Changes in the Mediterranean and related semi-arid climates are expected shortly, leading to temperature increases and more frequent and longer drought periods (Döll, 2002). These will increase crop water demand, while simultaneously reducing the availability of quality water (Schultz, 2017). Since in most grapevine-growing regions, freshwater is a scarce resource (Medrano et al., 2015), the use of alternative waters, such as wastewaters often high in salts, will be more and more needed to mitigate drought stress (Mirás-Avalos and Intrigliolo, 2017). Besides, conventional waters, such as underground water, can indeed be of low quality due to excessive concentrations of soluble salts (Cl^– and/or Na⁺), with an electrical conductivity over 3 dS m^–1 (Pérez-Pérez et al., 2015). This lack of water quality poses a challenge to the sustainability of deficit irrigation in viticulture, as this irrigation strategy could aggravate the effects of salinity (van Leeuwen et al., 2019).

Excessive soil salinity can cause water loss, nutrient deficiency, oxidative stress, photoinhibition, growth inhibition, and induce many metabolic and transcriptomic changes leading to physiological damage (Walker et al., 1997; Kumari et al., 2015; Saha et al., 2015; Upadhyay et al., 2018; Zhou-Tsang et al., 2021). Previous studies have demonstrated that among plant responses to salinity, mechanisms that control ion uptake, transport, and balance, as well as hydric regulation, photosynthesis, cell division, osmotic adjustment, enzymatic activities, antioxidant production, stress signaling, and regulation of root barriers play critical roles in plant tolerance to salinity (Gong et al., 2011; Shahid et al., 2020; Zhou-Tsang et al., 2021).

The Vitis vinifera L. is a crop classified as moderately sensitive to salinity (Maas and Hoffman, 1977; Cramer et al., 2007), with a soil saturation extract electrical conductivity at 25°C yield threshold (EC_t) of 2.6 dS m^–1 (Walker et al., 2002). The tolerance of grapevines to salinity depends on multiple factors and, particularly, on plant genetics, soil and climate characteristics, and the rate and length of the stress, to which vines are subjected (Maas and Hoffman, 1977; Zhang et al., 2002; Cramer et al., 2007; Chaves et al., 2009; Mirás-Avalos and Intrigliolo, 2017). Understanding the physiological and transcriptomic responses of grapevine to saline water is essential to prevent and mitigate potential negative effects on vine performance and grape composition (Ollat et al., 2016). Moreover, the contradictory effects of irrigation with saline or wastewater on vine performance and grape composition (Walker et al., 2004, 2007; Stevens et al., 2011; Mirás-Avalos and Intrigliolo, 2017) point toward the existence of important knowledge gaps regarding the effects of salinity and the salt tolerance mechanisms in Vitis spp. (Zhou-Tsang et al., 2021). Microarray studies of pot-grown own-rooted vines of CVS ‘Cabernet Sauvignon,’ ‘Razegui,’ and ‘Shiraz’ revealed that salinity stress impaired photosynthesis and increased the expression of some transcription factors and genes related to ROS scavenging, abscisic acid, and osmoprotectants such as various sugars and proline (Cramer et al., 2007; Daldoul et al., 2010). High throughput sequencing studies of potted cv. ‘Thompson Seedless’ and cv. ‘Summer Black’ under greenhouse conditions implicated the activity of genes involved in cell wall modulation, various cation and ABC transporters, signal transduction genes, HSPs, and biotic stress-related genes (Guan et al., 2018; Das and Majumder, 2019).

The ‘Tempranillo’ cultivar has been specifically classified as moderately salt-sensitive as well, showing growth decreases attributable to osmotic effects rather than to ion-specific toxicities (Urdanoz and Aragüés, 2009). Nonetheless, since grapevine yield potential under saline conditions is related to the root-zone salinity, the plant portion that primarily deals with soil salinity is not the scion, but the rootstock. Among the characteristics of the different rootstock that contribute to enhancing grapevine tolerance to salinity, there is its ability to exclude and not transport salt to the shoots; besides, there is also the vigor it confers to the scion (Walker et al., 2002, 2014; Munns et al., 2020). Additionally, rootstock can have a great influence on stomatal regulation in response to water and salinity stress, even more than the scion itself (Lavoie-Lamoureux et al., 2017). For instance, rootstock can affect the osmotic adjustment response, which is one of the main physiological processes, whereby the vine responds to salinity (Keller, 2010; Haider et al., 2019). This consists of the active accumulation of solutes, thus increasing leaf relative water content and turgor (Barrios-Masias et al., 2018). Regarding this, several studies are reporting that the rootstocks with lower osmotic adjustment capacity are those with greater capacity to restrict the leaf accumulation of Na⁺ and Cl^–, thus, preventing their possible phytotoxic effects (Stevens and Walker, 2002; Zhang et al., 2002), and minimizing their accumulation in the grape juice and wine in the long-term (Walker et al., 2004, 2014; Teakle and Tyerman, 2010).

American Vitis species, especially V. rupestris, V. riparia, and V. berlandieri are tolerant of saline and limestone soils (Williams et al., 1994; Ferlito et al., 2020). Some rootstocks derived from these species such as Ramsey (V. champini), 1103 Paulsen (1103P), 110 Richter, 140 Ruggeri, and 101–14 Mgt can exclude much salt (chiefly Na⁺ and Cl^–) from root uptake and root-to-shoot transport (Walker et al., 2004, 2010; Gong et al., 2011). For instance, some of the most salinity-tolerant rootstocks, such as 140 Ruggeri and 1103 Paulsen, have an EC_t value of up to 3.3 dS m^–1 (Walker et al., 2002; Zhang et al., 2002; Tregeagle et al., 2006). Conversely, rootstocks, such as SO4 and 3309C, are characterized by being very sensitive to salinity with an EC_t value below 1.8 dS m^–1 (Walker et al., 2010). Given the relatively narrow genetic pool within the commercial grapevine rootstocks and the significant genetic diversity of the genus Vitis, identifying salinity-tolerant grapevine rootstocks is a great opportunity to enhance viticulture sustainability (Schultz and Stoll, 2010). For instance, differential gene expression has been observed in potted Vitis vinifera L. ssp. sylvestris with different short-term salinity tolerance in greenhouse conditions (Askri et al., 2012). Therefore, a better understanding of the rootstock physiological, metabolomic, and transcriptomic mechanisms underlining salt stress tolerance is essential to improve breeding programs aimed at adapting to climate change (Ollat et al., 2016). In this sense, new information about salinity tolerance conferred by rootstocks is needed (Keller, 2010; Marín et al., 2021). Grapevine rootstock breeding programs, such as the one carried out by the University of Milan (Italy) with the M-series, are very promising for coping with water salinity (Meggio et al., 2014) and can benefit a lot from the results of field trials.

Therefore, the objective of the present research was to evaluate the physiology and transcriptomics underlying the performance against salinity of two new rootstocks, M1 and M4, compared to the well-known salinity-tolerant 1103P (Walker et al., 2010; Bianchi et al., 2020). In this work the experimental hypothesis was that the M-rootstocks may confer better salinity tolerance to the scion than the 1103P through enhanced uptake of salt-stress-contesting ions such as calcium, as well as vigor declining ability, in the case of the M1 (Porro et al., 2013; Vannozzi et al., 2017), and because of the leaf build-up of inorganic osmolytes and sodium-antagonists, such as potassium, in the case of the M4 (Meggio et al., 2014). In comparison to the M-rootstocks, the 1103P stands out for its ability to exclude Cl^– from uptake. Aiming at mimicking commercial conditions, the experiment was performed under field conditions and tried to isolate the salinity effect by fully irrigating the vines. Although the vineyard was under establishment, to our best knowledge, these grapevine rootstocks had not been previously tested against salinity under conditions so close to real practice. Besides, in contrast to previous comparative studies between these grapevine rootstocks in this work, all determinations were carried out directly in the scion. This was done considering that the scion is an integrator of rootstock-induced effects (Gambetta et al., 2012; Cookson et al., 2013). Finally, by assessing a young vineyard, i.e., one with a non-extensive root system, the physiological response to salinity could be studied ensuring that most of the roots were effectively under the intended salinity.

The effects of WQ and R on physiology and transcriptomics of cv. ‘Tempranillo’ vines were assessed indirectly because all determinations were carried out on the scion, not in the rootstock, which is the barrier against soil salinity. However, the scion cultivar is the genotype that ultimately bears fruit and ripens it and, therefore, confers economic value on the crop (Marguerit et al., 2012). Thus, in this approach, the scion is considered an integrator of the effects induced by the rootstock. It is important to bear this in mind when interpreting the results, especially the transcriptome analyses, because of the combination of two Vitis spp. Genotypes are studied by evaluating only one of them, i.e., Vitis vinifera L. In comparison, most of the grapevine transcriptomics responses reported in the literature have been assessed on a single genotype, i.e., directly in the own-rooted Vitis vinifera (Cramer et al., 2007; Guan et al., 2018; Das and Majumder, 2019; Lehr et al., 2022) or on the rootstock without grafting (Gong et al., 2011; Henderson et al., 2014; Meggio et al., 2014; Corso et al., 2015; Vannozzi et al., 2017; Fu et al., 2019; Çakır Aydemir et al., 2020), and if carried out in both the scion and the rootstock, they have been under highly controlled conditions (Upadhyay et al., 2018; Bianchi et al., 2020; Franck et al., 2020; Baggett et al., 2021), i.e., not under real field-grown conditions.

In the present trial, the water requirements of the grapevines were fully met trying to isolate the effect of WQ on the physiological and transcriptomic responses. When plant measurements and samplings were carried out, the water status experienced by the control vines grafted onto any of the rootstocks was indicative of very mild water stress according to Williams and Baeza (2007; Figure 1). This implies that irrigation largely met the evapotranspiration demand of the plants. However, it was not excessive, which would have resulted in irrigation water percolation and thus the washout of salts from the rooting depth. In fact, the ions’ concentration in the soil solution of Saline treatments caused vine water stress. This was observed in the general decrease of both Ψ_pre–dawn and Ψ_stem in the vines grafted onto all rootstocks under irrigation with saline water, which means a worsening of the plant water status (Figure 1). This physiological response is likely due to a reduction of the soil water potential by an osmotic effect (Tattersall et al., 2007), i.e., the so-called osmotic drought (Chaves et al., 2009). As expected, Ψ_pre–dawn was in line with Ψ_stem (Suter et al., 2019), although plants onto M4 tended to show less negative Ψ_stem values than those onto 1103P, with no difference in Ψ_pre–dawn (Table 1). These slight differences in Ψ_stem between M4 and 1103P agreed with what Frioni et al. (2020) observed in M4 under water shortage.

Plants react to salt stress and control their subsequent physiological responses using signals, which can be ionic, osmotic, hormonal, and/or reactive oxygen species regulation (Shahid et al., 2020; Zhou-Tsang et al., 2021). Concerning the ionic, in this work the leaf ion concentrations have been observed to differ among rootstocks, notably, Cl^–, Ca²⁺, Na⁺, and Mg²⁺ (Table 2). Regarding Cl^–, it usually builds up in the leaves of woody crops, and the plant’s ability to avoid accumulating Cl^– in leaves is considered directly proportional to its salinity tolerance. In this work the M-series rootstocks increased the leaf Cl^– twofold in the saline treatment compared to the control. In contrast, in the 1103P the leaf Cl^– increase in the saline treatment compared to the control was not significant. These results are in agreement, on the one hand, with Meggio et al. (2014), who also reported higher leaf Cl^– in vines onto M4 in comparison to the good salt excluder 101–14 Mgt (Walker et al., 2004, 2010) and, on the other hand, with Urdanoz and Aragüés (2009), who reported that the ‘Tempranillo’ cultivar grafted onto 1103P was able to exclude Cl^– from the leaves more efficiently than other cultivar-rootstock combinations.

The leaf Cl^– non-accumulation ability conferred by the 1103P could be due to (i) limited salt uptake, i.e., ion exclusion, and (ii) limited salt translocation from the root to the shoot. Abbaspour et al. (2013) suggested that 1103P contributes to reducing shoot Cl^– concentration by root efflux and vacuolar internalization. Besides, Henderson et al. (2014) suggested that transcriptional events contributing to the Cl^– exclusion mechanism in grapevine are not stress-inducible, but constitutively different between contrasting genotypes. Anyway, Cl^– exclusion factors are yet to be identified at the transcriptomic level, and are multigenic, including transport proteins (Gong et al., 2011; Das and Majumder, 2019; Zhou-Tsang et al., 2021). This genotype-dependent, though fuzzy, transcriptomic effects agree with our GSEA results, which identified much less statistically significantly (FDR q < 0.25) differentially expressed bins due to WQ in ‘Tempranillo’ grafted onto 1103P as compared to M4 and M1 (Figure 3). Baggett et al. (2021), also similarly observed that salinity affected transcript abundance more in salt-sensitive genotypes than in salt-tolerant ones. Importantly, the leaf Cl^– concentrations in our trial are higher than the ones reported by Urdanoz and Aragüés (2009) and Baggett et al. (2021), even though in the range of the ones found in ‘Cabernet Sauvignon’ onto 1103P by Dag et al. (2015) using similar WQ.

The capacity of rootstocks to restrict leaf salt buildup should not be the only parameter for rootstock selection (Zhou-Tsang et al., 2021). Regarding other criteria, several authors indicated the better M4 performance compared to other rootstocks because of an improved antioxidant capability (Meggio et al., 2014; Corso et al., 2015; Lucini et al., 2020; Prinsi et al., 2020). Furthermore, it is important to consider the likely accumulation of Cl^– and Na⁺ in the permanent instead of the short-lived organs of the vine (Stevens and Partington, 2013; Netzer et al., 2014), which may lead to salinity carry-over effects on the medium-to-long term. Based on our results, this would be a concern for rootstocks M1 and M4 and less for 1103P (Table 1), because of its possible detrimental effects on future bud fruitfulness (Walker et al., 2002). In fact, Dag et al. (2015) reported that irrigating the ‘Cabernet-Sauvignon’ scion grafted onto 1103P with water similar in salinity to the Saline treatment in this work, did not significantly affect vine performance in the first two seasons, but that Na⁺ and Cl^– accumulation in the wood eventually led to vine death in the third one.

Regarding Na⁺, it is less prone to build up in the leaves of grapevines than Cl^– (Henderson et al., 2018), which, given the Na⁺/Cl^– ratio of the waters applied in this work, was also observed here (Table 2). However, there were differences in salt-stress modulating ability among rootstocks with the M1 more liable to leaf Na⁺ accumulation as salinity increased than 1103P or M4. Regarding leaf Ca²⁺, it increased in the Saline treatments compared to the Controls (Table 2). That leaf Ca²⁺ increased in the ‘Tempranillo’ leaves as salinity grew regardless of the rootstock suggests that all three rootstocks can maintain high Ca²⁺/Na⁺ ratios and thus, efficiently exclude Na⁺ (Shahid et al., 2020). More interestingly, however, there were differences in leaf Ca²⁺ among the vines depending on the rootstock. Particularly, the M1 built up significantly more leaf Ca²⁺ than the 1103P and M4 (Table 2). Since Ca²⁺ can regulate plant signaling, enzyme activity, ion channel performance, and gene expression (Golldack et al., 2014), the higher leaf Ca²⁺ onto the M1 may be a positive plant adaptation as previously reported by Porro et al. (2013). Likewise, K⁺ is also key in maintaining the osmotic balance and thus the ionic homeostasis in plant cells (Kumari et al., 2015; Guan et al., 2018). However, in our work, leaf K⁺ decreased because of salinity, without differences among rootstocks (Table 2). Similarly, Guan et al. (2018) also found a decreasing trend in leaf K⁺ in ‘Summer Black’ cv. in response to NaCl irrigation, and Munns and Tester (2008) indicated that a strong relationship between leaf K⁺ and salt tolerance had not yet been reported. In our work, both the leaf K⁺/Ca²⁺ and K⁺/Na⁺ ratios were reduced by M1 compared to 1103P. This suggests that the 1103P conferred a greater salinity tolerance to the scion than the M1.

Concerning the osmolyte regulation signals, a tendency to a slight osmotic adjustment was observed in the leaves on all three rootstocks. This is because, independently of the leaf water status, i.e., Ψ_π ¹⁰⁰, the values of the saline treatments were significantly more negative (–0.16 MPa on average) than those of the Controls (Figure 1). Through osmotic adjustment plants cope with declining soil water potential mainly because increasing osmolyte concentrations decrease the water potential within plant cells, thus increasing the leaf relative water content and turgor for a given soil water potential (Barrios-Masias et al., 2018). These osmolytes can be inorganic, which are actively and passively taken from the same soil solution, or organic, which are obtained by biosynthesis of proline, glycine-betaine, etc. However, in our work, the expression of genes involved in amino acid metabolism was not altered in leaves in response to WQ (Figure 3), whereas the concentration of Cl^–, K⁺, and Ca²⁺ did increase in the leaves (Table 2). Accordingly, the slight observed osmotic adjustment was achieved through the build-up of inorganic osmolytes, and this was controlled by the rootstock because the root is the organ that regulates the entry of the soil solution ions into the plant. The mechanisms of ion exclusion and/or upward movement along the xylem should be genetically regulated at the root level, i.e., over-expression of the cation HKT transporters genes (Deinlein et al., 2014; Fu et al., 2019; Zhou-Tsang et al., 2021), and not at the scion level. However, despite occurring at the root level, the mechanisms may be genetically regulated in a scion-induced manner (Franck et al., 2020) and then, maybe, detected in the scion. Remarkably, among the 15 differentially expressed genes between the 1103P and M4, a lactoylglutathione lyase (Vitvi04g01424) and a Dof family transcription factor (Vitvi18g00858) were found. These genes have previously been implicated in response to abiotic stress in grapevine (Shangguan et al., 2020), as it was implicated in redox homeostasis in heat-stressed ‘Muscat Hamburg’ berries (Carbonell-Bejerano et al., 2013).

The generalized reduction found in net photosynthesis (A_N) under saline conditions, regardless of the rootstock (Figure 2), is related to stomatal and mesophyll conductance limitation, as there were no major differences in WUE_i beyond those expected, given the differences in water status (Flexas et al., 2004). Reductions are in line with those found by Flexas et al. (1999) in ‘Tempranillo’ and Baeza et al. (2007) and Baggett et al. (2021) in ‘Cabernet-Sauvignon’ cultivars. Moreover, no differences were detected in the ratio of internal to atmospheric CO₂ concentration (Ci/Ca) between treatments (0.76 and 0.75 in Control and Saline treatments, respectively; data not shown). This suggests that in this work salinity was not high enough to induce either toxic effects on the photosynthetic apparatus or cellular damage in the leaves, as confirmed using the leaf transcriptomic analysis (Figure 3), but rather that it simply increased water stress by lowering the soil water potential, which eventually showed up in g_s and, thus, A_N reduction (Figure 2). Interestingly, according to Bianchi et al. (2020), water shortcoming stress decreases stomatal conductance due to lower water potential, but the photosynthetic activity keeps high with bare differences among 1103P, M1, and M4. In contrast, in our trial, M1 performed differently from the other rootstocks by inducing an overall reduction in A_N. Moreover, Bianchi et al. (2020) did detect changes in the transcript abundances of key genes related to abscisic acid biosynthesis, but in the root, not in leaves, and studying only the wider Vitis spp. genotype.

The overall effects caused by salinity on decreasing leaf photosynthesis as well as LAI (Figure 2 and Table 2) should have led to reduced berry ripening (Cramer et al., 2007; Chaves et al., 2009; Liu et al., 2020; Zhou-Tsang et al., 2021). However, the opposite was observed. The Saline treatments increased TSS compared to the Control grapes. These results point toward the ability of all rootstocks to keep allocating energy resources to fruit ripening regardless of salt stress. Interestingly, Meggio et al. (2014) also highlighted the salt tolerance of these rootstocks regardless of their ability to limit specific ion accumulations in the scion, which was associated with a lower decrease in A_N and Ψ_leaf on M4 compared to 101–14 Mgt. This was not observed under salinity in this work, as it neither was an underwater shortage (Bianchi et al., 2020).

Effects of WQ and R on grape composition are usually not very conclusive according to studies where both factors are combined (Walker et al., 2007; Stevens et al., 2011; Hirzel et al., 2017; Mirás-Avalos and Intrigliolo, 2017). This is because there is a multitude of environmental factors that interact with rootstock response, most notably soil type (Ferlito et al., 2020). Specifically, the three rootstocks studied here perform well on soils high in calcium carbonate like the one used in this investigation because all three come from crossings with Vitis berlandieri, a species that evolved on calcareous soils (Harry, 1996). In this work, there was a salt-stress modulating effect by the rootstock on grape composition, primarily TSS and, secondarily, the phenolic composition as revealed by the R × WQ interactions (Table 3). Whereas barely anything was observed on T.A., and, specifically, pH, which did not change following the decrease in leaf K⁺ concentration due to salinity (Table 2) in accordance with Marín et al. (2021). Contrary to T.A., and pH, the TSS increased onto the M1 rootstock as salinity grew, whereas the other rootstocks did not respond in the same way. Moreover, the phenolic substances were also subjected to rootstock-specific modulating effects (Table 3). Despite these, the expected changes on gene expression of CHS and PAL pathways were not observed (Figure 4). This is, the significant reduction in anthocyanins content found in 1103P vines and polyphenols found in 1103P and M1 vines in response to salinity (Table 3) could not be related to the transcriptomic changes observed, nor to differences in berry size (Table 3).

Several studies have linked ultraviolet light to the induction of phenolic compound synthesis, specifically the expression of the CHS gene, a key enzyme in flavonoid biosynthesis (Merkle et al., 1994; Hernández et al., 2009; Wang et al., 2016; Reshef et al., 2018). However, these putative changes, which are related to berry exposure to sunlight in response to the saline effect on the vine leaf area (Zarrouk et al., 2016; Torres et al., 2020), would have been offset by the slight increase in the leaf area-to-production ratio (Walker et al., 2000; Bobeica et al., 2015). Moreover, flavonoid synthase is also involved in drought and osmotic stress tolerance and is controlled by rootstocks (Dal Santo et al., 2018; Bianchi et al., 2020; Zombardo et al., 2020). For instance, Zombardo et al. (2020), also in grape skin during ripening, reported some differentially expressed genes mainly involved in the synthesis and transport of phenylpropanoids (e.g., flavonoids) in response to rootstock effects. Besides, the most prominent differences in gene expression of the anthocyanin pathway usually occur during veraison, together with the differences of anthocyanin content and profile in the berry and begin to faint as the berry reaches final maturity (Castellarin et al., 2006; Castellarin and Di Gaspero, 2007). All of this highlights the complexity of relating phenotypic observations to changes in gene expression (Fu et al., 2019; Haider et al., 2019). In this regard, the next generation of omics is expected to help to identify gene function, speeding up the rootstock breeding programs for enhancing resilience to climate change in future viticulture (Marín et al., 2021).

The results of this work have shown how the grapevine M-rootstock’s physiological and transcriptomic responses integrate at the scion level because of the irrigation with saline water under real field-grown conditions for the first time. The determinations carried out in the scion (i.e., cv. ‘Tempranillo’) permitted us to obtain some insight into the possible mechanisms developed by the rootstocks in response to water salinity, and the differences between the three that were tested in this work. In the short period of this trial, and a vineyard under establishment, all three rootstocks similarly adjusted osmotic potential to cope with osmotic stress, and then, vine water status declined in response to irrigation with saline water compared to non-saline water. Regarding the differential response among rootstocks, based on, on the one hand, grapevine physiology and grape must composition and, on the other hand, salt accumulation in leaves and transcriptomic changes, there were differences worth highlighting. First, the M1 rootstock was the one that responded the most to salinity by reducing A_N and LAI, whereas the M4 rootstock was the one that buffered the best the effects of salinity on TSS and grape phenolic composition. Second, the 1103P rootstock was the one able to reduce the leaf Cl^– and Na⁺ build-up the most and affected transcriptomic expression the least, which might have positive effects on the long-term vine performance and grape composition. Longer-term studies are needed to unravel the molecular responses occurring in mature vineyards at both the scion and rootstock levels.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ebi.ac.uk/ena, PRJEB44658.

IB, JP-P, FV, DI, LB, MP-N, and JP contributed to the conception and design of the study. IB, JP-P, and RS acquired the data. IB, JP-P, FV, DI, KG, and MP-N performed the data analysis and interpretation. IB and RS prepared the first-draft. IB, JP-P, FV, RS, DI, MP-N, and JP reviewed and edited the manuscript. DI, LB, and JP supervised the work. DI, LB, MP-N, and JP acquired the funding. All authors read and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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