Tissue-specific signatures of metabolites and proteins in asparagus roots and exudates

Abstract

Comprehensive untargeted and targeted analysis of root exudate composition has advanced our understanding of rhizosphere processes. However, little is known about exudate spatial distribution and regulation. We studied the specific metabolite signatures of asparagus root exudates, root outer (epidermis and exodermis), and root inner tissues (cortex and vasculature). The greatest differences were found between exudates and root tissues. In total, 263 non-redundant metabolites were identified as significantly differentially abundant between the three root fractions, with the majority being enriched in the root exudate and/or outer tissue and annotated as ‘lipids and lipid-like molecules’ or ‘phenylpropanoids and polyketides’. Spatial distribution was verified for three selected compounds using MALDI-TOF mass spectrometry imaging. Tissue-specific proteome analysis related root tissue-specific metabolite distributions and rhizodeposition with underlying biosynthetic pathways and transport mechanisms. The proteomes of root outer and inner tissues were spatially very distinct, in agreement with the fundamental differences between their functions and structures. According to KEGG pathway analysis, the outer tissue proteome was characterized by a high abundance of proteins related to ‘lipid metabolism’, ‘biosynthesis of other secondary metabolites’ and ‘transport and catabolism’, reflecting its main functions of providing a hydrophobic barrier, secreting secondary metabolites, and mediating water and nutrient uptake. Proteins more abundant in the inner tissue related to ‘transcription’, ‘translation’ and ‘folding, sorting and degradation’, in accord with the high activity of cortical and vasculature cell layers in growth- and development-related processes. In summary, asparagus root fractions accumulate specific metabolites. This expands our knowledge of tissue-specific plant cell function.

References

[1] Canarini, A., Kaiser, C., Merchant, A., Richter, A. & Wanek, W. Root exudation of primary metabolites: mechanisms and their roles in plant responses to environmental stimuli. Front Plant Sci. 10, 157 (2019).

[2] Sasse, J., Martinoia, E. & Northen, T. Feed your friends: do plant exudates shape the root microbiome? Trends Plant Sci. 23, 25-41 (2018).

[3] Oburger, E. & Schmidt, H. New methods to unravel rhizosphere processes. Trends Plant Sci. 21, 243-255 (2016).

[4] van Dam, N. M. & Bouwmeester, H. J. Metabolomics in the rhizosphere: tapping into belowground chemical communication. Trends Plant Sci. 21, 256-265 (2016).

[5] Kong, C. H., Xuan, T. D., Khanh, T. D., Tran, H. D. & Trung, N. T. Allelochemicals and signaling chemicals in plants. Molecules 24, 15 (2019).

[6] Schmid, N. B. et al. Feruloyl-CoA 6‘-hydroxylase1-dependent coumarins mediate iron acquisition from alkaline substrates in Arabidopsis. Plant Physiol. 164, 160-172 (2014).

[7] Zhang, Y., Ruyter-Spira, C. & Bouwmeester, H. J. Engineering the plant rhizosphere. Curr. Opin. Biotechnol. 32, 136-142 (2015).

[8] Massalha, H., Korenblum, E., Tholl, D. & Aharoni, A. Small molecules belowground: the role of specialized metabolites in the rhizosphere. Plant J. 90, 788-807 (2017).

[9] Wurtzel, E. T. & Kutchan, T. M. Plant metabolism, the diverse chemistry set of the future. Science 353, 1232-1236 (2016).

[10] Lee, J. W. et al. Bioactive compounds, antioxidant and binding activities and spear yield of Asparagus officinalis L. Plant Foods Hum. Nutr. 69, 175-181 (2014).

[11] Creydt, M., Arndt, M., Hudzik, D. & Fischer, M. Plant metabolomics: evaluation of different extraction parameters for nontargeted UPLC-ESI-QTOF-mass spectrometry at the example of white Asparagus officinalis . J. Agric. Food Chem. 66, 12876-12887 (2018).

[12] Jiménez-Sánchez, C., Lozano-Sánchez, J., Rodríguez-Pérez, C., Segura-Carretero, A. & Fernández-Gutiérrez, A. Comprehensive, untargeted, and qualitative RP-HPLC-ESI-QTOF/MS2 metabolite profiling of green asparagus (Asparagus officinalis) . J. Food Comp. Anal. 46, 78-87 (2016).

[13] Iqbal, M. et al. Review on therapeutic and pharmaceutically important medicinal plant Asparagus officinalis L. J. Plant. Biochem. Phys. 5, 1 (2017).

[14] Zhang, H., Birch, J., Pei, J., Ma, Z. F. & Bekhit, A. E.-D. Phytochemical compounds and biological activity in asparagus roots: a review. Int. J. Food Sci. Tech. 54, 966-977 (2019).

[15] Onlom, C. et al. Quantification of saponins in Asparagus racemosus by HPLC-Q-TOF-MS/MS. Nat. Prod. Commun. 12, 7-10 (2017).

[16] Jaiswal, Y., Liang, Z., Ho, A., Chen, H. & Zhao, Z. A comparative tissue-specific metabolite analysis and determination of protodioscin content in asparagus species used in traditional Chinese medicine and Ayurveda by use of laser microdissection, UHPLC-QTOF/MS and LC-MS/MS. Phytochem. Anal. 25, 514-528 (2014).

[17] Srivastava, P. L., Shukla, A. & Kalunke, R. M. Comprehensive metabolic and transcriptomic profiling of various tissues provide insights for saponin biosynthesis in the medicinally important Asparagus racemosus . Sci. Rep. 8, 13 (2018).

[18] Shiomi, N., Yamada, J. & Izawa, M. Isolation and identification of fructooligosaccharides in roots of asparagus (Asparagus officinalis L.) . Agric. Biol. Chem. 40, 567-575 (1976).

[19] Suzuki, T. et al. Rapid analysis of fructans and comparison of fructan profiles in several different types of asparagus storage roots using MALDI-TOF MS. J. Hortic. Sci. Biotechnol. 86, 210-216 (2011).

[20] Zhang, H. et al. Identification of six phytochemical compounds from Asparagus officinalis L. root cultivars from New Zealand and China using UAE-SPE-UPLC-MS/MS: effects of extracts on H₂O₂ induced oxidative stress. Nutrients 11, 1 (2019).

[21] Rieger, M. & Litvin, P. Root system hydraulic conductivity in species with contrasting root anatomy. J. Exp. Bot. 50, 201-209 (1999).

[22] Upadhyay, S., Phukan, U. J., Mishra, S. & Shukla, R. K. De novo leaf and root transcriptome analysis identified novel genes involved in steroidal sapogenin biosynthesis in Asparagus racemosus . BMC Genomics 15, 13 (2014).

[23] Yi, T. G., Yeoung, Y. R., Choi, I.-Y. & Park, N.-I. Transcriptome analysis of Asparagus officinalis reveals genes involved in the biosynthesis of rutin and protodioscin. PLoS ONE 14, e0219973 (2019).

[24] Libault, M., Pingault, L., Zogli, P. & Schiefelbein, J. Plant systems biology at the single-cell level. Trends Plant Sci. 22, 949-960 (2017).

[25] Delude, C., Moussu, S., Joubes, J., Ingram, G. & Domergue, F. Lipids in Plant and Algae Development Vol. 86 Subcellular Biochemistry (eds Nakamura, Y & LiBeisson, Y) 287-313 (Springer, 2016).

[26] Colin, L. A. & Jaillais, Y. Phospholipids across scales: lipid patterns and plant development. Curr. Opin. Plant Biol. 53, 1-9 (2020).

[27] Girke, C., Daumann, M., Niopek-Witz, S. & Mohlmann, T. Nucleobase and nucleoside transport and integration into plant metabolism. Front. Plant Sci. 5, 12 (2014).

[28] Riegler, H., Geserick, C. & Zrenner, R. Arabidopsis thaliana nucleosidase mutants provide new insights into nucleoside degradation . N. Phytol. 191, 349-359 (2011).

[29] Jeter, C. R., Tang, W. Q., Henaff, E., Butterfield, T. & Roux, S. J. Evidence of a novel cell signaling role for extracellular adenosine triphosphates and diphosphates in Arabidopsis . Plant Cell 16, 2652-2664 (2004).

[30] Tanaka, K., Choi, J. M., Cao, Y. R. & Stacey, G. Extracellular ATP acts as a damage-associated molecular pattern (DAMP) signal in plants. Front. Plant Sci. 5, 9 (2014).

[31] Wang, L. M. et al. Early extracellular ATP signaling in Arabidopsis root epidermis: a multi-conductance process. Front. Plant Sci. 10, 8 (2019).

[32] Kato-Noguchi, H., Nakamura, K. & Okuda, N. Involvement of an autotoxic compound in asparagus decline. J. Plant Physiol. 224-225, 49-55 (2018).

[33] Miller, H. G., Ikawa, M. & Peirce, L. C. Caffeic acid identified as an inhibitory compound in asparagus root filtrate. Hortscience 26, 1525-1527 (1991).

[34] Domergue, F. & Kosma, D. K. Occurrence and biosynthesis of alkyl hydroxycinnamates in plant lipid barriers. Plants-Basel 6, 17 (2017).

[35] Biala, W. & Jasiński, M. The phenylpropanoid case - It is transport that matters. Front Plant Sci. 9, 1610 (2018).

[36] de Brito Francisco, R. & Martinoia, E. The vacuolar transportome of plant specialized metabolites. Plant Cell Physiol. 59, 1326-1336 (2018).

[37] Moses, T., Papadopoulou, K. K. & Osbourn, A. Metabolic and functional diversity of saponins, biosynthetic intermediates and semi-synthetic derivatives. Crit. Rev. Biochem. Mol. Biol. 49, 439-462 (2014).

[38] Faizal, A. & Geelen, D. Saponins and their role in biological processes in plants. Phytochem. Rev. 12, 877-893 (2013).

[39] Zhang, R. et al. Natural compound methyl protodioscin protects against intestinal inflammation through modulation of intestinal immune responses. Pharmacol. Res. 3, 2 (2015).

[40] Wang, S. et al. MALDI imaging for the localization of saponins in root tissues and rapid differentiation of three Panax herbs. Electrophoresis 37, 1956-1966 (2016).

[41] Mylona, P. et al. Sad3 and Sad4 are required for saponin biosynthesis and root development in oat. Plant Cell 20, 201-212 (2008).

[42] Nestler, J., Schutz, W. & Hochholdinger, F. Conserved and unique features of the maize (Zea mays L.) root hair proteome . J. Proteome Res. 10, 2525-2537 (2011).

[43] Brechenmacher, L. et al. Establishment of a protein reference map for soybean root hair cells. Plant Physiol. 149, 670-682 (2009).

[44] Janiak, A. et al. A comparative analysis of proteins that accumulate during the initial stage of root hair development in barley root hair mutants and their parent varieties. J. Appl. Genet. 53, 363-376 (2012).

[45] Rich-Griffin, C. et al. Single-cell transcriptomics: a high-resolution avenue for plant functional genomics. Trends Plant Sci. 25, 186-197 (2020).

[46] Zhu, Y. et al. Development of a laser capture microscope-based single-cell-type proteomics tool for studying proteomes of individual cell layers of plant roots. Hort. Res. 3, 16026 (2016).

[47] Witzel, K. et al. Plasma membrane proteome analysis identifies a role of barley membrane steroid binding protein in root architecture response to salinity. Plant Cell Environ. 41, 1311-1330 (2018).

[48] Moussaieff, A. et al. High-resolution metabolic mapping of cell types in plant roots. Proc. Natl Acad. Sci. USA 110, E1232-E1241 (2013).

[49] Djalali Farahani-Kofoet, R., Witzel, K., Gräfe, J., Grosch, R. & Zrenner, R. Species-specific impact of Fusarium infection on the root and shoot characteristics of asparagus. Pathogens 9, 6 (2020).

[50] Xu, D. et al. Rhizosecretion of stele-synthesized glucosinolates and their catabolites requires GTR-mediated import in Arabidopsis . J. Exp. Bot. 68, 3205-3214 (2016).

[51] Böttcher, C. et al. The multifunctional enzyme CYP71B15 (PHYTOALEXIN DEFICIENT3) converts cysteine-indole-3-acetonitrile to camalexin in the indole-3-acetonitrile metabolic network of Arabidopsis thaliana . Plant Cell 21, 1830-1845 (2009).

[52] Afendi, F. M. et al. KNApSAcK family databases: integrated metabolite-plant species databases for multifaceted plant research. Plant Cell Physiol. 53, e1 (2012).

[53] Shahaf, N. et al. The WEIZMASS spectral library for high-confidence metabolite identification. Nat. Commun. 7, 12423 (2016).

[54] Sawada, Y. et al. RIKEN tandem mass spectral database (ReSpect) for phytochemicals: a plant-specific MS/MS-based data resource and database. Phytochemistry 82, 38-45 (2012).

[55] Gorzolka, K. et al. in Bruker Daltonics Application Note Vol. 1866243-LCMS-148 (2018).

[56] Hulsen, T., de Vlieg, J. & Alkema, W. BioVenn - a web application for the comparison and visualization of biological lists using area-proportional Venn diagrams. BMC Genomics 9, 488 (2008).

[57] Witzel, K. & Matros, A. Fructans are differentially distributed in root tissues of asparagus. Cells 9, 9 (2020).

[58] Kaspar, S., Weier, D., Weschke, W., Mock, H. P. & Matros, A. Protein analysis of laser capture micro-dissected tissues revealed cell-type specific biological functions in developing barley grains. Anal. Bioanal. Chem. 398, 2883-2893 (2010).

[59] Witzel, K., Abu Risha, M., Albers, P., Börnke, F. & Hanschen, F. S. Identification and characterization of three epithiospecifier protein isoforms in Brassica oleracea . Front Plant Sci. 10, 1552 (2019).

[60] Kanehisa, M., Sato, Y. & Morishima, K . BlastKOALA and GhostKOALA: KEGG tools for functional characterization of genome and metagenome sequences. J. Mol. Biol. 428, 726-731 (2016).