Thiophene synthesis and distribution in young developing plants of Tagetes patula and Tagetes erecta

Thiophene synthesis and accumulation were investigated in organsof Tagetes patula and T. erecta. Thiophene accumulation startedrapidly in germinating seedlings of both species. Roots andhypocotyls were the major thiophene accumulating organs and5-(3-buten-1-ynyl)-2, 2-bithienyl (BBT) and 5-(4-acetoxy-1-butynyl)-2,2 -bithienyl (BBTOAc) were the major accumulated compounds.Higher thiophene concentrations were reached in Tagetes patulathan in T. erecta. Accumulation patterns for individual thiopheneswere different within organs, between organs and between bothspecies. Within hypocotyls of Tagetes patula, thiophene concentrationswere high in the epidermis and the vascular tissue and low inthe parenchymatic tissues of cortex and pith. Synthesis of thiopheneswas high in the roots and hypocotyls and very low in the leaves.Transport of thiophenes from the roots into the shoot occurred,but the rate of transport was too low to explain the high concentrationsin the hypocotyl. It is concluded that for the main part thiophenesare accumulated where they are synthesized. Key words: Tagetes, hiophenest, synthesis, accumulation, secondary metabolites     

Thiophene interconversion in elicitor-treated roots ofTagetes patula L.

Thiophenes are polyacetylene-related heterocyclic metabolites. Some of these compounds are phototoxic, but the bithiophenes occurring inTagetes mainly accumulate in the root where photo-activation is not likely to occur. A cell-free extract from the fungusFusarium oxysporum induced biosynthesis of hydrophilic thiophenes in root cultures and roots of seedlings ofTagetes patula. The thiophenes formed were partially excreted into the culture medium. The excreted thiophenes inhibited fungal growth in the absence of light and thus may play a role in the biochemical defense against pathogens.Abbreviations BBT 5-(3-buten-1-ynyl)-2,2-bithienyl - BBTOAc 5-(4-acetoxy-1-butynyl)-2,2-bithienyl - BBT(OAc)₂ 5-(3,4-diacetoxy-1-butynyl)-2,2-bithienyl - BBTOH 5-(4-hydroxy-1-butynyl)-2,2-bithienyl - BBT(OH)₂ 5-(3,4-dihydroxy-1-butynyl)-2,2-bithienyl - HPLC high-performance liquid chromatography - UV ultraviolet     

Thiophene Content in Normal and Transformed Root Cultures of Tagetes erecta: A Comparison with Thiophene Content in Roots of Intact Plants

Transformed roots of Tagetes erecta were obtained followinginfection of stems of sterile plantlets with Agrobacterium rhizogenesstrain TR105. The thiophenes detected were 5-(4-hydroxy-l-butenyl)-2,2'-bithienyl,5-(4-acetoxy-l-butenyl)-2,2'-bithienyl, 5-(3-buten-l-enyl)-2,2',-bithienyl and 2.2': 5', 2'-terthienyl. The thiophene patternwas the same in normal root cultures and roots of the intactplant. Transformed roots showed a higher growth rate and a higherbiomass yield than normal root cultures on a hormone-free media. Key words: Transformed roots normal roots, Tagetes erecta, thiophenes, Agrobacterium rhizogenes     

Growth and thiophene accumulation by hairy root cultures of Tagetes patula in media of varying initial pH

Summary Hairy roots of Tagetes patula were grown for 24 days in modified Murashige and Skoog's liquid medium at different initial pH levels of 4.0, 5.0, 5.7, 6.0 and 7.0. Irrespective of the initial pH, after 12 days, the pH of the culture medium was approximately 4.5. However the final pH, after 24 days of growth, did depend weakly on the initial pH of the medium. The biomass yield was lowest at an initial pH of 4.0, possibly due to lower utilization of ammonium at this pH. Similar patterns of thiophene accumulation was observed at all pH levels tested. Maximum thiophene accumulation occurred in root cultures which were 12–16 days old.Abbreviations BBTOH 5-(4-hydroxy-1-butenyl)-2,2-bithienyl - BBTOAc 5-(4-acetoxy-1-butenyl)-2,2-bithienyl - BBT 5-(3-buten-1-ynyl)-2,2-bithienyl - MS Murashige and Skoog's nutrient medium - B5 Gamborg's B5 nutrient salts - HPLC High pressure liquid chromatography     

Plant regeneration and thiophene formation in tissue cultures of Tagetes mendocina

Plants of the wild species Tagetes mendocina were regenerated, via organogenesis, from explants and callus cultures, and the occurrence of biocidal metabolites, thiophenes, by such undifferentiated tissues and regenerated plants was investigated. Differences between explant sources were apparent in terms of their competence for dedifferentiation and subsequent organogenesis. Leaf-derived cultures were more responsive than the root-derived ones. Biochemical studies revealed that the plants produced were capable of synthezising thiophene like-compounds following in vitro regeneration.Abbreviations BA 6-benzyladenine - IBA indole-3-butyric acid - MS Murashige & Skoog (1962) - NAA naphthaleneacetic acid - BBT 5-(3-buten-1-enyl)-2,2-bithienyl - BBTOAc 5-(4-acetoxy-1-butenyl)-2,2-bithienyl - -T 2,2 5,2"-terthienyl - BBTOH 5-(4-hydroxy-1-butenyl)-2,2-bithienyl     

Biosynthesis of thiophenes in Tagetes patula.

The biosynthesis of 5-(3-buten-1-ynyl)-2,2'-bithiophene was studied in root cultures of Tagetes patula. Organ cultures were grown with [U-13C(6)]glucose or [1-13C]glucose. The bithiopene and amino acids from cell protein were isolated and analysed by quantitative NMR spectroscopy. Retrobiosynthetic analysis establish acetyl-CoA or a closely related compound (e.g. malonyl-CoA) as building blocks and their orientations in the bithiophene. The data confirm a previously suggested biosynthetic route via long-chain fatty acids and polyacetylenes. However, a polyketide-like biosynthesis via a carbocyclic intermediate cannot be excluded.     

Quantitative relationships between boron and mannitol concentrations in phloem exudates of Olea europaea leaves under contrasting boron supply conditions

Root exudates are implicated in the chemical defense of plants, but testing such hypotheses has been hindered by the difficulties of quantifying allelochemical concentrations in soil. Here we describe a new, simple method to quantify the dynamics of non-polar root exudates in soil. Novel soil probes were constructed using stainless steel wire inserted into polydimethylsiloxane (PDMS) tubing. Probes were inserted into soil for 24 h, removed and extracted, and analyzed by HPLC. Lipophilic thiophenes produced by roots of Tagetes and Rudbeckia species were chosen as candidate compounds to test the method. Probes recovered microgram quantities of the highly phytotoxic thiophenes 5-(3-buten-1-ynyl)-2,2′-bithienyl (BBT) and α-terthienyl per probe per day from the root zone of Tagetes patula, and distribution of thiophenes beneath plants was spatially and temporally heterogeneous. Flux-proportional sampling of soil provides a means to test hypotheses about the role of root exudates in plant–plant and other interactions.     

Production of thiophenes from callus cultures of Tagetes patula L. and its mosquito larvicidal activity

Calli were initiated from leaf tissues of T. patula, on Murashige and Skoog's medium supplemented with 2-4 dichlorophenoxacetic acid (2,4-D) and kinetin (Kn). The maximum accumulation of biomass was recorded on 6th week at 1.11 g. dry wt/culture. Maximum thiophene content was recorded on 4th week at 0.008% on dry wt basis in callus cultures of T. patula subjected to nutrient stress. Nitrogen stress induced 3-fold increase in thiophene production level in six weeks (0.024% on dry weight basis). The best hormonal supplementation required for thiophenes production was found to be 2,4-D (2.0 mg L(-1)) and kinetin (2.0 mg L(-1)). The thiophenes produced in callus cultures of T. patula showed larvicidal effect against mosquito larvae.     

Distribution of acetylenic thiophenes in the pectidinae

The distribution and quantitative significance of biosynthetically related di- and ter-thiophenes from 27 species representing seven genera of the Pectidinae (Heliantheae) was investigated by reverse-phase HPLC. Adenophyllum, Chrysactinia and Nicolletia, three previously unstudied genera, were found to contain thiophenes for the first time. Four derivatives, 5-(4-hydroxy-1-butenyl)-2,2′-bithiophene (1), 5-(4-acetoxy-1-butenyl)-2,2′-bithiophene (2), 5-(3-buten-1-ynyl)-2,2′-bithiophene (3) and 2,2′:5′,2″-terthiophene (4) were common constituents in most species of Adenophyllum, Chrysactinia, Dyssodia, Hymenatherum, Nicolletia and Porophyllum. One additional compound, 5-methyl-2,2′:5′,2″-terthiophene (5), was also present in extracts of Adenophyllum, Dyssodia and Hymenatherum, but was not detected in any other genus. Acetylenic thiophenes were not found in any of the 18 species of Pectis examined.     

Postharvest changes in endogenous ABA levels and ABA metabolism in relation to dormancy in potato tubers

In this paper the effects of indole-3-acetic acid (IAA) on growth of Tagetes patula hairy root cultures and secondary product formation are presented. The biosynthesis of thiophenes, sulfurous compounds with nematicidal activity, was inhibited by IAA application, as was evident from a decrease of [³⁵S] sulfur incorporation. The inhibition only occurred after the roots had developed numerous laterals as a result of auxin action. However, in roots cultured in the absence of IAA, there was no significant correlation between branching and thiophene accumulation. Therefore, development of lateral roots is not a sufficient condition for a low capacity to synthesize thiophenes. The highest rate of thiophene accumulation in the roots culture is at its maximum. Hence, growth and the production of thiophenes appear to be compatible in T. Patula hair roots.     

Ultraviolet-mediated antibiotic activity of thiophene compounds of Tagetes

Two intensely mauve UV fluorescent compounds isolated from Tagetes root were found to be phototoxic to Candida albicans. By chromatography on alumina followed by gel filtration on Sephadex LH-20, the compounds were identified as 5-(3-buten-1-ynyl)-2,2′-bithienyl and α-terthienyl.     

Isolation and Characterization of Mutants of Thiophene Synthesis in Tagetes erecta

Two mutants of Tagetes erecta displaying aberrant thiophene composition were identified by screening more than 300 plants from a mutagenized M2 population using high-performance liquid chromatography analysis of root extracts. Both mutants, which may have originated from the same mutational event, contained high amounts of the C13 monothiophene 2-(but-3-en-1-ynyl)-5-(penta-1,3-diynyl)-thiophene that was previously not found in T. erecta and also high amounts of two C13 bithienyls that were absent or present at low concentrations in the wild type. The mutant phenotype was also expressed in 21 Agrobacterium rhizogenes transformed root clones derived from both mutants. Feeding experiments with root cultures derived from one mutant and from the wild type indicated that the monothiophene accumulating in the mutant is the common precursor for all bithienyl thiophenes in wild-type and mutant Tagetes erecta. These experiments also showed that one mutant is deficient in demethylation of the monothiophene.     

Effect of fungal elicitor on thiophene production in hairy root cultures of Tagetes patula

Summary The effect of fungal elicitor, derived from mycelial extracts of Fusarium conglutinans, on thiophene production in hairy-root cultures of Tagetes patula was studied. Various concentrations of elicitor were added to the culture media. Time-course experiments were carried out using a defined concentration of elicitor. Thiophene production increased with the addition of elicitor. The major thiophenes produced were 5-(4-aceoxy-1-butenyl)-2,2-bithiophene and 5-(buten-1-enyl)-2,2'bithiophene.On leave from Department of Biological Sciences, R. J. College, Bombay 86, India Offprint requests to: M. A. Hjortso     

The synthesis and tubulin binding activity of thiophene-based analogues of combretastatin A-4

A number of analogues of combretastatin A-4 (1), containing a thiophene ring interposed between the two phenyl groups, have been prepared. The synthesis of these compounds employed a combination of palladium-mediated coupling and iodocyclization techniques. The thiophene compounds 11, 14, 18, and 19 also represent non-benzofused analogues of some recently described tubulin binding benzo[b]thiophenes 3-5. The most active thiophene compounds identified in this study were 11, 14, and 18. Overall they are less active than 1 but exhibit comparable activity to the most active of the benzo[b]thiophenes 3-5. A structure-activity relationship of these compounds is considered.     

Distribution and Accumulation of Thiophenes in Plants and Calli of Different Tagetes Species

The diversity of thiophenes (natural biocides) and the differencesbetween the concentrations of these compounds in the leavesand roots of Tagetes erecta L., T. patula L. cv. Nana furia,and T. minuta L. (marigolds) indicated the presence of at leasttwo different sites of accumulation: leaves and roots. Leafexplants of Tagetes, however, are used by preference to obtaincallus cultures. Once subcultured, secondary (C₂) calli of T.patula obtained from leaves of 4 to 7-week-old plants, containedhigher amounts of accumulated thiophenes (up to 80% of the amountsin the leaves) than original (C₁) or twice subcultured calli(C₃). The concentrations of thiophenes in C₂ calli of T. minutawere about half those of C₁ calli, while the concentrationsof thiophenes of C₁ calli amounted to 1-2% of the leaf values.Most of the C₃ calli of T. minuta did not contain thiophenesat all. Although C₁ calli of T. erecta also contained considerableamounts of thiophenes, the C₂ calli died, most likely owingto high levels of accumulated polyphenolic compounds. The combinationof species effects and the physiological state of plants andcalli provides adequate information to decide whether Tagetescalli are able to produce thiophenes or not. It is concludedthat the ability to produce thiophenes does not depend on theorgan used, but on the genetic information present in the species,and on the physiological state of plants and calli, particularlytheir age. Key words: Callus, explant selection, Tagetes erecta, Tagetes minuta, Tagetes patula, thiophenes     

Molecular and metabolic control of secondary metabolism

Secondary metabolism is restricted to specific places in the plant. The concentrations of precursors and end products are the determining factors in the metabolic control of synthesis and breakdown of the compounds involved. Molecular control operates at the level of enzyme amount and gene expression. If the secondary product contains an element in its molecule which is derived from a mineral nutrient in the environment, the operation of the control mechanisms can be studied by varying the concentration of that mineral. This is exemplified by thiophene metabolism in root cultures ofTagetes. The characteristic groups in the molecule are two five-membered rings with a sulphur atom. In the experiments, the rate of thiophene biosynthesis was manipulated by varying the sulphate concentration in the medium. Sulphur limitation led to preferential channeling of sulphur into primary metabolism and a concomitant drop in thiophene biosynthesis. The major part of the reduction was caused by a drop in enzyme activity. Substrate availability played a minor role. The results indicate that sulphur is involved in the molecular control of secondary metabolism inTagetes.Abbreviations BBT 5-(but-3-en-l-ynyl)-2,2-bithienyl - BPT 2-(but-3-en-l-ynyl)-5-(penta-1,3-diynyl)-thiophene - PYE trideca-3,5,7,9,11-pentaynene     

Biosynthesis of benzofuran derivatives in root cultures of Tagetes patula via phenylalanine and 1-deoxy-D-xylulose 5-phosphate

Root cultures of Tagetes patula L. cv. Carmen were grown with a mixture of unlabeled glucose and [U-(13)C(6)]glucose or [1-(13)C(1)]glucose as carbon source. Isoeuparin and (-)-4-hydroxytremetone were isolated by solvent extraction of the cultured tissue, purified by chromatography and analysed by (1)H and (13)C NMR spectroscopy. Amino acids obtained by hydrolysis of protein from the same experiments were used for the reconstruction of the labelling patterns in central metabolic intermediates. These labelling patterns were used for the prediction of isotopolog compositions in the benzofuranone derivatives via different hypothetical pathways. Comparison with the experimentally observed isotopolog distributions showed that the benzenoid ring and the acetoxy group are exclusively or predominantly (>98%) derived from phenylalanine and not from acetyl-CoA via a polyketide-type biosynthesis. The isopropylidene side chain and two carbon atoms of the furan and dihydrofuran moiety, respectively, originate from an isoprenoid building block obtained exclusively or predominantly (>98%) via the deoxyxylulose phosphate pathway. The exomethylene atom of the isopropylidene side chain is biosynthetically equivalent to the (Z)-methyl group of dimethylallyl diphosphate. The data indicate that isoeuparin and (-)-4-hydroxytremetone are assembled from 4-hydroxyacetophenone and dimethylallyl diphosphate via prenyl-substituted 4-hydroxyacetophenone and dihydrobenzofurans as intermediates.     

Aerobic microbial metabolism of some alkylthiophenes found in petroleum

Six alkylthiophenes, 2-hexadecyl-5-methylthiophene (I), 2-methyl-5-tridecylthiophene (II) and 2-butyl-5-tridecylthiophene (III), 2-(3,7-dimethyloctyl)-5-methylthiophene (IV), 2-methyl-5-(3,7,11,15-tetramethyl-hexadecyl)thiophene (V) and 2-ethyl-5-(3,7,11,15-tetramethylhexadecyl)thiophene (VI) were synthesized and used as substrates in biodegradation studies. The products of their aerobic metabolism by pure bacterial cultures were identified. In most cases, the long alkyl chains of these thiophenes were preferentially attacked and in pure cultures of alkane-degrading bacteria, the major metabolites that accumulated in the medium were 5-methyl-2-thiopheneacetic acid from (I), 5-methyl-2-thiophenecarboxylic acid from (II) and occasionally from (V), 5-butyl-2-thiophenecarboxylic acid from (III) and 5-ethyl-2-thiopheneacetic acid from (VI). These transformations are consistent with the metabolism of the alkyl side chains via the beta-oxidation pathway. In contrast, 5-(3,7-dimethyloctyl)-2-thiophenecarboxylic acid was produced from (IV). Because it was available in greatest supply, (I) was studied most thoroughly. It supported growth of the six n-alkanedegrading bacteria tested and (I) was degraded more quickly than pristance but not as quickly as n-hexadecance in mixtures of these three compounds. In the presence of Prudhoe Bay crude oil and a mixed culture of petroleum-degrading bacteria, the acid metabolites from (I), (II) and (III) underwent further biotransformations to products that were not detected by the analytical methods used. The addition of n-hexadecane to the mixed culture of petroleum-degrading bacteria also enhanced the further biotransformations of the metabolites from (I).     

Morphological differentiation and occurrence of thiophenes in leaf callus cultures from Tagetes species: Relation to the growth medium of the plants

The morphology, and the number and concentration of thiophene-like compounds were studied in leaves, roots and calli of Tagetes species grown with different nutrient solutions. The type of nutrient solution exerted no effect on the number of thiophene-like compounds, but altered the type of morphological differentiation and thiophene content of calli. Calli of T. minuta L. showed little differentiation and resulted in suspensible callus after two passages. Calli of T. erecta L. cv. Rose d'Inde differentiated rapidly and turned dark brown after one passage. The morphology of calli from T. patula L. cv. Nana furia was intermediate. Tertiary callus of T. patula contained more thiophene-like compounds and higher concentrations than did the corresponding calli of T. minuta . The content of thiophene-like compounds decreased after various callus passages, but the relative decrease varied between species.     

Toxicity of Thiophenes from Echinops transiliensis (Asteraceae) against Aedes aegypti (Diptera: Culicidae) Larvae

Structure? activity relationships of nine thiophenes, 2,2′: 5′,2″‐terthiophene ( 1 ), 2‐chloro‐4‐[5‐(penta‐1,3‐diyn‐1‐yl)thiophen‐2‐yl]but‐3‐yn‐1‐yl acetate ( 2 ), 4‐(2,2′‐bithiophen‐5‐yl)but‐3‐yne‐1,2‐diyl diacetate ( 3 ), 4‐[5‐(penta‐1,3‐diyn‐1‐yl)thiophen‐2‐yl]but‐3‐yne‐1,2‐diyl diacetate ( 4 ), 4‐(2,2′‐bithiophen‐5‐yl)‐2‐hydroxybut‐3‐yn‐1‐yl acetate ( 5 ), 2‐hydroxy‐4‐[5‐(penta‐1,3‐diyn‐1‐yl)thiophen‐2‐yl]but‐3‐yn‐1‐yl acetate ( 6 ), 1‐hydroxy‐4‐[5‐(penta‐1,3‐diyn‐1‐yl)thiophen‐2‐yl]but‐3‐yn‐2‐yl acetate ( 7 ), 4‐(2,2′‐bithiophen‐5‐yl)but‐3‐yne‐1,2‐diol ( 8 ), and 4‐[5‐(penta‐1,3‐diyn‐1‐yl)thiophen‐2‐yl]but‐3‐yne‐1,2‐diol ( 9 ), isolated from the roots of Echinops transiliensis, were studied as larvicides against Aedes aegypti. Structural differences among compounds 3, 5 , and 8 consisted in differing AcO and OH groups attached to C(3″) and C(4″), and resulted in variations in efficacy. Terthiophene 1 showed the highest activity (LC₅₀, 0.16 μg/ml) among compounds 1 – 9 , followed by bithiophene compounds 3 (LC₅₀, 4.22 μg/ml), 5 (LC₅₀, 7.45 μg/ml), and 8 (LC₅₀, 9.89 μg/ml), and monothiophene compounds 9 (LC₅₀, 12.45 μg/ml), 2 (LC₅₀, 14.71 μg/ml), 4 (LC₅₀, 17.95 μg/ml), 6 (LC₅₀, 18.55 μg/ml), and 7 (LC₅₀, 19.97 μg/ml). These data indicated that A. aegypti larvicidal activities of thiophenes increase with increasing number of thiophene rings, and the most important active site in the structure of thiophenes could be the tetrahydro‐thiophene moiety. In bithiophenes, 3, 5 , and 8 , A. aegypti larvicidal activity increased with increasing number of AcO groups attached to C(3″) or C(4″), indicating that AcO groups may play an important role in the larvicidal activity.