Caracterização bioquímica e cinética de lipoxigenases de folhas de soja submetidas à remoção dos primórdios florais

Ferreira, Cristiano César de Araújo; Oliveira, Maria Goreti de Almeida; Pires, Christiano Vieira; Oliveira, Joel Antônio de; Pilon, Anderson Martins; Almeida, Fabrício Tadeu de; Brumano, Maria Helena Nasser; Moreira, Maurilio Alves

doi:10.1590/S1413-70542004000200003

Resumos

O uso da soja [Glycine max (L.) Merrill] na alimentação humana tem encontrado dificuldades devido ao seu sabor característico (beany flavor), que é derivado da ação das lipoxigenases (EC 1.13.11.12). Ao contrário das lipoxigenases de sementes de soja, que têm sido extensivamente estudadas, a diversidade e características das lipoxigenases de folhas não estão totalmente elucidadas. Sabe-se que além de sua atividade catalítica, as lipoxigenases de folhas estão envolvidas no armazenamento temporário de nitrogênio e na resposta da planta ao estresse. Com o objetivo de investigar o efeito da remoção dos primórdios florais na atividade das lipoxigenases de folhas de soja, foram utilizados dois genótipos de soja: IAC-100, uma variedade com presença de lipoxigenases na semente e IAC-100 TN, uma linhagem com ausência completa de lipoxigenases na semente. Por meio de dados da cinética enzimática, foram constatados dois picos mais acentuados de atividade em pH 5,0 e 6,5, com temperatura ótima de 25ºC. Para ambos os genótipos, as atividades foram maiores nos tratamentos que nos respectivos controles. Os valores de KM app aos 16 dias, nas plantas-controle, foram semelhantes para os dois genótipos, sugerindo não haver influência da manipulação genética das lipoxigenases na semente em relação à expressão dos genes que codificam as lipoxigenases de folhas. No entanto, os valores de KM app foram diferentes entre a fase vegetativa e a reprodutiva, em ambos genótipos, indicando pools distintos de isoenzimas lipoxigenases entre as duas fases.

Folhas de soja; lipoxigenase; parâmetros cinéticos; proteína vegetativa; estresse; Glycine max

The use of soybean [Glycine max (L.) Merrill] as human food has been encountered difficulties due to beany flavor. The beany flavour is originated from lipoxygenases (EC 1.13.11.12). Soybean seed lipoxygenases have been extensively studied, however, the diversity and characteristics of lipoxygenases present in leaves are still being studied. Besides its catalytic activity, lipoxygenases from soybean leaves have been implicated in temporary nitrogen storage and response to stress. The primary objective of this research was to investigate the effect of floral primordial elimination (sink deprivation) on soybean leaves lipoxygenases activity. Two different genotypes differing in the presence (IAC-100) or absence (IAC-100 TN) of LOX in their seeds were used. The enzymatic kinetics analysis showed two pronounced peaks of activity at pH 5 and 6.5, with optimal temperature of 25ºC. The KM app values were similar for both genotypes suggesting that there was not effect of genetic manipulation of seeds lipoxygenases on genes expression that code for leave lipoxygenases. However, the KM app values were different between the vegetative and reproductive development stage, suggesting that there are differents pool of lipoxygenases between the two stages.

soybean leaves; lipoxygenases; kinetic parameters; vegetative protein; stress; Glycine max

a03

Caracterização bioquímica e cinética de lipoxigenases de folhas de soja submetidas à remoção dos primórdios florais

Biochemical and kinetic characterization of lipoxygenases from soybean leaves submitted to the removal of floral primordial

Cristiano César de Araújo Ferreira^I, Maria Goreti de Almeida Oliveira^II; Christiano Vieira Pires^III; Joel Antônio de Oliveira^IV; Anderson Martins Pilon^III; Fabrício Tadeu de Almeida^V; Maria Helena Nasser Brumano^VI; Maurilio Alves Moreira^II

^IPós-Graduado em Agroquímica, DBB/BIOAGRO/UFV, 36571-000 Viçosa, MG

^IIProfessor do Departamento de Bioquímica e Biologia Molecular/BIOAGRO, UFV, E-mail: malmeida@mail.ufv.br

^IIIPós-Graduando do Departamento de Bioquímica e Biologia Molecular, UFV

^IVProfessor do Departamento de Química, UFV

^VEstudante de Iniciação Científica, Departamento de Bioquímica e Biologia Molecular, UFV

^VIPesquisadora do Laboratório de Enzimologia, BIOAGRO/UFV

RESUMO

O uso da soja [Glycine max (L.) Merrill] na alimentação humana tem encontrado dificuldades devido ao seu sabor característico (beany flavor), que é derivado da ação das lipoxigenases (EC 1.13.11.12). Ao contrário das lipoxigenases de sementes de soja, que têm sido extensivamente estudadas, a diversidade e características das lipoxigenases de folhas não estão totalmente elucidadas. Sabe-se que além de sua atividade catalítica, as lipoxigenases de folhas estão envolvidas no armazenamento temporário de nitrogênio e na resposta da planta ao estresse. Com o objetivo de investigar o efeito da remoção dos primórdios florais na atividade das lipoxigenases de folhas de soja, foram utilizados dois genótipos de soja: IAC-100, uma variedade com presença de lipoxigenases na semente e IAC-100 TN, uma linhagem com ausência completa de lipoxigenases na semente. Por meio de dados da cinética enzimática, foram constatados dois picos mais acentuados de atividade em pH 5,0 e 6,5, com temperatura ótima de 25ºC. Para ambos os genótipos, as atividades foram maiores nos tratamentos que nos respectivos controles. Os valores de KM app aos 16 dias, nas plantas-controle, foram semelhantes para os dois genótipos, sugerindo não haver influência da manipulação genética das lipoxigenases na semente em relação à expressão dos genes que codificam as lipoxigenases de folhas. No entanto, os valores de KM app foram diferentes entre a fase vegetativa e a reprodutiva, em ambos genótipos, indicando pools distintos de isoenzimas lipoxigenases entre as duas fases.

Termos para indexação: Folhas de soja, lipoxigenase, parâmetros cinéticos, proteína vegetativa, estresse, Glycine max.

ABSTRACT

The use of soybean [Glycine max (L.) Merrill] as human food has been encountered difficulties due to beany flavor. The beany flavour is originated from lipoxygenases (EC 1.13.11.12). Soybean seed lipoxygenases have been extensively studied, however, the diversity and characteristics of lipoxygenases present in leaves are still being studied. Besides its catalytic activity, lipoxygenases from soybean leaves have been implicated in temporary nitrogen storage and response to stress. The primary objective of this research was to investigate the effect of floral primordial elimination (sink deprivation) on soybean leaves lipoxygenases activity. Two different genotypes differing in the presence (IAC-100) or absence (IAC-100 TN) of LOX in their seeds were used. The enzymatic kinetics analysis showed two pronounced peaks of activity at pH 5 and 6.5, with optimal temperature of 25ºC. The KM app values were similar for both genotypes suggesting that there was not effect of genetic manipulation of seeds lipoxygenases on genes expression that code for leave lipoxygenases. However, the KM app values were different between the vegetative and reproductive development stage, suggesting that there are differents pool of lipoxygenases between the two stages.

Index terms: soybean leaves, lipoxygenases, kinetic parameters, vegetative protein, stress, Glycine max.

Texto completo disponível apenas em PDF.

Full text available only in PDF format.

(Recebido para publicação em 18 de novembro de 2002 e aprovado em 25 de dezembro de 2002)

ANDERSON, J. M.; SILATRO, S. R.; KLAUER, S. F.; FRANCESCHI, V. R. Jasmonic acid-dependent increases in the level of vegetative storage proteins in soybean. Plant Science, Shannon, v. 62, p. 45-52, 1989.
AXELROD, B.; CHEESBROUGH, T. M.; LAASKO, S. Lipoxygenases from soybeans. Methods Enzymology, [S.l.], v. 71, p. 441-451, 1981.
BOHLAND, C.; BALKENHOHL, T.; LOERS G.; FEUSSNER, I.; GRAMBOW, H. J. Differential induction of lipoxygenases isoforms in wheat upon treatment with rust fungus elicitor, chitin oligossaccharides, chitosan, and methyl jasmonate. Plant Physiology, Washington, v. 114, p. 679-685, 1997.
BUNKER, T. W.; KOETJE, D. S.; STEPHENSON, L. C.; CREELMAN, R. A.; MULLET, J. E.; GRIMES, H. D. Sink limitation induces the expression of multiple soybean lipoxygenase mRNAs while the endogenous jasmonic acid level remains low. Plant Cell, Tissue and Organ Culture, Dordrecht, v. 7, p. 1319-1331, 1995.
CROFT, K. P. C.; JÜTTNER, F.; SLUSARENKO, A. J. Volatile products of the lipoxygenase pathway evolved from Phaseolus vulgaris (L.) leaves inoculated with Pseudomonas syringae pv phaseolicola Plant Physiology, Washington, v. 101, p. 13-24, 1993.
FARMER, E. E.; RYAN, C. A. Octadecanoid precursors of jasmonic acid activate the syntesis of wound-inducible proteinase inhibitors. Plant Cell, Tissue and Organ Culture, Dordrecht, v. 4, p. 129-134, 1992.
FEHR, W. R.; CAVINESS, C. E. Stages of soybean development Ames: Cooperative Extension Service-lowa State University, 1977. 11 p. (Special Report, 80).
HILDEBRAND, D. F. Lipoxygenases. Physiologia Plantarum, Copenhagen, v. 76, p. 249-253, 1989.
LEATHERBARROW, R. J. Enziffiter manual. Biosoftware, London, p. 13-42, 1987.
OHTA, H.; IDA, S.; MIKAMI, B.; MORITA, Y. Changes in lipoxygenase components of rice seedling during germination. Plant Cell Physiology, Washington, v. 22, p. 911-918, 1986.
PARÉ, P. W.; TUMLINSON, J. H. De novo biosyntesis of volatiles inducible by insect herbivory in cotton plants. Plant Physiology, Washington, v. 114, p. 1161-1167, 1997.
PARK, T. K.; HOLLAND, M. A.; LASKEY, J. G.; POLACCO, J. C. Germination - associated lipoxygenases transcripts persist in maturing soybean plants and are induced by jasmonate. Plant Science, Shannon, v. 96, p. 109-117, 1994.
ROUET-MAYER, M.; BUREAU, J.; LAURIERI, C. Identification and characterization of lipoxygenase isoforms in senescing carnation petals. Plant Physiology, Washington, v. 98, p. 971-978, 1992.
SARAVITZ, D. M.; SIEDOW, J. N. Changes during leaf development, after wounding, and following reproductive sink removal. Plant Physiology, Washington, v. 107, p. 535-543, 1995.
SIEDOW, J. N. Plant lipoxygenase: struture and function. Annual Review Plant Physiology Plant Molecular Biology, Washington, v. 42, p. 145-188, 1991.
SILVA, M. D.; OLIVEIRA, M. G. A.; LANNA, A. N.; PIRES, C. V.; PIOVESAN, N. D.; JOSE, I. C.; BATISTA, R. B.; BARROS, E. G.; MOREIRA, M. A. Caracterizaçăo da via das lipoxigenases em plantas de soja resitentes e succeptiveis a Diaphorte phaseolorum F. SP. Meridionalis, agente causal do cancro da haste. Brazilian Journal of Plant Physiology, Săo Paulo, v. 13, n. 3, p. 316-328, 2001.
STASWICK, P. E. Novel regulation of vegetative storage protein genes. The Plant Cell, [S.l.], v. 2, p. 1-6, 1990.
STEPHENSON, L. C.; BUNKER, T. W.; DUBBS, W. E.; GRIMES, H. D. Specifc soybean lipoxygenases localize to discrete subcellular compartments and their mRNAs are differentially regulated by source-sink status. Plant Physiology, Washington, v. 116, p. 923-933, 1998.
TRANBARGER, T. J.; FRANCESCHI, V. R.; HILDEBRAND, D. F.; GRIMES, H. D. The soybean 94-kilodalton vegetative storage protein is a lipoxygenase that is localized in paraveinal mesophyll cell vacuoles. Plant Cell, Tissue and Organ Culture, Dordrecht, v. 3, p. 973-987, 1991.
VICK, B.; ZIMMERMAN, D. C. Oxidative systems for modification of fatty acids: the lipoxygenase pathway. In: STUMPF, P. K.; CONN, E. E. (Eds.). The biochemistry of plants Orlando: Academic, 1987. v. 9, p. 53-97.
VIEIRA, A. A.; OLIVEIRA, M. G. A.; JOSE, I. C.; PIOVESAN, N. D.; REZENDE, S. T.; MOREIRA, M. A.; BARROS, E. G. Biochemical evaluation of lipoxygenase pathaway of soybean plants submitted to wounding. Brazilian Journal of Plant Physiology, Săo Paulo, v. 13, n. 1, p. 5-12, 2001.

Datas de Publicação

Publicação nesta coleção
22 Out 2010
Data do Fascículo
Abr 2004

Histórico

Aceito
25 Dez 2002
Recebido
18 Nov 2002

This work is licensed under a Creative Commons Attribution 4.0 International License.

[1] ANDERSON, J. M.; SILATRO, S. R.; KLAUER, S. F.; FRANCESCHI, V. R. Jasmonic acid-dependent increases in the level of vegetative storage proteins in soybean. Plant Science, Shannon, v. 62, p. 45-52, 1989.

[2] AXELROD, B.; CHEESBROUGH, T. M.; LAASKO, S. Lipoxygenases from soybeans. Methods Enzymology, [S.l.], v. 71, p. 441-451, 1981.

[3] BOHLAND, C.; BALKENHOHL, T.; LOERS G.; FEUSSNER, I.; GRAMBOW, H. J. Differential induction of lipoxygenases isoforms in wheat upon treatment with rust fungus elicitor, chitin oligossaccharides, chitosan, and methyl jasmonate. Plant Physiology, Washington, v. 114, p. 679-685, 1997.

[4] BUNKER, T. W.; KOETJE, D. S.; STEPHENSON, L. C.; CREELMAN, R. A.; MULLET, J. E.; GRIMES, H. D. Sink limitation induces the expression of multiple soybean lipoxygenase mRNAs while the endogenous jasmonic acid level remains low. Plant Cell, Tissue and Organ Culture, Dordrecht, v. 7, p. 1319-1331, 1995.

[5] CROFT, K. P. C.; JÜTTNER, F.; SLUSARENKO, A. J. Volatile products of the lipoxygenase pathway evolved from Phaseolus vulgaris (L.) leaves inoculated with Pseudomonas syringae pv phaseolicola Plant Physiology, Washington, v. 101, p. 13-24, 1993.

[6] FARMER, E. E.; RYAN, C. A. Octadecanoid precursors of jasmonic acid activate the syntesis of wound-inducible proteinase inhibitors. Plant Cell, Tissue and Organ Culture, Dordrecht, v. 4, p. 129-134, 1992.

[7] FEHR, W. R.; CAVINESS, C. E. Stages of soybean development Ames: Cooperative Extension Service-lowa State University, 1977. 11 p. (Special Report, 80).

[8] HILDEBRAND, D. F. Lipoxygenases. Physiologia Plantarum, Copenhagen, v. 76, p. 249-253, 1989.

[9] LEATHERBARROW, R. J. Enziffiter manual. Biosoftware, London, p. 13-42, 1987.

[10] OHTA, H.; IDA, S.; MIKAMI, B.; MORITA, Y. Changes in lipoxygenase components of rice seedling during germination. Plant Cell Physiology, Washington, v. 22, p. 911-918, 1986.

[11] PARÉ, P. W.; TUMLINSON, J. H. De novo biosyntesis of volatiles inducible by insect herbivory in cotton plants. Plant Physiology, Washington, v. 114, p. 1161-1167, 1997.

[12] PARK, T. K.; HOLLAND, M. A.; LASKEY, J. G.; POLACCO, J. C. Germination - associated lipoxygenases transcripts persist in maturing soybean plants and are induced by jasmonate. Plant Science, Shannon, v. 96, p. 109-117, 1994.

[13] ROUET-MAYER, M.; BUREAU, J.; LAURIERI, C. Identification and characterization of lipoxygenase isoforms in senescing carnation petals. Plant Physiology, Washington, v. 98, p. 971-978, 1992.

[14] SARAVITZ, D. M.; SIEDOW, J. N. Changes during leaf development, after wounding, and following reproductive sink removal. Plant Physiology, Washington, v. 107, p. 535-543, 1995.

[15] SIEDOW, J. N. Plant lipoxygenase: struture and function. Annual Review Plant Physiology Plant Molecular Biology, Washington, v. 42, p. 145-188, 1991.

[16] SILVA, M. D.; OLIVEIRA, M. G. A.; LANNA, A. N.; PIRES, C. V.; PIOVESAN, N. D.; JOSE, I. C.; BATISTA, R. B.; BARROS, E. G.; MOREIRA, M. A. Caracterizaçăo da via das lipoxigenases em plantas de soja resitentes e succeptiveis a Diaphorte phaseolorum F. SP. Meridionalis, agente causal do cancro da haste. Brazilian Journal of Plant Physiology, Săo Paulo, v. 13, n. 3, p. 316-328, 2001.

[17] STASWICK, P. E. Novel regulation of vegetative storage protein genes. The Plant Cell, [S.l.], v. 2, p. 1-6, 1990.

[18] STEPHENSON, L. C.; BUNKER, T. W.; DUBBS, W. E.; GRIMES, H. D. Specifc soybean lipoxygenases localize to discrete subcellular compartments and their mRNAs are differentially regulated by source-sink status. Plant Physiology, Washington, v. 116, p. 923-933, 1998.

[19] TRANBARGER, T. J.; FRANCESCHI, V. R.; HILDEBRAND, D. F.; GRIMES, H. D. The soybean 94-kilodalton vegetative storage protein is a lipoxygenase that is localized in paraveinal mesophyll cell vacuoles. Plant Cell, Tissue and Organ Culture, Dordrecht, v. 3, p. 973-987, 1991.

[20] VICK, B.; ZIMMERMAN, D. C. Oxidative systems for modification of fatty acids: the lipoxygenase pathway. In: STUMPF, P. K.; CONN, E. E. (Eds.). The biochemistry of plants Orlando: Academic, 1987. v. 9, p. 53-97.

[21] VIEIRA, A. A.; OLIVEIRA, M. G. A.; JOSE, I. C.; PIOVESAN, N. D.; REZENDE, S. T.; MOREIRA, M. A.; BARROS, E. G. Biochemical evaluation of lipoxygenase pathaway of soybean plants submitted to wounding. Brazilian Journal of Plant Physiology, Săo Paulo, v. 13, n. 1, p. 5-12, 2001.

Brasil

Brasil

Caracterização bioquímica e cinética de lipoxigenases de folhas de soja submetidas à remoção dos primórdios florais

Biochemical and kinetic characterization of lipoxygenases from soybean leaves submitted to the removal of floral primordial

Resumos

Datas de Publicação

Histórico