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).