is strictly regulated in cyanobacteria. In an inorganic nitrogen-deficient environment, some vegetative cells of the cyanobacterium Anabaena differentiate into heterocysts. We assessed the photosynthesis and nitrogen-fixing capacities of heterocysts and vegetative cells, respectively, at the transcriptome level. RNA extracted from nitrogen-replete vegetative cells (NVs), nitrogen-deprived vegetative cells (NDVs), and nitrogen-deprived heterocysts (NDHs) in Anabaena sp. strain PCC 7120 was evaluated by transcriptome sequencing. Paired comparisons of NVs vs. NDHs, NVs vs. NDVs, and NDVs vs. NDHs revealed 2,044 differentially expressed genes (DEGs). Kyoto Encyclopedia of Genes and Genomes enrichment analysis of the DEGs showed that carbon fixation in photosynthetic organisms and several nitrogen metabolism-related pathways were significantly enriched. Synthesis of Gvp (Gas vesicle synthesis protein gene) in NVs was blocked by nitrogen deprivation, which may cause Anabaena cells to sink and promote nitrogen fixation under anaerobic conditions; in contrast, heterocysts may perform photosynthesis under nitrogen deprivation conditions, whereas the nitrogen fixation capability of vegetative cells was promoted by nitrogen deprivation. Immunofluorescence analysis of nitrogenase iron protein suggested that the nitrogen fixation capability of vegetative cells was promoted by nitrogen deprivation. Our findings provide insight into the molecular mechanisms underlying nitrogen fixation and photosynthesis in vegetative cells and heterocysts at the transcriptome level. This study provides a foundation for further functional verification of heterocyst growth, differentiation, and water bloom control.

Identification of vital candidate microRNA/mRNA pairs regulating ovule development using high-throughput sequencing in hazel.

BACKGROUND: Hazels (Corylus spp.) are economically important nut-producing species in which ovule development determines seed plumpness, one of the key parameters reflecting nut quality. microRNAs (miRNAs) play important roles in RNA silencing and the post-transcriptional regulation of gene expression. However, very little is currently known regarding the miRNAs involved in regulating ovule growth and development. RESULTS: In this study, we accordingly sought to determine the important miRNAs involved in ovule development and growth in hazel. We examined ovules at four developmental stages, namely, ovule formation (Ov1), early ovule growth (Ov2), rapid ovule growth (Ov3), and ovule maturity (Ov4). On the basis of small RNA and mRNA sequencing using the Illumina sequencing platform, we identified 970 miRNAs in hazel, of which 766 and 204 were known and novel miRNAs, respectively. In Ov1-vs-Ov2, Ov1-vs-Ov3, Ov1-vs-Ov4, Ov2-vs-Ov3, Ov2-vs-Ov4, and Ov3-vs-Ov4 paired comparisons, 471 differentially expressed microRNAs (DEmiRNAs) and their 3117 target differentially expressed messenger RNAs (DEmRNAs) formed 11,199 DEmiRNA/DEmRNA pairs, with each DEmiRNA changing the expression of an average of 6.62 target mRNAs. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of all DEmRNAs revealed 29 significantly enriched KEGG pathways in the six paired comparisons, including protein export (ko03060), fatty acid elongation (ko00062), starch and sucrose metabolism (ko00500), fatty acid biosynthesis (ko00061), and amino sugar and nucleotide sugar metabolism (ko00520). Our results indicate that DEmiRNA/DEmRNA pairs showing opposite change trends were related to stress tolerance, embryo and seed development, cell proliferation, auxin transduction, and the biosynthesis of proteins, starch, and fats may participate in ovule growth and development. CONCLUSIONS: These findings contribute to a better understanding of ovule development at the level of post-transcriptional regulation, and lay the foundation for further functional analyses of hazelnut ovule growth and development.

Whole-Genome Re-Sequencing of Corylus heterophylla Blank-Nut Mutants Reveals Sequence Variations in Genes Associated With Embryo Abortion.

Yield loss in the economically important hazelnut (Corylus spp.) occurs through the frequent formation of blank nuts. Although the condition is associated with embryo abortion, we have not yet identified the regulatory genes involved. Therefore, this study aimed to determine the genes related to embryo abortion in hazel. We performed whole-genome re-sequencing and single-nucleotide polymorphism (SNP) analysis on four mutant hazelnut trees (Empty1 to Empty4, C. heterophylla) bearing blank nuts and four wild-type trees (Full1 to Full4, C. heterophylla). A paired comparison of Empty1 vs. Full1, Empty2 vs. Full2, Empty3 vs. Full3, and Empty4 vs. Full4, along with the intersection of Empty1 to Empty4, revealed 3 081 common SNPs in the four blank-nut mutants. Of these, 215 synonymous SNPs in exonic regions were distributed across 178 candidate genes. Heterozygosity analysis showed that average homozygous and heterozygous SNP ratios were respectively 0.409 and 0.591 in the samples. According to Gene Ontology classification, candidate genes were enriched in the categories of binding, catalysis, molecular transducer, transporter, and molecular function regulator. Among these, 18 of 178 genes had homozygous SNPs in Empty1-4. Cis elements in the promoter region of starch synthase 4 (SS4) contain the RY-element, implying seed-specific expression. Starch granules were absent from Empty1-4 cotyledon cells, but abundantly present in Full1-Full4 cotyledon cells. The blank-nut phenotype has heavier nut shells. Overall, we conclude that single-nucleotide variants of Acetyl-CoA carboxylase 1 (ACC1), intracellular sodium/hydrogen exchanger 2 (NHX2), UDP-glycosyltransferase 74E2 (UGT74E2), DEFECTIVE IN MERISTEM SILENCING 3 (DMS3), DETOXIFICATION 43 (FRD3), and SS4 may induce embryo abortion, leading to blank-nut formation. Our results will benefit future research on how the gain or loss of candidate genes influences seed development. Moreover, our study provides novel prospects for seedless cultivar development.

Identification of genes regulating ovary differentiation after pollination in hazel by comparative transcriptome analysis.

BACKGROUND: Hazel (Corylus spp.) exhibits ovary differentiation and development that is initiated from the ovary primordium after pollination, conferring the plant with a unique delayed fertilization. Failure of development of the ovary and ovule after pollination can lead to ovary abortion and blank fruit formation, respectively, with consequent yield loss. However, the genes involved in ovary and ovule differentiation and development are largely unknown. RESULTS: In unpollinated pistillate inflorescences (stage F), the stigma shows an extension growth pattern. After pollination, a rudimentary ovary begins to form (stage S), followed by ovule differentiation (stage T) and growth (stage FO). Total RNA was obtained from pistillate inflorescences or young ovaries at stage F, S, T and FO, and sequencing was carried out on a HiSeq 4000 system. De novo assembly of sequencing data yielded 62.58 Gb of nucleotides and 90,726 unigenes; 5524, 3468, and 8714 differentially expressed transcripts were identified in F-vs-S, S-vs-T, and T-vs-FO paired comparisons, respectively. An analysis of F-vs-S, S-vs-T, and T-vs-FO paired comparisons based on annotations in the Kyoto Encyclopedia of Genes and Genomes revealed six pathways that were significantly enriched during ovary differentiation, including ko04075 (Plant hormone signal transduction). Auxin level increased after pollination, and an immunohistochemical analysis indicated that auxin was enriched at the growth center of pistillate inflorescences and young ovaries. These results indicate that genes related to auxin biosynthesis, transport, signaling, the floral quartet model, and flower development may regulate ovary and ovule differentiation and development in hazel. CONCLUSIONS: Our findings provide insight into the molecular mechanisms of ovary differentiation and development after pollination in this economically valuable plant.

Comparative Transcriptome Analysis Reveal Candidate Genes Potentially Involved in Regulation of Primocane Apex Rooting in Raspberry (Rubus spp.).

Raspberries (Rubus spp.) exhibit a unique rooting process that is initiated from the stem apex of primocane, conferring an unusual asexual mode of reproduction to this plant. However, the full complement of genes involved in this process has not been identified. To this end, the present study analyzed the transcriptomes of the Rubus primocane and floricane stem apex at three developmental stages by Digital Gene Expression profiling to identify genes that regulate rooting. Sequencing and de novo assembly yielded 26.82 Gb of nucleotides and 59,173 unigenes; 498, 7,346, 4,110, 7,900, 9,397, and 4,776 differently expressed genes were identified in paired comparisons of SAF1 (floricane at developmental stage 1) vs. SAP1 (primocane at developmental stage 1), SAF2 vs. SAP2, SAF3 vs. SAP3, SAP1 vs. SAP2, SAP1 vs. SAP3, and SAP2 vs. SAP3, respectively. SAP1 maintains an extension growth pattern; SAP2 then exhibits growth arrest and vertical (downward) gravitropic deflection; and finally, short roots begin to form on the apex of SAP3. The Kyoto Encyclopedia of Genes and Genomes enrichment analysis of SAP1 vs. SAP2 revealed 12 pathways that were activated in response to shoot growth arrest and root differentiation, including circadian rhythm-plant (ko04712) and plant hormone signal transduction (ko04075). Our results indicate that genes related to circadian rhythm, ethylene and auxin signaling, shoot growth, and root development are potentially involved in the regulation of primocane apex rooting in Rubus. These findings provide a basis for elucidating the molecular mechanisms of primocane apex rooting in this economically valuable crop.

Construction of an RNAi vector for knockdown of GM-ACS genes in the cotyledonary nodes of soybean

Background: Ethylene plays an important role in the regulation of floral organ development in soybean, and 1-aminocyclopropane-1-carboxylate synthase (ACS) is a rate-limiting enzyme for ethylene biosynthesis. However, whether ACS also regulates floral organ differentiation in soybean remains unknown. To address this, we constructed an RNAi vector to inhibit ACS expression in cotyledonary nodes. Linear DNA cassettes of RNAi-ACS obtained by PCR were used to transform soybean cotyledonary nodes. Results: In total, 131 of 139 transiently transformed plants acquired herbicide resistance and displayed GUS activities in the new buds. In comparison to untransformed seedling controls, a greater number of flower buds were differentiated at the cotyledonary node; GM-ACS1 mRNA expression levels and ethylene emission in the transformed buds were reduced. Conclusion: These results indicate that the cotyledonary node transient transformation system may be suitable for stable transformation and that the inhibition of ACS expression may be an effective strategy for promoting floral organ differentiation in soybean.

Evaluation of Pathogenicity of the Fungi Metarhizium anisopliae and Beauveria bassiana in Hazelnut Weevil (Curculio nucum L., Coleoptera, Curculionidae) Larvae.

The nut weevil (Curculio nucum) is one of the most important and widespread pests in hazelnut orchards. In order to screen entomopathogenic fungal strains with high virulence against C. nucum, the growth rate, sporulation, and cumulative mortality of different Metarhizium anisopliae and Beauveria bassiana strains were investigated, and the process by which M. anisopliae CoM 02 infects C. nucum larvae was observed using scanning electron microscopy. The results indicated that the growth rate and sporulation of different fungal strains significantly differed. Thirteen days after inoculation with M. anisopliae CoM 02, the cumulative mortality of C. nucum larvae reached 100 %, which was considerably higher than that of the other five strains. As the most virulent of the six test strains, the cadaver rate, LT50, and LT90 of M. anisopliae CoM 02 were 93.4 %, 7.05 and 11.90 days, respectively. Analysis of the infection process by scanning electron microscopy showed that the spore attachment, hyphal germination, hyphal rapid growth, and sporulation of M. anisopliae CoM 02 occurred on the 3rd, 5th, 7th, and 11th day after inoculation, respectively, indicating that the infection cycle takes approximately 11 days. This finding suggests that the highly virulent M. anisopliae plays an important role in the biocontrol of C. nucum in China.

The effects of ethylene on the HCl-extractability of trace elements during soybean seed germination

Background Ethylene is capable of promoting seed germination in some plant species. Mobilization of metals such as Fe, Cu, Mn, and Zn in mature seeds takes place when seeds are germinating. However, whether ethylene is involved in the regulation of soybean seed germination and metal element mobilization during early seed germination stage remains unknown. In the present study, seeds were treated with ethylene synthesis inhibitor aminoethoxyvinylglycine (AVG) and ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), and double distilled H2O (ddH(2)0) treatment was used as control. Ethylene emission, ACC synthase (ACS) expression, ACS enzyme activity and Ca, Zn, Mn, Cu and Fe content in hypocotyls were qualified to analyze the relationship between ethylene and mobilization of these elements. Results The results showed that ACS expression, ACS enzyme activity and ethylene emission peaked at 1 and 7 d after sowing. AVG inhibited ethylene production, promoted the hypocotyls length, ACS expression and its activity, concentrations of total and HCl-extractable Zn, and HCl-extractable Fe in hypocotyls, while ACC caused opposite effects. AVG and ACC treatment had no significantly effects on total and HCl-extractable Ca, Cu and HCl-extractable Mn. Total Mn concentration was promoted by AVG at 1, 3, and 5 d significantly, while ACC treatment tended to have no significantly effects on Mn concentration. Conclusion These findings suggested that ethylene is at least partly involved in the regulation of soybean seed germination. Remobilization of Zn and Fe may be negatively regulated by ethylene.

Transcriptome analysis and gene expression profiling of abortive and developing ovules during fruit development in hazelnut.

BACKGROUND: A high ratio of blank fruit in hazelnut (Corylus heterophylla Fisch) is a very common phenomenon that causes serious yield losses in northeast China. The development of blank fruit in the Corylus genus is known to be associated with embryo abortion. However, little is known about the molecular mechanisms responsible for embryo abortion during the nut development stage. Genomic information for C. heterophylla Fisch is not available; therefore, data related to transcriptome and gene expression profiling of developing and abortive ovules are needed. METHODOLOGY/PRINCIPAL FINDINGS: In this study, de novo transcriptome sequencing and RNA-seq analysis were conducted using short-read sequencing technology (Illumina HiSeq 2000). The results of the transcriptome assembly analysis revealed genetic information that was associated with the fruit development stage. Two digital gene expression libraries were constructed, one for a full (normally developing) ovule and one for an empty (abortive) ovule. Transcriptome sequencing and assembly results revealed 55,353 unigenes, including 18,751 clusters and 36,602 singletons. These results were annotated using the public databases NR, NT, Swiss-Prot, KEGG, COG, and GO. Using digital gene expression profiling, gene expression differences in developing and abortive ovules were identified. A total of 1,637 and 715 unigenes were significantly upregulated and downregulated, respectively, in abortive ovules, compared with developing ovules. Quantitative real-time polymerase chain reaction analysis was used in order to verify the differential expression of some genes. CONCLUSIONS/SIGNIFICANCE: The transcriptome and digital gene expression profiling data of normally developing and abortive ovules in hazelnut provide exhaustive information that will improve our understanding of the molecular mechanisms of abortive ovule formation in hazelnut.