The OsMYC2-JA feedback loop regulates diurnal flower-opening time via cell wall loosening in rice.

Diurnal flower-opening time (DFOT), the time of spikelet opening during the day, is an important trait for hybrid rice (Oryza sativa L.) seed production. Hybrids between indica and japonica rice varieties have strong heterosis, but the parental lines usually have different, nonoverlapping DFOTs. This reduces the success of hybrid seed production in crosses between indica and japonica subspecies, thus hindering the utilization of indica and japonica inter-subspecies heterosis. However, little is known about the molecular mechanisms regulating DFOT in rice. Here, we obtained japonica rice lines with a DFOT 1.5 h earlier than the wild type by overexpressing OsMYC2, a gene encoding a key transcription factor in the jasmonate (JA) signaling pathway. OsMYC2 is activated by JA signaling and directly regulates the transcription of genes related to JA biosynthesis and cell wall metabolism. Overexpressing OsMYC2 led to significantly increased JA contents and decreased cellulose and hemicellulose contents in lodicule cells, as well as the softening of lodicule cell walls. This may facilitate the swelling of lodicules, resulting in early diurnal flower-opening. These results suggest that the OsMYC2-JA feedback loop regulates DFOT in rice via cell wall remodeling. These findings shed light on the understanding of regulatory mechanism of the DFOT of plants, which should promote the development of indica and japonica varieties suitable for hybrid rice breeding.

Genome-wide QTL and eQTL mapping reveal genes associated with growth rate trait of the Pacific white shrimp (Litopenaeus vannamei).

BACKGROUND: Growth rate is a crucial economic trait for farmed animals, but the genetic regulation of this trait is largely unknown in non-model organisms such as shrimp. RESULTS: In this study, we performed genome-wide phenotypic quantitative trait loci (QTL) and expression quantitative trait loci (eQTL) mapping analyses to identify genes affecting the growth rate of Pacific white shrimp (Litopenaeus vannamei), which is the most commercially-farmed crustacean worldwide. We used RNA-sequencing of 268 individuals in a mapping population, and subsequently validated our findings through gene silencing and shrimp growth experiments. We constructed a high-density genetic linkage map comprising 5533 markers spanning 44 linkage groups, with a total distance of 6205.75 cM and an average marker interval of 1.12 cM. Our analyses identified 11 QTLs significantly correlated with growth rate, and 117,525 eQTLs. By integrating QTL and eQTL data, we identified a gene (metalloreductase STEAP4) highly associated with shrimp growth rate. RNA interference (RNAi) analysis and growth experiments confirmed that STEAP4 was significantly correlated with growth rate in L. vannamei. CONCLUSIONS: Our results indicate that the comprehensive analysis of QTL and eQTL can effectively identify genes involved in complex animal traits. This is important for marker-assisted selection (MAS) of animals. Our work contributes to the development of shrimp breeding and available genetic resources.

Controlling diurnal flower-opening time by manipulating the jasmonate pathway accelerates development of indica-japonica hybrid rice breeding.

Inter-subspecific indica-japonica hybrid rice (Oryza sativa) has the potential for increased yields over traditional indica intra-subspecies hybrid rice, but limited yield of F1 hybrid seed production (FHSP) hinders the development of indica-japonica hybrid rice breeding. Diurnal flower-opening time (DFOT) divergence between indica and japonica rice has been a major contributing factor to this issue, but few DFOT genes have been cloned. Here, we found that manipulating the expression of jasmonate (JA) pathway genes can effectively modulate DFOT to improve the yield of FHSP in rice. Treating japonica cultivar Zhonghua 11 (ZH11) with methyl jasmonate (MeJA) substantially advanced DFOT. Furthermore, overexpressing the JA biosynthesis gene OPDA REDUCTASE 7 (OsOPR7) and knocking out the JA inactivation gene CHILLING TOLERANCE 1 (OsHAN1) in ZH11 advanced DFOT by 1- and 2-h respectively; and knockout of the JA signal suppressor genes JASMONATE ZIM-DOMAIN PROTEIN 7 (OsJAZ7) and OsJAZ9 resulted in 50-min and 1.5-h earlier DFOT respectively. The yields of FHSP using japonica male-sterile lines GAZS with manipulated JA pathway genes were significantly higher than that of GAZS wildtype. Transcriptome analysis, cytological observations, measurements of elastic modulus and determination of cell wall components indicated that the JA pathway could affect the loosening of the lodicule cell walls by regulating their composition through controlling sugar metabolism, which in turn influences DFOT. This research has vital implications for breeding japonica rice cultivars with early DFOT to facilitate indica-japonica hybrid rice breeding.

OmpU and OmpC are the key OMPs for Litopenaeus vannamei hemocyanin recognizes Vibrio parahaemolyticus.

Hemocyanin is a multifunctional protein present in arthropods and mollusks, responsible for oxygen transport and participating in multiple roles of immune defense including antibacterial activity. However, the molecular basis of how hemocyanin recognizes pathogens and exerts antibacterial activity remains poorly understood. In the present study, the pull-down assay was used to isolate Vibrio parahaemolyticus outer membrane proteins (OMPs) that bind to Litopenaeus vannamei hemocyanin. Two interacting OMPs bands were determined as OmpC and OmpU, and the heterogeneous interaction between hemocyanin and the two OMPs was further confirmed by far-Western blot. After construction of ompC and ompU deletion mutants, we found that the agglutinating activity and antibacterial activity of hemocyanin significantly decreased compared to the wild-type strain. After hemocyanin treatment, we identified four intracellular proteins of V. parahaemolyticus, including fructose-bisphosphate aldolase and ribosomal proteins could interact with rOmpC and rOmpU, respectively. Furthermore, we found that the mRNA levels of ompC, ompU, fbaA, rpsB and rpsC significantly decreased after hemocyanin treatment. These findings indicated that OmpC and OmpU are the key targets for L. vannamei hemocyanin recognize pathogens and exert its antibacterial activity.

Function of hemocyanin-mediated succinate dehydrogenase in glucose metabolism and immunity of Penaeus vannamei.

Succinate dehydrogenase (SDH) is a crucial enzyme in the tricarboxylic acid cycle (TCA) and has established roles in immune function. However, the understanding of SDH in Penaeus vannamei, particularly its involvement in immune responses, is currently limited. Through affinity proteomics, a potential interaction between hemocyanin (HMC) and SDH in shrimp has been identified. The successful cloning of PvSDH in this study has revealed a high degree of evolutionary conservation. Additionally, it has been found that hemocyanin regulates SDH not only at the transcriptional and enzymatic levels but also through confirmed protein-protein interactions observed via Co-immunoprecipitation (CoIP) assay. Moreover, by combining PvHMC knockdown and Vibrio parahaemolyticus challenge, it was demonstrated that fumaric acid, a product of SDH, enhances the host's immune resistance to pathogen infection by modulating the expression of antimicrobial peptides. This research provides new insights into HMC as a crucial regulator of SDH, potentially impacting glycometabolism and the dynamics of immune responses.

MicroRNA sequencing analysis reveals immune responses in hepatopancreas of Fenneropenaeus penicillatus under white spot syndrome virus infection.

White Spot Disease is one of the most harmful diseases of the red tail shrimp, which can cause devastating economic losses due to the highest mortality up to 100% within a few days. MicroRNAs (miRNAs) are large class of small noncoding RNAs with the ability to post-transcriptionally repress the translation of target mRNAs. MiRNAs are considered to have a significant role in the innate immune response of crustaceans, particularly in relation to antiviral defense mechanisms. Numerous crustacean miRNAs have been verified to be required in host immune defense against viral infection, however, till present, the miRNAs functions of F. penicillatus defense WSSV infection have not been studied yet. Here in this study, for the first time, miRNAs involved in the F. penicillatus immune defense against WSSV infection were identified using high-throughput sequencing platform. A total of 432 miRNAs were obtained including 402 conserved miRNAs and 30 novel predicted miRNAs. Comparative analysis between the WSSV-challenged group and the control group revealed differential expression of 159 microRNAs in response to WSSV infection. Among these, 48 were up-regulated and 111 were down-regulated. Ten candidate MicroRNAs associated with immune activities were randomly selected for qRT-PCR analysis, which confirming the expression profiling observed in the MicroRNA sequencing data. As a result, most differentially expressed miRNAs were down-regulated lead to increase the expression of various target genes that mediated immune reaction defense WSSV infection, including genes related to signal transduction, Complement and coagulation cascade, Phagocytosis, and Apoptosis. Furthermore, the genes expression of the key members in Toll and Imd signaling pathways and apoptotic signaling were mediated by microRNAs to activate host immune responses including apoptosis against WSSV infection. These results will help to understand molecular defense mechanism against WSSV infection in F. penicillatus and to develop an effective WSSV defensive strategy in shrimp farming.

Dynamic Alternative Polyadenylation during Litopenaeus Vannamei Metamorphosis Development.

As an important mechanism in the post-transcriptional regulation of eukaryotic gene expression, alternative polyadenylation (APA) plays a key role in biological processes such as cell proliferation and differentiation. However, the role and dynamic pattern of APA during Litopenaeus vannamei metamorphosis are poorly understood. Here, RNA-seq data covering from the embryo to the maturation (16 time points) of L. vannamei were utilized. We identified 247 differentially expressed APA events between early and adult stages, and through fuzzy mean clustering analysis, we discovered five dynamic APA patterns. Among them, the gradual elongation of the 3'UTR is the major APA pattern that changes over time, and its genes are enriched in the pathways of protein and energy metabolism. Finally, we constructed mRNA-miRNA and PPI networks and detected several central miRNAs that may regulate L. vannamei development. Our results revealed the complex APA mechanisms in L. vannamei metamorphosis, shedding new light on post-transcriptional regulation of crustacean metamorphosis.

Chromosomal-level genome assembly and annotation of the tropical sea cucumber Holothuria scabra.

Holothuria scabra, a commercially valuable yet ecologically vulnerable tropical holothuroid, has experienced a severe decline in its wild populations, especially in China. Genomic resources are crucial for the development of effective genomic breeding projects and stock conservation strategies to restore these natural populations. Until now, a high-quality, chromosome-level reference genome for H. scabra has not been available. Here, we employed Oxford Nanopore and Hi-C sequencing technologies to assemble and annotate a high-quality, chromosome-level reference genome of H. scabra. The final genome comprised 31 scaffolds with a total length of 1.19 Gb and a scaffold N50 length of 53.52 Mb. Remarkably, 1,191.67 Mb (99.95%) of the sequences were anchored to 23 pseudo-chromosomes, with the longest one spanning 79.75 Mb. A total of 34,418 protein-coding genes were annotated in the final genome, with BUSCO analysis revealing 98.01% coverage of metazoa_odb10 genes, marking a significant improvement compared to the previous report. These chromosome-level sequences and annotations will provide an essential genomic basis for further investigation into molecular breeding and conservation management of H. scabra.

Biochemical indicators, cell apoptosis, and metabolomic analyses of the low-temperature stress response and cold tolerance mechanisms in Litopenaeus vannamei.

The cold tolerance of Litopenaeus vannamei is important for breeding in specific areas. To explore the cold tolerance mechanism of L. vannamei, this study analyzed biochemical indicators, cell apoptosis, and metabolomic responses in cold-tolerant (Lv-T) and common (Lv-C) L. vannamei under low-temperature stress (18 °C and 10 °C). TUNEL analysis showed a significant increase in apoptosis of hepatopancreatic duct cells in L. vannamei under low-temperature stress. Biochemical analysis showed that Lv-T had significantly increased levels of superoxide dismutase (SOD) and triglycerides (TG), while alanine aminotransferase (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH-L), and uric acid (UA) levels were significantly decreased compared to Lv-C (p < 0.05). Metabolomic analysis displayed significant increases in metabolites such as LysoPC (P-16:0), 11beta-Hydroxy-3,20-dioxopregn-4-en-21-oic acid, and Pirbuterol, while metabolites such as 4-Hydroxystachydrine, Oxolan-3-one, and 3-Methyldioxyindole were significantly decreased in Lv-T compared to Lv-C. The differentially regulated metabolites were mainly enriched in pathways such as Protein digestion and absorption, Central carbon metabolism in cancer and ABC transporters. Our study indicate that low temperature induces damage to the hepatopancreatic duct of shrimp, thereby affecting its metabolic function. The cold resistance mechanism of Lv-T L. vannamei may be due to the enhancement of antioxidant enzymes and lipid metabolism.

Genome-wide analysis of ATP-binding cassette (ABC) transporter in Penaeus vannamei and identification of two ABC genes involved in immune defense against Vibrio parahaemolyticus by affecting NF-κB signaling pathway.

The ATP-binding cassette (ABC) transporters have crucial roles in various biological processes such as growth, development and immune defense in eukaryotes. However, the roles of ABC transporters in the immune system of crustaceans remain elusive. In this study, 38 ABC genes were systematically identified and characterized in Penaeus vannamei. Bioinformation analysis revealed that PvABC genes were categorized into ABC A-H eight subfamilies with 17 full-transporters, 11 half transporters and 10 soluble proteins, and multiple immunity-related cis-elements were found in gene promoter regions. Expression analysis showed that most PvABC genes were widely and highly expressed in immune-related tissues and responded to the stimulation of Vibrio parahaemolyticus. To investigate whether PvABC genes mediated innate immunity, PvABCC5, PvABCF1 and PvABCB4 were selected for dsRNA interference experiment. Knockdown of PvABCF1 and PvABCC5 not PvABCB4 increased the cumulative mortality of P. vannamei and bacterial loads in hepatopancreas after infection with V. parahaemolyticus. Further analysis showed that the PvABCF1 and PvABCC5 knockdown decreased expression levels of NF-κB pathway genes and antimicrobial peptides (AMPs). Collectively, these findings indicated that PvABCF1 and PvABCC5 might restrict V. parahaemolyticus challenge by positively regulating NF-κB pathway and then promoting the expression of AMPs, which would contribute to overall understand the function of ABC genes in innate immunity of invertebrates.

Cloning, Identification, and Functional Analysis of the Foxl2 Gene in Procambarus clarkii.

Procambarus clarkii is the most widely distributed freshwater shrimp in China, with important economic value and great potential for development. The forkheadboxL2 (Foxl2) gene has been found to be involved in the reproductive development of many crustaceans. To understand the role of the Foxl2 gene in the gonad development of P. clarkii, we designed CDS-specific primers for the P. clarkii Foxl2 (PcFoxl2) gene and cloned its CDS sequence using RT-PCR. The nucleotide and protein sequence information was then analyzed through bioinformatics analysis. The expression and subcellular localization of PcFoxl2 in various tissues were detected using qRT-PCR and in situ hybridization. The effects of PcFoxl2 knockdown on gonad development were investigated using RNA interference. The results showed that the CDS length of the PcFoxl2 gene was 1614 bp and encoded 537 amino acids. Protein sequence comparison and phylogenetic analysis showed that PcFoxl2 was the closest relative to Crayfish. qRT-PCR analysis indicated that the expression level of PcFoxl2 in the testis was significantly higher (>40 fold) than that in the ovary (p < 0.01). The in situ hybridization results showed that PcFoxl2 was expressed in both the cytoplasm and the nucleus of egg cells, and that the expression was strongest in egg cells at the early stage of yolk synthesis, while weak in the secondary oocytes. The positive signal was strongest in the spermatocyte nucleolus, while only a trace signal was observed in the cytoplasm. After interfering with the PcFoxl2 gene using dsRNA, the expression of PcFoxl2 in the RNA interference group was significantly lower than that in the control group, and this interference effect lasted for one week. Moreover, the gonad index of the experimental group was significantly lower than that of the control group (p < 0.05) after 10 days of P. clarkii cultivation following PcFoxl2 knockdown. The expression levels of the nanos and S3a genes, which are related to gonad development, decreased significantly after PcFoxl2 gene interference. The results suggest that the Foxl2 gene is involved in the growth and development of gonads, particularly in the development of testis, and is related to the early development of oocytes. This study provides a theoretical basis for the artificial breeding of P. clarkii.

Effect of mild hypothermia on renal ischemia/reperfusion injury after cardiopulmonary resuscitation in a swine model

Abstract Purpose: To investigate the effect of intravascular cooling on renal function after resuscitation. Methods: Twenty four pigs were randomized into three groups (n=8 in each group): therapeutic hypothermia group (TH group), normothermia group (NH group) and sham operation group (SHAM group). After 6 minutes of untreated VF, CPR was performed. Upon ROSC, the TH group received the intravascular cooling. The NH and SHAM group did not undergo therapeutic hypothermia. Haemodynamic parameters were recorded. The bloods were analyzed for serum creatinine (sCr), CysC and NGAL. The kidney was surgically removed observe pathologic changes under a light microscope. Results: The sCr increased in both TH and NH groups after ROSC, compared to baseline. Between two groups, the sCr and creatinine clearance (Cc) showed lower level in the TH group. The urine volume per hour in the TH group were higher during cooling. After resuscitation, NGAL and CysC in the NH group were higher than in the TH group. Under the light microscope, compared with the TH group, the renal injury was prominent in the NH group. Conclusion: Mild hypothermia had a protection to renal ischemia reperfusion injury after resuscitation.