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The occurrence of whole-genome duplication or polyploidy may promote plant adaptability to harsh environments. Here, we clarify the evolutionary relationship of eight GhCIPK6 homologous genes in upland cotton (Gossypium hirsutum). Gene expression and interaction analyses indicate that GhCIPK6 homologous genes show significant functional changes after polyploidy. Among these, GhCIPK6D1 and GhCIPK6D3 are significantly up-regulated by drought stress. Functional studies reveal that high GhCIPK6D1 expression promotes cotton drought sensitivity, while GhCIPK6D3 expression promotes drought tolerance, indicating clear functional differentiation. Genetic and biochemical analyses confirm the synergistic negative and positive regulation of cotton drought resistance through GhCBL1A1-GhCIPK6D1 and GhCBL2A1-GhCIPK6D3, respectively, to regulate stomatal movement by controlling the directional flow of K+ in guard cells. These results reveal differentiated roles of GhCIPK6 homologous genes in response to drought stress in upland cotton following polyploidy. The work provides a different perspective for exploring the functionalization and subfunctionalization of duplicated genes in response to polyploidization.
Assuntos
Secas , Evolução Molecular , Regulação da Expressão Gênica de Plantas , Gossypium , Proteínas de Plantas , Poliploidia , Gossypium/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Estresse Fisiológico/genética , Genes de Plantas , Filogenia , Duplicação Gênica , Plantas Geneticamente Modificadas/genética , Estômatos de Plantas/genética , Estômatos de Plantas/fisiologia , Resistência à SecaRESUMO
In order to understand the molecular mechanism of cotton's response to drought during the flowering and boll stage, transcriptomics and metabolomics were carried out for two introgression lines (drought-tolerant line: T307; drought-sensitive line: S48) which were screened from Gossypium hirsutum cv. 'Emian22' with some gene fragments imported from Gossypium barbadense acc. 3-79, under drought stress by withdrawing water at flowering and boll stage. Results showed that the basic drought response in cotton included a series of broad-spectrum responses, such as amino acid synthesis, hormone (abscisic acid, ABA) signal transduction, and mitogen-activated protein kinases signal transduction pathway, which activated in both drought-tolerant and drought-sensitive lines. However, the difference of their imported fragments and diminished sequences triggers endoplasmic reticulum (ER) protein processing, photosynthetic-related pathways (in leaves), and membrane solute transport (in roots) in drought-tolerant line T307, while these are missed or not activated in drought-sensitive line S48, reflecting the different drought tolerance of the two genotypes. Virus-induced gene silencing assay of drought-tolerant differentially expressed heat shock protein (HSP) genes (mainly in leaf) and ATP-binding cassette (ABC) transporter genes (mainly in roots) indicated that those genes play important role in cotton drought tolerant. Combined analysis of transcriptomics and metabolomics highlighted the important roles of ER-stress-related HSP genes and root-specific ABC transporter genes in plants drought tolerance. These results provide new insights into the molecular mechanisms underlying the drought stress adaptation in cotton.
Assuntos
Secas , Gossypium , Transportadores de Cassetes de Ligação de ATP/genética , Ácido Abscísico , Trifosfato de Adenosina , Aminoácidos , Regulação da Expressão Gênica de Plantas , Gossypium/genética , Proteínas de Choque Térmico/genética , Hormônios , Metaboloma , Proteínas Quinases Ativadas por Mitógeno/genética , Estresse Fisiológico/genética , Transcriptoma , ÁguaRESUMO
HuR (human antigen R), an mRNA-binding protein responsible for poor prognosis in nearly all kinds of malignancies, is a potential anti-tumor target for drug development. While screening HuR inhibitors with a fluorescence polarization (FP) based high-throughput screening (HTS) system, the clinically used drug eltrombopag was identified. Activity of eltrombopag on molecular level was verified with FP, electrophoretic mobility shift assay (EMSA), simulation docking and surface plasmon resonance (SPR). Further, we showed that eltrombopag inhibited in vitro cell proliferation of multiple cancer cell lines and macrophages, and the in vivo anti-tumor activity was also demonstrated in a 4T1 tumor-bearing mouse model. The in vivo data showed that eltrombopag was efficient in reducing microvessels in tumor tissues. We then confirmed the HuR-dependent anti-angiogenesis effect of eltrombopag in 4T1 cells and RAW264.7 macrophages with qRT-PCR, HuR-overexpression and HuR-silencing assays, RNA stability assays, RNA immunoprecipitation and luciferase assays. Finally, we analyzed the in vitro anti-angiogenesis effect of eltrombopag on human umbilical vein endothelial cells (HUVECs) mediated by macrophages with cell scratch assay and in vitro Matrigel angiogenesis assay. With these data, we revealed the HuR-dependent anti-angiogenesis effect of eltrombopag in breast tumor, suggesting that the existing drug eltrombopag may be used as an anti-cancer drug.
RESUMO
Transcriptional factors are the major regulators of plant signaling pathways in response to environmental stresses i.e., drought, salinity and cold. Hereby, the GhMYB108-like was characterized to determine whether it regulate these stresses. The GhMYB108-like cDNA consisted of 1107 base pairs (bp) with 807 open reading frame encoded a protein of 268 amino acids. Its isoelectric point and molecular weight are 5.51 and 30.3 kDa respectively. Phylogenetic analysis and online databases revealed that GhMYB108-like proteins are closely related with the Arabidopsis thaliana MYB2. Important cis-elements were detected in the promotor region of GhMYB108-like responding to stresses and phytohormones. The 3D structure of GhMYB108-like protein has been predicted. In addition, various physico-chemical properties of GhMYB108-like have been determined. Subcellular localization confirmed that GhMYB108-like are nuclear localized protein. Quantitative expression analysis showed that polyethylene glycol and salt treatments significantly induced the expression of GhMYB108-like. Overall, our findings suggest that GhMYB108-like is an important gene that would plays important regulatory role in response to drought and salt stresses.
Assuntos
Regulação da Expressão Gênica de Plantas/genética , Gossypium/genética , Proteínas de Plantas/genética , Proteínas Proto-Oncogênicas c-myb/genética , Elementos Reguladores de Transcrição/genética , Estresse Fisiológico , Sequência de Aminoácidos , Sequência de Bases , Secas , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Gossypium/metabolismo , Filogenia , Fitocromo/farmacologia , Proteínas de Plantas/classificação , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Proteínas Proto-Oncogênicas c-myb/classificação , Proteínas Proto-Oncogênicas c-myb/metabolismo , Salinidade , Homologia de Sequência de Aminoácidos , Cloreto de Sódio/farmacologiaRESUMO
BACKGROUND AND AIMS: "Shexiang Baoxin Pill" (SBP), a commonly used traditional Chinese medicine, has been used to treat angina, myocardial infarction and coronary heart disease in China for thirty years. SBP has been proven to promote angiogenesis in a rat model of myocardial infarction (MI). The aim of the present study was to determine the pro-angiogenic effects and mechanism of SBP during inflammation or ischemic pathological conditions and elucidate its regulatory effects on endothelial cell function and signaling pathways mediated by macrophages. METHODS: We used a polyvinyl alcohol (PVA) sponge implantation mouse model as an inflammatory angiogenesis model and utilized a mouse femoral artery ligation model as a hind limb ischemia model. We also performed cell proliferation, cell migration and tubule formation in vitro experiments to assess the effects of SBP on endothelial cell function and signaling pathways by stimulating macrophage activity. RESULTS: The in vitro experiment results showed that SBP could significantly increase the expression of mRNAs and proteins associated with angiogenesis in endothelial cells by activating macrophages to release pro-angiogenic factors such as Vegf-a. Activation of macrophages by SBP eventually led to endothelial cell proliferation, migration and tubule formation and increased the expression of p-Akt and p-Erk1/2 proteins in the downstream PI3K/Akt and MAPK/Erk1/2 signaling pathways related to angiogenesis, respectively. The in vivo experiment results indicated that SBP had angiogenesis effects in both inflammatory and ischemic angiogenesis models with dose- and time-dependent effects. CONCLUSION: Shexiang Baoxin Pills can promote angiogenesis by activating macrophages to regulate endothelial cell function and signal transduction pathways.
Assuntos
Indutores da Angiogênese/farmacologia , Medicamentos de Ervas Chinesas/farmacologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/fisiologia , Macrófagos/fisiologia , Neovascularização Fisiológica/efeitos dos fármacos , Animais , Células Cultivadas , Camundongos , Camundongos Endogâmicos C57BL , Transdução de SinaisRESUMO
Effective delivery of chemotherapeutic agents to tumors is a critical objective of improved cancer therapy. Traditional antiangiogenic therapy aims at eradicating tumor blood vessels, but the subsequently reduced blood perfusion may limit the drug amount delivered into the tumor and potentially lead to tumor hypoxia, which has been proved to be unable to meet the therapeutic expectations. "Shexiang Baoxin Pill" (SBP) is a well-known traditional Chinese medicine (TCM) used in clinical treatment of cardiovascular diseases, which has the pharmacological effect of pro-angiogenesis demonstrated recently. In this study, we disclosed our finding that SBP could enhance the effective treatment performance of gemcitabine (GEM) while minimizing the toxic side effects caused by GEM. Mechanistically, SBP increased tumor angiogenesis, blood perfusion, vascular permeability, and vessel dilation, which subsequently favored the delivery of GEM to the tumor lesion. Moreover, combined treatment with SBP and GEM could modify tumor microenvironment and consequently overcome multidrug resistance, and this combination therapy is also suitable for combination of SBP with some other chemotherapeutic drugs as well. These results suggest that combining SBP with chemotherapeutic agents achieves better treatment efficiency, which can open an avenue for expanding the combined treatment of anti-cancer chemotherapeutic drugs with TCM.
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Cotton, a natural fiber producing crop of huge importance, is often prone to attack of Verticillium dahliae. Papain-like cysteine proteases (PLCPs) constitute a large family in plants and were proposed to involve in plant defense against pathogen attack in a number of studies. However, there is no detailed characterization of PLCP genes in cotton against infection of V. dahliae. In this study, we carried out a genome-wide analysis in cotton and identified seventy-eight PLCPs, which were divided into nine subfamilies based on their evolution phylogeny: RD21 (responsive to desiccation 21), CEP (cysteine endopeptidase), XCP (xylem cysteine peptidase), XBCP3 (xylem bark cysteine peptidase 3), THI, SAG12 (senescence-associated gene 12), RD19 (responsive to desiccation 19), ALP (aleurain-like protease) and CTB (cathepsin B-like). Genes in each subfamily exhibit a similar structure and motif composition. The expression patterns of these genes in different organs were examined, and subfamily RD21 was the most abundant in these families. Expression profiles under abiotic stress showed that thirty-five PLCP genes were induced by multiple stresses. Further transcriptome analysis showed that sixteen PLCP genes were up-regulated in response to V. dahliae in cotton. Among those, GhRD21-7 showed a higher transcription level than most other PLCP genes. Additionally, over-expression of GhRD21-7 led to enhanced resistance and RNAi lines were more susceptible to V. dahliae in cotton. Our results provide valuable information for future functional genomic studies of PLCP gene family in cotton.
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Abscission is a process that allows plants to shed tissues or organs via cell separation, and occurs throughout the life cycle. Removal of leaves through the use of chemical defoliants is very important for mechanical harvesting of cotton (Gossypium hirsutum). However, our knowledge of the molecular mechanisms of the defoliation response involved is limited. In this study, RNA-seq was conducted in order to profile the differentially expressed genes (DEGs) between cultivars X50 (sensitive to chemical defoliants) and X33 (relatively insensitive) at different time points after treatment with thidiazuron and ethephon (TE). A total of 2434 DEGs were identified between the two cultivars across the different time-points. Functional categories according to GO and KEGG analyses revealed that plant hormone signal transduction and zeatin biosynthesis were involved in the response to TE. Cytokinin oxidase/dehydrogenase (CKX) genes and ethylene-related genes were up-regulated following TE treatment, and were associated with increased level of ethylene, especially in cultivar X50. Down-regulation of GhCKX3 resulted in delayed defoliation and a reduced ethylene response. The results show that crosstalk between cytokinin and ethylene regulates cotton defoliation, and provide new insights into the molecular mechanisms underlying the mode of action of defoliants in cotton.
Assuntos
Citocininas/metabolismo , Desfolhantes Químicos/administração & dosagem , Etilenos/metabolismo , Gossypium/fisiologia , Folhas de Planta/crescimento & desenvolvimento , Gossypium/efeitos dos fármacos , Gossypium/genética , Compostos Organofosforados/administração & dosagem , Compostos de Fenilureia/administração & dosagem , Reguladores de Crescimento de Plantas/metabolismo , Folhas de Planta/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Tiadiazóis/administração & dosagemRESUMO
Allotetraploid cotton species (Gossypium hirsutum and Gossypium barbadense) have long been cultivated worldwide for natural renewable textile fibers. The draft genome sequences of both species are available but they are highly fragmented and incomplete1-4. Here we report reference-grade genome assemblies and annotations for G. hirsutum accession Texas Marker-1 (TM-1) and G. barbadense accession 3-79 by integrating single-molecule real-time sequencing, BioNano optical mapping and high-throughput chromosome conformation capture techniques. Compared with previous assembled draft genomes1,3, these genome sequences show considerable improvements in contiguity and completeness for regions with high content of repeats such as centromeres. Comparative genomics analyses identify extensive structural variations that probably occurred after polyploidization, highlighted by large paracentric/pericentric inversions in 14 chromosomes. We constructed an introgression line population to introduce favorable chromosome segments from G. barbadense to G. hirsutum, allowing us to identify 13 quantitative trait loci associated with superior fiber quality. These resources will accelerate evolutionary and functional genomic studies in cotton and inform future breeding programs for fiber improvement.
Assuntos
Genoma de Planta/genética , Gossypium/genética , Mapeamento Cromossômico/métodos , Cromossomos de Plantas/genética , Fibra de Algodão , Variação Genética/genética , Filogenia , Melhoramento Vegetal/métodos , Locos de Características Quantitativas/genéticaRESUMO
BACKGROUND: Transcription factors operate as important switches of transcription networks, and NAC (NAM, ATAF, and CUC) transcription factors are a plant-specific family involved in multiple biological processes. However, this gene family has not been systematically characterized in cotton. RESULTS: Here we identify a large number of genes with conservative NAC domains in four cotton species, with 147 found in Gossypium arboreum, 149 in G. raimondii, 267 in G. barbadense and 283 in G. hirsutum. Predicted membrane-bound NAC genes were also identified. Phylogenetic analysis showed that cotton NAC proteins clustered into seven subfamilies and homologous protein pairs showed similar characteristics. Evolutionary property analysis revealed that purifying selection of NAC genes occurred between diploid and polyploid cotton species, and variation analysis showed GhNAC genes may have been subjected to selection and domestication. NAC proteins showed extensive transactivation and this was dependent on the C-terminus. Some development and stress related cis-elements were enriched in the promoters of GhNAC genes. Comprehensive expression analysis indicated that 38 GhNAC genes were candidates for involvement in fiber development, and 120 in stress responses. Gene co-expression network analysis revealed relationships between fiber-associated NAC genes and secondary cell wall (SCW) biosynthesis genes. CONCLUSIONS: NAC genes were identified in diploid and tetraploid cotton, revealing new insights into their evolution, variation and homology relationships. Transcriptome analysis and co-expression network indicated roles for GhNAC genes in cotton fiber development and stress response, and NAC genes may prove useful in molecular breeding programmes.
Assuntos
Fibra de Algodão , Gossypium/metabolismo , Fatores de Transcrição/fisiologia , Sequência Conservada/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Genes de Plantas/fisiologia , Variação Genética , Genoma de Planta/genética , Estudo de Associação Genômica Ampla , Gossypium/genética , Gossypium/crescimento & desenvolvimento , Filogenia , Estresse Fisiológico , Sintenia/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional/genética , Ativação Transcricional/fisiologiaRESUMO
MicroRNAs (miRNAs) modulate many biological processes through inactivation of specific mRNA targets such as those encoding transcription factors. A delicate spatial/temporal balance between specific miRNAs and their targets is central to achieving the appropriate biological outcomes. Somatic embryogenesis in cotton (Gossypium hirsutum), which goes through initial cellular dedifferentiation, callus proliferation, and somatic embryo development, is of great importance for both fundamental research and biotechnological applications. In this study, we characterize the function of the GhmiR157a-GhSPL10 miRNA-transcription factor module during somatic embryogenesis in cotton. We show that overexpression of GhSPL10, a target of GhmiR157a, increases free auxin and ethylene content and expression of associated signaling pathways, activates the flavonoid biosynthesis pathway, and promotes initial cellular dedifferentiation and callus proliferation. Inhibition of expression of the flavonoid synthesis gene F3H in GhSPL10 overexpression lines (35S:rSPL10-7) blocked callus initiation, while exogenous application of several types of flavonol promoted callus proliferation, associated with cell cycle-related gene expression. Inhibition of ethylene synthesis by aminoethoxyvinylglycine treatment in the 35S:rSPL10-7 line severely inhibited callus initiation, while activation of ethylene signaling through 1-aminocyclopropane 1-carboxylic acid treatment, EIN2 overexpression, or inhibition of the ethylene negative regulator CTR1 by RNA interference promoted flavonoid-related gene expression and flavonol accumulation. These results show that an up-regulation of ethylene signaling and the activation of flavonoid biosynthesis in GhSPL10 overexpression lines were associated with initial cellular dedifferentiation and callus proliferation. Our results demonstrate the importance of a GhmiR157a-GhSPL10 gene module in regulating somatic embryogenesis via hormonal and flavonoid pathways.
Assuntos
Desdiferenciação Celular/genética , Proliferação de Células/genética , Etilenos/farmacologia , Flavonoides/biossíntese , Gossypium/fisiologia , Proteínas de Plantas/genética , RNA de Plantas/genética , Regulação da Expressão Gênica de Plantas , Redes Reguladoras de Genes/genética , Gossypium/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Reguladores de Crescimento de Plantas/farmacologia , Proteínas de Plantas/metabolismo , Técnicas de Embriogênese Somática de Plantas , RNA de Plantas/metabolismoRESUMO
Given both smoking and vitamin D are associated with coronary heart disease (CHD) via inflammation and smoking may interfere with the local antiinflammatory effects of vitamin D. We hypothesized that the relationship between smoking and severity of CHD may be modified by vitamin D.A cross-sectional study was conducted. 25-OH vitamin D values were determined in 348 consecutive patients (mean age 62.4â±â10.5 years; 56.3% male) undergoing coronary angiography at the Heart Center of Chaoyang Hospital affiliated to Capital Medical University between the period of September 2014 and May 2015. We categorized the patients into 2 groups based on 25-OH vitamin D levels, that is, severe hypovitaminosis D (25-OH vitamin Dâ<â10âng/mL) and higher vitamin D (25-OH vitamin Dâ> = â10âng/mL). Multivariable logistic regression models were used to estimate odds ratios (ORs) of severe coronary stenosis or higher Gensini score across three smoking status, that is, never smokers, former smokers, and current smokers in severe hypovitaminosis D and higher vitamin D groups, respectively.Of these patients, we identified 212 (60.9%) cases of severe CHD and 161 (46.3%) cases of severe hypovitaminosis D. Multivariable logistic regression model showed the ORs of severe CHD were 1.94 (95% confidence interval [CI]: 0.47, 7.98) for former smokers and 2.62 (95% CI: 0.83, 8.24) for current smokers, compared with never smokers in group with severe hypovitaminosis D (P-trend = 0.005). In contrast, smoking was not found to be significantly associated with severe CHD in group with higher 25-OH vitamin D (P-trend = 0.115). We found a significant interaction between smoking status and vitamin D on presence of severe CHD (P-interaction = 0.015). In terms of Gensini score as a dependent variable, similar results were identified.Our finding indicated the association between smoking and severity of CHD appeared to be substantially stronger among patients with severe hypovitaminosis D as compared with those with higher vitamin D levels. This suggests vitamin D sufficiency may have a protective effect against the damaging effects of smoking on coronary artery. Future cohort studies are warranted to confirm this finding.
Assuntos
Estenose Coronária/sangue , Estenose Coronária/etiologia , Fumar/efeitos adversos , Deficiência de Vitamina D/complicações , Vitamina D/análogos & derivados , Adulto , Idoso , Idoso de 80 Anos ou mais , Angiografia Coronária , Estenose Coronária/diagnóstico por imagem , Estudos Transversais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Índice de Gravidade de Doença , Vitamina D/sangue , Deficiência de Vitamina D/sangueRESUMO
Gossypium hirsutum contributes the most production of cotton fibre, but G. barbadense is valued for its better comprehensive resistance and superior fibre properties. However, the allotetraploid genome of G. barbadense has not been comprehensively analysed. Here we present a high-quality assembly of the 2.57 gigabase genome of G. barbadense, including 80,876 protein-coding genes. The double-sized genome of the A (or At) (1.50 Gb) against D (or Dt) (853 Mb) primarily resulted from the expansion of Gypsy elements, including Peabody and Retrosat2 subclades in the Del clade, and the Athila subclade in the Athila/Tat clade. Substantial gene expansion and contraction were observed and rich homoeologous gene pairs with biased expression patterns were identified, suggesting abundant gene sub-functionalization occurred by allopolyploidization. More specifically, the CesA gene family has adapted differentially temporal expression patterns, suggesting an integrated regulatory mechanism of CesA genes from At and Dt subgenomes for the primary and secondary cellulose biosynthesis of cotton fibre in a "relay race"-like fashion. We anticipate that the G. barbadense genome sequence will advance our understanding the mechanism of genome polyploidization and underpin genome-wide comparison research in this genus.
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Fibra de Algodão , Genoma de Planta , Gossypium/genética , Poliploidia , Sequência de Bases , Mapeamento Cromossômico , Cromossomos de Plantas/genética , Sequenciamento de Nucleotídeos em Larga Escala , Anotação de Sequência Molecular , FilogeniaRESUMO
Male reproduction in flowering plants is highly sensitive to high temperature (HT). To investigate molecular mechanisms of the response of cotton (Gossypium hirsutum) anthers to HT, a relatively complete comparative transcriptome analysis was performed during anther development of cotton lines 84021 and H05 under normal temperature and HT conditions. In total, 4,599 differentially expressed genes were screened; the differentially expressed genes were mainly related to epigenetic modifications, carbohydrate metabolism, and plant hormone signaling. Detailed studies showed that the deficiency in S-adenosyl-L-homocysteine hydrolase1 and the inhibition of methyltransferases contributed to genome-wide hypomethylation in H05, and the increased expression of histone constitution genes contributed to DNA stability in 84021. Furthermore, HT induced the expression of casein kinasei (GhCKI) in H05, coupled with the suppression of starch synthase activity, decreases in glucose level during anther development, and increases in indole-3-acetic acid (IAA) level in late-stage anthers. The same changes also were observed in Arabidopsis (Arabidopsis thaliana) GhCKI overexpression lines. These results suggest that GhCKI, sugar, and auxin may be key regulators of the anther response to HT stress. Moreover, phytochrome-interacting factor genes (PIFs), which are involved in linking sugar and auxin and are regulated by sugar, might positively regulate IAA biosynthesis in the cotton anther response to HT. Additionally, exogenous IAA application revealed that high background IAA may be a disadvantage for late-stage cotton anthers during HT stress. Overall, the linking of HT, sugar, PIFs, and IAA, together with our previously reported data on GhCKI, may provide dynamic coordination of plant anther responses to HT stress.
Assuntos
Metabolismo dos Carboidratos/genética , Flores/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Gossypium/genética , Temperatura Alta , Ácidos Indolacéticos/metabolismo , Transdução de Sinais/genética , Metilação de DNA/genética , Flores/citologia , Flores/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Genoma de Planta/genética , Glucose/metabolismo , Gossypium/citologia , Gossypium/crescimento & desenvolvimento , Histonas/metabolismo , Cinética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Processamento de Proteína Pós-Traducional/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Estresse Fisiológico/genética , Sacarose/metabolismoRESUMO
As a second messenger, Ca(2+) plays a major role in cold induced transduction via stimulus-specific increases in [Ca(2+)]cyt, which is called calcium signature. During this process, CAXs (Ca(2+)/H(+) exchangers) play critical role. For the first time, a putative Ca(2+)/H(+) exchanger GhCAX3 gene from upland cotton (Gossypium hirsutum cv. 'YZ-1') was isolated and characterized. It was highly expressed in all tissues of cotton except roots and fibers. This gene may act as a regulator in cotton's response to abiotic stresses as it could be up-regulated by Ca(2+), NaCl, ABA and cold stress. Similar to other CAXs, it was proved that GhCAX3 also had Ca(2+) transport activity and the N-terminal regulatory region (NRR) through yeast complementation assay. Over-expression of GhCAX3 in tobacco showed less sensitivity to ABA during seed germination and seedling stages, and the phenotypic difference between wild type (WT) and transgenic plants was more significant when the NRR was truncated. Furthermore, GhCAX3 conferred cold tolerance in yeast as well as in tobacco seedlings based on physiological and molecular studies. However, transgenic plant seeds showed more sensitivity to cold stress compared to WT during seed germination, especially when expressed in N-terminal truncated version. Finally, the extent of sensitivity in transgenic lines was more severe than that in WT line under sodium tungstate treatment (an ABA repressor), indicating that ABA could alleviate cold sensitivity of GhCAX3 seeds, especially in short of its NRR. Meanwhile, we also found that overexpression of GhCAX3 could enhance some cold and ABA responsive marker genes. Taken together, these results suggested that GhCAX3 plays important roles in the cross-talk of ABA and cold signal transduction, and compared to full-length of GhCAX3, the absence of NRR could enhance the tolerance or sensitivity to cold stress, depending on seedling's developmental stages.
Assuntos
Ácido Abscísico/farmacologia , Gossypium/metabolismo , Proteínas de Plantas/metabolismo , Temperatura Baixa , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/genética , Gossypium/efeitos dos fármacos , Gossypium/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Compostos de Tungstênio/farmacologiaRESUMO
Plant CIPKs were specific Ser/Thr protein kinases, which were activated through interaction with calcineurin B-like protein (CBL) containing four EF hands for Ca(2+) binding. The CBL/CIPK complexes play an important role in signal transduction in biotic and abiotic stresses, as well as developmental processes. Here a Ser/Thr protein kinase gene (defined as GhCIPK6), which was isolated from RNA-Seq profile during cotton somatic embryogenesis in our previous research was characterized. The GhCIPK6 gene contains an ORF of 1296 bp that putatively encodes a polypeptide of 431 amino acids with a predicted molecular mass of 48.46 kDa and isoelectric point of 9.12. Sequence alignment analysis confirmed that GhCIPK6 has no intron, and it was homologous to AtCIPK6. Expression analysis of the GhCIPK6 suggested that they might function in diverse tissues, including styles and anthers but not fibers. In addition, expression of the GhCIPK6 gene was induced by salt, drought and ABA treatments. Overexpression of GhCIPK6 significantly enhances the tolerance to salt, drought and ABA stresses in transgenic Arabidopsis, indicating that GhCIPK6 acts as a positive regulator in response to salt and drought stress, and is supposed to be a potential candidate gene to improve stress tolerance by genetic manipulation in cotton and other crops.