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1.
J Food Sci ; 85(2): 349-357, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31957892

RESUMO

Gluten-free products from rice are gaining popularity because of its hypoallergenic characteristic. The absence of gluten results in inferior bread qualities such as hard texture, reduced volume, and shorter shelf-life. Hydrolytic enzymes are activated during germination to stimulate plant growth, and germinated brown rice (GBR) has been shown to improve gluten-free bread properties. However, the changes in hydrolytic enzyme activities under different germination conditions and their relationship with the properties of germinated rice flour and bread have not been reported. Therefore, the objectives of this work were to investigate the activities of amylases and protease in GBR under aerobic and anaerobic germination for 2 and 4 days and their impacts on starch hydrolysis, flour properties, and bread qualities. Greater enzyme activities were observed in GBR germinated under aerobic condition and a longer time, and correlated with increased sugar content and foaming capacity. Breads were prepared from GBR along with brown rice (control). GBR breads showed a greater specific volume (4% to 10%), a reduced hardness (34% to 90%), and a lower starch retrogradation (66% to 90%) compared with the control. Bread prepared from 4-day aerobic GBR had the largest reduction in starch molecular size and displayed the lowest hardness and starch retrogradation. After stored for 5 days, GBR breads exhibited no change in specific volume and less hardness and retrogradation than the control bread. In conclusion, greater activities of protease and amylases in GBR significantly increased foaming capacity and reduced starch molecule size, respectively, which were responsible for the improved GBR bread qualities. PRACTICAL APPLICATION: Rice flour is widely used as the main ingredient in gluten-free breads, which however tend to have poor texture and reduced shelf-life due to the absence of gluten. The qualities of gluten-free breads are usually improved by the addition of many ingredients such as tapioca and potato starches. Germination process naturally produces bioactive compounds and activates enzymes. Germination conditions that produce greater activities of amylases and protease can be used to produce gluten-free breads with better qualities and longer shelf-life without the addition of starch.


Assuntos
Amilases/análise , Pão/análise , Oryza/química , Peptídeo Hidrolases/análise , Sementes/crescimento & desenvolvimento , Amido/química , Amilases/metabolismo , Farinha/análise , Manipulação de Alimentos , Germinação , Dureza , Humanos , Hidrólise , Oryza/enzimologia , Oryza/crescimento & desenvolvimento , Peptídeo Hidrolases/metabolismo , Sementes/química , Sementes/enzimologia , Amido/metabolismo , Paladar
2.
Plant Mol Biol ; 102(4-5): 373-388, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31872309

RESUMO

KEY MESSAGE: CRISPR-edited variants at the 3'-end of OsLOGL5's coding sequence (CDS), significantly increased rice grain yield under well-watered, drought, normal nitrogen, and low nitrogen field conditions at multiple geographical locations. Cytokinins impact numerous aspects of plant growth and development. This study reports that constitutive ectopic overexpression of a rice cytokinin-activation enzyme-like gene, OsLOGL5, significantly reduced primary root growth, tiller number, and yield. Conversely, mutations at the 3'-end of OsLOGL5 CDS resulted in normal rice plant morphology but with increased grain yield under well-watered, drought, normal nitrogen, and low nitrogen field conditions at multiple geographical locations. Six gene edited variants (Edit A to F) were created and tested in the field. Edit-B and Edit-F plants increased, but Edit-D and Edit-E plants decreased, the panicle number per plant. All OsLOGL5-edited plants significantly increased seed setting rate, total grain numbers, full-filled grain numbers per panicle, and thousand seed weight under drought conditions, suggesting that OsLOGL5 is likely involved in the regulation of both seed development and grain filling processes. Our results indicate that the C-terminal end of OsLOGL5 protein plays an important role in regulating rice yield improvement under different abiotic stress conditions, and OsLOGL5 is important for rice yield enhancement and stability.


Assuntos
Citocininas/metabolismo , Grão Comestível/genética , Oryza/genética , Proteínas de Plantas/metabolismo , Sistemas CRISPR-Cas , Secas , Grão Comestível/enzimologia , Edição de Genes , Regulação da Expressão Gênica de Plantas , Nitrogênio , Oryza/enzimologia , Proteínas de Plantas/genética , Raízes de Plantas/fisiologia , Domínios Proteicos , Sementes/fisiologia , Estresse Fisiológico
3.
Proc Natl Acad Sci U S A ; 117(1): 271-277, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31848246

RESUMO

Brown planthopper (BPH) is one of the most destructive insects affecting rice (Oryza sativa L.) production. Phenylalanine ammonia-lyase (PAL) is a key enzyme involved in plant defense against pathogens, but the role of PAL in insect resistance is still poorly understood. Here we show that expression of the majority of PALs in rice is significantly induced by BPH feeding. Knockdown of OsPALs significantly reduces BPH resistance, whereas overexpression of OsPAL8 in a susceptible rice cultivar significantly enhances its BPH resistance. We found that OsPALs mediate resistance to BPH by regulating the biosynthesis and accumulation of salicylic acid and lignin. Furthermore, we show that expression of OsPAL6 and OsPAL8 in response to BPH attack is directly up-regulated by OsMYB30, an R2R3 MYB transcription factor. Taken together, our results demonstrate that the phenylpropanoid pathway plays an important role in BPH resistance response, and provide valuable targets for genetic improvement of BPH resistance in rice.


Assuntos
Hemípteros/efeitos dos fármacos , Oryza/enzimologia , Oryza/metabolismo , Fenilalanina Amônia-Liase/metabolismo , Fenilalanina Amônia-Liase/farmacologia , Doenças das Plantas/imunologia , Fatores de Transcrição/metabolismo , Animais , DNA de Plantas/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Técnicas de Silenciamento de Genes , Genes de Plantas , Interações Hospedeiro-Parasita/genética , Interações Hospedeiro-Parasita/fisiologia , Lignina/metabolismo , Oryza/genética , Oryza/imunologia , Fenilalanina Amônia-Liase/genética , Doenças das Plantas/genética , Doenças das Plantas/parasitologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Ácido Salicílico/metabolismo
4.
Genes (Basel) ; 11(1)2019 12 24.
Artigo em Inglês | MEDLINE | ID: mdl-31878175

RESUMO

Glutathione S-transferases (GSTs)-an especially plant-specific tau class of GSTs-are key enzymes involved in biotic and abiotic stress responses. To improve the stress resistance of crops via the genetic modification of GSTs, we predicted the amino acids present in the GSH binding site (G-site) and hydrophobic substrate-binding site (H-site) of OsGSTU17, a tau class GST in rice. We then examined the enzyme activity, substrate specificity, enzyme kinetics and thermodynamic stability of the mutant enzymes. Our results showed that the hydrogen bonds between Lys42, Val56, Glu68, and Ser69 of the G-site and glutathione were essential for enzyme activity and thermal stability. The hydrophobic side chains of amino acids of the H-site contributed to enzyme activity toward 4-nitrobenzyl chloride but had an inhibitory effect on enzyme activity toward 1-chloro-2,4-dinitrobenzene and cumene hydroperoxide. Different amino acids of the H-site had different effects on enzyme activity toward a different substrate, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. Moreover, Leu112 and Phe162 were found to inhibit the catalytic efficiency of OsGSTU17 to 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, while Pro16, Leu112, and Trp165 contributed to structural stability. The results of this research enhance the understanding of the relationship between the structure and function of tau class GSTs to improve the abiotic stress resistance of crops.


Assuntos
Glutationa Transferase/química , Glutationa Transferase/metabolismo , Nitrobenzenos/metabolismo , Oryza/enzimologia , Derivados de Benzeno/farmacologia , Sítios de Ligação , Dinitroclorobenzeno/farmacologia , Estabilidade Enzimática , Glutationa Transferase/efeitos dos fármacos , Ligação de Hidrogênio , Oryza/química , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Ligação Proteica , Especificidade por Substrato , Termodinâmica
5.
PLoS One ; 14(12): e0226543, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31856232

RESUMO

Reactive oxygen species (ROS) are produced by plants. Hydrogen peroxide (H2O2) is one important component of ROS and able to modulate plant growth and development at low level and damage plant cells at high concentrations. Ascorbate peroxidase (APX) shows high affinity towards H2O2 and plays vital roles in H2O2-scavenging. In order to explore the differences of APXs from selected plant species, bioinformatics methods and public databases were used to evaluate the physicochemical properties, conserved motifs, potential modifications and cis-elements in all the APXs, and protein-protein network and expression profiles of rice APXs. The results suggested that APXs in the selected plant species showed high evolutionary conservation and were able to divide into seven groups, group I to VII. Members in the groups contained abundant phosphorylation sites. Interestingly, group I and VII had only PKC site. Additionally, promoters of the APXs contained abundant stress-related cis-elements. APXs in rice plant were able to interact with dehydroascorbate reductase 2. The eight APXs expressed differently in root, leaf, panicle, anther, pistil and seed. Drought, Pi-free, Cd and Xanthomonas oryzae pv. oryzicola B8-12 treatments were able to significantly alter the expression profiles of rice APXs. This study increases our knowledge to further explore functions and mechanisms of APXs and also guides their applications.


Assuntos
Ascorbato Peroxidases/metabolismo , Bases de Dados Factuais , Oryza/enzimologia , Motivos de Aminoácidos , Ascorbato Peroxidases/química , Ascorbato Peroxidases/genética , Sequência Conservada , Regulação da Expressão Gênica de Plantas , Modelos Moleculares , Oryza/genética , Filogenia , Mapeamento de Interação de Proteínas
6.
BMC Plant Biol ; 19(1): 472, 2019 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-31694539

RESUMO

BACKGROUND: Isocitrate lyase (ICL) is a key enzyme in the glyoxylate cycle. In a previous study in rice, the expression of the ICL-encoding gene (OsICL) was highly induced by salt stress and its expression was enhanced in transgenic rice lines overexpressing OsCam1-1, a calmodulin (CaM)-encoding gene. CaM has been implicated in salt tolerance mechanisms in plants; however, the cellular mechanisms mediated by CaM are not clearly understood. In this study, the role of OsICL in plant salt tolerance mechanisms and the possible involvement of CaM were investigated using transgenic plants expressing OsICL or OsCam1-1. RESULTS: OsICL was highly expressed in senesced leaf and significantly induced by salt stress in three OsCam1-1 overexpressing transgenic rice lines as well as in wild type (WT). In WT young leaf, although OsICL expression was not affected by salt stress, all three transgenic lines exhibited highly induced expression levels. In Arabidopsis, salt stress had negative effects on germination and seedling growth of the AtICL knockout mutant (Aticl mutant). To examine the roles of OsICL we generated the following transgenic Arabidopsis lines: the Aticl mutant expressing OsICL driven by the native AtICL promoter, the Aticl mutant overexpressing OsICL driven by the 35SCaMV promoter, and WT overexpressing OsICL driven by the 35SCaMV promoter. Under salt stress, the germination rate and seedling fresh and dry weights of the OsICL-expressing lines were higher than those of the Aticl mutant, and the two lines with the icl mutant background were similar to the WT. The Fv/Fm and temperature of rosette leaves in the OsICL-expressing lines were less affected by salt stress than they were in the Aticl mutant. Finally, glucose and fructose contents of the Aticl mutant under salt stress were highest, whereas those of OsICL-expressing lines were similar to or lower than those of the WT. CONCLUSIONS: OsICL, a salt-responsive gene, was characterized in the transgenic Arabidopsis lines, revealing that OsICL expression could revert the salt sensitivity phenotypes of the Aticl knockout mutant. This work provides novel evidence that supports the role of ICL in plant salt tolerance through the glyoxylate cycle and the possible involvement of OsCam1-1 in regulating its transcription.


Assuntos
Isocitrato Liase/metabolismo , Oryza/enzimologia , Plantas Tolerantes a Sal/enzimologia , Arabidopsis/genética , Calmodulina/genética , Calmodulina/metabolismo , Isocitrato Liase/genética , Oryza/genética , Plantas Geneticamente Modificadas/genética , Plantas Tolerantes a Sal/genética
7.
BMC Plant Biol ; 19(1): 502, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31730480

RESUMO

BACKGROUND: Selenium (Se) is a beneficial element for higher plants and essential for mammals. To study the effect of the foliar application of sodium selenate on fragrant rice performance, a pot experiment was conducted in Guangdong, China. At the initial heading stage, one-time foliar application of sodium selenate with concentrations of 0, 10, 20, 30, 40 and 50 µmol·L- 1 (named CK, Se1, Se2, Se3, Se4 and Se5, respectively) were foliar applied on two fragrant rice varieties, 'Meixiangzhan-2' and 'Xiangyaxiangzhan'. RESULTS: Selenate application at the initial heading stage not only improved the grain yield of fragrant rice by increasing the seed-setting rate and grain weight, but also promoted the grain quality by increasing crude protein contents and lowering the chalky rice rate. Furthermore, Se applications enhanced the biosynthesis of 2-acetyl-1- pyrroline (2-AP), the main aromatic compound, by increasing the contents of precursors (△1- pyrroline, proline and pyrroline-5-carboxylic acid (P5C)) and the activities of enzymes (proline dehydrogenase (PRODH), △1-pyrroline-5-carboxylic acid synthetase (P5CS), and ornithine aminotransferase (OAT)) in fragrant rice. The results also showed that foliar application of sodium selenate enhanced the antioxidant system of both varieties by promoting the activities of peroxidase (POD), superoxide dismutase (SOD), catalase (CAT) and reducing the contents of malondialdehyde (MDA). Furthermore, the real-time PCR analyses depicted that foliar application of selenate up-regulated the GPX1, GPX4 and CATC transcripts. The higher antioxidative enzymatic activities might strength the stress resistant to ensure the stability of yield in fragrant rice form abiotic stress. CONCLUSIONS: Foliar applications of sodium selenate at the initial heading stage increased the grain 2-AP content by enhancing the biosynthesis-related enzymes and precursors. The grain yield and quality of fragrant rice also increased due to selenate application. Furthermore, foliar application of selenate promoted the activities of enzymes such as POD, SOD and CAT and up-regulated the expression of gene GPX4, GPX1 and CATC.


Assuntos
Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Oryza/efeitos dos fármacos , Pirróis/metabolismo , Ácido Selênico/administração & dosagem , Antioxidantes/metabolismo , Biomassa , Catalase/metabolismo , Grão Comestível/efeitos dos fármacos , Grão Comestível/enzimologia , Grão Comestível/genética , Grão Comestível/crescimento & desenvolvimento , Malondialdeído/metabolismo , Oryza/enzimologia , Oryza/genética , Oryza/crescimento & desenvolvimento , Peroxidases/metabolismo , Proteínas de Plantas/metabolismo , Prolina/metabolismo , Superóxido Dismutase/metabolismo
8.
Environ Pollut ; 255(Pt 2): 113289, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31606664

RESUMO

Rice consumption is one of the primary sources of arsenic (As) exposure as the grains contain relatively higher concentration of inorganic As. Abundant studies on the ability of iron (Fe) plaque in hampering As uptake by plants has been reported earlier. However, little is known about its role in the mitigation of As mediated oxidative damage in rice plants. The present study highlights the effect of As and Fe co-supplementation on growth response, oxidative stress, Fe uptake related enzymes and nutrient status in rice varieties. Eight different Indica rice varieties were screened and finally four varieties (Varsha, Jaya, PB-1 and IR-64) were selected for detailed investigations. Improved germination and chlorophyll/protein levels during As+Fe co-exposure indicate healthier plants than As(III) treated ones. Interestingly Fe was found act both as an antagonist and also as a synergist of As treatments. It acted by reducing As translocation and improving the nutritional levels and enhancing the oxidative stress. Fe uptake related enzymes (nitrite reductase and ferric chelate reductase) and phytosiderophores analysis revealed that Fe supplementation can reduce its deficiency in rice plants. Morpho-biochemical, oxidative stress and nutrient analysis symbolizes higher tolerance of PB-1 towards As, while Varsha being most sensitive, efficiently combated the As(III) stress in the presence of Fe.


Assuntos
Arsênico/metabolismo , Ferro/metabolismo , Oryza/metabolismo , Poluentes do Solo/metabolismo , Arsênico/análise , Transporte Biológico/efeitos dos fármacos , Clorofila , FMN Redutase , Oryza/efeitos dos fármacos , Oryza/enzimologia , Oxirredução , Raízes de Plantas/química , Poluentes do Solo/análise
9.
Mol Cells ; 42(10): 711-720, 2019 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-31607684

RESUMO

Sink strength optimizes sucrose import, which is fundamental to support developing seed grains and increase crop yields, including those of rice (Oryza sativa). In this regard, little is known about the function of vacuolar invertase (VIN) in controlling sink strength and thereby seed size. Here, in rice we analyzed mutants of two VINs, OsVIN1 and OsVIN2, to examine their role during seed development. In a phenotypic analysis of the T-DNA insertion mutants, only the OsVIN2 mutant osvin2-1 exhibited reduced seed size and grain weight. Scanning electron microscopy analysis revealed that the small seed grains of osvin2-1 can be attributed to a reduction in spikelet size. A significant decrease in VIN activity and hexose level in the osvin2-1 spikelets interfered with spikelet growth. In addition, significant reduction in starch and increase in sucrose, which are characteristic features of reduced turnover and flux of sucrose due to impaired sink strength, were evident in the pre-storage stage of osvin2-1 developing grains. In situ hybridization analysis found that expression of OsVIN2 was predominant in the endocarp of developing grains. A genetically complemented line with a native genomic clone of OsVIN2 rescued reduced VIN activity and seed size. Two additional mutants, osvin2-2 and osvin2-3 generated by the CRISPR/Cas9 method, exhibited phenotypes similar to those of osvin2-1 in spikelet and seed size, VIN activity, and sugar metabolites. These results clearly demonstrate an important role of OsVIN2 as sink strength modulator that is critical for the maintenance of sucrose flux into developing seed grains.


Assuntos
Oryza/enzimologia , Sementes/anatomia & histologia , Sementes/enzimologia , Vacúolos/enzimologia , beta-Frutofuranosidase/metabolismo , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Metaboloma , Mutação/genética , Tamanho do Órgão , Fotossíntese , Plantas Geneticamente Modificadas , Amido/metabolismo , Sacarose/metabolismo
10.
BMC Plant Biol ; 19(1): 426, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31615413

RESUMO

BACKGROUND: Chemically inducible systems that provide both spatial and temporal control of gene expression are essential tools, with many applications in plant biology, yet they have not been extensively tested in monocotyledonous species. RESULTS: Using Golden Gate modular cloning, we have created a monocot-optimized dexamethasone (DEX)-inducible pOp6/LhGR system and tested its efficacy in rice using the reporter enzyme ß-glucuronidase (GUS). The system is tightly regulated and highly sensitive to DEX application, with 6 h of induction sufficient to induce high levels of GUS activity in transgenic callus. In seedlings, GUS activity was detectable in the root after in vitro application of just 0.01 µM DEX. However, transgenic plants manifested severe developmental perturbations when grown on higher concentrations of DEX. The direct cause of these growth defects is not known, but the rice genome contains sequences with high similarity to the LhGR target sequence lacO, suggesting non-specific activation of endogenous genes by DEX induction. These off-target effects can be minimized by quenching with isopropyl ß-D-1-thiogalactopyranoside (IPTG). CONCLUSIONS: Our results demonstrate that the system is suitable for general use in rice, when the method of DEX application and relevant controls are tailored appropriately for each specific application.


Assuntos
Dexametasona/administração & dosagem , Perfilação da Expressão Gênica/métodos , Expressão Gênica , Glucuronidase/genética , Oryza/genética , Proteínas de Plantas/genética , Redes Reguladoras de Genes/efeitos dos fármacos , Genes de Plantas/efeitos dos fármacos , Genes Reporter , Glucuronidase/metabolismo , Oryza/enzimologia , Oryza/metabolismo , Proteínas de Plantas/metabolismo
11.
J Agric Food Chem ; 67(41): 11436-11443, 2019 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-31553599

RESUMO

Phospholipids and phytic acid are important phosphorus (P)-containing compounds in rice grains. Phytic acid is considered as a major antinutrient, because the negatively charged phytic acid chelates cations, including essential micronutrients, and decreases their bioavailability to human beings and monogastric animals. To gain an insight into the interplay of these two kinds of phosphorus-containing metabolites, we used the CRISPR/Cas9 system to generate mutants of a phospholipase D gene (OsPLDα1) and analyzed the mutational effect on metabolites, including phytic acid in rice grains. Metabolic profiling of two ospldα1 mutants revealed depletion in the phosphatidic acid production and lower accumulation of cytidine diphosphate diacylglycerol and phosphatidylinositol. The mutants also showed significantly reduced phytic acid content as compared to their wild-type parent, and the expression of the key genes involved in the phytic acid biosynthesis was altered in the mutants. These results demonstrate that OsPLDα1 not only plays an important role in phospholipid metabolism but also is involved in phytic acid biosynthesis, most probably through the lipid-dependent pathway, and thus revealed a potential new route to regulate phytic acid biosynthesis in rice.


Assuntos
Oryza/genética , Fosfolipase D/genética , Ácido Fítico/biossíntese , Proteínas de Plantas/genética , Análise Mutacional de DNA , Regulação da Expressão Gênica de Plantas , Oryza/enzimologia , Oryza/metabolismo , Fosfolipase D/metabolismo , Fosfolipídeos/metabolismo , Proteínas de Plantas/metabolismo , Sementes/enzimologia , Sementes/genética , Sementes/metabolismo
12.
Int J Mol Sci ; 20(19)2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31557947

RESUMO

The effector proteins secreted by a pathogen not only promote virulence and infection of the pathogen, but also trigger plant defense response. Therefore, these proteins could be used as important genetic resources for transgenic improvement of plant disease resistance. Magnaporthe oryzae systemic defense trigger 1 (MoSDT1) is an effector protein. In this study, we compared the agronomic traits and blast disease resistance between wild type (WT) and MoSDT1 overexpressing lines in rice. Under control conditions, MoSDT1 transgenic lines increased the number of tillers without affecting kernel morphology. In addition, MoSDT1 transgenic lines conferred improved blast resistance, with significant effects on the activation of callose deposition, reactive oxygen species (ROS) accumulation and cell death. On the one hand, overexpression of MoSDT1 could delay biotrophy-necrotrophy switch through regulating the expression of biotrophy-associated secreted protein 4 (BAS4) and Magnaporthe oryzaecell death inducing protein 1 (MoCDIP1), and activate plant defense response by regulating the expression of Bsr-d1, MYBS1, WRKY45, peroxidase (POD), heat shock protein 90 (HSP90), allenoxide synthase 2 (AOS2), phenylalanine ammonia lyase (PAL), pathogenesis-related protein 1a (PR1a) in rice. On the other hand, overexpression of MoSDT1 could increase the accumulation of some defense-related primary metabolites such as two aromatic amino acids (L-tyrosine and L-tryptohan), 1-aminocyclopropane carboxylic acid, which could be converted to ethylene, vanillic acid and L-saccharopine. Taken together, overexpression of MoSDT1 confers improved rice blast resistance in rice, through modulation of callose deposition, ROS accumulation, the expression of defense-related genes, and the accumulation of some primary metabolites.


Assuntos
5'-Nucleotidase/genética , Resistência à Doença/genética , Expressão Gênica , Magnaporthe/genética , Oryza/genética , Oryza/microbiologia , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , 5'-Nucleotidase/química , 5'-Nucleotidase/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Magnaporthe/enzimologia , Oryza/enzimologia , Fenótipo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Espécies Reativas de Oxigênio/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
13.
J Agric Food Chem ; 67(42): 11607-11615, 2019 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-31560536

RESUMO

ζ-carotene desaturase (ZDS) is a key enzyme in carotenoid biosynthesis and plays an important role in plant photosynthesis. We characterized an albino leaf-color mutant obtained from ethyl methanesulfonate treatment: albino and seedling lethality 1 (ale1). The material contains a chloroplast thylakoid defect where photosynthetic pigments declined and reactive oxygen species accumulated resulting in ale1 death within 3 weeks. Positional cloning and sequencing revealed that there was a single base substitution in ALE1, which encoded a ZDS involved in carotenoid biosynthesis. RNAi and complementation tests confirmed the identity of ALE1. Subcellular localization showed that the ALE1 protein is localized in the chloroplast. Expression analysis indicated that the genes involved in chlorophyll and carotenoid biosynthesis were downregulated. We conclude that ALE1 plays an important role in chloroplast and plant growth in rice.


Assuntos
Cloroplastos/enzimologia , Oryza/crescimento & desenvolvimento , Oxirredutases/genética , Proteínas de Plantas/genética , Clorofila/metabolismo , Cloroplastos/genética , Regulação da Expressão Gênica de Plantas , Oryza/enzimologia , Oryza/genética , Oxirredutases/metabolismo , Fotossíntese , Proteínas de Plantas/metabolismo , Interferência de RNA , Plântula/enzimologia , Plântula/genética , Plântula/crescimento & desenvolvimento
14.
Plant Sci ; 287: 110188, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31481229

RESUMO

9-cis-epoxycarotenoid dioxygenase (NCED) is a rate-limiting enzyme for abscisic acid (ABA) biosynthesis. However, the molecular mechanisms of NCED5 that modulate plant development and abiotic stress tolerance are still unclear, particular in rice. Here, we demonstrate that a rice NCED gene, OsNCED5, was expressed in all tissues we tested, and was induced by exposure to salt stress, water stress, and darkness. Mutational analysis showed that nced5 mutants reduced ABA level and decreased tolerance to salt and water stress and delayed leaf senescence. However, OsNCED5 overexpression increased ABA level, enhanced tolerance to the stresses, and accelerated leaf senescence. Transcript analysis showed that OsNCED5 regulated ABA-dependent abiotic stress and senescence-related gene expression. Additionally, ectopic expression of OsNCED5 tested in Arabidopsis thaliana altered plant size and leaf morphology and delayed seed germination and flowering time. Thus, OsNCED5 may regulate plant development and stress resistance through control of ABA biosynthesis. These findings contribute to our understanding of the molecular mechanisms by which NCED regulates plant development and responses to abiotic stress in different crop species.


Assuntos
Dioxigenases/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Oryza/enzimologia , Proteínas de Plantas/metabolismo , Arabidopsis/genética , Arabidopsis/fisiologia , Desidratação , Dioxigenases/genética , Oryza/genética , Oryza/fisiologia , Folhas de Planta/enzimologia , Folhas de Planta/genética , Folhas de Planta/fisiologia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Estresse Fisiológico , Água/metabolismo
15.
Elife ; 82019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31535976

RESUMO

Plant nucleotide binding, leucine-rich repeat (NLR) receptors detect pathogen effectors and initiate an immune response. Since their discovery, NLRs have been the focus of protein engineering to improve disease resistance. However, this approach has proven challenging, in part due to their narrow response specificity. Previously, we revealed the structural basis of pathogen recognition by the integrated heavy metal associated (HMA) domain of the rice NLR Pikp (Maqbool et al., 2015). Here, we used structure-guided engineering to expand the response profile of Pikp to variants of the rice blast pathogen effector AVR-Pik. A mutation located within an effector-binding interface of the integrated Pikp-HMA domain increased the binding affinity for AVR-Pik variants in vitro and in vivo. This translates to an expanded cell-death response to AVR-Pik variants previously unrecognized by Pikp in planta. The structures of the engineered Pikp-HMA in complex with AVR-Pik variants revealed the mechanism of expanded recognition. These results provide a proof-of-concept that protein engineering can improve the utility of plant NLR receptors where direct interaction between effectors and NLRs is established, particularly where this interaction occurs via integrated domains.


Assuntos
Proteínas NLR/metabolismo , Proteínas de Plantas/metabolismo , Receptores Imunológicos/metabolismo , Antígenos de Bactérias/metabolismo , Proteínas NLR/genética , Oryza/enzimologia , Proteínas de Plantas/genética , Ligação Proteica , Engenharia de Proteínas , Receptores Imunológicos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
16.
Int J Mol Sci ; 20(15)2019 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-31382584

RESUMO

Low temperature is an environmental stress factor that is always been applied in research on improving crop growth, productivity, and quality of crops. Polyunsaturated fatty acids (PUFAs) play an important role in cold tolerance, so its genetic manipulation of the PUFA contents in crops has led to the modification of cold sensitivity. In this study, we over-expressed an ω-3 fatty acid desaturase from Glycine max (GmFAD3A) drove by a maize ubiquitin promoter in rice. Compared to the wild type (ZH11), ectopic expression of GmFAD3A increased the contents of lipids and total PUFAs. Seed germination rates in GmFAD3A transgenic rice were enhanced under low temperature (15 °C). Moreover, cold tolerance and survival ratio were significantly improved in GmFAD3A transgenic seedlings. Malondialdehyde (MDA) content in GmFAD3A transgenic rice was lower than that in WT under cold stress, while proline content obviously increased. Meanwhile, the activities of superoxide dismutase (SOD), hydroperoxidase (CAT), and peroxidase (POD) increased substantially in GmFAD3A transgenic rice after 4 h of cold treatment. Taken together, our results suggest that GmFAD3A can enhances cold tolerance and the seed germination rate at a low temperature in rice through the accumulation of proline content, the synergistic increase of the antioxidant enzymes activity, which finally ameliorated the oxidative damage.


Assuntos
Ácidos Graxos Dessaturases/genética , Oryza/genética , Plântula/genética , Estresse Fisiológico/genética , Catalase/genética , Regulação da Expressão Gênica de Plantas/genética , Germinação/genética , Metabolismo dos Lipídeos/genética , Oryza/enzimologia , Oryza/crescimento & desenvolvimento , Peroxidase/genética , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plântula/crescimento & desenvolvimento , Sementes/genética , Sementes/crescimento & desenvolvimento , Soja/enzimologia , Soja/genética , Superóxido Dismutase/genética
17.
Food Chem ; 301: 125271, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31376690

RESUMO

Mature endosperm was separated regionally into different parts in three rice cultivars, Te-qing (TQ), Wu-xiang 9915 (WX9915) and Guang-ling-xiang-nuo (GLXN), and their transgenic lines with inhibition of starch branching enzyme I and IIb (SBEI/IIb-). Within the three wild-type cultivars, starches from endosperm different regions showed similar molecular and crystalline structures. However, in rices with inhibition of SBEs, amylopectin short branch-chain content and branching degree gradually decreased, but amylopectin B3+ chain content and average chain length increased gradually from the interior to exterior of endosperm. The amylose content gradually increased from the interior to exterior of endosperm in TQ- and WX9915-SBEI/II- lines. From the interior to exterior of endosperm, starch changed gradually from CC- to CB-type in TQ-SBEI/II- line and from CA- to CC-type in GLXN-SBEI/II- line, and remained CA-type in WX9915-SBEI/II- line. These results provided some information for quality breeding and utilizations of rice with inhibition of SBE.


Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana/antagonistas & inibidores , Endosperma/enzimologia , Oryza/enzimologia , Amido/química , Amilopectina/química , Amilopectina/metabolismo , Amilose/análise , Endosperma/efeitos dos fármacos , Oryza/química , Oryza/efeitos dos fármacos , Oryza/metabolismo , Amido/metabolismo
18.
Bioelectrochemistry ; 130: 107336, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31377396

RESUMO

Cytokinin oxidase from Nipponbare (OsCKX4) was successfully displayed on the surface of E. coli cells by an ice nucleation protein from Pseudomonas borealis DL7 as an anchoring motif and a maltodextrin-binding protein(MBP) from E. coli as a solubility enhancer. The OsCKX4-displayed bacteria can be directly immobilized onto an electrode to selectively detect cytokinins, thus eliminating the need for enzyme extraction and purification. Direct electrochemistry of the cofactor FADH2 in OsCKX4 has been achieved on an edge-plane pyrolytic graphite electrode (PGE) with a formal potential (E0') of -0.45 V at pH 7.0 in phosphate buffer. With the addition of isopentenyladenine, the reduction peak current for FADH2 decreased, and the oxidative peak current increased gradually. Therefore, a bacteria-OsCKX4-modified PGE has been developed for the detection of isopentenyladenine with a linear range of 1.0-11.0 µM and a lower limit of detection of 0.7 µM (S/N = 3). Slight interference was observed in the presence of other phytohormones, including brassinosteroid, abscisic acid, methylene jasminate and gibberellin. The proposed bacterial biosensor is stable, specific and simple and has great potential for applications that require the detection of cytokinins.


Assuntos
Técnicas Biossensoriais/métodos , Citocininas/análise , Enzimas Imobilizadas/química , Oryza/enzimologia , Oxirredutases/química , Proteínas da Membrana Bacteriana Externa/química , Escherichia coli/química , Proteínas de Escherichia coli/química , Proteínas Periplásmicas de Ligação/química , Pseudomonas/química , Proteínas Recombinantes de Fusão/química
19.
J Biotechnol ; 305: 11-17, 2019 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-31430497

RESUMO

Fatty aldehydes are among the most important flavor and fragrance compounds. Most biotechnological production approaches make use of the one step conversion of fatty acids from renewable sources by the enzymes α-dioxygenase (αDox) or carboxylic acid reductase (CAR). Their reaction mechanisms and cofactor dependencies are very different. In contrast to heme-containing αDox which requires only oxygen as cosubstrate, CAR needs NADPH and ATP, which is a clear argument for the application of a whole cell catalyst. Therefore we compared fatty acid biotransformations with growing Escherichia coli cells expressing αDox or CAR to investigate their suitability for fatty aldehyde and also fatty alcohol production. Our results show the main product of fatty acid conversions with αDox-expressing cells to be the expected Cn-1 aldehyde. However, 14% of the products consist of the corresponding alcohol, but in addition, 17% of the products consist of further shortened aldehydes, alcohols and acids that result from the consecutive activity of αDox and a putative endogenous fatty aldehyde dehydrogenase activity in E. coli. Conversely, CAR-expressing cells produced only the unshortened fatty aldehyde and alcohol, whereby the latter surprisingly accounts for at least 80% of the products. The considerably higher extend of aldehyde reduction of CAR-expressing cells was shown to be causally connected to the CAR-mediated fatty acid conversion. Our study provides an overview about the applicability of αDox- or CAR-based whole cell catalysts and gives a detailed description of side products as well as suggestions for tailored strain engineering.


Assuntos
Dioxigenases/metabolismo , Escherichia coli/crescimento & desenvolvimento , Ácidos Graxos/biossíntese , Álcoois Graxos/metabolismo , Oxirredutases/metabolismo , Trifosfato de Adenosina/metabolismo , Aldeídos , Catálise , Dioxigenases/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Ácidos Graxos/metabolismo , Engenharia Genética , NADP/metabolismo , Oryza/enzimologia , Oryza/genética , Oxirredução , Oxirredutases/genética
20.
Org Biomol Chem ; 17(30): 7204-7214, 2019 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-31317164

RESUMO

A series of analogs of the iminosugars 1-deoxynojirimycin (DNJ) and 1-deoxymannojirimycin (DMJ), in which an extra five or six-membered ring has been fused to the C1-C2 bond have been prepared. The synthetic strategy exploits a key 2-keto-C-allyl iminosugar, easily accessible from gluconolactam, which upon Grignard addition and RCM furnishes a bicyclic scaffold that can be further hydroxylated at the C[double bond, length as m-dash]C bond. This strategy furnished DNJ mimics with the piperidine ring locked in a 1C4 conformation with all substituents in axial orientation when fused to a six-membered ring. Addition of an extra ring to DNJ and DMJ motif proved to strongly modify the glycosidase inhibition profile of the parent iminosugars leading to modest inhibitors. The 2-keto-C-allyl iminosugar scaffold was further used to access N-acetylglycosamine analogs via oxime formation.


Assuntos
1-Desoxinojirimicina/farmacologia , Inibidores de Glicosídeo Hidrolases/farmacologia , alfa-Glucosidases/metabolismo , beta-Glucosidase/antagonistas & inibidores , 1-Desoxinojirimicina/síntese química , 1-Desoxinojirimicina/química , Animais , Bovinos , Café/enzimologia , Relação Dose-Resposta a Droga , Inibidores de Glicosídeo Hidrolases/síntese química , Inibidores de Glicosídeo Hidrolases/química , Fígado/enzimologia , Conformação Molecular , Oryza/enzimologia , Relação Estrutura-Atividade , beta-Glucosidase/metabolismo
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