Metabolite profiling of rye samples
Untargeted LC-MS analysis detected 11670 ion features, of which 369 were annotated by spectrum matching with the reference mass spectra in the public and licensed libraries. The PCA score plot of the raw data acquired by HHPLC-QTOF-MS showed the accuracy and robustness of the instrument based on the grouping of QC and Blank samples (Figure S2).
Metabolites present in roots of rye plants growing alone were removed from the feature list, and only 58 metabolites originating from neighboring plants were kept. Figure 2 shows the classification of absorbed metabolites at superclass and class levels. Nearly 60% of the metabolites belonged to the phenylpropanoids and polyketides superclass, and flavonoids were the most abundant compounds taken up by rye root at the class level.
The PCA score plots (Figure 3) revealed the grouping of the samples based on the taxonomic relatedness of the neighboring species. Rye root samples growing with Alexandrian clover and subterranean clover that belongs to the same genus (Trifolium) were grouped. Root samples from rye plants growing together with fodder radish and Sinapis, two species belonging to the Brassicaceae family, were also grouped. Root samples of rye plants growing with oat, pea and hairy vetch were separated and did not group with other samples. The roots of the rye plant growing with Lolium did not have a distinct separation from rye plants growing alone. This observation suggests that Lolium and rye have similar metabolomes, and many of the metabolites exudated by Lolium plants and absorbed by rye were already present in rye roots and, hence, excluded in the data filtering process.
Figure 4 shows the hierarchical clustering heatmap of the metabolites absorbed by rye roots from plants growing alone or together with the eight other plant species. The intensity of all annotated metabolites in rye roots from plants growing alone was nearly zero. Therefore, we assumed that all the annotated metabolites were taken up from neighboring plants. Like the PCA analysis, clustering of heatmap indicated that rye root samples from plants grown with two clover species (Alexandrian and subterranean) clustered together and, similarly, samples from rye plants growing with fodder radish and Sinapis were clustered together. Malonylglycitin, wogonoside, and sissotrin were the annotated metabolites with the highest peak intensity in rye plant roots growing with legumes.
4-Methoxyglucobrassicin was the dominant annotated metabolite in roots of rye plants growing together with fodder radish and Sinapis (Figure S3).
Untargeted LC-MS analysis of rye shoot samples detected 5637 ion features, of which 176 features were annotated by spectrum matching with the reference mass spectra in the public and licensed libraries. The PCA score plot of 176 annotated metabolites in rye shoot showed no clear grouping of samples (Figure S4). Among all the annotated metabolites in the shoot, wogonin was the secondary plant metabolite found in the shoots of rye plants growing with hairy vetch, Alexandrian clover, and subterranean clover. Figure 5 shows the intensity of wogonin in rye root (A) and rye shoot (B). Wogonin was present in the roots of rye plants growing with Austrian pea, but it was absent in the shoots.
Discussion
A previous study confirmed that root-exuded BXs could be taken up by hairy vetch (Vicia vilosa ) roots and translocated to the shoots (Hazrati et al., 2020). Nevertheless, it was unclear if the uptake of root-exuded BXs varied among plant species. This study is the first to compare the uptake and translocation of root-exuded BXs in several plant species. Several studies show the uptake root uptake of secondary metabolite and their transformation in roots (Buer et al., 2007, Selmar et al., 2019). In addition, recent studies have shown BXs taken up by root can be translocated to the shoots (Hazrati et al., 2020). However, no studies have intended to unravel and provide a complete picture of the uptake of exuded/decomposed organic compounds present in the rhizosphere using an untargeted metabolomics approach. Here we applied mass spectrometry-based metabolomics and observed that roots of rye plants growing with other plant species contain numerous compounds that most likely have been exuded by neighboring plant species.
Our results showed that neighboring plant species could absorb all 12 BXs. This is in accordance with a previous study by Hazrati et al. (2020), which reported root uptake of 12 BXs by hairy vetch plants growing together with one rye plant. The concentration and composition of absorbed BXs varied between neighboring plant species. For instance, Sinapis and subterranean clover roots absorbed the lowest concentrations of BXs while oat took up the highest amount of BXs. This could be due to differences in the architecture and volume of neighboring plant roots. Roots of Sinapis and subterranean clover were denser and occupied a smaller volume than the roots of the other neighboring plant species (based on visual observations at harvest). Less root volume may reduce the exposure of roots to BXs present in the rhizosphere. On the other hand, oat roots have a root architecture very similar to rye. Their roots grew alongside the roots of the rye plants, which was expected to increase the exposure of their roots to BXs exuded from rye roots.
Differences in the composition and concentration of BXs in the roots of neighboring plant species may be related to microbial degradation. Burns et al. (2015) suggested that plant species identity was the main determinator of microbial community composition in the rhizosphere. Moreover, plant-associated microorganisms have been shown to degrade organic compounds in the rhizosphere (McGuinness and Dowling, 2009). For instance, Friebe et al. (1996) found that root colonized bacteria converted BOA to 2-amino-H-phenoxazin-3-one and 2-acetylamino-H-phenoxazin-3-one in Avena sativa . In the present study, each of the neighboring plants may have shaped the microbial communities in the rhizosphere differently, resulting in different degradation patterns and, hence, different BX compositions and availability in the rhizosphere.
Simple diffusion across biomembranes has been suggested as a mechanism for the uptake of xenobiotics (Trapp and Legind, 2011). Similarly, it was shown that plant uptake of alkaloids is by simple diffusion through the plasmalemma of the root cells (Nowak et al., 2016, Yahyazadeh et al., 2017). Simple diffusion requires specific physicochemical properties of organic compounds, such as an appropriate Log Pvalue (Inoue et al., 1998). Previous studies demonstrated that compounds having a Log P value of -1 to 3 could be taken up by roots through diffusion (Limmer and Burken, 2014, Trapp, 2000). Our observations are in accordance with this conclusion. Log P values for the BXs taken up by neighboring plant roots and many flavonoids, which were the dominant class of secondary plant metabolite absorbed by rye roots, are within the range of -1.6 to 3 (https://pubchem.ncbi.nlm.nih.gov/). Log P values for the BXs studies in this experiment are shown in Table 2.
Only four BXs were translocated into the shoots of neighboring plants, and only one metabolite (wogonin) was translocated into the shoot of rye plants. This may be due to high limits of detection, soil degradation, and/or metabolization of metabolites in neighboring plants. Rapid metabolism in plants may decrease the concentration of BXs in the target plants. It is demonstrated that contaminants such as pesticides may be transformed inside the plant or on the leaf surface by the plants or microorganisms living on the plants (Trapp and Legind, 2011). Secondary metabolites in the plants may face a similar fate and, therefore, not reach the shoots. Briggs et al. (1982) showed that translocation of non-ionic pesticides from root to shoot is related to their polarity by a Gaussian curve distribution. They concluded that the translocation of pesticides to shoots is most efficient for pesticides with intermediate polarities (log P =1-3). In the present study, Log P for all the tested BXs was between -1.6 and 1, and we could not find a significant correlation between the polarity of BXs and their translocation to shoot. Lolium’s shoot contained the highest amount of BXs, whereas shoots of Austrian pea, fodder radish, and Sinapis contained the lowest concentration of BXs. The aboveground biomass of Lolium was lower than of all the other neighboring plants (data not shown). Hence, it cannot be excluded that the lower biomass was partly the cause of the higher accumulation of BXs in Lolium shoots. Wogonin and its glucuronide, wogonoside, have various biological effects such as anti-cancerogenic, anti-inflammatory, and anti-angiogenesis in humans (Wang et al., 2018, Kim et al., 2018, Lin et al., 2012). It is, therefore, very likely that they act as a defense compound against biotic and abiotic stresses in plants. In the rye root, wogonoside was found in a much higher concentration than wogonin. However, wogonin was detected in the rye shoot, and wogonoside was not detected. We assume that wogonin found in rye shoot originates from the wogonoside present in the root. Wogonoside likely undergoes deglycosylation through enzymatic reactions or microbiome metabolism and covert into wogonin in the rye shoot. It has been reported that deglycosylation of wogonoside enhances its bioactivity by inhibiting the growth of cancer cells (Wang et al., 2018, Yu et al., 2013). Conversion of wogonoside to wogonin in plant shoot may increase their growth inhibitory effects.
Conclusion
In the present study, extensive transfer of bioactive compounds between rye and several crops and weed species as neighboring plants has been revealed by applying mass spectrometry-based metabolomic. The results from targeted metabolomics demonstrated that all the neighboring plants in this experiment absorbed BXs by their root and translocated them to shoots. Meanwhile, we showed that the composition and concentration of absorbed BXs in tested plants were varied among different plant species. Furthermore, the results from untargeted metabolomics indicated that the rye plant roots took up compounds originating from neighboring plants. Wogonin, which is a phytotoxic flavonoid, was detected in both rye root and shoot. Elucidating the root absorption and root-to-shoot translocation mechanisms is essential for future utilization of the bioactive compounds as natural agrochemicals.
Funding
This study was funded by a project (28180) at the Graduate School of Science and Technology, Aarhus University (GSST, AU), Denmark.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that may influence the work reported in this paper.
Acknowledgments
We would also like to thank Kirsten Heinrichson and Bente Birgitte Laursen for their excellent laboratory technical assistance. The authors wish to thank Dr. Aaron John Christian Andersen, Mette Amfelt, and Xinhui Wang from the Department of Biotechnology and Biomedicine, Technical University of Denmark, for their valuable support.
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Table 1. Optimized Compound dependent MS parameters for BXs