Interaction with Soil Bacteria Affects the Growth and Amino Acid Content of Piriformospora indica.

Exploration of the effect of soil bacteria on growth and metabolism of beneficial root endophytic fungi is relevant to promote favorable associations between microorganisms of the plant rhizosphere. Hence, the interaction between the plant-growth-promoting fungus Piriformospora indica and different soil bacteria was investigated. The parameters studied were fungal growth and its amino acid composition during the interaction. Fungus and bacteria were confronted in dual cultures in Petri dishes, either through agar or separated by a Perspex wall that only allowed the bacterial volatiles to be effective. Fungal growth was stimulated by Azotobacter chroococcum, whereas Streptomyces anulatus AcH 1003 inhibited it and Streptomyces sp. Nov AcH 505 had no effect. To analyze amino acid concentration data, targeted metabolomics was implemented under supervised analysis according to fungal-bacteria interaction and time. Orthogonal partial least squares-discriminant analysis (OPLS-DA) model clearly discriminated P. indica-A. chroococcum and P. indica-S. anulatus interactions, according to the respective score plot in comparison to the control. The most observable responses were in the glutamine and alanine size groups: While Streptomyces AcH 1003 increased the amount of glutamine, A. chroococcum decreased it. The fungal growth and the increase of alanine content might be associated with the assimilation of nitrogen in the presence of glucose as a carbon source. The N-fixing bacterium A. chroococcum should stimulate fungal amino acid metabolism via glutamine synthetase-glutamate synthase (GS-GOGAT). The data pointed to a stimulated glycolytic activity in the fungus observed by the accumulation of alanine, possibly via alanine aminotransferase. The responses toward the growth-inhibiting Streptomyces AcH 1003 suggest an (oxidative) stress response of the fungus.

Streptomyces AcH 505 triggers production of a salicylic acid analogue in the fungal pathogen Heterobasidion abietinum that enhances infection of Norway spruce seedlings.

The necrotrophic fungus Heterobasidion spp. is the causal agent of 'annosum root rot' of Norway spruce. In the presence of the rhizosphere bacterium Streptomyces AcH 505, enhanced colonization of Norway spruce roots with Heterobasidion abietinum 331 has previously been observed. By analyzing dual cultures of H. abietinum 331 and Streptomyces AcH 505 with HPLC, a fungal metabolite was identified that was increased in the presence of Streptomyces AcH 505. Likewise, challenge of H. abietum 331 with common antifungals produced by soil streptomycetes rendered the same effect. The structure of the compound, 5-formylsalicylic acid (5-FSA), was elucidated by HPLC-HR-ESI-Orbitrap-mass spectrometry and NMR spectroscopy. Based on in vivo measurements of maximum photosystem II efficiency of Norway spruce seedlings, 5-FSA did not influence plant vitality. However, when challenged with H. abietinum 331, ergosterol amounts in infected roots increased significantly for 5-FSA pre-treated seedlings. The severity of the infection was comparable to that observed in the presence of Streptomyces AcH 505. 5-FSA is a structural analogue of salicylic acid, an important signalling molecule active in plant defence. Thus, the expression of two defence-response related marker genes (PR1, Hel) was analysed in 5-FSA treated Arabidopsis thaliana seedlings by Northern blot analysis. The transcription of both marker genes was altered, indicating that 5-FSA is perceived by Arabidopsis in a similar manner to salicylic acid and is able to interfere with Arabidopsis defence signalling. The role of 5-FSA as a potential virulence factor of H. abietinum 331 in the presence of Streptomyces AcH 505 is discussed.

A whole-genome microarray study of Arabidopsis thaliana semisolid callus cultures exposed to microgravity and nonmicrogravity related spaceflight conditions for 5 days on board of Shenzhou 8.

The Simbox mission was the first joint space project between Germany and China in November 2011. Eleven-day-old Arabidopsis thaliana wild type semisolid callus cultures were integrated into fully automated plant cultivation containers and exposed to spaceflight conditions within the Simbox hardware on board of the spacecraft Shenzhou 8. The related ground experiment was conducted under similar conditions. The use of an in-flight centrifuge provided a 1 g gravitational field in space. The cells were metabolically quenched after 5 days via RNAlater injection. The impact on the Arabidopsis transcriptome was investigated by means of whole-genome gene expression analysis. The results show a major impact of nonmicrogravity related spaceflight conditions. Genes that were significantly altered in transcript abundance are mainly involved in protein phosphorylation and MAPK cascade-related signaling processes, as well as in the cellular defense and stress responses. In contrast to short-term effects of microgravity (seconds, minutes), this mission identified only minor changes after 5 days of microgravity. These concerned genes coding for proteins involved in the plastid-associated translation machinery, mitochondrial electron transport, and energy production.

Growth inhibition of an Araucaria angustifolia (Coniferopsida) fungal seed pathogen, Neofusicoccum parvum, by soil streptomycetes.

BACKGROUND: Araucariaceae are important forest trees of the southern hemisphere. Life expectancy of their seedlings can largely be reduced by fungal infections. In this study we have isolated and characterized such a fungus and investigated the potential of Streptomyces Actinobacteria from the respective rhizosphere to act as antagonists. RESULTS: The pathogenic fungus from Araucaria angustifolia seeds was identified by morphological markers (pore-associated Woronin-bodies) as belonging to the Pezizomycotina. Molecular data identified the fungus as Neofusicoccum parvum (Botryosphaeriaceae). Co-cultures on agar of this fungus with certain streptomycete isolates from the rhizosphere, and from the surface of Araucaria roots significantly reduced the growth of the fungus. HPLC analysis of the agar yielded streptomycete-specific exudate compounds which were partly identified. There were differences in compounds between single (bacteria, fungus) and dual cultures (bacteria + fungus). CONCLUSION: Streptomycetes from the rhizosphere of Araucariaceae produce exudates which can suppress the development of pathogenic fungi in their seeds.

Biotransformation of the fungal phytotoxin fomannoxin by soil streptomycetes.

Rhizosphere-associated Streptomyces sp. AcH 505 (AcH 505) promotes infection of Norway spruce (Picea abies) with the pathogenic fungus Heterobasidion abietinum 331, while Streptomyces sp. GB 4-2 (GB 4-2) enhances spruce defense against the fungus. To identify whether these bacteria influence the availability of the fungal phytotoxin fomannoxin and hence spruce infection, we analyzed the fomannoxin yield in H. abietinum 331-AcH 505 dual cultures. Further, the fate of fomannoxin was studied by adding the compound to cultures of AcH 505, GB 4-2 and nine other soil streptomycetes. Culture filtrates were extracted with ethyl acetate and analyzed by HPLC. Structures of novel compounds were elucidated by HPLC-HR-ESI-Orbitrap-MS and NMR spectroscopy. Phytotoxicity of the compounds was determined by in vivo measurement of maximum photosystem II efficiency of Arabidopsis thaliana seedlings. The amount of fomannoxin in H. abietinum 331-AcH 505 dual cultures was reduced compared to axenic fungus cultures and fungus-plant dual cultures. Following addition of fomannoxin to AcH 505 cultures, the compound disappeared and three novel fomannoxin derivatives without phytotoxic activity were detected. Another novel compound, fomannoxin amide, was discovered following fomannoxin addition to GB 4-2 cultures. Nine other streptomycetes converted fomannoxin into fomannoxin acid or fomannoxin amide. Both compounds exhibit the same phytotoxicity as fomannoxin. We, thus, conclude that the streptomycete-mediated modulation of spruce infection with H. abietinum 331 does not depend on the availability of fomannoxin. We further add evidence to the observation that the lipophilic side chain of fomannoxin is an important structural element for its phytotoxicity.

Production of fungal and bacterial growth modulating secondary metabolites is widespread among mycorrhiza-associated streptomycetes.

BACKGROUND: Studies on mycorrhiza associated bacteria suggest that bacterial-fungal interactions play important roles during mycorrhiza formation and affect plant health. We surveyed Streptomyces Actinobacteria, known as antibiotic producers and antagonists of fungi, from Norway spruce mycorrhizas with predominantly Piloderma species as the fungal partner. RESULTS: Fifteen Streptomyces isolates exhibited substantial variation in inhibition of tested mycorrhizal and plant pathogenic fungi (Amanita muscaria, Fusarium oxysporum, Hebeloma cylindrosporum, Heterobasidion abietinum, Heterobasidion annosum, Laccaria bicolor, Piloderma croceum). The growth of the mycorrhiza-forming fungus Laccaria bicolor was stimulated by some of the streptomycetes, and Piloderma croceum was only moderately affected. Bacteria responded to the streptomycetes differently than the fungi. For instance the strain Streptomyces sp. AcM11, which inhibited most tested fungi, was less inhibitory to bacteria than other tested streptomycetes. The determined patterns of Streptomyces-microbe interactions were associated with distinct patterns of secondary metabolite production. Notably, potentially novel metabolites were produced by strains that were less antagonistic to fungi. Most of the identified metabolites were antibiotics (e.g. cycloheximide, actiphenol) and siderophores (e.g. ferulic acid, desferroxiamines). Plant disease resistance was activated by a single streptomycete strain only. CONCLUSIONS: Mycorrhiza associated streptomycetes appear to have an important role in inhibiting the growth of fungi and bacteria. Additionally, our study indicates that the Streptomyces strains, which are not general antagonists of fungi, may produce still un-described metabolites.

Extending SILAC to proteomics of plant cell lines.

Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) is a widespread method for metabolic labeling of cells and tissues in quantitative proteomics; however, incomplete incorporation of the label has so far restricted its wider use in plants. Here, we argue that differential labeling by two different versions of the labeled amino acids renders SILAC fully applicable to dark-grown plant cell lines. By comparing Arabidopsis thaliana cell cultures labeled with two versions of heavy Lys (Lys-4 and Lys-8), we show that this simple modification of the SILAC protocol enables similar quantitation accuracy, precision, and reproducibility as conventional SILAC in animal cells.

Carbon allocation in ectomycorrhizal plants at limited optimal N supply: an attempt aat unraveling conflicting theories.

With regard to mycorrhiza, conflicting theories try to explain how the balance between fungal demand for carbohydrates and the plant's needs for nutrients varies, resulting in conflicting predictions. In order to evaluate current concepts, we investigated some metabolic parameters, which are indicative for plant carbon allocation in response to mycorrhization at limited and optimal N supply. Pinus pinaster seedlings were inoculated with living or dead (control) cultures of Pisolithus tinctorius, supplied with ammonium at 4 (limiting) or 7% d−1 (non-limiting) N relative addition rate (RARN), and followed development for 29 days. Mycorrhizal colonization of roots was quantified by the determination of ergosterol. A series of enzymes (sucrose and trehalose metabolism, anaplerosis) and metabolites (soluble carbohydrate, including trehalose; fructose 2,6 bisphosphate, free amino acids) relevant in the C/N exchange between symbionts, and in the carbon allocation and sink strength within the plant were assayed for 2-day-intervals for up to 14 days, and at 5-day-intervals for the rest of the experiment. The first 10 days reflected the establishment of mycorrhizal interaction, and the carbon allocation to the root was higher in M plants independent of N supply. Following this period, carbon allocation became N-related, higher at low, and lower at high N supply. The belowground C investment of M plants was dependent on N availability, but not on N gain. Finally, increased belowground C allocation was accompanied by a shift from plant to fungal metabolism.

RNAi-mediated suppression of isoprene emission in poplar transiently impacts phenolic metabolism under high temperature and high light intensities: a transcriptomic and metabolomic analysis.

In plants, isoprene plays a dual role: (a) as thermo-protective agent proposed to prevent degradation of enzymes/membrane structures involved in photosynthesis, and (b) as reactive molecule reducing abiotic oxidative stress. The present work addresses the question whether suppression of isoprene emission interferes with genome wide transcription rates and metabolite fluxes in grey poplar (Populus x canescens) throughout the growing season. Gene expression and metabolite profiles of isoprene emitting wild type plants and RNAi-mediated non-isoprene emitting poplars were compared by using poplar Affymetrix microarrays and non-targeted FT-ICR-MS (Fourier transform ion cyclotron resonance mass spectrometry). We observed a transcriptional down-regulation of genes encoding enzymes of phenylpropanoid regulatory and biosynthetic pathways, as well as distinct metabolic down-regulation of condensed tannins and anthocyanins, in non-isoprene emitting genotypes during July, when high temperature and light intensities possibly caused transient drought stress, as indicated by stomatal closure. Under these conditions leaves of non-isoprene emitting plants accumulated hydrogen peroxide (H(2)O(2)), a signaling molecule in stress response and negative regulator of anthocyanin biosynthesis. The absence of isoprene emission under high temperature and light stress resulted transiently in a new chemo(pheno)type with suppressed production of phenolic compounds. This may compromise inducible defenses and may render non-isoprene emitting poplars more susceptible to environmental stress.

WS-5995 B, an antifungal agent inducing differential gene expression in the conifer pathogen Heterobasidion annosum but not in Heterobasidion abietinum.

The mycorrhization helper bacterium Streptomyces sp. AcH 505 inhibits Norway spruce root infection and colonisation by the root and butt rot fungus Heterobasidion annosum 005 but not by the congeneric strain Heterobasidion abietinum 331 because of higher sensitivity of H. annosum 005 towards the AcH 505-derived naphthoquinone antibiotic WS-5995 B. Differences in antibiotic sensitivity between two isolates belonging to two species, H. annosum 005 and H. abietinum 331, were investigated by comparative gene expression analysis using macroarrays and quantitative RT-PCR after WS-5995 B, structurally related mollisin and unrelated cycloheximide application. Treatment with 25 microM WS-5995 B for 2 h resulted in a significant up-regulation of expression of inosine-5'-monophosphate dehydrogenase, phosphoglucomutase and GTPase genes, while the expression of genes encoding for thioredoxin and glutathione dependent formaldehyde dehydrogenase was down-regulated in the sensitive fungal strain. No differential expression in the tolerant strain was detected. Application of WS-5995 B at higher concentrations over a time course experiment revealed that H. annosum 005 and H. abietinum 331 responded differently to WS-5995 B. The fungal gene expression levels depended on both the concentration of WS-5995 B and the duration of its application. The WS-5995 B-unrelated cycloheximide caused highly specific changes in patterns of gene expression. Our findings indicate considerable variations in response to bacterial metabolites by the isolates of the conifer pathogen.

Changes in the effective gravitational field strength affect the state of phosphorylation of stress-related proteins in callus cultures of Arabidopsis thaliana.

In a recent study it was shown that callus cell cultures of Arabidopsis thaliana respond to changes in gravitational field strengths by changes in protein expression. Using ESI-MS/MS for proteins with differential abundance after separation by 2D-PAGE, 28 spots which changed reproducibly and significantly in amount (P <0.05) after 2 h of hypergravity (18 up-regulated, 10 down-regulated) could be identified. The corresponding proteins were largely involved in stress responses, including the detoxification of reactive oxygen species (ROS). In the present study, these investigations are extended to phosphorylated proteins. For this purpose, callus cell cultures of Arabidopsis thaliana were exposed to hypergravity (8 g) and simulated weightlessness (random positioning; RP) for up to 30 min, a period of time which yielded the most reliable data. The first changes, however, were visible as early as 10 min after the start of treatment. In comparison to 1 g controls, exposure to hypergravity resulted in 18 protein spots, and random positioning in 25, respectively, with increased/decreased signal intensity by at least 2-fold (P <0.05). Only one spot (alanine aminotransferase) responded the same way under both treatments. After 30 min of RP, four spots appeared, which could not be detected in control samples. Among the protein spots altered in phosphorylation, it was possible to identify 24 from those responding to random positioning and 12 which responded to 8 g. These 12 proteins (8 g) are partly (5 out of 12) the same as those changed in expression after exposure to 2 h of hypergravity. The respective proteins are involved in scavenging and detoxification of ROS (32%), primary metabolism (20.5%), general signalling (14.7%), protein translation and proteolysis (14.7%), and ion homeostasis (8.8%). Together with our recent data on protein expression, it is assumed that changes in gravitational fields induce the production of ROS. Our data further indicate that responses toward RP are more by post-translational protein modulation (most changes in the degree of phosphorylation occur under RP-treatment) than by protein expression (hypergravity).

Plant behavior upon contact with Streptomycetes.

Plants have a variety of chemical and anatomical defences, whose strengths depend on biotic and environmental influences. We show here that root inoculation with belowground bacteria, filamentous gram-positive streptomycetes, can induce plant defence responses. Such induced plant responses can occur belowground in the roots, but also aboveground, in the leaves, and include priming (sensitizing) like characters. Streptomycetes have also evolved mechanisms to facilitate plant root symbioses, mycorrhiza and root nodulation. By promoting fungal growth and by decreasing plant defence responses, these bacteria promote mycorrhiza formation. This minireview covers our current knowledge on the complex interactions that take place between streptomycetes, plants and rhizosphere microbes.

Root inoculation with a forest soil streptomycete leads to locally and systemically increased resistance against phytopathogens in Norway spruce.

Soil streptomycetes are commonly antagonistic against plant pathogens. However, interactions involving increased defense responses in the host plant, leading to suppression of plant disease development, have not yet been detailed. Here, the mechanisms were studied of disease suppression by Streptomyces sp. GB 4-2 against Heterobasidion root and butt rot in Norway spruce (Picea abies) seedlings. GB 4-2 promoted mycelial growth of the phytopathogenic fungus, germination rate of fungal spores, extension of germ tubes and early colonization of outer cortical layers of the plant root. Reduced colonization of the inner cortical cell layers was accompanied by the induction of cell wall appositions, and increased xylem formation in the vascular cylinder emerged after bacterium-fungus coinoculation. Bacterial treatment led to decreased water content in roots and needles and increased photosynthetic yield (F(v)/F(m)) and peroxidase activities in needles. The infection of needles by Botrytis cinerea was reduced by bacterial pretreatment. Complex interactions of GB 4-2 with Norway spruce and Heterobasidion abietinum were discovered. The bacterium promoted the growth of the phytopathogenic fungus but induced plant defense responses. Host responses indicate that GB 4-2 induces both local and systemic defense responses in Norway spruce.

Interaction with mycorrhiza helper bacterium Streptomyces sp. AcH 505 modifies organisation of actin cytoskeleton in the ectomycorrhizal fungus Amanita muscaria (fly agaric).

The actin cytoskeleton (AC) of fungal hyphae is a major determinant of hyphal shape and morphogenesis, implicated in controlling tip structure and secretory vesicle delivery. Hyphal growth of the ectomycorrhizal fungus Amanita muscaria and symbiosis formation with spruce are promoted by the mycorrhiza helper bacterium Streptomyces sp. AcH 505 (AcH 505). To investigate structural requirements of growth promotion, the effect of AcH 505 on A. muscaria hyphal morphology, AC and actin gene expression were studied. Hyphal diameter and mycelial density decreased during dual culture (DC), and indirect immunofluorescence microscopy revealed that the dense and polarised actin cap in hyphal tips of axenic A. muscaria changes to a loosened and dispersed structure in DC. Supplementation of growth medium with cell-free bacterial supernatant confirmed that reduction in hyphal diameter and AC changes occurred at the same stage of growth. Transcript levels of both actin genes isolated from A. muscaria remained unaltered, indicating that AC changes are regulated by reorganisation of the existing actin pool. In conclusion, the AC reorganisation appears to result in altered hyphal morphology and faster apical extension. The thus improved spreading of hyphae and increased probability to encounter plant roots highlights a mechanism behind the mycorrhiza helper effect.

Trehalose turnover during abiotic stress in arbuscular mycorrhizal fungi.

Trehalose is a common reserve carbohydrate in fungi, whose role has been recently extended to other cellular functions, such as stress tolerance, glycolysis control, sporulation and infectivity of some pathogenic strains. To gain some insight into the role of trehalose during abiotic stress in arbuscular mycorrhizal (AM) fungi, we assessed trehalose content as well as transcriptional regulation and enzyme activity of neutral trehalase and trehalose-6-phosphate phosphatase in Glomus intraradices in response to heat shock, chemical or osmotic stress. Prolonged or intensive exposure to heat or chemical stress, but not osmotic stress, caused an increase of trehalose in the cell. We found this associated with transient up-regulation of the trehalose-6-P phosphatase (GiTPS2) transcript that coincided with moderate increases in enzyme activity. By contrast, there were no changes in neutral trehalase (GiNTH1) RNA accumulation in response to stress treatments, while they promoted, in most cases, an increase in activity. After stress had ceased, trehalose returned to basal concentrations, pointing to a role of neutral trehalase activity in heat shock recovery. A yeast complementation assay confirmed the role of neutral trehalase in thermotolerance. Taken together, these results indicate that trehalose could play a role in AM fungi during the recovery from certain stresses such as heat shock and chemical treatment.

Suppression of plant defence response by a mycorrhiza helper bacterium.

The aim of the present study was to determine whether the mycorrhiza helper bacterium Streptomyces sp. AcH 505 could serve as a biocontrol agent against Heterobasidion root and butt rot. Bacterial influence on mycelial growth of Heterobasidion sp. isolates, on the colonization of wood discs and Norway spruce (Picea abies) roots was determined. The effect of AcH 505 on plant photosynthesis, peroxidase activity and gene expression, and needle infections were investigated. AcH 505 was antagonistic to 11 of 12 tested fungal Heterobasidion isolates. The antagonism resulted in a suppression of fungal colonization of Norway spruce roots and wood discs. Mycelial growth rate of the 12th strain, Heterobasidion abietinum 331 was not affected by AcH 505, and colonization of roots by this fungal strain was promoted by AcH 505. Bacterial inoculation led to decreased peroxidase activities and gene expression levels in roots. AcH 505 promotes plant root colonization by Heterobasidion strains that are tolerant to antifungal metabolites produced by the bacterium. This may result from unknown bacterial factors that suppress the plant defence response.

Increased trehalose biosynthesis in Hartig net hyphae of ectomycorrhizas.

To obtain photoassimilates in ectomycorrhizal symbiosis, the fungus has to create a strong sink, for example, by conversion of plant-derived hexoses into fungus-specific compounds. Trehalose is present in large quantities in Amanita muscaria and may thus constitute an important carbon sink. In Amanita muscaria-poplar (Populus tremula x tremuloides) ectomycorrhizas, the transcript abundances of genes encoding key enzymes of fungal trehalose biosynthesis, namely trehalose-6-phosphate synthase (TPS), trehalose-6-phosphate phosphatase (TPP) and trehalose phosphorylase (TP), were increased. When mycorrhizas were separated into mantle and Hartig net, TPS, TPP and TP expression was specifically enhanced in Hartig net hyphae. Compared with the extraradical mycelium, TPS and TPP expression was only slightly increased in the fungal sheath, while the increase in the expression of TP was more pronounced. TPS enzyme activity was also elevated in Hartig net hyphae, displaying a direct correlation between transcript abundance and turnover rate. In accordance with enhanced gene expression and TPS activity, trehalose content was 2.7 times higher in the Hartig net. The enhanced trehalose biosynthesis at the plant-fungus interface indicates that trehalose is a relevant carbohydrate sink in symbiosis. As sugar and nitrogen supply affected gene expression only slightly, the strongly increased expression of the investigated genes in mycorrhizas is presumably developmentally regulated.

Development of bioinformatic tools to support EST-sequencing, in silico- and microarray-based transcriptome profiling in mycorrhizal symbioses.

The great majority of terrestrial plants enters a beneficial arbuscular mycorrhiza (AM) or ectomycorrhiza (ECM) symbiosis with soil fungi. In the SPP 1084 "MolMyk: Molecular Basics of Mycorrhizal Symbioses", high-throughput EST-sequencing was performed to obtain snapshots of the plant and fungal transcriptome in mycorrhizal roots and in extraradical hyphae. To focus activities, the interactions between Medicago truncatula and Glomus intraradices as well as Populus tremula and Amanita muscaria were selected as models for AM and ECM symbioses, respectively. Together, almost, 20.000 expressed sequence tags (ESTs) were generated from different random and suppressive subtractive hybridization (SSH) cDNA libraries, providing a comprehensive overview of the mycorrhizal transcriptome. To automatically cluster and annotate EST-sequences, the BioMake and SAMS software tools were developed. In connection with the eNorthern software SteN, plant genes with a predicted mycorrhiza-induced expression were identified. To support experimental transcriptome profiling, macro- and microarray tools have been constructed for the two model mycorrhizae, based either on PCR-amplified cDNAs or 70mer oligonucleotides. These arrays were used to profile the transcriptome of AM and ECM roots under different conditions, and the data obtained were uploaded to the ArrayLIMS and EMMA databases that are designed to store and evaluate expression profiles from DNA arrays. Together, the EST- and transcriptome databases can be mined to identify candidate genes for targeted functional studies.

Time-course of changes in amounts of specific proteins upon exposure to hyper-g, 2-D clinorotation, and 3-D random positioning of Arabidopsis cell cultures.

In previous studies it has been shown that callus cell cultures of Arabidopsis thaliana respond to changes in gravitational field strengths by altered gene expression. In this study an investigation was carried out into how different g conditions affect the proteome of such cells. For this purpose, callus cells were exposed to 8 g (centrifugation) and simulated microgravity (2-D clinorotation: fast rotating clinostat, yielding 0.0016 g at maximum; and 3-D random positioning) for up to 16 h. Extracts containing total soluble protein were subjected to 2-D SDS-PAGE. Image analysis of Sypro Ruby-stained gels showed that approximately 28 spots reproducibly and significantly (P <0.05) changed in amount after 2 h of hypergravity (18 up- and 10 down-regulated). These spots were analysed by electrospray ionization tandem mass spectrometry (ESI-MS/MS). In the case of 2-D clinorotation, 19 proteins changed in a manner similar to hypergravity, while random positioning affected only eight spots. Identified proteins were mainly stress related, and are involved in detoxification of reactive oxygen species, signalling, and calcium binding. Surprisingly, centrifugation and clinorotation showed homologies which were not detected for random positioning. The data indicate that simulation of weightlessness is different between clinorotation and random positioning.

Phosphoenolpyruvate carboxylase genes in C3, crassulacean acid metabolism (CAM) and C3/CAM intermediate species of the genus Clusia: rapid reversible C3/CAM switches are based on the C3 housekeeping gene.

The genus Clusia includes species that exhibit either the C3 or crassulacean acid metabolism (CAM) mode of photosynthesis, or those that are able to switch between both modes according to water availability. In order to screen for species-specific genetic variability, we investigated the key carboxylase for CAM, phosphoenolpyruvate carboxylase (PEPC). Sequence analysis of DNA isolated from the obligate CAM species, Clusia hilariana, the obligate C3 species, Clusia multiflora, and an intermediate species that can switch between C3 and CAM photosynthesis, Clusia minor, revealed three different isoforms for C. hilariana and one each for the other two species. Sequence alignments indicated that PEPC from the intermediate species had high homology with the C3 protein and with one of CAM plant proteins. These were assumed to constitute 'housekeeping' proteins, which can also support CAM in intermediate species. The other two isoforms of the CAM plant C. hilariana were either CAM-specific or showed homologies with PEPC from roots. Phylogenetic trees derived from neighbour-joining analysis of amino acid sequences from 13 different Clusia species resulted in two distinct groups of plants with either 'housekeeping' PEPC only, or additionally CAM-related isoforms. Only C. hilariana showed the third, probably root-specific isoform. The high homology of the PEPC from the intermediate species with the C3 protein indicates that for the reversible transition from the C3 to CAM mode of photosynthesis, the C3 type of PEPC is sufficient. Its expression, however, is strongly increased under CAM-inducing conditions. The use of the C3 isoform could have facilitated the evolution of CAM within the genus, which occurred independently for several times.