The enrichment and isolation of proteins are considered limiting steps in proteomic studies. Identification of proteins whose expression is transient, those that are of low-abundance, and of natural peptides not described in databases, is still a great challenge. Plant extracts are in general complex, and contaminants interfere with the identification of proteins involved in important physiological processes, such as plant defense against pathogens. This review discusses the challenges and strategies of separomics applied to the identification of low-abundance proteins and peptides in plants, especially in plants challenged by pathogens. Separomics is described as a group of methodological strategies for the separation of protein molecules for proteomics. Several tools have been used to remove highly abundant proteins from samples and also non-protein contaminants. The use of chromatographic techniques, the partition of the proteome into subproteomes, and an effort to isolate proteins in their native form have allowed the isolation and identification of rare proteins involved in different processes.

Este trabalho teve como objetivos a quantificação de proteínas e da atividade da enzima α-galactosidase, no eixo embrionário e nos cotilédones, de sementes de Dalbergia nigra (jacarandá-da-bahia) durante a germinação. As sementes foram colocadas para embeber em água por sete dias, sendo retiradas amostras para a avaliação bioquímica e cinética da enzima. A atividade da enzima α-galactosidase aumenta com a embebição das sementes nos dois compartimentos, embora não esteja presente no eixo embrionário de sementes secas. A diferença na atividade da enzima entre os cotilédones e o eixo embrionário foi significativa. O pH 5,5 foi o de máxima atividade para as enzimas de ambos os compartimentos. A temperatura que mais estimulou a atividade da enzima nos cotilédones foi 50 ºC e de 50 a 60 ºC no eixo embrionário. A atividade da α-galactosidase foi inibida por β-mercaptoetanol e cobre, em ambos os compartimentos, enquanto a lactose e o cloreto de sódio estimularam a atividade tanto nos cotilédones como no eixo embrionário. Os valores de K M para enzimas do eixo embrionário e dos cotilédones foram de 0,239 e 0,228 mM, respectivamente.

This work aimed to quantify the protein content and the α-galactosidase activity in the embryonic axis and in the cotyledons of Dalbergia nigra seeds during the imbibition period. The seeds were submitted to water imbibition during seven days. Biochemical and kinetic characterization of the enzyme were done from samples taken during the imbibition period. The activity of the α-galactosidase increased in the two compartments with the soaking of the seeds, although, the enzyme activity was not detected in the embryonic axis of dry seeds. The difference in the activity of the enzyme between cotyledons and embryonic axis was significant. The pH of maximum activity was 5.5 for the enzyme of both compartments. In the cotyledons the higher activity of the enzyme was obtained at 50 ºC. In the embryonic axis the activity was higher at temperatures ranging from 50 to 60 ºC. The activity of the α-galactosidase was inhibited by β-mercaptoethanol and CuSO4 in both compartments, while lactose and sodium chloride stimulated the activity in the cotyledons and in the embryonic axis. The values of K M for the enzymes of the embryonic axis and cotyledons were respectively 0.239 and 0.228 mM.

Substâncias complexas são convertidas, pela ação de enzimas, em moléculas solúveis, durante o processo de germinação, as quais são translocadas para a plântula em crescimento, servindo como fonte de energia ou estrutura física. Com o objetivo de quantificar a atividade enzimática da α-galactosidase e a mobilização de mono e oligossacarídeos durante o período de germinação, foi conduzido este estudo utilizando-se sementes de jacarandá-da-bahia. As sementes foram mantidas em germinador a 25ºC sob luz contínua, sendo avaliada a protrusão da radícula, pelo período de 10 dias. Foram quantificados os teores de mono e de oligossacarídeos, assim como a atividade específica da enzima α-galactosidase nos tempos zero, um, três e cinco dias. Houve mobilização das reservas de glicose e manose nos cotilédones e xilose neste e no embrião nos três primeiros dias de germinação. A ramnose teve os teores aumentados nos cotilédones e no eixo embrionário. A rafinose foi o oligossacarídeo utilizado inicialmente, tanto no eixo embrionário, quanto nos cotilédones, enquanto a sacarose acumulou-se em ambos. A atividade da enzima variou no período estudado, com maior atividade específica no primeiro dia de embebição, tanto nos cotilédones, quanto no eixo embrionário.

Complex substances are converted by enzyme action into soluble molecules during the germination process, and these are in turn translocated to the growing plant embryo, serving as an energy source or physical structure. With the objective of quantifying α-galactosidase enzyme activity as well as mono-and oligosaccharide mobilization during germination, this study was conducted using Brazilian rosewood seeds. Seeds were kept in a germinator at 25ºC under continuous light for evaluation of radicle emergence (protrusion) over a period of 10 days. Mono-and oligosaccharide contents and also the specific activity of α-galactosidase enzyme were quantified on days zero, one, three and five. There was mobilization of glucose and mannose stored in cotyledons, and also of xylose in cotyledons and in the embryo in the first three days of germination. Rhamnose contents increased in cotyledons and in the embryonic axis. Raffinose was the initially used oligosaccharide, both in the embryonic axis and in cotyledons, while sucrose accumulated in both. Enzyme activity varied throughout, with greater specific activity on day one of imbibition, both in cotyledons and in the embryonic axis.