Stimulation of root hair elongation in Arabidopsis thaliana by low phosphorus availability

Low phosphorus availability stimulates root hair elongation in many plants, which may have adaptive significance in soil phosphorus acquisition. We investigated the effect of low phosphorus on the elongation of Arabidopsis thaliana root hairs. Arabidopsis thaliana plants were grown in plant culture containing high (1000 mmol m^?3) or low (1 mmol m^?3) phosphorus concentrations, and root hair elongation was analysed by image analysis. After 15d of growth, low-phosphorus plants developed root hairs averaging 0.9 mm in length while high-phosphorus plants of the same age developed root hairs averaging 0.3 mm in length. Increased root hair length in low-phosphorus plants was a result of both increased growth duration and increased growth rate. Root hair length decreased logarithmically in response to increasing phosphorus concentration. Local changes in phosphorus availability influenced root hair growth regardless of the phosphorus status of the plant. Low phosphorus stimulated root hair elongation in the hairless axr2 mutant, exogenously applied IAA stimulated root hair elongation in wild-type high-phosphorus plants and the auxin antagonist CM PA inhibited root hair elongation in low-phosphorus plants. These results indicate that auxin may be involved in the low-phosphorus response in root hairs.     

Metabolite profiling of Arabidopsis seedlings in response to exogenous sinalbin and sulfur deficiency

In order to determine how plant uptake of a sulfur-rich secondary metabolite, sinalbin, affects the metabolic profile of sulfur-deficient plants, gas chromatography time-of-flight mass spectrometry (GC-TOF-MS), in combination with liquid chromatography-mass spectrometry (LC-MS), was used to survey the metabolome of Arabidopsis seedlings grown in nutrient media under different sulfur conditions. The growth media had either sufficient inorganic sulfur for normal plant growth or insufficient inorganic sulfur in the presence or absence of supplementation with organic sulfur in the form of sinalbin (p-hydroxybenzylglucosinolate). A total of 90 metabolites were identified by GC-TOF-MS and their levels were compared across the three treatments. Of the identified compounds, 21 showed similar responses in plants that were either sulfur deficient or sinalbin supplemented compared to sulfur-sufficient plants, while 12 metabolites differed in abundance only in sulfur-deficient plants. Twelve metabolites accumulated to higher levels in sinalbin-supplemented than in the sulfur-sufficient plants. Secondary metabolites such as flavonol conjugates, sinapinic acid esters and glucosinolates, were identified by LC-MS and their corresponding mass fragmentation patterns were determined. Under sinalbin-supplemented conditions, sinalbin was taken up by Arabidopsis and contributed to the endogenous formation of glucosinolates. Additionally, levels of flavonol glycosides and sinapinic acid esters increased while levels of flavonol diglycosides with glucose attached to the 3-position were reduced. The exogenously administered sinalbin resulted in inhibition of root and hypocotyl growth and markedly influenced metabolite profiles, compared to control and sulfur-deficient plants. These results indicate that, under sulfur deficient conditions, glucosinolates can be a sulfur source for plants. This investigation defines an opportunity to elucidate the mechanism of glucosinolate degradation in vivo.     

Production of Strigolactones by Arabidopsis thaliana responsible for Orobanche aegyptiaca seed germination

The germination stimulants produced by Arabidopsis thaliana, a host of root parasitic plants Orobanche spp. but not of arbuscular mycorrhizal (AM) fungi were examined. Root exudates from the hydroponically grown A. thaliana plants were subjected to reverse phase high performance liquid chromatography (HPLC) and retention times of germination stimulants inducing O. aegyptiaca seed germination were compared with those of strigolactone standards. In addition, the root exudates were analyzed by using HPLC linked with tandem mass spectrometry (LC/MS/MS). A. thaliana was found to exude at least three different germination stimulants of which one was identified as orobanchol. This is the first report of strigolactone production by a non-mycotrophic plant. These results together with recent knowledge imply that strigolactones have other unrevealed functions in plant growth and development.     

Carnitine is associated with fatty acid metabolism in plants

The finding of acylcarnitines alongside free carnitine in Arabidopsis thaliana and other plant species, using tandem mass spectrometry coupled to liquid chromatography shows a link between carnitine and plant fatty acid metabolism. Moreover the occurrence of both medium- and long-chain acylcarnitines suggests that carnitine is connected to diverse fatty acid metabolic pathways in plant tissues. The carnitine and acylcarnitine contents in plant tissues are respectively a hundred and a thousand times lower than in animal tissues, and acylcarnitines represent less than 2% of the total carnitine pool whereas this percentage reaches 30% in animal tissues. These results suggest that carnitine plays a lesser role in lipid metabolism in plants than it does in animals.     

Cytokinin-mediated cell cycling arrest of pericycle founder cells in lateral root initiation of Arabidopsis

In Arabidopsis, lateral root formation is a post-embryonic developmental event, which is regulated by hormones and environmental signals. In this study, via analyzing the expression of cyclin genes during lateral root (LR) formation, we report that cytokinins (CTKs) inhibit the initiation of LR through blocking the pericycle founder cells cycling at the G(2) to M transition phase, while the promotion by CTK of LR elongation is due to the stimulation of the G(1) to S transition. No significant difference was detected in the inhibitory effect of CTK on LR formation between wild-type plants and mutants defective in auxin response or transport. In addition, exogenously applied auxin at different concentrations could not rescue the CTK-mediated inhibition of LR initiation. Our data suggest that CTK and auxin might control LR initiation through two separate signaling pathways in Arabidopsis. The CTK-mediated repression of LR initiation is transmitted through the two-component signal system and mediated by the receptor CRE1.     

Identification of tryptophan in leachate of oat hulls (Avena sativa) as a mediator of root growth regulation

Leachate of oat hulls ( Avena sativa L. cv. Sang) inhibits root elongation and causes swollen roots and abundant root hair formation. The active substance was isolated by column chromatography and thin layer chromatography (TLC) systems. High performance liquid chromatography (HPLC) revealed 3 peaks, one of which corresponded to the substance responsible. The latter was identified as tryptophan by means of its UV spectrum, amino acid analysis, nuclear magnetic resonance spectrometry (NMR) and mass spectrometry (MS).     

Oil content of Arabidopsis seeds: the influence of seed anatomy, light and plant-to-plant variation

Arabidopsis thaliana is frequently used as a model for the study of oilseed biology and metabolism. However, the very small seeds of Arabidopsis can complicate analysis of their oil content and influence the application of results to larger-seeded plants. Here, we describe how seed anatomy, light, and plant-to-plant variation influence the content and measurement of oil in Arabidopsis seeds. The anatomy of Arabidopsis and Brassica napus seeds were compared and the distribution of mass, oil and the fatty acid composition of different seed parts were determined. In Brassica, 90% of the seed oil resides in the cotyledons that contribute 74% of seed mass. By contrast, the values for Arabidopsis are 60% and 45%, respectively, with a higher fraction of the oil deposited in the radicle, hypocotyl, endosperm and seed coat. Growth of Arabidopsis plants with 600 micromol m(-2) s(-1) light resulted in a two-fold higher seed yield, a 40% increase in mass per seed and a 60% increase in oil per seed compared to growth at 100 micromol m(-2) s(-1). Factors that influence the analysis of oil content were evaluated. Intact-seed transmethylation followed by gas chromatography (GC) analysis provided reproducible analysis of Arabidopsis seed oil. However, plant-to-plant variation in oil content is large and we analyzed how this influences the ability to detect statistically valid changes in oil between different genotypes. These observations establish a reference data set on the fatty acid composition and distribution of mass and oil between tissues of Arabidopsis seeds that should help to predict the applicability of results obtained with Arabidopsis to other oilseeds.     

Roles of phosphatidylinositol 3-kinase in root hair growth

The root hair is a model system for understanding plant cell tip growth. As phosphatidylinositol 3-phosphate [PtdIns(3)P] has been shown in other plant cell types to regulate factors that affect root hair growth, including reactive oxygen species (ROS) levels, cytoskeleton, and endosomal movement, we hypothesized that PtdIns(3)P is also important for root hair elongation. The enzyme that generates PtdIns(3)P, phosphatidylinositol 3-kinase (PI3K), was expressed in root hair cells of transgenic plants containing the PI3K promoter:beta-glucuronidase reporter construct. To obtain genetic evidence for the role of PtdIns(3)P in root hair elongation, we attempted to isolate Arabidopsis (Arabidopsis thaliana) mutant plants that did not express the gene VPS34 encoding the PI3K enzyme. However, the homozygous mutant was lethal due to gametophytic defects, and heterozygous plants were not discernibly different from wild-type plants. Alternatively, we made transgenic plants expressing the PtdIns(3)P-binding FYVE domain in the root hair cell to block signal transduction downstream of PtdIns(3)P. These transgenic plants had shorter root hairs and a reduced hair growth rate compared with wild-type plants. In addition, LY294002, a PI3K-specific inhibitor, inhibited root hair elongation but not initiation. In LY294002-treated root hair cells, endocytosis at the stage of final fusion of the late endosomes to the tonoplast was inhibited and ROS level decreased in a dose-dependent manner. Surprisingly, the LY294002 effects on ROS and root hair elongation were similar in rhd2 mutant plants, suggesting that RHD2 was not the major ROS generator in the PtdIns(3)P-mediated root hair elongation process. Collectively, these results suggest that PtdIns(3)P is required for maintenance of the processes essential for root hair cell elongation.     

Trehalose determination in linseed subjected to osmotic stress. HPAEC‐PAD analysis: an inappropriate method

Trehalose is a non‐reducing disaccharide involved in stress tolerance in plants. To understand better the role of trehalose in the osmotic stress response in linseed (Linum usitatissimum), trehalose content in leaves was studied. First, the method commonly used for sugar determination, high performance anion exchange chromatography with pulsed amperometric detection (HPAEC‐PAD), gave unsatisfactory results and the separation efficiency could not be improved by varying the elution conditions. The same problem was also found in the model plant: Arabidopsis thaliana. After clearly highlighting a co‐elution of trehalose in these two species by a trehalase assay and liquid chromatography‐high resolution mass spectrometry analysis, gas chromatography–mass spectrometry (GC‐MS) was used as the analytical method instead. These results confirmed that trehalose content is currently overestimated by HPAEC‐PAD analysis, approximately 7 and 13 times for A. thaliana and linseed respectively. Thus GC‐MS gave more satisfactory results for trehalose quantification in plants. With this method, trehalose accumulation was observed in linseed during an osmotic stress (?0.30 MPa), the quantity (31.49 nmol g^–1 dry weight after 48 h) appears too low to assign an osmoprotector or osmoregulator role to trehalose in stressed linseed.     

Metabolome Analysis of Arabidopsis thaliana Roots Identifies a Key Metabolic Pathway for Iron Acquisition

Fe deficiency compromises both human health and plant productivity. Thus, it is important to understand plant Fe acquisition strategies for the development of crop plants which are more Fe-efficient under Fe-limited conditions, such as alkaline soils, and have higher Fe density in their edible tissues. Root secretion of phenolic compounds has long been hypothesized to be a component of the reduction strategy of Fe acquisition in non-graminaceous plants. We therefore subjected roots of Arabidopsis thaliana plants grown under Fe-replete and Fe-deplete conditions to comprehensive metabolome analysis by gas chromatography-mass spectrometry and ultra-pressure liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry. Scopoletin and other coumarins were found among the metabolites showing the strongest response to two different Fe-limited conditions, the cultivation in Fe-free medium and in medium with an alkaline pH. A coumarin biosynthesis mutant defective in ortho-hydroxylation of cinnamic acids was unable to grow on alkaline soil in the absence of Fe fertilization. Co-cultivation with wild-type plants partially rescued the Fe deficiency phenotype indicating a contribution of extracellular coumarins to Fe solubilization. Indeed, coumarins were detected in root exudates of wild-type plants. Direct infusion mass spectrometry as well as UV/vis spectroscopy indicated that coumarins are acting both as reductants of Fe(III) and as ligands of Fe(II).     

3-Phenyllactic acid is converted to phenylacetic acid and induces auxin-responsive root growth in Arabidopsis plants

Many microorganisms have been reported to produce compounds that promote plant growth and are thought to be involved in the establishment and maintenance of symbiotic relationships. 3-Phenyllactic acid (PLA) produced by lactic acid bacteria was previously shown to promote root growth in adzuki cuttings. However, the mode of action of PLA as a root-promoting substance had not been clarified. The present study therefore investigated the relationship between PLA and auxin. PLA was found to inhibit primary root elongation and to increase lateral root density in wild-type Arabidopsis, but not in an auxin signaling mutant. In addition, PLA induced IAA19 promoter fused β-glucuronidase gene expression, suggesting that PLA exhibits auxin-like activity. The inability of PLA to promote degradation of Auxin/Indole-3-Acetic Acid protein in a yeast heterologous reconstitution system indicated that PLA may not a ligand of auxin receptor. Using of a synthetic PLA labeled with stable isotope showed that exogenously applied PLA was converted to phenylacetic acid (PAA), an endogenous auxin, in both adzuki and Arabidopsis. Taken together, these results suggest that exogenous PLA promotes auxin signaling by conversion to PAA, thereby regulating root growth in plants.     

Characterization of strigolactones exuded by Asteraceae plants

Strigolactones (SLs), originally characterized as germination stimulants for root parasitic weeds, are now recognized as hyphal branching factors for symbiotic arbuscular mycorrhizal fungi and as a novel class of plant hormones inhibiting shoot branching. In the present study, SLs in root exudates of 13 Asteraceae plants including crops, a weed, and ornamental plants were characterized. High performance liquid chromatography/tandem mass spectrometry (LC–MS/MS) analyses revealed that all the Asteraceae plants examined exuded known SLs and, except for sunflower (Helianthus annuus), high germination stimulant activities at retention times corresponding to these SLs were confirmed. The two major SLs exuded by these Asteraceae plants were orobanchyl acetate and orobanchol. 5-Deoxystrigol and 7-hydroxyorobanchyl acetate were detected in root exudates from several Asteraceae species examined in this study.     

If the antibody fails--a mass western approach

SUMMARY: Sucrose-phosphate synthase (SPS) has attracted the interest of plant scientists for decades. It is the key enzyme in sucrose metabolism and is under investigation in various plant species, e.g. spinach, tobacco, poplar, resurrection plants, maize, rice, kiwi and Arabidopsis thaliana. In A. thaliana, there are four distinct SPS isoforms. Their expression is thought to depend on environmental conditions and plant tissue. However, these data were derived from mRNA expression levels only. No data on SPS protein identification from crude extracts have been available until now. An antibody approach failed to distinguish the four isoforms. Therefore, we developed a method for SPS quantification and isoform-specific identification in A. thaliana complex protein samples. Samples were separated on SDS-PAGE, digested and directly applied to liquid chromatography/triple-stage quadrupole mass spectrometry (LC/TSQ-MS). In this approach, known as mass Western, samples were analysed in multi-reaction monitoring (MRM) mode, so that all four SPS isoforms could be measured in one experiment. In addition to the relative quantification, stable isotope-labelled internal peptide standards allowed absolute quantification of SPS proteins. Protein extracts from various plant tissues, samples harvested during the day or the night, and cold-stressed plants were analysed. The stress-specific SPS5a isoform showed increased concentrations in cold-stressed leaf material.     

Auxin distribution and transport during embryogenesis and seed germi-nation of Arabidopsis

INTRODUCTIONAuxin plays an important role in regu1ating celldivision, e1ongation and differentiatiou, vascular tis-sue fOrmation[1], pollen deve1opment[2] and 1eafyhead fOrmation[3]. Adrin polar transport is be-1ieved to invohe in a variety of important growthand developmenial processes, including the patternfOrmation of eInbryO, leaf morphogenesis and theroot gravity response[4--8]. Auxin po1ar transportinhibitor has been proved essential illterference ofataln transport leading to patte…     

Nitrate, a signal relieving seed dormancy in Arabidopsis

Nitrate is an important nitrogen source for plants, but also a signal molecule that controls various aspects of plant development. In the present study the role of nitrate on seed dormancy in Arabidopsis was investigated. The effects of either mutations affecting the Arabidopsis nitrate reductase genes or of different nitrate regimes of mother plants on the dormancy of the seeds produced were analysed. Altogether, data show that conditions favouring nitrate accumulation in mother plants and in seeds lead to a lower dormancy of seeds with little other morphological or biochemical differences. Analysis of germination during seed development indicated that nitrate does not prevent the onset of dormancy but rather its maintenance. The effect of an exogenous supply of nitrate on seed germination was tested: nitrate in contrast to glutamine or potassium chloride clearly stimulated the germination of dormant seeds. Data show, moreover, that the Arabidopsis dual affinity nitrate transporter NRT1.1 (CHL1) may be involved in conveying the nitrate signal into seeds. Thus, nitrate provided exogenously or by mother plants to the produced seeds, acts as a signal molecule favouring germination in Arabidopsis. This signalling may involve interaction with the abscisic acid or gibberellin pathway.     

Impact of galactoglucomannan oligosaccharides on elongation growth in intact mung bean plants

The impact of exogenously applied galactoglucomannan oligosaccharides (GGMOs) and their structurally modified forms (GGMOs-r—galactoglucomannosyl alditols, GGMOs-g—with reduced galactose content) on the growth of mung bean (Vigna radiata (L.) Wilczek) intact plants cultured in hydroponics has been determined. GGMOs alone or in combination with exogenously added IBA have influenced (with stimulation and/or inhibition effect) hypocotyl and seminal root elongation, adventitious and lateral roots formation and elongation in dependency on their concentration used. The inhibition of elongation growth in hypocotyls as well as in roots was connected with changes of cell wall-associated peroxidases activity and is probably associated with the beginning of cell wall rigidification. Data presented in this paper confirm the hypothesis that exogenously added GGMOs may have antiauxin activity and may interact also with endogenous growth regulators. Certain monosaccharide sequences with terminal galactose in the side chain of GGMOs probably play important role in their biological activity in intact plants as it was demonstrated previously in individual parts of plants.     

Strigolactones are transported through the xylem and play a key role in shoot architectural response to phosphate deficiency in nonarbuscular mycorrhizal host Arabidopsis

The biosynthesis of the recently identified novel class of plant hormones, strigolactones, is up-regulated upon phosphate deficiency in many plant species. It is generally accepted that the evolutionary origin of strigolactone up-regulation is their function as a rhizosphere signal that stimulates hyphal branching of arbuscular mycorrhizal fungi. In this work, we demonstrate that this induction is conserved in Arabidopsis (Arabidopsis thaliana), although Arabidopsis is not a host for arbuscular mycorrhizal fungi. We demonstrate that the increase in strigolactone production contributes to the changes in shoot architecture observed in response to phosphate deficiency. Using high-performance liquid chromatography, column chromatography, and multiple reaction monitoring-liquid chromatography-tandem mass spectrometry analysis, we identified two strigolactones (orobanchol and orobanchyl acetate) in Arabidopsis and have evidence of the presence of a third (5-deoxystrigol). We show that at least one of them (orobanchol) is strongly reduced in the putative strigolactone biosynthetic mutants more axillary growth1 (max1) and max4 but not in the signal transduction mutant max2. Orobanchol was also detected in xylem sap and up-regulated under phosphate deficiency, which is consistent with the idea that root-derived strigolactones are transported to the shoot, where they regulate branching. Moreover, two additional putative strigolactone-like compounds were detected in xylem sap, one of which was not detected in root exudates. Together, these results show that xylem-transported strigolactones contribute to the regulation of shoot architectural response to phosphate-limiting conditions.     

Quantitative determination of indole-3-acetic acid in yeasts using high performance liquid chromatography—tandem mass spectrometry

High performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) was applied to the comprehensive analysis of the ability of yeast strains to synthesize a plant hormone indole-3-acetic acid (IAA). A total of 124 strains (37 species) of yeasts isolated from various regions and substrates were studied. Testing of IAA production showed that 92% strains were capable of IAA synthesis. The results indicated that, in general, ascomycetous yeasts were more active auxin producers than basidiomycetous ones. Geographically, strains from tropical regions were the most active IAA producers. Analysis of the substrate variability of the strains showed higher auxin production (on average) by the yeasts isolated from plants compared to the soil isolates, indicating a specific regulatory role of the plant yeast population.