Exogenous tryptophan increases soybean yield by enhancing sucrose-starch metabolism in leaves and seeds at the R6 stage under salt-alkali stress

Abstract

Background

Saline-alkali stress (SA) can significantly limit the growth and yield of soybean. The grain filling stage (R6) is a crucial growth period that determines the yield of soybeans and is also the most complex stage of sucrose-starch metabolism. Tryptophan (Trp) is an essential amino acid for protein synthesis and also an important signaling molecule in plants, plays an important role in maintaining osmotic regulation in plants and resisting adverse external environments. However, the mechanism of Trp regulation on sucrose-starch metabolism in R6-stage soybean leaves and seeds under SA is still unclear. This study investigated the effects of different Trp concentrations (100mg·L⁻¹、200mg·L⁻¹ and 300mg·L⁻¹) on sucrose-starch metabolism in soybean under SA (NaCl: Na₂SO₄: Na₂CO₃: NaHCO₃ = 1:9:1:9).

Results

The results showed that exogenous tryptophan alleviated the growth inhibition of R6 soybean under SA treatment. Exogenous Trp could enhance the photosynthetic capacity of soybean by increasing photosynthetic pigment content, net photosynthetic rate (Pn), intercellular carbon dioxide concentration (Ci), stomatal conductance (Gs), and the transpiration rate (Tr) of soybean leaves under SA. Exogenous Trp affected the balance of sucrose-starch metabolism in soybean leaves and seeds under SA by changing the activities of key enzymes in sucrose metabolism(SPS, SuSy, A-INV and N-INV) and expression levels of related genes. Meanwhile, exogenous Trp promoted the transport of sucrose equivalents from the source to the sink by increasing sucrose transport-related genes (GmSUC2, GmSWEET6, and GmSWEET15) under SA.

Conclusions

These results showed that exogenous Trp could improve the photosynthesis of leaves, regulate the metabolic balance of starch-sucrose, and increase the node number, pod number, and seed number, ultimately affecting high soybean yield at maturity and enhancing the saline-alkali tolerance of plants. These results can provide a new direction and theoretical basis for improving saline-alkali soil and tolerant crop breeding.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-025-07277-0.

Introduction

Soybean (Glycine max L.) is one of the most important oil and economic crops in the world. Today, soil salinization is the main limiting factor of soybean and legume production [1]. Saline-alkali stress causes physiological and biochemical metabolism disorders and excessive plant accumulation of reactive oxygen species (ROS). The result is oxidative damage to the plasma membrane and chloroplast membrane system and destruction of the photosynthetic electron transport chain and photoinhibition [2]. Additionally, saline-alkali stress increases the salt concentration in the root zone, causing water imbalance and ion toxicity and inhibiting plant growth [3], shortened roots, and plant dwarfness. Eventually, plant leaves turn yellow or even fall off, and biomass accumulation drops sharply, thus affecting crop yield [1]. Saline-alkali stress restricts soybean growth at the R6 stage, affecting carbon assimilation and changing the rules of carbon accumulation, distribution, and transport. The R6 stage is the most important stage for soybean yield and quality and the most vigorous and complex stage of carbon metabolism [4]. Therefore, understanding the saline-alkali stress mechanism on soybean yield helps improve seed yield and food security.

Photosynthesis is crucial in sucrose-starch metabolism, directly affecting the formation and transformation of photosynthetic products and crop yield [5]. Saline-alkali stress can reduce the Pn, Ci, Gs, and Tr of plant leaves, interfere with photosynthetic electron transport and photosynthetic phosphorylation, and affect plant quality and yield [6]. Through leaves and other photosynthetic organs, plants can assimilate inorganic carbon in the environment into organic carbon, providing the necessary energy for plant growth and development by synthesizing, degrading, and transforming carbohydrates such as sucrose and starch [7, 8]. Sucrose is the main product of photosynthesis in higher plants, the main substrate in carbon metabolism, and the key signal molecule coordinating the source-sink relationship in plants [9]. Soluble sugar accumulation (mainly sucrose) increases plant tolerance to abiotic stress [10]. Saline-alkali stress can promote starch hydrolysis in plant leaves, accelerate transport to sink organs, and change the intracellular sugar content [11].Moreover, phosphosucrose synthetase (SPS), sucrose synthase (SuSy), acid invertase (A-INV) and neutral invertase (N-INV) co-regulate the sucrose synthesis and degradation in plants. SPS and SS are key enzymes in the sucrose anabolic pathway, sucrose synthesis and decomposition in plant cells are synergistic with SPS and SuSy [12, 13]. Convertases (AI and NI) hydrolyze sucrose to UDPG, F-6-P by SuSy, glucose, and fructose in higher plants [14]. Starch is the main storage form of carbohydrates in plants. During adversity, plants use their starch stores to maintain normal physiological metabolism. Two key amylolytic enzymes, AMY and BAM, break down starch into soluble sugars [15, 16]. Abiotic stress increases the activity of starch metabolizing enzymes in the leaves of soybean seedlings, promoting starch transformation to soluble sugar and sucrose accumulation in the leaves [17].

Seed is a vital plant sink organ and the carrier of plant generations [18]. Promoting the transport and distribution of carbohydrates in plants to sink organs is key for coordinating source-sink relations and increasing yield [19, 20]. Photo-assimilates (sucrose) are usually loaded in the phloem and transported through the vascular tissue for metabolism and growth at the sink (young leaves, roots, and seeds) [9]. Symplast and apoplast are the main loading pathways in plants [21]. Sucrose transport in the symplast pathway is mainly dependent on the plasma. In the apoplast pathway, sucrose can diffuse to parenchyma cells through the symplast in the plasma. The SWEET proteins transport sucrose out of the parenchyma cells, and the sucrose transporter (SUC) transports the sucrose across the membrane to the sieve element-companion cell complex [22]. Furthermore, studies have demonstrated that sucrose transporter genes can regulate the distribution of soluble sugars in plants to help them adapt to abiotic stress conditions. For example, following salt and drought treatments, the expression level of OsSUC2 in rice was significantly upregulated, suggesting that SUC1 and SUC2 are involved in the plant’s response to salt and drought stress [23, 24]. Ectopic expression of the apple gene MdSWEET1 in tomatoes has been shown to enhance salt tolerance [25]. Similarly, MaSWEETs in bananas were found to be upregulated under cold, salt, and osmotic stress conditions, indicating their significant role in the plant’s abiotic stress response [26].

Trp is an essential amino acid for protein synthesis. The nitrogen in tryptophan can be absorbed and utilized by plants via transamination and deamination processes, thereby helping to maintain the carbon-nitrogen balance within plant systems, and plays a vital role in plant growth and development [27–29]. Research has found that foliar application of exogenous tryptophan can reduce the demand of kidney beans for nitrogen fertilizer and promote the growth and development of plants [30]. In addition, Trp enhances the stability of cell membranes by maintaining the balance of ions and hormones within the plant, thereby improving the plant’s tolerance to abiotic stress to a certain extent [31]. Exogenous Trp application can promote the production of K⁺, IAA, Trp, and other defense substances, maintaining osmoregulation in plants and resisting the damage of the adverse environment [31, 32]. Exogenous Trp application can promote IAA synthesis in quinoa, improving its growth and development, photosynthetic pigment and free amino acid contents, and enhancing drought tolerance [33]. Exogenous Trp application can alleviate the effects of salt stress on potato yield [34]. IAA signal transduction also plays a key role in regulating glucose metabolism and carbon partitioning [35]. Additionally, altering IAA biosynthesis can regulate glucose concentration, thus regulating seed germination and seedling morphogenesis in maize during germination [36]. Exogenous IAA can improve the activity of starch metabolism enzymes in soybean seedlings, increasing the content of sucrose, soluble sugar, and soluble solids, and promoting soybean seedling growth [37]. Additionally, exogenous Trp can promote the accumulation of anthocyanins, flavonoids, and phenols, improving the tolerance of plants to various environmental stresses. For instance, exogenous Trp improves the tolerance of lupinus albus to water deficiency by increasing photosynthetic pigments, indole acetic acid, phenolic compounds, and antioxidant activity [38]. Exogenous Trp increased the content of ascorbic acid, flavonoid, anthocyanin, and total phenol in sunflower plants, reducing the degree of membrane lipid peroxidation and the effect of cadmium stress on sunflower growth [39].

Saline-alkali stress inhibited the photosynthetic carbon assimilation capability of plants and seriously affected growth, development, and yield. As a signal molecule, Trp plays an essential role in promoting plant growth and development and resisting stress. However, how Trp alleviates saline-alkali stress in soybean and the role of Trp in the distribution, transport, and metabolism of sucrose-starch between source-sink under saline-alkali stress is unclear. Therefore, this study used two soybean varieties, Heihe 49 (HH49, saline-alkali tolerant) and Henong 95 (HN95, saline-alkali sensitive) as test materials. The study hypothesized that: (1) Exogenous Trp increased the plant height, stem diameter, number of main stem nodes, and dry matter accumulation of soybean, alleviating the growth inhibition of saline-alkali stressed soybean; (2) Exogenous Trp promoted the photosynthetic assimilate production by increasing the photosynthetic pigment content and photosynthetic capacity of soybean leaves under saline-alkali stress; (3) Exogenous Trp regulates the activities of sucrose-starch accumulation related enzymes and the relative expression of related genes in soybean leaves, seeds, and pod skins under saline-alkali stress, promoting the photosynthetic product transport and accumulation from source to sink, and improving soybean yield.

Materials and methods

Plant materials and growth conditions

The experiment was conducted in 2023 in a dry greenhouse at the potted planting field of Heilongjiang Bayi Agricultural University in Daqing City, Heilongjiang Province, China (45°46 ‘N, 124°19’ E), using the bucket planting method. The two soybean varieties, Heihe 49 (HH49, saline-alkali tolerant variety) and Henong 95 (HN95, saline-alkali sensitive variety), were provided by the Crop Germplasm Resources Innovation Laboratory of Heilongjiang Bayi Agricultural University.

Full, uniform-size seeds with no disease and insect spots were selected and sown evenly in the cultivation barrel. The sowing involved six soybean seeds in each barrel at a depth of 3 cm. The soil was packed in 30 cm barrels measuring 33 cm in height and 20 cm in diameter. After the soybean grew true leaves, the seedlings were thinned, and three seedlings with similar growth parameters were retained in each barrel.

At the V1 stage, soybean was subjected to saline-alkali stress following the 1:9:1:9 ratio of NaCl: Na₂SO₄: Na₂CO₃: NaHCO₃, Referring to the previous experimental results of the salinity ratio of saline-alkali soil in Daqing area, Heilongjiang Province, China by the laboratory [40], the final concentration of Na⁺ in the soil was Made to 2.76‰ (W/W), and the pH was 8.67. All the salt was mixed in 3 L of water to avoid triggering the salt stress reaction, and plants received a daily ration of 600 mL of treatment solution at intervals of 1 d, and the irrigation was completed in 5 portions. Meanwhile, the control group was irrigated with 600 mL of distilled water each time. Since then, with the use of conventional management, each treatment has had the same amount of water.

Foliar spraying treatments were carried out three times on soybean plants at the V3 stage, with a total spraying duration of three days, including five treatments: ①Spray water in control treatment (CK); ② Spray water in saline-alkali treatment (SA); ③ Spray 100 mg·L⁻¹ Trp in saline-alkali treatment (T1); ④ Spray 200 mg·L⁻¹ Trp in saline-alkali treatment (T2); ⑤ Spray 300 mg·L⁻¹ Trp in saline-alkali treatment (T3). The Trp concentration was obtained through the screening of the preliminary pre-experiment. The solutions were sprayed evenly to the leaf surfaces (front and back) until wet, with 10 mL per barrel and then managed conventionally. The R6 samples of soybean were used to determine various indexes. Soybean leaves (three inverted leaves), seeds, and pod skins were separated. The samples for physiological indexes and gene expression detection were frozen in Liquid nitrogen for 5 min and stored at −80 ℃. Other plants were managed normally, and the yield traits of soybean were measured at natural maturity. Three soybean plants in each barrel were treated as one biological replicate, and each treatment was repeated nine times.

Determination of soybean aboveground morphology

The plant height (cotyledon node to growing point) was measured using a ruler, and stem diameter (cotyledon node) was measured using a Vernier caliper. The number of nodes, pods, and soybean seeds was counted manually.

Determination of photosynthetic gas exchange parameters

Before sampling, the photosynthetic rate of the three leaves (functional leaves) on the Main stem of soybean During 3–5 h (9:00–11:30) after the photoperiod began was measured using the LI-6400 photosynthetic apparatus (LI-COR Inc., Lincoln, USA). The parameters of the photosynthesis measuring instrument are set as follows: The artificial Light source is 1200µmol·m⁻²s⁻¹; The temperature in the leaf chamber is 25 ℃−30 ℃. The relative humidity is 60%−65%. The concentration of CO₂ is 380 µmol·mol⁻¹. The air flow rate is 500µmol·s⁻¹. Five plants from each treatment were subjected to Pn, Gs, Tr, and Ci. measurements.

Determination of chlorophyll fluorescence parameters

The chlorophyll fluorescence parameters were determined using Multispe Q, a portable plant measuring instrument. The measurements include photosystem Ⅱ actual photochemical quantum yield (ΦPSⅡ), electron transfer rate (ETR), potential photochemical efficiency of photosystem Ⅱ (F_V/Fo), and maximum photochemical efficiency of photosystem Ⅱ (F_V/F_m).

Measurement of soybean plant biomass accumulation

The leaves, seeds, and pod skins of soybean were separated, and a one-thousandth balance determined the fresh weight of each sample. Then, the samples were put into a brown paper envelope and placed in the oven at 105 ℃ for 30 min, and then baked at 85 ℃ to constant weight. The dry weight of each sample was determined using a 1/1000 th balance. Each treatment was repeated 3 times.

Determination of photosynthetic pigment contents in leaves

Here, 0.1 g of frozen soybean functional leaves (three inverted leaves) were soaked in 10 mL absolute ethanol for 24 h in the dark, shaking them thrice until the leaves completely faded. The Light absorption values were measured at 665 nm, 649 nm, and 470 nm. The ethanol colorimetric method was adopted by Dhindsa [41], to calculate the photosynthetic pigment content using the following formula:

1. Chlorophyll a (Chl a) = 13.95OD₆₆₅−6.88OD₆₄₉

2. Chlorophyll b (Chl b) = 24.96OD₆₄₉−7.32OD₆₆₅

3. Total chlorophyll = Chl a + Chl b

4. Carotenoid (Car) = (1,000OD₄₇₀−2.05Chl a-111.48Chl b)/245

Determination of soluble sugar, starch, and sucrose content

The improved Xu et al. [42] method was used to extract and determine the contents of soluble sugar and sucrose in soybean leaves, seeds, and pod skins. Next, a 0.1 g grinding sample was placed in 80% (V/V) ethanol and extracted in a water bath at 80 °C for 30 min, then centrifuged at 10,000×g for 10 min. The residue was extracted twice using 80% ethanol. The three kinds of supernatant were mixed and topped with 80% ethanol to Make 5 mL. Finally, the OD values were determined at 620 nm and 480 nm, and the soluble sugar and sucrose contents were calculated accordingly.

The ethanol-insoluble residue of the above step was used for starch extraction by the Kuai et al. [43], method. After the ethanol was completely evaporated, 2 mL of distilled water was added to the mixture and incubated in a water bath at 100 ℃ for 15 min. The starch was hydrolyzed to the solution using 9.2 M HClO₄ and 4.6 M HClO_4, respectively. The OD value was determined using an anthrone reagent at 620 nm wavelength, and the starch content was calculated accordingly.

Determination of starch metabolism-related enzyme activities

A slightly modified starch metabolizing enzymes were extracted, and their activities were determined following the Kishorekumar et al. [44], method. Next, 1.5 mL of pre-cooled distilled water was added to a 0.15 g sample and ground into a homogenate. The extracts were centrifuged at 12,000×g, at 4 ℃ for 30 min. The activities of α-amylase and β-amylase were determined by collecting supernatant.

Determination of sucrose metabolism-related enzyme activities

The enzymes related to sucrose metabolism were extracted following the Liu et al. [45], method and used to determine the activities of SPS, SuSy, and INV.

RNA extraction, cDNA synthesis, and gene expression analysis

Total RNA from soybean leaves, seeds, and pod skins was extracted using the TransZol Plant Kit (TRAN, Beijing, China). The RNA purity and concentration were analyzed using a NanoDrop 2000 C ultra-micro spectrophotometer. The RNA integrity was detected by 1% agar-gel electrophoresis at 180 V constant pressure for 10 min. Single-strand cDNA was synthesized using the Perfect Start UniRT&qPCR Kit (TRAN, Beijing, China). The reaction was performed with Hieff UNICON^® Universal Blue qPCR SYBR Green Master Mix (Yeasen, Shanghai, China). Real-time quantitative PCR experiments were performed on the Step One real-time PCR system (Applied Biosystems). The relative expression of each gene was quantified by the comparative threshold cycle method (Primer sequence is shown in Table. S1) using GmEF1a as the internal reference. The qRT-PCR reaction included three biological and three technical replicates per treatment. The thermal cycle conditions involved 95 ℃ for 1 min, followed by 39 cycles at 95 ℃ for 5 s, 58 ℃ for 20 s, and 60 ℃ for 20 s. The relative expression was determined according to the 2^−∆∆t method.

Yield measurement

The yield was measured at the maturity stage (R8), and the number of pods per plant, number of seeds per plant, weight of seeds per plant, and hundred-seed weight were measured in three pots (totaling nine plants) per treatment.

Statistical analysis

SPSS 26.0 (SPSS Inc.) was used to analyze the data, GraphPad Prism 8.0 (GraphPad Inc.) was used to plot the histogram, Origin 2021 (Origin Inc.) was used to plot the heat map, and biorender was maked the sucrose-starch metabolism regulation model (https://www.biorender.com/library). Differences between the means were determined using Duncan multiple comparison (P < 0.05), and significant results were indicated by the different letters.

Results

Effects of exogenous trp on soybean growth and yield under saline-alkali stress

Compared with the control treatment, saline-alkali stress significantly decreased (P < 0.05) the plant height, stem diameter, leaf dry weight, and stem dry weight of HH49 and HN95 in the R6 stage (Fig. 1A-E). Exogenous Trp could increase the plant height, stem diameter, leaf dry weight, and stem dry weight of two soybean varieties under saline-alkali stress, and the effect was positively correlated with the concentration of Trp. Compared with saline-alkali treatment, T3 treatment increased the plant height, stem diameter, leaf dry weight, and stem dry weight of HH49 and HN95 by 59.02%, 47.26%, 119.01%, and 98.06%, and 35.00%, 54.61%, 76.07%, and 79.43%, respectively.

Fig. 1.

The effect of different Trp concentrations on the growth of saline-alkali stressed. HH49 and HN95 soybean in stage R6. (A) phenotypic picture, (B) plant height, (C) stem diameter, (D) leaf dry weight, (E) stem dry weight. Different letters above the error lines indicate a significant difference between the means, P < 0.05

Compared with the control treatment, saline-alkali stress significantly decreased (P < 0.05) the number of main stem nodes, number of pods per section of the main stem, and total pod number of main stems of HH49 and HN95 in stage R6 (Fig. 2A-D). Exogenous Trp significantly promoted the formation of main stems and pods of two saline-alkali-stressed soybean varieties. For example, T3 increased the number of main stem nodes, pod numbers per pod of the main stem, and total pods on the main stem in HH49 and HN95 by 30.30%, 16.26%, and 51.39%, 24.39%, 44.76%, and 83.87%, respectively. Additionally, saline-alkali stress significantly decreased the branch and total pod numbers of HH49 and HN95 in stage R6 (Table. S2). Exogenous Trp could increase the branch number and total pod numbers of the two soybean varieties under saline-alkali stress, and the effect was positively correlated with the concentration of Trp. However, T3 treatment increased the number of branches, and total pod numbers per branch of saline-alkali treated HH49 and HN95 by 75.00% and 64.29% and 88.89% and 93.33%, respectively.

Fig. 2.

Effect of different Trp concentrations on the yield of saline-alkali stressed HH49 and HN95 soybean at stage R6. (A) pod number per main stem, the horizontal coordinate represents the different treatment conditions, and the vertical coordinate represents the number of main stem nodes, (B) main stem nodes, (C) pod number per main stem, (D) total pod numbers of the main stem, (E) seed dry weight, (F) pod skin dry weight, and (G) pod dry weight. Different letters above the error lines indicate significantly different means, P < 0.05

Compared with the control treatment, saline-alkali stress significantly decreased (P < 0.05) the seed dry weight, pod skin dry weight, and pod dry weight of HH49 and HN95 in stage R6 (Fig. 2E-G). Exogenous Trp could increase the seed dry weight, pod skin dry weight, and pod dry weight of two saline-alkali stressed soybean varieties, the effect was positively correlated with the concentration of Trp. Compared with saline-alkali treatment, the seed dry weight, pod skin dry weight, and pod dry weight of T3 treated HH49 and HN95 increased by 55.92%, 49.51%, and 53.24%, and 41.84%, 51.75%, and 47.99%, respectively. Exogenous Trp significantly increased the yield and yield components of HH49 and HN95 at stage R8 under saline-alkali stress. However, exogenous Trp at the T3 concentration had a more significant promoting effect on yield (Table. S3).

Effects of exogenous trp on the photosynthetic capacity of saline-alkali stressed soybean

Compared with the control treatment, saline-alkali stress significantly decreased (P < 0.05) the contents of Chl a, Chl b, Car, and total Chl in HH49 and HN95 (Fig. 3A-D). Exogenous Trp could increase the photosynthetic pigment content of the two soybean varieties under saline-alkali stress, the effect was positively correlated with the concentration of Trp. T3 treatment increased the contents of Chl a, Chl b, Car, and total Chl in HH49 and HN95 by 31.57%, 83.41%, 88.14%, and 56.08% and 44.72%, 111.45%, 144.45%, and 75.48%, respectively.

Fig. 3.

Effects of different Trp concentrations on the photosynthetic characteristics of saline-alkali stressed HH49 and HN95 soybean in stage R6. (A-D) leaf photosynthetic pigment contents, (E-H) leaf gas exchange parameters, and (I-L) chlorophyll fluorescence parameters. * and ** indicate significant differences at the P<0.05 and P<0.01 level, respectively

Compared with the control treatment, saline-alkali stress significantly decreased (P < 0.05) the gas exchange parameters (Pn, Gs, Ci, and Tr) of HH49 and HN95 (Fig. 3E-H). Pn, Gs, Ci, and Tr decreased by 43.27%, 26.35%, 74.67%, and 68.86% and 35.76%, 30.92%, 102.47%, and 110.25% in HH49 and HN95, respectively. In conclusion, saline-alkali stress has a greater inhibition on the photosynthetic gas exchange and net photosynthetic rates of HN95 than HH49. Exogenous Trp could increase the Pn, Gs, Ci, and Tr of the two soybean varieties under saline-alkali stress, the effect was positively correlated with the concentration of Trp. Moreover, the effect of the T3 concentration of Trp was more significant. Compared with saline-alkali treatment, Pn, Gs, Ci, and Tr in HH49 under T3 treatment increased by 37.00%, 121.90%, 45.67%, and 71.42%, and in HN95 increased by 35.22%, 118.75%, 76.87%, and 119.24%, respectively.

Saline-alkali stress significantly inhibited (P < 0.05) chlorophyll fluorescence (Fv/Fm, Fv/Fo, ΦPSⅡ, and ETR) in HH49 and HN95 (Fig. 3I-L). The Fv/Fm, Fv/Fo, ΦPSⅡ, and ETR of HH49 and HN95 decreased by 15.23%, 23.59%, 11.65%, and 15.16%, and 35.01%, 21.68%, 12.50%, and 34.71%, respectively. Exogenous Trp significantly increased the Fv/Fm, Fv/Fo, ΦPSⅡ, and ETR of the two soybean varieties under saline-alkali stress, the effect was positively correlated with the concentration of Trp. The Fv/Fm, Fv/Fo, ΦPSⅡ, and ETR of T3 treated HH49 and HN95 increased by 21.95%, 24.15%, 121.22%, and 21.87%, and 42.63%, 22.09%, 105.61%, and 42.23%, respectively.

Effects of exogenous trp on carbon accumulation in saline-alkali stressed soybean

Trp significantly decreased (P < 0.05) the fructose content in the leaves, seeds, and pod skins, and starch contents in the leaves and seeds of saline-alkali stressed HH49 and HN95 but significantly increased the sucrose content (Fig. 4). Exogenous Trp could increase the contents of soluble sugar, sucrose, and starch in the leaves, seeds, and pod skins of the two soybean varieties under saline-alkali stress, the effect was positively correlated with the concentration of Trp. Furthermore, T3 treatment increased the contents of soluble sugar, sucrose, fructose, and starch in the leaves, seeds, and pod skins of HH49 by 32.02%, 38.00%, 54.68%, and 33.80%, 33.12%, 18.69%, 23.50%, and 25.39%, and 40.23%, 74.06%, 29.57%, and 27.39%, respectively. In HN95, T3 treatment increased the contents of soluble sugar, sucrose, fructose, and starch in the leaves, seeds, and pod skins by 26.62%, 48.18%, 53.95%, and 29.96%, 31.96%, 16.85%, 33.89%, and 22.99%, and 37.36%, 70.12%, 45.72%, and 33.03%, respectively.

Fig. 4.

Effects of different Trp concentrations on the sugar content in soybean leaves, seeds, and pod skins under saline-alkali stress. (A-C) soluble sugar content, (D-F) sucrose content, (G-I) fructose content, and (J-L) starch content in the leaves, seeds, and pod skins of HH49 and HN95 in phase R6. * and ** indicate significant differences at the P<0.05 and P<0.01 level, respectively

Effects of exogenous Trp on the activities of enzymes related to sucrose-starch metabolism in saline-alkali stressed soybean at R6

Trp significantly increased the SPS and SuSy-S enzyme activities in the leaves, seeds, and pod skins of HH49 and HN95 (P < 0.05). In contrast, Trp significantly decreased the SuSy-C, A-INV, and N-INV enzyme activities under saline-alkali stress (Fig. 5). Saline-alkali treatment increased the SPS activity of HH49 and HN95 leaves, seeds, and pod skins by 4.07%−22.10% and the SuSy-S activity by 10.72%−30.53%. In contrast, it decreased the SuSy-C activity by 61.84%−313.58%, AI activity by 109.75%−239.25%, and NI activity by 235.57%−454.96%. Exogenous Trp could increase the activities of sucrose metabolizing enzymes of leaves, seeds, and pod skins of the two soybean varieties under saline-alkali stress, the effect was positively correlated with the concentration of Trp. Moreover, T3 treatment increased the sucrose metabolic enzyme activity in the leaves, seeds, and pod skins by 34.50%−323.56%, 33.03%−296.93%, and 41.18%−410.35%, respectively.

Fig. 5.

Effects of different Trp concentrations on the activities of enzymes related to carbon metabolism in saline-alkali stressed soybean. In the heat map, the horizontal coordinate represents the different treatment conditions of the two soybean varieties, and the vertical coordinate represents the different parts of the soybean

Saline-alkali stress significantly increased the amylase activity in the leaves, seeds, and pod skins of HH49 and HN95 (P < 0.05); the activities of AMY and BAM increased by 38.07%−50.50% and 28.88%−37.46%, respectively (Fig. 5). Exogenous Trp could decrease the amylase activity of two soybean varieties under saline-alkali stress, the effect was positively correlated with the concentration of Trp. However, T3 treatment decreased the activities of AMY and BAM in HH49 and HN95 leaves, seeds, and pod skins by 36.46%, 30.42%, and 40.62%, 35.25%, 15.53%, and 35.41%, 37.03%, 30.04%, and 39.41%, and 32.91%, 18.91%, and 29.43%, respectively. However, the T3 values were higher than the T1 and T2 treatments.

Effects of exogenous Trp on the expression of genes related to starch metabolism in soybean under saline-alkali stress

Saline-alkali stress significantly decreased (P < 0.05) the relative expression of GmAMY3 and GmSS in the leaves of HH49 and HN95 and of GmAMY3 in the pods of HH49 (Fig. 6A-C). In contrast, saline-alkali stress significantly increased the relative expression of GmBAM1 in the leaves, seeds, and pod skins of HH49 and HN95. Exogenous Trp significantly increased the relative expression of GmSS in the leaves and pod skins of HH49 and HN95 and the relative expression of GmAMY3 in HN95 leaves. On the contrary, Trp significantly decreased the relative expression of GmBAM1, GmAMY3, and GmSS in HH49 and HN95 seeds, GmBAM1 in HH49 pod skins, and GmAMY3 in HN95 pod skins. Compared with T1 and T2 treatments, T3 had a more significant adjustment effect.

Fig. 6.

Effects of different Trp concentrations on the expression of genes related to starch metabolism in soybean under saline-alkali stress. Relative expression genes related to starch metabolism in HH49 and HN95 in R6 stage (A) leaves, (B) grains, and (C) pod skins levels. In the heat map, the horizontal coordinate represents the different treatment conditions of the two soybean varieties, and the vertical coordinate represents the different parts of the two soybean varieties

Effect of exogenous trp on the expression of sucrose transporter in saline-alkali stressed soybean

Saline-alkali stress significantly decreased (P < 0.05) the relative expression of GmC-INV in the leaves, seeds, and pod skins of HH49 and HN95. The relative expression of GmSuSy and GmSPS in the leaves and pod skins of HH49 and GmA-INV in the leaves and pod skins of HN95 were significantly decreased (Fig. 7A-C). Compared with saline-alkali treatment, exogenous Trp significantly increased the relative expression of GmA-INV, GmC-INV, GmSuSy, and GmSPS in the leaves, seeds, and pod skins of HH49 and HN95, the effect was positively correlated with the concentration of Trp. T3 treatment increased the relative expression of genes by 66.44%−655.31%, significantly higher than T1 (0.29%−290.55%) and T2 (38.29%−503.74%).

Fig. 7.

Effect of different Trp concentrations on the expression of sucrose transporter-related genes in soybean under salt-alkali stress. Relative expression related to sucrose metabolism genes in the (A) leaves, (B) seeds, and (C) pod skins of HH49 and HN95 at the R6 stage. Relative expression of genes related to sucrose metabolism in HH49 and HN95 (D) leaves, (E) seeds, and (F) pod skins in the R6 stage. In the heat map, the horizontal coordinate represents the different conditions of the two soybean varieties, and the vertical coordinate represents the different parts of the two soybean varieties

Further, saline-alkali significantly decreased (P < 0.05) the relative expression of GmSUC2 and GmSWEET6 in the pod skins of two soybean varieties, and the relative expression levels of GmSUC2 and GmSWEET6 in the seeds of HH49 and HN95 (Fig. 7D-F). Compared with saline-alkali treatment, exogenous Trp significantly decreased the relative expression of GmSUC2, GmSWEET6, and GmSWEET15 in the leaves of HH49 and HN95 but increased the relative expression of sucrose transport-related genes in seeds and pod skins. T3 treatment decreased the relative expression of genes in the leaves by 40.93%−68.23%, significantly higher than that under T1 (15.36%−56.26%) and T2 (9.75%−55.59%).

Discussion

Plant organ biomass is significantly positively correlated with the increase in yield [46]. Furthermore, the increase of soybean dry matter accumulation inevitably increases yield [47]. Egli showed that dry matter accumulation is an important determinant of soybean yield during the podding and seed-filling stages [48]. As a signal molecule, Trp mediates the physiological responses of plants to various biotic and abiotic stresses [49]. However, there are few studies on how Trp alleviates saline-alkali stress during the growth and development of soybean at the R6 stage.

Exogenous Trp promotes the production of sucrose equivalents by improving the photosynthetic capacity of soybean leaves; By regulating the relative expression of sucrose-starch metabolic enzymes and genes related to sucrose and starch metabolism, mediates the balance of sucrose and starch metabolism in soybean leaves and grains under salt-alkali stress and improves the salt-alkali tolerance of soybeans.

The synergistic interaction between soybean source and sink, the number of pods per plant, the number of seeds per plant, and the weight of hundred-seed directly affect soybean yield [50]. High dry matter accumulation, pod number, and seed number are among the most significant characteristics of high-yield soybean [51, 52]. In this study, saline-alkali treatment significantly inhibited (P < 0.05) the plant growth and dry matter accumulation of HH49 and HN95 in the R6 stage (Fig. 1B-F, Table. S2). These results are consistent with previous studies [53, 54], further verifying that saline-alkali stress inhibits soybean plant growth and affects soybean yield formation. Exogenous Trp treatment significantly increased the plant height, stem diameter, dry leaf weight, and dry stem weight of soybean. Higher Trp concentrations could significantly alleviate the growth inhibition of soybean under saline-alkali stress. Subfinite pod varieties usually start from the bottom of the main stem, moving up to the top [55, 56]. The pod distribution is relatively uniform [57], and the HH49 and HN95 soybean varieties selected in this study are subfinite pod varieties. In the pod stage, soybean copes with adversity and saline-alkali stress by reducing the photosynthetic capacity of leaves, photosynthetic product accumulation, and blocking carbohydrate transport. At maturity, the development on the top of the soybean plant slowed down, the number of main stem nodes, the number of pods per node of the main stem, and the total number of pods of the main stem decreased. Besides, soybean branching and differentiation are inhibited, and the number of branches and pods has decreased. The number of seeds per plant and the weight of hundred-seed also decreased at the maturation stage, seriously affecting soybean yield (Figs. 1D and 2A-B, Table S3). External Trp improved the photosynthetic capacity of soybean leaves and the accumulation of photosynthetic products, alleviating the growth inhibition at the top of saline-alkali-stressed soybean. The number of the main stem nodes, pod number per pod of the main stem, total pod number of the main stem, branch number, and the total pod number of branches of soybean increased. Nonetheless, yield composition factors such as the number of seeds per plant and hundred-seed weight of soybean at maturity also increased, the effect was positively correlated with the concentration of Trp.

The photosynthetic capacity during the reproductive growth period directly affects the seed yield of crops [58]. Moreover, the photosynthetic system of plants is very sensitive to saline-alkali stress. Excessive Na⁺ accumulation inhibits the chlorophyll synthesis and destroys the chlorophyll structure. Therefore, CO₂ diffuses through the stomata and mesophyll, inhibiting photosynthesis [59]. Under low nitrogen stress, exogenous Trp increases the chlorophyll content and photosynthetic rate of sorghum leaves, maintaining the balance of carbon and nitrogen physiology [60]. In rape, spraying proper Trp concentrations on the leaves can activate the auxin synthesis pathway, promote IAA synthesis, increase the chlorophyll content, and enhance the resistance to Cd [61]. In this study, saline-alkali significantly reduced Pn, Gs, Ci, and Tr in HH49 and HN95 at stage R6 (Fig. 3E-H). Additionally, saline-alkali stress also inhibited the contents of photosynthetic pigments (Chl a, Chl b, Car, and total Chl) (Fig. 3A-D) and blocked chlorophyll biosynthesis, further affecting the normal operation of photosynthesis in soybean. Exogenous Trp increased the Pn, Gs, Ci and Tr of the two soybean varieties and alleviated the decreasing photosynthetic pigment contents. Moreover, higher concentrations of Trp showed more significant mitigation effects. Chlorophyll fluorescence is closely related to photosynthetic efficiency. Thus, this study showed that the high pH induced by saline-alkali stress can inhibit the photosynthetic electron transport and PSⅡ photosynthesis and damage the PSⅡ reaction center in plants [62], reducing Fv/Fm, Fv/Fo, ΦPSⅡ, and ETR in soybean leaves. In this study, saline-alkali stress significantly reduced the Fv/Fm, Fv/Fo, ΦPSⅡ, and ETR in HH49 and HN95 soybean leaves. Exogenous Trp significantly increased the Fv/Fm, Fv/Fo, ΦPSⅡ, and ETR of the two soybean varieties, the effect was positively correlated with the concentration of Trp. These results indicate that exogenous Trp maintains the stability of the PSⅡ reaction center under saline-alkali stress and alleviates the damage of soybean under saline-alkali stress (Fig. 8).

Fig. 8.

Sucrose-starch metabolism regulation model of soybean under saline-alkali stress induced by exogenous Trp

Plant carbohydrate metabolism is more sensitive to saline-alkali stress than photosynthesis. Saline-alkali stress decreases the activity of Rubisco, the key enzyme of carbon assimilation, inhibiting the regeneration of inorganic phosphorus and affecting its photosynthetic capacity [58]. Plants can rely on the Calvin cycle increase to produce more photosynthetic products (such as sucrose) to improve their tolerance to abiotic stresses [63]. Under low nitrogen stress, exogenous Trp could increase the content of starch and soluble sugar in the roots of sorghum seedlings, activating SuSy, SPS, and INV and improving the sucrose synthesis and conversion ability of sorghum seedlings roots. Meanwhile, sorghum seedling roots could increase the auxin synthesis ability through the Trp pathway and activate the plasma membrane H⁺-ATPase [64]. In this study, saline-alkali stress affected carbohydrate distribution in the leaves, seeds, and pod skins of HH49 and HN95 at the R6 stage, the contents of fructose and starch in soybean leaves and grains were significantly reduced, while the contents of soluble sugar and sucrose in leaves, grains and pod skins were significantly increased (Fig. 4). These results suggest that saline-alkali stress promotes the conversion of starch into soluble sugar to maintain the intracellular osmotic balance. Furthermore, Trp increased the contents of soluble sugar, sucrose, fructose, and starch in each part of the two soybean varieties, the effect was positively correlated with the concentration of Trp, indicating that Trp could improve the saline-alkali tolerance of soybean by regulating the content of soybean carbohydrates. The added sugar can function as a source of carbon and energy for plant cell activities and as an important solute to maintain the balance of cell osmosis (Fig. 8).

The balance of sucrose synthesis and decomposition in plant cells mainly depends on the synergistic effect of SPS, SuSy, and INV [64]. In this study, saline-alkali stress increased the activities of SPS and SuSy in the leaves, seeds, and pod skins of HH49 and HN95 but decreased the activities of INV (Fig. 5). Additionally, saline-alkali stress decreased the relative expression of sucrose metabolism-related genes GmSPS, GmSuSy, GmC-INV, and GmA-INV in the leaves of two soybean varieties. On the contrary, saline-alkali stress increased the relative expression of GmSPS, GmSuSy and GmA-INV in the seeds and pod skins (Fig. 7A-C). These results suggest that saline-alkali stress can enhance sucrose utilization efficiency and promote soluble sugar accumulation in plants by increasing the activity and expression of sucrose metabolism enzymes. At the same time, the decreased activity of invertase genes in the leaves and seeds under saline-alkali stress can increase the sucrose content, thus enhancing cell osmoregulation. Starch synthesis and decomposition are mainly regulated by metabolic enzymes such as AMY and BAM. Under stress, plants consume their starch to maintain their normal physiological and metabolic functions [65]. Salt stress causes distinct differential expression of the key genes of the starch metabolism pathway of different salt-tolerant rice varieties, thus reducing the starch content [66]. Saline-alkali stress increased the relative expression of GmBAM1, GmSS, and GmAMY3 in the leaves, seeds, and pod skins of HH49 and HN95. However, it decreased the relative expression of GmSS and GmAMY3 in the leaves and pod skins (Fig. 6A-C). Saline-alkali stress could induce starch degradation in the leaves, seeds, and pod skins by enhancing the activities of AMY and BAM and promoting soluble sugar accumulation in plants (Fig. 5). Furthermore, Trp can promote plant growth and yield by regulating sucrose-starch metabolism [67]. For example, Trp and IAA can increase the total sugar content of strawberry fruits. After spraying IAA, the activities of sucrose invertase and amylase decreased at first and then increased slightly [68]. In this study, exogenous Trp significantly increased the activity of sucrose metabolic enzymes and related genes in various parts of the two soybean varieties. In contrast, Trp decreased the activity of starch metabolic enzymes and the expression of related genes and significantly increased the contents of soluble sugar, sucrose, fructose, and starch in leaves, seeds, and pod skins of both varieties at the pelleting stage, the effect was positively correlated with the concentration of Trp. Exogenous Trp promotes sucrose metabolism and starch accumulation in soybean under saline-alkali stress (Fig. 8).

Symplast or apoplast pathways transport sucrose equivalents in plants from source organs (mature leaves) to sink organs (seeds) to provide energy for growth and development [69]. The apoplast pathway is the main route for sucrose transport over long distances in plants and relies on specific sugar transporters (such as SWEET and SUT/SUC proteins) to actively transport sucrose into and out of cells through membranes [70]. Arabidopsis thaliana can unload the sucrose secreted from mesophyll or phloem parenchyma into the apoplast by regulating AtSWEET11 and AtWEET12, and the AtSUC2 protein loads sucrose into the phloem [71]. Additionally, knocking down AtSUC2 causes stunted growth, developmental retardation, and sterility [72]. In this study, saline-alkali stress increased the relative expression of GmSUC2, GmSWEET6, and GmSWEET15 in the leaves and seeds of HH49 and HN95. However, it decreased the relative expression of GmSUC2, GmSWEET6, and GmSWEET15 in pod skins (Fig. 7D-F). Nonetheless, Trp application increased the relative expression of GmSUC2, GmSWEET6, and GmSWEET15 in seed and pod skins. TI and T2 treatments also increased the relative expression of GmSUC2, GmSWEET6, and GmSWEET15 in leaves. These results showed that exogenous Trp promoted photosynthesis in the leaves, thus facilitating sucrose transport from source to sink in soybean. Even so, T3 treatment decreased the relative expression of GmSUC2, GmSWEET6, and GmSWEET15 in the leaves, probably due to the inhibition of sucrose transport-related genes by higher Trp concentrations. Low Trp concentrations can promote the expression of Trp synthesis genes, promoting Trp synthesis. In contrast, high Trp concentration has a feedback inhibition effect on endogenous Trp synthesis. For example, an optimal Trp concentration (100 mg·L⁻¹) can effectively alleviate the chlorophyll degradation and membrane lipid peroxidation caused by CdCl₂, thereby improving the resistance of rape seedlings to CdCl₂ [73]. A high Trp concentration (500 mg·L⁻¹) can also promote the growth and development of rape, but the effect is less than for low Trp [74].

Conclusions

Sucrose-starch distribution, transport, and metabolism can regulate soybean growth under saline-alkali stress. Saline-alkali stress decreases the photosynthetic capacity of soybean leaves, the photosynthetic products, and the activity of INV. However, it increased the activities of SPS, SuSy, AMY, and BAM, inhibiting sucrose and starch metabolism. Exogenous Trp treatment can increase the photosynthetic pigment content of soybean leaves, improve the photosynthetic capacity of soybean leaves, and promote the distribution, transport, and metabolism of sucrose and starch. Thus, it alleviates the growth inhibition of soybeans under saline-alkali stress and improves yield-related indicators such as the number of pods per plant, the number of seeds per plant, and the hundred-seed weight of soybeans. These findings will improve our understanding of the physiological mechanism of exogenous Trp alleviation of saline-alkali stress on soybean growth and development.

Abbreviations

SA

Saline-alkali stress

R6 stage

The grain filling stage

Trp

Tryptophan

ROS

Reactive oxygen species

SPS

Phosphosucrose synthetase

SuSy

Sucrose synthase

A-INV

Acid invertase

N-INV

Neutral invertase

UDPG

UDP-glucose

F-6-P

Fructose-6-phosphate

SPP

Sucrose phosphatase

SUC

Sucrose transporter

Authors’ contributions

W.W., J.D., and Y.D. designed this study. W.W., R.F., J.Z., Y.C., J.F., and Y.Z. executed the experiments; W.W., R.F., J.Z., Y.C., J.F., and Y.Z. contributed to the data collection; W.W., R.F., J.D., and Y.D. analyzed the data and wrote manuscript. J.D., and Y.D. revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by Application of Heilongjiang Natural Science Foundation Joint Guidance Project (LH2024C077), Saline-alkali Tolerant Soybean Germplasm and Demonstration of Saline-alkali Resistant Cultivation Technology (LJGXCG2022-111).

Data availability

The authors declare that the data supporting the findings of this study are available within the paper and its Supplementary Information files. Should any raw data files be needed in another format they are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Competing interests

The authors declare no competing interests.