Dual column chromatography combined with high-resolution mass spectrometry improves coverage of non-targeted analysis of plant root exudates.

BACKGROUND: Within the plant kingdom, there is an exceptional amount of chemical diversity that has yet to be annotated. It is for this reason that non-targeted analysis is of interest for those working in novel natural products. To increase the number and diversity of compounds observable in root exudate extracts, several workflows which differ at three key stages were compared: 1) sample extraction, 2) chromatography, and 3) data preprocessing. RESULTS: Plants were grown in Hoagland's solution for two weeks, and exudates were initially extracted with water, followed by a 24-h regeneration period with subsequent extraction using methanol. Utilizing the second extraction showed improved results with less ion suppression and reduced retention time shifting compared to the first extraction. A single column method, utilizing a pentafluorophenyl column, paired with high-resolution mass spectrometry ionized and correctly identified 34 mock root exudate compounds, while the dual column method, incorporating a pentafluorophenyl column and a porous graphitic carbon column, retained and identified 43 compounds. In a pooled quality control sample of exudate extracts, the single column method detected 1,444 compounds. While the dual method detected fewer compounds overall (1,050), it revealed a larger number of small polar compounds. Three preprocessing methods (targeted, proprietary, and open source) successfully identified 43, 31, and 38 mock root exudate compounds to confidence level 1, respectively. SIGNIFICANCE: Enhancing signal strength and analytical method stability involves removing the high ionic strength nutrient solution before sampling root exudate extracts. Despite signal intensity loss, a dual column method enhances compound coverage, particularly for small polar metabolites. Open-source software proves a viable alternative for non-targeted analysis, even surpassing proprietary software in peak picking.

Neighbour-induced changes in root exudation patterns of buckwheat results in altered root architecture of redroot pigweed.

Roots are crucial in plant adaptation through the exudation of various compounds which are influenced and modified by environmental factors. Buckwheat root exudate and root system response to neighbouring plants (buckwheat or redroot pigweed) and how these exudates affect redroot pigweed was investigated. Characterising root exudates in plant-plant interactions presents challenges, therefore a split-root system which enabled the application of differential treatments to parts of a single root system and non-destructive sampling was developed. Non-targeted metabolome profiling revealed that neighbour presence and identity induces systemic changes. Buckwheat and redroot pigweed neighbour presence upregulated 64 and 46 metabolites, respectively, with an overlap of only 7 metabolites. Root morphology analysis showed that, while the presence of redroot pigweed decreased the number of root tips in buckwheat, buckwheat decreased total root length and volume, surface area, number of root tips, and forks of redroot pigweed. Treatment with exudates (from the roots of buckwheat and redroot pigweed closely interacting) on redroot pigweed decreased the total root length and number of forks of redroot pigweed seedlings when compared to controls. These findings provide understanding of how plants modify their root exudate composition in the presence of neighbours and how this impacts each other's root systems.

Fagopyrum esculentum Alters Its Root Exudation after Amaranthus retroflexus Recognition and Suppresses Weed Growth.

Weed control by crops through growth suppressive root exudates is a promising alternative to herbicides. Buckwheat (Fagopyrum esculentum) is known for its weed suppression and redroot pigweed (Amaranthus retroflexus) control is probably partly due to allelopathic root exudates. This work studies whether other weeds are also suppressed by buckwheat and if the presence of weeds is necessary to induce growth repression. Buckwheat and different weeds were co-cultivated in soil, separating roots by a mesh allowing to study effects due to diffusion. Buckwheat suppressed growth of pigweed, goosefoot and barnyard grass by 53, 42, and 77% respectively without physical root interactions, probably through allelopathic compounds. Root exudates were obtained from sand cultures of buckwheat (BK), pigweed (P), and a buckwheat/pigweed mixed culture (BK-P). BK-P root exudates inhibited pigweed root growth by 49%. Characterization of root exudates by UHPLC-HRMS and principal component analysis revealed that BK and BK-P had a different metabolic profile suggesting that buckwheat changes its root exudation in the presence of pigweed indicating heterospecific recognition. Among the 15 different markers, which were more abundant in BK-P, tryptophan was identified and four others were tentatively identified. Our findings might contribute to the selection of crops with weed suppressive effects.

Metabolomics and food processing: from semolina to pasta.

The objective of this study was to investigate the metabolite variations during industrial pasta processing (from semolina to dried pasta) for five different commercial products. Up to 76 metabolites were detected. Significant differences were observed between wholemeal and refined pasta samples, with the wholemeal pasta richer in many classes of compounds such as phytosterols, policosanols, unsaturated fatty acids, amino acids, carotenoids, minerals, and so on. Significant differences were also observed between samples of refined pasta apparently similar for the actual parameters used for the assessment of pasta quality. The results indicated that a number of metabolites undergo a transformation during the pasta-making process depending on the processing conditions adopted. The approach used in this work shows the high potential of metabolite profiling for food investigations with regard to process-related transformation, safety, and nutrition.

Rice endosperm iron biofortification by targeted and synergistic action of nicotianamine synthase and ferritin.

Nearly one-third of the world's population, mostly women and children, suffer from iron malnutrition and its consequences, such as anaemia or impaired mental development. Iron fortification of food is difficult because soluble iron is either unstable or unpalatable, and non-soluble iron is not bioavailable. Genetic engineering of crop plants to increase iron content has therefore emerged as an alternative for iron biofortification. To date, strategies to increase iron content have relied on single genes, with limited success. Our work focuses on rice as a model plant, because it feeds one-half of the world's population, including the majority of the iron-malnourished population. Using the targeted expression of two transgenes, nicotianamine synthase and ferritin, we increased the iron content of rice endosperm by more than six-fold. Analysis of transgenic rice lines confirmed that, in combination, they provide a synergistic effect on iron uptake and storage. Laser ablation-inductively coupled plasma-mass spectrometry showed that the iron in the endosperm of the transgenic rice lines accumulated in spots, most probably as a consequence of spatially restricted ferritin accumulation. Agronomic evaluation of the high-iron rice lines did not reveal a yield penalty or significant changes in trait characters, except for a tendency to earlier flowering. Overall, we have demonstrated that rice can be engineered with a small number of genes to achieve iron biofortification at a dietary significant level.

Oxidative pentose phosphate pathway-dependent sugar sensing as a mechanism for regulation of root ion transporters by photosynthesis.

Root ion transport systems are regulated by light and/or sugars, but the signaling mechanisms are unknown. We showed previously that induction of the NRT2.1 NO(3)(-) transporter gene by sugars was dependent on carbon metabolism downstream hexokinase (HXK) in glycolysis. To gain further insights on this signaling pathway and to explore more systematically the mechanisms coordinating root nutrient uptake with photosynthesis, we studied the regulation of 19 light-/sugar-induced ion transporter genes. A combination of sugar, sugar analogs, light, and CO(2) treatments provided evidence that these genes are not regulated by a common mechanism and unraveled at least four different signaling pathways involved: regulation by light per se, by HXK-dependent sugar sensing, and by sugar sensing upstream or downstream HXK, respectively. More specific investigation of sugar-sensing downstream HXK, using NRT2.1 and NRT1.1 NO(3)(-) transporter genes as models, highlighted a correlation between expression of these genes and the concentration of glucose-6-P in the roots. Furthermore, the phosphogluconate dehydrogenase inhibitor 6-aminonicotinamide almost completely prevented induction of NRT2.1 and NRT1.1 by sucrose, indicating that glucose-6-P metabolization within the oxidative pentose phosphate pathway is required for generating the sugar signal. Out of the 19 genes investigated, most of those belonging to the NO(3)(-), NH(4)(+), and SO(4)(2-) transporter families were regulated like NRT2.1 and NRT1.1. These data suggest that a yet-unidentified oxidative pentose phosphate pathway-dependent sugar-sensing pathway governs the regulation of root nitrogen and sulfur acquisition by the carbon status of the plant to coordinate the availability of these three elements for amino acid synthesis.

Flavonoid profiling among wild type and related GM wheat varieties.

Pleiotropic effects are one of the main concerns regarding genetically modified organisms (GMOs). This includes unintended side effects of the transgene or its genome insertion site on the regulation of other endogenous genes, which could potentially cause the accumulation of different secondary metabolites that may have not only an impact on diet as repeatedly worried by the public but also on the environment. Regarding amount and possible environmental effects, flavonoids represent the most prominent group of secondary metabolites in wheat. Many flavonoids function as signalling or defence molecules. We used a robust and reproducible analytical method to compare the flavonoid content of genetically modified (GM) wheat (Triticum aestivum L., Gramineae) expressing genes that confer increased fungal resistance with their non-GM siblings. The transgenes provide either a broad-spectrum fungal defence (chitinase/glucanase from barley) or bunt-specific resistance by a viral gene (KP4). Significant differences in flavonoid composition were found between different wheat varieties whereas different lines of GM wheat with increased antifungal resistance showed only minor differences in their flavonoid composition relative to their non-GM siblings. In a field test, no significant differences were detectable between infected and non-infected wheat of the same variety regardless of the presence of the transgene. Our results are in agreement with the hypothesis that the transgenes we used to increase wheat defence to fungal pathogens do not interfere with the flavonoid biosynthesis pathway. More significantly, the genetic background resulting from conventional breeding has a direct impact on the biological composition of flavonoids, and thus possibly on the environment.

Identification of a 150 bp cis-acting element of the AtNRT2.1 promoter involved in the regulation of gene expression by the N and C status of the plant.

The Arabidopsis thaliana AtNRT2.1 gene, which encodes a NO(3)(-) transporter involved in high-affinity uptake by the roots, is a molecular target of several mechanisms responsible for the regulation of root NO(3)(-) acquisition by the N status of the plant. All levels of AtNRT2.1 expression (promoter activity, transcript level, protein accumulation, transport activity) are coordinately up-regulated in the presence of NO(3)(-), and repressed by downstream N metabolites. Transgenic plants expressing the GUS reporter gene under the control of upstream sequences of AtNRT2.1 have been studied to identify elements targeted by these two regulatory mechanisms. A 150 bp sequence located upstream of the TATA box that is required for both stimulation by NO(3)(-) and repression by N metabolites of the promoter has been identified. This sequence is able to confer these two regulations to a minimal promoter. Split-root experiments indicate that the stimulation of the chimaeric promoter by NO(3)(-) occurs only at the local level, whereas its repression by N metabolites is mediated by a systemic signal spread to the whole plant. The activity of the cis-acting 150 bp element is also regulated by sucrose supply to the roots, suggesting a possible interaction between N and C signalling within this short region. Accordingly, multiple motifs potentially involved in regulations by N and/or C status are identified within this sequence by bioinformatic approaches. This is the first report of such a cis-acting element in higher plants.

Regulation of root nitrate uptake at the NRT2.1 protein level in Arabidopsis thaliana.

In Arabidopsis the NRT2.1 gene encodes a main component of the root high-affinity nitrate uptake system (HATS). Its regulation has been thoroughly studied showing a strong correlation between NRT2.1 expression and HATS activity. Despite its central role in plant nutrition, nothing is known concerning localization and regulation of NRT2.1 at the protein level. By combining a green fluorescent protein fusion strategy and an immunological approach, we show that NRT2.1 is mainly localized in the plasma membrane of root cortical and epidermal cells, and that several forms of the protein seems to co-exist in cell membranes (the monomer and at least one higher molecular weight complex). The monomer is the most abundant form of NRT2.1, and seems to be the one involved in NO(3)(-) transport. It strictly requires the NAR2.1 protein to be expressed and addressed at the plasma membrane. No rapid changes in NRT2.1 abundance were observed in response to light, sucrose, or nitrogen treatments that strongly affect both NRT2.1 mRNA level and HATS activity. This suggests the occurrence of post-translational regulatory mechanisms. One such mechanism could correspond to the cleavage of NRT2.1 C terminus, which results in the presence of both intact and truncated proteins in the plasma membrane.

The Arabidopsis nitrate transporter AtNRT2.1 is targeted to the root plasma membrane.

Arabidopsis AtNRT2.1 protein is the best characterized high affinity nitrate transporter in higher plants. However, nothing is known about its sub-cellular localization. In this work, we used GFP imaging to follow the targeting of the AtNRT2.1 protein to the different cell membranes. A polyclonal antibody was also raised against a peptide derived from the AtNRT2.1 sequence. Comparison of wild type and mutant plant extracts showed that this antibody recognized specifically the AtNRT2.1 protein. Microsomal membranes were fractionated on sucrose gradients and immunological detections were performed on the different fractions. Altogether, our results demonstrate that the AtNRT2.1 protein is located in the plasma membrane of the root cells.

Additive contribution of AMT1;1 and AMT1;3 to high-affinity ammonium uptake across the plasma membrane of nitrogen-deficient Arabidopsis roots.

In Arabidopsis four root-expressed AMT genes encode functional ammonium transporters, which raises the question of their role in primary ammonium uptake. After pre-culturing under nitrogen-deficiency conditions, we quantified the influx of (15)N-labeled ammonium in T-DNA insertion lines and observed that the loss of either AMT1;1 or AMT1;3 led to a decrease in the high-affinity ammonium influx of approximately 30%. Under nitrogen-sufficient conditions the ammonium influx was lower in Columbia glabra compared with Wassilewskija (WS), and AMT1;1 did not contribute significantly to the ammonium influx in Col-gl. Ectopic expression of AMT1;3 under the control of a 35S promoter in either of the insertion lines amt1;3-1 or amt1;1-1 increased the ammonium influx above the level of their corresponding wild types. In transgenic lines carrying AMT-promoter-GFP constructs, the promoter activities of AMT1;1 and AMT1;3 were both upregulated under nitrogen-deficiency conditions and were localized to the rhizodermis, including root hairs. AMT gene-GFP fusions that were stably expressed under the control of their own promoters were localized to the plasma membrane. The double insertion line amt1;1-1amt1;3-1 showed a decreased sensitivity to the toxic ammonium analog methylammonium and a decrease in the ammonium influx of up to 70% relative to wild-type plants. These results suggest an additive contribution of AMT1;1 and AMT1;3 to the overall ammonium uptake capacity in Arabidopsis roots under nitrogen-deficiency conditions.