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.