We observed that soybean is sensitive to salt stress induced by KCl, especially at osmotic potential from –0.79 MPa, as it impaired root protrusion and germination, in addition to indicators of seedling growth and biomass production. Conversely, we found that seed priming with Parachlorella sp. microalgae mitigated the stressful effect, resulting in seedlings with greater vigor, indicating its role in the inducing tolerance, confirming our initial hypotheses.
How does salinity become a limiting factor on the germination and vigor of soybean seedlings? In this stressful condition, the seed has difficulty uptake water and starting the germination phases, hindering the formation of normal seedlings. This is because excess chloride and potassium causes ionic, osmotic and oxidative stress, negatively affecting root protrusion as it has effects similar to water deficit (Zhang et al. 2018; Singh et al. 2022; Xiao and Zhou 2023), as observed in our study.
Under salt stress, water potential and turgor pressure are reduced, promoting water and osmotic stress (Arif et al. 2020; Muhammad et al. 2024), harming water relations, the driving force for energy production, in addition to causing toxicity in the process of embryogenesis and protein synthesis (Stofberg et al. 2015; Sabagh et al. 2021; Zhou et al. 2024). It is suggested that this condition may affect the metabolism of reserves for the embryonic axis, resulting in lower biomass accumulation, as observed in our study, especially at –1.2 MPa.
Under salinity conditions, toxic ions accumulate, causing ionic and oxidative stress, which alters the flow and influx of mineral elements and electrons, favoring the excessive increase in reactive oxygen species (ROS), such as superoxide anions and hydrogen peroxide (Saini et al. 2018; Zhao et al. 2024), not determined in our study, affecting cellular homeostasis. The presence of ROS promotes lipid peroxidation and triggers changes in cellular structures, resulting in damage to membranes, leaking electrolytes from cellular contents (Isayenkov and Maathius 2019; İbrahimova et al. 2021), as observed in our study with the reduction in osmotic potential, especially at –1.2 MPa, reinforcing that it a stressful condition.
Conversely, moderate salinity, represented here at values close to –0.8 MPa, stimulated the growth of soybean seedlings, and we suggest this response to the role of K+ from the saline source, since this element in small or ideal doses contributes on cellular structure and osmotic adjustments (Kumar et al. 2020; Johnson et al. 2022) relieving ionic stress by excess Cl– ions, but above –0.79 MPa, K+ also became a stressor.
We suggest that the cultivar NEO 610 IPRO presents tolerance to osmotic potentials ≤ –0.8 MPa. According to Zhou et al. (2024), salt tolerance occurs after hormonal signaling and gene transcription that activate different protective pathways through physiological or biochemical adjustments to restore osmotic homeostasis. However, the response to salt stress level varies depending on the genotype. For example, Alves et al. (2022) observed that the genotypes FTR-4389 and BRS Sambaíba RR are sensitive to salinity (–0.3 MPa, NaCl).
Conversely, under severe salt stress, in our study represented by –1.2 MPa, there was a decrease in germination and seedling vigor, demonstrating sensitivity of soybeans to this condition, but when treated with Parachlorella sp. microalgae, especially at a dose of 10 mL, we verified a beneficial effect on growth and biomass accumulation.
How can we explain that soybean seedlings from seeds that were primed with Parachlorella sp. microalgae even under salt stress did show better vigor? We associate these responses with the molecular composition present in the Parachlorella sp. biomass (Table 1), rich in amino acids, nutrients, and phytohormones. These new technological discoveries are promising, as in addition to alleviating the stress effect on soybeans and being low-cost, they also contribute to the SDG.
Although SP with microalgae did not have a direct positive effect on root protrusion and normal seedlings under salt stress, we noticed that the seeds that received 5 and 10 mL of Parachlorella sp. in non-stressful conditions they had higher protrusion values compared to those without priming, demonstrating a beneficial effect on the physiological quality of seeds for radicle emission. This result can be explained by the fact that microalgae contain gibberellic acid (GA).
In the physiology of germination, GA is fundamental, as in addition to participating in the balance of abscisic acid (Kozaki and Aoyanagi 2022), acts on the aleurone layer, activating hydrolytic enzymes such as amylases, releasing the nutritional reserves necessary for the growth of the embryo (Lovegrove and Hooley 2000; Alybayev et al. 2024), favoring germination, as observed in our study. This phenotypic response was especially observed in the first count of normal soybean seedlings. It is important, as it contributes to the start of plant development, being a possible mechanism for escaping stress. In addition, GA acts on cell growth, favoring radicle elongation (Shah et al. 2023).
The increase in length of both the root and shoot of seedlings from seeds with Parachlorella sp. is due to the presence of aspartate, glutamine, glutamate and tryptophan in its composition. The first three amino acids are closely linked to nitrogen assimilation routes by the plant, which contribute to vegetative growth (McAllister et al. 2016; Liao et al. 2022; Yin et al. 2022). Tryptophan is a precursor to auxin, a hormone that stimulates plant growth and rooting (Cohen et al. 2003; Tivendale et al. 2014). Additionally, the product also contains readily available mineral nutrients, especially N and P2O5, which can aid mineral metabolism, resulting in higher seedlings.
The highest values of biomass production using especially 10 mL of microalgae are associated with energy performance due to the participation of adenosine, ketoglutarate, and pyruvate. These compounds participate in respiratory and photosynthetic metabolism, which act in the production of energy substrates and to help in respiration (Tanaka et al. 2010; Legendre et al. 2020; Pietrowska-Borek et al. 2020; Le et al. 2022), reflecting in carbohydrate accumulation.
In addition, better responses of soybean seedlings previously treated with the Parachlorella sp. under salt stress are owing to the various quantified amino acids, which act in protective pathways against osmotic and oxidative stress, participating in the detoxification of ROS, relieving the stressor. In general, amino acids such as proline, phenylalanine, glutamate, methionine, lysine and serine, among others, play a role in the regulatory complexes of enzymatic and non-enzymatic protective metabolism, in gene transcription and the synthesis of chlorophylls and proteins (Forde and Lea 2007; Kishor et al. 2020; Yang et al. 2020).
Proline and phenylalanine, for example identified in the Parachlorella sp. biomass, act in redox buffering and combat ROS, in addition to being sources of other protective compounds, they favor water balance, the integrity of membranes, becoming osmoregulatory solutes (Hossain et al. 2021; Kumari et al. 2023). It is noteworthy that priming facilitated this mitigating potential, in which the seeds absorb and metabolize the constituents present in the studied microalgae in advance of exposure to salt stress.
Based on our results, we accept the presupposed hypotheses, in which priming with Parachlorella sp. microalgae is a promising microorganism in the inducing soybean tolerance to severe salinity. In future perspectives, we suggest work evaluating enzymatic activity under these tested conditions, verifying increasing information about the protective mechanisms induced by microalgae in soybean.