Metabolomics-oriented isolation and structure elucidation of 37 compounds including two anthocyanins from Arabidopsis thaliana

In order to conduct metabolomic studies in a model plant for genome research, such as Arabidopsis thaliana (Arabidopsis), it is a prerequisite to obtain structural information for the isolated metabolites from the plant of interest. In this study, we isolated metabolites of Arabidopsis in a relatively non-targeted way, aiming at the construction of metabolite standards and chemotaxonomic comparison. Anthocyanins (5 and 7) called A8 and A10 were isolated and their structures were elucidated as cyanidin 3-O-[2-O-(β-d-xylopyranosyl)-6-O-(4-O-(β-d-glucopyranosyl)-E-p-coumaroyl)-β-d-glucopyranoside]-5-O-[6-O-(malonyl)-β-d-glucopyranoside] and cyanidin 3-O-[2-O-(2-O-(E-sinapoyl)-β-d-xylopyranosyl)-6-O-(4-O-(β-d-glucopyranosyl)-E-p-coumaroyl)-β-d-glucopyranoside]-5-O-[β-d-glucopyranoside] from analyses of 1D NMR, 2D NMR (¹H NMR, NOE, ¹³C NMR, HMBC and HMQC), HRFABMS, FT-ESI-MS and GC-TOF-MS data. In addition, 35 known compounds, including six anthocyanins, eight flavonols, one nucleoside, one indole glucosinolate, four phenylpropanoids and a derivative, together with three indoles, one carotenoid, one apocarotenoid, three galactolipids, two chlorophyll derivatives, one steroid, one hydrocarbon, and two dicarboxylic acids, were also isolated and identified from their spectroscopic data.     

Proteome analysis differentiates between two highly homologues germin-like proteins in Arabidopsis thaliana ecotypes Col-0 and Ws-2

A proteome approach based on 2-D gel electrophoresis (2-DE) was used to compare the protein patterns of the Arabidopsis ecotypes Col-0 and Ws-2. In leaf extracts a pair of protein spots were found to be diagnostic for each of the lines. Both pairs of spots were identified as closely related germin-like proteins differing in only one amino acid by using peptide mass finger printing of tryptic digests and by gaining additional data from post-source decay spectra in the MALDI-TOF analysis. Western blot analysis after separation of protein extracts by 2-DE confirmed results from Coomassie blue-stained gels and revealed additional immunoreactive spots for both ecotypes most likely representing dimers of the spots first identified. Western blot analysis and mass spectrometrical identification of the corresponding weakly stained protein in Coomassie blue-stained gels of the ecotype Col-0 also demonstrated for the first time the occurrence of AtGER3 protein in root extracts. Our results demonstrate the capacity of proteome analysis to analyse and distinguish closely related members of large protein families.     

Integration of XAS techniques and genetic methodologies to explore Cs-tolerance in Arabidopsis

We have characterised genetically and phenotypically a T-DNA insertion mutant line of A. thaliana (L.) Heynh. selected for Cs resistance when germinating and growing on Cs concentrations up to 600microM, lethal for the wild type. Measures of concentration and localisation of Cs, K, and Ca have been conducted on plants grown in vivo also utilising synchrotron light-based techniques as micro-SRXF (synchrotron radiation induced X-ray micro-fluorescence) and micro-XANES (micro X-ray absorption near edge structure) spectroscopy. We report here an attempt to apply micro-XANES to investigate Cs speciation and to measure the Cs content of living plants. The results obtained with micro-SRXF and micro-XANES spectroscopy complemented the genetic and physiological analyses: a comparison between wild type and mutant plants led to the conclusion that in our case a single gene mutation impairs Cs uptake and translocation, K and Ca homeostasis and plant biomass production.     

Boron transport in plants: co-ordinated regulation of transporters

Background

The essentiality of boron (B) for plant growth was established >85 years ago. In the last decade, it has been revealed that one of the physiological roles of B is cross-linking the pectic polysaccharide rhamnogalacturonan II in primary cell walls. Borate cross-linking of pectic networks serves both for physical strength of cell walls and for cell adhesion. On the other hand, high concentrations of B are toxic to plant growth. To avoid deficiency and toxicity problems, it is important for plants to maintain their tissue B concentrations within an optimum range by regulating transport processes. Boron transport was long believed to be a passive, unregulated process, but the identification of B transporters has suggested that plants sense and respond to the B conditions and regulate transporters to maintain B homeostasis.

Scope

Transporters responsible for efficient B uptake by roots, xylem loading and B distribution among leaves have been described. These transporters are required under B limitation for efficient acquisition and utilization of B. Transporters important for tolerating high B levels in the environment have also been identified, and these transporters export B from roots back to the soil. Two types of transporters are involved in these processes: NIPs (nodulin-26-like intrinsic proteins), boric acid channels, and BORs, B exporters. It is demonstrated that the expression of genes encoding these transporters is finely regulated in response to B availability in the environment to ensure tissue B homeostasis. Furthermore, plants tolerant to stress produced by low B or high B in the environment can be generated through altered expression of these transporters.

Conclusions

The identification of the first B transporter led to the discovery that B transport was a process mediated not only by passive diffusion but also by transporters whose activity was regulated in response to B conditions. Now it is evident that plants sense internal and external B conditions and regulate B transport by modulating the expression and/or accumulation of these transporters. Results obtained in model plants are applicable to other plant species, and such knowledge may be useful in designing plants or crops tolerant to soils containing low or high B.     

3,4-Dichloroaniline is detoxified and exported via different pathways in Arabidopsis and soybean

The metabolic fate of [UL-14C]-3,4-dichloroaniline (DCA) was investigated in Arabidopsis root cultures and soybean plants over a 48 h period following treatment via the root media. DCA was rapidly taken up by both species and metabolised, predominantly to N-malonyl-DCA in soybean and N-glucosyl-DCA in Arabidopsis. Synthesis occurred in the roots and the respective conjugates were largely exported into the culture medium, a smaller proportion being retained within the plant tissue. Once conjugated, the DCA metabolites in the medium were not then readily taken up by roots of either species. The difference in the routes of DCA detoxification in the two plants could be explained partly by the relative activities of the respective conjugating enzymes, soybean containing high DCA-N-malonyltransferase activity, while in Arabidopsis DCA-N-glucosyltransferase activity predominated. A pre-treatment of plants with DCA increased DCA-N-malonyltransferase activity in soybean but not in Arabidopsis, indicating differential regulation of this enzyme in the two plant species. This study demonstrates that DCA can undergo two distinct detoxification mechanisms which both lead to the export of conjugated metabolites from roots into the surrounding medium in contrast to the vacuolar deposition more commonly associated with the metabolism of xenobiotics in plants.     

A Mutant of Arabidopsis thaliana lpar;L.) Heynh. with Modified Control of Aspartate Kinase by Threonine

Mutagenesis and subsequent selection of Arabidopsis thaliana plantlets on a growth inhibitory concentration of lysine has led to the isolation of lysine-resistant mutants. The ability to grow on 2 m M lysine has been used to isolate mutants that may contain an aspartate kinase with altered regulatory-feedback properties. One of these mutants (RL 4) was characterized by a relative enhancement of soluble lysine. The recessive monogenic nuclear transmission of the resistance trait was established. It was associated with an aspartate kinase less sensitive to feedback inhibition by threonine. Two mutants (RLT 40 and RL 4) in Arabidopsis, characterized by an altered regulation of aspartate kinase, were crossed to assess the effects of the simultaneous presence of these different aspartate kinase forms. A double mutant (RLT40 × RL4) was isolated and characterized by two feedback-desensitized isozymes of aspartate kinase to, respectively, lysine and threonine but no threonine and/or lysine overproduction was observed. Genetical analysis of this unique double aspartate kinase mutant indicated that both mutations were located on chromosome 2, but their loci (ak1and ak2) were found to be unlinked.     

Female sterility associated with increased clonal propagation suggests a unique combination of androdioecy and asexual reproduction in populations of Cardamine amara (Brassicaceae)

Background and Aims The coexistence of hermaphrodites and female-sterile individuals, or androdioecy, has been documented in only a handful of plants and animals. This study reports its existence in the plant species Cardamine amara (Brassicaceae), in which female-sterile individuals have shorter pistils than seed-producing hermaphrodites.Methods Morphological analysis, in situ manual pollination, microsatellite genotyping and differential gene expression analysis using Arabidopsis microarrays were used to delimit variation between female-sterile individuals and hermaphrodites.Key Results Female sterility in C. amara appears to be caused by disrupted ovule development. It was associated with a 2.4- to 2.9-fold increase in clonal propagation. This made the pollen number of female-sterile genets more than double that of hermaphrodite genets, which fulfils a condition of co-existence predicted by simple androdioecy theories. When female-sterile individuals were observed in wild androdioecious populations, their ramet frequencies ranged from 5 to 54 %; however, their genet frequencies ranged from 11 to 29 %, which is consistent with the theoretically predicted upper limit of 50 %.Conclusions The results suggest that a combination of sexual reproduction and increased asexual proliferation by female-sterile individuals probably explains the invasion and maintenance of female sterility in otherwise hermaphroditic populations. To our knowledge, this is the first report of the coexistence of female sterility and hermaphrodites in the Brassicaceae.     

Tryptophan-dependent indole-3-acetic acid biosynthesis by ‘IAA-synthase’ proceeds via indole-3-acetamide

Plants are suggested to produce their major growth promoting phytohormone, indole-3-acetic acid (IAA), via multiple redundantly operating pathways. Although great effort has been made and plenty of possible routes have been proposed based on experimental evidence, a complete pathway for IAA production has yet to be demonstrated. In this study, an in-vitro approach was taken to examine the conversion of l-tryptophan (l-trp) to IAA by gas chromatography-mass spectrometry (GC-MS). Especially the influence of putative reaction intermediates on the enzymatic conversion of l-trp to IAA was analyzed. Among the substances tested only indole-3-acetamide (IAM) showed a pronounced effect on the l-trp conversion. We additionally report that IAM is synthesized from l-trp and that it is further converted to IAA by the utilized cell free Arabidopsis extract. Together, our results underscore the functionality of an IAM-dependent auxin biosynthesis pathway in Arabidopsis thaliana.     

Effect of temperature stress on the endogenous cytokinin content in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> (L.) Heynh plants

The levels of three endogenous cytokinin equivalents: zeatin (Z), iso-pentenyladenine (iP) and dihydrozeatin (dZ) in two Arabidopsis thaliana (L.) Heynh genotypes — wild type (wt) and ethylene-insensitive mutant (eti5), were compared using enzyme immunoassay (ELISA). Cytokinin content was measured after exposure to low (4 °C for 24 h in darkness) or high temperature (38 °C for 24 h in darkness). Measurements were performed immediately and 24, 48 and 120 h after treatments. It was found that at normal growth conditions eti5 plants contained more endogenous cytokinins compared to the wild type. At both temperature treatments mutant plants had decreased total cytokinin levels. Wild-type plants treated with high temperature (HT) exhibited reduced total cytokinins (with the exception of rates at 48 h), while low temperature (LT) treatment resulted in elevated total amount of the studied equivalents (except at 24 h). The obtained results suggested that HT had greater effect on cytokinin levels than LT since it caused more profound changes in the total content. We assume that this was due to the natural chilling tolerance of Arabidopsis plants.     

Perspectives on Systematic Analyses of Gene Function in Arabidopsis thaliana: New Tools, Topics and Trends

Since the sequencing of the nuclear genome of Arabidopsis thaliana ten years ago, various large-scale analyses of gene function have been performed in this model species. In particular, the availability of collections of lines harbouring random T-DNA or transposon insertions, which include mutants for almost all of the ~27,000 A. thaliana genes, has been crucial for the success of forward and reverse genetic approaches. In the foreseeable future, genome-wide phenotypic data from mutant analyses will become available for Arabidopsis, and will stimulate a flood of novel in-depth gene-function analyses. In this review, we consider the present status of resources and concepts for systematic studies of gene function in A. thaliana. Current perspectives on the utility of loss-of-function and gain-of-function mutants will be discussed in light of the genetic and functional redundancy of many A. thaliana genes.     

Gene dosage effect of WEE1 on growth and morphogenesis from arabidopsis hypocotyl explants

Background and Aims

How plant cell-cycle genes interface with development is unclear. Preliminary evidence from our laboratory suggested that over-expression of the cell cycle checkpoint gene, WEE1, repressed growth and development. Here the hypothesis is tested that the level of WEE1 has a dosage effect on growth and development in Arabidospis thaliana. To do this, a comparison was made of the development of gain- and loss-of-function WEE1 arabidopsis lines both in vivo and in vitro.

Methods

Hypocotyl explants from an over-expressing Arath;WEE1 line (WEE1^oe), two T-DNA insertion lines (wee1-1 and wee1-4) and wild type (WT) were cultured on two-way combinations of kinetin and naphthyl acetic acid. Root growth and meristematic cell size were also examined.

Key Results

Quantitative data indicated a repressive effect in WEE1^oe and a significant increase in morphogenetic capacity in the two T-DNA insertion lines compared with WT. Compared with WT, WEE1^oe seedlings exhibited a slower cell-doubling time in the root apical meristem and a shortened primary root, with fewer laterals, whereas there were no consistent differences in the insertion lines compared with WT. However, significantly fewer adventitious roots were recorded for WEE1^oe and significantly more for the insertion mutant wee1-1. Compared with WT there was a significant increase in meristem cell size in WEE1^oe for all three ground tissues but for wee1-1 only cortical cell size was reduced.

Conclusions

There is a gene dosage effect of WEE1 on morphogenesis from hypocotyls both in vitro and in vivo.     

Early Zn2+-induced effects on membrane potential account for primary heavy metal susceptibility in tolerant and sensitive Arabidopsis species

Background and Aims

Uptake of heavy metals by plant root cells depends on electro-physiological parameters of the plasma membrane. In this study, responses of the plasma membrane in root cells were analysed where early reactions to the metal ion-induced stress are localized. Three different Arabidopsis species with diverse strategies of their adaptation to heavy metals were compared: sensitive Arabidopsis thaliana and tolerant A. halleri and A. arenosa.

Methods

Plants of A. thaliana Col-0 ecotype and plants of A. arenosa and A. halleri originating from natural metallicolous populations were exposed to high concentrations of Zn²⁺. Plants were tested for root growth rate, cellular tolerance, plant morphology and cell death in the root apex. In addition, the membrane potential (E_M) of mature cortical root cells and changes in the pH of the liquid culture media were measured.

Key Results

Primary roots of A. halleri and A. arenosa plants grew significantly better at increased Zn²⁺ concentrations than A. thaliana plants. Elevated Zn²⁺ concentrations in the culture medium induced rapid changes in E_M. The reaction was species-specific and concentration-dependent. Arabidopsis halleri revealed the highest insensitivity of the plasma membrane and the highest survival rate under prolonged treatment with extra-high concentrations. Plants were able to effectively adjust the pH in the control, but much less at Zn²⁺-induced lower pH.

Conclusions

The results indicate a similar mode of early reaction to Zn²⁺, but with different extent in tolerant and sensitive species of Arabidopsis. The sensitivity of A. thaliana and a high tolerance of A. halleri and A. arenosa were demonstrated. Plasma membrane depolarization was lowest in the hyperaccumulator A. halleri and highest in A. thaliana. This indicates that rapid membrane voltage changes are an excellent tool to monitor the effects of heavy metals.     

Purification and characterization of three neutral extracellular isoperoxidases from rye leaves

Rye (Secale cereale L.) seedlings; contain two major flavone glucuronides, luteolin 7-O-diglucuronyl-4'-O-glucuronide (L3GlcUA) (1) and luteolin 7-O-diglucuronide (L2GlcUA) (2) in abundance in the apoplast of primary leaves; express a large number of peroxidase isoenzymes; and release H(2)O(2) into the apoplast during primary leaf development. We purified and characterized three neutral extracellular peroxidase isoenzymes (rPOXs N1, N2, and N3) that can oxidize L2GlcUA as a natural substrate. The isoelectric points and molecular weights of rPOXs N1, N2, and N3 were 6.1, 7.2, and 6.3, and 42, 37, and 51 kDa, respectively. The optimum pH of the rPOXs N1, N2, and N3 were 5.5, 5.5, and 8.5, respectively, and their optimum temperatures ranged from 45 to 50 degrees C for all isoenzymes. rPOXs N1, N2, and N3 recognized flavonoids with 3', 4'-OH groups as potential substrates, but not flavonoids with a glycosylated 4'-OH group or those without a 3'-OH group. The activities on phenol-type substrates were high in the order of guaiacol>catechol>o-cresol for all isoenzymes. rPOXs N1, N2, and N3 exhibited broad reactivity with endogenous hydrogen donors including luteolin glucuronides derived from the apoplast of rye primary leaves.     

Changes in homologous recombination frequency in Arabidopsis thaliana plants exposed to stress depend on time of exposure during development and on duration of stress exposure

In the past, we showed that exposure to abiotic and biotic stresses changes the homologous recombination frequency (HRF) in somatic tissue and in the progeny. In current work we planned to answer the following question: do stress intensity/duration and time during exposure influence changes in somatic HRF and transgenerational changes in HRF? Here, we tested the effects of exposure to UV-C, cold and heat on HRF at 7, 14, 21 and 28 days post germination (dpg). We found that exposure at 14 and 21 dpg resulted in a higher increase in HRF as compared to exposure at 7 dpg; longer exposure to UV-C resulted in a higher frequency of HR, whereas prolonged exposure to cold or heat, especially at later developmental stages, had almost no effect on somatic HRF. Exposure at 7 dpg had a positive effect on somatic growth of plants; plants exposed to stress at this age had larger leaves. The analysis of HRF in the progeny showed that the progeny of plants exposed to stress at 7 dpg had an increase in somatic HRF and showed larger sizes of recombination spots on leaves. The progeny of plants exposed to UV-C at 7 dpg and the progeny of plants exposed to cold or heat at 28 dpg had larger leaves as compared to control plants. To summarize, our experiments showed that changes in somatic and transgenerational HRF depend on the type of stress plants are exposed to, time of exposure during development and the duration of exposure.

Electronic supplementary material

The online version of this article (doi:10.1007/s12298-013-0197-z) contains supplementary material, which is available to authorized users.     

Assessing the regulation of leaf redox status under water stress conditions in Arabidopsis thaliana: Col-0 ecotype (wild-type and vtc-2), expressing mitochondrial and cytosolic roGFP1

Using Arabidopsis plants Col-0 and vtc2 transformed with a redox sensitive green fluorescent protein, (c-roGFP) and (m-roGFP), we investigated the effects of a progressive water stress and re-watering on the redox status of the cytosol and the mitochondria. Our results establish that water stress affects redox status differently in these two compartments, depending on phenotype and leaf age, furthermore we conclude that ascorbate plays a pivotal role in mediating redox status homeostasis and that Col-0 Arabidopsis subjected to water stress increase the synthesis of ascorbate suggesting that ascorbate may play a role in buffering changes in redox status in the mitochondria and the cytosol, with the presumed buffering capacity of ascorbate being more noticeable in young compared with mature leaves. Re-watering of water-stressed plants was paralleled by a return of both the redox status and ascorbate to the levels of well-watered plants. In contrast to the effects of water stress on ascorbate levels, there were no significant changes in the levels of glutathione, thereby suggesting that the regeneration and increase in ascorbate in water-stressed plants may occur by other processes in addition to the regeneration of ascorbate via the glutathione. Under water stress in vtc2 lines it was observed stronger differences in redox status in relation to leaf age, than due to water stress conditions compared with Col-0 plants. In the vtc2 an increase in DHA was observed in water-stressed plants. Furthermore, this work confirms the accuracy and sensitivity of the roGFP1 biosensor as a reporter for variations in water stress-associated changes in redox potentials.     

MIZ1-regulated hydrotropism functions in the growth and survival of Arabidopsis thaliana under natural conditions

Background and Aims

Root hydrotropism is a response to water-potential gradients that makes roots bend towards areas of higher water potential. The gene MIZU-KUSSEI1 (MIZ1) that is essential for hydrotropism in Arabidopsis roots has previously been identified. However, the role of root hydrotropism in plant growth and survival under natural conditions has not yet been proven. This study assessed how hydrotropic response contributes to drought avoidance in nature.

Methods

An experimental system was established for the study of Arabidopsis hydrotropism in soil. Characteristics of hydrotropism were analysed by comparing the responses of the miz1 mutant, transgenic plants overexpressing MIZ1 (MIZ1OE) and wild-type plants.

Key Results

Wild-type plants developed root systems in regions with higher water potential, whereas the roots of miz1 mutant plants did not show a similar response. This pattern of root distribution induced by hydrotropism was more pronounced in MIZ1OE plants than in wild-type plants. In addition, shoot biomass and the number of plants that survived under drought conditions were much greater in MIZ1OE plants.

Conclusions

These results show that hydrotropism plays an important role in root system development in soil and contributes to drought avoidance, which results in a greater yield and plant survival under water-limited conditions. The results also show that MIZ1 overexpression can be used for improving plant productivity in arid areas.     

\Cytokinin oxidase/dehydrogenase (CKX) activity in wild and ethylene-insensitive mutant eti5 type of Arabidopsis thaliana (L.) Heynh plants and the effect of cytokinin N1-(2-chloro-4-pyridyl)-N2-phenylurea on enzymatic activity and leaf morphology

The specific activity of cytokinin oxidase/dehydrogenase (EC 1.5.99.12) (CKX) was determined in leaves of wild type (wt) and ethylene-insensitive mutant (eti5) of Arabidopsis thaliana (L.) Heynh plants. Comparative studies showed that this mutation has lower basal CKX activity than wt. Application of 4PU-30 (N¹-(2-chloro-4-pyridyl)-N²-phenylurea) resulted in decreased CKX activity in both wt and mutant plants. The treatment increased leaf blade thickness and the volume of chlorophyll-containing cells per unit leaf area in wt but these changes were not observed in the eti5 mutant. The reduction in chlorophyll “a” and “b”, as well as in carotenoids content in the treated wt tissues resulting from altered leaf morphology was not detected in eti5 plants.     

Generalist insects behave in a jasmonate-dependent manner on their host plants,leaving induced areas quickly and staying longer on distant parts

Plants are sessile, so have evolved sensitive ways to detect attacking herbivores and sophisticated strategies to effectively defend themselves. Insect herbivory induces synthesis of the phytohormone jasmonic acid which activates downstream metabolic pathways for various chemical defences such as toxins and digestion inhibitors. Insects are also sophisticated animals, and many have coevolved physiological adaptations that negate this induced plant defence. Insect behaviour has rarely been studied in the context of induced plant defence, although behavioural adaptation to induced plant chemistry may allow insects to bypass the host''s defence system. By visualizing jasmonate-responsive gene expression within whole plants, we uncovered spatial and temporal limits to the systemic spread of plant chemical defence following herbivory. By carefully tracking insect movement, we found induced changes in plant chemistry were detected by generalist Helicoverpa armigera insects which then modified their behaviour in response, moving away from induced parts and staying longer on uninduced parts of the same plant. This study reveals that there are plant-wide signals rapidly generated following herbivory that allow insects to detect the heterogeneity of plant chemical defences. Some insects use these signals to move around the plant, avoiding localized sites of induction and staying ahead of induced toxic metabolites.