Photosynthetic Pigments and Photochemical Efficiency in Soursop under Saline Water Irrigation and Nitrogen Sources

The Studies have shown that nitrogen fertilization can mitigate the effects of saline stress on plants, however, the efficiency may vary according to the source of nitrogen used, specifically on the photosynthetic activity. In this context, the objective of this work was to evaluate the effect of different nitrogen sources on photosynthetic pigments and photochemical efficiency in soursop plants irrigated with distinct salinities. A randomized complete block design was used in a 4 × 4 factorial scheme, with the treatments corresponding to four levels of irrigation water salinity ECw (0.5, 1.5, 2.5 and 3.5 dS m) and four sources of nitrogen (urea, ammonium sulfate, calcium nitrate and potassium nitrate), with three replications. The irrigation with saline water from 0.5 dS m inhibits the concentration of photosynthetic pigments and promotes damages in the photochemical efficiency of photosynthesis at 110 days after transplanting. The different sources of nitrogen do not cause changes in the levels of photosynthetic pigments, however, the fertilization with potassium nitrate mitigates the effect of saline stress on the photochemical efficiency, mainly in the water salinity of 3.5 dS m.


Introduction
Soursop (Annona muricata L.) is one of the most cultivated species of the Annonaceae family in regions of tropical climate, where it adapts well to the edaphoclimatic conditions (Cavalcanti Junior et al., 2010), and the increasing demand for the pulp its fruit in internal and external markets has stimulated its exploitation by fruit growers (Rozane & Natale, 2014).
In semi-arid regions, crop management depends on irrigation, in many cases with high-salinity water, which consists in the greatest limitation to the establishment of this fruit crop, compromising its nutritional balance, physiology, growth and production (Távora et al., 2004).
Evaluation of photosynthetic pigments and fluorescence parameters allows to assess the effect of various types of stress on photosynthesis, including salinity, which reduces photosynthetic rate and electron transport, disorders driven by light through the emission of heat or fluorescence (Azevedo Neto et al., 2011;Baker, 2008), which are related to chlorophyll degradation by salt stress (Munns & Tester, 2008) and reduction in the absorption and use of light energy through photosystem II, during the photochemical reactions of photosynthesis (Baker, 2008).Nitrogen (N) has structural function in the plant, constituting amino acids, N bases, many enzymes and energy-transfer materials such as chlorophyll and proteins of the electron transport chain in chloroplasts (Taiz & Zeiger, 2013).In soursop, N deficiency directly affects physiology, with gradual loss of green color and premature fall of leaves, which often negatively affect crop growth and development (São José et al., 2014).
Ammonium sulfate and urea are the most used N sources in the Brazilian agriculture, possibly because of their lower cost and greater availability in the market (Binotti et al., 2010).However, there are reports that the fertilization with calcium nitrate and potassium nitrate reduces salt stress effects on plants (Andrade Júnior et al., 2011;Fernandes et al., 2010).
Hence, this study aimed to evaluate the effect of different N sources on the photosynthetic pigments and photochemical efficiency in soursop plants irrigated with solutions of different saline levels.

Experiment Localization and Treatments
The experiment was carried out from August to December 2016 in a greenhouse, arc model (width = 12 m, length = 25 m and right-foot height = 4 m), covered with a 100 micron plastic film, having on the sides a shading screen of 50% luminosity, at the Center of Technology and Natural Resources (CTRN) of the Federal University of Campina Grande (UFCG), in Campina Grande-PB, Brazil (7°12′88″ S, 35°54′40″ O; 532 m).According to the climatic classification of Köeppen, adapted to Brazil (Coelho & Soncin, 1982), the climate of the region is of type Csa, which represents mesothermal climate, subhumid; average annual precipitation of 802.7 mm and average temperature of 23.5 °C (IMMET, 2017).
Were studied 16 treatments arranged in a 4 × 4 factorial scheme, corresponding to four levels of irrigation water salinity -ECw (0.5; 1.5; 2.5 and 3.5 dS m -1 ) and four N sources (urea, ammonium sulfate -AS, calcium nitrate -CN and potassium nitrate -KN), distributed in randomized blocks, with three replicates, and the experimental unit consisted in a pot with one plant, totalizing 48 plots in the experiment.
The N dose applied through the different fertilizers, as well as phosphate and potassium fertilizations, were based on the recommendations for pot experiments (Novais et al., 1991), using 100, 300 and 150 mg kg -1 of N, P 2 O 5 and K 2 O, respectively.Phosphate fertilization was performed by incorporating single superphosphate to the soil in the planting hole, whereas N and K (potassium chloride) fertilizations were split into six applications in equal parts, by the dilution of each fertilizer in 150 ml of rainwater, manually applied in soil surface, starting 20 days after transplantation (DAT), followed by applications every 10 days.
Saline solutions with ECw of 0.5 and 1.5 dS m -1 were prepared by mixing rainwater (0.02 dS m -1 ) with water from the public supply system (1.7 dS m -1 ), whereas solutions with ECw of 2.5 and 3.5 dS m -1 were prepared through the addition of commercial iodine-free NaCl salts.

Plant Material and Management of the Experiment
The soursop variety 'Nordestina' was used in the experiment and its seedlings were obtained by sowing in 288 cm 3 plastic tubes containing substrate composed of soil and humus (2:1 proportion).The seedlings were transplanted 86 days after sowing, when they had four true leaves, fully expanded, to 22 dm 3 pots containing 20 kg of soil, whose physical and chemical characteristics (Table 1) were analyzed according to Claessen (1997).The planting pots had a layer of crushed stone (n o 0) at the bottom, involved by a nonwoven geotextile (Bidim OP 30), connected to a drain and a hose (20 mm diameter), which allow to monitor the drained volume and estimate water consumption by the crop.
Saline solutions began to be applied at 10 DAT and the different treatments were irrigated considering the estimate of crop water consumption, based on the drainage lysimetry principle (Bernardo et al., 2006), using as lysimeters, the pots of the experimental plots.At 60 DAT, a leaching fraction of 0.15 was applied based on the volume applied in this period, in order to minimize the accumulation of salts in the soil.
Photosynthetic pigments were determined in the fifth fully expanded leaf, from the apical bud to the base of the plant.A cork borer was used to collect a plant tissue disc from the middle third of the leaf blade, with area of 3.14 cm 2 .After that, the material was chopped and immersed in 6 cm 3 of acetone at 80% in 10 cm 3 glass containers, where the samples remained in full darkness for 48 hours in a refrigerator at temperature of 8 °C to extract the pigments from the supernatant.Subsequently, contents of chlorophyll a, chlorophyll b, total chlorophyll and carotenoids were quantified through spectrophotometry with absorbance (A) readings respectively at the wavelengths of 663, 646 and 470 nm, using 80% acetone as blank, according to Equations 1, 2, 3 and 4, following the methodology of Lichtenthäler (1987): The data were obtained in mg L -1 and, based on the leaf disc area (3.14 cm 2 ) and on extract dilution in 6 cm 3 (6 mL) of 80% acetone, CHLa, CHLb and CAR contents were transformed to units of mass per m 2 (g m -2 ).
Photochemical efficiency was measured through chlorophyll a fluorescence parameters, using the Handy PEA device (Hansatech), by attaching leaf clips to the third fully expanded leaf, from the apex to the base of the plant, which remained for 30 minutes to dark adapt the leaves before the readings.Basal quantum yield of photosystem II (Fo/Fm) was established by the ratio between Fo and Fm (Rohácek, 2002).

Statistical Analysis
The obtained data were subjected to analysis of variance by F test at 0.05 and 0.01 probability levels.In cases of significance, regression analysis was used for the factor saline levels and means comparison test (Tukey) for N sources, using the statistical software SISVAR/UFLA (Ferreira, 2011).The regression model was selected considering the best fit based on the coefficient of determination (R 2 ).

Results and Discussion
Irrigation water salinity had significant effect (p < 0.01) on the contents of CHLa, CHLb, CHLtotal, CAR, CHLa/CHLb and CHLtotal/CAR (Table 2).However, there was no significance of N sources or significant interaction (p > 0.05) between the factors irrigation water salinity and N sources for the studied variables.
Under these conditions, light harvesting efficiency may be reduced because these pigments capture energy at different wavelengths and transfer it to the reaction center, which effectively acts in the photochemical reactions of photosynthesis (Freire et al., 2013).In addition, plant vulnerability to oxidative stress increases, because carotenoids also act as antioxidant agents, protecting membrane lipids of reactive oxygen species in plants exposed to salinity (Falk & Munné-Bosch, 2010).
Deleterious effect of salt stress on the contents of CHLa, CHLb, CHLtotal and CAR at ECw levels from 0.5 to 4.5 dS m -1 have been observed in passion fruit (Cavalcante et al., 2011;Freire et al., 2013) and tomato (Tatagiba et al., 2014).
According to the analysis of variance summary (Table 3), there was significant interaction (p < 0.05) between the factors irrigation water salinity and N sources for Fm, Fv, Fv/Fm and Fo/Fm.Note.ns, non-significant; *, ** significant at p ≤ 0.05 and p ≤ 0.01; SV = Sources of variation; FD = Freedom degree; CV = coefficient of variation.
In the follow-up analysis of water salinity levels for each N source (Figure 2A), Fm linearly decreased by 5.32 and 5.61% per unit increase in ECw in plants fertilized with urea and ammonium sulfate (AS), respectively.For plants fertilized with calcium nitrate (CN), Fm data fitted best to the quadratic model, with maximum value (772.1 electrons quantum -1 ) obtained at ECw of 1.2 dS m -1 .In plants fertilized with potassium nitrate (KN), the Fm of chlorophyll a was not affected by the increment in irrigation water salinity, showing mean value of 746.5 electrons quantum -1 .
According to the follow-up analysis of N sources for each level of irrigation water salinity (Figure 2B), significant difference was found in Fm between the different sources only in plants irrigated using solutions with higher saline levels (2.5 and 3.5 dS m -1 ).For the irrigation with ECw of 2.5 dS m -1 , highest Fm values (750 and 740 electrons quantum -1 ) were caused by the fertilization with calcium nitrate (CN) and potassium nitrate (KN), whereas plants subjected to ECw of 3.5 dS m -1 showed maximum Fm (709.7 electrons quantum -1 ) under KN fertilization. Figure Figur ammon nitrogen s

Table 3 .
Summary of the analysis of variance analysis of variance for maximum fluorescence (Fm), variable fluorescence (Fv), quantum efficiency of photosystem II (Fv/Fm) and basal quantum yield of photosystem II (Fo/Fm) in soursop plants, 'Nordestina' variety, irrigated with solutions of different saline levels and fertilized with different nitrogen sources at 110 days after transplantation