lac‐1 and lag‐1 with ras‐1 affect aging and the biological clock in Neurospora crassa

Using an automated cell counting technique developed previously (Case et al., Ecology and Evolution 2014; 4: 3494), we explore the lifespan effects of lac‐1, a ceramide synthase gene paralogous to lag‐1 in Neurospora crassa in conjunction with the band bd (ras‐1) gene. We find that the replicative lifespan of a lac‐1^KO bd double mutants is short, about one race tube cycle, and this double mutant lacks a strong ~21‐hr clock cycle as shown by race tube and fluorometer analysis of fluorescent strains including lac‐1^KO. This short replicative lifespan phenotype is contrasted with a very long estimated chronological lifespan for lac‐1^KO bd double mutants from 247 to 462 days based on our regression analyses on log viability, and for the single mutant lac‐1^KO, 161 days. Both of these estimated lifespans are much higher than that of previously studied WT and bd single mutant strains. In a lac‐1 rescue and induction experiment, the expression of lac‐1⁺ as driven by a quinic acid‐dependent promoter actually decreases the median chronological lifespan of cells down to only 7 days, much lower than the 34‐day median lifespan found in control bd conidia also grown on quinic acid media, which we interpret as an effect of balancing selection acting on ceramide levels based on previous findings from the literature. Prior work has shown phytoceramides can act as a signal for apoptosis in stressed N. crassa cells. To test this hypothesis of balancing selection on phytoceramide levels, we examine the viability of WT, lag‐1^KO bd, and lac‐1^KO bd strains following the dual stresses of heat and glycolysis inhibition, along with phytoceramide treatments of different dosages. We find that the phytoceramide dosage–response curve is altered in the lag‐1^KO bd mutant, but not in the lac‐1^KO bd mutant. We conclude that phytoceramide production is responsible for the previously reported longevity effects in the lag‐1^KO bd mutant, but a different ceramide may be responsible for the longevity effect observed in the lac‐1^KO bd mutant.     

ICAM‐1 and MKL‐1 polymorphisms impose considerable impacts on coronary heart disease occurrence

This study was aimed to explore the correlation of intercellular adhesion molecule‐1 (ICAM‐1) K469E and megakaryoblastic leukaemia factor‐1 (MKL‐1) ?184C/T polymorphisms with the susceptibility to coronary heart disease (CHD) in the Chinese Han population. 100 CHD patients and 91 healthy people that had no blood connection with each other were enrolled in this case‐control study. ICAM‐1 and MKL‐1 polymorphisms were genotyped by polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) approach. Multiple logistic regression was used to analyse the correlation between polymorphisms of ICAM‐1 and MKL‐1 and CHD susceptibility. Differences of genotype and allele frequencies of the two SNPs between case and control groups were analysed by chi‐square test. Odds ratios (ORs) and 95% confidence intervals (CIs) were indicated relative susceptibility of CHD. The distributions of ICAM‐1 and MKL‐1 polymorphisms in each group conformed to Hardy‐Weinberg equilibrium (HWE). After adjusting for traditional risk factors, the TT genotype frequency of MKL‐1 ?184C/T polymorphism was found significantly higher in case group than in control group (P < .05). Meanwhile, T allele frequency increased in case group compared with control group, and the differences had statistical significance (P = .04, OR = 2.34, 95% CI = 1.34‐5.26). Logistic regression analysis in this study proved that smoking, hypertension, diabetes and triglyceride (TG) were all risk factors for CHD ICAM‐1 K469E polymorphism has no association with the onset of CHD. But MKL‐1 ?184C/T polymorphism is associated with the risk of CHD and T allele might be a susceptibility factor for CHD.     

Sphingosine‐1‐phosphate (S1P) enhances glomerular endothelial cells activation mediated by anti‐myeloperoxidase antibody‐positive IgG

Cumulating evidences suggested an important role of sphingosine‐1‐phosphate (S1P) and its receptors in regulating endothelial barrier integrity. Our previous study revealed that the circulating S1P levels and renal expression of S1PRs correlated with disease activity and renal damage in patients with antineutrophil cytoplasmic antibody (ANCA)‐associated vasculitis (AAV). This study investigated the role of S1P and its receptors in myeloperoxidase (MPO)‐ANCA‐positive IgG‐mediated glomerular endothelial cell (GEnC) activation. The effect of S1P on morphological alteration of GEnCs in the presence of MPO‐ANCA‐positive IgG was observed. Permeability assay was performed to determine endothelial monolayer activation in quantity. Both membrane‐bound and soluble ICAM‐1 and VCAM‐1 levels were measured. Furthermore, antagonists and/or agonists of various S1PRs were employed to determine the role of different S1PRs. S1P enhanced MPO‐ANCA‐positive IgG‐induced disruption of tight junction and disorganization of cytoskeleton in GEnCs. S1P induced further increase in monolayer permeability of GEnC monolayers in the presence of MPO‐ANCA‐positive IgG. S1P enhanced MPO‐ANCA‐positive IgG‐induced membrane‐bound and soluble ICAM‐1/VCAM‐1 up‐regulation of GEnCs. Soluble ICAM‐1 levels in the supernatants of GEnCs stimulated by S1P and MPO‐ANCA‐positive IgG increased upon pre‐incubation of S1PR1 antagonist, while pre‐incubation of GEnCs with the S1PR1 agonist down‐regulated sICAM‐1 level. Blocking S1PR2‐4 reduced sICAM‐1 levels in the supernatants of GEnCs stimulated by S1P and MPO‐ANCA‐positive IgG. Pre‐incubation with S1PR5 agonist could increase sICAM‐1 level in the supernatants of GEnC stimulated by S1P and MPO‐ANCA‐positive IgG. S1P can enhance MPO‐ANCA‐positive IgG‐mediated GEnC activation through S1PR2‐5.     

Tumour cell‐intrinsic CTLA4 regulates PD‐L1 expression in non‐small cell lung cancer

Cytotoxic T lymphocyte antigen 4 (CTLA4) and programmed cell death protein 1 (PD‐1) are immune checkpoint proteins expressed in T cells. Although CTLA4 expression was found in multiple tumours including non‐small cell lung cancer (NSCLC) tissues and cells, its function in tumour cells is unknown. Recently, PD‐1 was found to be expressed in melanoma cells and to promote tumorigenesis. We found that CTLA4 was expressed in a subset of NSCLC cell lines and in a subgroup of cancer cells within the lung cancer tissues. We further found that in NSCLC cells, anti‐CTLA4 antibody can induce PD‐L1 expression, which is mediated by CTLA4 and the EGFR pathway involving phosphorylation of MEK and ERK. In CTLA4 knockout cells, EGFR knockout cells or in the presence of an EGFR tyrosine kinase inhibitor, anti‐CTLA4 antibody was not able to induce PD‐L1 expression in NSCLC cells. Moreover, anti‐CTLA4 antibody promoted NSCLC cell proliferation in vitro and tumour growth in vivo in the absence of adaptive immunity. These results suggest that tumour cell‐intrinsic CTLA4 can regulate PD‐L1 expression and cell proliferation, and that anti‐CTLA4 antibody, by binding to the tumour cell‐intrinsic CTLA4, may result in the activation of the EGFR pathway in cancer cells.     

Long non‐coding RNA NR2F1‐AS1 promoted proliferation and migration yet suppressed apoptosis of thyroid cancer cells through regulating miRNA‐338‐3p/CCND1 axis

Thyroid cancer (TC) is a prevalent endocrine malignant cancer whose pathogenic mechanism remains unclear. The aim of the study was to investigate the roles of long non‐coding RNA (lncRNA) NR2F1‐AS1/miRNA‐338‐3P/CCND1 axis in TC progression. Differentially expressed lncRNAs and mRNAs in TC tissues were screened out and visualized by R program. Relative expression of NR2F1‐AS1, miRNA‐338‐3p and cyclin D1 (CCND1) was determined by quantitative real time polymerase chain reaction. In addition, Western blot analysis was adopted for evaluation of protein expression of CCND1. Targeted relationships between NR2F1‐AS1 and miRNA‐338‐3p, as well as miRNA‐338‐3p and CCND1 were predicted using bioinformatics analysis and validated by dual‐luciferase reporter gene assay. Besides, tumour xenograft assay was adopted for verification of the role of NR2F1‐AS1 in TC in vivo. NR2F1‐AS1 and CCND1 were overexpressed, whereas miRNA‐338‐3p was down‐regulated in TC tissues and cell lines. Down‐regulation of NR2F1‐AS1 and CCND1 suppressed proliferation and migration of TC cells yet greatly enhanced cell apoptotic rate. Silence of NR2F1‐AS1 significantly suppressed TC tumorigenesis in vivo. NR2F1‐AS1 sponged miRNA‐338‐3p to up‐regulate CCND1 expression to promote TC progression. Our study demonstrated that up‐regulation of NR2F1‐AS1 accelerated TC progression through regulating miRNA‐338‐3P/CCND1 axis.     

PTRN‐1/CAMSAP promotes CYK‐1/formin‐dependent actin polymerization during endocytic recycling

Cargo sorting and membrane carrier initiation in recycling endosomes require appropriately coordinated actin dynamics. However, the mechanism underlying the regulation of actin organization during recycling transport remains elusive. Here we report that the loss of PTRN‐1/CAMSAP stalled actin exchange and diminished the cytosolic actin structures. Furthermore, we found that PTRN‐1 is required for the recycling of clathrin‐independent cargo hTAC‐GFP. The N‐terminal calponin homology (CH) domain and central coiled‐coils (CC) region of PTRN‐1 can synergistically sustain the flow of hTAC‐GFP. We identified CYK‐1/formin as a binding partner of PTRN‐1. The N‐terminal GTPase‐binding domain (GBD) of CYK‐1 serves as the binding interface for the PTRN‐1 CH domain. The presence of the PTRN‐1 CH domain promoted CYK‐1‐mediated actin polymerization, which suggests that the PTRN‐1‐CH:CYK‐1‐GBD interaction efficiently relieves autoinhibitory interactions within CYK‐1. As expected, the overexpression of the CYK‐1 formin homology domain 2 (FH2) substantially restored actin structures and partially suppressed the hTAC‐GFP overaccumulation phenotype in ptrn‐1 mutants. We conclude that the PTRN‐1 CH domain is required to stimulate CYK‐1 to facilitate actin dynamics during endocytic recycling.     

Endothelin‐1 increases the expression of VEGF‐R1/Flt‐1 receptors in rat cultured astrocytes through ETB receptors

Expressions of vascular endothelial growth factor (VEGF) receptors in astrocytes are increased in damaged brains. To clarify the regulatory mechanisms of VEGF receptors, the effects of endothelin‐1 (ET‐1) were examined in rat cultured astrocytes. Expressions of VEGF‐R1 and ‐R2 receptor mRNA were at similar levels, whereas the mRNA expressions of VEGF‐R3 and Tie‐2, a receptor for angiopoietins, were lower. Placenta growth factor, a selective agonist of the VEGF‐R1 receptor, induced phosphorylation of focal adhesion kinase (FAK) and extracellular signal regulated kinase 1/2 (ERK1/2). Phosphorylations of FAK and ERK 1/2 were also stimulated by VEGF‐E, a selective VEGF‐R2 agonist. Increased phosphorylations of FAK and ERK1/2 by VEGF₁₆₅ were reduced by selective antagonists for VEGF‐R1 and ‐R2. Treatment with ET‐1 increased VEGF‐R1 mRNA and protein levels. The effects of ET‐1 on VEGF‐R1 mRNA were mimicked by Ala^1,3,11,15‐ET‐1, a selective agonist for ET_B receptors, and inhibited by BQ788, an ET_B antagonist. ET‐1 did not affect the mRNA levels of VEGF‐R2, ‐R3, and Tie‐2. Pre‐treatment with ET‐1 potentiated the effects of placenta growth factor on phosphorylations of FAK and ERK1/2. These findings suggest that ET‐1 induces up‐regulation of VEGF‐R1 receptors in astrocytes, and potentiates VEGF signals in damaged nerve tissues.

     

Jasmonoyl‐l‐isoleucine hydrolase 1 (JIH1) regulates jasmonoyl‐l‐isoleucine levels and attenuates plant defenses against herbivores

For most plant hormones, biological activity is suppressed by reversible conjugation to sugars, amino acids and other small molecules. In contrast, the conjugation of jasmonic acid (JA) to isoleucine (Ile) is known to enhance the activity of JA. Whereas hydroxylation and carboxylation of JA‐Ile permanently inactivates JA‐Ile‐mediated signaling in plants, the alternative deactivation pathway of JA‐Ile by its direct hydrolysis to JA remains unstudied. We show that Nicotiana attenuata jasmonoyl‐l ‐isoleucine hydrolase 1 (JIH1), a close homologue of previously characterized indoleacetic acid alanine resistant 3 (IAR3) gene in Arabidopsis, hydrolyzes both JA‐Ile and IAA‐Ala in vitro. When the herbivory‐inducible NaJIH1 gene was silenced by RNA interference, JA‐Ile levels increased dramatically after simulated herbivory in irJIH1, compared with wild‐type (WT) plants. When specialist (Manduca sexta) or generalist (Spodoptera littoralis) herbivores fed on irJIH1 plants they gained significantly less mass compared with those feeding on wild‐type (WT) plants. The poor larval performance was strongly correlated with the higher accumulation of several JA‐Ile‐dependent direct defense metabolites in irJIH1 plants. In the field, irJIH1 plants attracted substantially more Geocoris predators to the experimentally attached M. sexta eggs on their leaves, compared with empty vector plants, which correlated with higher herbivory‐elicited emissions of volatiles known to function as indirect defenses. We conclude that NaJIH1 encodes a new homeostatic step in JA metabolism that, together with JA and JA‐Ile‐hydroxylation and carboxylation of JA‐Ile, rapidly attenuates the JA‐Ile burst, allowing plants to tailor the expression of direct and indirect defenses against herbivore attack in nature.     

The impact of Arabidopsis thaliana SNF1‐related‐kinase 1 (SnRK1)‐activating kinase 1 (SnAK1) and SnAK2 on SnRK1 phosphorylation status: characterization of a SnAK double mutant

Arabidopsis thaliana SNF1‐related‐kinase 1 (SnRK1)‐activating kinase 1 (AtSnAK1) and AtSnAK2 have been shown to phosphorylate in vitro and activate the energy signalling integrator, SnRK1. To clarify this signalling cascade in planta, a genetic‐ and molecular‐based approach was developed. Homozygous single AtSnAK1 and AtSnAK2 T‐DNA insertional mutants did not display an apparent phenotype. Crossing of the single mutants did not allow the isolation of double‐mutant plants, whereas self‐pollinating the S1?/? S2+/? sesquimutant specifically gave approximatively 22% individuals in their offspring that, when rescued on sugar‐supplemented media in vitro, were shown to be AtSnAK1 AtSnAK2 double mutants. Interestingly, this was not obtained in the case of the other sesquimutant, S1+/? S2?/?. Although reduced in size, the double mutant had the capacity to produce flowers, but not seeds. Immunological characterization established the T‐loop of the SnRK1 catalytic subunit to be non‐phosphorylated in the absence of both SnAKs. When the double mutant was complemented with a DNA construct containing an AtSnAK2 open reading frame driven by its own promoter, a normal phenotype was restored. Therefore, wild‐type plant growth and development is dependent on the presence of SnAK in vivo, and this is correlated with SnRK1 phosphorylation. These data show that both SnAKs are kinases phosphorylating SnRK1, and thereby they contribute to energy signalling in planta.     

Modification,Biological Evaluation and SAR Studies of Novel 1H‐Pyrazol Derivatives Containing N,N′‐Disubstituted Urea Moiety as Potential Anti‐melanoma Agents

Malignant melanomas are amongst the most aggressive cancers. BRAF Inhibitors have exhibited therapeutic effects against BRAF‐mutant melanoma. In continuation of our earlier studies on anti‐melanoma agents based on 1H‐pyrazole skeleton, two sets of novel compounds that include 1H‐pyrazole‐4‐amines FA 1 – FA13 and corresponding urea derivatives FN 1 – FN13 have been synthesized and evaluated for their BRAF^V600E inhibitory and antiproliferation activities. Compound FN 10 displayed the most potent biological activity against BRAF^V600E (IC₅₀ = 0.066 μm ) and the A375 human melanoma cell line (GI₅₀ = 0.81 μm ), which was comparable to the positive control vemurafenib, and more potent than our previously reported 1H‐pyrazole‐3‐amines and their urea derivatives. The results of SAR studies and molecular docking can guide further optimization and may help to improve potency of these pyrazole‐based anti‐melanoma agents.     

Mutant Alleles at the Brown Locus Encoding Tyrosinase‐Related Protein‐1 (TRP‐1) Affect Proliferation of Mouse Melanocytes in Culture *

Tyrosinase related protein‐1 (TRP‐1) is a melanocyte‐specific gene product involved in eumelanin synthesis. Mutation in the Tyrp1 gene is associated with brown pelage in mouse and oculocutaneous albinism Type 3 in humans (OCA3). It has been demonstrated that TRP‐1 expresses DHICA oxidase activity in the murine system. However, its actual function in the human system is still unclear. The study was designed to determine the effects of mutation at two Typr1 alleles, namely the Tyrp1^b (brown) and Tyrp1^b‐cj (cordovan) compared with wild type Tyrp1^B (black) on melanocyte function and melanin biosynthesis. The most significant finding was that both of the Tyrp1 mutations (i.e. brown expressing a point mutation and cordovan expressing decreased amount of TRP‐1 protein) resulted in attenuation of cell proliferation rates. Neither necrosis nor apoptosis was responsible for the observed decrease in cell proliferation rates of the brown and cordovan melanocytes. Ultrastructural evaluation by electron microscopic analysis revealed that both mutations in Tyrp1 affected melanosome maturation without affecting its structure. These observations demonstrate that mutation in Tyrp1 compromised tyrosinase activity within the organelle. DOPA histochemistry revealed differences in melanosomal stages between black and brown melanocytes but not between black and cordovan melanocytes. There were no significant differences in tyrosine hydroxylase activities of tyrosinase and TRP‐1 in wild type black, brown and cordovan melanocyte cell lysates. We conclude that mutations in Tyrp1 compromise cell proliferation and melanosomal maturation in mouse melanocyte cultures.     

PTEN regulates IGF‐1R‐mediated therapy resistance in melanoma

Inhibition of the mitogen‐activated protein kinase (MAPK) pathway is a major advance in the treatment of metastatic melanoma. However, its therapeutic success is limited by the rapid emergence of drug resistance. The insulin‐like growth factor‐1 receptor (IGF‐1R) is overexpressed in melanomas developing resistance toward the BRAF^V600 inhibitor vemurafenib. Here, we show that hyperactivation of BRAF enhances IGF‐1R expression. In addition, the phosphatase activity of PTEN as well as heterocellular contact to stromal cells increases IGF‐1R expression in melanoma cells and enhances resistance to vemurafenib. Interestingly, PTEN‐negative melanoma cells escape IGF‐1R blockade by decreased expression of the receptor, implicating that only in melanoma patients with PTEN‐positive tumors treatment with IGF‐1R inhibitors would be a suitable strategy to combat therapy resistance. Our data emphasize the crosstalk and therapeutic relevance of microenvironmental and tumor cell‐autonomous mechanisms in regulating IGF‐1R expression and by this sensitivity toward targeted therapies.     

Che‐1‐induced inhibition of mTOR pathway enables stress‐induced autophagy

Mammalian target of rapamycin (mTOR) is a key protein kinase that regulates cell growth, metabolism, and autophagy to maintain cellular homeostasis. Its activity is inhibited by adverse conditions, including nutrient limitation, hypoxia, and DNA damage. In this study, we demonstrate that Che‐1, a RNA polymerase II‐binding protein activated by the DNA damage response, inhibits mTOR activity in response to stress conditions. We found that, under stress, Che‐1 induces the expression of two important mTOR inhibitors, Redd1 and Deptor, and that this activity is required for sustaining stress‐induced autophagy. Strikingly, Che‐1 expression correlates with the progression of multiple myeloma and is required for cell growth and survival, a malignancy characterized by high autophagy response.     

LncRNA SNHG1 functions as a ceRNA to antagonize the effect of miR‐145a‐5p on the down‐regulation of NUAK1 in nasopharyngeal carcinoma cell

How lncRNA SNHG1 influences the aggressiveness of nasopharyngeal carcinoma cells as well as the underlying mechanism was studied. The lncRNA differences were analysed by GSE12452 gene microarray. The expression of SNHG1, MiR‐145‐5p and NUAK1 was identified by qRT‐PCR and western blot. Transfection was conducted to construct nasopharyngeal carcinoma cells with different expressions of SNHG1, miR‐145‐5p and NUAK1. Dual‐luciferase reporter assay was performed to explore the relationship between SNHG1, miR‐145‐5p and NUAK1. Wound‐healing assay and transwell invasion experiments were employed to study changes in cell migration capacity and cell invasion, respectively. Tumour xenografts were performed to observe lung metastasis of nude mice inoculated with transfected CNE cells. SNHG1 is highly expressed in nasopharyngeal carcinoma tissues and in cell lines. Down‐regulation of SNHG1 facilitated the expression of miR‐145‐5p and further suppressed the level of NAUK1 in CNE and HNE‐1 cells. Silencing of SNHG1, up‐regulation of miR‐145‐5p and inhibition of NAUK1 by relative transfection all attenuated the aggressiveness of CNE and HNE‐1 cells both in vivo and in vitro. Moreover, the impaired cell migration and invasion by SNHG1 siRNA could be rescued by cotransfection of miR‐145‐5p in CNE and HNE‐1 cells. LncRNA SNHG1 promoted the expression of NUAK1 by down‐regulating miR‐145‐5p and thus promoted the aggressiveness of nasopharyngeal carcinoma cells through AKT signalling pathway and induced epithelial‐mesenchymal transition (EMT).     

The dependence of leaf senescence on the balance between 1‐aminocyclopropane‐1‐carboxylate acid synthase 1 (ACS1)‐catalysed ACC generation and nitric oxide‐associated 1 (NOS1)‐dependent NO accumulation in Arabidopsis

• Ethylene and nitric oxide (NO) act as endogenous regulators during leaf senescence. Levels of ethylene or its precursor 1‐aminocyclopropane‐1‐carboxylate acid (ACC) depend on the activity of ACC synthases (ACS), and NO production is controlled by NO‐associated 1 (NOA1). However, the integration mechanisms of ACS and NOA1 activity still need to be explored during leaf senescence.
• Here, using experimental techniques, such as physiological and molecular detection, liquid chromatography‐tandem mass spectrometry and fluorescence measurement, we investigated the relevant mechanisms.
• Our observations showed that the loss‐of‐function acs1‐1 mutant ameliorated age‐ or dark‐induced leaf senescence syndrome, such as yellowing and loss of chlorophyll, that acs1‐1 reduced ACC accumulation mainly in mature leaves and that acs1‐1‐promoted NOA1 expression and NO accumulation mainly in juvenile leaves, when compared with the wild type (WT). But the leaf senescence promoted by the NO‐deficient noa1 mutant was not involved in ACS1 expression. There was a similar sharp reduction of ACS1 and NOA1 expression with the increase in WT leaf age, and this inflection point appeared in mature leaves and coincided with the onset of leaf senescence.
• These findings suggest that NOA1‐dependent NO accumulation blocked the ACS1‐induced onset of leaf senescence, and that ACS1 activity corresponds to the onset of leaf senescence in Arabidopsis.

     

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.