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1.
J Agric Food Chem ; 68(8): 2366-2372, 2020 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-32017555

RESUMO

Spermidine possesses multiple healthy functions, and soybeans contain the most abundant spermidine. In this study, spermidine contents of soybeans from different varieties and production regions in China were evaluated, and a spermidine synthase gene (speE) was identified by recombinant expression, transcriptional verification, and sequence analysis. Spermidine contents of soybean samples from 18 varieties ranged 72.38-228.82 mg/kg, and those from 19 production regions ranged 134.64-242.32 mg/kg. The highest-spermidine sample GZ was used to clone four predicted speE genes. Expressing the gene speE5 improved the spermidine titer by 54% in Bacillus amyloliquefaciens, confirming that speE5 was involved in spermidine synthesis. Transcriptional verification was performed through a soybean germination model. Germination for 48 h led to a onefold increase of spermidine in samples SHX and HB, and corresponding speE5 transcriptional levels were improved by 26-fold and 18-fold, respectively, further verifying the function of speE5. Finally, the sequences of the speE5 gene and deduced amino acids were analyzed, and the conserved sites and catalysis mechanisms were presented. This study identified an active spermidine synthase gene from soybean for the first time, which provided an important gene resource for genetic breeding of spermidine-rich soybean or microbial cell factory.


Assuntos
Proteínas de Plantas/genética , Soja/enzimologia , Espermidina Sintase/genética , Sequência de Aminoácidos , Bacillus amyloliquefaciens/genética , Bacillus amyloliquefaciens/metabolismo , Germinação , Dados de Sequência Molecular , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sementes/enzimologia , Sementes/genética , Sementes/crescimento & desenvolvimento , Sementes/metabolismo , Alinhamento de Sequência , Soja/genética , Soja/crescimento & desenvolvimento , Soja/metabolismo , Espermidina/metabolismo , Espermidina Sintase/química , Espermidina Sintase/metabolismo , Transcrição Genética
2.
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
3.
Artigo em Inglês | MEDLINE | ID: mdl-31896032

RESUMO

OsERdj7 is one of six endoplasmic reticulum (ER)-resident J-domain-containing proteins (J-proteins) encoded by the rice genome that acts as a co-chaperone for Hsp70 and is characterized by the presence of two transmembrane domains. It is N-glycosylated and primarily exists in a dimeric form with a molecular mass of 64 kDa. When the microsomal fraction of maturing seeds was treated with alkaline, high salt or detergent compounds, OsERdj7 was solubilized, even in alkaline and high salt environments, indicating that it is not tightly integrated in the ER membrane. Next, to investigate its role during seed maturation, expression of OsERdj7 was specifically downregulated using RNA interference (RNAi) under the control of the endosperm-specific 16 kDa prolamin promoter in transgenic rice. As a result, the unfolded protein response (UPR) was induced in maturing seeds via activation of OsIRE1/OsbZIP50 and ATF6 orthologs, such as OsbZIP39 and OsbZIP60, leading to upregulation of several chaperones and folding enzymes. Furthermore, some prolamins (RM4 and RM9) were retained in the ER lumen in the form of a mesh-like structure without deposition to the inherent ER-derived protein bodies (PB-Is), although major storage protein glutelins were normally transported to protein storage vacuoles (PB-IIs). On the other hand, induction of ER associated degradation (ERAD) increased OsERdj7 expression in transgenic rice seeds in which ERAD related genes were highly expressed. Due to PDIL2-3 and OsHard3 co-immunoprecipitating with OsERdj7 in rice protoplasts, this result implicates OsERdj7 in the translocation of some seed proteins within the ER lumen and in the degradation of misfolded or unfolded proteins.


Assuntos
Estresse do Retículo Endoplasmático/genética , Degradação Associada com o Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Endosperma/metabolismo , Chaperonas Moleculares/metabolismo , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Fator 6 Ativador da Transcrição/genética , Fator 6 Ativador da Transcrição/metabolismo , Endosperma/enzimologia , Endosperma/genética , Regulação da Expressão Gênica de Plantas/genética , Glutens/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Oryza/genética , Plantas Geneticamente Modificadas/genética , Prolaminas/metabolismo , Domínios Proteicos , Sementes/enzimologia , Sementes/genética , Sementes/crescimento & desenvolvimento , Vacúolos/metabolismo
4.
Artigo em Inglês | MEDLINE | ID: mdl-31862648

RESUMO

Indole-3-acetic acid (IAA) conjugation is one of the mechanisms responsible for auxin homeostasis. IAA ester conjugates biosynthesis has been studied during development of maize seeds where IAA-inositol (IAInos) and its glycosidic forms make up about 50 % of its ester conjugates pool. 1-O-indole-3-acetyl-ß-d-glucose (IAGlc) synthase and indole-3-acetyl transferase (IAInos synthase) are key enzymes in a two-step pathway of IAInos synthesis. In the first reaction, IAA is glucosylated to a high energy acetal, 1-O-indole-3-acetyl-ß-d-glucose by IAGlc synthase, whereas in the second step, IAInos synthase transfers IAA moiety to myo-inositol forming a stable auxin ester, indole-3-acetyl-myo-inositol (IAInos). It should be mentioned that IAGlc synthase catalyzes a reversible reaction with unfavourable equilibrium that delivers IAGlc for favourable transacylation to IAInos. This is the first study where IAGlc synthase and IAInos synthase are simultaneously analyzed by enzymatic activity assay and quantitative RT-PCR in maize seeds at four stages of development (13, 26, 39 and 52 Days After Flowering). Activity of IAGlc/IAInos synthases as well as their expression profiles during seed development were different. While both enzymatic activities and ZmIAIn expression were the highest in seeds at 26 DAF, the highest expression of ZmIAGlc was observed at 13 DAF. Protein gel blot analysis showed that IAInos synthase exists as a mixture of several isoforms at a similar protein level at particular stages of seed development. Neither of other ester conjugates of IAA (IAA-mannose) nor IAA-amino acids were detected at the stages studied. Catalytic activity of l-tryptophan aminotransferase involved in IAA biosynthesis as well as UDPG pyrophosphorylase, synthesizing UDPG as a substrate for IAGlc synthase, were also analyzed. l-tryptophan aminotransferase activity was the highest at 26 DAF. Changes in enzyme activity of UDPG pyrophosphorylase are difficult to interpret. Expression levels of ZmIPS and ZmIPP encoding two enzymes of myo-inositol biosynthesis pathway: inositol-x-phosphate synthase (IPS) and inositol-x-phosphate phosphatase (IPP), respectively, were analyzed. 26 DAF seeds displayed the highest expression level of ZmIPS, whereas transcription of ZmIPP was the highest at 13 DAF.


Assuntos
Glucosiltransferases/metabolismo , Ácidos Indolacéticos/metabolismo , Sementes/enzimologia , Sementes/crescimento & desenvolvimento , Zea mays/enzimologia , Zea mays/crescimento & desenvolvimento , Vias Biossintéticas/genética , Vias Biossintéticas/fisiologia , Catálise , Glucosiltransferases/genética , Homeostase/genética , Homeostase/fisiologia , Indóis/metabolismo , Inositol/metabolismo , Inositol Polifosfato 5-Fosfatases/metabolismo , Liases Intramoleculares/metabolismo , Reguladores de Crescimento de Planta/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Triptofano Transaminase/metabolismo , UTP-Glucose-1-Fosfato Uridililtransferase/metabolismo , Uridina Difosfato Glucose/metabolismo , Zea mays/metabolismo
5.
Planta ; 251(1): 29, 2019 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-31802247

RESUMO

MAIN CONCLUSION: A novel allele of the sugary-1 rice mutant was isolated. The single amino acid change led to isoamylase activity reduction and accumulation of high-molecular-weight phytoglycogen in seeds. A new sugary rice variety with an improved seed appearance has been isolated and designated Hemisugary1. This mutant, which was derived from Japonica-type cultivar Tsugaruroman treated with sodium azide, has about half the isoamylase activity of seeds in the original Tsugaruroman. The mutant also accumulates significant phytoglycogen, albeit approximately 40% of the total phytoglycogen in the existing sugary cultivar Ayunohikari which is defective in its most isoamylase activity. The site of mutation was identified using a re-sequence of the whole genome and a cleaved amplified polymorphic sequence (CAPS) marker. The hemisugary phenotypes of the F2 progeny were entirely consistent with the results of genotyping using the CAPS marker. Segregation analysis of the F2 population showed that the hemisugary phenotype was controlled by a single recessive gene, which was produced by a G → A single nucleotide polymorphism in the sugary-1 gene, resulting in a missense mutation from glycine to aspartic acid at amino acid position 333. Zymogram showed that this amino acid replacement resulted in a decrease in isoamylase activity with a concomitant reduction in the formation of isoamylase complexes. Phytoglycogen molecules from Hemisugary1 seeds were 3.5 times larger and contained more short glucan chains than did Ayunohikari seeds. Our data provide new insights into the relationship between isoamylase structure and phytoglycogen formation.


Assuntos
Alelos , Genes de Plantas , Mutação/genética , Oryza/genética , Açúcares/metabolismo , Sequência de Bases , Segregação de Cromossomos/genética , Glucanos/metabolismo , Glucose/metabolismo , Modelos Moleculares , Oryza/enzimologia , Fenótipo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Polimorfismo de Nucleotídeo Único/genética , Sementes/anatomia & histologia , Sementes/enzimologia , Solubilidade , Água/química
6.
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
7.
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
8.
Int J Mol Sci ; 20(15)2019 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-31370140

RESUMO

The drought is a crucial environmental factor that determines yielding of many crop species, e.g., Fabaceae, which are a source of valuable proteins for food and feed. Herein, we focused on the events accompanying drought-induced activation of flower abscission zone (AZ)-the structure responsible for flower detachment and, consequently, determining seed production in Lupinus luteus. Therefore, detection of molecular markers regulating this process is an excellent tool in the development of improved drought-resistant cultivars to minimize yield loss. We applied physiological, molecular, biochemical, immunocytochemical, and chromatography methods for a comprehensive examination of changes evoked by drought in the AZ cells. This factor led to significant cellular changes and activated AZ, which consequently increased the flower abortion rate. Simultaneously, drought caused an accumulation of mRNA of genes inflorescence deficient in abscission-like (LlIDL), receptor-like protein kinase HSL (LlHSL), and mitogen-activated protein kinase6 (LlMPK6), encoding succeeding elements of AZ activation pathway. The content of hydrogen peroxide (H2O2), catalase activity, and localization significantly changed which confirmed the appearance of stressful conditions and indicated modifications in the redox balance. Loss of water enhanced transcriptional activity of the abscisic acid (ABA) and ethylene (ET) biosynthesis pathways, which was manifested by elevated expression of zeaxanthin epoxidase (LlZEP), aminocyclopropane-1-carboxylic acid synthase (LlACS), and aminocyclopropane-1-carboxylic acid oxidase (LlACO) genes. Accordingly, both ABA and ET precursors were highly abundant in AZ cells. Our study provides information about several new potential markers of early response on water loss, which can help to elucidate the mechanisms that control plant response to drought, and gives a useful basis for breeders and agronomists to enhance tolerance of crops against the stress.


Assuntos
Produtos Agrícolas/genética , Secas , Flores/genética , Regulação da Expressão Gênica de Plantas , Lupinus/genética , Proteínas de Plantas/genética , Sementes/genética , Ácido Abscísico/metabolismo , Adaptação Fisiológica/genética , Catalase/genética , Catalase/metabolismo , Produtos Agrícolas/enzimologia , Produtos Agrícolas/crescimento & desenvolvimento , Etilenos/biossíntese , Flores/enzimologia , Flores/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Peróxido de Hidrogênio , Ligases/genética , Ligases/metabolismo , Lupinus/enzimologia , Lupinus/crescimento & desenvolvimento , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Oxirredução , Oxirredutases/genética , Oxirredutases/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Sementes/enzimologia , Sementes/crescimento & desenvolvimento , Estresse Fisiológico/genética
9.
Planta ; 250(5): 1655-1670, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31407031

RESUMO

MAIN CONCLUSION: The transfer of polyunsaturated fatty acids from phosphatidylcholine to other lipids involves several enzymes. In Camelina sativa seeds, acyl-CoA:lysophosphatidylcholine acyltransferases could be one of the most important players in this process. The transfer of polyunsaturated fatty acids from the location of their synthesis (phosphatidylcholine) to other lipids, e.g., triacylglycerol, remains insufficiently understood. Several enzymes could be involved in this process. One of these enzymes is acyl-CoA:lysophosphatidylcholine acyltransferases (LPCATs). In Camelina sativa seeds, LPCATs could be one of the most important players in this process. Our data clearly indicate that the CsLPCATs present in developing seeds have the potential to transfer almost all polyunsaturated fatty acids synthesised on phosphatidylcholine to the acyl-CoA pool. CsLPCAT activity is the highest at 30 °C, and the enzymes operate well at a pH of 7.0-11.0, with the best activity at a pH of 9.0. The activity of CsLPCATs was inhibited by calcium and magnesium ions at a concentration of 0.05-2 mM. In the forward reaction, CsLPCATs preferentially utilise 18:2-CoA; however, other C18 unsaturated fatty acids are also well accepted. In the backward reactions, there is no clear discrimination between the C18 unsaturated fatty acids utilised by the enzymes for phosphatidylcholine remodelling. The activity of CsLPCATs does not differ much between the stages of seed development.


Assuntos
1-Acilglicerofosfocolina O-Aciltransferase/metabolismo , Brassicaceae/enzimologia , 1-Acilglicerofosfocolina O-Aciltransferase/genética , Brassicaceae/genética , Brassicaceae/crescimento & desenvolvimento , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Sementes/enzimologia , Sementes/genética , Sementes/crescimento & desenvolvimento
10.
Int J Biol Macromol ; 138: 1044-1055, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31348972

RESUMO

In the present study, out of three isoforms of α-mannosidase identified in the crude extract of defatted Custard apple seed powder, isoform III has been purified to homogeneity by two-step chromatography: hydrophobic interaction and gel filtration. The purified Custard apple α-mannosidase isoform III (CAM) hydrolyzed both chromogenic (p-nitrophenyl-α-D-mannopyranoside) and fluorescent (4-methylumbelliferyl α-D-mannopyranoside) substrates. Custard apple α-mannosidase migrated as a single band in native PAGE, showed about 220 kDa molecular mass in gel filtration and in SDS PAGE, dissociated into four bands (Mr ~ 75, 68, 56 and 50 kDa respectively). Temperature and pH optima were found to be 50 °C and 4.0-5.0 respectively and CAM was stable up to 60-70 °C. The enzymatic activity of CAM was inhibited by EDTA, Ag+, Hg2+, Ni2+ and swainsonine (IC50 value of 1.5 µM). CAM was observed to be a metallo enzyme requiring zinc for its activity. Kinetic parameters KM and Vmax were found to be 1.75 mM and 0.068 U/mL respectively. The CD spectral analysis at far UV region (190-300 nm) shows that purified CAM exists as helix (30.4%), ß turns (18%) and random coils (29.7%) in its secondary structure. Chemical modification studies with N-Bromosuccinimide revealed the presence of tryptophan in its active site.


Assuntos
Annona/enzimologia , Sementes/enzimologia , Zinco/química , alfa-Manosidase/química , alfa-Manosidase/isolamento & purificação , Cromatografia , Ativação Enzimática , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Isoenzimas , Temperatura
11.
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
12.
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
13.
Int J Mol Sci ; 20(13)2019 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-31284614

RESUMO

We examined the substrate preference of Cuphea paucipetala acyl-ACP thioesterases, CpFatB4 and CpFatB5, and gene expression changes associated with the modification of lipid composition in the seed, using Brassica napus transgenic plants overexpressing CpFatB4 or CpFatB5 under the control of a seed-specific promoter. CpFatB4 seeds contained a higher level of total saturated fatty acid (FA) content, with 4.3 times increase in 16:0 palmitic acid, whereas CpFatB5 seeds showed approximately 3% accumulation of 10:0 and 12:0 medium-chain FAs, and a small increase in other saturated FAs, resulting in higher levels of total saturated FAs. RNA-Seq analysis using entire developing pods at 8, 25, and 45 days after flowering (DAF) showed up-regulation of genes for ß-ketoacyl-acyl carrier protein synthase I/II, stearoyl-ACP desaturase, oleate desaturase, and linoleate desaturase, which could increase unsaturated FAs and possibly compensate for the increase in 16:0 palmitic acid at 45 DAF in CpFatB4 transgenic plants. In CpFatB5 transgenic plants, many putative chloroplast- or mitochondria-encoded genes were identified as differentially expressed. Our results report comprehensive gene expression changes induced by alterations of seed FA composition and reveal potential targets for further genetic modifications.


Assuntos
Brassica napus/enzimologia , Brassica napus/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Sementes/enzimologia , Sementes/genética , Tioléster Hidrolases/genética , Brassica napus/crescimento & desenvolvimento , Ontologia Genética , Genes de Plantas , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , Tioléster Hidrolases/metabolismo , Transcriptoma/genética
14.
New Phytol ; 224(1): 336-351, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31211859

RESUMO

Nitrogen (N) deficiency triggers an accumulation of a storage lipid triacylglycerol (TAG) in seed plants and algae. Whereas the metabolic pathway and regulatory mechanism to synthesize TAG from diacylglycerol are well known, enzymes involved in the supply of diacylglycerol remain elusive under N starvation. Lysophosphatidic acid acyltransferase (LPAT) catalyzes an important step of the de novo phospholipid biosynthesis pathway and thus has a strong flux control in the biosynthesis of phospholipids and TAG. Five LPAT isoforms are known in Arabidopsis; however, the functions of LPAT4 and LPAT5 remain elusive. Here, we show that LPAT4 and LPAT5 are functional endoplasmic-reticulum-localized LPATs. Seedlings of the double knockout mutant lpat4-1 lpat5-1 showed reduced content of phospholipids and TAG under normal growth condition. Under N starvation, lpat4-1 lpat5-1 seedlings showed severer growth defect than the wild-type in shoot. The phenotype was similar to dgat1-4, which affects a major TAG biosynthesis pathway and showed similarly reduced TAG content as the lpat4-1 lpat5-1. We suggest that LPAT4 and LPAT5 may redundantly function in endoplasmic-reticulum-localized de novo glycerolipid biosynthesis for phospholipids and TAG, which is important for the N starvation response in Arabidopsis.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/metabolismo , Nitrogênio/deficiência , Triglicerídeos/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , DNA Bacteriano/genética , Ácidos Graxos/metabolismo , Regulação da Expressão Gênica de Plantas , Mutação/genética , Especificidade de Órgãos/genética , Fenótipo , Ácidos Fosfatídicos/metabolismo , Folhas de Planta/metabolismo , Folhas de Planta/ultraestrutura , Brotos de Planta/anatomia & histologia , Brotos de Planta/crescimento & desenvolvimento , Proteínas Recombinantes/metabolismo , Plântula/crescimento & desenvolvimento , Sementes/enzimologia , Sementes/crescimento & desenvolvimento
15.
Plant Signal Behav ; 14(8): 1620059, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31131700

RESUMO

Fertilization-related kinase (FRK) is a group of the mitogen-activated protein kinase kinase kinase (MAP3K or MEKK) that has proliferated in Solanaceae species. Studies on the wild potato Solanum chacoense have shown that three ScFRKs are directly involved in female gametophyte development. Decreasing the expression of ScFRK1 and ScFRK3 by RNA interference lead to embryonic sac development arrest at the functional megaspore (FM) stage. As for ScFRK2, the first FRK studied, antisense and co-suppression lines showed no abnormality, while overexpression lines lead to a drastic decrease in seed numbers, presumably caused by a conversion of the ovule into a carpel-like structure. Here we show that in ScFRK2 overexpression lines, carpel-like structures from the ovule cannot explain the drastic decrease in seeds considering the low percentage of these carpel-like structures but occurs in early ovule development as observed in Scfrk1 and Scfrk3 knockdown mutants were most ovules are arrested at the FM stage. The highly similar phenotype from knockdown mutants (Scfrk1 and Scfrk3) and ScFRK2 overexpression lines suggests that these MAP kinases could operate antagonistically through a balance between ScFRK1 and 3 on one side and ScFRK2 on the other. This study strongly suggests the importance of the FRK family expression levels during early stages of ovule development in Solanum chacoense embryo sac.


Assuntos
MAP Quinase Quinase Quinases/metabolismo , Plantas Geneticamente Modificadas/embriologia , Plantas Geneticamente Modificadas/enzimologia , Solanum/embriologia , Solanum/enzimologia , Regulação da Expressão Gênica de Plantas , MAP Quinase Quinase Quinases/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Sementes/embriologia , Sementes/enzimologia , Sementes/metabolismo , Solanum/metabolismo
16.
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
17.
BMC Biotechnol ; 19(1): 24, 2019 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-31035982

RESUMO

BACKGROUND: Increasing the content of oleic acid in peanut seeds is one of the major goals in peanut breeding due to consumer and industry benefits, such as anti-oxidation and long shelf-life. Homeologous ahFAD2A and ahFAD2B genes encode fatty acid desaturases, which are the key enzymes for converting oleic acid to linoleic acid that oxidizes readily. To date, all high oleic acid peanut varieties result from natural mutations occurred in both genes. A method to induce mutations in the genes of other elite cultivars could speed introgression of this valuable trait. The gene-editing approach utilizing CRISPR/Cas9 technology was employed to induce de novo mutations in the ahFAD2 genes using peanut protoplasts and hairy root cultures as models. RESULTS: The hot spot of natural mutation in these genes was selected as the target region. Appropriate sgRNAs were designed and cloned into a CRISPR/Cas9 expression plasmid. As a result of CRISPR/Cas9 activity, three mutations were identified - G448A in ahFAD2A, and 441_442insA and G451T in ahFAD2B. The G448A and 441_442insA mutations are the same as those seen in existing high oleate varieties and the G451T is new mutation. Because natural mutations appear more often in the ahFAD2A gene than in the ahFAD2B gene in subspecies A. hypogaea var. hypogaea, the mutations induced in ahFAD2B by gene editing may be useful in developing high oleate lines with many genetic backgrounds after validation of oleic acid content in the transformed lines. The appearance of the G448A mutation in ahFAD2A is a further benefit for high oleic acid oil content. CONCLUSIONS: Overall, these results showed that mutations were, for the first time, induced by CRISPR-based gene editing approach in peanut. This research demonstrated the potential application of gene editing for mutagenesis in peanut and suggested that CRISPR/Cas9 technology may be useful in the peanut breeding programs.


Assuntos
Arachis/genética , Sistemas CRISPR-Cas , Ácidos Graxos Dessaturases/genética , Edição de Genes/métodos , Mutagênese , Proteínas de Plantas/genética , Arachis/enzimologia , Sequência de Bases , Ácidos Graxos Dessaturases/metabolismo , Ácido Linoleico/metabolismo , Ácido Oleico/metabolismo , Melhoramento Vegetal/métodos , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Sementes/enzimologia , Sementes/genética
18.
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
19.
J Sci Food Agric ; 99(12): 5384-5391, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31077382

RESUMO

BACKGROUND: Soybean seeds contain 18-24% lipids, which are made up of 85% polyunsaturated fatty acids. Two of these (linoleic and linolenic acids) comprise essential fatty acids that are not synthesized in humans and animals. Linolenic acid plays a vital role in the maintenance of brain function and is a source of docosahexaenoic acid for retinal and nerve tissue, with its physiological functions being a focus of attention. RESULTS: We developed mutant soybean populations via gamma irradiation of Korean cultivars Danbaek and Daepung and evaluated the linolenic acid content of 78 and 154 M9 mutant progenies. We selected the four mutant lines with the highest linolenic acid contents based on 2 years of investigation of fatty acids. The selected mutant lines had linolenic acid contents that were 33.9% to 67.7% higher than those of the original cultivars and exhibited increased fatty acid desaturase (FAD) gene expression levels during seed development. We also identified nucleotide polymorphisms of FAD genes in the four mutant lines. CONCLUSION: The present study found that linolenic acid content is related to significantly increased expression levels of the FAD3C and FAD3D genes in the endoplasmic reticulum, which was uncovered by radiation mutation breeding of soybean. © 2019 Society of Chemical Industry.


Assuntos
Ácidos Graxos Dessaturases/genética , Proteínas de Plantas/genética , Sementes/crescimento & desenvolvimento , Soja/enzimologia , Soja/genética , Ácido alfa-Linoleico/análise , Ácidos Graxos Dessaturases/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Mutação , Proteínas de Plantas/metabolismo , Polimorfismo de Nucleotídeo Único , Sementes/química , Sementes/enzimologia , Sementes/genética , Soja/química , Soja/crescimento & desenvolvimento , Ácido alfa-Linoleico/metabolismo
20.
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
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