Physiological and biochemical responses of Annona squamosa genotypes to progressive salinity stress levels

Introduction

Salinity is a major setback in most of the fruit crops. Over 20 percent of the world's irrigated land is already compromised by salinity, and this number is rising at a rate of 10 percent annually (Shrivastava and Kumar, 2015) [30] . There are more underutilized fruit crops that possess a high level of health benefits. Among those, custard apple is one that belongs to the Annona genus, and it is widely distributed in the wild worldwide. Native to the Caribbean, Central America, and northern South America, this fruit has gained global recognition not only for its sweet, creamy pulp but also for its numerous health benefits and medicinal properties. (Martínez et al., 2013; Gautam et al., 2024) [22,13] . It is also a powerhouse of essential nutrients, including vitamins, minerals, and antioxidants. It is particularly rich in vitamin C, a potent antioxidant that supports the immune system, helps combat free radicals, and promotes skin health. The fruit also contains significant amounts of dietary fibre, which aids in digestion and helps prevent constipation. The mineral content of custard apple is also noteworthy, with high levels of potassium, magnesium, and calcium, all of which are essential for maintaining heart health, bone density, and muscle function. Moreover, the fruit contains beneficial compounds such as polyphenols, flavonoids, and carotenoids, which contribute to its antioxidant and anti-inflammatory properties (Gautam et al., 2024) [13] . Annonaceae is found in the tropics and subtropics of both the Old and New Worlds, well-adapted to altitudes below 1,000 msl and to annual rainfall of more than 1,000 mm, with a dry period during flowering. It is adapted to both tropical and subtropical areas. Fruits of all Annona spp. are climacteric with a rapid ripening after harvest. Besides their use in fruit production, Annona spp. are valued for their essential oils, insecticidal compounds, medicinal compounds, and wood (Leal and Paull, 2023) [17] . Salinity stress drastically reduces crop yields, affecting plant growth and restricting land use. As the world population is increasing at an alarming rate, agricultural land is shrinking due to industrialization and habitat use.

Hence, there is a need to utilize salt-affected land to meet food requirements. Excessive salt above what plants need limits plant growth and productivity and can lead to plant death (Safdar et al., 2019) [27] . Increased chloride concentration and decreased nitrogen concentration near the root zone. The sodium response indicated that some form of exclusion or compartmentation occurred. Salinity reduced the osmotic potential of root tissue but did not influence foliar osmotic or predawn xylem potential. These results indicate that A. squamosa is sensitive to salinity stress and that its responses to salinity are consistent with those of other salt-sensitive woody perennial species (Marler et al., 1996) [21] . Even though Annona is a popular dryland fruit crop, little attention has been given to the physiological response under abiotic stress conditions, particularly salinity. The present study aimed to evaluate the effects of salt stress on physiological and growth parameters of Annona squamosa, a locally available species in India. This may help breeders to develop abiotic stress tolerance in Annona squamosa varieties/hybrids.

Materials and Methods

Experimental Location

The research study was conducted on "Physiological and biochemical responses of Annona squamosa genotypes to progressive salinity stress levels" during 2025-2026 at the North Farm Polyhouse, Karunya Institute of Technology and Sciences, Coimbatore, Tamil Nadu, India, from December to February.

Collection of plants

A salinity tolerance screening experiment will be conducted using selected genotypes of Annona, obtained from diverse agro-climatic regions, to ensure genetic variability. Seedlings will be procured from certified nurseries located in and around Tamil Nadu to ensure genetic authenticity and quality of planting material. Healthy, uniform seedlings of each genotype will be transplanted into plastic bags filled with a well-drained potting mixture of soil, sand, and farmyard manure in a 2:1:1 ratio.

Application of Treatment

Five salinity levels were established using sodium chloride (NaCl) solutions at EC values of 3.5, 4.0, 4.5, 5.0, and 5.5 dS m⁻¹, with the 3.5 dS m⁻¹ treatment serving as the reference benchmark, representing a mildly saline condition. Salinity stress was imposed 60 days after transplanting by irrigating with NaCl-supplemented water to achieve these predetermined electrical conductivity (EC) levels, as measured using an EC meter, and maintained consistently throughout the experimental period. The physiological and biochemical responses of Annona squamosa genotypes were then evaluated against progressively elevated salinity stress conditions.

Plant height was measured using a measuring scale from the soil level to the tip of the largest leaf. The average is taken from three replications and expressed in cm.

Chlorophyll Concentration

The amount of chlorophyll concentration in leaves after the 60th day of treatment imposition is measured using a digital SPAD meter (SPAD-502 Plus).

A measure of membrane injury via ion leakage from tissue (Lutts et al., 1996) [19] . Twenty discs (1 cm²) of different Annona genotype leaves were put into flasks, each containing 25 mL of deionized water. The samples were shaken for 20h at ambient temperature. First, electrical conductivity (C 1 ) was recorded for each vial. Flasks were then submerged in a hot water bath at 80 °C for 1 hour. The vials were again shaken for 20 hours at 21 °C. Last, conductivity (C 2 ) was measured for each flask (Dionisio-Sese and Tobita, 1998) [10] . It was then calculated by using the formula.

conductivity after boiling

Relative water content (RWC) is a critical physiological trait that indicates a plant's ability to maintain water balance und er stress conditions. Different Annona genotypes of leaf discs (1 cm in diameter) were taken from the fully expanded leaves. The fresh weight (FW) was recorded, and then the discs were incubated in distilled water for 4 h. Turgid weight was recorded, and the discs were put in an oven at 70 °C for 24 h to calculate the dry weight (DW). Relative water content was measured by the methodology of (Sanchez et al., 2004) [28] . Then it was calculated by using the formula. The measure of quantification of free proline as an osmoprotectant under stress. To measure proline accumulation, a solution of glacial acetic acid, proline, and ninhydrin acid (1:1:1) was incubated for 1 h at 90 °C. Then, the samples were cooled in an ice bath. The extraction was done using 2 mL of toluene, and the absorbance was determined at 520nm using a spectrophotometer as µg g⁻¹ FW (Bates et al., 1973) [4] . It was then calculated by using the formula.

Abs 520 × ml toluene 115.5 × g sample

Statistical Analysis

The experimental design followed was Factorial Randomized Block Design (FRBD) with genotypes and salinity levels as factors, replicated three times. Consisting of four genotypes (Balanagar, Golden Sita, Red Sita, Nattu Sita) and five salinity levels (3.5, 4.0, 4.5, 5.0 dS m -1 ) respectively. All obtained data were subjected to an ANOVA analysis using OPSTAT, and significant differences between treatments were indicated by p<0.05.

Results and Discussion

Chlorophyll Concentration 60 days after the imposition of salinity, the concentration of chlorophyll was measured. Table 1 shows that NMK Golden (V 3 ) outperformed all other Annona squamosa varieties, with the highest chlorophyll concentration (39.619) and the lowest (27.999) recorded in Nattu Sita. According to Table 2, the low EC concentration of 3.5 dS m⁻¹ (T 1 ) had the highest chlorophyll concentration (78.698), while the high EC concentration of 5.5 dS m⁻¹ (T 5 ) had the lowest (66.933). Table 3 depicts the combination effects of Annona varieties with different levels of EC concentrations, and the rate of reduction was calculated from the treatment combinations. Table 4 shows that a lower rate of reduction percentage of chlorophyll concentration (3.31%) is obtained in the treatment combinations of the NMK Golden variety compared to all other combinations. The above findings show that an increase in the EC concentrations led to a decrease in the chlorophyll concentration, thus proving that salinity affects the chlorophyll concentration. The presence of salts in the soil solution may favour increased chlorophyll content in salt-tolerant plants and decreased chlorophyll content in more susceptible plants, and the chlorophyll index reduction may be associated with reactive oxygen species formation and with the photoinhibition of photosynthesis (Heidari, 2012) [14] . These results are in line with Figueiredo et al. (2019) [12] , who reported higher chlorophyll indexes at 0.5 dS m⁻¹ and lowest values at 4.0 dS m⁻¹ in sugar-apple plants, and Ferreira et al. (2021) [11] , who observed the highest Chlorophyll total concentration (2.74 mg g⁻¹ FM) at the lowest electrical conductivity (0.8 dS m⁻¹). The reduction in chloroplast pigment concentrations occurs due to degradation of pigment molecules and reduction in the chlorophyll synthesis process induced by high salinity conditions, which was further corroborated by Oliveira et al. (2018) [25] , who reported reductions of up to 27.43% in total Chlorophyll concentrations in jackfruit plants irrigated with waters of EC 4.0 dS m⁻¹.

According to Table 1, NMK Golden (V 3 ) outperformed all other Annona squamosa types, with the lowest electrolyte leakage (32.074%) and the greatest (78.124%) recorded in Nattu Sita. Table 2 demonstrates that a rise in EC concentrations caused an increase in electrolyte leakage, with the lowest electrolyte leakage (56.040%) observed in low EC concentration of 3.5 dS m⁻¹ (T 1 ) and the highest electrolyte leakage (66.943%) observed in high EC concentration of 5.5 dS m⁻¹ (T 5 ). The combination effects of Annona types with varying EC concentrations are shown in Table 3, and the rate of reduction was determined from the treatment combinations. Table 4 demonstrates that, in comparison to all other combinations, the Red Sita variety treatment combinations yield a reduced rate of increase in the percentage of electrolyte leakage (5.28%). The above findings show that an increase in the EC concentrations led to an increase in the electrolyte leakage, thus proving that salinity results in increased membrane damage. Plasma membranes are the primary site of ion-specific salt injury (Mansour and Salama, 2004) [20] , and electrolyte leakage is reported as one of the most important selection criteria for identification of salt-tolerant plants (Ashraf and Ali, 2008) [2] , with a cell deemed damaged when electrolyte leakage surpasses 50%. It was also observed that relative water content correlated positively with all variables, except electrolyte leakage (93%), further confirming the inverse relationship between water retention and membrane damage under salinity stress (Edmilson Júnio Medeiros Caetano et al., 2024) [5] . These findings are in line with Santos et al.

(2023) [29] and Lima et al. (2023) [9] , who reported increased electrolyte leakage with increasing salinity levels in passion fruit plants, and similarly, Hniličková et al. (2019) [15] observed that electrolyte leakage progressively increased with increasing NaCl concentrations in lettuce and other plant species.

Table 1 shows that Red Sita (V 4 ) outperformed all other Annona squamosa varieties, with the highest relative water content (83.456%) and the lowest (58.840%) recorded by Nattu Sita. According to Table 2, the low EC concentration of 3.5 dS m⁻¹ (T 1 ) had the highest relative water content (78.698%), while the high EC concentration of 5.5 dS m⁻¹ (T 5 ) had the lowest (66.933%). Table 4 demonstrates that treatment combinations of the NMK Golden variety had the highest rate of reduction percentage of relative water content (3.11%) when compared to all other combinations. This suggests that an increase in EC concentrations caused a decrease in relative water content, demonstrating that salinity has an impact on plants' relative water content. These findings are in line with Santos et al. (2023) [29] , who reported that irrigation with water having an electrical conductivity of 0.6 dS m⁻¹ induced a reduction in relative water content and gas exchange in sour passion fruit plants, and Lima et al. (2023) [9] , who highlighted that irrigation with water EC of 0.3 dS m⁻¹ or higher reduces the relative water content in passion fruit plants. It was also observed that relative water content significantly decreased (62 and 50%) at salinity levels of 1500 and 3000 ppm of NaCl compared to controls (82%) in sweet pepper plants (Abdelaal et al., 2020) [1] . The decreased relative water content occurs as a result of the negative effect of salinity on water absorption capacity from the soil to the root system, reducing the internal water supply and negatively affecting plant organs (Liang et al., 2002; Muneer et al., 2014) [18,23] .

Proline Accumulation (μmolg -1 FW) Table 1 shows that NMK Golden (V 3 ) outperformed all other Annona squamosa varieties, with the highest proline accumulation (0.143 μmol g⁻¹ FW) and the lowest (0.044 μmol g⁻¹ FW) observed in Nattu Sita (V 1 ). According to Table 2, the low EC concentration of 3.5 dS m⁻¹ (T 1 ) had the lowest proline accumulation (0.075 μmol g⁻¹ FW), whereas the high EC concentration of 5.5 dS m⁻¹ (T 5 ) had the highest proline accumulation (106 μmol g⁻¹ FW). Table 4 shows that the highest rate of increase in the percentage of proline accumulation (23.06%) was obtained in treatment combinations of NMK Golden variety compared to all other combinations, indicating that an increase in EC concentrations led to an increase in proline accumulation, thus proving the positive effects of proline accumulation under salinity conditions. Proline plays multiple roles as an osmolyte, scavenging reactive oxygen species (ROS), controlling cell redox homeostasis, and supplying energy (Dawood et al., 2014) [8] , and this increment of proline is one of the typical responses to stress factors, particularly salinity stress, which plays a pivotal role in protecting chlorophyll pigments from degradation (Yin et al., 2013) [33] . These findings are in line with Abdelaal et al. (2020) [1] , who reported that proline concentrations (18 and 24 µg g⁻¹ FW) significantly increased in sweet pepper plants under salinity conditions compared to controls (11 µg g⁻¹ FW), further corroborating the present findings.

Conclusion

The study on physiological and biochemical responses of Annona squamosa genotypes to progressive salinity stress levels revealed that salinity stress significantly influenced all the biochemical and physiological parameters assessed. An increase in EC concentrations consistently led to a decrease in plant height, chlorophyll concentration, and relative water content, while proline accumulation increased with increasing salinity levels, proving that proline plays a positive and protective role in plants against salinity stress conditions. Among all the genotypes evaluated, NMK Golden performed the best under salinity stress, exhibiting higher proline accumulation and better chlorophyll retention compared to Balanagar, Red Sita, and Nattu Sita, indicating a stronger osmotic adjustment mechanism and superior salinity tolerance. These findings confirm that genotypic variation exists among Annona squamosa varieties in their response to salinity stress. Therefore, future research can focus on utilizing NMK Golden as a potential parent in breeding programs aimed at developing salinity-tolerant Annona squamosa varieties, as well as exploring its use as a suitable rootstock in propagation studies to confer salinity tolerance to other genotypes. Such studies will help in utilizing salt-affected lands for Annona cultivation, ultimately contributing to enhanced fruit production and improved livelihood of farmers.

Acknowledgement

AcknowledgementThe authors express their gratitude to the School of Agricultural Science, Karunya Institute of Technology and Sciences, for their assistance and provision of the facilities needed to complete this research work.

References

1. Abdelaal KA, Mazrou YS, Hafez YM. Silicon foliar application mitigates salt stress in sweet pepper plants by enhancing water status, photosynthesis, antioxidant enzyme activity and fruit yield. Plants. 2020;9(6):733733.
2. Ashraf MA, Ashraf M. Growth stage-based modulation in physiological and biochemical attributes of two genetically diverse wheat (Triticum aestivum L.) cultivars grown in salinized hydroponic culture. Environmental Science and Pollution Research. 2016;23(7):6227-6243.
3. Ashraf M, Ali Q. Relative membrane permeability and activities of some antioxidant enzymes as the key determinants of salt tolerance in canola (Brassica napus L.). Environmental and Experimental Botany. 2008;63(1-3):266-273.
4. Bates LS, Waldren RP, Teare ID. Rapid determination of free proline for water-stress studies. Plant and Soil. 1973;39:205-207.
5. Caetano EJM, Silva AARD, Lima GSD, Azevedo CAVD, Veloso LLDSA, Arruda TFDL, et al. Application techniques and concentrations of ascorbic acid to reduce saline stress in passion fruit. Plants. 2024;13(19):2718-2718.
6. Chima UD, Fredrick C, Amaogu DC. Salinity tolerance of two seedling growth stages of Annona muricata L. in Port Harcourt, Nigeria. African Journal of Agriculture Technology and Environment. 2019;8(1):164-176.
7. Dantas BF, Ribeiro LDS, Aragão CA. Physiological response of cowpea seeds to salinity stress. Revista Brasileira de Sementes. 2005;27(1):144-148.
8. Dawood MG, Taie HAA, Nassar RMA, Abdelhamid MT, Schmidhalter U. The changes induced in the physiological, biochemical and anatomical characteristics of Vicia faba by the exogenous application of proline under seawater stress. South African Journal of Botany. 2014;93:54-63.
9. de Lima LA, Machado DV, e Silva CR, de Rezende CE, Marques ED, Silva-Filho EV. Using geochemical and isotopic signature to investigate the salinity origins and groundwater evolution in aquifers from the emerged portion of the Campos Basin, Brazil. Environmental Earth Sciences. 2025;84(21):634-634.
10. Dionisio-Sese ML, Tobita S. Antioxidant responses of rice seedlings to salinity stress. Plant Science. 1998;135:1-9.
11. Ferreira FN, Lima GSD, Gheyi HR, Sá FVDS, Dias AS, Pinheiro FW. Photosynthetic efficiency and production of Annona squamosa L. under salt stress and fertilization with NPK. Revista Brasileira de Engenharia Agrícola e Ambiental. 2021;25(7):446-452.
12. Figueiredo FRA, de Melo Gonçalves AC, da Silva Ribeiro JE, da Silva TI, Nóbrega JS, Dias TJ, et al. Gas exchanges in sugar apple (Annona squamosa L.) subjected to salinity stress and nitrogen fertilization. Australian Journal of Crop Science. 2019;13(12):19591966.
13. Gautam DK, Singh V, Singh SK, Tiwari P, Verma S, Tripathy S, et al. Custard apple (Annona squamosa L.): Exploring its health benefits and medicinal properties. European Journal of Nutrition and Food Safety. 2024;16(10):129-140.
14. Heidari M. Effects of salinity stress on growth, chlorophyll content and osmotic components of two basil (Ocimum basilicum L.) genotypes. African Journal of Biotechnology. 2012;11(2):379-384.
15. Hniličková H, Hnilička F, Orsák M, Hejnák V. Effect of salt stress on growth, electrolyte leakage, Na⁺ and K⁺ content in selected plant species. Plant, Soil and Environment. 2019;65(2):90-96.
16. Jamil M, Bashir S, Anwar S, Bibi S, Bangash A, Ullah F, et al. Effect of salinity on physiological and biochemical characteristics of different varieties of rice. Pakistan Journal of Botany. 2012;44(1):7-13.
17. Leal F, Paull RE. The genus Annona: Botanical characteristics, horticultural requirements and uses. Crop Science. 2023;63(3):1030-1049.
18. Liang S, Zhou R, Dong S, Shi S. Adaptation to salinity in mangroves: Implication on the evolution of salttolerance. Chinese Science Bulletin. 2008;53(11):17081715.
19. Lutts S, Kinet JM, Bouharmont J. NaCl-induced senescence in leaves of rice (Oryza sativa L.) cultivars differing in salinity resistance. Annals of Botany. 1996;78(3):389-398.
20. Mansour MMF, Salama KH. Cellular basis of salinity tolerance in plants. Environmental and Experimental 2004;52(2):113-122.
21. Marler TE, Zozor Y. Salinity influences photosynthetic characteristics, water relations, and foliar mineral composition of Annona squamosa L. Journal of the American Society for Horticultural Science. 1996;121(2):243-248.
22. Martínez F, Miranda D, Magnitskiy S. Anatomy of sugar apple (Annona squamosa L.) seeds (Annonaceae). Agronomía Colombiana. 2013;31(3):279-287.
23. Muneer S, Park YG, Manivannan A, Soundararajan P, Jeong BR. Physiological and proteomic analysis in chloroplasts of Solanum lycopersicum L. under silicon efficiency and salinity stress. International Journal of Molecular Sciences. 2014;15(12):21803-21824.
24. Nawaz K, Hussain K, Majeed A, Khan F, Afghan S, Ali K. Fatality of salt stress to plants: Morphological, physiological and biochemical aspects. African Journal of Biotechnology. 2010;9(34):5475-5480.
25. Oliveira CRDR, De Oliveira VQ, Pimentel OALF, Dos Santos EP, de Oliveira Filho PRC, Galvez AO, et al. Growth performance and proximate composition of Penaeus vannamei reared in low-salinity water with different ionic compositions in a synbiotic system. Aquaculture International. 2022;30(6):3123-3141.
26. Rawat KS, Mishra AK, Sehgal VK, Tripathi VK. Spatial variability of ground water quality in Mathura district (Uttar Pradesh, India) with geostatistical method. International Journal of Remote Sensing Applications. 2012;2(1):1-9.
27. Safdar H, Amin A, Shafiq Y, Ali A, Yasin R, Shoukat A, et al. A review: Impact of salinity on plant growth. Natural Science. 2019;17(1):34-40.
28. Sanchez FJ, De Andres EF, Tenorio JL, Ayerbe L. Growth of epicotyls, turgor maintenance and osmotic adjustment in pea plants (Pisum sativum L.) subjected to water stress. Field Crops Research. 2004;86(1):8190.
29. Santos LFS, de Lima GSD, de Lima VLA, da Silva AAR, Soares LADA, Nóbrega AS, et al. Foliar application of proline on the mitigation of salt stress in the physiological indices of sour passion fruit. Plants. 2023;12.
30. Shrivastava P, Kumar R. Soil salinity: A serious environmental issue and plant growth promoting bacteria as one of the tools for its alleviation. Saudi Journal of Biological Sciences. 2015;22(2):123-131.
31. Taffouo VD, Nouck AH, Dibong SD, Amougou A. Effects of salinity stress on seedlings growth, mineral nutrients and total chlorophyll of some tomato (Lycopersicum esculentum L.) cultivars. African Journal of Biotechnology. 2010;9(33):5366-5371.
32. Wanderley JAC, Brito MEB, Azevedo CAVD, Silva FDC, Ferreira FN, Lima RFD. Cell damage and biomass of yellow passion fruit under water salinity and nitrogen fertilization. Revista Caatinga. 2020;33:757765.
33. Yin L, Wang S, Li J, Tanaka K, Oka M. Application of silicon improves salt tolerance through ameliorating osmotic and ionic stresses in the seedling of Sorghum bicolor. Acta Physiologiae Plantarum. 2013;35(11):3099-3107.