Rapamycin and Starvation Mitigate Indomethacin -Induced Intestinal Damage through Preservation of Lysosomal Vacuolar ATPase Integrity.

Nonsteroidal anti-inflammatory drugs (NSAIDs) possess anti-inflammatory, antipyretic, and analgesic properties and are among the most commonly used drugs. Although the cause of NSAID-induced gastric ulcers is well-understood, the mechanism behind small intestinal ulcers remains elusive. In this study, we examined the mechanism through which indomethacin (IM), a prominent NSAID, induces small intestinal ulcers, both in vitro and in vivo In IEC6 cells, a small intestinal epithelial cell line, IM treatment elevated levels of LC3-Ⅱ and p62. These expression levels remained unaltered after treatment with chloroquine or bafilomycin, which are vacuolar ATPase (V-ATPase) inhibitors. IM treatment reduced the activity of cathepsin B, a lysosomal protein hydrolytic enzyme, and increased the lysosomal pH. There was a notable increase in subcellular co-localization of LC3 with Lamp2, a lysosome marker, post-IM treatment. The increased lysosomal pH and decreased cathepsin B activity were reversed by pretreatment with rapamycin (Rapa) or glucose starvation, both of which stabilize V-ATPase assembly. To validate the in vitro findings in vivo, we established an IM-induced small intestine ulcer mouse model. In this model, we observed multiple ulcerations and heightened inflammation following IM administration. However, pretreatment with Rapa or fasting, which stabilize V-ATPase assembly, mitigated the IM-induced small intestinal ulcers in mice. Co-immunoprecipitation studies demonstrated that IM binds to V-ATPase in vitro and in vivo These findings suggest that IM induces small intestinal injury through lysosomal dysfunction, likely due to the disassembly of lysosomal V-ATPase caused by direct binding. Moreover, Rapa or starvation can prevent this injury by stabilizing the assembly. Significance Statement This study elucidates the largely unknown mechanisms behind small intestinal ulceration induced by indomethacin and reveals the involvement of lysosomal dysfunction via V-ATPase disassembly. The significance lies in identifying potential preventative interventions, such as rapamycin treatment or glucose starvation, offering pivotal insights that extend beyond NSAID-induced ulcers to broader gastrointestinal pathologies and treatments, thereby providing a foundation for novel therapeutic strategies aimed at a wide array of gastrointestinal disorders.

Petal abscission is promoted by jasmonic acid-induced autophagy at Arabidopsis petal bases.

In angiosperms, the transition from floral-organ maintenance to abscission determines reproductive success and seed dispersion. For petal abscission, cell-fate decisions specifically at the petal-cell base are more important than organ-level senescence or cell death in petals. However, how this transition is regulated remains unclear. Here, we identify a jasmonic acid (JA)-regulated chromatin-state switch at the base of Arabidopsis petals that directs local cell-fate determination via autophagy. During petal maintenance, co-repressors of JA signaling accumulate at the base of petals to block MYC activity, leading to lower levels of ROS. JA acts as an airborne signaling molecule transmitted from stamens to petals, accumulating primarily in petal bases to trigger chromatin remodeling. This allows MYC transcription factors to promote chromatin accessibility for downstream targets, including NAC DOMAIN-CONTAINING PROTEIN102 (ANAC102). ANAC102 accumulates specifically at the petal base prior to abscission and triggers ROS accumulation and cell death via AUTOPHAGY-RELATED GENEs induction. Developmentally induced autophagy at the petal base causes maturation, vacuolar delivery, and breakdown of autophagosomes for terminal cell differentiation. Dynamic changes in vesicles and cytoplasmic components in the vacuole occur in many plants, suggesting JA-NAC-mediated local cell-fate determination by autophagy may be conserved in angiosperms.

Visualization of root extracellular traps in an ectomycorrhizal woody plant (Pinus densiflora) and their interactions with root-associated bacteria.

MAIN CONCLUSION: Extracellular traps in the primary root of Pinus densiflora contribute to root-associated bacterial colonization. Trapped rhizobacteria induce the production of reactive oxygen species in root-associated, cap-derived cells. Ectomycorrhizal (ECM) woody plants, such as members of Pinaceae and Fagaceae, can acquire resistance to biotic and abiotic stresses through the formation of mycorrhiza with ECM fungi. However, germinated tree seedlings do not have mycorrhizae and it takes several weeks for ectomycorrhizae to form on their root tips. Therefore, to confer protection during the early growth stage, bare primary roots require defense mechanisms other than mycorrhization. Here, we attempted to visualize root extracellular traps (RETs), an innate root defense mechanism, in the primary root of Pinus densiflora and investigate the interactions with root-associated bacteria isolated from ECM and fine non-mycorrhizal roots. Histological and histochemical imaging and colony-forming unit assays demonstrated that RETs in P. densiflora, mainly consisting of root-associated, cap-derived cells (AC-DCs) and large amounts of root mucilage, promote bacterial colonization in the rhizosphere, despite also having bactericidal activity via extracellular DNA. Four rhizobacterial strains retarded the mycelial growth of a pathogenic strain belonging to the Fusarium oxysporum species complex in dual culture assay. They also induced the production of reactive oxygen species (ROS) from host tree AC-DCs without being excluded from the rhizosphere of P. densiflora. Applying three Paraburkholderia strains, especially PM O-EM8 and PF T-NM22, showed significant differences in the ROS levels from the control group. These results reveal the indirect contributions of rhizobacteria to host root defense and suggest that root-associated bacteria could be a component of RETs as a first line of defense against root pathogens in the early growth stage of ECM woody plants.

Successful surgical experience for acute severe aortic valve regurgitation with acquired Gerbode defect: A case report.

INTRODUCTION AND IMPORTANCE: The incidence of acquired Gerbode defect has been increasing due to advances in cardiac imaging technology, and some closure methods have been introduced. PRESENTATION OF CASE: A 58-year-old man developed cardiogenic shock due to acute severe aortic valve regurgitation with an acquired Gerbode defect caused by infective endocarditis. Emergency surgery was performed. A large patch with a 0.4 mm extended-polytetrafluoroethylene (e-PTFE) sheet covered with autologous pericardium was used to close the Gerbode defect, and a bioprosthetic valve was used for aortic valve replacement. CLINICAL DISCUSSION: Large patch closure with 0.4 mm e-PTFE sheet and autologous pericardium for fragile Gerbode defect caused by infective endocarditis might be effective with regard to sturdiness, good fitting to the tissue, and excellent resistance to bacteria. CONCLUSION: We encountered a rare case of cardiogenic shock due to acute severe aortic valve regurgitation and acquired Gerbode defect caused by infective endocarditis. In our case, large-patch closure for perforation in a fragile membranous septum was effective.

LysM-mediated signaling in Marchantia polymorpha highlights the conservation of pattern-triggered immunity in land plants.

Pattern-recognition receptor (PRR)-triggered immunity (PTI) wards off a wide range of pathogenic microbes, playing a pivotal role in angiosperms. The model liverwort Marchantia polymorpha triggers defense-related gene expression upon sensing components of bacterial and fungal extracts, suggesting the existence of PTI in this plant model. However, the molecular components of the putative PTI in M. polymorpha and the significance of PTI in bryophytes have not yet been described. We here show that M. polymorpha has four lysin motif (LysM)-domain-containing receptor homologs, two of which, LysM-receptor-like kinase (LYK) MpLYK1 and LYK-related (LYR) MpLYR, are responsible for sensing chitin and peptidoglycan fragments, triggering a series of characteristic immune responses. Comprehensive phosphoproteomic analysis of M. polymorpha in response to chitin treatment identified regulatory proteins that potentially shape LysM-mediated PTI. The identified proteins included homologs of well-described PTI components in angiosperms as well as proteins whose roles in PTI are not yet determined, including the blue-light receptor phototropin MpPHOT. We revealed that MpPHOT is required for negative feedback of defense-related gene expression during PTI. Taken together, this study outlines the basic framework of LysM-mediated PTI in M. polymorpha and highlights conserved elements and new aspects of pattern-triggered immunity in land plants.

Stomatal regulators are co-opted for seta development in the astomatous liverwort Marchantia polymorpha.

The evolution of special types of cells requires the acquisition of new gene regulatory networks controlled by transcription factors (TFs). In stomatous plants, a TF module formed by subfamilies Ia and IIIb basic helix-loop-helix TFs (Ia-IIIb bHLH) regulates stomatal formation; however, how this module evolved during land plant diversification remains unclear. Here we show that, in the astomatous liverwort Marchantia polymorpha, a Ia-IIIb bHLH module regulates the development of a unique sporophyte tissue, the seta, which is found in mosses and liverworts. The sole Ia bHLH gene, MpSETA, and a IIIb bHLH gene, MpICE2, regulate the cell division and/or differentiation of seta lineage cells. MpSETA can partially replace the stomatal function of Ia bHLH TFs in Arabidopsis thaliana, suggesting that a common regulatory mechanism underlies setal and stomatal formation. Our findings reveal the co-option of a Ia-IIIb bHLH TF module for regulating cell fate determination and/or cell division of distinct types of cells during land plant evolution.

A Novel Amphotericin B Hydrogel Composed of Poly(Vinyl Alcohol)/Borate Complex for Ophthalmic Formulation.

In this study, we developed a water-soluble complex-hydrogel viscosity-controlled formulation of amphotericin B (AmB). AmB is insoluble in water, but borax makes it soluble by forming a complex with AmB. Borax also forms complexes with poly(vinyl alcohol) (PVA) to produce viscous hydrogels. Furthermore, boric acid interacts with mucin expressed in corneal epithelial cells. Accordingly, by utilizing these properties of borax simultaneously, we prepared a water-soluble AmB complex-hydrogel with poly(vinyl alcohol)/borate (PVA-B-AmB), which is suitable for eye drops. PVA-B-AmB was easily prepared by simply mixing aqueous AmB solution dissolved in borax, PVA solution, and water. The 11B-NMR results suggested that PVA-B-AmB existed by bonding PVA and AmB via boronic acid. PVA-B-AmB (gel ratio = 0.55) has a viscosity of 18.3 ± 0.5 mPa·s and is suitable for ophthalmic formulations. This formulation exhibited sustained release of AmB of approximately 45% at 24 h. It was also shown that this formulation interacts with mucin. These results suggest that PVA-B-AmB can be used as a water-soluble AmB preparation suitable for ophthalmic use.

Arrest, Senescence and Death of Shoot Apical Stem Cells in Arabidopsis thaliana.

Shoot stem cells act as the source of the aboveground parts of flowering plants. A precise regulatory basis is required to ensure that plant stem cells show the right status during the stages of proliferation, senescence and cell death. Over the past few decades, the genetic circuits controlling stem cell fate, including the regulatory pathways of establishment, maintenance and differentiation, have been largely revealed. However, the morphological changes and molecular mechanisms of the final stages of stem cells, which are represented by senescence and cell death, have been less studied. The senescence and death of shoot stem cells are under the control of a complex series of pathways that integrate multiple internal and external signals. Given the crucial roles of shoot stem cells in influencing plant longevity and crop yields, researchers have attempted to uncover details of stem cell senescence and death. Recent studies indicate that stem cell activity arrest is controlled by the FRUITFULL-APETALA2 pathway and the plant hormones auxin and cytokinin, while the features of senescent and dead shoot apical stem cells have also been described, with dynamic changes in reactive oxygen species implicated in stem cell death. In this review, we highlight the recent breakthroughs that have enriched our understanding of senescence and cell death processes in plant stem cells.

I-Composite Graft with Right Internal Thoracic Artery and Right Gastroepiploic Artery in Coronary Artery Bypass Grafting.

PURPOSE: When added to the internal thoracic artery (ITA), the right gastroepiploic artery (GEA) has been used as an in-situ graft or an I-composite right ITA-right GEA graft in coronary artery bypass grafting (CABG). We aimed to verify its potential. METHODS: We evaluated 104 patients who underwent first isolated CABG with this I-composite graft. The number of distal anastomoses, graft flow (GF) and pulsatility index (PI) during surgery, and graft patency in the early term regarding this I-composite graft were evaluated. RESULTS: The number of total distal anastomoses and distal anastomoses with arterial grafts were 4.17 ± 0.81 and 3.63 ± 0.81, respectively. This I-composite graft achieved 2.38 ± 0.69 distal anastomoses. GF tended to increase according to the increased number of distal anastomoses (p = 0.241), and the PI maintained a low score regardless of the number of distal anastomoses (p = 0.834). Graft patency was 95.5%; moreover, the number of distal anastomoses with this I-composite graft did not affect early-term graft patency. CONCLUSION: Right GEA utility was expanded as this I-composite graft in addition to in-situ graft. This I-composite graft has an adequate flow capacity for revascularization in non-left anterior descending coronary artery lesions.

Single-Cell RNA Sequencing of Arabidopsis Leaf Tissues Identifies Multiple Specialized Cell Types: Idioblast Myrosin Cells and Potential Glucosinolate-Producing Cells.

The glucosinolate-myrosinase defense system (GMDS), characteristic of Brassicales, is involved in plant defense. Previous single-cell transcriptomic analyses have reported the expression profiles of multiple GMDS-related cell types (i.e. myrosinase-rich myrosin idioblasts and multiple types of potential glucosinolate synthetic cells as well as a candidate S-cell for glucosinolate accumulation). However, differences in plant stages and cell-type annotation methods have hindered comparisons among studies. Here, we used the single-cell transcriptome profiles of extended Arabidopsis leaves and verified the distribution of previously used markers to refine the expression profiles of GMDS-associated cell types. Moreover, we performed beta-glucuronidase promoter assays to confirm the histological expression patterns of newly obtained markers for GMDS-associated candidates. As a result, we found a set of new specific reporters for myrosin cells and potential glucosinolate-producing cells.

Histone Demethylases JMJ30 and JMJ32 Modulate the Speed of Vernalization Through the Activation of FLOWERING LOCUS C in Arabidopsis thaliana.

Vernalization is the promotion of flowering after prolonged exposure to cold. In Arabidopsis thaliana, vernalization induces epigenetic silencing of the floral repressor gene FLOWERING LOCUS C (FLC). Among the repressive epigenetic marks, the trimethylation of lysine 27 on histone H3 proteins (H3K27me3) is a critical contributor to the epigenetic silencing of FLC. The deposition of H3K27me3 is mediated by Polycomb Repressive Complex 2 (PRC2). Conversely, the elimination of H3K27me3 is mediated by histone demethylases, Jumonji-C domain-containing protein JMJ30 and its homolog JMJ32. However, the role of JMJ30 and JMJ32 in vernalization is largely unknown. In this study, we found that cold treatment dramatically reduced the expression levels of JMJ30 and did not reduce those of JMJ32. Next, by using the genetic approach, we found that the flowering of jmj30 jmj32 was accelerated under moderate vernalized conditions. Under moderate vernalized conditions, the silencing of FLC occurred more quickly in jmj30 jmj32 than in the wild type. These results suggested that the histone demethylases JMJ30 and JMJ32 brake vernalization through the activation of FLC. Our study suggested that PRC2 and Jumonji histone demethylases act in an opposing manner to regulate flowering time via epigenetic modifications.

Dynamic Changes in Reactive Oxygen Species in the Shoot Apex Contribute to Stem Cell Death in Arabidopsis thaliana.

In monocarpic plants, stem cells are fated to die. However, the potential mechanism of stem cell death has remained elusive. Here, we reveal that the levels of two forms of reactive oxygen species (ROS), superoxide anion free radical (O2·-) and hydrogen peroxide (H2O2), show dynamic changes in the shoot apex during the plant life cycle of Arabidopsis thaliana. We found that the level of O2·- decreased and disappeared at four weeks after bolting (WAB), while H2O2 appeared at 3 WAB and showed a burst at 5 WAB. The timing of dynamic changes in O2·- and H2O2 was delayed for approximately three weeks in clv3-2, which has a longer lifespan. Moreover, exogenous application of H2O2 inhibited the expression of the stem cell determinant WUSCHEL (WUS) and promoted the expression of the developmentally programmed cell death (dPCD) marker gene ORESARA 1 (ORE1). These results indicate that H2O2 triggers an important signal inducing dPCD in stem cells. Given that O2·- plays roles in maintaining WUS expression and stem cell activity, we speculate that the dynamic shift from O2·- to H2O2 in the shoot apex results in stem cell death. Our findings provide novel insights for understanding ROS-mediated regulation during plant stem cell death.

The David Operation Offers Shorter Hemostasis Time Than the Bentall in Case of Acute Aortic Dissection Type A.

Background The aim of the present study was to compare the clinical outcome of the David operation and the Bentall operation in patients with Stanford type A acute aortic dissection (AADA) from the viewpoint of hemostasis. Methods Between April 2016 and April 2020, 235 patients underwent emergent surgery for AADA. Of them, 38 patients required aortic root replacement (ARR: The David operation 17, the Bentall operation 21). The mean age was 59.3±12.6 years. In the present series, the David operation was the first choice for relatively young people, and the Bentall operation was performed for relatively elderly patients and cases in which valve-sparing seemed impossible. Results Between the David and the Bentall group, the 30-day mortality rate did not differ significantly. However, hemostasis time (144.6±50.3 vs. 212.5±138.1 min, p=0.047), defined as the interval from the cessation of cardio-pulmonary bypass (CPB) to the end of the operation, and total operation time (477.8±85.7 vs. 578.3±173.6 min, p=0.027) were significantly shorter in the David group than in the Bentall group, and the amount of blood transfusion was less in the David group than in the Bentall group (red blood cells: 3.5±3.6 vs. 9.2±5.9 units, p=0.013; fresh frozen plasma: 4.1±4.7 vs 9.4±5.1 units, p=0.002; platelet concentrate: 33.2±11.3 vs 42.2±12.0 units, p=0.025). Conclusion David operation offers a shorter hemostasis time and consequently shorter operation time than the Bentall operation in the setting of AADA, probably due to double suture lines, despite its surgical complexity.

Recanalized saphenous vein graft once occluded in postoperative acute period.

A 69-year-old female with diabetes mellitus underwent off-pump coronary artery bypass grafting for old myocardial infarction and unstable angina with reduced left ventricular wall motion due to triple vessel disease. Although the saphenous vein graft was confirmed to be patent during surgery, it developed occlusion at the distal anastomotic site on postoperative first day. However, recanalization was achieved for this saphenous vein graft following the administration of direct oral anticoagulants in addition to antiplatelet therapy. Anticoagulant therapy, in addition to antiplatelet therapy, should be considered for preventing and dissolving thrombus in postoperative acute period, especially in high-risk patients for thrombotic graft occlusion.

A Novel Boron Lipid to Modify Liposomal Surfaces for Boron Neutron Capture Therapy.

Boron neutron capture therapy (BNCT) is a cancer treatment with clinically demonstrated efficacy using boronophenylalanine (BPA) and sodium mercaptododecaborate (BSH). However, tumor tissue selectivity of BSH and retention of BPA in tumor cells is a constant problem. To ensure boron accumulation and retention in tumor tissues, we designed a novel polyethylene glycol (PEG)-based boron-containing lipid (PBL) and examined the potency of delivery of boron using novel PBL-containing liposomes, facilitated by the enhanced permeability and retention (EPR) effect. PBL was synthesized by the reaction of distearoylphosphoethanolamine and BSH linked by PEG with Michael addition while liposomes modified using PBL were prepared from the mixed lipid at a constant molar ratio. In this manner, novel boron liposomes featuring BSH in the liposomal surfaces, instead of being encapsulated in the inner aqueous phase or incorporated in the lipid bilayer membrane, were prepared. These PBL liposomes also carry additional payload capacity for more boron compounds (or anticancer agents) in their inner aqueous phase. The findings demonstrated that PBL liposomes are promising candidates to effect suitable boron accumulation for BNCT.

Postoperative Changes in Pulmonary Function after Valve Surgery: Oxygenation Index Early after Cardiopulmonary Is a Predictor of Postoperative Course.

OBJECTIVE: To determine pulmonary functional changes that predict early clinical outcomes in valve surgery requiring long cardiopulmonary bypass (CPB). METHODS: This retrospective study included 225 consecutive non-emergency valve surgeries with fast-track cardiac anesthesia between January 2014 and March 2020. Blood gas analyses before and 0, 2, 4, 8, and 14 h after CPB were investigated. RESULTS: Median age and EuroSCORE II were 71.0 years (25-75 percentile: 59.5-77.0) and 2.46 (1.44-5.01). Patients underwent 96 aortic, 106 mitral, and 23 combined valve surgeries. The median CPB time was 151 min (122-193). PaO2/FiO2 and AaDO2/PaO2 significantly deteriorated two hours, but not immediately, after CPB (both p < 0.0001). Decreased PaO2/FiO2 and AaDO2/PaO2 were correlated with ventilation time (r2 = 0.318 and 0.435) and intensive care unit (ICU) (r2 = 0.172 and 0.267) and hospital stays (r2 = 0.164 and 0.209). Early and delayed extubations (<6 and >24 h) were predicted by PaO2/FiO2 (377.2 and 213.1) and AaDO2/PaO2 (0.683 and 1.680), measured two hours after CPB with acceptable sensitivity and specificity (0.700-0.911 and 0.677-0.859). CONCLUSIONS: PaO2/FiO2 and AaDO2/PaO2 two hours after CPB were correlated with ventilation time and lengths of ICU and hospital stays. These parameters suitably predicted early and delayed extubations.

Identification of a Devernalization Inducer by Chemical Screening Approaches in Arabidopsis thaliana.

Vernalization is the promotion of flowering after prolonged exposure to cold. In Arabidopsis thaliana, vernalization induces epigenetic silencing of the floral repressor gene FLOWERING LOCUS C (FLC). The repressive epigenetic mark trimethylation of lysine 27 on histone H3 proteins (H3K27me3) is a critical contributor to the epigenetic silencing of FLC. Interestingly, the deposited H3K27me3 in the FLC locus can be erased by short-term high-temperature treatment. This is referred to as devernalization. In this study, we identified a novel chemical compound, 4-Isoxazolecarboxylic acid, 3,5-dimethyl-2-(4-fluorophenyl)-4-isoxazole carboxylic acid 1-methyl-2-oxoethyl ester named as DEVERNALIZER01 (DVR01), which induces devernalization in Arabidopsis seedlings, by an FLC-luciferase reporter-based high-throughput screening assay. DVR01 decreased the amount of H3K27me3 in the FLC locus in vernalized plants, resulting in the upregulation of FLC in the whole plant, including the vasculature and meristem, where FLC represses floral induction genes. We also showed that a 2-week treatment with DVR01 reverted plants with a vernalized status back to a fully non-vernalized status. Collectively, this study provides a novel structure of DVR01, which modulates devernalization via demethylation of H3K27me3 in the FLC locus.

The Cell Differentiation of Idioblast Myrosin Cells: Similarities With Vascular and Guard Cells.

Idioblasts are defined by abnormal shapes, sizes, and contents that are different from neighboring cells. Myrosin cells are Brassicales-specific idioblasts and accumulate a large amount of thioglucoside glucohydrolases (TGGs, also known as myrosinases) in their vacuoles. Myrosinases convert their substrates, glucosinolates, into toxic compounds when herbivories and pests attack plants. In this review, we highlight the similarities and differences between myrosin cells and vascular cells/guard cells (GCs) because myrosin cells are distributed along vascular cells, especially the phloem parenchyma, and myrosin cells share the master transcription factor FAMA with GCs for their cell differentiation. In addition, we analyzed the overlap of cell type-specific genes between myrosin cells and GCs by using published single-cell transcriptomics (scRNA-seq) data, suggesting significant similarities in the gene expression patterns of these two specialized cells.

Optimization of preparation methods for high loading content and high encapsulation efficiency of BSH into liposomes.

To optimize the preparation methods for liposomes encapsulating mercaptoundecahydrododecaborate (BSH), we examined BSH and lipid concentrations that increased the boron content in liposomes. We improved the BSH encapsulation efficiency and boron content of the liposomes from 4.2 to 45.9 % and 9.5-54.3 µg, respectively, by changing the lipid concentration from 10 to 150 mg/mL. Notably, the boron content increased significantly from 26.2 µg to 326.3 µg at a constant lipid concentration of 30 mg/mL with increased BSH concentrations.

Morphological and Physiological Framework Underlying Plant Longevity in Arabidopsis thaliana.

Monocarpic plants have a single reproductive phase, in which their longevity is developmentally programmed by molecular networks. In the reproductive phase of Arabidopsis thaliana, the inflorescence meristem (IM) maintains a central pool of stem cells and produces a limited number of flower primordia, which result in seed formation and the death of the whole plant. In this study, we observed morphological changes in the IM at cellular and intracellular resolutions until the end of the plant life cycle. We observed four biological events during the periods from 1 week after bolting (WAB) till the death of stem cells: (1) the gradual reduction in the size of the IM, (2) the dynamic vacuolation of IM cells, (3) the loss of the expression of the stem cell determinant WUSCHEL (WUS), and (4) the upregulation of the programmed cell death marker BIFUNCTIONAL NUCLEASE1 (BFN1) in association with the death of stem cells. These results indicate that the stem cell population gradually decreases in IM during plant aging and eventually is fully terminated. We further show that the expression of WUS became undetectable in IM at 3 WAB prior to the loss of CLAVATA3 (CLV3) expression at 5 WAB; CLV3 is a negative regulator of WUS. Moreover, clv3 plants showed delayed loss of WUS and lived 6 weeks longer compared with wild-type plants. These results indicated that the prolonged expression of CLV3 at 4-5 WAB may be a safeguard that inhibits the reactivation of WUS and promotes plant death. Finally, through transcriptome analysis, we determined that reactive oxygen species (ROS) are involved in the control of plant longevity. Our work presents a morphological and physiological framework for the regulation of plant longevity in Arabidopsis.