Deoxyribonuclease I Alleviates Septic Liver Injury in a Rat Model Supported by Venoarterial Extracorporeal Membrane Oxygenation.

Sepsis is an unusual systemic reaction with high mortality and secondary septic liver injury is proposed to be the major cause of mortality. Extracorporeal membrane oxygenation (ECMO) can enhance terminal organ perfusion by elevating circulatory support which is used in severe sepsis patients. However, the interaction of blood components with the biomaterials of the extracorporeal membrane elicits a systemic inflammatory response. Besides, inflammation and apoptosis are the main mediators in the pathophysiology of septic liver injury. Therefore, we investigated the protective effect of Deoxyribonuclease I (DNase I) against septic liver injury supported by ECMO in rats. Sepsis was induced by lipopolysaccharide (LPS) and 24 hours after the administration, the rats were treated with ECMO. Then blood samples and liver tissues were collected. DNase I significantly attenuated the level of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and significantly decreased hepatic levels of NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome, myeloperoxidase (MPO), downstream inflammatory factor interleukin-1ß (IL-1ß) and interleukin-18 (IL-18), and improved neutrophil infiltration. Additionally, DNase I significantly reduced the expression of apoptosis key protein and terminal-deoxynucleotidyl transferase-mediated nick end labeling (TUNEL)-labeled apoptotic hepatocytes. In summary, our findings demonstrated that DNase I alleviates liver injury in ECMO-supported septic rats by reducing the inflammatory and apoptotic responses.

LPA2 Alleviates Septic Acute Lung Injury via Protective Endothelial Barrier Function Through Activation of PLC-PKC-FAK.

Background: Increased endothelial permeability of pulmonary vessels is a primary pathological characteristic of septic acute lung injury (ALI). Previously, elevated lysophosphatidic acid (LPA) levels and LPA2 (an LPA receptor) expression have been found in the peripheral blood and lungs of septic mice, respectively. However, the specific role of LPA2 in septic ALI remains unclear. Methods: A lipopolysaccharide (LPS)-induced model of sepsis was established in wild-type (WT) and global LPA2 knockout (Lpar2-/-) mice. We examined mortality, lung injury, assessed endothelial permeability through Evans blue dye (EBD) assay in vivo, and transendothelial electrical resistance (TEER) of mouse lung microvascular endothelial cells (MLMECs) in vitro. Enzyme-linked immunosorbent assay (ELISA), histopathological, immunofluorescence, immunohistochemistry, and Western blot were employed to investigate the role of LPA2 in septic ALI. Results: Lpar2 deficiency increased vascular endothelial permeability, impaired lung injury, and increased mortality. Histological examination revealed aggravated inflammation, edema, hemorrhage and alveolar septal thickening in the lungs of septic Lpar2-/- mice. In vitro, loss of Lpar2 resulted in increased permeability of MLMECs. Pharmacological activation of LPA2 by the agonist DBIBB led to significantly reduced inflammation, edema and hemorrhage, as well as increased expression of the vascular endothelial tight junction (TJ) protein zonula occludens-1 (ZO-1) and claudin-5, as well as the adheren junction (AJ) protein VE-cadherin. Moreover, DBIBB treatment was found to alleviate mortality by protecting against vascular endothelial permeability. Mechanistically, we demonstrated that vascular endothelial permeability was alleviated through LPA-LPA2 signaling via the PLC-PKC-FAK pathway. Conclusion: These data provide a novel mechanism of endothelial barrier protection via PLC-PKC-FAK pathway and suggest that LPA2 may contribute to the therapeutic effects of septic ALI.

Transcriptomic Profiling of circRNAs in rat Hippocampus after Deep Hypothermic Circulatory Arrest.

Neurologic abnormalities occurring after deep hypothermic circulatory arrest (DHCA) remain a significant concern. However, molecular mechanisms leading to DHCA-related cerebral injury are still ill-defined. Circular RNAs (circRNAs) are a class of covalently closed non-coding RNAs and can play important roles in different types of cerebral injury. This study aimed to investigate circRNAs expression profiles in rat hippocampus after DHCA and explore the potential functions of circRNAs in DHCA-related cerebral injury. Hence, the DHCA procedure in rats was established and a transcriptomic profiling of circRNAs in rat hippocampus was done. As a result, a total of 35192 circRNAs were identified. Among them, 339 circRNAs were dysregulated, including 194 down-regulated and 145 up-regulated between DHCA and sham group. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were performed based on the host genes of all dysregulated circRNAs. Also, 4 circRNAs were validated by RT-qPCR (rno_circ_0028462, rno_circ_0037165, rno_circ_0045161 and rno_circ_0019047). Then a circRNA-microRNA (miRNA) interaction network involving 4 candidate circRNAs was constructed. Furthermore, functional enrichment analysis of the miRNA-targeting mRNAs of every candidate circRNA was conducted to gain insight into each of the 4 circRNAs. Our study provided a better understanding of circRNAs in the mechanisms of DHCA-related cerebral injury and some potential targets for neuroprotection.

Neutrophil extracellular traps are increased after extracorporeal membrane oxygenation support initiation and present in thrombus: A preclinical study using sheep as an animal model.

BACKGROUND: The balance between thrombosis and hemostasis is a difficult issue during extracorporeal membrane oxygenation (ECMO) support. The pathogenesis leading to thrombotic complications during ECMO support is not well understood. Neutrophil extracellular traps (NETs) were reported to participate in thrombosis and have a key role in inflammation. This study aimed to explore the role of NETs in thrombosis during ECMO support and investigate NETs as a predictive biomarker for thrombotic complications during ECMO assistance. METHODS: Ten ovine models of ECMO support were established. Animals were then randomly divided into 2 groups (5 sheep/group): venoarterial (VA) ECMO group and venovenous (VV) ECMO group. The venous blood samples were collected at different time points. Markers of NETs were detected in plasma, neutrophils, and thrombi from the vessels and membrane. Moreover, circulating NETs levels in 8 adults treated in the intensive care unit (ICU) who received VA-ECMO and 8 healthy controls were detected; patient survival was also recorded. RESULTS: In vivo study showed that neutrophils and NETs markers (dsDNA and citH3) levels were significantly elevated 6 h after ECMO compared to baseline. Isolated neutrophils from fresh blood at 6 h could release more NETs. dsDNA and citH3 levels were significantly higher in the VA mode than in the VV mode. NETs were found in thrombi from the vessel and membrane. Clinical data further revealed that dsDNA, citH3, and nucleosomes were higher in patients who received ECMO than in healthy controls. CONCLUSIONS: These data suggest NETs might be associated with thrombus during ECMO support, especially in the VA mode. These findings provide new insight into preventing thrombotic complications by targeting NETs. Also, NETs may potentially become an early warning biomarker for thrombosis under ECMO assistance.

LPA2 Contributes to Vascular Endothelium Homeostasis and Cardiac Remodeling After Myocardial Infarction.

RATIONALE: Myocardial infarction (MI) is one of the most dangerous adverse cardiovascular events. Our previous study found that lysophosphatidic acid (LPA) is increased in human peripheral blood after MI, and LPA has a protective effect on the survival and proliferation of various cell types. However, the role of LPA and its receptors in MI is less understood. OBJECTIVES: To study the unknown role of LPA and its receptors in heart during MI. METHODS AND RESULTS: In this study, we found that mice also had elevated LPA level in peripheral blood, as well as increased cardiac expression of its receptor LPA2 in the early stages after MI. With adult and neonate MI models in global Lpar2 knockout (Lpar2-KO) mice, we found Lpar2 deficiency increased vascular leak leading to disruption of its homeostasis, so as to impaired heart function and increased early mortality. Histological examination revealed larger scar size, increased fibrosis, and reduced vascular density in the heart of Lpar2-KO mice. Furthermore, Lpar2-KO also attenuated blood flow recovery after femoral artery ligation with decreased vascular density in gastrocnemius. Our study revealed that Lpar2 was mainly expressed and altered in cardiac endothelial cells during MI, and use of endothelial-specific Lpar2 knockout mice phenocopied the global knockout mice. Additionally, adenovirus-Lpar2 and pharmacologically activated LPA2 significantly improved heart function, reduced scar size, increased vascular formation, and alleviated early mortality by maintaining vascular homeostasis owing to protecting vessels from leakage. Mechanistic studies demonstrated that LPA-LPA2 signaling could promote endothelial cell proliferation through PI3K-Akt/PLC-Raf1-Erk pathway and enhanced endothelial cell tube formation via PKD1-CD36 signaling. CONCLUSIONS: Our results indicate that endothelial LPA-LPA2 signaling promotes angiogenesis and maintains vascular homeostasis, which is vital for restoring blood flow and repairing tissue function in ischemic injuries. Targeting LPA-LPA2 signal might have clinical therapeutic potential to protect the heart from ischemic injury.

Lysophosphatidic Acid Receptor 3 Suppress Neutrophil Extracellular Traps Production and Thrombosis During Sepsis.

Sepsis consists of life-threatening organ dysfunction resulting from a dysregulated response to infection. Recent studies have found that excessive neutrophil extracellular traps (NETs) contribute to the pathogenesis of sepsis, thereby increasing morbidity and mortality. Lysophosphatidic acid (LPA) is a small glycerophospholipid molecule that exerts multiple functions by binding to its receptors. Although LPA has been functionally identified to induce NETs, whether and how LPA receptors, especially lysophosphatidic acid receptor 3 (LPA3), play a role in the development of sepsis has never been explored. A comprehensive understanding of the impact of LPA3 on sepsis is essential for the development of medical therapy. After intraperitoneal injection of lipopolysaccharide (LPS), Lpar3-/-mice showed a substantially higher mortality, more severe injury, and more fibrinogen content in the lungs than wild-type (WT) mice. The values of blood coagulation markers, plasma prothrombin time (PT) and fibrinogen (FIB), indicated that the Lpar3-/- mice underwent a severe coagulation process, which resulted in increased thrombosis. The levels of NETs in Lpar3-/- mice were higher than those in WT mice after LPS injection. The mortality rate and degree of lung damage in Lpar3-/- mice with sepsis were significantly reduced after the destruction of NETs by DNaseI treatment. Furthermore, in vitro experiments with co-cultured monocytes and neutrophils demonstrated that monocytes from Lpar3-/- mice promoted the formation of NETs, suggesting that LPA3 acting on monocytes inhibits the formation of NETs and plays a protective role in sepsis. Mechanistically, we found that the amount of CD14, an LPS co-receptor, expressed by monocytes in Lpar3-/-mice was significantly elevated after LPS administration, and the MyD88-p65-NFκB signaling axis, downstream of toll-like receptor 4 signaling, in monocytes was overactivated. Finally, after an injection of the LPA3 agonist (2S)-1-oleoyl-2-methylglycero-3-phosphothionate (OMPT), the survival rate of mice with sepsis was improved, organ damage was reduced, and the production of NETs was decreased. This suggested the possible translational value and application prospects of (2S)-OMPT in the treatment of sepsis. Our study confirms an important protective role of LPA3 in curbing the development of sepsis by suppressing NETs production and thrombosis and provides new ideas for sepsis treatment strategies.

A DE1 BINDING FACTOR 1-GLABRA2 module regulates rhamnogalacturonan I biosynthesis in Arabidopsis seed coat mucilage.

The mucilage surrounding hydrated Arabidopsis thaliana seeds is a specialized extracellular matrix composed mainly of the pectic polysaccharide rhamnogalacturonan I (RG-I). Although, several genes responsible for RG-I biosynthesis have been identified, the transcriptional regulatory mechanisms controlling RG-I production remain largely unknown. Here we report that the trihelix transcription factor DE1 BINDING FACTOR 1 (DF1) is a key regulator of mucilage RG-I biosynthesis. RG-I biosynthesis is significantly reduced in loss-of-function mutants of DF1. DF1 physically interacts with GLABRA2 (GL2) and both proteins transcriptionally regulate the expression of the RG-I biosynthesis genes MUCILAGE MODIFIED 4 (MUM4) and GALACTURONOSYLTRANSFERASE-LIKE5 (GATL5). Through chromatin immunoprecipitation-quantitative PCR and transcriptional activation assays, we uncover a cooperative mechanism of the DF1-GL2 module in activating MUM4 and GATL5 expression, in which DF1 binds to the promoters of MUM4 and GATL5 through interacting with GL2 and facilitates the transcriptional activity of GL2. The expression of DF1 and GL2 is directly regulated by TRANSPARENT TESTA GLABRA2 (TTG2) and, in turn, DF1 directly represses the expression of TTG2. Taken together, our data reveal that the transcriptional regulation of mucilage RG-I biosynthesis involves a regulatory module, comprising DF1, GL2, and TTG2.

HOMEODOMAIN GLABROUS2 regulates cellulose biosynthesis in seed coat mucilage by activating CELLULOSE SYNTHASE5.

Numerous proteins involved in cellulose biosynthesis and assembly have been functionally characterized. Nevertheless, we have a limited understanding of the mechanisms underlying the transcriptional regulation of the genes that encode these proteins. Here, we report that HOMEODOMAIN GLABROUS2 (HDG2), a Homeobox-Leucine Zipper IV transcription factor, regulates cellulose biosynthesis in Arabidopsis (Arabidopsis thaliana) seed coat mucilage. HDG2 is a transcriptional activator with the transactivation domain located within its Leucine-Zipper domain. Transcripts of HDG2 were detected specifically in seed coat epidermal cells with peak expression at 10 d postanthesis. Disruptions of HDG2 led to seed coat mucilage with aberrant morphology due to a reduction in its crystalline cellulose content. Electrophoretic mobility shift and yeast one-hybrid assays, together with chromatin immunoprecipitation and quantitative PCR, provided evidence that HDG2 directly activates CELLULOSE SYNTHASE5 (CESA5) expression by binding to the L1-box cis-acting element in its promoter. Overexpression of CESA5 partially rescued the mucilage defects of hdg2-3. Together, our data suggest that HDG2 directly activates CESA5 expression and thus is a positive regulator of cellulose biosynthesis in seed coat mucilage.

Hydrogen Sulfide Promotes Cardiomyocyte Proliferation and Heart Regeneration via ROS Scavenging.

Neonatal mouse hearts can regenerate completely in 21 days after cardiac injury, providing an ideal model to exploring heart regenerative therapeutic targets. The oxidative damage by Reactive Oxygen Species (ROS) is one of the critical reasons for the cell cycle arrest of cardiomyocytes (CMs), which cause mouse hearts losing the capacity to regenerate in 7 days or shorter after birth. As an antioxidant, hydrogen sulfide (H2S) plays a protective role in a variety of diseases by scavenging ROS produced during the pathological processes. In this study, we found that blocking H2S synthesis by PAG (H2S synthase inhibitor) suspended heart regeneration and CM proliferation with ROS deposition increase after cardiac injury (myocardial infarction or apex resection) in 2-day-old mice. NaHS (a H2S donor) administration improved heart regeneration with CM proliferation and ROS elimination after myocardial infarction in 7-day-old mice. NaHS protected primary neonatal mouse CMs from H2O2-induced apoptosis and promoted CM proliferation via SOD2-dependent ROS scavenging. The oxidative DNA damage in CMs was reduced with the elimination of ROS by H2S. Our results demonstrated for the first time that H2S promotes heart regeneration and identified NaHS as a potent modulator for cardiac repair.

Lysophosphatidic acid promotes thrombus stability by inducing rapid formation of neutrophil extracellular traps: A new mechanism of thrombosis.

BACKGROUND: Lysophosphatidic acid (LPA), a bioactive phospholipid released by activated platelets, can induce platelet shape changes and aggregation, which may play an important role in thrombosis. In contrast, the interaction of LPA with neutrophils in thrombosis has not been studied. Recently, neutrophil extracellular traps (NETs) have been shown to bind plasma proteins and activate platelets, which promotes thrombosis. OBJECTIVES: To investigate whether LPA could activate neutrophils to release NETs, predisposing to thrombosis and promoting thrombus stability. METHODS: Levels of neutrophils, NETs, and LPA were detected in 56 participants. Immunofluorescence of NETs and autotaxin, the LPA-producing ectoenzyme, were performed. Induction of NETs and signaling pathways were explored in vitro. RESULTS: Patients with acute pulmonary embolism showed elevated levels of neutrophils, NETs (dsDNA, MPO-DNA, citrullinated histone H3, and nucleosomes), LPA18:1, and LPA20:4. NETs were present in human intrapulmonary thrombi and were surrounded by autotaxin. LPA18:1 induced rapid release of NETs from human neutrophils via a peptidylarginine deiminase 4-dependent pathway. LPA-induced NETs provided a scaffolding for plasma protein binding and generated a tissue plasminogen activator (tPA)-resistant blood clot. Addition of deoxyribonuclease I to tPA significantly accelerated the lysis of clots and human intrapulmonary thrombi. Furthermore, LPA-induced NETs could activate platelets to release LPA. CONCLUSION: This is the first study to implicate LPA in regulating the stability of thrombi by inducing rapid release of NETs in vitro and ex vivo, which could be a new mechanism of thrombosis. These findings provide new insight into the prevention and therapy of venous thromboembolic disease by targeting the LPA-NET signaling pathway.

KNAT7 regulates xylan biosynthesis in Arabidopsis seed-coat mucilage.

As a major hemicellulose component of plant cell walls, xylans play a determining role in maintaining the wall structure. However, the mechanisms of transcriptional regulation of xylan biosynthesis remain largely unknown. Arabidopsis seed mucilage represents an ideal system for studying polysaccharide biosynthesis and modifications of plant cell walls. Here, we identify KNOTTED ARABIDOPSIS THALIANA 7 (KNAT7) as a positive transcriptional regulator of xylan biosynthesis in seed mucilage. The xylan content was significantly reduced in the mucilage of the knat7-3 mutant and this was accompanied by significantly reduced expression of the xylan biosynthesis-related genes IRREGULAR XYLEM 14 (IRX14) and MUCILAGE MODIFIED 5/MUCILAGE-RELATED 21 (MUM5/MUCI21). Electrophoretic mobility shift assays, yeast one-hybrid assays, and chromatin immunoprecipitation with quantitative PCR verified the direct binding of KNAT7 to the KNOTTED1 (KN1) binding site [KBS,TGACAG(G/C)T] in the promoters of IRX7, IRX14, and MUM5/MUCI21 in vitro, in vivo, and in planta. Furthermore, KNAT7 directly activated the expression of IRX14 and MUM5/MUCI21 in transactivation assays in mesophyll protoplasts, and overexpression of IRX14 or MUM5/MUCI21 in knat7-3 partially rescued the defects in mucilage adherence. Taken together, our results indicate that KNAT7 positively regulates xylan biosynthesis in seed-coat mucilage via direct activation of the expression of IRX14 and MUM5/MUCI21.

Transcription Factors BLH2 and BLH4 Regulate Demethylesterification of Homogalacturonan in Seed Mucilage.

The polysaccharide pectin is a major component of the plant cell wall. The pectic glycan homogalacturonan (HG) is a proportionally small but important component of a specialized seed cell wall called mucilage. HG is synthesized in a highly methylesterified form, and, following secretion, is de-methylesterified by pectin methylesterases (PMEs). The degree of methylesterification of HG determines the structural and functional properties of pectin, but how methylesterification is regulated remains largely unknown. Here, we identified two BEL1-Like homeodomain (BLH) transcription factors, BLH2 and BLH4, as positive regulators of HG de-methylesterification in Arabidopsis (Arabidopsis thaliana) seed coat mucilage. BLH2 and BLH4 were significantly expressed in mucilage secretory cells during seed mucilage production. BLH2 and BLH4 single mutants exhibited no obvious mucilage phenotype, but the blh2 blh4 double mutant displayed significantly reduced mucilage adherence to the seed. Reduced mucilage adherence in blh2 blh4 was caused by decreased PME activity in the seed coat, which increased the degree of methylesterification of HG in mucilage. The expression of several PME metabolism-related genes, including PME58, PECTIN METHYLESTERASE INHIBITOR6, SEEDSTICK, and MYB52 was significantly altered in blh2 blh4 seeds. BLH2 and BLH4 directly activated PME58 expression by binding to its TGACAGGT cis-element. Moreover, pme58 mutants exhibited reduced mucilage adherence similar to that of blh2 blh4, and the blh2 blh4 pme58 triple mutant exhibited no additional mucilage adherence defects. Furthermore, overexpression of PME58 in blh2 blh4 rescued the mucilage adherence defect. Together, these results demonstrate that BLH2 and BLH4 redundantly regulate de-methylesterification of HG in seed mucilage by directly activating PME58.

Sex-related differences in the association between plasma fibrinogen and non-calcified or mixed coronary atherosclerotic plaques.

BACKGROUND: Plasma fibrinogen (FIB) has been demonstrated to be a risk factor for cardiovascular disease. Patients with non-calcified plaque (NCP) or mix plaque (MP) have a higher risk of poor outcomes. However, the association between FIB and the presence of NCP or MP (NCP/MP) remains unclear, and if present, whether sex has any impact on this association remains unknown. The aim of this study was to investigate the role of FIB in predicting the presence of NCP/MP and evaluate whether sex has any impact on this association. METHODS: A total of 329 subjects were recruited, and the clinical and laboratory data were collected. Plasma FIB was detected by enzyme-linked immunosorbent assay. According to whether they had coronary atherosclerotic plaques and the characteristics of the most stenotic plaque, we divided them into three groups: no plaque (NP), calcified plaque (CP), and NCP/MP. RESULTS: Patients with NCP/MP had significantly higher FIB level in females, but not in males. Multiple logistic regression analysis showed that FIB was an independent risk factor for the presence of NCP/MP (odds ratio [OR] = 3.677, 95% CI 1.539-8.785, P = 0.003) in females. Receiver operating characteristic (ROC) curve analysis showed that the optimal cut-off value FIB for predicting the presence of NCP/MP was 3.41 g/L (area under curve [AUC] = 0.73, 95% CI 0.63-0.82, P <  0.001) in females. CONCLUSIONS: FIB is independently associated with the presence of NCP/MP in females, but not in males. These results suggest that the potential significance of FIB-lowering regimens in females with NCP/MP.

De novo Transcriptome Analysis of Miscanthus lutarioriparius Identifies Candidate Genes in Rhizome Development.

HIGHLIGHTDe novo transcriptome profiling of five tissues reveals candidate genes putatively involved in rhizome development in M. lutarioriparius. Miscanthus lutarioriparius is a promising lignocellulosic feedstock for second-generation bioethanol production. However, the genomic resource for this species is relatively limited thus hampers our understanding of the molecular mechanisms underlying many important biological processes. In this study, we performed the first de novo transcriptome analysis of five tissues (leaf, stem, root, lateral bud and rhizome bud) of M. lutarioriparius with an emphasis to identify putative genes involved in rhizome development. Approximately 66 gigabase (GB) paired-end clean reads were obtained and assembled into 169,064 unigenes with an average length of 759 bp. Among these unigenes, 103,899 (61.5%) were annotated in seven public protein databases. Differential gene expression profiling analysis revealed that 4,609, 3,188, 1,679, 1,218, and 1,077 genes were predominantly expressed in root, leaf, stem, lateral bud, and rhizome bud, respectively. Their expression patterns were further classified into 12 distinct clusters. Pathway enrichment analysis revealed that genes predominantly expressed in rhizome bud were mainly involved in primary metabolism and hormone signaling and transduction pathways. Noteworthy, 19 transcription factors (TFs) and 16 hormone signaling pathway-related genes were identified to be predominantly expressed in rhizome bud compared with the other tissues, suggesting putative roles in rhizome formation and development. In addition, a predictive regulatory network was constructed between four TFs and six auxin and abscisic acid (ABA) -related genes. Furthermore, the expression of 24 rhizome-specific genes was further validated by quantitative real-time RT-PCR (qRT-PCR) analysis. Taken together, this study provide a global portrait of gene expression across five different tissues and reveal preliminary insights into rhizome growth and development. The data presented will contribute to our understanding of the molecular mechanisms underlying rhizome development in M. lutarioriparius and remarkably enrich the genomic resources of Miscanthus.

The Miscanthus NAC transcription factor MlNAC9 enhances abiotic stress tolerance in transgenic Arabidopsis.

NAC (NAM, ATAF1/2, and CUC2) transcription factors are known to play important roles in responses to abiotic stresses in plants. Currently, little information regarding the functional roles of NAC genes in stress tolerance is available in Miscanthus lutarioriparius, a promising bioenergy plant for cellulosic ethanol production. In this study, we carried out the functional characterization of MlNAC9 in abiotic stresses. MlNAC9 was shown to act as a nuclear localized transcription activator with the activation domain in its C-terminus. The overexpression of MlNAC9 in Arabidopsis conferred hypersensitivity to abscisic acid (ABA) at seed germination and root elongation stages. In addition, the overexpression of MlNAC9 led to increased seed germination rate and root growth under salt (NaCl) treatment. Meanwhile, the transgenic Arabidopsis overexpressing MlNAC9 showed enhanced tolerance to drought and cold stresses. The expression of stress-responsive marker genes was significantly increased in MlNAC9 overexpression lines compared to that of WT under ABA, drought, salt, and cold stresses. Correspondingly, the activities of antioxidant enzymes superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were significantly increased and the malondialdehyde (MDA) content was lower accumulated in MlNAC9 overexpression lines under drought and salt treatments. These results indicated that the overexpression of MlNAC9 improved the tolerance to abiotic stresses via an ABA-dependent pathway, and the enhanced tolerance of transgenic plants was mainly attributed to the increased expression of stress-responsive genes and the enhanced scavenging capability of reactive oxygen species (ROS).