Senescence-associated Genes Research Articles

TNF-α promotes α-synuclein propagation through stimulation of senescence-associated lysosomal exocytosis

Cell-to-cell propagation of α-synuclein is thought to be the underlying mechanism of Parkinson’s disease progression. Recent evidence suggests that inflammation plays an important role in the propagation of protein aggregates. However, the mechanism by which inflammation regulates the propagation of aggregates remains unknown. Here, using in vitro cultures, we found that soluble factors secreted from activated microglia promote cell-to-cell propagation of α-synuclein and further showed that among these soluble factors, TNF-α had the most robust stimulatory activity. Treatment of neurons with TNF-α triggered cellular senescence, as shown by transcriptomic analyses demonstrating induction of senescence-associated genes and immunoanalysis of senescence phenotype marker proteins. Interestingly, secretion of α-synuclein was increased in senescent neurons, reflecting acquisition of a senescence-associated secretory phenotype (SASP). Using vacuolin-1, an inhibitor of lysosomal exocytosis, and RNAi against rab27a, we demonstrated that the SASP was mediated by lysosomal exocytosis. Correlative light and electron microscopy and immunoelectron microscopy confirmed that propagating α-synuclein aggregates were present in electron-dense lysosome-like compartments. TNF-α promoted the SASP through stimulation of lysosomal exocytosis, thereby increasing the secretion of α-synuclein. Collectively, these results suggest that TNF-α is the major inflammatory factor that drives cell-to-cell propagation of α-synuclein by promoting the SASP and subsequent secretion of α-synuclein.

WATER-SOAKED SPOT1 Controls Chloroplast Development and Leaf Senescence via Regulating Reactive Oxygen Species Homeostasis in Rice.

Transmembrane kinases (TMKs) play important roles in plant growth and signaling cascades of phytohormones. However, its function in the regulation of early leaf senescence (ELS) of plants remains unknown. Here, we report the molecular cloning and functional characterization of the WATER-SOAKED SPOT1 gene which encodes a protein belongs to the TMK family and controls chloroplast development and leaf senescence in rice (Oryza sativa L.). The water-soaked spot1 (oswss1) mutant displays water-soaked spots which subsequently developed into necrotic symptoms at the tillering stage. Moreover, oswss1 exhibits slightly rolled leaves with irregular epidermal cells, decreased chlorophyll contents, and defective stomata and chloroplasts as compared with the wild type. Map-based cloning revealed that OsWSS1 encodes transmembrane kinase TMK1. Genetic complementary experiments verified that a Leu396Pro amino acid substitution, residing in the highly conserved region of leucine-rich repeat (LRR) domain, was responsible for the phenotypes of oswss1. OsWSS1 was constitutively expressed in all tissues and its encoded protein is localized to the plasma membrane. Mutation of OsWSS1 led to hyper-accumulation of reactive oxygen species (ROS), more severe DNA fragmentation, and cell death than that of the wild-type control. In addition, we found that the expression of senescence-associated genes (SAGs) was significantly higher, while the expression of genes associated with chloroplast development and photosynthesis was significantly downregulated in oswss1 as compared with the wild type. Taken together, our results demonstrated that OsWSS1, a member of TMKs, plays a vital role in the regulation of ROS homeostasis, chloroplast development, and leaf senescence in rice.

Role of MicroRNAs in acceleration of vascular endothelial senescence

BackgroundsMany factors are involved in cellular aging, and senescence induction requires complex regulation of various signaling networks and processes. Specifically, in the area of aging-related vascular cognitive impairment, laboratory-based findings have not yet yielded agents of practical use for clinical settings. One possible reason is that the physiologic elements of aging have been insufficiently considered. We sought to establish techniques to better model cellular aging using modulation of microRNAs, aiming to identify key microRNAs capable of fine-tuning aging-associated genes, and thereby regulating the senescence of vascular endothelial cells. MethodsWe utilized expression microRNA arrays to evaluate control and senescent vascular endothelial cells in order to identify testable candidates. Bioinformatic analysis was used to select key microRNAs. These candidates were then modulated in vitro using microRNA mimics and inhibitors in endothelial cells, and senescence-associated gene expression patterns were evaluated by qPCR. ResultsSeventeen microRNAs were found to be significantly increased more than 2-fold in senescent cells. Of those, bioinformatic analysis concluded that miR-181a-5p, miR-30a-5p, miR-30a-3p, miR-100-5p, miR-21-5p, and miR-382-5p were likely associated with regulation of cellular senescence. We evaluated the potential targets of these six microRNAs by comparing them with cell-cycling and apoptosis-related genes from published mRNA transcriptional array data from aged tissues, and found that miR-181a-5p, miR-30a-5p and miR-30a-3p were enriched in overlapping targets compared with the other candidates. Modulation of these microRNAs in vascular endothelial cells revealed that over-expression of miR-30a-5p, and inhibition of both miR-30a-3p and miR-181a-5p, induced senescence. Conclusion:miR-181a-5p, miR-30a-5p and miR-30a-3p likely contribute to aging-associated vascular endothelial cell senescence.

Non-coding RNAs in photoaging-related mechanisms: a new paradigm in skin health.

The aging of skin is a biological process affected by environmental or genetic factors. Exposure to ultraviolet (UV) radiation is the main environmental factor causing skin aging. Cumulative UV-induced photodamage of the skin tissue is associated with premature cellular senescence, extracellular degradation, and inflammatory responses in photoaging processes. Non-coding RNAs (ncRNAs) are untranslated transcripts and master regulators of protein-coding genes. ncRNAs have a critical regulatory role in maintaining skin structure, skin barrier function, morphogenesis, and development. Altered ncRNA expression has been reported in various skin disorders such as photoaging and skin cancers. ncRNAs contribute to the suppression and promotion of photoaging by modulating signaling pathways such as mitogen-activated protein kinase (MAPK) pathway and regulating inflammatory cytokines, matrix metalloproteinases (MMPs), and senescence-associated genes. Elucidation of the functions of ncRNAs will improve the identification of molecular mechanisms underlying photoaging, and can be used in the development of therapeutic approaches in skin health and prevention of sun-induced aging. This review summarized the currently described ncRNAs and their functions in photoaging.

Overexpression of AHL9 accelerates leaf senescence in Arabidopsis thaliana

BackgroundLeaf senescence, the final stage of leaf growth and development, is regulated by numerous internal factors and environmental cues. Ethylene is one of the key senescence related hormones, but the underlying molecular mechanism of ethylene-induced leaf senescence remains poorly understood.ResultsIn this study, we identified one AT-hook like (AHL) protein, AHL9, as a positive regulator of leaf senescence in Arabidopsis thaliana. Overexpression of AHL9 significantly accelerates age-related leaf senescence and promotes dark-induced leaf chlorosis. The early senescence phenotype observed in AHL9 overexpressing lines is inhibited by the ethylene biosynthesis inhibitor aminooxyacetic acid suggesting the involvement of ethylene in the AHL9-associated senescence. RNA-seq and quantitative reverse transcription PCR (qRT-PCR) data identified numerous senescence-associated genes differentially expressed in leaves of AHL9 overexpressing transgenic plants.ConclusionsOur investigation demonstrates that AHL9 functions in accelerating the leaf senescence process via ethylene synthesis or signalling.

Moderate DNA methylation changes associated with nitrogen remobilization and leaf senescence in Arabidopsis.

The lifespan of plants is restricted by environmental and genetic components. Following the transition to reproductive growth, leaf senescence ends cellular life in monocarpic plants to remobilize nutrients to storage organs. In Arabidopsis, we initially observed altered leaf to seed ratios, faster senescence progression, altered leaf nitrogen recovery after transient nitrogen removal, and ultimately enhanced nitrogen remobilization from the leaves in two methylation mutants (ros1 and the triple dmr1/2 cmt3 knockout). Analysis of the DNA methylome in wild type Col-0 leaves identified an initial moderate decline of cytosine methylation with progressing leaf senescence, predominantly in the CG context. Late senescence was associated with moderate de novo methylation of cytosines, primarily in the CHH context. Relatively few differentially methylated regions, including one in the ROS1 promoter linked to down-regulation of ROS1, were present, but these were unrelated to known senescence-associated genes. Differential methylation patterns were identified in transcription factor binding sites, such as the W-boxes that are targeted by WRKYs. Methylation in artificial binding sites impaired transcription factor binding in vitro. However, it remains unclear how moderate methylome changes during leaf senescence are linked with up-regulated genes during senescence.

Identification and Validation of Aging-Related Genes in Alzheimer's Disease.

Aging is recognized as the key risk factor for Alzheimer’s disease (AD). This study aimed to identify and verify potential aging-related genes associated with AD using bioinformatics analysis. Aging-related differential expression genes (ARDEGs) were determined by the intersection of limma test, weighted correlation network analysis (WGCNA), and 1153 aging and senescence-associated genes. Potential biological functions and pathways of ARDEGs were determined by GO, KEGG, GSEA, and GSVA. Then, LASSO algorithm was used to identify the hub genes and the diagnostic ability of the five ARDEGs in discriminating AD from the healthy control samples. Further, the correlation between hub ARDEGs and clinical characteristics was explored. Finally, the expression level of the five ARDEGs was validated using other four GEO datasets and blood samples of patients with AD and healthy individuals. Five ARDEGs (GFAP, PDGFRB, PLOD1, MAP4K4, and NFKBIA) were obtained. For biological function analysis, aging, cellular senescence, and Ras protein signal transduction regulation were enriched. Diagnostic ability of the five ARDEGs in discriminating AD from the control samples demonstrated a favorable diagnostic value. Eventually, quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) validation test revealed that compared with healthy controls, the mRNA expression level of PDGFRB, PLOD1, MAP4K4, and NFKBIA were elevated in AD patients. In conclusion, this study identified four ARDEGs (PDGFRB, PLOD1, MAP4K4, and NFKBIA) associated with AD. They provide an insight into potential novel biomarkers for diagnosing AD and monitoring progression.

Selective Growth Suppressive Effect of Pravastatin on Senescent Human Lung Fibroblasts.

Various chemical reagents containing inhibitors of mitochondrial activity, antioxidants, nuclear factor-kappa B (NF-kB) inhibitor, mammalian target of rapamycin (mTOR) inhibitor and other clinical therapeutics were screened in order to identify those that selectively decrease the viability of senescent human lung fibroblasts. Cell viability was measured using the CCK-8 assay. The results showed that pravastatin, a drug for hyperlipidemia, decreased the viability of senescent cells but not non-senescent cells. The effect of pravastatin on senescent cells is thought to be due to the inhibition of cell proliferation, rather than cell death. The effect of pravastatin was further investigated using the glucose metabolism assay, which showed that glucose consumption was inhibited both in non-senescent and senescent cells and intracellular nicotinamide adenine dinucleotide (NAD) was decreased in senescent cells. Changes to the mRNA expression levels of senescence-associated genes in response to pravastatin treatment were quantified by real-time-qPCR. There were no significant changes in the relative mRNA expression levels of IL-1β, p16, p21, and p53 in pravastatin-treated non-senescent cells, whereas the expression of IL-1β and p16 were increased by pravastatin only in senescent cells. The results of this study suggest that pravastatin does not induce senolysis, but rather selectively inhibits the proliferation of senescent cells and that cellular senescence is enhanced by decreasing intracellular NAD and promoting IL-1β production.

Transcriptome Analysis of Leaf Senescence Regulation Under Alkaline Stress in Medicago truncatula.

In plants, the leaf is an essential photosynthetic organ, and is the primary harvest in forage crops such as alfalfa (Medicago sativa). Premature leaf senescence caused by environmental stress can result in significant yield loss and quality reduction. Therefore, the stay-green trait is important for improving the economic value of forage crops. Alkaline stress can severely damage leaf cells and, consequently, cause leaf senescence. To understand the molecular regulatory mechanisms and identify vital senescence-associated genes under alkaline stress, we used high-throughput sequencing to study transcriptional changes in Medicago truncatula, a model plant for forage crops. We identified 2,165 differentially expressed genes, 985 of which were identical to those in the dark-induced leaf senescence group. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses showed that the 985 genes were mainly enriched in nutrient cycling processes such as cellular amino acid metabolic processes and organic substance catabolic processes, indicating nutrient redistribution. The other 1,180 differentially expressed genes were significantly enriched in the oxidoreductase complex, aerobic respiration, and ion transport. Our analysis showed the two gene sets guiding the coupled physiological and biochemical alterations play different roles under alkaline stress with a coordinated and integrated way. Many transcription factor families were identified from these differentially expressed genes, including MYB, WRKY, bHLH, and NAC which have particular preference involved in stress resistance and regulation of senescence. Our results contribute to the exploration of the molecular regulatory mechanisms of leaf senescence in M. truncatula under alkaline stress and provide new candidate genes for future breeding to improve the biomass and quality of forage crops.

Genome-wide H3K9 acetylation level increases with age-dependent senescence of flag leaf in rice.

Flag leaf senescence is an important biological process that drives the remobilization of nutrients to the growing organs of rice. Leaf senescence is controlled by genetic information via gene expression and histone modification, but the precise mechanism is as yet unclear. Here, we analysed genome-wide acetylated lysine residue 9 of histone H3 (H3K9ac) enrichment by chromatin immunoprecipitation-sequencing (ChIP-seq), and examined its association with transcriptomes by RNA-seq during flag leaf aging in rice (Oryza sativa). We found that genome-wide H3K9 acetylation levels increased with age-dependent senescence in rice flag leaf, and there was a positive correlation between the density and breadth of H3K9ac with gene expression and transcript elongation. During flag leaf aging, we observed 1249 up-regulated differentially expressed genes (DEGs) and 996 down-regulated DEGs, showing a strong relationship between temporal changes in gene expression and gain/loss of H3K9ac. We produced a landscape of H3K9 acetylation-modified gene expression targets that include known senescence-associated genes, metabolism-related genes, as well as miRNA biosynthesis-related genes. Our findings reveal a complex regulatory network of metabolism- and senescence-related pathways mediated by H3K9ac, and elucidate patterns of H3K9ac-mediated regulation of gene expression during flag leaf aging in rice.

An Olive Oil Mill Wastewater Extract Improves Chemotherapeutic Activity Against Breast Cancer Cells While Protecting From Cardiotoxicity.

Cardiovascular toxicity in cancer patients receiving chemotherapy remains one of the most undesirable side effects, limiting the choice of the most efficient therapeutic regimen, including combinations of different anticancer agents. Anthracyclines (doxorubicin) and antimetabolites (5-fluorouracil (5-FU), capecitabine) are among the most known agents used in breast cancer and other neoplasms and are associated with cardiotoxic effects. Extra-virgin olive oil (EVOO) is rich in polyphenols endowed with antioxidant cardioprotective activities. Olive mill wastewater (OMWW), a waste product generated by EVOO processing, has been reported to be enriched in polyphenols. In this study, we investigated the activities of polyphenol-rich extract from OMWW, A009, in cooperation with chemotherapy on two breast cancer cell lines, namely, BT459 and MDA-MB-231, in a cardio-oncology perspective. The effects of A009 on cardiac cells were also investigated with and without chemotherapeutic agents. Cell viability was determined on BT459 and MDA-MB-231 (i.e., breast cancer cells) and H9C2 (i.e., rat cardiomyocytes) cells, using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. A spheroids assay was used as a 3D in vitro model on BT459 and MDA-MB-231 cells. For in vivo studies, the murine sponge assay of angiogenesis was used as a model of breast cancer-associated vascularization. The embryo of Danio rerio (zebrafish) was used to detect the cardioprotective activities of the OMWW. We found that the A009 extract exhibited antiangiogenic activities induced by breast cancer cell supernatants and increased T-cell recruitment in vivo. The combination of the OMWW extracts with doxorubicin or 5-FU limited BT459 and MDA-MB-231 cell viability and the diameter of 3D spheroids, while mitigating their toxic effects on the rat H9C2 cardiomyocytes. Cardioprotective effects were observed by the combination of OMWW extracts with doxorubicin in zebrafish embryos. Finally, in human cardio myocytes, we observed 5-FU-induced upregulation of the inflammatory, senescence-associated cytokine IL6 and p16 genes, which expression was reduced by OMWW treatment. Our study demonstrates that the polyphenol-rich purified OMWW extract A009 combined with cancer chemotherapy could represent a potential candidate for cardiovascular protection in breast cancer patients, while increasing the effects of breast cancer chemotherapy.

Regulation of Arabidopsis Matrix Metalloproteinases by Mitogen-Activated Protein Kinases and Their Function in Leaf Senescence

Leaf senescence is a developmentally programmed cell death process that is influenced by a variety of endogenous signals and environmental factors. Here, we report that MPK3 and MPK6, two Arabidopsis mitogen-activated protein kinases (MAPKs or MPKs), and their two upstream MAPK kinases (MAPKKs or MKKs), MKK4 and MKK5, are key regulators of leaf senescence. Weak induction of constitutively active MAPKKs driven by steroid-inducible promoter, which activates endogenous MPK3 and MPK6, induces leaf senescence. This gain-of-function phenotype requires functional endogenous MPK3 and MPK6. Furthermore, loss of function of both MKK4 and MKK5 delays leaf senescence. Expression profiling leads to the identification of matrix metalloproteinases (MMPs), a family of zinc- and calcium-dependent endopeptidases, as the downstream target genes of MPK3/MPK6 cascade. MPK3/MPK6 activation-triggered leaf senescence is associated with rapid and strong induction of At3-MMP and At2-MMP. Expression of Arabidopsis MMP genes is strongly induced during leaf senescence, qualifying them as senescence-associated genes (SAGs). In addition, either constitutive or inducible overexpression of At3-MMP is sufficient to trigger leaf senescence. Based on these findings, we conclude that MPK3/MPK6 MAPK cascade and MMP target genes further downstream are involved in regulating leaf senescence in Arabidopsis.

Stem cell-homing hydrogel-based miR-29b-5p delivery promotes cartilage regeneration by suppressing senescence in an osteoarthritis rat model.

Osteoarthritis (OA) is a common joint disease characterized by progressive loss of cartilage and reduction in lubricating synovial fluid, which lacks effective treatments currently. Here, we propose a hydrogel-based miRNA delivery strategy to rejuvenate impaired cartilage by creating a regenerative microenvironment to mitigate chondrocyte senescence that mainly contributes to cartilage breakdown during OA development. An aging-related miRNA, miR-29b-5p, was first found to be markedly down-regulated in OA cartilage, and their up-regulation suppressed the expression of matrix metalloproteinases and senescence-associated genes (P16INK4a/P21) via ten-eleven-translocation enzyme 1 (TET1). An injectable bioactive self-assembling peptide nanofiber hydrogel was applied to deliver agomir-29b-5p, which was functionalized by conjugating a stem cell–homing peptide SKPPGTSS for endogenous synovial stem cell recruitment simultaneously. Sustained miR-29b-5p delivery and recruitment of synovial stem cells and their subsequent differentiation into chondrocytes led to successful cartilage repair and chondrocyte rejuvenation. This strategy enables miRNA-based therapeutic modality to become a viable alternative for surgery in OA treatment.

Preharvest Hydrogen Peroxide Treatment Delays Leaf Senescence of Chinese Flowering Cabbage During Storage by Reducing Water Loss and Activating Antioxidant Defense System.

Leaf yellowing, an indicator of senescence, reduces commercial value of Chinese flowering cabbage after harvest. Hydrogen peroxide (H2O2) plays a dual role in mediating plant stress responses, but it is not clear whether and how it affects leaf senescence when exogenously stimulating the plants before harvest. Here, we found that preharvest application with low concentrations of H2O2 to root delays leaf senescence. Around 10 mM H2O2 reduced leaf yellowing rate by 8.2 and 26.4% relative to the control following 4 and 8 days storage, respectively. The H2O2-treated cabbages showed higher chlorophyll and lower relative expression of senescence-associated gene (SAG) BrSAG12 than the control. Proteomic analysis revealed 118 and 204 differentially expressed proteins (DEPs) in H2O2-treated plants at 4 and 8 days of storage, respectively. The main DEPs are involved in chlorophyll degradation and synthesis, water deprivation, antioxidant activity, and protections on chloroplast membranes. Decline of water loss in H2O2-treated cabbages was coincide with increase of proline contents and modulation of leaf stomatal aperture. Alleviation of oxidative stress was indicated by suppression of respiratory burst oxidase homolog and upregulation of reactive oxygen species (ROS) scavenging-related genes. These results were also supported by the alleviation of lipid peroxidation and the protections on cell integrity and photochemical efficiency in H2O2-treated group. Collectively, preharvest H2O2 treatment alleviates water loss and activates antioxidant defense system, protects chloroplast membrane from oxidative damage, and ultimately delays leaf senescence during storage. This study provides novel insights into the roles of H2O2 for regulating leaf senescence of Chinese flowering cabbage.

Gingerenone A Induces Antiproliferation and Senescence of Breast Cancer Cells.

Ginger is a popular spice and consists of several bioactive antioxidant compounds. Gingerenone A (Gin A), a novel compound isolated from Zingiber officinale, is rarely investigated for its anti-breast-cancer properties. Some ginger extracts have been reported to initiate senescence, an anticancer strategy. However, the anticancer effects of Gin A on breast cancer cells remain unclear. The present study aims to assess the modulating impact of Gin A acting on proliferation and senescence to breast cancer cells. Gin A diminished the cellular ATP content and decreased the cell viability of the MTS assay in several breast cancer cell lines. It also showed a delayed G2/M response to breast cancer cells (MCF7 and MDA-MB-231). N-acetylcysteine (NAC), an oxidative stress inhibitor, can revert these responses of antiproliferation and G2/M delay. The oxidative stress and senescence responses of Gin A were further validated by increasing reactive oxygen species, mitochondrial superoxide, and β-galactosidase activity, which were reverted by NAC. Gin A also upregulated senescence-associated gene expressions. In addition to oxidative stress, Gin A also induced DNA damage responses by increasing γH2AX level and foci and generating 8-hydroxyl-2′-deoxyguanosine in breast cancer cells, which were reverted by NAC. Therefore, Gin A promotes antiproliferation and senescence of breast cancer cells induced by oxidative stress.

Lnc-IL7R alleviates PM2.5-mediated cellular senescence and apoptosis through EZH2 recruitment in chronic obstructive pulmonary disease.

Long-term exposure to PM2.5 (particulate matter with an aerodynamic diameter of ≤ 2.5μm) is associated with pulmonary injury and emphysema in patients with chronic obstructive pulmonary disease (COPD). We investigated mechanisms through which the long noncoding RNA lnc-IL7R contributes to cellular damage by inducing oxidative stress in COPD patients exposed to PM2.5. Associations of serum lnc-IL7R levels with lung function, emphysema, and previous PM2.5 exposure in COPD patients were analyzed. Reactive oxygen species and lnc-IL7R levels were measured in PM2.5-treated cells. The levels of lnc-IL7R and cellular senescence-associated genes, namely p16INK4a and p21CIP1/WAF1, were determined through lung tissue section staining. The effects of p16INK4a or p21CIP1/WAF1 regulation were examined by performing lnc-IL7R overexpression and knockdown assays. The functions of lnc-IL7R-mediated cell proliferation, cell cycle, senescence, colony formation, and apoptosis were examined in cells treated with PM2.5. Chromatin immunoprecipitation assays were conducted to investigate the epigenetic regulation of p21CIP1/WAF1. Lnc-IL7R levels decreased in COPD patients and were negatively correlated with emphysema or PM2.5 exposure. Lnc-IL7R levels were upregulated in normal lung epithelial cells but not in COPD cells exposed to PM2.5. Lower lnc-IL7R expression in PM2.5-treated cells induced p16INK4a and p21CIP1/WAF1 expression by increasing oxidative stress. Higher lnc-IL7R expression protected against cellular senescence and apoptosis, whereas lower lnc-IL7R expression augmented injury in PM2.5-treated cells. Lnc-IL7R and the enhancer of zeste homolog 2 (EZH2) synergistically suppressed p21CIP1/WAF1 expression through epigenetic modulation. Lnc-IL7R attenuates PM2.5-mediated p21CIP1/WAF1 expression through EZH2 recruitment, and its dysfunction may augment cellular injury in COPD.

Rapid Investigation of Functional Roles of Genes in Regulation of Leaf Senescence Using Arabidopsis Protoplasts.

Leaf senescence is the final stage of leaf development preceding death, which involves a significant cellular metabolic transition from anabolism to catabolism. Several processes during leaf senescence require coordinated regulation by senescence regulatory genes. In this study, we developed a rapid and systematic cellular approach to dissect the functional roles of genes in senescence regulation through their transient expression in Arabidopsis protoplasts. We established and validated this system by monitoring the differential expression of a luciferase-based reporter that was driven by promoters of SEN4 and SAG12, early and late senescence-responsive genes, depending on effectors of known positive and negative senescence regulators. Overexpression of positive senescence regulators, including ORE1, RPK1, and RAV1, increased the expression of both SEN4- and SAG12-LUC while ORE7, a negative senescence regulator decreased their expression. Consistently with overexpression, knockdown of target genes using amiRNAs resulted in opposite SAG12-LUC expression patterns. The timing and patterns of reporter responses induced by senescence regulators provided molecular evidence for their distinct kinetic involvement in leaf senescence regulation. Remarkably, ORE1 and RPK1 are involved in cell death responses, with more prominent and earlier involvement of ORE1 than RPK1. Consistent with the results in protoplasts, further time series of reactive oxygen species (ROS) and cell death assays using different tobacco transient systems reveal that ORE1 causes acute cell death and RPK1 mediates superoxide-dependent intermediate cell death signaling during leaf senescence. Overall, our results indicated that the luciferase-based reporter system in protoplasts is a reliable experimental system that can be effectively used to examine the regulatory roles of Arabidopsis senescence-associated genes.

Transcriptome Analysis of the Molecular Patterns of Pear Plants Infected by Two Colletotrichum fructicola Pathogenic Strains Causing Contrasting Sets of Leaf Symptoms.

Colletotrichum fructicola infects pear leaves, resulting in two major symptoms: tiny black spots (TS) followed by severe early defoliation and big necrotic lesions (BnL) without apparent damage depending on the pathotypes. How the same fungal species causes different symptoms remains unclear. To understand the molecular mechanism underlying the resulting diseases and the diverse symptoms, two C. fructicola pathogenetic strains (PAFQ31 and PAFQ32 responsible for TS and BnL symptoms, respectively) were inoculated on Pyrus pyrifolia leaves and subjected to transcriptome sequencing at the quiescent stage (QS) and necrotrophic stage (NS), respectively. In planta, the genes involved in the salicylic acid (SA) signaling pathway were upregulated at the NS caused by the infection of each strain. In contrast, the ethylene (ET), abscisic acid (ABA), and jasmonic acid (JA) signaling pathways were specifically related to the TS symptoms caused by the infection of strain PAFQ31, corresponding to the yellowish and early defoliation symptoms triggered by the strain infection. Correspondingly, SA was accumulated in similar levels in the leaves infected by each strain at NS, but JA was significantly higher in the PAFQ31-infected as measured using high-performance liquid chromatography. Weighted gene co-expression network analysis also reveals specific genes, pathways, phytohormones, and transcription factors (TFs) associated with the PAFQ31-associated early defoliation. Taken together, these data suggest that specific metabolic pathways were regulated in P. pyrifolia in response to the infection of two C. fructicola pathotypes resulting in the diverse symptoms: JA, ET, and ABA accumulated in the PAFQ31-infected leaves, which negatively affected the chlorophyll metabolism and photosynthesis pathways while positively affecting the expression of senescence-associated TFs and genes, resulted in leaf yellowing and defoliation; whereas SA inhibited JA-induced gene expression in the PAFQ32-infected leaves, which led to hypersensitive response-like reaction and BnL symptoms.

A unique miR775-GALT9 module regulates leaf senescence in Arabidopsis during post-submergence recovery by modulating ethylene and the abscisic acid pathway.

The submergence-induced hypoxic condition negatively affects the plant growth and development, and causes early onset of senescence. Hypoxia alters the expression of a number of microRNAs (miRNAs). However, the molecular function of submergence stress-induced miRNAs in physiological or developmental changes and recovery remains poorly understood. Here, we show that miR775 is an Arabidopsis thaliana-specific young and unique miRNA that possibly evolved non-canonically. miR775 post-transcriptionally regulates GALACTOSYLTRANSFERASE 9 (GALT9) and their expression is inversely affected at 24 h of complete submergence stress. The overexpression of miR775 (miR775-Oe) confers enhanced recovery from submergence stress and reduced accumulation of RBOHD and ROS, in contrast to wild-type and MIM775 Arabidopsis shoot. A similar recovery phenotype in the galt9 mutant indicates the role of the miR775-GALT9 module in post-submergence recovery. We predicted that Golgi-localized GALT9 is potentially involved in protein glycosylation. The altered expression of senescence-associated genes (SAG12, SAG29 and ORE1), ethylene signalling (EIN2 and EIN3) and abscisic acid (ABA) biosynthesis (NCED3) pathway genes occurs in miR775-Oe, galt9 and MIM775 plants. Thus, our results indicate the role for the miR775-GALT9 module in post-submergence recovery through a crosstalk between the ethylene signalling and ABA biosynthesis pathways.

Effects of different photoperiods on flower opening, flower closing and circadian expression of clock-related genes in Iris domestica and I. dichotoma.

The circadian clock can entrain to forced light-dark cycles by adjusting the phases and periods of flower opening and closing in ephemeral flowers. The responses of circadian rhythms to the same light conditions differ from species. However, the differences in internal genetic mechanisms underlying the different responses between species remain unclear. Iris domestica and I. dichotoma have ephemeral flowers and significantly divergent flower opening and closing times. The effects of different photoperiods (continuous darkness, 4L20D, 8L16D, 12L12D, 16L8D, 20L4D and continuous white light) on flower opening and closing, and expression patterns of seven genes (CRYPTOCHROME 1, PHYTOCHROME B, LATE ELONGATED HYPOCOTYL, PSEUDO RESPONSE REGULATOR 95, PHYTOCHROME INTERACTING FACTOR 4-like, SMUX AUXIN UP RNA 64-like and senescence-associated gene 39-like) involved in the circadian regulation of flower opening and closing were compared between I. domestica and I. dichotoma. Flower opening and closing in the two species exhibited circadian rhythms under continuous darkness (DD), but showed arrhythmia in continuous white light (LL). In the two species, keeping robust rhythms, strong synchronicity, rapid progressions of flower opening and closing and reaching full opening stage required a dark period longer than 4h. In light-dark cycles with dark periods longer than 4h, flower opening and closing times of the two species delayed with the delay of dawn, and the degree to which flower opening time varies with the time of dawn was greater in I. dichotoma than in I. domestica. The arrhythmia of flower opening and closing under 20L4D and LL would result from the arrhythmic output signals rather than arrhythmia of oscillators and photoreceptors. The different responses of the two species to the change of photoperiods would be caused by the transcriptional differences of genes in the output pathway of circadian clock system rather than in the input pathway or oscillators.