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1.
Plant Physiol Biochem ; 141: 164-171, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31170640

RESUMO

For a comprehensive understanding of gene expression, enzyme activity and sugar concentrations in response to short-term water deficit in apple (Greensleeves), sugar-modulated gene expression and enzyme activities were analyzed. Water stress resulted in the accumulation of sorbitol, glucose, fructose, galactose and starch, accompanied by a significant reduction in photosynthesis and sucrose concentration. In response to short-term water deficits, the activities of aldose-6-phosphate reductase (A6PR; EC 1.1.1.200), sorbitol dehydrogenase (SDH; EC 1.1.1.14), neutral invertase (NINV; EC 3.2.1.26), sucrose synthase (SUSY; EC 2.4.1.13), and fructokinase (FK; EC 2.7.1.4) were higher, whereas cell wall invertase (CWINV; EC 3.2.1.26) and hexokinase (HK; EC 2.7.1.1) activities were lower. In addition, sucrose phosphate synthase (SPS; EC 2.4.1.14) activity increased during the initial stages of dehydration and then decreased as the drought strengthened. Transcript levels of MdA6PR, MdSDH1/2, MdNINV1/2, MdSUSY3, MdFK1/2/4, MdSOT1/2, MdSUC1-3, MdTMT2/3, MdvGT1, MdpGlcT1-4 were upregulated, whereas transcript levels of MdCWINV1/2, MdHK1/2/3/5, and MdTMT1 were downregulated after 6 days of water stress. These findings suggest that the sorbitol metabolism pathway is induced and high levels of hexose derived from photosynthetic products are transported into vacuoles for adjustment to the water deficit. Our results provide insights into the relationships between sugar levels and sugar-modulated gene and enzyme activity in response to the imposition of short-term water stress.


Assuntos
Metabolismo dos Carboidratos/genética , Secas , Regulação da Expressão Gênica de Plantas , Malus/fisiologia , Folhas de Planta/fisiologia , Parede Celular/metabolismo , Frutas/metabolismo , Glucosiltransferases/metabolismo , Malus/genética , Fotossíntese , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/fisiologia , Sorbitol/metabolismo , Estresse Fisiológico , Sacarose/metabolismo , beta-Frutofuranosidase/metabolismo
2.
J Environ Sci Health B ; 54(6): 449-458, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30939985

RESUMO

Fipronil is a broad-spectrum insecticide that has a good control effect on pests of commercial poultry. Although many studies have reported the environmental fate of fipronil, the influence of residual fipronil in poultry waste on biogas production has not been further explored yet. In this article, an experimental comparative study on anaerobic digestion (AD) of chicken manure (CM) and corn straw (CS) with different fipronil concentrations (FCs) was carried at 8% of total solid (TS) and mid-temperature (35 ± 1)°C. The results showed that fipronil had a significant effect on biogas production during AD of CM and CS. When the FC is at a low level (≤10 mg·kg-1), the biogas production rate is increased and the digestion period was shortened, while higher FC (≥ 20 mg·kg-1) showed an inhibitory effect. During the monitoring of enzyme activity, low FC showed no significant effect on cellulase and saccharase, but the urease activity increased in the early stage. High FC showed inhibition of activity of cellulase and urease, but the saccharase activity was significantly inhibited until FC reached 40 mg·kg-1. This study also confirms that the environment in anaerobic digester is favorable for the degradation of fipronil, and its half-life is about 15.83 days.


Assuntos
Biocombustíveis , Esterco , Pirazóis , Eliminação de Resíduos Líquidos/métodos , Zea mays , Anaerobiose/efeitos dos fármacos , Animais , Celulase/antagonistas & inibidores , Celulase/metabolismo , Galinhas , Meia-Vida , Inseticidas/metabolismo , Inseticidas/farmacologia , Metano , Caules de Planta/metabolismo , Pirazóis/metabolismo , Pirazóis/farmacologia , Temperatura Ambiente , Eliminação de Resíduos Líquidos/instrumentação , beta-Frutofuranosidase/antagonistas & inibidores , beta-Frutofuranosidase/metabolismo
3.
Environ Sci Pollut Res Int ; 26(13): 13630-13648, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30919191

RESUMO

The objective of the present investigation was to consider the effectiveness of exogenous silicate supplementation in reviving the arsenate imposed alterations on pigment content, Hill activity, photosynthetic parameters, sugar metabolism, polyamine, and ion contents in wheat (Triticum aestivum L. cv. PBW-343) seedlings. Experiments were conducted under different levels of arsenate (0, 25 µM, 50 µM, and 100 µM) in combination with silicate (0, 5 mM) in a hydroponic environment with modified Hoagland's solution for 21 days to determine the ameliorative role of silicon (Si). Arsenate exposure led to a decline in chlorophyll content by 28% and Hill activity by 30% on an average along with photosynthetic parameters. Activity of starch phosphorylase increased causing a subsequent decrease in starch contents by 26%. Degradation of starch enhanced sugar contents by 61% in the test cultivar. Dose-dependant increments in the activities of carbohydrate metabolizing enzymes viz., sucrose synthase, sucrose phosphate synthase, and acid invertase were also noted. Putrescine content was significantly enhanced along with a consequent decline in spermidine and spermine contents. The macro- and micronutrient contents declined proportionally with arsenate imposition. Conversely, silicate amendments irrespective of all arsenate concentrations brought about considerable alterations in all parameters tested with respect to arsenate treatment alone. Marked improvement in pigment content and Hill activity also improved the gas exchange parameters. Soluble sugar contents decreased and starch contents were enhanced. Increase in polyamine contents improved the ionic balance in the test cultivar as well. This study highlights the potentiality of silicon in ameliorating the ecotoxicological risks associated with arsenic pollution and the probable ability of silicon to offer an approach in mitigating arsenate-induced stress leading to restoration of growth and metabolism in wheat seedlings.


Assuntos
Arsênico/metabolismo , Clorofila/metabolismo , Glucosiltransferases/metabolismo , Hidroponia/métodos , Poliaminas/metabolismo , Plântula/metabolismo , Silício/química , Açúcares/metabolismo , Triticum/crescimento & desenvolvimento , beta-Frutofuranosidase/metabolismo , Arsênico/química , Metabolismo dos Carboidratos , Clorofila/química , Glucosiltransferases/química , Fotossíntese , Poliaminas/química , Plântula/química , Silício/farmacologia , Açúcares/química , beta-Frutofuranosidase/química
4.
Enzyme Microb Technol ; 125: 53-62, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30885325

RESUMO

The thermostable ß-fructosidase (BfrA) from the bacterium Thermotoga maritima converts sucrose into glucose, fructose, and low levels of short-chain fructooligosaccharides (FOS) at high substrate concentration (1.75 M) and elevated temperatures (60-70 °C). In this research, FOS produced by BfrA were characterized by HPAE-PAD analysis as a mixture of 1-kestotriose, 6G-kestotriose (neokestose), and to a major extent 6-kestotriose. In order to increase the FOS yield, three BfrA mutants (W14Y, W14Y-N16S and W14Y-W256Y), designed from sequence divergence between hydrolases and transferases, were constructed and constitutively expressed in the non-saccharolytic yeast Pichia pastoris. The secreted recombinant glycoproteins were purified and characterized. The three mutants synthesized 6-kestotriose as the major component of a FOS mixture that includes minor amounts of tetra- and pentasaccharides. In all cases, sucrose hydrolysis was the predominant reaction. All mutants reached a similar overall FOS yield, with the average value 37.6% (w/w) being 3-fold higher than that of the wild-type enzyme (12.6%, w/w). None of the mutations altered the enzyme thermophilicity and thermostability. The single mutant W14Y, with specific activity of 841 U mg-1, represents an attractive candidate for the continuous production of FOS-containing invert syrup at pasteurization temperatures.


Assuntos
Proteínas de Bactérias/metabolismo , Oligossacarídeos/biossíntese , Thermotoga maritima/enzimologia , beta-Frutofuranosidase/metabolismo , Proteínas de Bactérias/genética , Domínio Catalítico , Expressão Gênica , Concentração de Íons de Hidrogênio , Cinética , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Mutação , Oligossacarídeos/química , Pichia/genética , Pichia/metabolismo , Engenharia de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Sacarose/metabolismo , Temperatura Ambiente , Thermotoga maritima/genética , beta-Frutofuranosidase/genética
5.
ScientificWorldJournal ; 2019: 6956202, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30728756

RESUMO

ß-fructofuranosidase (invertase) and ß-D-fructosyltransferase (FTase) are enzymes used in industrial processes to hydrolyze sucrose aiming to produce inverted sugar syrup or fructooligosaccharides. In this work, a black Aspergillus sp. PC-4 was selected among six filamentous fungi isolated from canned peach syrup which were initially screened for invertase production. Cultivations with pure carbon sources showed that invertase and FTase were produced from glucose and sucrose, but high levels were also obtained from raffinose and inulin. Pineapple crown was the best complex carbon source for invertase (6.71 U/mL after 3 days of cultivation) and FTase production (14.60 U/mL after 5 days of cultivation). Yeast extract and ammonium chloride nitrogen sources provided higher production of invertase (6.80 U/mL and 6.30 U/mL, respectively), whereas ammonium nitrate and soybean protein were the best nitrogen sources for FTase production (24.00 U/mL and 24.90 U/mL, respectively). Fermentation parameters for invertase using yeast extract were Y P/S = 536.85 U/g and P P = 1.49 U/g/h. FTase production showed values of Y P/S = 2,627.93 U/g and P P = 4.4 U/h using soybean protein. The screening for best culture conditions showed an increase of invertase production values by 5.10-fold after 96 h cultivation compared to initial experiments (fungi bioprospection), while FTase production increased by 14.60-fold (44.40 U/mL) after 168 h cultivation. A. carbonarius PC-4 is a new promising strain for invertase and FTase production from low cost carbon sources, whose synthesized enzymes are suitable for the production of inverted sugar, fructose syrups, and fructooligosaccharides.


Assuntos
Aspergillus/enzimologia , Alimentos em Conserva/microbiologia , Proteínas Fúngicas/metabolismo , Hexosiltransferases/metabolismo , beta-Frutofuranosidase/metabolismo , Aspergillus/efeitos dos fármacos , Carbono/metabolismo , Carbono/farmacologia , Meios de Cultura/química , Meios de Cultura/metabolismo , Meios de Cultura/farmacologia , Fermentação , Proteínas Fúngicas/isolamento & purificação , Hexosiltransferases/isolamento & purificação , Xarope de Milho Rico em Frutose , Microbiologia Industrial/métodos , Nitrogênio/metabolismo , Nitrogênio/farmacologia , Prunus persica/química , Prunus persica/microbiologia , beta-Frutofuranosidase/isolamento & purificação
6.
Amino Acids ; 51(4): 599-610, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30673850

RESUMO

Knowing the key features of the structure and the biochemistry of proteins is crucial to improving enzymes of industrial interest like ß-fructofuranosidase. Gene sacA from Bacillus licheniformis ATCC 14580 codifies a sucrose-6-phosphate hydrolase, a ß-fructofuranosidase (E.C. 3.1.2.26, protein BlsacA), which has no crystallographic structure available. In this study, we report the results from numerous biochemical and biophysical techniques applied to the investigation of BlsacA in solution. BlsacA was successfully expressed in E. coli in soluble form and purified using affinity and size-exclusion chromatographies. Results showed that the optimum activity of BlsacA occurred at 30 °C around neutrality (pH 6.0-7.5) with a tendency to alkalinity. Circular dichroism spectrum confirmed that BlsacA contains elements of a ß-sheet secondary structure at the optimum pH range and the maintenance of these elements is related to BlsacA enzymatic stability. Dynamic light scattering and small-angle X-ray scattering measurements showed that BlsacA forms stable and elongated homodimers which displays negligible flexibility in solution at optimum pH range. The BlsacA homodimeric nature is strictly related to its optimum activity and is responsible for the generation of biphasic curves during differential scanning fluorimetry analyses. The homodimer is formed through the contact of the N-terminal ß-propeller domain of each BlsacA unit. The results presented here resemble the key importance of the homodimeric form of BlsacA for the enzyme stability and the optimum enzymatic activity.


Assuntos
Bacillus licheniformis/enzimologia , Sacarose/análogos & derivados , Fosfatos Açúcares/metabolismo , beta-Frutofuranosidase/química , beta-Frutofuranosidase/metabolismo , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Cinética , Estrutura Secundária de Proteína , Espalhamento a Baixo Ângulo , Especificidade por Substrato , Sacarose/metabolismo , Difração de Raios X
7.
Mar Pollut Bull ; 138: 598-603, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30660311

RESUMO

Seagrass meadows are experiencing worldwide declines mainly because of nutrient enrichment, which always result in macroalgae bloom and consequently periodic collapse and decomposition. However, effects of macroalgae decay on the sediment organic carbon (SOC) sequestration capacity remain unknown. Depending on the macroalgae biomass in eutrophic seagrass meadows of South China Sea, we carried out a laboratory chamber experiment to investigate the sediment labile organic carbon (OC) compositions and the influencing SOC transformation enzyme activity variations of seagrass meadows in response to common macroalgae bloom species (Cladophora spp.) decomposition. Although the dehydrogenase and ß-glucosidase activities were not affected by macroalgae decomposition, the macroalgae decomposition significantly elevated the salt-extractable carbon (SEC) content, SEC/SOC, levels of invertase and polyphenol oxidase activities, and the CO2 release. Overall, this study indicates that macroalgae decomposition stimulates the SOC transformation, and therefore, it is not benefit for SOC sequestration within seagrass meadows of the South China Sea.


Assuntos
Sequestro de Carbono , Alga Marinha/fisiologia , Carbono/metabolismo , Dióxido de Carbono/metabolismo , Catecol Oxidase/metabolismo , Celulases/metabolismo , China , Ecossistema , Eutrofização , Sedimentos Geológicos , Oxirredutases/metabolismo , Alga Marinha/crescimento & desenvolvimento , beta-Frutofuranosidase/metabolismo
8.
Int J Biol Macromol ; 125: 1103-1111, 2019 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-30578899

RESUMO

It has been thought that when different strains of Aureobasidium spp. were grown in sucrose, the produced fructooligosaccharides (FOSs) by ß-d-fructofuranosidase were beneficial for their cell growth and pullulan biosynthesis. However, it is still unknown about how ß-d-fructofuranosidases activity and synthesized FOSs influence on pullulan biosynthesis. It was found that the genomic DNA of Aureobasidium melanogenum P16, a high pullulan producing yeast, contained three genes encoding ß-d-fructofuranosidase1, ß-d-fructofuranosidase2 and ß-d-fructofuranosidase3. The FTR1 factor, a transcriptional activator, activated expression of the three ß-d-fructofuranosidase genes and invertase gene. Disruption of the FTR1 gene rendered a disruptant DF3 to produce less FOSs (12.1 ±â€¯0.4 g/L), less ß-d-fructofuranosidase activity (1.1 ±â€¯0.2 U/mL), lower Mw (3.8 × 105) of the pullulan and more pullulan titer (77.0 ±â€¯2.6 g/L) than the yeast strain P16. Similarly, removal of both the two genes encoding ß-d-fructofuranosidase1 and ß-d-fructofuranosidase3 resulted in a double mutant DF4-7 producing 77.5 ±â€¯3.1 g/L pullulan with Mw of 3.4 × 105, 0.2 ±â€¯0.0 U/mL of ß-d-fructofuranosidase activity and the trace amount of FOSs while its wild type strain P16 yielded 65.7 ±â€¯3.5 g/L pullulan with Mw of 4.4 × 105, 6.8 ±â€¯0.0 U/mL of ß-d-fructofuranosidase activity and 6.2 ±â€¯0.5 g/L of FOSs. These confirmed that high ß-d-fructofuranosidase activity, the presence of high level of FOSs negatively influenced pullulan biosynthesis, but positively increased Mw of the produced pullulan. However, the ß-d-fructofuranosidase2 had no such function. Furthermore, complementation of the FTR1 gene, ß-d-fructofuranosidase1 gene and ß-d-fructofuranosidase3 gene enabled the corresponding transformants to restore ß-d-fructofuranosidase activity, FOSs and pullulan biosynthesis and Mw of the pullulan.


Assuntos
Ascomicetos/metabolismo , Metabolismo dos Carboidratos , Glucanos/biossíntese , Oligossacarídeos/biossíntese , beta-Frutofuranosidase/metabolismo , Ascomicetos/enzimologia , Ascomicetos/genética , Clonagem Molecular , Expressão Gênica , Sacarose/metabolismo , Fatores de Transcrição/metabolismo , beta-Frutofuranosidase/genética
9.
BMC Plant Biol ; 18(1): 228, 2018 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-30309330

RESUMO

BACKGROUND: Vacuolar invertases (VINs) have been reported to regulate plant growth and development and respond to abiotic stresses such as drought and cold. With our best knowledge, the functions of VIN genes little have been reported in tea plant (Camellia sinensis L.). Therefore, it is necessary to develop research in this field. RESULTS: Here, we identified a VIN gene, CsINV5, which was induced by cold acclimation and sugar treatments in the tea plant. Histochemical assays results showed that the 1154 bp 5'-flanking sequence of CsINV5 drove ß-glucuronidase (GUS) gene expression in roots, stems, leaves, flowers and siliques of transgenic Arabidopsis during different developmental stages. Moreover, promoter deletion analysis results revealed that an LTRE-related motif (CCGAAA) and a WBOXHVISO1 motif (TGACT) within the promoter region of CsINV5 were the core cis-elements in response to low temperature and sugar signaling, respectively. In addition, overexpression of CsINV5 in Arabidopsis promoted taproot and lateral root elongation through glucose-mediated effects on auxin signaling. Based on physiological and RNA-seq analysis, we found that overexpression of CsINV5 improved cold tolerance in transgenic Arabidopsis mainly by increasing the contents of glucose and fructose, the corresponding ratio of hexose to sucrose, and the transcription of osmotic-stress-related genes (P5CS1, P5CS2, AtLEA3, COR413-PM1 and COR15B) to adjust its osmotic potential. CONCLUSIONS: Comprehensive experimental results suggest that overexpression of CsINV5 may enhance the cold tolerance of plant through the modification of cellular sugar compounds contents and osmotic regulation related pathways.


Assuntos
Arabidopsis/metabolismo , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Chá/enzimologia , beta-Frutofuranosidase/metabolismo , Arabidopsis/genética , Temperatura Baixa , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , beta-Frutofuranosidase/genética
10.
Int J Mol Sci ; 19(8)2018 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-30110937

RESUMO

Invertases are essential enzymes that irreversibly catalyze the cleavage of sucrose into glucose and fructose. Cell wall invertase (CWI) and vacuolar invertase (VI) are glycosylated proteins and exert fundamental roles in plant growth as well as in response to environmental cues. As yet, comprehensive insight into invertase encoding genes are lacking in Glycine max. In the present study, the systematic survey of gene structures, coding regions, regulatory elements, conserved motifs, and phylogenies resulted in the identification of thirty⁻two putative invertase genes in soybean genome. Concomitantly, impacts on gene expression, enzyme activities, proteins, and soluble sugar accumulation were explored in specific tissues upon stress perturbation. In combination with the observation of subcellular compartmentation of the fluorescent fusion protein that indeed exported to apoplast, heterologous expression, and purification in using Pichia pastoris system revealed that GmCWI4 was a typical extracellular invertase. We postulated that GmCWI4 may play regulatory roles and be involved in pathogenic fungi defense. The experimental evaluation of physiological significance via phenotypic analysis of mutants under stress exposure has been initiated. Moreover, our paper provides theoretical basis for elucidating molecular mechanisms of invertase in association with inhibitors underlying the stress regime, and will contribute to the improvement of plant performance to a diverse range of stressors.


Assuntos
Proteínas Fúngicas , Genes Fúngicos , Doenças das Plantas/microbiologia , Soja/microbiologia , beta-Frutofuranosidase , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Estudo de Associação Genômica Ampla , beta-Frutofuranosidase/genética , beta-Frutofuranosidase/metabolismo
11.
Plant Sci ; 274: 261-270, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30080612

RESUMO

Plant growth and reproduction are both energy-requiring processes; the necessary energy is supplied by the products of photosynthesis. Both the vegetative growth and reproductive success of rice are compromised by the absence of a functional copy of the gene OsHAK1. Here, a comparison between wild type rice and OsHAK1 knockout mutants not only confirmed the known detrimental effect of the absence of OsHAK1 on root growth, pollen viability and fertility, but also showed that sucrose phosphate synthase activity was lowered, and the sucrose content of the leaves was markedly increased, due to a partial block on the up-loading of sucrose into the phloem. The impaired allocation of sugar to the roots and spikelets caused by the knocking out of OsHAK1 was accompanied by a down-regulation in the leaf sheaths and panicle axes of genes encoding sucrose transporters (SUT genes), which are active in the phloem, as well as in the roots and spikelets of those encoding monosaccharide transporters (MST genes), which transport hexose sugars across the plant plasma membrane. The activity of sucrose synthase, acid invertase and neutral invertase in the roots of mutant plants assayed at the tillering stage, and in their spikelets, assayed during grain-filling, was significantly lower than in the equivalent organs of wild type plants. As a result, the supply of total soluble sugar, glucose and fructose to sink organs was reduced, consistent with the effect of the mutation on root growth and panicle fertility. Compared to wild type plants, the mutants accumulated less potassium (K) throughout the plant. The conclusion was that the failure to fully supply the demand of the mutant's sink organs for assimilate was responsible for its compromised phenotype, and that the deficiency in K uptake induced by the loss of OsHAK1 functionality was responsible for the disruption of sugar metabolism.


Assuntos
Metabolismo dos Carboidratos/efeitos dos fármacos , Proteínas de Transporte de Cátions/metabolismo , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Potássio/metabolismo , Transporte Biológico , Proteínas de Transporte de Cátions/genética , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Oryza/genética , Proteínas de Plantas/genética , Amido/metabolismo , Sacarose/metabolismo , beta-Frutofuranosidase/genética , beta-Frutofuranosidase/metabolismo
12.
Plant Sci ; 274: 476-484, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30080637

RESUMO

The mechanism underlying internal browning (IB), or brown discoloration, of the central region of tuberous roots of sweet potato (Ipomoea batatas) was examined. IB disorder begins in roots from approx. 90 days after transplanting, and the severity increases significantly with time. IB damage initially occurs in cells around the secondary vascular tissue, and the area per cell occupied by starch grains in this region was larger than in the unaffected region. High levels of reducing sugars, polyphenol oxidase (PPO) activities, chlorogenic acid, and hydrogen peroxide (H2O2) were detected in cells from the IB damaged regions. The content of sugar and polyphenols was higher in disks (transverse sections) with larger amounts of damaged tissues than in disks of sound root. The transcript levels of acid invertase (IbAIV) tended to be higher with greater IB severity, whereas fluctuation patterns of ADP-glucose pyrophosphorylase (IbAGPase), granule bound starch synthase (IbGBSS), and starch branching enzyme 1 (IbSBE1) were lower with higher IB severity. These observations suggest that the incidence of IB disorder in sweet potato is largely dependent on the excessive generation of reactive oxygen species (ROS) in cells around the secondary vascular tissues due to the abundant accumulation of sugar and/or starch grains during the root maturation period.


Assuntos
Ipomoea batatas/fisiologia , Proteínas de Plantas/metabolismo , Tubérculos/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Amido/metabolismo , Açúcares/metabolismo , Enzima Ramificadora de 1,4-alfa-Glucana/genética , Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Glucose-1-Fosfato Adenililtransferase/genética , Glucose-1-Fosfato Adenililtransferase/metabolismo , Ipomoea batatas/enzimologia , Ipomoea batatas/genética , Tubérculos/enzimologia , Tubérculos/genética , Feixe Vascular de Plantas/enzimologia , Feixe Vascular de Plantas/genética , Feixe Vascular de Plantas/fisiologia , Sintase do Amido/genética , Sintase do Amido/metabolismo , beta-Frutofuranosidase/genética , beta-Frutofuranosidase/metabolismo
13.
Ying Yong Sheng Tai Xue Bao ; 29(7): 2211-2216, 2018 Jul.
Artigo em Chinês | MEDLINE | ID: mdl-30039658

RESUMO

To understand the effects of climate warming and vegetation disturbance on soil ecological process during different stages of growing season in the alpine scrub ecosystem, the responses of soil invertase and urease activities to warming (0.6-1.3 ℃) and plant removal were investigated in a Sibiraea angustata scrubland on the eastern Qinghai-Tibetan Plateau, China. The results showed that experimental warming significantly increased soil invertase activity by 3.7%-13.3% in the removal- and unremoval-plant plots throughout the entire growing season. Warming significantly increased soil urease activity by 10.8%-56.3% in the removal- and unremoval-plant plots, except the late growing stage, during which warming had no significant effect on soil urease activity in the unremoval-plant plots. The effects of plant removal treatments on soil invertase and urease activities varied with warming and growing stages. Plant removal significantly decreased soil invertase activity of the warmed plots during the entire growing season and the unwarmed plots during the early and late growing stages, but did not affect soil invertase activity in the unwarmed plots during the mid-growing stage. Plant removal only significantly decreased soil urease activity by 10.5% in the unwarmed plots during the late growing stage. However, in the warmed plots, plant removal significantly decreased soil urease activity by 16.0%-18.7% during the early and mid growing stages. The results would increase our understanding of soil carbon and nitrogen cycling process in the alpine scrub ecosystems.


Assuntos
Ecossistema , Solo/química , Urease/metabolismo , beta-Frutofuranosidase/metabolismo , Altitude , China , Estações do Ano , Microbiologia do Solo
14.
Int J Mol Sci ; 19(8)2018 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-30042352

RESUMO

Abnormally developed endosperm strongly affects rice (Oryza sativa) appearance quality and grain weight. Endosperm formation is a complex process, and although many enzymes and related regulators have been identified, many other related factors remain largely unknown. Here, we report the isolation and characterization of a recessive mutation of White Belly 1 (WB1), which regulates rice endosperm development, using a modified MutMap method in the rice mutant wb1. The wb1 mutant develops a white-belly endosperm and abnormal starch granules in the inner portion of white grains. Representative of the white-belly phenotype, grains of wb1 showed a higher grain chalkiness rate and degree and a lower 1000-grain weight (decreased by ~34%), in comparison with that of Wild Type (WT). The contents of amylose and amylopectin in wb1 significantly decreased, and its physical properties were also altered. We adopted the modified MutMap method to identify 2.52 Mb candidate regions with a high specificity, where we detected 275 SNPs in chromosome 4. Finally, we identified 19 SNPs at 12 candidate genes. Transcript levels analysis of all candidate genes showed that WB1 (Os04t0413500), encoding a cell-wall invertase, was the most probable cause of white-belly endosperm phenotype. Switching off WB1 with the CRISPR/cas9 system in Japonica cv. Nipponbare demonstrates that WB1 regulates endosperm development and that different mutations of WB1 disrupt its biological function. All of these results taken together suggest that the wb1 mutant is controlled by the mutation of WB1, and that the modified MutMap method is feasible to identify mutant genes, and could promote genetic improvement in rice.


Assuntos
Endosperma/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas , Oryza/crescimento & desenvolvimento , Proteínas de Plantas/genética , beta-Frutofuranosidase/genética , Amilopectina/análise , Amilose/análise , Sistemas CRISPR-Cas , Endosperma/genética , Qualidade dos Alimentos , Biblioteca Gênica , Mutação , Oryza/genética , Proteínas de Plantas/metabolismo , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA , Amido/metabolismo , Grãos Integrais/metabolismo , beta-Frutofuranosidase/metabolismo
15.
Plant Mol Biol ; 97(4-5): 385-406, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29948658

RESUMO

KEY MESSAGE: The recent release of the maize genome (AGPv4) contains annotation errors of invertase genes and therefore the enzymes are bestly curated manually at the protein level in a comprehensible fashion The synthesis, transport and degradation of sucrose are determining factors for biomass allocation and yield of crop plants. Invertase (INV) is a key enzyme of carbon metabolism in both source and sink tissues. Current releases of the maize genome correctly annotates only two vacuolar invertases (ivr1 and ivr2) and four cell wall invertases (incw1, incw2 (mn1), incw3, and incw4). Our comprehensive survey identified 21 INV isogenes for which we propose a standard nomenclature grouped phylogenetically by amino acid similarity: three vacuolar (INVVR), eight cell wall (INVCW), and ten alkaline/neutral (INVAN) isogenes which form separate dendogram branches due to distinct molecular features. The acidic enzymes were curated for the presence of the DPN tripeptide which is coded by one of the smallest exons reported in plants. Particular attention was placed on the molecular role of INV in vascular tissues such as the nodes, internodes, leaf sheath, husk leaves and roots. We report the expression profile of most members of the maize INV family in nine tissues in two developmental stages, R1 and R3. INVCW7, INVVR2, INVAN8, INVAN9, INVAN10, and INVAN3 displayed the highest absolute expressions in most tissues. INVVR3, INVCW5, INVCW8, and INVAN1 showed low mRNA levels. Expressions of most INVs were repressed from stage R1 to R3, except for INVCW7 which increased significantly in all tissues after flowering. The mRNA levels of INVCW7 in the vegetative stem correlated with a higher transport rate of assimilates from leaves to the cob which led to starch accumulation and growth of the female reproductive organs.


Assuntos
Biologia Computacional , Genoma de Planta/genética , Zea mays/enzimologia , beta-Frutofuranosidase/genética , Sequência de Aminoácidos , Hidrolases/genética , Hidrolases/metabolismo , Isoenzimas , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Alinhamento de Sequência , Zea mays/genética , beta-Frutofuranosidase/metabolismo
16.
Biosci Biotechnol Biochem ; 82(9): 1599-1605, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29873621

RESUMO

1-Kestose is a key prebiotic fructooligosaccharide (FOS) sugar. Some ß-fructofuranosidases (FFases) have high transfructosylation activity, which is useful for manufacturing FOS. Therefore, obtaining FFases that produce 1-kestose efficiently is important. Here, we established a rapid FFase evaluation method using Escherichia coli that display different FFases fused to a PgsA anchor protein from Bacillus subtilis. E. coli cell suspensions expressing the PgsA-FFase fusion efficiently produce FOS from sucrose. Using this screening technique, we found that the E. coli transformant expressing Aspergillus kawachii FFase (AkFFase) produced a larger amount of 1-kestose than those expressing FFases from A. oryzae and A. terreus. Saturation mutagenesis of AkFFase was performed, and the mutant G85W was obtained. The E. coli transformant expressing AkFFase G85W markedly increased production of 1-kestose. Our results indicate that the surface display technique using PgsA is useful for screening of FFases, and AkFFase G85W is likely to be suitable for 1-kestose production. ABBREVIATIONS: AkFFase: Aspergillus kawachii FFase; AoFFase: Aspergillus oryzae FFase; AtFFase: Aspergillus terreus FFase; FFase: ß-fructofuranosidase; FOS: fructooligosaccharide; fructosylnystose: 1F-ß-fructofuranosylnystose.


Assuntos
Aspergillus/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Trissacarídeos/metabolismo , beta-Frutofuranosidase/biossíntese , Aspergillus/enzimologia , Escherichia coli/genética , Mutagênese , beta-Frutofuranosidase/genética , beta-Frutofuranosidase/metabolismo
17.
J Appl Microbiol ; 125(2): 441-456, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29663625

RESUMO

AIMS: To screen and identify a potential extracellular ß-d-fructofuranosidase or invertase-producing bacterium from soil, and comparatively evaluate the enzyme biosynthesis under submerged and solid-state fermentation (SSF). METHODS AND RESULTS: Extracellular invertase-producing bacteria were screened from soil. Identification of the potent bacterium was performed based on microscopic examinations and 16S rDNA molecular sequencing. Bacillus subtilis LYN12 invertase secretion was surplus with wheat bran humidified with molasses medium (70%), with elevated activity at 48 h and 37°C under SSF, whereas under submerged conditions, increased activity was observed at 24 h and 45°C in the molasses medium. The study revealed a simple fermentative medium for elevated production of extracellular invertase from a fast growing Bacillus strain. CONCLUSIONS: Bacterial invertases are scarce and limited reports are available. By far, this is the first report on the comparative analysis of optimization of extracellular invertase synthesis from B. subtilis strain by submerged and SSF. The use of agricultural residues increased yields resulting in the development of a cost-effective and stable approach. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacillus subtilis LYN12 invertase possesses excellent fermenting capability to utilize agro-industrial residues under submerged and solid-state conditions. This could be a beneficial candidate in food and beverage processing industries.


Assuntos
Bacillus subtilis/enzimologia , Bacillus subtilis/metabolismo , Fermentação , beta-Frutofuranosidase/metabolismo , Reatores Biológicos/microbiologia , Biotecnologia/métodos , Fibras na Dieta , Melaço
18.
Ecotoxicol Environ Saf ; 158: 182-186, 2018 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-29689495

RESUMO

A field-scale trial was conducted to investigate the remediation effects of sepiolite on Cd-polluted paddy soils under different moisture managements, using a series of variables (pH and extractable Cd of soil, plant Cd concentration, plant nutrition and enzyme activity in soil). Results revealed that soil pH increased significantly after sepiolite addition, which promoted the reduction in extractable Cd in soil. After applying 0.5-2.5% sepiolite into soil, due to higher pH and lower TCLP Cd concentration, brown rice Cd reduced by 17-67% under continuous flooding, 14-62% under conventional irrigation, and 13-61% under wetting irrigation (p < 0.05). The activities of phosphatase and invertase increased compared with unamended soil (p < 0.05). The available phosphorus in clay treated soil showed a remarkable raise, with a maximum increase by 14.5%, 16.9% and 18.1% under three moisture managements (p < 0.05). The increasing values of enzyme activity and then plant nutrition in soil revealed that clay application improved the ecological condition of Cd-contaminated paddy soil. The sepiolite application in combination with continuous flooding provided an efficient and safe remediation technology for Cd-polluted rice field.


Assuntos
Silicatos de Alumínio/análise , Cádmio/análise , Silicatos de Magnésio/química , Oryza/química , Poluentes do Solo/análise , Argila , Recuperação e Remediação Ambiental , Inundações , Concentração de Íons de Hidrogênio , Nitrogênio/análise , Monoéster Fosfórico Hidrolases/metabolismo , Fósforo/análise , Potássio/análise , Solo/química , beta-Frutofuranosidase/metabolismo
19.
Biochim Biophys Acta Gene Regul Mech ; 1861(6): 543-553, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29660529

RESUMO

Sucrose is the most commonly transported sugar in plants and is easily assimilated by insects to fulfill the requirement of physiological metabolism. BmSuc1 is a novel animal ß-fructofuranosidase (ß-FFase, EC 3.2.1.26)-encoding gene that was firstly cloned and identified in silkworm, Bombyx mori. BmSUC1 was presumed to play an important role in the silkworm-mulberry enzymatic adaptation system by effectively hydrolyzing sucrose absorbed from mulberry leaves. However, this has not been proved with direct evidence thus far. In this study, we investigated sucrose hydrolysis activity in the larval midgut of B. mori by inhibition tests and found that sucrase activity mainly stemmed from ß-FFase, not α-glucosidase. Next, we performed shRNA-mediated transgenic RNAi to analyze the growth characteristics of silkworm larvae and variations in glycometabolism in vivo in transgenic silkworms. The results showed that in the RNAi-BmSuc1 transgenic line, larval development was delayed, and their body size was markedly reduced. Finally, the activity of several disaccharidases alone in the midgut and the sugar distribution, total sugar and glycogen in the midgut, hemolymph and fat body were then determined and compared. Our results demonstrated that silencing BmSuc1 significantly reduced glucose and apparently activated maltase and trehalase in the midgut. Together with a clear decrease in both glycogen and trehalose in the fat body, we conclude that BmSUC1 acts as an essential sucrase by directly modulating the degree of sucrose hydrolysis in the silkworm larval midgut, and insufficient sugar storage in the fat body may be responsible for larval malnutrition and abnormal petite phenotypes.


Assuntos
Bombyx/enzimologia , Metabolismo dos Carboidratos/fisiologia , Proteínas de Insetos/metabolismo , beta-Frutofuranosidase/metabolismo , Animais , Bombyx/genética , Hidrólise , Proteínas de Insetos/química , Proteínas de Insetos/genética , Morus/metabolismo , Especificidade de Órgãos/fisiologia , Folhas de Planta/metabolismo , beta-Frutofuranosidase/química , beta-Frutofuranosidase/genética
20.
Int J Mol Sci ; 19(3)2018 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-29495594

RESUMO

ß-fructofuranosidase (ß-FFase) belongs to the glycosyl-hydrolase family 32 (GH32), which can catalyze both the release of ß-fructose from ß-d-fructofuranoside substrates to hydrolyze sucrose and the synthesis of short-chain fructooligosaccharide (FOS). BmSuc1 has been cloned and identified from the silkworm Bombyx mori as a first animal type of ß-FFase encoding gene. It was hypothesized that BmSUC1 plays an important role in the silkworm-mulberry adaptation system. However, there is little information about the enzymatic core sites of BmSUC1. In this study, we mutated three amino acid residues (D63, D181, and E234) that represent important conserved motifs for ß-FFase activity in GH32 to alanine respectively by using site-directed mutagenesis. Recombinant proteins of three mutants and wild type BmSUC1 were obtained by using a Bac-to-Bac/BmNPV expression system and BmN cells. Enzymatic activity, kinetic properties, and substrate specificity of the four proteins were analyzed. High Performance Liquid Chromatography (HPLC) was used to compare the hydrolyzing and transfructosylating activities between D181A and wtBmSUC1. Our results revealed that the D63A and E234A mutations lost activity, suggesting that D63 and E234 are key amino acid residues for BmSUC1 to function as an enzyme. The D181A mutation significantly enhanced both hydrolyzing and transfructosylating activities of BmSUC1, indicating that D181 may not be directly involved in catalyzation. The results provide insight into the chemical catalyzation mechanism of BmSUC1 in B. mori. Up-regulated transfructosylating activity of BmSUC1 could provide new ideas for using B. mori ß-FFase to produce functional FOS.


Assuntos
Substituição de Aminoácidos , Bombyx/enzimologia , Bombyx/genética , Códon , Mutagênese Sítio-Dirigida , beta-Frutofuranosidase/genética , beta-Frutofuranosidase/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Catálise , Sequência Conservada , Ativação Enzimática , Hidrólise , Ligação Proteica , Especificidade por Substrato , beta-Frutofuranosidase/química
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