Elevated CO2 improves the growth of grapevine young plants under salinity favoring ionic homeostasis and decreasing oxidative damages

doi:10.21203/rs.3.rs-2978660/v1

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Elevated CO₂ improves the growth of grapevine young plants under salinity favoring ionic homeostasis and decreasing oxidative damages

https://doi.org/10.21203/rs.3.rs-2978660/v1

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This study tested the hypothesis that elevated CO₂ improves the growth of grapevine seedlings, achieving greater vigor and shortening the production time under salinity due to the best ionic homeostasis and oxidative protection. Seedlings were exposed to isolate and combined effects of CO₂ (ambient – 400 and elevated – 800 µmol mol^− 1) and salinity (absence – 0 and presence – 80 mM NaCl) in a growth chamber. In these conditions, two-old-month grapevine (Vitis ssp.) plants were grown and defoliated after 20 days, followed by more than 60 days of growth to the emission of new shoots (buds, leaves, and stems). Salinity delays and decreases the emission of new shoots, while elevated CO₂ improves the growth of new shoots. Elevated CO₂ promoted the leaf dry matter (DM) contends and shoot/root ratio in the absence and presence of salt, and the partial recovery of losses leaf DM under salt. High CO₂ mitigated ionic toxicity under salt stress through lower leaf Cl⁻ and Na⁺ accumulation with a preferential allocation of these ions in stems and roots, relative to plants under salt alone. Elevated CO₂ stimulated the K⁺ content in the whole plant (leaves/stems/roots), in salt absence and presence, with increases for K⁺/Na⁺ ratios in leaves and roots under salinity. High CO₂ favored the water potential (Ψ_W) reduction under salinity and increase water relative content (RWC) of plants under two salt levels. Under salt absence, photochemical efficiency indicators quenching photochemical (qP), electron transport rate (ETR), and non-photochemical quenching (NPQ) showed that photosynthesis was significantly favored by high CO₂ and the effect was also important for mitigating the salt impact for CO₂ uptake. Taken together, data show that elevated CO₂ has the potential to improve grapevine seedlings’ production system, mainly under salt stress.

Elevated CO2

combined factors

grapevine

salinity

Soil salinity is among the main abiotic stresses that affect and threaten the sustainability of crops in the world. Disturbances triggered by salt stress initially affect the plant’s hydraulic conductivity, followed by ionic toxicity and other cell-specific damages, as oxidative stress (Haider et al. 2019). These affect several key metabolic processes in cell, tissue, and organ levels related to water relations, nutrient balance and uptake, and photosynthesis, with negative impacts on plant yield (Brito et al. 2020). Thus, in addition to using the most salt-resistant crop species/varieties and soil correction practical, some salt-stress-mitigation treatments in plants also have been proposed. These include plant growth promoter factors like bacteria (Kumar et al. 2020), phytohormones (Dehnavi et al. 2022), and atmospheric carbon dioxide elevated [eCO₂] concentration (Brito et al. 2020; Melgar et al. 2008; Pérez-López et al. 2009).

The atmospheric [CO₂] is expected to reach levels of 420 ppm (RCP2.6) to 1300 ppm (RCP8.5) by the end of this century (IPCC, 2013), associated with a possible rise in mean global temperature of about 2 ^◦C by 2050 (IPCC, 2014). This heat wave associated with rising [CO₂] can potentially alter the water regime in some parts of the world, impacting agricultural activity. Several studies have already demonstrated the occurrence of interactive responses to the combined effects of elevated CO₂ and heat in crops different (Wang and Liu 2021; Abdelhakim et al. 2022; Thomey et al. 2019), including grapevine plants (Greer 2019; Morales et al. 2014; Arrizabalaga-Arriazu et al. 2020). On the other hand, elevated CO₂ (600–700 µmol mol^− 1) can stimulate the growth of C3 plants by 30%, and fruit trees are generally beneficial by the increase in photosynthesis, water use efficiency, growth, and biomass yield (Fischer et al. 2022).

Because stimulate photosynthesis and growth an increase in atmospheric [CO₂] could positively affect crop sustainability, mainly in areas with the occurrence of abiotic stresses such as drought and salinity. In fact, studies employed approaches different for the application of elevated CO₂ including Growth Chambers (GC), Free Air CO₂ Enrichment (FACE), and Open Top Chambers (OTC), have already demonstrated its beneficial effects on plant growth. For grapevine-specific studies, long-term FACE experiments (two growing seasons) under field conditions have demonstrated a significant increase in production (total and fruit biomass) in response to elevated CO₂ alone (Bindi et al. 2021). In this study, elevated [CO₂] also improved the grape quality for wine production by increasing sugars and organic acids contents. The increased growth of grapevine plants in response to high CO₂ also was demonstrated in greenhouse experiments under controlled conditions (Kizildeniz et al. 2015).

Increases in plant growth stimulated by elevated CO₂ have already been proposed and demonstrated for more than four decades for different species. However, the set of specific mechanisms in cell and tissue levels responsible for this phenomenon (high CO₂ growth stimulation) is still little known. In plant shoots, high CO₂ can stimulate both the induction of leaf primordia and leaves individual expansion, while that leaf lamina growth should be more due to increasing cell expansion rather than cell division (Taylor et al. 1994). In the shoot of grapevine plants is a common occurrence of primary and secondary buds. Primary buds are mainly responsible for bud fruitfulness and their fertility is associated with grape yield potential under field conditions (May 2000). Recently, a study performed by microscopic bud dissection in grapevine plants demonstrated that elevated CO₂ (applied by the FACE method) significantly improves bud fertility indicated by the higher number of inflorescence primordia, a response apparently genotype-dependent (Wohlfahrt et al. 2019).

Plants’ growth stimulation in response to elevated CO₂ has been attributed to improving photosynthesis and other beneficial biochemical and physiologic processes. High CO₂ pressure in leaf mesophyll can favor the net CO₂ assimilation through higher carboxylation efficiency, mitigating the generation of reactive species oxygen (ROS), a feature that should occur mainly in C3 plants. Moreover, eCO₂ also may lead to oxidative protection by lower photorespiration (Souza et al. 2019), increase water use efficiency (Hatfield and Dold 2019), and promote the contents of carbohydrates and organic solutes (Brito et al. 2020). Under salinity, elevated CO₂ stimulates photosynthesis associated with higher growth performance in tomato plants (Brito et al. 2020). On the other hand, the role of elevated CO₂ on protection relative to growth and metabolic disturbances in plants under abiotic stresses like salinity still needs to be better characterized, especially in young plants during the autotrophic establishment phase.

We have demonstrated that elevated CO₂ improves ionic homeostasis (K⁺/Na⁺ ratio) and photoprotection in cashew young plants subjected to salt stress, traits that are compatible with salt resistance (Souza et al. 2019). In olive young plants, elevated CO₂ can promote water use efficiency (WUE) and lower toxic saline ions uptake, but these responses unaffected direct growth under salinity (Melgar et al. 2008). For grapevine plants, have been demonstrated that elevated CO₂ increases drought resistance through both the best WUE and Rubisco carboxylation rates as well as lower cell respiration rates (Silva et al. 2017). However, to the best of our knowledge, studies that demonstrated high CO₂ attenuate effects on salt-induced metabolic disturbances in grapevine plants are still scarce. In this study, was tested the hypothesis that eCO₂ could improve the production of vine young seedlings under salinity leading to higher vigor and shortening the growth time through the favoring ionic homeostasis and higher oxidative protection.

Plant material and treatments

Grapevine (Vitis ssp) grafted seedlings were obtained from a commercial nursery, where were produced with scions of the ‘Nubia’ variety grafted onto the ‘IAC 572’ rootstock (101 − 14 MGT (Vitis Riparia × Vitis rupestris) × Vitis caribaea)). Seedlings were grown in a substrate composed of a mix of sand and vermiculite (v/v) in 5 L plastic pots. The substrate humidity was maintained near 100% of the holding capacity by irrigation with a quarter-strength nutrient solution (Hoagland and Arnon, 1950). This scion/rootstock combination was chosen for the study it is widely cultivated in the Brazilian semiarid and has a good ability to acclimatize to these edaphoclimatic conditions. The experiment was performed in a growth chamber (Fitotron SGC 120) under controlled conditions at a temperature of 28°C during the day and 22°C at night, relative humidity (RU) of 55% during the day and 65% at night, photosynthetic photons flux density (PPFD) of 350 µmol m^-2 s^-1, and 12 h photoperiod. This chamber is accoupled to a CO₂ container and for the treatments the gas (CO₂) concentration inside was adjusted for ambient (aCO₂ − 400 µmol mol^-1) or elevated (eCO₂ − 800 µmol mol^-1).

In these conditions, two-month-old seedlings with averages of 4–5 fully expanded leaves were divided into two groups and irrigated every 2 days with a nutrient solution without (absence) or added 80 mM NaCl (salinity). Thus, plants were subjected to effects isolate and combined of CO₂ and salinity as follows: control – (aCO₂ + 0 mM NaCl); salinity (aCO₂ + 80 mM NaCl); elevated CO₂ (eCO₂ + 0 mM NaCl) and elevated CO₂ and salinity combined (eCO₂ + 80 mM NaCl), during 20 days. After this period, plants were fully defoliated for the emission of new shoots (leaf buds, leaves, and stems) when grown under four treatments for more than 60 days. In this phase, the plant development was registered through photography at 7, 14, and 21 days, aiming to characterize the dynamics of emission of new shoots (leaf buds, leaves, and stems). The first visual record was made at 7 days since the control plants already had developed true leaves. The new leaves produced after defoliation were harvested and frozen in nitrogen liquid for biochemical analysis. The CO₂ treatments were applied using the same growth chamber aim minimized the inter-chamber effects (Alonso et al. 2009; Souza et al. 2019). The environmental gradient effects inside the growth chamber were also minimized by randomly repositioning the pots inside the chamber every five days.

Measurements of parameters of chlorophyll fluorescence

Measurements of chlorophyll fluorescence were carried out with a modulated photosynthesis yield analyzer (MINI-PAM II – Walz - Germany), by the saturation pulse method as described by Schreiber et al. (1994). This analyzer (Modulated fluorometer) automatically provides the parameters of maximum photochemical yield (F_V/F_M), electron transport rate (ETR) as well as photochemical (qP), and non-photochemical quenching (NPQ), which were the efficiency photochemical indicators here evaluated. All measurements were realized in leaves fully expanded, that were previously exposed to 30 min dark-adapted conditions. For the measurements, light intensity was increased (0–1800 µmol m^− 2 s^− 1) and the parameters (F_V/F_M; ETR; qP and NPQ) responses to increasing light were evaluated.

Measurements of sodium, chloride, and potassium contents

For the ion’s extraction samples of leaves, stems and roots were dried in an oven with forced air ventilation at 70°C for 3 days. Dry matter (DM) was ground in a mortar with a pestle and an aqueous extraction was performed by incubating ground samples (0.2 g) in test tubes with 25 ml of distilled water and boiled at 100°C for 1 h. Readings for Na⁺ and K⁺ were performed in a flame photometer and contents were estimated from a standard curve of KCl (for K⁺) and NaCl (for Na⁺), ranging from 0 at 1000 µmol L^− 1. The chloride (Cl⁻) content was measured by titration of 20 ml aqueous extract with a silver nitrate (AgNO₃) solution (28 mmol L^− 1) using 5% potassium chromate (K₂CrO₄) as an indicator of the end of the reaction. The ion’s contents were calculated and results were expressed as µmol g^− 1 dry matter (DM).

Measurements of water potential, water relative content, and stomatal conductance

Leaf water potential (Ψ_W) was determined at the end of the experiment in leaves from the middle third of the plants. For measurements, leaves were harvested at 4 am, and readings immediately were performed using the Scholander pressure chamber (Model 1505D-EXP - PMS Instrument Company), as described by Scholander et al. (1964). Membrane damage (MD) and relative water content (RWC) were determined by Ferreira-Silva et al. (2008). For RWC twenty leaf discs (10 mm) were collected and fresh weight (FW) was immediately determined. Then, these discs were incubated in deionized water for 6 h for determination of turgid weight (TW), and after drying at 70°C for 72 h the dry weight (DW) was accessed. The RWC was estimated as follows: [RWC = (FW – DW)/(TW – DW) x 100]. The measurements of stomatal conductance (g_S) were accessed between 10:00 and 11:00 hours using a portable leaf promoter model SC-1 (DECAGON DEVICE – USA). Fully expanded leaves and healthy were chosen from the plants' middle third mean for measurements.

Lipid peroxidation and contents of glutathione and ascorbate, and hydrogen peroxide

For extraction of reduced glutathione (GSH) and ascorbate (AsA), thiobarbituric acid-reactive substances (TBARS), and hydrogen peroxide, fresh leaf samples (0.2 g) were powered in a mortar in the presence of liquid nitrogen and extracted in a 5% (w/v) trichloroacetic acid (TCA) solution, followed by centrifugation at 10,000 g for 30 min at 4°C. Lipid peroxidation was based on the formation of thiobarbituric acid-reactive substances (TBARS) in accordance with Heath and Packer (1968). For the reaction, aliquots (0.5 mL) of the extract were added in 2.0 mL of the 20% TCA solution and 0.5% thiobarbituric acid (TBA) and heated in a water bath at 95°C in hermetically closed tubes for 1 h. The reaction was stopped in an ice bath and the readings absorbances were performed on a spectrophotometer at 532 and 600 nm. Liquid readings obtained by subtracting the valours (600 nm – 532 nm) were used for estimating the TBARS content, using an extinction coefficient of 155 mM^− 1 cm^− 1, and results were expressed as ηmol TBARS g^− 1 fresh weight (FW).

The AsA content was assayed according to Kampfenkel et al. (1995). The assay is based on the reduction of Fe³⁺ to Fe²⁺ by AsA and the spectrophotometric detection of Fe²⁺ complexed with 2,2′-bipyridyl (pink color). For reaction, 0.1 mL aliquots extract was added to a medium with 0.3 ml 200 mM potassium phosphate buffer (pH 7.4), 0.1 mL deionized water, 0.5 mL 1% (w/v) TCA, 0.4 mL 42% H₃PO₄, 0.4 mL 4% (w/v) 2,2′-bipyridyl and 0.2 mL 3% (w/v) FeCl₃. The mix was heated at 42°C in a water bath for 30 min and readings were then taken at 525 nm. The GSH content was measured by reaction with 5,5′-dithiobis (2-nitro-benzoic acid), DTNB, according to Griffith (1980). For reaction, 0.2 mL extract was added to a medium containing 2.6 mL 150 mM potassium phosphate buffer (pH 7.4) and 0.2 mL 6 mM DTNB prepared in 100 mM potassium phosphate buffer (pH 7.0). Test tubes were led to a water bath at 30°C for 10 min and absorbances were measured at 412 nm. Reduced GSH and AsA contents were calculated from the standard curve and results were expressed as µmol g^− 1 FW.

Hydrogen peroxide (H₂O₂) content was measured by direct reaction of H₂O₂ with iodide (I) according to Velikova et al. (2020). This methodology is based on potassium iodide (KI) oxidation by H₂O₂ in an acidic medium, as described by Junglee et al., (2014). For the reaction, 0.5 ml aliquots supernatant, obtained by extraction in 5% (w/v) TCA, was added to the reaction medium containing 0.5 mL 100 mM potassium phosphate buffer (pH 7.0) and 1 mL 1 M KI. Absorbances were measured at 390 nm and H₂O₂ contents were estimated from a standard curve and results were expressed as mmol H₂O₂ g^− 1 FW.

Protein extraction and enzymatic activities

For protein extraction lyophilized leaf samples (0.1 g) were powdered in the presence of liquid nitrogen in a mortar and pestle and extracted in 100 mM Tris–HCl buffer (pH 8.0), with 20% (v/v) glycerol, 0.1% (w/v) triton X100, and 3% (w/v) Polyethylene glycol − 6000. For ascorbate peroxidase (APX) was added 2 mM reduced ascorbate (AsA) to buffer extraction to avoid APX denaturation. The soluble protein content was measured by Bradford (1976), with bovine serum albumin (BSA) as standard.

Superoxide dismutase (SOD; EC: 1.15.1.1) activity was determined by adding leaf extract to a mixture containing 50 mM potassium phosphate buffer (pH 7.8), 0.1 mM EDTA, 13 mM L-methionine, 2 µM riboflavin, and 75 µM p-nitro blue tetrazolium chloride (NBT) in under darkness. The reaction was performed under illumination (30W fluorescent lamp) at 25°C for 6 min and readings absorbance was measured at 540 ηm (Giannopolitis and Ries 1977). One SOD activity unit (AU) was defined as the amount of enzyme required to inhibit NBT photoreduction by 50% (Beauchamp and Fridovich 1971), and SOD activity was expressed as AU g^− 1 DW min^− 1.

Ascorbate peroxidases (APX; EC: 1.11.1.11) activity was assayed after the reaction in the presence of 50 mM potassium phosphate buffer (pH 6.0) and 0.5 mM ascorbic acid and enzymatic extract. The reaction started when 0.1 mL of 30 mM H₂O₂ was added, and the decreasing absorbance at 290 ηm was monitored over 300 s with successive readings every 30 s (Nakano and Asada 1981). APX activity was estimated by utilizing the molar extinction coefficient of ascorbate (2.8 mM^− 1 cm^− 1) and expressed as µmol AsA g^− 1 DW min^− 1.

Catalase (EC: 1.11.1.6) activity was determined after the reaction of enzymatic extract in the presence of 50 mM potassium phosphate buffer (pH 7.0) containing 20 mM H₂O₂. The reaction took place at 30°C, monitoring the absorbance at 240 ηm over 300 s with readings every 30 s (Havir et al. 1987). CAT activity was calculated according to the molar extinction coefficient of H₂O₂ (36 M^− 1 cm^− 1) and was expressed as µmol H₂O₂ g^− 1 DW min^− 1.

Glycolate oxidase (GO; EC: 1.1.3.15) activity was assayed by the formation of the complex glyoxylate–phenylhydrazone read at 324 nm (Baker and Tolbert 1966). The GO assay mixture was a 100 mM phosphate buffer (pH 8.3), 100 mM L-cysteine, 40 mM glycolic acid, and 100 mM phenylhydrazine. The reaction started by adding FMN 1 mM. The GO activity was calculated by the molar extinction coefficient of the glyoxylate–phenylhydrazone complex (17 mM^− 1 cm^− 1) and expressed as µmol glycolate g^− 1 FW min^− 1.

Contents of carbohydrates and chlorophylls and carotenoids pigments

The soluble sugar (carbohydrates) content was measured by the phenol-sulfuric method (Dubois et al. 1956). For aqueous extraction, samples (0.2 g FW) of leaves, stems, and roots were incubated in 5 mL of deionized water in test tubes and boiled for 1 h. Then, 0.5 mL aliquots were added to the medium with 0.5 mL 5% (w/v) phenol and sulfuric acid, and absorbances were read at 490 nm. Soluble sugar content was estimated from a standard curve elaborated with aqueous solutions of D-glucose and the results were expressed in µmol g^− 1 DW.

The measurements of chlorophyll and carotenoid contents were performed according to Lichtenthaler and Buschmann (2001). Extraction was performed by incubation of leaf samples (0.2 g) in test tubes with 20 mL of 80% acetone for 48 hours. After extraction absorbances at 470, 664, and 648 nm were read and used for estimated the pigments content, as follows: Chl a = [13.36 x A664–5.19 x A648]; Chl b = [27.43 x A648–8.12 x A664]; Chl total = [Chl a + Chl b]; and Car = [(1000 x A470–2.13 x (Chl a) – 97.64 x (Chl b))/209], where “A” represents the absorbance at the respective wavelengths. Results were expressed as mg g^− 1 FW.

Stomatal image/density

Images were obtained from stomata impressions by pressing the abaxial (AB) sides of the leaf against cyanoacrylate (C₅H₅NO₂) drops on glass slides. Imaging of stomata was performed by an optical microscope (LX 500 type) coupled to a digital camera. The stomata density (De, est./mm²) was determined using the Image-Pro Plus 4.5.0.29 software. The readings were taken at a resolution of 150 µm with a 10x lens.

Statistical analysis

The experiment was performed in a completely randomized design in a factorial arrangement of 2 x 2; two (0 and 80 mM) NaCl levels, and two (400 and 800 µmol mol^− 1) CO₂ concentrations. All treatments were represented by four replicates and each consisting of a pot containing a plant. The data were submitted for analysis of variance, and the means were compared by the Tukey test at 5% probability (p < 0.05).

Changes in growth and physiological indicators of salt resistance in grapevine plants

In order to evaluate the phenotypic development of the seedlings exposed to the different treatments, they were registered through photographs until the 21st day after removing the leaves. This record made it possible to visualize the differences in shoots’ growth (phenotypic visual stages) in terms of the emission of new leaves and stems, and elongation (Fig. S1). Visual results show that up to 7 days, occurred the emission of true leaves (young) in the control plants. In plants subjected to eCO₂ and eCO₂ + NaCl combination were observed only buds, and in the salt-stress plants in aCO₂ the emission of the new shoot (leaves and stems) was fully impaired. The emission of new shoots was significantly higher at 14 and 21 days in the non-salinized plants under eCO₂, if compared to aCO₂ plants. The emission of the new shoot in the aCO₂ plants was severely impaired by salinity during the period of evaluation (21 days), but in the eCO₂ plants already was observed emission of new buds at 7 days, and this eCO₂ beneficial effect was maintained for 14, and 21 days under salt stress (Fig. S1).

The beneficial effects of eCO₂ on plants’ phenotype had also impact significative in the dry matter (DM) yield. The DM content of plants was severely reduced by salinity, an effect that was partially recovered by elevated CO₂. Under ambient CO₂, the DM content of leaves, stems, and roots in plants subjected to salt stress was reduced by 75%, 37%, and 55%, respectively, relative to control plants (Table 1). However, for plants grown under elevated CO₂ exposed to salinity, these reductions were only about 37%, 23%, and 45%, respectively, if compared to controls (Table 1). On the other hand, the leaf DM of plants grown under high CO₂ in salt stress absence showed an increase of 27%, while the stems and roots were unaltered, relative to control plants. Relative whole plant, the shoot/root ratio did not was affected by salinity but was significantly higher (~ 23%) in the plants grown under high CO₂ in the salt absence and presence.

Table 1

Changes of dry matter (DM) content of leaves, stems, and roots and shoot/root ratio of the grapevine plants grown under isolated and combined conditions of salinity and elevated CO₂.
[CO₂] (µmol mol^− 1)	NaCl (mM)	Leaf	Stem	Root	Shoot/Root
[CO₂] (µmol mol^− 1)	NaCl (mM)	g DM. plant^− 1
400	0	8.94 Ba	12.18 Aa	8.79 Aa	2.49 Ba
400	80	2.32 Bb	7.66 Ab	3.89 Ab	2.64 Ba
800	0	11.44 Aa	13.25 Aa	7.92 Aa	3.32 Aa
800	80	5.56 Ab	9.34 Ab	4.74 Ab	3.27 Aa
Values are the averages of four replicates. Similar lowercase letters between salt levels and uppercase letters between CO₂ concentrations do not differ according to Tukey’s test (p < 0.05).

Salinity and elevated CO₂ alone and/or combined affected different growth and some physiological indicators of salt resistance in the plants. The water potential (Ψ_W) was unaffected by high CO₂ but was reduced under salinity isolate and more intensively by high CO₂ + NaCl, relative to control plants (Fig. 1A). Leaf relative water content (RWC) was unaltered under the salinity alone or combined with high CO₂, but increased significantly in the plants exposed to elevated CO₂, if compared to controls (Fig. 1B). Stomatal conductance (g_S) was significantly reduced by elevated CO₂ and more severely under salinity isolate and combined to high CO₂ (similarly), relative to controls (Fig. 1C).

Contents of chloride, sodium, and potassium and K⁺/Na⁺ ratio

Ions contents were differently affected in plants by salt stress and elevated CO₂ as well as by the combination of these factors. Under salinity, the Cl⁻ content in leaves, stems and roots of plants grown in ambient CO₂ increased by 437%, 82%, and 67%, respectively, while under elevated CO₂ these increases were about 72%, 270%, and 98%, relative to controls (Table 2). In salt absence, plants grown under elevated CO₂ presented only a slight reduction in root Cl⁻ content, relative to controls. When grown under ambient CO₂, plants showed an increase of about 520%, 357%, and 208% of Na⁺ content for leaves, stems, and roots, respectively, compared to controls (Table 2). Moreover, for plants under high CO₂ more NaCl these increases were by 297%, 457%, and 310%, for leaves, stems, and roots, respectively, relative to controls (Table 2). These data showed that elevated CO₂ attenuated significantly the leaf Cl⁻ and Na⁺ accumulation of plants subjected to salinity.

Table 2

Contents of Cl⁻ and Na⁺ in tissues of leaves, stems, and roots in grapevine plants grown under isolated and combined conditions of salinity and elevated CO₂
[CO₂] (µmol mol^− 1)	NaCl (mM)	Leaf	Stem	Root	Leaf	Stem	Root
[CO₂] (µmol mol^− 1)	NaCl (mM)	(µmol Cl⁻ g^− 1 DM)			(µmol Na⁺ g^− 1 DM)
400	0	206 Ab	107Ab	228Ab	40Ab	75Ab	151Ab
400	80	1107 Aa	195Ba	382Ba	261Aa	343 Ba	463Ba
800	0	182 Ab	102Ab	168Bb	34Ab	81 Ab	92Bb
800	80	767 Ba	401Aa	455Aa	161Ba	425Aa	628Aa
Values are the averages of four replicates. Similar lowercase letters between salt levels and uppercase letters between CO₂ concentrations do not differ according to Tukey’s test (p < 0.05).

Leaf K⁺ content in the plants exposed to salinity grown in ambient CO₂ increased (~ 25%), but was severely reduced (~ 50%) in stems and roots, relative to control plants (Table 3). Under elevated CO₂ alone, K⁺ content was significantly higher in leaves, stems, and roots, relative to controls. Moreover, leaf K⁺ content in the plants exposed to the combination of NaCl + high CO₂ increased (~ 20%), but was reduced by 30% in stems and roots, relative to controls. Changes in Na⁺ and K⁺ contents in response to NaCl and CO₂ treatments affected significantly the K⁺/Na⁺ ratio in the plant (Table 3). The K⁺/Na⁺ ratio was severely reduced (> 80%) in leaves, stems, and roots by salinity isolate, but increased by about 40% and 97% in leaves and roots, respectively, in the plants subjected only to elevated CO₂. K⁺/Na⁺ ratio decreased by 71% in leaves and about 80% in stems and roots of the plants exposed to combined treatments (NaCl + high CO₂), if compared to controls.

Table 3

Contents of K⁺ and K⁺/Na⁺ ratio in tissues of leaves, stems, and roots in grapevine plants subjected to effects isolated and combined of salinity and elevated CO₂ grown under controlled conditions
[CO₂] (µmol mol^− 1)	NaCl (mM)	Leaf	Stem	Root	Leaf	Stem	Root
[CO₂] (µmol mol^− 1)	NaCl (mM)	(µmol K⁺ g^− 1 DM)			(K⁺/Na⁺ ratio)
400	0	826 Bb	712 Ba	632 Ba	21.43 Ba	9.44 Aa	4.14 Ba
400	80	1034 Aa	312 Bb	297 Bb	3.98 Bb	0.91 Ab	0.64 Ab
800	0	1018 Aa	795 Aa	722 Aa	29.92 Aa	9.95 Aa	8.07 Aa
800	80	986 Aa	488 Ab	405 Ab	6.18 Ab	1.15 Ab	0.65 Ab
Values are the averages of four replicates. Similar lowercase letters between salt levels and uppercase letters between CO₂ concentrations do not differ according to Tukey’s test (p < 0.05).

Changes in indicators of photochemical activity and damages and protection oxidative

A detailed analysis of in vivo photochemical activity through parameters quenching photochemical (qP), electron transport rate (ETR), and non-photochemical quenching (NPQ) in response to an increase in light intensity (PPFD) was performed. The qP was relatively lower in the plants under salt isolate in response to increase light intensity, if compared to controls (Fig. 2A). In the plants grown in elevated CO₂ alone, the qP was significantly higher, and for those subjected to combined treatments, it was unaltered in response to increasing PPFD, if compared to control plants. Similarly, the ETR shown increases significantly lowers in response to an increase of PPFD in plants under salinity isolate, but this parameter was relatively higher in plants grown in elevated CO₂ isolate and was maintained unaffected in the plants subjected to combination (elevated CO₂ + NaCl), relative to controls (Fig. 2B). On the other hand, the NPQ was increased in the plants exposed to salinity, but was relatively lower in plants subjected to combination (high CO₂ + NaCl) and severely reduced in the plants grown under high CO₂ alone, when compared to control plants (Fig. 2C). The plants did not present apparent photoinhibition, as was indicated by Fv/Fm ratio (Fig. 2C)

Stress-induced photochemical changes can lead to metabolic disturbances associated with cell oxidative damage. Data showed that the leaf lipid peroxidation (an indicator of membrane oxidative damage) was unaltered in response isolate effects of salinity and elevated CO₂, but was lower under the combination of NaCl + high CO₂ (Fig. 3A). Leaf hydrogen peroxide (H₂O₂) content showed a slight increase (~ 10%) under elevated CO₂ alone but was significantly reduced under salinity and by the combination of salt + high CO₂ (Fig. 3B). Analysis of the two main non-enzymatic antioxidants displayed that reduced glutathione (GSH) content increased under salinity but was significantly reduced by the high CO₂ in the absence and presence of salt (Fig. 3C). Moreover, plants showed a slight increase of reduced ascorbate (Asc) content under salinity isolate and this was even greater in the combination of salt + high CO₂ (Fig. 3D).

Relative to enzymatic antioxidants, superoxide dismutase (SOD) activity presented a slight reduction similarly under salinity isolate and high CO₂ in the absence and presence of salt stress, if compared to control plants (Fig. 4A). The catalase (CAT) activity was reduced by salinity and maintained unaltered in response to elevated CO₂ and the combination of high CO₂ + salt, relative to controls (Fig. 4B). Moreover, the ascorbate peroxidase (APX) activity was significantly increased only in plants subjected to combined factors, if compared to controls (Fig. 4C). The glycolate oxidase (GO) activity was significantly reduced in plants treated with high CO₂ combined with salt, relative to control plants (Fig. 4D).

Stomatal pattern and contents of carbohydrates and photosynthetic pigments

An analysis of abaxial stomatal density revealed that leaf anatomy was influenced by elevated CO₂ in the absence and presence of salinity. Under ambient CO₂ concentration, the plants displayed a stomatal density mean of about 65 mm^− 2 in the absence and presence of salinity (Fig. 5A and 5B). On the other hand, stomatal density was reduced by 65% and 44% in response to the high CO₂ alone and combined with salt stress, respectively, if compared to control plants (Fig. 5C and 5D). A study of carbohydrate partitioning in whole plants showed an effect predominant of high CO₂ relative to salinity. Salinity alone did not affect the carbohydrate content in leaves, stems, and roots, if compared to controls (Table 4). However, under high CO₂ in the absence and presence of salt, carbohydrates contents in leaves and roots increased by about 51% and 59%, respectively, while that was reduced by 15% in stems, relative to control plants.

Table 4

Carbohydrates content in leaves, stems, and roots of grapevine plants grown under isolated and combined conditions of salinity and elevated CO₂
[CO₂] (µmol mol^− 1)	NaCl (mM)	Leaf	Stem	Root
[CO₂] (µmol mol^− 1)	NaCl (mM)	(µmol g^− 1 DM)
400	0	516.28 Ba	226.06 Aa	140.52 Ba
400	80	508.82 Ba	207.92 Aa	128.36 Ba
800	0	777.57 Aa	187.34 Ba	226.52 Aa
800	80	745.13 Aa	175.00 Ba	201.25 Aa
Values are the averages of four replicates. Similar lowercase letters between salt levels and uppercase letters between CO₂ concentrations do not differ according to Tukey’s test (p < 0.05).

Data shows that the salinity and high CO₂ affected different content of photosynthetic pigments chlorophylls (chl) and carotenoids (car). Under ambient CO₂, plants presented significant increases of the chlorophyll fractions a and b (by 62%) as well as total chlorophyll (by 95%) content, relative to controls (Table 5). Under elevated CO₂, chl a content increased by about 95% and 75% in the absence and presence of salt, respectively, while that chl b was similarly reduced (by 31%) independent salinity, relative to control. Moreover, chl b content increased by 72% and 45% in response to high CO₂ isolate and combined with NaCl, respectively, if compared to the control. The chl a/chl b ratio did not was affected by salt in the plants grown in ambient CO₂, but was similarly increased by 171% in response to elevated CO₂ in the absence and presence of salt, if compared to respective controls (Table 5). Leaf carotenoid content was significantly lower only in the plants subjected to the combination of elevated CO₂ + salinity (Table 5).

Table 5

Contents of photosynthetic pigments (chlorophylls and carotenoids) and Chl a/Chl b ratio in leaf tissues of grapevine plants grown under isolated and combined conditions of salinity and elevated CO₂
[CO₂] (µmol mol^− 1)	NaCl (mM)	Chl a	Chl b	Chl total	Car	Chl a/Chl b
[CO₂] (µmol mol^− 1)	NaCl (mM)	(mg g^− 1 FW)				Chl a/Chl b
400	0	0.328Bb	0.217Ab	0.474Bb	0.163Aa	1.451 Ba
400	80	0.528Aa	0.408Aa	0.937Aa	0.183Aa	1.301 Ba
800	0	0.646Aa	0.170Ba	0.816Aa	0.154Aa	3.903 Aa
800	80	0.567Ab	0.140Ba	0.695Ba	0.109Bb	4.208 Aa
Values are the averages of four replicates. Similar lowercase letters between salt levels and uppercase letters between CO₂ concentrations do not differ according to Tukey’s test (p < 0.05).

In this study, the grapevine young plants were defoliated to allow the emission of new shoots (buds, leaves, and stems) under ambient and elevated CO₂ concentrations combined with the absence and presence of salinity. This approach was employed aim to maximize the effect of elevated CO₂ in leaf morphophysiological features effectively related to acclimation to CO₂ under salinity. Relative to growth, our results show that elevated CO₂ significantly improved the leaf growth (dry matter content) of plants in both the absence and presence of salinity. This beneficial effect attributed to elevated CO₂ led to an expressive increase in the shoot/root ratio, favoring the production of photosynthetic tissues. Moreover, shoots growth stimulates by elevated CO₂ occurred associated with the major precocity emission news buds and leaves, in both salinized and non-salinized conditions. Thus, elevated CO₂ apparently has the potential to shorten the production time of grapevine seedlings and could be considered as a tool technology to improve the production system of this specie, mainly under salt stress.

Plants’ growth increase under elevated CO₂ concentrations is associated with significant increases in net carbon assimilation and higher control of cell processes relative to the energy and carbon losses, like photorespiration that occurs mainly in C3 species. In grapevine plants already have been demonstrated that high CO₂ concentrations can stimulate growth and yield under field conditions, with beneficial effects including for plants subjected to drought stress (Bindi et al. 2021; Kizildeniz et al. 2015). In grapevine plants, elevated CO₂ delayed drought effects on net photosynthesis and Rubisco carboxylase activity as well as reduced the Rubisco oxygenation activity, mitigating deleterious effects of water stress (Silva et al. 2017). Overall, the protection conferred by high CO₂ should be able to suppress or mitigate stress-induced disturbances. In the present study, data showed that the elevated CO₂ triggered some cell and tissue responses in the plants under salinity that are compatible with salt resistance.

Relative to the ionic protection, plants exposed to salinity grown under eCO₂ presented a lower leaf Na⁺ and Cl⁻ accumulation associated with higher content of these ions in stems and roots, if compared with plants subjected to salinity, indicating lower ionic toxicity conferred by eCO₂ under salt stress. The K⁺ content in stems and roots was significantly reduced under salinity, both this salt-stress effect was partially recovered by elevated CO₂. Moreover, the leaf K⁺/Na⁺ ratio between salinized plants was significantly favored by eCO₂. This result suggests that eCO₂ may partially change the pattern of toxic ion allocation in the grapevine under salinity, favoring ionic homeostasis and lower shoot toxicity. This lower Na⁺ and Cl⁻ content in the shoot grapevine plants may have been due to the higher stomatal control exercised by eCO₂. In fact, plants’ hydraulic conductance is strictly associated with ionic flux from the root system for the shoot under both saline and non-saline conditions.

Our data reveal that leaf water potential (Ψ_W) was reduced by salinity and more severely by salt combined with eCO₂, while that stomatal conductance (g_S) was reduced by eCO₂ isolated and more intensively by salinity under two CO₂ levels. This g_S lower under high CO₂ alone occurred associated with higher relative water content (RWC) in leaves, suggesting a high CO₂-induced stomatal control for avoiding loss of water. The stomatal closing of plants is a recurring response to elevated CO₂ concentration. This change can improve water use efficiency (WUE) since the maintenance of CO₂ uptake occurs associated with lower loss of water by transpiration (Hatfield and Dold, 2019). Under the absence of salt stress, data show that grapevine plants grown in eCO₂ presented the best photochemical efficiency for CO₂ assimilation, indicated by higher valours of photochemical quenching (qP) and electron transport rate (ETR) associated with lower energy dissipation by non-photochemical quenching (NPQ).

Under salinity, eCO₂ did not increase RWC, but results show that had an apparent effect on stimulating photochemical activity in this condition. The qP and ETR parameters were relatively higher for plants subjected to salt stress combined with eCO₂ if compared to salinized plants alone. These photochemical parameters (qP and ETR) are indicators of flux electrons for the synthesis of energy (ATP and NADPH) during the photophosphorylation reactions in chloroplasts (Khatri and Rathore 2019). Thus, results suggest that eCO₂ potentially increased WUE (best CO₂ uptake under stomatal conductance reduced) in both the non-salinized and salinized conditions. Moreover, data on stomatal density revealed that stomata number was unaltered by salinity isolated, but was significantly reduced by eCO₂ in two salt levels. These data show that g_S reduction under eCO₂ should be had occurred by both closing and lower stomata number, while under salinity this reduction was mainly due to stomatal closing.

Salinity-induced photochemical disturbances in plants may lead to cell oxidative damage (Amorin et al. 2023; Raja et al. 2022). However, data show that the oxidative damages indicators here evaluated were unaffected. The TBARS content (an indicator of lipid peroxidation) was unaltered by salinity and eCO₂ and even presented a slight reduction under combinations of Salt + eCO₂, while the H₂O₂ content was reduced by two treatments. These results suggest an apparent absence of cell oxidative damage severe. But is a need to highlight the effect potential of eCO₂ in mitigating the membrane lipid peroxidation in plants under salinity. In fact, the absence of damages severe here observed could be attributed to the induction of non-enzymatic and enzymatic protection systems, mainly in response to eCO₂.

Plants subjected to a combination of eCO₂ and NaCl presented an increase in reduced ascorbate (AsA) content, that occurred associated with significant increases in the activities of ascorbate peroxidase (APX) and catalase (CAT) enzymes if compared to salinized plants. Moreover, glycolate oxidase (GO) activity was reduced by eCO₂ in the presence of salt stress. The AsA is a key non-enzymatic antioxidant for oxidative protection in plants subjected to salt stress (Raja et al. 2022). This antioxidant act as an electron-specific donor for APX enzymes and by direct reaction with some reactive species of oxygen (Hasanuzzaman et al. 2019). The APX and CAT enzymes are the main peroxidases involved in plant cell oxidative protection and are present in different cell sitios related to ROS generation, like cytosol, chloroplast, and mitochondria (Moradbeygi et al. 2020).

Another physiological factor apparently conferred by eCO₂ that may have contributed to oxidative protection and the possible increase in photosynthetic efficiency was a significant reduction of GO activity. This enzyme is considered a biochemical marker of photorespiratory activity in C3 plants (Souza et al. 2019). Thus, data suggest that eCO₂ could be an attenuating factor of this process in grapevine plants, mainly under salinity. In the photorespiratory cycle, the GO enzyme is in peroxisomes and acts in the reaction of glyoxylate for glycolate, with equimolar production of hydrogen peroxide (H₂O₂) molecules (Dellero et al. 2016). This H₂O₂ generated in the photorespiratory process may lead to cell damage and is scavenged by catalase-peroxidase into plant cell peroxisomes, avoiding excessive H₂O₂ accumulation (Hasanuzzaman et al. 2019). Here the CAT activity in the salt-stressed plants under eCO₂ was compatible with oxidative protection. This cell and tissue protection conferred by eCO₂ also should have contributed for improve other physiological attributes related to salt resistance.

Our data show that plants exposed to eCO₂ presented higher carbohydrate contents in leaves and roots in both the absence and presence of salt. Increases in carbohydrate levels favor salt resistance mechanisms since these organic solutes are involved with the osmotic adjustment and may act as energy and carbon skeletons sources necessary for the growth and synthesis of biomolecules (Li et al. 2020). Moreover, higher leaf and root carbohydrate levels may contribute to the best growth of grapevine plants, a response that was here observed mainly for leaves in the plants under salinity. In addition, high CO₂ favored the synthesis of chlorophyll a and total in salt stress absence, pigments that can help in the stability of the photochemical apparatus and carbon assimilation process.

Our findings reveal results effectively related to protection mechanisms conferred by elevated CO₂ in grapevine plants growing under salinity. Taken together, data show that eCO₂ promotes a lower leaf Na⁺ and Cl⁻ accumulation along with the best K⁺ nutrition and ionic homeostasis (K⁺/Na⁺ ratio) under salt stress. Elevated CO₂ favored indicators of photochemical efficiency qP and ETR and partially recovered the salt effects on these parameters, suggesting the potential to mitigate salinity damages in the photosynthesis of grapevine young plants. High CO₂ also attenuated photorespiration and triggered non-enzymatic and enzymatic antioxidants, achieving better oxidative protection under salinity. Treatment with eCO₂ stimulates the shoot’s growth and precocity emission of buds and leaves. Among future perspectives, eCO₂ has the potential to shorten the production time of grapevine seedlings and could be considered as a technology able to improve the production system of this specie, mainly under salt stress.

Funding/Acknowledgements

The authors are grateful to Coordination for the Improvement of Higher Education Personnel (CAPES), National Council for Scientific and Technological Development (CNPq - Processes —423583/2018-2; 311429/2018-1 and 435508/2018-0) - and Foundation for the Support of Science and Technology of the State of Pernambuco (FACEPE – Proc. 88887.136369/2017-00) for financial support. HRBS is supported by FACEPE/CAPES (Proc 0300-5.01/19).

Author contribution

PJH: Investigation, Conceptualization, Formal Analysis. HRBS: Supervision, Writing - Review & Editing. JBN: Investigation, Formal analysis. TGFS: Investigation, Formal analysis. ANS: Writing - Review & Editing. ES: Writing - Review & Editing, Funding acquisition. SLFS: Conceptualization, Methodology, Funding acquisition, Project administration.

Conflicts of interest/Competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Availability of data and material

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Supplementaryfigure1.tif
Figure Supplementary 1 Visual aspects with the highlight for emission of new shoots (buds, stems, and leaves) of grapevine plants at 7, 14, and 21 days grown in chamber growth under controlled conditions subjected to effects of isolated and combined salinity and elevated CO₂.

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Elevated CO₂ improves the growth of grapevine young plants under salinity favoring ionic homeostasis and decreasing oxidative damages

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Abstract

Figures

Introduction

Materials and methods

Plant material and treatments

Measurements of parameters of chlorophyll fluorescence

Measurements of sodium, chloride, and potassium contents

Measurements of water potential, water relative content, and stomatal conductance

Lipid peroxidation and contents of glutathione and ascorbate, and hydrogen peroxide

Protein extraction and enzymatic activities

Contents of carbohydrates and chlorophylls and carotenoids pigments

Stomatal image/density

Statistical analysis

Results

Changes in growth and physiological indicators of salt resistance in grapevine plants

Contents of chloride, sodium, and potassium and K⁺/Na⁺ ratio

Changes in indicators of photochemical activity and damages and protection oxidative

Stomatal pattern and contents of carbohydrates and photosynthetic pigments

Discussion

Conclusion

Declarations

References

Supplementary Files

Status:

Version 1

Elevated CO2 improves the growth of grapevine young plants under salinity favoring ionic homeostasis and decreasing oxidative damages

Status:

Version 1

Abstract

Figures

Introduction

Materials and methods

Plant material and treatments

Measurements of parameters of chlorophyll fluorescence

Measurements of sodium, chloride, and potassium contents

Measurements of water potential, water relative content, and stomatal conductance

Lipid peroxidation and contents of glutathione and ascorbate, and hydrogen peroxide

Protein extraction and enzymatic activities

Contents of carbohydrates and chlorophylls and carotenoids pigments

Stomatal image/density

Statistical analysis

Results

Changes in growth and physiological indicators of salt resistance in grapevine plants

Contents of chloride, sodium, and potassium and K+/Na+ ratio

Changes in indicators of photochemical activity and damages and protection oxidative

Stomatal pattern and contents of carbohydrates and photosynthetic pigments

Discussion

Conclusion

Declarations

References

Supplementary Files

Status:

Version 1

Elevated CO₂ improves the growth of grapevine young plants under salinity favoring ionic homeostasis and decreasing oxidative damages

Contents of chloride, sodium, and potassium and K⁺/Na⁺ ratio