Drought, heat, or combined stress differently affect the root exudate composition
In field conditions, plants are regularly subjected to co-occurring abiotic stress factors, especially due to climate change (Zandalinas, Mittler, Balfagón, Arbona & Gómez-Cadenas 2018). In such adverse growing conditions root exudates are crucial in allowing plants to survive (Mimmo et al. 2018; Vives-Peris et al. 2019). Here we show for the first time distinctive responses of maize root exudation to drought and heat stress, as well as to their combination.
Our results indicate that abiotic stresses modified the root exudates composition, in a more pronounced manner for heat and combined stress, but also evident for drought-stressed plants (second latent vector in figure 4). With this regard, phenylpropanoids and amino acids were the main metabolites involved in the stress-changed root exudation profiles. In this sense, literature indicates that root exudates could be modulated by external factors such as abiotic stress, confirming our results. Indeed, it is known that drought affects the quantity and quality of root exudation, with a prevalence of secondary metabolites (Williams and de Vries, 2020; Gargallo-Garriga et al. 2018). Similarly, several authors also reported a tailored exudation when plants are exposed to high temperature (Pramanik et al., 2000; Vives-Peris et al., 2020).
Our results corroborate previous findings and indicate that both shared and specific metabolites were modulated between the single and combined stress. Among the shared metabolites, citramalic, malonic acid, and 4-hydroxycoumarin increased in presence of all stress treatments, compared to the control. Citramalic acid seems to be involved in soil phosphorus solubilization (Khorassani et al. 2011). Since both drought and heat decrease the water content in the rhizosphere, mobility of nutrients (including phosphorus) may be reduced. Thus, the increase of citramalic acid exudation could be a plant mechanism to enhance the solubilization of the drought-induced mobility-limited nutrients, and consequently their uptake. (Zhang, Chen, Zhao, Zhou & Zhao 2017) observed that citramalic acid was sharply reduced in the root collar tissue of Caragana korshinskii during drought stress, a highly drought tolerant plant, but not in the root system. This result could indicate the involvement of citramalic acid exudation in drought-adaptation in Caragana korshinskii , although further confirmation is needed. Nonetheless, citramalic acid was found to be accumulated in leaves of soybean seedling under salt stress (Zhang, Yang, Li & Shi 2016), further indicating that it might play a role in plant response to abiotic stresses that involve osmotic imbalance. Another organic acid that was found in the root exudome of all stressed maize plants is malonic acid. Indeed, the malonic acid content increased in exudates derived from stressed plants such as wheatgrass (Henry A., Doucette W., Norton J. & Bugbee B., 2007) and has been associated with osmotic adjustment and stress response (Li & Copeland 2000). Moreover, malonic acid is a major competitive inhibitor of succinate dehydrogenase (Li & Copeland 2000) and thus is involved in plant stress signalingvia mitochondrial succinate dehydrogenase (Belt et al.2017). However, Festuca arundinacea as well as sevenTriticeae species decreased their malonic acid leave content under heat stress (Yu, Du, Xu & Huang; Ullah, Yüce, Neslihan Öztürk Gökçe & Budak 2017). Finally, 4-hydroxycoumarin was another up-exuded metabolite observed in all stressed maize plants. This compound belongs to the coumarins, known also as allelopathic compounds which interact with the root microbiota for improving nutrient acquisition (Harbort et al., 2020) and, in general, for the assembly of the root microbiome (Stringlis et al., 2019).
Differently to the up-exuded compounds, the amino acids L-serine and threonine and flavones, (especially hispidulin) were the down-exuded metabolites being shared in all stress treatments (Supplementary material 5 and Table 1). In particular, L-serine was clearly down-exuded under both heat and combined stress, suggesting a role of temperature in its production. The release of threonine instead was repressed by both drought and combined stress. The reduction of the amino acid exudation like serine and threonine could be a plant mechanism to preserve important elements, e.g. C and N, within the plant in presence of abiotic stress conditions. Further, plants might compete with microorganisms for carbon within the rhizosphere since they are also able to reacquire root exudates (Tiziani et al 2020). Thus, plants might activate a so-called ‘energy saving strategy’ modifying their exudation pattern reducing the microbial competition (Doornbos, van Loon & Bakker 2012). This may indicate that plants provide a fine tuning of the exudation process under heat and drought stress, even more than under normal growing conditions. Hispidulin was among the metabolite suppressed in all stress treatments respect than control (Supplementary 5 and Table 1). Hispidulin is a flavone with antioxidant activity and belongs to the flavonoids commonly present in root exudates (Lucini et al. 2019). Usually, under heat and/or drought stress, shoots increase the production of antioxidant compounds to counteract the oxidative imbalance generated by increased production of reactive oxygen species (Fahad et al. 2017). The strong repression of hispidulin together with the modulation of other flavonoids might indicate that the maize plant focuses on protecting the shoots rather than prioritizing metabolites for root exudation.
Interestingly, specific stress-related exudation responses could be also observed. Targeted metabolomics indicated that the release of aconitic acid and total phenols was significantly higher in heat-treated plants (Figure 2), and a specific phenolic, namely 4-vinylphenol, was observed to increase by untargeted metabolomics (Table 1). Among phenolics, benzoic acid and its derivatives (i.e. 3,5-dihydroxybenzoic acid, 2,4-dihydroxybenzoic acid, benzoic acid) were specifically exuded under heat stress conditions (Table 1). In agreement with our results, benzoic acid and hydroxybenzoic acid were modulated as a response to alteration in temperature in root exudates (Pramanik et al., 2000). It is also known that benzoic acid released by root exudates plays a role in plant-microorganisms interaction (Liu et al., 2015). Malate, another up-exuded compound observed in maize root exudates, has been demonstrated to recruit beneficial microbes like Bacillus subtilis (Thimmaraju et al., 2008). Indeed, this specific bacterium is involved in the enhancement of plant stress tolerance specifically in dry and salty environments (Bokhari et al., 2019). On the other hand, L-ascorbate was specifically down-exuded in heat-treated maize exudates (supplementary material 5 and Table 1). In planta, ascorbic acid is a well-known key antioxidant and signaling molecule involved in mitigating excessive activities of the reactive oxygen species determined by several abiotic stresses (Venkatesh and Park, 2014). It can be postulated that heat-stressed maize plants preserve the presence of this essential compounds within the heat-induced stressed root cells, rather than exude it into rhizosphere.
Among the specific compounds up-exuded by maize roots subjected to drought stress, we observed resveratrol. This compound belongs to the stilbenes, natural phenolic phytoalexin metabolites elicited by abiotic stress (Chung et al., 2003). Resveratrol has also been observed in the root exudate metabolome of Quercus ilex under drought stress (Gargallo-Garriga et al., 2018), yet at very low concentrations since it was most likely readily degraded by bacteria (Kurt et al., 2018). In the present study, we detected resveratrol because probably we collected the exudates in axenic conditions suggesting hence its ecological role in the rhizosphere.
Interestingly, multiple stress exhibited a distinct and chemically diverse exudate profile compared to the individual stresses, with 13 metabolites being specifically up-exuded by maize root under combined stress (Table 1). Besides phenolics, which appeared as a main class of compounds generally modulated by stress, the combined stress triggered the specific exudation of tryptophan and palmitic acid. In this regard, it is known that the exudation of tryptophan could be stimulated by the plant beneficial Bacillus amyloliquefaciens (Liu et al., 2016). Moreover, the exudation of tryptophan-derived secondary metabolites are involved in the response to biotic stress (Baetz and Martinoia (2014). Analogously, palmitic acid was considered as metabolite recruiting beneficial rhizosphere bacteria in the halophyte Limonium sinense(Xiong et al, 2020) suggesting an ecological role in the soil salinization, an abiotic stress derived by a combination of salt, drought and heat. The specificity of exudation response to the combined stress, here observed, highlight the non-additive effects of the co-occurence of abiotic stresses. This is in agreement with previous findings observed for other maize traits such as root morphology (Vescio et al., 2021b), root microbiota (Vescio et al., 2021a), leaf proteome (Zhao et al., 2016), and yield and nutrient uptake (Hussain et al., 2019). Our results, further, could be interesting considering that the exudation pattern probably was a depiction of what took place in the whole plant as pointed out by Gargallo-Garriga et al. (2018).