Made available in DSpace on 2015-03-26T13:36:47Z (GMT). No. of bitstreams: 1 texto completo.pdf: 436143 bytes, checksum: 16cb464b668a51c499aa49ff3f90d796 (MD5) Previous issue date: 2007-07-23 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / The arsenic (As) influence on the relative growth rate (TCR) and on the antioxidant system of four leguminous species: Leucocephala leucocephala, Sesbania grandiflora (woody), Cajanus cajan and Crotalaria spectabilis (shrub), it was evaluated, as well as, the accumulation of this element for these species. After the exhibition of these species to the As, in nutritious solution, they were appraised TCR and the enzymatic activity of the enzymes: superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), ascorbate peroxidase (APX) and glutathione reductase (GR) and the accumulation of As for the same ones. Of the woody species, S. grandiflora showed minor influences of the As on TCR, besides presenting larger accumulation of this element than L. leucocephala. The largest tolerance presented by S. grandiflora can be related to efficient of the antioxidant system of the same. S. grandiflora, when cultivated in nutritious solution without addition of As, it presented the largest activities of the enzymes SOD, CAT, POX and GR than L. leucocephala. In the presence of As, S. grandiflora presented increment in the activity of SOD, of CAT and of POX and, L. leucocephala presented increment in the activity of SOD and of GR. Of the shrub species, C. spectabilis introduced minor influences of the As on TCR, same presenting larger concentration of this element, that C. cajan. The largest tolerance presented by C. spectabilis to the As, it should be related to the efficient antioxidant system presented by this species. C. spectabilis, when cultivated in solution containing As, it presented increment in the activity of the enzymes SOD, CAT, POX and APX, while C. cajan presented reduction in the activity of these same enzymes. In spite of other studies they be necessary to know better the mechanisms of tolerance to the As, S. grandiflora (woody) and C. spectabilis (shrub) they seem to have larger potentials for use in revegetation programs of polluted areas with As. / A influência do As sobre a taxa de crescimento relativo (TCR) e sobre o sistema antioxidante de quatro espécies vegetais leguminosas: Leucaena leucocephala e Sesbania grandiflora (arbóreas) e Cajanus cajan e Crotalaria spectabilis (arbustivas), foi avaliada, assim como, o acúmulo deste elemento por estas espécies. Após a exposição destas espécies ao As, em solução nutritiva, foram avaliadas a TCR e a atividade enzimática das enzimas: dismutase do superóxido (SOD), da catalase (CAT), da peroxidase (POX), da peroxidase do ascorbato (APX) e da redutase da glutationa (GR) e o acúmulo de As pelas mesmas. Das espécies arbóreas, S. grandiflora apresentou menor influencia do As sobre sua TCR, além de apresentar maior acúmulo deste elemento do que L. leucocephala. A maior tolerância apresentada por S. grandiflora pode estar relacionada às repostas do sistema antioxidante da mesma. S. grandiflora, quando cultivada em solução nutritiva sem adição de As, apresentou as maiores atividades das enzimas SOD, CAT, POX e GR do que L. leucocephala. Na presença de As, S. grandiflora apresentou incremento na atividade da SOD, da CAT e da POX e, L. leucocephala apresentou incremento na atividade da SOD e da GR. Das espécies arbustivas, C. spectabilis apresentou menor influencia do As sobre sua TCR, mesmo apresentando maior concentração deste elemento, do que C. cajan. A maior tolerância apresentada por C. spectabilis ao As, deve estar relacionada ao eficiente sistema antioxidante apresentado por esta espécie. C. spectabilis, quando cultivada em solução contendo As, apresentou incremento na atividade das enzimas SOD, CAT, POX e APX, enquanto que C. cajan apresentou redução na atividade destas mesmas enzimas. Apesar de outros estudos serem necessários para conhecer melhor os mecanismos de tolerância ao As, S. grandiflora (arbórea) e C. spectabilis (arbustiva) parecem ter maiores potenciais para utilização em programas de revegetação de áreas contaminadas com As.

Assembly of virus capsid protein (CP) into icosahedrally symmetric particles is an intriguing and elegant process. In most cases of virus assembly, a large number of identical protein subunits self-assemble to generate a shell that protects the viral genome. Studies on virus assembly have resulted in a new scientific technique that uses these proteinaceous shells as nano-particles for a variety of biological applications. The current thesis deals with understanding the factors that govern the assembly of the Sesbania mosaic virus (SeMV) and a pleomorphic virus, Tobacco streak virus (TSV). CP of SeMV, a T=3 plant virus, consists of a disordered N-terminal R-domain and an ordered S-domain. The importance of the R-domain in the assembly was probed by replacement with polypeptides such as the B-domain of Staphylococcus aureus protein A and polypeptides P10 and P8 of SeMV. These chimera assembled into T=3 or larger virus like particles (VLPs). Addition of divalent cations resulted in the formation of heterogeneous nucleoprotein complexes that disappeared upon treatment with EDTA/RNAse. One of the chimeras (N∆65-B) purified in a dimeric form by affinity chromatography assembled into T=1 VLPs during crystallization. The three dimensional structure of these VLPs showed that they were devoid of divalent ions and the B-domain was disordered. These studies demonstrate the importance of N-terminal residues, metal ions in virus assembly and robustness of the assembly process. Also, the B-domain was functional in N∆65-B VLPs, suggesting possible biotechnological applications. Tobacco streak virus (TSV) is a polymorphic virus and a major plant pathogen. TSV capsids encapsidate the tri-partite ss-RNA genome of the virus in three spheroidal particles of diameters 27, 30 and 33 nm, respectively. CPs of ilarviruses are also involved in genome activation. The labile nature of ilarviruses has posed difficulties in their structure determination. This thesis describes the first crystal structure of truncated TSV-CP. The core of TSV CP conforms to the canonical β-barrel jelly roll tertiary structure found in other viral coat proteins. Dimers of CP with swapped C-terminal arms (C-arm) were observed in the two crystal structures determined. The C-arm was found to be flexible and responsible for the polymorphic and pleomorphic nature of TSV capsids. Mutations in the hinge region of the C-arm that reduce the flexibility resulted in the formation of more uniform particles. TSV CP was also found to be structurally similar to that of Alfalfa mosaic virus (AMV) accounting for similar mechanism of genome activation in alfamo and ilar viruses.

No description available.

1. Viruses are obligate parasites that hijack the host cell machinery to synthesize their own gene products and for their propagation. In order to establish a successful viral infection, viruses have evolved different strategies to evade host check points. Further more, their success also relies in employing varied strategies to express maximum number of functional proteins from their small constrained genome. Polyprotein processing is a widely used strategy of expression by many plant viruses. With limited information available on this aspect for sobemoviruses, the present study was undertaken. 2. The present thesis deals with the mechanism of Sesbania mosaic virus (SeMV) polyprotein processing and functional characterization of the polyprotein domains. SeMV infects Sesbania grandiflora that belongs to the Fabaceae family. It is a positive sense ssRNA virus with a genome length of 4149 nucleotides. The genome encodes four potential overlapping open reading frames (ORFs). ORF1 codes for an 18 kDa protein that is proposed to be involved in the movement of the virus. ORF 3 codes for the coat protein (CP) that encapsidates the viral genomic RNA to form the viral particles. The central ORF codes for polyprotein that has a serine protease domain at its Nterminus that cleaves the polyprotein at specific E-T/S sites to release the functional domains. So far only in SeMV, the E. coli expressed polyprotein, Protease-VPg-RdRp was shown to undergo processing at E325-T326, E402-T403 and E498-S499 releasing protease, VPg, P10 and RdRp domains respectively. 3. Based on the arrangement of the central ORF, the genome organization of SeMV was earlier shown to be like that of SCPMV type. However, recent sequencing data from the laboratory showed that the organization of SeMV gRNA was like that of CfMV type. This would imply that in SeMV the central two ORFs will be translated to give two polyproteins, 2a (Protease-VPg-C-terminal domain) and 2ab (Protease-VPg-RdRp) the C-terminus of 2a and N-terminus of RdRp being different from what was reported previously. Therefore, in the light of the new genome organization for SeMV, the mechanism of processing of polyprotein 2a and 2ab needs to be revisited. 4. SeMV protease domain was shown to require natively unfolded VPg at its Cterminus for its activity. Aromatic stacking interactions between protease and VPg (via W43 residue) were shown to confer the activity to the protease. However, the residues in the protease domain involved in these interactions have not been identified. 5. The objectives of the present studies are • To elucidate the mechanism of processing of polyproteins 2a and 2ab in E. coli and in planta. • To identify residues in the protease domain involved in mediating aromatic stacking interactions with VPg. • To functionally characterize the C-terminal domain of polyprotein 2a. 6. Polyprotein 2a when expressed in E. coli, from the new cDNA clone, got cleaved at the earlier identified sites E325-T326, E402-T403 and E498-S499 to release protease, VPg, P10 and P8 respectively. The specificities of the cleavage sites were established by mutational analysis. 7. Additionally, a novel cleavage was identified within the protease domain at position E132-S133. The polyprotein 2a that was mutated for this site (ΔN70 2a-E132A) showed no release of P8 protein though the polyprotein was intact for E498-S499 site. Unlike other cleavage site mutants, ΔN70 2a-E132A mutant also revealed large accumulation of intact polyprotein, again implying that the mutation not only abolished the proteolytic cleavage at that site but hampered the processing at other sites. The results confirmed that the cleavage at N-terminus of the protease/polyprotein is crucial for an efficient processing in particular for the cleavage between P10-P8. 8. Interestingly, though the sites in polyprotein 2ab are exactly the same as identified in polyprotein 2a, the former got cleaved between Protease-VPg but not between VPg-RdRp. This cleavage site appeared to be rather masked in polyprotein 2ab. Also, the cleavage at E132-S133 site appeared to be rather slow. These results indicate to a differential cleavage pattern, governed probably by the conformation of 2ab. In other words, the local context of the cleavage site and just not the sequence per se could be playing a key role in 2ab polyprotein processing. 9. Products, corresponding to all cleavages identified in E. coli (E132-S133, E325-T326, E402-T403 and E498-S499) were also detected in infected Sesbania leaves. Products corresponding to the sizes of ΔN132 Protease and ΔN132 Protease-VPg were detected suggesting that the removal of the membrane anchoring domain from the protease does occur in planta. Also, detection of band corresponding P8, confirmed that the cleavage between P10-P8 indeed occured in planta too. 10. The trans cleavage experiments suggested that not all of the four cleavages in polyprotein 2a occur in trans (intermolecular). Cleavages at E132-S133 and E498-S499 do not occur in trans impling that cleavages at these sites could only occur in cis (intramolecular) by auto-proteolysis of the polyprotein. 11. The Thr at P1’ did not make a site trans cleavable. Interestingly, SeMV protease was found to cleave even an E-S site in trans but only when present at positions 324-325 and 402-403, suggesting that trans cleavage in SeMV is governed by the context rather than the Thr at P1’position of the cleavage site. The E498-S499 site was found to be highly stringent not only for the mode of its cleavage (cis cleavage) but also for its sequence (E-S only). A Thr substitution for Ser at this site, made it non cleavable in cis. 12. The results reveal that the polyprotein processing in SeMV is regulated by a number of strategies, viz. a) availability of the cleavage site depending on the conformation of the flanking domains (E132-S133 and E402-T403 cleavages in 2ab). b) Mode of recognition (cis or trans). c) Context/position of the cleavage site. 13. Based on the sequences of all four cleavage sites identified, a consensus has been drawn for SeMV serine protease cleavage site, i.e., N/Q-E-T/S-X (where X is an aliphatic residue) at P2-P1-P1’-P2’ position respectively. 14. With a view to understand the structural reasons for such high specificity, the residues in the S1 and S2 binding pocket, that recognize the substrate P1 and P2 residues respectively, were identified based on the structural comparison of SeMV protease with other Glu/Gln specific proteases. Mutational analysis of these residues clearly demonstrated that H298, T279 and N308 of the S1-binding pocket that would bind the substrate glutamate are crucial for the protease activity. R309 that forms the S2 binding pocket is also crucial for protease activity. 15. Also, the P2 (Asn/Gln) residue recognized by R309 plays an important role in determining the substrate specificity. A positively charged residue Lys was not tolerated at this position. SeMV protease was also shown to efficiently cleave the peptide bond C-terminus to an uncharged Gln in vivo suggesting that it is a Glu/Gln specific protease. 16. An interesting feature of the SeMV protease domain is the presence of a disulphide bond that holds the S1-binding pocket. However, unlike for the cellular counterparts like trypsin, the disulphide was found to be not essential for either the SeMV protease activity or structural stability. 17. Protease and VPg domains were proposed to be involved in aromatic interactions that conferred activity to the protease. The structure of protease revealed a stack of aromatic residues (W271, F269. Y315 and Y319) exposed to the solvent. Mutational analysis was performed to identify their role in mediating the interactions and hence the activity of protease. H275, though not a part of exposed aromatic stack in the protease, was chosen for mutational analysis as it lies close to the W271 in sequence and is conserved in the protease domain across all the known sobemoviruses. The in vivo and trans cleavage assays suggested that residues W271 and H275 but not Y315 or Y319 are crucial for protease activity. 18. The Far-UV CD spectrum of protease-VPg is characterized by a positive peak at 230 nm, signifying the aromatic interactions. Far-UV CD spectral analysis of the aromatic mutants showed that W271 and H275, but not F269 and Y319 are the major contributors of the 230 nm positive peak, confirming the direct involvement of these residues in the stacking interactions with W43 of VPg. Thermal stability studies, fluorescence spectroscopy and 1D-NMR spectroscopy studies also confirmed the histidine aromatic interactions between W271, H275 of protease with W43 of VPg. 19. The loss in aromatic interactions in the mutants caused Protease-VPg to aggregate, suggesting that the aromatic interactions between protease and VPg not only conferred activity to the protease but also the active oligomeric status. 20. In silico analysis of the C-terminal domain showed that it has no significant similarities with any known functional proteins. The region corresponding to P8 was amplified and cloned in pRSET C vector, over-expressed and purified. 21. The purified His-tagged P8 showed mass abnormality on the SDS-PAGE. However, the mass spectrometric analysis of the purified protein showed that it had a molecular mass of 9.766 kDa as is expected for a His-tagged P8. P8 is highly basic, which could possibly explain its anomalous behaviour on the SDS-PAGE. The purified recombinant P8 protein was found to be natively unfolded. In vitro binding studies revealed that P8 had nucleic acid binding property. The protein was also found to be phosphorylated both in vitro and in vivo conditions. 22. Interestingly, P18, (a precursor of P8) but not P8, was found to possess an inherent ATP hydrolyzing property. Optimum conditions for the ATPase assay were found to be Tris HCl pH 8.0, 37 ºC, 5 mM MgCl2. The activity was linear upto 20 mins. P18 could utilize all NTPs and dNTPs. Studies revealed that ATPase activity resided in the P10 domain of P18, though P8 region could enhance the activity. Conclusively, the results demonstrate that the C-terminal domains of polyprotein 2a have ATPase and nucleic acid binding activity and could therefore have possible roles in movement and replication.

4 pp. / Summer cover crops can add nitrogen to the soil, build up and maintain soil organic matter, suppress pest populations, mitigate soil erosion, and reduce nutrient leaching when they are used in Arizona vegetable systems. However, careful management is required since cover crops can modify the availability of soil nitrogen and other critical nutrients. The ratio between carbon to nitrogen (C:N) in decomposing cover crop biomass is a critical indicator of the overall process of breakdown and eventual release of nutrients. This article introduces five cover crops that could improve vegetable systems in Arizona. The mixtures of a legume and a non-legume cover crop species can also be planted to obtain desired C:N ratios to optimize the benefits of cover crops.

Leucaena leucocephala and Sesbania sesban in dry form were fed as supplements to goats feeding on a low quality basal diet of barley straw. Each browse supplement was fed at four levels: 0 %, 0.83 % liveweight (LW), 1.66 % LW and at ad libitum. The intakes of dry matter (DM) and organic matter (OM), DM and OM digestibility, nitrogen (N) digestibility and balance, and liveweight gain were evaluated during a 5 week trial. For goats offered both leucaena and sesbania there was no significant (p<0.05) difference in DM or OM intakes between 0.83 % LW and 1.66 % LW levels of browse supplementation. When both lecaena and sesbania were offered ad libitum the DM and OM intakes were significantly (p<0.05) lower than for either 0.83 % LW or 1.66 % LW level of supplementation. Feeding both leucaena and sesbania increased the DM and OM digestibility coefficients significantly over thos of the controls. The growth rates for goats supplemented with leucaena at various levels were not significantly different from each other. For the goats offered sesbania at ad libitum, their growth rates were significantly lower than for those fed sesbania at either 0.83 % LW or 1.66 % LW. The apparent digestibility of nitrogen (ADN) was not significantly different among leucaena supplemented treatments. For goats offered sesbania, the ADN was significantly higher than for the controls. The ability of browse supplements to increase intakes and digestibility of both dry matter and organic matter is discussed.

Diss. (sammanfattning). Umeå : Sveriges lantbruksuniv. / Härtill 3 uppsatser.

Sesbania drummondii seedlings were grown in a medium to which lead (Pb), copper (Cu), nickel (Ni) and zinc (Zn) were added singly and in combinations in order to assess the effects of metal interactions on seedling growth, metal accumulation and anti-oxidative system. S. drummondii growth was significantly inhibited with metal treatments. S. drummondii accumulated substantially higher concentrations of metals in the roots than shoots. The uptake of metals followed the order Pb > Cu > Zn > Ni in roots and Pb > Zn > Cu > Ni in shoots. In addition, uptake of a single metal by S. drummondii was affected by the presence of a second metal, suggesting an antagonistic effect or competition between metals at the plant uptake site. A significant increase in both enzymatic [superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR)] and non-enzymatic (glutathione) antioxidant was observed in the S. drummondii seedlings exposed to different metal treatments. The enhancement in enzyme activities followed the order of Cu > Ni > Pb > Zn. However, compared to the effect of individual metal, metals in combination increased the enzyme activities to varying degrees.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal. / Une plante tropicale de la famille des Fabaceae, Sesbania pachycarpa, a fait l'objet d'une évaluation insecticide et/ou insectifuge sur le charançon de la carotte, Listronotus oregonensis (Lec.), et sur le doryphore de la pomme de terre, Leptinotarsa decanlineata (Say). Dans une première phase des travaux, le plus polaire de deux extraits obtenus par séparation chimique de la poudre de la plante a démontré, sur le charançon de la carotte, une activité 3 à 4 fois supérieure à celle de l'huile de neem, ou Azadirachta indica, plante tropicale de référence pour ses propriétés insecticides. Le second extrait, faiblement polaire, n'a démontré aucun effet après 72 heures, à une concentration de 4 %. L'action répulsive d'une concentration de 1 % de l'extrait polaire assure une protection foliaire significative contre la larve LT et l'adulte du doryphore de la pomme de terre ; il faut doubler la quantité d'extrait moins polaire pour assurer une protection équivalente à celle obtenue avec l'extrait de neem. Dans une seconde phase de l'étude, les 17 fractions qui sont obtenues par chromatographie "flash" des deux extraits sont testées sur la larve L^ du doryphore de la pomme de terre. La mortalité après 72 h donne l'ordre d'activité décroissante suivant : 84, B5, AI i, Ag et 85. Les extraits Ag, A^, 84 et 85 ont une activité antiappétante équivalente. Les autres fractions sont peu actives ou en proportion insuffisante pour entreprendre des tests biologiques. L'étude des fractions plus actives 84 et 85, sans le synergiste piperonyl butoxide (PBO), a permis de démontrer qu'une concentration de 0,3125 % assure une protection foliaire significative après 24 h et qu'une augmentation atteignant 8 fois cette concentration n'améliore pas cette protection. Après 24 et 48 h, la mortalité n'est pas significative ; mais elle le devient après 72 h, à la concentration de 2,5 %. En présence du PBO (1 % de la fraction), les 2 fractions, à une concentration de 0,3125 %, assurent une protection foliaire de la plante et une mortalité des insectes significatives. Les résultats indiquent que la fraction 84, après 24 heures, possède une action précoce plus marquée que la fraction B5. Après 72 heures, les deux fractions ont une activité semblable. L'étude chimique a permis de déterminer qu'une monoamine tertiaire, l'hydrochlorure de méthylamine, était le composé le plus abondant de la fraction 84. Dans les conditions expérimentales, cette molécule pourrait plutôt provenir des solvants utilisés que d'une origine végétale ; néanmoins, une petite ouverture sur cette dernière est envisageable, en référence aux études antérieures qui montrent une diversité des monoamines aliphatiques chez diverses espèces végétales.

Sementes de Sesbania virgata (Cav.) Pers. acumulam suas reservas de carbono no endosperma na forma de um polissacarídeo de parede celular, o galactomanano. Os galactomananos são polissacarídeos constituídos de uma cadeia principal de resíduos de D-manose ligadas &beta;-1,4, ramificada por resíduos de D-galactose &alpha;-1,6 ligados. A mobilização deste ocorre após a germinação e envolve três enzimas hidrolíticas (&alpha;-galactosidase, endo-&beta;-mananase e exo-&beta;-manosidase). A &alpha;-galactosidase é a primeira enzima atuar sobre o galactomanano hidrolisando as ligações &alpha;-1,6 das galactoses ramificadas a cadeia principal de manano (ligados &beta;-1,4), permitindo a ação da endo-&beta;-mananase, que hidrolisará o polissacarídeo a oligossacarídeos, onde a &beta;-manosidase atuará (ligações &beta;-1,4), transformando oligossacarídeos a monossacarídeos a serem utilizados no desenvolvimento do embrião. Buscando a compreensão das características da &alpha;-galactosidase e modo de ação sobre o galactomanano, procedeu-se com a purificação, em três etapas,e caracterização bioquímica (pH ótimo, temperatura ótima e aspectos cinéticos) da &alpha;-galactosidase de sementes de Sesbania virgata (Cav.) Pers. Além disso, visando evidenciar a modulação da enzima endo-&beta;-mananase pela distribuição de ramificações de galactose no galactomanano (estrutura fina do galactomanano), procedeu-se com hidrólises enzimáticas do galactomanano de Sesbania virgata (Cav.) Pers. utilizando a enzima endo-&beta;-mananase de Aspergillus niger (Megazyme&reg;) somente ou em conjunto com a &alpha;-galactosidase semipurificada de Sesbania virgata (Cav.) Pers. (Capítulo 1) ou com a &alpha;-galactosidase comercial de Cyamopsis tetragonoloba (Megazyme&reg;), seguido de análise dos oligossacarídeos por HPAEC-PAD (High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection). Também procedeu-se com hidrólises enzimáticas de galactomananos de 6 espécies com razão manose:galactose variando de 1:1 a 150:1 com endo-&beta;-mananase de Aspergillus niger (Megazyme&reg;) e análise dos oligossacarídeos produzidos por HPAEC-PAD. A &alpha;-galactosidase semipurificada possui, aproximadamente, 42 kDa de peso molecular em condições desnaturantes e, aproximadamente 72 kDa de peso molecular na forma nativa, sugerindo que a enzima assuma estrutura quartenária. A temperatura ótima apresentada se encontra na faixa de 50°C a 55°C, pH ótimo na faixa de 4,4 a 5,4, Km= 1,8276 mM e a velocidade máxima de 0,5024 &mu;molGal.min-1.mgprot-1. A espectrometria de massas gerou os fragmentos: ALADYV-HSK-RMPGSLGHEE-QDAK-TT-GDIEDNWNSM-TSIADS NDKW-ASYAGPGGWN-DPDMLEVGNG-GMTTEEYR-AP-LLVGCDIR-VAVIL-WNR, estando a proteína referente a esta sequência relacionada à mobilização de reserva. Durante a purificação e sequenciamento interno da &alpha;-galactosidase e demais proteínas foram detectadas isoformas da &alpha;-galactosidase de pesos moleculares variados (42 kDa a 20 kDa). Sugere-se que estas isoformas encontradas inicialmente na purificação estejam relacionadas com outras funções da &alpha;-galactosidase, enquanto as isoformas encontradas após todas as etapas de purificação e identificação por espectrometria de massas estejam relacionadas com ativação e adaptação da &alpha;-galactosidase durante todo o processo de mobilização de reservas. Os dados gerados das comparações dos oligossacarídeos produzidos em cada hidrólise sugerem que as ramificações do galactomanano podem modular o reconhecimento de sítios de clivagem pela endo-&beta;-mananase: (1) existe a produção de oligossacarídeos limites de digestão F1, F2 e F3 após hidrólise do galactomanano com endo-&beta;-mananase, como demonstrado para xiloglucanos; (2) os oligossacarídeos F1 possuem proporções distintas quando da hidrólise do galactomanano com endo-&beta;-mananase em diferentes concentrações (ExP I e EXP IV), evidenciando preferência por sítios com menor grau de galactosilação; (3) a presença da &alpha;-galactosidase diminui a produção dos oligossacarídeos F2 e F3, mostrando que estes não possuem resistência intrínseca a hidrólise e que a reação atinge o equilíbrio mesmo quando ainda existem sítios de clivagem ainda disponíveis (EXP III); (4) polissacarídeos com estruturas diferentes, razão manose:galactose variando entre 150:1 a 1:1, são digeridos em diferentes taxas de hidrólise pela mesma enzima, evidenciando que a ramificação com galactose dificulta a ação da endo-&beta;-mananase. Dessa forma, sugere-se que a estrutura do polissacarídeo galactomanano também contenha, pelo menos, parte da informação requerida para seu próprio metabolismo, código para a sua degradação, estando esta informação contida na distribuição das ramificações com resíduos de D-galactose. Sendo assim, sugere-se que as diferentes isoformas da &alpha;-galactosidase relacionadas à degradação da reserva de galactomanano de sementes de Sesbania virgata (Cav.) Pers. seriam produto da ação proteolítica da própria enzima a fim de melhorar a afinidade da &alpha;-galactosidase ao substrato durante o processo de mobilização de reserva. O aumento da afinidade da &alpha;-galactosidase ao substrato durante todo o processo de mobilização garantiria a liberação das ramificações com galactose de forma contínua, permitindo e aumentando a eficiência da ação da enzima endo-&beta;-mananase aos sítios de clivagem, garantindo a degradação do polissacarídeo a oligossacarídeos de forma regulada, passível de bloqueio, pelo acúmulo de oligossacarídeos e galactose livre que inibem a ação das enzimas endo-&beta;-mananase e &alpha;-galactosidase, respectivamente, e dificultando a ação de microorganismos, propiciando ao embrião a maior quantidade de açúcares para o seu desenvolvimento, aumentando as chances de sucesso no estabelecimento da plântula / The seeds of Sesbania virgata (Cav.) Pers. have an endosperm which accumulates galactomannan as a storage polysaccharide in the cell walls. Galactomannans are composed of a linear backbone of &beta;-(1,4)-linked D-mannose residues with D-galactose &alpha;-(1,6)-linkages substitutions. The galactomannans are hydrolysed after protrusion of the radicle. This process is perfomed by three enzymes (&alpha;-galactosidase, endo-&beta;-mannanase and exo-&beta;-manosidase). The &alpha;-galactosidase is the first enzyme to cleave the polysaccharides, removing the D-galactose residues, allowing the performance of the endo-&beta;-mannanase, which hydrolyses the mannan backbone to mannan oligosaccharides. The last part of the process includes exo-&beta;-manoside, that cleaves the mannan oligosaccharides to mannose residues, which could be used by the embryo during growth. Aiming at understanding the function of ?-galactosidase in the process of galatomanannan degradation, we studied its mode of action on mannans and galactomannans. The &alpha;-galactosidase of Sesbania virgata (Cav.) Pers. was purified and characterized (pH and temperature optimum and the enzyme kinetics). We found that the semipurified &alpha;-galactosidase molecular weight was 42kDa at denaturating conditions, but in native conditions was 72kDa, suggesting that the enzyme has a quaternary structure. The enzyme optimum pH was between 4,4-5,4, optimum temperature between 50°C-55°C, Km= 1,8276 mM and Vmáx= 0,5024 &mu;molGal.min-1.mgprot-1. Mass spectrometry measures resulted the following fragments: ALADYV-HSK-RMPGSLGHEE-QDAK-TT-GDIEDNWNSMTSIADS-NDKW-ASYAGPGGWN-DPDMLEVGNG-GMTTEEYR-AP-LLVGCDIR-VAVIL-WNR, being the protein from this sequence related with storage mobilization. Possible &alpha;-galactosidase isoforms were detected during the purification, suggesting other functions for the enzyme. The &alpha;-galactosidase isoforms detected after all purification steps and with measured mass spectrometry (from 42kDa to 20kDa) should be related to the storage mobilization. We suggest that the &alpha;-galactosidase isoforms in Sesbania virgata (Cav.) Pers. seeds represents products of the enzyme self-digestion, this process being correlated with the enzyme/polysaccharide affinity and at last, correlated to the galactomannan mobilization. An extract semipurified from Sesbania virgata (Cav.) Pers. and enriched with &alpha;-galactosidase activity, was used along with endo-&beta;-mannanase from Aspergillus niger (Megazyme&reg;) or both endo-&beta;-mannanase and &alpha;-galactosidase (semipurified from Sesbania virgata seeds - Chapter 1- or commercial enzyme from Cyamopsis tetragonoloba - Megazyme&reg;) were used to study the fine structure of galactomannans. Hydrolysis of galactomannans from six species with different mannose:galactose (1:1 to 150:1) ratio were performed with endo-&beta;-mananase from Aspergillus niger. The oligosaccharides from all hydrolysis were analyzed by HPAEC-PAD (High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection). The hydrolysis fragments data (HPAEC-PAD) suggest that the side-chains of the polysaccharides can modulate the hydrolytic sites recognition on the galactomannan by the endo-&beta;-mannanase. This conclusion is supported by: (1) the presence of limited digest oligosaccharides F1 and dimmers (F2) and trimers (F3) of the F1 oligosaccharides; (2) the presence of different F1 oligosaccharides proportions after hydrolysis with endo-&beta;-mannanase at different concentrations, showing preference on less-branched hydrolytic sites; (3) the &alpha;-galactosidase digestion avoided the accumulation of oligosaccharides F2 and F3, showing that these oligosaccharides do not present intrinsic resistance to hydrolysis and that the reaction reaches an equilibrium even when sites of hydrolysis are still available; (4) polymers with different fine structure (ratio mannose:galactose 1:1 to 150:1) were hydrolysed at different rates by the endo-&beta;-mannanase, showing that galactose branching interferes on the enzyme action. Considering that, the branching pattern of the polysaccharide seems to have direct influence on the interaction of the enzyme with substrate; we suggest that the structure of the galactomannan holds part of information required for its own degradation. The higher enzyme x substrate affinity, ensure the galactose branches digestion, improving the endo-&beta;-mannanase action, ensuring the degradation of the polysaccharides to oligosaccharides. This highly regulated degradation process prevents microorganisms predation and increases the plantlet establishement