Physiological and transcriptomic analyses reveal the cadmium tolerance mechanism of Miscanthus lutarioriparia.

Miscanthus lutarioriparia is a promising energy crop that is used for abandoned mine soil phytoremediation because of its high biomass yield and strong tolerance to heavy metals. However, the biological mechanism of heavy metal resistance is limited, especially for applications in the soil restoration of mining areas. Here, through the investigation of soil cadmium(Cd) in different mining areas and soil potted under Cd stress, the adsorption capacity of Miscanthus lutarioriparia was analyzed. The physiological and transcriptional effects of Cd stress on M. lutarioriparia leaves and roots under hydroponic conditions were analyzed. The results showed that M. lutarioriparia could reduce the Cd content in mining soil by 29.82%. Moreover, different Cd varieties have different Cd adsorption capacities in soils with higher Cd concentration. The highest cadmium concentrations in the aboveground and belowground parts of the plants were 185.65 mg/kg and 186.8 mg/kg, respectively. The total chlorophyll content, superoxide dismutase and catalase activities all showed a trend of increasing first and then decreasing. In total, 24,372 differentially expressed genes were obtained, including 7735 unique to leaves, 7725 unique to roots, and 8912 unique to leaves and roots, which showed differences in gene expression between leaves and roots. These genes were predominantly involved in plant hormone signal transduction, glutathione metabolism, flavonoid biosynthesis, ABC transporters, photosynthesis and the metal ion transport pathway. In addition, the number of upregulated genes was greater than the number of downregulated genes at different stress intervals, which indicated that M. lutarioriparia adapted to Cd stress mainly through positive regulation. These results lay a solid foundation for breeding excellent Cd resistant M. lutarioriparia and other plants. The results also have an important theoretical significance for further understanding the detoxification mechanism of Cd stress and the remediation of heavy metal pollution in mining soil.

Sema4C Is Required for Vascular and Primary Motor Neuronal Patterning in Zebrafish.

Endothelial cells (ECs) and neurons share a number of common signaling pathways and molecular mediators to orchestrate directional migration and guide the pattern of the vascular network and nervous system. So far, research concerning the functional coupling between vascular and neuronal pathfinding remains insufficient. Semaphorin4C (sema4C), a member of class 4 semaphorins, is initially described in the nervous system, whose role has been demonstrated in diverse biological developments. The present study focused on the role of sema4C in the vascular and neural development process in zebrafish embryos. It confirmed that sema4C is expressed in both the nervous system and intersegmental vessels (ISVs) in zebrafish embryos by diverse expression analysis. It also showed that the knockdown of sema4C caused a serious pathfinding anomaly both in the ISVs and primary motor neurons (PMNs) of zebrafish embryos. In addition, overexpressing exogenous sema4C mRNA in sema4C morphants remarkably neutralized the defective pattern of the vascular and neural system. Collectively, this report suggests that sema4C acts as a dual guiding factor regulating vascular and neuronal development. These findings elucidate a new molecular mechanism underlying blood vessel and nerve development and might serve as groundwork for future research on functional coupling between both systems.

Aquaporin 8ab is required in zebrafish embryonic intestine development.

The aquaporin 8 (AQP8) is a small integral membrane protein that selectively transports water and other small uncharged solutes across cell plasma membranes. It has been demonstrated that AQP8 is ubiquitously present in various tissues and organs of mammals, and participates in many physiological and pathological processes. Recent studies showed that AQP8 is highly expressed in the columnar epithelial cells of mammalian colonic mucosa facing lumen, indicating that AQP8 plays potential roles in the physiology and pathophysiology of gastrointestinal tract. However, the role of AQP8 during gastrointestinal tract development is unclear. In the present study, RT-PCR results reveal that the zebrafish genome encodes three kinds of aqp8s ( aqp8aa, aqp8ab, and aqp8b). We use whole mount in situ hybridization to describe aqp8 genes spatiotemporal expression pattern, and the results show that aqp8ab mRNA is detectable mainly in the zebrafish embryonic intestine. To reveal the details of aqp8ab distribution, histological sections are employed. Transverse sections indicate that aqp8ab mRNA expression is more intense in the layer lining the intestinal cavity. Knockout of aqp8ab using the CRISPR/Cas9 system induces intestine development defects and abnormal formation of intestinal lumen. In addition, aqp8ab mRNA significantly rescues the intestine defects in the aqp8ab mutant. These results indicate that aqp8ab is required in the intestine development of zebrafish.

Foxc1a regulates zebrafish vascular integrity and brain vascular development through targeting amotl2a and ctnnb1.

Accumulating evidences have pointed that foxc1a is essential for vascular development and integrity maintenance through regulating the expression of downstream genes and interacting with signaling pathways. However, the underling cellular and molecular mechanisms of foxc1a in regulating vascular development remain undetermined. Based on two different foxc1a mutant zebrafish lines (foxc1anju18 and foxc1anju19 which generated predicted truncated foxc1a proteins with 50aa and 315aa respectively), we found that around 30 % of foxc1anju18 zebrafish exhibited severe vascular developmental defects with obvious hemorrhage in hindbrain and trunk at embryonic stages. Confocal imaging analysis revealed that the formation of middle cerebral vein (MCeV), intra-cerebral central arteries (CtAs) and dorsal longitudinal vein (DLV) of brain vessels was significantly blocked in foxc1anju18enbryos. Injection of exogenous full length and foxc1anju19 truncated foxc1a mRNA both rescued the deficiency of foxc1anju18 embryos. Transcriptome analysis revealed 186 DEGs in foxc1anju18 zebrafish among which amotl2a and ctnnb1 expression were reduced and functionally associated with adherens junctions. Dual-Luciferase assays validated amotl2a and ctnnb1 were both directly transactivated by foxc1a. Rescue experiments demonstrated that amotl2a was mainly responsible for the vascular integrity caused by foxc1a mutation and also coordinated with ctnnb1 to regulate brain vascular development. Our data point to a novel clue that foxc1a regulates vascular integrity and brain vascular development through targeting amotl2a and ctnnb1.

[Establishment of a microtubule-fluorescent fusion protein mosaically labeled zebrafish motor neuron system].

Motor neurons are an important type of neurons that control movement. The transgenic fluorescent protein (FP)-labeled motor neurons of zebrafish line is disadvantageous for studying the morphogenesis of motor neurons. For example, the individual motor neuron is indistinguishable in this transgenic line due to the high density of the motor neurons and the interlaced synapses. In order to optimize the in vivo imaging methods for the analysis of motor neurons, the present study was aimed to establish a microtubule-fluorescent fusion protein mosaic system that can label motor neurons in zebrafish. Firstly, the promotor of mnx1, which was highly expressed in the spinal cord motor neurons, was subcloned into pDestTol2pA2 construct combined with the GFP-α-Tubulin fusion protein sequence by Gateway cloning technique. Then the recombinant constructs were co-injected with transposase mRNA into the 4-8 cell zebrafish embryos. Confocal imaging analysis was performed at 72 hours post fertilization (hpf). The results showed that the GFP fusion protein was expressed in three different types of motor neurons, and individual motor neurons were mosaically labeled. Further, the present study analyzed the correlation between the injection dose and the number and distribution of the mosaically labeled neurons. Fifteen nanograms of the recombinant constructs were suggested as an appropriate injection dose. Also, the defects of the motor neuron caused by the down-regulation of insm1a and kif15 were verified with this system. These results indicate that our novel microtubule-fluorescent fusion protein mosaic system can efficiently label motor neurons in zebrafish, which provides a more effective model for exploring the development and morphogenesis of motor neurons. It may also help to decipher the mechanisms underlying motor neuron disease and can be potentially utilized in drug screening.

Sema6D Regulates Zebrafish Vascular Patterning and Motor Neuronal Axon Growth in Spinal Cord.

Vessels and nerves are closely associated in anatomy as well as functions. Accumulating evidences have demonstrated that axon-guiding signals may affect endothelial cells migration and path finding, which is crucial for the patterning of both the complex vascular network and neural system. However, studies regarding the functional overlap between vascular and neuronal orchestrating are still incomplete. Semaphorin6D (Sema6D) belongs to the Semaphorin family and has been identified as an important regulating factor in diverse biological processes. Its roles in vascular development are still unclear. Here, we confirmed that sema6D is enriched in neural system and blood vessels of zebrafish embryos by in situ hybridization. Then, the deficiency of sema6D caused by specific antisense morpholino-oligonucleotides (MO) led to dramatic path finding defects in both intersegmental vessels (ISVs) and primary motor neurons (PMNs) of spinal cord in zebrafish embryos. Furthermore, these defective phenotypes were confirmed in F0 generation of sema6D knockouts and rescue experiments by overexpression of sema6D mRNA in sema6D morphants. These data collectively indicate that sema6D regulates zebrafish vascular patterning and motor neuronal axon growth in the spinal cord, which might be of great therapeutical use to regulate vessel and nerve guidance in the relevant diseases that affect both systems.

MicroRNA-22 coordinates vascular and motor neuronal pathfinding via sema4 during zebrafish development.

A precise guiding signal is crucial to orchestrate directional migration and patterning of the complex vascular network and neural system. So far, limited studies have reported the discovery and functions of microRNAs (miRNAs) in guiding vascular and neural pathfinding. Currently, we showed that the deficiency of miRNA-22a, an endothelial-enriched miRNA, caused dramatic pathfinding defects both in intersegmental vessels (ISVs) and primary motor neurons (PMNs) in zebrafish embryos. Furthermore, we found the specific inhibition of miR-22a in endothelial cells (ECs) resulted in patterning defects of both ISVs and PMNs. Neuronal block of miR-22a mainly led to axonal defects of PMN. Sema4c was identified as a potential target of miR-22a through transcriptomic analysis and in silico analysis. Additionally, a luciferase assay and EGFP sensor assay confirmed the binding of miR-22a with 3'-UTR of sema4c. In addition, downregulation of sema4c in the miR-22a morphants significantly neutralized the aberrant patterning of vascular and neural networks. Then we demonstrated that endothelial miR-22a regulates PMNs axonal navigation. Our study revealed that miR-22a acted as a dual regulatory cue coordinating vascular and neuronal patterning, and expanded the repertoire of regulatory molecules, which might be of use therapeutically to guide vessels and nerves in the relevant diseases.

Dual-Specificity Phosphatase 14 Regulates Zebrafish Hair Cell Formation Through Activation of p38 Signaling Pathway.

Most cases of acquired hearing loss are due to degeneration and subsequent loss of cochlear hair cells. Whereas mammalian hair cells are not replaced when lost, in zebrafish, they constantly renew and regenerate after injury. However, the molecular mechanism among this difference remains unknown. Dual-specificity phosphatase 14 (DUSP14) is an important negative modulator of mitogen-activated protein kinase (MAPK) signaling pathways. Our study was to investigate the effects of DUSP14 on supporting cell development and hair cell regeneration and explore the potential mechanism. Our results showed that dusp14 gene is highly expressed in zebrafish developing neuromasts and otic vesicles. Behavior analysis showed that dusp14 deficiency resulted in hearing defects in zebrafish larvae, which were reversed by dusp14 mRNA treatment. Moreover, knockdown of dusp14 gene caused a significant decrease in the number of neuromasts and hair cells in both neuromast and otic vesicle, mainly due to the inhibition of the proliferation of supporting cells, which results in a decrease in the number of supporting cells and ultimately in the regeneration of hair cells. We further found significant changes in a series of MAPK pathway genes through transcriptome sequencing analysis of dusp14-deficient zebrafish, especially mapk12b gene in p38 signaling. Additionally, inhibiting p38 signaling effectively rescued all phenotypes caused by dusp14 deficiency, including hair cell and supporting cell reduction. These results suggest that DUSP14 might be a key gene to regulate supporting cell development and hair cell regeneration and is a potential target for the treatment of hearing loss.

FGF binding protein 3 is required for spinal cord motor neuron development and regeneration in zebrafish.

Fibroblast growth factor binding protein 3 (Fgfbp3) have been known to be crucial for the process of neural proliferation, differentiation, migration, and adhesion. However, the specific role and the molecular mechanisms of fgfbp3 in regulating the development of motor neurons remain unclear. In this study, we have investigated the function of fgfbp3 in morphogenesis and regeneration of motor neuron in zebrafish. Firstly, we found that fgfbp3 was localized in the motor neurons and loss of fgfbp3 caused the significant decrease of the length and branching number of the motor neuron axons, which could be partially rescued by fgfbp3 mRNA injection. Moreover, the fgfbp3 knockdown (KD) embryos demonstrated similar defects of motor neurons as identified in fgfbp3 knockout (KO) embryos. Furthermore, we revealed that the locomotion and startle response of fgfbp3 KO embryos were significantly restricted, which were partially rescued by the fgfbp3 overexpression. In addition, fgfbp3 KO remarkably compromised axonal regeneration of motor neurons after injury. Lastly, the malformation of motor neurons in fgfbp3 KO embryos was rescued by overexpressing drd1b or neurod6a, respectively, which were screened by transcriptome sequencing. Taken together, our results provide strong cellular and molecular evidence that fgfbp3 is crucial for the axonal morphogenesis and regeneration of motor neurons in zebrafish.

SARS-CoV-2 spike protein causes blood coagulation and thrombosis by competitive binding to heparan sulfate.

Thrombotic complication has been an important symptom in critically ill patients with COVID-19. It has not been clear whether the virus spike (S) protein can directly induce blood coagulation in addition to inflammation. Heparan sulfate (HS)/heparin, a key factor in coagulation process, was found to bind SARS-CoV-2 S protein with high affinity. Herein, we found that the S protein can competitively inhibit the bindings of antithrombin and heparin cofactor II to heparin/HS, causing abnormal increase in thrombin activity. SARS-CoV-2 S protein at a similar concentration (~10 µg/mL) as the viral load in critically ill patients can cause directly blood coagulation and thrombosis in zebrafish model. Furthermore, exogenous heparin/HS can significantly reduce coagulation caused by S protein, pointing to a potential new direction to elucidate the etiology of the virus and provide fundamental support for anticoagulant therapy especially for the COVID-19 critically ill patients.

Comparative analysis of codon usage patterns in chloroplast genomes of five Miscanthus species and related species.

Miscanthus is not only a perennial fiber biomass crop, but also valuable breeding resource for its low-nutrient requirements, photosynthetic efficiency and strong adaptability to environment. In the present study, the codon usage patterns of five different Miscanthus plants and other two related species were systematically analyzed. The results indicated that the cp genomes of the seven representative species were preference to A/T bases and A/T-ending codons. In addition, 21 common high-frequency codons and 4-11 optimal codons were detected in the seven chloroplast genomes. The results of ENc-plot, PR2-plot and neutrality analysis revealed the codon usage patterns of the seven chloroplast genomes are influenced by multiple factors, in which nature selection is the main influencing factor. Comparative analysis of the codon usage frequencies between the seven representative species and four model organisms suggested that Arabidopsis thaliana, Populus trichocarpa and Saccharomyces cerevisiae could be considered as preferential appropriate exogenous expression receptors. These results might not only provide important reference information for evolutionary analysis, but also shed light on the way to improve the expression efficiency of exogenous gene in transgenic research based on codon optimization.

Comparative transcriptome analysis and identification of candidate adaptive evolution genes of Miscanthus lutarioriparius and Miscanthus sacchariflorus.

Miscanthus species are perennial C4 grasses that are considered promising energy crops because of their high biomass yields, excellent adaptability and low management costs. Miscanthus lutarioriparius and Miscanthus sacchariflorus are closely related subspecies that are distributed in different habitats. However, there are only a few reports on the mechanisms by which Miscanthus adapts to different environments. Here, comparative transcriptomic and morphological analyses were used to study the evolutionary adaptation of M. lutarioriparius and M. sacchariflorus to different habitats. In total, among 7586 identified orthologs, 2060 orthologs involved in phenylpropanoid biosynthesis and plant hormones were differentially expressed between the two species. Through an analysis of the Ka/Ks ratios of the orthologs, we estimated that the divergence time between the two species was approximately 4.37 Mya. In addition, 37 candidate positively selected orthologs (PSGs) that played important roles in the adaptation of these species to different habitats were identified. Then, the expression levels of 20 PSGs in response to flooding and drought stress were analyzed, and the analysis revealed significant changes in their expression levels. These results facilitate our understanding of the evolutionary adaptation to habitats and the speciation of M. lutarioriparius and M. sacchariflorus. We hypothesise that lignin synthesis genes are the main cause of the morphological differences between the two species. In summary, the plant nonspecific phospholipase C gene family and the receptor-like protein kinase gene family played important roles in the evolution of these two species. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01030-1.

Genetic, transcriptional, and regulatory landscape of monolignol biosynthesis pathway in Miscanthus × giganteus.

BACKGROUND: Miscanthus × giganteus is widely recognized as a promising lignocellulosic biomass crop due to its advantages of high biomass production, low environmental impacts, and the potential to be cultivated on marginal land. However, the high costs of bioethanol production still limit the current commercialization of lignocellulosic bioethanol. The lignin in the cell wall and its by-products released in the pretreatment step is the main component inhibiting the enzymatic reactions in the saccharification and fermentation processes. Hence, genetic modification of the genes involved in lignin biosynthesis could be a feasible strategy to overcome this barrier by manipulating the lignin content and composition of M. × giganteus. For this purpose, the essential knowledge of these genes and understanding the underlying regulatory mechanisms in M. × giganteus is required. RESULTS: In this study, MgPAL1, MgPAL5, Mg4CL1, Mg4CL3, MgHCT1, MgHCT2, MgC3'H1, MgCCoAOMT1, MgCCoAOMT3, MgCCR1, MgCCR2, MgF5H, MgCOMT, and MgCAD were identified as the major monolignol biosynthetic genes in M. × giganteus based on genetic and transcriptional evidence. Among them, 12 genes were cloned and sequenced. By combining transcription factor binding site prediction and expression correlation analysis, MYB46, MYB61, MYB63, WRKY24, WRKY35, WRKY12, ERF021, ERF058, and ERF017 were inferred to regulate the expression of these genes directly. On the basis of these results, an integrated model was summarized to depict the monolignol biosynthesis pathway and the underlying regulatory mechanism in M. × giganteus. CONCLUSIONS: This study provides a list of potential gene targets for genetic improvement of lignocellulosic biomass quality of M. × giganteus, and reveals the genetic, transcriptional, and regulatory landscape of the monolignol biosynthesis pathway in M. × giganteus.

The Requirement of Sox2 for the Spinal Cord Motor Neuron Development of Zebrafish.

Sex-determining region Y box 2 (Sox2), expressed in neural tissues, plays an important role as a transcription factor not only in the pluripotency and proliferation of neuronal cells but also in the opposite function of cell differentiation. Nevertheless, how Sox2 is linked to motor neuron development remains unknown. Here, we showed that Sox2 was localized in the motor neurons of spinal cord by in situ hybridization and cell separation, which acted as a positive regulator of motor neuron development. The deficiency of Sox2 in zebrafish larvae resulted in abnormal PMN development, including truncated but excessively branched CaP axons, loss of MiP, and increase of undifferentiated neuron cells. Importantly, transcriptome analysis showed that Sox2-depleted embryos caused many neurogenesis, axonogenesis, axon guidance, and differentiation-related gene expression changes, which further support the vital function of Sox2 in motor neuron development. Taken together, these data indicate that Sox2 plays a crucial role in the motor neuron development by regulating neuron differentiation and morphology of neuron axons.

Selection of suitable reference genes for quantitive real-time PCR normalization in Miscanthus lutarioriparia.

Miscanthus lutarioriparia, which is found widespread in China, has attracted great attention as a most potential bioenergy plant for years. The quantitative real time PCR (RT-qPCR) has appeared as a sensitive and powerful technique to measure gene expression in living organisms during different development stages. In this study, we evaluated ten candidate genes, including 25S ribosomal RNA gene (25S rRNA), actin1 gene (ACT1), carotenoid-binding protein 20 gene (CBP20), glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH), Ubiquitin gene (UBQ), eukaryotic elongation factor 1-αgene (eEF-1α), α-tubulin gene (α-TUB), ß-tubulin gene (ß-TUB), eukaryotic translation initiation factor 4α-1 gene (eIF-4α) and NAC domain protein gene(NAC) in a series of 30 M. lutarioriparia samples followed by statistical algorithms geNorm and Normfinder to analyze the gene expression stability. The results indicated that eIF-4αand UBQ were the most stable expressed genes while CBP20 showed as the least stable among all the samples. Based on above research, we recommend that at least two top-ranked reference genes should be employed for expression data normalization. The best genes selected in this study will provide a starting point to select reference genes in the future in other tissues and under other experimental conditions in this energy crop candidate.

Confounding effect of benign pulmonary diseases in selecting volatile organic compounds as markers of lung cancer.

Lung cancer (LC) is a leading cause of cancer-related morbidity and mortality globally, and exhaled breath testing has been considered as a fast, convenient and non-invasive way to diagnose LC in its early stages. Volatile organic compounds (VOCs), as markers of LC in exhaled breath, have been widely investigated for cancer diagnosis. However, few studies have reported on the interference of benign pulmonary diseases (BPD) in the selection of VOC markers for LC. During this study, 207 samples were analyzed using thermal desorption instrumentation/gas chromatography/mass spectrometry (TD-GCMS) to detect C6-C30 VOCs, and all samples were divided into four groups: LC group, BPD group, lung disease (LD) group (including LC group and BPD group) and healthy group. To make up for the deficiency of detecting low carbon hydrocarbons (

Transcriptomics and proteomics reveal genetic and biological basis of superior biomass crop Miscanthus.

Miscanthus is a rhizomatous C4 grass which is considered as potential high-yielding energy crop with the low-nutrient requirements, high water-use efficiency, and capability of C mitigation. To better understand the genetic basis, an integrative analysis of the transcriptome and proteome was performed to identify important genes and pathways involved in Miscanthus leaves. At the transcript level, 64,663 transcripts in M. lutarioriparius, 97,043 in M. sacchariflorus, 97,043 in M. sinensis, 67,323 in M. floridulus and 70,021 in M. × giganteus were detected by an RNA sequencing approach. At the protein level, 1964 peptide-represented proteins were identified and 1933 proteins differed by 1.5-fold or more in their relative abundance, as indicated by iTRAQ (isobaric tags for relative and absolute quantitation) analysis. Phylogenies were constructed from the nearly taxa of Miscanthus. A large number of genes closely related to biomass production were found. And SSR markers and their corresponding primers were derived from Miscanthus transcripts and 90% of them were successfully detected by PCR amplification among Miacanthus species. These similarities and variations on the transcriptional and proteomic level between Miscanthus species will serve as a resource for research in Miscanthus and other lignocellulose crops.

Nuclear DNA content in Miscanthus sp. and the geographical variation pattern in Miscanthus lutarioriparius.

The genome sizes of five Miscanthus species, including 79 accessions of M. lutarioriparius, 8 of M. floridulus, 6 of M. sacchariflorus, 7 of M. sinensis, and 4 of M. × giganteus were examined using flow cytometry. The overall average nuclear DNA content were 4.256 ± 0.6 pg/2C in M. lutarioriparius, 5.175 ± 0.3 pg/2C in M. floridulus, 3.956 ± 0.2 pg/2C in M. sacchariflorus, 5.272 ± 0.2 pg/2C in M. sinensis, and 6.932 ± 0.1 pg/2C in M. × giganteus. Interspecific variation was found at the diploid level, suggesting that DNA content might be a parameter that can be used to differentiate the species. Tetraploid populations were found in M. lutarioriparius, M. sacchariflorus, and M. sinensis, and their DNA content were 8.34 ± 1.2, 8.52, and 8.355 pg, respectively. The association between the DNA content of M. lutarioriparius, collected from representative ranges across the Yangtze River, and its geographic distribution was statistically analyzed. A consistent pattern of DNA content variation in 79 M. lutarioriparius accessions across its entire geographic range was found in this study. Along the Yangtze River, the DNA content of M. lutarioriparius tended to increase from the upstream to the downstream areas, and almost all tetraploids gathered in the upstream area extended to coastal regions.