Endo-beta-mannanase from the endosperm of seeds of Sesbania virgata (Cav.) Pers. (Leguminosae): purification, characterisation and its dual role in germination and early seedling growth

Carrara

Buckeridge

2010

The endosperm of seeds of Sesbania virgata (Cav.) Pers. accumulates galactomannan as a cell wall storage polysaccharide. It is hydrolysed by three enzymes, one of them being a-galactosidase. A great amount of protein bodies is found in the cytoplasm of endospermic cells, which are thought to play the major role as a nitrogen reserve in this seed. The present work aimed at understanding how the production of enzymes that degrade storage compounds is controlled. We performed experiments with addition of inhibitors of transcription (actinomycin-D and a-amanitin) and translation (cycloheximide) during and after germination. In order to follow the performance of storage mobilisation, we measured fresh mass, protein contents and a-galactosidase activity. All the inhibitors tested had little effect on seed germination and seedling development. Actinomycin-D and cycloheximide provoked a slight inhibition of the storage protein degradation and concomitantly lead to an elevation of the a-galactosidase activity. Although a-amanitin showed some effect on seedling development at latter stages, it presented the former effect and did not change galactomannan degradation performance. Our data suggest that some of the proteases may be synthesised de novo, whereas a-galactosidase seems to be present in the endosperm cells probably as an inactive polypeptide in the protein bodies, being probably activated by proteolysis when the latter organelle is disassembled. These evidences suggest the existence of a connection between storage proteins and carbohydrates mobilisation in seeds of S. virgata, which would play a role by assuring a balanced afflux of the carbon and nitrogen to the seedling development.

Section: Galactomannan Mobilisationsupporting

confidence: 84%

Storage proteins and cell wall mobilisation in seeds of Sesbania virgata (Cav.) Pers. (Leguminosae)

Carrara

Buckeridge

2010

“…The membranes were incubated overnight in a solution containing anti-endo-β-mannanase antibody diluted 1:200 in PBS, prepared in rabbits against endo-β-mannanase purified from coffee seeds, gently provided by Prof. Dr. J. Giorgini from the Department of Biology of University of São Paulo, Brazil. Specificity of the antibody to S. virgata endo-β-mannanase was shown by Lisboa et al (2006) and Tonini et al (2006). The membranes were washed with 50 mM Tris-HCl pH 7.4 and subsequently incubated for 1 h in a solution containing goat anti-rabbit IgG conjugated to peroxidase diluted 1:200 in PBS.…”

Section: Tissue Printingmentioning

confidence: 99%

Effect of abscisic acid on galactomannan degradation and endo-β-mannanase activity in seeds of Sesbania virgata (Cav.) Pers. (Leguminosae)

Lisboa

Freschi

et al. 2006

Seeds of Sesbania virgata (Cav.) Pers. (Leguminosae) have an endosperm which accumulates galactomannan as a storage polysaccharide in the cell walls. After germination, it is hydrolysed by three enzymes: α-galactosidase (EC 3.2.1.22), endo-β-mannanase (EC 3.2.1.78) and β-mannosidase (EC 3.2.1.25). This work aimed at studying the effect of abscisic acid (ABA) on galactomannan degradation during and after germination. Seeds were imbibed in water or in 10 −4 M ABA, and used to evaluate the effect of exogenous and endogenous ABA. Tissue printing was used to follow biochemical events by detecting and localising endo-β-mannanase in different tissues of the seed. The presence of exogenous ABA provoked a delay in the cellular disassembly of the endosperm and disappearance of endo-β-mannanase in the tissue. This led to a delay in galactomannan degradation. The testa (seed coat) of S. virgata contains endogenous ABA, which decreases ca. fourfold during storage mobilisation after germination, permitting the galactomannan degradation in the endosperm. Furthermore, endo-β-mannanase was immunolocalised in the testa, which has a living cell layer. The ABA appears to modulate storage mobilisation in the legume seed of S. virgata, and a cause-effect relationship between Communicated by U. Lüttge ABA (probably through testa) and activities of hydrolases is proposed.

“…The purified endo-β-mannanase, obtained as described by Lisboa et al (2006), after separation by SDS-PAGE (Laemmli 1970, Hames 1990, was transferred to a nitrocelulose membrane, for 2 h in 0.4 M glycine-20% methanol buffer using a constant current of 400 mA, which was subsequently incubated for 1 h in 100 mM potassium ferricyanide, to inhibit peroxidase seed activity (Guéra and Sabater 2002). The membrane was blocked for 3 h in a solution containing 0.1% gelatine, 1% BSA in 0.1 M phosphate buffer saline (PBS) pH 7.0, with some drops of Tween 20.…”

Section: Western Immunoblottingmentioning

confidence: 99%

“…According to Lisboa et al (2006), the action of the α-galactosidase from S. virgata on galactomannan is a necessary condition to grant access of endo-β-mannanase to the main chain of galactomannan polymer in seeds of S. virgata. This explains the appearance of α-galactosidase activity prior to endo-β-mannanase ( Fig.…”

Section: Galactomannan Hydrolases Activities and Localisation Of Endomentioning

confidence: 99%

Testa is involved in the control of storage mobilisation in seeds of Sesbania virgata (Cav.) Pers., a tropical legume tree from of the Atlantic Forest

Lisboa

Silva

et al. 2006

Seeds of Sesbania virgata (Cav.) Pers. (Leguminosae) contain galactomannan as a cell wall storage polysaccharide in the endosperm. After germination, it is hydrolysed by three enzymes: α-galactosidase (EC 3.2.1.22), endo-β-mannanase (EC 3.2.1.78) and β-mannosidase (EC 3.2.1.25). This work aimed at studying the role of the testa (seed coat) on galactomannan degradation during and after germination. Seeds were imbibed in water, with and without the testa, and used to evaluate the effect of this tissue on storage mobilisation, as well as its possible role in the galactomannan hydrolases activities. Immunocytochemistry and immunodotblots were used to follow biochemical events by detecting and localising endo-β-mannanase in different tissues of the seed. Endo-β-mannanase and α-galactosidase activities were found in the testa and latter in the endosperm during galactomannan degradation. The former enzyme was immunologically detected in the testa, mainly during germination. The absence of the testa during imbibition led to the anticipation of protein mobilisation and increased of the α-galactosidase activity and galactomannan degradation. Thus, the testa appears to play a role during storage mobilisation in the legume seed of S. virgata probably by participating in the control of the production, modification and/or storage of the hydrolases.