RESUMO
Glucosinolate transporters (GTRs) are part of the nitrate/peptide transporter (NPF) family, members of which also transport specialized secondary metabolites as substrates. Glucosinolates are defense compounds derived from amino acids. We selected 4-methylthiobutyl (4MTB) and indol-3-ylmethyl (I3M) glucosinolates to study how GTR1 from Arabidopsis thaliana transports these substrates in computational simulation approaches. The designed pipeline reported here includes massive docking of 4MTB and I3M in an ensemble of GTR1 conformations (in both inward and outward conformations) extracted from molecular dynamics simulations, followed by clustered and substrate-protein interactions profiling. The identified key residues were mutated, and their role in substrate transport was tested. We were able to identify key residues that integrate a major binding site of these substrates, which is critical for transport activity. In silico approaches employed here represent a breakthrough in the plant transportomics field, as the identification of key residues usually takes a long time if performed from a purely wet-lab experimental perspective. The inclusion of structural bioinformatics in the analyses of plant transporters significantly speeds up the knowledge-gaining process and optimizes valuable time and resources.
Assuntos
Arabidopsis/metabolismo , Glucosinolatos/metabolismo , Proteínas de Transporte de Monossacarídeos/química , Proteínas de Transporte de Monossacarídeos/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Transporte Biológico , Butiratos/metabolismo , Indóis/metabolismo , Modelos Moleculares , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica , Tioglucosídeos/metabolismoRESUMO
In traditional, small-scale agriculture in the Andes, potatoes are frequently co-cultivated with the Andean edible tuber Tropaeolum tuberosum, commonly known as mashua, which is believed to exert a pest and disease protective role due to its content of the phenylalanine-derived benzylglucosinolate (BGLS). We bioengineered the production of BGLS in potato by consecutive generation of stable transgenic events with two polycistronic constructs encoding for expression of six BGLS biosynthetic genes from Arabidopsis thaliana. First, we integrated a polycistronic construct coding for the last three genes of the pathway (SUR1, UGT74B1 and SOT16) into potato driven by the cauliflower mosaic virus 35S promoter. After identifying the single-insertion transgenic event with the highest transgene expression, we stacked a second polycistronic construct coding for the first three genes in the pathway (CYP79A2, CYP83B1 and GGP1) driven by the leaf-specific promoter of the rubisco small subunit from chrysanthemum. We obtained transgenic events producing as high as 5.18 pmol BGLS/mg fresh weight compared to the non-transgenic potato plant producing undetectable levels of BGLS. Preliminary bioassays suggest a possible activity against Phytophthora infestans, causing the late blight disease and Premnotrypes suturicallus, referred to as the Andean potato weevil. However, we observed altered leaf morphology, abnormally thick and curlier leaves, reduced growth and tuber production in five out of ten selected transgenic events, which indicates that the expression of BGLS biosynthetic genes has an undesirable impact on the potato. Optimization of the expression of the BGLS biosynthetic pathway in potato is required to avoid alterations of plant development.
Assuntos
Solanum tuberosum , Bioengenharia , Resistência à Doença/genética , Doenças das Plantas/genética , Plantas Geneticamente Modificadas/genética , Solanum tuberosum/genética , Tiocianatos , TioglucosídeosRESUMO
Tropaeolum tuberosum Ruiz & Pavon, along with other several species, is an edible-tuber crop that grows in the Andean region. Folk medicine describes the use of mashua to reduce reproductive function in men. The present study aimed to evaluate the effect of mashua (1 g kg(-1)) on sperm production in rats during 7, 12, 21 and 42 days of treatment. The following parameters were assessed: reproductive organ weights, spermatid count and daily sperm production (DSP), sperm count in epididymis and sperm transit and serum testosterone levels. Freeze-dried extract of mashua had 3.7 g 100 g(-1) of benzyl glucosinolate. Mashua-treated rats showed a reduction in testicular spermatid number and DSP from day 12 to day 42; meanwhile, the effect of mashua was noted in epididymal sperm count after 12 and 42 days of treatment. In addition, epididymal sperm transit time was delayed at day 7 and it was accelerated on days 12 and 21 of treatment. No differences in serum testosterone levels were found between rats treated with vehicle and mashua after 42 days of treatment. Finally, mashua reduces testicular function after one spermatogenic cycle by reducing spermatid and sperm number, DSP and epididymal sperm transit time.
Assuntos
Anticoncepcionais Masculinos/farmacologia , Extratos Vegetais/farmacologia , Espermatogênese/efeitos dos fármacos , Testículo/efeitos dos fármacos , Tropaeolum/química , Administração Oral , Animais , Peso Corporal/efeitos dos fármacos , Anticoncepcionais Masculinos/administração & dosagem , Esquema de Medicação , Masculino , Tamanho do Órgão/efeitos dos fármacos , Extratos Vegetais/administração & dosagem , Ratos , Contagem de Espermatozoides , Testosterona/sangue , Tiocianatos/análise , Tioglucosídeos/análiseRESUMO
Papaya is a climacteric fruit that has high amounts of benzylglucosinolates (BG) and benzylisothiocyanates (BITC), but information regarding levels of BG or BITC during fruit development and ripening is limited. Because BG and BITC are compounds of importance from both a nutritional and a crop yield standpoint, the aim of this work was to access data on the distribution and changes of BG and BITC levels during fruit development and ripening. BG and BITC levels were quantified in peel, pulp, and seeds of papaya fruit. Volatile BITC was also verified in the internal cavity of the fruit during ripening. The influence of the ethylene in BG and BITC levels and mirosinase activity was tested by exposing mature green fruits to ethylene and 1-methylcyclopropene (1-MCP). The highest BG levels were detected in seeds, followed by the peel and pulp being decreased in all tissues during fruit development. Similarly, the levels of BITC were much higher in the seeds than the peel and pulp. The levels of BG for control and ethylene-treated fruit were very similar, increasing in the pulp and peel during late ripening but not changing significantly in seeds. On the other hand, fruit exposed to 1-MCP showed a decrease in BG amount in the pulp and accumulation in seed. The treatments did not result in clear differences regarding the amount of BITC in the pulp and peel of the fruit. According to the results, ethylene does not have a clear effect on BITC accumulation in ripening papaya fruit. The fact that BG levels in the pulp did not decrease during ripening, regardless of the treatment employed, and that papaya is consumed mainly as fresh fruit, speaks in favor of this fruit as a good dietary source for glucosinolate and isothiocyanates.
Assuntos
Carica/química , Carica/crescimento & desenvolvimento , Frutas/crescimento & desenvolvimento , Glicosídeo Hidrolases/genética , Isotiocianatos/análise , Proteínas de Plantas/genética , Tiocianatos/análise , Tioglucosídeos/análise , Sequência de Aminoácidos , Carica/enzimologia , Carica/genética , Ciclopropanos/metabolismo , Etilenos/metabolismo , Frutas/química , Frutas/enzimologia , Frutas/fisiologia , Expressão Gênica , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Dados de Sequência Molecular , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Alinhamento de SequênciaRESUMO
AIM: To determine the effect of two different extracts of red maca in male rats. METHODS: Prostatic hyperplasia was induced in male rats with testosterone enanthate (TE). The study comprised six groups: one control group (group 1), one group treated with TE (group 2), two groups treated with TE and aqueous extract of red maca (groups 3 and 4), one group treated with hydroalcoholic extract of red maca (group 5) and one group treated with finasteride (0.1 mg, group 6). Differences in the aqueous extract dependent on the length of time of boiling, whether for 2 or 3 hours, for groups 3 and 4 was assessed. Extracts of red maca contained 0.1 mg of benzylglucosinolate. Thereafter, a dose-response effect of different doses of benzylglucosinolates (0.02-0.08 mg) in red maca extracts was assessed. RESULTS: Prostate weight was similar in rats treated with freeze-dried aqueous extract of red maca prepared after 2 and 3 hours of boiling. Freeze-dried aqueous extract of red maca, hydroalcoholic extract of red maca and finasteride reduced prostate weight in rats with prostatic hyperplasia. No difference was observed between the data obtained from aqueous extract or hydroalcoholic extract of red maca. A dose dependent reduction of prostate weight was observed with the increase of the dose of benzylglucosinolates in red maca extracts. CONCLUSION: The present study showed that hydroalcoholic or aqueous extract of red maca containing 0.1 mg of benzylglucosinolate can reduce prostate size in male rats in which prostatic hyperplasia had been induced by TE.