RESUMO
Legume nodulation requires light perception by plant shoots and precise long-distance communication between shoot and root. Recent studies have revealed that TGACG-motif binding factors (GmSTFs) integrate light signals to promote root nodulation; however, the regulatory mechanisms underlying nodule formation in changing light conditions remain elusive. Here, we applied genetic engineering, metabolite measurement, and transcriptional analysis to study soybean (Glycine max) nodules. We clarify a fine-tuning mechanism in response to ultraviolet B (UV-B) irradiation and rhizobia infection, involving GmUVR8-dependent UV-B perception and GmSTF3/4-GmMYB12-GmCHS-mediated (iso)flavonoid biosynthesis for soybean nodule formation. GmUVR8 receptor-perceived UV-B signal triggered R2R3-MYB transcription factors GmMYB12-dependent flavonoid biosynthesis separately in shoot and root. In shoot, UV-B-triggered flavonoid biosynthesis relied on GmUVR8a, b, c receptor-dependent activation of GmMYB12L-GmCHS8 (chalcone synthase) module. In root, UV-B signaling distinctly promotes the accumulation of the isoflavones, daidzein, and its derivative coumestrol, via GmMYB12B2-GmCHS9 module, resulting in hypernodulation. The mobile transcription factors, GmSTF3/4, bind to cis-regulatory elements in the GmMYB12L, GmMYB12B2, and GmCHS9 promoters, to coordinate UV-B light perception in shoot and (iso)flavonoid biosynthesis in root. Our findings establish a novel shoot-to-root communication module involved in soybean nodulation and reveal an adaptive strategy employed by soybean roots in response to UV-B light.
Assuntos
Glycine max , Transdução de Sinais , Glycine max/genética , Transdução de Sinais/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regiões Promotoras Genéticas/genética , Comunicação , Nodulação/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMO
BACKGROUND: Stalk lodging is one of the main factors affecting maize (Zea mays L.) yield and limiting mechanized harvesting. Developing maize varieties with high stalk lodging resistance requires exploring the genetic basis of lodging resistance-associated agronomic traits. Stalk strength is an important indicator to evaluate maize lodging and can be evaluated by measuring stalk rind penetrometer resistance (RPR) and stalk buckling strength (SBS). Along with morphological traits of the stalk for the third internodes length (TIL), fourth internode length (FIL), third internode diameter (TID), and the fourth internode diameter (FID) traits are associated with stalk lodging resistance. RESULTS: In this study, a natural population containing 248 diverse maize inbred lines genotyped with 83,057 single nucleotide polymorphism (SNP) markers was used for genome-wide association study (GWAS) for six stalk lodging resistance-related traits. The heritability of all traits ranged from 0.59 to 0.72 in the association mapping panel. A total of 85 significant SNPs were identified for the association mapping panel using best linear unbiased prediction (BLUP) values of all traits. Additionally, five candidate genes were associated with stalk strength traits, which were either directly or indirectly associated with cell wall components. CONCLUSIONS: These findings contribute to our understanding of the genetic basis of maize stalk lodging and provide valuable theoretical guidance for lodging resistance in maize breeding in the future.
Assuntos
Locos de Características Quantitativas , Zea mays , Zea mays/anatomia & histologia , Estudo de Associação Genômica Ampla , Genes de Plantas , Melhoramento VegetalRESUMO
Glioma originated from excessively proliferative and highly invaded glial cells is a common intracranial malignant tumor with poor prognosis. Resistance to temozolomide (TMZ) is a clinical challenge in glioma treatment due to the fact that chemoresistance remains a main obstacle in the improvement of drug efficacy. Salvianolic acid A (Sal A), originated from traditional Chinese herbal medicine Salvia miltiorrhiza, possesses anti-tumor effects and could facilitate the delivery of drugs to brain tumor tissues. In the present work, effects of Sal A on the viability, proliferation, migration, invasion and apoptosis of human glioma cell line U87 cells as well as influence of Sal A on TMZ resistance were measured, so as to identify the biological function of Sal A in the malignant behaviors and chemoresistance of glioma cells. Additionally, activation of TAGLN2/PI3K/Akt pathway in glioma cells was also detected to investigate whether Sal A could regulate TAGLN2/PI3K/Akt to manipulate the progression of glioma and TMZ resistance. Results discovered that Sal A treatment reduced the viability, repressed the proliferation, migration and invasion of glioma cells as well as promoted the apoptosis of glioma cells. Besides, Sal A treatment suppressed TAGLN2/PI3K/Akt pathway in glioma cells. Sal A treatment strengthened the suppressing effect of TMZ on glioma cell proliferation and reinforced the promoting effect of TMZ on glioma cell apoptosis, which were abolished by upregulation of TAGLN2. To conclude, Sal A treatment could suppress the malignant behaviors of glioma cells and improve TMZ sensitivity through inactivating TAGLN2/PI3K/Akt pathway.
Assuntos
Neoplasias Encefálicas , Glioma , Neoplasias Encefálicas/patologia , Ácidos Cafeicos , Linhagem Celular Tumoral , Glioma/metabolismo , Humanos , Lactatos , Proteínas dos Microfilamentos , Proteínas Musculares , Fosfatidilinositol 3-Quinase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositóis/farmacologia , Fosfatidilinositóis/uso terapêutico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Temozolomida/farmacologiaRESUMO
Crop breeding during the Green Revolution resulted in high yields largely due to the creation of plants with semi-dwarf architectures that could tolerate high-density planting. Although semi-dwarf varieties have been developed in rice, wheat and maize, none was reported in soybean (Glycine max), and few genes controlling plant architecture have been characterized in soybean. Here, we demonstrate that the auxin efflux transporter PINFORMED1 (GmPIN1), which determines polar auxin transport, regulates the leaf petiole angle in soybean. CRISPR-Cas9-induced Gmpin1abc and Gmpin1bc multiple mutants displayed a compact architecture with a smaller petiole angle than wild-type plants. GmPIN1 transcripts and auxin were distributed asymmetrically in the petiole base, with high levels of GmPIN1a/c transcript and auxin in the lower cells, which resulted in asymmetric cell expansion. By contrast, the (iso)flavonoid content was greater in the upper petiole cells than in the lower cells. Our results suggest that (iso)flavonoids inhibit GmPIN1a/c expression to regulate the petiole angle. Overall, our study demonstrates that a signal cascade that integrates (iso)flavonoid biosynthesis, GmPIN1a/c expression, auxin accumulation, and cell expansion in an asymmetric manner creates a desirable petiole curvature in soybean. This study provides a genetic resource for improving soybean plant architecture.
Assuntos
Glycine max , Ácidos Indolacéticos , Proteínas de Membrana Transportadoras , Folhas de Planta , Proteínas de Plantas , Flavonoides/biossíntese , Ácidos Indolacéticos/metabolismo , Proteínas de Membrana Transportadoras/genética , Folhas de Planta/anatomia & histologia , Folhas de Planta/citologia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Glycine max/anatomia & histologia , Glycine max/genética , TranscriptomaRESUMO
Microbial contamination and nitrite accumulation are the two major concerns in the quality control of fermented vegetables. In the present study, a lactic acid bacteria strain Lactobacillus plantarum ZJ316 (ZJ316) was inoculated during Stachys sieboldii Miq. (SSM) fermentation, and the effects of ZJ316 on the quality and bacterial community of SSM during fermentation were investigated. It was observed that ZJ316 could avoid the occurrence of the nitrite peak and maintain the nitrite content of fermented SSM at a low level. Gas chromatography-mass spectrometry (GC-MS) results suggested that ZJ316 gave good flavor to the fermented SSM. 16S rDNA sequencing showed that Firmicutes was the dominant flora after ZJ316 inoculation, and the abundance of Proteobacteria decreased at the same time. At the level of the genus, SSM fermented by ZJ316 had a more obvious inhibitory effect on Pseudomonas on the 7th day compared with the naturally fermented SSM. Additionally, the effect of ZJ316-fermented SSM on gut microbiota modulation was also evaluated using an in vitro fecal fermentation system. The results revealed that ZJ316 had a relatively subtle influence on intestinal communities with a potentially positive impact on probiotics such as Lactobacillus and Bifidobacterium and a negative impact on Enterobacteriaceae. Furthermore, SSM fermented by ZJ316 promoted the production of short-chain fatty acids (SCFAs) in the human intestine. These results demonstrate that L. plantarum ZJ316 can be used as a good starter in the fermentation process of pickles.
Assuntos
Alimentos Fermentados , Lactobacillus plantarum , Probióticos , Stachys , Manipulação de Alimentos , Microbiologia de Alimentos , Microbioma Gastrointestinal/efeitos dos fármacos , HumanosRESUMO
Maize (Zea mays) endosperm filling is coordinated with cell expansion to enlarge the grain size, but the mechanism coupling the two processes is poorly understood. Starchy endosperm cells basically contain no visible vacuoles for cell expansion. During grain filling, efficient synthesis of storage compounds leads to reduced cytoplasm and thus lowered cell turgor pressure. Although bioactive gibberellins (GAs) are essential for cell expansion, they accumulate at a low level at this stage. In this study, we identified an endosperm-specific GRAS domain-containing protein (ZmGRAS11) that lacks the DELLA domain and promotes cell expansion in the filling endosperm. The zmgras11 loss-of-function mutants showed normal grain filling but delayed cell expansion, thereby resulting in reduced kernel size and weight. Overexpression of ZmGRAS11 led to larger endosperm cells and therefore increased kernel size and weight. Consistent with this, ZmGRAS11 positively regulates the expression of ZmEXPB12, which is essential for cell expansion, at the endosperm filling stage. Moreover, we found that Opaque2 (O2), a central transcription factor that regulates endosperm filling, could directly bind to the promoter of ZmGRAS11 and activate its expression. Taken together, these results suggest that endosperm cell expansion is coupled with endosperm filling, which is orchestrated by the O2-ZmGRAS11-centered transcriptional regulatory network. Our findings also provide potential targets for maize yield improvement by increasing the storage capacity of endosperm cells.
Assuntos
Endosperma , Zea mays , Endosperma/genética , Endosperma/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Redes Reguladoras de Genes , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Amido/metabolismo , Zea mays/genética , Zea mays/metabolismoRESUMO
General combining ability (GCA) is an important index for inbred lines breeding of maize. To identify the genetic loci of GCA and associated agronomic traits, an association analysis with 195 SSRs was made in phenotypic traits of 240 F1 derived from 120 elite inbred lines containing current breeding resources of maize crossed with 2 testers (Zheng58 and Chang7-2) in two places in 2018. All of the 20 association loci detected for grain yield (GY), plant height (PH), ear height (EH) and GCA for the three traits in two places could explain a phenotypic variation range of 7.31%-9.29%. Among the 20 association loci, 9 (7.31%-9.04%) were associated with GY, 4 (7.22%-8.91%) were related to GCA of GY, 1 (7.56%) was associated with PH, and 3 (7.53%-8.96%) were related to EH. In addition, 3 loci (9.14%-9.29%) were associated with GCA of PH whereas no locus was identified for GCA of EH. In the comparison of the association loci detected in Baoding and Handan, interestingly, one locus (7.69% and 8.11%) was identified in both environments and one locus (7.52% and 7.82%) was identified for yield and GCA of yield. Therefore, the identification of GY-, PH-, EH- and GCA-related association loci could not only provide references for high yield breeding of maize, but also help us comprehend the relationships among GY, agricultural traits and GCA.
Assuntos
Mapeamento Cromossômico , Cruzamentos Genéticos , Zea mays/anatomia & histologia , Zea mays/genética , Marcadores Genéticos , Variação Genética , Endogamia , Desequilíbrio de Ligação/genética , Fenótipo , Locos de Características Quantitativas/genéticaRESUMO
BACKGROUND: Nuclear factor-κB (NF-κB) plays a prominent role in promoting inflammation and resistance to DNA damaging therapy. We searched for proteins that modulate the NF-κB response as a prerequisite to identifying novel factors that affect sensitivity to DNA damaging chemotherapy. RESULTS: Using streptavidin-agarose pull-down, we identified the DExD/H-box RNA helicase, DDX39B, as a factor that differentially interacts with κB DNA probes. Subsequently, using both RNA interference and CRISPR/Cas9 technology, we demonstrated that DDX39B inhibits NF-κB activity by a general mechanism involving inhibition of p65 phosphorylation. Mechanistically, DDX39B mediates this effect by interacting with the pattern recognition receptor (PRR), LGP2, a pathway that required the cellular response to cytoplasmic double-stranded RNA (dsRNA). From a functional standpoint, loss of DDX39B promoted resistance to alkylating chemotherapy in glioblastoma cells. Further examination of DDX39B demonstrated that its protein abundance was regulated by site-specific sumoylation that promoted its poly-ubiquitination and degradation. These post-translational modifications required the presence of the SUMO E3 ligase, PIASx-ß. Finally, genome-wide analysis demonstrated that despite the link to the PRR system, DDX39B did not generally inhibit interferon-stimulated gene expression, but rather acted to attenuate expression of factors associated with the extracellular matrix, cellular migration, and angiogenesis. CONCLUSIONS: These results identify DDX39B, a factor with known functions in mRNA splicing and nuclear export, as an RNA-binding protein that blocks a subset of the inflammatory response. While these findings identify a pathway by which DDX39B promotes sensitization to DNA damaging therapy, the data also reveal a mechanism by which this helicase may act to mitigate autoimmune disease.
Assuntos
RNA Helicases DEAD-box/genética , NF-kappa B/metabolismo , Receptores de Reconhecimento de Padrão/genética , Transdução de Sinais , Alquilação , Animais , RNA Helicases DEAD-box/metabolismo , Sondas de DNA , Tratamento Farmacológico , Humanos , Camundongos , Receptores de Reconhecimento de Padrão/metabolismoRESUMO
Enhancing broad-spectrum resistance is a major goal of crop breeding. However, broad-spectrum resistance has not been thoroughly investigated, and its underlying molecular mechanisms remain elusive. In the model plant Arabidopsis (Arabidopsis thaliana), ACCELERATED CELL DEATH6 (ACD6) is a key component of broad-spectrum resistance that acts in a positive feedback loop with salicylic acid (SA) to regulate multiple pattern recognition receptors. However, the role of ACD6 in disease resistance in crop plants is unclear. Here, we show that the transcript of ANK23, one of the 15 ACD6-like genes in maize (Zea mays), is induced by SA and by infection with the pathogenic fungus Ustilago maydis. Heterologous expression of ANK23 restored disease resistance in the Arabidopsis mutant acd6-2. We show that ANK23 is a maize ortholog of ACD6 and therefore rename ANK23 as ZmACD6. Furthermore, using CRISPR/Cas9, we generated ZmACD6 knockout maize plants, which are more susceptible to U. maydis than wild-type plants. We also identified a maize line (SC-9) with relatively high ZmACD6 expression levels from a diverse natural maize population. SC-9 has increased disease resistance to U. maydis and defense activation, suggesting a practical approach to cultivate elite varieties with enhanced disease resistance.
Assuntos
Resistência à Doença , Genes de Plantas , Doenças das Plantas/microbiologia , Ustilago/fisiologia , Zea mays/genética , Zea mays/microbiologia , Sequência de Bases , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Mutação com Perda de Função/genética , Filogenia , Doenças das Plantas/genética , Ácido Salicílico/farmacologia , Fatores de Tempo , Ustilago/efeitos dos fármacos , Zea mays/efeitos dos fármacosRESUMO
The deregulated breakpoint cluster region (Bcr)-Abelson tyrosine kinase (Abl) fusion protein represents an attractive pharmacological target for the treatment of chronic myeloid leukemia (CML). The high affinity of monobody AS25 was designed to target the Src homology 2 (SH2) domain of Bcr-Abl, leading to allosteric inhibition of Bcr-Abl through formation of protein-protein interactions. An I164E mutation in the SH2 domain disrupts AS25 binding to the SH2 domain of Bcr-Abl. The detailed mechanisms, however, remain to be unresolved. Here, molecular dynamics (MD) simulations and binding free energy calculations were performed to explore the conformational and energetic differences between the wild-type (WT) complexes of Bcr-Abl SH2 domain and AS25 (SH2WT-AS25) as well as the mutated complexes (SH2I164E-AS25). The results revealed that I164E mutation not only caused an increase in the conformational flexibility of SH2-AS25 complexes, but also weakened the binding affinity of AS25 to SH2. The comparative binding modes of SH2-AS25 complexes between WT and the I164E mutant were comprehensively analyzed to unravel the disruption of hydrophobic and hydrogen bonding interactions in the interface of the SH2-AS25 complex triggered by the I164E mutation. The results obtained may help to design the next generation of higher affinity Bcr-Abl SH2-specific peptide inhibitors.
Assuntos
Proteínas de Fusão bcr-abl/química , Inibidores de Proteínas Quinases/química , Anticorpos de Cadeia Única/química , Domínios de Homologia de src , Regulação Alostérica , Proteínas de Fusão bcr-abl/genética , Humanos , Estrutura Quaternária de ProteínaRESUMO
In plants, cell surface pattern recognition receptors (PRRs) provide a first line of defense against pathogens. Although each PRR recognizes a specific ligand, they share common signaling outputs, such as callose and other cell wall-based defenses. Several PRRs are also important for callose induction in response to the defense signal salicylic acid (SA). The extent to which common components are needed for PRR signaling outputs is not known. The gain-of-function Arabidopsis mutant of ACCELERATED CELL DEATH6 (ACD6) acd6-1 shows constitutive callose production that partially depends on PRRs. ACD6-1 (and ACD6) forms complexes with the PRR FLAGELLIN SENSING2, and ACD6 is needed for responses to several PRR ligands. Thus, ACD6-1 could serve as a probe to identify additional proteins important for PRR-mediated signaling. Candidate signaling proteins (CSPs), identified in our proteomic screen after immunoprecipitation of hemagglutinin (HA)-tagged ACD6-1, include several subfamilies of receptor-like kinase (RLK) proteins and a MECHANO-SENSITIVE CHANNEL OF SMALL CONDUCTANCE-LIKE 4 (MSL4). In acd6-1, CSPs contribute to autoimmunity. In wild type, CSPs are needed for defense against bacteria and callose responses to two or more microbial-derived patterns and an SA agonist. CSPs may function to either i) promote the assembly of signaling complexes, ii) regulate the output of known PRRs, or both.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/imunologia , Autoimunidade , Membrana Celular/metabolismo , Mecanotransdução Celular , Proteínas Serina-Treonina Quinases/metabolismo , Arabidopsis/genética , Arabidopsis/microbiologia , Regulação da Expressão Gênica de Plantas , Mutação/genética , Fenótipo , Pseudomonas syringae/crescimento & desenvolvimento , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ácido Salicílico/metabolismo , Regulação para Cima/genéticaRESUMO
The Arabidopsis membrane protein ACCELERATED CELL DEATH 6 (ACD6) and the defense signal salicylic acid (SA) are part of a positive feedback loop that regulates the levels of at least 2 pathogen-associated molecular patterns (PAMP) receptors, including FLAGELLIN SENSING 2 (FLS2) and CHITIN ELICITOR RECEPTOR (LYSM domain receptor-like kinase 1, CERK1). ACD6- and SA-mediated regulation of these receptors results in potentiation of responses to FLS2 and CERK1 ligands (e.g. flg22 and chitin, respectively). ACD6, FLS2 and CERK1 are also important for callose induction in response to an SA agonist even in the absence of PAMPs. Here, we report that another receptor, EF-Tu RECEPTOR (EFR) is also part of the ACD6/SA signaling network, similar to FLS2 and CERK1.
Assuntos
Anquirinas/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Resistência à Doença , Doenças das Plantas , Receptores de Reconhecimento de Padrão/metabolismo , Ácido Salicílico/metabolismo , Morte Celular , Glucanos/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de SinaisRESUMO
In Arabidopsis thaliana, responses to pathogen-associated molecular patterns (PAMPs) are mediated by cell surface pattern recognition receptors (PRRs) and include the accumulation of reactive oxygen species, callose deposition in the cell wall, and the generation of the signal molecule salicylic acid (SA). SA acts in a positive feedback loop with ACCELERATED CELL DEATH6 (ACD6), a membrane protein that contributes to immunity. This work shows that PRRs associate with and are part of the ACD6/SA feedback loop. ACD6 positively regulates the abundance of several PRRs and affects the responsiveness of plants to two PAMPs. SA accumulation also causes increased levels of PRRs and potentiates the responsiveness of plants to PAMPs. Finally, SA induces PRR- and ACD6-dependent signaling to induce callose deposition independent of the presence of PAMPs. This PAMP-independent effect of SA causes a transient reduction of PRRs and ACD6-dependent reduced responsiveness to PAMPs. Thus, SA has a dynamic effect on the regulation and function of PRRs. Within a few hours, SA signaling promotes defenses and downregulates PRRs, whereas later (within 24 to 48 h) SA signaling upregulates PRRs, and plants are rendered more responsive to PAMPs. These results implicate multiple modes of signaling for PRRs in response to PAMPs and SA.
Assuntos
Anquirinas/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Ácido Salicílico/metabolismo , Anquirinas/genética , Arabidopsis/genética , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Flagelina/farmacologia , Regulação da Expressão Gênica de Plantas , Glucanos/metabolismo , Interações Hospedeiro-Patógeno , Immunoblotting , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutação , Ligação Proteica/efeitos dos fármacos , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Pseudomonas syringae/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Receptores de Reconhecimento de Padrão/genética , Receptores de Reconhecimento de Padrão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ácido Salicílico/agonistas , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Tiadiazóis/farmacologiaRESUMO
ACCELERATED CELL DEATH6 (ACD6) is a multipass membrane protein with an ankyrin domain that acts in a positive feedback loop with the defense signal salicylic acid (SA). This study implemented biochemical approaches to infer changes in ACD6 complexes and localization. In addition to forming endoplasmic reticulum (ER)- and plasma membrane (PM)-localized complexes, ACD6 forms soluble complexes, where it is bound to cytosolic HSP70, ubiquitinated, and degraded via the proteasome. Thus, ACD6 constitutively undergoes ER-associated degradation. During SA signaling, the soluble ACD6 pool decreases, whereas the PM pool increases. Similarly, ACD6-1, an activated version of ACD6 that induces SA, is present at low levels in the soluble fraction and high levels in the PM. However, ACD6 variants with amino acid substitutions in the ankyrin domain form aberrant, inactive complexes, are induced by a SA agonist, but show no PM localization. SA signaling also increases the PM pools of FLAGELLIN SENSING2 (FLS2) and BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1). FLS2 forms complexes ACD6; both FLS2 and BAK1 require ACD6 for maximal accumulation at the PM in response to SA signaling. A plausible scenario is that SA increases the efficiency of productive folding and/or complex formation in the ER, such that ACD6, together with FLS2 and BAK1, reaches the cell surface to more effectively promote immune responses.
Assuntos
Anquirinas/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Arabidopsis/metabolismo , Ácido Salicílico/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Anquirinas/química , Anquirinas/genética , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Ativação Enzimática , Proteínas de Choque Térmico HSP70/metabolismo , Dados de Sequência Molecular , Mutação , Complexo de Endopeptidases do Proteassoma/metabolismo , Estrutura Terciária de Proteína , Transporte Proteico , ProteóliseRESUMO
OBJECTIVE: The objective of this study was to investigate the reversal effects of 5,5'-dimethoxylariciresinol-4'-O-ß-D-glucoside (DMAG) extracted from traditional Chinese medicines Mahonia on multidrug resistance (MDR) of human leukemia cells to chemotherapeutic agents. MATERIALS AND METHODS: MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed to determine the effect of DMAG on doxorubicin sensitivity to K562/DOX cells. Propidium iodide /Hoechst 33342 double staining assay was used to investigate the effect of DMAG on doxorubicin-induced cellular apoptosis. Intracellular accumulation of doxorubicin and rhodamine 123 assay were performed to evaluate the effect of DMAG on drugs efflux activity of P-glycoprotein. RESULTS: DMAG significantly enhanced the doxorubicin cytotoxicity to K562/DOX cells. In the presence of 1.0 µM of DMAG, the IC50 of doxorubicin decreased from 34.93 ± 1.37 µM to 12.51 ± 1.28 µM. DMAG of 1.0 µM significantly enhanced doxorubicin-induced cell apoptosis in K562/DOX cells and the enhancement was time-dependent. A significant increase in accumulation of doxorubicin in the presence of DMAG was observed. After treatment of the K562/DOX cells for 1 h with 15.0 µM doxorubicin alone, the fluorescence intensity was 33093.12. With the addition of 1.0 µM of DMAG, the fluorescence intensity of doxorubicin was 2.3-fold higher. A significant increase of accumulation of rhodamine 123 in the presence of DMAG was also observed. With the addition of 1.0 µM of DMAG, the fluorescence intensity was increased by 49.11% compared with rhodamine 123 alone. CONCLUSION: DMAG was shown to effectively enhance chemosensitivity of resistant cells, which makes it might be a suitable candidate for potential MDR-reversing agents.
Assuntos
Antibióticos Antineoplásicos/uso terapêutico , Doxorrubicina/uso terapêutico , Resistência a Múltiplos Medicamentos , Glucosídeos/uso terapêutico , Leucemia Eritroblástica Aguda/tratamento farmacológico , Lignanas/uso terapêutico , Humanos , Células K562RESUMO
The cross-talk between plant disease resistance and development is fundamental to understanding systemic physiological processes during pathogen attack. Our previous study showed that the Arabidopsis GH3.5 gene acts as a bifunctional modulator of the salicylic acid (SA)-mediated resistance and the auxin-mediated susceptibility during the Arabidopsis-Pseudomonas syringae interaction as well as development. Here, we further study the role and mechanism of GH3.5 involved in the SA-dependent defense pathway. Transcript and histochemical analysis of the GH3.5 promoter::GUS reporter expression indicate that GH3.5 is expressed with a strong temporal and spatial manner with predominant expression in the divisional tissues. Upon bacterial challenge, GUS activity is induced in the junction tissue around the infiltrated zone with higher levels in the vasculature with a pattern different between the incompatible and compatible interactions. Exogenous SA application enhances disease resistance in the activation-tagged mutant gh3.5-1D, while the GH3.5-mediated defense enhancement is depleted in the SA deficient gh3.5-1D/NahG double mutant, indicating that GH3.5 modulates defense response through the SA-dependent pathway. Furthermore, bacterial growth in the gh3.5-1D/npr1 double mutant treated with SA indicates that GH3.5 enhances the SA-mediated defense response through both NPR1-dependent and independent pathways.
RESUMO
Salicylic acid (SA) plays a central role in plant disease resistance, and emerging evidence indicates that auxin, an essential plant hormone in regulating plant growth and development, is involved in plant disease susceptibility. GH3.5, a member of the GH3 family of early auxin-responsive genes in Arabidopsis (Arabidopsis thaliana), encodes a protein possessing in vitro adenylation activity on both indole-3-acetic acid (IAA) and SA. Here, we show that GH3.5 acts as a bifunctional modulator in both SA and auxin signaling during pathogen infection. Overexpression of the GH3.5 gene in an activation-tagged mutant gh3.5-1D led to elevated accumulation of SA and increased expression of PR-1 in local and systemic tissues in response to avirulent pathogens. In contrast, two T-DNA insertional mutations of GH3.5 partially compromised the systemic acquired resistance associated with diminished PR-1 expression in systemic tissues. The gh3.5-1D mutant also accumulated high levels of free IAA after pathogen infection and impaired different resistance-gene-mediated resistance, which was also observed in the GH3.6 activation-tagged mutant dfl1-D that impacted the auxin pathway, indicating an important role of GH3.5/GH3.6 in disease susceptibility. Furthermore, microarray analysis showed that the SA and auxin pathways were simultaneously augmented in gh3.5-1D after infection with an avirulent pathogen. The SA pathway was amplified by GH3.5 through inducing SA-responsive genes and basal defense components, whereas the auxin pathway was derepressed through up-regulating IAA biosynthesis and down-regulating auxin repressor genes. Taken together, our data reveal novel regulatory functions of GH3.5 in the plant-pathogen interaction.