RESUMO
The endopeptidases (EPs) in wheat endosperm during seed germination and subsequent seedling growth were characterized by gradient-polyacrylamide gel electrophoresis with gelatin copolymerized into the gel. Four cysteine EPs (EP1, EP2, EP3 and EP4) were detected in wheat endosperm during the 7 d growth after seed imbibition. The results also showed that the activities of all of these EPs increased continuously, and EP2 first appeared and had the highest proteolytic activity among the four EPs in this experimental process. The optimum pH and temperature of all four EPs were 4.0 and 40.0 degrees C. All EPs were completely inhibited by 25 micromol/L E-64 and had no good thermal stabilities, especially EP1. In addition, these EPs had different substrate specificities to albumins, globulins, gliadins and glutenins; the main storage proteins of mature wheat endosperm. Among them, EP2 had the highest proteolytic activities on globulins, gliadins and glutenins, and might be the most important and specific EP with potential to be tightly correlated with seedling development.
Assuntos
Cisteína Endopeptidases/metabolismo , Germinação/fisiologia , Plântula/enzimologia , Plântula/crescimento & desenvolvimento , Sementes/enzimologia , Triticum/embriologia , Triticum/enzimologia , Estabilidade Enzimática/efeitos dos fármacos , Germinação/efeitos dos fármacos , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Inibidores de Proteases/farmacologia , Proteínas de Armazenamento de Sementes/metabolismo , Plântula/efeitos dos fármacos , Sementes/efeitos dos fármacos , Especificidade por Substrato/efeitos dos fármacos , Temperatura , Triticum/efeitos dos fármacosRESUMO
The degradation of large subunit (LSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in wheat (Triticum aestivum L. cv. Yangmai 158) leaves was studied. A novel 51-kDa fragment was detected in leaf crude extracts and in chloroplast lysates from leaves with dark-induced senescence. Further studies showed that the 51-kDa fragment was found in the reaction solution with stroma fraction but not in that with the chloroplast membrane fraction and in the chloroplast lysates from mature wheat leaves. The reaction of producing the 51-kDa fragment was inhibited by 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF), 1,10-phenanthroline and EDTA. The N-terminal sequence analysis indicated that the LSU was cleaved at the peptide bond between Lys-14 and Ala-15. In addition, a 50-kDa fragment of LSU formed obviously at pH 6.0-6.5 was detected in the crude extracts of leaves with dark-induced senescence but was not found in lysates of chloroplasts. The degradation was prevented by AEBSF, leupeptin and transepoxysuccinyl-l-leucylamido (4-guanidino) butane (E-64). The results obtained in this study imply that the appearance of the 51-kDa fragment could be because of the involvement of a new senescence-associated protease that is located in the stroma of chloroplasts in senescing wheat leaves.
Assuntos
Cloroplastos/enzimologia , Folhas de Planta/enzimologia , Ribulose-Bifosfato Carboxilase/metabolismo , Triticum/enzimologia , Cloroplastos/metabolismo , Escuridão , Ácido Egtázico/farmacologia , Eletroforese em Gel de Poliacrilamida , Estabilidade Enzimática/efeitos dos fármacos , Estabilidade Enzimática/efeitos da radiação , Leupeptinas/farmacologia , Pepstatinas/farmacologia , Fenantrolinas/farmacologia , Folhas de Planta/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Prótons , Ribulose-Bifosfato Carboxilase/química , Sulfonas/farmacologia , Temperatura , Triticum/metabolismoRESUMO
The role of serine endopeptidase in cucumber leaf senescence was studied by using the inhibitor of serine endopeptidase and plant growth regulators (6-BA and ABA) on darkness-induced cucumber leaves. The results showed that the senescence of cucumber leaves were delayed by AEBSF [4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride], an inhibitor of serine-type endopeptidase, or 6-BA treatment. The chlorophyll contents increased by AEBSF (Fig.3) and the protein degradation of leaves under AEBSF treatment declined more slowly than in the control or under ABA 50 micromol/L treatment (Fig.4), partly because the activities of serine endopeptidases became lower during senescence. However, the activities of endopeptidase in cucumber leaf were increased by ABA 50 micromol/L (Fig.2A), furthermore, the MDA content were also influenced by AEBSF and plant growth regulators (Fig.5). Native gradient PAGE showed that six bands of isoenzymes were detected in cucumber leaves and four bands of which were the type of serine-endopeptidase (Fig.1), and proved that the activities of serine-endopeptidase were inhibited by AEBSF, but enhanced by ABA (Fig.2B) in the leaves. It implies that serine endopeptidases might play an important role in cucumber leaf senescence.
Assuntos
Cucumis sativus/enzimologia , Folhas de Planta/enzimologia , Proteínas de Plantas/metabolismo , Serina Endopeptidases/metabolismo , Ácido Abscísico/farmacologia , Clorofila/metabolismo , Cucumis sativus/efeitos dos fármacos , Cucumis sativus/fisiologia , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/fisiologia , Proteínas de Plantas/fisiologia , Inibidores de Serina Proteinase/farmacologiaRESUMO
Human brain natriuretic peptide (hBNP) was used clinically for the treatment of acute decompensated congestive heart failure. In this paper, hBNP was expressed as a fusion protein with a histidine tag and Ssp dnaB mini-intein which was capable of self-cleavage. After affinity chromatography with Ni-Sepharose and renaturation, the fusion protein was enriched with CM-cellulose. Ssp dnaB mini-intein mediated peptide-bond hydrolysis was triggered by shifting the pH and temperature in the CM-cellulose column, which let to the release of hBNP from the fusion protein and the separation of hBNP from His-DnaB. The hBNP sample was further purified by C4 reverse phase HPLC, and 2.8mg of the peptide with homogeneity of 97% was obtained from one liter of culture medium. The biological activity was assayed in vitro, which indicated that hBNP had a potent vasodilatory effect on rabbit aortic strips with an EC50 of 1.94 x 10(-6) mg/mL.
Assuntos
DnaB Helicases/genética , Inteínas/genética , Peptídeo Natriurético Encefálico/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Animais , DnaB Helicases/biossíntese , Escherichia coli/genética , Escherichia coli/metabolismo , Vetores Genéticos , Peptídeo Natriurético Encefálico/genética , Engenharia de Proteínas/métodos , Processamento de Proteína , Coelhos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologiaRESUMO
Results in this paper showed that the effect of nitric oxide (NO) donor, sodium nitroprusside (SNP), which could promote the germination of wheat seeds under osmotic stress, was through enhancing imbibition using water content determination and imbibition analysis of wheat seed. SNP could also enhance amylase isozyme I activities, accelerate the liquefaction of endosperms, while have no effect on esterase. Moreover, SNP could strongly increase the sugars, such as the fructose, glucose and sucrose contents in wheat seeds during early germination (12 h) under normal conditions. When the seeds were treated with SNP with or without PTIO, an NO scavenger, and different exogenous sugars, i.e. fructose, glucose and sucrose, it was found that these sugars might activate amylase isozyme I activity mediated by the signal molecule NO to start the early stage of germination.
Assuntos
Amilases/metabolismo , Germinação/efeitos dos fármacos , Doadores de Óxido Nítrico/farmacologia , Proteínas de Plantas/metabolismo , Sementes/efeitos dos fármacos , Triticum/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Frutose/metabolismo , Germinação/fisiologia , Glucose/metabolismo , Nitroprussiato/farmacologia , Pressão Osmótica , Sementes/crescimento & desenvolvimento , Sementes/metabolismo , Sacarose/metabolismo , Triticum/crescimento & desenvolvimento , Triticum/metabolismoRESUMO
The effects of nitric oxide (NO) and gibberellic acid (GA(3)) on the responses of amylases in wheat (Triticum aestivum L.) seeds (caryopses) were investigated during the first 12 h of germination. GA(3) had no effects on the activities of alpha-amylase (EC 3.2.1.1) or beta-amylase (EC 3.2.1.2), either in intact seeds or embryoless halves within 12 h. In contrast, addition of sodium nitroprusside (SNP), an NO donor, was able to induce a rapid increase in beta-amylase activity without affecting alpha-amylase. Furthermore, the rapid response of beta-amylase to SNP in wheat seeds could be attributed to NO and was approximately dose-dependent. Some other aspects of SNP induction of amylase isozymes were also characterized. Further investigations showed that SNP might play an interesting role in the dissociation of free beta-amylase from small homopolymers or heteropolymers. Furthermore, SNP also directly induced the release of bound beta-amylase from glutenin and its crude enzyme preparation. However, the slight increase in protease also induced by SNP might not be responsible for this action. Interestingly, based on the fact that the rapid response of beta-amylase to NO also existed in seeds of other species, such as barley, soybean, rice and watermelon, it might be a universal event in early seed germination.
Assuntos
Giberelinas/fisiologia , Óxido Nítrico/fisiologia , Triticum/enzimologia , beta-Amilase/biossíntese , Benzoatos/farmacologia , Relação Dose-Resposta a Droga , Germinação/efeitos dos fármacos , Germinação/fisiologia , Imidazóis/farmacologia , Nitroprussiato/farmacologia , Sementes/efeitos dos fármacos , Sementes/fisiologia , Fatores de Tempo , Triticum/efeitos dos fármacos , alfa-Amilases/biossínteseRESUMO
When wheat (Triticum aestivum L. Yangmai 158) seedling (with three fully expanded leaves) roots were treated with 15% PEG-6000 in combination with different concentrations (0.1 and 0.5 mmol/L) of exogenous nitric oxide donor sodium nitroprusside (SNP) and NO(-)(3)/NO(-)(2) (control), the enhancement of lipoxygenase (LOX) activity in wheat seedling leaves under osmotic stress was delayed at the lower concentration of SNP treatment (0.1 mmol/L), while the generation rate of O(-.)(2), the enhancement of membrane permeability and the accumulation of MDA and H(2)O(2)were also alleviated. SNP 0.1 mmol/L significantly enhanced the activity of superoxide dismutase (SOD), and accelerated the accumulation of proline. In comparison with these changes, the effects of higher concentration of SNP (0.5 mmol/L) and corresponding control (NO(-)(3)/NO(-)(2)) were not so obvious. The above results indicate that lower concentrations of in vitro nitric oxide (NO) can protect wheat seedling leaves from membrane lipid peroxidation caused by osmotic stress.
Assuntos
Peroxidação de Lipídeos/efeitos dos fármacos , Lipídeos de Membrana/metabolismo , Óxido Nítrico/farmacologia , Folhas de Planta/metabolismo , Plântula/metabolismo , Triticum/metabolismo , Peróxido de Hidrogênio/metabolismo , Lipoxigenase/metabolismo , Pressão Osmótica , Prolina/análise , Superóxidos/metabolismoRESUMO
Effects of nitric oxide (NO), a substance newly found to have protective functions in plants, on root growth of wheat (Triticum aestivum L. Yangmai 158) seedlings under salt stress were studied. Sodium nitroprusside (SNP), an NO donor, markedly alleviated the inhibitory effect of salt on root elongation at salt concentrations around 150 mmol/L, but was ineffective when NaCl concentration was at 300 mmol/L or higher. It was most effective at 0.05-0.1 mmol/L, and had harmful effect at 0.30-5 mmol/L. Furthermore, when wheat seedling roots were treated with 150 mmol/L NaCl in combination with 0.05 mmol/L SNP and NO scavenger (hemoglobin, Hb), NO(-)(2)/NO(-)(3) and K(3)Fe(CN)(6) as controls, both of which were the two other major products besides NO when SNP dissolved in water, respectively, activities of superoxide dismutase (SOD), peroxidase (POD) and ascorbate peroxidase (APX) were enhanced to some extent. While, the generation of O(-.)(2) and the accumulation of MDA and H(2)O(2) were alleviated. DNA laddering was observed when wheat seedling roots exposed to 150 mmol/L NaCl for 6 d, also suggesting that salt stress might induce oxidative damage in root-tips. In combination with hemoglobin, NO(-)(2)/NO(-)(3) and K(3)Fe(CN)(6) as controls, the results showed that NO could block DNA ladders. Above results suggest that it is NO rather than any substances which is protecting root-tip cells of wheat seedlings from oxidative damage caused by salt stress.
Assuntos
Óxido Nítrico/farmacologia , Raízes de Plantas/crescimento & desenvolvimento , Plântula/crescimento & desenvolvimento , Cloreto de Sódio/farmacologia , Triticum/crescimento & desenvolvimento , Apoptose , Fragmentação do DNA , Peróxido de Hidrogênio/análise , Malondialdeído/análise , Nitroprussiato/farmacologia , Oxirredução , Plântula/metabolismo , Superóxidos/metabolismo , Triticum/metabolismoRESUMO
The contribution of rice as a protein source is important. Rice seed protein can be divided into four forms, glutelin (57 kDa, 37-39 kDa, 22-23 kDa), prolamine (13 kDa), albumin (16 kDa) and globulin (10 kDa, 26 kDa) on its solubility. Glutelin is the major storage protein of rice and accounted for 80% of total protein found in the rice grain, the mature glutelin comprises an acidic (37-39 kDa) and an basic subunit (22-23 kDa) coming from a common precursor (57 kDa) by post-transcriptional hydrolytic cleavage. Prolamine is the second important. Rice seed proteins localize in two types of protein bodies, PB-I, PB-II. PB-I containing prolamine is indigestible, whereas PB-II being rich in glutelin is digestible. The nutritional value of rice could thus be raised by improving its digestible protein glutelin content. On the other hand, the character of low digestible protein is also an important target of rice breeding. Low protein rice is required for the diet of patients with kidney disease. Three glutelin mutants, W3660, W204, W379, were found by screening 168 rice varieties through SDS-PAGE analysis of the seeds total proteins. The amounts of 37-39 kDa and 22-23 kDa glutelin subunits were much lower and that of 13 kDa prolamine polypeptide was higher in W3660 seeds than in ordinary rice; The amounts of 37-39 kDa and 22-23 kDa glutelin subunits in W204 or W379 seeds were between those in W3660 and ordinary rice. Especially, in W379 seed, there was a large quantity of 57 kDa polypeptide. For characterizing the genetics of the glutelin mutant, the cross population between W3660 and Otorokimochi was constructed. SDS-PAGE analysis of the progeny seed total proteins showed, low glutelin content was always accompanied by high prolamine content; all F1 seeds had low glutelin and high prolamine content; the segregation of low glutelin and normal type in F2 seeds was 3:1; the genotypes of F2 plants were deduced by the analysis of F3 seeds, and among F2 plants the ratio of homozygotes of low glutelin, heterozygotes of low glutelin and homozygotes of normal type was about 1:2:1. These results proved that the trait of low glutelin and high prolamine was controlled by a single dominant gene and could be inherited by its progeny.
Assuntos
Glutens/genética , Mutação , Oryza/genética , Glutens/análise , Peso Molecular , Fenilpropanolamina/análiseRESUMO
Aluminum-induced exudation of organic acids from roots has been proposed as a mechanism for Al tolerance in plants. To better understand the regulatory process leading to efflux of organic acids, the possible involvement of salicylic acid (SA) in regulating Al-induced citrate release in Cassia tora L. was identified. The response of citrate efflux to exogenous SA was concentration-dependent. Application of SA at 5 microM in solution containing 20 microM Al increased citrate efflux to levels 1.76-fold higher than in controls (20 microM Al alone). However, inhibition of citrate release was observed when SA concentrations increased to more than 20 microM. Increased citrate efflux due to the SA treatment was associated with decreased inhibition of root growth and Al content in root tips, suggesting that exogenous SA could confer Al tolerance by increasing citrate efflux. We also examined citrate synthase activities (EC 4.1.3.7) and citrate concentrations in root tips exposed to Al and/or SA. However, both citrate synthase activities and citrate accumulation remained unaffected. These results indicate that SA-promotion of Al-induced citrate efflux is not correlated with increase in citrate production. Total endogenous SA concentrations were measured in root tips and the SA concentrations were significantly enhanced by Al at levels of 10-50 microM.