DISCUSSION
Plants are sessile organisms responding to environmental stimuli and stresses with consequent rearrangement of the metabolism and an impact on the root-associated and rhizosphere microbiota. These two aspects are tightly connected and can be considered two perspectives of the same response since specific plant metabolites like phenolics, glucosinolates, and strigolactones can shape soil microbes and their interactions (Hartman et al. , 2017; Jacoby et al. , 2021).
A distinctive adjustment of the specialised metabolism was observed depending on the stress, with more pronounced effects in roots subjected to thermal stress (H). Under limited water conditions (D), oxidative stress may induce the synthesis of phenolic compounds and flavonoids via the increase in the activity of phenylalanine ammonia-lyase (PAL), the key upstream enzyme of the phenylpropanoids pathway (Jun et al.2018). Despite showing a general down accumulation of phenolics during thermal stress, our results indicate some sub-classes being enhanced, thus suggesting a specific modulation of this class of metabolites. Nonetheless, accumulating other low molecular weight antioxidants such as ascorbate and glutathione may help the plant mitigate ROS-mediated damage related to limited transpiration related to drought. Overall, changes in phenolic acids and flavonoids also depend on the species, the intensity of the stress and its duration. Recent work demonstrated that different wheat genotypes under water scarcity did not exhibit any significant change in phenolic acids and flavonoids, while others had higher concentrations than non-stressed ones (Laddomada et al.2021). In addition, a higher phenolic acids content was detected under severe drought conditions, while moderate drought and severe heat stress did not lead to their accumulation (Shamloo et al. 2017).
A general down accumulation was observed in both the single stress conditions concerning N-containing compounds. However, glucosinolates and related compounds were accumulated. These compounds have been reported to potentially affect the rhizosphere communities (Jacobyet al. 2021). Interestingly, the production of aliphatic glucosinolates is induced by drought in Arabidopsis thaliana , in parallel with phytoalexins repression (Kliebenstein 2004). Together with glucosinolates, a slight modulation of amino acids synthetic pathways was observed in response to single stresses. Under abiotic stress, amino acids may accumulate as precursors for synthesising specialised metabolites and signaling molecules or as substrates for protein synthesis to promote rapid plant metabolism recovery from stress. Among them, L-methionine was reported to be an effective regulator of plant growth under environmental stress such as drought (Mehak et al.2021).
Several phytohormones such as jasmonic acid (JA), abscisic acid (ABA), brassinosteroids (BRs), cytokinins (CKs), and gibberellic acid (GA) have been shown to enhance abiotic stress tolerance (Sharma et al.2019). In fact, under high temperature and/or water deprivation conditions, plant responses are mediated by phytohormones, which coordinate complex stress-adaptive signaling cascades (Vile et al. 2012) through a complex cross-talk between the different signaling pathways (Verma, Ravindran & Kumar 2016). Several studies focused on hormonal changes during a combination of high temperatures and salinity or high light intensity, agreeing that a coordinated hormonal response to each specific stress combination is essential to trigger the proper acclimation responses. In our conditions, ABA was involved in the plant response to heat and drought stress. Zandalinas et al. , 2016 revealed that although ABA is required for the acclimation of Arabidopsis, stomatal closure may also be regulated by H2O2. A cross-talk mechanism between gibberellin and abscisic acid during limited water conditions was also reported, in which ABA biosynthesis and the control of stomatal conductance were regulated by the receptor for gibberellin under water stress (Gaion et al. 2018). These latter are involved in the adaptive response to various abiotic stresses such as cold, salinity, heat, flooding, and drought, despite their role in drought stress adaptation is still unclear. A cross-talk between ABA and BRs has also been reported (Ha, Shang & Nam 2016). However, decreasing CKs (negative regulators of plant root growth and branching) under H stress can improve plant survival rate by reducing the expression of stress response genes (Liu, Zhang, Meng, Wang & Chen 2020). Similarly, also jasmonates have a significant role in abiotic stress tolerance because of their linkage with other growth regulators, antioxidants and osmoprotectants, especially its conjugate isoleucine-JA, the most active form of JAs (Sharma et al. 2019). Notably, phytohormones may also be related to microbial colonization and play a pivotal role in the assembly of plant microbiota (Eichmann, Richards & Schäfer 2021). In turn, the ability of beneficial microorganisms to directly produce CKs, GAs, and ABA, rather than aminocyclopropane-1-carboxylase (ACC) deaminase that cleaves the ethylene precursor, can support plant growth under stress (Pascale, Proietti, Pantelides & Stringlis 2020; Vishwakarma et al. 2020; Eichmann et al. 2021).
Plants’ acclimation to a particular abiotic stress condition involves tailored responses to their specific environmental conditions. Previous literature indicates that a moderate overlap could be observed among transcriptomic responses to abiotic stressors such as drought (Rizhskyet al. 2004), cold (Kreps et al. 2002), salinity (Yonget al. 2002) and light excess (Rossel, Wilson & Pogson 2002). Similarly, despite being recognised as a common response to abiotic stressors, the shift in gene expression patterns related to ROS-triggered responses was differently modulated by stress treatments (Mittler, Vanderauwera, Gollery & Van Breusegem 2004). As in our case, several studies have shown that combined stress responses are mostly related to non-additive effects. Our findings revealed that H and D stress largely modulated root metabolism and triggered different plant responses, while the combined stress did not imply a sum of both stresses but presented a metabolic profile closer to those plants exposed to heat stress. These findings agree with previous studies and confirm that the combination of abiotic stresses is rarely the sum of the single stresses (Fahad et al. 2017; Lamaoui et al.2018; Zandalinas, Mittler, Balfagón, Arbona & Gómez-Cadenas 2018; Laddomada et al. 2021). Indeed, the review by Mittler on abiotic stress combination reports how unique stresses cannot be used to extrapolate combined stresses (Mittler et al. 2004). To date, little information is available on the molecular mechanisms underlying the interaction between abiotic stresses, even though the comprehension of such mechanisms is important to facilitate the development of crops with enhanced stress tolerance (Rizhsky et al. 2004).
Regarding the specific stress combination in our experiments, the interaction between drought and high temperature is realistic in the context of a climate change scenario, and it is also relevant in terms of stress interaction. During heat stress, stomata are opened to increase transpiration and cool leaves; however, the combination with drought hampers this heat dissipation mechanism and represents a clear example of positive interaction. Surprisingly, our results indicate the hierarchically prevalent effect of heat while confirming the positive interaction. Consistently with our findings on multivariate modelling of the plant metabolome, the interaction between heat and drought is believed to provide unique responses (Rizhsky, Liang & Mittler 2002) and should be considered an independent stress condition. Indeed, tolerance to combined stresses involves the cross-talk among different signal transduction processes that requires multiple controls. This latter point, reflecting synergistic relationships among stresses, has been defined as “cross-hardening”(Bowler & Fluhr 2000). As highlighted by our data, the integration between stresses is rather complex; it involves signaling processes like hormones, mitogen-activated protein kinases (MAPK), and calcium and ROS species (Bowler & Fluhr 2000). Moreover, plants employ complex “stress sensing” mechanisms to detect stresses, depending on the species, organ, and type of stress (Kranner, Minibayeva, Beckett & Seal 2010) and the cross-talk between receptors may also be involved (Casal 2002).
Together with highlighting the non-additive effect of multiple stresses, our results strengthen the concept of whole holobiont response to stress where a coordinated plant-microbiome modulation could be observed. Accordingly, the combined effect of heat and drought stress on the rhizosphere microbiome produced a different outcome compared to single stressors. Concerning Rhizobacteria, the literature on their role in plant abiotic stress mitigation is vast, as recently reviewed (Navarroet al. 2006; Dimkpa, Weinand & Asch 2009). At the molecular level, plant perception of eubacterial flagellins can activate plant responses at the gene expression level. Xu et al., suggested that at the earlier development stage, the roots bacterial community is more susceptible than at older plant stages (Xu et al. 2018), although mitigation features are stress-dependent and not a per se feature of the strains (Rolli et al. 2015). While heat affects the rhizosphere microbiome via the host plant (indirectly), drought shapes the bacteria community, directly promoting the enrichment of bacteria belonging to Firmicutes and Actinobacteria (Simmons et al. 2020), which are known to be physiologically adapted to drought conditions and that their abundance is positively correlated to plant drought resistance (Naylor, Degraaf, Purdom & Coleman-Derr 2017; Fitzpatricket al. 2018; Hartman & Tringe 2019). Moreover, in accordance with our results, it has been recently reported that under drought stress, endophytic actinobacteria induced artemisinin biosynthesis, which accumulation is known to be involved in modulating drought tolerance (Li et al. 2012).
Plants actively exudate compounds that act as attractants forRhizobium species and that may be used as carbon sources by other species, including Burkholderia , and their breakdown products might modify the microbial biodiversity and the species abundance (Schütz et al. 2021). These rhizhobiales-plants interactions significantly mitigate abiotic stresses (Munir et al. 2022). Our results on beta diversity highlighted the involvement of Proteobacteria, known to be the main members of Arabidopsis’ root microbiota both in the roots and in the soil, followed by Bacteoroidetes and Actinobacteria, as a function of the stress applied. As expected, the magnitude of bacterial microbiota shifts was consistently lower in soil than in root-associated compartments.
The involvement of the root endophyte Enterobacteriales , stimulated by both H and H+D treatments, in mediating plant thermotolerance has been recently described (Shekhawat et al.2021). They reported that Enterobacter sp. SA187 enhanced the H3K4me3 levels at heat stress memory gene loci, which was mediated by ethylene signaling. Similarly, Proteobacteria such as Aeromonassp., which use flavonoids-mediated signaling for host recognition, have been proven to be an enhancer of plant dehydration resistance (Heet al ., 2022).
Actinobacteria possess a drought-tolerant nature and, under stress, increase the transcription of specific genes and the production of spores highly tolerant to dehydration (Omae & Tsuda 2022). Consistently, we observed an increase in Actinobacteria , especially under H treatment. Among monoderm lineages,Actinobacteria have been reported to exhibit the strongest enrichment under abiotic stress and to support plant carbohydrate and amino acid transport and metabolism, as well as to positively modulate plant secondary metabolism (Ngumbi & Kloepper 2016; Xu et al.2018). The mechanisms by which Actinobacteria mitigate abiotic stress in plants include the production of osmolytes to maintain osmotic balance, the synthesis of plant hormones, and enhanced availability of nutrients (Fitzpatrick et al. 2018).
Proteobacteria , given their high ability to utilize root exudates, are fast-growing rhizosphere and root colonizers that respond rapidly to organic carbon sources (Bulgarelli, Schlaeppi, Spaepen, Van Themaat & Schulze-Lefert 2013). Despite having a relatively superior colonization ability within the root and rhizosphere under well-watered conditions, diderm bacteria are less suited to survive the selective pressures caused by drought (Xu et al. 2018). However, significant differences are present at the genus level, where the structure of the peptidoglycan cell wall, rather than the presence or absence of an outer membrane, can determine significant differences among microorganisms (Sutcliffe 2010; Xu et al. 2018). Interestingly, the thickness and composition of the cell wall have been linked to a different tolerance to ROS species, one of the mechanisms proposed in the differences observed at the rhizomicrobiota level under abiotic stress (Shade et al. 2012; Fitzpatrick et al.2018). Consistently, proteobacteria were extensively altered by the different stress conditions, including the genera Pseudomonas(decreasing), Azospirillium , Rhizobium ,Enterobacter , and Burkholderia (all increasing in both roots and soil). Beneficial bacteria like Azospirillales ,Rhizobiales and Burkholderia (all involved in beta-diversity shifts following H and H+D) also led to the production of osmoprotectants like proline, betaine, trehalose, and glycine (Zulfiqar, Akram & Ashraf 2020). Similarly, Enterobacter can promote stress tolerance, likely because of its phosphate-solubilizing ability (Dolkar, Dolkar, Angmo, Chaurasia & Stobdan 2018). In our experiment,Enterobacterales were predominant in H+D stressed roots, whileRhizobiales characterized H conditions. At the molecular level, these microorganisms can increase plant biomass under abiotic stress by shaping the phytohormone profile and improving the antioxidant machinery (Brilli et al. 2019).
Despite the evident coordinated modulation of plant metabolome and metabolome in root-associated compartments, our results must be considered in the framework of a strong niche differentiation during plant development. Indeed, the root and rhizosphere communities undergo an initial period of dynamic recruitment followed by a later period of relative stability. The roots grown under pre-flowering drought treatment showed a more pronounced reshape of microbiota than post-flowering drought treatments (Xu et al. 2018; Francioliet al. 2021; Lewin et al. 2021; Francioli et al.2022). While confirming the orchestrated response of the whole holobiont to a combination of abiotic stressors, based on the studies mentioned above and our work, it would be very promising to investigate dynamics occurring as a function of plant stage at root and rhizosphere microbiota level.