Volume 381, Issue 8

Plant peroxisomes can be isolated by Percoll density gradient centrifugation at high purity and metabolic competence as well as in relatively large quantities. According to biochemical and electrophysiological analyses, plant peroxisomes have recently been shown to differ from other cell organelles in essential structural properties. Unlike mitochondria or plastids, compartmentalization of plant peroxisomal metabolism is in major parts not caused by a boundary function of the membrane but is primarily due to the specific structure of the protein matrix. The enzymes of the photorespiratory C 2 cycle of leaf peroxisomes are arranged as multienzyme complexes that allow efficient metabolic channelling with high flux rates and minimum leakage of reactive oxygen species from the organelle. Transfer of metabolites, such as carboxylates, proceeds across the peroxisomal membrane via a porin-like channel, which represents a relatively unspecific but highly efficient transport system. Because all variants of peroxisomes, which all contain only a single boundary membrane, are confronted with the task of transporting a large group of metabolites while preventing the escape of reactive intermediates, it is reasonable to speculate that the unique compartmentalization feature of leaf peroxisomes also applies to peroxisomes from fungi and mammals.

A burst of active oxygen species (AOS) is known to be involved in local cell death as part of plant defence against pathogens. It is, however, under dispute to what extent AOS can induce pathogen resistance and immunity throughout the plant. Three experimental strategies that reveal a primary role for AOS and a surprisingly low chemical and spatial specificity are now described for tobacco and Arabidopsis thaliana plants. Ozone is a gaseous AOS that was applied to non-transgenic plants. Hydrogen peroxide or singlet oxygen are AOS that were induced by high-light treatment of transgenic plants that contained antisense constructs inhibiting catalase activity or chlorophyll biosynthetic enzymes. In all cases, activated oxygen species, cellular lesions, ethylene and salicylic acid, and components of major plant defence systems (systemic acquired resistance, hypersensitive response) were induced, as was resistance towards pathogens (tobacco mosaic virus, Pseudomonas syringae or Peronospora parasitica ). It is concluded that active oxygen species can act as mediators of plant immunity so that new non-pesticidal plant protection strategies could be developed.

Introduction of a bean phenylalanine ammonia-lyase (PAL) transgene into tobacco plants results in epigenetic post-transcriptional gene silencing which is unstable, such that after self-pollination first generation progeny may become PAL over-expressors. The change from gene silencing to PAL over-expression is accompanied by a loss of cytosine methylation of the PAL transgene and reduced methylation of the endogenous tobacco PAL2 gene, but not the PAL1 gene. These changes are associated with the appearance of high levels of bean PAL and tobacco PAL2 transcripts in the total RNA fraction from PAL over-expressing plants. However, tobacco PAL2 transcripts are inefficiently recruited into polysomes, and tobacco PAL2 protein is not detected in leaves of PAL over-expressing or wild-type lines. Thus, in spite of the post-transcriptionally controlled increase in tobacco PAL2 transcripts in PAL over-expressors, the increased PAL activity is primarily the result of the increase in bean PAL transcripts and corresponding enzymatic activity. These results reveal a complex cross-talk between expression of the PAL transgene and the corresponding endogenous PAL genes at the levels of transcription, transcript stability and polysomal recruitment during sense transgene-mediated silencing and subsequent over-expresson of PAL in tobacco.

Two distinct cDNA clones, Pc CHI1 and Pc CHI2, with high sequence similarity to plant chitinases were isolated from parsley ( Petroselinum crispum ), expressed in Escherichia coli , and the encoded proteins functionally identified as endochitinases. Different expression patterns of the corresponding mRNAs and proteins in infected and uninfected parsley plants indicated distinct roles of the two isoforms in both pathogen defense and plant development. Infection of parsley leaf buds with Phytophthora sojae resulted in the rapid, transient and highly localized accumulation of Pc CHI1 mRNA and protein around infection sites, whereas Pc CHI2 mRNA and protein were systemically induced at later infection stages. Similar differences in the timing of induction were observed in elicitor-treated, suspension-cultured parsley cells. In uninfected plants, Pc CHI1 mRNA was particularly abundant in the transmitting tract of healthy flowers, suggesting a role in the constitutive protection of susceptible transmitting tissue of the style against pathogen ingress and/or in the fertilization process, possibly by affecting pollen tube growth. Localization of Pc CHI2 mRNA and protein in the parenchymatic collenchyme of young pedicels may indicate a function in the constitutive protection of this tissue. In addition to such distinct roles of Pc CHI1 and Pc CHI2 in preformed and induced pathogen defense, both chitinases may have endogenous regulatory functions in plant development.

An enzyme complex was isolated from Arabidopsis thaliana that catalyzes the entire pathway of biosynthesis of the major plant growth hormone, indole-3-acetic acid (IAA), from ( S )-tryptophan. The 160–180 kDa, soluble complex catalyzes a strictly O 2 -dependent reaction which requires no further added factors and is stereospecific for the substrate ( S )-tryptophan (app. K m = 120μM). H 2 18 O labeling proved that both oxygen atoms of IAA were delivered via H 2 O. This, as well as immunological evidence for the presence of a nitrilase-like protein in the complex, suggests the reaction to proceed via the intermediate indole-3-acetonitrile. IAA-synthase forms a tight metabolite channel committed to IAA production and occurs in shoots, roots and cell cultures of A. thaliana.

A major goal in understanding protein transport across membranes is the investigation of the structure and regulation of the translocon subunits. We analysed Toc75, a pore-forming subunit of the translocon of the outer envelope of chloroplasts. Toc75 was over-expressed and reconstituted into liposomes. Immunoprecipitation of liposome-reconstituted Toc75 indicates an N in -C in orientation of Toc75. Limited proteolytic digestion of Toc75 present in outer envelope vesicles with specific proteases combined with amino acid sequencing was used to study the topology of Toc75. Finally, computer modelling based on known protein structures indicates that Toc75 traverses the outer envelope with 16 amphiphilic beta sheets and the topology model is presented.

We investigated the enzyme methionine synthase (MSY) in Catharanthus roseus . The properties were characterized with purified protein isolated either from plant cell cultures or after heterologous expression in Escherichia coli . The protein was a monomer and accepted both the triglutamate (CH 3 -H 4 PteGlu3, apparent K m = 80μM) and the monoglutamate (CH 3 -H 4 PteGlu1, apparent K m = 350μM) of methyl-5,6,7,8- tetrahydropteroate as methyl donor, with a ratio of approximately 90 :1 in favor of the triglutamate. Both activities required inorganic phosphate, but with different kinetics, and both were dependent on reducing agents. The activity required zinc, as shown by depletion and reconstitution experiments. Mg 2+ had no effect on the activity. Two MSY isoforms purified from parsley cell cultures revealed the same properties as the C . roseus enzyme, however, the parsley proteins had no detectable activity with the monoglutamate substrate. The second part of the work compared the expression of the three enzymes of the methyl cycle (MSY, S-adenosyl-L-methionine synthetase, S-adenosyl- L-homocysteine hydrolase). In cell cultures, all three enzymes were present under all conditions investigated, with small changes at the protein level and more pronounced changes at the RNA level. Studies with seedlings revealed a low expression of all three enzymes in cotyledons, when compared to hypocotyls and radiculas. Immunohistochemical experiments indicated that MSY expression in cotyledons is cell-type specific, with the strongest signals detected in the upper epidermis.

The ability of legumes to recognize and respond to β-glucan elicitors by synthesizing phytoalexins is consistent with the existence of a membrane-bound β-glucan-binding site. Related proteins of approximately 75 kDa and the corresponding mRNAs were detected in various species of legumes which respond to β-glucans. The cDNAs for the β-glucan-binding proteins of bean and soybean were cloned. The deduced 75-kDa proteins are predominantly hydrophilic and constitute a unique class of glucan-binding proteins with no currently recognizable functional domains. Heterologous expression of the soybean β-glucan-binding protein in tomato cells resulted in the generation of a high-affinity binding site for the elicitor-active hepta-β-glucoside conjugate ( K d = 4.5nM). Ligand competition experiments with the recombinant binding sites demonstrated similar ligand specificities when compared with soybean. In both soybean and transgenic tomato, membrane-bound, active forms of the glucan-binding proteins coexist with immunologically detectable, soluble but inactive forms of the proteins. Reconstitution of a soluble protein fraction into lipid vesicles regained β-glucoside-binding activity but with lower affinity ( K d = 130 nM). We conclude that the β-glucan elicitor receptors of legumes are composed of the 75 kDa glucan-binding proteins as the critical components for ligand-recognition, and of an as yet unknown membrane anchor constituting the plasma membrane-associated receptor complex.

Jasmonates and their precursors, the octadecanoids, are signals in stress-induced alteration of gene expression. Several mRNAs coding for enzymes of jasmonic acid (JA) biosynthesis are up-regulated upon JA treatment or endogenous increase of the JA level. Here we investigated the positive feedback of endogenous JA on JA formation, as well as its β-oxidation steps. JA-responsive gene expression was recorded in terms of proteinase inhibitor2 (pin2) mRNA accumulation. JA formed upon treatment of tomato ( Lycopersicon esculentum cv. Moneymaker) leaves with JA derivatives carrying different lengths of the carboxylic acid side chain was quantified by gas chromatography-mass spectrometry (GC-MS). The data revealed that β-oxidation of the side chain occurs up to a butyric acid moiety. The amount of JA formed from side-chain modified JA derivatives correlated with pin2-mRNA accumulation. JA derivatives with a carboxylic side chain of 3, 5 or 7 carbon atoms were unable to form JA and to express on pin2, whereas evennumbered derivatives were active. After treatment of tomato leaves with (10- 2 H)-(–)-12-oxophytoenoic acid, (4- 2 H)-(–)-JA and its methyl ester were formed and could be quantified separately from the endogenously nonlabeled JA pool by GC-MS analysis via isotopic discrimination. The level of 8 nmol per g fresh weight JA and its methyl ester originated exclusively from labeled 12-oxophytoenic acid. This and further data indicate that endogenous synthesis of the JA precursor 12-oxophytodienoic acid, as well as of JA and its methyl ester, are not induced in tomato leaves, suggesting that positive feedback in JA biosynthesis does not function in vivo .

Plants lack specialised organs and circulatory systems, and oxygen can fall to low concentrations in metabolically active, dense or bulky tissues. In animals that tolerate hypoxia or anoxia, low oxygen triggers an adaptive inhibition of respiration and metabolic activity. Growing potato tubers were used to investigate whether an analogous response exists in plants. Oxygen concentrations fall below 5% in the centre of growing potato tubers. This is accompanied by a decrease of the adenylate energy status, and alterations of metabolites that are indicative of a decreased rate of glycolysis. The response to low oxygen was investigated in more detail by incubating tissue discs from growing tubers for 2 hours at a range of oxygen concentrations. When oxygen was decreased in the range between 21% and 4% there was a partial inhibition of sucrose breakdown, glycolysis and respiration. The energy status of the adenine, guanine and uridine nucleotides decreased, but pyrophosphate levels remained high. The inhibition of sucrose breakdown and glycolysis was accompanied by a small increase of sucrose, fructose, glycerate-3-phosphate, phosphenolpyruvate, and pyruvate, a decrease of the acetyl-coenymeA:coenzymeA ratio, and a small increase of isocitrate and 2-oxoglutarate. These results indicate that carbon fluxes are inhibited at several sites, but the primary site of action of low oxygen is probably in mitochondrial electron transport. Decreasing the oxygen concentration from 21% to 4% also resulted in a partial inhibition of sucrose uptake, a strong inhibition of amino acid synthesis, a decrease of the levels of cofactors including the adenine, guanine and uridine nucleotides and coenzymeA, and attenuated the wounding-induced increase of respiration and invertase and phenylalanine lyase activity in tissue discs. Starch synthesis was maintained at high rates in low oxygen. Anoxia led to a diametrically opposed response, in which glycolysis rose 2-fold to support fermentation, starch synthesis was strongly inhibited, and the level of lactate and the lactate:pyruvate ratio and the triose-phosphate:glycerate-3-phosphate ratio ratio increased dramatically. It is concluded that low oxygen triggers (i) a partial inhibition of respiration leading to a decrease of the cellular energy status and (ii) a parallel inhibition of a wide range of energy-consuming metabolic processes. These results have general implications for understanding the regulation of glycolysis, starch synthesis and other biosynthetic pathways in plants, and reveal a potential role for pyrophosphate in conserving energy and decreasing oxygen consumption.

A facile test system based on the accumulation of benzo[ c ]phenanthridine alkaloids in Eschscholzia californica cell suspension culture (an indicator of defense gene activation) has been used to analyze a series of synthetic compounds for elicitor-like activity. Of the 200 jasmonic acid and coronatine analogs tested with this system, representative results obtained with 49 of them are presented here. The following can be summarized concerning structure-activity relationships: there is a large degree of plasticity allowed at the C-3 of jasmonic acid in the activation of defense genes. The carbonyl moiety is not strictly required, but exocyclic double bond character appears necessary. The pentenyl side chain at C-2 cannot tolerate bulky groups at the terminal carbon and still be biologically active. Substitutions to the C-1′ position are tolerated if they can potentially undergo β-oxidation. Either an alkanoic acid or methyl ester is required at C-1, or a side chain that can be shortened by β-oxidation or by peptidase hydrolysis. Coronatine and various derivatives thereof are not as effective as jasmonic acid, and derivatives in inducing benzo[ c ]phenanthridine alkaloid accumulation. Jasmonic acid rather than the octadecanoic precursors is therefore considered to be a likely signal transducer of defense gene activation in planta .

The phenylpropanoid pathway results in the synthesis of thousands of compounds, including flavonoids like flavonols, anthocyanidins and tannins. In Arabidopsis thaliana , the lack of tannins in the seed coat ( testa ) causes the transparent testa ( tt ) phenotype. In the present study, we identified the gene responsible for the tt 7 mutation. We show that TT7 encodes the enzyme flavonoid 3′-hydroxylase (F3′H), and demonstrate that this P450-dependent monooxygenase has F3′H activity. The availability of the AtF3′H gene and promoter sequence will allow us to study the coregulation of a complete set of flavonol and anthocyanidin biosynthesis genes in A. thaliana , and makes in vitro synthesis of hydroxylated flavonoids more feasible.

N -Acylamino acids are dominant and widespread constituents of insect oral secretions (regurgitants), serving the insect as biosurfactants in the digestive process. During feeding the conjugates may be introduced into damaged leaves and contribute there to the elicitation of plant defenses such as the induction of volatile biosynthesis. From gut segments of Spodoptera exigua, Mamestra brassicae and Agrotis segetum 23 bacterial strains were isolated, ten of which were able to synthesise typical lepidopteran N -acylamino acids from externally added precursors. Four strains, Providencia rettgeri , Ochrobactrum spec. , Myroides odoratus and Acinetobacter sp. genospecies 11 were identified on the basis of their 16 S rDNA. The organisms displayed a very broad substrate tolerance, since fatty acids of different chain length and different degree of saturation were converted into N -acylamino acids. Moreover, most of the proteinogenic amino acids, but not glutamic and aspartic acid, were used as substrates. The dominant occurrence of fatty acids conjugated with glutamine may result from a preferred transport of glutamine from the hemolymph into the gut of the insects. The involvement of bacteria in the biosynthesis of compounds which play a pivotal role in the interaction of plants, herbivores and their predators adds a new trophic level to this complex network of interactions. Due to their short generation cycle and the ease of adaptation endosymbiontic bacteria may have an outstanding importance for the coevolution of plant-insect interactions.

Recently, the involvement of the MAP kinase ERK in mitogenic signaling of cholecystokinin B (CCK B ) receptors has been shown. However, the intracellular effector systems involved in this signaling pathway are poorly defined. In this study, we used COS-7 cells transiently transfected with the human CCK B receptor to investigate cholecystokinin-induced MAP kinase activation. CCK-8 induced activation of ERK2 which is associated with its phosphorylation and localization in the nucleus. The CCK-8-dependent ERK stimulation is sensitive to wortmannin an inhibitor of phosphoinositide 3-kinases (PI3Ks) indicating the involvement of PI3K activity. To identify the PI3K species involved in mitogenic signaling of the CCK B receptor several dominant-negative mutants of PI3K regulatory and catalytic subunits were transiently expressed. Surprisingly, different catalytically inactive mutants of the G protein-sensitive PI3Kγ did not affect ERK stimulation induced by CCK, whereas a dominant-negative mutant of the regulatory p85 subunit induced significant inhibition of CCK-dependent ERK activity. These results indicate an involvement of PI3K class 1A species α, β or/and δ in signal transduction via CCK B receptors. In addition, protein kinase C (PKC)-dependent signaling pathways contribute to CCK B -mediated MAP kinase signaling as shown by inhibition of CCK-8-induced ERK activation by the PKC inhibitor bisindolylmaleimide.

Among five ribosome-inactivating proteins tested only saporin-S6 could efficiently release the adenine from adenosine 20 of the synthetic oligoribonucleotide (SRD RNA) mimic of the sarcin/ricin domain of rat 28S rRNA with a K m of 9μM and a k cat of approximately 0.4 min −1 at pH 7.6. The optimal pH for the depurination activity of saporin-S6 is 5.0. However, saporin-S6 lost its site-specificity of depurination on SRD RNA around the optimal pH. The non-specific depurination activity of saporin-S6 was dependent on the enzyme concentration and pH conditions. These results are valuable to understand the diversity and the depurination mechanism of ribosome-inactivating proteins.

Sequences of fetuin cDNA and its deduced amino acid residues from the African green monkey cell line Vero were found to differ by 7.3% and 12.9%, respectively, from the corresponding human sequences. Most amino acid substitutions were clustered within a small segment of the third domain (D3). Calculations of nonsynonymous and synonymous nucleotide substitution rates suggest that this small segment was mutated under positive selection. cDNAs encoding α 1 -antitrypsin, β-actin and the sequences of intron 4 of α 1 -antitrypsin gene in human liver and Vero cells were also investigated. The results substantiated the positive selection imposed on the D3 segment.