Introduction

The Eucalyptus genus is frequently cultivated in regions with low precipitation, inducing plants to drought stress, which is considered the main environmental factor limiting plant development1. A range of important adaptive mechanisms of drought tolerance, intended to limit water loss, are well documented. Such adaptations include reduction of the leaf area; biochemical strategies like as osmotic changes to maintain cell turgor; physiological adjustments, such as fast stomatal closure followed by stomatal conductance (gs) reduction and, hence, a drop in transpiration rate (E)2. Furthermore, tolerant genotypes alleviate stress by lowering their osmotic potential and adjusting their cell elasticity3. Along with drought stress, potassium (K) availability is an important issue to Eucalyptus development and it has been broadly influencing photosynthetic attributes and promoting environmental stress tolerance4. Potassium is known for its contribution as an osmoticum, playing an essential role in plant metabolism, maintaining cell growth and turgor, protein synthesis, enzymatic activation, stomata adjustments, electron transport rate through photosystem II, and nitrogen (N) assimilation5,6. In this context, an adequate nutrient management can reduce drought stress impacts by increasing plant water use efficiency (WUE)7.

Sodium (Na) is beneficial for plants at low concentrations8, replacing K to a certain degree mainly by reducing K requirements9. However, the life cycle of non-halophytes (found only in non-saline soils) is inhibited by Na concentrations above 100 mM of NaCl2. The Na supplementation seems to enhance leaf gas exchanges10, reduce reactive oxygen species (ROS)11 and photoinhibition in K-deficient plants12. Despite the well-known positive effects of Na supply when K is limiting4,7,10,12,13,14, the potential role of Na in the stomatal movement is not well determined15 and may offer a way to improve plants’ leaf gas exchange parameters. Nonetheless, since solutes accumulation reduce leaf osmotic potential9, Na supply may also enhance drought tolerance by maintaining water uptake during drought periods7 and improving plant’s water balance16. However, the physiological mechanisms underlying a partial K-replacement by Na in Eucalyptus under drought conditions remain unclear, being extremely important to enhance management practices in order to face future climate changes. Moreover, due to the well-known importance of K fertilizer for crop yield, potash consumption has increased dramatically in most regions of the world17, justifying the great interest in understanding K and Na dualism for the improvement of plant yield with cheaper fertilizers, as NaCl. Positive effects in growth stimulation due to Na supply in place of the more expensive K fertilizer were observed both in natrophilic and glycophytic plant species grown under (i) soil with low concentration of available K or Na, (ii) highly K-fixing soils and (iii) in irregular rainfall areas2. According to the authors, despite the genotypic differences, a huge amount of the expensive K supply would be necessary to stimulate plant growth in these situations, supporting the fertilizer strategy with NaCl.

The influence of K on CO2 assimilation (A) is major linked to mesophyll diffusion conductance18, while changes in environmental conditions such as drought stress, affect greatly gs, and therefore the internal to ambient CO2 concentration ratio (Ci/Ca)19, leading to alterations in photosynthetic discrimination in C3 leaves. The carbon (C) isotope fractionation method (δ13C) can be divided in two main discriminating steps during CO2 fixation in C3 plants: 1) discrimination during CO2 diffusion from the ambient air into the leaves through the stomata and 2) discrimination by the RuBisCO carboxylation, which preferentially uses 12CO2 relative to 13CO220. By contrast the N isotope composition (δ15N) is a good predictor of the plant growth status and N metabolism21. Plants with higher photosynthetic capacity and transpiration efficiency accumulate more N, the most growth-limiting nutrient element for plants. In this context, the δ13C and δ15N have been used to gain insights into the effect of growing conditions on C and N metabolism, as also the mechanisms responsible for plants responses to nutritional status and abiotic stress throughout its whole cycle, since WUE, N acquisition and C uptake may be correlated22,23,24,25. However, studies regarding the interactive effect of partial K substitution by Na on the δ13C and δ15N of the Eucalyptus seedlings grown under drought condition are scarce.

Despite the similarities between the Na+ and K+ hydrated ionic radii, the extent of K replacement by Na in plant nutrition not only varies among species, but can also be harmful. Most of agriculturally crops are intolerant to salt, being inhibited by high Na concentrations15. Under salinity conditions, which affect more than 20% of the global agriculture lands, the Na exceeds the cells’ ability to compartmentalize Na+ in the vacuole, increase the osmotic potential and disrupt cell membrane integrity21. As a result, Na accumulates in the cytoplasm, leading to enzyme activity inhibition26, and sudden death of entire shoots27. Currently, studies regarding Na in plants mainly focus on salinity conditions, i.e., > 100 mM of NaCl, and despite recent advances in understanding drought stress and K supply, little attention has been paid to the combined use of K and Na as an agricultural practice to optimize productivity and efficiency of resources in Eucalyptus under drought conditions. Therefore, the effect of partial K-replacement by Na on leaf 13C, 15N compositions and photosynthesis-related parameters remains unclear, especially in plants with different drought tolerances when subjected to soil–water availabilities. We hypothesized that (i) the partial K substitution by Na has the potential to alleviate the drought-induced stress in Eucalyptus by up-regulating growth, physiological and biochemical parameters and (ii) confirm the use of natural isotopic abundance (δ13C and δ15N) as a reliable indicator of the positive effects of partial K substitution by Na on C and N metabolism throughout plants metabolism cycle. Thus, we applied a multitiered approach at a range of leaf and plant levels to (i) characterize leaf C and N isotope compositions, leaf water potentials, oxidative stress; (ii) quantify the long-term plants water use efficiency; and (iii) evaluate K- and Na-uptake as well as eucalyptus species growth.

Results

The relative effects (increase or decrease) of partial K replacement by Na (50/50% of K/Na) and exclusive Na-supply (0/100% of K/Na) compared to the well K-supply (100/0% of K/Na) in E. saligna, E. urophylla and E. camaldulensis grown under W+ and W− condition can be observed in Supplementary Table 1S.

Growth parameters

A significant decrease was observed in the growth parameters of plants grown under 0/100% of K/Na (exclusive Na-supplied plants) relative to 100/0% of K/Na (well K-supplied plants) or 50/50% of K/Na (partial K replacement by Na) (Fig. 1).

Figure 1
figure 1

Height (a–c), collar diameter (d–f) and total dry mass (g–i) of E. saligna, E. urophylla and E. camaldulensis seedlings under three soil levels of K/Na in well-watered (W+) and water-stressed (W−) condition. Different uppercase letters indicate difference between water conditions and different lowercase letters indicate differences between the treatments (% of K/Na). Vertical bars indicate standard errors between blocks (n = 4).

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Regardless plant species and water condition, the exclusive Na-supply decreased the height (20%), collar diameter (30%) and total dry mass (TDM) (45%) when compared to well K-supplied plants. Moreover, the partial K replacement by Na decreased the TDM of E. saligna (up to 30%) compared to well K-supplied plants under both water conditions, while no effects was observed in the drought tolerant species, such as E. urophylla and E. camaldulensis (Fig. 1g–i).

Drought had no effect on height and collar diameter, while decreased the TDM of E. saligna (20%) under well K-supply, as also of E. urophylla (20%) and E. camaldulensis (25%) under well K-supply and partial K replacement by Na.

Leaf gas exchange

Overall, the CO2 assimilation (A), transpiration rate (E) and stomatal conductance (gs) were significantly improved by the partial K replacement by Na (Fig. 2).

Figure 2
figure 2

Photosynthetic rate-A (a–c), leaf transpiration rate-E (d-f) and stomatal conductance-gs (g–i), in leaves of E. saligna, E. urophylla and E. camaldulensis seedlings under three soil levels of K/Na in well-watered (W+) and water-stressed (W−) condition. Different uppercase letters indicate difference between water conditions and different lowercase letters indicate differences between % of K replacement by Na. Vertical bars indicate standard errors between blocks (n = 4).

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Regardless water condition, the partial K replacement by Na increased the A in all plant species by 45%, while the exclusive Na-supply decreased its values by 40%, relative to well K-supplied plants. Drought-induced stress increased the A of E. saligna (100%), while decreased of E. urophylla (35%) and E. camaldulensis (25%) grown under exclusive Na-supply (Fig. 2a–c and Supplementary Table 1S).

The E was higher in E. saligna and E. urophylla (200%) with partial K replacement by Na compared to well K-supplied plants, while the exclusive Na-supply increased its values only in E. camaldulensis (55%). The drought decreased the E of E. saligna (57%) and E. urophylla (67%) grown both under partial K replacement by Na and exclusive Na-supply, compared to plants grown in W+ condition (Fig. 2d–f).

The partial K replacement by Na also increased the gs of E. saligna (190%) and E. urophylla under W− (45%), relative to well K-supplied plants in the same water condition. Moreover, the exclusive Na-supply increased the gs of E. saligna under W+, while decreased the gs of E. urophylla under W+ and E. camaldulensis under both water condition, compared to 100/0% of K/Na treatment. The drought also decreased the gs of E. saligna and E. camaldulensis grown under partial K replacement by Na and exclusive Na-supply, and of E. urophylla under the three soil levels of K/Na (Fig. 2g–i).

Water use efficiency (WUEI, WUET, WUEL)

In general, a significant decrease was observed in WUE of exclusive Na-supplied plants when compared to well K-supplied eucalyptus plants or under partial K replacement by Na, which did not differ from each other, except in the WUEL of E. saligna subjected to W−condition (Fig. 3).

Figure 3
figure 3

Intrinsic (a–c), instantaneous (d–f) and long-term (g–i) water use efficiencies in leaves of E. saligna, E. urophylla and E. camaldulensis seedlings under three soil levels of K/Na in well-watered (W+) and water-stressed (W−) condition. Different uppercase letters indicate difference between water conditions and different lowercase letters indicate differences between % of K replacement by Na. Vertical bars indicate standard errors between blocks (n = 4).

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Regardless water condition, the exclusive Na-supply dramatically decreased the WUEI, WUET and WUEL of E. saligna, E. urophylla and E. camaldulensis, while the partial K replacement by Na also decreased the WUEL of E. saligna (W−: 25%), relative to well K-supplied plants.

The drought effects on WUE varied among the measurement methods (leaf level or plant scale). Under W− condition, the WUEI was higher in E. saligna (65%) under exclusive Na-supply, whereas the WUET decreased in E. saligna (45%) under the three levels of K/Na rates and in E. urophylla (15%) at exclusive Na-supply, relative to W+ condition. Moreover, the WUEL was also higher in E. saligna, E. urophylla and E. camaldulensis, under well K-supply, partial K replacement by Na and exclusive Na-supply (Fig. 3 and Supplementary Table 1S).

Stable isotope natural abundances (δ13C, δ15N and C/N ratio)

Regardless of the water condition, exclusive Na-supplied plants showed the lowest results to δ13C (except E. saligna under W −) and C/N ratio, as well as the highest results to δ15N (Fig. 4). Moreover, plants grown under W −  condition showed the highest δ13C and the lowest C/N ratio in all species.

Figure 4
figure 4

Leaf carbon δ13C (a–c), and nitrogen (δ15N) isotope composition (d–f) and C/N ratio (g–i) of E. saligna, E. urophylla and E. camaldulensis seedlings under three soil levels of K/Na in well-watered (W+) and water-stressed (W−) condition. Different uppercase letters indicate difference between water conditions and different lowercase letters indicate differences between % of K replacement by Na. Vertical bars indicate standard errors between blocks (n = 4).

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The exclusive Na-supply significantly decreased the δ13C to the lowest values in E. saligna under W+ condition (− 32.0 ± 0.3‰), and for E. urophylla and E. camaldulensis in both water regimes. In contrast, the values of δ13C% for E. saligna under W− condition were the highest at 0/100% of K/Na (− 29.5 ± 0.3‰). Drought stress increased the mean δ13C values of E. saligna (3%), E. urophylla (1.5%) and E. camaldulensis (2.3%) under the three soil levels of K/Na, compared to W+ condition (Fig. 4a–c and Supplementary Table 1S).

The highest 15N was found in the exclusive Na-supplied plants of E. saligna, E. urophylla and E. camaldulensis under both water conditions. The drought had no effects on plants15N values (Fig. 4d–f).

Under W+ condition, the exclusive Na-supply decreased the C/N ratio of E. saligna (60%), E. urophylla (20%) and E. camaldulensis (15%), while the partial K replacement by Na decreased its values for E. saligna (27%), and increased for E. urophylla and E. camaldulensis by 20%, relative to well K-supplied plants. Moreover, under W− condition, the exclusive Na-supply decreased the C/N ratio of E. saligna (30%), E. urophylla (20%) and E. camaldulensis (20%), relative to well K-supplied plants at the same water condition. Drought stress decreased the C/N ratio of E. saligna (40%) at the treatment of well K-supplied plants and partial K replacement by Na, as also of E. urophylla (30%) and E. camaldulensis (15%) at the three soil levels of K/Na, compared to W+ condition (Fig. 4g–i).

Soluble proteins, H2O2 and MDA

The well K-supply and partial K replacement by Na showed similar values in the soluble proteins, H2O2 and MDA concentration in all species, whereas the exclusive Na supply down-regulated soluble proteins, thus increasing H2O2 and MDA concentration in all species (Fig. 5).

Figure 5
figure 5

Soluble protein (a–c), hydrogen peroxide—H2O2 (d–f) and malondialdehyde—MDA (g–i) concentrations in the shoots of E. saligna, E. urophylla and E. camaldulensis seedlings under three soil levels of K/Na in well-watered (W+) and water-stressed (W−) condition. Different uppercase letters indicate difference between water conditions and different lowercase letters indicate differences between % of K replacement by Na. Vertical bars indicate standard errors between blocks (n = 4).

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The exclusive Na-supply decreased the soluble proteins of E. saligna, E. urophylla and E. camaldulensis relative to well K-supplied plants. The partial K replacement by Na had no effects in soluble proteins content of E. urophylla and E. camaldulensis under both water conditions, while increased its values in E. saligna under W+ condition, whereas reduced under W− condition, relative to well K-supplied plants. Drought stress increased the soluble protein content of E. saligna at well K-supply and exclusive Na-supply, as also for E. urophylla at well K-supply and partial K replacement by Na. However, the W− condition decreased its values in exclusive Na-supply in E. urophylla and E. camaldulensis, compared to W+ condition (Fig. 5a–c and Supplementary Table 1S).

Plants of E. saligna showed the lowest H2O2 concentration when grown under partial K replacement by Na, reducing its values by 40% under both water conditions, compared to well K-supplied plants at the same water condition. In addition, the exclusive Na-supply significantly increased the H2O2 concentration of E. saligna, E. urophylla and notably in E. camaldulensis. Drought stress showed a trend to increase the H2O2 concentration of all species: in E. urophylla by 60% at well K-supplied plants and partial K replacement by Na, and for E. camaldulensis (75%) at exclusive Na-supply, relative to W+ condition (Fig. 5d–f).

The exclusive Na-supply increased MDA of E. saligna, E. urophylla and E. camaldulensis, relative to well K-supplied plants at the same water condition. Moreover, the partial K replacement by Na increased the MDA of E. urophylla. Drought stress increased the MDA of E. urophylla and E. camaldulensis at well K-supplied plants and partial K replacement by Na, relative to plants under W+ condition (Fig. 5g–i).

Stomatal density (Std), stomatal pore area and total stomatal pore area (TSPA)

Overall, the partial K replacement by Na increased the Std, while the exclusive Na-supplied plants reduced the Std for adaxial and abaxial sides, as also for TSPA under both water conditions, relative to either well K-supplied plants or the partial K replacement by Na (Table 1). Besides, no stomata were found in the adaxial side of E. saligna and E. urophylla (hypostomatic leaves, i.e. the Std was lower than 5 mm−2)28, while the leaves of E. camaldulensis were amphistomatic (occurred on both abaxial and adaxial sides, Supplementary Fig. 2S).

Table 1 Stomatal density, stomatal area and total stomatal pore area (adaxial plus abaxial surface of leaves) of E. saligna, E. urophylla and E. camaldulensis seedlings under three soil levels of K/Na in well-watered (W+) and water-stressed (W−) condition.
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There were no significant differences between well K-supplied plants and the partial K replacement by Na in E. saligna and E. camaldulensis under both water conditions, and E. urophylla under W+ condition. However, the exclusive Na-supply decreased the Std of E. saligna, E. urophylla and E. camaldulensis compared to well K-supplied plants at the same water condition (Table 1). The adaxial Std in E. camaldulensis also had no significant difference among the three soil levels of K/Na. Drought stress decreased the Std of E. saligna, E. urophylla and E. camaldulensis in partial K replacement by Na, and of E. saligna in well K-supplied plants. Otherwise, an increase in Std was observed in E. saligna and E. urophylla with exclusive Na-supply, when compared to W+ condition.

The partial K replacement by Na increased the stomatal pore area of E. urophylla under W+ condition and E. camaldulensis in abaxial and adaxial side relative to well K-supplied plants (Table 1 and Supplementary Table 1S). However, the exclusive Na-supply decreased the stomatal pore area of E. urophylla and E. camaldulensis under W− condition in abaxial and adaxial side, compared to well K-supplied plants. Drought stress decreased the stomatal pore area of E. saligna grown under the three soil levels of K/Na and E. camaldulensis in abaxial side at exclusive Na-supply. Otherwise, the drought significantly increased the stomatal pore area in abaxial of E. urophylla and adaxial side of E. camaldulensis grown under well K-supply.

The exclusive Na-supply decreased the TSPA of E. saligna, E. urophylla and E. camaldulensis relative to well K-supplied plants, meanwhile the partial K replacement by Na reduced its values for E. urophylla under W− condition. Otherwise, an increase was observed for E. urophylla and E. camaldulensis at partial K replacement by Na under W+ condition (Table 1). Drought stress decreased the TSPA of E. saligna at well K-supply, partial K replacement by Na and exclusive Na-supply, and increased its values by 60% in E. urophylla at well K-supply and exclusive Na-supply and in E. camaldulensis at well K-supply, relative to W+ condition.

Chlorophyll and flavonoids content, Na and K accumulation in plants and leaf water potential

A significant decrease was observed in chlorophyll content (Chl) and leaf water potential (Ψw) of plants under drought stress, relative to W+ condition, as also of exclusive Na-supplied plants and those under partial K replacement by Na, when compared to well K-supplied plants. The Na accumulation in plants increased by augmenting Na rates, while the K accumulation decreased by reducing K supply, according to the K/Na combination rates (Table 2 and Supplementary Table 1S).

Table 2 Chlorophyll content, flavonoids (Dualex units), Na and K accumulation and leaf water potential (Ψw) of E. saligna, E. urophylla and E. camaldulensis seedlings under three soil levels of K/Na in well-watered (W+) and water-stressed (W −) condition.
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The partial K replacement by Na and exclusive Na-supply treatments decreased the Chl of E. saligna, E. urophylla, and E. camaldulensis relative to well K-supply (Table 2). The drought stress decreased the Chl of E. saligna at well K-supply and exclusive Na-supply, of E. urophylla at the three soil levels of K/Na, compared to W+ condition, and E. camaldulensis at partial K replacement by Na and exclusive Na-supply. The flavonoids content (Flav) decreased at partial K replacement by Na in E. saligna, whereas increased in E. urophylla, and at exclusive Na-supply in E. saligna and E. urophylla under W− condition (Table 2). Flavonoids content on E. saligna subjected to drought-induced stress increased by 22% at exclusive Na-supply, relative to plants under W+ condition.

The Na accumulation in plants augmented at partial K replacement by Na and at exclusive Na-supply relative to well K-supply. The drought stress increased the Na accumulation in plants of E. saligna at exclusive Na-supply, and of E. urophylla at partial K replacement by Na, compared to W+ condition (Table 2).

The K accumulation in plants decreased at partial K replacement by Na and at exclusive Na-supply relative to well K-supplied plants. K accumulation in E. saligna under drought-induced stress increased at partial K replacement by Na and exclusive Na-supply and of E. camaldulensis at exclusive Na-supply, compared to plants under to W+ condition (Table 2).

The partial K replacement by Na and exclusive Na-supply treatments decreased the Ψw of all eucalyptus species relative to well K-supply, at both water conditions. In addition, the drought-induced stress decreased the Ψw by 30% and 60%, respectively, of E. saligna and E. camaldulensis, at the three soil levels of K/Na compared to W+ condition (Table 2).

Multivariate analysis: linking carbon–nitrogen isotope compositions, physiological and nutritional responses

The PC1 and PC2 explained 41% and 23% of the total variance (Fig. 6a), respectively. The PC1 was characterized by D, TDM, TSPA, WUET, K and Na accumulation and PC2 by the isotopic parameters (δ13C and δ15N), WUEL, WUEI, Flav, and E. Regardless of water condition, well K-supplied plants were characterized by high growth responses (H, D, TDM), WUET, TSPA, A, Chl, K accumulation, as well as by low δ15N, Na accumulation, H2O2 and MDA values. Plants under partial K replacement by Na showed similar trends to the well K-supplied plants, while those under exclusive Na-supply were characterized by high δ15N, Na accumulation, MDA and H2O2 values.

Figure 6
figure 6

Principal component analysis—PCA with confidence ellipses (95%) (a) and hierarchically clustered heat map (b) of E. saligna, E. urophylla and E. camaldulensis seedlings under three soil levels of K/Na in well-watered (W+) and water-stressed (W−) condition. The heatmap indicates values close (blue tones) and distant (red tones). Four biological replicates were analyzed per sample (n = 4). Abbreviations: height (H), collar diameter (D), CO2 assimilation (A), stomatal conductance (gs), transpiration rate (E), intrinsic water use efficiency (WUEI), instantaneous water use efficiency (WUET), long-term water use efficiency (WUEL), relative leaf chlorophyll content (Chl), flavonoids content (Flav), carbon and nitrogen isotope composition (δ13C and δ15N) of leaves, leaf concentration of malondialdehyde (MDA), hydrogen peroxide (H2O2), total stomatal pore area (TSPA), K and Na accumulation in plants (K and Na), total dry mass (TDM).

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The hierarchically clustered heat map indicated the formation of two main groups among evaluated traits and treatments (Fig. 6b). Traits were grouped into group 1, composed by growth responses (H, D and TDM), Chl, WUE, TSPA, A, δ13C, and K accumulation; and group 2, formed by Flav, H2O2, MDA, E, gs, Na and δ15N. The treatments were grouped into group 1, comprised by plants grown under well K-supply and partial K replacement by Na, irrespective of species and water condition, except E. saligna at partial K replacement by Na under W−; and group 2 was formed by plants under well K-supply, regardless species and water condition, and E. saligna at partial K replacement by Na under W− condition. These findings are supported by the confidence ellipse, which clearly allow the visualization of differential behavior in these two main groups of treatments, as observed by the high correlation between plants under well K-supply and partial K replacement by Na, and the higher variance in the exclusive Na-supply, irrespective of water condition and species (Fig. 6a). The δ13C and WUEL was negatively correlated to δ15N in a linear relationship (p < 0.01, Supplementary Fig. 1S) under the three species.

Discussion

The data obtained revealed that the partial K replacement by Na (50/50% of K/Na) up-regulated the leaf gas exchanges (A, E and gs), boosting WUEL to maximum values in both water conditions tested, compared to the well K-supplied plants (100/0% of K/Na). The highest levels of WUEL observed in plants grown under partial K replacement by Na is probably due to the ability of Na accumulation to promote not only osmotic adjustments, such as better stomatal control and lower leaf osmotic potential9, but also mesophyll cell enlargement, enhancing water uptake and storage11, thus, alleviating drought impacts in Eucalyptus species.

Moreover, drought decreased the Std of E. saligna—the drought sensitive species—grown under 100/0% and 50/50% of K/Na, which also had two-fold higher Std values than E. urophylla and E. camaldulensis, suggesting lower Std value as an effective adaptation tool for survival during dry seasons, as also observed by Frank et al.29. The stomatal density and its distribution at each leaf side have a clear impact on carbon physiology30, as observed by the positively correlation of TSPA with A and gs, being strongly driven by historic drought regime of the species31. In E. saligna and E. urophylla, stomata were located on the abaxial side, which is commonly observed in plants grown under mesophytic areas. However, the E. camaldulensis showed even Std in abaxial and adaxial side, which is common for plants grown under drought (Supplementary Fig. 2S). These findings explain the greater values of A, E and gs observed in E. camaldulensis, the drought tolerant species, compared to E. saligna and E. urophylla.

The similar plant yields observed in the treatments with K/Na rates of 100/0% and 50/50% confirm the possibility of using Na to promote CO2 assimilation and WUE4, regardless the water status. The notably positive effects of K replacement by Na in plant growth parameters of E. urophylla and E. camaldulensis than E. saligna are linked to the higher K-use efficiency32 in drought tolerant species, stimulating greater K uptake of plants grown under stress condition7. This mechanism may occur due to the higher ability to sequester Na into vacuoles, while sensitive species allocate Na mainly into cell cytosol33.

The stable isotope natural abundances (δ15N and δ13C) have been utilized as powerful tools to evaluate genotypic variation in WUE and integrate physiological responses to stress due to their sensitivity to environmental factors, delivering useful information about leaf gas exchange19, plant yield34 and drought-tolerant crops selection35. However, the WUE measurement is methodologically challenging since it can be determined via several methods, providing information on different spatial and temporal scales36. On a short time/leaf level scale, the WUEI can be measured through the ratio of A by gs or WUET, through the ratio of A by E, producing accurate data of a specific time36. On the long time/whole plant scale, the WUEL can also be determined by the relationship between plant dry mass yield and water consumption throughout the experimental period37. As observed in this study, the large increases in leaf level WUE (WUEI and WUET) were not reflected in significant increases in CO2 assimilation over the life of the plant, concealing the long-term adjustments in leaf level. This must carefully be considered when integrating these values to create more accurate and complex models for predicting WUE31. Thus, the WUE at the whole plant level becomes more reliable when studying gas exchanges and changes in the environmental conditions that occur during the cultivation period.

The magnitude of stomatal and/or non-stomatal limitations, that play an important role in photosynthesis, depends on the severity of the imposed stress. Under mild drought, where stomatal factors are dominant, the decline in A occurs as a consequence of the decrease in gs, and the expected trend is the increase in WUE and δ13C and a decrease in Ci. However, under severe drought, non-stomatal limitations are preponderant, leading to constraints in mesophyll resistance, ribulose phosphate regeneration, rubisco activity and photochemical activity. Thus, the decrease in A does not occur due to a decrease in gs, and the expected consequence is the decrease in WUE and δ13C and an increase in Ci38,39. As observed in our study, a highest A, δ13C and WUE was observed in plants grown under well K-supply and partial K replacement by Na, leading to a more conservative water use strategy40. In this context, we can assume that the stomata of plants under partial K replacement by Na were partially closed, optimizing stomatal movements to avoid water loss of Eucalyptus during photosynthesis. In contrast, the exclusive Na supply down-regulated stomata movements and decreased plant’s TSPA, A, δ13C and WUE while increased the E, due to non-stomatal factors, which may have arisen from increased affinity of RuBisCO in capturing the CO2 delivered by gs and mesophyll conductance and isotope discrimination39. Regarding water condition, the drought decreased considerably the gs, while the WUE and δ13C increased, indicating that stomatal factors were responsible for the decline in A of Eucalyptus species.

The δ13C at 100/0% and 50/50% of K/Na increased up to 3%, 1.7% and 2% due to drought in E. saligna (drought sensitive), E. urophylla (moderate drought tolerance) and E. camadulensis (drought tolerance), reflecting an increase in WUEL of 50%, 20% and 25%, respectively. It may be expected that even more significant increases in δ13C and WUEL correlation would be attained for longer drought periods41. Thus, the δ13C provided a reliable estimation of the K substitution by Na effect on water status of leaves, acting as a long-term indicator of plant metabolism and integrating CO2 assimilation and WUE throughout the plant life. The exclusive Na supply led to the appearance of leaf chlorosis and necrosis, visual symptoms of K deficiency (Supplementary Fig. 3S), and consequently to the lower plant yield, since K is an essential element that cannot be fully replaced42. Meanwhile, the K-deficiency also affected plants natural profusion of 15N, which higher δ15N values indicate lower N uptake and accumulation in plant tissues, affecting plants carbon–nitrogen balance (leaf C/N ratio)43. As observed by the negative correlation of growth parameters and leaf gas exchange (Fig. 6a), δ13C and WUEL (Fig. 1S) with δ15N, our results corroborate the utilization of δ15N for crop screening on N metabolism and growth conditions44.

The elevated ROS concentrations (H2O2), harm the cellular membranes and other cellular components, resulting in oxidative stress and cell death45. Potassium is responsible for stomatal movement and its deficiency may lead to stomatal closure, which causes lower light energy absorption during CO2 fixation and consequently intensify the electron transport chain, resulting in ROS accumulation and cell membrane oxidation46. As also observed for well K-supplied plants, the partial K replacement by Na maintained high values of soluble proteins, indicating higher detoxifying enzyme activities to reduce oxidative damage (lowest values of H2O2)18. Furthermore, the partial Na supply also relieved MDA concentration11 probably by entering into vacuole of stomatal cells and enabling stomatal aperture and CO2 fixation, decreasing therefore H2O2 synthesis47. Thus, partial Na supply appears to avoid oxidative stress (indicated by H2O2) and, consequently, cell membrane disruption (indicated by MDA) in plants grown under limited K supply. The Chl levels reduced with Na supply under both water conditions in all species (Table 2). These findings provide an interesting insight: partial Na supply significantly improved A, gs, and WUEL, besides decreasing the Chl pigment content, possibly due an efficient regulation of the available amount of light and lower ROS formation by enhanced soluble proteins.

Otherwise, the exclusive Na supply may have exceeded the capacity of H2O2 scavenging enzymes, as indicated by the low soluble protein values, depressing the photosystem activity and electron transport rate48 and therefore resulting in elevated concentration of H2O2 and MDA (final product of lipid peroxidation). Consequently, as typical symptom of oxidative stress, the K deficiency led to photo-oxidation of chlorophyll pigments of all Eucalyptus species, whose low values indicate hampered photosynthetic capacity49. Thus, Eucalyptus plants receiving less than 50% of their critical K supply could not be fully compensated by Na supply, suggesting that at least half the K requirements are necessary for functions that is poorly substituted by Na.

As mentioned, the K-deficient plants were characterized by high Na accumulation, MDA and H2O2 values. Nevertheless, the novelty was the similarity of these values with high δ15N and low δ13C and WUEL (Fig. 6a). Thus, the data presented in this study confirms the potential of partial K replacement by Na in Eucalyptus plants with genotypes adapted to drought, such as E.urophylla e E.camaldulensis. Nonetheless, according to our hypothesis, we validate the use of natural isotopic abundance (δ13C and δ15N) as a useful sensitive indicator of WUE, stress responses and growth parameters, representing the long-term metabolism of plant life.

Methods

Plant material and growth conditions

The experiment was performed during the spring of 2018 with three commercial and widely cultivated Eucalyptus species contrasting in drought tolerance: E. saligna Sm. (sensitive); E. urophylla S.T. Blake (moderate) and E. camaldulensis Dehn (tolerant)50. Healthy and uniform 12 weeks old seedlings with 30 cm in height were obtained in a seedling nursery from Institute of Forest Research and Studies (Piracicaba city, Brazil) and transferred into pots containing 5 kg of homogenized topsoil (Oxisol soil with pH = 4.2; K = 0.3 mmolc dm−3; and Na = 0.1 mmolc dm−3). Seedlings were grown in a greenhouse (mean temperature of 26 °C and relative humidity of 65%) for 120 days, at the Center for Nuclear Energy in Agriculture at University of São Paulo, Piracicaba, Brazil. The soil composition was: pH = 4.0; organic matter = 15 g dm−3; P = 5 mg dm−3; S = 6 mg dm−3; K = 0.25 mmolc dm−3; Ca = 1 mmolc dm−3; Mg = 0.8 mmolc dm−3; Al = 3 mmolc dm−3; H + Al = 25 mmolc dm−3; B = 0.1 mg dm−3; Cu = 0.7 mg dm−3; Fe = 30 mg dm−3; Mn = 0.4 mg dm−3; and Zn = 0.3 mg dm−3.

Experimental design

The experiment was performed in randomized blocks with four replicates per treatment, in a 3 × 2 factorial design: three rates of K replacement by Na and two water conditions. Based on soil earlier trials15, the K replacement by Na was applied (as KCl and NaCl) in three soil levels: 100/0 (well K-supplied plants), 50/50 (partial K replacement by Na) and 0/100 (exclusive Na-supplied plants) % of K / % of Na, containing, respectively, 0/0.90, 0.44/0.44 and 0.90/0 mmolc dm−3 of Na/mmolc dm−3 of K, reaching the K level required for forest species development (< 1.20 mmolc dm−3 of K)7.

In addition, the seedlings were fertilized with basal nutrients (nitrogen, phosphorus, calcium, magnesium, sulfur, copper, zinc, iron, boron, manganese and molybdenum), as defined by Novais et al.51. Sixty days after the onset of the treatments, the seedlings were watered with two different regimes: well-watered (W+) and water stressed (W −) condition. The pots were daily weighed and watered with deionized water until their respective soil relative water content reached W+ : 80% and W−: 35% respectively, accordingly to the gravimetric method52 to replace the evaporated and transpired water.

To analyze the influence of Na supply in extractable cations levels (K, Ca, Mg and Al), another supplementary soil incubation experiment was performed. Pots with same treatments and basal nutrients fertilization were watered daily to W+ soil relative water content and the soil was mixed monthly for 120 days and then analyzed. There were not any significant effects of Na supply (50/50 and 0/100% of K/Na) on exchangeable K, Ca, Mg and Al levels compared to the well K-supplied soil (100/0% of K/Na, data not shown).

Leaf gas exchange

Pior to harvesting, gas exchange measures on fully expanded leaves were performed (between 9 to 11 am), with a Li-Cor 6400XT (Li-Cor, Inc., Lincoln, NE, USA). Cuvette temperature was set to 25 °C and relative humidity to 65% maintaining vapor pressure deficit in the cuvette at around 1.1 kPa. The CO2 concentration was kept at 380 ppm throughout all measurements. A LED array provided a photosynthetic photon flux density (PPFD) of 1,200 μmol m−2 s−1. CO2 assimilation (A), stomatal conductance (gs), and transpiration (E) were measured and averaged over 5–10 min after sample stabilization. Intrinsic water use efficiency (WUEI) and instantaneous water use efficiency (WUET) of the leaf were determined by dividing the values of A by gs and A by E, respectively36. Long-term water use efficiency (WUEL) was calculated by dividing the total dry mass value (belowground plus aboveground) by water use throughout the experiment (g dry mass/kg H2O)37. The leaf water potential (Ψw) was measured in the same leaves at noon (12 p.m) with a Scholander Pressure Chamber.

Chlorophyll and flavonoids content, stomatal density and total stomatal pore area

Relative leaf chlorophyll content and flavonoids content were expressed in Dualex units, and estimated using a portable Dualex-DX4 (FORCE-A, Orsay, France). Stomatal density (Std; stomates mm−2) was calculated using two fully expanded leaves per plant counting both abaxial and adaxial surfaces9. The number of stomata was counted using ImageJ program (https://imagej.nih.gov/ij/).

The stomatal pore area (µm2) was considered as an ellipse (π × 0.25 × stomatal length × stomatal width)28, which observations were made at 1000 × magnification by scanning electron microscopy (JEOL JSM-IT300 LV, Tokyo-Japan) at 20 kV. The length and width of stomates of four randomly selected fields of view of each leaf surface were measured per plant. The total stomatal pore area (TSPA; 10−2 mm mm−2) was calculated as the product of stomatal pore area and Std.

Growth measurements

A graduated ruler and a digital pachymeter were used to measure the growth in height (cm/plant) and collar diameter (mm/plant), respectively. Plants were harvested on the 120th day of the experiment and their leaves, stems, and roots were separated. The dry mass production of each part was calculated by drying the samples in a forced air ventilation oven at 60 °C for 72 h and then weighing them.

Elementary and isotopic analyses

Subsequently, the plant material was ground in a Wiley type mill to quantify the K and Na concentration by nitric-perchloric digestion53. The accumulated K and Na (g plant−1) were calculated as the product of the concentration of each element in the plant part by the dry mass production of the respective tissue. To determine the leaf isotope composition (δ13C and δ15N) and the total leaf C and N, used to calculate the leaf C/N ratio, aliquots of different mass were packed into tin capsules and analyzed with an automatic nitrogen carbon analyzer (ANCA-GLS) interfaced to a continuous-flow isotope ratio mass spectrometer (Hydra 20–20, Sercon Ltd, Crewe, UK)54 with an analytical precision of ± 0.3‰. The isotopes composition were calculated as the following equation20: δ = (R(sample)/R(standard) − 1); where R is the isotope ratio of 13C/12C or 15N/14N. The δ13C results were reported relative to the Vienna Pee Dee Belemnite (VPDB standard) and δ15N is relative to the standard atmospheric N2. Measurements were carried out at Isotope Ratio Mass Spectrometry Core Facility (CM-IRMS CENA) of the University of São Paulo.

Protein extraction

The samples (0.25 g of frozen leaves) were homogenized with a mortar and pestle to 2.5 mL of 100 mmol L−1 phosphate buffer (pH 7.8) containing 0.04 g of PVPP and 2% Triton X-100. The homogenate compound was centrifuged at 10,000 rpm for 30 min at 4 °C, and the supernatant was stored in 0.2 mL aliquots at − 80 °C. The soluble protein concentration was determined by Bradford55 method using bovine serum albumin as a standard in a spectrophotometer at 595 nm (Genesys 10S UV–VIS,Thermo Fisher Scientific, Walthan, USA).

Determination of hydrogen peroxide (H2O2) and lipid peroxidation

The leaf concentration of hydrogen peroxide (H2O2, µmol g−1 fresh mass−1) was determined according to the method described by Alexieva et al.56. Lipid peroxidation was estimated by the leaf concentration of malondialdehyde (MDA, nmol g−1 fresh mass−1), according to the thiobarbituric acid (TBA) test57. The samples (0.2 g of frozen fully-expanded leaves) were ground in 2 mL of 0.1% (w/v) trichloroacetic acid (TCA) and 0.04 g of polyvinyl polypyrrolidone (PVPP) and centrifuged at 10,000 rpm for 10 min at 4 °C. For H2O2, 0.20 mL of supernatant was added to 0.2 mL of 100 mmol L−1 potassium phosphate buffer (pH 7.5) and 0.8 mL of 1 mmol L−1 potassium iodide. The tubes remained at room temperature in the dark for 1 h in continuous dark. Readings at 390 nm were measured using a spectrophotometer (Genesys 10S UV–VIS,Thermo Fisher Scientific, Walthan, USA). Three independent replicates of each plant were used. For MDA determination, the initial procedures were the same for H2O2 measurements as described above. Following centrifugation, 0.25 mL of supernatant was added to 1 mL of 20% (w/v) TCA containing 0.5% TBA and heated in a water bath for 30 min at 95 °C. After cooling for 20 min on ice, the precipitate was removed by centrifugation at 10,000 rpm for 10 min and then, its absorbance read with a spectrophotometer (Genesys 10S UV–Vis, Thermo Fisher Scientific, Walthan, USA) at 532 and 600 nm. Three independent replicates of each plant were used. The leaf concentration of MDA was calculated as the following equation58:

$$C = \left[ {{\text{ABS}}\left( {535{-}600} \right) \div 155,000} \right] \times 10^{6} .$$

Statistical procedures

The K replacement by Na and water regimes were statistically analyzed by two-way ANOVA followed by post-hoc Tukey test (p < 0.05) using the software SAS version 9.1 (SAS Institute Inc, 2012). The original data were standardized to be analyzed via principal components analysis (PCA), integrating the measured variables in each treatment, genotype and water condition. For the PCA, we used the treatments with Na supply for the first two principal components (PC1 and PC2) and 95% confidence ellipses to visualize the multivariate trends of Na application under W+ and W− conditions using R package (R Development Core Team, 2018). The results are indicated as mean ± standard error of four independent biological replicates and graphically visualized using SigmaPlot 11.0 (Systat Software Inc., San Jose, CA, USA).

Research involving plants

The use of plants parts in the present study complies with international guidelines (IUCN Policy Statement on Research Involving Species at Risk of Extinction and the Convention on the Trade in Endangered Species of Wild Fauna and Flora).