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While uniparental transmission of mtDNA is widespread and dominating in eukaryotes leaving mutation as the major source of genotypic diversity, recently, biparental inheritance of mitochondrial genes has been demonstrated in reciprocal crosses of Pelargonium zonale and P. inquinans. The thereby arising heteroplasmy carries the potential for recombination between mtDNAs of different descent, i.e. between the parental mitochondrial genomes. We have analyzed these Pelargonium hybrids for mitochondrial intergenomic recombination events by examining differences in DNA blot hybridization patterns of the mitochondrial genes atp1 and cob. Further investigation of these genes and their flanking regions using nucleotide sequence polymorphisms and PCR revealed DNA segments in the progeny, which contained both P. zonale and P. inquinans sequences suggesting an intergenomic recombination in hybrids of Pelargonium. This turns Pelargonium into an interesting subject for studies of recombination and evolutionary dynamics of mitochondrial genomes.

A specific signaling role for H(2)O(2) in Chlamydomonas reinhardtii was demonstrated by the definition of a promoter that specifically responded to this ROS. Expression of a nuclear-encoded reporter gene driven by this promoter was shown to depend not only on the level of exogenously added H(2)O(2) but also on light. In the dark, the induction of the reporter gene by H(2)O(2) was much lower than in the light. This lower induction was correlated with an accelerated disappearance of H(2)O(2) from the culture medium in the dark. Due to a light-induced reduction in catalase activity, H(2)O(2) levels in the light remained higher. Photosynthetic electron transport mediated the light-controlled down-regulation of the catalase activity since it was prevented by 3-(3'4'-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of photosystem II. In the presence of light and DCMU, expression of the reporter gene was low while the addition of aminotriazole, a catalase inhibitor, led to a higher induction of the reporter gene by H(2)O(2) in the dark. The role of photosynthetic electron transport and thioredoxin in this regulation was investigated by using mutants deficient in photosynthetic electron flow and by studying the correlation between NADP-malate dehydrogenase and catalase activities. It is proposed that, contrary to expectations, a controlled down-regulation of catalase activity occurs upon a shift of cells from dark to light. This down-regulation apparently is necessary to maintain a certain level of H(2)O(2) required to activate H(2)O(2)-dependent signaling pathways.

Gravitropism is explained by the Cholodny-Went hypothesis: the basipetal flow of auxin is diverted laterally. The resulting lateral auxin gradient triggers asymmetric growth. However, the Cholodny-Went hypothesis has been questioned repeatedly because the internal auxin gradient is too small to account for the observed growth asymmetry. Therefore, an additional gradient in indolyl-3-acetic acid (IAA) sensitivity has been suggested (Brauner and Hager in Planta 51:115-147, 1958). We challenged the Cholodny-Went hypothesis for gravitropism of rice coleoptiles (Oryza sativa L.) and found it to be essentially true. However, we observed, additionally, that the two halves of gravitropically stimulated coleoptiles responded differentially to the same amount of exogenous auxin: the auxin response is reduced in the upper flank but normal in the lower flank. This indicates that the auxin-gradient is amplified by a gradient of auxin responsiveness. Hormone contents were measured across the coleoptile by a GC-MS/MS technique and a gradient of jasmonate was detected opposing the auxin gradient. Furthermore, the total content of jasmonate increased during the gravitropic response. Jasmonate gradient and increase persist even when the lateral IAA gradient is inhibited by 1-N-naphtylphtalamic acid. Flooding with jasmonate delays the onset of gravitropic bending. Moreover, a jasmonate-deficient rice mutant bends more slowly and later than the wild type. We discuss a role of jasmonate as modulator of auxin responsiveness in gravitropism.

Hydroxyl radicals (*OH), produced in the cell wall, are capable of cleaving wall polymers and can thus mediate cell wall loosening and extension growth. It has recently been proposed that the biochemical mechanism responsible for *OH generation in the cell walls of growing plant organs represents an enzymatic reaction catalyzed by apoplastic peroxidase (POD). This hypothesis was investigated by supplying cell walls of maize ( Zea mays L.) coleoptiles and sunflower ( Helianthus annuus L.) hypocotyls with external NADH, an artificial substrate known to cause *OH generation by POD in vitro. The effects of NADH on wall loosening, growth, and *OH production in vivo were determined. NADH mediates cell wall extension in vitro and in vivo in an H2O2-dependent reaction that shows the characteristic features of POD. NADH-mediated production of *OH in vivo was demonstrated in maize coleoptiles using electron paramagnetic resonance spectroscopy in combination with a specific spin-trapping reaction. Kinetic properties and inhibitor/activator sensitivities of the *OH-producing reaction in the cell walls of coleoptiles resembled the properties of horseradish POD. Apoplastic consumption of external NADH by living coleoptiles can be traced back to the superimposed action of two enzymatic reactions, a KCN-sensitive reaction mediated by POD operating in the *OH-forming mode, and a KCN-insensitive reaction with the kinetic properties of a superoxide-producing plasma-membrane NADH oxidase the activity of which can be promoted by auxin. Under natural conditions, i.e. in the absence of external NADH, this enzyme may provide superoxide (O2*-) (and H2O2 utilized by POD for) *OH production in the cell wall.

Mechanical and chemical properties as well as microfibril angles of wood tissues from different ontogenetic stages are determined for the neotropical lianas Bauhinia guianensis and Condylocarpon guianense. The mechanical properties include the elastic moduli under bending and under dynamic torsion. The chemical analyses cover (i) the content of cellulose, lignin and hemicelluloses fractions, (ii) the monomeric composition of the uncondensed lignin, and (iii) the composition of the hemicelluloses with respect to neutral monosaccharides. By comparing the wood properties of these lianas with the corresponding properties of wood from self-supporting deciduous trees, common characteristics and differences are revealed. Additionally, the changes in the lignin and polysaccharides fractions as well as the variations in microfibril orientation that occur during ontogeny of the two liana species are discussed with regard to their implications for the mechanical properties of wood.

'Coat-imposed' seed dormancy of many non-endospermic and endospermic species is released during after-ripening. After-ripening-mediated promotion of tobacco ( Nicotiana tabacum L.) seed germination is mainly due to a promotion of testa rupture and a similar promotion of subsequent endosperm rupture. Treatment of after-ripened or freshly harvested mature seeds with abscisic acid (ABA) delays endosperm rupture and inhibits the induction of class I beta-1,3-glucanase (betaGlu I) in the micropylar endosperm, but does not affect the kinetics of testa rupture. After-ripening-mediated release of photodormancy is correlated with a decreased gibberellin (GA) requirement for testa rupture during dark-imbibition. Reciprocal crosses between wild-type tobacco and sense-betaGlu I transformant lines showed that betaGlu I over-expression in the seed covering layers can replace the promoting effect of after-ripening on testa rupture in light, but only if the mother plant is a sense-betaGlu I line. This maternal effect supports the model of two sites for betaGlu I action: (i) betaGlu I contribution to the after-ripening-mediated release of dormancy in the dry seed state, which is manifested in the promotion and ABA-insensitivity of testa rupture during imbibition. (ii) ABA-sensitive expression of betaGlu I in the micropylar endosperm, which contributes to endosperm rupture. The importance of GA-signaling and testa characteristics appear to be a common feature during the after-ripening-mediated release of coat-imposed dormancy in endospermic and non-endospermic seeds.

We investigated the relation between cell wall structure and the resulting mechanical characteristics of different plant tissues. Special attention was paid to the mechanical behaviour beyond the linear-elastic range, the underlying micromechanical processes and the fracture characteristics. The previously proposed model of reorientation and slippage of the cellulose microfibrils in the cell wall [H.-CH. Spatz et al. (1999) J Exp Biol 202:3269-3272) was supported and is here refined, using measurements of the changes in microfibrillar angle during straining. Our model explains the widespread phenomenon of stress-strain curves with two linear portions of different slope and sheds light on the micromechanical processes involved in viscoelasticity and plastic yield. We also analysed the velocity dependence of viscoelasticity under the perspective of the Kelvin model, resolving the measured viscoelasticity into functions of a velocity-dependent and a velocity-independent friction. The influence of lignin on the above-mentioned mechanical properties was examined by chemical lignin extraction from tissues of Aristolochia macrophylla Lam. and by the use of transgenic plants of Arabidopsis thaliana (L.) Heynh. with reduced lignin content. Additionally, the influence of extraction of hemicelluloses on the mechanical properties was investigated as well as a cell wall mutant of Arabidopsis with an altered configuration of the cellulose microfibrils.

Reactive oxygen intermediates, i.e. the superoxide radical (O*-)(2), hydrogen peroxide (H2O2) and the hydroxyl radical (*OH), are generally regarded as harmful products of oxygenic metabolism causing cell damage in plants, animals and microorganisms. However, oxygen radical chemistry may also play a useful role in polymer breakdown leading to wall loosening during extension growth of plant cells controlled by the phytohormone auxin. Backbone cleavage of cell wall polysaccharides can be accomplished in vitro by (*OH) produced from H2O2 in a Fenton reaction or in a reaction catalyzed by peroxidase supplied with O2 and NADH. Here, we show that coleoptile growth of maize seedlings is accompanied by the release of reactive oxygen intermediates in the cell wall. Auxin promotes release of (O*-)(2) and subsequent generation of (*OH)when inducing elongation growth. Experimental generation of (*OH) in the wall causes an increase in wall extensibility in vitro and replaces auxin in inducing growth. Auxin-induced growth can be inhibited by scavengers of (O*-)(2), H2O2 or (*OH), or inhibitors interfering with the formation of these molecules in the cell wall. These results provide the experimental background for a novel hypothesis on the mechanism of plant cell growth in which (*OH), produced from (O*-)(2) and H2O2 by cell wall peroxidase, acts as a wall-loosening agent.

The frequency of free oscillations of plant stems with apical loads, as found in some cereals, is different depending on whether the stems are oriented vertically or horizontally. Neglecting the stem's own weight the differential equations describing the oscillation can be solved for both cases, although in the vertical orientation only for a limited set of conditions including constant bending stiffness along the stem. Comparison with experimental data shows that the difference between the oscillation frequencies in vertical and horizontal orientations can be attributed to the fact that in the vertical orientation the top load due to gravity induces a bending moment varying with the oscillation, while in the horizontal case this bending moment is nearly constant.

Seed germination of Nicotiana tabacum L. cv. Havana 425 is determined by the balance of forces between the growth potential of the embryo and the mechanical restraint of the micropylar endosperm. In contrast to the gibberellin GA4, the brassinosteroid (BR) brassinolide (BL) did not release photodormancy of dark-imbibed photodormant seeds. Brassinolide promoted seedling elongation and germination of non-photodormant seeds, but did not appreciably affect the induction of class I beta-1,3-glucanase (betaGLU I) in the micropylar endosperm. Brassinolide, but not GA4, accelerated endosperm rupture of tobacco seeds imbibed in the light. Brassinolide and GA4 promoted endosperm rupture of dark-imbibed non-photodormant seeds, but only GA4 enhanced betaGLU I induction. Promotion of endosperm rupture by BL was dose-dependent and 0.01 microM BL was most effective. Brassinolide and GA4 promoted abscisic acid (ABA)-inhibited dark-germination of non-photodormant seeds, but only GA4 replaced light in inducing betaGLU I. These results indicate that BRs and GAs promote tobacco seed germination by distinct signal transduction pathways and distinct mechanisms. Gibberellins and light seem to act in a common pathway to release photodormancy, whereas BRs do not release photodormancy. Induction of betaGLU I in the micropylar endosperm and promotion of release of 'coat-enhanced' dormancy seem to be associated with the GA-dependent pathway, but not with BR signalling. It is proposed that BRs promote seed germination by directly enhancing the growth potential of the emerging embryo in a GA- and betaGLU I-independent manner.

Genes that are expressed upon a shift to nitrogen-free medium, an event that initiates gametogenesis, were identified in Chlamydomonas reinhardtii by using the differential display technique. Ten different cDNAs were isolated and shown to have increased levels of their transcripts upon removal of the nitrogen source. The initial kinetics of RNA accumulation allowed an ordering of the genes with respect to the timing of their expression, with individual genes being expressed very early, early, intermediately, or late after induction. For very early genes, significantly increased transcript levels were detected within 30 min. This fast response suggests that gene expression is rapidly activated after removal of the nitrogen source. The accumulation of transcripts from the very early, early, and intermediate genes preceded the appearance of mating competence. Though transcript levels of several very early genes fluctuated during subsequent incubation in nitrogen-free medium, most of them exhibited maxima when the highest level of mating competence was attained. One of these very early genes was shown to encode a urate oxidase type-II enzyme.

Endogenous extractable factors associated with auxin action in plant tissues were investigated, especially their effects on elongation of 1-mm coleoptile segments of maize (Zea mays L.), in the presence of saturating 10 microM indole-3-acetic acid (IAA). The relative growth response, to auxin alone, was much smaller in segments shorter than 2-3 mm compared to 10-mm segments. Fusicoccin-induced elongation, however, was less affected by shortening the segments. A reduced auxin response may result from the depletion through cut surfaces of a substance required for IAA-mediated growth. Sucrose, phenolics like flavonoids, and vitamins were ruled out as the causal factors. A partially purified methanol extract of maize coleoptiles supported longterm, auxin-controlled elongation. The active material was also found among substances bleeding from scrubbed maize coleoptiles. The active factor from maize was further purified by HPLC and characterised by the UV spectrum and its pH shift. This factor was identified as 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) by mass spectroscopy. Activity tests confirmed that pure DIMBOA from other sources sustained auxin-induced elongation of short maize coleoptile segments. However, DIMBOA only partially restored the activity lost from short segments. This indicates that an additional factor, other than DIMBOA, is required. Extracts from Avena or Cucurbita did not contain the factor DIMBOA; it was active on maize elongation, but not on Avena coleoptiles or Cucurbita hypocotyls. This narrow specificity and the lack of DIMBOA action in short-term tests with maize indicate that DIMBOA is not the general auxin cofactor but may specifically "spare" the co-auxin in maize.

The causal relationship between gravitropic growth responses and microtubule reorientation has been studied. Growth and microtubule reorientation have been uncoupled during the gravitropic response of maize (Zea mays L.) coleoptiles. Microtubule orientation and growth were measured under three different conditions: (i) a gravitropic stimulation where the growth response was allowed to be expressed (intact seedlings were displaced from the vertical position by 90 degrees), (ii) a gravitropic stimulation where the growth response was suppressed (coleoptiles were attached to microscope slides and kept in a horizontal position), (iii) suppression of growth in the absence of gravitropic stimulation (coleoptiles were attached to microscope slides and kept in a vertical position). It was found that (i) gravitropic stimulation can induce a microtubular reorientation from transverse to longitudinal in the upper (slower growing) flank of the coleoptile, and an inhibition of growth; (ii) the reorientation of microtubules precedes the inhibition of growth; (iii) the gravitropic response of microtubules is weaker, not elevated, when the inhibition of growth is artificially enhanced by attaching the coleoptiles to a slide; and (iv) artificial inhibition of growth in the absence of gravitropic stimulation cannot induce a microtubular response. Thus, the extent of microtubule reorientation is not correlated with the extent of growth inhibition. Moreover, these findings demonstrate that microtubules do not reorient passively after growth changes, but actively in response to gravitropic stimulation.

Using the tetrazolium salt XTT (Na,3'-[(phe-nylamino)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzenesulfonic acid hydrate) as a sensitive and physiologically compatible probe for the determination of superoxide (O2*-) production in vivo, we have shown that maize (Zea mays L.) coleoptiles possess the capacity of generating O2*- in the apoplastic space. Our results are in agreement with the notion that this activity is localized at the plasma membrane and can be attributed to an O2*--synthesizing enzyme with catalytic and kinetic properties similar to that of the NADPH oxidase of mammalian phagocytes, with the important exception that it utilizes NADH instead of NADPH as electron donor. When applied to the apoplastic space, NADH strongly increased the O2*--producing activity of coleoptiles. The maize NADH-dependent O2*--synthase activity could clearly be differentiated from peroxidase-mediated O2*--synthesizing activity by its insensitivity to cyanide and azide, as well as by its much higher affinity to O2. Formation of O2*-, and concomitantly appearing H2O2, was preferentially localized in the outer epidermis of the coleoptile. The physiological significance of O2*- and H2O2 production in relation to the growth-controlling function of the epidermal cell wall is discussed.

The mechanical properties of young stems of Aristolochia macrophylla Lam. and Aristolochia brasiliensis Mart. et Zucc. were studied during elongation growth and primary differentiation. Data for the modulus of elasticity, for the viscoelastic behaviour caused by longitudinal tension and for the shear modulus resulting from torsion around a longitudinal axis were related to the underlying structural changes by quantitative analysis of stem anatomy, tissue distribution, ultrastructure, and cell wall biochemistry. The orientation of cellulose microfibrils was determined by light microscopy and small-angle X-ray diffraction, and the lignin content was determined by thioglycolic acid derivatization and spectroscopic quantification. It was demonstrated that the increase in stability during early development is due to the complementary effects of increase in cell wall material, lignification, and cellulose microfibril alignment. A detailed micromechanical model, considering internal prestresses, is proposed to explain the characteristic biphasic stress-strain behaviour as well as the strain-hardening observed.

In protonemal tip cells of the moss Ceratodon purpureus (Hedw.) Brid., phototropism and chlorophyll accumulation are regulated by the photoreceptor phytochrome. The mutant ptr116 lacks both responses as a result of a defect in the biosynthesis of phytochromobilin, the chromophore of phytochrome, at the point of biliverdin formation. The rescue of the phototropic response and of chlorophyll synthesis were tested by injecting different substances into tip cells of ptr116. Microinjection was first optimised with the use of fluorescent dyes and an expression plasmid containing a green fluorescent protein (GFP) gene. Injected phycocyanobilin, which substitutes for phytochromobilin, rescued both the phototropic response and light-induced chlorophyll accumulation in ptr116. The same results were obtained when expression plasmids with heme oxygenase genes of rat (HO-1) and Arabidopsis thaliana (L.) Heynh. (HY1) were injected. Heme oxygenase catalyses the conversion of heme into biliverdin. Whereas HY1 has a plastid target sequence and is presumably transferred to plastids, HO-1 is proposed to be cytosolic. The data show that ptr116 lacks heme oxygenase enzyme activity and indicate that heme oxygenases of various origin are active in Ceratodon bilin synthesis. In addition, it can be inferred from the data that the intracellular localisation of the expressed heme oxygenase is not important since the plastid enzyme can be replaced by a cytosolic one.

In order to test the interaction of different phytochromes and blue-light receptors, etiolated seedlings of wild-type Arabidopsis thaliana (L.) Heynh., a phytochrome (phy) B-overexpressor line (ABO), and the photoreceptor mutants phyA-201, phyB-5, hy4-2.23n, fha-1, phyA-201/phyB-5, and phyA-201/hy4-2.23n were exposed to red and far-red light pulses after various preirradiations. The responsiveness to the inductive red pulses is primarily mediated by phyB which is rather stable in its far-red-absorbing form as demonstrated by a very slow loss of reversibility. Without preirradiation the red pulses had an impact on hypocotyl elongation only in PHYA mutants but not in the wild type. This indicates a suppression of phyB function by the presence of phyA. Preirradiation with either far-red or blue light resulted in an inhibition of hypocotyl elongation by red pulses in the wild type. Responsiveness amplification by far-red light is mediated by phyA and disappears slowly in the dark. The extent of responsiveness amplification by blue light was identical in the wild type and in the absence of phyA, or the cryptochromes cryl (hy4-2.23n) or cry2 (fha-1). Therefore, we conclude that stimulation of phyB by blue light preirradiation is either mediated by an additional still-unidentified blue-light-absorbing pigment or that phyA, cry1 and cry2 substitute for each other completely. Both blue and red preirradiation established responsiveness to red pulses in phyA-201/phyB-5 double mutants. These results demonstrate that inhibition of hypocotyl elongation by red pulses is not only mediated by phyB but also by a phytochrome(s) other than phyA and phyB.

Pathogenesis-related expression of the two antifungal hydrolases beta-1,3-glucanase (EC 3.2.1.39) and chitinase (EC 3.2.1.14) was studied in wheat (Triticum aestivum L.) as part of the defence response to stem rust (Puccinia graminis f.sp. tritici, Pgt), mediated by the semi-dominantly acting resistance genes Sr5 and Sr24. Complete resistance (infection type 0), mediated by the Sr5 gene in cultivar Pre-Sr5, closely correlates with the hypersensitive response of penetrated cells at early stage of the interaction, when the first haustorium is formed. In contrast, cultivar Pre-Sr24 shows intermediate resistance (infection type 2-3) which is not directly linked to cell death. In both cases, the plant response included a rapid increase in beta-1,3-glucanase activity between 24 and 48 h after inoculation. One main extracellular 30-kDa isform of beta-1,3-glucanase was present in both lines, as shown by polyacrylamide-gel electrophoresis. Two additional minor isoforms (32 and 23 kDa) were detected only in Pre-Sr24, and only at later time points. Increased enzme activity and the appearance of new isoforms in the resistance lines was preceded by accumulation of mRNAs encoding beta-1,3-glucanase and chitinases. However, there were no changes in chitinase activity or isoforms. A high constitutive level of chitinase activity was observed in all wheat genotypes. Serological studies indicated the presence of a class II chitinase of 26 kDa. Accumulation of beta-1,3-glucanase and chitinase transcripts was detected before the pathogen penetrated the leaves through stomata and approximately 16 h before the typical hypersensitive response was observed, indicating that signal(s) for defense gene activation were recognised by the host plant long before a tight contact between the pathogen and a host cell is established. A glycoprotein (Pgt elicitor) derived from hyphal walls, strongly induced beta-1,3-glucanase. We discuss the possible role of the elicitor in the early signalling mediating Sr5- and Sr24-specified resistance in wheat.

Hypocotyls of dark-grown seedlings of Arabidosis thaliana exhibit a strong negative gravitropism, which is reduced by red and also by long-wavelength, far-red light treatments. Light treatments using phytochrome A (phyA)- and phytochrome B (phyB)-deficient mutants showed that this response is controlled by phyB in a red/far-red reversible way, and by phyA in a non-reversible, very-low-fluence response. Crosses of the previously analyzed phyB-1 allele (in the ecotype Landsberg erecta background) to the ecotype Nossen wild-type (WT) background resulted in a WT-like negative gravitropism in darkness, indicating that the previously described gravitropic randomization observed with phyB-1 in the dark is likely due to a second mutation independent of that in the PHYB gene.

Overexpression of phytochrome A results in an increased inhibition of hypocotyl elongation under red and far-red light. We used this approach to assay for the function of N-terminal mutations of rice (Oryza sativa L.) phytochrome A. Transgenic tobacco seedlings that express the wild-type rice phytochrome A (RW), a rice phytochrome A lacking the first 80 amino acids (NTD) or a rice phytochrome A with a conversion of the first 10 serines into alanine residues (S/A) were compared with untransformed wild-type tobacco (Nicotiana tabacum L. cv. Xanthi) seedlings. Experiments under different fluence rates showed that RW and, even more strongly, S/A increased the response under both red and far-red light, whereas NTD decreased the response under far-red light but hardly altered the response under red light. These results indicate that NTD not only lacks residues essential for an increased response under red light but also distorts the wild-type response under far-red light. Wild-type rice phytochrome A and, even more so, S/A mediate an enhanced phytochrome A as well as phytochrome B function, whereas NTD interferes with the function of endogenous tobacco phytochrome A as well as that of rice phytochrome A when co-expressed in a single host. Experiments with seedlings of different ages and various times of irradiation under far-red light demonstrated that the effect of NTD is dependent on the stage of development. Our results suggest that the lack of the first 80 amino acids still allows a rice phytochrome A to interact with the phytochrome transduction pathway, albeit non-productively in tobacco seedlings.

We examined the chalcone synthase (chs) promoter from parsley [Petroselinum crispum Miller (A.W. Hill)] for the existence of separate promoter elements responsible for transcriptional activation of the chs gene by UV-B and by blue light. A combination of in-vivo foot-printing in parsley cells and light-induced transient expression assays with different chs promoter constructs in parsley protoplasts was used. Dark controls and blue-light-irradiated cells gave identical in-vivo footprints on the chs promoter. Pre-irradiation with blue light prior to a UV-B-light pulse is known to cause a shift in the timing of UV-B-light-induced increase in chs transcription rates. This shift was also manifested on the DNA template, since UV-B-light-induced in-vivo footprints in cells pretreated with blue light were detected earlier than in cells which had been irradiated with a UV-B-light pulse only. Although there was a clear shift in the timing of footprint appearance, the patterns of footprinting did not change. Light-induced transient-expression assays revealed that the shortest tested chs promoter which retained any light responsiveness, was sufficient for mediating both induction by UV light and the blue-light-mediated kinetic shift. These findings argue against a spatial separation of UV-B- and blue-light-responsive elements on the chs promoter. We interpret these data by postulating that the signal transduction pathways originating from the excitation of UV-B- and blue-light receptors merge at the chs promoter, or somewhere between light perception and protein-DNA interaction.

The 3-kb external spacer from a maize (Zea mays L. cv. A619) nuclear rRNA gene unit which contains nine highly homologous 200-bp repeat elements was found to include a region with DNA-curvature properties. The centre of curvature was localized within repeats 5 and 6 using a circular permutation assay. A 60-bp-long subfragment of this region was found to interact with nuclear proteins, including high-mobility-group (HMG) proteins, and with the maize HMGa protein synthesized in Escherichia coli from a recombinant plasmid. The potential influence of the binding of the HMG proteins on the conformation of this subfragment was studied with a permutation assay based on a bending vector.

Two properties of phytotropins, their ability to bind to 1-N-naphthylphthalamic acid (NPA) receptors located on microsomal vesicles isolated from Cucurbita pepo L. hypocotyls, and to stimulate auxin (indol-3-yl acetic acid, IAA) accumulation into such vesicles by blocking its efflux from them, were assessed in double labelling experiments using [2,3,4,5-(3)H]1-N-naphthylphthalamic acid and 3-indolyl-[2-(14)C]acetic acid. Two sites of differing affinities and activities on IAA accumulation were found. 1-N-Naphthylphthalamic acid was found to have high affinity (KD at 10(-8)mol·l(-1)) for one site and low affinity (KD at 10(-6) mol·l(-1)) for the other, whereas 2-(1-pyrenoyl)benzoic acid displaced NPA with high efficiency (KD below 10(-8) mol·l(-1)) from both sites. Other phytotropins had intermediate affinities for either site. Occupation of the site with low affinity for NPA stimulated auxin accumulation, while occupation of the high-affinity site with a phytotropin did not interfere with auxin accumulation into vesicles.

Auxin causes elongation growth of plant cells by increasing the plastic extensibility of the cell wall. Putative cellular events involved in this hormone action were studied using maize (Zea mays L.) coleoptiles with the following results: (i) Auxin enhances membrane flow from the endoplasmic reticulum to the plasma membrane (PM). This effect was demonstrated by pulse-labeling of the endoplasmic reticulum with myo-[(3)H]inositol in coleoptile segments and by measuring the distribution of the label within isolated and separated microsomal membrane fractions, (ii) Auxin rapidly increases the amount of antibody-detectable H(+)-ATPase in the PM. This augmentation is already significant 10 min after the addition of indole-3-acetic acid (IAA) and reaches a new higher steady-state level after about 30 min. (iii) Cycloheximide, a potent inhibitor of both protein synthesis and extension growth, quickly diminishes the auxin-enhanced level of the PM H(+)-ATPase, indicating an apparent half-life of the enzyme of around 12 min. (iv) Cordycepin, which blocks the synthesis of mRNAs, reduces the auxin-elevated level of the H(+)-ATPase similar to cycloheximide. (v) Changes in the growth rate of coleoptile segments in response to IAA, cycloheximide, and cordycepin exactly reflect the changes of the H(+)-ATPase level in the PM. (vi) The elongation growth induced by fusicoccin, or ester compounds, or by an elevated CO2 concentration in the incubation medium, is not related to an increased number of H(+)-ATPase molecules within the PM. (vii) The necessity of H(+) for cell-wall-loosening processes is again demonstrated by growth experiments with abraded coleoptile segments. The adjustment of the cell wall to a pH of ≥6.5 completely abolishes the auxin-induced elongation growth; no inhibition occurs with non-abraded segments. Buffer solutions of pH ≤6.0 induce "acid growth" of abraded segments for several hours. It is suggested that auxin activates a cluster of genes responsible (i) for the induction and acceleration of exocytotic processes (e.g. by the synthesis of either proteins, necessary for the fusion of membranes, or of other effectors); (ii) for the synthesis of PM H(+)-ATPases, increasing the capacity for H(+)-extrusion into the apoplast as a precondition for wall enlargement ("acid growth"); (iii) for a supposed synthesis and exocytosis of certain proteins, enzymes and wall precursors necessary for wall metabolism and the "repair" of the proton-loosened and turgor-stretched cell wall. Both, fusicoccin and auxin affect cell-wall plasticity according to the "acid-growth" theory. However, the mechanisms leading to this event are completely different; the auxinenhanced H(+)-extrusion is a gene-controlled process.

Chromosomal nonhistone high-mobility-group (HMG) proteins were purified from nuclei of maize (Zea mays L. cv. A619) endosperm and leaf tissue. Tissuespecific differences were observed in their polypeptide patterns, in in-vitro phosphorylation experiments with a casein-kinase type II, and by Western blot analysis with antisera against different HMG proteins. Gelfiltration chromatography demonstrated that maize HMG proteins occur as monomers. By measuring the capacity of the HMG proteins to bind to the 5' flanking region of a zein gene, the sensitivity of the proteins to different temperatures, salt concentrations and pH values was determined.

The hypothesis was tested that reversible riboflavin (RF)-binding sites are part of the photoreceptor in Euglena gracilis. Published evidence shows that the phototactic stimulus - with a flavin-type action spectrum - is perceived at the paraflagellar body (PFB). Flagella with PFBs were isolated from Euglena gracilis by a combined cold and Ca(2+) shock. Saturable binding of [(14)C]RF was demonstrated with such preparations, in the oxidized state as well as under reducing conditions in the presence of dithionite. Affinities for RF were high: K D (oxidized)=0.08 μM, and K D (reduced)=0.7 μM. Flavin mononucleotide and flavin adenine dinucleotide showed lower binding affinities. The in vitro RF binding per unit of protein was enriched approximately tenfold in the flagellar preparations when compared with homogenates of whole cells. The number of (reduced) binding sites per entire flagellum was determined to be in the order of 10(6). This number is in line with published estimates of chromophores bound in or at the PFB.

Two different chalcone synthase (CHS) transcripts show similar expression characteristics under different light regimes in cotyledons of mustard (Sinapis alba L.). Etiolated seedlings show an increase in dark-expression 36-42 h after sowing. Under continuous red or far-red irradiation both CHS transcripts start to accumulate to levels above those of the dark control at 24-27 h after sowing. This time point can therefore be considered as the starting (or competence) point for phytochrome control of CHS. Continuous far-red irradiation stimulates transcript accumulation more than red light, indicating the involvement of a high-irradiance response (HIR). Irradiation of etiolated seedlings with 5 min long-wavelength far-red light (RG9) at 6-21 h after sowing decreases CHS-mRNA levels below those of the dark control. It is concluded that CHS dark-expression in etiolated seedlings is controlled by a pool of stabletype phytochrome which is derived from seed tissue. By contrast, an RG9-light pulse given to etiolated seedlings 30 h after sowing causes accumulation of CHS-mRNA above the dark-control level. This response and the HIR are attributed to the action of labile phytochrome for which the seedling becomes competent at the starting point 24-27 h after sowing. The different starting points for CHS-mRNA expression in darkness and in light (36 h and 24 h, respectively, after sowing) also indicate that the tested CHS genes in mustard are under the photocontrol of two distinct phytochrome pools. Northern analysis shows that both CHS-mRNAs are expressed in primary leaves, epicotyls and young flower buds. In-situ hybridization with gene-specific CHS probes reveals similar expression patterns for both transcripts in cotyledons of seedlings grown under 42 h continuous far-red light.

Sequestered particles of phytochrome (SAPs) were partially purified from red-light-irradiated oat coleoptiles. Phytochrome pelletability was enhanced by using buffers containing 10 mM Mg(2+) or high concentrations (0.6-0.8 M) of orthophosphate (Pi). Combining the pelletability of phytochrome in the presence of Mg(2+) with that in the presence of 0.6 Pi resulted in a strong enrichment (about 100-fold) of pelletable phytochrome. Antisera were raised against Mg(2+)-Pi-pellets from darkgrown seedlings. Using these antisera, no evidence was found by Western blotting and immunocytochemistry that SAPs contain major proteins other than phytochrome. The major contamination of these enriched SAP preparations consisted of protein crystals which are probably catalase. The preparations contained methyltransferase and protein-kinase activities which were not associated with SAPs. Phytochrome purified from SAPs served as a substrate for protein-kinase activity but not for the methyltransferase activity. Phytochrome itself did not show any kinase activity.

We have recently reported the isolation and characterization of a glycoprotein (Mr 67 000) from germ-tube walls of Puccinia graminis f. sp. tritici which elicits the cellular hypersensitive lignification response in wheat (G. Kogel et al., 1988, Physiol. Mol. Plant Pathol. 33, 173-185). The present study uses this glycoprotein, referred to as Pgt elicitor, to identify putative elicitor targets in wheat cell membranes. In enzyme-linked immunosorbent assays using anti-Pgt elicitor antibodies, specific binding sites for Pgt elicitor were detected in highly purified plasma-membrane vesicles of wheat (Triticum aestivum L.) primary leaf cells. Binding proved to be independent of the presence or absence in wheat of the Sr5 gene for rust resistance, and also occurred on barley (Hordeum vulgare L.) plasma membrane. The binding sites have an Mr of 30 000 and 33 000, respectively, and binding activity was not lost in the presence of sodium dodecyl sulfate. [(14)C]imido-Pgt elicitor was used to determine the apparent K d value for specific binding, found to be 2.0 μM, and the maximum content of binding sites, found to be 250 pmol per mg of plasma-membrane protein. The relevance of the elicitor binding for the outcome of the interaction of P. graminis and wheat is discussed.

For a comprehensive study of phototropism in sporangiophores of the fungus Phycomyces, quantitative treatment of spatial aspects is necessary. The first step in quantifying spatial factors of phototropic signal processing is the elucidation of the non-uniform light profile, predominantly caused by a lens effect in the cylindrical body of the sporangiophore. Herein we compare recently presented theoretical and experimental studies of light profiles. Errors and ambiguities arising from instrumental limitations and arbitrary assumptions are revealed. On the other hand, by combining theoretical and experimental results we have been able to select out the reliable information, which can now be applied in phototropic studies.

The intracellular localization of prenyltransferases involved in the biosynthesis of the phytoalexins glyceollin in soybean (Glycine max L.) and phaseollin in French bean (Phaseolus vulgaris L.) has been investigated. By sucrose- and Percoll-gradient centrifugation of microsomes of an elicitor-challenged soybean cell culture, the membranes containing prenyltransferase were separated from the endoplasmic reticulum and shown to be lighter in density. In a continuous Percoll gradient the peak of prenyltransferase activity coincided with the peak of galactolipid synthesis, as determined by incorporation of uridine 5'-diphospho-[(14)C]galactose (UDP-[(14)C]galactose). Intact chloroplasts isolated from cupricchloride-treated bean leaves contained both prenyltransferase and UDP-galactose transferase activity. Both activities increased during chloroplast isolation. Fractionation of swollen chloroplasts on a discontinuous sucrose gradient showed prenyltransferase and UDP-galactose transferase activity in the envelope membrane subfraction. It is concluded that in both plants prenyltransferase is located in the envelope membrane of plastids.

A gentle procedure allowed the isolation of intact and highly active chloroplasts from the unicellular green algaAcetabularia mediterranea. These chloroplasts incorporated carbon from NaH(14)CO3 into fatty acids and prenyl lipids at a rate of about 20-50 nmol carbon· (mg chlorophyll)(-1)·h(-1). Most of the fatty acids formed in vitro were esterified in galactolipids. The main prenyl lipids synthesized were the chlorophyll side chain, intermediates of the carotenogenic path, α-and β-carotene, as well as lutein. Large amounts of [1-(14)C]acetate were incorporated, but exclusively into fatty acids.Isopentenyl diphosphate was a good substrate for prenyl-lipid formation in hypotonically treated chloroplasts. The envelope of intact chloroplasts, however, was impermeable to this compound. Intermediates of the mevalonate pathway were not accepted as precursors under conditions whereisopentenyl diphosphate was well incorporated. The results show that the lipid biosynthetic pathways in the plastids ofAcetabularia, a member of the ancient family of Dasycladaceae, are very similar to those in higher-plant plastids.

Resveratrol synthase (RS; EC 2.3.1.-) catalyzes the formation of the phytoalexin resveratrol from 4-coumaroyl-CoA and malonyl-CoA. We present the characterization of new genomic RS sequences (RS3, RS4), and describe studies with gene-specific oligonucleotides on the expression of four different RS sequences (RScDNA, RS1, RS2, RS3) during growth of a cell culture fromArachis hypogaea L. and after application of various inducers (elicitor fromPhytophtora megasperma, yeast extract, and dilution of the cultures). Transcripts from RScDNA were induced by all of the factors tested, and they represented the majority of all identified RS RNAs. Expression from RS1 and RS3 was much lower than from RScDNA, and transcripts from RS2 were never detected. Both RS1 and RS3 were induced by elicitor, but they reacted differently from the other inducers: RS1 was induced by yeast extract, but RS3 was not, and RS3 was induced by dilution of the cultures, but RS1 was not. The results indicate that the RS genes inA. hypogaea represent a gene family, and that some of the members are regulated by different signals. The quantitative data also show that the sum of the transcripts identified with gene-specific oligonucleotides was lower than the total amount of RS-specific transcripts, indicating that the cells contain active genes which have not yet been identified.

The intracellular localisation of phytochrome in oat (Avena sativa L. cv. Garry Oat) coleoptiles was analysed by electron microscopy. Serial ultrathin sections of resin-embedded material were indirectly immunolabeled with polyclonal antibodies against phytochrome together with a gold-coupled second antibody. The limits of detectability of sequestered areas of phytochrome (SAPs) were analysed as a function of light pretreatments and amounts of the far-red absorbing form of phytochrome (Pfr) established. In 5-d-old dark-grown Avena coleoptiles SAPs were not detectable if less than 13 units of Pfr - compared with 100 units total phytochrome of 5-d-old dark-grown seedlings - were established by a red light pulse. In other sets of experiments, seedlings were preirradiated either with a non-saturating red light pulse to allow destruction to occur or with a saturating red followed by a far-red light pulse to induce first SAP formation and then its disaggregation. These preirradiations resulted in an increase of the limit of detectability of SAP formation after a second red light pulse to 38-41 and 19-23 units Pfr, respectively. We conclude that with respect to Pfr-induced SAP formation an adaptation process exists and that our data indicate that SAP formation is not a simple self-aggregation of newly formed Pfr.

The fluence dependence of the time course of accumulation of chalcone synthase mRNA in ultraviolet (UV)-light-irradiated cell suspension cultures of parsley (Petroselinum crispum) and the additional effects of blue and far-red light have been investigated. Variations of the UV fluence had no detectable influence on the initial rate of increase in mRNA amount or translational activity, nor on the preceding lag period of approximately 3 h, but strongly influenced the duration of the transient increase. The effects were the same whether the fluence rate or the time of irradiation was varied to obtain a given fluence. Blue-light pretreatment of the cells resulted in increased amounts of mRNA and abolished the apparent lag period. This effect remained cryptic without the subsequent UV-light treatment. Irradiation with long-wavelength far-red light following UV-light pulses shortened the duration of the mRNA accumulation period. This effect was not altered by a preceding blue-light treatment. Thus, three photoreceptors, a UV-B receptor, a blue-light receptor and phytochrome, participate in the regulation of chalcone synthase mRNA accumulation in this system.

Two sesquiterpene lactones belonging to the germacranolides were isolated from the leaves and stems of Helianthus annuus L. Their formation in the plant is light-dependent. Both sesquiterpene lactones (SL) strongly inhibit indole-3-acetic acid (IAA)-induced elongation growth of Avena sativa L. coleoptile segments and Helianthus annuus L. hypocotyl segments. Both SL do not, however, inhibit acid-induced growth nor growth triggered by fusicoccin at all. In the presence of dithiothreitol (DTT), the inhibitory effect of SL in the Avena-segment-test can be completely neutralized. This can be attributed to the binding of DTT to both SL. Using thin-layer-chromatography it could be shown that the inhibitors build adducts with SH-rich compounds, e.g., cysteine, glutathione, mercapto-ethanol, and DTT, whose Rf-value significantly differs from those of the primary substances. If the coleoptile segments are first treated with an inhibitor and the inhibitor is subsequently washed out, close to normal elongation growth can be induced by adding an IAA-solution. If the segments are simultaneously treated with inhibitor and IAA, no notable growth can be initiated for an extended amount of time, after the removal of both substances and the anewed addition of IAA. Fusicoccin, however, can immediately neutralize the induced growth inhibition. The same irreversible inhibition is observed when 2,4-dichlorophenoxyacetic acid (2,4-D) is used: If coleoptile segments are treated with an inhibitor plus 2,4-D or an inhibitor plus 3,5-dichlorophenoxyacetic acid (3,5-D), respectively, IAA-induced growth after removal of the substances can only be observed by those coleoptiles which had previously been treated with the non-auxin, 3,5-D plus an inhibitor. Based on these results, a possible mechanism describing how the inhibitor functions is discussed. The binding of an auxin to an auxin receptor sets a SH-group free (possibly due to a change in the conformation of the receptor); a site is given to which the inhibitor can bind irreversibly (via a S-bond). The IAA-receptor-inhibitor-complex is then no longer able to initiate elongation growth. If auxin is not present, no lasting bond between the inhibitor and the receptor can occur, since the essential SH-group remains masked. The inhibitor can be washed out again. Consequently, the SL's have to be able to intervene at the beginning of the IAA-induced reaction sequence, while the following steps remain uninfluenced, i.e. namely, the active excretion of protons into the cell wall compartments, which is directly induced by fusicoccin and causes elongation growth.

β-Glucosidase activity for coniferin (coniferyl alcohol β-D-glucoside) is not present in spruce (Picea abies L. Karst.) seeds but appears in the young seedlings. Lignification starts at ca. day 9 of germination in the vascular bundles. An antiserum against glucosidase 1 isolated from spruce seedlings (Marcinowski and Grisebach, Eur J. Biochem. 1978) was employed for the localization of the enzyme in cross sections of hypocotyls using immunofluorescent technique. The results indicate that at this stage of development the glucosidase is localized at the inner layer of the secondary cell wall. Glucosidase activity was present in all cells of the investigated hypocotyl tissue.

Standard stereologic methods have been applied to electron micrographs of Daucus carota L. suspension culture cells. The relative frequencies of the different membrane systems within the cells have been determined and compared with published data form mature leaf cells.

Seedlings from the white mustard, Sinapis alba, grown under continuous far-red light exhibit enhanced plastid enzyme activities when compared with dark-grown seedlings (for review, see Mohr 1981). These activities are even more pronounced upon illumination with white light during the etioplast/chloroplast transformation. Etioplasts and etiochloroplasts from the cotyledons of such seedlings show high prenyl-lipid-synthesizing activities when [1-(14)C]isopentenyl diphosphate is used as the precursor. They lack, however, any enzymatic activities for the formation of isopentenyl diphosphate via the mevalonate pathway, i.e. hydroxymethylglutaryl-CoA reductase, mevalonate kinase, phosphomevalonate kinase and diphosphomevalonate decarboxylase, which are present and easily detectable within the endoplasmic reticulum and cytoplasm. These results corroborate the view that the cytoplasm of the plant cell is the only site of isopentenyl-diphosphate formation via the mevalonate pathway.

The laminar pulvinus of primary leaves of Phaseolus coccineus L. was investigated with respect to the total K(+) content, the apoplastic K(+) content, and the water potential of extensor and flexor sections in relation to the leaf positions in a circadian leaf-movement cycle, as well as the cation-exchange properties of isolated extensor- and flexor-cell walls. Turgid tissue showed a high total but low apoplastic K(+) content, shrunken tissue a low total but high apoplastic K(+) content. Thus, part of the K(+) transported into and out of the swelling or shrinking protoplasts is shuttled between the protoplasts and the surrounding walls, another part between different regions of the pulvinus. The K(+) fraction shuttled between protoplasts and walls was found to be 30-40% of the total transported K(+) fraction. Furthermore, 15-20% of the total K(+) content of the tissue is located in the apoplast when the apoplastic reservoir is filled, 5-10% when the apoplastic reservoir is depleted. The ion-exchange properties of walls of extensor and flexor cells appear identical in situ and in isolated preparations. The walls behave as cation exchangers of hhe weak-acid type with a strong dependence of the activity of fixed negative charges as well as of the K(+)-storing capacity on pH and [K(+)] of the equilibration solution. The high apoplastic K(+) contents of freshly cut tissues reflect the cation-storing capacity of the isolated walls. We suggest that K(+) ions of the Donnan free space are used for the reversible volume changes (mediating the leaf movement) mainly by an electrogenic proton pump which changes the pH and-or the [K(+)] in the water free space of the apoplast.

Cell suspension cultures of parsley and soybean were incubated for 38 h with (14)C-labeled benzo[a]pyrene; autoclaved cultures were used as controls. Metabolites were isolated by a sequential extraction procedure and further studied by chromatography or by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The soluble metabolites amounted to 1-2.2% in the case of parsley cells, and 19-28% in the case of soybean cells. These metabolites varied in polarity, some being soluble in organic solvent or aqueous buffer while other metabolite fractions were soluble only in hot aqueous sodium dodecylsulphate. In addition, a significant amount of an insoluble metabolite fraction was isolated from the culture fluid as well as the cellular material of soybean suspension cultures.

The specific activities of the enzymes cinnamic acid 4-hydroxylase and cytidine-5'-diphospho-choline: 1,2-diacylglycerol cholinephosphotransferase (E.C. 2.7.8.2) were determined in microsomal fractions from a) dark-grown and b) irradiated parsley (Petroselinum hortense Hoffm.) cell suspension cultures. Cinnamic acid 4-hydroxylase activity was increased 4-fold after light treatment, while cholinephosphotransferase activity was decreased by about one third of its original activity. It can be concluded that increased microsomal lecithin biosynthesis is not required for the light-induced increase of cinnamic acid 4-hydroxylase activity, although the latter enzyme has previously been found to require a lipid cofactor for activity.

Mitochondria from etiolated corn possess a much greater Ca(2+) uptake capacity per mg protein than microsomes from the same source. Differences in energy requirements, sensitivity to specific inhibitors, and sedimentation properties enabled us to study both Ca(2+) uptake mechanisms without mutual contamination. The microsomal Ca(2+) uptake does not vary much among different plants as compared to the mitochondrial Ca(2+) uptake; this is also true for different organs of the same plant. Mitochondrial Ca(2+) uptake is more dependent on the age of the seedlings than microsomal uptake, because of changes in active Ca(2+) uptake activity rather than of changes in efflux. Intactness and the oxidative and phosphorylative properties of the mitochondria remained unchanged during this time period. Na(+) and Mg(2+) do not induce Ca(2+) release from mitochondria.

Evacuolated mesophyll protoplasts from oat (Avena sativa L.) were fractionated by a membrane-filtration technique. This method of rapid quenching of metabolic reactions permitted estimation of the in-vivo pools of fructose 2,6-bisphosphate (Fru2,6bisP) in the cytosol, chloroplasts and mitochondria. Vacuolar Fru2,6bisP was calculated as the difference between control protoplasts and evacuolated ones. The results indicate that Fru2,6bisP is exclusively cytosol-located in oat mesophyll protoplasts. Assuming a cytosolic volume of about 2 pl per evacuolated protoplast, the cytosolic concentration there was 11 μM if protoplasts were in darkness. Illumination of either control or evacuolated protoplasts resulted in a significant decrease in the Fru2,6bisP content within 5 min.

Transport of (86)Rb(+)/K(+), (22)Na(+), (36)Cl(-), and [(3)H]indole acetic acid (IAA) has been studied on suspension-cultured cells of the parsley, Petroselinum crispum (Mill) Nym. By compartmental analysis two intracellular compartments of K(+), Na(+), and Cl(-) have been identified and ascribed to the cytoplasm and vacuole; half-times of exchange were around 200 s and 5 h, respectively. According to the Ussing-Teorell flux equation, active transport is required for the influx into the cytoplasm at the plasmalemma (K(+), Cl(-)) and the tonoplast (K(+), Na(+), Cl(-)). The plasmalemma permeability pattern, PK:PNa:PCl=1.00:0.24:0.38, features an increased chloride permeability compared with cells from higher plant tissues. IAA uptake showed an exponential timecourse, was half-maximal after 10 min, and a linear function of the IAA concentration from 10(-9) to 10(-5) M. IAA and 2,4-dichlorophenoxy acetic acid reduce the apparent influx of K(+), Na(+), Cl(-) during the initial 30 min after addition and subsequently accelerate both in- and efflux of these ions. We discuss that auxins could affect the ion fluxes in a complex way, e.g. by protonophorous activity and by control of the hypothetical proton pump.

The existence of specific binding sites for a β-glucan elicitor of phytoalexin synthesis derived from the fungus Phytophthora megasperma f.sp. glycinea at the plasma membrane of soybean (Glycine max L.) tissues (W.E. Schmidt, J. Ebel (1987) Proc. Natl. Acad. Sci. USA 84, 4117-4121) might imply that stimulation of phytoalexin formation by the elicitor is a membrane-mediated process. Addition of the β-glucan elicitor to soybean cellsuspension cultures, which has previously been shown to induce phytoalexin accumulation, also results in rapid changes in the phosphate turnover of several phosphoproteins. The effect of the elicitor on protein phosphorylation was tested after labeling of the cells with [(32)P]orthophosphate. As shown by analysis using one-and two-dimensional gel electrophoresis, decreases as well as increases in the labeling of several phosphoroteins occurred rapidly, being detectable within 5 min after elicitor application, and persisted for at least 15 min. As judged by their relative molecular masses (Mr) and isoelectric points (pI), a number of proteins which were radioactively labeled in vivo were also phosphorylated in vitro by endogenous protein-kinase activity in the presence of Ca(2+). The most pronounced effect was observed with a protein substrate with Mr=69000 and pI=5.7 (pp69) whose phosphate labeling markedly decreased in response to elicitor treatment in vivo. Phosphorylation of pp69 in vitro in the presence of γ-[(32)P]ATP was strongly enhanced by a phosphorylation-stimulating factor (effector) derived from soybean cell cultures and occurred predominantly at serine residues. The effector possessed a low apparent Mr (≤1000), was negatively charged at pH 7.3, and was relatively heat stable. The effector was inactivated by treatment with alkaline phosphatase from calf intestine. Phosphorylation of pp69 was only slightly stimulated by Ca(2+), and was insensitive to cAMP, cGMP, calmodulin, a lipid mixture, a ganglioside mixture, or spermine under the assay conditions used. A 10 mM concentration of 3-phosphoglycerate increased pp69 phosphorylation to the extent of about 50% of that induced by the soybean effector. There was no evidence, however, that such concentrations of 3-phosphoglycerate occurred in effector preparations. The results are discussed in relation to hypothetical signal transduction during elicitor action on soybean cells.

Protein-kinase activity has been found to be associated with a membrane fraction obtained from dark-grown zucchini (Cucurbita pepo L., cv. Senator) hypocotyl hooks. Proteins of this membrane fraction were used as protein substrates. The effects of Mg(2+), Na(+) and K(+) on phosphorylation, measured as (32)P incorporation, was investigated. The kinetics of phosphorylation of the individual protein peptides indicate the presence of specific phosphatase activity. Phosphorylation activity is strongly influenced by Ca(2+). One peptide (relative molecular weight: 180,000) exhibits strong inhibition of (32)P incorporation at physiological Ca(2+) concentrations between 0.1 and 1 μM. Phosphorylation of about 10 other proteins was enhanced by Ca(2+), being maximal in most cases at a concentration of about 3 μM free Ca(2+). Five out of these 10 peptides show increased phosphorylation in the presence of 1 μM calmodulin. This calmodulin-dependent enhancement of phosphorylation could be completely inhibited by the calmodulin antagonist fluphenazine. Cyclic AMP was found to have no stimulating effect on protein phosphorylation.

The influence of nucleoside triphosphates in relation to divalent cations on RNA synthesis of cells from a suspension culture from parsley was investigated. The data obtained from experiments with isolated nuclei and with an in vitro system with highly purified RNA polymerase I were compared with a chromatin-bound nucleoside triphosphatase activity within the nucleus. The results might suggest a regulatory role of the nucleoside triphosphatase activity in RNA synthesis.

Purified spinach chloroplast and daffodil chromoplast preparations do not use mevalonate, phosphomevalonate, and diphosphomevalonate for the synthesis of isopentenyl diphosphate. Isopentenyl diphosphate, on the other hand, is incorporated into plastidal polyprenoids in large amounts. In the presence of a cytoplasmic supernatant, however, mevalonate and the phosphomevalonates were incorporated into the plastidal polyprenoids in equally large amounts, which demonstrates that the enzymes mevalonate kinase (EC 2.7.1.36), phosphomevalonate kinase (EC 2.7.4.2), and diphosphomevalonate decarboxylase (EC 4.1.1.33) are soluble cytoplasmic enzymes and that they apparently do not occur as isoenzymes within the plastids. The concept is developed that isopentenyl diphosphate is a central intermediate in plant polyprenoid formation which is channeled into several compartment for different biosynthetic pathways.