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1.
BMC Plant Biol ; 19(1): 308, 2019 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-31299895

RESUMO

BACKGROUND: Land preparation is an important component of fragrant rice production. However, the effect of tillage on fragrant rice production is unclear, especially regarding the biosynthesis of 2-acetyl-1-pyrroline (2-AP), which is the main compound of the unique aroma of fragrant rice. This study aimed to explore 2-AP biosynthesis in fragrant rice under different tillage regimes. Three tillage methods were applied in the present study: conventional rotary tillage (CK) as the control, plough tillage (PT), and no-tillage (NT). RESULT: Compared with CK, the PT treatment increased 2-AP content in grain, upregulated the activity of ornithine aminotransferase (OAT) and increased contents of 1-pyrroline and pyrroline-5-carboxylic acid (P5C). Furthermore, the PT treatment increased the grain yield and nitrogen accumulation of fragrant rice. Meanwhile, the 2-AP content in the grain produced under the NT treatment was significantly higher than that in the grain produced under both the PT and CK treatments due to the enhancement of proline content and the activities of proline dehydrogenase (PDH) and △1-pyrroline-5-carboxylic acid synthetase (P5CS). However, the present study observed that the overall production of fragrant rice under NT conditions was inferior due to lower yield, nitrogen accumulation, and anti-oxidative enzymatic activities. Moreover, the organic matter content and soil microorganism quantity increased due to PT and NT treatments. CONCLUSIONS: Compared to CK, PT improved fragrant rice grain yield and nitrogen accumulation and induced an increase in OAT activity and led to an increase in 2-AP concentration. No-tillage also produced increased 2-AP content in grain by enhancing PDH and P5CS activities but limited yields and nitrogen accumulation in fragrant rice.


Assuntos
Oryza/fisiologia , Prolina Oxidase/metabolismo , Pirróis/metabolismo , Odorantes , Oryza/enzimologia , Oryza/genética , Oryza/crescimento & desenvolvimento , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Prolina/análise , Prolina Oxidase/genética , Sementes/enzimologia , Sementes/genética , Sementes/crescimento & desenvolvimento , Sementes/fisiologia
2.
Plant Mol Biol ; 101(1-2): 183-202, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31286324

RESUMO

KEY MESSAGE: Isoforms of 2-OGDH E1 subunit are not functionally redundant in plant growth and development of A. thaliana. The tricarboxylic acid cycle enzyme 2-oxoglutarate dehydrogenase (2-OGDH) converts 2-oxoglutarate (2-OG) to succinyl-CoA concomitant with the reduction of NAD+. 2-OGDH has an essential role in plant metabolism, being both a limiting step during mitochondrial respiration as well as a key player in carbon-nitrogen interactions. In Arabidopsis thaliana two genes encode for E1 subunit of 2-OGDH but the physiological roles of each isoform remain unknown. Thus, in the present study we isolated Arabidopsis T-DNA insertion knockout mutant lines for each of the genes encoding the E1 subunit of 2-OGDH enzyme. All mutant plants exhibited substantial reduction in both respiration and CO2 assimilation rates. Furthermore, mutant lines exhibited reduced levels of chlorophylls and nitrate, increased levels of sucrose, malate and fumarate and minor changes in total protein and starch levels in leaves. Despite the similar metabolic phenotypes for the two E1 isoforms the reduction in the expression of each gene culminated in different responses in terms of plant growth and seed production indicating distinct roles for each isoform. Collectively, our results demonstrated the importance of the E1 subunit of 2-OGDH in both autotrophic and heterotrophic tissues and suggest that the two E1 isoforms are not functionally redundant in terms of plant growth in A. thaliana.


Assuntos
Arabidopsis/enzimologia , Carbono/metabolismo , Complexo Cetoglutarato Desidrogenase/metabolismo , Nitrogênio/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Dióxido de Carbono/metabolismo , Clorofila/metabolismo , Complexo Cetoglutarato Desidrogenase/genética , Mitocôndrias/enzimologia , Mutagênese Insercional , Nitratos/metabolismo , Fenótipo , Filogenia , Folhas de Planta/enzimologia , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Isoformas de Proteínas , Subunidades Proteicas , Plântula/enzimologia , Plântula/genética , Plântula/crescimento & desenvolvimento , Sementes/enzimologia , Sementes/genética , Sementes/crescimento & desenvolvimento
3.
Plant Sci ; 283: 195-201, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31128689

RESUMO

Methionine and threonine are two essential amino acids whose low levels limit the nutritional quality of seeds. The current objective was to define factors that regulate and might increase their levels in seeds. Feeding experiments carried out on receptacles of developing tobacco (Nicotiana tabacum) capsules showed that 1 mM of S-methylmethionine increased the level of methionine to contents similar to 2.5 mM of homoserine, an intermediate metabolite of the aspartate family of amino acids. The latter also increased the level of threonine. Based on these findings, we generated tobacco seeds that expressed a combination of bacterial feedback-insensitive aspartate kinase (bAK), which was previously reported to have a high level of threonine/methionine, and feedback-insensitive cystathionine γ-synthase (CGS), the regulatory enzyme of the methionine biosynthesis pathway. Plants expressing this latter gene previously showed having higher levels of methionine. The results of total amino acids analysis showed that the level of threonine was highest in the bAK line, which has moderate levels of methionine and lysine, while the highest level of methionine was found in seeds expressing both heterologous genes. The results suggest that the level of threonine in tobacco seeds is limited by the substrate, while that of methionine is limited also by the activity of CGS.


Assuntos
Carbono-Oxigênio Liases/metabolismo , Metionina/metabolismo , Proteínas de Plantas/metabolismo , Sementes/metabolismo , Treonina/metabolismo , Tabaco/metabolismo , Aminoácidos/metabolismo , Homosserina/metabolismo , Redes e Vias Metabólicas , Plantas Geneticamente Modificadas , Sementes/enzimologia , Tabaco/enzimologia
4.
J Agric Food Chem ; 67(22): 6432-6444, 2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-31095381

RESUMO

Liquid feeding strategies have been devised with the aim of enhancing grain nutrient availability for livestock. It is characterized by a steeping/soaking period that softens the grains and initiates mobilization of seed storage reserves. The present study uses 2D gel-based proteomics to investigate the role of proteolysis and reduction by thioredoxins over a 48 h steeping period by monitoring protein abundance dynamics in barley-based liquid feed samples supplemented with either protease inhibitors or NADPH-dependent thioredoxin reductase/thioredoxin (NTR/Trx). Several full-length storage proteins were only identified in the water-extractable fraction of feed containing protease inhibitors, illustrating significant inhibition of proteolytic activities arising during the steeping period. Application of functional NTR/Trx to liquid feed reductively increased the solubility of known and potentially new Trx-target proteins, e.g., outer membrane protein X, and their susceptibility to proteolysis. Thus, the NTR/Trx system exhibits important potential as a feed additive to enhance nutrient digestibility in monogastric animals.


Assuntos
Ração Animal/análise , Hordeum/enzimologia , Proteínas de Plantas/química , Tiorredoxina Dissulfeto Redutase/química , Tiorredoxinas/química , Eletroforese em Gel Bidimensional , Manipulação de Alimentos , Hordeum/química , Extratos Vegetais/química , Extratos Vegetais/metabolismo , Proteínas de Plantas/metabolismo , Inibidores de Proteases/química , Inibidores de Proteases/metabolismo , Proteômica , Sementes/química , Sementes/enzimologia , Tiorredoxina Dissulfeto Redutase/metabolismo , Tiorredoxinas/metabolismo
5.
DNA Cell Biol ; 38(6): 510-520, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31017480

RESUMO

Plants, being sessile organisms, have evolved several dynamic mechanisms of gene regulation. Epigenetic modification especially cytosine methylation and demethylation actively regulates the expression of genes. To understand the role of cytosine methylation during isoflavonoid biosynthesis and accumulation, we performed cytosine methylation analysis in the coding region of two isoforms IFS1 and IFS2 gene, in two contrasting soybean genotypes differing in total isoflavone content (NRC37: high isoflavone; and NRC7: low isoflavone). The results indicated increased 5-mC in both the isoforms in NRC37 (∼20.51% in IFS2 and ∼85% in IFS1) compared with NRC7 (∼7.8% in IFS2 and ∼2.5% in IFS1) genotype, which signifies the positive role of 5-mC in the coding region of the gene leading to enhanced expression. In addition, temporal expression profiling [35 days after flowering (DAF), 45, 55, and 65 DAF] of both the isoforms showed increasing trend of accumulation in both the genotypes with maximum in NRC37 at 65 DAF. To further establish a correlation between methylation and expression of transcripts, we quantified the different isoforms of isoflavone in both the genotypes across all the stages. Therefore, the finding of this study would certainly increase our understanding of epigenetic regulation of isoflavone biosynthetic pathway mediated by the cytosine methylation that would assist molecular breeders to get high-performing soybean genotypes with better isoflavone yield.


Assuntos
Citosina/metabolismo , Metilação de DNA , Regulação da Expressão Gênica de Plantas , Oxigenases/genética , Soja/genética , Simulação por Computador , Genótipo , Isoenzimas/genética , Isoenzimas/metabolismo , Isoflavonas/biossíntese , Oxigenases/metabolismo , Sementes/enzimologia , Sementes/genética , Sementes/crescimento & desenvolvimento , Soja/embriologia , Soja/enzimologia
6.
J Sci Food Agric ; 99(11): 5176-5186, 2019 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-31021402

RESUMO

BACKGROUND: The literature on the role of calcium ion (Ca2+ ) in relation to phenolic compounds metabolism and related enzymes activities remains controversial. It is still unclear whether Ca2+ affects phenolic compounds content of barley sprouts. This study investigated the role and function of Ca2+ in phenolic compound metabolism of barley (Hordeum vulgare L.) sprouts under sodium chloride (NaCl) stress. RESULTS: Calcium chloride (CaCl2 ) significantly improved total calcium and calmodulin (CaM) contents as well as Ca2+ concentration, and enhanced phenolic compound accumulation by regulating the gene, protein expression and the activities of enzymes related to phenolics biosynthesis. Specifically, CaCl2 significantly increased the activities of phenylalanine ammonia lyase (PAL), cinnamic acid 4-hydroxylase (C4H), 4-coumarate coenzyme A ligase (4CL) and ferulic acid 5-hydroxylase (F5H) by up-regulating the corresponding protein expression. The activity of p-coumaric acid 3-hydroxylase (C3H) decreased during germination while caffeic acid O-methyltransferase (COMT) increased initially and then decreased, which was consistent with the changes in gene and protein expression under CaCl2 treatment. Conversely, lanthanum(III) chloride (LaCl3 ), ethylene glycol tetraacetic acid (EGTA) and 2-aminoethoxydiphenyl borate (2-APB) induced opposite effects. Decreased calcium and CaM contents and Ca2+ concentration were observed, and fluctuation change of relevant gene and protein expressions and PAL, C4H, 4CL, C3H, COMT and F5H activitives were also detected. CONCLUSION: Calcium ion played an important role for mediating NaCl stress-induced phenolics accumulation in barley sprouts. It required both Ca2+ influx and release from apoplast and intracellular stores, respectively. © 2019 Society of Chemical Industry.


Assuntos
Hordeum/metabolismo , Fenóis/metabolismo , Sementes/crescimento & desenvolvimento , Cloreto de Sódio/metabolismo , Cloreto de Cálcio/metabolismo , Coenzima A Ligases/metabolismo , Germinação , Hordeum/enzimologia , Hordeum/crescimento & desenvolvimento , Metiltransferases/metabolismo , Sementes/enzimologia , Sementes/metabolismo
7.
J Food Sci ; 84(5): 946-953, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30947368

RESUMO

Germination of wheat maximizes phytochemical content and antioxidant activity while altering chemical composition, gluten content, and pasting properties. This study investigated the effect of short-term imbibition on gene expression profiles and the physical and functional characteristics of wheat. Changes in gene expression profiles of wheat during short-term imbibition (0, 16, and 24 hr) were evaluated by DNA microarray analysis. Gene Ontology (GO) analysis was carried out to categorize the function of genes with altered expression. Genes related to cellulose and cell wall synthesis were upregulated by imbibition for 16 hr, whereas those associated with polysaccharide catabolism and nucleosome assembly were upregulated in the subsequent 8 hr. The genes related to proteases and gluten were expressed in dry seeds but disappeared after 16 hr of imbibition. Genes encoding α-amylase were not expressed in dry seeds whereas those encoding ß-amylase were expressed in dry seeds and downregulated by imbibition. According to quantitative real-time PCR and enzymatic activity assay, α-Amylase expression increased by imbibition and reached a maximum 24 hr after imbibition, with a corresponding increase in enzymatic activity. Pasting properties of flour made from wheat seeds imbibed for different times were decreased when seeds were imbibed for over 16 hr, by examination with Rapid Visco Analyzer. Gluten content did not significantly change until 24-hr imbibition, although expression of genes encoding gliadin and glutenin disappeared by 16-hr imbibition. The data indicated that it was possible to use 16-hr imbibed wheat, with up to the 50% w/w replacement of nonimbibed wheat.


Assuntos
Farinha/análise , Regulação da Expressão Gênica de Plantas , Germinação/genética , Sementes , Triticum/genética , Água , alfa-Amilases/metabolismo , Antioxidantes , Grão Comestível , Qualidade dos Alimentos , Perfilação da Expressão Gênica , Genes de Plantas , Gliadina/metabolismo , Glutens/análise , Glutens/metabolismo , Humanos , Análise em Microsséries , Plântula/metabolismo , Sementes/enzimologia , Triticum/enzimologia , Triticum/metabolismo
8.
Planta ; 249(6): 1823-1836, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30847571

RESUMO

MAIN CONCLUSION: The enzymes HaKCS1 and HaKCS2 are expressed in sunflower seeds and contribute to elongation of C18 fatty acids, resulting in the C20-C24 fatty acids in sunflower oil. Most plant fatty acids are produced by plastidial soluble fatty acid synthases that produce fatty acids of up to 18 carbon atoms. However, further acyl chain elongations can take place in the endoplasmic reticulum, catalysed by membrane-bound synthases that act on acyl-CoAs. The condensing enzymes of these complexes are the ketoacyl-CoA synthase (KCSs), responsible for the synthesis of very long chain fatty acids (VLCFAs) and their derivatives in plants, these including waxes and cuticle hydrocarbons, as well as fatty aldehydes. Sunflower seeds accumulate oil that contains around 2-3% of VLCFAs and studies of the fatty acid elongase activity in developing sunflower embryos indicate that two different KCS isoforms drive the synthesis of these fatty acids. Here, two cDNAs encoding distinct KCSs were amplified from RNAs extracted from developing sunflower embryos and named HaKCS1 and HaKCS2. These genes are expressed in developing seeds during the period of oil accumulation and they are clear candidates to condition sunflower oil synthesis. These two KCS cDNAs complement a yeast elongase null mutant and when expressed in yeast, they alter the host's fatty acid profile, proving the encoded KCSs are functional. The structure of these enzymes was modelled and their contribution to the presence of VLCFAs in sunflower oil is discussed based on the results obtained.


Assuntos
Acetiltransferases/metabolismo , Helianthus/enzimologia , Modelos Estruturais , Óleo de Girassol/metabolismo , Acetiltransferases/química , Acetiltransferases/genética , Acil Coenzima A/metabolismo , Aldeídos/metabolismo , Sequência de Aminoácidos , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , DNA Complementar/genética , Ácido Graxo Sintases/química , Ácido Graxo Sintases/genética , Ácido Graxo Sintases/metabolismo , Ácidos Graxos/metabolismo , Helianthus/genética , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sementes/enzimologia , Sementes/genética , Alinhamento de Sequência
9.
Plant Sci ; 280: 408-415, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30824019

RESUMO

Using various inhibitors and scavengers we took advantage of the size of sunflower (Helianthus annuus) seeds to investigate in vivo the effects of hormones, namely abscisic acid (ABA) and ethylene (ET), and reactive oxygen species (ROS) on the polarization of dormant (D) and non-dormant (ND) embryonic seed cells using microelectrodes. Our data show that D and ND seed cells present different polarization likely due to the regulation of plasma membrane (PM) H+-ATPase activity. The data obtained after addition of hormones or ROS scavengers further suggest that ABA dependent inhibition of PM H+-ATPases could participate in dormancy maintenance and that ET-and ROS-dependent PM H+-ATPase stimulation could participate in dormancy release in sunflower seeds.


Assuntos
Helianthus/enzimologia , Dormência de Plantas , Reguladores de Crescimento de Planta/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ácido Abscísico/metabolismo , Membrana Celular/enzimologia , Etilenos/metabolismo , Germinação , Helianthus/genética , Helianthus/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , ATPases Translocadoras de Prótons/genética , Sementes/enzimologia , Sementes/genética , Sementes/fisiologia
10.
Plant Mol Biol ; 100(1-2): 181-197, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30868545

RESUMO

KEY MESSAGE: The knowledge of substrate specificity of XET enzymes is important for the general understanding of metabolic pathways to challenge the established notion that these enzymes operate uniquely on cellulose-xyloglucan networks. Xyloglucan xyloglucosyl transferases (XETs) (EC 2.4.1.207) play a central role in loosening and re-arranging the cellulose-xyloglucan network, which is assumed to be the primary load-bearing structural component of plant cell walls. The sequence of mature TmXET6.3 from Tropaeolum majus (280 residues) was deduced by the nucleotide sequence analysis of complete cDNA by Rapid Amplification of cDNA Ends, based on tryptic and chymotryptic peptide sequences. Partly purified TmXET6.3, expressed in Pichia occurred in N-glycosylated and unglycosylated forms. The quantification of hetero-transglycosylation activities of TmXET6.3 revealed that (1,3;1,4)-, (1,6)- and (1,4)-ß-D-glucooligosaccharides were the preferred acceptor substrates, while (1,4)-ß-D-xylooligosaccharides, and arabinoxylo- and glucomanno-oligosaccharides were less preferred. The 3D model of TmXET6.3, and bioinformatics analyses of identified and putative plant xyloglucan endotransglycosylases (XETs)/hydrolases (XEHs) of the GH16 family revealed that H94, A104, Q108, K234 and K237 were the key residues that underpinned the acceptor substrate specificity of TmXET6.3. Compared to the wild-type enzyme, the single Q108R and K237T, and double-K234T/K237T and triple-H94Q/A104D/Q108R variants exhibited enhanced hetero-transglycosylation activities with xyloglucan and (1,4)-ß-D-glucooligosaccharides, while those with (1,3;1,4)- and (1,6)-ß-D-glucooligosaccharides were suppressed; the incorporation of xyloglucan to (1,4)-ß-D-glucooligosaccharides by the H94Q variant was influenced most extensively. Structural and biochemical data of non-specific TmXET6.3 presented here extend the classic XET reaction mechanism by which these enzymes operate in plant cell walls. The evaluations of TmXET6.3 transglycosylation activities and the incidence of investigated residues in other members of the GH16 family suggest that a broad acceptor substrate specificity in plant XET enzymes could be more widespread than previously anticipated.


Assuntos
Glicosiltransferases/metabolismo , Proteínas de Plantas/metabolismo , Engenharia de Proteínas , Sementes/enzimologia , Tropaeolum/enzimologia , Sequência de Aminoácidos , Sequência de Bases , DNA Complementar/genética , Germinação , Glicosilação , Glicosiltransferases/química , Modelos Moleculares , Petroselinum/enzimologia , Filogenia , Proteínas de Plantas/química , Homologia Estrutural de Proteína , Especificidade por Substrato
11.
Food Chem Toxicol ; 126: 313-321, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30831153

RESUMO

Examining tissue-specific expression and the measurement of protein abundance are important steps when assessing the performance of genetically engineered crops. Liquid chromatography-mass spectrometry offers many advantages over traditional methods for protein quantitation, especially when dealing with transmembrane proteins that are often difficult to express or generate antibodies against. In this study, discovery proteomics was used to detect the seven transgenic membrane-bound enzymes from the docosahexaenoic acid (DHA) biosynthetic pathway that had been engineered into canola. Subsequently, a targeted LC-MS/MS method for absolute quantitation was developed and applied to the simultaneous measurement of the seven DHA biosynthetic pathway enzymes in genetically modified canola grown across three sites. The results of this study demonstrated that the enzymatic proteins that drive the production of DHA using seed-specific promoters were detected only in mature and developing seed of DHA canola. None of the DHA biosynthesis pathway proteins were detected in wild-type canola planted in the same site or in the non-seed tissues of the transgenic canola, irrespective of the sampling time or the tissues tested. This study describes a streamlined approach to simultaneously measure multiple membrane-bound proteins in planta.


Assuntos
Brassica rapa/enzimologia , Ácidos Docosa-Hexaenoicos/biossíntese , Espectrometria de Massas/métodos , Proteínas de Membrana/química , Proteínas de Plantas/química , Plantas Geneticamente Modificadas/enzimologia , Brassica rapa/química , Brassica rapa/genética , Brassica rapa/metabolismo , Engenharia Genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/química , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Sementes/química , Sementes/enzimologia , Sementes/genética , Sementes/metabolismo
12.
J Agric Food Chem ; 67(15): 4214-4223, 2019 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-30915847

RESUMO

Raffinose, an oligosaccharide found in many seeds, plays an important role in seed vigor; however, the regulatory mechanism governing raffinose biosynthesis remains unclear. We report here that maize W22 wild type (WT) seeds, but not W22 viviparous1 ( zmvp1) mutant seeds, start accumulating galactinol and raffinose 28 days after pollination (DAP). Transcriptome analysis of the zmvp1 embryo showed that the expression of GALACTINOL SYNTHASE2 ( GOLS2) was down-regulated relative to WT. Further experiments showed that the expression of ZmGOLS2 was up-regulated by ZmABI5 but not by ZmVP1, and it was further increased by the coexpression of ZmABI5 and ZmVP1 in maize protoplasts. ZmABI5 interacted with ZmVP1, while ZmABI5, but not ZmVP1, directly binds to the ZmGOLS2 promoter. Together, all of the findings suggest that ZmVP1 interacts with ZmABI5 and regulates ZmGOLS2 expression and raffinose accumulation in maize seeds.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Galactosiltransferases/metabolismo , Proteínas de Plantas/metabolismo , Rafinose/metabolismo , Sementes/metabolismo , Zea mays/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/genética , Galactosiltransferases/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Regiões Promotoras Genéticas , Ligação Proteica , Sementes/enzimologia , Sementes/genética , Zea mays/enzimologia , Zea mays/genética
13.
Ecotoxicol Environ Saf ; 174: 637-648, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-30875557

RESUMO

Gamma irradiation has been reported to modulate the biochemical and molecular parameters associated with the tolerance of plant species under biotic/ abiotic stress. Wheat is highly sensitive to heat stress (HS), as evident from the decrease in the quantity and quality of the total grains. Here, we studied the effect of pre-treatment of wheat dry seeds with different doses of gamma irradiation (0.20, 0.25 and 0.30 kGy) on tolerance level and quality of developing wheat endospermic tissue under HS (38 °C, 1 h; continuously for three days). Expression analysis of genes associated with defence and starch metabolism in developing grains showed maximum transcripts of HSP17 (in response to 0.25 kGy + HS) and AGPase (under 0.30 kGy), as compared to control. Gamma irradiation was observed to balance the accumulation of H2O2 by enhancing the activities of SOD and GPx in both the cvs. under HS. Gamma irradiation was observed to stabilize the synthesis of starch and amylose by regulating the activities of AGPase, SSS and α-amylase under HS. The appearance of isoforms of gliadins (α, ß, γ, ω) were observed more in gamma irradiated seeds (0.20 kGy), as compared to control. Gamma irradiation (0.25 kGy in HD3118 & 0.20 kGy in HD3086) was observed to have positive effect on the width, length and test seed weight of the grains under HS. The information generated in present investigation provides easy, cheap and user-friendly technology to mitigate the effect of terminal HS on the grain-development process of wheat along with development of robust seeds with high nutrient density.


Assuntos
Grão Comestível/efeitos da radiação , Endosperma/efeitos da radiação , Raios gama , Estresse Oxidativo/efeitos da radiação , Triticum , Grão Comestível/enzimologia , Grão Comestível/fisiologia , Endosperma/enzimologia , Endosperma/fisiologia , Irradiação de Alimentos , Resposta ao Choque Térmico/efeitos da radiação , Peróxido de Hidrogênio/metabolismo , Sementes/enzimologia , Sementes/fisiologia , Sementes/efeitos da radiação , Amido/biossíntese
14.
BMC Plant Biol ; 19(1): 27, 2019 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-30646852

RESUMO

BACKGROUND: Seed germination is a crucial process in the plant life cycle when a dramatic variation of type and sugar content occurs just as the seed is hydrated. The production of hexose 6 phosphate is a key node in different pathways that are required for a successful germination. Hexokinase (HXK) is the only plant enzyme that phosphorylates glucose (Glc), so it is key to fueling several metabolic pathways depending on their substrate specificity, metabolite regulatory responses and subcellular localization. In maize, the HXK family is composed of nine genes, but only six of them (ZmHXK4-9) putatively encode catalytically active enzymes. Here, we cloned and functionally characterized putative catalytic enzymes to analyze their metabolic contribution during germination process. RESULTS: From the six HXKs analyzed here, only ZmHXK9 has minimal hexose phosphorylating activity even though enzymatic function of all isoforms (ZmHXK4-9) was confirmed using a yeast complementation approach. The kinetic parameters of recombinant proteins showed that ZmHXK4-7 have high catalytic efficiency for Glc, fructose (Fru) and mannose (Man), ZmHXK7 has a lower Km for ATP, and together with ZmHXK8 they have lower sensitivity to inhibition by ADP, G6P and N-acetylglucosamine than ZmHXK4-6 and ZmHXK9. Additionally, we demonstrated that ZmHXK4-6 and ZmHXK9 are located in the mitochondria and their location relies on the first 30 amino acids of the N-terminal domain. Otherwise, ZmHXK7-8 are constitutively located in the cytosol. HXK activity was detected in cytosolic and mitochondrial fractions and high Glc and Fru phosphorylating activities were found in imbibed embryos. CONCLUSIONS: Considering the biochemical characteristics, location and the expression of ZmHXK4 at onset of germination, we suggest that it is the main contributor to mitochondrial activity at early germination times, at 24 h other ZmHXKs also contribute to the total activity. While in the cytosol, ZmHXK7 could be responsible for the activity at the onset of germination, although later, ZmHXK8 also contributes to the total HXK activity. Our observations suggest that the HXKs may be redundant proteins with specific roles depending on carbon and ATP availability, metabolic needs, or sensor requirements. Further investigation is necessary to understand their specific or redundant physiological roles.


Assuntos
Citosol/fisiologia , Germinação/fisiologia , Hexoquinase/metabolismo , Sementes/fisiologia , Zea mays/enzimologia , Zea mays/fisiologia , Citosol/enzimologia , Citosol/metabolismo , Germinação/genética , Hexoquinase/genética , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Sementes/enzimologia , Sementes/metabolismo , Zea mays/metabolismo
15.
Plant Cell Rep ; 38(3): 345-359, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30649573

RESUMO

KEY MESSAGE: FLO15encodes a plastidic glyoxalase I protein, OsGLYI7, which affects compound starch granule formation and starch synthesis in rice endosperm. Starch synthesis in rice (Oryza sativa) endosperm is a sophisticated process, and its underlying molecular machinery still remains to be elucidated. Here, we identified and characterized two allelic rice floury endosperm 15 (flo15) mutants, both with a white-core endosperm. The flo15 grains were characterized by defects in compound starch granule development, along with decreased starch content. Map-based cloning of the flo15 mutants identified mutations in OsGLYI7, which encodes a glyoxalase I (GLYI) involved in methylglyoxal (MG) detoxification. The mutations of FLO15/OsGLYI7 resulted in increased MG content in flo15 developing endosperms. FLO15/OsGLYI7 localizes to the plastids, and the in vitro GLYI activity derived from flo15 was significantly decreased relative to the wild type. Moreover, the expression of starch synthesis-related genes was obviously altered in the flo15 mutants. These findings suggest that FLO15 plays an important role in compound starch granule formation and starch synthesis in rice endosperm.


Assuntos
Endosperma/enzimologia , Regulação da Expressão Gênica de Plantas , Lactoilglutationa Liase/metabolismo , Oryza/enzimologia , Amido/metabolismo , Grânulos Citoplasmáticos/metabolismo , Endosperma/citologia , Endosperma/genética , Genes Reporter , Lactoilglutationa Liase/genética , Mutação , Oryza/citologia , Oryza/genética , Fenótipo , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plastídeos/enzimologia , Sementes/citologia , Sementes/enzimologia , Sementes/genética , Técnicas do Sistema de Duplo-Híbrido
16.
Planta ; 249(5): 1285-1299, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30610363

RESUMO

MAIN CONCLUSION: In vivo and in vitro analyses of Euphorbiaceae species' triacylglycerol assembly enzymes substrate selectivity are consistent with the co-evolution of seed-specific unusual fatty acid production and suggest that many of these genes will be useful for biotechnological production of designer oils. Many exotic Euphorbiaceae species, including tung tree (Vernicia fordii), castor bean (Ricinus communis), Bernardia pulchella, and Euphorbia lagascae, accumulate unusual fatty acids in their seed oils, many of which have valuable properties for the chemical industry. However, various adverse plant characteristics including low seed yields, production of toxic compounds, limited growth range, and poor resistance to abiotic stresses have limited full agronomic exploitation of these plants. Biotechnological production of these unusual fatty acids (UFA) in high yielding non-food oil crops would provide new robust sources for these valuable bio-chemicals. Previous research has shown that expression of the primary UFA biosynthetic gene alone is not enough for high-level accumulation in transgenic seed oils; other genes must be included to drive selective UFA incorporation into oils. Here, we use a series of in planta molecular genetic studies and in vitro biochemical measurements to demonstrate that lysophosphatidic acid acyltransferases from two Euphorbiaceae species have high selectivity for incorporation of their respective unusual fatty acids into the phosphatidic acid intermediate of oil biosynthesis. These results are consistent with the hypothesis that unusual fatty acid accumulation arose in part via co-evolution of multiple oil biosynthesis and assembly enzymes that cooperate to enhance selective fatty acid incorporation into seed oils over that of the common fatty acids found in membrane lipids.


Assuntos
Aciltransferases/metabolismo , Euphorbiaceae/enzimologia , Euphorbiaceae/metabolismo , Ácidos Graxos/metabolismo , Óleos Vegetais/metabolismo , Sementes/enzimologia , Sementes/metabolismo , Regulação da Expressão Gênica de Plantas , Ácidos Ricinoleicos/metabolismo
17.
Virology ; 526: 91-98, 2019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-30388630

RESUMO

Highly pathogenic alphaviruses display complex glycans on their surface. These glycans play a crucial role in viral pathogenesis by facilitating glycan-host interaction during viral entry which can be targeted. Various studies have reported antiviral activity of lectins that bind to the glycans present on the surface of enveloped viruses. This study evaluates the antiviral potential of a chitinase (chi)-like lectin from Tamarind (TCLL) having specificity for N-acetylglucosamine (NAG). Thus, TCLL might bind to N-glycan rich surface of alphavirus and inhibit the entry of virus into the host cells. The direct treatment of TCLL with virus reduced the virus infection. Remarkably, the addition of NAG to TCLL abolished antiviral activity confirming that NAG binding property of TCLL is accountable for its antiviral activity. Further, an ELISA assay confirmed the binding of TCLL to alphaviruses. Taken together, this study will prove to be beneficial in developing lectin therapeutics targeting alphavirus glycan.


Assuntos
Acetilglucosamina/metabolismo , Antivirais/farmacologia , Vírus Chikungunya/efeitos dos fármacos , Quitinases/farmacologia , Lectinas de Plantas/farmacologia , Polissacarídeos/metabolismo , Tamarindus/enzimologia , Animais , Antivirais/isolamento & purificação , Antivirais/metabolismo , Antivirais/uso terapêutico , Linhagem Celular , Febre de Chikungunya/tratamento farmacológico , Febre de Chikungunya/virologia , Vírus Chikungunya/crescimento & desenvolvimento , Vírus Chikungunya/metabolismo , Quitinases/isolamento & purificação , Quitinases/metabolismo , Relação Dose-Resposta a Droga , Lectinas de Plantas/isolamento & purificação , Lectinas de Plantas/metabolismo , Ligação Proteica , RNA Viral/metabolismo , Sementes/enzimologia , Tamarindus/química , Proteínas do Envelope Viral/metabolismo , Ensaio de Placa Viral , Internalização do Vírus/efeitos dos fármacos
18.
Food Chem ; 274: 422-428, 2019 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-30372960

RESUMO

The objective of this study was to characterize a plant origin ß-glucosidase from black plum seeds and identify its conformational changes in twenty-six imidazolium- and amino acid-based ionic liquids (ILs). The results revealed that the purified 60 kDa enzyme was monomeric in nature, maximally active at 55 °C and pH 5.0, and nearly completely inhibited by Hg2+ and Ag+. Attractive peculiarities of the relative low kinetic and higher glucose inhibition constants (Km = 0.58 mM [pNPG]; Ki = 193.5 mM [glucose]) demonstrated its potential applications in food industry. Circular dichroism studies showed that the secondary structural changes of the enzyme depended not only on the anions, but also on the cations of the assayed ILs. Interestingly, no corresponding relations were observed between the changes in enzyme structure induced by ILs and its catalytic activities, suggesting that the influences of ILs on enzymatic processes don't rely simply on enzyme conformational changes.


Assuntos
Glucose/farmacologia , Líquidos Iônicos/farmacologia , Prunus domestica/enzimologia , Sementes/enzimologia , beta-Glucosidase/química , beta-Glucosidase/isolamento & purificação , Biocatálise , Cinética , beta-Glucosidase/metabolismo
19.
Food Chem ; 274: 480-486, 2019 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-30372968

RESUMO

The present research studied the thermal pre-treatment of non-soaked and soaked black seed (BS) by microwave radiation (1-3.5 min) in comparison with conventional roasting (2-8 min, 180 °C). As BS was treated for a longer time, a significant increase (p < 0.05) was observed in total chlorophylls, carotenoids and phenolic contents in the extracted oils. The oils obtained from the soaked seeds (to 8%) had significantly higher oil quality properties. The optimum extraction conditions were: irradiation time of 3.5 min and BS moisture content of 8%, which resulted in an extraction yield of 35.76%, 10643.730 caffeic acid equivalents/100 g, PV of 9.63 meq O2/kg and 18.88 h of Rancimat oxidative stability. The microwave pre-treatment was more effective than conventional roasting in reducing lipase activity. Based on the results obtained, it was advisable to treat BS with microwaves and soak them before extracting the oil in a cold press at room temperature.


Assuntos
Lipase/metabolismo , Micro-Ondas , Óleos Vegetais/análise , Sementes/enzimologia , Carotenoides/química , Clorofila/química , Temperatura Alta , Nigella sativa/crescimento & desenvolvimento , Fenóis/química , Óleos Vegetais/química , Sementes/química , Sementes/metabolismo
20.
Phytochemistry ; 157: 121-127, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30399494

RESUMO

Ribosome-inactivating proteins (RIPs) are enzymes with N-glycosylase activity that remove adenine bases from the ribosomal RNA. In theory, one single RIP molecule internalized into a cell is sufficient to induce cell death. For this reason, RIPs are of high potential as toxic payload for anti-tumor therapy. A considerable number of RIPs are synthesized by plants that belong to the carnation family (Caryophyllaceae). Prominent examples are the RIPs saporin from Saponaria officinalis L. or dianthin from Dianthus caryophyllus L. In this study, we have isolated and characterized a novel RIP (termed gypsophilin-S) from the tiny seeds of Gypsophila elegans M. Bieb. (Caryophyllaceae). It is noteworthy that this is the first study presenting the complete amino acid sequence of a RIP from a Gypsophila species. Gypsophilin-S was isolated from the defatted seed material following ammonium sulphate precipitation and HPLC-based ion exchange chromatography. Gypsophilin-S-containing fractions were analysed by SDS-PAGE and mass spectrometry. The full amino acid sequence of gypsophilin-S was assembled by MALDI-TOF-MS-MS and PCR. Gypsophilin-S exhibited strong adenine releasing activity and its cytotoxicity in human glioblastoma cells was investigated using an impedance-based real-time assay in comparison with recombinant saporin and dianthin.


Assuntos
Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacologia , Caryophyllaceae/enzimologia , Saporinas/química , Saporinas/farmacologia , Sementes/enzimologia , Sequência de Aminoácidos , Antineoplásicos Fitogênicos/isolamento & purificação , Linhagem Celular Tumoral , Humanos , Modelos Moleculares , Conformação Proteica , Saporinas/isolamento & purificação
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