ABSTRACT

In semi-arid regions, the use of saline water for irrigation has become an increasingly frequent reality due to the qualitative and quantitative scarcity of water sources occurring most of the year. Thus, the use of hydrogen peroxide can be a strategy capable of mitigating the deleterious effects of salt stress on plants and guaranteeing the agricultural production of crops such as soursop. Thus, the objective of this study was to evaluate the effects of foliar application of hydrogen peroxide on the physiological and growth indices of soursop plants cv. Morada Nova irrigated with waters of different salinity levels. The treatments were distributed in a randomized block design, in a 4 × 4 factorial scheme, corresponding to four levels of electrical conductivity of irrigation water and four concentrations of hydrogen peroxide, with three replicates. Foliar application of hydrogen peroxide at concentrations of 12, 18 and 15 µM, respectively, attenuated the effects of salt stress on stomatal conductance, CO₂ assimilation rate and chlorophyll a synthesis of soursop, at 780 days after transplanting. The 30 µM hydrogen peroxide concentration intensified salt stress on gas exchange, variable fluorescence and electrolyte leakage in the leaf blade of soursop plants cv. Morada Nova, 780 days after transplanting.

Keywords:
Annona muricata L; Salinity; Reactive oxygen species; Oxidative stress

RESUMO

Em regiões semiáridas, a utilização de água salina para irrigação, tem se tornado uma realidade cada vez mais frequente em função da escassez qualitativa e quantitativa das fontes hídricas que ocorre na maior parte do ano. Assim, a utilização de peróxido de hidrogênio pode ser uma estratégia capaz de amenizar os efeitos deletérios do estresse salino sobre as plantas e garantir a produção agrícola de culturas, como a gravioleira. Deste modo, objetivou-se com esta pesquisa avaliar os efeitos da aplicação foliar de peróxido de hidrogênio sobre os índices fisiológicos e de crescimento de plantas de graviola cv. Morada Nova irrigadas com águas de diferentes níveis salinos. Os tratamentos foram distribuídos em um delineamento de blocos casualizados, em esquema fatorial 4 × 4, correspondendo a quatro níveis de condutividade elétrica da água de irrigação e quatro concentrações de peróxido de hidrogênio, com três repetições. A aplicação foliar de peróxido de hidrogênio nas concentrações de 12, 18 e 15µM, respectivamente, atenuaram os efeitos do estresse salino sobre a condutância estomática, a taxa de assimilação de CO₂ e a síntese de clorofila a de gravioleira, aos 780 dias após o transplantio. A concentração de peróxido de hidrogênio de 30 µM intensificou o estresse salino sobre as trocas gasosas, a fluorescência variável e o extravasamento de eletrólitos no limbo foliar de plantas de graviola cv. Morada Nova, aos 780 dias após o transplantio.

Palavras-chave:
Annona muricata L; Salinidade; Espécie reativa de oxigênio; Estresse oxidativo

INTRODUCTION

Belonging to the Annonaceae family, soursop (Annona muricata L.) is a fruit tree cultivated in Brazil, mainly in the semi-arid region of the Northeast, due to the multifunctionality of its production, standing out as a good source of vitamins B and C (HASMILA; NATSIR; SOEKAMTO, 2019HASMILA, I.; NATSIR, H.; SOEKAMTO, N. H. Análise fitoquímica e atividade antioxidante do extrato da folha de graviola (Annona muricata L.). Journal of Physics: Conference Series, 1341:1-6, 2019.). It is a source of carbohydrates, minerals, antioxidant substances and important bioactive compounds, such as acetogenins, which act in the prevention of degenerative diseases (MUTAKIN et al., 2022MUTAKIN, M. et al. Pharmacological activities of Soursop (Annona muricata Lin.), Molecules, 27: e1201, 2022.).

Although the semi-arid region of Northeastern Brazil has edaphoclimatic conditions for soursop cultivation, but the scarcity of rainfall and the high evapotranspiration that occur in most months of the year contribute for the sources of water, whether underground (wells) and/or superficial (small and medium-sized dams and lagoons), to contain high levels of salts, limiting its use for irrigation (BRITO et al., 2020BRITO, Y. J. V et al. Estudo experimental de um dessalinizador solar do tipo bandeja com dupla inclinação para potabilização de água no semiárido paraibano. Águas Subterrâneas, 34:156-165, 2020.).

Excess salts in the water reduce the osmotic potential of the soil solution, affecting water availability and causing ionic effect and changes in soil physical and chemical properties (SOARES et al., 2018SOARES, L. A. A. et. al. Growth and fiber quality of colored cotton under salinity management strategies. Revista Brasileira de Engenharia Agrícola e Ambiental, 22: 332-337, 2018.). In addition, excessive presence of salts in the root zone of plants can negatively affect cell membrane integrity, limiting the photosynthetic process due to the partial stomatal closure, resulting in damage to the photosynthetic apparatus and/or the enzymatic system of CO₂ fixation, regardless of the nature of the salts (VELOSO et al., 2020aVELOSO, L. L. S. A. et al. Physiological changes and growth of soursop plants under irrigation with saline water and H2O2 in post-grafting phase. Semina: Ciências Agrárias, 41: 3023-3038, 2020a.).

However, salt stress effects on plants can be mitigated with the use of elicitors, such as hydrogen peroxide (H₂O₂) (CAPITULINO et al., 2022CAPITULINO, J. D et al. Gas exchange and growth of soursop under salt stress and H2O2 application methods. Brazilian Journal of Biology, 84: e261312, 2022.). When plants are subjected to pre-treatment with adequate concentrations of H₂O₂, they undergo changes in their metabolism, through the activation of the enzymatic and non-enzymatic antioxidant defense system, which confers greater tolerance to abiotic stresses such as salinity (VELOSO et al., 2020bVELOSO, L. L. S. A. et al. Application methods of hydrogen peroxide in soursop seedlings irrigated with saline water. Comunicata Scientiae, 11: 3288, 2020b.).

In this context, some studies have reported that H₂O₂ application can increase tolerance to salt stress, as observed in passion fruit plants (RAMOS et al., 2022RAMOS, J. G. et al. Hydrogen peroxide as salt stress attenuator in sour passion fruit. Revista Caatinga, 35: 412-422, 2022.) and in soursop in the seedling formation phase (CAPITULINO et al., 2022CAPITULINO, J. D et al. Gas exchange and growth of soursop under salt stress and H2O2 application methods. Brazilian Journal of Biology, 84: e261312, 2022.). In view of the above, the objective of this study was to evaluate the effects of foliar application of H₂O₂ on the physiological indices and growth of soursop under irrigation with saline waters.

MATERIAL AND METHODS

The experiment was carried out between April 2020 and May 2022 in a greenhouse, located at the Academic Unit of Agricultural Engineering - UAEA of the Federal University of Campina Grande - UFCG, in Campina Grande, Paraíba, at geographic coordinates 7°15’18” South latitude, 35°52’28” West longitude and average altitude of 550 m. Temperature (maximum and minimum) and relative humidity data measured inside the greenhouse are shown in Figure 1.

The treatments consisted of four levels of electrical conductivity of irrigation water - ECw (0.8, 1.6, 2.4 and 3.2 dS m^-1) and four concentrations of hydrogen peroxide - H₂O₂ (0, 10, 20 and 30 μM), in a 4 × 4 factorial arrangement, distributed in randomized blocks, with three replicates. Water electrical conductivity levels were based on a previous study conducted by Silva et al. (2019a)SILVA, A. A. R. et al. Salt stress and exogenous application of hydrogen peroxide on photosynthetic parameters of soursop. Revista Brasileira de Engenharia Agrícola e Ambiental, 23: 257-263, 2019a., while H₂O₂ concentrations were established based on a study conducted by Veloso et al. (2020b)VELOSO, L. L. S. A. et al. Application methods of hydrogen peroxide in soursop seedlings irrigated with saline water. Comunicata Scientiae, 11: 3288, 2020b..

Soursop seedlings cv. Morada Nova were obtained in a commercial nursery accredited by the Registry of Seeds and Seedlings, in the District of São Gonçalo, Sousa, PB, produced in polyethylene bags with dimensions of 10 × 20 cm. The Morada Nova cultivar was chosen due to its greater use in commercial orchards in Brazil, as well as its production potential and the size of its fruits, which can weigh up to 15 kg (SÃO JOSÉ et al., 2014SÃO JOSÉ, A. R. et al. Atualidades e perspectivas das Anonáceas no mundo. Revista Brasileira de Fruticultura, 36: 86-93, 2014.).

The experiment was conducted using plastic pots adapted as drainage lysimeters, with 211.5 L (depth of 0.75 m and area of 0.2115 m²) and filled with a 1.0-kg layer of crushed stone followed by 230 kg of soil classified as Neossolo Regolítico (Entisol) of clayey loam texture, collected at 0-30 cm depth, from the municipality of Riachão do Bacamarte, PB, Brazil, whose physicochemical characteristics (Table 1) were determined according to Teixeira et al. (2017)TEIXEIRA, P. C. et al. Manual de métodos de análise de solo. 3. ed. Brasília, DF: Embrapa, 2017. 573 p..

The saline waters were prepared by the addition of NaCl, CaCl₂.2H₂O and MgCl₂.6H₂O salts, in the equivalent ratio of 7:2:1, which prevails in the main sources of water available for irrigation in the Brazilian Northeast (MEDEIROS, 1992MEDEIROS, J. F. Qualidade da água de irrigação utilizada nas propriedades assistidas pelo “GAT” nos Estados do RN, PB, CE e avaliação da salinidade dos solos. 1992. 173 f. Dissertação (Mestrado em Engenharia Agrícola: Área de Concentração Irrigação e Drenagem) - Universidade Federal de Campina Grande, Campina Grande, 1992.), following the relationship between ECw and the concentration of salts (RICHARDS, 1954RICHARDS, L. A. Diagnosis and improvement of saline and alkali soils. Washington: U.S, Department of Agriculture. 1954. 160 p.), according to Equation 1:

Q ≈ 640 × ECw (1)

where:

Q = Quantity of salts to be applied (mg L ^-1);

ECw = Electrical conductivity of water (dS m^-1)

At 60 days after transplanting, irrigation with saline water began to be performed, applying water to each lysimeter according to treatment, in order to keep soil moisture close to field capacity and avoid excessive accumulation of salts in the soil. The volume to be applied was determined according to the water needs of the plants. Every day, at 5 p.m., the volume corresponding to that obtained by the water balance was manually applied to each container, according to Equation 2:

where:

VI = volume of water to be used in the next irrigation event (mL);

Va = volume applied in the previous irrigation event (mL);

Vd = volume drained (mL); and

LF = leaching fraction of 0.10, applied every 30 days.

NPK fertilization was carried out as recommended by Cavalcante et al. (2008)CAVALCANTE, F. J. A. Recomendação de adubação para o Estado de Pernambuco: 2. Aproximação. 3. ed. Recife, PE: IPA. 2008. 212 p., applying 40 g of N, 60 g of K₂O and 40 g of P₂O₅ per plant year . Fertilization was split into 24 portions and applied every 15 days. Urea (45% N), potassium sulfate (50% K₂O and 17% S) and monoammonium phosphate (12% N and 54% P₂O₅) were used as sources of nitrogen, potassium and phosphorus, respectively.

Micronutrients were applied through the leaves from 60 days after transplanting and 2.5 g L¹ of a Dripsol® Micro solution with the following composition continued to be applied fortnightly: N (15%); P₂O₅ (15%); K₂O (15%); Ca (1%); Mg (1.4%); S (2.7%); Zn (0.5%); B (0.05%); Fe (0.5%); Mn (0.05%); Cu (0.5%); Mo (0.02%).

The different concentrations of H₂O₂ were obtained by dilution in distilled water, with subsequent calibration using a spectrophotometer at an absorbance wavelength of 240 nm. Foliar applications began 45 days after transplanting (DAT) of seedlings grown in lysimeters and were performed at 30-day intervals. To ensure that the leaves were completely wet, a knapsack sprayer with an adjustable conical 1 cm metal nozzle, service pressure of 2.07 MPa and flow rate of 1.1 L min^-1 was used to spray the solution on the abaxial and adaxial sides of the leaves, between 5 and 6 p.m. The volume of H₂O₂ solution per plant was approximately 400 mL. In addition, to control the drift of the solution between treatments, a plastic tarpaulin curtain was placed around each plant during the application of the H₂O₂ solution.

Formative pruning was carried out when the plant reached 60 cm height, when the apical meristem was cut. From the shoots that emerged, three well-distributed and equidistant branches were selected and then pruned when they reached 40 cm in length (SILVA et al., 2020SILVA, A. A. R. et al. Salicylic acid as an attenuator of salt stress in soursop. Revista Caatinga, 33: 1092-1101, 2020.). During the experiment, the appearance of pests and diseases was monitored and, when the incidence was observed, these were eradicated with the use of pesticides.

At 780 DAT, gas exchange, chlorophyll a fluorescence, photosynthetic pigments, electrolyte leakage, saturation deficit in the leaf blade and relative growth rate in stem diameter of soursop cv. Morada Nova were evaluated. Gas exchange was evaluated in leaves of the middle third of the plants, using a portable infrared gas analyzer – IRGA (Infrared Gas Analyser, LCpro-SD model, from ADC BioScientific, UK). The variables analyzed were CO₂ assimilation rate - A (µmol CO₂ m^-2 s^-1), transpiration - E (mmol H₂O m^-2 s^-1), stomatal conductance - gs (mol H₂O m^-2 s^-1) and internal CO₂ concentration - Ci (μmol CO₂ m^-2 s^-1).

A/gs and A/Ci ratios were used to determine water use efficiency – WUEi [(μmol CO₂ m^-2 s^-1) (mmol H₂O m^-2 s^-1)^-1] and carboxylation efficiency – CEi [(μmol CO₂ m^-2 s^-1) (μmol CO₂ m^-2 s^-1)^-1], respectively. The readings were taken between 7:00 and 10:00 a.m., on the third fully expanded leaf counted from the apical bud, under natural conditions of air temperature, atmospheric CO₂ concentration, using constant light of 1,200 (μmol m^-2 s^-1 established through the light-photosynthesis response curve (FERNANDES et al., 2021FERNANDES, E. A. et al. Cell damage, gas exchange, and growth of Annona squamosa L. under saline water irrigation and potassium fertilization. Semina: Ciências Agrárias, 42: 999-1018, 2021.).

Chlorophyll a fluorescence was measured on the same leaves using a pulse-modulated fluorometer, OS5p model from Opti Science. Initial (F₀), maximum (Fm) and variable (Fv) fluorescence and quantum efficiency of photosystem II (Fv/Fm) were measured; this protocol was performed after adaptation of the leaves to the dark for a period of 30 min, between 6:00 and 9:00 am, using a clip of the device, to ensure that all primary acceptors were fully oxidized.

Contents of chlorophyll a, chlorophyll b, total chlorophyll and carotenoids were also determined at 780 DAT, according to the methodology of Arnon (1949)ARNON, D. I. Copper enzymes in isolated cloroplasts: polyphenoloxidases in Beta vulgaris. Plant Physiology, 24:1-15, 1949., using 5 leaf discs obtained from the third mature leaf from the apex, immersed in 80% acetone and stored in the dark for 48 hours. The extracts obtained were subjected to spectrophotometer readings at wavelengths of 470, 647 and 663 nm. The values observed in the readings were applied in the following equations (Equations 3, 4, 5):

The values obtained for the contents of chlorophyll a, chlorophyll b, total chlorophyll and carotenoids in the leaves were expressed in mg g^-1 FM (fresh matter).

Electrolyte leakage (%EL) in the leaf blade was determined using a copper hole punch to obtain five leaf discs with area of 1.54 cm² each, per experimental unit, which were washed and placed in an Erlenmeyer flask containing 50 mL of distilled water. After being closed with aluminum foil, the Erlenmeyer flasks were stored at a temperature of 25 °C for 24 hours, and then the initial conductivity of the medium (Xi) was measured using a benchtop conductivity meter (MB11, MS Techonopon®). Then, the Erlenmeyer flasks were subjected to a temperature of 90 °C for 120 minutes in a drying oven (SL100/336, SOLAB®) and, after their contents cooled down, the final conductivity (Xf) was measured. Electrolyte leakage was expressed as the percentage of initial electrical conductivity relative to electrical conductivity after treatment for 90 minutes at 90 ºC, according to the methodology described by Scotti-Campos et al. (2013)SCOTTI-CAMPOS, P. et al. Physiological responses and membrane integrity in three Vigna genotypes with contrasting drought tolerance. Emirates Journal of Food and Agriculture, 25: 1002-1013, 2013., as shown in Equation 6:

where:

%EL = electrolyte leakage (%);

Xi = initial conductivity of the medium;

Xf = final conductivity.

To determine the water saturation deficit (WSD), two leaves were collected from the middle third of the main branch and four 12-mm-diameter discs were obtained from each leaf. Immediately after collection, the discs were weighed, avoiding moisture loss, and fresh mass (FM) was obtained; then, these samples were placed in beaker, immersed in 50 mL of distilled water, and kept at rest for 24 hours. After this period, excess water was removed from the resting discs using paper towels and the turgid mass (TM) of the samples was obtained. These samples were then dried in an oven at a temperature of ≈ 65 ± 3 ºC until reaching constant weight to obtain the dry mass (DM). WSD was determined according to Lima et al. (2015)LIMA, G. S. et al. Water relations and gas exchange in castor bean irrigated with saline water of distinct cationic nature. African Journal of Agricultural Research, 10: 1581-1594, 2015., using Equation 7:

where:

WSD = water saturation deficit (%);

FM = leaf fresh mass (g);

TM = leaf turgid mass (g);

DM = leaf dry mass (g).

Soursop growth was evaluated based on stem diameter (SD) and on the relative growth rates of stem diameter (RGRsd) following the methodology of Benincasa (2003)BENINCASA, M. M. P. Análise de crescimento de plantas: noções básicas. 2. ed. Jaboticabal, SP: FUNEP, 2003. 42 p., according to Equation 8:

where:

RGRsd = Relative growth rate of stem diameter (mm day^-1),

A₂ = stem diameter at 780 DAT;

A₁ = stem diameter at 210 DAT;

t₂ – t₁ = time difference between assessments; and

ln = natural logarithm.

The collected data were subjected to the distribution normality test (Shapiro-Wilk test). Then, analysis of variance was performed at 0.05 probability level and, in cases of significance of individual factors, regression analysis was performed, using the statistical program SISVAR-ESAL (FERREIRA, 2019FERREIRA, D. F. Sisvar: A computer analysis system to fixed effects split-plot type designs. Revista Brasileira de Biometria, 37: 529-535, 2019.). In case of significance of the interaction between factors, SigmaPlot v.12.5 software was used to construct the response surfaces.

RESULTS AND DISCUSSION

There were significant effects (Table 2) of the interaction between water salinity levels and H₂O₂ concentrations on the internal CO₂ concentration (Ci) CO₂ assimilation rate (A), transpiration (E) and instantaneous water use efficiency (WUEi) of soursop plants. The water salinity levels alone influenced the water saturation deficit. On the other hand, H₂O₂ concentrations alone influenced electrolyte leakage (%EL), stomatal conductance (gs) and instantaneous carboxylation efficiency (CEi).

Water saturation deficit in the leaf blade increased linearly (Figure 2A) by 7.80% per unit increase in ECw. Soursop plants irrigated with water of highest salinity (3.2 dS m^-1) showed an increase of 17.61% (2.84) compared to those irrigated under ECw of 0.8 dS m^-1. Increase in WSD is a consequence of the lower relative water content due to the restriction in the absorption of water and nutrients imposed by salt stress. On the other hand, soursop plants subjected to H₂O₂ concentration of 13 μM (Figure 2B) obtained lower % EL (47.19%), with an increase in electrolyte leakage of soursop plants from this concentration. This increase in %EL of plants grown under H₂O₂ concentration of 30 μM may be r elate d to the fact that H₂O₂ is the most stable reactive oxygen species in cells and, at high concentrations, can spread rapidly across the subcellular membrane, resulting in oxidative damage to the membrane (FAROUK; AMIRA, 2018FAROUK, S.; AMIRA, M. S. A. Q. Enhancing seed quality and productivity as well as physio-anatomical responses of pea plants by folic acid and or hydrogen peroxide application. Scientia Horticulturae, 240: 29-37, 2018.). Veloso et al. (2020b)VELOSO, L. L. S. A. et al. Application methods of hydrogen peroxide in soursop seedlings irrigated with saline water. Comunicata Scientiae, 11: 3288, 2020b. also observed that foliar application of H₂O₂ (0 and 20 μM) to soursop (Anonna muricata L.) plants irrigated with saline waters (ECw ranging from 1.6 to 4.0 dS m^-1) reduced electrolyte leakage at 150 days after transplanting.

H₂O₂ concentrations significantly influenced the stomatal conductance (Figure 3A) of soursop plants, whose maximum estimated value (0.05325 mol H₂O m^-2 s^-1) was obtained under foliar application of H₂O₂ at 13 μM. On the other hand, the lowest value (0.03880 mol H₂O m^-2 s^-1) was observed in soursop plants cultivated under foliar application of H₂O₂ at 30 μM. For the internal CO₂ concentration (Figure 3B), irrigation with 1.32 dS m^-1 water associated with foliar application of H₂O₂ at 12.19 μM promoted the highest estimated value (313.916 μmol CO₂ m^-2 s^-1), being limited by the ECw of 3.2 dS m^-1 and the highest concentrations of H₂O₂ (30 μM), with reduction of 109.74 μmol CO₂ m^-2 s^-1 (34.96%). The reductions in Ci with the increase of ECw in soursop plants treated with H₂O₂ concentrations above 12 μM probably occurred due to the lower CO₂ diffusion in the substomatal chamber, as a consequence of stomatal closure.

Stomatal closure can be considered a strategy to reduce water losses to the atmosphere and, consequently, maintain the turgidity of guard cells, directly affecting the formation of carbohydrates in photosynthesis and production (LIMA et al., 2020LIMA, G. S. et al. Water status, cell damage and gas exchanges in West Indian cherry (Malpighia emarginata) under salt stress and nitrogen fertilization. Australian Journal Crop Science, 14: 319-324, 2020.). The beneficial effect of H₂O₂ at low concentrations was also observed by Silva et al. (2019a)SILVA, A. A. R. et al. Salt stress and exogenous application of hydrogen peroxide on photosynthetic parameters of soursop. Revista Brasileira de Engenharia Agrícola e Ambiental, 23: 257-263, 2019a., who evaluated the gas exchange and photosynthetic pigments of soursop cv. Morada Nova irrigated with saline water (ECw ranging from 0.7 to 3.0 dS m^-1) and subjected to the application of H₂O₂ (H₂O₂ ranging from 0 to 100 μM) by seed soaking and foliar spraying and observed that the H₂O₂ concentration of 25 μM mitigated the deleterious effects of salt stress on stomatal conductance and CO₂ assimilation rate at 120 days after sowing. However, it is important to emphasize that the application of H₂O₂ at inadequate concentrations can cause oxidative stress and, consequently, damage the cell membrane and other cellular structures, compromising the photosynthetic efficiency of the plant.

As observed for Ci, foliar application of H₂O₂ concentrations increased the CO₂ assimilation rate (Figure 3C) and transpiration (Figure 3D), with the highest values of 3.83 μmol CO₂ m^-2 s^-1 and 1.6404 mmol H₂O m^-2 s^-1 obtained in the treatments with ECw of 1.6 dS m^-1 and H₂O₂ concentration of 18 μM for A and 11 μM for E. The lowest A value (2.49 μmol CO₂ m^-2 s^-1) was observed in plants that did not receive H₂O₂ associated with ECw of 3.2 dS m^-1. On the other hand, the lowest E value was found when the plants were subjected to a H₂O₂ concentration of 30 μM associated with ECw of 0.8 dS m^-1, which was 0.6754 mol H₂O m^-2 s^-1. Reduction in transpiration rate is directly linked to the decrease in gs, because with less opening of the stomata there will be a decline in transpiration, restricting the loss of water from the leaf to the atmosphere in the form of vapor, thus reducing the transpiration rate of the plant (LIMA et al., 2017LIMA, G. S. de et al. Gas exchanges and production of colored cotton under salt stress and nitrogen fertilization. Bioscience Journal, 33:1495-1505, 2017.).

Capitulino et al. (2022)CAPITULINO, J. D et al. Gas exchange and growth of soursop under salt stress and H2O2 application methods. Brazilian Journal of Biology, 84: e261312, 2022., in a study evaluating the gas exchange and growth of soursop seedlings under salt stress (ECw ranging from 0.6 to 3.0 dS m^-1) and application of H₂O₂ (0 and 20 μM), found that the application of H₂O₂ at concentration of 20 μM attenuated the deleterious effects of irrigation water salinity on gas exchange at 85 days after sowing.

Figure 4A shows that the highest value for instantaneous water use efficiency – WUEi (2.69 [(μmol CO₂ m^-2 s^-1) (mmol H₂O m^-2 s^-1)^-1])^-1]) was obtained in plants irrigated using water with ECw of 1.75 dS m^-1 and under H₂O₂ concentration of 25 μM. On the other hand, irrigation with water of 3.2 dS m^-1 in the absence of H₂O₂ application led to the minimum estimated value for WUEi (1.62 [(μmol CO₂ m^-2 s^-1) (mmol H₂O m^-2 s^-1)^-1]). With the increase in the concentration of salts in water and/or soil, plants have greater difficulty absorbing water and nutrients. Consequently, to prevent excessive water loss, they close their stomata, restricting the entry of CO₂ into the substomatal chamber, compromising instantaneous carboxylation and water use efficiency (SILVA et al., 2019bSILVA, S. S. da et al. Gas exchanges and production of watermelon plant under salinity management and nitrogen fertilization. Pesquisa Agropecuária Tropical, 49: e54822, 2019b.).

Foliar application of H₂O₂ increased the instantaneous carboxylation efficiency - CEi of soursop plants at 780 DAT (Figure 4B), with the highest value (0.01435 [(μmol CO₂ m^-2 s^-1 ) (μmol ]) observed in plants subjected to H₂O₂ application of 17 μM. It was also observed that the lowest CEi value was obtained when the plants were cultivated in the absence of H₂O₂ application (0.01246 [(μmol CO₂ m^-2 s^-1) (mmol H₂O m^-2 s^-1)^-1]). Thus, pretreatment of plants with H₂O₂ may cause a metabolic signaling in the cell, inducing the antioxidant defense system and/or the increase of a metabolite, minimizing the negative effects caused by salinity, which may favor the physiological performance of plants exposed to subsequent conditions of more severe stress (PANHWAR; KEERIO; ROBERT, 2017PANHWAR, M.; KEERIO, M. I; ROBERT, M. R. Evaluating changes in wheat genotypes caused by hydrogen peroxide during seed treatment and their involvement in salt tolerance. Pakistan Journal of Agriculture, Agricultural Engineering and Veterinary Sciences, 33: 23-36, 2017.). Silva et al. (2021)SILVA, A. A. R. et al. Hydrogen peroxide in the acclimation of yellow passion fruit seedlings to salt stress. Revista Brasileira de Engenharia Agrícola e Ambiental, 25: 116-123, 2021., when evaluating the effect of exogenous application of H₂O₂ on emergence, growth and gas exchange of yellow passion fruit subjected to salt stress, observed that the H₂O₂ concentration of 10 μM induces the acclimatization of passion fruit plants to salt stress, mitigating the deleterious effects of salinity on CO₂ assimilation rate and instantaneous carboxylation efficiency.

There was a significant effect of the interaction between water salinity levels and H₂O₂ concentrations on the variable fluorescence, chlorophyll b and carotenoids of soursop plants (Table 3). The salinity levels of irrigation water significantly influenced the initial and maximum fluorescence, the quantum efficiency of photosystem II and the total chlorophyll contents of soursop. H₂O₂ concentrations, as individual factor, did not influence the initial fluorescence, chlorophyll a and total chlorophyll.

Irrigation with saline water linearly increased the initial fluorescence of soursop plants (Figure 5A), by 1.97% per unit increment in ECw, i.e., plants grown under water salinity of 3.2 dS m^-1 reduced their F₀ by 4.65% (557.059) compared to those under irrigation with the lowest salinity level (0.8 dS m^-1) (532.265). F₀ expresses the loss of photochemical energy released by the chlorophyll a molecules of the photosystem antenna, so the F₀ value may increase when the reaction centers of photosystem II are compromised or the transfer of excitation energy from the antenna to the reaction centers is impaired, i.e., when there is a reduction in the electron transport capacity (CINTRA et al., 2020CINTRA, P. H. N. et al. Análise de fluorescência da clorofila a em mudas de cafeeiro sob estresse hídrico. Brazilian Journal of Development, 6: 28006-28014, 2020.), which may have occurred in the present study, as the water electrical conductivity levels increased.

It was also observed that the highest value of F₀ (Figure 5B) was obtained in soursop plants subjected to the application of 21 μM of H₂O₂ (547.631), while the lowest value was observed in the absence of H₂O₂ application (0 μM) (539.5). As already mentioned, an increase in F₀ is indicative of damage to the reaction center of photosystem II or reduction in the ability to transfer energy from the excitation of the light-harvesting system to the reaction center of PSII (FAROUK; AMIRA, 2018FAROUK, S.; AMIRA, M. S. A. Q. Enhancing seed quality and productivity as well as physio-anatomical responses of pea plants by folic acid and or hydrogen peroxide application. Scientia Horticulturae, 240: 29-37, 2018.), so the increase of F₀ in soursop cv. Morada Nova is probably related to the high H₂O₂ concentration used and/or the time of exposure of the plants, since H₂O₂ is a more stable reactive oxygen species in cells and, at high concentrations, can spread rapidly through the subcellular membrane, resulting in oxidative damage to the membrane cell.

For maximum fluorescence (Figure 5C), the highest estimated value was observed in plants subjected to irrigation with ECw of 1.9 dS m^-1 (2233.107). From this ECw level, there was a reduction, and the lowest Fm was obtained in plants cultivated under ECw of 3.2 dS m^-1 (2158.599). Andrade et al. (2022)ANDRADE, E. M. G. et al. Hydrogen peroxide as attenuator of salt stress effects on the physiology and biomass of yellow passion fruit. Revista Brasileira de Engenharia Agrícola e Ambiental, 28: 571-578, 2022., when evaluating the physiology of passion fruit under salt stress (ECw ranging from 0.7 to 2.8 dS m^-1), also found that the increase in salt stress inhibited the photochemical activity of photosystem II, observed by the reduction of maximum fluorescence at 205 days after transplanting (DAT).

Soursop plants irrigated with ECw of 2.9 dS m^-1 and subjected to H₂O₂ concentration of 13 μM (Figure 5D) stood out with the highest Fv value (1671.27). The lowest Fv value (1575.86) was obtained in plants irrigated with ECw of 0.8 dS m^-1 and subjected to a H₂O₂ concentration of 30 μM. At high concentrations, H₂O₂ can cause changes in plant metabolism, mainly as a consequence of oxidative stress, limiting photosynthetic activities; however, when applied at low concentrations, it can induce the action of antioxidant enzymes, reducing the deleterious effects of salt stress (CATTIVELLI et al., 2008CATTIVELLI, L. et al. Drought tolerance improvement in crop plants: An integrated view from breeding to genomics. Field Crops Research, 105: 1-14, 2008.).

Veloso et al. (2020a)VELOSO, L. L. S. A. et al. Physiological changes and growth of soursop plants under irrigation with saline water and H2O2 in post-grafting phase. Semina: Ciências Agrárias, 41: 3023-3038, 2020a., when evaluating the photosynthetic pigment contents and photochemical efficiency of soursop plants under irrigation with saline water (ECw ranging from 0.6 to 3.0 dS m^-1) and different methods of H₂O₂ application (0 and 20 μM), found that the application of 20 μM of H₂O₂ via seed imbibition resulted in increments in maximum and variable fluorescence.

Water salinity linearly reduced the quantum efficiency of photosystem II (Fv/Fm) (Figure 5E), by 0.55% per unit increment in ECw. When comparing the Fv/Fm of plants grown under 3.2 dS m^-1 to the values of those grown under water salinity of 0.8 dS m^-1, a decrease of 1.35% was observed.

Foliar application of H₂O₂ promoted a quadratic effect on the contents of chlorophyll a (Figure 6A), with a maximum value of 1.5839 mg g^-1 FM obtained in plants subjected to H₂O₂ concentration of 15 μM, while the minimum value was observed in the absence of H₂O₂ application (1.0769 mg g^-1 FM). At low concentrations, H₂O₂ can stimulate antioxidant enzyme activity, promoting less pigment degradation and increase in photosynthetic activity, which results in greater plant growth and development (VELOSO et al., 2022VELOSO, L. L. S. A. et al. Growth and gas exchange of soursop under salt stress and hydrogen peroxide application. Revista Brasileira de Engenharia Agrícola e Ambiental, 26: 119-125, 2022.).

In a study evaluating the effect of the application of H₂O₂ (0, 15, 30 and 45 μM) to mitigate salt stress (ECw from 0 . 6 to 3.0 dS m^-1) on the growth of sour passion fruit (Passiflora edulis Sims) at 240 days after transplanting, Ramos et al. (2022)RAMOS, J. G. et al. Hydrogen peroxide as salt stress attenuator in sour passion fruit. Revista Caatinga, 35: 412-422, 2022. also found a beneficial effect of the application of 15 μM of H₂O₂ on the contents of photosynthetic pigments.

Foliar application of 17 μM of H₂O₂ increased the chlorophyll b contents of soursop cv. Morada Nova (Figure 6B), irrigated with water of 1.1 dS m^-1 (0.5217 mg g^-1 FM). In the absence of H₂O₂ (0 μM) and subjected to the highest salinity level (3.2 dS m^-1), soursop plants obtained the lowest value of Chl b (0.2236 mg g^-1 FM). This was possibly due to the multiple physiological functions performed by H₂O₂ in the plant, such as the ability to increase chlorophyll biosynthesis, since the use of signaling agents in plants, such as H₂O₂, can promote metabolic alteration in the cell and activation of antioxidant enzymes such as superoxide dismutase, catalase, guaiacol peroxidase and ascorbate peroxidase, resulting in a decrease in oxidative stress (RAMOS et al., 2022RAMOS, J. G. et al. Hydrogen peroxide as salt stress attenuator in sour passion fruit. Revista Caatinga, 35: 412-422, 2022.).

The increase in the electrical conductivity of irrigation water reduced the synthesis of total chlorophyll (Figure 6C), which decreased by 11.15% per unit increase in ECw. When comparing the Chl t contents of plants irrigated with water of 3.2 dS m^-1 with the values of those cultivated under water salinity of 0.8 dS m^-1, a reduction of 41.63% (0.635 mg g^-1 FM) was observed. Decrease in chlorophyll contents is usually accompanied by the inactivation of photochemical reactions, especially those mediated by photosystem II (PSII) in plants under salt stress (ZHAO et al., 2019ZHAO, H. et al. Effects of salt stress on chlorophyll fluorescence and the antioxidant system in Ginkgo biloba L. Seedlings. HortScience, 54: 2125-2133, 2019.). Irrigation with saline water (ECw from 0.6 to 3.0 dS m^-1) also reduced the contents of chlorophyll a and total chlorophyll of sour passion fruit cv. BRS Rubi do Cerrado, at 240 days after transplanting (RAMOS et al., 2022RAMOS, J. G. et al. Hydrogen peroxide as salt stress attenuator in sour passion fruit. Revista Caatinga, 35: 412-422, 2022.).

As observed for chlorophyll a synthesis, soursop plants obtained the highest total chlorophyll content (Figure 6D) when subjected to a H₂O₂ concentration of 15 μM (2.293 mg

g^-1 FM). On the other hand, the lowest Chl t value was obtained at H₂O₂ concentration of 30 μM (1.4779 mg g^-1 FM). At low concentrations, H₂O₂ helps plants acclimatize to the deleterious effects of salt stress, due to metabolic changes that are responsible for increasing their tolerance to stress, thus enabling the use of waters with higher concentrations of salts (ANDRADE et al., 2019ANDRADE, E. M. G. et al. Gas exchanges and growth of passion fruit under saline water irrigation and H2O2 application. Revista Brasileira de Engenharia Agrícola e Ambiental, 23: 945-951, 2019.).

The carotenoid contents of soursop plants cv. Morada Nova were significantly affected by the interaction between ECw and H₂O₂ concentrations (Figure 6E). Plants cultivated under ECw of 2.1 dS m^-1 obtained the maximum estimated value of 0.4213 mg g^-1 FM under foliar application of 12 μM of H₂O₂. On the other hand, plants grown in water with ECw of 0.8 dS m^-1 and that received the highest concentration of H₂O₂ (30 μM) had the lowest value of Car (0.2711 mg g^-1 FM). Thus, it is suggested that, for the carotenoid content, H₂O₂ at concentration of 12 μM may act as an abiotic stress-signaling molecule, through the action of enzymatic and non-enzymatic components, promoting an increase in carotenoid content, induced by the production of β-carotenes, which are integrated components of thylakoids, involved in the absorption and transfer of light to chlorophyll (SILVA et al., 2017SILVA, A. A. R. et al. Pigmentos fotossintéticos e potencial hídrico foliar em plantas jovens de coqueiro sob estresses hídrico e salino. Revista Agro@mbiente, 10: 317-325, 2017.).

There was a significant effect of the interaction between water salinity levels and H₂O₂ concentrations on stem diameter (SD) (Table 4). Water salinity levels significantly affected the relative growth rate of stem diameter (RGRsd). H₂O₂ concentrations alone did not influence any of the variables analyzed.

The stem diameter (Figure 7A) of soursop plants was favored by the foliar application of H₂O₂ at an estimated concentration of 24 μM and ECw of 1.86 dS m^-1. On the other hand, the lowest SD was obtained in the absence of H₂O₂ (0 μM) and when the plants were irrigated with the highest ECw (3.2 dS m^-1). The higher estimated value of SD in plants subjected to H₂O₂ concentration of 24 μM may be associated with the ability of the H₂O₂ molecule to stimulate plant growth, promoting cell division and elongation (ORABI; DAWOOD; SALMAM, 2015ORABI, S. A.; DAWOOD, M. G.; SALMAN, S. R. Comparative study between the physiological role of hydrogen peroxide and salicylic acid in alleviating the harmful effect of low temperature on tomato plants grown under sand-ponic culture. Scientia Agriculturae, 9: 49-59, 2015.). Veloso et al. (2022)VELOSO, L. L. S. A. et al. Growth and gas exchange of soursop under salt stress and hydrogen peroxide application. Revista Brasileira de Engenharia Agrícola e Ambiental, 26: 119-125, 2022., when s tu d ying the growth of soursop seedlings under salt stress (ECw from 0.7 to 3.7 dS m^-1) and foliar application of H₂O₂ (0, 25, 50 and 75 μM), also observed that the application of H₂O₂ favored growth in stem diameter and plant height.

Regarding the relative growth rate of stem diameter (RGRsd), the maximum estimated value (0.00230 mm day^-1) was obtained in plants irrigated with ECw of 2.0 dS m^-1 (Figure 7B). From this salinity level, there was a reduction and RGRsd reached 0.002156 mm day^-1 at ECw of 3.2 dS m^-1. The reduction in RGRsd may have occurred due to the decrease in the osmotic potential of the soil solution caused by the excess of soluble salts in the root zone, which limits the absorption of water by the roots, causing the plant to reduce stomatal opening, as a mechanism to reduce water loss, which leads to the reduction of cell turgor and, consequently, to reductions in cell expansion and plant growth (LIMA et al., 2020LIMA, G. S. et al. Water status, cell damage and gas exchanges in West Indian cherry (Malpighia emarginata) under salt stress and nitrogen fertilization. Australian Journal Crop Science, 14: 319-324, 2020.), which may have occurred in this study, causing reductions in the RGRsd of soursop plants.

CONCLUSIONS

Foliar application of hydrogen peroxide at concentrations ranging from 11 to 13 μM attenuates the effects of salt stress up to ECw of 1.6 dS m^-1 on the stomatal conductance and CO₂ assimilation rate of soursop cv. Morada Nova, respectively.

Hydrogen peroxide concentration of 30 μM intensifies salt stress effects on gas exchange, variable fluorescence and electrolyte leakage in the leaf blade of soursop cv. Morada Nova, at 780 days after transplantation.

Foliar application of hydrogen peroxide at concentrations of up to 15 μM stimulates the biosynthesis of chlorophyll a and total chlorophyll and increases the instantaneous carboxylation efficiency and, at concentration of 11 μM, alleviates the effect of water salinity up to 2.1 dS m ^-1 on the carotenoid contents of soursop plants cv. Morada Nova.

ACKNOWLEDGMENTS

To CNPq (National Council for Scientific and Technological Development) (Proc. 306245/2017-5 and 430525/2018-4), CAPES (Coordination for the Improvement of Higher Education Personnel), and UFCG (Federal University of Campina Grande).

REFERENCES

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