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Metabolomics

, Volume 6, Issue 4, pp 497–510 | Cite as

Targeted metabolomics in an intrusive weed, Rumex obtusifolius L., grown under different environmental conditions reveals alterations of organ related metabolite pathway

  • Atsuko Miyagi
  • Kentaro Takahara
  • Hideyuki Takahashi
  • Maki Kawai-Yamada
  • Hirofumi Uchimiya
Original Article

Abstract

This study was intended to analyze the metabolic pathway of Rumex obtusifolius L. (Broad-leaved dock), destructive weeds worldwide, in relation to major environmental factors (light and temperature). It was found that R. obtusifolius can be classified as plants in accumulating major organic acids such as oxalate in leaves and citrate in stems (Miyagi et al., Metabolomics 6:146–155 2010). The organ specific accumulation of certain metabolites was dissected by metabolomics approach in relation to metabolic pathway. Light or dark experiments showed that in the case of the oxalate accumulation, the major or the most dominated pathway was found to be the citrate-isocitrate-oxalate shunt. Furthermore, under the dark and/or low temperature (5°C) leaves showed sustainable growth with normal accumulation of TCA metabolites. Unlike leaves, there was a different pattern of metabolite accumulation in stems. Other metabolites such as amino acids also showed the organ specific alterations under the different ambient environments.

Keywords

Rumex obtusifolius New leaves Stems Oxalate Citrate Organ specific metabolites Capillary electrophoresis mass spectrometry Principal component analysis Hierarchical clustering analysis 

1 Introduction

In plants, studies employing metabolomics in relation to environmental constrains revealed changes in carbohydrates and a broad range of primary metabolites (Foyer et al. 2002; Guy et al. 2008; Sanchez et al. 2008; Shulaev et al. 2008). The principal component analysis revealed that the metabolite patterns changed progressively through a diurnal period in potato leaves, suggesting strict temporal regulation of metabolite compositions (Urbanczyk-Wochniak et al. 2005). Furthermore, thermotolerance acquired in response to heat and freezing resulted in temporal changes in metabolite compositions in Arabidopsis thaliana (Kaplan et al. 2004). These results contributed to elucidate the roles of signaling molecules and protectants in relationships between heat- and cold-shock responses.

In the previous study, we reported metabolic alterations among polygonaceous plants including eight Rumex species and Fallopia japonica (Miyagi et al. 2010). We measured primary metabolites (such as organic acids, sugar phosphates and amino acids) and metabolites in oxalate pathways. Results concluded that accumulated amounts of certain organic acids in R. obtusifolius are much higher than other polygonaceous plants. It was shown that polygonaceous plants accumulate oxalate in leaves and citrate in stems as one of the most abundant metabolite. Our unpublished data showed that the content of oxalate (70 μmol/gFW) in leaves grown under light for 4 weeks was 7 times higher than that of sucrose (11 μmol/gFW). In stems, the content of citrate (8 μmol/gFW) was about one-sixth of sucrose (45 μmol/gFW). C reserves in the root tend to lower than those in the stem (unpublished data).

Rumex (Polygonaceae; C3 plant) is composed of several hundred species around the world (Löve and Kapoor 1967). Among them Rumex obtusifolius L. (common name; Broad-leaved dock) is known as a perennial plant. This species is one of problematic weeds infesting agricultural lands (Holm et al. 1977). Plants were brought to Japan in the early 1900s as an intruder, a contaminant in grasses imported from Europe (Makuchi and Sakai 1984). The occurrence of R. obtusifolius was first reported in parts of the northern island of Hokkaido. Today, R. obtusifolius is found throughout the whole country (Hongo 1986). One of the reasons for this rapid spread is its ability to produce over 60,000 seeds per plant (Cavers and Harper 1964; Holm et al. 1977; Hongo 1989). Moreover, the seeds are long lived. For example, 83% can still germinate after burial for 21 years (Toole and Brown 1946). R. obtusifolius has also a high capacity for vegetative propagation from stem/roots after cutting or grazing (Cavers and Harper 1964; Hongo 1989; Pino et al. 1995).

Our primarily interest in present study was to investigate the major metabolite pathway in R. obtusifolius under different ambient conditions such as light/dark and low temperature because R. obtusifolius are able to sustain growth even under severe winter. Thus, experiments were carried out using “new leaves” and stems. Namely, all leaves removed from 2 month-old-plants were grown under different ambient environment. Two organ samples, i.e., generated leaves (“new leaves”) and stems were collected, and used for metabolome analysis.

As reported previously, we employed capillary electrophoresis mass spectrometry (CE-MS) (Takahashi et al. 2006a, b; Miyagi et al. 2010) to quantify various metabolites, which were then subjected to both principal component and hierarchical clustering analysis. Comparison was made on light and temperature with respect to their effects on metabolite accumulation in R. obtusifolius. Here, we demonstrate that metabolomics approach was powerful tool for the visualization of metabolite pathway in different organs under certain environmental conditions. The objective of this study was to assess the alteration of metabolites in R. obtusifolius under different light and temperature, the most conceivable factors. Results indicated that R. obtusifolius showed stable level of oxalate (in leaves) and citrate (in stems) regardless of environmental constrains.

2 Materials and methods

2.1 Plant materials and treatments

A single plant (R. obtusifolius L.) was grown in a pot containing Jiffy-7 (70 g in 4.0 cm × 4.0 cm × 4.5 cm pot; Jiffy Products International AS, Norway). Plants were cultivated in a climate-controlled growth room (22°C, 70% RH) under continuous light [60 μmol m−2 s−1]. For light and temperature experiments, all leaves were removed from 2 month-old-plants grown in Jiffy soil. Plants were then cultivated under either continuous light or dark conditions at 22°C. Samples were collected every week till 4 weeks. For low temperature experiment, plants were held at 5°C (in the dark) for 3 weeks. All the collected leaves and stems were immediately frozen in liquid nitrogen and then stored at −80°C. In present study, we denoted generated leaves to “new leaves” (the term “new leaves” will be used thereafter). Three to six plants were used in each experiment. In some cases, experiments were repeated twice.

2.2 Metabolite analysis with capillary electrophoresis-mass spectrometry (CE-MS)

Metabolites were quantified by the described method (Miyagi et al. 2010). Fifty milligrams samples of leaves/stems frozen in liquid nitrogen were ground and homogenised in 50% (v/v) methanol solution, which contains 50 μM 1,4-piperazine diethane sulfonic acid (PIPES) and 50 μM methionine sulfone (MeS) as the internal standard. Thus, all metabolites we analysed are soluble. The homogenate (supernatant) was centrifuged (15,000 rpm, 5 min at 4°C), and transferred to a 3 kDa cutoff filter (Millipore, Japan). After centrifugation (13,000 rpm, 100 min at 4°C), filtrate (13 μl) was subjected to CE-MS. Further determination of compounds follows the previous methods (Miyagi et al. 2010). Using the Agilent ChemStation software (Rev.A.10.01), the quantitative accuracy of corresponding compounds was determined by the measurement of known concentrations of selected compounds.

2.3 Metabolome data analysis

For visualization of the difference among the metabolite data set, principal component analysis and hierarchical clustering analysis with the squared Euclidean distance and the average linkage method (between groups) employed the Statistical Package for the Social Sciences (SPSS v10.0). In both analyses the normalization of metabolites data was represented by Z score. Using Microsoft Excel 2007, the heatmap was developed. Correlation analysis within the metabolites was used by Pearson’s correlation coefficient. Correlation significances were obtained by SPSS. Significance (P < 0.05, P < 0.01 level) by the student t-test was added in each data.

3 Results

3.1 Effects of light on growth and metabolite levels

Light is an essential environmental factor for the sustainable growth of photosynthetic plants. In this experiment, all leaves were removed from 2-month-old R. obtusifolius. Plants were then subjected to either light or dark conditions so as to monitor the accumulation of metabolites in the newly developed leaves (“new leaves”). Figure 1 shows that total leaves (gram fresh weight per plant) increased parallel to time progression under the continuous light or dark condition. There was no statistical difference in weight increase in both conditions. In the case of stems, unlike leaves, the fresh weight was not changed in either the light or dark during 4 weeks.
Fig. 1

Effects of light or dark on the growth of R. obtusifolius. The fresh weight of new leaves and stems of plants grown under either light (open box) or dark (shaded box) conditions at 22°C for 4 weeks was recorded. Bar; S.D.

Quantitative changes of the amount of major metabolites in new leaves are presented in Fig. 2. Under the light or dark condition, new leaves contained oxalate as the most abundant metabolite, followed by citrate. Amounts of oxalate increased as leaf grew from 1 to 2 weeks. Similar trend was seen in amino acids such as glutamate and aspartate. In the dark, new leaves contained more glutamine than those grown in the light. Figure 3 shows that citrate was seen as the most abundant metabolite in stems regardless of light or dark conditions. In stems, amino acid levels were not changed by the light or dark.
Fig. 2

Summary of metabolite contents in new leaves of R. obtusifolius grown under either light or dark at 22°C. Quantitative comparison of organic acids and amino acids (vertical axis: μmol gFW−1) in new leaves of R. obtusifolius grown under either light or dark. Horizontal axis; 1, 2, 3 and 4 weeks. Metabolites colored in the map are presented in each figure. Bar; S.D. * P < 0.05, ** P < 0.01

Fig. 3

Summary of metabolite contents in stems of R. obtusifolius grown under either light or dark at 22°C. Quantitative comparison of organic acids and amino acids in stems of R. obtusifolius grown under either light or dark. Each axis shown is same as in the caption for Fig. 2. Bar; S.D. * P < 0.05, ** P < 0.01

To visualize the difference among the metabolite data set, we carried out principal component analysis. Analysis of 44 metabolites in new leaves shows that two principal components representing about 52% of the observed variance in the sample set (Fig. 4a–c) were extracted. Plots of the principal component scores revealed differences in the metabolic profiles of new leaves grown in the light or dark. There were two distinct clusters; one was new leaves from plants grown under the light. Another was those grown under the dark (Fig. 4a). These clusters were separated by the first component, namely 34.3% of the total variance. Metabolites in new leaves (the light condition) contributing to the first component were mainly organic acids and sugar phosphates, whereas those under the dark condition were amino acids (Fig. 4b). Additionally, the second component seems to be the response to the growth of new leaves under the dark (Fig. 4c). In the case of metabolite profiles in stems, the principal component analysis revealed two clusters by the first component (31.0% of total variance); the first cluster was the stems in the light condition, the second one was those grown under the dark (Fig. 4d). Metabolites contributing to positive direction of first component were mainly amino acids, whereas those to negative direction were organic acids and sugar phosphate (Fig. 4e).
Fig. 4

Principal component analysis of metabolites in new leaves or stems of R. obtusifolius. Scores of principal component analysis are presented in a (new leaves) and d (stems) based on a combination of 2 components (PC1 and 2). Variances (new leaves: 34.3% for PC1 and 17.9% for PC2; stems: 31.0% for PC1 and 18.3% for PC2) were recorded in each component. Loadings score of metabolites is presented in PC1 (b and e) and PC2 (c and f). The vertical axis shows each PC loading value (b, c, e and f). The order of listed metabolites was obtained by the SPSS software as described in Sect. 2. w week(s)

To visualize the relationships and differences in levels of metabolites among each sample, we used the hierarchical cluster analysis and heatmap for metabolite profiles in new leaves (Fig. 5a). One group (left) consisted mainly of amino acids, whereas the other was mainly of organic acids and sugar phosphates. The heatmap presented that the majority of the amino acids were stimulated response to the growth under the dark whereas organic acids and sugar phosphates were likely to be up-regulated in the light (Fig. 5b). Hierarchical cluster analysis of metabolites in stems showed that there were two clusters, with the left hand, larger one divided into two sub-clusters (Fig. 6a). The group consisted mainly of amino acids such as glutamate and asparagine, the second was mainly of organic acids such as citrate, oxalate and ascorbate and few amino acids (e.g. glutamine, aspartate), and the third was mainly of sugar phosphate. The heatmap of metabolites in stems revealed that majority of amino acids were stimulated under the dark as those in new leaves (Fig. 6b). On the other hand, amounts of amino acids decreased in response to growth, whereas those increased in 0 to 1 week. Organic acids decreased in 0–1 week and did not change after 1 week regardless of light or dark. Sugar phosphates such as fructose-1,5-bisphosphate (FBP), 3-phosphoglycerate (3PGA), and phosphoenolpyruvate (PEP) in stems of plants grown in the light were higher than those grown in the dark (up to 1 week).
Fig. 5

Metabolite profiles in new leaves of R. obtusifolius grown under either light or dark. a Hierarchical classification of metabolites was done according to dissimilarity scale. The order of metabolites is corresponding to the order of Fig. 4b and c. b Heatmap of metabolites in new leaves of R. obtusifolius is presented as colour in the bottom of this figure (see Sect. 2). w week(s)

Fig. 6

Metabolite profiles in stems of R. obtusifolius grown under either light or dark. a Hierarchical classification of metabolites was done according to dissimilarity scale. The order of metabolites is corresponding to the order of Fig. 4e and f. b Heatmap of metabolites in stems of R. obtusifolius is added in the bottom. w week(s)

To clear the relationships within the metabolites, we analysed the correlation among metabolites. The results showed that there was a significant relationship between oxalate and organic acids (citrate, ascorbate), and also between oxalate and amino acids (glutamate, aspartate) in new leaves (Fig. 7). Citrate showed negative correlation to oxalate in new leaves grown under the light, whereas positive correlation was noted between citrate and oxalate in those grown under the dark. Similar phenomena were observed between oxalate and glutamine or asparagine. In stems, there was positive correlation between citrate and oxalate, and between citrate and ascorbate regardless of light or dark (Supplementary data). Furthermore, there was also high correlation between citrate and amino acids such as aspartate and glutamine.
Fig. 7

Relationship between oxalate and organic acids or amino acids in new leaves of R. obtusifolius. Data were taken from Fig. 5. w week(s). * P < 0.05, ** P < 0.01

3.2 Effects of low temperature on the accumulation of metabolites in the dark

Rumex obtusifolius is perennial weeds, which can survive under harsh winter. Thus, we were interested to analyse metabolites in plants held under low temperature (5°C) in the dark. To avoid the effects of photosynthesis, we used plants whose leaves had been removed and grown in the dark. Surprisingly, new leaves were able to generate under low temperature. Figure 8 shows the amounts of the major metabolites in new leaves of plants grown for 3 weeks at 22°C or at 5°C. In new leaves, the amount of oxalate was unchanged. On the other hand, most leaf metabolites (including those of the TCA cycle and of the amino acid pathway) were up-regulated under the cold conditions. In stems, the amount of citrate was not changed by low temperature (Fig. 9). The metabolite of TCA cycle such as malate and fumarate and amino acids in stems were also up-regulated at 5°C.
Fig. 8

Summary of metabolite contents in new leaves from R. obtusifolius grown in different temperature. Each plant was kept under the dark for 3 weeks. Amounts of organic acids and amino acids (expressed as μmol gFW−1) in new leaves from R. obtusifolius grown at either 5 or 22°C are presented. Bar; S.D. * P < 0.05, ** P < 0.01

Fig. 9

Summary of metabolite contents in stems from R. obtusifolius grown at different temperature. As described in Fig. 8, plants were kept under the dark for 3 weeks. Amounts of organic acids and amino acids (expressed as μmol gFW−1) in stems from R. obtusifolius grown at either 5 or 22°C are presented. Bar; S.D. * P < 0.05, ** P < 0.01

The principal component analysis showed distinct discrimination between the new leaves grown at 22°C and those grown at 5°C by the first component (59.8% of total variance) (Fig. 10a). Metabolites positively contributing to this component were mainly from the amino acids such as aspartate and glutamate, the sugar phosphates (the metabolites in glycolysis), and organic acids such as malate and fumarate (Fig. 10b). In stems, the similar results were obtained (Fig. 10d). The hierarchical clustering analysis of the metabolites in new leaves exhibited that there are several clusters with the left hand, larger one divided into two sub-clusters and a cluster of a few metabolites from the dendrogram (Fig. 11a). The heatmap of respective metabolites corresponding to each new leaves is also presented (Fig. 11b). One group contained fumarate, malate, dihydroxyacetone phosphate (DHAP), pyruvate, PEP, citrate and majority of amino acids (except for lysine, alanine, glycine and methionine). Another group contained mainly of organic acids such as oxalate and ascorbate, 3PGA and glucose-6-phosphate (G6P). The smallest group, which was distinct in respect to almost all metabolites, contained isocitrate, lysine, alanine, glycine, succinate and methionine. These revealed that new leaves grown at 5°C accumulated high amounts of most metabolites except for oxalate, cinnamate, 2-oxoglutarate (2OG), isocitrate, lysine, alanine, glycine succinate and methionine. In stems, the hierarchical clustering analysis revealed two major clusters (Fig. 11c). One group consisted of amino acids such as glutamate and aspartate, organic acids such as oxalate, malate and fumarate, and sugar phosphate in glycolysis. Another one consisted of mainly amino acids such as alanine and glycine, citrate and ribulose-5-phosphate (Ru5P). Amino acids as glutamate and aspartate, organic acids such as oxalate, malate and fumarate were up-regulated by low temperature (Fig. 11d).
Fig. 10

Principal component analysis of metabolites in new leaves or stems of R. obtusifolius grown at different temperature. Scores of principal component analysis are presented in a (new leaves) and d (stems) based on a combination of 2 components (PC1 and 2). Variances (new leaves: 59.8% for PC1 and 17.9% for PC2; stems: 58.3% for PC1 and 23.7% for PC2) were recorded in each component. Loading score of metabolites is presented in PC1 (b, e) and PC2 (c, f). The vertical axis shows each PC loading value (b, c, e and f)

Fig. 11

Metabolite profiles in new leaves or stems of R. obtusifolius grown at different temperature. Hierarchical classification of metabolites in new leaves (a) or stems (c) was done according to dissimilarity scale. The order of metabolites is corresponding to the order of Fig. 10b or e. Heatmap of metabolites in new leaves (b) or stems (d) of R. obtusifolius grown at either 5 or 22°C is presented

4 Discussion

Several physiological studies have been carried out on Rumex plants grown under different constraints such as submergence (Cox et al. 2004; Voesenek et al. 2006), extreme soil pH and low nitrogen (Hongo 1989) or low phosphorous (Horie and Nemoto 1990) and metallic ion stress such as from aluminum and copper (Tolrà et al. 2005; Ke et al. 2007). Nevertheless, a comprehensive metabolome analysis on the metabolites accumulation in Rumex plants under different environmental stresses has not yet been presented.

We compared metabolites in new leaves of Rumex plants under light or dark conditions. Results indicated that the oxalate level was not reduced by the lack of light. This suggests that light did not stimulate the carbon compound pool in Rumex plants which, in turn, is used actively for the production of oxalate even under the dark. Thus, for the accumulation of oxalate in leaves, light is not essential. Namely, absence of Calvin cycle does not detrimental to the oxalate accumulation in R. obtusifolius. Furthermore, glycolate pathway (blocked by dark treatment) is not essential for oxalate production. Dark condition stimulated the content of certain amino acids (glutamine and asparagine), whereas glutamate, aspartate and serine were reduced by dark in new leaves. Thus, it can be concluded that these differences of metabolite profiles in each condition did not depended on developmental stages due to no statistical significance among fresh weight of leaves regardless of presence or absence of light. In contrast to leaves, there were no significant changes in most metabolites in stems regardless of presence or absence of light.

Hierarchical clustering analysis also confirmed the similarity of compounds in the proposed metabolic pathway. In stems, there was no significant accumulation of oxalate, whereas citrate levels were highest. Apparently, there was an organ related metabolite alterations in R. obtusifolius. In this respect, comparison of effects of light or dark may provide some clue to answer how specific organic acid is accumulated via photosynthetic pathway. Oxalate seems to be synthesized through multiple biochemical pathways in plants. At least three distinct pathways have been reported. One is mediated by the glycolate pathway via photorespiration, the second is through the TCA-glyoxylate cycle and the third is a pathway via ascorbate synthesis (Franceschi and Nakata 2005). However, it is still unclear how these pathways are orchestrated and regulated. Our results indicate that major pathway for the production of oxalate can be the isocitrate-oxalate shunt in new leaves. Mclaren and Smith (1978) reported the significance of phytochrome in growth and development of R. obtusifolius. Thus, it may be necessary to analyze precise relationship between light quality and metabolite accumulation.

Significance of temperature on seed dormancy of Rumex crispus L. and R. obtusifolius has been described (Van Assche and Vanlerberghe 1989; Totterdell and Roberts 1979). However, the influence of temperature on the metabolite accumulation has not been studied in Rumex species. Thus, we compared metabolites in R. obtusifolius grown under different temperature (22 or 5°C). Results indicated that leaves can grow even at 5°C, where accumulation of oxalate as the most abundant metabolite was sustained. In spinach the content of oxalate remained unchanged at 10 or 25°C (Proietti et al. 2009). Maize leaves are extremely sensitive to chilling injury (below 15°C), which usually results in premature leaf senescence (Foyer et al. 2002). Similar cases are also known in mung bean (Yang et al. 2005), where incapability of leaf greening is the only failure event for the de-etiolation of mung bean seedlings at low temperature. We showed here that the low temperature (5°C) did not lower leaf growth or the level of metabolites in TCA cycle. Chilling during imbibitions causes mitochondrial damage at metabolic level in soybean seed axis (Yin et al. 2009). Unlike soybean, R. obtusifolius may possess an active respiratory function, possibly from active mitochondria even at low temperature. In stems, high level of citrate was seen, but oxalate level was low. This result suggests that citrate stored in stems can serve as the carbon source for oxalate synthesis in “new leaves”. In our unpublished experiments, we studied relationships between oxalate contents and leaves at different stages. Leaves at different days (0, 3, 7, and 14) of 2-month-old-plants kept under light condition were used to compare metabolites. Results indicated that amounts of oxalate were parallel to expansion of leaves. Since the cell number of leaves is likely kept constant, increments of oxalate accumulation may occur in expanded cells. Taking such data into consideration, it can be concluded that oxalate accumulation in leaves of Rumex species is stable regardless of presence or absence of light even under low temperature. Hence, the carbon source stored in chloroplasts may not be the sole source for oxalate in Rumex plants. In potato tubers, cold-stress affected the expression and activities of plant uncoupling mitochondrial protein and alternative oxidase, enabling similar extents of mitochondrial respiration (Calegario et al. 2003). Therefore, Rumex plant may possess similar respiratory potential as potato tuber in maintaining normal metabolic capacity even under low temperature.

In conclusion, it was shown that application of metabolomics is a powerful tool for the visualization of major metabolite pathway in R. obtusifolius under different environmental conditions. Further analyses would be needed with respect to the effect of other environmental factors (such as osmotic stress, submergence, high CO2 and others) on metabolite accumulation and balance of carbon and nitrogen compounds in R. obtusifolius.

Notes

Acknowledgements

This research was supported by a grant from the Program for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry (BRAIN) and the CREST, JST, Japan.

Supplementary material

11306_2010_220_MOESM1_ESM.pdf (24 kb)
Supplementary data Relationship between citrate and organic acids or amino acids in stems of R. obtusifolius grown under either light or dark. Data were taken from Fig. 6. W; week(s). *; P < 0.05, **; P < 0.01. (PDF 25 kb)

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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Atsuko Miyagi
    • 1
    • 2
  • Kentaro Takahara
    • 2
  • Hideyuki Takahashi
    • 3
  • Maki Kawai-Yamada
    • 1
    • 4
    • 5
  • Hirofumi Uchimiya
    • 1
    • 2
    • 3
  1. 1.Institute for Environmental Science and TechnologySaitama UniversitySaitama CityJapan
  2. 2.Institute of Molecular and Cellular BiosciencesThe University of TokyoTokyoJapan
  3. 3.Iwate Biotechnology Research CenterIwateJapan
  4. 4.Graduate School of Science and EngineeringSaitama UniversitySaitama CityJapan
  5. 5.Core Research for Evolutional Science and Technology (CREST)Japan Science and Technology Agency (JST)SaitamaJapan

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