RESUMO
BACKGROUND AND AIMS: Proteases are responsible for protein degradation during leaf senescence, allowing nutrients to be redirected to sink tissues. In a previous work, we reported that sulphur deficiency produced a delay in the leaf senescence of barley (Hordeum vulgare L.) plants, at both vegetative and reproductive stages. In this work, we analyse the effect of sulphur deficiency on the expression of several genes coding for proteases of different catalytic groups, which have been strongly associated with leaf senescence. METHODS: Four independent experiments were performed in order to impose low sulphur availability conditions: one of steady-state sulphur deficiency during the vegetative stage and three of sulphur starvation during vegetative and reproductive stages. KEY RESULTS: Sulphur deficiency inhibited or reduced the senescence-associated induction of seven of the eight proteases analysed. Their induction, as well as senescence and phloem amino acid remobilization, could be achieved with senescence inducers such as methyl-jasmonate (a hormonal stimulus) and darkness, but with different rates of induction dependent on each gene. Sulphur deficiency also exerted an opposite effect on the expression of two cysteine-protease genes (HvSAG12 and HvLEGU) as well as on one serine-protease gene (HvSUBT) according to leaf age and plant phenological stages. All three genes were induced in green leaves but were repressed in senescent leaves of sulphur-deficient plants at the vegetative stage. At the reproductive stage, both cysteine-proteases were only repressed in senescent leaves, while the serine-protease was induced in green and senescent leaves by sulphur deficiency. CONCLUSIONS: Our results highlight the relevance of adequate sulphur nutrition in order to ensure leaf senescence onset and induction of protease genes, which will consequently impact on grain protein composition and quality. In addition, our results provide evidence that leaf age, plant developmental stage and the nature of the stress modulate the sulphur responses.
Assuntos
Hordeum/genética , Proteínas de Plantas/genética , Regulação da Expressão Gênica de Plantas , Peptídeo Hidrolases , Folhas de Planta/genética , EnxofreRESUMO
Proteases play a main role in the mobilization of storage proteins during seed germination. Until today, there is little information about the involvement of serine proteases, particularly subtilases, in the germination of barley grains. The aims of the present work were to study the contribution of serine proteases to the total proteolytic activity induced during germination of barley grains and evaluate the specific involvement of subtilases in this process. Proteolytic activity assayed against azocasein in the presence of specific inhibitors, showed that serine proteases contributed between 10 and 20% of total activity along germination. Subtilase activity increased from day 1 after imbibition with a peak between days 4-5. Moreover, in vivo determination of subtilase activity in germinating grains revealed increasing activity along germination mainly localized in the seed endosperm and developing rootlets. Finally, the expression of 19 barley genes encoding subtilases was measured by real time PCR during germination. Three of the analyzed genes increased their expression along germination, five showed a transient induction, one was down-regulated, nine remained unchanged and one was not expressed. The present work demonstrates the involvement of subtilases in germination of barley grains and describes the positive association of eight subtilase genes to this process.
Assuntos
Germinação , Hordeum/crescimento & desenvolvimento , Proteínas de Plantas/metabolismo , Plântula/crescimento & desenvolvimento , Subtilisinas/metabolismo , Aminoácidos/metabolismo , Regulação da Expressão Gênica de Plantas , Hordeum/enzimologia , Hordeum/metabolismo , Proteólise , Reação em Cadeia da Polimerase em Tempo Real , Plântula/enzimologia , Plântula/metabolismoRESUMO
Senescence is the final developmental stage of every plant organ, which leads to cell death. It is a highly regulated process, involving differential gene expression and outstanding increment in the rate of protein degradation. Senescence-associated proteolysis enables the remobilization of nutrients, such as nitrogen (N), from senescent tissues to developing organs or seeds. In addition to the nutrient recycling function, senescence-associated proteases are also involved in the regulation of the senescence process. Nearly, all protease families have been associated with some aspects of plant senescence, and numerous reports addressing the new identification of senescence-associated proteases are published every year. Here, we provide an updated report with the most recent information published in the field, focusing on senescence-associated proteases presumably involved in N remobilization.
Assuntos
Nitrogênio/metabolismo , Peptídeo Hidrolases/metabolismo , Proteínas de Plantas/metabolismo , Plantas/enzimologia , Morte Celular , Proteínas de Cloroplastos/metabolismo , Cloroplastos/enzimologia , Ativação Enzimática , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Peptídeo Hidrolases/genética , Desenvolvimento Vegetal , Folhas de Planta/enzimologia , Folhas de Planta/genética , Proteínas de Plantas/genética , Plantas/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Estresse Fisiológico , Especificidade por SubstratoRESUMO
The influence of different storage conditions (temperature, illumination, brand of mineral water and storage time) on growth of mould spores was studied. Alternaria alternata, Penicillium citrinum and Cladosporium cladosporioides spores were inoculated in bottles of mineral and mineralised water, packaged in polyethylene terephtalate (PET). The bottles were incubated under different storage conditions. The strains had been isolated from bottled mineral water in a previous study. Storage time was the parameter that had the most important influence in mould growth. The spores grew into visible colonies after 5 month of incubation in bottles just filled, and in a month in bottles that had been stored for 5 month. This could be due to the migration of compounds from PET packaging material into mineral water. This compounds could be used as nutrients (organic matter) for mould growth. The plasticizer additive di-n-butyl phthalate (DBP) concentration in recently bottled mineral water and in 5-month stored bottles was measured. An increase of 20% of DBP concentration was observed. A. alternata and P. citrinum strains were toxicological characterised. Both strains produced mycotoxins in vitro, and P. citrinum produced citrinin in mineral water, posing a potential health risk for consumers.