A UPLC-MS/MS method for quantification of metabolites in the ethylene biosynthesis pathway and its biological validation in Arabidopsis.

The plant hormone ethylene is of vital importance in the regulation of plant development and stress responses. Recent studies revealed that 1-aminocyclopropane-1-carboxylic acid (ACC) plays a role beyond its function as an ethylene precursor. However, the absence of reliable methods to quantify ACC and its conjugates malonyl-ACC (MACC), glutamyl-ACC (GACC), and jasmonyl-ACC (JA-ACC) hinders related research. Combining synthetic and analytical chemistry, we present the first, validated methodology to rapidly extract and quantify ACC and its conjugates using ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS). Its relevance was confirmed by application to Arabidopsis mutants with altered ACC metabolism and wild-type plants under stress. Pharmacological and genetic suppression of ACC synthesis resulted in decreased ACC and MACC content, whereas induction led to elevated levels. Salt, wounding, and submergence stress enhanced ACC and MACC production. GACC and JA-ACC were undetectable in vivo; however, GACC was identified in vitro, underscoring the broad applicability of the method. This method provides an efficient tool to study individual functions of ACC and its conjugates, paving the road toward exploration of novel avenues in ACC and ethylene metabolism, and revisiting ethylene literature in view of the recent discovery of an ethylene-independent role of ACC.

Glutathione Is Required for the Early Alert Response and Subsequent Acclimation in Cadmium-Exposed Arabidopsis thaliana Plants.

Pollution by cadmium (Cd) is a worldwide problem, posing risks to human health and impacting crop yield and quality. Cadmium-induced phytotoxicity arises from an imbalance between antioxidants and pro-oxidants in favour of the latter. The Cd-induced depletion of the major antioxidant glutathione (GSH) strongly contributes to this imbalance. Rather than being merely an adverse effect of Cd exposure, the rapid depletion of root GSH levels was proposed to serve as an alert response. This alarm phase is crucial for an optimal stress response, which defines acclimation later on. To obtain a better understanding on the importance of GSH in the course of these responses and how these are defined by the rapid GSH depletion, analyses were performed in the GSH-deficient cadmium-sensitive 2-1 (cad2-1) mutant. Cadmium-induced root and leaf responses related to oxidative challenge, hydrogen peroxide (H2O2), GSH, ethylene, and 1-aminocyclopropane-1-carboxylic acid (ACC) were compared between wild-type (WT) and mutant Arabidopsis thaliana plants. Although the cad2-1 mutant has significantly lower GSH levels, root GSH depletion still occurred, suggesting that the chelating capacity of GSH is prioritised over its antioxidative function. We demonstrated that responses related to GSH metabolism and ACC production were accelerated in mutant roots and that stress persisted due to suboptimal acclimation. In general, the redox imbalance in cad2-1 mutant plants and the lack of proper transient ethylene signalling contributed to this suboptimal acclimation, resulting in a more pronounced Cd effect.

Identifying the Pressure Points of Acute Cadmium Stress Prior to Acclimation in Arabidopsis thaliana.

The toxic metal cadmium (Cd) is a major soil pollutant. Knowledge on the acute Cd-induced stress response is required to better understand the triggers and sequence of events that precede plant acclimation. Therefore, we aimed to identify the pressure points of Cd stress using a short-term exposure set-up ranging from 0 h to 24 h. Acute responses related to glutathione (GSH), hydrogen peroxide (H2O2), 1-aminocyclopropane-1-carboxylic acid (ACC), ethylene and the oxidative challenge were studied at metabolite and/or transcript level in roots and leaves of Arabidopsis thaliana either exposed or not to 5 µM Cd. Cadmium rapidly induced root GSH depletion, which might serve as an alert response and modulator of H2O2 signalling. Concomitantly, a stimulation of root ACC levels was observed. Leaf responses were delayed and did not involve GSH depletion. After 24 h, a defined oxidative challenge became apparent, which was most pronounced in the leaves and concerted with a strong induction of leaf ACC synthesis. We suggest that root GSH depletion is required for a proper alert response rather than being a merely adverse effect. Furthermore, we propose that roots serve as command centre via a.o. root-derived ACC/ethylene to engage the leaves in a proper stress response.

Starch biosynthesis contributes to the maintenance of photosynthesis and leaf growth under drought stress in maize.

To understand the growth response to drought, we performed a proteomics study in the leaf growth zone of maize (Zea mays L.) seedlings and functionally characterized the role of starch biosynthesis in the regulation of growth, photosynthesis and antioxidant capacity, using the shrunken-2 mutant (sh2), defective in ADP-glucose pyrophosphorylase. Drought altered the abundance of 284 proteins overrepresented for photosynthesis, amino acid, sugar and starch metabolism, and redox-regulation. Changes in protein levels correlated with enzyme activities (increased ATP synthase, cysteine synthase, starch synthase, RuBisCo, peroxiredoxin, glutaredoxin, thioredoxin and decreased triosephosphate isomerase, ferredoxin, cellulose synthase activities, respectively) and metabolite concentrations (increased ATP, cysteine, glycine, serine, starch, proline and decreased cellulose levels). The sh2 mutant showed a reduced increase of starch levels under drought conditions, leading to soluble sugar starvation at the end of the night and correlating with an inhibition of leaf growth rates. Increased RuBisCo activity and pigment concentrations observed in WT, in response to drought, were lacking in the mutant, which suffered more oxidative damage and recovered more slowly after re-watering. These results demonstrate that starch biosynthesis contributes to maintaining leaf growth under drought stress and facilitates enhanced carbon acquisition upon recovery.

Does long-term cadmium exposure influence the composition of pectic polysaccharides in the cell wall of Medicago sativa stems?

BACKGROUND: The heavy metal cadmium (Cd) accumulates in the environment due to anthropogenic influences. It is unessential and harmful to all life forms. The plant cell wall forms a physical barrier against environmental stress and changes in the cell wall structure have been observed upon Cd exposure. In the current study, changes in the cell wall composition and structure of Medicago sativa stems were investigated after long-term exposure to Cd. Liquid chromatography coupled to mass spectrometry (LC-MS) for quantitative protein analysis was complemented with targeted gene expression analysis and combined with analyses of the cell wall composition. RESULTS: Several proteins determining for the cell wall structure changed in abundance. Structural changes mainly appeared in the composition of pectic polysaccharides and data indicate an increased presence of xylogalacturonan in response to Cd. Although a higher abundance and enzymatic activity of pectin methylesterase was detected, the total pectin methylation was not affected. CONCLUSIONS: An increased abundance of xylogalacturonan might hinder Cd binding in the cell wall due to the methylation of its galacturonic acid backbone. Probably, the exclusion of Cd from the cell wall and apoplast limits the entry of the heavy metal into the symplast and is an important factor during tolerance acquisition.

Study of cytokinin transport from shoots to roots of wheat plants is informed by a novel method of differential localization of free cytokinin bases or their ribosylated forms by means of their specific fixation.

The aim of the present report was to demonstrate how a novel approach for immunohistochemical localization of cytokinins in the leaf and particularly in the phloem may complement to the study of their long-distance transport. Different procedures of fixation were used to conjugate either cytokinin bases or their ribosides to proteins of cytoplasm to enable visualization and differential localization of these cytokinins in the leaf cells of wheat plants. In parallel to immunolocalization of cytokinins in the leaf cells, we immunoassayed distribution of free bases of cytokinins, their nucleotides and ribosides between roots and shoots of wheat plants as well as their presence in phloem sap after incubation of leaves in a solution supplemented with either trans-zeatin or isopentenyladenine. The obtained data show ribosylation of the zeatin applied to the leaves and its elevated level in the phloem sap supported by in vivo localization showing the presence of ribosylated forms of zeatin in leaf vessels. This suggests that conversion of zeatin to its riboside is important for the shoot-to-root transport of zeatin-type cytokinins in wheat. Exogenous isopentenyladenine was not modified, but diffused from the leaves as free base. These metabolic differences may not be universal and may depend on the plant species and age. Although the measurements of cytokinins in the phloem sap and root tissue is the most defining for determining cytokinin transport, study of immunolocalization of either free cytokinin bases or their ribosylated forms may be a valuable source of information for predicting their transport in the phloem and to the roots.

Early pathologic amyloid induces hypersynchrony of BOLD resting-state networks in transgenic mice and provides an early therapeutic window before amyloid plaque deposition.

INTRODUCTION: In Alzheimer's disease (AD), pathologic amyloid-beta (Aß) is synaptotoxic and impairs neuronal function at the microscale, influencing brain networks at the macroscale before Aß deposition. The latter can be detected noninvasively, in vivo, using resting-state functional MRI (rsfMRI), a technique used to assess brain functional connectivity (FC). METHODS: RsfMRI was performed longitudinally in TG2576 and PDAPP mice, starting before Aß deposition to determine the earliest FC changes. Additionally, the role of pathologic Aß on early FC alterations was investigated by treating TG2576 mice with the 3D6 anti-Aß-antibody. RESULTS: Both transgenic models showed hypersynchronized FC before Aß deposition and hyposynchronized FC at later stages. Early anti-Aß treatment in TG2576 mice prevented hypersynchronous FC and the associated synaptic impairments and excitatory/inhibitory disbalances. DISCUSSION: Hypersynchrony of FC may be used as a new noninvasive read out of early AD and can be recovered by anti-Aß treatment, encouraging preventive treatment strategies in familial AD.

Ultraviolet-B radiation stimulates downward leaf curling in Arabidopsis thaliana.

Plants are very well adapted to growth in ultraviolet-B (UV-B) containing light. In Arabidopsis thaliana, many of these adaptations are mediated by the UV-B receptor UV resistance locus 8 (UVR8). Using small amounts of supplementary UV-B light, we observed changes in the shape of rosette leaf blades. Wild type plants show more pronounced epinasty of the blade edges, while this is not the case in uvr8 mutant plants. The UVR8 effect thus mimics the effect of phytochrome (phy) B in red light. In addition, a meta-analysis of transcriptome data indicates that the UVR8 and phyB signaling pathways have over 70% of gene regulation in common. Moreover, in low levels of supplementary UV-B light, mutant analysis revealed that phyB signaling is necessary for epinasty of the blade edges. Analysis of auxin levels and the auxin signal reporter DR5::GUS suggest that the epinasty relies on altered auxin distribution, keeping auxin at the leaf blade edges in the presence of UV-B. Together, our results suggest a co-action of phyB and UVR8 signaling, with auxin as a downstream factor.

Cadmium-induced ethylene production and responses in Arabidopsis thaliana rely on ACS2 and ACS6 gene expression.

BACKGROUND: Anthropogenic activities cause metal pollution worldwide. Plants can absorb and accumulate these metals through their root system, inducing stress as a result of excess metal concentrations inside the plant. Ethylene is a regulator of multiple plant processes, and is affected by many biotic and abiotic stresses. Increased ethylene levels have been observed after exposure to excess metals but it remains unclear how the increased ethylene levels are achieved at the molecular level. In this study, the effects of cadmium (Cd) exposure on the production of ethylene and its precursor 1-aminocyclopropane-1-carboxylic acid (ACC), and on the expression of the ACC Synthase (ACS) and ACC Oxidase (ACO) multigene families were investigated in Arabidopsis thaliana. RESULTS: Increased ethylene release after Cd exposure was directly measurable in a system using rockwool-cultivated plants; enhanced levels of the ethylene precursor ACC together with higher mRNA levels of ethylene responsive genes: ACO2, ETR2 and ERF1 also indicated increased ethylene production in hydroponic culture. Regarding underlying mechanisms, it was found that the transcript levels of ACO2 and ACO4, the most abundantly expressed members of the ACO multigene family, were increased upon Cd exposure. ACC synthesis is the rate-limiting step in ethylene biosynthesis, and transcript levels of both ACS2 and ACS6 showed the highest increase and became the most abundant isoforms after Cd exposure, suggesting their importance in the Cd-induced increase of ethylene production. CONCLUSIONS: Cadmium induced the biosynthesis of ACC and ethylene in Arabidopsis thaliana plants mainly via the increased expression of ACS2 and ACS6. This was confirmed in the acs2-1acs6-1 double knockout mutants, which showed a decreased ethylene production, positively affecting leaf biomass and resulting in a delayed induction of ethylene responsive gene expressions without significant differences in Cd contents between wild-type and mutant plants.

Analysis of genome sequences from plant pathogenic Rhodococcus reveals genetic novelties in virulence loci.

Members of Gram-positive Actinobacteria cause economically important diseases to plants. Within the Rhodococcus genus, some members can cause growth deformities and persist as pathogens on a wide range of host plants. The current model predicts that phytopathogenic isolates require a cluster of three loci present on a linear plasmid, with the fas operon central to virulence. The Fas proteins synthesize, modify, and activate a mixture of growth regulating cytokinins, which cause a hormonal imbalance in plants, resulting in abnormal growth. We sequenced and compared the genomes of 20 isolates of Rhodococcus to gain insights into the mechanisms and evolution of virulence in these bacteria. Horizontal gene transfer was identified as critical but limited in the scale of virulence evolution, as few loci are conserved and exclusive to phytopathogenic isolates. Although the fas operon is present in most phytopathogenic isolates, it is absent from phytopathogenic isolate A21d2. Instead, this isolate has a horizontally acquired gene chimera that encodes a novel fusion protein with isopentyltransferase and phosphoribohydrolase domains, predicted to be capable of catalyzing and activating cytokinins, respectively. Cytokinin profiling of the archetypal D188 isolate revealed only one activate cytokinin type that was specifically synthesized in a fas-dependent manner. These results suggest that only the isopentenyladenine cytokinin type is synthesized and necessary for Rhodococcus phytopathogenicity, which is not consistent with the extant model stating that a mixture of cytokinins is necessary for Rhodococcus to cause leafy gall symptoms. In all, data indicate that only four horizontally acquired functions are sufficient to confer the trait of phytopathogenicity to members of the genetically diverse clade of Rhodococcus.

Perturbation of cytokinin and ethylene-signalling pathways explain the strong rooting phenotype exhibited by Arabidopsis expressing the Schizosaccharomyces pombe mitotic inducer, cdc25.

BACKGROUND: Entry into mitosis is regulated by cyclin dependent kinases that in turn are phosphoregulated. In most eukaryotes, phosphoregulation is through WEE1 kinase and CDC25 phosphatase. In higher plants a homologous CDC25 gene is unconfirmed and hence the mitotic inducer Schizosaccharomyces pombe (Sp) cdc25 has been used as a tool in transgenic plants to probe cell cycle function. Expression of Spcdc25 in tobacco BY-2 cells accelerates entry into mitosis and depletes cytokinins; in whole plants it stimulates lateral root production. Here we show, for the first time, that alterations to cytokinin and ethylene signaling explain the rooting phenotype elicited by Spcdc25 expression in Arabidopsis. RESULTS: Expressing Spcdc25 in Arabidopsis results in increased formation of lateral and adventitious roots, a reduction of primary root width and more isodiametric cells in the root apical meristem (RAM) compared with wild type. Furthermore it stimulates root morphogenesis from hypocotyls when cultured on two way grids of increasing auxin and cytokinin concentrations. Microarray analysis of seedling roots expressing Spcdc25 reveals that expression of 167 genes is changed by > 2-fold. As well as genes related to stress responses and defence, these include 19 genes related to transcriptional regulation and signaling. Amongst these was the up-regulation of genes associated with ethylene synthesis and signaling. Seedlings expressing Spcdc25 produced 2-fold more ethylene than WT and exhibited a significant reduction in hypocotyl length both in darkness or when exposed to 10 ppm ethylene. Furthermore in Spcdc25 expressing plants, the cytokinin receptor AHK3 was down-regulated, and endogenous levels of iPA were reduced whereas endogeous IAA concentrations in the roots increased. CONCLUSIONS: We suggest that the reduction in root width and change to a more isodiametric cell phenotype in the RAM in Spcdc25 expressing plants is a response to ethylene over-production. The increased rooting phenotype in Spcdc25 expressing plants is due to an increase in the ratio of endogenous auxin to cytokinin that is known to stimulate an increased rate of lateral root production. Overall, our data reveal important cross talk between cell division and plant growth regulators leading to developmental changes.

Unraveling tobacco BY-2 protein complexes with BN PAGE/LC-MS/MS and clustering methods.

To understand physiological processes, insight into protein complexes is very important. Through a combination of blue native gel electrophoresis and LC-MS/MS, we were able to isolate protein complexes and identify their potential subunits from Nicotiana tabacum cv. Bright Yellow-2. For this purpose, a bioanalytical approach was used that works without a priori knowledge of the interacting proteins. Different clustering methods (e.g., k-means and hierarchical clustering) and a biclustering approach were evaluated according to their ability to group proteins by their migration profile and to correlate the proteins to a specific complex. The biclustering approach was identified as a very powerful tool for the exploration of protein complexes of whole cell lysates since it allows for the promiscuous nature of proteins. Furthermore, it searches for associations between proteins that co-occur frequently throughout the BN gel, which increases the confidence of the putative associations between co-migrating proteins. The statistical significance and biological relevance of the profile clusters were verified using functional gene ontology annotation. The proof of concept for identifying protein complexes by our BN PAGE/LC-MS/MS approach is provided through the analysis of known protein complexes. Both well characterized long-lived protein complexes as well as potential temporary sequential multi-enzyme complexes were characterized.

UV radiation reduces epidermal cell expansion in Arabidopsis thaliana leaves without altering cellular microtubule organization.

Upon chronic UV treatment pavement cell expansion in Arabidopsis leaves is reduced, implying alterations in symplastic and apoplastic properties of the epidermal cells. In this study, the effect of UV radiation on microtubule patterning is analysed, as microtubules are thought to serve as guiding rails for the cellulose synthase complexes depositing cellulose microfibrils. Together with hemicelluloses, these microfibrils are regarded as the load-bearing components of the cell wall. Leaves of transgenic plants with fluorescently tagged microtubules (GFP-TUA6) were as responsive to UV as wild type plants. Despite the UV-induced reduction in cell elongation, confocal microscopy revealed that cellular microtubule arrangements were seemingly not affected by the UV treatments. This indicates an unaltered deposition of cellulose microfibrils in the presence of UV radiation. Therefore, we surmise that the reduction in cell expansion in UV-treated leaves is most probably due to changes in cell wall loosening and/or turgor pressure.   

UV radiation reduces epidermal cell expansion in leaves of Arabidopsis thaliana.

Plants have evolved a broad spectrum of mechanisms to ensure survival under changing and suboptimal environmental conditions. Alterations of plant architecture are commonly observed following exposure to abiotic stressors. The mechanisms behind these environmentally controlled morphogenic traits are, however, poorly understood. In this report, the effects of a low dose of chronic ultraviolet (UV) radiation on leaf development are detailed. Arabidopsis rosette leaves exposed for 7, 12, or 19 d to supplemental UV radiation expanded less compared with non-UV controls. The UV-mediated decrease in leaf expansion is associated with a decrease in adaxial pavement cell expansion. Elevated UV does not affect the number and shape of adaxial pavement cells, nor the stomatal index. Cell expansion in young Arabidopsis leaves is asynchronous along a top-to-base gradient whereas, later in development, cells localized at both the proximal and distal half expand synchronously. The prominent, UV-mediated inhibition of cell expansion in young leaves comprises effects on the early asynchronous growing stage. Subsequent cell expansion during the synchronous phase cannot nullify the UV impact established during the asynchronous phase. The developmental stage of the leaf at the onset of UV treatment determines whether UV alters cell expansion during the synchronous and/or asynchronous stage. The effect of UV radiation on adaxial epidermal cell size appears permanent, whereas leaf shape is transiently altered with a reduced length/width ratio in young leaves. The data show that UV-altered morphogenesis is a temporal- and spatial-dependent process, implying that common single time point or single leaf zone analyses are inadequate.

Arabidopsis thaliana plants acclimated to low dose rates of ultraviolet B radiation show specific changes in morphology and gene expression in the absence of stress symptoms.

Ultraviolet B (UV-B) acclimation comprises complex and poorly understood changes in plant metabolism. The effects of chronic and ecologically relevant UV-B dose rates on Arabidopsis thaliana were determined. The UV-B acclimation process was studied by measuring radiation effects on morphology, physiology, biochemistry and gene expression. Chronic UV-B radiation did not affect photosynthesis or the expression of stress responsive genes, which indicated that the UV-acclimated plants were not stressed. UV-induced morphological changes in acclimated plants included decreased rosette diameter, decreased inflorescence height and increased numbers of flowering stems, indicating that chronic UV-B treatment caused a redistribution rather than a cessation of growth. Gene expression profiling indicated that UV-induced morphogenesis was associated with subtle changes in phytohormone (auxins, brassinosteroids and gibberellins) homeostasis and the cell wall. Based on the comparison of gene expression profiles, it is concluded that acclimation to low, chronic dose rates of UV-B is distinct from that to acute, stress-inducing UV-B dose rates. Hence, UV-B-induced morphogenesis is functionally uncoupled from stress responses.

Cytokinin import rate as a signal for photosynthetic acclimation to canopy light gradients.

Plants growing in dense canopies are exposed to vertical light gradients and show photosynthetic acclimation at the whole-plant level, resulting in efficient photosynthetic carbon gain. We studied the role of cytokinins transported through the transpiration stream as one of probably multiple signals for photosynthetic acclimation to light gradients using both tobacco (Nicotiana tabacum) and Arabidopsis (Arabidopsis thaliana). We show that substantial variation in leaf transpiration parallels the light gradient in tobacco canopies and experimental reduction of the transpiration rate of a leaf, independent of light, is sufficient to reduce photosynthetic capacity in both species, as well as transcript levels of the small subunit of Rubisco (rbcS) gene in Arabidopsis. Mass spectrometric analysis of xylem sap collected from intact, transpiring tobacco plants revealed that shaded leaves import less cytokinin than leaves exposed to high light. In Arabidopsis, reduced transpiration rate of a leaf in the light is associated with lower cytokinin concentrations, including the bioactive trans-zeatin and trans-zeatin riboside, as well as reduced expression of the cytokinin-responsive genes ARR7 and ARR16. External application of cytokinin to shaded leaves rescued multiple shade effects, including rbcS transcript levels in both species, as did locally induced cytokinin overproduction in transgenic tobacco plants. From these data, we conclude that light gradients over the foliage of a plant result in reduced cytokinin activity in shaded leaves as a consequence of reduced import through the xylem and that cytokinin is involved in the regulation of whole-plant photosynthetic acclimation to light gradients in canopies.

A hormone and proteome approach to picturing the initial metabolic events during Plasmodiophora brassicae infection on Arabidopsis.

We report on the early response of Arabidopsis thaliana to the obligate biotrophic pathogen Plasmodiophora brassicae at the hormone and proteome level. Using a CYCB1;1::GUS construct, the re-initiation of infection-related cell division is shown from 4 days after inoculation on. Sensitivity to cytokinins and auxins as well as the endogenous hormone levels are evaluated. Both an enhanced cytokinin gene response and an accumulation of isopentenyl adenine and adenosine precede this re-initiation of cell division, whereas an enhanced auxin gene response is observed from 6 days after inoculation on. The alhl mutant, impaired in the cross talk between ethylene and auxins, is resistant to P. brassicae. A differential protein analysis of infected versus noninfected roots and hypocotyls was performed using two-dimensional gel electrophoresis and quantitative image analysis, coupled to matrix-assisted laser desorption ionization time of flight-time of flight mass spectrometry-based protein identification. Of the visualized proteins, 12% show altered abundance compared with the noninfected plants, including proteins involved in metabolism, cell defense, cell differentiation, and detoxification. Combining the hormone and proteome data, we postulate that, at the very first stages of Plasmodiophora infection, plasmodial-produced cytokinins trigger a local re-initiation of cell division in the root cortex. Consequently, a de novo meristematic area is established that acts as a sink for host-derived indole-3-acetic acid, carbohydrates, nitrogen, and energy to maintain the pathogen and to trigger gall development.

Cytokinin-induced hypocotyl elongation in light-grown Arabidopsis plants with inhibited ethylene action or indole-3-acetic acid transport.

Cytokinins inhibit hypocotyl elongation in darkness but have no obvious effect on hypocotyl length in the light. However, we found that cytokinins do promote hypocotyl elongation in the light when ethylene action is blocked. A 50% increase in Arabidopsis thaliana (L.) Heynh. hypocotyl length was observed in response to N6-benzyladenine (BA) treatment in the presence of Ag+. The level of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid was strongly increased, indicating that ethylene biosynthesis was up-regulated by treatment with cytokinin. Furthermore, the effects of cytokinins on hypocotyl elongation were also tested using a series of mutants in the cascade of the ethylene-signal pathway. In the ethylene-insensitive mutants etr1-3 and ein2-1, cytokinin treatment resulted in hypocotyl lengths comparable to those of wild-type seedlings treated with both Ag+ and BA. A similar phenotypical response to cytokinin was observed when auxin transport was blocked by alpha-naphthylphthalamic acid (NPA). Applied cytokinin largely restored cell elongation in the basal and middle parts of the hypocotyls of NPA-treated seedlings and at the same time abolished the NPA-induced decrease in indole-3-acetic acid levels. Our data support the hypothesis that, in the light, cytokinins interact with the ethylene-signalling pathway and conditionally up-regulate ethylene and auxin synthesis.

Biosynthesis of auxin by the gram-positive phytopathogen Rhodococcus fascians is controlled by compounds specific to infected plant tissues.

The role and metabolism of indole-3-acetic acid in gram-negative bacteria is well documented, but little is known about indole-3-acetic acid biosynthesis and regulation in gram-positive bacteria. The phytopathogen Rhodococcus fascians, a gram-positive organism, incites diverse developmental alterations, such as leafy galls, on a wide range of plants. Phenotypic analysis of a leafy gall suggests that auxin may play an important role in the development of the symptoms. We show here for the first time that R. fascians produces and secretes the auxin indole-3-acetic acid. Interestingly, whereas noninfected-tobacco extracts have no effect, indole-3-acetic acid synthesis is highly induced in the presence of infected-tobacco extracts when tryptophan is not limiting. Indole-3-acetic acid production by a plasmid-free strain shows that the biosynthetic genes are located on the bacterial chromosome, although plasmid-encoded genes contribute to the kinetics and regulation of indole-3-acetic acid biosynthesis. The indole-3-acetic acid intermediates present in bacterial cells and secreted into the growth media show that the main biosynthetic route used by R. fascians is the indole-3-pyruvic acid pathway with a possible rate-limiting role for indole-3-ethanol. The relationship between indole-3-acetic acid production and the symptoms induced by R. fascians is discussed.

IAA production during germination of Orobanche spp. seeds.

Broomrapes (Orobanche spp.) are parasitic plants, whose growth and development fully depend on the nutritional connection established between the parasite and the roots of the respective host plant. Phytohormones are known to play a role in establishing the specific Orobanche-host plant interaction. The first step in the interaction is seed germination triggered by a germination stimulant secreted by the host-plant roots. We quantified indole-3-acetic acid (IAA) and abscisic acid (ABA) during the seed germination of tobacco broomrape (Orobanche ramosa) and sunflower broomrape (O. cumana). IAA was mainly released from Orobanche seeds in host-parasite interactions as compared to non-host-parasite interactions. Moreover, germinating seeds of O. ramosa released IAA as early as 24 h after the seeds were exposed to the germination stimulant, even before development of the germ tube. ABA levels remained unchanged during the germination of the parasites' seeds. The results presented here show that IAA production is probably part of a mechanism triggering germination upon the induction by the host factor, thus resulting in seed germination.