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Wuhua Long - One of the best experts on this subject based on the ideXlab platform.
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floury shrunken Endosperm1 connects phospholipid metabolism and amyloplast development in rice
Plant Physiology, 2018Co-Authors: Wuhua Long, Yunlong Wang, Susong Zhu, Wen Jing, Yihua Wang, Yulong Ren, Yunlu Tian, Shijia Liu, Xi LiuAbstract:Starch synthesized and stored in amyloplasts serves as the major energy storage molecule in cereal Endosperm. To elucidate the molecular mechanisms underlying amyloplast development and starch synthesis, we isolated a series of floury Endosperm mutants in rice (Oryza sativa). We identified the rice mutant floury shrunken Endosperm1 (fse1), which exhibited obvious defects in the development of compound starch grains, decreased starch content, and altered starch physicochemical features. Map-based cloning showed that FSE1 encodes a phospholipase-like protein homologous to phosphatidic acid-preferring phospholipase A1. FSE1 was expressed ubiquitously with abundant levels observed in developing seeds and roots. FSE1 was localized to both the cytosol and intracellular membranes. Lipid profiling indicated that total extra-plastidic lipids and phosphatidic acid were increased in fse1 plants, suggesting that FSE1 may exhibit in vivo phospholipase A1 activity on phosphatidylcholine, phosphatidylinositol, phosphatidyl-Ser, phosphatidylethanolamine, and, in particular, phosphatidic acid. Additionally, the total galactolipid content in developing fse1 Endosperm was significantly reduced, which may cause abnormal amyloplast development. Our results identify FSE1 as a phospholipase-like protein that controls the synthesis of galactolipids in rice Endosperm and provide a novel connection between lipid metabolism and starch synthesis in rice grains during Endosperm development.
Yihua Wang - One of the best experts on this subject based on the ideXlab platform.
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floury shrunken Endosperm1 connects phospholipid metabolism and amyloplast development in rice
Plant Physiology, 2018Co-Authors: Wuhua Long, Yunlong Wang, Susong Zhu, Wen Jing, Yihua Wang, Yulong Ren, Yunlu Tian, Shijia Liu, Xi LiuAbstract:Starch synthesized and stored in amyloplasts serves as the major energy storage molecule in cereal Endosperm. To elucidate the molecular mechanisms underlying amyloplast development and starch synthesis, we isolated a series of floury Endosperm mutants in rice (Oryza sativa). We identified the rice mutant floury shrunken Endosperm1 (fse1), which exhibited obvious defects in the development of compound starch grains, decreased starch content, and altered starch physicochemical features. Map-based cloning showed that FSE1 encodes a phospholipase-like protein homologous to phosphatidic acid-preferring phospholipase A1. FSE1 was expressed ubiquitously with abundant levels observed in developing seeds and roots. FSE1 was localized to both the cytosol and intracellular membranes. Lipid profiling indicated that total extra-plastidic lipids and phosphatidic acid were increased in fse1 plants, suggesting that FSE1 may exhibit in vivo phospholipase A1 activity on phosphatidylcholine, phosphatidylinositol, phosphatidyl-Ser, phosphatidylethanolamine, and, in particular, phosphatidic acid. Additionally, the total galactolipid content in developing fse1 Endosperm was significantly reduced, which may cause abnormal amyloplast development. Our results identify FSE1 as a phospholipase-like protein that controls the synthesis of galactolipids in rice Endosperm and provide a novel connection between lipid metabolism and starch synthesis in rice grains during Endosperm development.
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floury Endosperm7 encodes a regulator of starch synthesis and amyloplast development essential for peripheral Endosperm development in rice
Journal of Experimental Botany, 2016Co-Authors: Long Zhang, Yunlong Wang, Yulong Ren, Chunyan Yang, Zhiming Feng, Zhou Liu, Jun Chen, Ying Wang, Wenwei Zhang, Yihua WangAbstract:In cereal crops, starch synthesis and storage depend mainly on a specialized class of plastids, termed amyloplasts. Despite the importance of starch, the molecular machinery regulating starch synthesis and amyloplast development remains largely unknown. Here, we report the characterization of the rice (Oryza sativa) floury Endosperm7 (flo7) mutant, which develops a floury-white Endosperm only in the periphery and not in the inner portion. Consistent with the phenotypic alternation in flo7 Endosperm, the flo7 mutant had reduced amylose content and seriously disrupted amylopectin structure only in the peripheral Endosperm. Notably, flo7 peripheral Endosperm cells showed obvious defects in compound starch grain development. Map-based cloning of FLO7 revealed that it encodes a protein of unknown function. FLO7 harbors an N-terminal transit peptide capable of targeting functional FLO7 fused to green fluorescent protein to amyloplast stroma in developing Endosperm cells, and a domain of unknown function 1338 (DUF1338) that is highly conserved in green plants. Furthermore, our combined β-glucuronidase activity and RNA in situ hybridization assays showed that the FLO7 gene was expressed ubiquitously but exhibited a specific expression in the Endosperm periphery. Moreover, a set of in vivo experiments demonstrated that the missing 32 aa in the flo7 mutant protein are essential for the stable accumulation of FLO7 in the Endosperm. Together, our findings identify FLO7 as a unique plant regulator required for starch synthesis and amyloplast development within the peripheral Endosperm and provide new insights into the spatial regulation of Endosperm development in rice.
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floury Endosperm6 encodes a cbm48 domain containing protein involved in compound granule formation and starch synthesis in rice Endosperm
Plant Journal, 2014Co-Authors: Cheng Peng, Yihua Wang, Long Zhang, Kunneng Zhou, Jia Lv, Ming Zheng, Shaolu Zhao, Chunming Wang, Ling Jiang, Xin ZhangAbstract:Summary Starch is the most widespread form of energy storage in the plant kingdom. Although many enzymes and related factors have been identified for starch biosynthesis, unknown players remain to be identified, given that it is a complicated and sophisticated process. The Endosperm of rice (Oryza sativa) has been used for the study of starch synthesis. Here, we report the cloning and characterization of the FLOURY Endosperm6 (FLO6) gene in rice. In the flo6 mutant, the starch content is decreased and the normal physicochemical features of starch are changed. Significantly, flo6 mutant Endosperm cells show obvious defects in compound granule formation. Map-based cloning showed that FLO6 encodes a protein of unknown function. It harbors an N–terminal transit peptide that ensures its correct localization and functions in the plastid, and a C–terminal carbohydrate-binding module 48 (CBM48) domain that binds to starch. Furthermore, FLO6 can interact with isoamylase1 (ISA1) both in vitro and in vivo, whereas ISA1 does not bind to starch directly. We thus propose that FLO6 may act as a starch-binding protein involved in starch synthesis and compound granule formation through a direct interaction with ISA1 in developing rice seeds. Our data provide a novel insight into the role of proteins with the CBM48 domain in plant species.
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floury Endosperm6 encodes a cbm48 domain containing protein involved in compound granule formation and starch synthesis in rice Endosperm
Plant Journal, 2014Co-Authors: Cheng Peng, Yihua Wang, Yulong Ren, Long Zhang, Kunneng Zhou, Ming Zheng, Shaolu Zhao, Chunming Wang, Feng Liu, Ling JiangAbstract:Starch is the most widespread form of energy storage in the plant kingdom. Although many enzymes and related factors have been identified for starch biosynthesis, unknown players remain to be identified, given that it is a complicated and sophisticated process. The Endosperm of rice (Oryza sativa) has been used for the study of starch synthesis. Here, we report the cloning and characterization of the FLOURY Endosperm6 (FLO6) gene in rice. In the flo6 mutant, the starch content is decreased and the normal physicochemical features of starch are changed. Significantly, flo6 mutant Endosperm cells show obvious defects in compound granule formation. Map-based cloning showed that FLO6 encodes a protein of unknown function. It harbors an N-terminal transit peptide that ensures its correct localization and functions in the plastid, and a C-terminal carbohydrate-binding module 48 (CBM48) domain that binds to starch. Furthermore, FLO6 can interact with isoamylase1 (ISA1) both in vitro and in vivo, whereas ISA1 does not bind to starch directly. We thus propose that FLO6 may act as a starch-binding protein involved in starch synthesis and compound granule formation through a direct interaction with ISA1 in developing rice seeds. Our data provide a novel insight into the role of proteins with the CBM48 domain in plant species.
Tai Wang - One of the best experts on this subject based on the ideXlab platform.
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proteomic dissection of Endosperm starch granule associated proteins reveals a network coordinating starch biosynthesis and amino acid metabolism and glycolysis in rice Endosperms
Frontiers in Plant Science, 2016Co-Authors: Tai WangAbstract:Starch biosynthesis and starch granule packaging in cereal Endosperms involve a coordinated action of starch biosynthesis enzymes and coordination with other metabolisms. Because directly binding to starch granules, starch granule-associated proteins (SGAPs) are essential to understand the underlying mechanisms, however the information on SGAPs remains largely unknown. Here, we dissected developmentally changed SGAPs from developing rice Endosperms from 10 to 20 days after flowering (DAF). Starch granule packaging was not completed at 10 DAF, and was finished in the central Endosperm at 15 DAF and in the whole Endosperm at 20 DAF. Proteomic analysis with two-dimensional differential in-gel electrophoresis and mass spectrometry revealed 115 developmentally changed SGAPs, representing 37 unique proteins. 65% of the unique proteins had isoforms. 39% of the identified SGAPs were involved in starch biosynthesis with main functions in polyglucan elongation and granule structure trimming. Almost all proteins involved in starch biosynthesis, amino acid biosynthesis, glycolysis, protein folding, and PPDK pathways increased abundance as the Endosperm developed, and were predicted in an interaction network. The network represents an important mechanism to orchestrate carbon partitioning among starch biosynthesis, amino acid biosynthesis and glycolysis for efficient starch and protein storage. These results provide novel insights into mechanisms of starch biosynthesis and its coordination with amino acid metabolisms and glycolysis in cereal Endosperms.
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comparative proteomic study reveals the involvement of diurnal cycle in cell division enlargement and starch accumulation in developing Endosperm of oryza sativa
Journal of Proteome Research, 2012Co-Authors: Can Hui Zheng, Tai WangAbstract:The development and starch accumulation of cereal Endosperms rely on the sugar supply of leaves, which is subject to diurnal cycles, and the Endosperm itself also experiences a light/dark switch. However, revealing how the cereal Endosperm responds to diurnal input remains a major challenge. We used comparative proteo- mic approaches to probe diurnally affected processes in rice Endosperm (Oryza sativa) 10 days after flowering under 12-h light/12-h dark. StarchgranulesinriceEndospermshowedagrowthringstructureunder a normal light/dark cycle but not under constant light. Sucrose showed a high level in light and low level in dark. Two-dimensional (2-D) differentialin-gelelectrophoresis-basedproteomicanalysisrevealed101 protein spots diurnally changed and 91 identities, which were involved in diverse processes with preferred distribution in stress response, protein synthesis/destination and metabolism. Proteins involved in cell division showed high expression in light and those in cellenlargementandcellwallsynthesishighindark,whilestarchsynthesisproteinswerelight-downregulatedanddark-upregulated. Redox homeostasis-associated proteins showed in-phase peaks under light and dark. These data demonstrate diurnal input- regulated diverse cellular and metabolic processes in rice Endosperm, and coordination among these processes is essential for development and starch accumulation with diurnal input.
Yasunori Nakamura - One of the best experts on this subject based on the ideXlab platform.
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roles of isoamylase and adp glucose pyrophosphorylase in starch granule synthesis in rice Endosperm
Plant Journal, 2005Co-Authors: Yasushi Kawagoe, Hikaru Satoh, Akiko Kubo, Fumio Takaiwa, Yasunori NakamuraAbstract:Summary Amyloplast-targeted green fluorescent protein (GFP) was used to monitor amyloplast division and starch granule synthesis in the developing Endosperm of transgenic rice. Two classical starch mutants, sugary and shrunken, contain reduced activities of isoamylase1 (ISA1) and cytosolic ADP-glucose pyrophosphorylase, respectively. Dividing amyloplasts in the wild-type and shrunken Endosperms contained starch granules, whereas those in sugary Endosperm did not contain detectable granules, suggesting that ISA1 plays a role in granule synthesis at the initiation step. The transition from phytoglycogen to sugary-amylopectin was gradual in the boundary region between the inner and outer Endosperms of sugary. These results suggest that the synthesis of sugary-amylopectin and phytoglycogen involved a stochastic process and that ISA1 activity plays a critical role in the stochastic process in starch synthesis in rice Endosperm. The reduction of cytosolic ADP-glucose pyrophosphorylase activity in shrunken Endosperm did not inhibit granule initiation but severely restrained the subsequent enlargement of granules. The shrunken Endosperm often developed pleomorphic amyloplasts containing a large number of underdeveloped granules or a large cluster of small grains of amyloplasts, each containing a simple-type starch granule. Although constriction-type divisions of amyloplasts were much more frequent, budding-type divisions were also found in the shrunken Endosperm. We show that monitoring GFP in developing amyloplasts was an effective means of evaluating the roles of enzymes involved in starch granule synthesis in the rice Endosperm.
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the starch debranching enzymes isoamylase and pullulanase are both involved in amylopectin biosynthesis in rice Endosperm
Plant Physiology, 1999Co-Authors: Akiko Kubo, Hikaru Satoh, Kyuya Harada, Toshiaki Matsuda, Naoko Fujita, Yasunori NakamuraAbstract:The activities of the two types of starch debranching enzymes, isoamylase and pullulanase, were greatly reduced in Endosperms of allelic sugary-1 mutants of rice ( Oryza sativa ), with the decrease more pronounced for isoamylase than for pullulanase. However, the decrease in isoamylase activity was not related to the magnitude of the sugary phenotype (the proportion of the phytoglycogen region of the Endosperm), as observed with pullulanase. In the moderately mutated line EM-5, the pullulanase activity was markedly lower in the phytoglycogen region than in the starch region, and isoamylase activity was extremely low or completely lost in the whole Endosperm tissue. These results suggest that both debranching enzymes are involved in amylopectin biosynthesis in rice Endosperm. We presume that isoamylase plays a predominant role in amylopectin synthesis, but pullulanase is also essential or can compensate for the role of isoamylase in the construction of the amylopectin multiple-cluster structure. It is highly possible that isoamylase was modified in some sugary-1 mutants such as EM-273 and EM-5, since it was present in significant and trace amounts, respectively, in these mutants but was apparently inactive. The results show that the Sugary-1 gene encodes the isoamylase gene of the rice genome.
Lisboa, César Gustavo Serafim - One of the best experts on this subject based on the ideXlab platform.
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Endo-beta-mananase de Endosperma de Sesbania virgata (Cv.) Pers. : purificação, caracterização e importancia na germinação e desenvolvimento da plantula
[s.n.], 2018Co-Authors: Lisboa, César Gustavo SerafimAbstract:Orientador: Marcos Silveira BuckeridgeDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: As sementes de muitas plantas possuem Endosperma, um tecido especializado com funções tanto de reserva como de constrição mecânica do embrião nas primeiras etapas de desenvolvimento. Em muitas espécies de leguminosas, estes tecidos especializados armazenam como polissacarídeo de reserva, o galactomanano. Este polímero apresenta um cadeia central de manose ( ß-1,4 ligadas) e ramificações de galactose (?-l,6 ligadas ao esqueleto central). Três enzimas estão envolvidas na hidrólise do galactomanano: a endo-ß- mananase, a ?-galactosidase e a ß-manosidase. Da forma com que a endo-mananase ataca o polímero, inicialmente produz oligossacarídeos galactosilados que, são posteriormente hidrolisados pelas duas últimas exo-enzimas (?-galactosidase e ß-mananase) até galactose e manose livres. Neste trabalho, uma endo-ß-mananase foi purificada do Endosperma das sementes de Sesbania virgata, uma leguminosa nativa. Uma curva de tempo de germinação mostrou que a atividade da mananase aparece inicialmente na região do Endosperma próxima à área de protrusão da radícula e, subseqüentemente, aumenta na região lateral do Endosperma da semente seguindo o desenvolvimento da plântula. Medindo a atividade em pH 5 a 45°C, o máximo da atividade catalítica correspondeu a 120h de desenvolvimento para, em seguida, decair. Para a purificação da enzima, utilizou-se uma coluna de troca iônica DEAE-celulose seguida de uma coluna de afinidade biológica em Sepharose Concanavalina A. A mananase purificada é uma glicoproteína de peso molecular de cerca de 30KDa, com pH ótimo entre 3,5 e 5, temperatura ótima de 45°C e ponto isoelétrico 4,5. A caracterização da atividade da endo-mananase sobre diferentes galactomananos com diferentes razões manose:galactose, indicou que a ramificação do polissacarídeo com galactose é o "fator-chave" na modulação da própria ação catalítica dessa enzima. Com essas observações, foi concluído que, a atividade da endo-mananase, além de estar relacionada com a mobilização de reservas, facilita a emersão da radícula agindo na área próxima à protrusão. Entendendo que a mananase pura não foi capaz de degradar o galactomanano da Trigonellafoenum-graecum (razão 1:1 de manose/galactose), sugere-se que exista uma relação inversa entre o grau de ramificação e a porcentagem de hidrólise da endo-mananaseAbstract; Many plant seeds have an Endosperm, a tissue specialised either as storage or mechanical constraint to embryo growth. In many legume species, Endosperms contain galactomannan. This polyssacharide is composed of a main chain of 1,4-ß-mannan branched with variable amounts of 1,6-?-linked galactosyl residues. Three enzymes produced by an aleurone layer are involved in galactomannan hydrolysis: endo-ß-mannanase, ?-galactosidase and ß- mannosidase. The former with mannanase attacks the polymer producing galactosylated oligossacharides which are further hydrolysed by the two last exo-enzymes to free mannose and galactose. In the present work, an endo-ß-mannanase was purified from the endosp erm of Sesbanía vírgata, a Brazilian native legume. The time-course shows that the activity of mannanase appears initially in the Endosperm cap of seeds prior to radicle emergence and subsequently increases in the remaining lateral Endosperm following seedling growth. Measuring the activity at pH 5 and 45°C, endo-mannanase peaked at 120h and then decreased. We used these conditions to perform purification procedures in DEAE-cellulose followed by affinity chromatography with Sepharose Concanavaline-A. The enzyme is a glycoprotein with an apparent molecular weight of 30Kda, optimum pH at 4.5 and temperature at 45°C, pI at 4.5. The mode of action of the enzyme on different galactomannans indicates the modulation of mannanase activity by the branching pattems of the polymers with galactose. From these observations it was concluded that mannanase activity facilitates radicle protrusion through the surrounding Endosperm by weakening it in the area close to the radicle tip. On the basis that the pure enzyme can not degrade the fenugreek galactomannan (a fully substituted polysaccharide), we suggest that there is an inverse relationship between galactose branching and the degree of hydrolysisMestradoBiologia CelularMestre em Biologia Celular e Estrutura
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Endo-beta-mananase do Endosperma de sementes de Sesbania virgata (Cav.) Pers. (Leguminosae): purificação, caracterização e seu duplo papel na germinação e crescimento inicial da plântula
Brazilian Journal of Plant Physiology, 2015Co-Authors: Lisboa, César Gustavo Serafim, Tonini, Patrícia Pinho, Tiné, Marco Aurélio Silva, Buckeridge, Marcos SilveiraAbstract:Galactomannans are storage cell wall polysaccharides present in seeds of some legumes. Their degradation is carried out by three hydrolases (alpha-galactosidase (EC 3.2.1.22), endo-beta-mannanase (EC 3.2.1.78) and ß-mannosidase (EC 3.2.1.25)). In the present study we purified and characterised an endo-beta-mannanase from seeds of Sesbania virgata and addressed its role in germination and seedling development. The polypeptide purified by Ion Exchange Chromatography and Affinity Chromatography on Sepharose-Concanavalin A, showed a pH optimum between 3.5 and 5 at 45ºC and high stability at pH 7.8. The low stability at pH 5 appears to be associated with isoelectric precipitation, in view of the pI of the enzyme being 4.5. The purified enzyme is a glycoprotein with a molecular mass of 26 KDa by SDS-PAGE and 36 KDa by gel chromatography. The purified polypeptide attacked galactomannan from different sources, being more effective on polymers with a lower degree of galactosylation (from carob gum), in comparison with medium or highly galactosylated galactomannans (from guar, S. virgata and fenugreek), respectively. A peak of endo-beta-mannanase activity was detected during radicle protrusion in the Endosperm tissue surrounding the radicle and later on in the lateral Endosperm. This second peak was associated with the period of reserve mobilisation. Using an antibody raised against coffee endo-beta-mannanase, the enzyme could be detected in immunodot-blots performed with extracts of S. virgata Endosperms. The results are consistent with the hypothesis that the peak of endo-mannanase during germination facilitates radicle protrusion through the surrounding Endosperm by weakening it in the region close to the radicle tip.Galactomananos são polissacarídeos de reserva de parede celular presentes em sementes de leguminosas. Sua degradação é efetuada por três hidrolases (alfa-galactosidase (EC 3.2.1.22), endo-beta-mananase (EC 3.2.1.78) e manosidase (EC 3.2.1.25)). No presente estudo, nós purificamos e caracterizamos uma endo-beta-mananase de sementes de Sesbania virgata e focamos no seu papel na germinação e no desenvolvimento da plântula. A enzima foi purificada por cromatografia de troca iônica e cromatografia de afinidade em sepharose-concanavalina A, mostrando um pH ótimo entre 3,5 e 5 a 45 ºC e alta estabilidade em pH 7,8. A baixa estabilidade em pH 5 parece estar associada à precipitação isoelétrica, pois o pI da enzima é 4,5. O polipeptídeo purificado é uma glicoproteína com massa molecular de 26 KDa em SDS-PAGE e 36 KDa em cromatografia em gel. O polipeptídeo purificado atacou galactomamano de diferentes fontes, sendo efetivo sobre polímeros com grau de galactosilação mais baixo (goma caroba), em comparação com galactomamanos com médio e alto graus de galactosilação (guar, S. virgata e feno grego), respectivamente. Um pico de atividade de endo-beta-mananase foi detectado durante a protrusão da radícula no tecido endospérmico ao redor da radícula e mais tarde nas porções laterais do Endosperma. O segundo pico foi inversamente associado à mobilização de reservas. Usando um anticorpo feito contra endo-beta-mananase de café, a enzima foi detectada por immunodot-blots feitos com extratos de Endosperma de S. virgata. Os resultados são consistentes com a hipótese de que o pico de endo-mananase durante a germinação facilita a protrusão da radícula através do enfraquecimento do Endosperma que circunda a área próxima à ponta da radícula
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Endo-beta-mananase do Endosperma de sementes de Sesbania virgata (Cav.) Pers. (Leguminosae): purificação, caracterização e seu duplo papel na germinação e crescimento inicial da plântula
Brazilian Journal of Plant Physiology, 2015Co-Authors: Lisboa, César Gustavo Serafim, Tonini, Patrícia Pinho, Tiné, Marco Aurélio Silva, Buckeridge, Marcos SilveiraAbstract:Galactomannans are storage cell wall polysaccharides present in seeds of some legumes. Their degradation is carried out by three hydrolases (alpha-galactosidase (EC 3.2.1.22), endo-beta-mannanase (EC 3.2.1.78) and ß-mannosidase (EC 3.2.1.25)). In the present study we purified and characterised an endo-beta-mannanase from seeds of Sesbania virgata and addressed its role in germination and seedling development. The polypeptide purified by Ion Exchange Chromatography and Affinity Chromatography on Sepharose-Concanavalin A, showed a pH optimum between 3.5 and 5 at 45ºC and high stability at pH 7.8. The low stability at pH 5 appears to be associated with isoelectric precipitation, in view of the pI of the enzyme being 4.5. The purified enzyme is a glycoprotein with a molecular mass of 26 KDa by SDS-PAGE and 36 KDa by gel chromatography. The purified polypeptide attacked galactomannan from different sources, being more effective on polymers with a lower degree of galactosylation (from carob gum), in comparison with medium or highly galactosylated galactomannans (from guar, S. virgata and fenugreek), respectively. A peak of endo-beta-mannanase activity was detected during radicle protrusion in the Endosperm tissue surrounding the radicle and later on in the lateral Endosperm. This second peak was associated with the period of reserve mobilisation. Using an antibody raised against coffee endo-beta-mannanase, the enzyme could be detected in immunodot-blots performed with extracts of S. virgata Endosperms. The results are consistent with the hypothesis that the peak of endo-mannanase during germination facilitates radicle protrusion through the surrounding Endosperm by weakening it in the region close to the radicle tip.Galactomananos são polissacarídeos de reserva de parede celular presentes em sementes de leguminosas. Sua degradação é efetuada por três hidrolases (alfa-galactosidase (EC 3.2.1.22), endo-beta-mananase (EC 3.2.1.78) e manosidase (EC 3.2.1.25)). No presente estudo, nós purificamos e caracterizamos uma endo-beta-mananase de sementes de Sesbania virgata e focamos no seu papel na germinação e no desenvolvimento da plântula. A enzima foi purificada por cromatografia de troca iônica e cromatografia de afinidade em sepharose-concanavalina A, mostrando um pH ótimo entre 3,5 e 5 a 45 ºC e alta estabilidade em pH 7,8. A baixa estabilidade em pH 5 parece estar associada à precipitação isoelétrica, pois o pI da enzima é 4,5. O polipeptídeo purificado é uma glicoproteína com massa molecular de 26 KDa em SDS-PAGE e 36 KDa em cromatografia em gel. O polipeptídeo purificado atacou galactomamano de diferentes fontes, sendo efetivo sobre polímeros com grau de galactosilação mais baixo (goma caroba), em comparação com galactomamanos com médio e alto graus de galactosilação (guar, S. virgata e feno grego), respectivamente. Um pico de atividade de endo-beta-mananase foi detectado durante a protrusão da radícula no tecido endospérmico ao redor da radícula e mais tarde nas porções laterais do Endosperma. O segundo pico foi inversamente associado à mobilização de reservas. Usando um anticorpo feito contra endo-beta-mananase de café, a enzima foi detectada por immunodot-blots feitos com extratos de Endosperma de S. virgata. Os resultados são consistentes com a hipótese de que o pico de endo-mananase durante a germinação facilita a protrusão da radícula através do enfraquecimento do Endosperma que circunda a área próxima à ponta da radícula.269280Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq
