Aberrant TCF21 upregulation in adenomyosis impairs endometrial decidualization by increasing PDE4C expression.

BACKGROUND: Impaired decidualization is a major cause of infertility in patients with adenomyosis (AM). However, the effect of transcription factor 21 (TCF21) on AM and the underlying mechanism of associated-impaired decidualization remain unclear. The aim of this study was to investigate the expression of TCF21 in endometrial tissues of AM patients and the specific mechanisms by which it impairs the decidualization of human endometrial stromal cells (HESCs), with a view to improving the reproductive outcome of AM infertile patients. METHODS: We compared gene expressions via transcriptomics between the control and AM-associated recurrent implantation failure (RIF) groups. qRT-PCR, western blot, and IHC were performed to confirm the expression and location of TCF21 in the endometrium. Furthermore, we confirmed that high expression of TCF21 impairs decidualization by qRT-PCR, immunofluorescence, and western blot. RNA-seq following overexpression of TCF21 in HESCs was conducted to identify TCF21-related molecular changes during in vitro decidualization. Then we performed ChIP-seq/qPCR and dual-luciferase reporter assay to explore the exact interaction between TCF21 and PDE4C. The related downstream mechanisms were further proved using IHC, qRT-PCR, western blot, and ELISA. RESULTS: According to the RNA-seq analysis, TCF21 expression was remarkably higher in the endometrium of the AM-related RIF group compared to the control group. We confirmed the same results using samples from patients with AM and controls. TCF21 overexpression in HESCs impaired decidualization through suppression of decidual markers and cytoskeleton alterations. The mechanistic analysis revealed that TCF21 inhibited intracellular cAMP levels by directly increasing PDE4C expression and suppressing FOXO1 expression. CONCLUSIONS: TCF21 compromises decidualization in patients with AM via the PDE4C/cAMP-FOXO1 axis, which offers valuable insights on the pathology of decidualization-related infertility and indicates a potential treatment to improve endometrial receptivity in AM.

Study on the mechanism of Huangqi Guizhi Wuwu Decoction in treating diabetes peripheral neuropathy based on network pharmacology and molecular docking.

This study aimed to explore the effective substances and mechanism network of Huangqi Guizhi Wuwu Decoction in treating diabetes peripheral neuropathy. Based on the TCM systemic pharmacological analysis platform (TCMP) and UniProt database, the database of active Huqarqu Decoction was constructed, and the related targets of diabetic peripheral neuropathy were collected through the OMIM, CTD, DisGeNET, TTD and GeneCards databases. The intersection targets were obtained to construct the network diagram of Huangqi dis Guizhi Wuwu Decoction-Active Through the String database, the interaction between target proteins was analyzed, and molecular docking between active components and potential targets was carried out. Combined with the DAVID v6.8 database, GO function analysis and KEGG pathway analysis were performed on the targets. Guizhi Wuwu Decoction mainly acts on core targets such as IL6, MAPK3, VE GFA, JUN and ESR1 through quercetin, kaempferol and naringin and regulates the TNF signaling pathway, estrogen signaling pathway and MAPK signaling pathway, thus achieving the effect of treating diabetes peripheral neuropathy. Huangqi Guizhi Wu has multiple targets and regulates multiple signaling pathways in neuropathy, which lays a foundation for future pharmacological research.

Expression and clinical significance of tumor necrosis factor receptor-associated factor 1 in peripheral blood of patients with advanced lung cancer.

BACKGROUND: To assess the serum level of tumor necrosis factor receptor related factor 1 (TRAF1) in advanced lung cancer patients and its clinical significance. METHODS: In this retrospective study, the serum level of TRAF1 in 50 patients with stage III-IV lung cancer and 50 healthy people who received physical examination during the same period were detected and compared. The differences in serum TRAF1 level in patients with lung cancer in terms of gender, age, smoking status, pathological type, tumor location, TNM stage and other clinicopathological features were analyzed. The 50 patients with lung cancer were treated with conventional chemotherapy for 2 cycles, and serum TRAF1 level was tested. The area under the curve (AUC) was calculated to evaluate the diagnostic value of serum TRAF1 for advanced lung cancer. RESULTS: The serum level of TRAF1 in lung cancer patients was significantly higher than that of healthy controls (P < 0.05). The serum level of TRAF1 in patients with stage IV lung cancer was significantly higher than that in patients with stage III lung cancer (P < 0.05). Serum level of TRAF1 in lung adenocarcinoma and lung squamous cell carcinoma group after chemotherapy was significantly lower than that before chemotherapy (P < 0.05); However, the serum level of TRAF1 in small cell lung cancer group after chemotherapy had no significant change compared with that before chemotherapy (P > 0.05). The AUC of serum TRAF1 for the diagnosis of lung cancer was 0.903, and the yoden index was 0.668. The best cut-off value of serum TRAF1 for the diagnosis of lung cancer was 113.87 pg/ml, with a sensitivity of 90.6% and a specificity of 78.57%. CONCLUSION: Serum level of TRAF1 has potential diagnostic value for advanced lung cancer. TRAF1 could assist clinicians to diagnose lung cancer patient and assess patient's condition.

Self-Assembled BODIPY Derivative with A-D-A Structure as Organic Nanoparticles for Photodynamic/Photothermal Cancer Therapy.

Organic nanomaterials have attracted considerable attention in the area of photodynamic and photothermal therapy, owing to their outstanding biocompatibility, potential biodegradability, well-defined chemical structure, and easy functionalization. However, it is still a challenge to develop a single organic molecule that obtains both photothermal and photodynamic effects. In this contribution, we synthesized a new boron-dipyrromethene (BODIPY)-based derivative (DPBDP) with an acceptor-donor-acceptor (A-D-A) structure by coupling 3,6-di(2-thienyl)-2,5-dihydropyrrolo [3,4-c] pyrrole-1,4-dione (DPP) and BODIPY. To enhance the hydrophilicity of the BODIPY derivative, the polyethylene glycol (PEG) chains were introduced to the meso- position of BODIPY core. The amphiphilic DPBDP was then self-assembled into related nanoparticles (DPBDP NPs) with improved hydrophilicity and enhanced absorbance in the NIR region. DPBDP NPs could simultaneously generate the singlet oxygen (1O2) and heat under the irradiation of a single laser (690 nm). The 1O2 quantum yield and photothermal conversion efficiency (PCE) of DPBDP NPs were calculated to be 14.2% and 26.1%, respectively. The biocompatibility and phototherapeutic effect of DPBDP NPs were evaluated through cell counting kit-8 (CCK-8) assay. Under irradiation of 690 nm laser (1.0 W/cm2), the half maximal inhibitory concentration (IC50) of DPBDP NPs was calculated to be 16.47 µg/mL. Thus, the as-prepared DPBDP NPs could be acted as excellent candidates for synergistic photodynamic/photothermal therapy.

Short- and long-term efficacy of sustained-release chemotherapy in tumor bed interstitium combined with surgical resection for recurrent malignant glioma.

OBJECTIVE: To discuss and analyze the short- and long-term curative effect of sustained-release chemotherapy combined with surgical resection on recurrent malignant glioma. METHODS: The clinical data of 137 patients with recurrent glioma admitted to our hospital from March 2016 to July 2018 were retrospectively analyzed. Among them, 67 patients who received local chemotherapy with cisplatin slow-release polymer after surgical resection were included in the observation group, and the other 70 patients who did not receive chemotherapy after surgical resection were regarded as the control group. The short-term therapeutic efficacy, quality of life score before and after surgery, incidence of toxic and side effects, long-term recurrence rate and survival were compared between the two groups. RESULTS: The total remission rate of clinical treatment in observation group was remarkably higher than that in control group (P<0.05). There was no statistically significant difference in quality of life score between the two groups before and after surgery (P>0.05). There was no significant difference in the incidence of postoperative side effects between the two groups (P>0.05). While the recurrence rates of the observation group at 12 and 24 months after surgery were significantly lower than those in the control group (P<0.05). The overall postoperative survival of observation group was obviously superior to that of control group (P<0.05). In patients who received sustained-release chemotherapy in tumor bed interstitium combined with surgical resection, older ones and the ones with partial surgical resection or pathological grade IV had worse long-term survival (P<0.05). CONCLUSION: The combined treatment of sustained release chemotherapy in tumor bed interstitium and surgical resection for recurrent malignant glioma can effectively improve the clinical efficacy, reduce postoperative recurrence rate and prolong the survival time of the patients.

Geriatric Nutritional Risk Index as a Prognostic Factor of Patients with Non-Small Cell Lung Cancer: A Meta-Analysis.

Geriatric nutritional risk index (GNRI), a newly developed indicator of nutritional status retrieved by serum albumin concentration and ideal body weight, has been suggested as a prognostic factor for various malignancies. The aim of the study was to summarize the prognostic role of GNRI for patients with non-small cell lung cancer (NSCLC) in a meta-analysis. Cohort studies evaluating the relationship between GNRI at baseline and survival OF NSCLC were retrieved by search of PubMed, Embase, and Web of Science databases from inception to January 12, 2022. A conservative random-effect model incorporating the possible influence of between-study heterogeneity was used to pool the results. Eleven cohorts including 2865 patients with NSCLC were included. Compared to those with higher GNRI, NSCLC patients with lower GNRI were associated with poorer overall survival [OS, hazard ratio (HR): 2.39, 95% CI: 1.97-2.91, p<0.001; I2=29%), progression-free survival (HR: 1.94, 95% CI: 1.52-2.47, p<0.001; I2=29%), and cancer-specific survival (HR: 2.59, 95% CI: 1.55-4.35, p<0.001; I2=0%). Subgroup analyses showed that the significant association between lower GNRI and worse OS in patients with NSCLC was not affected by study characteristics including study location, design, cancer stage, treatment, or follow-up durations (p for subgroup effects all<0.001). In conclusion, a lower GNRI in patients with NSCLC may be a predictor of poor survival. Nutritional status indicated by GNRI may be important for the prognostic prediction of patients with NSCLC.

Endometrial stromal cell ferroptosis promotes angiogenesis in endometriosis.

Endometriosis, a chronic disorder characterised by the presence of endometrial-like tissue outside the uterus, is associated with iron overload and oxidative stress in the lesion. Although it is well established that iron overload can trigger ferroptosis, the results of previous studies on ferroptosis resistance and ferroptosis in endometriotic lesions are paradoxical. Here, we found that some stromal cells of the cyst walls that were in contact with the cyst fluid underwent ferroptosis. Surprisingly, endometrial stromal cell ferroptosis triggered the production of angiogenic, inflammatory and growth cytokines. In particular, angiogenic cytokines, such as vascular endothelial growth factor A (VEGFA) and interleukin 8 (IL8), promoted human umbilical vein endothelial cell (HUVEC) vascular formation in vitro. Moreover, we found that inhibition of p38 mitogen-activated protein kinase/signal transducer and activator of transcription 6 (p38 MAPK/STAT6) signalling represses VEGFA and IL8 expression when endometrial stromal cells undergo ferroptosis. Notably, VEGFA and IL8 showed localised expression and were significantly upregulated in ectopic lesions compared to control and eutopic endometrium samples from patients with endometriosis. Thus, our study reveals that endometrial stromal cell ferroptosis in the ovarian endometrioma may trigger cytokine secretion and promote angiogenesis of adjacent lesions via paracrine actions to drive the development of endometriosis, providing a rationale for translation into clinical practice and developing drugs for endometriosis.

MicroRNA-21 regulate the cell apoptosis and cell proliferation of polycystic ovary syndrome (PCOS) granulosa cells through target toll like receptor TLR8.

Polycystic ovary syndrome (PCOS) is a complex reproductive endocrine disease characterized by polycystic ovary. The aim of the study was to assess microRNA-21 regulates granulosa cell apoptosis and proliferation in polycystic ovary syndrome through target toll-like receptor 8. Granulosa cells were collected from 30 PCOS patients and 30 normal patients with tubal or male factor infertility (control) during in vitro fertilization-Embryo Transfer (IVF-ET) and were flash frozen with liquid nitrogen for storage for subsequent use. PCOS diagnosis was based on the revised standards of the American Society of Reproductive Medicine (ASRM) and the Rotterdam criteria PCOS granulosa cells and control granulosa cells were cultured in DMEM/F12 medium containing 10% fetal bovine serum and 1% antibiotic. After this RT-PCR, Western blot assessment and Detection of apoptosis by flow cytometry were conducted. The results of qPCR showed that the mRNA and protein expression of TLR8 in PCOS granulosa cells were significantly increased compared with the normal group. The results of Western blot also showed that the expression of TLR8, IFN-γ, TNF-α and IL-12 gene protein in the transfected cells was significantly higher than that in the control cells. Here, we show that miR-21 and TLR8 significantly increased in PCOS granulosa cell as compared with normal granulosa cells, and miR-21 enhances the TLR8 mRNA translation and then promotes the IFN-γ, TNF-α, and IL-12 secretion. Our study demonstrates that miR-21/ TLR8 involved in the PCOS inflammation, it provides profound insights into pathogenesis of PCOS.

Hypo-Expression of Tuberin Promotes Adenomyosis via the mTOR1-Autophagy Axis.

Adenomyosis (AM) is a disease in which endometrial tissue invades the myometrium and has a 10-60% prevalence in reproductive-aged women. TSC2 regulates autophagy via mTOR1 signalling in colorectal cancer and endometrial carcinoma. Dysregulation of autophagy is implicated in adenomyosis pathogenesis. However, whether TSC2 participates in adenomyosis via autophagy remains obscure. Here, we found that the expression of TSC2 in adenomyosis was significantly decreased than that in normal endometrium during the secretory phase. Moreover, TSC2 and autophagy marker expression was significantly lower in ectopic lesions than in eutopic samples. TSC2 downregulation inhibited autophagy through mTOR1 signalling pathway activation in endometrial cells, leading to excessive proliferation, migration, and EMT; TSC2 overexpression induced the opposite effects. Rapamycin treatment suppressed cell proliferation, migration and EMT in the absence of TSC2. In parallel, an autophagy-specific inhibitor (SAR-405) restored migration and EMT under rapamycin treatment in TSC2-knockdown Ishikawa cells. Finally, SAR-405 treatment promoted EMT and migration of overexpressing cells. Collectively, our results suggest that TSC2 controls endometrial epithelial cell migration and EMT by regulating mTOR1-autophagy axis activation and that hypo-expression of TSC2 in the endometrium might promote adenomyosis.

Assay Methods for ACS Activity and ACS Phosphorylation by MAP Kinases In Vitro and In Vivo.

Ethylene, a gaseous phytohormone, has profound effects on plant growth, development, and adaptation to the environment. Ethylene-regulated processes begin with the induction of ethylene biosynthesis. There are two key steps in ethylene biosynthesis. The first is the biosynthesis of 1-aminocyclopropane-1-carboxylic acid (ACC) from S-Adenosyl-Methionine (SAM), a common precursor in many metabolic pathways, which is catalyzed by ACC synthase (ACS). The second is the oxidative cleavage of ACC to form ethylene under the action of ACC oxidase (ACO). ACC biosynthesis is the committing and generally the rate-limiting step in ethylene biosynthesis. As a result, characterizing the cellular ACS activity and understanding its regulation are important. In this chapter, we detail the methods used to measure, (1) the enzymatic activity of both recombinant and native ACS proteins, and (2) the phosphorylation of ACS protein by mitogen-activated protein kinases (MAPKs) in vivo and in vitro.

Nucleocytoplasmic trafficking is essential for BAK1- and BKK1-mediated cell-death control.

BRI1-ASSOCIATED KINASE 1 (BAK1) was initially identified as a co-receptor of the brassinosteroid (BR) receptor BRI1. Genetic analyses also revealed that BAK1 and its closest homolog BAK1-LIKE 1 (BKK1) regulate a BR-independent cell-death control pathway. The double null mutant bak1 bkk1 displays a salicylic acid- and light-dependent cell-death phenotype even without pathogen invasion. Molecular mechanisms of the spontaneous cell death mediated by BAK1 and BKK1 remain unknown. Here we report our identification of a suppressor of bak1 bkk1 (sbb1-1). Genetic analyses indicated that cell-death symptoms in a weak double mutant, bak1-3 bkk1-1, were completely suppressed by the loss-of-function mutation in SBB1, which encodes a nucleoporin (NUP) 85-like protein. Genetic analyses also demonstrated that individually knocking out three other nucleoporin genes from the SBB1-located sub-complex was also able to rescue the cell-death phenotype of bak1-3 bkk1-1. In addition, a DEAD-box RNA helicase, DRH1, was identified in the same protein complex as SBB1 via a proteomic approach. The drh1 mutation also rescues the cell-death symptoms of bak1-3 bkk1-1. Further analyses indicated that export of poly(A)(+) RNA was greatly blocked in the nup and drh1 mutants, resulting in accumulation of significant levels of mRNAs in the nuclei. Over-expression of a bacterial NahG gene to inactivate salicylic acid also rescues the cell-death phenotype of bak1-3 bkk1-1. Mutants suppressing cell-death symptoms always showed greatly reduced salicylic acid contents. These results suggest that nucleocytoplasmic trafficking, especially of molecules directly or indirectly involved in endogenous salicylic acid accumulation, is critical in BAK1- and BKK1-mediated cell-death control.

Development of an agroinoculation system for full-length and GFP-tagged cDNA clones of cucumber green mottle mosaic virus.

The complete 6243-nucleotide sequence of a cucumber green mottle mosaic virus (CGMMV) isolate from bottle gourd in Zhejiang province, China, was determined. A full-length cDNA clone of this isolate was constructed by inserting the cDNA between the 35S promoter and the ribozyme in the binary plasmid pCB301-CH. A suspension of an Agrobacterium tumefaciens EHA105 clone carrying this construct was highly infectious in Nicotiana benthamiana and bottle gourd. Another infectious clone containing the green fluorescence protein (GFP) reporter gene was also successfully constructed. This study is the first report of the efficient use of agroinoculation for generating CGMMV infections.

Arabidopsis cysteine-rich receptor-like kinase 45 positively regulates disease resistance to Pseudomonas syringae.

Arabidopsis cysteine-rich receptor-like protein kinase 45 (CRK45) was found to be involved in ABA signaling in Arabidopsis thaliana previously. Here, we reported that it also positively regulates disease resistance. The CRK45 overexpression plants increased expression of the defense genes, and enhanced resistance to Pseudomonas syringae whereas the crk45 mutant were more sensitive to P. syringae and weakened expression of the defense genes, compared to the wild type. We also found that treatment with P. syringae leads to a declined expression of CRK45 in the npr1 mutant and the NahG transgenic plants. At the same time, significantly decreased expression of CRK45 transcript in the wrky70 mutant than that in the wild type was also detected. Our results suggested that CRK45 acted as a positive regulator in Arabidopsis disease resistance, and was regulated downstream of NPR1 and WRKY70 at the transcriptional level.

Arabidopsis cysteine-rich receptor-like kinase 45 functions in the responses to abscisic acid and abiotic stresses.

The phytohormone abscisic acid (ABA) regulates seed germination, plant growth and development, and response to abiotic stresses such as drought and salt stresses. Receptor-like kinases are well known signaling components that mediate plant responses to developmental and environmental stimuli. Here, we characterized the biological function of an ABA and stress-inducible cysteine-rich receptor-like protein kinase, CRK45, in ABA signaling in Arabidopsis thaliana. The crk45 mutant was less sensitive to ABA than the wild type during seed germination and early seedling development, whereas CRK45 overexpression plants were more sensitive to ABA compared to the wild type. Furthermore, overexpression of CRK45 led to hypersensitivity to salt and glucose inhibition of seed germination, whereas the crk45 mutant showed the opposite phenotypes. In addition, CRK45 overexpression plants had enhanced tolerance to drought. Gene expression analyses revealed that the expression of representative stress-responsive genes was significantly enhanced in CRK45 overexpression plants in response to salt stress. ABA biosynthetic genes such as NCED3,(2)NCED5,(3)ABA2,(4) and AAO3(5) were also constitutively elevated in the CRK45 overexpression plants. We concluded that CRK45 plays an important role in ABA signaling that regulates Arabidopsis seeds germination, early seedling development and abiotic stresses response, by positively regulating ABA responses in these processes.

Dual-level regulation of ACC synthase activity by MPK3/MPK6 cascade and its downstream WRKY transcription factor during ethylene induction in Arabidopsis.

Plants under pathogen attack produce high levels of ethylene, which plays important roles in plant immunity. Previously, we reported the involvement of ACS2 and ACS6, two Type I ACS isoforms, in Botrytis cinerea-induced ethylene biosynthesis and their regulation at the protein stability level by MPK3 and MPK6, two Arabidopsis pathogen-responsive mitogen-activated protein kinases (MAPKs). The residual ethylene induction in the acs2/acs6 double mutant suggests the involvement of additional ACS isoforms. It is also known that a subset of ACS genes, including ACS6, is transcriptionally induced in plants under stress or pathogen attack. However, the importance of ACS gene activation and the regulatory mechanism(s) are not clear. In this report, we demonstrate using genetic analysis that ACS7 and ACS11, two Type III ACS isoforms, and ACS8, a Type II ACS isoform, also contribute to the B. cinerea-induced ethylene production. In addition to post-translational regulation, transcriptional activation of the ACS genes also plays a critical role in sustaining high levels of ethylene induction. Interestingly, MPK3 and MPK6 not only control the stability of ACS2 and ACS6 proteins via direct protein phosphorylation but also regulate the expression of ACS2 and ACS6 genes. WRKY33, another MPK3/MPK6 substrate, is involved in the MPK3/MPK6-induced ACS2/ACS6 gene expression based on genetic analyses. Furthermore, chromatin-immunoprecipitation assay reveals the direct binding of WRKY33 to the W-boxes in the promoters of ACS2 and ACS6 genes in vivo, suggesting that WRKY33 is directly involved in the activation of ACS2 and ACS6 expression downstream of MPK3/MPK6 cascade in response to pathogen invasion. Regulation of ACS activity by MPK3/MPK6 at both transcriptional and protein stability levels plays a key role in determining the kinetics and magnitude of ethylene induction.

Genotypic differences in pod wall and seed growth relate to invertase activities and assimilate transport pathways in asparagus bean.

BACKGROUND AND AIMS: Coordination of sugar transport and metabolism between developing seeds and their enclosing fruit tissues is little understood. In this study the physiological mechanism is examined using two genotypes of asparagus bean (Vigna unguiculata ssp. sesquipedialis) differing in pod wall and seed growth rates. Pod growth dominates over seed growth in genotype 'Zhijiang 121' but not in 'Zhijiang 282' in which a 'bulging pod' phenotype is apparent from 8 d post-anthesis (dpa) onward. METHODS: Seed and pod wall growth rates and degree of pod-bulging were measured in the two genotypes together with assays of activities of sucrose-degrading enzymes and sugar content in pod wall and seed and evaluation of cellular pathways of phloem unloading in seed coat using a symplasmic fluorescent dye, 5(6)-carboxyfluorescein (CF). KEY RESULTS: Activities of cell wall, cytoplasmic and vacuolar invertases (CWIN, CIN and VIN) were significantly smaller in pod walls of '282' than in '121' at 10 dpa onwards. Low INV activities were associated with weak pod wall growth of '282'. In seed coats, CF was confined within the vasculature in '282' but moved beyond the vasculature in '121', indicating apoplasmic and symplasmic phloem unloading, respectively. Higher CWIN activity in '282' seed coats at 6-8 dpa correlated with high hexose concentration in embryos and enhanced early seed growth. However, CWIN activity in '282' decreased significantly compared with '121' from 10 dpa onwards, coinciding with earlier commencement of nuclei endoreduplication in their embryos. CONCLUSIONS: The study shows genotypic differences between 'bulging pod' and 'non-bulging' phenotypes of asparagus bean in sucrose metabolism in relation to the pathway of phloem unloading in developing seed coats, and to pod and seed growth. Low INV activity in pod wall corresponds to its shortened and weak growth period; by contrast, the apoplasmic path in the seed coat is associated with high CWIN activity and strong early seed growth.

An AP2 domain-containing gene, ESE1, targeted by the ethylene signaling component EIN3 is important for the salt response in Arabidopsis.

Accumulating investigations reveal that ethylene signaling is involved in the salt response in Arabidopsis (Arabidopsis thaliana), and it has been reported that overexpression of a number of ethylene response factor (ERF) genes enhances salt tolerance; however, transcriptional regulation of the ethylene signal component ETHYLENE INSENSITIVE3 (EIN3) in the salt response has not been clearly defined. Consulting microarray data and transcriptional confirmations showed that three of the ERF genes were ethylene and salt inducible, named ESE1 to ESE3. Additionally, the expression of one of the ESE genes (ESE1) was suppressed in ein2, ein3-1, eil1-3, and ein3 eil1 but enhanced in EIN3-overexpressing (EIN3ox) lines. Inhibitors of ethylene biosynthesis, aminoethoxyvinylglycine, and ethylene action, AgNO3, reduced the expression of ESE1, while ethylene overproduction eto mutants enhanced the expression of ESE1, indicating that ESE1 is an ethylene-modulated gene downstream of EIN3/EIL1. Further analyses with biochemical and molecular approaches revealed that EIN3 physically binds to the ESE1 promoter, demonstrating that ESE1 was one target of EIN3. ESE1 in turn binds to promoters of salt-related genes, such as RD29A and COR15A. Moreover, either EIN3ox or ESE1ox was sufficient to enhance transcript levels of salt-related genes and salt tolerance. In addition, ESE1ox in ein3 enhanced the salt response during seed germination and seedling development, demonstrating that ESE1 is genetically downstream of EIN3. Thus, the evidence in this report reveals that the transcriptional complex of EIN3-ESE1 is a crucial event in the salt response, thereby connecting the transcriptional regulation of EIN3 and the downstream ERF protein ESE1 in the salt response.

Sugar input, metabolism, and signaling mediated by invertase: roles in development, yield potential, and response to drought and heat.

Invertase (INV) hydrolyzes sucrose into glucose and fructose, thereby playing key roles in primary metabolism and plant development. Based on their pH optima and sub-cellular locations, INVs are categorized into cell wall, cytoplasmic, and vacuolar subgroups, abbreviated as CWIN, CIN, and VIN, respectively. The broad importance and implications of INVs in plant development and crop productivity have attracted enormous interest to examine INV function and regulation from multiple perspectives. Here, we review some exciting advances in this area over the last two decades, focusing on (1) new or emerging roles of INV in plant development and regulation at the post-translational level through interaction with inhibitors, (2) cross-talk between INV-mediated sugar signaling and hormonal control of development, and (3) sugar- and INV-mediated responses to drought and heat stresses and their impact on seed and fruit set. Finally, we discuss major questions arising from this new progress and outline future directions for unraveling mechanisms underlying INV-mediated plant development and their potential applications in plant biotechnology and agriculture.

Mitogen-activated protein kinase 3 and 6 regulate Botrytis cinerea-induced ethylene production in Arabidopsis.

Plants challenged by pathogens, especially necrotrophic fungi such as Botrytis cinerea, produce high levels of ethylene. At present, the signaling pathways underlying the induction of ethylene after pathogen infection are largely unknown. MPK6, an Arabidopsis stress-responsive mitogen-activated protein kinase (MAPK) was previously shown to regulate the stability of ACS2 and ACS6, two type I ACS isozymes (1-amino-cyclopropane-1-carboxylic acid synthase). Phosphorylation of ACS2 and ACS6 by MPK6 prevents rapid degradation of ACS2/ACS6 by the 26S proteasome pathway, resulting in an increase in cellular ACS activity and ethylene biosynthesis. Here, we show that MPK3, which shares high homology and common upstream MAPK kinases with MPK6, is also capable of phosphorylating ACS2 and ACS6. In the mpk3 mutant background, ethylene production in gain-of-function GVG-NtMEK2(DD) transgenic plants was compromised, suggesting that MPK6 and MPK3 function together to stabilize ACS2 and ACS6. Using a liquid-cultured seedling system, we found that B. cinerea-induced ethylene biosynthesis was greatly compromised in mpk3/mpk6 double mutant seedlings. In contrast, ethylene production decreased only slightly in the mpk6 single mutant and not at all in the mpk3 single mutant, demonstrating overlapping roles for these two highly homologous MAPKs in pathogen-induced ethylene induction. Consistent with the role of MPK3/MPK6 in the process, mutation of ACS2 and ACS6, two genes encoding downstream substrates of MPK3/MPK6, also reduced B. cinerea-induced ethylene production. The residual levels of ethylene induction in the acs2/acs6 double mutant suggest the involvement of additional ACS isoforms, possibly regulated by MAPK-independent pathway(s).

AtNUDT7, a negative regulator of basal immunity in Arabidopsis, modulates two distinct defense response pathways and is involved in maintaining redox homeostasis.

Plants have evolved complicated regulatory systems to control immune responses. Both positive and negative signaling pathways interplay to coordinate development of a resistance response with the appropriate amplitude and duration. AtNUDT7, a Nudix domain-containing protein in Arabidopsis (Arabidopsis thaliana) that hydrolyzes nucleotide derivatives, was found to be a negative regulator of the basal defense response, and its loss-of-function mutation results in enhanced resistance to infection by Pseudomonas syringae. The nudt7 mutation does not cause a strong constitutive disease resistance phenotype, but it leads to a heightened defense response, including accelerated activation of defense-related genes that can be triggered by pathogenic and nonpathogenic microorganisms. The nudt7 mutation enhances two distinct defense response pathways: one independent of and the other dependent on NPR1 and salicylic acid accumulation. In vitro enzymatic assays revealed that ADP-ribose and NADH are preferred substrates of NUDT7, and the hydrolysis activity of NUDT7 is essential for its biological function and is sensitive to inhibition by Ca(2+). Further analyses indicate that ADP-ribose is not likely the physiological substrate of NUDT7. However, the nudt7 mutation leads to perturbation of cellular redox homeostasis and a higher level of NADH in pathogen-challenged leaves. The study suggests that the alteration in cellular antioxidant status caused by the nudt7 mutation primes the cells for the amplified defense response and NUDT7 functions to modulate the defense response to prevent excessive stimulation.