Stigma receptors control intraspecies and interspecies barriers in Brassicaceae.

Flowering plants have evolved numerous intraspecific and interspecific prezygotic reproductive barriers to prevent production of unfavourable offspring1. Within a species, self-incompatibility (SI) is a widely utilized mechanism that rejects self-pollen2,3 to avoid inbreeding depression. Interspecific barriers restrain breeding between species and often follow the SI × self-compatible (SC) rule, that is, interspecific pollen is unilaterally incompatible (UI) on SI pistils but unilaterally compatible (UC) on SC pistils1,4-6. The molecular mechanisms underlying SI, UI, SC and UC and their interconnections in the Brassicaceae remain unclear. Here we demonstrate that the SI pollen determinant S-locus cysteine-rich protein/S-locus protein 11 (SCR/SP11)2,3 or a signal from UI pollen binds to the SI female determinant S-locus receptor kinase (SRK)2,3, recruits FERONIA (FER)7-9 and activates FER-mediated reactive oxygen species production in SI stigmas10,11 to reject incompatible pollen. For compatible responses, diverged pollen coat protein B-class12-14 from SC and UC pollen differentially trigger nitric oxide, nitrosate FER to suppress reactive oxygen species in SC stigmas to facilitate pollen growth in an intraspecies-preferential manner, maintaining species integrity. Our results show that SRK and FER integrate mechanisms underlying intraspecific and interspecific barriers and offer paths to achieve distant breeding in Brassicaceae crops.

Transcriptome analysis reveals the potential lncRNA-mRNA modules involved in genetic male sterility and fertility of Chinese cabbage (brassica rapa L. ssp. pekinensis).

BACKGROUND: Long non-coding RNAs (lncRNAs) play a crucial role in regulating gene expression vital for the growth and development of plants. Despite this, the role of lncRNAs in Chinese cabbage (Brassica rapa L. ssp. pekinensis) pollen development and male fertility remains poorly understood. RESULTS: In this study, we characterized a recessive genic male sterile mutant (366-2 S), where the delayed degradation of tapetum and the failure of tetrad separation primarily led to the inability to form single microspores, resulting in male sterility. To analyze the role of lncRNAs in pollen development, we conducted a comparative lncRNA sequencing using anthers from the male sterile mutant line (366-2 S) and the wild-type male fertile line (366-2 F). We identified 385 differentially expressed lncRNAs between the 366-2 F and 366-2 S lines, with 172 of them potentially associated with target genes. To further understand the alterations in mRNA expression and explore potential lncRNA-target genes (mRNAs), we performed comparative mRNA transcriptome analysis in the anthers of 366-2 S and 366-2 F at two stages. We identified 1,176 differentially expressed mRNAs. Remarkably, GO analysis revealed significant enrichment in five GO terms, most notably involving mRNAs annotated as pectinesterase and polygalacturonase, which play roles in cell wall degradation. The considerable downregulation of these genes might contribute to the delayed degradation of tapetum in 366-2 S. Furthermore, we identified 15 lncRNA-mRNA modules through Venn diagram analysis. Among them, MSTRG.9997-BraA04g004630.3 C (ß-1,3-glucanase) is associated with callose degradation and tetrad separation. Additionally, MSTRG.5212-BraA02g040020.3 C (pectinesterase) and MSTRG.13,532-BraA05g030320.3 C (pectinesterase) are associated with cell wall degradation of the tapetum, indicating that these three candidate lncRNA-mRNA modules potentially regulate pollen development. CONCLUSION: This study lays the foundation for understanding the roles of lncRNAs in pollen development and for elucidating their molecular mechanisms in regulating male sterility in Chinese cabbage.

Genome-Wide Identification, Expression, and Protein Analysis of CKX and IPT Gene Families in Radish (Raphanus sativus L.) Reveal Their Involvement in Clubroot Resistance.

Cytokinins (CKs) are a group of phytohormones that are involved in plant growth, development, and disease resistance. The isopentenyl transferase (IPT) and cytokinin oxidase/dehydrogenase (CKX) families comprise key enzymes controlling CK biosynthesis and degradation. However, an integrated analysis of these two gene families in radish has not yet been explored. In this study, 13 RsIPT and 12 RsCKX genes were identified and characterized, most of which had four copies in Brassica napus and two copies in radish and other diploid Brassica species. Promoter analysis indicated that the genes contained at least one phytohormone or defense and stress responsiveness cis-acting element. RsIPTs and RsCKXs were expanded through segmental duplication. Moreover, strong purifying selection drove the evolution of the two gene families. The expression of the RsIPT and RsCKX genes distinctly showed diversity in different tissues and developmental stages of the root. Expression profiling showed that RsCKX1-1/1-2/1-3 was significantly upregulated in club-resistant materials during primary infection, suggesting their vital function in clubroot resistance. The interaction network of CKX proteins with similar 3D structures also reflected the important role of RsCKX genes in disease resistance. This study provides a foundation for further functional study on the IPT and CKX genes for clubroot resistance improvement in Raphanus.

Comparative Metabolome and Transcriptome Analysis Reveals the Defense Mechanism of Chinese Cabbage (Brassica rapa L. ssp. pekinensis) against Plasmodiophora brassicae Infection.

Chinese cabbage (Brassica rapa L. ssp. pekinensis) ranks among the most cultivated and consumed vegetables in China. A major threat to its production is Plasmodiophora brassicae, which causes large root tumors, obstructing nutrient and water absorption and resulting in plant withering. This study used a widely targeted metabolome technique to identify resistance-related metabolites in resistant (DH40R) and susceptible (DH199S) Chinese cabbage varieties after inoculation with P. brassicae. This study analyzed disease-related metabolites during different periods, identifying 257 metabolites linked to resistance, enriched in the phenylpropanoid biosynthesis pathway, and 248 metabolites linked to susceptibility, enriched in the arachidonic acid metabolism pathway. Key metabolites and genes in the phenylpropanoid pathway were upregulated at 5 days post-inoculation (DPI), suggesting their role in disease resistance. In the arachidonic acid pathway, linoleic acid and gamma-linolenic acid were upregulated at 5 and 22 DPI in resistant plants, while arachidonic acid was upregulated at 22 DPI in susceptible plants, leading to the conclusion that arachidonic acid may be a response substance in susceptible plants after inoculation. Many genes enriched in these pathways were differentially expressed in DH40R and DH199S. The research provided insights into the defense mechanisms of Chinese cabbage against P. brassicae through combined metabolome and transcriptome analysis.

Cytological and transcriptomic analyses provide insights into the pollen fertility of synthetic allodiploid Brassica juncea hybrids.

KEY MESSAGE: Imbalanced chromosomes and cell cycle arrest, along with down-regulated genes in DNA damage repair and sperm cell differentiation, caused pollen abortion in synthetic allodiploid Brassica juncea hybrids. Interspecific hybridization is considered to be a major pathway for species formation and evolution in angiosperms, but the occurrence of pollen abortion in the hybrids is common, prompting us to recheck male gamete development in allodiploid hybrids after the initial combination of different genomes. Here, we investigated the several key meiotic and mitotic events during pollen development using the newly synthesised allodiploid B. juncea hybrids (AB, 2n = 2× = 18) as a model system. Our results demonstrated the partial synapsis and pairing of non-homologous chromosomes concurrent with chaotic spindle assembly, affected chromosome assortment and distribution during meiosis, which finally caused difference in genetic constitution amongst the final tetrads. The mitotic cell cycle arrest during microspore development resulted in the production of anucleate pollen cells. Transcription analysis showed that sets of key genes regulating cyclin (CYCA1;2 and CYCA2;3), DNA damage repair (DMC1, NBS1 and MMD1), and ubiquitin-proteasome pathway (SINAT4 and UBC) were largely downregulated at the early pollen meiosis stages, and those genes involved in sperm cell differentiation (DUO1, PIRL1, PIRL9 and LBD27) and pollen wall synthesis (PME48, VGDH11 and COBL10) were mostly repressed at the late pollen mitosis stages in the synthetic allodiploid B. juncea hybrids (AB). In conclusion, this study elucidated the related mechanisms affecting pollen fertility during male gametophyte development at the cytological and transcriptomic levels in the synthetic allodiploid B. juncea hybrids.

Integrative Transcriptome, miRNAs, Degradome, and Phytohormone Analysis of Brassica rapa L. in Response to Plasmodiophora brassicae.

Clubroot is an infectious root disease caused by Plasmodiophora brassicae in Brassica crops, which can cause immeasurable losses. We analyzed integrative transcriptome, small RNAs, degradome, and phytohormone comprehensively to explore the infection mechanism of P. brassicae. In this study, root samples of Brassica rapa resistant line material BrT24 (R-line) and susceptible line material Y510-9 (S-line) were collected at four different time points for cytological, transcriptome, miRNA, and degradome analyses. We found the critical period of disease resistance and infection were at 0-3 DAI (days after inoculation) and 9-20 DAI, respectively. Based on our finding, we further analyzed the data of 9 DAI vs. 20 DAI of S-line and predicted the key genes ARF8, NAC1, NAC4, TCP10, SPL14, REV, and AtHB, which were related to clubroot disease development and regulating disease resistance mechanisms. These genes are mainly related to auxin, cytokinin, jasmonic acid, and ethylene cycles. We proposed a regulatory model of plant hormones under the mRNA-miRNA regulation in the critical period of P. brassicae infection by using the present data of the integrative transcriptome, small RNAs, degradome, and phytohormone with our previously published results. Our integrative analysis provided new insights into the regulation relationship of miRNAs and plant hormones during the process of disease infection with P. brassicae.

bra-miR167a Targets ARF8 and Negatively Regulates Arabidopsis thaliana Immunity against Plasmodiophora brassicae.

Clubroot is a soil-borne disease caused by Plasmodiophora brassicae, which can seriously affect the growth and production of cruciferous crops, especially Chinese cabbage crops, worldwide. At present, few studies have been conducted on the molecular mechanism of this disease's resistance response. In this experiment, we analyzed the bioinformation of bra-miR167a, constructed a silencing vector (STTM167a) and an overexpression vector (OE-miR167a), and transformed them to Arabidopsis to confirm the role of miR167a in the clubroot resistance mechanism of Arabidopsis. Afterwards, phenotype analysis and expression level analysis of key genes were conducted on transgenic plants. From the result, we found that the length and number of lateral roots of silence transgenic Arabidopsis STTM167a was higher than that of WT and OE-miR167a. In addition, the STTM167a transgenic Arabidopsis induced up-regulation of disease resistance-related genes (PR1, PR5, MPK3, and MPK6) at 3 days after inoculation. On the other hand, the auxin pathway genes (TIR1, AFB2, and AFB3), which are involved in maintaining the balance of auxin/IAA and auxin response factor (ARF), were down-regulated. These results indicate that bra-miR167a is negative to the development of lateral roots and auxins, but positive to the expression of resistance-related genes. This also means that the STTM167a can improve the resistance of clubroot by promoting lateral root development and the level of auxin, and can induce resistance-related genes by regulating its target genes. We found a positive correlation between miR167a and clubroot disease, which is a new clue for the prevention and treatment of clubroot disease.

BrWAX2 plays an essential role in cuticular wax biosynthesis in Chinese cabbage (Brassica rapa L. ssp. pekinensis).

KEY MESSAGE: Map-based cloning was used to identify the BrWAX2 gene, which was involved in the cuticular wax biosynthesis. The malfunction of BrWAX2 together with other reduced expression of genes in alkane-forming pathway caused the glossy phenotype. Cuticular wax covering the outer plant surface plays various roles in protecting against biotic and abiotic stresses. Wax-less mutant shows glossy in stem and leaf surface and plays important roles in enriching Chinese cabbage germplasm resources for breeding brilliant green varieties. However, genes responsible for the glossy trait in Chinese cabbage are rarely reported. In this study, we identified a glossy Chinese cabbage line Y1211-1. Genetic analysis indicated that the glossy trait in Y1211-1 was controlled by a single recessive locus, BrWAX2 (Brassica rapa WAX 2). Using bulked segregant sequencing (BSA-Seq) and kompetitive allele-specific PCR (KASP) assays, BrWAX2 was fine-mapped to an interval of 100.78 kb. Functional annotation analysis, expression analysis, and sequence variation analysis revealed that Bra032670, homologous to CER1 in Arabidopsis, was the most likely candidate gene for BrWAX2. The gene Bra032670 was absent in glossy mutant. Cuticular wax composition analysis and RNA-Seq analysis suggested that the absence of BrWAX2 together with the decreased expression of other genes in alkane-forming pathway reduced the wax amount and caused the glossy phenotype. Furthermore, we developed and validated the functional marker BrWAX2-sp for BrWAX2. Overall, these results provide insight into the molecular mechanism underlying cuticular wax biosynthesis and reveal valuable information for marker-assisted selection (MAS) breeding in Chinese cabbage.

BrWAX3, Encoding a ß-ketoacyl-CoA Synthase, Plays an Essential Role in Cuticular Wax Biosynthesis in Chinese Cabbage.

In this study, we identified a novel glossy mutant from Chinese cabbage, named SD369, and all wax monomers longer than 26 carbons were significantly decreased. Inheritance analysis revealed that the glossy trait of SD369 was controlled by a single recessive locus, BrWAX3. We fine-mapped the BrWAX3 locus to an interval of 161.82 kb on chromosome A09. According to the annotated genome of Brassica rapa, Bra024749 (BrCER60.A09), encoding a ß-ketoacyl-CoA synthase, was identified as the candidate gene. Expression analysis showed that BrCER60.A09 was significantly downregulated in all aerial organs of glossy plants. Subcellular localization indicated that the BrCER60.A09 protein functions in the endoplasmic reticulum. A 5567-bp insertion was identified in exon 1 of BrCER60.A09 in SD369, which lead to a premature stop codon, thus causing a loss of function of the BrCER60.A09 enzyme. Moreover, comparative transcriptome analysis revealed that the 'cutin, suberine, and wax biosynthesis' pathway was significantly enriched, and genes involved in this pathway were almost upregulated in glossy plants. Further, two functional markers, BrWAX3-InDel and BrWAX3-KASP1, were developed and validated. Overall, these results provide a new information for the cuticular wax biosynthesis and provide applicable markers for marker-assisted selection (MAS)-based breeding of Brassica rapa.

A Role for MINIYO and QUATRE-QUART2 in the Assembly of RNA Polymerases II, IV, and V in Arabidopsis.

RNA polymerases IV and V (Pol IV and Pol V) are required for the generation of noncoding RNAs in RNA-directed DNA methylation (RdDM). Their subunit compositions resemble that of Pol II. The mechanism and accessory factors involved in their assembly remain largely unknown. In this study, we identified mutant alleles of MINIYO (IYO), QUATRE-QUART2 (QQT2), and NUCLEAR RNA POLYMERASE B11/D11/E11 (NRPB/D/E11) that cause defects in RdDM in Arabidopsis thaliana We found that Pol IV-dependent small interfering RNAs and Pol V-dependent transcripts were greatly reduced in the mutants. NRPE1, the largest subunit of Pol V, failed to associate with other Pol V subunits in the iyo and qqt2 mutants, suggesting the involvement of IYO and QQT2 in Pol V assembly. In addition, we found that IYO and QQT2 were mutually dependent for their association with the NRPE3 subassembly prior to the assembly of Pol V holoenzyme. Finally, we show that IYO and QQT2 are similarly required for the assembly of Pol II and Pol IV. Our findings reveal IYO and QQT2 as cofactors for the assembly of Pol II, Pol IV, and Pol V and provide mechanistic insights into how RNA polymerases are assembled in plants.

Non-homologous chromosome pairing during meiosis in haploid Brassica rapa.

KEY MESSAGE: Cytological observations of chromosome pairing showed that evolutionarily genome duplication might reshape non-homologous pairing during meiosis in haploid B. rapa. A vast number of flowering plants have evolutionarily undergone whole genome duplication (WGD) event. Typically, Brassica rapa is currently considered as an evolutionary mesohexaploid, which has more complicated genomic constitution among flowering plants. In this study, we demonstrated chromosome behaviors in haploid B. rapa to understand how meiosis proceeds in presence of a single homolog. The findings showed that a diploid-like chromosome pairing was generally adapted during meiosis in haploid B. rapa. Non-homologous chromosomes in haploid cells paired at a high-frequency at metaphase I, over 50% of examined meiocytes showed at least three pairs of bivalents then equally segregated at anaphase I during meiosis. The fluorescence immunostaining showed that the cytoskeletal configurations were mostly well-organized during meiosis. Moreover, the expressed genes identified at meiosis in floral development was rather similar between haploid and diploid B. rapa, especially the expression of known hallmark genes pivotal to chromosome synapsis and homologous recombination were mostly in haploid B. rapa. Whole-genome duplication evolutionarily homology of genomic segments might be an important reason for this phenomenon, which would reshape the first division course of meiosis and influence pollen development in plants.

Geminiviral V2 Protein Suppresses Transcriptional Gene Silencing through Interaction with AGO4.

In plants, RNA-directed DNA methylation (RdDM)-mediated transcriptional gene silencing (TGS) is a natural antiviral defense against geminiviruses. Several geminiviral proteins have been shown to target the enzymes related to the methyl cycle or histone modification; however, it remains largely unknown whether and by which mechanism geminiviruses directly inhibit RdDM-mediated TGS. In this study, we showed that Cotton leaf curl Multan virus (CLCuMuV) V2 directly interacts with Nicotiana benthamiana AGO4 (NbAGO4) and that the L76S mutation in V2 (V2L76S) abolishes such interaction. We further showed that V2, but not V2L76S, can suppresses RdDM and TGS. Silencing of NbAGO4 inhibits TGS, reduces the viral methylation level, and enhances CLCuMuV DNA accumulation. In contrast, the V2L76S substitution mutant attenuates CLCuMuV infection and enhances the viral methylation level. These findings reveal that CLCuMuV V2 contributes to viral infection by interaction with NbAGO4 to suppress RdDM-mediated TGS in plants.IMPORTANCE In plants, the RNA-directed DNA methylation (RdDM) pathway is a natural antiviral defense mechanism against geminiviruses. However, how geminiviruses counter RdDM-mediated defense is largely unknown. Our findings reveal that Cotton leaf curl Multan virus V2 contributes to viral infection by interaction with NbAGO4 to suppress RNA-directed DNA methylation-mediated transcriptional gene silencing in plants. Our work provides the first evidence that a geminiviral protein is able to directly target core RdDM components to counter RdDM-mediated TGS antiviral defense in plants, which extends our current understanding of viral counters to host antiviral defense.

Comparative transcriptome analysis in Chinese cabbage (Brassica rapa ssp. pekinesis) for DEGs of Ogura-, Polima-CMS and their shared maintainer.

Cytoplasmic male sterility (CMS) is maternally inherited trait, which hinders the ability to produce viable pollen in plants. It serves as a useful tool for hybrid seed production via exploiting heterosis in crops. The molecular mechanism of CMS and fertility restoration has been investigated in different crops. However, limited number of reports is available on comparison of Ogura- and Polima-CMS with their shared maintainer in Chinese cabbage. We performed transcript profiling of sterile Ogura CMS (Tyms), Polima CMS (22m2) and their shared maintainer line (231-330) with an aim to identify genes associated with male sterility. In this work, we identified 912, 7199 and 6381 DEGs (Differentially Expressed Genes) in 22m2 Vs Tyms, 231-330 VS 22m2 and 231-330 Vs Tyms, respectively. The GO (Gene Ontology) annotation and KEGG pathway analysis suggested that most of the DEGs were involved in pollen development, carbon metabolism, lipase activity, lipid binding, penta-tricopeptide repeat (PPR), citrate cycle and oxidative phosphorylation, which were down-regulated in both CMS lines. This result will provide an important resource for further understanding of functional pollen development, the CMS mechanism and to improve molecular breeding in Chinese cabbage.

The increased expression of GABA receptors within the arcuate nucleus is associated with high intraocular pressure.

Purpose: To investigate the relationship between intraocular pressure (IOP) and GABA receptors within the arcuate nucleus (ARC). Methods: In the chronic high IOP rat model, ibotenic acid (IBO) was injected to induce impairment of the ARC, and IOP was measured at the 0, 1, 2, 3, and 4 week time points with a Tono-Pen. To assess the expression of GABA-A/B receptors within the ARC under persistent high IOP, we performed immunofluorescence (IF) and immunohistochemical (IHC) staining at 2 weeks and 4 weeks. Furthermore, we treated the ARC with GABA-A/B receptor antagonists separately, and IOP was evaluated, as well as retinal ganglion cell apoptosis in the chronic high IOP rat model. In the following induced high IOP animal model, the expression of GABA-A/B receptors within the ARC was evaluated in DBA/2J mice which developed progressive eye abnormalities spontaneously that closely mimic human hereditary glaucoma. Results: Compared with the control group, statistically significant downregulation of IOP was noted due to the IBO injection into the ARC at the 2, 3, and 4 week time points (p<0.05). Persistent high IOP elicited increased expression of the GABA-A/B receptors in the ARC compared with the control group (p<0.01). In addition, treatment with GABA-A/B receptor antagonists separately caused a decrease in the IOP, along with reduced retinal ganglion cell apoptosis (p<0.01). In the DBA/2J mice, the expression of the GABA receptors was statistically significantly increased (p<0.01). Conclusions: GABA-A/B receptors in the ARC may be involved in regulation of IOP, and pathologically high IOP affects the expression of GABA-A/B receptors in the ARC.

Co-expression of vasoactive intestinal peptide and protein gene product 9.5 surrounding the lumen of human Schlemm's canal.

Previous studies of aqueous humor outflow have focused primarily on resistance at the trabecular meshwork (TM), and little is known about the function of Schlemm's canal (SC). Here, we aimed to investigate whether SC is innervated by the peripheral nervous system. Ten eye specimens from eight donors were processed for histological analysis. CD31 was used to identify SC, after which we used protein gene product (PGP) 9.5 as a marker to detect nerve fibers around SC. We then characterized the nerves by double staining for PGP9.5 and sympathetic nerve markers, such as tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DßH), or the parasympathetic marker vasoactive intestinal peptide (VIP), as well as sensory nerve marker calcitonin gene-related peptide (CGRP) and vesicular glutamate transporter 2 (VGLUT2). Immunohistochemistry and immunofluorescence were also performed to detect the expression of γ-epithelial Na+ channel (γ-ENaC) in SC. We found that different markers were expressed in the anterior chamber angle, with the luminal surface of SC were only positive stained for PGP9.5, VIP, and γ-ENaC. CGRP and VGLUT2 were expressed in TM and scleral spur (SS), whereas TH and DßH were absent in both TM and SC. Furthermore, PGP9.5 was co-expressed with VIP and γ-ENaC in the region surrounding the SC as well as in SS. Our findings indicate that the peripheral nerves anatomically spread in the tissues around the SC and the local nerve fibers may be parasympathetic or sensory rather than sympathetic.

Quercetin exerts cardiovascular protective effects in LPS-induced dysfunction in vivo by regulating inflammatory cytokine expression, NF-κB phosphorylation, and caspase activity.

Impaired myocardial contractile function, one of the well-documented features of sepsis, contributes greatly to the high rate of mortality. Quercetin is widely accepted as a potential antioxidant and free radical scavenger. Epidemiologic studies have suggested that an increase in the intake of dietary Quercetin can reduce the risk of cardiac disease. However, presently there is no report yet on the influence of Quercetin on LPS-induced myocardial dysfunction in vivo. Cardiovascular protective effects of Quercetin on LPS-induced sepsis in mice were measured after intragastric administration, using normal saline as a positive control. Quercetin pretreatment significantly alleviated LPS-induced cardiac abnormalities in mice. The histopathologic findings in the present study justify the findings reported from the biochemical analyses. Our observation from the present research work reveals that Quercetin suppressed the production of proinflammatory cytokines at different levels, such as TNF-α and IL-1ß, and inhibits the activation of I-κB phosphorylation, whereas the total content was not affected. Apoptotic pathways are related to Quercetin protection in the development of myocardial dysfunction. In conclusion, our findings demonstrate the adjuvant potentials of Quercetin for clinical sepsis treatment.

Gadolinium-enhanced 7.0 T magnetic resonance imaging assessment of the aqueous inflow in rat eyes in vivo.

The goal of this study was to calculate the anterior chamber volume and assess aqueous inflow in rat eyes in vivo, under anesthetic condition. Gadolinium-contrast agent (Gd-DTPA, 234.5 mg/ml) was administered to Sprague-Dawley rat eyes via anterior chamber injection or instillation of 234.5 or 117.25 mg/ml Gd-DTPA in 0.2% azone as eye drops, and changes of Gd signal visualized by 7.0 T magnetic resonance imaging (MRI). The safety of local application of Gd-DTPA and azone were performed after MRI scanning. The anterior chamber injection of Gd-DTPA (234.5 mg/ml) group was used for anterior chamber volume and aqueous inflow calculating. Serial changes in Gd-DTPA relative concentration in the anterior chamber was determined based on the initial Gd signal gray values and the initial relative concentration of Gd-DTPA after anterior chamber Gd-DTPA injection. The mean aqueous inflow in rat eyes in vivo was assessed based on changes in Gd-DTPA relative concentration and the anterior chamber volume. Eye drops of Gd-DTPA (234.5 mg/ml) in 0.2% azone readily allowed safe assessment of the aqueous inflow by 7.0 T MRI. Under anesthetic condition in vivo, the mean anterior chamber volume (ACV) in rats was 8493.6 ± 657.4 µm3, no differences were observed in the aqueous inflow measured by topical instillation of 234.5 mg/ml Gd-DTPA in 0.2% azone (0.182 ± 0.011 µl/min) between that measured by anterior chamber injection (0.165 ± 0.041 µl/min, P > 0.05), Timolol reduced aqueous inflow to 0.124 ± 0.020 µl/min (P < 0.05). Our results indicated that Gd-enhanced 7.0 T MRI allows evaluation of the Gd signal variation and anterior chamber volume in rats in vivo. The aqueous inflow calculation via non-invasive local application of 234.5 mg/ml Gd-DTPA can be assessed by the variability of relative concentration of Gd-DTPA in anterior chamber and ACV in vivo, under anesthetic condition.

Extracellular vesicles-mediated transfer of miR-208a/b exaggerate hypoxia/reoxygenation injury in cardiomyocytes by reducing QKI expression.

In this study, we tested the hypothesis that extracellular vesicles (EVs)-mediated transfer of miR-208a/b can exacerbate apoptosis of cardiomyocytes (CMs) induced by hypoxia/reoxygenation (H/R) injury by reducing the expression of the RNA-binding protein Quaking (QKI). EVs were isolated from culture medium of hypoxic H9c2 cells (EVs-H). In in vitro H9c2 cell model, the EVs-H could be taken up by normoxic CMs and exacerbated cell apoptosis induced by H/R injury. In addition, miR-208a and miR-208b were enriched in EVs-H. Suppression of miR-208a and miR-208b loading significantly suppressed the detrimental effect of EVs-H on H/R injury in H9c2 cells. Inhibition of endogenous miR-208a and miR-208b restored QKI5 and QKI6 after H/R treatment. Dual-luciferase assay confirmed direct bindings between miR-208a/b and QKI 3'UTR. Functionally, QKI5 overexpression significantly suppressed H/R-induced CM apoptosis and suppressed the enhancing effect of EVs-H on CM apoptosis. Therefore, we infer that EVs-mediated transfer of miR-208a/b can exaggerate H/R injury in CMs by reducing QKI expression. This represents a previously unrecognized pathway of H/R injury in CMs.

An RdDM-independent function of Pol V transcripts in gene regulation and plant defence.

RNA polymerase V (Pol V) and Pol IV are known to be specialized for RNA-directed DNA methylation (RdDM). Here we report that Pol V, but not Pol IV, regulates hundreds of genes including jasmonic acid-responsive genes and confers plant defence to Botrytis cinerea and Spodoptera exigua. About half of the Pol V-regulated genes are associated with Pol V transcripts (PVTs). We thus hypothesized that some PVTs could regulate gene expression in an RdDM-independent manner. To test this hypothesis, we studied three PVTs, PVT-ERF5a/b and PVT-ERF6, as models. PVT-ERF5a/b and PVT-ERF6 are transcribed from the upstream regions of ERF5 and ERF6 and positively regulate their transcription, thereby regulating plant defence. Such regulation involves PVT-dependent H3K4me3 deposition and requires the DRD1-DMS3-RDM1 complex that mediates Pol V recruitment to the target loci. These findings highlight an unprecedented role for PVTs in regulating gene transcription, apart from serving as scaffold RNAs to direct DNA methylation.