Riboflavin Attenuates Fluoride-Induced Testicular Injury via Interleukin 17A-Mediated Classical Pyroptosis.

Male reproductive toxicity of fluoride is of great concern worldwide, yet the underlying mechanism is unclear. Pyroptosis is a novel mode of inflammatory cell death, and riboflavin with anti-inflammatory properties has the potential to protect against fluoride damage. However, it is unknown whether pyroptosis is involved in fluoride-induced testicular injury and riboflavin intervention. Here, we first found that riboflavin could alleviate fluoride-caused lower sperm quality and damaged testicular morphology by reducing pyroptosis based on a model of ICR mice treated with NaF (100 mg/L) and/or riboflavin supplementation (40 mg/L) via drinking water for 13 weeks. And then, together with the results of in vitro Leydig cell modelsm it was confirmed that the pyroptosis occurs predominantly through classical NLRP3/Caspase-1/GSDMD pathway. Furthermore, our results reveal that interleukin-17A mediates the process of pyroptosis in testes induced by fluoride and riboflavin attenuation according to the results of our established models of riboflavin- and/or fluoride-treated IL-17A knockout mice. The results not only declare a new mechanism by which fluoride induces testicular injury via interleukin 17A-mediated classical pyroptosis but also provide evidence for the potential clinical application of riboflavin as an effective therapy for fluoride toxicity.

Fluoride induces pyroptosis via IL-17A-mediated caspase-1/11-dependent pathways and Bifidobacterium intervention in testis.

Fluoride, a ubiquitous environmental pollutant, poses a significant public health threat. Our previous study revealed a correlation between fluoride-induced testicular pyroptosis and male reproductive dysfunction. However, the underlying mechanism remains unclear. Wild-type and interleukin 17A knockout mice were exposed to sodium fluoride (100 mg/L) in deionized drinking water for 18 weeks. Bifidobacterium intervention (1 × 109 CFU/mL, 0.2 mL/day, administered via gavage) commenced in the 10th week. Sperm quality, testicular morphology, key pyroptosis markers, spermatogenesis key genes, IL-17A signaling pathway, and pyroptosis pathway related genes were determined. The results showed that fluoride reduced sperm quality, damaged testicular morphology, affected spermatogenesis, elevated IL-17A levels, and induced testicular pyroptosis. Bifidobacterium intervention alleviated adverse reproductive outcomes. Fluoride-activated testicular pyroptosis through both typical and atypical pathways, with IL-17A involvement. Bifidobacterium supplementation attenuated pyroptosis by downregulating IL-17A, inhibiting NLRP3 and PYRIN-mediated caspase-1 and caspase-11 dependent pathways in testis, thereby alleviating fluoride-induced male reproductive damage. In summary, this study uncovers the mechanism underlying fluorine-induced testicular pyroptosis and illustrates the novel protecting feature of Bifidobacterium against fluoride-induced harm to male reproduction, along with its potential regulatory mechanism. These results provide fresh perspectives on treating male reproductive dysfunction resulting from fluoride or other environmental toxins.

Riboflavin alleviates fluoride-induced ferroptosis by IL-17A-independent system Xc-/GPX4 pathway and iron metabolism in testicular Leydig cells.

Fluoride is widely found in groundwater, soil, animal and plant organisms. Excessive fluoride exposure can cause reproductive dysfunction by activating IL-17A signaling pathway. However, the adverse effects of fluoride on male reproductive system and the mechanisms remain elusive. In this study, the wild type and IL-17A knockout C57BL/6J mouse were treated with 24 mg/kg·bw·d sodium fluoride and/or 5 mg/kg·bw·d riboflavin-5'-phosphate sodium for 91 days. Results showed that fluoride caused dental fluorosis, increased the levels of ROS in testicular Leydig cells and GSSG in testicular tissue, and did not affect the iron and serum hepcidin levels in testicular tissue. Riboflavin alleviated above adverse changes, whereas IL-17A does not participate in the oxidative stress-mediated reproductive toxicity of fluoride. Based on this, TM3 cells were used to verify the injury of fluoride on Leydig cells. Results showed that fluoride increased mRNA levels of ferroptosis marker SLC3A2, VDAC3, TFRC, and SLC40A1 and decreased Nrf2 mRNA levels in TM3 cells. The ferroptosis inhibitor Lip-1 and DFO were used to further investigate the relationship between male reproductive toxicity and ferroptosis induced by fluoride. We found that the fluoride-induced decrease in cell viability, increase in xCT, TFRC, and FTH protein expression, and decrease in GPX4 protein expression, can all be rescued by Lip-1 and DFO. Similar results were also observed in the riboflavin treatment group. Moreover, riboflavin mitigated fluoride-induced increases in ROS levels and SLC3A2 protein levels. In all, our work revealed that riboflavin inhibited ferroptosis in testicular Leydig cells and improved the declined male reproductive function caused by fluoride. This study provides new perspectives for revealing new male reproductive toxicity mechanisms and mitigating fluoride toxicity damage.

Occurrence and Characterization of Sclerotinia sclerotiorum Causing Fruit Rot on Sweet Cherry in Southern China.

Sweet cherry (Prunus avium L.) is widely planted in northern China due to its high economic value, and its cultivation has gradually spread south to warm regions. However, fruit rot, observed on the young fruits, poses a considerable threat to the development of sweet cherry. To determine the causal agent, morphological observation, molecular identification, and pathogenicity tests were performed on isolates obtained from diseased fruits. As a result, Sclerotinia sclerotiorum was identified as the pathogen. Pathogenicity tests on different sweet cherry cultivars indicated that 'Summit' was highly sensitive to S. sclerotiorum, whereas 'Hongmi' showed significant resistance. Besides sweet cherry, S. sclerotiorum could also infect other vegetable crops we tested, such as cowpea, soybean, tomato, and chili. Fungicide sensitivity and efficacy assays showed that both fludioxonil and pyraclostrobin can effectively inhibit the mycelial growth of S. sclerotiorum and decrease disease incidences on the young fruits of sweet cherry. Furthermore, genome sequencing resulted in a 37.8 Mb assembly of S. sclerotiorum strain ScSs1, showing abundant SNPs, InDels, and SVs with the genome of S. sclerotiorum reference strain 1980 UF-70. The above results provide an important basis for controlling the fruit rot of sweet cherry caused by S. sclerotiorum in China.

The red/far-red light photoreceptor FvePhyB regulates tissue elongation and anthocyanin accumulation in woodland strawberry.

Light is an important environmental signal that influences plant growth and development. Among the photoreceptors, phytochromes can sense red/far-red light to coordinate various biological processes. However, their functions in strawberry are not yet known. In this study, we identified an EMS mutant, named P8, in woodland strawberry (Fragaria vesca) that showed greatly increased plant height and reduced anthocyanin content. Mapping-by-sequencing revealed that the causal mutation in FvePhyB leads to premature termination of translation. The light treatment assay revealed that FvePhyB is a bona fide red/far-red light photoreceptor, as it specifically inhibits hypocotyl length under red light. Transcriptome analysis showed that the FvePhyB mutation affects the expression levels of genes involved in hormone synthesis and signaling and anthocyanin biosynthesis in petioles and fruits. The srl mutant with a longer internode is caused by a mutation in the DELLA gene FveRGA1 (Repressor of GA1) in the gibberellin pathway. We found that the P8 srl double mutant has much longer internodes than srl, suggesting a synergistic role of FvePhyB and FveRGA1 in this process. Taken together, these results demonstrate the important role of FvePhyB in regulating plant architecture and anthocyanin content in woodland strawberry.

Transcriptome Analysis Reveals the Molecular Mechanism and Responsive Genes of Waterlogging Stress in Actinidia deliciosa Planch Kiwifruit Plants.

Waterlogging stress is one of the major natural issues resulting in stunted growth and loss of agricultural productivity. Cultivated kiwifruits are popular for their rich vitamin C content and unique flavor among consumers, while commonly sensitive to waterlogging stress. The wild kiwifruit plants are usually obliged to survive in harsh environments. Here, we carried out a transcriptome analysis by high-throughput RNA sequencing using the root tissues of Actinidia deliciosa (a wild resource with stress-tolerant phenotype) after waterlogging for 0 d, 3 d, and 7 d. Based on the RNA sequencing data, a high number of differentially expressed genes (DEGs) were identified in roots under waterlogging treatment, which were significantly enriched into four biological processes, including stress response, metabolic processes, molecular transport, and mitotic organization, by gene ontology (GO) simplify enrichment analysis. Among these DEGs, the hypoxia-related genes AdADH1 and AdADH2 were correlated well with the contents of acetaldehyde and ethanol, and three transcription factors Acc26216, Acc08443, and Acc16908 were highly correlated with both AdADH1/2 genes and contents of acetaldehyde and ethanol. In addition, we found that there might be an evident difference among the promoter sequences of ADH genes from A. deliciosa and A. chinensis. Taken together, our results provide additional information on the waterlogging response in wild kiwifruit plants.

Melatonin alleviated fluoride-induced impairment of spermatogenesis and sperm maturation process via Interleukin-17A.

Fluoride-induced male reproductive failure is a major environmental and human health concern, but interventions are still lacking. Melatonin (MLT) has potential functions in regulating testicular damage and interleukin-17 (IL-17) production. This study aims to explore whether MLT can mitigate fluoride-induced male reproductive toxicity through IL-17A, and screen the potential targets. So the wild type and IL-17A knockout mice were employed and treated with sodium fluoride (100 mg/L) by drinking water and MLT (10 mg/kg.BW, intraperitoneal injection per two days starting from week 16) for 18 weeks. Bone F- concentrations, grade of dental damage, sperm quality, spermatogenic cells counts, histological morphology of testis and epididymis, and the mRNA expression of spermatogenesis and maturation, classical pyroptosis related and immune factor genes were detected respectively. The results revealed that MLT supplementations alleviated fluoride-induced impairment of spermatogenesis and maturation process, protecting the morphology of testis and epididymis through IL-17A pathway, and Tesk1 and Pten were identified as candidate targets from 29 regulation genes. Taken together, this study demonstrated a new physiological role for MLT in the protection against fluoride-induced reproductive injury and possible regulation mechanisms, which providing a useful therapeutic strategy for male reproductive function failure caused by fluoride or other environmental pollutants.

Potential Protective Effect of Riboflavin Against Pathological Changes in the Main Organs of Male Mice Induced by Fluoride Exposure.

Long-term exposure to excessive fluorine could cause damage to various tissues and organs in human and animals. However, there is no effective antidote to prevent and cure fluorosis except for avoiding fluoride intake. As an essential nutrient, riboflavin (VB2) has been identified to relieve oxidative stress and inflammation in animal tissues caused by other toxic substances, whether it can alleviate the damage caused by fluoride is unknown. For this, 32 ICR male mice were allocated to four groups of eight each. They were treated with 0 (distilled water), 100 mg/L sodium fluoride (NaF), 40 mg/L VB2, and their combination (100 mg/L NaF plus 40 mg/L VB2) via the drinking water for 90 consecutive days, respectively. The content of bone fluoride and the histomorphology of the main organs including liver, kidney, cerebral cortex, epididymis, small intestine, and colon were evaluated and pathologically scored. The results found that fluoride caused the pathological changes in liver, kidney, cerebral cortex, epididymis, small intestine, and colon at varying degrees, while riboflavin supplementation reduced significantly the accumulation of fluoride in bone, alleviated the morphological damage to cerebral cortex, epididymis, ileum, and colon. This study provides new clues for deeply exploring the mechanism of riboflavin intervention in fluorosis.

Genetic modulation of RAP alters fruit coloration in both wild and cultivated strawberry.

Fruit colour affects consumer preference and is an important trait for breeding in strawberry. Previously, we isolated the Reduced Anthocyanins in Petioles (RAP) gene encoding a glutathione S-transferase (GST) that binds anthocyanins to facilitate their transport from cytosol to vacuole in the diploid strawberry Fragaria vesca. The parent of rap was the F. vesca variety 'Yellow Wonder' that develops white fruit due to a natural mutation in the FveMYB10 gene. Here, we investigated the application potential of RAP in modulating fruit colours by overexpression of RAP in F. vesca and knockout of RAP in the cultivated strawberry Fragaria × ananassa. Unexpectedly, the RAP overexpression in Yellow Wonder background caused formation of red fruit. In addition, the red coloration occurs precociously at floral stage 10 and continues in the receptacle during early fruit development. Transcriptome analysis revealed that the anthocyanin biosynthesis genes were not up-regulated in RAP-ox; rap myb10 flowers at anthesis and largely inhibited at the turning stage in fruit, suggesting a coloration mechanism independent of FveMYB10. Moreover, we used CRISPR/Cas9 to knockout RAP in cultivated strawberry which is octoploid. Six copies of RAP were simultaneously knocked out in the T0 generation leading to the green stem and white-fruited phenotype. Several T1 progeny have segregated away the CRISPR/Cas9 transgene but maintain the green stem trait. Our results indicate that enhancing the anthocyanin transport could redirect the metabolic flux from proanthocyanidin to anthocyanin production at early developmental stages of fruit and that RAP is one promising candidate gene in fruit colour breeding of strawberry.

Reporter gene expression reveals precise auxin synthesis sites during fruit and root development in wild strawberry.

The critical role of auxin in strawberry fruit set and receptacle enlargement was demonstrated previously. While fertilization is known to trigger auxin biosynthesis, the specific tissue source of fertilization-induced auxin is not well understood. Here, the auxin reporter DR5ver2::GUS was introduced into wild strawberry (Fragaria vesca) to reveal auxin distribution in the seed and fruit receptacle pre- and post-fertilization as well as in the root. In addition, the expression of TAR and YUCCA genes coding for enzymes catalysing the two-step auxin biosynthesis pathway was investigated using their respective promoters fused to the ß-glucuronidase (GUS) reporter. Two FveTARs and four FveYUCs were shown to be expressed primarily in the endosperm and embryo inside the achenes as well as in root tips and lateral root primordia. Expression of these reporters in dissected tissues provided more detailed and precise spatial (cell and tissue) and temporal (pre- and post-fertilization) information on where auxin is synthesized and accumulates than previous studies in strawberry. Moreover, we generated CRISPR-mediated knock-out mutants of FveYUC10, the most abundant YUC in seeds; the mutants had a lower free auxin level in young fruit, but displayed no obvious morphological phenotypes. However, overexpression of FveYUC10 resulted in elongated hypocotyls in Arabidopsis caused by elevated auxin level. Overall, the study revealed auxin accumulation in the chalazal seed coat, embryo, receptacle vasculature, root tip, and lateral root primordia and highlighted the endosperm as the main auxin biosynthesis site for fruit set.

Development and Validation of an Effective CRISPR/Cas9 Vector for Efficiently Isolating Positive Transformants and Transgene-Free Mutants in a Wide Range of Plant Species.

The CRISPR/Cas9 technique is a highly valuable tool in creating new materials for both basic and applied researches. Previously, we succeeded in effectively generating mutations in Brassica napus using an available CRISPR/Cas9 vector pKSE401, while isolation of Cas9-free mutants is laborious and inefficient. Here, we inserted a fluorescence tag (sGFP) driven by the constitutive 35S promoter into pKSE401 to facilitate a visual screen of mutants. This modified vector was named pKSE401G and tested in several dicot plant species, including Arabidopsis, B. napus, Fragaria vesca (strawberry), and Glycine max (soybean). Consequently, GFP-positive plants were readily identified through fluorescence screening in all of these species. Among these GFP-positive plants, the average mutation frequency ranged from 20.4 to 52.5% in Arabidopsis and B. napus with stable transformation, and was 90.0% in strawberry and 75.0% in soybean with transient transformation, indicating that the editing efficiency resembles that of the original vector. Moreover, transgene-free mutants were sufficiently identified in Arabidopsis in the T2 generation and B. napus in the T1 generation based on the absence of GFP fluorescence, and these mutants were stably transmissible to next generation without newly induced mutations. Collectively, pKSE401G provides us an effective tool to readily identify positive primary transformants and transgene-free mutants in later generations in a wide range of dicot plant species.

Reduced Anthocyanins in Petioles codes for a GST anthocyanin transporter that is essential for the foliage and fruit coloration in strawberry.

The red color of the foliage and fruit in strawberry comes from anthocyanins stored in the vacuole; however, how this anthocyanin accumulation is regulated remains unclear. A reduced anthocyanin in petioles (rap) mutant was identified in an N-ethyl-N-nitrosourea (ENU) mutagenized population of YW5AF7, a white-fruited variety of the wild strawberry Fragaria vesca. The causative mutation was identified to be a premature stop codon in a glutathione S-transferase (GST) gene. In addition to the foliage coloration, RAP also mediates fruit pigmentation and acts downstream of the fruit-specific transcription factor FvMYB10. Among all eight GST genes in the same subfamily, RAP is most abundantly expressed in the ripening fruit. Expression analysis and transient expression assays demonstrated that RAP is the principal transporter of anthocyanins among the paralogs. Moreover, domain-swap experiments showed that both the N- and C-terminals of RAP are essential for the binding capability of anthocyanins. In addition, transient knock-down of RAP resulted in reduced fruit coloration in cultivated strawberry. Collectively, our results demonstrate that RAP encodes the principal GST transporter of anthocyanins in the strawberry foliage and fruit, and it could be modified to alter the fruit color in strawberry.

Genome re-annotation of the wild strawberry Fragaria vesca using extensive Illumina- and SMRT-based RNA-seq datasets.

The genome of the wild diploid strawberry species Fragaria vesca, an ideal model system of cultivated strawberry (Fragaria × ananassa, octoploid) and other Rosaceae family crops, was first published in 2011 and followed by a new assembly (Fvb). However, the annotation for Fvb mainly relied on ab initio predictions and included only predicted coding sequences, therefore an improved annotation is highly desirable. Here, a new annotation version named v2.0.a2 was created for the Fvb genome by a pipeline utilizing one PacBio library, 90 Illumina RNA-seq libraries, and 9 small RNA-seq libraries. Altogether, 18,641 genes (55.6% out of 33,538 genes) were augmented with information on the 5' and/or 3' UTRs, 13,168 (39.3%) protein-coding genes were modified or newly identified, and 7,370 genes were found to possess alternative isoforms. In addition, 1,938 long non-coding RNAs, 171 miRNAs, and 51,714 small RNA clusters were integrated into the annotation. This new annotation of F. vesca is substantially improved in both accuracy and integrity of gene predictions, beneficial to the gene functional studies in strawberry and to the comparative genomic analysis of other horticultural crops in Rosaceae family.