Hypericum perforatum plant cells reduce Agrobacterium viability during co-cultivation.

Plant recalcitrance is the major barrier in developing Agrobacterium-mediated transformation protocols for several important plant species. Despite the substantial knowledge of T-DNA transfer process, very little is known about the factors leading to the plant recalcitrance. Here, we analyzed the basis of Hypericum perforatum L. (HP) recalcitrance to Agrobacterium-mediated transformation using cell suspension culture. When challenged with Agrobacterium, HP cells swiftly produced an intense oxidative burst, a typical reaction of plant defense. Agrobacterium viability started to decline and reached 99% mortality within 12 h, while the plant cells did not suffer apoptotic process. This is the first evidence showing that the reduction of Agrobacterium viability during co-cultivation with recalcitrant plant cells can affect transformation.

Identification of the substrate specificity-conferring amino acid residues of 4-coumarate:coenzyme A ligase allows the rational design of mutant enzymes with new catalytic properties.

4-Coumarate:coenzyme A ligases (4CLs) generally use, in addition to coumarate, caffeate and ferulate as their main substrates. However, the recently cloned Arabidopsis thaliana isoform At4CL2 is exceptional because it has no appreciable activity with ferulate. On the basis of information obtained from the crystal structure of the phenylalanine-activating domain of gramicidin S-synthetase, 10 amino acid residues were identified that may form the substrate binding pocket of 4CL. Among these amino acids, representing the putative "substrate specificity motif," only one residue, Met(293), was not conserved in At4CL2, compared with At4CL1 and At4CL3, two isoforms using ferulate. Substitution of Met(293) or Lys(320), another residue of the putative substrate specificity motif, which in the predicted three-dimensional structure is located in close proximity to Met(293), by smaller amino acids converted At4CL2 to an enzyme capable of using ferulate. The activity with caffeate was not or only moderately affected. Conversely, substitution of Met(293) by bulky aromatic amino acids increased the apparent affinity (K(m)) for caffeate up to 10-fold, whereas single substitutions of Val(294) did not affect substrate use. The results support our structural assumptions and suggest that the amino acid residues 293 and 320 of At4CL2 directly interact with the 3-methoxy group of the phenolic substrate and therefore allow a first insight into the structural principles determining substrate specificity of 4CL.

Two differentially regulated class II chitinases from parsley.

Two distinct cDNA clones, PcCHI1 and PcCHI2, 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 PcCHI1 mRNA and protein around infection sites, whereas PcCHI2 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, PcCHI1 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 PcCHI2 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 PcCHI1 and PcCHI2 in preformed and induced pathogen defense, both chitinases may have endogenous regulatory functions in plant development.

Mutational analysis of 4-coumarate:CoA ligase identifies functionally important amino acids and verifies its close relationship to other adenylate-forming enzymes.

4-Coumarate:coenzyme A ligase (4CL) is a key enzyme of general phenylpropanoid metabolism which provides the precursors for a large variety of important plant secondary products, such as lignin, flavonoids, or phytoalexins. To identify amino acids important for 4CL activity, eight mutations were introduced into Arabidopsis thaliana At4CL2. Determination of specific activities and K(m) values for ATP and caffeate of the heterologously expressed and purified proteins identified four distinct classes of mutants: enzymes with little or no catalytic activity; enzymes with greatly reduced activity but wild-type K(m) values; enzymes with drastically altered K(m) values; and enzymes with almost wild-type properties. The latter class includes replacement of a cysteine residue which is strictly conserved in 4CLs and had previously been assumed to be directly involved in catalysis. These results substantiate the close relationship between 4CL and other adenylate-forming enzymes such as luciferases, peptide synthetases, and fatty acyl-CoA synthetases.

Three 4-coumarate:coenzyme A ligases in Arabidopsis thaliana represent two evolutionarily divergent classes in angiosperms.

The enzyme 4-coumarate:CoA ligase (4CL) plays a key role in channelling carbon flow into diverse branch pathways of phenylpropanoid metabolism which serve important functions in plant growth and adaptation to environmental perturbations. Here we report on the cloning of the 4CL gene family from Arabidopsis thaliana and demonstrate that its three members, At4CL1, At4CL2 and At4CL3, encode isozymes with distinct substrate preference and specificities. Expression studies revealed a differential behaviour of the three genes in various plant organs and upon external stimuli such as wounding and UV irradiation or upon challenge with the fungus, Peronospora parasitica. Phylogenetic comparisons indicate that, in angiosperms, 4CL can be classified into two major clusters, class I and class II, with the At4CL1 and At4CL2 isoforms belonging to class I and At4CL3 to class II. Based on their enzymatic properties, expression characteristics and evolutionary relationships, At4CL3 is likely to participate in the biosynthetic pathway leading to flavonoids whereas At4CL1 and At4CL2 are probably involved in lignin formation and in the production of additional phenolic compounds other than flavonoids.

A distinct member of the basic (class I) chitinase gene family in potato is specifically expressed in epidermal cells.

We have isolated cDNA clones encoding class I chitinase (ChtC) from potato leaves which share a high degree of nucleotide and amino acid sequence similarity to other, previously described basic (class I) chitinases (ChtB) from potato. Despite this similarity, characteristic features distinguish ChtC from ChtB, including an extended proline-rich linker region between the hevein and catalytic domains and presence of a potential glycosylation site (NDT) in the deduced protein. These differences are in accordance with the properties of purified chitinase C which is glycosylated and hence has a higher molecular mass in comparison to chitinase B. In contrast to the coding sequences, the 3'-untranslated regions of ChtC and ChtB exhibited a low degree of similarity, which allowed us to generate gene-specific probes to study the genomic organization and expression of both types of gene. Genomic DNA blots suggest that ChtC and ChtB are each encoded by one or two genes per haploid genome. RNA blot analysis showed that in healthy potato plants ChtC mRNA is most abundant in young leaves, the organs which also contain high levels of chitinase C. By contrast, ChtB mRNA abundance is highest in old leaves, which accumulate chitinase B. By in situ RNA hybridization with gene-specific probes we could demonstrate that ChtC mRNA in leaves is restricted to epidermal cells, whereas ChtB mRNA showed no distinct pattern of cell-type-specific localization. Infection of potato leaves with Phytophthora infestans, or treatment with fungal elicitor, ethylene, or wounding resulted in accumulation of both ChtC and ChtB mRNAs; however, for ChtC, in contrast to ChtB, no corresponding accumulation of the encoded protein could be detected, suggesting a post-transcriptional mechanism of regulation. Salicylic acid treatment did not induce accumulation of either mRNA. The possible functional implications of these findings for pathogen defence and developmental processes are discussed.

Primary structure and expression of acidic (class II) chitinase in potato.

Infection of potato (Solanum tuberosum) leaves by the late blight fungus Phytophthora infestans or treatment with fungal elicitor leads to a strong increase in chitinase activity. We isolated cDNAs encoding acidic (class II) chitinases (ChtA) from potato leaves and determined their structures and expression patterns in healthy and stressed plants. From the total number of cDNAs and the complexity of genomic DNA blots we conclude that acidic chitinase in potato is encoded by a gene family which is considerably smaller than that encoding basic (class I) chitinase (ChtB). The deduced amino acid sequences show 78 to 96% identity to class II chitinases from related plant species tomato, tobacco) whereas the identity to basic chitinases of potato is in the range of 60%. RNA blot analysis revealed that both acidic and basic chitinases were strongly induced by infection or elicitor treatment and that the induction occurred both locally at the site of infection and systemically in upper uninfected leaves. In contrast, a differential response to other types of stress was observed. Acidic chitinase mRNA was strongly induced by salicylic acid, whereas basic chitinase mRNA was induced by ethylene or wounding. In healthy, untreated plants, acidic chitinase mRNA accumulated also in an organ-, cell-type- and development-specific manner as revealed by RNA blot analysis and in situ RNA hybridization. Relatively high transcript levels were observed in old leaves and young internodes as well as in vascular tissue and cells constituting the stomatal complex in leaves and petioles. Lower, but appreciable mRNA levels were also detectable in roots and various flower organs, particularly in sepals and stamens. The possible implications of these findings in pathogen defense, development and growth processes are discussed.

Primary structure and expression of mRNAs encoding basic chitinase and 1,3-beta-glucanase in potato.

Infection of potato leaves (Solanum tuberosum L. cv. Datura) by the late blight fungus Phytophthora infestans, or treatment with fungal elicitor leads to a strong increase in chitinase and 1,3-beta-glucanase activities. Both enzymes have been implicated in the plant's defence against potential pathogens. In an effort to characterize the corresponding genes, we isolated complementary DNAs encoding the basic forms (class I) of both chitinase and 1,3-beta-glucanase, which are the most abundant isoforms in infected leaves. Sequence analysis revealed that at least four genes each are expressed in elicitor-treated leaves. The structural features of the potato chitinases include a hydrophobic signal peptide at the N-terminus, a hevein domain which is characteristic of class I chitinases, a proline- and glycine-rich linker region which varies among all potato chitinases, a catalytic domain, and a C-terminal extension. The potato 1,3-beta-glucanases also contain a N-terminal hydrophobic signal peptide and a C-terminal extension, the latter comprising a potential glycosylation site. RNA blot hybridization experiments showed that basic chitinase and 1,3-beta-glucanase are strongly and coordinately induced in leaves in response to infection, elicitor treatment, ethylene treatment, or wounding. In addition to their activation by stress, both types of genes are regulated by endogenous factors in a developmental and organ-specific manner. Appreciable amounts of chitinase and 1,3-beta-glucanase mRNAs were found in old leaves, stems, and roots, as well as in sepals of healthy, untreated plants, whereas tubers, root tips, and all other flower organs (petals, stamen, carpels) contained very low levels of both mRNAs. In young leaves and stems, chitinase and 1,3-beta-glucanase were differentially expressed. While chitinase mRNA was abundant in these parts of the plant, 1,3-beta-glucanase mRNA was absent. DNA blot analysis indicated that in potato, chitinase and 1,3-beta-glucanase are encoded by gene families of considerable complexity.

Purification and characterization of extracellular, acidic chitinase isoenzymes from elicitor-stimulated parsley cells.

Treatment of cultured parsley cells (Petroselinum crispum) with fungal elicitor caused large increases in the activities of chitinase and 1,3-beta-glucanase. Chitinase activity accumulated predominantly in the culture medium, whereas 1,3-beta-glucanase activity was located almost exclusively intracellularly. Extracellular chitinase activity was resolved into six different isoenzymes, all of which were purified and characterized. All six isoforms were acidic proteins (pI 3.8-5.3), with molecular mass 30-38 kDa. Four were exochitinases and two were endochitinases. The most abundant isoform also showed lysozyme activity. Three of the exochitinases were glycoproteins and two of these were reactive with an antiserum specific for xylose in complex glycosidic structures. The exochitinases constituted relatively small proportions of the total chitinase activity and may serve a different function in cellular metabolism compared to the more abundant endochitinases.

Structural analysis and activation by fungal infection of a gene encoding a pathogenesis-related protein in potato.

The structure, genomic organization, and temporal pattern of activation of a gene encoding a pathogenesis-related protein (PR1) in potato (Solanum tuberosum) have been analyzed. The gene is rapidly activated in leaves from the potato cultivar Datura, containing the resistance gene R1, in both compatible and incompatible interactions with appropriate races of the late-blight fungus Phytophthora infestans. Activation is also observed in leaves treated with fungal elicitor. The gene occurs in multiple, very similar copies and encodes a polypeptide (Mr = 25,054; pI = 5.5) that is classified as a PR protein by several criteria. Small fragments with great sequence similarity to portions of the two exons were found closely linked to the expressed gene, which altogether represents a simple case of genome organization in potato. The coding sequence of the prp1 gene and the deduced amino acid sequence are strikingly similar to the corresponding sequences of a 26-kDa heat shock protein from soybean.

Rapid, systemic repression of the synthesis of ribulose 1,5-bisphosphate carboxylase small-subunit mRNA in fungus-infected or elicitor-treated potato leaves.

The levels of ribulose 1,5-bisphosphate carboxylase small-subunit (SSU) mRNA and protein decreased considerably in potato (Solanum tuberosum L.) leaves upon infection with the pathogenic fungus,Phytophthora infestans, or upon treatment with an elicitor preparation from the fungal culture fluid. This effect occurred systemically throughout the affected leaf, regardless of whether the interaction withP. infestans was compatible or incompatible. Using the comparatively drastic and synchronous response to fungal elicitor, we demonstrated that the repression of SSU synthesis was caused by rapid gene inactivation. The timing of repression was similar to that observed previously for the transcriptional activation of various pathogen defense reactions. This supports the hypothesis that induction of the extensive, multi-component defense response requires repression of other cellular functions to ensure metabolic balance.

Differential early activation of defense-related genes in elicitor-treated parsley cells.

A cDNA library from cultured parsley (Petroselinum crispum) cells was differentially screened using labeled run-off transcripts derived from nucleic of elicitor-treated and untreated cells. This resulted in the isolation of 18 independent cDNA families representing putative defense-related genes. All genes are rapidly and transiently activated after elicitor application, but the time courses of transcriptional activity exhibit considerable variations, indicating differences in the mechanisms of gene regulation.

Several "pathogenesis-related" proteins in potato are 1,3-beta-glucanases and chitinases.

Chitinase {poly[1,4-(N-acetyl-beta-D-glucosaminide)]glycanohydrolase, EC 3.2.1.14} and 1,3-beta-glucanase (1,3-beta-D-glucan 3-glucanohydrolase, EC 3.2.1.6) activities increased rapidly in potato (Solanum tuberosum) leaves inoculated with the pathogenic fungus Phytophthora infestans or treated with fungal elicitor. The enzyme activities were resolved into a total of two distinct 1,3-beta-glucanases and six proteins with chitinase activity. By several criteria, all of these proteins are classified as "pathogenesis-related" proteins whose biochemical functions have so far been unknown. Some of them constitute a major portion of the proteins accumulating in the intercellular space of infected potato leaves and are assumed to play an important role in pathogen defense.

Transient Induction of Phenylalanine Ammonia-Lyase and 4-Coumarate: CoA Ligase mRNAs in Potato Leaves Infected with Virulent or Avirulent Races of Phytophthora infestans.

Infection of potato leaves with the fungal pathogen Phytophthora infestans (Pi) resulted in the rapid stimulation of phenylpropanoid metabolism. Increases in the activities of several mRNAs, including those encoding phenylalanine ammonia-lyase (PAL) and 4-coumarate:CoA ligase (4CL), were detectable within a few hours postinoculation, as demonstrated by two-dimensional gel electrophoresis of proteins synthesized in vitro. This effect was closely mimicked by application of Pi culture filtrate through cut leaf stems. PAL and 4CL mRNA activities were also rapidly and transiently induced in potato cell suspension cultures by treatments with Pi culture filtrate or arachidonic acid. This induction was exploited to generate cDNA probes complementary to PAL and 4CL mRNAs. Blot hybridizations using these probes revealed almost immediate, transient and coordinate increases in the transcription rates and subsequent changes in the amounts of PAL and 4CL mRNAs in leaves treated with Pi culture filtrate. Similar changes in the mRNA amounts were found in infected leaves of potato cultivars carrying resistance genes R1 (cv Datura) or R4 (cv Isola), independent of whether a virulent or an avirulent Pi pathotype was used for inoculation. These results are discussed in relation to recent cytological observations with the same potato cultivars and Pi pathotypes.

Occurrence of phytoalexins and other putative defense-related substances in uninfected parsley plants.

Considerable amounts of the following substances were found in uninfected parsley (Petroselinum crispum) cotyledons: furanocoumarins, the putative phytoalexins of this and some related plant species, two enzymes of the furanocoumarin pathway (S-adenosyl-L-methionine: xanthotoxol and S-adenosyl-L-methionine: bergaptol O-methyltransferases), two hydrolytic enzymes (1,3-ß-glucanase, EC 3.2.1.39, and chitinase, EC 3.2.1.14), and 'pathogenesis-related' proteins. The furanocoumarins and the methyltransferase activities reached their highest levels at the onset of cotyledon senescence as the hydrolytic enzymes increased from low to relatively high activity values. The relative amounts of pathogenesis-related proteins 1 and 2, as well as the corresponding mRNAs, also increased markedly. Two enzymes of general phenylpropanoid metabolism, L-phenylalanine ammonia-lyase and 4-coumarate: CoA ligase, decreased in activity in a biphasic fashion during cotyledon development. At all developmental stages, the levels of these putative defense-related agents in total cotyledon extracts were too high to enable detection of, possibly, additional changes upon infection with zoospores of Phytophthora megasperma f. sp. glycinea, a fungal pathogen to which parsley shows a non-host, hypersensitive resistance response.

Responses of cultured parsley cells to elicitors from phytopathogenic fungi : timing and dose dependency of elicitor-induced reactions.

Cultured parsley cells (Petroselinum crispum) responded to treatment with heat-released soluble cell-wall fragments (elicitors) from several different phytopathogenic fungi by forming coumarin derivatives (phytoalexins). This response was preceded in all cases by large but transient increases in the activities of two enzymes of general phenylpropanoid metabolism, phenylalanine ammonia-lyase (PAL) and 4-coumarate:CoA ligase (4CL). The activities of two hydrolytic enzymes, chitinase and 1,3-beta-glucanase, also increased strongly in elicitor-treated cells, whereas the activities of three enzymes participating in primary metabolism were affected differently by the elicitor treatment. Glucose-6-phosphate dehydrogenase increased, phosphofructokinase remained almost constant, and pyrophosphate:fructose-6-phosphate phosphotransferase declined sharply in activity. Different amounts of cell-wall preparations from various phytopathogenic fungi were required for maximum elicitor activity. While three oomycetes (Phytophthora spp.) yielded the most active elicitors studied (maximum coumarin accumulation at concentrations of about 10 microgram per milliliter), cell-wall preparations from an ascomycete and three deuteromycetes gave comparable results only at 10 to 100 times higher concentrations. Optimal induction of PAL, 4CL, and chitinase with Phytophthora elicitor required only about 1 microgram per milliliter, whereas 1,3-beta-glucanase induction showed a dose dependence similar to that observed for coumarins. The elicitor concentration had pronounced effects not only on the extent, but also on the timing of all induced reactions.

Dependence of the level of phytoalexin and enzyme induction by fungal elicitor on the growth stage of Petroselinum crispum cell cultures.

The extent of induction of some metabolic activities in cultured parsley cells (Petroselinum crispum) by an elicitor preparation from Phytophthora megasperma f. sp. glycinea varied with the growth stage of the cell culture. On the basis of cell fresh weight, the induction of phytoalexin accumulation was high until cell mass reached a maximum, and then declined to a low level which was indistinguishable from a level caused by an endogenous mechanism operating at this late growth stage. The induction of phenylalanine ammonia-lyase and 4-coumarate:CoA ligase activities by the elicitor showed a high degree of coordination and a sharp maximum preceding the stage of maximal cell mass. 1,3-ß-Glucanase activity was induced to about the same level throughout all growth stages, with a large contribution by an endogenous mechanism at late stages.

Kinetic properties of pyrophosphate:fructose-6-phosphate phosphotransferase from germinating castor bean endosperm.

Pyrophosphate:fructose-6-phosphate phosphotransferase (PFP) was purified over 500-cold from endosperm of germinating castor bean (Ricinus commiunis L. var. Hale). The kinetic properties of the purified enzyme were studied. PFP was specific for pyrophosphate and had a requirement for a divalent metal ion. The pH optimum for activity was 7.3 to 7.7. The enzyme had similar activities in the forward and reverse directions and exhibited hyperbolic kinetics with all substrates. Kinetic constants were determined in the presence of fructose 2,6-bisphosphate, which stimulated activity about 20-fold and increased the affinity of the enzyme for fructose 6-phosphate, fructose 1,6-bisphosphate, and pyrophosphate up to 10-fold. Half-maximum activation of PFP by fructose 2,6-bisphosphate was obtained at 10 nanomolar. The affinity of PFP for this activator was reduced by decreasing the concentration of fructose 6-phosphate or increasing that of phosphate. Phosphate inhibited PFP when the reaction was measured in the reverse direction, i.e. fructose 6-phosphate production. In the presence of fructose 2,6-bisphosphate, phosphate was a mixed inhibitor with respect to both fructose 6-phosphate and pyrophosphate when the reaction was measured in the forward direction, i.e. fructose 1,6-bisphosphate production. The possible roles of fructose 2,6-bisphosphate, fructose 6-phosphate, and phosphate in the control of PFP are discussed.

Fructose 2,6-bisphosphate as a contaminant of commercially obtained fructose 6-phosphate: effect on PPi:fructose 6-phosphate phosphotransferase.

Fructose 6-phosphate from several commercial sources was shown to be contaminated with fructose 2,6-bisphosphate. This contaminant was identified by its activation of PPi:fructose 6-phosphate phosphotransferase, extreme acid lability and behaviour on ion-exchange chromatography. The apparent kinetic properties of PPi:fructose 6-phosphate phosphotransferase from castor bean endosperm were considerably altered when contaminated fructose 6-phosphate was used as a substrate. Varying levels of fructose 2,6-bisphosphate in the substrate may account for differences that have been observed in the properties of the above enzyme from several plant sources.

Transport of purine and pyrimidine bases and nucleosides from endosperm to cotyledons in germinating castor bean seedlings.

During germination and early growth of castor bean (Ricinus communis), all cellular constituents of the endosperm are eventually transferred to the growing embryo. The present results bear on the transport of breakdown products of nucleic acids. The total content of nucleic acids and nucleotides declines rapidly between day 4 and day 8 of seedling development. Concomitant with this decline, a secretion of adenosine, guanosine, and adenine from excised endosperms into the incubation medium takes place, accompanying a much more extensive release of sucrose and amino acids. Release of nucleotides could not be detected. The rates of release were linear for at least 5 hours for all compounds measured, indicating that they were liberated due to a coordinated metabolism. Uptake studies with cotyledons removed from the seedling showed that these have the ability to absorb all the substances released from the endosperm. Besides sucrose and amino acids, both nucleosides and free purine and pyrimidine bases were taken up by the cotyledons with high efficiency. AMP was also transported whereas ATP was not. Kinetic analyses were carried out to estimate the maximal uptake capacities of the cotyledons. Rates of uptake were linear for at least 1 to 2 hours and saturation kinetics were observed for all substances investigated. It is concluded that nucleosides can serve best as transport metabolites of nucleic acids, inasmuch as they are taken up by the cotyledons with the highest efficiency, the V(max)/K(m) ratios being considerably higher than those found for free purine and pyrimidine bases. For both adenosine and adenine transport, the V(max) was about 2 micromoles per hour per gram fresh weight, and the K(m) values were 0.12 and 0.37 millimolar, respectively. The rates of metabolite release from the endosperm and the capacity of the absorption system in the cotyledons are shown to account for the observed rates of disappearance of nucleic acids from the endosperm and efficient transport to the growing embryo.