Jasmonate-induced changes in flavonoid metabolism in barley (Hordeum vulgare) leaves

The effects of jasmonic acid (JA) on secondary metabolism in barley (Hordeum vulgare L.) were investigated. A reversed-phase HPLC analysis revealed that the amount of a particular compound increased in excised barley leaf segments that had been treated with JA. This compound was purified and identified as 6'-feruloylsaponarin (1) by spectroscopic analyses and alkaline hydrolysis. A related compound, 6'-sinapoylsaponarin (2), was also found to accumulate in excised leaves independently of the JA treatment. The accumulation of these compounds was accompanied by a decrease in the saponarin (3) content. [8,9-(13)C]p-Coumaric acid and [2,3,4,5,6-(2)H]L-phenylalanine were effectively incorporated into the hydroxycinnamoyl moieties in 1 and 2, while the degree of incorporation of the labeled precursors into the saponarin part was small. These findings indicate that the hydroxycinnamoyl moieties of 1 and 2 are synthesized de novo from phenylalanine via the phenylpropanoid pathway, and that the saponarin part is mainly provided by the constitutive pool of 3.     

Transport of [2-14C]jasmonic acid from leaves to roots mimics wound-induced changes in endogenous jasmonic acid pools in Nicotiana sylvestris

Jasmonic acid (JA) is part of a long-distance signal-transduction pathway that effects increases in de-novo nicotine synthesis in the roots of Nicotiana sylvestris Speg et Comes (Solanaceae) after leaf wounding. Elevated nicotine synthesis increases whole-plant nicotine pools and makes plants more resistant to herbivores. Leaf wounding rapidly increases JA pools in damaged leaves, and after a 90-min delay, root JA pools also increase. The systemic response in the roots could result from either: (i) the direct transport of JA from wounded leaves, or (ii) JA synthesis or its release from conjugates in roots in response to a second, systemic signal. We synthesized [2-¹⁴C]JA, and applied it to a single leaf in a quantity (189 μg) known to elicit both a whole-plant nicotine and root JA response equivalent to that found in plants subjected to leaf wounding. We quantified radioactive material in JA, and in metabolites both more and less polar than JA, from treated and untreated leaves and roots of plants in eight harvests after JA application. [2-¹⁴C]Jasmonic acid was transported from treated leaves to roots at rates and in quantities equivalent to the wound-induced changes in endogenous JA pools. The [2-¹⁴C]JA that had been transported to the roots declined at the same rate as endogenous JA pools in the roots of plants after leaf wounding. Most of the labeled material applied to leaves was metabolized or otherwise immobilized at the application site, and the levels of [2-¹⁴C]JA in untreated leaves did not increase over time. We measured the free JA pools before and after four different hydrolytic extractions of root and shoot tissues to estimate the size of the potential JA conjugate pools, and found them to be 10% or less of the free JA pool. We conclude that the direct transport of wound-induced JA from leaves to roots can account for the systemic increase in root JA pools after leaf wounding, and that metabolism into less polar structures determines the duration of this systemic increase. However, the conclusive falsification of this hypothesis will require the suppression of all other signalling pathways which could have shoot-to-root transport kinetics similar to that of endogenous JA. Received: 14 April 1997 / Accepted: 9 June 1997     

Action spectra for C-glucosylflavone accumulation in Hordeum vulgare plumules

Five-day-old etiolated barley plumules contain the C-glucosylflavones saponarin, lutonarin, and lutonarin 3′-methyl ether. When harvested 24 hr after illumination, increased flavonoid levels were essentially linear with increased energies of monochromatic light at seven wavelengths between 450 and 750 nm. Action spectra for saponarin and for a mixture of lutonarin and its 3′-methyl ether were determined between 380 and 760 nm at 6.6 kerg·cm^?2. The saponarin action spectrum showed distinct peaks at 620 and at 660 nm. These two peaks were similar in their photoreversibility when followed by either 6·6 or 34 kerg·cm^?2 of far-red light. Phytochrome is apparently the photoreceptor for the saponarin action spectrum. Lutonarin and its 3′-methyl ether showed peaks at 520 580, 620 and near 660 nm. The 660 nm peak was not photoreversible by 6·6 kerg·cm^?1, but was by 34 kerg·cm_?2, of far-red light. Phytochrome and protochlorophyll are the likely photoreceptors for these 3′-substituted flavonoids.     

Quantification, correlations and manipulations of wound-induced changes in jasmonic acid and nicotine in Nicotiana sylvestris

Jasmonic acid (JA) is thought to be part of a signal-transduction pathway which dramatically increases de-novo nicotine synthesis in the roots and increases whole-plant (WP) nicotine pools in response to the wounding of the leaves in Nicotiana sylvestrisSpegazzini and Comes (Solanaceae). We report the synthesis of a doubly labeled JA ([1, 2-¹³C]JA) and use it as an internal standard to quantify by gas chromatography-mass spectrometry the changes in root and shoot JA pools in plants subjected to differing amounts of standardized leaf wounding. Wounding increased JA pools 10-fold locally in damaged leaves within 90 min and systemically in the roots (3.5-fold) 180 min after wounding. If JA functions as an intermediary between stimulus and response, quantitative relationships among the stimulus, JA, and the response should exist. To examine these relationships, we varied the number of punctures in four leaves and quantified both the resulting JA in damaged leaves after 90 min and the resulting WP nicotine concentration after 5 d. We found statistically significant, positive relationships among number of leaf punctures, endogenous JA, and WP nicotine accumulation. We used two inhibitors of wound-induced nicotine production, methyl salicylate and indole-3-acetic acid, to manipulate the relationships between wound-induced changes in JA and WP nicotine accumulation. Since wounding and the response to wounding occur in widely separated tissues, we applied inhibitors to different plant parts to examine their effects on the local and systemic components of this response. In all experiments, inhibition of the wound-induced increase in leaf JA 90 min after wounding was associated with the inhibition of the nicotine response 5 d after wounding. We conclude that wound-induced increases in leaf JA are an important component of this long-distance signal-transduction pathway. Received: 24 April 1996 / Accepted: 18 July 1996     

Uptake, Cellular Distribution, and Metabolism of 14C-picloram by Excised Plant Tissues

Excised soybean (Glycine max (L.) Merr.) hypocotyls, barley (Hordeum vulgare L.) coleoptiles, barley leaf sections, and Canada thistle (Cirsium arvense (L.) Scop.) leaf discs continuously absorbed ¹⁴C-picloram from a buffered solution over a 24 h period. After an 8 h uptake period excised sections released up to 30 % of the absorbed picloram into ‘clean’ buffer solution in a 4 h period. Leaf sections released less than did the other tissues. Uptake of ¹⁴C-picloram by soybean hypocotyl and barley coleoptile sections increased with an increase in temperature from 5 to 35°C. Uptake was promoted by added ATP and sucrose but inhibited by actinomycin D, cycloheximide, and DNP. After differential centrifugation of aqueous extracts of ¹⁴C-picloram-treated excised tissues more than 95 % of the radioactivity was in the soluble fraction. In excised barley and Canada thistle leaf tissues, 3 days after treatment, part of the ¹⁴C-picloram was conjugated with plant constituents, largely with sugar(s). After acid hydrolysis of ethanol extracts of such tissues only unaltered picloram was detected. In barley coleoptile and soybean hypocotyl sections no conjugation products of ¹⁴C-picloram were detected.     

Perennial peanut (Arachis glabrata Benth.) leaves contain hydroxycinnamoyl-CoA:tartaric acid hydroxycinnamoyl transferase activity and accumulate hydroxycinnamoyl-tartaric acid esters

Many plants accumulate hydroxycinnamoyl esters to protect against abiotic and biotic stresses. Caffeoyl esters in particular can be substrates for endogenous polyphenol oxidases (PPOs). Recently, we showed that perennial peanut (Arachis glabrata Benth.) leaves contain PPO and identified one PPO substrate, caftaric acid (trans-caffeoyl-tartaric acid). Additional compounds were believed to be cis- and trans-p-coumaroyl tartaric acid and cis- and trans-feruloyl-tartaric acid, but lack of standards prevented definitive identifications. Here we characterize enzymatic activities in peanut leaves to understand how caftaric acid and related hydroxycinnamoyl esters are made in this species. We show that peanut leaves contain a hydroxycinnamoyl-CoA:tartaric acid hydroxycinnamoyl transferase (HTT) activity capable of transferring p-coumaroyl, caffeoyl, and feruloyl moieties from CoA to tartaric acid (specific activities of 11 ± 2.8, 8 ± 1.8, 4 ± 0.8 pkat mg^?1 crude protein, respectively). The HTT activity was used to make cis- and trans-p-coumaroyl- and -feruloyl-tartaric acid in vitro. These products allowed definitive identification of the corresponding cis- and trans-hydroxycinnamoyl esters extracted from leaves. We tentatively identified sinapoyl-tartaric acid as another major phenolic compound in peanut leaves that likely participates in secondary reactions with PPO-generated quinones. These results suggest hydroxycinnamoyl-tartaric acid esters are made by an acyltransferase, possibly a BAHD family member, in perennial peanut. Identification of a gene encoding HTT and further characterization of the enzyme will aid in identifying determinants of donor and acceptor substrate specificity for this important class of biosynthetic enzymes. An HTT gene could also provide a means by genetic engineering for producing caffeoyl- and other hydroxycinnamoyl-tartaric acid esters in forage crops that lack them.     

Jasmonic acid and abscisic acid in shoots,coleoptiles, and roots of wheat seedlings

The endogenous levels of abscisic acid (ABA) and jasmonic acid (JA) were analyzed in wheat seedlings grown in water, a system which in the past has been used to test the effects of these plant growth inhibitors. The levels in different plant parts and in the medium were measured by gas chromatography-mass spectrometry-selected ion monitoring, using [²H₃]ABA and [²H₆]JA as internal standards. In every plant part, JA levels were about 2 orders of magnitude greater than those of ABA. The exudation of JA from roots per seedling was about 14,000-fold greater than that of ABA, although the roots contained only about 170 times more JA than ABA. It is suggested that JA is a possible allelopathic compound.     

Amino acid conjugates as metabolites of the plant growth regulator dihydrojasmonic acid in barley (Hordeum vulgare)

The biotransformation of [2-¹⁴C](±)9, 10-dihydrojasmonic acid (DJA) was studied in excised shoots of 6-day-old barley seedlings after 72 h. From the ethyl acetate extract, some minor metabolites were isolated and purified by DEAE-Sephadex A-25 chromatography, thin-layer chromatography (TLC), C₁₈-cartridges, and high-performance liquid chromatography (HPLC). The structural identification of these metabolites was performed by gas chromatography-mass spectrometry (GC-MS), circular dichroism (CD), and amino acid analysis, and the following amino acid conjugates were found:N-[(–)9,10-dihydrojasmonoyl]valine,N-[(–)9,10-dihydrojasmonoyl]isoleucine,N-[9,10-dihydrojasmonoyl]leucine,N-[11-hydroxy-9,10-dihydrojasmonoyl]valine,N-[11-hydroxy-9,10-dihydrojasmonoyl]isoleucine,N-[12-hydroxy-9,10-dihydrojasmonoyl]isoleucine; and the cucurbic acid-related compoundsN-{[3-hydroxy-2(4-hydroxypentyl)-cyclopent-1-yl]-acetyl}isoleucine andN-{[3-hydroxy-2(5-hydroxypentyl)-cyclopent-1-yl]-acetyl}isoleucine. The results suggest conjugation with isoleucine and valine, as well as preferential hydroxylation at position C-11 or hydrogenation at position C-6, as being important steps in the metabolism of (±)DJA in barley shoots.     

Biosynthesis of withanolides in Acnistus breviflorus: biogenetic relationships among the main withanolides

To excised leaves and 15-day-old seedlings of Acnistus breviflorus sodium [1-¹⁴C]acetate, [2-¹⁴C]mevalonolactone and [¹⁴C-methyl]methionine were administered in separate experiments. From the absolute incorporation values of withaferin A (1), jaborosalactone A (2) and jaborosalactone D (3) isolated at different times after administration of the tracers, it was deduced that compound 2 is a precursor of both 1 and 3 and that the withanolides are later biodegraded to unknown products. Inoculation of [¹⁴C]jaborosalactone A confirmed its transformation into 1 and 3.     

Removal of the acetate-moiety of 2,4-dichlorophenoxyacetic acid in Ribes sativum

Ten minutes after uptake of 2,4-dichlorophenoxyacetic acid-1-¹⁴C(2,4-D-1-¹⁴C) by excised Ribes sativum leaves, 37·8 % of the radioactivity in water-soluble metabolites was in glyoxylic acid. When 2,4-D- 2-¹⁴C was supplied under the same conditions, 23·0 % of the radioactivity of the water-soluble rnetabolites was in glyoxylic acid. Radioactive glycine and glyoxylic acid, isolated from Ribes sativum 6 hr after uptake of 2,4-D-1-¹⁴C, contained essentially all of the ¹⁴C in the carboxyl-carbon atoms. When 2,4-D-2-¹⁴C was the precursor, the glycine isolated contained 64·8 % of its radioactivity in C₂, while 60·0 % of the radioactivity in glyoxylic acid was in C₂. The side-chain label of 2,4-D-2-¹⁴C-4-³⁶Cl was more efficiently incorporated into ethanol-insoluble plant residue than the ring-label. The metabolism of glyoxylic acid-1-¹⁴C and 2,4-D-1-¹⁴C in excised Ribes sativum leaves were compared. The data suggest a cleavage of the acetate-moiety of 2,4-D resulting in a C₂ compound, perhaps glyoxylate.     

Jasmonic acid inhibition of in vitro shoot organogenesis in Pinus radiata cotyledons

The effect of jasmonic acid (JA) onde novo shoot formation in excised cotyledons of radiata pine (Pinus radiata D. Don), was examined. JA had no effect on shoot-forming (SF) tissues at concentrations up to 10⁻⁶ mol · L⁻¹. At concentrations greater than that, JA caused a reduction in the number of shoots formed, as well as the lengths of the shoots and fresh and dry masses of the tissues. Reciprocal transfer of excised radiata pine cotyledons from a SF medium with 10⁻⁵ mol · L⁻¹ JA to a SF medium without JA and vice versa showed that any exposure of the cotyledons to JA either during the shoot induction phase (days 0-21) or the shoot development phase (beyond day 21) led to a reduction in shoot formation. However, the JA effect was significantly less if the cotyledons were not exposed to JA during the initial 10 days in culture; indicating that the JA effect was mainly during shoot primordia formation and the subsequent development into shoots.     

Biosynthesis of sitosterol in barley seedlings

A method is described for the chemical synthesis of stigmasta-5,24-dien-3β-ol-[26-¹⁴C] and (24S)-24-ethylcholesta-5,25-dien-3β-ol-[26-¹⁴C] (clerosterol). 28-Isofucosterol-[7-³H₂] fed to developing barley seedlings (Hordeum vulgare) was incorporated into sitosterol and stigmasterol confirming the utilisation of a 24-ethylidene sterol intermediate in 24α-ethyl sterol production in this plant. Also, the use of mevalonic acid-[2-¹⁴C(4R)-4-³H₁] verified the loss of the C-25 hydrogen of 28-isofucosterol during its conversion into sitosterol and stigmasterol in agreement with the previously postulated isomerisation of the 24-ethylidene sterol to a Δ²⁴⁽²⁵⁾-sterol prior to reduction. However, feeding stigmasta-5,24-dien-3β-ol [26-¹⁴C] to barley seedlings gave very low incorporation into sitosterol. Attempts to trap radioactivity from mevalonic-[2-¹⁴C(4R)-4-³H₁] in stigmasta-5,24-dien-3β-ol when this unlabelled sterol was administered to barley seedlings gave only a very small incorporation although both 28-isofucosterol and sitosterol were labelled.     

Photogeneration of singlet oxygen (1O2) and free radicals (Sen·-, O·-2) by tetra-brominated hypocrellin B derivative

To improve photodynamic activity of the parent hypocrellin B (HB), a tetra-brominated HB derivative (compound 1) was synthesized in high yield. Compared with HB, compound 1 has enhanced red absorption and high molar extinction coefficients. The photodynamic action of compound 1, especially the generation mechanism and efficiencies of active species (Sen^·-, O^·-₂ and ¹O₂) were studied using electron paramagnetic resonance (EPR) and spectrophotometric methods. In the deoxygenated DMSO solution of compound 1, the semiquinone anion radical of compound 1 is photogenerated via the self-electron transfer between the excited and ground state species. The presence of electron donor significantly promotes the reduction of compound 1. When oxygen is present, superoxide anion radical (O^·-₂) is formed via the electron transfer from Sens^·- to the ground state molecular oxygen. The efficiencies of Sens^·- and O^·-₂ generation by compound 1 are about three and two times as much as that of HB, respectively. Singlet oxygen (¹O₂) can be produced via the energy transfer from triplet compound 1 to ground state oxygen molecules. The quantum yield of singlet oxygen (¹O₂) is 0.54 in CHCl₃ similar to that of HB. Furthermore, it was found that the accumulation of Sens^·- would replace that of O^·-₂ or ¹O₂ with the depletion of oxygen in the sealed system.     

Characterization of a novel ginsenoside-hydrolyzing α-l-arabinofuranosidase, AbfA, from Rhodanobacter ginsenosidimutans Gsoil 3054T

The gene encoding an α-l-arabinofuranosidase that could biotransform ginsenoside Rc {3-O-[β-d-glucopyranosyl-(1–2)-β-d-glucopyranosyl]-20-O-[α-l-arabinofuranosyl-(1–6)-β-d-glucopyranosyl]-20(S)-protopanaxadiol} to ginsenoside Rd {3-O-[β-d-glucopyranosyl-(1–2)-β-d-glucopyranosyl]-20-O-β-d-glucopyranosyl-20(S)-protopanaxadiol} was cloned from a soil bacterium, Rhodanobacter ginsenosidimutans strain Gsoil 3054^T, and the recombinant enzyme was characterized. The enzyme (AbfA) hydrolyzed the arabinofuranosyl moiety from ginsenoside Rc and was classified as a family 51 glycoside hydrolase based on amino acid sequence analysis. Recombinant AbfA expressed in Escherichia coli hydrolyzed non-reducing arabinofuranoside moieties with apparent K _m values of 0.53 ± 0.07 and 0.30 ± 0.07 mM and V _max values of 27.1 ± 1.7 and 49.6 ± 4.1 μmol min⁻¹ mg⁻¹ of protein for p-nitrophenyl-α-l-arabinofuranoside and ginsenoside Rc, respectively. The enzyme exhibited preferential substrate specificity of the exo-type mode of action towards polyarabinosides or oligoarabinosides. AbfA demonstrated substrate-specific activity for the bioconversion of ginsenosides, as it hydrolyzed only arabinofuranoside moieties from ginsenoside Rc and its derivatives, and not other sugar groups. These results are the first report of a glycoside hydrolase family 51 α-l-arabinofuranosidase that can transform ginsenoside Rc to Rd.     

The biosynthesis and conjugation of indole-3-acetic acid in germinating seed and seedlings ofDalbergia dolichopetala

Germinating seed ofDalbergia dolichopetala converted both [²H₅]l-tryptophan and [²H₅]indole-3-ethanol to [²H₅]indole-3-acetic acid (IAA). Metabolism of [2-¹⁴C]IAA resulted in the production of indole-3-acetylaspartic acid (IAAsp), as well as several unidentified components, referred to as metabolites I, II, IV and V. Re-application of [¹⁴C]IAAsp to the germinating seed led to the accumulation of the polar, water-soluble compound, metabolite V, as the major metabolite, together with a small amount of IAA. Metabolites I, II and IV were not detected, nor were these compounds associated with the metabolism of [2-¹⁴C]IAA by shoots and excised cotyledons and roots from 26-d-oldD. dolichopetala seedlings. Both shoots and cotyledons converted IAA to IAAsp and metabolite V, while IAAsp was the only metabolite detected in extracts from excised roots. The available evidence indicates that inDalbergia, and other species, IAAsp may not act as a storage product that can be hydrolysed to provide the plant with a ready supply of IAA.Abbreviations HPLC-RC high-performance liquid chromatography-radiocounting - IAA indole-3-acetic acid - IAAsp indole-3-acetylaspartic acid - IAlnos 2-O-indole-3-acetyl-myo-inositol - IEt indole-3-ethanol     

C-glycosylflavone accumulation in Sandoz 6706-treated barley seedlings is a photocontrolled response

Sandoz 6706 pretreatment of white light grown barley seedlings causes a 60% increase in saponarin (6-C-glucosyl-7-O-glucosylapigenin) but a 300% increase in lutonarin (3′-hydroxysaponarin). Norflurazon has little effect on saponarin levels but is almost as effective as Sandoz 6706 in enhancing lutonarin net synthesis. Barley roots contain saponarin and lutonarin only after herbicide treatment. Mung bean seedlings respond to Sandoz 6706 by accumulating higher levels of rutin and delphinidin 3-glucoside. The results are discussed in relation to the site of action of the herbicides, the High Energy photoresponse, and control of flavonoid 3′-hydroxylation.     

Evidence for intra- and extra-protoplasmic feruloylation and cross-linking in wheat seedling roots

The sub-cellular feruloylation and oxidative coupling sites of cell wall polysaccharides were investigated in planta by monitoring the kinetics of appearance of arabinosyl- and feruloyl-radiolabelled polysaccharides in the protoplasmic compartment and their secretion in the wall either in the presence or absence of brefeldin A (BFA). By using root apical segments excised from wheat seedlings (Triticum durum Desf.), incubated with trans-[U-¹⁴C]cinnamic acid, we demonstrated that [¹⁴C]ferulate, likely [¹⁴C]diferulate, as well as trimers and larger products of ferulate are incorporated into the protoplasmic polysaccharides very rapidly within 1–3 min of [¹⁴C]cinnamate feeding. This agrees with the assumption that (glucurono)arabinoxylans [(G)AX] feruloylation and oxidative coupling occur intracellularly, likely in the Golgi apparatus. Simultaneously, polymer bound radioactive hydroxycinnamic acids appeared to be incorporated into the cell wall of root apical segments as early as 2 min after trans-[U-¹⁴C]cinnamic acid feeding. On the contrary, starting from l-[1-¹⁴C]arabinose as tracer, the secretion of the pentose-containing polymers into the wall was between 5 to 10 min. These results indicated that (G)AX feruloylation and oxidative coupling occur both intra-protoplasmically and in muro. The occurrence of in muro feruloylation and oxidative coupling was confirmed by the use of BFA a well known inhibitor of secretion. The drug caused a strong inhibition of the synthesis and secretion into the wall of the ¹⁴C-pentosyl-labelled polymers as well as of ¹⁴C-feruloyl-polymers. In spite of this, the total amount of ¹⁴C-feruloyl-polymers incorporated into the wall was only slightly affected by BFA. This indicates the existence of a mechanism involved into secretion of the activated hydroxycinnamoyl precursors to the wall, alternative to that involved in polysaccharide secretion. Lucia Ilenia Mastrangelo and Marcello Salvatore Lenucci equally contributed to this work.     

Glycerolipid synthesis by homogenate and oil bodies from developing mustard (Sinapis alba L.) seed

[1-¹⁴C]Oleic acid and [14-¹⁴C]erucic acid were converted to their acyl-CoA derivatives and incorporated into acyl lipids by a homogenate from developing mustard (Sinapis alba L.) seed and oil bodies, as well as supernatant isolated by centrifugation at 20000 g. In both homogenate and oil bodies, the oleoyl moieties from exogenous [1-¹⁴C]oleoyl-CoA were most extensively incorporated into phosphatidic acids, but very little into phosphatidylcholines. The pattern of labelling of acyl lipids by oleoyl versus erucoyl moieties from either of the corresponding fatty acids, added individually or as a mixed substrate, indicates that oleoyl-CoA directly acylates sn-glycerol-3-phosphate to yield lysophosphatidic acids and phosphatidic acids that are subsequently converted to mono- and diacylglycerols. In contrast, erucoyl-CoA predominantly acylates preformed mono-and diacylglycerols containing oleoyl moieties to yield triacylglycerols containing erucoyl moieties.     

Biosynthesis of oleanolic acid glycosides in protoplasts isolated from Calendula officinalis L. roots

Many medicinal plants contain oleanane saponins in roots, however, only scarce data on their biosynthesis in this organ are available so far, including our previous results concerning Calendula officinalis plant. Thus, the purpose of the present work was to confirm the presumable biosynthetic pathway of oleanolic acid glycosides in roots of young C. officinalis plants. First of all, the effective method of isolation of protoplasts from C. officinalis roots was established. Then, isolated root protoplasts were supplied with radioactive precursors, [2-¹⁴C] mevalonate (MVA) and [3-³H] oleanolic acid (OL) and their transformations were studied with comparison to results obtained with excised roots. The penetration of both precursors into protoplasts was more rapid and effective than in the case of excised roots. The labeling of sterols and OL during the incubation with MVA showed that the isoprenoid pathway leading to triterpenoids was operative in excised roots as well as isolated root protoplasts. Moreover, the transformations of OL into two series of its glycosides, i.e. glucosides and glucuronides were investigated. It has been shown that both series of OL glycosides are synthesized in isolated root protoplasts in the same way as in excised roots of young marigold plants.     

The occurrence and photoregulation of flavonoids in barley plastids

Whole-leaf extracts of etiolated or light-grown barley shoots contain the C-glycosylflavones saponarin, lutonarin and lutonarin 3′-methyl ether. Plastids isolated by aqueous techniques contain only saponarin. Contamination experiments using foreign flavonoids indicate that saponarin recovered from plastids is not a contaminant from other cellular fractions. In response to brief red light treatment 24 hr before harvest, saponarin levels are approximately doubled in whole-shoot extracts, but increased about 3.5 fold in plastids. This photocontrolled increase is far-red reversible. Thus saponarin is selectively accumulated in barley plastids and this accumulation is controlled by phytochrome.