Whole-leaf Extracts Research Articles

Flavonoid Versus Artemisinin Anti-malarial Activity in Artemisia annua Whole-Leaf Extracts.

Artemisinin, a sesquiterpene lactone produced by Artemisia annua glandular secretory trichomes, is the active ingredient in the most effective treatment for uncomplicated malaria caused by Plasmodium falciparum parasites. Other metabolites in A. annua or related species, particularly flavonoids, have been proposed to either act as antimalarials on their own or act synergistically with artemisinin to enhance antimalarial activity. We identified a mutation that disrupts the CHALCONE ISOMERASE 1 (CHI1) enzyme that is responsible for the second committed step of flavonoid biosynthesis. Detailed metabolite profiling revealed that chi1-1 lacks all major flavonoids but produces wild-type artemisinin levels, making this mutant a useful tool to test the antiplasmodial effects of flavonoids. We used whole-leaf extracts from chi1-1 and mutant lines impaired in artemisinin production in bioactivity in vitro assays against intraerythrocytic P. falciparum Dd2. We found that chi1-1 extracts did not differ from wild-type extracts in antiplasmodial efficacy nor initial rate of cytocidal action. Furthermore, extracts from the A. annua cyp71av1-1 mutant and RNAi lines impaired in amorpha-4,11-diene synthase gene expression, which are both severely compromised in artemisinin biosynthesis but unaffected in flavonoid metabolism, showed very low or no antiplasmodial activity. These results demonstrate that in vitro bioactivity against P. falciparum of flavonoids is negligible when compared to that of artemisinin.

A role for beta-sitosterol to stigmasterol conversion in plant-pathogen interactions.

Upon inoculation with pathogenic microbes, plants induce an array of metabolic changes that potentially contribute to induced resistance or even enhance susceptibility. When analysing leaf lipid composition during the Arabidopsis thaliana-Pseudomonas syringae interaction, we found that accumulation of the phytosterol stigmasterol is a significant plant metabolic process that occurs upon bacterial leaf infection. Stigmasterol is synthesized from beta-sitosterol by the cytochrome P450 CYP710A1 via C22 desaturation. Arabidopsis cyp710A1 mutant lines impaired in pathogen-inducible expression of the C22 desaturase and concomitant stigmasterol accumulation are more resistant to both avirulent and virulent P. syringae strains than wild-type plants, and exogenous application of stigmasterol attenuates this resistance phenotype. These data indicate that induced sterol desaturation in wild-type plants favours pathogen multiplication and plant susceptibility. Stigmasterol formation is triggered through perception of pathogen-associated molecular patterns such as flagellin and lipopolysaccharides, and through production of reactive oxygen species, but does not depend on the salicylic acid, jasmonic acid or ethylene defence pathways. Isolated microsomal and plasma membrane preparations exhibited a similar increase in the stigmasterol/beta-sitosterol ratio as whole-leaf extracts after leaf inoculation with P. syringae, indicating that the stigmasterol produced is incorporated into plant membranes. The increased contents of stigmasterol in leaves after pathogen attack do not influence salicylic acid-mediated defence signalling but attenuate pathogen-induced expression of the defence regulator flavin-dependent monooxygenase 1. P. syringae thus promotes plant disease susceptibility through stimulation of sterol C22 desaturation in leaves, which increases the stigmasterol to beta-sitosterol ratio in plant membranes.

Tannin Composition Affects the Oxidative Activities of Tree Leaves

We examined whether tannin composition plays an important role in explaining the oxidative activities of tree leaves of Acer saccharum (sugar maple) and Quercus rubra (red oak). Sugar maple leaves contained substantial amounts of ellagitannins, condensed tannins, and galloyl glucoses, whereas red oak leaves contained almost exclusively condensed tannins. Oxidative activities of the crude phenolics from both species, and the phenolic fractions from sugar maple, were measured with electron paramagnetic resonance (EPR) spectrometry and UV-visible spectrophotometry. The two assays produced similar results: (1) sugar maple phenolics produced larger semiquinone radical concentrations,and higher semiquinone decay rates and browning rates than did red oak phenolics; (2) ellagitannin levels were positively associated with the three measures of oxidative activity; and (3) condensed tannin and galloyl glucose levels were negatively associated with these measures. The negative relationship between condensed tannin levels and oxidative activity resulted from the antioxidant effects of condensed tannins on hydrolyzable tannins; several purified condensed tannins significantly decreased the concentrations of semiquinone radicals and browning rates of pedunculagin (an ellagitannin) and pentagalloyl glucose. As expected, whole-leaf extracts from sugar maple produced elevated levels of semiquinone radicals, but none were observed in red oak extracts when the two species were compared with an EPR time-course assay. We conclude that the oxidative activities of tree leaves may be affected by tannin composition, and that the prooxidant activity of ellagitannins may be decreased by co-occurring condensed tannins.

Compartment-specific role of the ascorbate-glutathione cycle in the response of tomato leaf cells to Botrytis cinerea infection

Changes in AA-GSH cycle activity following Botrytis cinerea infection were studied in tomato whole-leaf extracts as well as in chloroplasts, mitochondria, and peroxisomes. The oxidative effect of infection affected all cellular compartments although mitochondria and peroxisomes underwent the most pronounced changes. Apart from organelle-specific variations, a general shift of the cellular redox balance towards the oxidative state was found. It was manifested by the significant decline in concentrations and redox ratios of the ascorbate and glutathione pools as well as by the insufficient activity of MDHAR, DHAR, and GR needed for antioxidant regeneration. There was no compatibility between the ascorbate- and glutathione-mediated changes in different compartments. It was concluded that B. cinerea was able to break down the protective antioxidant barrier of the AA-GSH cycle at both the cellular and organellar levels. The changes in the AA-GSH cycle activity could partly be related to the B. cinerea-induced promotion of senescence that favoured disease progress.

The Identification and Properties of Apoplastic Carboxylesterases from Wheat That Catalyse Deesterification of Herbicides

Formulated herbicides are frequently applied as carboxylesters. The first step in the metabolism of these compounds in plants is enzymatic deesterification, which generally leads to their bioactivation and increases their mobility within the plant. In apoplastic extracts from wheat devoid of cytoplasmic contamination, we have identified a pool of nitrophenyl acetate and naphthyl acetate esterases. These esterases were resolved by isoelectric focusing and activity staining into at least 11 proteins. Furthermore, the apoplastic pool of esterases could be distinguished from soluble esterases in whole-leaf extracts by two-dimensional electrophoresis and their affinity for concanavalin A. The apoplastic fraction deesterified some but not all members of the arylphenoxypropionate family of herbicides tested. Deesterification was inhibited by phenylmethylsulfonyl fluoride. We conclude that there is a pool of soluble carboxylesterases in the leaf apoplast of wheat seedlings that are largely both glycoproteins and serine hydrolases. These apoplastic esterases catalyze the hydrolysis of herbicide esters and are substrate specific. We speculate that the apoplastic esterases contribute to the bioavailability of herbicides in planta.

Early H(2)O(2) accumulation in mesophyll cells leads to induction of glutathione during the hyper-sensitive response in the barley-powdery mildew interaction.

H(2)O(2) production and changes in glutathione, catalase, and peroxidase were followed in whole-leaf extracts from the susceptible (AlgS [Algerian/4* (F14) Man.(S)]; ml-a1 allele) and resistant (AlgR [Algerian/4* (F14) Man.(R)]; Ml-a1 allele) barley (Hordeum vulgare) isolines between 12 and 24 h after inoculation with powdery mildew (Blumeria graminis [DC]. Speer [syn. Erysiphe graminis DC] f.sp hordei Marchal). Localized papilla responses and cell death hypersensitive responses were not observed within the same cell. In hypersensitive response sites, H(2)O(2) accumulation first occurred in the mesophyll underlying the attacked epidermal cell. Subsequently, H(2)O(2) disappeared from the mesophyll and accumulated around attacked epidermal cells. In AlgR, transient glutathione oxidation coincided with H(2)O(2) accumulation in the mesophyll. Subsequently, total foliar glutathione and catalase activities transiently increased in AlgR. These changes, absent from AlgS, preceded inoculation-dependent increases in peroxidase activity that were observed in both AlgR and AlgS at 18 h. An early intercellular signal precedes H(2)O(2), and this elicits anti-oxidant responses in leaves prior to events leading to death of attacked cells.

Phosphoenolpyruvate carboxykinase is involved in the decarboxylation of aspartate in the bundle sheath of maize

We recently showed that maize (Zea mays L.) leaves contain appreciable amounts of phosphoenolpyruvate carboxykinase (PEPCK; R.P. Walker, R.M. Acheson, L.I. Tecsi, R.C. Leegood [1997] Aust J Plant Physiol 24: 459-468). In the present study, we investigated the role of PEPCK in C4 photosynthesis in maize. PEPCK activity and protein were enriched in extracts from bundle-sheath (BS) strands compared with whole-leaf extracts. Decarboxylation of [4-14C]aspartate (Asp) by BS strands was dependent on the presence of 2-oxoglutarate and Mn2+, was stimulated by ATP, was inhibited by the PEPCK-specific inhibitor 3-mercaptopicolinic acid, and was independent of illumination. The principal product of Asp metabolism was phosphoenolpyruvate, whereas pyruvate was a minor product. Decarboxylation of [4-14C]malate was stimulated severalfold by Asp and 3-phosphoglycerate, was only slightly reduced in the absence of Mn2+ or in the presence of 3-mercaptopicolinic acid, and was light dependent. Our data show that decarboxylation of Asp and malate in BS cells of maize occurs via two different pathways: Whereas malate is mainly decarboxylated by NADP-malic enzyme, decarboxylation of Asp is dependent on the activity of PEPCK.

Developmental and environmental effects on the expression of the C3-C4 intermediate phenotype in moricandia arvensis

Cellular anatomy and expression of glycine decarboxylase (GDC) protein were studied during leaf development of the C3-C4 intermediate species Moricandia arvensis. Leaf anatomy was initially C3-like and the number and profile area of mitochondria in the bundle-sheath cells were the same as those in adjacent mesophyll cells. Between a leaf length of 6 and 12 mm there was a bundle-sheath-specific, 4-fold increase in the number of mitochondrial profiles, followed by a doubling of their individual profile areas as the leaves expanded further. Subunits of GDC were present in whole-leaf extracts before the anatomical development of bundle-sheath cells. Whereas the GDC H-protein content of leaves increased steadily throughout development, the increase in GDC P-protein was synchronous with the development of mitochondria in the bundle sheath. The P-protein was confined to bundle-sheath mitochondria throughout leaf development, and its content in individual mitochondria increased before the anatomical development of the bundle sheath. Anatomical and biochemical attributes of the C3-C4 character were present in the cotyledons and sepals but not in other photosynthetic organs/tissues. In leaves and cotyledons that developed in the dark, the expression of the P-protein and the organellar development were reduced but the bundle-sheath cell specificity was retained.

Expression of tobacco carbonic anhydrase in the C4 dicot flaveria bidentis leads to increased leakiness of the bundle sheath and a defective CO2-concentrating mechanism

Flaveria bidentis (L.) Kuntze, a C4 dicot, was genetically transformed with a construct encoding the mature form of tobacco (Nicotiana tabacum L.) carbonic anhydrase (CA) under the control of a strong constitutive promoter. Expression of the tobacco CA was detected in transformant whole-leaf and bundle-sheath cell (bsc) extracts by immunoblot analysis. Whole-leaf extracts from two CA-transformed lines demonstrated 10% to 50% more CA activity on a ribulose-1,5-bisphosphate carboxylase/oxygenase-site basis than the extracts from transformed, nonexpressing control plants, whereas 3 to 5 times more activity was measured in CA transformant bsc extracts. This increased CA activity resulted in plants with moderately reduced rates of CO2 assimilation (A) and an appreciable increase in C isotope discrimination compared with the controls. With increasing O2 concentrations up to 40% (v/v), a greater inhibition of A was found for transformants than for wild-type plants; however, the quantum yield of photosystem II did not differ appreciably between these two groups over the O2 levels tested. The quantum yield of photosystem II-to-A ratio suggested that at higher O2 concentrations, the transformants had increased rates of photorespiration. Thus, the expression of active tobacco CA in the cytosol of F. bidentis bsc and mesophyll cells perturbed the C4 CO2-concentrating mechanism by increasing the permeability of the bsc to inorganic C and, thereby, decreasing the availability of CO2 for photosynthetic assimilation by ribulose-1,5-bisphosphate carboxylase/oxygenase.

Distribution of Folate Derivatives and Enzymes for Synthesis of 10-Formyltetrahydrofolate in Cytosolic and Mitochondrial Fractions of Pea Leaves.

Leaf extracts of 14-d-old pea (Pisum sativum L. cv Homesteader) seedlings were examined for folate derivatives and for 10-formyltetrahydrofolate synthetase (SYN), 5,10-methenyltetrahydrofolate cyclohydrolase (CYC), and 5,10-methylenetetrahydrofolate dehydrogenase (DHY) activities. Microbiological and enzyme assays showed that leaf folates SYN, CYC, and DHY were predominantly cytosolic. Extracts of Percoll gradient-purified mitochondria contained less than 1% of total leaf folate and less that 1% of each enzyme activity. Fractionation of whole-leaf homogenates resulted in the copurification of DHY and CYC (subunit 38 kD) and the isolation of a SYN protein (subunit 66 kD). Polyclonal antibodies were raised against purified cytosolic DHY-CYC (DHY-CYC-Ab) and cytosolic SYN (SYN-Ab), respectively. Immunoblots showed that DHY-CYC-Ab cross-reacted with a mitochondrial protein band (38 kD). Two mitochondrial protein bands (subunit Mr = 40,000 and 44,000) cross-reacted with SYN-Ab. Immunoaffinity chromatography (DHY-CYC-Ab as the immobile ligand) indicated that the bulk of mitochondrial SYN activity was not associated with mitochondrial DHY or CYC. When 9-d-old etiolated pea seedlings were exposed to light for up to 3 d, the specific enzyme activities of DHY-CYC in whole-leaf extracts rose 2-fold and more DHY-CYC-Ab cross-reacting protein was detected. In contrast, the specific activity of SYN fell from 5 to 1 [mu]mol min-1 mg-1 protein and less SYN-Ab cross-reacting protein was detected. The data suggest that in pea leaves, the bulk of one-carbon-substituted tetrahydrofolates and enzymes for the generation of 10-formyltetrahydrofolate are extra-mitochondrial.

In barley leaf cells, jasmonates do not act as a signal during compatible or incompatible interactions with the powdery mildew fungus ( Erysiphe graminis f. sp. hordei)

Summary We have studied a possible function of jasmonates as mediators in the host-pathogen interaction of barley (Hordeum vulgare L.) with the powdery mildew fungus Egh (Erysiphe graminis f. sp. hordei). Previous findings from whole-leaf extracts demonstrated that (i) extracts from infected barley leaves did not contain enhanced levels of jasmonates, (ii) transcripts of jasmonate-inducible genes were not expressed upon infection, and (iii) exogenous application of jasmonates did not induce resistance to Egh (Kogel et al., 1995). Nevertheless, the question arises whether or not jasmonates are involved in the interaction of barley with the powdery mildew fungus at the local site of infection. Using an immunocytological approach the analysis of leaf cross-sections from a susceptible barley cultivar and its near-isogenic mlo5-resistant line revealed no accumulation of JIP-23, the most abundant jasmonate inducible protein, neither in epidermal cells attacked by the pathogen nor in adjacent mesophyll cells. As a positive control, cross-sections from methyl jasmonate-treated leaf segments showed a strong signal for JIP-23 accumulation. Because the presence of the jasmonate-inducible protein is highly indicative for an already low threshold level of endogenous jasmonate (Lehmann et al., 1995), the lack of JIP-23 accumulation at the sites of attempted fungal infection clearly demonstrates the absence of enhanced levels of jasmonates. This excludes even a local rise of jasmonate confined to those single cells penetrated (Mlo genotype) or attacked (mlo5 genotype) by the fungus.

Elicitor-Induced Cinnamyl Alcohol Dehydrogenase Activity in Lignifying Wheat (Triticum aestivum L.) Leaves.

The substrate-specific induction of wheat (Triticum aestivum L. cv Fenman) leaf cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.195) was examined in relation to its role in regulating the composition of defensive lignin induced at wound margins. Treatment of wounds with a partially acetylated chitosan hydrolysate or spores of the nonpathogen Botrytis cinerea elicited lignification at wound margins and invoked significant increases in phenylalanine ammonia-lyase (EC 4.3.1.5), peroxidase (EC 1.11.1.7), and CAD activities. The substrate-specific induction of CAD with time was determined in elicitor-treated leaves and in excised lignifying wounds. In whole leaf extracts no significant increases in p-cou-maryl and coniferyl alcohol dehydrogenase activities were detectable, but a significant 5-fold increase in sinapyl alcohol dehydrogenase activity was evident 32 h after elicitor treatment. Similarly, fungal challenge resulted in elevated levels of only sinapyl alcohol dehydrogenase in whole-leaf extracts. In excised lignifying tissues p-coumaryl alcohol dehydrogenase levels were similar to those observed in healthy tissue. A small yet significant increase in coniferyl alcohol dehydrogenase was apparent, but the most dramatic increase occurred in sinapyl alcohol dehydrogenase activity, which increased to values approximately 10 times higher than the untreated controls. Our results show for the first time that CAD induction in lignifying tissues of wheat is predominantly attributable to highly localized increases in sinapyl alcohol dehydrogenase activity.

Enzymic and Photosynthetic Characteristics of Reciprocal F1 Hybrids of Flaveria pringlei (C3) and Flaveria brownii (C4-Like Species)

The activities of key C(4) enzymes in gel-filtered, whole-leaf extracts and the photosynthetic characteristics for reciprocal F(1) hybrids of Flaveria pringlei (C(3)) and F. brownii (C(4)-like species) were measured to determine whether any inherited C(4)-photosynthetic traits are responsible for their reduced CO(2) compensation concentration values (AS Holaday, S Talkmitt, ME Doohan Plant Sci 41: 31-39). The activities of phosphoenolpyruvate carboxylase, pyruvate, orthophosphate dikinase, and NADP-malic enzyme (ME) for the reciprocal hybrids are only about 7 to 17% of those for F. brownii, but are three- to fivefold greater than the activities for F. pringlei. The low activities of these enzymes in the hybrids appear to be the result of a partial dominance of F. pringlei genes over certain F. brownii genes. However, no such dominance occurs with respect to the expression of genes for NADP-malate dehydrogenase, which is as active in the hybrids as in F. brownii. In contrast to the situation with the enzymes above, cytoplasmic factors appear to determine the inheritance of NAD-ME. The NAD-ME activity in each hybrid is comparable to that in the respective maternal parent. Pulse-chase (14)CO(2) incorporation analyses at ambient CO(2) levels indicate that the hybrids initially assimilate 7 to 9% of the total assimilated CO(2) into C(4) acids as compared to 3.5% for F. pringlei. In the hybrids, the percentage of (14)C in malate decreases from an average of 6.5 to 2.1% after a 60-second chase in (12)CO(2)/air. However, this apparent C(4)-cycle activity is too limited or inefficient to substantially alter CO(2) exchange from that in F. pringlei, since the values of net photosynthesis and O(2) inhibition of photosynthesis are similar for the hybrids and F. pringlei. Also, the ratio of the internal to the external CO(2) concentration and the initial slopes of the plot of CO(2) concentration versus net photosynthesis are essentially the same for the hybrids and F. pringlei. At 45 micromoles CO(2) per mole and 0.21 mole O(2) per mole, the hybrids assimilate nearly fivefold more CO(2) into C(4) acids than does F. pringlei. Some turnover of the malate pool occurs in the hybrids, but the labelling of the photorespiratory metabolites, glycine and serine, is the same in these plants as it is in F. pringlei. Thus, although limited C(4)-acid metabolism may operate in the hybrids, we conclude that it is not effective in altering O(2) inhibition of CO(2) assimilation. The ability of the hybrids to assimilate more CO(2) via phosphoenolpyruvate carboxylase at low levels of CO(2) than does F. pringlei may result in an increased rate of reassimilation of photorespiratory CO(2) and CO(2) compensation concentrations below that of their C(3) parent. If the hybrids do possess a limited C(4) cycle, it must operate intracellularly. They are not likely to have inherited an intercellular compartmentation of C(4) enzymes, since F. brownii has incomplete compartmentation of key C(3) and C(4) enzymes.

One-Carbon Substituted Folylpolyglutamates of Isolated Bundle Sheath Cells

Corn ( Zea mays L. cv. Gills Early Market) and sorghum ( Sorghum bicolor L. cv. P 130) seedlings were grown for 14 days under controlled environmental conditions. Bundle sheath cells were isolated from fully expanded leaves by cellulase treatment and endogenous folates were extracted in the presence of ascorbate. Total folate concentrations were determined by microbiological assay before and after conjugase treatment of the extracts. A differential microbiological assay using Lactobacillus casei (ATCC 7469) and Pediococcus acidilactici (ATCC 8081) was employed to determine the nature of C-1 substituted folates present in whole-leaf and bundle sheath cell extracts. Individual derivatives were recovered by DEAE-cellulose chromatography. The bundle sheath cell folates of both species were mainly (70–83 %) formyland unsubstituted derivatives of tetrahydrofolate polyglutamate. In whole-leaf extracts these derivatives represented 35–40 % of the total folate pool. Bundle sheath cells contained only minor concentrations of 5-methyltetrahydrofolate polyglutamate and isolated cells had little ability to synthesize methionine from formate, glycine or serine. The possible significance of the formyltetrahydrofolate pool to the generation of f ormate by bundle sheath cells is discussed.

Distribution of enzymes related to C3 and C4 pathway of photosynthesis between mesophyll and bundle sheath cells of <italic>Panicum hians</italic> and <italic>Panicum milioides</italic><xref ref-type="fn" rid="fn1"><sup>1</sup></xref>

Enzymes of the C4 and C3 pathways and photorespiration were analyzed for P. hians and P. milioides, which have chlorenchymatous bundle sheath cells in the leaves. On whole-leaf extracts the levels of PEP carboxylase were relatively low compared to C4 species, RuDP carboxylase is typical of C3 species, and enzymes of photorespiratory metabolism appeared somewhat intermediate between C3 and C4. Substantial levels of PEP carboxylase, RuDP carboxylase, and photorespiratory enzymes were found in both mesophyll and bundle sheath cells. Low levels of C4-acid decarboxylating enzymes may limit the capacity for C4 photosynthesis in P. hians and P. milioides. The results on enzyme activity and distribution between mesophyll and bundle sheath cells were consistent with CO2 fixation via the C3 pathway in these 2 species

The distribution of flavonoids in chloroplasts of twenty five species of vascular plants

A survey was made of the major flavonoids in whole leaf extracts and in chloroplast preparations from twenty five species of vascular plants including Anthophyta (20), Coniferophyta (1), Ginkophyta (1), Pterophyta (2), and Arthrophyta (1). The chloroplasts variously contained derivatives of flavones, C-glycosylflavones, flavonols, flavanones, isoflavones, 3-deoxyanthocyanidins, and anthocyanins. Twenty three species contain one or more flavonoids in isolated chloroplast, usually in a pattern quite similar to that found in whole-leaf extracts but occasionally showing enrichment of one or more flavonoids in the chloroplasts. Flavonoids are apparently absent from chloroplasts of Phaseolus aureus and Morus alba although whole-leaf extracts of these species are rich in quercetin derivatives.

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.