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1.
BMC Plant Biol ; 19(1): 371, 2019 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-31438856

RESUMO

BACKGROUND: Propamocarb (PM) is one of the main pesticides used for controlling cucumber downy mildew. However, due to its volatility and internal absorption, PM can easily form pesticide residues on cucumber fruits that seriously endanger human health and pollute the environment. The breeding of new cucumber varieties with a low abundance of PM residues via genetic methods constitutes an effective strategy for reducing pesticide residues and improving cucumber safety and quality. To help elucidate the molecular mechanism resulting in a low PM residue abundance in cucumber, we used the cucumber cultivar 'D0351' (which has the lowest PM residue content) as the test material and identified genes related to low PM residue abundance through high-throughput tag-sequencing (Tag-Seq). RESULTS: CsMAPEG was constitutively expressed and showed both varietal and organizational differences. This gene was strongly expressed in 'D0351'. The expression levels of CsMAPEG in different cucumber tissues under PM stress were as follows: fruit>leaf>stem>root. CsMAPEG can respond to salicylic acid (SA), gibberellin (GA) and Corynespora cassiicola Wei (Cor) stress and thus plays an important regulatory role in plant responses to abiotic and biological stresses. The PM residue abundance in the fruits of CsMAPEG-overexpressing plants was lower than those found in antisense CsMAPEG plants and wild-type plants at all tested time points. The results revealed that CsMAPEG played a positive role in reducing the PM residue abundance. A CsMAPEG sense construct increased the contents of SOD, POD and GST in cucumber fruits, enhanced the degradation and metabolism of PM in cucumber, and thus effectively reduced the pesticide residue abundance in cucumber fruits. CONCLUSIONS: The expression patterns of CsMAPEG in cucumber cultivars with high and low pesticide residue abundances and a transgenic verification analysis showed that CsMAPEG can actively respond to PM stress and effectively reduce the PM residue abundance in cucumber fruits. The results of this study will help researchers further elucidate the mechanism responsible for a low PM residue abundance in cucumber and lay a foundation for the breeding of new agricultural cucumber varieties with low pesticide residue abundances.


Assuntos
Carbamatos/farmacologia , Cucumis sativus/genética , Fungicidas Industriais/farmacologia , Genes de Plantas , Resíduos de Praguicidas , Clonagem Molecular , Cucumis sativus/efeitos dos fármacos , Cucumis sativus/enzimologia , Cucumis sativus/fisiologia , Perfilação da Expressão Gênica , Vetores Genéticos , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Transformação Genética
2.
Plant Physiol Biochem ; 141: 431-445, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31238253

RESUMO

S-adenosylmethionine synthetase (SAMS) catalyzes methionine and ATP to generate S-adenosyl-L-methionine (SAM). In plants, accumulating SAMS genes have been characterized and the majority of them are reported to participate in development and stress response. In this study, two putative SAMS genes (CsSAMS1 and CsSAMS2) were identified in cucumber (Cucumis Sativus L.). They displayed 95% similarity and had a high identity with their homologous of Arabidopsis thaliana and Nicotiana tabacum. The qRT-PCR test showed that CsSAMS1 was predominantly expressed in stem, male flower, and young fruit, whereas CsSAMS2 was preferentially accumulated in stem and female flower. And they displayed differential expression profiles under stimuli, including NaCl, ABA, SA, MeJA, drought and low temperature. To elucidate the function of cucumber SAMS, the full-length CDS of CsSAMS1 was cloned, and prokaryotic expression system and transgenic materials were constructed. Expressing CsSAMS1 in Escherichia coli BL21 (DE3) improved the growth of the engineered strain under salt stress. Overexpression of CsSAMS1 significantly increased MDA content, H2O2 content, and POD activity in transgenic lines under non-stress condition. Under salt stress, however, the MDA content of transgenic lines was lower than that of the wild type, the H2O2 content remained high, the polyamine and ACC synthesis in transgenic lines exhibited a CsSAMS1-expressed dependent way. Taken together, our results suggested that both CsSAMS1 and CsSAMS2 were involved in plant development and stress response, and a proper increase of expression level of CsSAMS1 in plants is benificial to improving salt tolerance.


Assuntos
Cucumis sativus/enzimologia , Cucumis sativus/fisiologia , Metionina Adenosiltransferase/metabolismo , Tolerância ao Sal , Antioxidantes/metabolismo , Arabidopsis/metabolismo , Secas , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Peróxido de Hidrogênio/química , Metionina Adenosiltransferase/genética , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Poliaminas/química , Sais , Estresse Mecânico , Estresse Fisiológico/genética , Tabaco/metabolismo
3.
Int J Mol Sci ; 20(4)2019 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-30791389

RESUMO

Transglutaminase (TGase) is a regulator of posttranslational modification of protein that provides physiological protection against diverse environmental stresses in plants. Nonetheless, the mechanisms of TGase-mediated salt tolerance remain largely unknown. Here, we found that the transcription of cucumber TGase (CsTGase) was induced in response to light and during leaf development, and the CsTGase protein was expressed in the chloroplast and the cell wall. The overexpression of the CsTGase gene effectively ameliorated salt-induced photoinhibition in tobacco plants, increased the levels of chloroplast polyamines (PAs) and enhanced the abundance of D1 and D2 proteins. TGase also induced the expression of photosynthesis related genes and remodeling of thylakoids under normal conditions. However, salt stress treatment reduced the photosynthesis rate, PSII and PSI related genes expression, D1 and D2 proteins in wild-type (WT) plants, while these effects were alleviated in CsTGase overexpression plants. Taken together, our results indicate that TGase-dependent PA signaling protects the proteins of thylakoids, which plays a critical role in plant response to salt stress. Thus, overexpression of TGase may be an effective strategy for enhancing resistance to salt stress of salt-sensitive crops in agricultural production.


Assuntos
Cucumis sativus/genética , Expressão Gênica , Tabaco/genética , Transglutaminases/genética , Biomassa , Cloroplastos/genética , Cloroplastos/metabolismo , Cloroplastos/ultraestrutura , Cucumis sativus/enzimologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Imuno-Histoquímica , Fotossíntese , Folhas de Planta/genética , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , Tolerância ao Sal , Estresse Fisiológico , Tilacoides/metabolismo , Tabaco/metabolismo , Transglutaminases/metabolismo
4.
Plant Physiol Biochem ; 136: 118-126, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30660677

RESUMO

Azospirillum brasilense was reported to up-regulate iron (Fe) uptake mechanisms, such as Fe reduction and rhizosphere acidification, in both Fe sufficient and deficient cucumber plants (Cucumis sativus L.). Strategy I plants take up both Fe and copper (Cu) after their reduction mediated by the ferric-chelate reductase oxidase (FRO) enzyme. Interestingly, in cucumber genome only one FRO gene is reported. Thus, in the present study we applied a bioinformatics approach to identify the member of cucumber FRO gene family and allowed the identification of at least three CsFRO genes, one of which was the already identified, i.e. CsFRO1. The expression patterns of the newly identified transcripts were investigated in hydroponically grown cucumber plants treated with different Fe and Cu nutritional regimes. Gene expression was then correlated with morphological (i.e. root architecture) and physiological (Fe(III) reducing activity) parameters to shed light on: i) the CsFRO homologue responsible of the increased reduction activity in Fe-sufficient plants inoculated with A. brasilense cucumber plants, and ii) the possible effect of A. brasilense in ameliorating the symptoms of Cu toxicity in cucumber plants. The data obtained showed that all the CsFRO genes were expressed in the root tissues of cucumber plants and responded to Cu starvation, combined Cu/Fe deficiency and Cu toxicity. Only CsFRO3 was modulated by the A. brasilense in Fe-sufficient plants suggesting for the first time a different specificity of action of the three isoenzymes depending not only on the nutritional regime (either deficiency or toxicity) but also on the presence of the PGPR. Furthermore, results suggest that the PGPR could even ameliorate the stress symptoms caused by both the double (i.e. Cu and Fe) and Cu deficiency as well as Cu toxicity modulating, on one hand, the growth of the root system and, on the other hand, the root nutrient uptake.


Assuntos
Azospirillum brasilense/fisiologia , Cucumis sativus/microbiologia , FMN Redutase/metabolismo , Cobre/deficiência , Cobre/metabolismo , Cucumis sativus/enzimologia , Cucumis sativus/metabolismo , Ferro/deficiência , Ferro/metabolismo , Raízes de Plantas/metabolismo , Reação em Cadeia da Polimerase em Tempo Real
5.
Photochem Photobiol Sci ; 18(2): 424-433, 2019 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-30628617

RESUMO

Expression of cucumber (Cucumis sativus) genes encoding the phenylpropanoid and flavonoid biosynthetic enzymes phenylalanine ammonia lyase (PAL), cinnamic acid 4-hydroxylase (C4H), and chalcone synthase (CHS), was studied under control light conditions (photosynthetically active radiation, PAR) in root, stem, and leaf. Furthermore, the expression was quantified in leaves illuminated with PAR and supplemental ultraviolet-A (315-400 nm) or ultraviolet-B (280-315 nm) radiation. The expression patterns of all twelve CsPAL, three CsC4H, and three CsCHS genes were established. Among the genes regulated by UV two general expression patterns emerge. One pattern applies to genes primarily regulated by enriched UV-A illumination (pattern 1). Another pattern (pattern 2) was found for the genes regulated by enriched UV-B. Three of the pattern 2 genes (CsPAL4, CsPAL10, and CsCHS2) displayed a particular sub-pattern (pattern 2b) with transcription enriched by at least 30-fold. In contrast to the other genes studied, the promoters of the genes regulated according to pattern 2b contained a combination of a number of cis-acting regulatory elements (MREs, ACEs, and G-boxes) that may be of importance for the particularly high enhancement of expression under UV-B-containing light. The regulation of phenylpropanoid and flavonoid biosynthesis genes in cucumber resembles that of a number of other plants. However, cucumber, due to its greater size, is an attractive species for combining more detailed studies of the morphology, physiological parameters and fine regulation of spatial and temporal expression of key genes. This, in turn, can facilitate the quantitative investigation of the relationships among different promoter motifs, the expression levels of each of these three genes, and metabolite accumulation profiles.


Assuntos
Cucumis sativus/genética , Cucumis sativus/efeitos da radiação , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Genes de Plantas/genética , Propanóis/metabolismo , Raios Ultravioleta , Cucumis sativus/enzimologia , Cucumis sativus/metabolismo , Especificidade de Órgãos , Folhas de Planta/genética , Folhas de Planta/efeitos da radiação
6.
Sci Total Environ ; 649: 431-439, 2019 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-30176456

RESUMO

Many pharmaceutical and personal care products (PPCPs) enter agroecosystems during reuse of treated wastewater and biosolids, presenting potential impacts on plant development. Here, acetaminophen, one of the most-used pharmaceuticals, was used to explore roles of glutathione (GSH) conjugation in its biotransformation in crop plants. Acetaminophen was taken up by plants, and conjugated quickly with GSH. After exposure to 5 mg L-1 acetaminophen for 144 h, GSH-acetaminophen conjugates were 15.2 ±â€¯1.3 nmol g-1 and 1.2 ±â€¯0.1 nmol g-1 in cucumber roots and leaves, respectively. Glutathione-acetaminophen was also observed in common bean, alfalfa, tomato, and wheat. Inhibition of cytochrome P450 decreased GSH conjugation. Moreover, the GSH conjugate was found to further convert to cysteine and N-acetylcysteine conjugates. Glutathione S-transferase activity was significantly elevated after exposure to acetaminophen, while levels of GSH decreased by 55.4% in roots after 48 h, followed by a gradual recovery thereafter. Enzymes involved in GSH synthesis, regeneration and transport were consistently induced to maintain the GSH homeostasis. Therefore, GST-mediated conjugation likely played a crucial role in minimizing phytotoxicity of acetaminophen and other PPCPs in plants.


Assuntos
Acetaminofen/metabolismo , Cucumis sativus/metabolismo , Glutationa Transferase/metabolismo , Proteínas de Plantas/metabolismo , Poluentes do Solo/metabolismo , Poluentes Químicos da Água/metabolismo , Analgésicos não Entorpecentes/metabolismo , Antipiréticos/metabolismo , Biodegradação Ambiental , Cucumis sativus/enzimologia , Inativação Metabólica
7.
J Plant Physiol ; 228: 150-157, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29913429

RESUMO

Sucrose phosphate synthases (SPSs) are rate-limiting sucrose synthesis enzymes present in photosynthetic and non-photosynthetic tissues. The cucumber genome contains three SPSs that can be grouped into families A, B, and C. CsSPS1 and CsSPS2 are highly expressed in flowers and mature leaves, while the expression level of CsSPS4 increased gradually after leaf unfolding in our study and reached its peak after 20 days. In CsSPS4-overexpression tobacco plants, sucrose content and sucrose/starch ratio were increased significantly and resulted in improved leaf yield. By contrast, in CsSPS4-overexpression (CsSPS4-OE) cucumber lines, contents of sucrose and starch were unchanged, and raffinose was increased in transgenic cucumber leaves. The expression of cucumber raffinose family oligosaccharide (RFO)-synthesis-related genes increased obviously in cucumber CsSPS4-OE plants, and the sucrose, raffinose, and stachyose contents increased significantly in the petioles of CsSPS4-OE lines. In CsSPS4-antisense (CsSPS4-A) cucumber lines, decreases occurred in mRNA expression, enzyme activity, sucrose content, sucrose/starch ratio, and stachyose transport, but the RFO-synthesis-related genes were nearly unchanged. Together, these results suggest that overexpression of CsSPS4 can lead to carbon metabolism prioritizing sugar transport in cucumber, and suppression of CsSPS4 likely promotes carbon metabolism to accumulate starch, showing a more complicated carbon distribution model than in transgenic tobacco plants.


Assuntos
Carbono/metabolismo , Cucumis sativus/enzimologia , Cucumis sativus/metabolismo , Glucosiltransferases/metabolismo , Oligossacarídeos/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Tabaco/metabolismo , Metabolismo dos Carboidratos/genética , Metabolismo dos Carboidratos/fisiologia , Glucosiltransferases/genética , Plantas Geneticamente Modificadas/genética , Tabaco/genética
8.
Ying Yong Sheng Tai Xue Bao ; 29(3): 899-908, 2018 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-29722233

RESUMO

The effects of 0.2 µmol·L-1 exogenous 2,4-epibrassinolide (EBR) on the growth and reactive oxygen species metabolism of cucumber seedlings ('Jinyan 4' cucumber) under salt-alkaline stress (30 mmol·L-1 NaHCO3) were examined by hydroponics method. The results showed that NaHCO3 stress significantly induced production of O2-· and accumulation of H2O2 in leaves and roots, resulting in the increases of MDA content and electrolyte leakage. Under NaHCO3 stress, activities of superoxide dismutase, peroxidase, catalase, ascorbate peroxidase, dehydroascorbatereductase, monodehydrodroascorbate reductase, and glutathione reductase as well as contents of ascorbic acid and glutathione were firstly increased and then decreased with progress of stress time. Exogenous EBR application significantly increased the activities of antioxidant enzymes and contents of antioxidants as well as the ratio of AsA/DHA (dehydroascorbic acid) and GSH/GSSG (L-glutathione oxidized) in leaves and roots of cucumber under NaHCO3 stress. Such changes improved the redox hemostasis in plants, reduced the level of reactive oxygen species, and alleviated the membrane lipid peroxidation. Together, they increased the alkaline tolerance of cucumber seedlings.


Assuntos
Brassinosteroides/farmacologia , Cucumis sativus/crescimento & desenvolvimento , Esteroides Heterocíclicos/farmacologia , Antioxidantes , Ácido Ascórbico , Catalase , Cucumis sativus/enzimologia , Glutationa , Peróxido de Hidrogênio , Oxirredução , Plântula , Superóxido Dismutase
9.
J Plant Physiol ; 224-225: 112-120, 2018 May - Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29617631

RESUMO

Raffinose family oligosaccharides (RFOs) accumulate under stress conditions in many plants and have been suggested to act as stress protectants. To elucidate the metabolic process of RFOs under cold stress, levels of RFOs, and related carbohydrates, the expression and activities of main metabolic enzymes and their subcellular compartments were investigated during low-temperature treatment and during the recovery period in cucumber leaves. Cold stress induced the accumulation of stachyose in vacuoles, galactinol in vacuoles and cytosol, and sucrose and raffinose in vacuoles, cytosol, and chloroplasts. After cold stress removal, levels of these sugars decreased gradually in the respective compartments. Among four galactinol synthase genes (CsGS), CsGS1 was not affected by cold stress, while the other three CsGSs were up-regulated by low temperature. RNA levels of acid-α-galactosidase (GAL) 3 and alkaline-α-galactosidase (AGA) 2 and 3, and the activities of GAL and AGA, were up-regulated after cold stress removal. GAL3 protein and GAL activity were exclusively located in vacuoles, whereas AGA2 and AGA 3 proteins were found in cytosol and chloroplasts, respectively. The results indicate that RFOs, which accumulated during cold stress in different subcellular compartments in cucumber leaves, could be catabolized in situ by different galactosidases after stress removal.


Assuntos
Temperatura Baixa , Cucumis sativus/fisiologia , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Rafinose/metabolismo , alfa-Galactosidase/genética , Cucumis sativus/enzimologia , Folhas de Planta/enzimologia , Folhas de Planta/fisiologia , Proteínas de Plantas/metabolismo , Estresse Fisiológico , alfa-Galactosidase/metabolismo
10.
Theor Appl Genet ; 131(6): 1379-1389, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29541828

RESUMO

KEY MESSAGE: Two round-leaf mutants, rl-1 and rl-2, were identified from EMS-induced mutagenesis. High throughput sequencing and map-based cloning suggested CsPID encoding a Ser/Thr protein kinase as the most possible candidate for rl-1. Rl-2 was allelic to Rl-1. Leaf shape is an important plant architecture trait that is affected by plant hormones, especially auxin. In Arabidopsis, PINOID (PID), a regulator for the auxin polar transporter PIN (PIN-FORMED) affects leaf shape formation, but this function of PID in crop plants has not been well studied. From an EMS mutagenesis population, we identified two round-leaf (rl) mutants, C356 and C949. Segregation analysis suggested that both mutations were controlled by single recessive genes, rl-1 and rl-2, respectively. With map-based cloning, we show that CsPID as the candidate gene of rl-1; a non-synonymous SNP in the second exon of CsPID resulted in an amino acid substitution and the round leaf phenotype. As compared in the wild type plant, CsPID had significantly lower expression in the root, leaf and female flowers in C356, which may result in the less developed roots, round leaves and abnormal female flowers, respectively in the rl-1 mutant. Among the three copies of PID genes, CsPID, CsPID2 and CSPID2L (CsPID2-like) in the cucumber genome, CsPID was the only one with significantly differential expression in adult leaves between WT and C356 suggesting CsPID plays a main role in leaf shape formation. The rl-2 mutation in C949 was also cloned, which was due to another SNP in a nearby location of rl-1 in the same CsPID gene. The two round leaf mutants and the work presented herein provide a good foundation for understanding the molecular mechanisms of CsPID in cucumber leaf development.


Assuntos
Cucumis sativus/genética , Genes de Plantas , Folhas de Planta/crescimento & desenvolvimento , Proteínas Serina-Treonina Quinases/genética , Alelos , Mapeamento Cromossômico , Clonagem Molecular , Cucumis sativus/enzimologia , Genes Recessivos , Ligação Genética , Mutação , Fenótipo , Filogenia , Folhas de Planta/genética , Polimorfismo de Nucleotídeo Único
11.
Ecotoxicol Environ Saf ; 154: 171-179, 2018 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-29471279

RESUMO

During its life cycle, plant has to cope with a number of abiotic stresses including cadmium stress. Cadmium (Cd) is highly toxic to plant and greatly influences its growth and entire metabolism. Antioxidants have to enable plant to beat such stresses. Therefore, effects of ascorbate (AsA), proline (Pro) and glutathione (GSH) applied, as seed soaking solutions, singly or in a sequence on cucumber transplant growth, physio-biochemical attributes and antioxidant defense system activity were investigated under 2 mM Cd stress. Adding Cd to transplants in irrigation water reduced photosynthetic efficiency, and nutrient (K+ and Ca2+) contents, while increased the activity of defense systems (non-enzymatic and enzymatic antioxidants) and Cd2+ contents in roots and leaves. Exogenous AsA, Pro and GSH applied singly or in a sequence improved transplant growth (e.g., shoot length, leaf area, shoot fresh weight and shoot dry weight), photosynthetic efficiency (i.e., SPAD chlorophyll, Fv/Fm and PI), transplant health (i.e., increased leaf MSI and RWC, and decreased root and leaf Cd2+ contents), antioxidant defense systems activity (enzymatic; superoxide dismutase, catalase, glutathione reductase and ascorbate peroxidase, and non-enzymatic; Pro, AsA and GSH antioxidants) and nutrient (K+ and Ca2+) contents. These positive results were obtained under irrigation with or without Cd, AsA. Sequenced AsA-Pro-GSH was the best treatment of which this study recommends to use, followed by GSH treatment, for growing cucumber transplants under Cd stress.


Assuntos
Antioxidantes/farmacologia , Ácido Ascórbico/farmacologia , Cádmio/toxicidade , Cucumis sativus/efeitos dos fármacos , Glutationa/farmacologia , Prolina/farmacologia , Ascorbato Peroxidases/metabolismo , Cádmio/metabolismo , Catalase/metabolismo , Clorofila/metabolismo , Cucumis sativus/enzimologia , Cucumis sativus/crescimento & desenvolvimento , Cucumis sativus/metabolismo , Glutationa Redutase/metabolismo , Fotossíntese/efeitos dos fármacos , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Sementes/efeitos dos fármacos , Sementes/metabolismo , Estresse Fisiológico , Superóxido Dismutase/metabolismo
12.
Methods Mol Biol ; 1696: 205-215, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29086406

RESUMO

Plasma membrane (PM) H+-ATPase, which generates the proton gradient across the outer membrane of plant cells, plays a fundamental role in the regulation of many physiological processes fundamental for growth and development of plants. It is involved in the uptake of nutrients from external solutions, their loading into phloem and long-distance transport, stomata aperture and gas exchange, pH homeostasis in cytosol, cell wall loosening, and cell expansion. The crucial role of the enzyme in resistance of plants to abiotic and biotic stress factors has also been well documented. Such great diversity of physiological functions linked to the activity of one enzyme requires a suitable and complex regulation of H+-ATPase. This regulation comprises the transcriptional as well as post-transcriptional levels. Herein, we describe the techniques that can be useful for the analysis of the plasma membrane proton pump modifications at genetic and protein levels under environmental factors.


Assuntos
Membrana Celular/enzimologia , Cucumis sativus/citologia , ATPases Translocadoras de Prótons/genética , Estresse Fisiológico , Fracionamento Celular , Membrana Celular/genética , Células Cultivadas , Cucumis sativus/enzimologia , Cucumis sativus/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Processamento Pós-Transcricional do RNA
13.
Plant Sci ; 264: 37-47, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28969801

RESUMO

The present research aim was to define the role of brassinosteroids (BRs) in plant adaptation to cadmium stress. We observed a stimulating effect of exogenous BR on the activity of two plasma membrane enzymes which play a key role in plants adaptation to cadmium stress, H+-ATPase (EC 3.6.3.14) and NADPH oxidase (EC 1.6.3.1). Using anti-phosphothreonine antibody we showed that modification of PM H+-ATPase activity under BR action could result from phosphorylation of the enzyme protein. Also the relative expression of genes encoding both PM H+-ATPase and NADPH oxidase was affected by BR. To confirm the role of BR in the cadmium stimulating effect on activity of both studied plasma membrane enzymes, an assay in the presence of a BR biosynthesis inhibitor (propiconazole) was performed. Moreover, as a tool in our work we used commercially available plant mutants unable to BR biosynthesis or with dysfunctional BR signaling pathway, to further confirm participation of BR in plant adaptation to heavy metal stress. Presented results demonstrate some elements of the brassinosteroid-induced pathway activated under cadmium stress, wherein H+-ATPase and NADPH oxidase are key factors.


Assuntos
Brassinosteroides/metabolismo , Cádmio/toxicidade , Cucumis sativus/fisiologia , Regulação da Expressão Gênica de Plantas , NADPH Oxidases/efeitos dos fármacos , ATPases Translocadoras de Prótons/efeitos dos fármacos , Adaptação Fisiológica , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Cucumis sativus/efeitos dos fármacos , Cucumis sativus/enzimologia , Cucumis sativus/genética , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , ATPases Translocadoras de Prótons/genética , ATPases Translocadoras de Prótons/metabolismo , Transdução de Sinais , Estresse Fisiológico
14.
Anal Bioanal Chem ; 409(24): 5645-5654, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28730304

RESUMO

A recombinant ribonuclease, cusativin, was characterized for its cytidine-specific cleavage ability of RNA to map chemical modifications. Following purification of native cusativin protein as described before (Rojo et al. Planta 194:328, 17), partial amino acid sequencing was carried out to identify the corresponding protein coding gene in cucumber genome. Cloning and heterologous expression of the identified gene in Escherichia coli resulted in successful production of active protein as a C-terminal His-tag fusion protein. The ribonuclease activity and cleavage specificity of the fusion protein were confirmed with a variety of tRNA isoacceptors and total tRNA. Characterization of cusativin digestion products by ion-pairing reverse-phase liquid chromatography coupled with mass spectrometry (IP-RP-LC-MS) analysis revealed cleavage of CpA, CpG, and CpU phosphodiester bonds at the 3'-terminus of cytidine under optimal digestion conditions. Ribose methylation or acetylation of cytosine inhibited RNA cleavage. The CpC phosphodiester bond was also resistant to cusativin-mediated RNA cleavage; a feature to our knowledge has not been reported for other nucleobase-specific ribonucleases. Here, we demonstrate the analytical utility of such a novel feature for obtaining high-sequence coverage and accurate mapping of modified residues in substrate RNAs. Graphical abstract Cytidine-specific novel ribonuclease activity of cusativin.


Assuntos
Cucumis sativus/enzimologia , Citidina/metabolismo , Endorribonucleases/metabolismo , RNA de Transferência/metabolismo , Ribonucleases/metabolismo , Acetilação , Sequência de Bases , Cucumis sativus/metabolismo , Citidina/análise , Clivagem do RNA , RNA de Transferência/química
15.
Theor Appl Genet ; 130(8): 1549-1558, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28466109

RESUMO

KEY MESSAGE: Next-generation sequencing-aided map-based cloning delimited the cucumber tendril - less1 ( td - 1 ) locus into a 190.7-kb region in chromosome 6 harboring a putative, novel-function candidate gene encoding a histone acetyltransferase ( CsGCN5 ). The tendril initiated from the lateral meristem is an important and characteristic organ for the species in the Cucurbitaceae family including cucumber (Cucumis sativus L.). While the tendril has its evolutionary significance, it also poses a nuisance in cucumber cultivation under protected environments in which tendril-less cucumber has its advantages. From an EMS mutagenesis population, we identified a tendril-less mutant B007, which was controlled by a recessive gene td-1. Through next-generation sequencing-aided map-based cloning, we show CsGCN5 (Cucumis sativus GENERAL CONTROL NONDEREPRESSIBLE 5), a cucumber gene for a histone acetyltransferase as the most possible candidate for td-1. A non-synonymous SNP in the first exon of CsGCN5 resulted in an amino-acid substitution from Asp (D) in the wild type to Asn (N) in the tendril-less mutant. The candidacy of CsGCN5 was further confirmed by multiple lines of evidence in both biparental and natural cucumber populations. Non-significant expression of CsGCN5 in multiple organs was found between the wild type and the mutant. CsGCN5 exhibited strong expression in the tendril of wild-type plants suggesting its important roles in growth and development of plant tendrils. The identification and characterization of the td-1 mutant from the present study provided a useful tool in understanding the molecular mechanisms of tendril organogenesis and investigation of novel functions of the histone acetyltransferase in cucumber.


Assuntos
Cucumis sativus/genética , Genes Recessivos , Histona Acetiltransferases/genética , Mapeamento Cromossômico , Clonagem Molecular , Cucumis sativus/enzimologia , DNA de Plantas/genética , Genes de Plantas , Ligação Genética , Filogenia , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA
16.
Physiol Plant ; 161(2): 224-234, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28543370

RESUMO

Rubisco activase (RCA) is an important enzyme that can catalyze the carboxylation and oxygenation activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), which is involved in the photosynthetic carbon reduction cycle. Here, we studied the effects of changes in RCA activity on photosynthesis, growth and development, as well as the low temperature and weak light tolerance of RCA overexpressing transgenic cucumber (Cucumis sativus) plants. CsRCA overexpression increased the plant height, leaf area and dry matter, and decreased the root/top ratio in transgenic cucumber plants compared with the wild-type (WT) plants. Low temperature and low light stress led to decreases in the CsRCA expression and protein levels, the photosynthetic rate (Pn) and the stomatal conductance (Gs), but an increase in the intercellular CO2 (Ci) concentration in cucumber leaves. The actual photochemical efficiency and maximal photochemical efficiency of photosystem II in cucumber seedlings also declined, but the initial fluorescence increased during low temperature and weak light stress. Transgenic plants showed a lower decrease in the CsRCA expression level and actual and maximal photochemical efficiencies, as well as increases in the Ci and initial fluorescence relative to the WT plants. Low temperature and low light stress resulted in a significant increase in the malondialdehyde (MDA) content; however, this increase was reduced in transgenic plants compared with that in WT plants. Thus, the overexpression of CsRCA may promote the growth and low temperature and low light tolerance of cucumber plants in solar greenhouses.


Assuntos
Aclimatação , Cucumis sativus/enzimologia , Fotossíntese , Proteínas de Plantas/metabolismo , Metabolismo dos Carboidratos , Temperatura Baixa , Cucumis sativus/genética , Cucumis sativus/crescimento & desenvolvimento , Cucumis sativus/efeitos da radiação , Luz , Folhas de Planta/enzimologia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Ribulose-Bifosfato Carboxilase/metabolismo
17.
Theor Appl Genet ; 130(8): 1693-1703, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28516384

RESUMO

KEY MESSAGE: A novel dwarf cucumber mutant, scp-2, displays a typical BR biosynthesis-deficient phenotype, which is due to a mutation in CsDET2 for a steroid 5-alpha-reductase. Brassinosteroids (BRs) are a group of plant hormones that play important roles in the development of plant architecture, and extreme dwarfism is a typical outcome of BR-deficiency. Most cucumber (Cucumis sativus L.) varieties have an indeterminate growth habit, and dwarfism may have its value in manipulation of plant architecture and improve production in certain production systems. In this study, we identified a spontaneous dwarf mutant, super compact-2 (scp-2), that also has dark green, wrinkle leaves. Genetic analyses indicated that scp-2 was different from two previously reported dwarf mutants: compact (cp) and super compact-1 (scp-1). Map-based cloning revealed that the mutant phenotype was due to two single nucleotide polymorphism and a single-base insertion in the CsDET2 gene that resulted in a missense mutation in a conserved amino acid and thus a truncated protein lacking the conserved catalytic domains in the predicted steroid 5α-reductase protein. Measurement of endogenous hormone levels indicated a reduced level of brassinolide (BL, a bioactive BR) in scp-2, and the mutant phenotype could be partially rescued by the application of epibrassinolide (EBR). In addition, scp-2 mutant seedlings exhibited dark-grown de-etiolation, and defects in cell elongation and vascular development. These data support that scp-2 is a BR biosynthesis-deficient mutant, and that the CsDET2 gene plays a key role in BR biosynthesis in cucumber. We also described the systemic BR responses and discussed the specific BR-related phenotypes in cucumber plants.


Assuntos
3-Oxo-5-alfa-Esteroide 4-Desidrogenase/genética , Cucumis sativus/genética , Proteínas de Plantas/genética , Sequência de Aminoácidos , Brassinosteroides/farmacologia , Mapeamento Cromossômico , Cucumis sativus/enzimologia , Cucumis sativus/crescimento & desenvolvimento , Genes de Plantas , Fenótipo , Esteroides Heterocíclicos/farmacologia
18.
Plant Physiol Biochem ; 108: 323-327, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27497301

RESUMO

This study determined whether phosphoenolpyruvate carboxykinase (PEPCK) and phosphoenolpyruvate carboxylase (PEPC) are phosphorylated in the flesh of a range of fruits. This was done by incubating fruit flesh with 32P[P] (where 32P[P] = 32PO43-), then PEPCK and PEPC were immunoprecipitated from extracts using specific antisera. The incorporation of 32P[P] into these enzymes was then determined by autoradiography of SDS-PAGE gels. Both enzymes were subject to phosphorylation in vivo in the flesh of grape, tomato, cherry and plum. PEPCK was also subject to phosphorylation in vivo in developing grape seeds. Proteolytic cleavage of PEPCK showed that it was phosphorylated at a site(s) located on its N-terminal extension. Potentially phosphorylation of these enzymes could contribute to the coordinate regulation of their activities in the flesh of fruits and in developing seeds.


Assuntos
Frutas/enzimologia , Magnoliopsida/enzimologia , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Fosfoenolpiruvato Carboxilase/metabolismo , Proteínas de Plantas/metabolismo , Cucumis sativus/enzimologia , Cucumis sativus/metabolismo , Frutas/metabolismo , Lycopersicon esculentum/enzimologia , Lycopersicon esculentum/metabolismo , Magnoliopsida/metabolismo , Radioisótopos de Fósforo/farmacocinética , Fosforilação , Prunus/enzimologia , Prunus/metabolismo , Sementes/enzimologia , Sementes/crescimento & desenvolvimento , Distribuição Tecidual , Vitis/enzimologia , Vitis/metabolismo
19.
Theor Appl Genet ; 129(10): 1961-73, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27435733

RESUMO

KEY MESSAGE: The cucumber chlorophyll-deficient golden leaf mutation is due to a single nucleotide substitution in the CsChlI gene for magnesium chelatase I subunit which plays important roles in the chlorophyll biosynthesis pathway. The Mg-chelatase catalyzes the insertion of Mg(2+) into the protoporphyrin IX in the chlorophyll biosynthesis pathway, which is a protein complex encompassing three subunits CHLI, CHLD, and CHLH. Chlorophyll-deficient mutations in genes encoding the three subunits have played important roles in understanding the structure, function and regulation of this important enzyme. In an EMS mutagenesis population, we identified a chlorophyll-deficient mutant C528 with golden leaf color throughout its development which was viable and able to set fruits and seeds. Segregation analysis in multiple populations indicated that this leaf color mutation was recessively inherited and the green color showed complete dominance over golden color. Map-based cloning identified CsChlI as the candidate gene for this mutation which encoded the CHLI subunit of cucumber Mg-chelatase. The 1757-bp CsChlI gene had three exons and a single nucleotide change (G to A) in its third exon resulted in an amino acid substitution (G269R) and the golden leaf color in C528. This mutation occurred in the highly conserved nucleotide-binding domain of the CHLI protein in which chlorophyll-deficient mutations have been frequently identified. The mutant phenotype, CsChlI expression pattern and the mutated residue in the CHLI protein suggested the mutant allele in C528 is unique among mutations identified so far in different species. This golden leaf mutant not only has its potential in cucumber breeding, but also provides a useful tool in understanding the CHLI function and its regulation in the chlorophyll biosynthesis pathway as well as chloroplast development.


Assuntos
Clorofila/deficiência , Cucumis sativus/genética , Liases/genética , Sequência de Aminoácidos , Mapeamento Cromossômico , Cucumis sativus/enzimologia , DNA de Plantas/genética , Éxons , Mutação , Nucleotídeos/genética , Fenótipo , Filogenia , Folhas de Planta/genética , Alinhamento de Sequência , Análise de Sequência de DNA
20.
Mol Plant ; 9(9): 1315-1327, 2016 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-27403533

RESUMO

Sex determination in plants gives rise to unisexual flowers that facilitate outcrossing and enhance genetic diversity. In cucumber and melon, ethylene promotes carpel development and arrests stamen development. Five sex-determination genes have been identified, including four encoding 1-aminocyclopropane-1-carboxylate (ACC) synthase that catalyzes the rate-limiting step in ethylene biosynthesis, and a transcription factor gene CmWIP1 that corresponds to the Mendelian locus gynoecious in melon and is a negative regulator of femaleness. ACC oxidase (ACO) converts ACC into ethylene; however, it remains elusive which ACO gene in the cucumber genome is critical for sex determination and how CmWIP1 represses development of female flowers. In this study, we discovered that mutation in an ACO gene, CsACO2, confers androecy in cucumber that bears only male flowers. The mutation disrupts the enzymatic activity of CsACO2, resulting in 50% less ethylene emission from shoot tips. CsACO2 was expressed in the carpel primordia and its expression overlapped with that of CsACS11 in female flowers at key stages for sex determination, presumably providing sufficient ethylene required for proper CsACS2 expression. CmACO3, the ortholog of CsACO2, showed a similar expression pattern in the carpel region, suggesting a conserved function of CsACO2/CmACO3. We demonstrated that CsWIP1, the ortholog of CmWIP1, could directly bind the promoter of CsACO2 and repress its expression. Taken together, we propose a presumably conserved regulatory module consisting of WIP1 transcription factor and ACO controls unisexual flower development in cucumber and melon.


Assuntos
Aminoácido Oxirredutases/metabolismo , Cucumis sativus/enzimologia , Cucumis sativus/metabolismo , Flores/enzimologia , Flores/metabolismo , Proteínas de Plantas/metabolismo , Aminoácido Oxirredutases/genética , Cucumis sativus/genética , Etilenos/metabolismo , Flores/genética , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Proteínas de Plantas/genética , Regiões Promotoras Genéticas/genética
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