Seed Priming With an Animal-derived Protein Hydrolysate Improves Drought Tolerance of Tomato Seeds by Enhancing Reserve Mobilization, Osmotic Adjustment, and Antioxidant Mechanism


 Purpose Protein hydrolysates obtained from agro-industrial byproducts have received much attentions due to their positive roles in regulating plant responses to environmental stresses. However, little is known about the roles of animal protein hydrolysates in mediating seed drought tolerance and the underlying mechanism. Here, the effects of seed priming with pig blood protein hydrolysates (PP) on tomato seed germination and seedling growth under drought stress were investigated. Methods Tomato seeds were soaked with different concentrations of PP solutions for 24 h, and then transferred to filter paper moistened with distilled water or 10% PEG-6000 solution in Petri dish. The germination traits, seeding growth, reserve mobilization, osmolytes, and antioxidant system were determined.Results PP priming effectively alleviated the reduction in seed germination traits, resulting in improved tomato seedling growth under drought stress. PP priming enhanced amylase and sucrose synthase activities, soluble sugar, soluble protein, and free amino acid levels, thereby promoting reserve mobilization in seeds. Moreover, PP priming also reduces osmotic toxicity by increasing the accumulation of proline, soluble protein, and soluble sugar. Drought stress substantially enhanced the production of ROS and subsequent increases in MDA and Evans blue uptake, which were significantly alleviated after PP priming by improving the activities of SOD, POD, and CAT, and non-enzymatic antioxidants. Conclusion PP priming is a feasible method for improving tomato seed germination and seedling growth under drought stress by enhancing reserve mobilization, osmolyte accumulation, and antioxidant systems.


Introduction
Crops under natural conditions face various environmental constraints, including drought, salinity, temperature, and heavy metals (Anderson et (Biju et al., 2017;Sheteiwy et al., 2018;Yang et al., 2021). The primary reason for this decline in seedling emergence is the reduction in water uptake during the imbibition phase of germination, where all physiological and metabolic processes are inhibited (Fabregas and Fernie, 2019;. In general, carbohydrates and proteins are mobilized to provide substrates and energy for seed germination, where amylases are the key enzymes related to the hydrolysis of starch to soluble sugar, which was reduced by low water availability (Fabregas and Fernie, 2019; Lei et al. 2021; Saharan et al., 2016). The osmotic adjustment in seeds or seedlings during drought, by accumulation of osmolytes, such as sugars, amino acids, and soluble protein, is one of the major adaptive strategies for maintaining osmotic balance, thereby improving water uptake (Ozturk et   Tomato (Solanum lycopersicum L.) is an important economic crop, which is usually affected by drought stress and exhibits a reduction in physiological and biochemical processes (Elbadrawy and Sello, 2016). In our study, we evaluated (1) the feasibility of seed priming with pig blood-derived protein hydrolysates (PP) to enhance tomato seed germination and seedling growth; (2) the potential role of PP priming in regulating the reserve mobilization of seeds; and (3) the function of PP priming in preventing osmotic stress and oxidative damage under drought stress. Our results provide a new strategy for the agronomic application of protein hydrolysates in alleviating drought stress in the agriculture.

Plant materials and treatment
Tomato seeds were purchased from Shouguang City, China. PP was obtained from Win Plus Biotech Co., Ltd (Xiangyang, China), which is a complex mixture of peptides and free amino acids derived from pig blood protein by enzyme hydrolysis. The composition of the peptides and free amino acids is shown in Tomato seeds were surface-sterilized with 10% sodium hypochlorite for 10 min, and then washed three times with distilled water. The seeds were soaked with different concentrations of PP solutions (0, 1, 2, 3, and 5 g L − 1 ) at 20°C for 24 h in the dark. Then, 20 seeds were placed on lter paper moistened with distilled water or 10% PEG-6000 solution in each 12 cm diameter Petri dish. Seeds were cultured in an arti cial climate chamber with a photoperiod of 16/8 h (day/night), relative humidity of 60%, and temperature of 25/16°C (day/night). The treatment conditions were as follows: CK (seeds soaked and germinated in distilled water); PEG (seeds soaked in distilled water and germinated in 10% PEG-6000 solution); PP1 + PEG, PP2 + PEG, PP3 + PEG, and PP5 + PEG (seeds soaked in 1, 2, 3, and 5 g L − 1 of PP, respectively, and seed germinated in 10% PEG-6000 solution).
Non-germinated or irregularly germinated seeds were discarded, and then seeds were shelled for physiological and biochemical analysis at 0 h, 48 h, and 72 h. Germination rate (%) was equal to the number of germinated seeds on the 7th day of the germination. Germination potential (%) was equal to the number of germinated seeds on the 4th day of the germination. The germination index was equal to ∑(Gi/Ti) (where Gi is the germination percentage in times of Ti). After 7 days, fresh samples were collected, and fresh weight, root length, and seedling height were determined, and directly stored at -20°C until the completion of biochemical assays.
Analysis of α-amylase, β-amylase, total amylase, and sucrose synthase activities Seed samples were collected at 0 h, 48 h, and 72 h after treatment, and the following indices were determined: the α-amylase, β-amylase, and total amylase were measured using 3,5-dinitrosalicylic acid (DNS) method (Biju et al., 2017). For α-amylase determination, tomato seeds (0.1 g) were homogenized with 1.5 mL of distilled water, and centrifuged at 10 000 × g for at 4°C 10 min. The extract (0.2 mL) was mixed with 0.2 M citrate buffer (pH 5.6), and incubated at 70°C for 30 min. Then, 2 mL of DNS and 1 mL of soluble starch (1% v/v) were added to a boiling water bath for 5 min, and the absorbance was determined at 540 nm. β-amylase activity was determined as described above; however, the starch was replaced by amylopectin. The total amylase activity was calculated as the sum of α-amylase and βamylase activities.
Sucrose synthase activity was determined as described by Verma  Proline content was measured according to the method of Bates et al. (1973). Brie y, 0.2 g of samples were added into aqueous sulfosalicylic acid (3%) and kept in boiling water for 1 h. After the mixture was cooled to room temperature, the ninhydrin and acetic acid was added, and then the mixture was maintained in a boiling water bath for 30 min and cooled in an ice bath. Subsequently, 5 mL of toluene was added, and the mixture was placed in the dark for 5 h. Eventually, the absorbance was recorded at 520 nm. The soluble protein content was measured following the Bradford's method (Bradford, 1973

Analysis of non-enzymatic antioxidant content
Fresh samples (0.2 g) were homogenized with 1.5 mL of 0.5 M EDTA solution containing 3% trichloroacetic acid, and then centrifuged at 12 000 × g at 4°C for 10 min. The supernatant was used to assay the ascorbic acid (AsA) and glutathione (GSH) contents and calculated as described by Zhou et al. (2020).
The analysis of phenolic compounds was conducted using the method described by Zhou, et al. (2018).
Brie y, fresh samples were homogenized with 80% (v:v) methanol solution and then centrifuged at 10 000 × g at 4°C for 10 min. The supernatant was used to determine the total phenolic, avonoid, and anthocyanin content.

Analysis of antioxidant activities
The DPPH free radical scavenging capacity (DFRSC) was measured by recording the decrease of absorbance at 517 nm, and the results were expressed as percent scavenging of DPPH radicals (Zhang et al., 2013

Statistical analysis
All data were analyzed using the SPSS statistical software. Data are presented as the mean ± standard deviation (SD) of at least three independent experiments using one-way analysis of variance (ANOVA). The least signi cant difference (LSD) test was performed to determine signi cant differences among the treatments at P < 0.05.

Results
Tomato seed germination and seedling growth Compared to the control, the PEG treatment negatively affected tomato seed germination, as indicated by the substantial reduction in germination rate, germination potential, and germination index (Fig. 1a-c). However, PP priming positively stimulated seed germination, and the maximum enhancement of germination rate, germination potential, and germination index were observed in the PP2 treatment, which increased by 39.5%, 283.5%, and 103.5%, respectively, compared to the PEG treatment. In addition, PP2 treatment signi cantly alleviated the PEG-induced decrease in seedling growth to the maximum extent, and the seedling fresh weight, seedling height, and root length increased by 33.4%, 45.4%, and 22.9%, respectively, compared to the PEG treatment ( Fig. 1d-f).
Activity of α-amylase, β-amylase, total amylase, and sucrose synthase The activities of α-amylase, β-amylase, and total amylase in tomato seeds gradually increased with germination time (Fig. 2a- Starch, soluble sugar, soluble protein, and free amino acid content in seeds A gradual decrease in starch content was observed in the tomato seeds with germination time, and this trend was slowed down by the PEG treatment (Fig. 3a). However, PP2 priming signi cantly decreased the starch content of seeds by 18.2% and 10.5% at 48 h and 72 h, respectively, compared to the PEG treatment. After germination, the soluble sugar content exhibited an increasing trend at 48 h, followed by a decreasing trend at 72 h (Fig. 3b). Compared to the PEG treatment, PP2 priming signi cantly increased the soluble sugar content by 13.5% and 15.3% at 48 h and 72 h, respectively. In addition, the soluble protein and free amino acid content signi cantly decreased by 28.8% and 20.3% at 72 h in the PEGtreated seeds compared to those in the control (Fig. 3c and d) (Fig. 4a and b) (Fig. 4c and d). However, PP priming substantially decreased the MDA content and Evans blue uptake, with maximal reductions of 18.5% and 47.2%, respectively, observed in the PP2 treatment. This result was further veri ed by the root histochemical staining with Evans blue (Fig. 4g).
The activities of SOD, POD, and CAT were increased by the PEG treatment in tomato seedlings compared to the control (Fig. 5a). The PP2 and PP3 priming further signi cantly increased the activities of SOD (by 40.4% and 23.7%, respectively), POD (by 85.9% and 41.2%, respectively), and CAT (by 82.5% and 49.7%, respectively), whereas PP1 priming only enhanced the activities of POD and CAT by 18.3% and 27.9%, respectively, compared to the PEG treatment. Moreover, higher contents of non-enzymatic antioxidants, including total phenolics, avonoids, anthocyanins, ASA, and GSH, were also observed in the PEG-treated seedlings (Fig. 5b). PP priming further improved the total phenolics, avonoids, anthocyanins, ASA, and GSH contents, and their maximum enhancement was observed in PP2 treatment by 53.1%, 57.1%, 325.7%, 64.6%, and 33.6%, respectively, compared to the PEG treatment. As a result, compared to the PEG treatment, the highest increase in DFRSC and FRAP were found in the PP2 treatment by 28.8% and 58.0%, respectively (Fig. 6).
Soluble sugar, soluble protein, and proline in tomato seedlings Soluble sugar, soluble protein, and proline are important substances involved in the osmotic adjustment of plants under drought stress. Compared to the control, the PEG treatment increased the levels of soluble sugar and proline signi cantly by 14.7% and 27.0%, respectively, and decreased the content of soluble protein by 23.5% (Fig. 7). However, PP priming positively stimulated the accumulation of these osmolytes, and the maximum enhancement of soluble sugar, soluble protein, and proline were observed in the PP2 treatment, which increased by 170.4%, 15.9%, and 82.3%, respectively, compared to the PEG treatment.

Discussion
Drought is a main abiotic stress that threatens plant growth, development, and yield production by inhibiting various physiological processes (Fabregas and  ). In the present study, drought stress induced by the usage of 10% PEG signi cantly reduced the germination rate, germination potential, and germination index of tomato seeds, as well as the seedling fresh weight, seedling height, and root length (Fig. 1). In order to reduce the negative effects of drought stress on seed germination and seedling growth, a low-cost and feasible method known as seed priming has been widely used to improve drought stress tolerance (Marthandan et al., 2020;Salah Sheteiwy et al., 2018; Zul qar, 2021).
Remarkably, seed priming with PP signi cantly alleviated drought-induced inhibition of seed germination and seedling growth. Notably, this positive effect of PP application is perhaps due to the high supply of active peptides and amino acids, which confer crop tolerance to various abiotic stresses by regulating biochemical and physiological processes (Colla et al., 2015). Therefore, to the best of our knowledge, our study is the rst to verify that PP could alleviate the reduced tomato seed germination and seedling growth; thus, it could be a potential strategy to improve seed tolerance to drought stress.
Seed germination is a complex physical and chemical process, beginning with water uptake of dry seeds to radicle protrusion and growth, where the necessary energy is supplied by the degradation of storage substances, such as starch and protein (Lei et  The present study showed that drought stress remarkedly inhibited the activities of α-amylase, β-amylase, total amylase, and sucrose synthase, leading to a reduction in the hydrolysis of starch to soluble sugars ( Fig. 2 and Fig. 3). In addition, the PEG-induced water de ciency also adversely decreased the content of soluble protein and free amino acids, suggesting that drought stress inhibited the metabolism of sugar and degradation of storage proteins during germination. However, PP priming signi cantly increased the activities of amylase and sucrose synthase and the levels of soluble sugar, soluble protein, and free amino acids in tomato seeds. These results indicate that PP may enhance amylase activity and subsequent reserve mobilization to stimulate seed germination under drought stress. Previous ndings have revealed that protein hydrolysates are closely associated with the levels of endogenous hormones, as their biosynthesis, stimulating the activity of various enzymes, including amylase (Casadesús et al., 2020; Sorrentino et al., 2021).
Plants usually accumulate a variety of substances for osmotic regulation, such as proline, betaine, soluble protein, and soluble sugar, which help to maintain turgor pressure, promote water absorption and retention, and improve tolerance among plants against drought stress (Ozturk et al., 2021;Razi and Muneer, 2021). In the present study, rapid increases in soluble sugars, soluble protein, and proline accumulation in tomato seedlings were also observed under drought conditions. Seed priming with PP resulted in further improvements in soluble sugars, soluble protein, and proline accumulation ( Fig. 3 and Fig. 7), which may be bene cial for the osmotic homeostasis under drought stress. Similar to our results, exogenous application of silicon, quercetin, and melatonin partially improved drought resistance via . Proline, an amino acid act as an essential osmolyte, and its accumulation in drought-treated seedlings after PP priming might be due to the high proline supply of PP, which contained 2.45% of proline (Fig. S1). Therefore, it can be speculated that PP priming might play a key role in seed tolerance to drought stress by improving the accumulation of active osmolytes, thereby reducing osmotic toxicity.
Under normal growth conditions, the production and removal of ROS are in a dynamic balance, which does not cause damage to plants in general (Choudhury et al., 2017 Fig. 4a and b). Overproduction of ROS under abiotic stress usually leads to an oxidative damage to the cellular components, leading to membrane lipid peroxidation and cell membrane destruction (Kamal et al., 2021;Sasi et al., 2021;Sun et al., 2017). The MDA and Evans blue uptake are considered as key indicators of lipid peroxidation and integrity of the plasma membrane in plants to re ect the degree of membrane damage (Sun et al., 2017). Under drought stress, the augmented MDA and Evans blue uptake were observed compared to the control ( Fig. 4c and  resulting in a subsequent oxidative damage to the cell membrane in the root tips; however, PP priming could effectively reduce the widespread staining of root tips ( Fig. 4e-g). Altogether, these data indicate that seed priming with PP enables tomato seedlings to maintain ROS at an appropriate level, thereby contributing to enhanced drought tolerance. Patel and Paroda, 2021). It was noticed that the activities of SOD, POD, and CAT in tomato seedlings were largely upregulated after the PP priming, compared to the PEG treatment alone (Fig. 5a). The results in this study are in accordance with Sitohy et al. (2020), who indicated that pumpkin seed protein hydrolysate treatment could enhance CAT and SOD activities, which is positively related to salt tolerance. Moreover, other ROS scavenging antioxidants are a class of low molecular weight compounds such as phenolic compounds, carotenoids, GSH, and AsA, which can effectively scavenge the accumulation of ROS in plants, and thus protect them from oxidative damage under abiotic stresses Zhou, et al., 2018). In our study, drought stress substantially increased the contents of total phenolics, avonoids, anthocyanins, AsA, and GSH in tomato seedlings, whereas PP priming further improved these antioxidant contents (Fig. 5b). Consequently, this results in a considerable increase in antioxidant activities as indicated by DFRSC and FRAP in tomato seedlings (Fig. 6). In addition, a large number of antioxidant peptides have been identi ed from different sources of protein hydrolysates that exhibit strong antioxidant capacity (Bougatef et al., 2010;Wen et al., 2020). Therefore, the decreased ROS accumulation in tomato seedlings after PP priming might be partially due to the presence of some antioxidant peptides in PP. Subsequently, these results indicate that seed priming with PP effectively scavenge ROS through improving the activities of antioxidant enzymes and accumulation of antioxidant compounds, and consequently enhancing the tomato seed tolerance to drought stress.

Conclusions
In conclusion, exogenous PP could be a feasible priming agent for promoting seed germination and seedling growth of tomato plants under drought conditions (Fig. 8). First, PP priming may improve reserve mobilization by increasing amylase activity and soluble sugar, soluble protein, and free amino acid content; second, PP priming may reduce osmotic toxicity by enhancing the accumulation of osmolytes; and third, PP priming may decrease ROS generation via increasing antioxidant systems, thereby minimizing the oxidative damage under drought stress. Thus, our ndings provide a new and effective method for reducing drought stress and promoting plant growth.

Declarations
The authors declare no competing financial interest. 47. Zhou W, Lv T, Hu Y, Liu W, Bi Q, Jin C, Lu L, Lin X (2020) Effect of nitrogen limitation on antioxidant qualities is highly associated with genotypes of lettuce (Lactuca sativa L.  Figure 1 Effects of PP priming on the seed germination rate (a), generation potential (b), generation index (c), seedling fresh weight (d), seedling height (e), and root length (f) of tomato under drought stress. Different letters indicate a signi cant difference at P < 0.05.

Figure 2
Effects of PP priming on α-amylase (a), β-amylase (b), total amylase (c), and sucrose synthase activity (d) of tomato seeds under drought stress after germination. Different letters indicate a signi cant difference at P < 0.05.

Supplementary Files
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