Photosynthesis and water relations of sunflower cultivars under salinity conditions

The study of photosynthetic responses in plants provides useful information for understanding the physiological processes involved in the salinity tolerance and susceptibility mechanisms of sunflower cultivars. The aim of the present study was to evaluate the gas exchange responses, water relations (stress hydric), and growth characteristics of two sunflower cultivars, Agrobel 963 and Aguará 4, subjected to different salt concentrations. The study was conducted in a randomized block experimental design through a 2 x 5 factorial arrangement, with the factors being two sunflower cultivars (Agrobel 963 and Aguará 4) and five salt concentrations (0, 25, 50, 100 and 150 mM NaCl) applied in Hoagland nutrient solution, with five replicates. Gas exchange, water relations and growth characteristics were evaluated. The gas exchange measurements showed that the two sunflower cultivars maintained the photosynthetic activity per unit of leaf area even at the highest NaCl concentration tested (150 mM). With the increasing salinity in the nutrient solution, the leaf water potential decreased, while the concentration of optically active substances increased in both the leaves and roots, which helped maintain the plant’s water status. Reduction on dry mass of sunflowers was response to decreasing on the leaf total area and not as effect of salinity on the photosynthetic rate by leaf area unity.


INTRODUCTION
Agriculture in the midwest region of Brazil is mainly dedicated to grain production.Over the last few years, however, large-scale sugarcane plantations have been established in the agricultural areas of this region.This increase is mostly a consequence of incentives for the production of alcohol as biofuel by the Alcohol National Program (Programa Nacional do Álcool -Proálcool) (Rodrigues and Ortiz, 2006).
An increase in the production of vinasse, which is a byproduct of alcohol production, occurs concomitantly with the increase in sugarcane production.Vinasse is currently being used in irrigation as a fertilizer.However, if applied in excess, vinasse can cause damage to crops of commercial interest due to nutritional imbalances and *Corresponding author.E-mail: alcarcos@pq.cnpq.br.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License base saturation, leading to soil salinization (Lelis, 2008).The degree of salinization, however, depends on the type and physicochemical characteristics of the soil.
Salinity is one of the abiotic stresses that induce changes and responses at all functional levels of the organism.These changes can be reversible in early stages or become irreversible under extreme conditions (Larcher, 2004).Excess salt can damage plants by causing osmotic stress, which leads to difficulties in water absorption, or because of ionic effects associated with specific ion toxicities (Dias and Blanco, 2010).Salinity leads to osmotic stress, which disrupts water relations and reduces growth, leaf area and dry matter production (Hussain et al., 2012).The ionic effects, which are mostly promoted by Na+, are a consequence of the accumulation of ions in the plant tissues and cause nutritional imbalances, toxicity and metabolic changes (Munns and Tester, 2008;Silva et al., 2009).
Photosynthetic rates are also affected by salinity and may occur in response to stomatal closure, which is mediated by hormones, or by photochemical changes and changes in carbon metabolism (Chaves et al., 2009).Increasing, salinity results in plant growth changes with the leaf area being the most affected parameter.According to Steduto et al. (2000), leaf area modulation is the most important mechanism of stress avoidance in sunflower plants.This response suggests a strategy to reduce transpiration and, consequently, water loss by the plant.Morphological, anatomical and metabolic changes in sunflower plants depend on the genotype and salt content of the plant (Silva et al., 2009).
However, some plants have the capacity to prevent the entry of salts or to minimize their concentration in the cytoplasm via vacuolar compartmentalization, thus avoiding the toxic effects of salts on photosynthesis and other metabolic processes (Chaves et al., 2009).Different tolerance mechanisms, however, can occur in different species or in different cultivars of the same species.
The sunflower (Helianthus annuus L.) is a species originating from the southwestern USA and north of Mexico (Rossi, 1998).This crop has a high oleaginous potential and great economic and agricultural relevance.The sunflower is highly resistant to drought, cold and heat, and it is adaptable to soil-climate conditions (Gomes et al., 2006).However, the sunflower's tolerance or sensitivity to salinity varies with the cultivar.
Thus, understanding the physiological and morphological processes involved in the mechanisms of tolerance and susceptibility to salinity, such as gas exchange, water relations and growth, of sunflower plants is essential to elucidate the salt tolerance mechanisms and their use in saline soils.Therefore, the aim of the present study was to evaluate the gas exchange responses, water relations and growth characteristics of two sunflower cultivars, Agrobel 963 and Aguará 4, subjected to different salt concentrations.

MATERIALS AND METHODS
The experiment was conducted in a greenhouse at the Ecophysiology and Plant Productivity Laboratory of the Federal Institute of Goiás (Instituto Federal Goiano -IF Goiano) on the Rio Verde Campus (17°48'07"S, 50°54'20"W, 755-m altitude).

Generation of seedlings and plant acclimation
The sunflower seeds were germinated in Bioplant ® commercial substrate in expanded polystyrene trays.Fifteen days following germination, healthy and uniform seedlings were selected and transferred to trays containing Hoagland and Arnon (1950) nutrient solution.After six days of plant acclimation, the plants were transferred into Styrofoam boxes, with two plants per box, containing 2.5 L of full-strength Hoagland and Arnon (1950) nutrient solution, which was aerated every 15 min.

Treatment application and experimental conditions
The salinity levels were gradually increased by adding 25 mM NaCl to the nutrient solution every 24 h until 25, 50, 100 and 150 mM NaCl solution concentrations were reached.The electrical conductivity (EC) was monitored every 48 h with a conductivity meter, model Brazil), and maintained at 25% of the initial EC.The pH was maintained at approximately 6.0±0.05 and monitored with a pH meter, model 221 (Lutron, Taiwan).

Plant evaluation
Five gas exchange measurements were performed on fully expanded sunflower leaves.The water potential (ψw), refractive index (RI), and growth characteristics, were measured at the end of the experiment, which was 26 days after the beginning of the treatment application.

Gas exchange
The gas exchange measurements were always performed between 07:00 and 11:00 am.The photosynthetic rate (A, µmol m -2 s -1 ), respiration rate (E, mmol m -2 s -1 ), stomatal conductance (gs, mol H2O m -2 s -1 ) and the relationship between the internal and external CO2 concentrations (Ci/Ca) were measured.The measurements were performed using a portable photosynthesis system with an LCi Light Systems light source (ADC Bioscientific, Herts, England), consisting of a ventilated chamber containing a 20-W dichroic halogen lamp with 1,000-µmol m -2 s -1 photon flux density.

Water relations
Fully expanded leaves that were inserted at the third node from the shoot tip were collected before dawn and used for the determination of Water potential (Ψw, MPa) using a Scholander pressure chamber.The Refractive index (RI) was measured with a manual refractometer (Abbe Atago, Japan) in leaf-and root-cell sap, which was extracted with a hand press.

Growth characteristics
The number of leaves and nodes, leaf area (LA), aerial part dry matter (APDM), root dry matter (RDM), total dry matter (TDM), specific leaf area (SLA) and leaf area ratio (LAR) were measured.The dry matter mass (g plant‫)¹-‬ was obtained by drying the harvested material in a convection oven at 65°C until reaching a constant weight.The LA (cm 2 ) was measured by scanning the leaf and using image-analysis software.The SLA and LAR were calculated using the following equations: SLA= LA/LDM and LAR= LA/TDM, respectively.

Statistical analysis
An analysis of variance was performed for the data obtained, and the means for different cultivars were compared using an F-test.When necessary, regression models were adjusted to the NaCl concentration levels using the Software Analysis and Experimentation Group (SAEG) 9.1 software (UFV, Viçosa, Brazil).

Gas exchange
The increasing NaCl concentration did not result in significant changes in A (Figure 1A) or g s (Figure 1B) in either of the tested cultivars.However, a higher A (Figure 1A) and E (Figure 1C) and lower water-use efficiency (WUE) (Figure 1D) were observed for cultivar Agrobel 963 compared with Aguará 4. The Ci/Ca also did not change significantly with increasing salinity in the growth medium (Figure 2).A significant difference was only observed between cultivars, with Aguará 4 exhibiting the highest values for this ratio (Figure 2).

Water relations
The ψ w of both Aguará 4 and Agrobel 963 decreased with increasing salinity in the growth medium, especially at the highest NaCl concentration.This decrease reached up to 48% for Aguará 4 and up to 20% for Agrobel 963 compared with control plants (Figure 3).The RI of both the leaf and root increased with increasing NaCl concentrations (Figure 4A and B).There were increases of 102% in the leaf (Figure 4A) and 34% in the root (Figure 4B) in plants grown at 150-mM NaCl

Growth characteristics
The interaction between the NaCl concentration and the type of sunflower cultivar influenced the growth characteristics.
Increasing the NaCl concentration of the nutrient solution significantly reduced the number of leaves (Figure 5A) and nodes (Figure 5B), regardless of the cultivar.When evaluating the isolated effect of the cultivar on this characteristic, the reduction was more pronounced for the cultivar Agrobel 963 (Figure 6A and  B).The SLA (Figure 7A), LAR (Figure 7B), LA (Figure 8A) and RDM (Figure 8C) of the plants from both cultivars drastically decreased with increasing NaCl concentrations in the nutrient solution.Similar results were observed for the APDM (Figure 8B) and TDM (Figure 8D) of the plants from the Agrobel 963 cultivar.However, low NaCl concentrations (≤0.25 mM) led to significant increases in the APDM (Figure 8B) and TDM (Figure 8D) of the plants from the Aguará 4 cultivar.

DISCUSSION
The growth characteristics measured in the present study, namely the LA, SLA, LAR, APDM, TDM and RDM of the plants, for both tested cultivars decreased with increasing NaCl concentrations in the nutrient solution.Decreased in growth is one of the most commonly observed symptoms in plants grown in saline environments and has been well documented for Barbados nut (Silva et al., 2009) and some sunflower cultivars (Steduto et al., 2000;Hussain et al., 2012).The changes in plant growth may occur as a result of the external increase in osmotic pressure and the accumulation of Na + in the leaves (Munns and Tester, 2008).
In the present study, the decreased LA may be related to an adaptation mechanism to salinity, allowing water conservation by reducing transpiration area.The decrease in leaf area has been observed in several plant species in response to increasing NaCl concentrations (Medeiros, et al., 2012;Araújo, et al., 2010;Neto and Tabosa, 2000).The lowest RDM values were observed for estimated concentrations of 106.7 and 100 mM NaCl in the nutrient solution for the Aguará 4 and Agrobel 963 cultivars, respectively.For the Aguará 4 cultivar, a similar behavior was observed for the APDM and TDM, which increased with the NaCl content up to concentrations of 22 and 13 mM, respectively.Júnior et al. ( 2011 According to Nobre et al. (2010), salinity affects the plant dry matter production because the high salt concentrations at the root zone lead to decreased water availability.The growth decreases may therefore be associated with osmotic stress, resulting from an increase in solutes in the solution that hampers water absorption by the plant.The increase in solutes also led to a decreased leaf ψ w and increased RI of leaf-and rootcell sap.As the water content decreased, the turgor     pressure also decreased, resulting in a decrease in the number of elongating cells and, consequently, growth (Willadino and Camara, 2010).
Thus, the results obtained indicate that the tested sunflower cultivars, Aguará 4 and Agrobel 963, underwent an osmotic adjustment, accumulating compatible solutes in the interior of the cells due to the increased RI and consequent reduction in ψ w .This effect helped the maintenance of the ψ w gradient between the medium and the interior of the plant (Taiz and Zeiger, 2012).Increases in RI were also observed by Queiroz and Büll (2001) in cotton plants up to 24 dSm -1 soil salinity.
The increase in NaCl concentration in the present study led to a reduction in the LAR, indicating that most of the photoassimilates were not translocated to the leaf area.Similar results were reported by Garcia et al. (2010) for bean genotypes under increasing salt concentrations.Silva et al. (2009) reported an increase in the LAR only at the early stages of ornamental sunflower cultivation, after which there was a decrease in the LAR.
The lowest observed SLA values for both cultivars tested in the present study indicate a larger investment in leaf thickness than in leaf expansion.Similar results were observed by Silva et al. (2009) for leaves of ornamental sunflower plants subjected to 6.5 dSm -1 EC during fertigation and by Porto et al. (2006) for melon plants grown at 4.5 dSm -1 EC.The decrease in SLA observed in the present study resulted from the more pronounced effect on LA than on LDM, which is in accordance with Porto et al. (2006).Therefore, the growth of both cultivars was negatively affected by salinity.The reduction in leaf area decreased transpiration surface, photosynthetic area and dry matter production compared with the control plants.The higher growth of the control plants of both cultivars was likely due to higher turgor pressure compared to the plants subjected to high NaCl concentrations.
Those results are also confirmed by the fact that the RI of both the leaf and root increased with increasing NaCl concentrations, thus indicating the presence of optically active substances, such as glucose and fructose.The determination of the concentration of these substances is usually performed through the RI, which is based on how a given substance deflects polarized light (Bagatin et al., 2005).The presence of optically active substances contributes to osmotic adjustment, to the maintenance of cell hydration and, consequently, to the protection of cell structures (Ashraf andHarris, 2004, Marcondes andGarcia, 2009).
Changes to the metabolism, which are induced by excess ions, are consequence of several plant physiological responses, such as changes in ion balance, stomatal changes or changes to the photosynthetic capacity (Zanandrea et al., 2006).Although there was no alterations on photosynthetic rate by leaf area, reduction on total leaf area of sunflower plants as response to salinity was observed.As direct consequence, the small leaf area resulted to a small area water loss by transpiration, small area loss of carbon absorption on photosynthetic process, low values of dry mater mass and biometric measures of plants as observed in this study.
The increase in NaCl concentrations did not result in stomatal closure, as indicated by the g s , which indicates that both studied cultivars adopted metabolic strategies to minimize the effect of salt.Among these possible strategies, osmotic adjustment with synthesis of osmotically is compatible with organic solutes and ion compartmentalization at the vacuole or ion storage in older leaves (Silveira et al., 2009).Steduto et al. (2000) found no changes in A or g s in sunflower hybrids under salinity conditions.The authors considered that CO 2 assimilation in sunflower plants is controlled by leaf area modulation, that is, under saline conditions, sunflower plants adjust morphologically as opposed to physiologically.Conversely, the reduced LA led to a reduction in the total photosynthetic area, which negatively affected C accumulation, even though no decrease in the photosynthetic rate per leaf area was observed.
In addition to the reductions in leaf area and transpiration area, other mechanisms may have been used to avoid water loss and maintain the functionality of physiological activities.One of these mechanisms was the increase in WUE, as observed in the Aguará 4 cultivar for concentrations of up to 100 mM NaCl.The Aguará 4 cultivar exhibited better values than the Agrobel 963 cultivar for this parameter.In the Agrobel 963 cultivar, increasing the NaCl concentration led to decreased WUE and increased E at 50 mM NaCl, as compared with the control plants.By maintaining turgor, processes such as stomatal conductance, CO 2 assimilation rates and tissue expansion are also maintained for longer periods under stress conditions (Ludlow, 1987;Nepomuceno et al., 1998;Nepomuceno et al., 2001).In young Barbados nut plants, after seven days of treatment with 100 mM NaCl, significant decreases were observed in g s and E (Silva et al., 2011).Salt ions reached high concentrations in leaves after 14 days, leading to pronounced photosynthetic damage (Silva et al., 2011).

Conclusion
The photosynthetic rates and the remaining gas exchange parameters remained constant in both the Aguará 4 and Agrobel 963 cultivars.The greater capacity of the Aguará 4 cultivar to reduce E and increase WUE indicates a higher tolerance of this cultivar to salt stress conditions compared to the Agrobel 963 cultivar.The increase in concentrations of optically active substances in Aguará 4 and Agrobel 963 was associated with the greater osmotic adjustment capacity of these plants, favoring water absorption and the maintenance of the functionality of physiological activity.The decrease in dry matter accumulation in both cultivars was a result of the decrease in leaf area of the plants subjected to salt stress conditions and not due to a reduction in the photosynthetic activity by leaf area unity.

Figure 1 .
Figure 1.Photosynthetic rate (A) (A), stomatal conductance (gs) (B), transpiration rate (E) (C) and water-use efficiency (WUE) (D) of sunflower plants of the Aguará 4 and Agrobel 963 cultivars grown under different NaCl (mM) concentrations in the nutrient solution.Upper case letters in the figure stand for significant differences between cultivars, according to the Tukey's test (p≤0.05).The effect of salinity within each cultivar was better explained by a quadratic and a linear model in (C) and a quadratic and a square root model in (D).Significance: *p<0.05;**p<0.01.

Figure 2 .
Figure 2. Relationship between the internal and external CO2 concentrations (Ci/Ca) in sunflower plants of the Aguará 4 and Agrobel 963 cultivars grown under different NaCl (mM) concentrations in the nutrient solution.Means followed by the same letter are not significantly different according to the F-test (p≤0.05).

Figure 3 .
Figure 3. Leaf water potential (ψw) in sunflower plants of the Aguará 4 and Agrobel 963 cultivars grown under different NaCl (mM) concentrations in the nutrient solution.For each NaCl concentration tested, different upper case letters inside the figure indicate significant differences between the cultivars, according to the F-test (p≤0.05).The effect of salinity was better explained by a square root model for the Aguará cultivar 4 and by a linear model for the Agrobel 963 cultivar.Significance: *p<0.05;**p<0.01.

Figure 4 .
Figure 4. Refractive index of leaves (A) and roots (B) of sunflower plants of the Aguará 4 and Agrobel 963 cultivars grown under different NaCl (mM) concentrations in the nutrient solution.The effect of salinity was better explained by a square root model in (A) and by a quadratic model in (B).Significance: *p<0.05;**p<0.01.

Figure 5 .
Figure 5. Number of leaves (A) and nodes (B) of sunflower plants of the Aguará 4 and Agrobel 963 cultivars grown under different NaCl (mM) concentrations in the nutrient solution.The effect of salinity was better explained by a quadratic model.Significance: *p<0.05.

Figure 6 .
Figure 6.Number of leaves (A) and nodes (B) of sunflower plants of the Aguará 4 and Agrobel 963 cultivars grown under different NaCl (mM) concentrations in the nutrient solution.Means followed by the same letter were not significantly different according to the F-test (p≤0.05).

Figure 7 .
Figure 7. Specific leaf area (SLA) and leaf area ratio (LAR) of sunflower plants of the Aguará 4 and Agrobel 963 cultivars grown under different NaCl (mM) concentrations in the nutrient solution.For each NaCl concentration tested, different upper case letters inside the figure indicate significant differences between cultivars, according to the F-test (p≤0.05).The effect of salinity was better explained by a square root model in (A) and by a linear and a quadratic model in (B).Significance: **p<0.01.

Figure 8 .
Figure 8. Leaf area (LA), aerial part dry matter (APDM), root dry matter (RDM) and total dry matter (TDM) in sunflower plants of the Aguará 4 and Agrobel 963 cultivars grown under different NaCl (mM) concentrations in the nutrient solution.For each NaCl concentration tested, different upper case letters inside the figure indicate significant differences between cultivars, according to the F-test (p≤0.05).The effect of salinity was better explained by a linear model in (A), by a square root and a quadratic model in (B), by a quadratic model in (C) and by a square root and a quadratic model in (D).Significance: *p<0.05;**p<0.01.