Crude aqueous extracts from Arabidopsis leaves were subjected to chromatographic separations, after which the different fractions were monitored for antimicrobial activity using the fungus Neurospora crassa as a test organism. Two major fractions were obtained that appeared to have the same abundance in leaves from untreated plants versus leaves from plants challenge inoculated with the fungusAlternaria brassicicola. One of both major antimicrobial fractions was purified to homogeneity and identified by 1H nuclear magnetic resonance, gas chromatography/electron impact mass spectrometry, and gas chromatography/chemical ionization mass spectrometry as 4-methylsulphinylbutyl isothiocyanate (ITC). This compound has previously been described as a product of myrosinase-mediated breakdown of glucoraphanin, the predominant glucosinolate in Arabidopsis leaves. 4-Methylsulphinylbutyl ITC was found to be inhibitory to a wide range of fungi and bacteria, producing 50% growth inhibition in vitro at concentrations of 28 μm for the most sensitive organism tested (Pseudomonas syringae). A previously identified glucosinolate biosynthesis mutant, gsm1-1, was found to be largely deficient in either of the two major antimicrobial compounds, including 4-methylsulphinylbutyl ITC. The resistance ofgsm1-1 was compared with that of wild-type plants after challenge with the fungi A. brassicicola,Plectosphaerella cucumerina, Botrytis cinerea, Fusarium oxysporum, orPeronospora parasitica, or the bacteria Erwinia carotovora or P. syringae. Of the tested pathogens, only F. oxysporum was found to be significantly more aggressive on gsm1-1 than on wild-type plants. Taken together, our data suggest that glucosinolate-derived antimicrobial ITCs can play a role in the protection of Arabidopsis against particular pathogens.

A series of linear and angular prenylated furanocoumarins and a benzofuran derivative were isolated from leaves and twigs of Dorstenia gigas (Moraceae), a plant occurring endemically on Socotra Island (Yemen). The structures were elucidated by spectroscopic methods (NMR, MS, UV) and chemical derivatization.

Brassinosteroids are a class of steroidal phytohormones with high growth-promoting properties. The preferred side-chain conformations of 10 brassinosteroids were determined by means of detailed NMR investigations and molecular-modeling studies. Vacuum conformations obtained by simulated annealing calculations and Boltzmann statistical analysis were compared with solution conformations derived from NOE experiments and molecular dynamic simulations, and with X-ray structures. In general, results from simulated annealing calculations and NMR-supported molecular dynamics simulations are in good agreement. For some of the compounds investigated the conformation was less well-defined at the end of the side-chain. It could be shown that the energetically most favorable and most probable conformations also include the conformations obtained by NMR supported molecular-dynamics calculations and by X-ray analysis. For the most bioactive compound brassinolide (1) the majority of conformations show a side-chain bent towards the β-face of the steroid skeleton, whereas for the less bioactive brassinosteroids, conformations with straight side-chains or side-chains bent towards the α-face are more frequent.

Two novel alkaloidal glucosides derived from the recently discovered antidesmone (1 ), together with four known megastigmane and three lignan glucosides, two of which had not previously been described, were isolated from 1‐butanol extracts of Antidesma membranaceum (Euphorbiaceae). The structural elucidation of (17RS )‐17‐(β‐D ‐glucopyranosyloxy)antidesmone (2 ) and (17RS )‐8‐deoxo‐17‐(β‐D ‐glucopyranosyloxy)antidesmone (3 ) is based on 1H, 13C, COSY, NOESY, HMQC and HMBC NMR spectra, together with LC/ESI‐CIDMS and CD data. Determination of the absolute configuration at C‐17 was accomplished by comparison with 1H NMR spectroscopic data for alk‐2‐yl β‐D ‐glucopyranosides, an approach that also proved useful for the megastigmane glucosides blumenyl C β‐D ‐glucopyranoside (4 ), 3‐oxo‐α‐ionyl β‐D ‐glucopyranoside (5 ), blumenyl B β‐D ‐glucopyranoside (6 ) and blumenyl A β‐D ‐glucopyranoside (7 ). The lignan glucosides lyoniresin‐4‐yl β‐D ‐glucopyranoside (8 ), 4′‐O‐methyllyoniresin‐4‐yl β‐D ‐glucopyranoside (9 ) and secoisolariciresin‐4‐yl β‐D ‐glucopyranoside (10 ), featuring an unusual glucosylation position, were investigated with the aid of 1H and 2D NMR, CD and MS data.

Enzymatic and non-enzymatic lipid peroxidation has been implicated in programmed cell death, which is a major process of leaf senescence. To test this hypothesis we developed a high-performance liquid chromatography (HPLC) method for a simultaneous analysis of the major hydro(pero)xy polyenoic fatty acids. Quantities of lipid peroxidation products in leaves of different stages of development including natural senescence indicated a strong increase in the level of oxygenated polyenoic fatty acids (PUFAs) during the late stages of leaf senescence. Comprehensive structural elucidation of the oxygenation products by means of HPLC, gas chromatography/mass spectrometry and 1H nuclear magnetic resonance suggested a non-enzymatic origin. However, in some cases a small share of specifically oxidized PUFAs was identified suggesting involvement of lipid peroxidizing enzymes. To inspect the possible role of enzymatic lipid peroxidation in leaf senescence, we analyzed the abundance of lipoxygenases (LOXs) in rosette leaves of Arabidopsis. LOXs and their product (9Z,11E,13S,15Z)-13-hydroperoxy-9,11,15-octadecatrienoic acid were exclusively detected in young green leaves. In contrast, in senescing leaves the specific LOX products were overlaid by large amounts of stereo-random lipid peroxidation products originating from non-enzymatic oxidation. These data indicate a limited contribution of LOXs to total lipid peroxidation, and a dominant role of non-enzymatic lipid peroxidation in late stages of leaf development.

Crude aqueous extracts from Arabidopsis leaves were subjected to chromatographic separations, after which the different fractions were monitored for antimicrobial activity using the fungus Neurospora crassa as a test organism. Two major fractions were obtained that appeared to have the same abundance in leaves from untreated plants versus leaves from plants challenge inoculated with the fungusAlternaria brassicicola. One of both major antimicrobial fractions was purified to homogeneity and identified by 1H nuclear magnetic resonance, gas chromatography/electron impact mass spectrometry, and gas chromatography/chemical ionization mass spectrometry as 4-methylsulphinylbutyl isothiocyanate (ITC). This compound has previously been described as a product of myrosinase-mediated breakdown of glucoraphanin, the predominant glucosinolate in Arabidopsis leaves. 4-Methylsulphinylbutyl ITC was found to be inhibitory to a wide range of fungi and bacteria, producing 50% growth inhibition in vitro at concentrations of 28 μm for the most sensitive organism tested (Pseudomonas syringae). A previously identified glucosinolate biosynthesis mutant, gsm1-1, was found to be largely deficient in either of the two major antimicrobial compounds, including 4-methylsulphinylbutyl ITC. The resistance ofgsm1-1 was compared with that of wild-type plants after challenge with the fungi A. brassicicola,Plectosphaerella cucumerina, Botrytis cinerea, Fusarium oxysporum, orPeronospora parasitica, or the bacteria Erwinia carotovora or P. syringae. Of the tested pathogens, only F. oxysporum was found to be significantly more aggressive on gsm1-1 than on wild-type plants. Taken together, our data suggest that glucosinolate-derived antimicrobial ITCs can play a role in the protection of Arabidopsis against particular pathogens.

A series of linear and angular prenylated furanocoumarins and a benzofuran derivative were isolated from leaves and twigs of Dorstenia gigas (Moraceae), a plant occurring endemically on Socotra Island (Yemen). The structures were elucidated by spectroscopic methods (NMR, MS, UV) and chemical derivatization.

Brassinosteroids are a class of steroidal phytohormones with high growth-promoting properties. The preferred side-chain conformations of 10 brassinosteroids were determined by means of detailed NMR investigations and molecular-modeling studies. Vacuum conformations obtained by simulated annealing calculations and Boltzmann statistical analysis were compared with solution conformations derived from NOE experiments and molecular dynamic simulations, and with X-ray structures. In general, results from simulated annealing calculations and NMR-supported molecular dynamics simulations are in good agreement. For some of the compounds investigated the conformation was less well-defined at the end of the side-chain. It could be shown that the energetically most favorable and most probable conformations also include the conformations obtained by NMR supported molecular-dynamics calculations and by X-ray analysis. For the most bioactive compound brassinolide (1) the majority of conformations show a side-chain bent towards the β-face of the steroid skeleton, whereas for the less bioactive brassinosteroids, conformations with straight side-chains or side-chains bent towards the α-face are more frequent.

Two novel alkaloidal glucosides derived from the recently discovered antidesmone (1 ), together with four known megastigmane and three lignan glucosides, two of which had not previously been described, were isolated from 1‐butanol extracts of Antidesma membranaceum (Euphorbiaceae). The structural elucidation of (17RS )‐17‐(β‐D ‐glucopyranosyloxy)antidesmone (2 ) and (17RS )‐8‐deoxo‐17‐(β‐D ‐glucopyranosyloxy)antidesmone (3 ) is based on 1H, 13C, COSY, NOESY, HMQC and HMBC NMR spectra, together with LC/ESI‐CIDMS and CD data. Determination of the absolute configuration at C‐17 was accomplished by comparison with 1H NMR spectroscopic data for alk‐2‐yl β‐D ‐glucopyranosides, an approach that also proved useful for the megastigmane glucosides blumenyl C β‐D ‐glucopyranoside (4 ), 3‐oxo‐α‐ionyl β‐D ‐glucopyranoside (5 ), blumenyl B β‐D ‐glucopyranoside (6 ) and blumenyl A β‐D ‐glucopyranoside (7 ). The lignan glucosides lyoniresin‐4‐yl β‐D ‐glucopyranoside (8 ), 4′‐O‐methyllyoniresin‐4‐yl β‐D ‐glucopyranoside (9 ) and secoisolariciresin‐4‐yl β‐D ‐glucopyranoside (10 ), featuring an unusual glucosylation position, were investigated with the aid of 1H and 2D NMR, CD and MS data.

Enzymatic and non-enzymatic lipid peroxidation has been implicated in programmed cell death, which is a major process of leaf senescence. To test this hypothesis we developed a high-performance liquid chromatography (HPLC) method for a simultaneous analysis of the major hydro(pero)xy polyenoic fatty acids. Quantities of lipid peroxidation products in leaves of different stages of development including natural senescence indicated a strong increase in the level of oxygenated polyenoic fatty acids (PUFAs) during the late stages of leaf senescence. Comprehensive structural elucidation of the oxygenation products by means of HPLC, gas chromatography/mass spectrometry and 1H nuclear magnetic resonance suggested a non-enzymatic origin. However, in some cases a small share of specifically oxidized PUFAs was identified suggesting involvement of lipid peroxidizing enzymes. To inspect the possible role of enzymatic lipid peroxidation in leaf senescence, we analyzed the abundance of lipoxygenases (LOXs) in rosette leaves of Arabidopsis. LOXs and their product (9Z,11E,13S,15Z)-13-hydroperoxy-9,11,15-octadecatrienoic acid were exclusively detected in young green leaves. In contrast, in senescing leaves the specific LOX products were overlaid by large amounts of stereo-random lipid peroxidation products originating from non-enzymatic oxidation. These data indicate a limited contribution of LOXs to total lipid peroxidation, and a dominant role of non-enzymatic lipid peroxidation in late stages of leaf development.