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.