Peptides from Swine Blood Enhance Salinity Stress Tolerance in Sweet Potato (Ipomoea batatas (L.) Lam) Through Osmotic Adjustment and Maintenance of Cellular Redox Homeostasis

Abstract

Sweet potato (Ipomoea batatas (L.) Lam) is an important food and energy crop. Soil salinization is a major abiotic stress that limits agricultural productivity and severely reduces yield of crops. Protein hydrolysates, as a class of natural biostimulants, have gained increasing attention for their potential to improve crop yield, quality and stress tolerance. This study investigated the effects of peptides from swine blood (PSB) on high salinity stress tolerance in sweet potato. Application of PSB promoted the growth of both aerial and underground parts of sweet potato under normal and high-salinity conditions. Further analysis revealed that, under high salinity stress, exogenous PSB up-regulated the expression of genes associated with stress responses, increased the accumulation of organic osmotic adjustment compounds such as free amino acids, promoted K⁺ uptake to elevate the K⁺/Na⁺ ratio, and enhanced the activity of key antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) involved in the reactive oxygen species-scavenging system. These biochemical responses contributed to maintaining cellular osmotic balance and redox homeostasis, protecting the cell membrane from damage while preserving its structural integrity and normal physiological functions, and improving photosynthetic efficiency, thereby enhancing high salinity stress tolerance in sweet potato. Thus, PSB holds significant potential as an effective natural biostimulant for sweet potato cultivation in saline soils.

1. Introduction

Soil salinization, accounting for about 20% of the irrigated agricultural lands around the world, is a major environmental limitation to crop production [1]. Among all types of salinity, the most soluble and widespread salt is sodium chloride (NaCl). It is expected that, by the middle of the 21st century, the rate of lost arable land might rise to 50% [2]. On the other hand, the demand for food has increased sharply to meet the rapidly expanding human population [3]. Meanwhile, the climate changes have become particularly widespread [4]. Thus, high salinity stress will threaten the long-term food security in the future [5].

High salinity stress can induce two primary adverse influences including osmotic stress and ion toxicity [6]. This is due to the limited water and nutrient uptake caused by the high sodium concentrations in saline soils [7]. Meanwhile, high salinity stress can result in the excessive accumulation of reactive oxygenic species (ROS), which induce the oxidative damage of lipids, proteins and nucleic acids [8]. As a result, numerous plant growth processes such as seed germination, flowering and root development are damaged under salt stress conditions [9]. During the long period of evolution, crops have formed a series of mechanisms to ensure their survival in high salinity stress [8]. The molecular mechanisms of plant salt tolerance include several processes such as stress perception, signal transduction and metabolic responses [10]. Salt stress signals including excess Na⁺, intracellular Ca⁺ and reactive oxygen species (ROS) sense environmental stimuli and trigger downstream responses [11]. Subsequently, crops go through several physiological changes to enhance high salinity stress tolerance. Salt excretion, salt dilution, salt accumulation and salt exclusion are four major methods to protect crops from salt injury [12]. All higher plants can follow physiological regulation including osmotic adjustment and ROS scavenging to enhance high salinity stress tolerance [13]. Osmotic regulators include two main categories: inorganic ions and organic substances. Under high salinity stress, these osmotic regulators themselves can act as osmolytes or take part in stabilizing the structure of biological molecules to enhance osmotic stress tolerance [14]. ROS are common by-products of aerobic metabolism that stay at a low level under normal conditions. Under a stressful environment, the overgeneration of ROS may cause oxidative damage to plants [15]. Plants have developed two different kinds of ROS-scavenging systems called enzymatic and non-enzymatic antioxidants [16]. It can be speculated that the activation of osmotic adjustment and ROS scavenging will enhance high salinity stress tolerance in crops.

Amino acids play important roles in plant growth regulation as precursors for the synthesis of secondary metabolites and signaling molecules [17]. It has been demonstrated that both endogenous and exogenous amino acids could regulate the response to salt stress [18,19]. Although genetic engineering could change the level of endogenous amino acids to affect the salt tolerance in crops, more eco-friendly plant growth-regulating substances are required to achieve the goal of agricultural sustainability. Protein hydrolysates contain a high level of free amino acid and peptides that have positive effects on abiotic stress tolerance in crops [20]. Recently, several studies indicated that the application of protein hydrolysates could enhance the salt stress tolerance in different kinds of plants [21,22,23]. It has been observed that animal-derived protein hydrolysates containing high levels of active peptides and amino acids could enhance abiotic stress tolerance in plants [24,25]. The peptides from swine blood (PSB) isolated by the microbial fermentation of swine blood are a kind of animal-derived protein hydrolysates with many kinds of biological activities and have been reported as positive regulators of salt stress tolerance in tomato [23]. However, the salt stress tolerance-related studies of PSB are still limited in other plants.

Sweet potato (Ipomoea batatas (L.) Lam), ranking the seventh most important food crop in the world, is widely cultivated in China [26]. In addition, sweet potato is also regarded as an industrial and bio-energy crop due to its high yield and rich starch content [27,28]. Although sweet potato can tolerate abiotic stress to some degree, its growth and development is still limited by salinity stress [29]. Saline–alkali soil, which is one of the main soil types with a low yield, is widely distributed around the world, and large areas of saline–alkali land need to be developed and utilized urgently [30]. Therefore, it is necessary for ensuring food and energy security to explore new methods to enhance the salinity tolerance in sweet potato. In this study, we found that the foliar application of animal-derived protein hydrolysates from PSB enhanced the salt tolerance of sweet potato seedlings by decreasing the ROS accumulation and improving the root development under salt stress. Our study provides a more eco-friendly method of improving salinity tolerance and insights into the underlying mechanisms of PSB in enhancing crop tolerance to salt stress.

2. Materials and Methods

2.1. Plant Materials

The sweet potato cultivar Pushu 32 (bred by Puning academy of agricultural sciences in Guangdong) was used as the experimental material in this study. Four-week-old seedlings with uniform sizes were selected for the subsequent experiments. These seedlings were planted in pots (an inner diameter of 9 cm and a height of 8 cm with small holes at the bottom, one seedling/pot) filled with 200 g of garden soil each. The seedlings were then transferred to an artificial climate-controlled chamber with an air temperature of 25 °C, a light/dark cycle of 14/10 h and a humidity of 50%.

2.2. Experimental Design

The information of PSB (Wavne, Biotechnology, Inc. Shanghai, China) is as follows: the content of crude protein was more than 85% and rich in various amino acids, and the percentage of small peptides (molecular weight ≤ 1500) was more than 80%. All the selected seedlings were randomly divided into four groups (Group I–Group IV). Seedlings of Groups II and IV were foliar-sprayed with 10 g/L PSB solution twice with a 3-day interval. At the same time, seedlings of Groups I and III were treated with an equivalent amount of distilled water. A relatively moderate concentration of PSB at 10 g/L was most effective according to our previous experiments. Seven days later, seedlings of Groups III and IV were bottom-seepage-irrigated with 300 mM NaCl solution (100 mL per pot) three times with a 2-day interval (a concentration validated to yield ~120 mM salinity in the soil solution), while seedlings of Groups I and II were irrigated with an equivalent amount of distilled water. Physiological and biochemical parameters were determined at 7 days post-initiation of salinity treatment. The fourth fully expanded leaves from the top were selected for all the measurements. Three independent biological replicates were performed for each treatment, with each replicate comprising six sweet potato seedlings.

2.3. Determination of Gas Exchange Parameters, Chlorophyll Fluorescence and SPAD Values

Determination of net photosynthetic rate (Pn) was conducted between 9:00 am and 11:00 am using the portable photosynthesis system (Li-COR 6800, Lincoln, NE, USA). Chlorophyll fluorescence was measured with imaging pulse amplitude-modulated (PAM) fluorimeter (IMAG-MAXI; Heinz Walz, Effeltrich, Germany) according to the instructions after 30 min dark adaptation. The minimum fluorescence emission signal (Fo), maximal fluorescence (Fm), steady-state fluorescence yield (Fs), and light-adapted maximum fluorescence (Fm′) were measured with the compound leaves. Then, the maximal photochemical efficiency of photosystem II (PSII) (Fv/Fm), the quantum efficiency of PSII photochemistry (ΦPSII), the photochemical activity of PSII (Fv′/Fm′), and the non-photochemical quenching (NPQ) were calculated as described by Qu et al. [31]. The chlorophyll relative content (SPAD values) of the fourth fully expanded leaves was measured using the Minolta SPAD meter (SPAD-502 Plus, Konica Minolta Optics Inc., Tokyo, Japan).

2.4. Measurement of Relative Water Content, Electrolyte Leakage, and Lipid Peroxidation

For the measurement of relative water content (RWC), leaves were weighed (FW) and then soaked in distilled water for 4 h (TW). They were dried to constant weight at 80 °C (DW). The RWC was calculated according to the method of Toscano et al. [32]. RWC (%) = [(FW − DW)/(TW − DW)] × 100. The relative electrolyte conductivity (REC) of leaves under different conditions was measured using the conductivity bridge (DDS-307A, LEX Instruments Co., Ltd., Suzhou, China) according to the method of Wei et al. [33]. The extraction of malondialdehyde (MDA), an index of lipid peroxidation level, was conducted according to the instructions of the relevant kit (Grace Biotechnology, Suzhou, China). The content of MDA was determined by an enzyme-labeling measuring instrument (Infinite M Plex, Tecan Trading Co., Ltd., Shanghai, China).

2.5. Histochemical Staining and Quantitative Assay of H₂O₂ and O²⁻

The accumulation of hydrogen peroxide (H₂O₂) in leaves under different conditions was visually detected by histochemical staining with a 3,3’-diaminobenzidine (DAB) solution (1 mg/mL, pH 3.8). The accumulation of H₂O₂ was determined according to the staining condition after all chlorophyll was eliminated in 95% (v/v) ethanol [34]. The accumulation of superoxide anion (O²⁻) in leaves was visually detected using nitro blue tetrazolium (NBT) staining according to the method described by Alvarez et al. [34]. In brief, the leaves were covered with an NBT solution (1 mg/mL, pH 6.1), and then they were soaked in 95% (v/v) ethanol to eliminate chlorophyll. The quantitative analysis of H₂O₂ and O²⁻ was carried out with the relevant kits according to their instructions (Grace Biotechnology, Suzhou, China).

2.6. Content of Total Proteins, Starches, Soluble Sugars, Reducing Sugars, Free Amino Acids and Proline

To determine the content of total proteins, 0.1 g tissue was homogenized in an extraction buffer according to the kit manufacturer’s instructions (Grace Biotechnology, Suzhou, China), the supernatant was collected after centrifugation, and absorbance was measured at 600 nm using a spectrophotometer (UV-5800, Metash Instruments Co., Ltd., Shanghai, China). The protein concentration was calculated against a standard curve. Total starch was quantified using the anthrone-sulfuric acid method, 0.1 g tissue was homogenized in perchloric acid and heated at 95 °C, and the supernatant’s absorbance was measured at 620 nm against a glucose standard curve (Grace Biotechnology, Suzhou, China). To determine the content of soluble sugars in the samples, a total of 0.2 g well-ground plant materials were extracted with 80% (v/v) ethanol at 80 °C for 20 min. After centrifugation, the supernatant was collected and assessed with the relevant kits (Grace Biotechnology, Suzhou, China) according to their instructions. The absorbance of simples at 620 nm was measured by a spectrophotometer to calculate the content of total soluble sugars. Reducing sugar content was quantified using the 3,5-dinitrosalicylic acid (DNS) method, 0.2 g tissue was homogenized in 80% (v/v) ethanol, heated at 95 °C, and reacted with the DNS reagent, and absorbance was measured at 500 nm (Grace Biotechnology, Suzhou, China). To assess the content of free amino acids, a total of 0.2 g sample was treated with the solutions of the relevant kit (Grace Biotechnology, Suzhou, China) according to its instructions. The content of free amino acids was obtained after the absorbance was determined at 570 nm. The proline in plant materials was extracted using sulfosalicylic acid. After reaction with acid ninhydrin, the content of proline was calculated by measuring the absorbance at 520 nm by the colorimetric method according to the instructions of the relevant kit (Grace Biotechnology, Suzhou, China).

2.7. Measurement of Root Weight

In order to determine the dry weight of roots after different treatments, all fresh roots should be dissected and washed with distilled water two times. The dissected roots were initially oven-dried at 105 °C and subsequently maintained at 85 °C until a constant weight was achieved.

2.8. Measurement of Na⁺ and K⁺

For Na⁺ and K⁺ content measurement, 0.1 g leaf or root dry samples were powdered and immersed in concentrated sulfuric acid. The digestive solution after attenuation was used for Na⁺ and K⁺ determination with a TAS-990 atomic absorption spectrophotometer (Purkinje General Instrument Co., Ltd., Beijing, China) according to the previously described method [35].

2.9. Activity Analysis of Antioxidant Enzymes

For the activity analysis of antioxidant enzymes, leaves after different treatments were frozen in liquid nitrogen and stored at −80 °C. Catalase (CAT) activity was determined based on the oxidation decline of H₂O₂ at 240 nm. Peroxidase (POD) activity was assayed based on the oxidation degree of guaiacol measured at 470 nm. Superoxide dismutase (SOD) activity was determined according to the formazan measured at 450 nm, which was obtained by inhibiting the O²⁻ produced by WST-8. A total of 0.1 g of fresh plant samples was used for each enzyme activity determination with the corresponding kits, according to their instructions, and three independent biological replicates were performed for each assay.

2.10. Library Construction and Transcriptome Sequencing

For the transcriptome sequencing analysis, the leaf samples after different treatments were used for total RNA isolation. RNA sequencing libraries were constructed by the Novogene Bioinformatics Technology Co. Ltd., Beijing, China and sequenced using an Illumina NovaSeq 6000 PE150 (Illumina, CA, USA) following standard protocols. FastQC was used to perform quality control. Low quality reads and sequencing primers were filtered out or trimmed using TrimGalore. The clean data were mapped to the reference genome sequences of sweet potato (pasi3, https://sweetpotato-garden.kazusa.or.jp/ (accessed on 13 March 2021)) using Bowtie 2–2.2.3 with default parameters. Cufflinks were used to count the read numbers mapped to each gene and the FPKM of each gene.

2.11. Identification and Functional Annotation of Different Expression Genes

Differentially expressed genes (DEGs) were identified using the DESeq2 R package (version 1.32.0) with the default parameter. Genes with an adjusted p-value of < 0.05 and a log2FC of ≥0.6 were considered to be differentially expressed. The clusterProfiler (version 3.18.0) software was used to perform the GO enrichment analysis of DEGs with the following settings: p-value cutoff = 0.05; Q value cutoff = 0.05. The scatter plot of GO terms was produced by REVIGO (http://revigo.irb.hr/).

2.12. Expression Analysis of Genes Related to High Salinity Response

The same samples used for transcriptome analysis were employed for subsequent qRT-PCR-based gene expression validation. Total RNA extraction and first-strand cDNA synthesis were carried out in accordance with the instructions of the corresponding kits (Vazyme, Nanjing, China). qRT-PCR was performed with ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China), and the amplification reaction was completed on the Quant Studio 5 (Applied Biosystems, Waltham, MA, USA). The relative expression levels of the target genes were calculated using the 2^−ΔΔCT method, with the internal control gene being IbActin. The “Salt” treatment group was set as the control (with the expression level normalized to 1). The sequences of all qRT-PCR primers are detailed in Supplementary Table S8. Each treatment comprised three biological replicates, with four technical replicates conducted per biological replicate.

2.13. Statistical Analysis

Differences among the four treatment groups were assessed using one-way ANOVA with Excel 2010. Tukey’s honest significant difference (p < 0.05) was performed to evaluate the differences in each treatment. Comparisons between two groups were conducted using Student’s t test. Each treatment value is the average of three independent biological replicates.

3. Results

3.1. Effects of Exogenous PSB on Plant Growth Under Normal Conditions and Salinity Stress Treatment in Sweet Potato

The sweet potato seedlings with similar growth conditions were primed with distilled water or 10 g/L PSB for 7 days. Then, these seedlings were exposed to normal irrigation or high salinity stress. Under normal conditions, the PSB-treated seedlings showed an increased number of fully expanded leaves compared to water-treated seedlings. On the other hand, high salinity stresses severely stunted the growth of sweet potato seedlings and resulted in the senescence of old leaves. However, priming with PSB strikingly alleviated the damage caused by high-salinity treatment (Figure 1a). Consistent with the phenotypic changes in the sweet potato seedlings, the chlorophyll fluorescence parameters showed that Fv′/Fm′, Y(NPQ) and ΦPSII were all significantly lower under high salinity stress than those under normal conditions (Figure 1b–d). Meanwhile, the chlorophyll fluorescence parameters of seedlings primed with PSB showed significant increase when compared with those of seedlings without PSB treatment under high-salinity conditions (Figure 1b–d). However, exogenous PSB had no significant effects on the chlorophyll fluorescence parameters under normal conditions (Figure 1b–d).

There is a good correlation between the leaf chlorophyll content and the SPAD value. The SPAD value significantly decreased under high-salinity conditions without PSB priming; however, the application of PSB showed a significant increase in the SPAD value (Figure 2a). The further measurement of photosynthetic parameters showed that sweet potato seedlings without PSB treatment displayed a significant decrease in Pn under high-salinity conditions; however, exogenous PSB significantly reversed the deleterious effects (Figure 2b). Under normal conditions, exogenous PSB also lead to a significant increase in Pn (Figure 2b). The water-holding ability measurement results showed that high-salinity treatment significantly decreased the relative water content (RWC) of sweet potato seedlings, and priming with PSB could notably decrease water loss (Figure 2c). Nevertheless, the application of PSB had no effect on the RWC under normal conditions (Figure 2c).

Subsequently, the growth of underground parts was examined. The results revealed that, under normal conditions, root systems developed robustly, with some roots exhibiting early swelling (Figure 3a). In contrast, high-salinity treatment strongly suppressed root growth, but the application of PSB could partially alleviate the inhibitory effects of salt stress on root growth (Figure 3a). Root length and dry weight measurements further confirmed that PSB enhanced the development of underground parts across diverse growth environments (Figure 3b,c). In general, PSB could promote the growth of sweet potato seedlings to some extent under both normal and high-salinity stress conditions.

3.2. Effects of Exogenous PSB on Cell Membrane Stability and ROS Accumulation Under Normal and Salinity Stress Conditions in Sweet Potato

High salinity stress could cause lipid peroxidation, leading to the damage of cell membrane integrity and intracellular electrolyte leakage. The detection of cell membrane stability showed that the MDA content and REC significantly increased under high salinity stress; however, exogenous PSB could notably weaken the variation in MDA content and REC (Figure 4a,b). The results indicated that PSB treatment could alleviate the cell membrane damage caused by high salinity. The accumulation of two representative ROS, H₂O₂ and O²⁻, was analyzed using the method of histochemical allocation and quantitative determination. The results showed that the content of H₂O₂ was low under normal conditions, while it significantly increased under high salinity stress. However, exogenous PSB could reduce the accumulation of H₂O₂ under stress treatment (Figure 4c,e). The accumulation of O²⁻ showed a similar drift with H₂O₂ after different treatments (Figure 4d,f).

Consistent with the results of ROS accumulation, the determination of key antioxidant enzyme activities showed that, under high salinity stress, exogenous PSB significantly increased SOD, POD and CAT activities compared to the control without PSB application, suggesting enhanced oxidative resistance in PSB-treated seedlings under salinity conditions (Figure 5). These results indicated that the application of PSB could activate ROS-scavenging system key enzymes and reduce ROS accumulation under high salinity stress.

3.3. Effects of Exogenous PSB on Organic Compounds and Cation Content Under Normal Conditions and Salinity Stress in Sweet Potato

The content of macromolecular organisms such as proteins and starch in sweet potato leaves was measured under different conditions. The results showed that PSB treatment could reduce the protein content in sweet potato leaves under both normal and high-salinity conditions (Figure 6a). On the other hand, the accumulation of starch decreased after high salinity stress, and exogenous PSB did not affect the content of starch under normal or high-salinity conditions (Figure 6b). The content of low-molecular-weight organic compounds such as soluble sugars, reducing sugars and free amino acids of leaves after different treatments was further determined. The results showed that the exogenous application of PSB could significantly increase the content of soluble sugars and free amino acids under both normal and high-salinity conditions (Figure 6c,e). The content of proline was further determined, and the results showed that it had a similar changing profile to that of free amino acid content (Figure 6d). On the other hand, exogenous PSB did not affect the reducing sugar content under different conditions (Figure 6f). The cation content in sweet potato seedlings under different treatment conditions was determined. The results showed that PSB application increased the levels of K⁺, particularly under high salt stress, but it had no significant effect on Na⁺ content (Figure 6g,h). These findings suggested that PSB enhanced high salinity tolerance by promoting protein degradation, enhancing the accumulation of osmoregulatory substances and increasing K⁺ uptake.

3.4. Exogenous PSB Enhances Salt Tolerance in Sweet Potato Plants by Promoting the Expression of Genes Associated with Photosynthesis, Antioxidant Response and Stress Responses Under Salt Stress Conditions

To further investigate the effects of exogenous PSB on salt tolerance in sweet potato, transcriptome sequencing was performed under different treatment conditions (“Control”, “Salt”, and “PSB+Salt”), followed by a systematic analysis of the resulting data (Table S1). The differential expression gene (DEG) analysis revealed that, when compared with “Control”, “Salt” exhibited 3610 up-regulated and 3894 down-regulated genes (Figure 7a,b, Table S2). Furthermore, “PSB+Salt” exhibited 4036 up-regulated genes and 4254 down-regulated genes relative to “control” (Figure 7a,b, Table S3). Compared with “control”, a total of 2753 genes were up-regulated and 3177 genes were down-regulated in both “salt” and “PSB+Salt” (Figure 7a,b). The GO enrichment analysis of differentially expressed genes in “Salt” and “PSB+Salt” relative to “Control” revealed that multiple up-regulated genes were enriched in pathways such as stress response and protein conformation regulation in both groups (Figure 7c, Tables S4 and S5). In amino acid modification and metabolism pathways, the significant enrichment of up-regulated genes was observed exclusively in “Salt”, whereas in RNA splicing and modification pathways, enrichment was detected only in “PSB+Salt” (Figure 7d, Tables S4 and S5).

For down-regulated genes, significant enrichment in lignin synthesis and ABA metabolism pathways occurred solely in “Salt”, while cell wall synthesis and ion transmembrane transport pathways showed enrichment exclusively in “PSB+Salt”. The further analysis of “Salt” and “PSB+Salt” revealed that, compared with “Salt”, 648 genes were up-regulated and 684 genes were down-regulated in “PSB+Salt” (Figure 8a, Table S6). The GO enrichment analysis of these DEGs revealed that, compared with “salt”, genes associated with stress response, photosynthesis and protein structural organization were promoted, while genes involved in starch degradation, cell division and tricarboxylic acid cycle were suppressed (Figure 8a,b, Table S7). These findings suggested that PSB might enhance salt tolerance in sweet potato through the modulation of protein metabolism, regulation of stress-responsive gene expression and maintenance of photosynthetic capacity under high-salinity conditions. To validate the RNA-seq results, qRT-PCR was conducted on six representative DEGs selected from “Salt” versus “PSB+Salt”. The expression trends observed by qRT-PCR were fully consistent with those from RNA-seq, confirming the technical reliability and biological reproducibility of the transcriptomic dataset (Figure S1).

4. Discussion

High salinity is one of the major abiotic stresses limiting plant growth and development [35]. As climate change intensifies and challenges such as inadequate irrigation management persist, the increasing frequency of extreme weather events and growing scarcity of freshwater resources have further exacerbated the problem. Meanwhile, the continuous growth of the global population has led to the rising demand for agricultural output [1]. In this context, the scientific and rational development and utilization of high-salinity land are essential for ensuring food security and promoting sustainable agricultural development [36]. Protein hydrolysates are natural plant growth regulators composed of peptides and free amino acids, generated through the enzymatic or chemical hydrolysis of plant or animal proteins [37]. Accumulating evidence suggested that they played a potential role in promoting plant growth and abiotic stress tolerance [38]. Ertani et al. demonstrated that a growth regulator derived from alfalfa hydrolysate significantly promotes maize growth under both normal and salt-stressed conditions [21]. Moreover, Zhou et al. confirmed that pig blood-derived protein hydrolysates effectively alleviated salt stress-induced physiological impairments in tomato, such as growth inhibition and reduced photosynthetic efficiency [23]. Previous studies demonstrated that high salinity significantly affected key physiological parameters such as photosynthesis and RWC [13]. This study demonstrated that high salinity significantly inhibited the growth of sweet potato and reduced RWC and the photosynthetic rate; however, the exogenous application of PSB effectively alleviates these salt stress-induced adverse effects (Figure 1, Figure 2 and Figure 3). Under normal conditions, although PSB treatment did not significantly alter the SPAD value, it enhanced the photosynthetic rate and promoted the translocation of photosynthates from leaves to roots, thereby increasing biomass accumulation in roots (Figure 1, Figure 2 and Figure 3). These findings suggested that protein hydrolysates could promote plant growth and development across multiple species under diverse environmental conditions.

The membrane system, as a fundamental component of plant cells, played critical roles in key biological processes such as substance transport and signal transduction [39]. Salt stress affects plants primarily through two mechanisms: osmotic stress induced by high environmental salt concentrations and ion toxicity caused by the excessive accumulation of Na⁺ and Cl⁻ [14]. Subsequently, the accumulation of ROS is markedly increased, leading to oxidative stress and consequent damage to the membrane system, characterized by decreased membrane fluidity and elevated permeability, ultimately resulting in electrolyte leakage [14]. These combined effects disrupt multiple physiological and biochemical processes, leading to the significant inhibition of growth in plants. Throughout long-term evolution, plants have evolved antioxidant defense systems composed of enzymatic and non-enzymatic components that effectively mitigate the excessive accumulation of ROS under stress conditions [16]. MDA is a major product of membrane lipid peroxidation and serves as a key indicator for assessing the extent of membrane lipid damage [14]. Numerous studies demonstrated that high salinity induced elevated ROS and MDA levels in sweet potato [40,41]. Under high-salinity conditions, salt-tolerant seedlings exhibited the higher activity of scavenging system, reduced ROS accumulation and diminished membrane lipid peroxidation damage [40,41]. This study demonstrated that high salinity significantly promoted the accumulation of ROS, such as H₂O₂ and O²⁻, in sweet potato seedlings, leading to marked increases in the MDA content and REC (Figure 4). In contrast, the exogenous application of PSB effectively suppressed ROS accumulation and mitigated membrane lipid peroxidation damage (Figure 4). Further analysis of key enzyme activities in ROS-scavenging system revealed significantly enhanced antioxidant enzyme activities in sweet potato under high salinity following PSB treatment (Figure 5). Collectively, these results indicated that PSB enhanced salt tolerance by activating enzymatic antioxidant system in sweet potato.

In addition to scavenging ROS, higher plants enhanced their tolerance to high salinity through osmotic adjustment. The compounds involved in this process were broadly classified into two categories: organic solutes and inorganic ions [13]. Free amino acids, particularly proline, served as key organic osmolytes due to their strong hydration capacity protecting proteins from denaturation under dehydration conditions, and their antioxidant activity helped to reduce ROS accumulation [42]. Accumulating evidence has indicated that increased proline accumulation under high salinity is closely linked to enhanced salt tolerance in plants [26,35]. Moreover, several studies indicated that exogenous proline application effectively mitigates the adverse effects of high salinity in plants [43]. In this study, exogenous PSB was shown to significantly increase the levels of free amino acids under both normal and high-salinity conditions, with a particularly marked accumulation of proline, while concurrently decreasing protein content (Figure 6a,c,d). Given that PSB is inherently a protein hydrolysate rich in free amino acids, these findings suggested that exogenous protein hydrolysate could enhance the accumulation of free amino acids (including proline) under both non-stress and high-salinity conditions and contribute to enhanced salt tolerance in sweet potato. Soluble sugars represented another important kind of organic osmotic adjustment compounds. Growing evidence has indicated that, under abiotic stress conditions, plants could maintain elevated levels of soluble sugars by inhibiting starch synthesis or enhancing its degradation [13]. Sweet potato seedlings exhibited reduced starch content under high-salt conditions; however, exogenous PSB had no significant effect on starch content (Figure 6b). It was also found that the reducing sugar level remained unchanged, whereas soluble sugar content increased (Figure 6e,f). These findings indicated that exogenous protein hydrolysate did not promote soluble sugar accumulation through the modulation of starch metabolic pathway under either normal or high-salinity conditions, suggesting that its potential contribution to enhanced salt tolerance might be independent of this metabolic route in sweet potato. Inorganic ions also played crucial roles in osmotic regulation. Na⁺ and K⁺ uptakes were competitively regulated under high-salinity conditions. The excessive accumulation of Na⁺ disrupted intracellular ionic homeostasis, whereas enhanced K⁺ absorption and active Na⁺ exclusion represented a key mechanism underlying adaptation to high salinity stress in plants [44,45]. Under high-salinity conditions, exogenous PSB had no significant effect on Na+ content but significantly increased K⁺ content, indicating that exogenous protein hydrolysates maintained ionic homeostasis primarily by enhancing K⁺ uptake in sweet potato (Figure 6g,h). This finding supported the salt tolerance paradigm of maintaining a high cytosolic K⁺/Na⁺ ratio. The absence of a significant change in Na⁺ accumulation indicated the limited impact of PSB on canonical Na⁺ transporters, suggesting Na⁺ homeostasis is likely preserved through enhanced redistribution within different tissues. Transcriptome analysis indicated that exogenous PSB activated the expression of stress-responsive and photosynthesis-related genes under high salinity stress (Figure 7 and Figure 8). It was found that transcriptome profiling revealed a low differential expression of core K⁺/Na⁺ transporter genes, but instead showed the significant enrichment of genes involved in plant hormone signaling, ROS scavenging and homeostasis of other metallic cations, collectively indicating that PSB modulates K⁺/Na⁺ balance through upstream hormonal and redox-mediated control of cellular ion compartmentalization and membrane stability.

In conclusion, the results of this study confirmed that PSB could enhance salt stress tolerance by increasing the accumulation of osmotic adjustment substances through the promotion of protein metabolism and K⁺ uptake, reducing ROS levels, and improving photosynthetic performance in sweet potato. It could be concluded that protein hydrolysates rich in free amino acids held potential as broadly applicable biostimulants that promote plant growth and stress resistance.