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Heat stress (HS) adversely affects plant growth and productivity. The Class A1 HS transcription factors (HSFA1s) act as master regulators in the plant response to HS. However, how HSFA1-mediated transcriptional reprogramming is modulated during HS remains to be elucidated. Here, we report that a module formed by the microRNAs miR165 and miR166 and their target transcript, PHABULOSA (PHB), regulates HSFA1 at the transcriptional and translational levels to control plant HS responses. HS-triggered induction of MIR165/166 in Arabidopsis thaliana led to decreased expression of target genes including PHB. MIR165/166 overexpression lines and mutations in miR165/166 target genes enhanced HS tolerance, whereas miR165/166 knockdown lines and plants expressing a miR165/166-resistant form of PHB were sensitive to HS. PHB directly repressed the transcription of HSFA1s and globally modulated the expression of HS-responsive genes. PHB and HSFA1s share a common target gene, HSFA2, which is essential for activation of plant responses to HS. PHB physically interacted with HSFA1s and exerted an antagonistic effect on HSFA1 transcriptional activity. PHB and HSFA1s co-regulated transcriptome reprogramming upon HS. Together, these findings indicate that heat-triggered regulation of the miR165/166–PHB module controls HSFA1-mediated transcriptional reprogramming and plays a critical role during HS in Arabidopsis.

RCF1 is a highly conserved DEAD-box helicase found in yeast, plants and mammals. Studies about the functions of RCF1 in plants are limited. Here we uncovered the functions of RCF1 inArabidopsis thalianaas a player in pri-miRNA processing and splicing, as well as in pre-mRNA splicing. A mutant with miRNA biogenesis defects was isolated and the defect was traced to a recessive point mutation inRCF1(rcf1-4). We show that RCF1 promotes D-body formation and facilitates the interaction between pri-miRNAs and HYL1. Finally, we show that intron-containing pri-miRNAs and pre-mRNAs exhibit a global splicing defect inrcf1-4. Together, this work uncovers roles for RCF1 in miRNA biogenesis and RNA splicing inArabidopsis.One-sentence summaryRCF1 promotes not only the processing of pri-miRNAs, but also the splicing of intron-containing pri-miRNAs, therefore promotes miRNA biogenesis.

Epidemiological statistics showed that the proportion of erectile dysfunction in diabetic men was significantly higher than that in non diabetic men. In recent years, some studies have reported the prevalence of ED among people with diabetes in China, however, most of these studies are limited to certain area, small sample size, or specific age period. Therefore, it is particularly important to obtain national prevalence and related factors, which can help to improve health awareness and formulate appropriate public health policies. We searched China national knowledge internet (CNKI), Wanfang database, Pubmed, and Embase database until November 11, 2022. Pooled prevalence estimates are presented visually using forest plots. For analysis of relevant risk factors, the fixed-effect model was used to combine these ORs to obtain an overall OR when there was no significant heterogeneity between studies. Otherwise, the random effects model was applied. Finally, we found that the overall prevalence of ED among Chinese males with DM was 67.2% (95% CI: 62.0-72.4%). Sensitivity analysis indicated that there was no significant variation in pooled prevalence by excluding any of the study, confirming the stability of present results. In the present meta-analysis, we also evaluated the risk factors of ED among Chinese males with DM. We found that patients’ age (OR=1.06, 95% CI [1.03-1.10]) and DM duration (OR=1.32, 95% CI [1.18-1.47]) significantly affected the risk of ED among Chinese males with DM. In conclusions, the present meta-analysis has shown an alarmingly high prevalence of ED among Chinese males with DM. Patients’ age, and the duration of DM were significantly associated with ED risk. Therefore, it is essential to inform policy and practice to reduce the current and future burden of ED among Chinese males with DM.

Although phosphodiesterase type 5 inhibitors (PDE5i) have a therapeutic effect on diabetic erectile dysfunction, its therapeutic effect is not as good as that of non-diabetic population. In recent years, several randomized controlled trials(RCTs) found that the therapeutic effect of traditional Chinese medicine(TCM) combined with PDE5i on diabetic erectile dysfunction may be better than that of PDE5i alone, and we now conduct a systematic review and meta-analysis of these studies. We searched China national knowledge internet (CNKI), Wanfang database, Pubmed, and Embase database. Mean difference (MD) or pooled odds ratio (OR), as well as their 95% confidence intervals (CIs) were calculated. Risk bias tool was used to evaluate the quality of RCTs included in the meta-analysis. Publication bias was estimated by Begg’s funnel plots and Egger’s test. Results of this systematic review and meta-analysis revealed that the change in the IIEF-5 score of TCM + PDE5i group was significantly higher than that of PDE5i group(MD = 3.6, 95% CI [2.34, 4.86]). The clinical efficacy of TCM + PDE5i group was significantly higher than that of PDE5i group(OR = 2.86, 95% CI [1.83, 4.48]). Furthermore, the sexual satisfaction of TCM + PDE5i group was significantly higher than that of PDE5i group (OR = 3.07, 95% CI[1.63, 5.80]). There was no significant difference of the number of adverse events between TCM + PDE5i group and PDE5i group(OR = 0.93, 95% CI [0.49,1.78]). According to the meta-analysis, compared with using PDE5i alone, the regimen combined TCM plus PDE5i has more advantages in terms of IIEF-5, efficiency, and sexual satisfaction, with no increase in adverse effects for erectile dysfunction in patients with type 2 diabetes mellitus. More strict and larger sample size RCTs are needed to verify the findings of this meta-analysis.

Ban et al. (2014) proposed a nomenclature for ribosomal proteins (r-proteins) that reflects the current understanding of ribosomal protein evolution. In the past few years, this nomenclature has been widely adopted among biomedical researchers and microbiologists. This homology-based r-protein nomenclature has not been as widely adopted among plant biologists, however, presumably because r-protein nomenclature is much more complicated in plants due to gene duplication. Here, we propose compatible upgrades to the homology-guided nomenclature proposed by Ban et al. (2014) so that this naming system can be adopted for widespread use in the plant biology community. We note that Lan et al. (2022) recently proposed updated nomenclature for plant cytosolic ribosomal proteins, focused on Arabidopsis and rice. The nomenclature outlined here is an extension of that proposed by Lan et al. (2022), expanding to include organellar ribosomes and additional species, with the intent that this nomenclature can serve as a template to guide future plant genome annotations. A more detailed comparison highlighting how this naming system builds on the Ban et al. (2014) and Lan et al. (2022) nomenclatures is offered below.At this time, we request community feedback on this proposed nomenclature so that the naming system ultimately chosen represents a broad consensus. Feedback can be communicated to the this working group at plantribosome@gmail.com before July 25th, 2022. Coauthors of this letter and anyone in the scientific community expressing significant interest will then discuss this feedback as a group, reach a consensus agreement, and communicate the updated nomenclature rules through a letter to the editor (expected to be published at The Plant Cell) and the databases at TAIR and MaizeGDB.

Background Human endogenous retrovirus‐H long terminal repeat‐associating protein 2 (HHLA2) is a member of B7 family, which is upregulated in multiple tumors. However, its exact functions in non‐small cell lung cancer (NSCLC) have not been fully understood. This study aimed to investigate the biological roles of HHLA2 in human NSCLC and the relevant mechanisms. In addition, the effects of tumor cell‐derived HHLA2 on tumor‐associated macrophage (TAM) polarization were explored. Methods NSCLC cell growth, migration, and invasion were assessed by colony formation and modified Boyden chamber assays. Cell cycle and the CD163+ TAMs were examined by flow cytometry. A co‐culture model of THP‐1 macrophages and NSCLC cells was conducted to investigate the impacts of tumor cell‐derived HHLA2 on THP‐1 macrophage polarization. Moreover, a xenograft nude mouse model was established to explore the effects of HHLA2 on tumorigenesis in vivo. Results HHLA2 was upregulated in A549 and H1299 cells compared with the normal lung epithelial BEAS‐2B cells. HHLA2 deficiency inhibited NSCLC cell proliferation, migration, invasion, and induced G0/G1 phase arrest partially via inhibiting EGFR/MAPK/ERK signaling pathway. Furthermore, HHLA2 knockdown inhibited M2 polarization of TAMs via downregulating IL‐10. In addition, knockdown of HHLA2 inhibited tumor growth in vivo. Conclusion HHLA2 downregulation inhibited NSCLC growth and TAM M2 polarization. HHLA2 may serve as a therapeutic target and promising prognostic biomarker in NSCLC., HHLA2 deficiency inhibited the proliferation, migration, invasion, and induced G0/G1 arrest of A549 and H1299 cells by inactivating EGFR/MAPK/ERK signaling pathway. In addition, knockdown of HHLA2 in NSCLC cells inhibited M2 polarization of THP‐1 macrophages via decreasing the secretion of IL‐10.

Background: Recent advances in nanomedicine provided promising alternatives for tumor treatment to improve the survival and life quality of cancer patients. This study was designed to explore the insight mechanisms of the anti-tumor effects of the novel nanocomposites (NCs) MFP-FePt-GO with non-small cell lung cancer (NSCLC). Methods: A chemical co-reduction method was applied to the synthesis process of MFP-FePt-GO NCs. The chemical synthesis efficiency and morphology of the NCs were measured with spectroscope and transmission electron microscope. Colony formation assay and cell apoptosis were conducted to assess the radiosensitivity effect of NCs with radiation. Then, we detected cell mitochondrial membrane potential and reactive oxygen species (ROS) level by flow cytometry to further explore the cause of cell death. Immunofluorescence staining and Confocal were carried out to determine the DNA damage repair. A Lewis lung carcinoma animal model was used to measure safety and anti-tumor efficiency in vivo. Results: The novel NCs MFP-FePt-GO designed on a lamellar-structure magnetic graphene oxide and polyethylene glycol drug delivery system was synthesized and functionalized for co-delivery of metronidazole and 5-fluorouracil. While no severe allergies, liver and kidney damage, or drug-related deaths were observed, MFP-FePt-GO NCs promoted radiosensitivity of NSCLC cells both in vivo and in vitro. It improved the effects of radiation via activating intrinsic mitochondrial-mediated apoptosis and impairing DNA damage repair. This NCs also induced a ROS burst, which suppressed the antioxidant protein expression and induced cell apoptosis. Furthermore, MFP-FePt-GO NCs prevented NSCLC cell migration and invasion. Conclusion: MFP-FePt-GO NCs showed a synergistic anti-tumor effect with radiation to eliminate tumors. With good safety and efficacy, this novel NCs could be a potential radiosensitive agent for NSCLC patients.

The growing incidence of cancer raises an urgent need to develop effective diagnostic and therapeutic strategies. With the rapid development of nanomedicine, nanoscale metal-organic frameworks (NMOFs) presented promising potential in various biomedical applications in the last 2 decades, especially in cancer theranostics. Due to the unique features of NMOFs, including structural diversities, enormous porosity, multifunctionality and biocompatibility, they have been widely used to deliver imaging contrast agents and therapeutic drugs. Moreover, multiple types of contrast agents, anti-cancer drugs and targeting ligands could be co-delivered through one single NMOF to enable combination therapy. Co-delivering system using NMOFs helped to avoid multidrug resistance, to reduce adverse effects, to achieve imaging-guided precise therapy and to enhance anti-cancer efficacy. This review summarized the recent research advances on the application of NMOFs in biomedical imaging and cancer treatments in the last few years. The current challenges that impeding their translation to clinical practices and the perspectives for their future applications were also highlighted and discussed.

Standardized naming systems are essential to integrate and unify distinct research fields, and to link multi-species data within and across kingdoms. We conducted a comprehensive survey of cytoplasmic ribosomal proteins (CRPs) in the dicot model Arabidopsis thaliana and the monocot model rice, noting that the standardized naming system has not been widely adopted in the plant community. We generated a database linking the old classical names to their updated and compliant names. We also explored the sequences, molecular evolution, and structural and functional characteristics of all plant CRP families, emphasizing evolutionarily conserved and plant-specific features through cross-kingdom comparisons. Unlike fungal CRP paralogs that were mainly created by whole-genome duplication (WGD) or retroposition under a concerted evolution mode, plant CRP genes evolved primarily through both WGD and tandem duplications in a rapid birth-and-death process. We also provide a web-based resource (http://www.plantcrp.cn/) with the aim of sharing the latest knowledge on plant CRPs and facilitating the continued development of a standardized framework across the entire community.

The MIR2118 family has undergone tremendous expansion in the grass lineage, in which the miRNA targets numerous noncoding PHAS loci to produce 21-nt phased small interfering RNAs (phasiRNAs) involved in male fertility. However, the evolutionary trajectory of the grass MIR2118 genes and the functions of phasiRNAs have not yet been fully elucidated. We conducted comparative genomic, molecular evolution, expression and parallel analysis of RNA ends (PARE) analyses of MIR2118 and the miR2118-mediated regulatory pathway in grasses, focusing on Oryza sativa. In total, 617 MIR2118 and eight MIR1859 novel members were identified. Phylogenetic analyses showed that grass MIR2118 genes form a distinct clade from the MIR482/2118 genes of nongrass species. We reconstructed hypothetical evolutionary histories of the grass MIR2118 clusters and its MIR1859 variants, and examined the polycistronic composition and the differential expression of the osa-MIR2118 clusters. PARE data showed that osa-miR2118 might also direct the cleavage of some protein-coding gene transcripts. Importantly, we found that PARE analysis is inherently prone to false-positive target predictions when a large number of small RNAs, such as phasiRNAs, are analysed. Our results revealed the evolution and diversification of the MIR2118 family, and provide new insights into the functions of phasiRNAs in the grasses.

microRNAs (miRNAs) are small non-coding RNAs which play critical roles in growth, development and responses to abiotic and biotic stresses in plants and animals. Functional characterization of these miRNAs is important in understanding their roles and applications of the gained knowledge in crop improvement programs. Various approaches have been adopted to study functions of miRNAs in plants. These approaches involve overexpressing miRNAs for gain-of-function studies or their decoys/mimics in the form of target mimics (TMs), short tandem target mimics (STTMs) and sponges (SPs), for loss-of-function analyses. These decoys sequester and/or degrade mature target miRNAs, preventing the accumulation of miRNAs in sufficient amount to suppress their respective target genes. To suppress the expression of MIRNA genes or target genes, controlling agronomic traits, RNA knockdown techniques, such as RNAi or CRISPR/Cas13a can be used. Furthermore, gene-editing tools, such as CRISPR/Cas-based systems, can complement RNAi-based approaches to edit and/or alter expression of genes encoding miRNA or their targets. All constructs, targeting miRNAs or their target genes, can be expressed using constitutive, ubiquitous, tissue-specific or inducible promoters to achieve silencing in a desired manner, spatially or temporally. Altogether, modulating the expression of a miRNA, or of its target gene(s), can help manipulating their functions to engineer desirable traits in plants for crop improvement.

Improvements in plant agricultural productivity are urgently needed to reduce the dependency on limited natural resources and produce enough food for a growing world population. Human intervention over thousands of years has improved the yield of important crops; however, it is increasingly difficult to find new targets for genetic improvement. MicroRNAs (miRNAs) are promising targets for crop improvement, but their inactivation is technically challenging and has hampered functional analyses. We have produced a large collection of transgenic short tandem target mimic (STTM) lines silencing 35 miRNA families in rice as a resource for functional studies and crop improvement. Visual assessment of field-grown miRNA-silenced lines uncovered alterations in many valuable agronomic traits, including plant height, tiller number, and grain number, that remained stable for up to five generations. We show that manipulation of miR398 can increase panicle length, grain number, and grain size in rice. In addition, we discovered additional agronomic functions for several known miRNAs, including miR172 and miR156. Our collection of STTM lines thus represents a valuable resource for functional analysis of rice miRNAs, as well as for agronomic improvement that can be readily transferred to other important food crops.

Conidia and mycelia are two important developmental stages in the asexual life cycle of entomopathogenic fungus Metarhizium. Despite the crucial role that DNA methylation plays in many biological processes, its role in regulation of gene expression and development in fungi is not yet fully understood. We performed genome-wide analysis of DNA methylation patterns of an M. robertsii strain with single base pair resolution. Specifically, we examined for changes in methylation patterns between the conidia and mycelia stages. The results showed that approximately 0.38 % of cytosines are methylated in conidia, which is lower than the DNA methylation level (0.42 %) in mycelia. We found that DNA methylation undergoes genome-wide reprogramming during fungal development in M. robertsii. 132 differentially methylated regions (DMRs), which were mostly distributed in gene regions, were identified. KEGG analysis revealed that the DMR-associated genes belong to metabolic pathways. Intriguingly, in contrast to most other eukaryotes, promoter activities in M. robertsii seemed differentially modulated by DNA methylation levels. We found that transcription tended to be enhanced in genes with moderate promoter methylation, while gene expression was decreased in genes with high or low promoter methylation.

The growing incidence of cancer raises an urgent need to develop effective diagnostic and therapeutic strategies. With the rapid development of nanomedicine, nanoscale metal-organic frameworks (NMOFs) presented promising potential in various biomedical applications in the last 2 decades, especially in cancer theranostics. Due to the unique features of NMOFs, including structural diversities, enormous porosity, multifunctionality and biocompatibility, they have been widely used to deliver imaging contrast agents and therapeutic drugs. Moreover, multiple types of contrast agents, anti-cancer drugs and targeting ligands could be co-delivered through one single NMOF to enable combination therapy. Co-delivering system using NMOFs helped to avoid multidrug resistance, to reduce adverse effects, to achieve imaging-guided precise therapy and to enhance anti-cancer efficacy. This review summarized the recent research advances on the application of NMOFs in biomedical imaging and cancer treatments in the last few years. The current challenges that impeding their translation to clinical practices and the perspectives for their future applications were also highlighted and discussed.

Prostate cancer (PCa), an epithelial malignancy that occurs in the prostate, is the second leading cause of cancer death worldwide. MicroRNAs (miRs/miRNAs) are reported to have important applications in the field of cancer diagnosis and treatment. The present study aimed to investigate the function of miRNA-122 in the chemoresistance of PCa cells and the underlying mechanism. Significantly decreased miR-122 and increased pyruvate kinase (PKM2) levels were observed in docetaxel-resistant PCa cells, and PKM2 was negatively correlated with miR-122. MiR-122 mimic transfection in docetaxel-resistant LNCaP cells significantly inhibited cell proliferation, promoted apoptosis and decreased glucose uptake and lactate production, which was counteracted by PKM2 overexpression. Inhibition of miR-122 in LNCaP cells had an opposite effect to miR-122 mimic transfection. In addition, miR-122 mimic transfection significantly increased the sensitivity of docetaxel-resistant LNCaP cells to docetaxel, while inhibition of miR-122 significantly decreased the sensitivity of LNCaP cells to docetaxel. Luciferase reporter assays showed that miR-122 regulated PKM2 expression by binding to the 3'-untranslated region of PKM2. The results suggest that upregulation of miR-122 could enhance docetaxel sensitivity, inhibit cell proliferation and promote apoptosis in PCa cells,possibly through the downregulation of its target protein PKM2.

Background: Many studies have been reported that platelet-to-lymphocyte ratio (PLR) may be associated with the prognosis of bladder cancer, but the results are inconsistent. Therefore, we performed a meta-analysis to evaluate the effect of pretreatment PLR on the prognosis of bladder cancer. Methods: The databases PubMed, Embase, Cochrane Library, and Web of Science were searched. Pooled hazard ratios (HRs) and 95% confidence intervals (CIs) were used to analyze the relationship between PLR and prognosis. Pooled odds ratios (ORs) and 95% CIs were used to analyze the relationship between PLR and clinicopathological features. Publication bias was estimated using Begg's funnel plot asymmetry tests. Results: A total of 8 studies comprising 3,303 patients were included in this meta-analysis. An elevated PLR was significantly associated with poorer overall survival (OS) (HR = 1.26, 95% CI = 1.03–1.54, p = 0.026), but not with cancer-specific survival (CSS) (HR = 1.15, 95% CI = 0.95–1.38, p = 0.149), or recurrence-free survival (RFS) (HR = 1.72, 95% CI = 0.79–3.75, p = 0.175). In addition, high PLR was correlated with age ≥ 65 years (OR = 1.82, 95% CI = 1.24–2.67, p = 0.002), whereas was not significantly correlated with sex, tumor grade, tumor stage, distant metastasis, or tumor size. Conclusions: The pretreatment PLR could serve as a predicative biomarker of poor prognosis for patients with bladder cancer.

MicroRNAs (miRNAs) are small noncoding RNAs which regulate various functions related to growth, development, and stress responses in plants and animals. Rice, Oryza sativa, is one of the most important food crops of the world. In rice, a number of quantitative trait loci (QTL) controlling yield-related traits have been identified. Some of them are actually controlled by miRNAs, which control various yield-related quantitative traits in rice. On one hand, many of these miRNAs are found to regulate more than one yield-related traits, such as tillering, grain size, and branch number of a panicle. On the other hand, a rice yield-related trait is usually controlled by multiple miRNAs, for example, grain size being controlled by miR156, miR167, miR396, miR397, and miR1432. In rare case, a single miRNA may specifically regulate only one yield-related trait, such as, miR444 regulating rice tillering. In this review, we focus on the functions of miRNAs in controlling yield-related quantitative traits in rice, including panicle grain number, grain weight/size, panicle length and branching, tiller number per plant, spikelet number, seed setting rate, and leaf inclination, and discuss how to modulate the expression of these miRNAs using modern molecular biology tools to promote grain yield. This article is categorized under: RNA in Disease and Development > RNA in Development.

MicroRNAs (miRNAs) have been recognized as sequence-specific regulators of the genome, transcriptome, and proteome in eukaryotes. However, the functions and working mechanisms of hundreds of fungal miRNA-like (miR-like) RNAs are obscure. Here, we report that a short tandem target mimic (STTM) triggered the degradation of several fungal miR-like RNAs in two different fungal species, Metarhizium robertsii and Aspergillus flavus, and that small-RNA-degrading nucleases (SDNs) were indispensable for such degradation. STTMs were most effective when the fungal polymerase II (Pol II) promoter was used for their expression, while the Pol III promoter was less effective. The length of the STTM spacer, approximately 48 to 96 nucleotides, and the number of miR-like RNA binding sites, from 2 to 4 copies, showed no significant difference in the degradation of miR-like RNAs. STTMs modulated the miR-like RNA expression levels in at least two different fungal species, which further impacted fungal asexual growth and sporulation. Further analysis showed that the degraded miR-like RNAs in STTM mutants led to the upregulation of potential target genes involved in fungal development and conidial production, which result in different phenotypes in these mutants. The STTM technology developed in this study is an effective and powerful tool for the functional dissection of fungal miR-like RNAs. IMPORTANCE The development and application of STTM technology to block miR-like RNAs in M. robertsii and A. flavus may allow for efficient generation of miR-like RNA mutants in various fungi, providing a powerful tool for functional genomics of small RNA molecules in fungi.

Small RNA (sRNA) population in plants comprises of primarily micro RNAs (miRNAs) and small interfering RNAs (siRNAs). MiRNAs play important roles in plant growth and development. The miRNA-derived secondary siRNAs are usually known as phased siRNAs, including phasiRNAs and tasiRNAs. The miRNA and phased siRNA biogenesis mechanisms are highly conserved in plants. However, their functional conservation and diversification may differ in maize. In the past two decades, lots of miRNAs and phased siRNAs have been functionally identified for curbing important maize agronomic traits, such as those related to developmental timing, plant architecture, sex determination, reproductive development, leaf morphogenesis, root development and nutrition, kernel development and tolerance to abiotic stresses. In contrast to Arabidopsis and rice, studies on maize miRNA and phased siRNA biogenesis and functions are limited, which restricts the small RNA-based fundamental and applied studies in maize. This review updates the current status of maize miRNA and phased siRNA mechanisms and provides a survey of our knowledge on miRNA and phased siRNA functions in controlling agronomic traits. Furthermore, improvement of those traits through manipulating the expression of sRNAs or their targets is discussed.

Table S1. Composition of diets. (DOCX 109 kb)

Figure S1. Changes in blood glucose and insulin sensitivity in mice fed with LFD or LFD + B. a Weekly changes in blood glucose levels over 4–16 weeks. b Insulin tolerance test (ITT) was performed in 16-week-old mice and blood glucose levels were assessed at the indicated times following an intraperitoneal injection of insulin (0.5 U/kg body weight). Values were represented as the percent of t = 0 glucose levels. n = 4–8 mice per group. (PDF 35 kb)

Prostate cancer (PCa), an epithelial malignancy that occurs in the prostate, is the second leading cause of cancer death worldwide. MicroRNAs (miRs/miRNAs) are reported to have important applications in the field of cancer diagnosis and treatment. The present study aimed to investigate the function of miRNA-122 in the chemoresistance of PCa cells and the underlying mechanism. Significantly decreased miR-122 and increased pyruvate kinase (PKM2) levels were observed in docetaxel-resistant PCa cells, and PKM2 was negatively correlated with miR-122. MiR-122 mimic transfection in docetaxel-resistant LNCaP cells significantly inhibited cell proliferation, promoted apoptosis and decreased glucose uptake and lactate production, which was counteracted by PKM2 overexpression. Inhibition of miR-122 in LNCaP cells had an opposite effect to miR-122 mimic transfection. In addition, miR-122 mimic transfection significantly increased the sensitivity of docetaxel-resistant LNCaP cells to docetaxel, while inhibition of miR-122 significantly decreased the sensitivity of LNCaP cells to docetaxel. Luciferase reporter assays showed that miR-122 regulated PKM2 expression by binding to the 3'-untranslated region of PKM2. The results suggest that upregulation of miR-122 could enhance docetaxel sensitivity, inhibit cell proliferation and promote apoptosis in PCa cells,possibly through the downregulation of its target protein PKM2.

MicroRNA1917 (miR1917) is a newly identified miRNAs that regulate ethylene responses in tomato. However, evidence is still limited about its functions in fruit development and ripening. Here, we investigated the possible roles of miR1917-SlCTR4 module in tomato fruit development. We generated miR1917 knock-down mutants by expressing Short Tandem Target Mimic (STTM1917). qRT-PCR and northern-blot analyses suggested that the expression of miR1917 are down-regulated in STTM1917. Concomitantly, miR1917-targeted SlCTR4 gene was up-regulated. STTM1917 plants showed a series of developmental phenotypes, including larger biomass, longer terminal leaflet, bigger floral organ and enhanced fruit and seed size. RNA-seq and qRT-PCR analyses suggested that the expression levels of numerous miRNAs and genes in the transgenic line were significantly altered compared to the wild type. These miRNAs and genes include fruit development-related miRNAs, fruit ripening-related transcription factors and ethylene metabolism genes. Altogether, our results demonstrated that working in concert with ripening regulators, miR1917 might regulate multiple genes in ethylene pathway, thereby modulating fruit development. Our results further indicated that fine-tuning miRNAs expression via STTM can be deployed for agronomic improvement of tomato.

Background Blueberry is rich in bioactive substances and possesses powerful antioxidant potential, which can protect against oxidant-induced and inflammatory cell damage and cytotoxicity. The aim of this study was to determine how blueberry affects glucose metabolism and pancreatic β-cell proliferation in high fat diet (HFD)-induced obese mice. Methods Wild type male mice at age of 4 weeks received two different kinds of diets: high-fat diet (HFD) containing 60% fat or modified HFD supplemented with 4% (wt:wt) freeze-dried whole blueberry powder (HFD + B) for 14 weeks. A separate experiment was performed in mice fed with low-fat diet (LFD) containing 10% fat or modified LFD + B supplemented with 4% (wt:wt) freeze-dried whole blueberry powder. The metabolic parameters including blood glucose and insulin levels, glucose and insulin tolerances were measured. Results Blueberry-supplemented diet significantly increased insulin sensitivity and glucose tolerance in HFD + B mice compared to HFD mice. However, no difference was observed in blood glucose and insulin sensitivity between LFD + B and LFD mice. In addition, blueberry increased β-cell survival and prevented HFD-induced β-cell expansion. The most important finding was the observation of presence of small scattered islets in blueberry treated obese mice, which may reflect a potential role of blueberry in regenerating pancreatic β-cells. Conclusions Blueberry-supplemented diet can prevent obesity-induced insulin resistance by improving insulin sensitivity and protecting pancreatic β-cells. Blueberry supplementation has the potential to protect and improve health conditions for both type 1 and type 2 diabetes patients. Electronic supplementary material The online version of this article (10.1186/s12986-019-0363-6) contains supplementary material, which is available to authorized users.

Tumor necrosis factor‑related apoptosis‑inducing ligand (TRAIL) exhibits antitumor activity in various types of tumor cell and tumor‑bearing animals. However, acquired TRAIL resistance is a common issue that restricts its clinical application. Previous studies have revealed that β‑catenin is associated with TRAIL resistance in melanoma and colorectal tumors. In the present study, an acquired‑resistance non‑small‑cell lung cancer (NSCLC) cell line (H460‑TR) was established from parental TRAIL‑sensitive H460 cells using a gradient ascent model (8‑256 ng/ml TRAIL). Cellular FADD‑like interleukin‑1β converting enzyme inhibitory protein and Mcl‑1 were upregulated and the cell surface distribution of death receptor (DR)4 and DR5 was downregulated in H460‑TR cells compared with the parental H460 cells. The results of reverse transcription‑quantitative polymerase chain reaction and western blot analysis indicated that H460 cells expressed increased levels of β‑catenin and were more sensitive to TRAIL compared with H460‑TR cells. β‑catenin‑knockdown in H460 cells decreased their sensitivity to TRAIL, while upregulation of β‑catenin expression in H460‑TR cells increased their sensitivity to TRAIL, increased the cell surface distribution of DRs and activated caspase‑3/8. Taken together, the results of the present study suggest that β‑catenin impairs acquired TRAIL resistance in NSCLC cells by promoting the redistribution of DR4 and DR5 to the cytomembrane, and inducing TRAIL‑mediated cell apoptosis via caspase‑3/8 activation.

Leaf development is a sequential process that involves initiation, determination, transition, expansion and maturation. Many coding genes and a few non-coding small RNAs (sRNAs) have been identified as being involved in leaf development. sRNAs and their interactions not only determine gene expression and regulation, but also play critical roles in leaf development through their coordination with other genetic networks and physiological pathways. In this review, we first introduce the biogenesis pathways of sRNAs, mainly microRNAs (miRNAs) and trans-acting small interfering RNAs (ta-siRNAs), and then describe the function of miRNA-transcription factors in leaf development, focusing on guidance by interactive sRNA regulatory networks.

Background Photoperiod-sensitivity is a critical endogenous regulatory mechanism for plant growth and development under specific environmental conditions, while phosphate and sucrose signaling processes play key roles in cell growth and organ initiation. MicroRNA399 is phosphate-responsive, but, whether it has roles in other metabolic processes remains unknown. Results MicroRNA399 was determined to be sucrose-responsive through a microRNA array assay. High levels of sucrose inhibited the accumulation of microRNA399 family under phosphate starvation conditions in Arabidopsis thaliana. Similarly, exogenous sucrose supplementation also reduced microRNA399 expression in maize at developmental transition stages. RNA sequencing of a near-isogenic line(photoperiod-sensitive) line and its recurrent parent Huangzao4, a photoperiod-insensitive line, was conducted at various developmental stages. Members of microRNA399 family were down-regulated under long-day conditions in the photoperiod-sensitive near-isogenic line that accumulated more sucrose in vivo compared with the control line Huangzao4. Conclusion MicroRNA399s may play central roles in the integration of sucrose sensing and photoperiodic responses under long day conditions in maize. Electronic supplementary material The online version of this article (10.1186/s12870-018-1460-9) contains supplementary material, which is available to authorized users.

MicroRNA 165 and 166 (miR165/166) is composed of nine members and targets five members (PHB, PHV, REV, ATHB8 and ATHB15) of the HD-ZIP III transcription factor family. Mutants generated by traditional methods could hardly reveal the overall functions of miR165/166 in plant development. In this study, the expressions of all miR165/166 members were simultaneously blocked by over-expressing STTM165/166-31 in Arabidopsis and tomato for functional dissection of miR165/166 family. Following a down-regulation of over 90% endogenous miR165/166, the target HD-ZIP III genes were correspondingly up-regulated in the STTM transgenic Arabidopsis and tomato plants. Notably, the STTM165/166-31 over-expressed Arabidopsis and tomato displayed pleiotropic effects on development which were not frequently observed in previously identified genetic mutants of either individual miR165/166 gene or any of the five target genes. Furthermore, the transgenic Arabidopsis showed increased IAA content and decreased IAA sensitivity accompanied by enhanced expressions of genes responsible for auxin biosynthesis and signaling, suggesting possible roles of auxin in mediation of miR165/166-regulated processes. Importantly, the transgenic Arabidopsis exhibited the improved behavior under salt stress. Overall, such diverse variations in plant development and physiological process revealed by STTM165/166 demonstrate a key role of miR165/166-mediated network in regulating plant development and responses to abiotic stresses.

MicroRNAs (miRNAs) are a group of endogenous noncoding small RNAs frequently 21 nucleotides long. miRNAs act as negative regulators of their target genes through sequence-specific mRNA cleavage, translational repression, or chromatin modifications. Alterations of the expression of a miRNA or its targets often result in a variety of morphological and physiological abnormalities, suggesting the strong impact of miRNAs on plant development. Here, we review the recent advances on the functional studies of plant miRNAs. We will summarize the regulatory networks of miRNAs in a series of developmental processes, including meristem development, establishment of lateral organ polarity and boundaries, vegetative and reproductive organ growth, etc. We will also conclude the conserved and species-specific roles of plant miRNAs in evolution and discuss the strategies for further elucidating the functional mechanisms of miRNAs during plant development.

microRNAs (miRNAs) are endogenous small non-coding RNAs that bind to mRNAs and target them for cleavage and/or translational repression, leading to gene silencing. We previously developed short tandem target mimic (STTM) technology to deactivate endogenous miRNAs in Arabidopsis. Here, we created hundreds of STTMs that target both conserved and species-specific miRNAs in Arabidopsis, tomato, rice, and maize, providing a resource for the functional interrogation of miRNAs. We not only revealed the functions of several miRNAs in plant development, but also demonstrated that tissue-specific inactivation of a few miRNAs in rice leads to an increase in grain size without adversely affecting overall plant growth and development. RNA-seq and small RNA-seq analyses of STTM156/157 and STTM165/166 transgenic plants revealed the roles of these miRNAs in plant hormone biosynthesis and activation, secondary metabolism, and ion-channel activity-associated electrophysiology, demonstrating that STTM technology is an effective approach for studying miRNA functions. To facilitate the study and application of STTM transgenic plants and to provide a useful platform for storing and sharing of information about miRNA-regulated gene networks, we have established an online Genome Browser (https://blossom.ffr.mtu.edu/designindex2.php) to display the transcriptomic and miRNAomic changes in STTM-induced miRNA knockdown plants.

In plants, microRNAs (miRNAs) regulate more than hundred target genes comprising largely transcription factors that control growth and development as well as stress responses. However, the exact functions of miRNA families could not be deciphered because each miRNA family has multiple loci in the genome, thus are functionally redundant. Therefore, an ideal approach to study the function of a miRNA family is to silence the expression of all members simultaneously, which is a daunting task. However, this can be partly overcome by Target Mimic (TM) approach that can knockdown an entire miRNA family. STTM is a modification of TM approach and complements it. STTMs have been successfully used in monocots and dicots to block miRNA functions. miR159 has been shown to be differentially regulated by various abiotic stresses including ABA in various plant species. Here, we describe in detail the protocol for designing STTM construct to block miR159 functions in Arabidopsis, with the potential to apply this technique on a number of other stress-regulated miRNAs in plants.

Background The soybean cyst nematode (SCN), Heterodera glycines, is one of the most devastating diseases limiting soybean production worldwide. It is known that small RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), play important roles in regulating plant growth and development, defense against pathogens, and responses to environmental changes. Results In order to understand the role of soybean miRNAs during SCN infection, we analyzed 24 small RNA libraries including three biological replicates from two soybean cultivars (SCN susceptible KS4607, and SCN HG Type 7 resistant KS4313N) that were grown under SCN-infested and -noninfested soil at two different time points (SCN feeding establishment and egg production). In total, 537 known and 70 putative novel miRNAs in soybean were identified from a total of 0.3 billion reads (average about 13.5 million reads for each sample) with the programs of Bowtie and miRDeep2 mapper. Differential expression analyses were carried out using edgeR to identify miRNAs involved in the soybean-SCN interaction. Comparative analysis of miRNA profiling indicated a total of 60 miRNAs belonging to 25 families that might be specifically related to cultivar responses to SCN. Quantitative RT-PCR validated similar miRNA interaction patterns as sequencing results. Conclusion These findings suggest that miRNAs are likely to play key roles in soybean response to SCN. The present work could provide a framework for miRNA functional identification and the development of novel approaches for improving soybean SCN resistance in future studies. Electronic supplementary material The online version of this article (doi:10.1186/s12864-017-3963-4) contains supplementary material, which is available to authorized users.

Although the molecular basis of carpel fusion in maize ovary development remains largely unknown, increasing evidence suggests a critical role of microRNAs (miRNAs). In this study, a combination of miRNA sequencing, degradome and physiological analyses was used to characterize carpel fusion development in maize ovaries showing incompletely (IFC) and completely fused carpels (CFC). A total of 162 known miRNAs distributed across 33 families were identified, of which 20 were differentially expressed. In addition, 53 miRNA candidates were identified, of which 10 were differentially expressed in the IFC and CFC ovaries. In degradome analysis, a total of 113 and 11 target genes were predicted for the known and novel miRNAs, respectively. Moreover, 24 (60%) target genes of the differentially expressed known miRNAs were found to code transcription factors, including auxin response factor (ARF), TB1-CYC-PCFs (TCP), APETALA2 (AP2), growth regulating factor (GRF), MYB, NAC, and NF-YA, all of which have been shown to play a role in carpel fusion development. Correlation analysis of these differentially expressed known miRNAs and their targets with phytohormone signals revealed significant correlations with at least one phytohormone signal, the main regulator of carpel fusion development. These results suggest that incomplete carpel fusion is partly the result of differential expression of certain miRNAs and their targets. Overall, these findings improve our knowledge of the effect of miRNA regulation on target expression, providing a useful resource for further analysis of the interactions between miRNAs, target genes and phytohormones during carpel fusion development in maize.

The primers used in the paper. (DOCX 14 kb)

Small RNAs, including microRNAs (miRNAs), are abundant in plants and play key roles in controlling plant development and physiology. miRNAs regulate the expression of the target genes involved in key plant processes. Due to functional redundancy among miRNA family members in plants, an ideal approach to silence the expression of all members simultaneously, for their functional characterization, is desirable. Target mimic (TM) was the first approach to achieve this goal. Short tandem target mimic (STTM) is a potent approach complementing TM for silencing miRNAs in plants. STTMs have been successfully used in dicots to block miRNA functions. Here, we describe in detail the protocol for designing STTM construct to block miRNA functions in rice. Such approach can be applied to silence miRNAs in other monocots as well.

Predicted targets of OsmiR159 by psRobot and their expressions detected by RNAseq. (DOCX 15 kb)

In plants, microRNAs (miRNAs) regulate more than hundred target genes comprising largely transcription factors that control growth and development as well as stress responses. However, the exact functions of miRNA families could not be deciphered because each miRNA family has multiple loci in the genome, thus are functionally redundant. Therefore, an ideal approach to study the function of a miRNA family is to silence the expression of all members simultaneously, which is a daunting task. However, this can be partly overcome by Target Mimic (TM) approach that can knockdown an entire miRNA family. STTM is a modification of TM approach and complements it. STTMs have been successfully used in monocots and dicots to block miRNA functions. miR159 has been shown to be differentially regulated by various abiotic stresses including ABA in various plant species. Here, we describe in detail the protocol for designing STTM construct to block miR159 functions in Arabidopsis, with the potential to apply this technique on a number of other stress-regulated miRNAs in plants.

Phylogenetic analysis of the targets of OsmiR159 and AtmiR159. (DOCX 54 kb)

†SUMMARY The genus Phytophthora consists of many notorious pathogens of crops and forestry trees. At present, battling Phytophthora diseases is challenging due to a lack of understanding of their pathogenesis. We investigated the role of small RNAs in regulating soybean defense in response to infection by Phytophthora sojae, the second most destructive pathogen of soybean. Small RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), are universal regulators that repress target gene expression in eukaryotes. We identified known and novel small RNAs that differentially accumulated during P. sojae infection in soybean roots. Among them, miR393 and miR166 were induced by heat-inactivated P. sojae hyphae, indicating that they may be involved in soybean basal defense. Indeed, knocking down the level of mature miR393 led to enhanced susceptibility of soybean to P. sojae; furthermore, the expression of isoflavonoid biosynthetic genes was drastically reduced in miR393 knockdown roots. These data suggest that miR393 promotes soybean defense against P. sojae. In addition to miRNAs, P. sojae infection also resulted in increased accumulation of phased siRNAs (phasiRNAs) that are predominantly generated from canonical resistance genes encoding nucleotide binding-leucine rich repeat proteins and genes encoding pentatricopeptide repeat-containing proteins. This work identifies specific miRNAs and phasiRNAs that regulate defense-associated genes in soybean during Phytophthora infection.

Arabidopsis glycogen synthase kinase 3 (GSK3)-like kinases have versatile functions in plant development and in responding to abiotic stresses. Although physiological evidence suggested a potential role of GSK3-like kinases in abscisic acid (ABA) signaling, the underlying molecular mechanism was largely unknown. Here we identified members of Snf1-related kinase 2s (SnRK2s), SnRK2.2 and SnRK2.3, that can interact with and be phosphorylated by a GSK3-like kinase, brassinosteroid insensitive 2 (BIN2). bin2-3 bil1 bil2, a loss-of-function mutant of BIN2 and its two closest homologs, BIN2 like 1 (BIL1) and BIN2 like 2 (BIL2), was hyposensitive to ABA in primary root inhibition, ABA-responsive gene expression, and phosphorylating ABA Response Element Binding Factor (ABF) 2 fragment by in-gel kinase assays, whereas bin2-1, a gain-of-function mutation of BIN2, was hypersensitive to ABA, suggesting that these GSK3-like kinases function as positive regulators in ABA signaling. Furthermore, BIN2 phosphorylated SnRK2.3 on T180, and SnRK2.3(T180A) had decreased kinase activity in both autophosphorylation and phosphorylating ABFs. Bikinin, a GSK3 kinase inhibitor, inhibited the SnRK2.3 kinase activity and its T180 phosphorylation in vivo. Our genetic analysis further demonstrated that BIN2 regulates ABA signaling downstream of the PYRABACTIN RESISTANCE1/PYR1-LIKE/REGULATORY COMPONENTS OF ABA RECEPTORS receptors and clade A protein phosphatase 2C but relies on SnRK2.2 and SnRK2.3. These findings provide significant insight into the modulation of ABA signaling by Arabidopsis GSK3-like kinases.

MicroRNAs (miRNAs) are a population of highly conserved specific small ribo-regulators that negatively regulate gene expressions in both plants and animals. They play a key role in post-transcriptional gene regulation by destabilizing the target gene transcripts or blocking protein translation from them. Interestingly, these negative regulators are largely compromised by an upstream layer of negative regulators "target mimics" found in plants or "endogenous competing RNAs" revealed recently in animals. These endogenous regulatory mechanisms of "double negatives making a positive" have now been developed into a key strategy in the study of small RNA functions. This review presents some reflections on the long journey to the short tandem target mimic (STTM) for selective destruction/blockage of specific miRNAs in plants and animals, and the potential applications of STTM are discussed.

RNA-induced silencing complex (RISC) is composed of miRNAs and AGO proteins. AGOs use miRNAs as guides to slice target mRNAs to produce truncated 5' and 3' RNA fragments. The 5' cleaved RNA fragments are marked with uridylation for degradation. Here, we identified novel cofactors of Arabidopsis AGOs, named RICE1 and RICE2. RICE proteins specifically degraded single-strand (ss) RNAs in vitro; but neither miRNAs nor miRNA*s in vivo. RICE1 exhibited a DnaQ-like exonuclease fold and formed a homohexamer with the active sites located at the interfaces between RICE1 subunits. Notably, ectopic expression of catalytically-inactive RICE1 not only significantly reduced miRNA levels; but also increased 5' cleavage RISC fragments with extended uridine tails. We conclude that RICEs act to degrade uridylated 5’ products of AGO cleavage to maintain functional RISC. Our study also suggests a possible link between decay of cleaved target mRNAs and miRNA stability in RISC. DOI: http://dx.doi.org/10.7554/eLife.24466.001, eLife digest DNA contains all the information needed to build a body, yet molecules of RNA carry these instructions to the sites in the cell where they can be used. Cells must control how much RNA they produce in order to ensure that they develop properly and can respond well to their environment. RNA silencing refers to a collection of mechanisms that use smaller RNA molecules called microRNAs to incapacitate certain RNA molecules and selectively switch off the genes that encode them to stop more from being made. One key player in RNA silencing is the multi-protein complex called RISC, which contains microRNA and a group of proteins called AGOs. Once the microRNA has identified its RNA target, the AGOs cut the RNA into two pieces, known as the 5’ cleavage fragment and 3’ cleavage fragment. The two resulting fragments need to be cleared away swiftly, so that the RISC can move on to the next target. While it was known how the 3’ cleavage fragment was removed, it was less clear how the 5’ cleavage fragment was dealt with. Previous studies had shown that the 5’ cleavage fragment was marked with a chemical called uridine, which somehow signals to the RISC that this fragment needs to be destroyed. Now, using biochemical techniques, Zhang et al. have identified two new proteins in the model plant Arabidopsis that attach to the AGO proteins and degrade the 5’ cleavage fragments that are marked with uridine. The two proteins are named RICE1 and RICE2. Zhang et al. then analyzed the three-dimensional shape of RICE1 and identified the ‘active’ region that is responsible for degrading the RNA fragments. When these active regions were blocked, the microRNA levels were low, but the uridine-marked 5’ cleavage fragments were high. Also, the RISC complex could not work properly, which lead to problems during the development of the plant. These results suggest that RICE proteins degrade 5’ cleavage fragments modified with uridine to activate RISC. RICE proteins are conserved between plants and animals, and it is likely that their counterparts in humans will have a similar role to the plant proteins. The next challenge will be to explore how RICE proteins work in more details, which may lead to new ways to manipulate the levels of microRNAs to change the architecture of the plant and to improve their tolerance to various stress conditions. DOI: http://dx.doi.org/10.7554/eLife.24466.002

The function of miR165/166 in plant growth and development has been extensively studied, however, its roles in abiotic stress responses remain largely unknown. Here, we report that reduction in the expression of miR165/166 conferred a drought and cold resistance phenotype and hypersensitivity to ABA during seed germination and post-germination seedling development. We further show that the ABA hypersensitive phenotype is associated with a changed transcript abundance of ABA-responsive genes and a higher expression level of ABI4, which can be directly regulated by a miR165/166 target. Additionally, we found that reduction in miR165/166 expression leads to elevated ABA levels, which occurs at least partially through the increased expression of BG1, a gene that is directly regulated by a miR165/166 target. Taken together, our results uncover a novel role for miR165/166 in the regulation of ABA and abiotic stress responses and control of ABA homeostasis., Author Summary Functions of miRNAs in plant development and stress responses have been extensively studied. However, little is known about how a miRNA may perform critical functions in both plant development and abiotic stress responses. One well-known miRNA, miR165/166, has critical roles in plant development. In this study, we show that this miRNA also has important functions in ABA and abiotic stress responses. Since the expression level of miR165/166 can be reduced to different extents using short tandem target mimicry (STTM), in the present work, we used STTM165/166 transformants with moderate developmental phenotype to examine its potential role in abiotic stress responses. Our results show that miR165/166 plays critical roles in drought and cold stress resistance as well as in ABA responses. Our work reveals that miR165/166-mediated regulatory module is linked with ABA responses and homeostasis through ABI4 and BG1.

Animal and plant cells have repair capabilities to combat DNA damage. DNA damage and repair dynamics can be determined by technologies such as IDLV capture, BLESS, HTGTS, digenome-seq, and GUIDE-seq. Here we highlight GUIDE-seq, a technology used in therapeutics, and envision its application in plants.

Background Maize grain yield depends mainly on the photosynthetic efficiency of functional leaves, which is controlled by an array of gene networks and other factors, including environmental conditions. MicroRNAs (miRNAs) are small RNA molecules that play important roles in plant developmental regulation. A few senescence-associated miRNAs (SA-miRNAs) have been identified as important participants in regulating leaf senescence by modulating the expression levels of their target genes. Results To elucidate miRNA roles in leaf senescence and their underlying molecular mechanisms in maize, a stay-green line, Yu87-1, and an early leaf senescence line, Early leaf senescence-1 (ELS-1), were selected as experimental materials for the differential expression of candidate miRNAs. Four small RNA libraries were constructed from ear leaves at 20 and 30 days after pollination and sequenced by Illumina deep sequencing technology. Altogether, 81 miRNAs were detected in both lines. Of these, 16 miRNAs of nine families were differentially expressed between ELS-1 andYu87-1. The phenotypic and chlorophyll content analyses of both lines identified these 16 differentially expressed miRNAs as candidate SA-miRNAs. Conclusions In this study, 16 candidate SA-miRNAs of ELS-1 were identified through small RNA deep sequencing technology. Degradome sequencing results indicated that these candidate SA-miRNAs may regulate leaf senescence through their target genes, mainly transcription factors, and potentially control chlorophyll degradation pathways. The results highlight the regulatory roles of miRNAs during leaf senescence in maize. Electronic supplementary material The online version of this article (doi:10.1186/s12870-016-0755-y) contains supplementary material, which is available to authorized users.

Artificial microRNA (amiRNA) technology utilizes microRNA (miRNA) biogenesis pathway to produce artificially selected small RNAs using miRNA gene backbone. It provides a feasible strategy for inducing loss of gene function, and has been applied in functional genomics study, improvement of crop quality and plant virus disease resistance. A big challenge in amiRNA applications is the unpredictability of silencing efficacy of the designed amiRNAs and not all constructed amiRNA candidates would be expressed effectively in plant cells. We and others found that high efficiency and specificity in RNA silencing can be achieved by designing amiRNAs with perfect or almost perfect sequence complementarity to their targets. In addition, we recently demonstrated that Agrobacterium-mediated transient expression system can be used to validate amiRNA constructs, which provides a simple, rapid and effective method to select highly expressible amiRNA candidates for stable genetic transformation. Here, we describe the methods for design of amiRNA candidates with perfect or almost perfect base-pairing to the target gene or gene groups, incorporation of amiRNA candidates in miR168a gene backbone by one step inverse PCR amplification, construction of plant amiRNA expression vectors, and assay of transient expression of amiRNAs in Nicotiana benthamiana through agro-infiltration, small RNA extraction, and amiRNA Northern blot.

It is of great importance and interest to develop crop varieties with low polyphenol oxidase (PPO) activity for the food industry because PPO-mediated oxidative browning is a main cause of post-harvest deterioration and quality loss of fresh produce and processed foods. We recently demonstrated that potato tubers with reduced browning phenotypes can be produced by inhibition of the expression of several PPO gene isoforms using artificial microRNA (amiRNA) technology. The approach introduces a single type of 21-nucleotide RNA population to guide silencing of the PPO gene transcripts in potato tissues. Some advantages of the technology are: small RNA molecules are genetically transformed, off-target gene silencing can be avoided or minimized at the stage of amiRNA designs, and accuracy and efficiency of the processes can be detected at every step using molecular biological techniques. Here we describe the methods for transformation and regeneration of potatoes with amiRNA vectors, detection of the expression of amiRNAs, identification of the cleaved product of the target gene transcripts, and assay of the expression level of PPO gene isoforms in potatoes.