Natural variation in root exudate composition in the genetically structured Arabidopsis thaliana in the Iberian Peninsula

Introduction

Plant root exudates encompass a vast array of primary (e.g. carbohydrates, amino acids, and organic acids) and secondary metabolites (e.g. flavonoids, terpenoids, and alkaloids) that shape the physical, chemical, and biological properties of the soil (Oburger & Jones, 2018). They also facilitate nutrient cycling and mediate biotic interactions in the rhizosphere, thereby fostering a healthy soil ecosystem (Badri & Vivanco, 2009; Rasmann & Hiltpold, 2022). Despite the ecological relevance of root exudates, several factors, such as stress and developmental status, influence their chemical composition and challenge their quantification. For instance, stress by elevated phosphorus increases pthalic acid in Cyperus alternifolius (Duan et al., 2020), hydric stress induces various organic acids in Zea mays (Song et al., 2012), and pathogen infection in Arabidopsis thaliana stimulates long-chain fatty acids and amino acids that recruit protective Pseudomonas species (Wen et al., 2021). Furthermore, development also affects exudate profiles in A. thaliana with sugar alcohols decreasing and amino acids increasing over time in early developmental stages (Chaparro et al., 2013), whereas young fir trees exudate more carbohydrates and quercetin than older trees, which secrete more lipids and salicylic acids, shifting from nutrient acquisition to defense over-development (Chen et al., 2023). Given the influence of root exudates on plant–environment interactions and adaptive strategies (Novoplansky, 2019; Williams & de Vries, 2020; Subrahmaniam et al., 2023), unraveling the chemistry of root exudates may help decipher the complexity of plant metabolism but also the ecology of plant communities (Mommer et al., 2016; van Dam & Bouwmeester, 2017; McLaughlin et al., 2023).

However, our knowledge of natural variation in root exudate composition is rather scarce (Vives-Peris et al., 2020; Escolà Casas & Matamoros, 2021; Wang et al., 2021). This is a problem because understanding natural variation in plant traits is of paramount importance in different disciplines, as natural variation reflects long-term evolutionary dynamics, can reveal environmental factors driving this variation, and facilitates the exploration of the genetic basis of trait differences (Mitchell-Olds & Schmitt, 2006; Alonso-Blanco et al., 2009). One reason for the scarcity of studies on natural variation in plant root exudates has to do with the technical challenges for capturing and analyzing the complex chemical data from root exudates (van Dam & Bouwmeester, 2017; Oburger & Jones, 2018). In fact, few studies have described natural variation in chemical composition of root exudates in various plant species (Micallef et al., 2009; Biedrzycki et al., 2010; Badri et al., 2012; Houshyani et al., 2012; Chaparro et al., 2013; Fang et al., 2013; Strehmel et al., 2014; Mönchgesang et al., 2016; Kawasaki et al., 2018; Liu et al., 2020), all of them using low sample sizes (< 20 accessions in all cases). Interestingly, recent developments in mass spectrometry and nuclear magnetic resonance spectroscopy now enable the characterization and quantification of specific chemical compounds present in root exudates from a large number of samples (Pantigoso et al., 2021; Wang et al., 2022).

In this study, we took advantage of such recent technical advances, combined with the availability of dense collections of natural accessions exceptionally well characterized at the ecological, phenotypic, and genomic levels of the annual plant Arabidopsis thaliana, to conduct the first regional-scale assessment of natural variation in root exudate composition in plants with a large sample size. We analyzed root exudates from 105 distinct natural accessions of A. thaliana from the Iberian Peninsula. The Iberian collection of A. thaliana is geographically structured into four differentiated genetic clusters (Picó et al., 2008; Castilla et al., 2020), reflecting the complexity of the demographic and evolutionary history of this species across the region. Besides, the Iberian collection contains a remarkably high genetic and phenotypic diversity, including adaptive variation in life-history traits (Picó et al., 2008; Méndez-Vigo et al., 2011; Marcer et al., 2018; Tabas-Madrid et al., 2018; Castilla et al., 2020) and the highest genomic diversity from the species' native Eurasian range (The 1001 Genomes Consortium, 2016).

Here, we quantified the extent of chemical variation in root exudates across Iberian A. thaliana accessions by combining ultra-high-performance liquid chromatography with quadrupole time-of-flight mass spectrometry (Subrahmaniam et al., 2023). We estimated the broad-sense heritability values of root exudates, thereby assessing their degree of genetic determination. Furthermore, by conducting genome-wide association (GWA) analyses, we also provided insights into the genetic basis of regional-scale variation in root exudates. Finally, we examined the eco-evolutionary forces putatively driving natural variation in root exudates by correlating chemical variation among accessions with their geographic, environmental, life history, and genetic patterns of variation across the Iberian Peninsula.