GC-MS based metabolomic profiling of sandalwood induced systemic signalling in host plants through a split root system

Introduction

Indian sandalwood (Santalum album L.) is a central tropical hemiparasitic arboreal organism, which is very widespread in tropical and subtropical regions. Although the plant has photosynthetic capacities of its own, its growth pattern and developmental stages have been found to be partly dependent on the heterotrophic relationships with host flora, whereby it acquires water and mineral nutrients needed. The genus Santalum is placed taxonomically among the cadres of xylem tapping hemiparasites, with the expression of specialized invasive organelles located as haustoria (Mallaburn, 1992) [20] . The structures actively inch through host root tissues and establish vascular continuity that allows the free flow of resources between parasites and hosts (Tennakoon et al., 1997) [33] . In S. album, haustorial connections may also be established with nitrogen-fixing nodules in the case of leguminous hosts, thus improving nutrient uptake (Lu et al., 2014) [19] . Sandalwood has the ability to associate with a wide range of host species, however, it has been shown to have a particular association with some of the chosen hosts, which include Acacia, Casuarina, Dalbergia and Sesbania. Previous studies by Lu et al. (2014) [19] has determined that the relationship with nitrogen fixing hosts significantly elevates the amount of nitrogen in leaves, photosynthetic performance, and water-use performance in sandalwood. Besides, stable-isotope experiments have been used to support a two-way exchange of carbon and nitrogen between S. album and nitrogen-fixing hosts, thus showing complex physiological integration (Lu et al., 2014) [19] . Lastly, it has been shown that the strength of the hostparasite interaction varies with nitrogen availability, suggesting a central role of the nutritional condition of the host in regulating the strength of this interaction (Meng et al., 2021) [24] . The study by Anil and co-workers has shown that non-leguminous Casuarina is a superior host and induces enhanced sandalwood growth in plantations (Mohapatra and Anil, 2022) [26] . The early phases of host recognition and successful infection during the complex life cycles of parasitic angiosperms are largely mediated by chemotactic signals released by the roots of the host species (Kubo et al., 2009) [18] . Notably, the empirical evidence suggests that about 70% of the sandalwood seedlings can begin haustorial formation after about 30 days of germination, reflecting the extraordinarily early onset of host dependent morphogenesis (Nagaveni and Srimathi, 1985) [27] . Verma et al. (2024) [36] revealed that physiological processes and antioxidant systems in sandalwood were mainly governed by the host species. Even though the pathways of nutrient translocation during post attachment are relatively well understood, the complex processes that govern pre-attachment host detection and the early signalling interactions remain poorly characterized. The split root in vitro system (SRS), that physically partitions the root of a plant into two compartments, offers a complex methodological approach to the breakdown of local and systemic physiological activities in an individual plant root system. The method has been massively used to understand plant signalling networks (Torres et al., 2021) [34] , sectoriality, which is the compartmentalization of resource and hormonal fluxes within discrete physiological units in a plant (Marquis, 1996; Orians et al., 2004) [23,28] and water uptake (Ameglio et al., 1999), ion transport (Gloser et al., 2009b) and ectomycorrhizal interactions (Rose et al., 2023) [11] . In addition to the physiological research, the split-root system (SRS) method has been used in ecological research to investigate plant -plant interactions and plant -microbe interactions. The SRS method has been used to investigate the dynamics of interspecific competition (Marler, 2013) [22] as well as plant interactions with pathogenic organisms and mycorrhizal associations (Kennedy et al., 2009) [17] . The flexibility of the SRS allows for the distinction between contact and systemic signaling responses in the same plant. Although many studies have explained how nutrients move from the host to sandalwood after the haustorium was formed, it is still not understood whether chemical signals released by sandalwood can induce metabolic changes in the host plants before the formation of direct haustoria connection. This study aims to check whether the chemical signals perceived in one part of the root network of one host could be transmitted to spatially separate root compartments, and thereby, induce metabolic reconfiguration in the absence of direct physical contact with sandalwood root exudates

Materials and methods

The split root experiment was carried out at the Department of Plant Biotechnology, University of Agricultural Sciences (UAS), GKVK, Bengaluru, to assess the metabolite variation caused due to sandalwood-host root interactions. Sandalwood (Santalum album) plantlets and Casuarina seedlings were collected from Tapovana Sandalwood Nursery, Bengaluru, while Mulberry plants were purchased from the Department of Sericulture, GKVK. The experiment was set up in hydroponic systems using Hoagland's nutrient solution in a controlled growth chamber with temperature, light, and humidity controls. The plants were first grown in Hoagland's solution for a period of two months to allow them to establish properly and acclimatize to the hydroponic environment. After the acclimatization process, the root system of each host plant was carefully split into two equal parts and placed in two separate compartments labelled as left (L) and right (R) (Table 1, Fig 1). In treatment combinations, sandalwood seedlings were placed in the left compartment along with half of the host root system, while the right compartment had only host roots. Control treatments had host plants raised in split root systems without sandalwood. The root exudates were collected six days after treatment setup for GCMS analysis. Root exudates in the hydroponic system were collected and subjected to solvent extraction using ethyl acetate. The extracts were filtered and concentrated prior to analysis. The prepared samples were analysed using a GC-MS system (QP2030, Shimadzu) to identify the bioactive compounds present in root extracts. Separation was achieved using an SH-RXI-5SIL MS capillary column (30m×0.25mm internal diameter, 0.25μm film thickness). Helium was used as the carrier gas at a constant flow rate of 1.0ml min⁻¹. The injector temperature was maintained at 280 °C. The oven temperature started at 50 °C and was held for 5 minutes. It was then gradually raised to 280°C, with intermediate hold points listed in Table 1. The entire temperature program cycle took 45 minutes to complete. The detailed oven temperature program used during GC-MS analysis is presented in Table 1. The mass spectrometer was operated in electron ionization (EI) mode at 70eV, with the ion source temperature maintained at 230 °C (Table 2). Compounds were identified based on their retention time and mass spectral data.

Venn Diagram Analysis

To visualize the overlap and uniqueness of metabolites identified across different treatments, Venn diagrams were generated using the Venny 2.1 web tool. This tool enables rapid comparison of multiple datasets and facilitates identification of common and treatment specific compounds.

KEG pathway analysis

KEGG pathway analysis was carriedout on metabolites identified by GC-MS. This helps us to understand the biological significance of the detected compounds. Analysis was carriedout using MetaboAnalyst 6.0 to find the metabolic pathways that the target compounds in the root exudates affects. The compound's KEGG ID was mapped to the KEGG pathway database.

Results

GC-MS profiling of

Mulberry and Casuarina root exudates in the split root experiment showed significant metabolic differentiation between control and sandalwood associated treatments.

In control treatment, where Mulberry roots were evenly divided in both compartments without sandalwood (M(L) control vs M(R) control), the metabolic profiles were very similar. A total of 37 metabolites were common in both compartments, while only GC-MS analysis of Casuarina root exudates in the split-root experiment showed that, In the control treatment (C(L) control vs C(R) control), 39 metabolites were common to both, with only 9 unique metabolites found in each side (Fig 3B). This is a very high level of commonality and suggests a strong level of baseline metabolic homogeneity and exudation patterns in the absence of sandalwood interaction. After the addition of sandalwood to the left side (C+S(L)), the level of commonality in metabolite composition dropped significantly to 9 common compounds. Three-way comparison among C+S(L), C(R), and the control metabolite set showed that only 4 metabolites were common to all. Notably, 15 metabolites were found exclusively in the right compartment (C(R)) when sandalwood was restricted to the left compartment (Fig 3C). These included a hexahydropyridine derivative with known antioxidant activity; L-lysine, involved in protein synthesis and stress response; methoxyacetic acid, heptadecyl ester, with known herbicidal properties; hydrocarbons like 2-methyloctane and nonane derivatives, known to be involved in plant defense and cuticular wax composition; and 2-adamantanone oxime, a precursor of biologically active adamantane derivatives (Fig 3A ). [32] 7 Histamine, N-BOC-5-iodo-337. 16 8 Cetene (likely 1-tetradecene) 196.37 Note: M/Z value: Mass to charge ratio Cross-validation of the compound identity was done using KEGG, PubChem (CID: 3349), ChEBI (CHEBI: 18019), and CAS registry number (56-87-1). Pathway analysis of the enriched pathways revealed that Llysine is involved in a number of primary and secondary metabolic pathways. It is involved in lysine biosynthesis (map00300) and lysine degradation (map00310), in aminobiosynthesis (map01230) and general metabolism (map01100). Also, L-lysine is used in synthesizing secondary metabolites (map01110), such as the synthesis of alkaloids (map01064) as well as siderophores (map00975) and plant secondary metabolites (map01060). The module analysis shows that L -lysine is used by various lysinebiosynthetic pathways, which include the succinyl-DAP pathway (M00016), the acetyl-DAP pathway (M00525), and the AAA pathway (M00030), and degradation modules producing saccharopine and acetoacetyl-CoA (M00032) (Table 6). Enzyme mapping indicates that lysine is involved in reactions catalyzed by various enzyme familiesaminotransferases (EC 2.6.1.x), oxidoreductases (EC 1.4.x.x), decarboxylases (EC 4.1.1.x), and ligases (EC 6.3.x.x): this reflects its important position in metabolism.

The KEGG BRITE hierarchy combined with a functional classification proves that lysine is an amino acid that is common and has broad biological significance. The determination of L -lysine in the samples indicates that there is an active amino acid metabolism and can be associated with protein synthesis, nitrogen processing, and perhaps secondary pathways. Its discovery further indicates that Llysine is involved in central metabolism pathways and metabolism that are triggered by biotic and abiotic stress.

Discussion

The split-root experiment showed that sandalwood association causes a strong and systemic metabolic reprogramming response in both Mulberry, Casuarina and Alternanthera. The large overlap of metabolites between left and right compartments under control conditions confirms that the split-root system did not introduce any variability, and that root exudation was metabolically homogeneous in the absence of sandalwood. The composition of root exudates is relatively stable under controlled hydroponic conditions unless affected by biotic interactions (Bais et al., 2006; Badri and Vivanco, 2009).

In Mulberry, the dramatic reduction in shared metabolites (from 37 in the control to 5 in the sandalwood-treated samples) and the appearance of many unique metabolites in both compartments reflect a strong biochemical modulation. Notably, there were also large changes in the right compartment, which was not exposed to sandalwood, and this provides evidence for systemic root-to-root transmission of signals. Systemic transmission of signal between roots separated in space is well established and is typically mediated by vascular transport of hormones such as jasmonic acid, ethylene, auxins, and reactive oxygen species (Karban et al., 2006; Shah and Zeier, 2013). The sole detection of (S)-glyceraldehyde in right compartment indicates a modulation of primary carbon metabolism, which may be indicative of an elevated glycolytic flux or a redirection of carbon to facilitate stress adaptation (Bolton, 2009). Modulation of central carbon metabolism is a wellestablished response to biotic stress and resource allocation (Caretto et al., 2015). Casuarina and Alternanthera also showed a similar but more drastic metabolic reprogramming. The finding of Llysine in exudates of Casuarina indicates the activation of amino acid metabolism and possibly the activation of nitrogen remobilization pathways, which are usually activated under stress conditions (Veiga-da-Cunha et al., 2012). The finding of methoxyacetic acid esters and 2adamantanone oxime also indicates the activation of secondary metabolism and bioactive compound production, which are usually linked to plant-plant or plant-parasite interactions (Inderjit and Duke, 2003) [13] . The identification and KEGG based functional annotation of L-lysine (C00047) add further mechanistic insight. Lysine is a key player in nitrogen metabolism and is involved in lysine biosynthesis and degradation, amino acid biosynthesis, and secondary metabolite biosynthesis. Amino acids, such as lysine, are used as precursors for the synthesis of alkaloids and other nitrogenous secondary compounds that play a role in defense (Facchini, 2001) [10] . Lysine degradation is also a source of signaling molecules that play a role in stress adaptation (Arruda et al., 2000) [2] .

Conclusion

The split-root experiment shows that sandalwood (Santalum album) causes strong and systemic metabolic reprogramming in host plants even in the absence of direct exposure. Under normal conditions, the metabolite profiles of Mulberry, Casuarina and Alternanthera in the left and right root compartments were very similar, but when sandalwood was introduced into one of the compartments, there was a strong reduction in the shared metabolites and the appearance of a large number of unique metabolites in both the sandalwood-exposed and non-exposed compartments. The identification and metabolic pathway assignment of L-lysine (KEGG C00047) also emphasize the activation of amino acid metabolism and nitrogen redistribution, suggesting the regulation of primary and

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