Targeted metabolomics in an intrusive weed, Rumex obtusifolius L., grown under different environmental conditions reveals alterations of organ related metabolite pathway
- 266 Downloads
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.
KeywordsRumex obtusifolius New leaves Stems Oxalate Citrate Organ specific metabolites Capillary electrophoresis mass spectrometry Principal component analysis Hierarchical clustering analysis
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.
3.1 Effects of light on growth and metabolite levels
3.2 Effects of low temperature on the accumulation of metabolites in the dark
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.
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.
- Calegario, F. F., Cosso, R. G., Fagian, M. M., Almeida, V., Jardim, W. F., Ježek, P., et al. (2003). Stimulation of potato tuber respiration by cold stress is associated with an increased capacity of both plant uncoupling mitochondrial protein (PUMP) and alternative oxidase. Journal of Bioenergetics and Biomembranes, 35, 211–220.CrossRefPubMedGoogle Scholar
- Cox, M. C. H., Benschop, J. J., Vreeburg, R. A. M., Wagemaker, C. A. M., Moritz, T., Peeters, A. J. M., et al. (2004). The roles of ethylene, auxin, abscisic acid, and gibberellin in the hyponastic growth of submerged Rumex palustris petioles. Plant Physiology, 136, 2948–2960.CrossRefPubMedGoogle Scholar
- Holm, L. G., Plucknett, D. L., Pancho, J. V., & Herberger, J. P. (1977). Rumex crispus and Rumex obtusifolius. In L. G. Holm (Ed.), The world’s worst weeds: Distribution and biology (pp. 401–408). Honolulu: University Press of Hawaii.Google Scholar
- Hongo, A. (1986). Infestation of Rumex obtusifolius L., distribution pattern of its individual plants in sown grasslands in eastern Hokkaido. Weed Research, Japan, 31, 300–315.Google Scholar
- Horie, H., & Nemoto, M. (1990). Comparison of the growth response to phosphorus and aluminum concentrations in four Rumex species. Weed Research, Japan, 35, 340–345.Google Scholar
- Ke, W., Xiong, Z. T., Chen, S., & Chen, J. (2007). Effect of copper and mineral nutrition on growth, copper accumulation and mineral element uptake in two Rumex japonicus populations from a copper mine and an uncontaminated field sites. Environmental and Experimental Botany, 59, 59–67.CrossRefGoogle Scholar
- Löve, A., & Kapoor, B. M. (1967). A chromosome atlas of the collective genus Rumex. Cytologia, 32, 328–342.Google Scholar
- Makuchi, T., & Sakai, H. (1984). Seedling survival and flowering of Rumex obtusifolius L. in various habitats. Weed Research, Japan, 29, 123–130.Google Scholar
- Toole, E. H., & Brown, E. (1946). Final results of the Duvel buried seed experiment. Journal of Agricultural Research, 72, 201–206.Google Scholar