Volatiles of rhizobacteria Serratia and Stenotrophomonas alter growth and metabolite composition of Arabidopsis thaliana.

The emission of volatiles is a common, but mostly neglected, ability of bacteria that is important for inter- and intraspecific interactions. Currently, limited information is available on how the bacterial volatile (mVOC) signal is integrated into a plant's life at the physiological, transcriptional and metabolic level. Previous results provided evidence for volatile-dependent regulation of WRKY18, a pathogen-responsive transcription factor of Arabidopsis thaliana in co-culture with two rhizobacteria, Serratia plymuthica HRO-C48 and Stenotrophomonas maltophilia R3089. Dual cultures of these bacteria and A. thaliana; application of the common mVOC 2-phenyl-ethanol; extraction of metabolites of A. thaliana after exposure to bacterial volatiles; and analysis of the metabolomes (GC-TOF/MS) were carried out. The prominent microbial aromatic compound 2-phenyl-ethanol, emitted by both bacteria, negatively affects growth of A. thaliana wild type, whereas WRKY18 T-DNA insertion mutants were significantly more tolerant than wild-type seedlings. This paper also demonstrates for the first time the impact of the rhizobacterial volatiles on the metabolome of A. thaliana. Upon mVOC exposure the plants rearrange their metabolism by accumulation of e.g. amino acids and TCA intermediates that potentially allow plants to cope with and survive this stress. Our findings illustrate the high degree of complexity of metabolic rearrangements underlying the interactions of bacterial volatile elicitors and resulting plant responses. Furthermore, the impact of the volatile 2-phenyl-ethanol as a signal in the WRKY18-dependent pathway highlights this compound as an important molecular player.

Biogenic volatile emissions from the soil.

Volatile compounds are usually associated with an appearance/presence in the atmosphere. Recent advances, however, indicated that the soil is a huge reservoir and source of biogenic volatile organic compounds (bVOCs), which are formed from decomposing litter and dead organic material or are synthesized by underground living organism or organs and tissues of plants. This review summarizes the scarce available data on the exchange of VOCs between soil and atmosphere and the features of the soil and particle structure allowing diffusion of volatiles in the soil, which is the prerequisite for biological VOC-based interactions. In fact, soil may function either as a sink or as a source of bVOCs. Soil VOC emissions to the atmosphere are often 1-2 (0-3) orders of magnitude lower than those from aboveground vegetation. Microorganisms and the plant root system are the major sources for bVOCs. The current methodology to detect belowground volatiles is described as well as the metabolic capabilities resulting in the wealth of microbial and root VOC emissions. Furthermore, VOC profiles are discussed as non-destructive fingerprints for the detection of organisms. In the last chapter, belowground volatile-based bi- and multi-trophic interactions between microorganisms, plants and invertebrates in the soil are discussed.

Effects of phytoestrogen extracts isolated from flax on hormone production of trophoblast tumour cells Jeg 3 and BeWo.

UNLABELLED: AIM AND SETTING: To test the effects of crude extracts from flax (Linum usitatissimum) on progesterone and estradiol and ERα and ß/PR production in choriocarcinoma cell lines Jeg 3 and BeWo. Tumor trophoblast cells (Jeg 3 and BeWo) were incubated in the presence of different concentrations of the flax crude extracts. Estradiol and progesterone production was measured. Estrogen receptor α and ß as well as progesterone receptor expressions were also assessed. RESULTS: In Jeg 3 cells, progesterone production was downregulated by flax root and leaves extract, while in BeWo cells only flax root extract did manage to downregulate progesterone production. ERß expression was significantly downregulated by flax root and flax leaves extract in both cell lines; on the contrary, ERα expression was increased by flax leaves extract in BeWo cells. PR expression was downregulated by flax leaves extract in Jeg 3 and by flax root extract in BeWo cells. CONCLUSION: Flax extracts derived from leaves and especially from roots can modify progesterone and possibly estradiol production, while at the same time they seem to alter ERß expression. Further studies on animal models and adequately designed retrospective epidemiological studies are imperative to clarify this role upon progesterone.

Online monitoring of cellular metabolism in the MCF-7 carcinoma cell line treated with phytoestrogen extracts.

BACKGROUND: Phytoestrogens are naturally occurring, plant-derived, nonsteroidal phytochemicals with anticarcinogenic potential. The aim of this study was to isolate phytoestrogens from the flax root of Linum usitatissimum and to test their effect on cellular metabolism in the human mammalian carcinoma cell line MCF-7 using the Bionas 2500 analysis system. MATERIALS AND METHODS: Metabolically relevant parameters such as acidification, oxygen consumption and cell adhesion were registered continuously over 8 and 24 hours on six sensor chips in parallel at different concentrations of flax root extracts. RESULTS: The extracts from flax roots of L. usitatissimum reduced extracellular acidification, respiration and adhesion in a concentration-dependent manner. CONCLUSION: The Bionas 2500 analysis system allows multiparametric online monitoring of cellular processes and can be used to detect the mode of action of anticarcinogenic compounds in cellular metabolism.

Effects of phytoestrogen extracts isolated from flax on estradiol production and ER/PR expression in MCF7 breast cancer cells.

BACKGROUND: In this study, we tested the effects of crude extracts from flax (Linum usitatissimum) on the production of estradiol and expression of estrogen receptor (ER) and progesterone receptor (PR) in human breast cancer MCF7 cells. MATERIALS AND METHODS: Isoflavone and lignan extracts from flax plant Linum usitatissimum were obtained, using different extraction methods. Breast carcinoma cells (MCF7) were incubated with various concentrations of the isolated extracts. Untreated MCF7 cells were used as controls. Supernatants were removed at designated times and tested for estradiol with an ELISA method. Furthermore, the effect of phytoestrogen extracts on the production of ERa and ERbeta as well as on PR was examined. RESULTS AND CONCLUSION: Production of estradiol is elevated in MCF7 cells in a concentration-dependent manner after stimulation with isoflavone and lignan extracts from Linum usitatissimum. Expression of ERalpha is up-regulated after stimulation with lower concentrations of lignan extracts from flax plants, unchanged at median concentrations and down-regulated at high concentrations. Expression of ERbeta is down-regulated in a concentration-dependent manner.

Effects of phytoestrogen extracts from Linum usitatissimum on the Jeg3 human trophoblast tumour cell line.

BACKGROUND: Phytoestrogens are a diverse group of nonsteroidal plant compounds which have similar effects to endogenous estrogens in humans and have been ascribed potential anticarcinogenic activities. We tested the effects of phytoestrogen extracts from different plant organs of flax, Linum usitatissimum, on cell proliferation in trophoblast tumour cells of the cell line Jeg3. MATERIALS AND METHODS: Phytoestrogen extracts were prepared from leaves, stems and roots of L. usitatissimum using different extraction methods. The isolated phytoestrogens were identified using HPLC-MS analysis. The influence on cell proliferation (MTT test) was determined in the trophoblast tumour cells, Jeg3. RESULTS: Cell proliferation of trophoblast tumour Jeg3 cells was significantly affected by the phytoestrogens isolated from leaves, stems and roots of L. usitatissimum. Root extracts inhibited Jeg3 cell growth significantly. CONCLUSION: A cell culture model system of the human trophoblast tumour cell line, Jeg3, was established to test the effect of potential phytoestrogens on cell proliferation. It was shown that the roots of L. usitatissimum contain measurable concentrations of lignans and isoflavones.

Localization of methyl benzoate synthesis and emission in Stephanotis floribunda and Nicotiana suaveolens flowers.

The emission of fragrances can qualitatively and quantitatively differ in different parts of flowers. A detailed analysis was initiated to localize the floral tissues and cells which contribute to scent synthesis in STEPHANOTIS FLORIBUNDA (Asclepiadaceae) and NICOTIANA SUAVEOLENS (Solanaceae). The emission of scent compounds in these species is primarily found in the lobes of the corollas and little/no emission can be attributed to other floral organs or tissues. The rim and centre of the petal lobes of S. FLORIBUNDA contribute equally to scent production since the amount of SAMT (salicylic acid carboxyl methyltransferase) and specific SAMT activity compensate each other in the rim region and centre region. IN SITU immunolocalizations with antibodies against the methyl benzoate and methyl salicylate-synthesizing enzyme indicate that the adaxial epidermis with few subepidermal cell layers of S. FLORIBUNDA is the site of SAMT accumulation. In N. SUAVEOLENS flowers, the petal rim emits twice as much methyl benzoate due to higher total protein concentrations in the rim versus the petal centre; and, both the adaxial and abaxial epidermis house the BSMT (salicylic acid/benzoic acid carboxyl methyltransferase).

Effects of phytoestrogen extracts isolated from rye, green and yellow pea seeds on hormone production and proliferation of trophoblast tumor cells Jeg3.

BACKGROUND: Phytoestrogens are a diverse group of non-steroidal plant compounds. Because they have chemical structures similar to estrogens they are able to bind on estrogen receptors in humans. OBJECTIVES: In this study, we tested the effects of crude phytoestrogen extracts from rye (Secale cereale), green pea (Pisum sativum) and yellow pea seeds (Pisum sativum cv.) on cell proliferation and the production of progesterone in trophoblast tumor cells of the cell line Jeg3. METHODS: Isoflavone extracts from green and yellow pea seeds and lignan extracts from rye seeds were obtained, using different extraction methods. Isolated extracts were incubated in different concentrations with trophoblast tumor cells. Untreated cells were used as controls. At designated times, aliquots were removed and tested for estradiol and progesterone production. In addition, we tested the effects of the phytoestrogen extracts on cell proliferation. RESULTS: Cell proliferation is significantly inhibited by potential phytoestrogens isolated from rye, green and yellow pea seeds in trophoblast tumor cells of the cell line Jeg3. We found a correlation between the effects of proliferation and production of estradiol in isoflavone extracts from green and yellow pea seeds in Jeg3 cells. In addition, higher concentrations of isoflavones isolated from green pea seeds and lignans from rye showed also a inhibition of progesterone production whereas higher concentrations of rye lignans elevated estradiol production in Jeg3 cells. CONCLUSION: A useful indicator test system for potential phytoestrogens could be established. Based on the obtained results it is proposed that green and yellow pea seeds contain measurable concentrations of isoflavones and rye seeds contain lignans which can be isolated and used for special human diet programs.

Flax-seed extracts with phytoestrogenic effects on a hormone receptor-positive tumour cell line.

UNLABELLED: The higher soy intake in the Asian population compared to Europeans is believed to be an essential factor for the lower incidence of hormone-dependent tumours in Asia. It has already been shown that soya beans, with their ingredients genistein and daidzein from the isoflavonoid group, have protective effects on hormone-caused diseases. Lignans are another, less investigated, group of phytoestrogens. The aim of this study was to investigate the effects of flax-seed, which is typically found in Northern European diets, on the proliferation and hormone production of an estrogen receptor (ER)-positive trophoblast tumour cell line. MATERIALS AND METHODS: Trophoblast tumour cells of the cell line Jeg3 were incubated with 2 different concentrations of the isolated crude extract of flax-seed and 7 chemically partitioned extract fractions. Untreated cells were used as controls. After 48 h of stimulation, cell proliferation was measured using the BrdU method. The concentrations of hCG and progesterone produced by the trophoblast tumour cells were measured 48 h after stimulation. Extract fractions with antiproliferative effects in the BrdU- test were analysed by HPLC-MS. RESULTS: Our study showed an inhibitory influence of some of the isolated flax-seed fractions on the Jeg3 tumour cells. Proliferation of the Jeg3 cells was decreased by flax-seed fractions I, V, VI and VII in a dose-dependent manner. Inhibition of hCG production by flax-seed extracts III, V, VI and VII was also dose-dependent. Extract fractions V and VI decreased the production of progesterone by 58% to 86%. Some extract fractions showed a stimulating effect on hormone production and cell proliferation. HPLC-MS analysis showed the presence of matairesinol and biochanin A in flax-seed fraction VI. DISCUSSION: Flax-seed seems to have similar inhibitory effects to soya on hormone production and proliferation of hormone-sensitive tumour cells. Our results showed a dose-dependent inhibition by isolated flax-seed extracts on the Jeg3 cell line. Matairesinol and biochanin A seem to be useful candidates for extended tests on other tumour cell lines and normal tissues to evaluate the potential benefit of a lignan-containing therapy in hormone-dependent diseases.

Visual representation by atomic force microscopy (AFM) of tomato spotted wilt virus ribonucleoproteins.

Atomic force microscopy (AFM) allows the observation of biological material without fixation procedures. Here we present AFM images of ribonucleoproteins (nucleocapsids) derived from a plant infecting RNA virus (tomato spotted wilt virus, TSWV), which have been recorded in contact mode. The nucleocapsids, prepared from systemically infected leaves of tobacco, were spreaded on a glass surface and dried in air, and appeared as regularly formed rings, resembling the proposed pseudocircular and panhandle structure of encapsidated genomic RNA. Average values between 1300 and 2200 nm of nucleocapsid lengths could be related to dimensions estimated by electron microscopy, thereby validating a filamentous configuration of the TSWV ribonucleoproteins. However, to our knowledge regular, ring-like forms of ribonucleoproteins have not been obtained by electron microscopy, which rather showed an amorphous structure of the virus particles. Hence, the AFM approach provides a starting point for further detailed studies on TSWV ribonucleoprotein complexes.

Circadian expression of the light-harvesting complex protein genes in plants.

Photosynthesis is one of the important processes that enable life on earth. To optimize photosynthesis reactions during a solar day, most of them are timed to be active during the light phase. This includes the components of the thylakoid membranes in chloroplasts. Prominent representatives are the proteins of the light-harvesting complex (LHC). The synthesis of both the Lhc mRNA and the LHC protein occurs during the day and is regulated by the circadian clock, exhibiting the following pattern: increasing levels after sunrise, reaching a maximum around noon, and decreasing levels in the afternoon. To elucidate the involved control elements and regulatory circuits, the following strategies were applied: (1) analysis of promoters of Lhc genes, (2) analysis of DNA binding proteins, and (3) screening and investigation of mutants. The most promising elements found so far that may be involved in mediating the circadian rhythmicity of Lhc mRNA oscillations are a myb-like transcription factor CCA1 (Wang et al. 1997) and the corresponding DNA binding sequence (Piechulla et al. 1998).

Identification of tomato Lhc promoter regions necessary for circadian expression.

Expression of the light-harvesting complex protein genes (Lhc) is under the control of a circadian clock. To dissect the molecular regulatory components of the circadian clock a promoter deletion analysis of four tomato Lhc genes was performed in transgenic tobacco plants. The important 5'-upstream promoter regions are present at different positions relative to the transcription start site of Lhc b1*1, b1*2, Lhc a3 and Lhc a4. A short sequence of 47 nucleotides is necessary for conferring circadian Lhc mRNA oscillations. Sequence alignment of the specified promoter regions revealed a novel motif 'CAANNNNATC'. This motif is conserved in 5'-upstream regions of clock controlled Lhc genes and overlaps with a sequence relevant in phytochrome mediated gene expression.

Circadian oscillations of Lhc mRNAs in a photoautotrophic cell culture of Lycopersicon peruvianum.

Fourteen genes encoding proteins of the light harvesting complex (Lhc) are expressed in a photoautotrophic cell culture from the wild species of tomato (Lycopersicon peruvianum). For two genes, Lhca2 (cab7) and Lhcb2(*)1 (cab4), a rhythmic oscillation of the transcript accumulation is observed under light/dark and constant dark conditions indicating that gene expression is controlled by a circadian clock in the tomato cell culture. The circadian expression of the Lhc genes remains present after application of 2,2'-dipyridyl. However, the amplitude of Lhc mRNA oscillations and the photosynthetic capacity (Fmax/Fo) decrease significantly. The transcript accumulations of psbA, rbcS and rbcL are less or not at all affected by 2,2'-dipyridyl.

Diurnal Lhc gene expression is present in many but not all species of the plant kingdom.

The diurnal and circadian expression of light-harvesting genes (Lhc) is well documented for many plant species of the 'Angiospermae' division. Here we present the diurnal mRNA levels of species of the Gymnospermae, Pteridophyta, Bryophyta and Phycophyta divisions. Except for four Coniferophytina species, diurnal Lhc mRNA accumulation is detected in fern, moss and algae, supporting the idea that the concept of 'ciracadian clock'-controlled gene expression is an ancient process. Possible reasons why plants need the 'circadian clock' control mechanism are discussed.

Concerted circadian oscillations in transcript levels of nineteen Lha/b (cab) genes in Lycopersicon esculentum (tomato).

Steady-state mRNA levels of nineteen members of the Lha/b (cab) gene family of Lycopersicon esculentum, encoding nine different types of light-harvesting complex (LHC) polypeptides, were determined by primer extension analysis. Each Lha/b gene is expressed and individual mRNAs accumulate to distinct levels. The relative contribution of each Lha/b mRNA to the total Lha/b mRNA levels is very similar in different green organs (leaves, stems, fruits, sepals) and after light treatment of etiolated seedlings. Detailed analysis of Lha/b mRNA accumulation in leaves under light/dark conditions, continuous darkness and continuous light revealed diurnal and circadian oscillations of Lha/b mRNAs for all genes. Only minor instances of divergence from a general expression pattern are apparent. Together these results indicate a concerted expression of all genes, suggesting that similar or identical molecular mechanisms and signal transduction chain control the expression of all Lha/b genes.

Diurnal and Circadian Light-Harvesting Complex and Quinone B-Binding Protein Synthesis in Leaves of Tomato (Lycopersicon esculentum).

In leaves of tomato (Lycopersicon esculentum), the synthesis of a light-harvesting complex (LHC) polypeptide of photosystem II and the quinone B (Q(B))-binding protein varies at different time points during the day. In vivo labeling with [(35)S]methionine revealed diurnal oscillations of synthesis of these thylakoid membrane proteins. Both proteins are synthesized at elevated levels right after the transition from darkness to light, a maximum is reached around noon, and decreasing levels were measured during the afternoon and night. In addition, in constant darkness both proteins were also synthesized to varied extents at different diurnal time points. Together, these results indicate that the synthesis of a LHC II and the Q(B)-binding protein is under the control of the circadian clock. This circadian oscillation of LHC II protein synthesis correlates with the very well documented circadian Lhc a/b mRNA accumulation.

Determination of steady-state mRNA levels of individual chlorophyll a/b binding protein genes of the tomato cab gene family.

The steady-state levels of mRNA produced by 14 genes encoding members of the tomato chlorophyll a/b binding protein family were quantified. All genes were found to be expressed in leaf tissue, but the mRNAs accumulated to significantly different levels. The transcripts of cab 1A, cab 1B, cab 3A and cab 3B, encoding the Type I LHC proteins of photosystem II, are abundant, while low levels were measured for mRNAs encoding the Type II LHC II and the LHC I proteins. Sequences from the 5' upstream regions (-400 to translational start) of some cab genes were determined in this study, and a total of 16 tomato cab gene promoters for which sequences are now available were analyzed. Significant sequence conservation was found for those genes which are tandemly linked on the chromosome. However, the level of sequence conservation is different for the different cab subfamilies, e.g. 85% similarity between cab 1A and cab 1D vs. 45% sequence similarity between cab 3A and cab 3C upstream sequences. Characteristic GATA repeats with a conserved spacing were found in 5' upstream sequences of cab 1A-D, cab 3A-C, cab 11 and cab 12. The consensus sequence CCTTATCAT, which is believed to mediate light responsiveness, was found at different locations in the upstream sequences of cab 6B, cab 7, cab 8, cab 9, cab 10A, cab 10B and cab 11. In 11 out of 15 genes the transcription initiation site was found to center on the triplet TCA.

Effect of Temperature Alterations on the Diurnal Expression Pattern of the Chlorophyll a/b Binding Proteins in Tomato Seedlings.

In the leaves of plants that are grown in the natural environment, the accumulation of mRNAs encoding the chlorophyll a/b binding proteins (CAB) follow a circadian rhythm. It is generally accepted that the day/night (sunset, light/dark) or night/day (sunrise, dark/light) transitions play an important role in the synchronization of the rhythm and the determination of the accumulation amplitude. As the results of the experiments presented in this paper indicate, temperature alterations also support the setting and the arrangement of the rhythm. Apparently, simulating "day/night" temperature alternations influences the tomato (Lycopersicon esculentum) plants to express a typical circadian oscillation pattern of cab mRNAs. This rhythm was sustained in the plants after long-term exposure to an alternating temperature regime. In constant conditions, e.g. continuous illumination at either 18 degrees C or 24 degrees C or in continuous darkness at 24 degrees C, this diurnal fluctuation pattern with a period of about 24 hours remained present for at least 2 days.