Advances in the Study of the Transcriptional Regulation Mechanism of Plant miRNAs.

MicroRNAs (miRNA) are a class of endogenous, non-coding, small RNAs with about 22 nucleotides (nt), that are widespread in plants and are involved in various biological processes, such as development, flowering phase transition, hormone signal transduction, and stress response. The transcriptional regulation of miRNAs is an important process of miRNA gene regulation, and it is essential for miRNA biosynthesis and function. Like mRNAs, miRNAs are transcribed by RNA polymerase II, and these transcription processes are regulated by various transcription factors and other proteins. Consequently, the upstream genes regulating miRNA transcription, their specific expression, and the regulating mechanism were reviewed to provide more information for further research on the miRNA regulatory mechanism and help to further understand the regulatory networks of plant miRNAs.

Quercetin Alleviates Asthma-Induced Airway Inflammation and Remodeling through Downregulating Periostin via Blocking TGF-ß1/Smad Pathway.

INTRODUCTION: The aim of the study was to discuss whether the anti-asthmatic effect of quercetin is related to periostin and the downstream molecular pathway of quercetin's anti-asthmatic effect. METHODS: We constructed asthmatic mice, sensitized by ovalbumin, and administrated different treatments into mice according to the experimental design. In this study, we mainly observed the inflammatory response, airway fibrosis, and airway hyperresponsiveness in asthmatic mice. Pathological stains (H&E, PAS, and Masson) were performed. We also detected the inflammation factors and fibrosis-related cytokines by enzyme-linked immunosorbent serologic assay. In addition, we also explored the level of periostin by enzyme-linked immunosorbent serologic assay and Western blot. At the same time, TGF-ß1/Smad pathway was also determined by Western blot. RESULTS: A high expression of periostin was found in asthmatic mice, and quercetin decreases periostin content in bronchoalveolar lavage fluid. Quercetin and OC-20 inhibit airway inflammation response, airway fibrosis, and airway hyperreactivity. Quercetin downregulated TGF-ß1/Smad pathway in the lung tissues of asthmatic mice. Anti-asthma role of quercetin is related to periostin. Then deeper mechanical study revealed that inhibiting TGF-ß1 could improve asthmatic symptoms, and quercetin exerted the protective effect on asthmatic mice through inhibition of TGF-ß1/Smad pathway. CONCLUSION: Quercetin provided a protective role against asthma via periostin, manifested by mild inflammatory infiltration, reduced goblet cell proliferation, and reduced airway fibrosis. TGF-ß1/Smad pathway is an important transduction system, participating in the protective effect of quercetin on asthma.

Characteristics of photosynthesis and vertical canopy architecture of citrus trees under two labor-saving cultivation modes using unmanned aerial vehicle (UAV)-based LiDAR data in citrus orchards.

Analyzing and comparing the effects of labor-saving cultivation modes on photosynthesis, as well as studying their vertical canopy architecture, can improve the tree structure of high-quality and high-yield citrus and selection of labor-saving cultivation modes. The photosynthesis of 1080 leaves of two labor-saving cultivation modes (wide-row and narrow-plant mode and fenced mode) comparing with the traditional mode were measured, and nitrogen content of all leaves and photosynthetic nitrogen use efficiency (PNUE) were determined. Unmanned aerial vehicle (UAV)-based light detection and ranging (LiDAR) data were used to assess the vertical architecture of three citrus cultivation modes. Results showed that for the wide-row and narrow-plant and traditional modes leaf photosynthetic CO2 assimilation rate, stomatal conductance, and transpiration rate of the upper layer were significantly higher than those of the middle layer, and values of the middle layer were markedly higher than those of the lower layer. In the fenced mode, a significant difference in photosynthetic factors between the upper and middle layers was not observed. A vertical canopy distribution had a more significant effect on PNUE in the traditional mode. Leaves in the fenced mode had distinct photosynthetic advantages and higher PNUE. UAV-based LiDAR data effectively revealed the differences in the vertical canopy architecture of citrus trees by enabling calculating the density and height percentile of the LiDAR point cloud. The point cloud densities of three cultivation modes were significantly different for all LiDAR density slices, especially at higher canopy heights. The labor-saving modes, particularly the fenced mode, had significantly higher height percentile data.

Spatio-temporal evolution and driving factors of carbon storage in the Western Sichuan Plateau.

The carbon sequestration function of the ecosystem is one of the most important functions of ecosystem service, and it of great significance to study the spatio-temporal differentiation of carbon storage for promoting regional sustainable development. Ecosystems on the Western Sichuan Plateau are highly variable, but its spatio-temporal differentiation and driving factors are not yet clear. In this study, on the basis of land use monitoring data, meteorological and demographic data interpreted from Landsat remote sensing image, and through GIS analysis tools, the carbon storage module of InVEST (Integrated Valuation of Ecosystem Services and Trade-offs) model was used to estimate carbon storage and geodetector was used to detect the driving factors of carbon storage spatial differentiation. The results show that: (1) The carbon storage increased to 1.2455 × 1010 t from 1.2438 × 1010 t in the past 20 years, the ecosystem developed in a healthy way overall. (2) Carbon storage show High-High and Low-Low aggregation characteristics, but the area decreased by 1481.81 km2 and 311.11 km2 respectively, and the spatial cluster effect gradually weakened. (3) HAI is the leading factor causing the spatio-temporal differentiation of regional carbon storage, followed by temperature and NDVI; the interaction between factors significantly enhances the spatial differentiation of carbon storage, indicating that the change of carbon storage is the result of the joint action of natural and socioeconomic factors. The results of the study provide some theoretical basis for the development of differentiated ecological regulation models and strategies, and help to promote high-quality regional development.

The miR399-CsUBC24 Module Regulates Reproductive Development and Male Fertility in Citrus.

MicroRNA399 (miR399) regulates phosphate homeostasis in plants by down-regulating the expression of PHOSPHATE2 (PHO2, or UBC24 encoding the ubiquitin-conjugating E2 enzyme). We previously identified CsmiR399a.1 in a small RNA sequencing screen of a male-sterile somatic cytoplasmic hybrid (or cybrid) of pummelo (Citrus grandis). Here, we report that miR399 affects reproductive development and male fertility in citrus. Down-regulation of CsmiR399a.1 using a short tandem target mimic (STTM) led to abnormal floral development, inhibition of anther dehiscence, and decreased pollen fertility. When grown in inorganic phosphate (Pi)-sufficient conditions, CsmiR399a.1-STTM plants had lower total phosphorus content in their leaves than the wild type and showed typical symptoms of Pi deficiency. In CsmiR399a.1-STTM plants, the expression of genes involved in starch metabolism and Pi homeostasis was significantly different than in the wild type. Thus, we conclude that miR399-STTM mimicked Pi deficiency, disturbed starch metabolism, and was responsible for pollen grain collapse in the transgenic lines. We identified CsUBC24, a citrus homolog of Arabidopsis (Arabidopsis thaliana) AtUBC24 (PHO2), as a target of CsmiR399a.1 that physically interacts with the floral development regulators SEPALLATA family (CsSEP1.1, CsSEP1.2, and CsSEP3) and the anther dehiscence regulator INDUCER OF CBF EXPRESSION1 (CsICE1). We hypothesize that CsUBC24 downregulates the CsSEPs, which disrupts the floral meristem identity regulatory network and leads to developmental abnormalities in flowers. By interacting with CsICE1, CsUBC24 disturbs stomate function on the anther surface, which inhibits anther dehiscence. These findings indicate that a miR399-based mechanism influences both reproductive development and male fertility in citrus.

Genome-scale mRNA and small RNA transcriptomic insights into initiation of citrus apomixis.

Nucellar embryony (NE) is an adventitious form of apomixis common in citrus, wherein asexual embryos initiate directly from nucellar cells surrounding the embryo sac. NE enables the fixation of desirable agronomic traits and the production of clonal offspring of virus-free rootstock, but impedes progress in hybrid breeding. In spite of the great importance of NE in citrus breeding and commercial production, little is understood about the underlying molecular mechanisms. In this study, the stages of nucellar embryo initiation (NEI) were determined for two polyembryonic citrus cultivars via histological observation. To explore the genes and regulatory pathways involved in NEI, we performed mRNA-seq and sRNA-seq analyses of ovules immediately prior to and at stages during NEI in the two pairs of cultivars. A total of 305 differentially expressed genes (DEGs) were identified between the poly- and monoembryonic ovules. Gene ontology (GO) analysis revealed that several processes are significantly enriched based on DEGs. In particular, response to stress, and especially response to oxidative stress, was over-represented in polyembryonic ovules. Nearly 150 miRNAs, comprising ~90 conserved and ~60 novel miRNAs, were identified in the ovules of either cultivar pair. Only two differentially expressed miRNAs (DEMs) were identified, of which the novel miRN23-5p was repressed whereas the targets accumulated in the polyembryonic ovules. This integrated study on the transcriptional and post-transcriptional regulatory profiles between poly- and monoembryonic citrus ovules provides new insights into the mechanism of NE, which should contribute to revealing the regulatory mechanisms of plant apomixis.

High-throughput sequencing and degradome analysis reveal altered expression of miRNAs and their targets in a male-sterile cybrid pummelo (Citrus grandis).

BACKGROUND: G1 + HBP is a male sterile cybrid line with nuclear genome from Hirado Buntan pummelo (C. grandis Osbeck) (HBP) and mitochondrial genome from "Guoqing No.1" (G1, Satsuma mandarin), which provides a good opportunity to study male sterility and nuclear-cytoplasmic cross talk in citrus. High-throughput sRNA and degradome sequencing were applied to identify miRNAs and their targets in G1 + HBP and its fertile type HBP during reproductive development. RESULTS: A total of 184 known miRNAs, 22 novel miRNAs and 86 target genes were identified. Some of the targets are transcription factors involved in floral development, such as auxin response factors (ARFs), SQUAMOSA promoter binding protein box (SBP-box), MYB, basic region-leucine zipper (bZIP), APETALA2 (AP2) and transport inhibitor response 1 (TIR1). Eight target genes were confirmed to be sliced by corresponding miRNAs using 5' RACE technology. Based on the sequencing abundance, 42 differentially expressed miRNAs between sterile line G1 + HBP and fertile line HBP were identified. Differential expression of miRNAs and their target genes between two lines was validated by quantitative RT-PCR, and reciprocal expression patterns between some miRNAs and their targets were demonstrated. The regulatory mechanism of miR167a was investigated by yeast one-hybrid and dual-luciferase assays that one dehydrate responsive element binding (DREB) transcription factor binds to miR167a promoter and transcriptionally repress miR167 expression. CONCLUSION: Our study reveals the altered expression of miRNAs and their target genes in a male sterile line of pummelo and highlights that miRNA regulatory network may be involved in floral bud development and cytoplasmic male sterility in citrus.

Genomewide analysis of small RNAs in nonembryogenic and embryogenic tissues of citrus: microRNA- and siRNA-mediated transcript cleavage involved in somatic embryogenesis.

Somatic embryogenesis (SE) is a process of somatic cells becoming dedifferentiated and generating embryos. SE has been widely used in biotechnology as a powerful way of regeneration and a model system for studying plant embryogenesis, but the controlling mechanisms of SE are far from clear. Here, we show the genomewide profiles of miRNAs/siRNAs and their target genes in nonembryogenic and embryogenic tissues of 'Valencia' sweet orange. By high-throughput sequencing (HTS) of small RNAs and RNA degradome tags, we identified 50 known and 45 novel miRNAs, 130 miniature inverted-repeat transposable elements (MITEs) derived, 94 other and 235 phased small interfering RNAs (siRNAs), as well as 203 target genes. The majority of the abundantly expressed miRNAs/siRNAs exhibit lower expression levels in embryogenic callus (EC) or during SE process than in nonembryogenic callus (NEC), which is supposed to derepress the target genes that are involved in development and stress response, thus to activate the biological processes required for cell differentiation. However, the conserved csi-miR156a/b, miR164b and 171c directed suppression of specific transcription factors (TFs) are supposed to inactivate the postembryonic growth thus to maintain normal SE. In this study, miRNA- and siRNA-mediated silencing of target genes was found under sophisticated regulation in citrus SE system; the enhancement effect of specific conserved miRNAs on SE was discussed, providing new clues for future investigation of mechanisms that control SE.

iTRAQ-based quantitative proteomics analysis revealed alterations of carbohydrate metabolism pathways and mitochondrial proteins in a male sterile cybrid pummelo.

Comprehensive and quantitative proteomic information on citrus floral bud is significant for understanding male sterility of the cybrid pummelo (G1+HBP) with nuclear genome of HBP and foreign mitochondrial genome of G1. Scanning electron microscopy and transmission electron microscopy analyses of the anthers showed that the development of pollen wall in G1+HBP was severely defective with a lack of exine and sporopollenin formation. Proteomic analysis was used to identify the differentially expressed proteins between male sterile G1+HBP and fertile type (HBP) with the aim to clarify their potential roles in anther development and male sterility. On the basis of iTRAQ quantitative proteomics, we identified 2235 high-confidence protein groups, 666 of which showed differentially expressed profiles in one or more stages. Proteins up- or down-regulated in G1+HBP were mainly involved in carbohydrate and energy metabolism (e.g., pyruvate dehydrogenase, isocitrate dehydrogenase, ATP synthase, and malate dehydrogenase), nucleotide binding (RNA-binding proteins), protein synthesis and degradation (e.g., ribosome proteins and proteasome subunits). Additionally, the proteins located in mitochondria also showed changed expression patterns. These findings provide a valuable inventory of proteins involved in floral bud development and contribute to elucidate the mechanism of cytoplasmic male sterility in the cybrid pummelo.

[Antifibrotic effects of oxymatrine in mice with chronic viral myocarditis].

OBJECTIVE: To compare the antifibrotic effect of oxymatrine and captopril in mice with chronic viral myocarditis (CVMC) and determine the possible antifibrotic mechanism of oxymatrine in CVMC. METHODS: Ninety Balb/c mice were randomly divided into normal control group 1 (n = 10), normal control group 2(n = 10) and CVMC model group (n = 70). The mice in CVMC model group were infected with coxsackievirus B(3) (CVB(3)) on days 0, 14 and 28 to establish CVMC model. The volume of CVB(3) suspension was 0.20 ml, 0.25 ml and 0.30 ml, whose 50% tissue culture infection dose was 10(9) respectively. The mice in the normal control group 1 and 2 were given normal saline of volumes equal to those of viral suspension given to the model group at the same time points. Echocardiography and collagen specific picrosirius red staining were performed to evaluate the CVMC model on day 42 for the mice of the normal control group 1 and 8 mice of CVMC model group. The remaining mice in CVMC model group were randomly divided into CVMC control group, captopril group and oxymatrine group on day 42. From then on, the mice in captopril group and oxymatrine group were treated with captopril or oxymatrine at the dose of 100 mg/kg, by gavage once a day for 28 days, and meanwhile the mice in CVMC control group and the normal control group were given equal-volume normal saline by gastric gavage every day, for 28 days successively. All these mice were sacrificed on day 70. Heart tissue slices were stained with collagen specific picrosirius red and the collagen volume fraction (CVF) was calculated with image analysis software. The expressions of AngII and TGF-beta1 were determined by immunohistochemistry and Western blotting. RESULTS: Compared with normal group 1, the left ventricular end-diastolic internal diameters, left ventricular end-systolic internal diameters and heart rates were significantly increased in CVMC model group (P < 0.05, P < 0.01, P < 0.05, respectively), ejection fractions, fractional shortenings and peak velocity of aorta were all significantly decreased in CVMC model group (P < 0.01 for all comparisons), and CVF levels were significantly increased in CVMC group (P < 0.01) on day 42. Compared with normal control group 2, captopril group and oxymatrine group, CVF levels and the expressions of TGF-beta1 were significantly increased in CVMC control group (P < 0.01 for all comparisons) on day 70. The expressions of AngII in CVMC control group were higher than those in normal control group and captopril group (P < 0.01 for all comparisons), but there were no significant difference between oxymatrine group and CVMC control group (P > 0.05) on day 70. CONCLUSION: Oxymatrine can inhibit myocardial fibrosis in CVMC, and the mechanisms of its antifibrotic effects might be related with the down-regulation of TGF-beta1 expression.