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Key message This study showed the systematic identification of long non-coding RNAs (lncRNAs) involving in flag leaf senescence of rice, providing the possible lncRNA-mRNA regulatory relationships and lncRNA-miRNA-mRNA ceRNA networks during leaf senescence. Abstract LncRNAs have been reported to play crucial roles in diverse biological processes. However, no systematic identification of lncRNAs associated with leaf senescence in plants has been studied. In this study, a genome-wide high throughput sequencing analysis was performed using rice flag leaves developing from normal to senescence. A total of 3953 lncRNAs and 38757 mRNAs were identified, of which 343 lncRNAs and 9412 mRNAs were differentially expressed. Through weighted gene co-expression network analysis (WGCNA), 22 continuously down-expressed lncRNAs targeting 812 co-expressed mRNAs and 48 continuously up-expressed lncRNAs targeting 1209 co-expressed mRNAs were considered to be significantly associated with flag leaf senescence. Gene Ontology results suggested that the senescence-associated lncRNAs targeted mRNAs involving in many biological processes, including transcription, hormone response, oxidation–reduction process and substance metabolism. Additionally, 43 senescence-associated lncRNAs were predicted to target 111 co-expressed transcription factors. Interestingly, 8 down-expressed lncRNAs and 29 up-expressed lncRNAs were found to separately target 12 and 20 well-studied senescence-associated genes (SAGs). Furthermore, analysis on the competing endogenous RNA (CeRNA) network revealed that 6 down-expressed lncRNAs possibly regulated 51 co-expressed mRNAs through 15 miRNAs, and 14 up-expressed lncRNAs possibly regulated 117 co-expressed mRNAs through 21 miRNAs. Importantly, by expression validation, a conserved miR164-NAC regulatory pathway was found to be possibly involved in leaf senescence, where lncRNA MSTRG.62092.1 may serve as a ceRNA binding with miR164a and miR164e to regulate three transcription factors. And two key lncRNAs MSTRG.31014.21 and MSTRG.31014.36 also could regulate the abscisic-acid biosynthetic gene BGIOSGA025169 (OsNCED4) and BGIOSGA016313 (NAC family) through osa-miR5809. The possible regulation networks of lncRNAs involving in leaf senescence were discussed, and several candidate lncRNAs were recommended for prior transgenic analysis. These findings will extend the understanding on the regulatory roles of lncRNAs in leaf senescence, and lay a foundation for functional research on candidate lncRNAs.

Anaerobic digestion of common rural wastes (human feces (HF), food waste (FW) and lawn grass (LG)) were studied considering the specific methane yield, process parameters and microbial characteristics (mainly microbial community and pathogenic bacteria). The results showed that co-digestion of multiple substrates obtained high digestion performance when the total solid (TS) was 4%. The optimal co-digestion ratio of HF, FW, and LG was 33–56%, 21–38% and 20–40%, respectively. The digestion system containing HF underwent ammonia inhibition, which leads to the succession of the methanogenesis pathway from the acetoclastic pathway to the hydrogenotrophic pathway. Simultaneously, the dominant methanogenic archaea changed from Methanosaeta to Methanobacterium and Methanosarcina. Co-digestion reduced Salmonella's absolute concentration. The recovered energy and nitrogen could meet 52–109 % energy demand of rural community and all nitrogen demand in lawn fertilization, respectively. The main rural organic wastes could be recycled by anaerobic digestion, considering the flexibility of substrate ratio.

N-Glycosylation is one of the most ubiquitous protein modifications. However, the qualitative analysis of N-glycoproteome during leaf senescence has not been studied yet. In this study, N-glycoproteins were identified in senescent flag leaf of rice through hydrophilic interaction chromatography enrichment and liquid chromatography–tandem mass spectrometry strategy. A total of 381 N-glycoproteins and 479 N-glycosylated sites were identified. In which, 40% of glycoproteins contained signal peptide and 40% had transmembrane domain. Meanwhile, α helix and coil/loop accounted for 1.5% and 98.5% of all N-glycosylated sites. Motif-X analysis suggested that [NxT], [NxS] and [NS] were significantly conserved and enriched. During leaf senescence, proteolysis-related proteins were predominately N-glycosylated by gene ontology analysis. Meanwhile, most identified glycoproteins were enriched in photosynthesis and N-glycan biosynthesis pathways by Kyoto Encyclopedia of Genes and Genomes analysis. Protein–protein interaction analysis showed that N-glycoproteins formed interaction networks to function in the metabolism process of energy substances (carbohydrates, lipids and amino acids), the proteolysis process, and the protein glycosylation process. Among 381 N-glycoproteins, 183 proteins were involved in various and famous senescence-related biological processes including energy substance metabolism (70), proteolysis (44), photosynthesis (17), protein glycosylation (24), reactive oxygen species scavenging (14), transcriptional regulation (6), senescence-associated genes (2) and hormone response (6), suggesting the important role of protein N-glycosylation in leaf senescence. Additionally, ten glycoproteins were found to have been well-studied in senescent process of rice. The results provided a novel insight into the significant involvement of protein N-glycosylation in leaf senescence.

Functional inactivation of UDP-N-acetylglucosamine pyrophosphorylase 1 (UAP1) induces defense response-related lesion-mimic spots and subsequent early senescence in every newly grown leaf of the rice mutant uap1 after a short period's normal growth. However, the molecular mechanism of these leaves sustaining the short period's survival is still unknown. Phenotypic and molecular studies show that defense response-related lesion-mimic spots and early leaf senescence appear on the normally grown uap1 leaf and aggravate with the growth time. Bioinformatic analysis reveals that UAP proteins are evolutionarily conserved among eukaryotes, and there exists UAP2 protein except UAP1 protein in many higher organisms, including rice. Rice UAP2 and UAP1 proteins present high sequence identities and very similar predicted 3D structures. Transcriptional expression profile of the UAP2 gene decreases with the appearance and aggravating of leaf spots and early senescence of uap1, implying the role of the UAP2 gene in maintaining the initial normal growth of uap1 leaves. Enzymatic experiments verified that the UAP2 protein performs highly similar UAP enzymatic activity with the UAP1 protein, catalyzing the biosynthesis of UDP-GlcNAc. And these two UAP proteins are found to have the same subcellular localization in the cytoplasm, where they most presumably perform their functions. Overexpression of the UAP2 gene in uap1 plants succeeds to rescue their leaf mutant phenotype to normal, providing direct evidence for the similar function of the UAP2 gene as the UAP1 gene. The UAP2 gene is mainly expressed in the young leaf stage for functions, while the UAP1 gene is highly expressed during the whole leaf developmental stages. Based on these findings, it is suggested that UAP2 and UAP1 play key roles in rice leaf survival during its development in a synergetic manner, protecting the leaf from early senescence.

Global warming-associated increases in temperature, particularly at nighttime, are detrimental to rice yield and quality. Metabolomic profiling was used to examine and compare the short-term extreme high nighttime temperature-induced molecular perturbations in rice ( Oryza sativa) coisogenic strains with contrasting heat-tolerances at the first stage of seed ripening. Compared to the heat-sensitive strain, antioxidant molecules were higher in abundance in the heat-tolerant strain, whereas the abundances of molecules involved in photosynthesis, nucleotide catabolism, and the S-adenosylmethionine (SAM) cycle varied only slightly. Thus, we proposed that the high abundance of antioxidant molecules in the heat-tolerant strain alleviated cellular oxidative stress, which protected photosynthesis, nucleotide catabolism, and the SAM cycle, leading to good grain filling.

Ovarian cancer is one of the most common types of gynecological malignancy worldwide, and is the fourth leading cause of cancer-associated mortality among women. Despite improvements in therapeutic treatments, the prognosis for epithelial ovarian cancer (EOC) remains poor, mainly due to the rapid growth and metastasis of ovarian cancer tumors. An increasing number of studies have indicated that microRNAs (miRNAs) are involved in the carcinogenesis and progression of human cancer, suggesting that miRNAs may be used in clinical prognosis and as a therapeutic target in EOC. The aim of the present study was to investigate the expression levels of miRNA-494 in EOC tissues and cell lines. The clinical significance of miRNA-494 in patients with EOC was also evaluated. The results demonstrated that miRNA-494 was significantly downregulated in EOC tissues and cell lines. Low expression levels of miRNA-494 were associated with poor prognostic features, including International Federation of Gynecology and Obstetrics stage, tumor size and lymph node metastasis. In vitro functional studies demonstrated that overexpression of miRNA-494 inhibited proliferation, migration and invasion in EOC cells. By contrast, knockdown of miRNA-494 enhanced cell growth, migration and invasion in EOC cells. Notably, sirtuin 1 (SIRT1) was identified as a direct target of miRNA-494 in EOC. Furthermore, MTT, cell migration and invasion assays verified that EOC cell proliferation, migration and invasion were completely restored with forced miRNA-494 expression and SIRT1 restoration. Together, these findings suggest that miRNA-494 is a potential prognostic marker, and may provide novel therapeutic regimens of targeted therapy for EOC.

Methylation plays an important role in the regulation of gene expression in many cancer tissues. RUNX3 is an important tumor suppressor gene located on human chromosome 1p36.1, and many tumors do not express it due to methylation of the promoter region of the CpG island. The molecular mechanisms involved in RUNX3 gene expression and epithelial ovarian cancer are not fully understood. This study investigates the relationship between RUNX3 methylation and expression in ovarian cancer. The methylation of the RUNX3 gene promoter region was measured in 32 primary epithelial ovarian cancer samples and corresponding nonmalignant ovarian tissues, 36 benign epithelial ovarian tumor tissues, and 10 normal ovarian tissues by methylation-specific PCR (MSP) and RT-PCR. The relationships between RUNX3 methylation status, expression, and clinicopathologic characteristics were analyzed. RUNX3 methylation was further assessed by MSP and RT-PCR before and after 5-aza-2'-deoxycytidine (5-aza-dc) treatment in normal and cancer cell lines. We detected RUNX3 methylation in 53.1% of primary ovarian cancer tumors, 16.7% of benign ovarian tumors, and 28% of nonmalignant tissues surrounding ovarian cancers. No methylation was detected in normal ovarian tissues. No significant correlation between RUNX3 methylation and clinicopathological characteristics was observed. The RT-PCR results found RUNX3 expression in all normal ovarian tissues (10/10) and in most of the unmethylated ovarian cancer tissues (12/15); in contrast, it was not detected in most of the RUNX3-methylated ovarian cancer tissues (16/17). Our data suggest that methylation plays a critical role in the regulation of RUNX3 repression, and that it is significantly correlated with RUNX3 mRNA expression in ovarian cancer tissues (p = 0.006).

RUNX3 is one of the suppressor gene in ovarian cancer, it is not clear whether the expression of RUNX3 is regulated by its promoter methylation or the demethylating agent 5-aza-2'-deoxycytidine. We carried out methylation — specific PCR (MSP) to investigate the methylation status of RUNX3 promoter region in human ovarian cancer cell line 3AO, KOV3 and normal ovarian epithelial cell NOEC. The methylation of RUNX3 promoter was found in tumor cells but not in NOEC. When treated with 5-aza-2'-deoxycytidine, the methylation was induced or completely reversed, the growth of tumor cells were notably inhibited, however, the agent did not affect the growth of NOEC. In conclusion, methylation of RUNX3 may be involved in the carcinogenesis of ovarian cancer and may serve as a predictive biomarker. The reversion of methylation by 5-aza-2'-deoxycytidine offers a novel therapeutic strategy for ovarian cancer.