INTRODUCTION
The reduction of water availability (i.e ., drought stress) and
increase in global temperature (i.e . heat stress) are among the
major challenges we are going to face in the next few decades (IPCC
2018). These abiotic stresses generated by climate change significantly
impact plants (Pandey, Ramegowda & Senthil-Kumar 2015), causing
substantial agronomical and economic losses every year on a global scale
(Lipiec, Doussan, Nosalewicz & Kondracka 2013). The effect of abiotic
stresses on plants has been widely dissected over the years. However,
plants are rarely exposed to single stresses but rather to a combination
of multiple stresses, requiring plants to finely tune their
physiological responses. Several studies have shown that combined stress
responses provide non-additive effects (i.e. , synergistic or
antagonistic), and therefore the combined effects cannot be predicted
based on results from single-stress studies (Rivero et al. 2014).
Hence, it is of huge importance to understand how plants respond to the
combination of heat and drought stress.
One of the most important physiological traits that plants evolved to
interact with the environment is the modulation of their root exudation
pattern (Vives-Peris, de Ollas, Gómez-Cadenas & Pérez-Clemente 2019).
Root exudates include primary and secondary metabolites of both low
(<1000 Da) and high molecular weight (>1000 Da)
(Oburger & Jones 2018) that plants produce to directly influence the
chemical, physical and biological characteristics of the rhizosphere
(Mommer, Hinsinger, Prigent-Combaret & Visser 2016). Examples include
carboxylates, amino acids, carbohydrates, phenols, proteins, and fatty
acids. Plant age, genotype, biotic or abiotic stresses, environmental
factors, as well as the interaction with living organisms in the
rhizosphere can potentially affect the quantity and quality of root
exudation (Czarnota, Rimando & Weston 2003; Pii et al. 2015;
Valentinuzzi et al. 2015; Oburger & Jones 2018; Tiziani et
al. 2020a; Tiziani, Pii, Celletti, Cesco & Mimmo 2020b). Despite the
large interest in drought- and heat-tolerant crops in the last years,
only a few studies focused on the root exudation responses to these
abiotic stresses (Williams & de Vries 2020).
In the presence of water deficit it has been suggested that plants
allocate a higher fraction of carbon to exudates at the expenses of
biomass production, although it seems that total exuded carbon is
limited during the drought stress (Karst, Gaster, Wiley & Landhäusser
2017; Preece, Farré-Armengol, Llusià & Peñuelas 2018). A recent study
investigated drought effects on root exudate quality and quantity in
sunflower and soybean. Sunflower plants exposed to drought increased the
exudation rates after rewetting (+330% in terms of carbon), but the
composition of metabolites remained unchanged compared to not-stressed
plants. In soybean, it has been observed that plants did not modify
exudation rates, but rather the profile of metabolites exuded, with
increased concentrations of osmolytes (proline and pinitol) (Canarini,
Merchant & Dijkstra 2016). Another study performed on Quercus
ilex showed that under normal growing conditions, the plant mainly
exuded primary metabolites, whereas under drought stress, the exudation
was shifted irreversibly to secondary metabolites (Gargallo-Garrigaet al. 2018). In contrast, Henry, Doucette, Norton & Bugbee
(2007) showed that drought stress in wheatgrass increased carboxylate
exudation, especially malate, compared to control plants. Taken
together, the plant responses to drought often seem to be species- or
even genotype-specific. Research on the effects of heat stress on root
exudation is extremely scarce, while effects like decreased root growth
and function, including nutrient and water uptake, have been widely
described (Heckathorn, Giri, Mishra & Bista 2013). Under salt or heat
stress, citrus plants have been shown to release proline in larger
quantities from roots during heat stress (Vives-Peris, Molina, Segura,
Gómez-Cadenas & Pérez-Clemente 2018). Furthermore, the exudation of the
phytohormones salicylic acid, indole acetic acid, abscisic acid, and
jasmonic acid generally increased under elevated temperatures
(Vives-Peris et al. 2018a). Another recent study examined the
effect of heat on Sorghum bicolor root exudation, highlighting
that heat-stressed plants exuded large amounts of compounds, including
ascorbate, carotene, glutathione, or flavonoids (Yaqoob, Bhatti, Sultana
& Shahid 2020). Considering that the effects of the single stress, as
water deficit and high temperatures, on the root exudation have been
reported, we still know little about this essential physiological
process to the combination of these abiotic stress despite that they
co-occur in nature and determine a unique plant response that is
different from each single stress.
Most studies regarding the root exudation process have been focused on
exudate profiles of the whole root system. However, the plant root
system comprises different root classes or types, such as embryonic
(primary and seminal) and post-embryonic roots (nodal and lateral),
which are characterized by distinct developmental, physiological, and
functional signatures (Bengough 2003). For example, several studies
reported different morpho-physiological responses among root types to
drought stress (Zhan, Schneider & Lynch 2015), allelochemicals
(Abenavoli, Sorgonà, Albano & Cacco 2004; Lupini, Sorgonà, Princi,
Sunseri & Abenavoli 2016), P deficiency (Rubio, Sorgonà & Lynch 2004;
Tiziani et al. 2020a) and combined nitrogen deficiency/drought
stress (Lynch 2013). In addition to this diversification between root
classes/types, roots also show differences in functionality along their
axis (Rubio et al. 2004; Sorgonà et al. 2011; Tizianiet al. 2020a). Despite this functional complexity of the root
system, very few studies considered the within-root variability of the
exudation processes. Specifically, the carbon efflux is generally highest
at the root tips (McCully and Canny, 1985; Iijima et al., 2000).
However, it has been observed that branch roots released more flavonoids
in white clover (Mathesius et al., 2000) and tryptophan in annual grass,
although the apical region of the primary root emitted more sucrose
(Jaeger et al., 1999). Understanding the variation of exudate profiles
among and along the diverse root types will allow predicting the impact
of the root architecture on biophysical and edaphic properties
(aggregation, structure, pH, moisture, temperature, and nutrient
stoichiometry) in different soil locations.
In addition to the effects on the physio-chemical proprieties of the
rhizosphere, root exudation is a key process through which plants
interact with soil microbes and, consequently, influencing several other
rhizosphere processes. Despite the recent increase of research on root
exudation and their link with rhizosphere microbial communities (de
Vries et al. 2018; Gargallo-Garriga et al. 2018; Karlowskyet al. 2018; Vescio et al., 2021), until now the relationship
between exudation pattern and microbial responses under drought and heat
stress, in single and combination, has been largely unexplored.
In this framework, in the present study we test the effect of drought,
heat stress, and their combination on the root exudation profile of
maize plants, also considering the differences that might exist between
root types (seminal and primary) and root zones (apical and sub-apical).
According to previous studies, we hypothesize that (i) drought and heat
stress will induce a differential response in the blend of root
exudates; (ii) the combination of the two stresses will generate a
different exudation profile than each single stress; (iii) diverse root
types and zones will point out a different exudate pattern for each
single or combine stress. Furthermore, to provide a mechanistic overview
of the role of root exudates in rhizosphere processes, here we leverage
data generated by our previous study (Vescio et al. 2021). In
particular, we aim at unraveling the interaction between root exudates
and the rhizosphere bacterial microbiota when maize plants are exposed
to abiotic stress. According to previous studies suggesting a role of
root exudates in recruiting beneficial microbes, we hypothesize that
(iv) stress-specific root exudates will influence the relative abundance
of microbial taxa that have a beneficial effect on maize plants.