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.
References
BAIS, H. P., WEIR, T. L., PERRY, L. G., GILROY, S. & VIVANCO, J. M.
2006. The role of root exudates in rhizosphere interactions with plants
and other organisms. Annu Rev Plant Biol, 57, 233-66.
BRIGGS, G. G., BROMILOW, R. H. & EVANS, A. A. 1982. Relationships
between lipophilicity and root uptake and translocation of non-ionised
chemicals by barley. Pesticide Science, 13, 495-504.
BUER, C. S., MUDAY, G. K. & DJORDJEVIC, M. A. 2007. Flavonoids are
differentially taken up and transported long distances in Arabidopsis.Plant physiology, 145, 478-490.
BURNS, J. H., ANACKER, B. L., STRAUSS, S. Y. & BURKE, D. J. 2015. Soil
microbial community variation correlates most strongly with plant
species identity, followed by soil chemistry, spatial location and plant
genus. AoB PLANTS, 7.
CEVALLOS-CEVALLOS, J. M., REYES-DE-CORCUERA, J. I., ETXEBERRIA, E.,
DANYLUK, M. D. & RODRICK, G. E. 2009. Metabolomic analysis in food
science: a review. Trends in Food Science & Technology,20, 557-566.
CHIAPUSIO, G., PELLISSIER, F. & GALLET, C. 2004. Uptake and
translocation of phytochemical 2-benzoxazolinone (BOA) in radish seeds
and seedlings. Journal of Experimental Botany, 55,1587-1592.
CHONG, J., SOUFAN, O., LI, C., CARAUS, I., LI, S., BOURQUE, G., WISHART,
D. S. & XIA, J. 2018. MetaboAnalyst 4.0: towards more transparent and
integrative metabolomics analysis. Nucleic acids research,46, W486-W494.
COTTON, T. E. A., PÉTRIACQ, P., CAMERON, D. D., MESELMANI, M. A.,
SCHWARZENBACHER, R., ROLFE, S. A. & TON, J. 2019. Metabolic regulation
of the maize rhizobiome by benzoxazinoids. The ISME Journal,13, 1647-1658.
DAVIES, J. & CASELEY, J. C. 1999. Herbicide safeners: a review.Pesticide Science, 55, 1043-1058.
DEFELICE, B. C., MEHTA, S. S., SAMRA, S., ČAJKA, T., WANCEWICZ, B.,
FAHRMANN, J. F. & FIEHN, O. 2017. Mass Spectral Feature List Optimizer
(MS-FLO): A Tool To Minimize False Positive Peak Reports in Untargeted
Liquid Chromatography–Mass Spectroscopy (LC-MS) Data Processing.Analytical Chemistry, 89, 3250-3255.
DJOUMBOU FEUNANG, Y., EISNER, R., KNOX, C., CHEPELEV, L., HASTINGS, J.,
OWEN, G., FAHY, E., STEINBECK, C., SUBRAMANIAN, S., BOLTON, E., GREINER,
R. & WISHART, D. S. 2016. ClassyFire: automated chemical classification
with a comprehensive, computable taxonomy. Journal of
Cheminformatics, 8, 61.
DUNN, W. B., ERBAN, A., WEBER, R. J. M., CREEK, D. J., BROWN, M.,
BREITLING, R., HANKEMEIER, T., GOODACRE, R., NEUMANN, S., KOPKA, J. &
VIANT, M. R. 2013. Mass appeal: metabolite identification in mass
spectrometry-focused untargeted metabolomics. Metabolomics,9, 44-66.
FREY, M., SCHULLEHNER, K., DICK, R., FIESSELMANN, A. & GIERL, A. 2009.
Benzoxazinoid biosynthesis, a model for evolution of secondary metabolic
pathways in plants. Phytochemistry, 70, 1645-1651.
FRIEBE, A., WIELAND, I. & SCHULZ, M. 1996. Tolerance of Avena sativa to
the allelochemial benzoxazolinone. Degradation of BOA by root-colonizing
bacteria. Angewandte Botanik, 70, 150-154.
HAZRATI, H., FOMSGAARD, I. S. & KUDSK, P. 2020. Root-Exuded
Benzoxazinoids: Uptake and Translocation in Neighboring Plants.Journal of Agricultural and Food Chemistry, 68,10609-10617.
HAZRATI, H., FOMSGAARD, I. S. & KUDSK, P. 2021. Targeted metabolomics
unveil alteration in accumulation and root exudation of flavonoids as a
response to interspecific competition. Journal of Plant
Interactions, 16, 53-63.
HAZRATI, H., FOMSGAARD, I. S., MELANDER, B. & KUDSK, P. 2019. Role of
natural products in belowground interactions between plant species.Planta Med, 85, P-396.
HU, L., ROBERT, C. A. M., CADOT, S., ZHANG, X., YE, M., LI, B., MANZO,
D., CHERVET, N., STEINGER, T., VAN DER HEIJDEN, M. G. A., SCHLAEPPI, K.
& ERB, M. 2018. Root exudate metabolites drive plant-soil feedbacks on
growth and defense by shaping the rhizosphere microbiota. Nature
Communications, 9, 2738.
INOUE, J., CHAMBERLAIN, K. & BROMILOW, R. H. 1998. Physicochemical
factors affecting the uptake by roots and translocation to shoots of
amine bases in barley. Pesticide science, 54, 8-21.
KIM, K. A., JUNG, J. H., CHOI, Y. S., KANG, G. & KIM, S. T. 2018.
Anti-inflammatory effect of wogonin on allergic responses in
ovalbumin-induced allergic rhinitis in the mouse. Allergy &
rhinology (Providence, R.I.), 9,2152656718764145-2152656718764145.
KRAUSS, M., SINGER, H. & HOLLENDER, J. 2010. LC–high resolution MS in
environmental analysis: from target screening to the identification of
unknowns. Analytical and Bioanalytical Chemistry, 397,943-951.
KUDJORDJIE, E. N., SAPKOTA, R., STEFFENSEN, S. K., FOMSGAARD, I. S. &
NICOLAISEN, M. 2019. Maize synthesized benzoxazinoids affect the host
associated microbiome. Microbiome, 7, 59.
LEE, D. Y., BOWEN, B. P. & NORTHEN, T. R. 2010. Mass spectrometry-based
metabolomics, analysis of metabolite-protein interactions, and imaging.BioTechniques, 49, 557-565.
LEWERENZ, L., HIJAZIN, T., ABOUZEID, S., HÄNSCH, R. & SELMAR, D. 2020.
Pilot study on the uptake and modification of harmaline in acceptor
plants: An innovative approach to visualize the interspecific transfer
of natural products. Phytochemistry, 174, 112362.
LIMMER, M. A. & BURKEN, J. G. 2014. Plant translocation of organic
compounds: molecular and physicochemical predictors. Environmental
Science & Technology Letters, 1, 156-161.
LIN, C. M., CHEN, Y. H., ONG, J. R., MA, H. P., SHYU, K. G. & BAI, K.
J. 2012. Functional role of wogonin in anti-angiogenesis. Am J
Chin Med, 40, 415-27.
LU, W., BENNETT, B. D. & RABINOWITZ, J. D. 2008. Analytical strategies
for LC–MS-based targeted metabolomics. Journal of Chromatography
B, 871, 236-242.
MCGUINNESS, M. & DOWLING, D. 2009. Plant-associated bacterial
degradation of toxic organic compounds in soil. International
journal of environmental research and public health, 6,2226-2247.
MISRA, B. B. & VAN DER HOOFT, J. J. 2016. Updates in metabolomics tools
and resources: 2014-2015. Electrophoresis, 37, 86-110.
NOWAK, M., WITTKE, C., LEDERER, I., KLIER, B., KLEINWÄCHTER, M. &
SELMAR, D. 2016. Interspecific transfer of pyrrolizidine alkaloids: An
unconsidered source of contaminations of phytopharmaceuticals and plant
derived commodities. Food Chemistry, 213, 163-168.
PEDERSEN, H. A., STEFFENSEN, S. K., HEINRICHSON, K. & FOMSGAARD, I. S.
2017. Biphenyl Columns Provide Good Separation of the Glucosides of
DIMBOA and DIM2BOA. Natural Product Communications, 12,1934578X1701200708.
PEZZATTI, J., GONZÁLEZ-RUIZ, V., CODESIDO, S., GAGNEBIN, Y., JOSHI, A.,
GUILLARME, D., SCHAPPLER, J., PICARD, D., BOCCARD, J. & RUDAZ, S. 2019.
A scoring approach for multi-platform acquisition in metabolomics.Journal of Chromatography A, 1592, 47-54.
ROBERTS, L. D., SOUZA, A. L., GERSZTEN, R. E. & CLISH, C. B. 2012.
Targeted metabolomics. Current Protocols in Molecular Biology.
SCHULZ, M., MAROCCO, A., TABAGLIO, V., MACIAS, F. A. & MOLINILLO, J. M.
G. 2013. Benzoxazinoids in Rye Allelopathy - From Discovery to
Application in Sustainable Weed Control and Organic Farming.Journal of Chemical Ecology, 39, 154-174.
SELMAR, D., WITTKE, C., BECK-VON WOLFFERSDORFF, I., KLIER, B., LEWERENZ,
L., KLEINWÄCHTER, M. & NOWAK, M. J. E. P. 2019. Transfer of
pyrrolizidine alkaloids between living plants: A disregarded source of
contaminations. 248, 456-461.
SIKDER, M. M., VESTERGÅRD, M., KYNDT, T., FOMSGAARD, I. S., KUDJORDJIE,
E. N. & NICOLAISEN, M. 2021. Benzoxazinoids selectively affect maize
root-associated nematode taxa. Journal of Experimental Botany .
THEODORIDIS, G. A., GIKA, H. G., WANT, E. J. & WILSON, I. D. J. A. C.
A. 2012. Liquid chromatography–mass spectrometry based global
metabolite profiling: a review. 711, 7-16.
TRAPP, S. 2000. Modelling uptake into roots and subsequent translocation
of neutral and ionisable organic compounds. Pest Management
Science: formerly Pesticide Science, 56, 767-778.
TRAPP, S. & LEGIND, C. N. 2011. Uptake of organic contaminants from
soil into vegetables and fruits. Dealing with contaminated sites.Springer.
TSUGAWA, H., CAJKA, T., KIND, T., MA, Y., HIGGINS, B., IKEDA, K.,
KANAZAWA, M., VANDERGHEYNST, J., FIEHN, O. & ARITA, M. 2015. MS-DIAL:
data-independent MS/MS deconvolution for comprehensive metabolome
analysis. Nature methods, 12, 523-526.
VINAYAVEKHIN, N. & SAGHATELIAN, A. 2010. Untargeted metabolomics.Curr Protoc Mol Biol, Chapter 30, Unit 30.1.1-24.
VRHOVSEK, U., MASUERO, D., GASPEROTTI, M., FRANCESCHI, P., CAPUTI, L.,
VIOLA, R. & MATTIVI, F. 2012. A versatile targeted metabolomics method
for the rapid quantification of multiple classes of phenolics in fruits
and beverages. J Agric Food Chem, 60, 8831-40.
WANG, C.-Z., WAN, J.-Y., ZHANG, C.-F., LU, F., CHEN, L. & YUAN, C.-S.
2018. Deglycosylation of wogonoside enhances its anticancer potential.Journal of cancer research and therapeutics, 14,S594-S599.
WEIR, T. L., PARK, S.-W. & VIVANCO, J. M. J. C. O. I. P. B. 2004.
Biochemical and physiological mechanisms mediated by allelochemicals.
7, 472-479.
WOUTERS, F. C., BLANCHETTE, B., GERSHENZON, J. & VASSÃO, D. G. 2016.
Plant defense and herbivore counter-defense: benzoxazinoids and insect
herbivores. Phytochemistry Reviews, 15, 1127-1151.
YAHYAZADEH, M., NOWAK, M. & KIMA, H. 2017. Horizontal natural product
transfer: A potential source of alkaloidal contaminants in
phytopharmaceuticals. Phytomedicine, 34, 21.
YU, C., ZHANG, Z., ZHANG, H., ZHEN, Z., CALWAY, T., WANG, Y., YUAN, C.
S. & WANG, C. Z. 2013. Pretreatment of baicalin and wogonoside with
glycoside hydrolase: a promising approach to enhance anticancer
potential. Oncol Rep, 30, 2411-8.
ZHANG, C., FENG, C., ZHENG, Y., WANG, J. & WANG, F. 2020. Root Exudates
Metabolic Profiling Suggests Distinct Defense Mechanisms Between
Resistant and Susceptible Tobacco Cultivars Against Black Shank Disease.Frontiers in Plant Science, 11, 1352.
Table 1. Optimized Compound dependent MS parameters for BXs