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The search for antifungal secondary metabolites from cruciferous plants exhibiting resistance to pathogenic fungi led to the investigation of <i>Eruca sativa </i>(rocket). Chemical analysis of extracts showed arvelexin (51) as the only inducible component. Bioassay guided isolation (FCC, PTLC) and characterization (NMR, MS) led to the identification of two phytoanticipins, 4-methylthiobutyl isothiocyanate (166) and bis(4-isothiocyanatobutyl)disulfide (167). Compounds 166 and 167 inhibited the germination of spores of <i>Cladosporium cucumerinum</i> in TLC biodetection assays.<p>Next, isotopically labeled compounds containing 2H and 34S at specific sites were synthesized for use in studying of the biosynthetic pathway of crucifer phytoalexins and indolyl glucosinolates. Among the synthesized precursors, [4',5',6',7'-2H4]indolyl-3-[34S]acetothiohydroxamic acid (174a), the first sulfur-34 containing indolyl derivative was synthesized. In addition, non-isotopically labeled compounds (containing 1-methyl, 1-boc and 1-acetyl groups), that is, substrates used for precursor-directed biosynthesis, were also prepared.<p>With the precursors in hand, the biosynthetic pathway(s) and biogenetic relationship between phytoalexins was investigated using the tuberous crucifers, <i>Brassica napus </i> L. ssp rapifera (rutabaga) and <i>B. rapa </i> (turnip), and detached leaves of <i>Erucastrum gallicum </i> (dog mustard). The biosynthetic relationship between indolyl glucosinolates and phytoalexins was investigated in rutabaga and turnip. The indolyl moiety of the phytoalexins cyclobrassinin (28), rutalexin (33), spirobrassinin (34), brassicanate A (43), and rapalexin A (53), as well as indolyl glucosinolates glucobrassicin (70), 4-methoxyglucobrassicin (156), and neoglucobrassicin (199) was confirmed to derive from L-tryptophan (78). The 1-methoxy-containing phytoalexins, erucalexin (38) and 1-methoxyspirobrassinin (35) were shown to derive from indolyl-3-acetaldoxime (112) through 1-methoxyindolyl-3-acetaldoxime (116). The 1-methoxy substituent of neoglucobrassicin was also shown to derive from 1-methoxyindolyl-3-acetaldoxime (116).<p>The incorporation of indolyl-3-acetothiohydroxamic acid (174) into the phytoalexins cyclobrassinin, rutalexin, brassicanate A, rapalexin A, and spirobrassinin, and into the glucosinolate glucobrassicin is reported for the first time. On the other hand, incorporation of 174 into 4-methoxyglucobrassicin and neoglucobrassicin was not detected under current experimental conditions. Cyclobrassinin was incorporated into spirobrassinin among the NH-containing phytoalexins, whereas sinalbin B (31) [biosynthesized from 1-methoxybrassinin (18)] was incorporated into erucalexin and 1-methoxyspirobrassinin. The efficient metabolism of [SC2H3]brassicanal A into [SC2H3]brassicanate A suggested a biogenetic relationship between these two phytoalexins, whereas absence of incorporation of indolyl-3-acetonitrile (49) into rutabaga phytoalexins or indolyl glucosinolates indicated that 49 is not a precursor of these secondary metabolites under the current experimental conditions.<p>The rutabaga and turnip tubers separately metabolized 1-methylindolyl-3-acetaldoxime (170) and 1-methylindolyl-3-acetothiohydroxamic acid (178) into 1-methylglucobrassicin (201); however, no 1-methyl-containing phytoalexins were detected in the extracts. Rutabaga tissues metabolized 1-(tert-butoxycarbonyl)indolyl-3-methylisothiocyanate (180) into 1-(tert-butoxycarbonyl)brassinin (181) and 1-(tert-butoxycarbonyl)spirobrassinin (196), whereas 1-acetylbrassinin (184) was the only detectable metabolic product of 1-acetylindolyl-3-methylisothiocyanate (183) in both rutabaga and turnip root tissues.<p>In conclusion, indolyl-3-acetothiohydroxamic acid (174) seems to be the branching point between brassinin and glucobrassicin. The biosynthetic pathway of NH-containing crucifer phytoalexins was mapped and follows the sequence L-tryptophan, indolyl-3-acetaldoxime, indolyl-3-acetothiohydroxamic acid, brassinin (possibly through indolyl-3-methylisothiocyanate), and other phytoalexins. The biosynthetic pathway of 1-methoxy-containing phytoalexins follows a similar sequence through 1-methoxyindolyl-3-acetaldoxime (biosynthesized from indolyl-3-acetaldoxime).

The search for antifungal secondary metabolites from cruciferous plants exhibiting resistance to pathogenic fungi led to the investigation of <i>Eruca sativa </i>(rocket). Chemical analysis of extracts showed arvelexin (51) as the only inducible component. Bioassay guided isolation (FCC, PTLC) and characterization (NMR, MS) led to the identification of two phytoanticipins, 4-methylthiobutyl isothiocyanate (166) and bis(4-isothiocyanatobutyl)disulfide (167). Compounds 166 and 167 inhibited the germination of spores of <i>Cladosporium cucumerinum</i> in TLC biodetection assays.<p>Next, isotopically labeled compounds containing 2H and 34S at specific sites were synthesized for use in studying of the biosynthetic pathway of crucifer phytoalexins and indolyl glucosinolates. Among the synthesized precursors, [4',5',6',7'-2H4]indolyl-3-[34S]acetothiohydroxamic acid (174a), the first sulfur-34 containing indolyl derivative was synthesized. In addition, non-isotopically labeled compounds (containing 1-methyl, 1-boc and 1-acetyl groups), that is, substrates used for precursor-directed biosynthesis, were also prepared.<p>With the precursors in hand, the biosynthetic pathway(s) and biogenetic relationship between phytoalexins was investigated using the tuberous crucifers, <i>Brassica napus </i> L. ssp rapifera (rutabaga) and <i>B. rapa </i> (turnip), and detached leaves of <i>Erucastrum gallicum </i> (dog mustard). The biosynthetic relationship between indolyl glucosinolates and phytoalexins was investigated in rutabaga and turnip. The indolyl moiety of the phytoalexins cyclobrassinin (28), rutalexin (33), spirobrassinin (34), brassicanate A (43), and rapalexin A (53), as well as indolyl glucosinolates glucobrassicin (70), 4-methoxyglucobrassicin (156), and neoglucobrassicin (199) was confirmed to derive from L-tryptophan (78). The 1-methoxy-containing phytoalexins, erucalexin (38) and 1-methoxyspirobrassinin (35) were shown to derive from indolyl-3-acetaldoxime (112) through 1-methoxyindolyl-3-acetaldoxime (116). The 1-methoxy substituent of neoglucobrassicin was also shown to derive from 1-methoxyindolyl-3-acetaldoxime (116).<p>The incorporation of indolyl-3-acetothiohydroxamic acid (174) into the phytoalexins cyclobrassinin, rutalexin, brassicanate A, rapalexin A, and spirobrassinin, and into the glucosinolate glucobrassicin is reported for the first time. On the other hand, incorporation of 174 into 4-methoxyglucobrassicin and neoglucobrassicin was not detected under current experimental conditions. Cyclobrassinin was incorporated into spirobrassinin among the NH-containing phytoalexins, whereas sinalbin B (31) [biosynthesized from 1-methoxybrassinin (18)] was incorporated into erucalexin and 1-methoxyspirobrassinin. The efficient metabolism of [SC2H3]brassicanal A into [SC2H3]brassicanate A suggested a biogenetic relationship between these two phytoalexins, whereas absence of incorporation of indolyl-3-acetonitrile (49) into rutabaga phytoalexins or indolyl glucosinolates indicated that 49 is not a precursor of these secondary metabolites under the current experimental conditions.<p>The rutabaga and turnip tubers separately metabolized 1-methylindolyl-3-acetaldoxime (170) and 1-methylindolyl-3-acetothiohydroxamic acid (178) into 1-methylglucobrassicin (201); however, no 1-methyl-containing phytoalexins were detected in the extracts. Rutabaga tissues metabolized 1-(tert-butoxycarbonyl)indolyl-3-methylisothiocyanate (180) into 1-(tert-butoxycarbonyl)brassinin (181) and 1-(tert-butoxycarbonyl)spirobrassinin (196), whereas 1-acetylbrassinin (184) was the only detectable metabolic product of 1-acetylindolyl-3-methylisothiocyanate (183) in both rutabaga and turnip root tissues.<p>In conclusion, indolyl-3-acetothiohydroxamic acid (174) seems to be the branching point between brassinin and glucobrassicin. The biosynthetic pathway of NH-containing crucifer phytoalexins was mapped and follows the sequence L-tryptophan, indolyl-3-acetaldoxime, indolyl-3-acetothiohydroxamic acid, brassinin (possibly through indolyl-3-methylisothiocyanate), and other phytoalexins. The biosynthetic pathway of 1-methoxy-containing phytoalexins follows a similar sequence through 1-methoxyindolyl-3-acetaldoxime (biosynthesized from indolyl-3-acetaldoxime).

Thesis (Ph. D.)--University of Wisconsin--Madison, 1958. / Typescript. Abstracted in Dissertation abstracts, v. 19 (1958) no. 4, p. 627. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves [60]-63).

Made available in DSpace on 2014-06-11T19:25:18Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-05-19Bitstream added on 2014-06-13T18:53:11Z : No. of bitstreams: 1 otuka_ak_me_jabo.pdf: 1152577 bytes, checksum: 8e07d08c9d9ef5d2ba47566363186697 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O trabalho foi desenvolvido no Laboratório de Biologia e Criação de Insetos (LBCI) da FCAV-Unesp, para avaliar a influência de diferentes temperaturas e períodos de exposição no desenvolvimento de Plutella xylostella, para viabilizar o armazenamento de ovos, lagartas e pupas e facilitar o manejo da criação em laboratório. Avaliou-se a biologia de P. xylostella armazenando ovos e lagartas nas temperaturas de 3, 5, 8, 10, 12, 16, 20 e 24°C por 0, 5, 10, 15, 20, 25 e 30 dias de exposição, sendo “0” a testemunha, mantida em 25±1°C, 70±10% UR e 12 horas de fotofase. Avaliou-se também a biologia de P. xylostella armazenando pupas nas temperaturas de 3, 5 e 8°C por 0, 5, 10, 15, 20, 25 e 30 dias de exposição. O armazenamento de ovos e pupas de P. xylostella pode ser realizado por até 15 dias a 8ºC sem que ocorram perdas consideráveis nas suas características biológicas. O armazenamento de lagartas de 1° ínstar de P. xylostella pode ser realizado até 10 dias a 20ºC, já as de 2° ínstar podem ser armazenadas por até 15 dias a 16ºC, as de 3º ínstar por até 20 dias a 20ºC e as de 4º ínstar por até 5 dias a 20ºC, acima desses períodos e abaixo dessas temperaturas ocorrem reduções significativas no número de ovos colocados por fêmea / The work was conducted in the Laboratory of Insect Biology and Rearing (LBCI), FCAV-Unesp, to evaluate the influence of different temperatures and exposure periods on development of Plutella xylostella, trying to enable the storage of eggs, larvae and pupae to facilitate the management of laboratory rearing. For this, we evaluated the biology of P. xylostella larvae and storing eggs at temperatures of 3, 5, 8, 10, 12, 16, 20 and 24°C for 0, 5, 10, 15, 20, 25 and 30 days of exposure, and 0 the check, maintained at 25±1°C, 70±10% RH and 12 hours photophase. We also evaluated the biology of P. xylostella storing pupae at temperatures of 3, 5 and 8°C for 0, 5, 10, 15, 20, 25 and 30 days of exposure. The storage of eggs and pupae of P. xylostella can be done 15 days at 8°C without significant losses that occur in biological characteristics. The storage of a larval first instar of P. xylostella can be performed up to 10 days at 20°C, already the second instar can be stored for 15 days at 16°C, third instar for up to 20 days at 20°C and fourth instar for 5 days at 20°C, above this periods and below these temperatures are reduced significantly the number of eggs per female

Orientador: Sergio Antonio De Bortoli / Banca: Antonio Sergio Ferraudo / Banca: Rogéria Inês Rosa Lara / Resumo: O trabalho foi desenvolvido no Laboratório de Biologia e Criação de Insetos (LBCI) da FCAV-Unesp, para avaliar a influência de diferentes temperaturas e períodos de exposição no desenvolvimento de Plutella xylostella, para viabilizar o armazenamento de ovos, lagartas e pupas e facilitar o manejo da criação em laboratório. Avaliou-se a biologia de P. xylostella armazenando ovos e lagartas nas temperaturas de 3, 5, 8, 10, 12, 16, 20 e 24°C por 0, 5, 10, 15, 20, 25 e 30 dias de exposição, sendo "0" a testemunha, mantida em 25±1°C, 70±10% UR e 12 horas de fotofase. Avaliou-se também a biologia de P. xylostella armazenando pupas nas temperaturas de 3, 5 e 8°C por 0, 5, 10, 15, 20, 25 e 30 dias de exposição. O armazenamento de ovos e pupas de P. xylostella pode ser realizado por até 15 dias a 8ºC sem que ocorram perdas consideráveis nas suas características biológicas. O armazenamento de lagartas de 1° ínstar de P. xylostella pode ser realizado até 10 dias a 20ºC, já as de 2° ínstar podem ser armazenadas por até 15 dias a 16ºC, as de 3º ínstar por até 20 dias a 20ºC e as de 4º ínstar por até 5 dias a 20ºC, acima desses períodos e abaixo dessas temperaturas ocorrem reduções significativas no número de ovos colocados por fêmea / Abstract: The work was conducted in the Laboratory of Insect Biology and Rearing (LBCI), FCAV-Unesp, to evaluate the influence of different temperatures and exposure periods on development of Plutella xylostella, trying to enable the storage of eggs, larvae and pupae to facilitate the management of laboratory rearing. For this, we evaluated the biology of P. xylostella larvae and storing eggs at temperatures of 3, 5, 8, 10, 12, 16, 20 and 24°C for 0, 5, 10, 15, 20, 25 and 30 days of exposure, and "0" the check, maintained at 25±1°C, 70±10% RH and 12 hours photophase. We also evaluated the biology of P. xylostella storing pupae at temperatures of 3, 5 and 8°C for 0, 5, 10, 15, 20, 25 and 30 days of exposure. The storage of eggs and pupae of P. xylostella can be done 15 days at 8°C without significant losses that occur in biological characteristics. The storage of a larval first instar of P. xylostella can be performed up to 10 days at 20°C, already the second instar can be stored for 15 days at 16°C, third instar for up to 20 days at 20°C and fourth instar for 5 days at 20°C, above this periods and below these temperatures are reduced significantly the number of eggs per female / Mestre

Made available in DSpace on 2016-01-26T18:56:21Z (GMT). No. of bitstreams: 1 Dissertacao.pdf: 149957 bytes, checksum: d219d7c38eca166d0eac355501df82ee (MD5) Previous issue date: 2009-08-28 / The project goal was to study the productivity and quality of forage produced bromatological from monoculture of sorghum and maize and intercropping of them with dwarf pea, sunn hemp, white lupine, sunflower and radish. The experiment was installed in the area of agricultural production of the Campus II Unoeste in Presidente Prudente-SP, on April 3rd, 2008. The experimental design was a split plot with four replications, with the following treatments: monoculture of sorghum (Sorghum bicolor) and maize (Zea mays) and sorghum consortium + pigeon pea (Cajanus cajan var. Dwarf) sorghum + sunn hemp (Crotalaria juncea), Sorghum + Sunflower (Helianthus annuus), sorghum fodder radish (Raphanus sativus), sorghum + white lupine (Lupinus albus), maize + pigeon pea dwarf, corn / sunn hemp, corn / sunflower corn + radish and corn + white lupine. The plots were scaled with eight rows of sowing of six meters. Biomass were collected at 60, 90 and 120 days after sowing for the quantification of plant material and chemical analysis. Statistical analysis consists of analysis of variance and Tukey test at 5% probability to compare treatment means. The single or intercropped sorghum was more efficient during low nutritive value, especially for syndications with sunflower, hemp and pigeon, which may have occurred because plants are more adapted to the dry climate of the region. Since the time of cutting that stood out with a productive balance and quality of the composition was 90 DAS. / O objetivo do projeto foi estudar a produtividade e a qualidade bromatológica de forragem produzida a partir de monocultivos de sorgo e milho, bem como a consorciação dos mesmos com guandu-anão, crotalária juncea, tremoço branco, girassol e nabo forrageiro. O experimento foi instalado na área de produção agrícola do Campus II da Unoeste, em Presidente Prudente-SP, no dia 03 de abril de 2008. O delineamento experimental foi em parcelas sub-divididas, com quatro repetições, com os seguintes tratamentos: monocultivos de sorgo (Sorghum bicolor) e milho (Zea mays), e consórcios de sorgo + guandu-anão (Cajanus cajan, var. anão), sorgo + crotalária juncea (Crotalaria juncea), sorgo + girassol (Helianthus annuus), sorgo + nabo forrageiro (Raphanus sativus), sorgo + tremoço branco (Lupinus albus), milho + guandu-anão, milho + crotalária juncea, milho + girassol, milho + nabo forrageiro e milho + tremoço branco. As parcelas foram dimensionadas com oito linhas de semeadura de seis m de comprimento. Foram coletadas fitomassas aos 60, 90 e 120 dias após a semeadura para quantificação da material vegetal e análises bromatológicas. O estudo estatístico consta de análise de variância e teste Tukey a 5% de probabilidade para comparar as médias dos tratamentos. O sorgo solteiro ou consorciado mostrou-se mais eficiente na maioria das avaliações bromatológicas, com destaque para as consorciações com girassol, crotalária e guandu, fato que pode ter ocorrido por serem plantas mais adaptadas ao clima seco da região. Sendo que a época de corte que se sobressaiu com um equilíbrio produtivo e qualidade bromatológica foi a de 90 DAS.

Made available in DSpace on 2016-07-18T17:51:05Z (GMT). No. of bitstreams: 1 Dissertacao.pdf: 149957 bytes, checksum: d219d7c38eca166d0eac355501df82ee (MD5) Previous issue date: 2009-08-28 / The project goal was to study the productivity and quality of forage produced bromatological from monoculture of sorghum and maize and intercropping of them with dwarf pea, sunn hemp, white lupine, sunflower and radish. The experiment was installed in the area of agricultural production of the Campus II Unoeste in Presidente Prudente-SP, on April 3rd, 2008. The experimental design was a split plot with four replications, with the following treatments: monoculture of sorghum (Sorghum bicolor) and maize (Zea mays) and sorghum consortium + pigeon pea (Cajanus cajan var. Dwarf) sorghum + sunn hemp (Crotalaria juncea), Sorghum + Sunflower (Helianthus annuus), sorghum fodder radish (Raphanus sativus), sorghum + white lupine (Lupinus albus), maize + pigeon pea dwarf, corn / sunn hemp, corn / sunflower corn + radish and corn + white lupine. The plots were scaled with eight rows of sowing of six meters. Biomass were collected at 60, 90 and 120 days after sowing for the quantification of plant material and chemical analysis. Statistical analysis consists of analysis of variance and Tukey test at 5% probability to compare treatment means. The single or intercropped sorghum was more efficient during low nutritive value, especially for syndications with sunflower, hemp and pigeon, which may have occurred because plants are more adapted to the dry climate of the region. Since the time of cutting that stood out with a productive balance and quality of the composition was 90 DAS. / O objetivo do projeto foi estudar a produtividade e a qualidade bromatológica de forragem produzida a partir de monocultivos de sorgo e milho, bem como a consorciação dos mesmos com guandu-anão, crotalária juncea, tremoço branco, girassol e nabo forrageiro. O experimento foi instalado na área de produção agrícola do Campus II da Unoeste, em Presidente Prudente-SP, no dia 03 de abril de 2008. O delineamento experimental foi em parcelas sub-divididas, com quatro repetições, com os seguintes tratamentos: monocultivos de sorgo (Sorghum bicolor) e milho (Zea mays), e consórcios de sorgo + guandu-anão (Cajanus cajan, var. anão), sorgo + crotalária juncea (Crotalaria juncea), sorgo + girassol (Helianthus annuus), sorgo + nabo forrageiro (Raphanus sativus), sorgo + tremoço branco (Lupinus albus), milho + guandu-anão, milho + crotalária juncea, milho + girassol, milho + nabo forrageiro e milho + tremoço branco. As parcelas foram dimensionadas com oito linhas de semeadura de seis m de comprimento. Foram coletadas fitomassas aos 60, 90 e 120 dias após a semeadura para quantificação da material vegetal e análises bromatológicas. O estudo estatístico consta de análise de variância e teste Tukey a 5% de probabilidade para comparar as médias dos tratamentos. O sorgo solteiro ou consorciado mostrou-se mais eficiente na maioria das avaliações bromatológicas, com destaque para as consorciações com girassol, crotalária e guandu, fato que pode ter ocorrido por serem plantas mais adaptadas ao clima seco da região. Sendo que a época de corte que se sobressaiu com um equilíbrio produtivo e qualidade bromatológica foi a de 90 DAS.

Phytoalexins and phytoanticipins are antimicrobial natural products involved in plant defence pathways against plant pathogens and other stresses. Most cruciferous phytoalexins are indole containing compounds with various side chains (dithiocarbamates, isothiocyanate, isonitrile, acetonitriles etc.). Many phytoanticipins of crucifers are glucosinolates and their metabolites, which have diverse structures and precursors, including aliphatic, phenyl or indolyl containing amino acids. Indole glucosinolates are derived from tryptophan, which is also a biosynthetic precursor to cruciferous phytoalexins, however the biosynthetic relationship between cruciferous phytoalexins and indole glucosinolates has not been clarified. In this work, investigation of antifungal metabolites from wild crucifers, synthesis of antifungal metabolites and potential perdeuterated biosynthetic precursors, biosynthesis of metabolites of salt cress and that of rutabaga will be described. Investigation of the wild crucifers Brassica tournefortii, Crambe abyssinica, Diplotaxis tenuifolia and Diplotaxis tenuisiliqua for production of elicited antifungal metabolites, resulted in the discovery of a new phytoalexin, 1ꞌ,4ꞌ-dimethoxyindolyl-3ꞌ-acetonitrile, from D. tenuisiliqua. 1ꞌ,4ꞌ-dimethoxyindolyl-3ꞌ-acetonitrile is the first dimethoxy substituted phytoalexin with strong antifungal activity against plant fungal pathogens. The remaining plant species produced known phytoalexins which were initially discovered in wild and cultivated species; all of them produced arvelexin. A novel phytoalexin isocyalexin A, was isolated from rutabaga roots irradiated with UV-light; this is the first isocyanide of plant origin. The second section of the thesis deals with the biosynthesis of metabolites of salt cress (T. salsuginea) and their biosynthetic relationships with indole glucosinolates. In that regard, non-isotopically labeled compounds and perdeuterated biosynthetic intermediates such as [2,2,4ꞌ,5ꞌ,6ꞌ,7ꞌ-2H6]glucobrassicin, [2H3CS;4ꞌ,5ꞌ,6ꞌ,7ꞌ-2H4]-1ꞌ-methoxybrassinin, L-[2ꞌ,4ꞌ,5ꞌ,6ꞌ,7ꞌ-2H5]tryptophan, [2H3CO]-1ꞌ-methoxyindolyl-3ꞌ-acetaldoxime, L-[2H3CS]methionine, [4ꞌ,5ꞌ,6ꞌ,7ꞌ-2H4]brassinin and 1ꞌ-methoxy-2ꞌ-methylbrassinin were administered to salt cress leaves. For the first time, the biosynthetic relationship between indole glucosinolates and cruciferous phytoalexins was established. Intact incorporations of hexadeuterated glucobrassicin ([2,2,4ꞌ,5ꞌ,6ꞌ,7ꞌ-2H6]glucobrassicin) into wasalexins A, B and biswasalexins A1 and A2 were observed. Based on the feeding experiment results, for the first time a biosynthetic route that includes both indole glucosinolates (glucobrassicin and 1ꞌ-methoxyglucobrassicin) and 1ꞌ-methoxybrassinin was proposed. The third section of the thesis is about biosynthesis of metabolites of rutabaga (Brassica napus). Rutabaga produces phytoalexins that differ on their side chains. Biosynthetic origin of their side chains was investigated by administering fully labeled tryptophan (L-[U-13C11,U-15N2]Trp) and other perdeuterated precursors to rutabaga roots which revealed that the carbon and nitrogen atoms of cyclobrassinin, rapalexin A, isocyalexin A and spirobrassinin are fully derived from tryptophan, and also both rapalexin A and isocyalexin A incorporated deuterium from glucobrassicin. [4',5',6',7'-2H4]-4'-Methoxybrassinin was incorporated into 4ꞌ-methoxycyclobrassinin and 4ꞌ-methoxydehydrocyclobrassinin but not into rapalexin A, isocyalexin A and isalexin. The biosynthetic pathway that leads to isalexin, rapalexin A and isocyalexin A was further investigated using perdeuterated biosynthetic precursors such as (R,S)-[2H3CO,5',6',7'-2H3]-4'-methoxyindolyl-3'-glycine, [2H3CO,5',6',7'-2H3]-4'-methoxyindole-3'-carboxaldehyde oxime, [2H3CO,5',6',7'-2H3]desulfoglucorapassicin and etc. It has been confirmed that the pathway involves series of rearrangements that allow transformation of side chain of tryptophan into the side chains of rapalexin A and isocyalexin A without any degradations. In conclusion, cruciferous phytoalexins are derived from glucobrassicin which is a precursor for 1ꞌ- and 4ꞌ-methoxyglucobrassicins. 1-Methoxylated phytoalexins are biosynthesized through 1ꞌ-methoxyglucobrassicin via 1ꞌ-methoxybrassinin. Similarly, 4-methoxy phytoalexins are derived from 4ꞌ-methoxyglucobrassicin through two distinct pathways: via 4ꞌ-methoxybrassinin and 4ꞌ-methoxyindolyl-3ꞌ-glycine.