Plant Physiology and Biochemistry 225 (2025) 110050

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Plant Physiology and Biochemistry

journal homepage: www.elsevier.com/locate/plaphy

Starch-sucrose metabolic homeostasis in germinating soybean reserve

mobilization with different levels of salt stress
Zhaoning Zhang a,b , Qiang Zhao a,b , Weiyu Wang a,b, Ruiqi Feng a,b, Yu Cao a ,
Guangda Wang a,b , Jidao Du a,b,c,*, Yanli Du a,b,c,**
a
College of Agriculture, Heilongjiang Bayi Agricultural University, Daqing, 163319, China
b
Crop Germplasm Resource Innovation Laboratory, National Coarse Cereals Engineering Research Center, Daqing, 163319, China
c
Research Center of Saline and Alkali Land Improvement Engineering Technology in Heilongjiang Province, Daqing, 163319, China

A R T I C L E I N F O A B S T R A C T

Keywords: Salt stress is one of the abiotic factors limiting crop growth. However, the mechanisms regulating starch-sucrose
Soybean (Glycine max L.) metabolism in the mobilization of soybean (Glycine max L.) reserves at different levels of salt stress during
Germination stage germination remain unknown. This study evaluated the effects of different levels of salt stress [0 mmol L− 1 NaCl
Salt stress
(CK), 75 mmol L− 1 NaCl (S), and 150 mmol L− 1 NaCl (SS)] on germinating seeds of three soybean varieties. Salt
Starch-sucrose metabolism
stress, especially the SS treatment, significantly reduced the radicle length, radicle fresh weight, and total fresh
Reserve mobilization
weight. Salt stress had different effects on the starch accumulation of cotyledons and radicles, while significantly
increased the sucrose accumulation through several mechanisms. The regulatory mechanisms governing starch-
sucrose metabolism in cotyledon and radicle during the germination stage exhibit distinct differences. In coty­
ledons, the decrease of starch content under S and SS treatment was due to the decrease of sucrose decompo­
sition, which inhibited the metabolic cycle of starch. The up-regulated expression of GmSWEET6/15 under
different levels of salt stress can promote the accumulation of sucrose in radicles. However, different salt stress
levels have different response mechanisms to sucrose and starch metabolism in radicles. The activities of sucrose-
metabolizing enzymes sucrose synthase (cleavage) and invertase in the radicle of S treatment were significantly
reduced, while SS treatment could activate the sucrose metabolic cycle and increase the efficiency of starch-
sucrose conversion. Compared with S treatment, the accumulation of starch content in SS treatment was due
to the increase of SSS, α-amylase and β-amylase activities and the up-regulation of GmAMY3 and GmBAM1
expression levels. Based on the results of this study, in order to promote soybean germination in saline soils,
rationalized soybean seed compositions can be designed according to the degree of salinity in each region, which
can provide a reference to the future breeding of salt-tolerant soybeans.

et al., 2020). Reclaiming salinized land is a worthwhile challenge in

order to further increase soybean production without taking land away
1. Introduction from staple grain cultivation. Soybeans in salt-affected soil must suc­
cessfully germinate from sowing to harvest, which is the first stage to
Soil salinization has affected more than 800 million hm2 of land, cross. Although studies have shown that various salt stress levels affect
approximately 7 % of the total land area globally (Flowers and Flowers, soybean plants differently, the physiological and molecular response
2005; van Zelm et al., 2020). However, salt concentrations may also mechanisms of germinating soybean to different levels of salt stress are
vary from one site to another due to geographic or anthropogenic fac­ unknown, especially starch-sucrose metabolism in sprouted soybean
tors. Salt stress can cause an osmotic potential imbalance between plant reserve mobilization.
tissues and the environment, leading to physiological drought, osmotic Starch production in the cotyledons of soybeans during the early
stress, and material transport issues, thus slowing plant growth (Nicotra stages of growth (within 5 days of sowing) is converted from soluble
et al., 2010; Zhu, 2016). Soybean (Glycine max L.) is a major source of sugar (mainly sucrose), due to mobilization of protein and lipid reserves
protein, oil, and trace elements in the human diet and animal feed (Yan

* Corresponding author. College of Agriculture, Heilongjiang Bayi Agricultural University, Daqing 163319, China.
** Corresponding author. College of Agriculture, Heilongjiang Bayi Agricultural University, Daqing 163319, China.
E-mail addresses: zhangzhaoning@yeah.net (Z. Zhang), djdbynd@163.com (J. Du), dyl0305@sina.cn (Y. Du).

https://doi.org/10.1016/j.plaphy.2025.110050
Received 12 December 2024; Received in revised form 24 April 2025; Accepted 19 May 2025
Available online 19 May 2025
0981-9428/© 2025 Elsevier Masson SAS. All rights are reserved, including those for text and data mining, AI training, and similar technologies.
Z. Zhang et al. Plant Physiology and Biochemistry 225 (2025) 110050

invertase (INV), including acid invertase (AI) and neutral/alkaline

Abbreviation invertase (NI), can hydrolyze sucrose (Hendrix and Huber, 1986). Os­
motic stress up-regulated GmSPS, GmSuSy, and GmINV in soybean leaves
AGPP ADP-Glc pyrophosphorylase under drought conditions. Furthermore, elevated activities of SPS and
GBSS granule-bound starch synthase SuSy can promote sucrose accumulation and by osmotic stress resistance
SSS soluble starch synthase in soybean plants (Du et al., 2020a). Under 80 mmol L− 1 NaCl, Enhanced
SBE starch branching enzymes SuSy and INV activities promoted sucrose metabolism in cucumber
INV Invertase leaves, thus alleviating salt damage (Li et al., 2020). Sucrose content and
NI neutral/alkaline invertase the SPS and SuSy activities increased in the leaves subtending cotton
AI Acid invertase bolls at the seedling stage with increasing salinity (Ju et al., 2021).
SPS Sucrose phosphate synthetase When sweet sorghum (Sorghum bicolor L.) was subjected to varying
SUC Sucrose transporter levels of salt treatment, the number of leaves remained unchanged at 50
SuSy Sucrose synthase mmol L− 1 NaCl, while leaf weight significantly decreased and sugar
SuSy-S Sucrose synthase (synthetic) content increased. Under 150 mmol L− 1 NaCl treatment, both leaf
SuSy-C Sucrose synthase (cleavage) number and leaf weight were significantly suppressed, while sugar
SWEET Sugars Will Eventually be Exported Transporters. content varied among individuals. KEGG (Kyoto Encyclopedia of Genes
and Genomes) pathway analysis revealed that severe salt stress dis­
rupted starch and sucrose metabolism. However, it is noteworthy that
the study lacks in-depth analysis of the 50 mmol L− 1 NaCl treatment and
had not yet begun, this is different from the starch catabolic stage of the comparative differences between 50 and 150 mmol L− 1 NaCl levels (Sui
wheat and rice seed germination process (Hildebrand and Hymowitz, et al., 2015).
1981; Brown and Huber, 1988; Moreira et al., 2019). The key enzymes Sucrose plays a key role in biomass partitioning between sources
involved in starch synthesis are ADP-Glc pyrophosphorylase (AGPP), (leaves) and reservoirs (roots) (Cai et al., 2017). Sucrose is transported
granule-bound starch synthase (GBSS), soluble starch synthase (SSS) through the phloem of plant tissues, and its transport is achieved via the
and starch branching enzymes (SBE), where GBSS and SSS are collec­ plastid extracellular or coplasmic pathways (Mišić et al., 2012; Ruan,
tively referred to as starch synthases (SS), while those that breakdown 2012). Sucrose transport proteins, such as the SWEET (Sugars Will
starch into soluble sugars include α/β-amylase (Stitt and Zeeman, 2012). Eventually be Exported Transporters) protein family and SUC (Sucrose
Several studies have indicated that salt stresses can affect starch transporter), are crucial for the transmembrane transport of sucrose
metabolism (Chen et al., 2008; Farooq et al., 2022). In rice (Oryza sativa) (Chen et al., 2010, 2015; Sosso et al., 2015; Abelenda et al., 2019). In
leaves, 200 mmol L− 1 NaCl reduced GBSS activity (Chen et al., 2008). In Arabidopsis, the SWEET proteins are divided into four subfamilies,
Arabidopsis leaves, 150 mmol L− 1 NaCl increases AGPP activity (Kempa among which AtSWEET11/12 belongs to the third subfamily and pref­
et al., 2008). β-Amylase activity was enhanced in Ricinus communis erentially transports sucrose across the membrane (Le Hir et al., 2015).
plants under 150 mmol L− 1 NaCl, promoting the breakdown of starch for AtSWEET11/12 was up-regulated in plants under drought stress,
sucrose synthesis (Li et al., 2023). Furthermore, sucrose accumulation possibly owing to increased carbon export from leaves to the root system
can alleviate salt stress damage in plants. Genes encoding starch (Fatima et al., 2022). Water deficit significantly up-regulated GmSUC2
metabolizing enzymes also have important roles in regulating plant (homolog of AtSUC2), GmSWEET6 (homolog of AtSWEET11), and
stress responses. Exposure to 150 mM NaCl significantly upregulated the GmSWEET15 (homolog of AtSWEET12) in soybean leaves and roots,
expression of α-amylase (AMY2) and β-amylase (BAM) in alfalfa (Med­ promoting sucrose transport from leaves to roots and root development,
icago sativa L.), which enhanced salt stress tolerance by stimulating thus alleviating water deficit-induced damage (Du et al., 2020a). Plant
starch hydrolysis into sucrose (Li et al., 2022). This enzymatic regulation SUC also redistributes sucrose in source reservoirs under salt stress
mechanism provides an adaptive strategy for osmotic adjustment under (Wang et al., 2020). For example, AtSUC2/4, located in the plasma
saline conditions through enhanced carbohydrate metabolism. Under membrane of Arabidopsis, can improve sucrose loading in the phloem.
250 mmol L− 1 NaCl treatment, barley (Hordeum vulgare L.) seeds Furthermore, sucrose content increased in the aboveground parts but
exhibited down-regulated expression of Amy1/2/3, which inhibited decreased in the root system of AtSUC2/4 knockout seedlings. Mutant
α-amylase activity, thereby reducing starch conversion efficiency and seedlings are highly sensitive to abiotic stresses, such as salt stress (Gong
ultimately suppressing seed germination under salt stress (Xiong et al., et al., 2015). However, a few studies have assessed the mechanisms
2024). Although numerous studies have investigated starch metabolism regulating sucrose transport in germinating soybean under different
under salt stress, there remains a lack of comparative analysis on starch levels of salt stress, and mechanisms of differential regulation of
metabolic variations across different levels of salt stress conditions. starch-sucrose metabolism in soybean during germination under
Sucrose is a key carbon source for plant growth, development, and different levels of salt stress are unclear.
resistance to stresses at the cellular level (Ruan, 2014). Sucrose and In the present study, three soybean varieties, with similar germina­
sucrose metabolism are crucial for signal transduction, cellular biosyn­ tion rates (GR) under normal conditions, were used to investigate the
thesis, and biosynthesis of osmoprotective substances in plants (Wind effects of different levels of salt stress on growth, starch-sucrose meta­
et al., 2010). Salt stress alters the distribution and metabolism of sugars bolism, and sucrose transport in germinating soybean seeds. The aim of
in plants, thus reducing both energy expenditure and growth rate (Feng this study was to elucidate the homeostatic shifts of starch-sucrose
et al., 2019). Sucrose phosphate synthase (SPS) and sucrose synthase metabolism during reserve mobilization in soybean at the germination
(SuSy) regulate sugar metabolism in plants (Fisher and Wang, 1995; stage (the during early growth) under different levels of salt stress.
Wang et al., 2000).
Specifically, SPS mainly regulates sucrose synthesis, and salt stress 2. Materials and methods
increases sucrose accumulation by increasing SPS activity (Peng et al.,
2016). SPS activity was higher in mature leaves of tomato (Lycopersicon) 2.1. Experimental design and treatment
under 70 mmol L− 1 NaCl, salt stress increased the sucrose content, thus
enhancing sucrose transfer to reservoir organs to ensure source–sink This experiment was conducted at the College of Agriculture, Hei­
balance (Balibrea et al., 2003). Sucrose synthase (synthetic) (SuSy-S) longjiang Bayi Agricultural University, and the National Coarse Cereals
and sucrose synthase (cleavage) (SuSy-C) are crucial in sucrose synthesis Engineering Research Center, Heilongjiang Province, China. The three
and conversion to starch in the starch storage organs. SuSy-C and soybean varieties Heike68 (HK68), Longken330 (LK330), and

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Z. Zhang et al. Plant Physiology and Biochemistry 225 (2025) 110050

Mengdou46 (MD46) were sourced from the Crop Germplasm Resource 2.4.1.21) and SBE (EC 2.4.1.18) activity determination; the precipitate
Innovation Laboratory, National Coarse Cereals Engineering Research was suspended with 1 mL of extraction buffer and used for GBSS (EC
Center, Heilongjiang, China. The experiment involved three treatments: 2.4.1.242) activity determination (Nakamura and Yuki, 1992).
0 mmol L− 1 NaCl [distilled water as control (CK)], 75 mmol L− 1 NaCl Pipet 20 μL of enzyme crude extract to a 1.5 mL centrifuge tube, and
solution [moderate salt stress (S)], and 150 mmol L− 1 NaCl solution then 110 μL of reaction solution [100 mmol L− 1 Hepes-NaOH (pH =
[severe salt stress (SS)]. Soybean seeds were soaked in 5 % sodium hy­ 7.4), 1.2 mmol L− 1 ADPG, 3 mmol L− 1 PPi; 5 mmol L− 1 MgC12, 4 mmol
pochlorite solution for 5 min and then rinsed thrice with distilled water. L− 1 DTT (dithiothreitol)] was added to the centrifuge tube. dithio­
Fifteen seeds were evenly placed on a double layer of filter paper in Petri threitol)]. After mixing with an oscillator, the reaction was carried out in
dishes (Φ = 90 mm) and covered with another layer of filter paper. The a water bath at 30 ◦ C for 20 min, and then terminated in a boiling water
different concentrations of NaCl solution or distilled water (8 mL) were bath for 2 min. Centrifugation was carried out at 15,000 rpm/min for 10
then added to the Petri dishes. The Petri dishes were incubated in the min, and then 100 μL of the supernatant was extracted, followed by
dark at 25 ◦ C for four days, and seed germination was recorded. Soybean adding 5.2 μL of the colorimetric solution [5.76 mmol L− 1 NADP+, 0.08
cotyledons and radicles were collected and divided into three portions. IU PGM (Phosphoglucose Glycosylmutase), 0.07 IU G6PDH (Glucose-6-
The first portion was stored in the refrigerator at − 20 ◦ C for determining phosphate dehydrogenase)] was added)]. The reaction was carried out
the physiological indexes, while the second portion was stored at − 80 ◦ C in a water bath at 30 ◦ C for 10 min. A volume of 60 μL aliquot of the
after being snap frozen in liquid nitrogen for RNA extraction. The assay solution was loaded into a 50 μL microcuvette, with 100 mmol L− 1
remaining portion was dried and stored at room temperature for starch HEPES buffer serving as the reference solution, and 20 μL of the boiled
and sucrose content analysis. Physiological and molecular indexes were crude enzyme solution was used as a control. The activity of AGPP
analyzed using three technical replicates. pyrophosphorylase was calculated from the increase in OD value at 340
nm using the NADPH standard curve.
2.2. Germination rate, radicle length, radicle fresh weight, and total fresh In a 1.5 mL centrifuge tube, 36 μL of reaction solution I [50 mmol L− 1
weight Hepes-NaOH, pH 7.4, 1.6 mmol L− 1 ADPG, 0.7 mg of branched-chain
starch, and 15 mmol L− 1 DTT] was added, and 20 μL of the crude
Germination events were recorded based on the observation of soy­ enzyme solution were mixed. The reaction was carried out in a water
bean seeds with normal young roots and at least one cotyledon attached bath at 30 ◦ C for 20 min and terminated in a boiling water bath for 2
to the young roots or two cotyledons retained for more than two-thirds. min. It was cooled in an ice bath. Then 20 μL of reaction solution II [50
The GR was calculated as follows: (number of seeds germinated at day mmol L− 1 Hepes-NaOH (pH 7.4), 4 mmol L− 1 PEP (enolpyruvate phos­
4/number of seeds tested) *100 %. The radicle length (RL), radicle fresh phate), 200 mmol L− 1 KCl, 10 mmol L− 1 MgCl2, 1.2 IU PK (pyruvate
weight (RFW), and total fresh weight (TFW) were measured using three kinase)] were mixed. Reaction was carried out at 30 ◦ C for 20 min and
selected samples. terminated by centrifugation at 4 ◦ C, 15,000 rpm/min for 13 min. The
reaction was terminated by centrifugation at 15,000 rpm/min for 13
2.3. Starch and sucrose content min at 4 ◦ C. A volume of 48 μL of supernatant was removed and 34.4 μL
of reaction solution III [50 mmol L− 1 Hepes-NaOH (pH 7.4), 10 mmol
The sucrose content of soybean cotyledons and radicles was deter­ L− 1 glucose, 20 mmol L− 1 MgCl2, 2 mmol L− 1 NADP+, 1.4 IU Hexokinase
mined based on the modified method reported by Xu et al. (2015). (hexokinase), 0.35 IU G6PDH (glucose 6-phosphate dehydrogenase)]
Briefly, 80 % (V/V) ethanol was added to 0.1 g of ground sample and were mixed. The reaction was carried out at 30 ◦ C for 10 min. A volume
incubated in a water bath at 80 ◦ C for 30 min. The sample was centri­ of 60 μL of the assay solution was taken into a 50 μL microcuvette with
fuged at 10,000×g for 10 min, and the residue was extracted twice with 50 mmol L− 1 HEPES buffer as a reference and 20 μL of boiled crude
80 % ethanol. The three supernatants were mixed, then 80 % ethanol enzyme solution as a control. The viability of SSS and GBSS enzymes was
was added, and the volume was fixed to 5 mL. Used for the determi­ calculated from the increase on OD value at 340 nm using the NADPH
nation of sucrose and starch content. standard curve, respectively.
After aspirating 0.4 mL of the supernatant, add 0.2 mL of 2 M NaOH, Add 50 μL of the above crude enzyme solution to 1.45 mL of reaction
incubate in a boiling water bath for 5 min, and cool to room tempera­ solution [50 mM Hepes-NaOH (pH 7.5), 7.5 g L− 1 soluble starch]. The
ture. Subsequently, add 2.8 mL of 30 % HCl and 0.8 mL of 0.1 % reaction was carried out in a water bath at 37 ◦ C for 40 min, terminated
phloroglucinol, mix thoroughly, react in a water bath at 80 ◦ C for 10 by boiling water for 3 min, cooled in an ice bath. Thirty microliters of 10
min, and finally cool to room temperature. The optical density (OD) % HCI was added, 150 μL of 0.2 % I2-KI solution, and 0.32 mL of ul­
value was measured at 480 nm using a spectrophotometer, and the value trapure water for a total volume of 2.0 mL. The color was developed for
was used to calculate the sucrose content (Institute of Plant Physiology 10 min, and measured absorbance at 660 nm. The reference solution is
and EcologyChinese Academy of Sciences, 1999). prepared by mixing 150 μL of 0.2 % I2-KI solution with 1.85 mL of ul­
The ethanol-insoluble residue obtained after residue extraction was trapure water. A control is set up using 50 μL of boiled crude enzyme
used for starch extraction, as described by Kuai et al. (2014). Ethanol solution. The activity of SBE is calculated with one unit (U) defined as a
was completely evaporated, and then 2 mL of distilled water was added 1 % decrease in the iodine blue value per unit time under specified
to the sample before incubation in a water bath at 100 ◦ C for 15 min. conditions.
Starch was hydrolyzed using 9.2 mol L− 1 HClO4 and 4.6 mol L− 1 HClO4. The extraction and analysis of starch metabolizing-enzyme activity
Anthrone reagent was used to quantify starch content and the absor­ were conducted as described by Kishorekumar et al. (2007) with slight
bance of samples were recorded at 620 nm. modifications. Briefly, weigh 0.15 g of cotyledons and radicles of
sprouting soybean into a pre-cooled mortar, add 1.5 mL of cold distilled
2.4. Starch metabolizing-enzyme activity water, grind to homogenate on an ice bath, and pour into a centrifuge
tube. Then centrifuged at 12,000×g for 30 min; the resulting superna­
Weigh 0.25 g of cotyledons and radicles of sprouting soybean into a tant was collected for analysis of α-amylase (EC 3.2.1.1) and β-amylase
pre-cooled mortar, add 1 mL of extraction buffer (containing 100 mmol (EC 3.2.1.2) activities.
L− 1 Tricine-NaOH, pH 7.5, 8 mmol L− 1 MgC12, 2 mmol L− 1 EDTA, 12.5 Before α-amylase detection, β-amylase was first inactivated at 70 ◦ C
% glycerol, 1 % PVP-40, 50 mmol L− 1 β-mercaptoethanol), grind to for 5 min. Briefly, the enzyme extract (0.22 mL) was mixed with 0.13 mL
homogenate on an ice bath, pour into a centrifuge tube, add 1 mL of of 3 mmol L− 1 CaCl2, incubated at 70 ◦ C for 15 min to inactivate
extraction buffer, wash the mortar once, and pour into a centrifuge tube. β-amylase, and then cooled immediately. The α-amylase reaction solu­
The supernatant was collected and used for AGPP (EC 2.7.7.27), SSS (EC tion (0.65 mL) containing 0.1 mmol L− 1 citrate buffer (pH 5.0) and 2 %

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Z. Zhang et al. Plant Physiology and Biochemistry 225 (2025) 110050

soluble starch solution was then incubated at 30 ◦ C for 10 min before the standard curve. Finally, the enzyme activity (μmol Glucose
addition of 1 mL of 3,5-ditrisalicylic acid (DNS) solution. The mixture mg− 1⋅Pr⋅min− 1) was calculated.
was heated in boiling water for 5 min and cooled immediately. The
α-amylase activity was then measured based on OD value at 540 nm 2.6. RNA extraction, complementary DNA synthesis, and qRT-PCR
using a spectrophotometer. analysis
Before β-amylase activity detection, α-amylase was inactivated with
0.1 mol L− 1 EDTA (pH 3.4) α-amylase was inactivated with 0.1 mol L− 1 Total RNA was isolated from cotyledons and radicles using TransZol
EDTA (pH 3.4). The β-amylase reaction buffer containing 0.1 mmol L− 1 Plant reagent (TransGen Biotech, Beijing, China). RNA quantity and
citrate buffer (pH 3.4) and 2 % soluble starch was added to 0.7 mL of the integrity were assessed by measuring the OD at 260 nm and through gel
enzyme extract to form a reaction volume of 2 mL and incubated at 30 ◦ C electrophoresis on 1.0 % agar gel, respectively. Hifair® III 1st Strand
for 5 min. DNS (2 mL) was added to terminate the reaction, and cDNA Synthesis Kit (Yeasen, Shanghai, China) was used to synthesize
β-amylase activity was then measured based on OD value at 540 nm cDNA. Finally, Hieff UNICON® Universal Blue qPCR SYBR Green Master
using a spectrophotometer. Mix (Yeasen. Shanghai, China) was used for the real-time quantitative
PCR experiments on a StepOne real-time PCR system (Applied Bio­
2.5. Sucrose metabolizing-enzyme activity systems, USA). The relative expression of each gene was quantified via
the comparative threshold cycling method with based on the stable and
Sucrose metabolism-related enzymes were extracted as described by reliable GmEF1a of our laboratory as an internal reference (Du et al.,
Liu et al. (2013) to analyze SPS (EC 2.4.1.14), SuSy (EC 2.4.1.13), and 2020a, 2020b). Each sample had three technical replicates. The thermal
INV (EC 3.1.2.1.26) activities. Briefly, weigh 0.2 g of frozen cotyledon or cycling conditions were as follows: 95 ◦ C for 1 min, followed by 39
radicle samples, add 1.8 mL of extraction buffer (50 mmol L− 1 Tris-HCl cycles of 95 ◦ C for 5 s, 58 ◦ C for 20 s, and 60 ◦ C for 20 s. The expression
(pH 7.5), 1 mmol L− 1 EDTA, 1 mmol L− 1 MgCl2, 12.5 % (V/V) glycerol, levels were determined via the 2-ΔΔt method. The gene-specific p rimers
10 % polyvinylpyrrolidone (PVP), and 10 mmol L− 1 mercaptoethanol), are listed in S1.
and quickly grind into a homogenate in a mortar. Transfer the homog­
enate into a tube, and centrifuged at 15,000×g for 20 min at 4 ◦ C. 2.7. Statistical analysis
A reaction mixture containing 200 μL of the extract, 12 mmol L− 1
UDP glucose, 200 mmol L− 1 Tris-HCl (pH 7.0), 40 mmol L− 1 fructose-6- SPSS 26.0 (SPSS Inc., Chicago, IL, USA) was used for all data analysis.
p, and 40 mmol L− 1 MgCl2 was incubated in a water bath at 30 ◦ C for 30 GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA) was used
min. The reaction was terminated by adding 0.1 mL of 2 mol L− 1 NaOH to plot the bar graphs. For statistical analysis, three biological replicates
and heating the mixture in a boiling water bath for 10 min. The samples were randomly selected from each group. Duncan’s multiple range test
were cooled to room temperature before the addition of 1 mL of 0.1 % was used to determine differences between means, and P < 0.05 indi­
(W/V) resorcinol [dissolved in 95 % (V/V) ethanol] and 3.5 mL of 30 % cated significant differences.
(W/V) HCl. The mixture was incubated in a water bath at 80 ◦ C for 10
min and cooled, and the OD values were measured at 480 nm. The su­ 3. Results
crose content (mg− 1⋅Pr⋅min− 1) of the SPS reaction was determined
based on a standard curve. The enzyme activity was then calculated 3.1. Effect of different levels of salt stress on the germination rate, radicle
(μmol sucrose mg− 1⋅Pr⋅min− 1). length, and radicle dry weight of germinating soybean seeds
SuSy-S activity was determined using a reaction medium containing
100 mmol L− 1 Tris-HCl (pH 7.2), 5 mmol L− 1 UDPG, 10 mmol L− 1 To investigate the effects of different levels of salt stress on the
MgCl2, and 5 mmol L− 1 D-fructose. The mixture was incubated in a water soybean phenotype at the germination stage, we measured the changes
bath at 30 ◦ C for 30 min and then boiled for 5 min. The reaction was in GR, RL, RFW, and TFW of the germinating seeds. The effects of the
terminated by adding 0.1 mL of 2 mol L− 1 NaOH and boiling the mixture different levels of salt stress on the GR of HK68 were not significantly
for another 10 min. The mixture was immediately cooled, and 1 mL of different (Fig. 1a). Compared with the control, the S and SS treatments
0.1 % resorcinol and 3.5 mL of 30 % HCl were added. The mixture was significantly reduced the GR of LK330, with decreases of 13.6 % and
shaken well, incubated in the water bath at 80 ◦ C for 10 min, and then 22.7 %, respectively (Fig. 1a). Unlike the S treatment, the SS treatment
cooled. The OD value was measured at 480 nm, and the sucrose content significantly reduced the GR of MD46 by 47.7 % (Fig. 1a). Compared
of the SuSy-S reaction was calculated based on a standard curve. Finally, with the control, salt stress significantly decreased the RL, RFW, and
the enzyme activity was calculated (μmol sucrose mg− 1⋅Pr⋅min− 1). TFW of the three soybean varieties. Overall, the phenotypic decline in
SuSy-C activity was determined using a reaction medium containing soybean growth at the germination stage was greater under SS than
100 mmol L− 1 (pH 7.2), Tris-HCl, 5 mmol L− 1 UDP, 10 mmol L− 1 MgC12, under S treatment (Fig. 1b–d).
and 50 mmol L− 1 sucrose. The mixture was incubated in a water bath at
30 ◦ C for 30 min and then boiled for 5 min. The reaction was terminated 3.2. Effect of different levels of salt stress on starch and sucrose content of
using 0.1 mL of 2 mol L− 1 NaOH. DNS (0.5 mL; 5 mmol L− 1) was added cotyledons and radicles
to the mixture, which was heated in a boiling water bath for 5 min and
then cooled. Distilled water was added, and the OD value was measured To investigate the distribution of starch-sucrose in soybean during
at 540 nm. The fructose content of the SuSy-C reaction was calculated germination under different levels of salt stress, we measured the
based on a standard curve, and the enzyme activity (μmol Fructose changes in starch and sucrose content in soybean cotyledons and radi­
mg− 1⋅Pr⋅min− 1) was calculated. cles. Two-way ANOVA showed that soybean variety and salt stress levels
The activity of INV, including AI and NI, was determined as had significant effects on starch and sucrose accumulation and were
described by Hu et al. (2018). Briefly, a reaction medium containing significantly affected by variety × salt stress levels interactions (S2).
100 μL of extract, 200 μL of 1 mol L− 1 sucrose, and either 2.2 mL of 100 Compared with the control, the S and SS treatments significantly
mmol L− 1 sodium acetate-acetic acid (pH 7.5) (for NI assay) or 2.2 mL of decreased the starch content in the cotyledons, with decreases of 15.4
200 mmol L− 1 acetic acid-sodium hydroxide (pH 5.0) (for AI assay) was %–45.5 % and 18.6 %–55.6 %, respectively. The S treatment signifi­
incubated in a water bath at 30 ◦ C for 30 min. Thereafter, the mixture cantly decreased the starch content of HK68 radicles by 37.0 %, but did
was boiled for 5 min, and the reaction was stopped using 1 mL of DNS. not significantly affect the starch content of LK330 and MD46 radicles.
The OD value was measured at 540 nm after cooling. The glucose con­ However, the SS treatment significantly increased the starch content in
tent of the AI and NI activity reactions was calculated based on a the radicles of three soybean varieties by 17.6 %–24.4 % (Fig. 2a and b).

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Z. Zhang et al. Plant Physiology and Biochemistry 225 (2025) 110050

Fig. 1. Effect of different levels of salt stress on germination rate, radicle length, radicle fresh weight and total fresh weight of HK68, LK330, and MD46 soybean
seeds at 4 days of germination. (a), GR, germination rates; (b), RL, radicle length; (c), RFW, radicle fresh weight; (d), TFW, total fresh weight; (e), Morphological
characteristics of HK68, LK330, and MD46 soybean seeds at 4 days of germination. CK, control condition; S, 75 mmol L− 1 NaCl; SS, 150 mmol L− 1 NaCl. Data shown
are the means ± standard deviation of three independent samples. Different letters on vertical bars indicate significant differences between means at the P <
0.05 level.

Fig. 2. Effect of different levels of salt stress on the starch and sucrose contents of HK68, LK330, and MD46 soybean seeds at 4 days of germination. (a) and (b), starch
content; (c) and (d), sucrose content. CK, control condition; S, 75 mmol L− 1 NaCl; SS, 150 mmol L− 1 NaCl. Data shown are the means ± standard deviation of three
independent samples. Different letters on vertical bars indicate significant differences between means at the P < 0.05 level.

Compared with the control, salt stress significantly increased the sucrose related to starch metabolism and sucrose metabolism.
content in cotyledons and radicles, especially the radicles (Fig. 2c and
d). Sucrose content was significantly higher under the SS treatment than
under the S treatment in the radicles by 3142.8 %–9708.1 %. In order to 3.3. Effect of different levels of salt stress on the activity of starch
further elucidate the changes in starch and sucrose content mainly metabolism-related enzymes
caused by the changes in the activities of specific enzymes, we con­
ducted subsequent assays on the changes in the activities of enzymes In order to investigate the changes in starch metabolism in response
to different levels of salt stress during the germination period of

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Z. Zhang et al. Plant Physiology and Biochemistry 225 (2025) 110050

soybean, we determined changes in the activities of starch metabolism-

related enzymes in soybean cotyledons and radicles. GBSS was not
affected by soybean variety, and the rest of the starch metabolizing
enzymes were significantly affected by soybean variety, salt stress levels
and both interactions (S2). Compared with the control, in cotyledons,
starch metabolizing enzymes showed an overall decrease in salt stress
compared to the control, with no significant change in α-amylase ac­
tivity under S conditions, no significant change in SBE and AGPP ac­
tivities in HK68, and no significant change in SSS activity in MD46 under
SS conditions, and the rest of the starch metabolizing enzyme activities
were significantly decreased in three genotypes of soybeans. Under
different levels of salt stress, Under the SS treatment compared to the S
condition, the activities of GBSS and SSS were significantly upregulated
by 23.1 %–28.3 % and 25 %–150 %, respectively, while α-amylase ac­
tivity in cotyledons exhibited a 12.4 %–22.7 % reduction only under SS
conditions (Fig. 3a–c, e, g, i and k). In the radicle, amylase activity was
decreased overall in the S condition compared to the control, but SBE
activity was significantly decreased only in HK68 by 20.4 %. Under the
SS condition, SBE and GBSS activities varied among soybean genotypes
in the radicle, the SSS activity was significantly increased by 10.5 %–
11.2 % except for no significant change in LK330, the AGPP activity was
significantly decreased by 17.4 %–22.6 % except for no significant
change in MD46, and α/β-amylase activities were significantly increased
by 32.9 %–73.2 % and 11.0 %–56.7 % (Fig. 3b–d, f, h, j and l). These
results indicate that the starch metabolizing enzyme activities of soy­
bean cotyledons were reduced under salt stress, similar to those of the
radicle under moderate salt stress, whereas the radicle under severe salt
stress were increased some of the starch synthases and starch catabo­
lizing enzymes.

3.4. Effect of different levels of salt stress on the activity of sucrose

metabolism-related enzymes

To investigate the changes in sucrose metabolism during soybean

germination under different levels of salt stress, we determined changes
in the activities of related sucrose metabolizing enzymes in soybean
cotyledons and radicles. Sucrose metabolizing enzymes were signifi­
cantly affected by soybean variety and salt stress levels, sucrose syn­
thesizing enzyme classes were significantly affected by the interaction
between the two, whereas sucrose catabolic enzyme classes varied in
different sites in the interaction between soybean variety and salt stress
levels, with SuSy-C and AI having significant effect only in the cotyledon
had significant effects, and NI had significant effects only in radicle (S2).
Compared with the control, the S and SS treatments significantly
increased the SPS and SuSy-S activities in the cotyledons. In contrast, the
S treatment did not significantly change the SPS and SuSy-S activities in
the radicles of the three varieties, while the SS treatment significantly
Fig. 3. Effect of different levels of salt stress on the activities of enzymes related
increased the SPS and SuSy-S activities by 98.59 %–115.14 % and 36.06
to starch metabolism in HK68, LK330, and MD46 soybean seeds at 4 days of
%–80.35 %, respectively (Fig. 4a–d). Compared with the control, the S germination. (a) and (b), SBE, starch branching enzymes; (c) and (d), GBSS,
and SS treatments significantly decreased the SuSy-C activity in the granule-bound starch synthase; (e) and (f), SSS, soluble starch synthase; (g) and
cotyledons. Furthermore, the S treatment decreased, while the SS (h), AGPP, ADP-Glc pyrophosphorylase; (i) and (j), α-amylase; (k) and (l),
treatment significantly increased the SuSy-C activity in the radicles by β-amylase. CK, control condition; S, 75 mmol L− 1 NaCl; SS, 150 mmol L− 1 NaCl.
17.57 %–54.86 % (Fig. 4e and f). Compared with the control, salt stress Data shown are the means ± standard deviation of three independent samples.
significantly decreased the AI and NI activities in cotyledons by 23.0 %– Different letters on vertical bars indicate significant differences between means
58.7 % and 38.8 %–64.3 %, respectively. Furthermore, the S treatment at the P < 0.05 level.
significantly decreased AI (by 10.8 %–28.4 %) and NI (by 11.1 %–28.1
%) activities, while the SS treatment significantly increased NI activities 3.5. Effect of different levels of salt stress on the expression of starch
by 10.7–22.9 % in the radicles (Fig. 4g–j). Overall, the germinated seeds metabolism-related genes
under the SS treatment, in most cases, showed a greater magnitude of
change in the activity of sucrose metabolizing enzymes relative to the S Changes in the expression of genes related to starch metabolism in
treatment. Sucrose catabolic enzyme activity was stronger in soybean cotyledons and radicles of germinating soybean under different levels of
radicles than in cotyledons, whereas sucrose synthase activity was salt stress were analyzed by qRT-PCR. We measured the relative
stronger in cotyledons than in radicles. expression of GmSS, GmAMY3, and GmBAM1. Compared with the con­
trol, both S and SS treatments significantly enhanced GmSS expression,
inducing 1.1− 1.3-fold (S) and 1.0− 1.4-fold (SS) increases in cotyledons,
whereas radicles demonstrated differential responses with 0.9–1.7-fold

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Z. Zhang et al. Plant Physiology and Biochemistry 225 (2025) 110050

Fig. 4. Effect of different levels of salt stress on the activities of enzymes related to sucrose metabolism in HK68, LK330, and MD46 soybean seedlings at 4 days of
germination. (a) and (b), SPS, sucrose phosphate synthase; (c) and (d), SuSy-S, sucrose synthase (synthesis); (e) and (f), SuSy-C, sucrose synthase (cleavage); (g) and
(h), AI, acid invertase; (i) and (j), NI, neutral/alkaline invertase. CK, control condition; S, 75 mmol L− 1 NaCl; SS, 150 mmol L− 1 NaCl. Data shown are the means ±
standard deviation of three independent samples. Different letters on vertical bars indicate significant differences between means at the P < 0.05 level.

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Z. Zhang et al. Plant Physiology and Biochemistry 225 (2025) 110050

(S) and substantially stronger 1.9− 9.0-fold up-regulation (SS) (Fig. 5a under different levels of salt stress, we measured the relative expression
and b). The S treatment did not significantly alter GmAMY3 expression of GmSPS, GmSuSy GmA-INV, and GmC-INV. Compared with the control,
in cotyledons, while the SS treatment significantly up-regulated the S and SS treatments significantly down-regulated GmSPS in cotyle­
GmAMY3 in cotyledons compared with the control by 0.5–1.2-fold. dons by 0.3− 0.7-fold, while up-regulating GmSPS in radicles by 1.2–8.9-
Furthermore, the S and SS treatments significantly up-regulated fold and 1.7− 30.6-fold, respectively (Fig. 6a and b). The S and SS
GmAMY3 in the radicles by 0.7–1.1-fold and 2.4− 7.8-fold, respec­ treatments significantly increased GmSuSy expression in cotyledons and
tively (Fig. 5c and d). Additionally, salt stress significantly down- radicles compared with the control. Moreover, GmSuSy expression was
regulated GmBAM1 in the cotyledons by 0.3− 0.6-fold, while the S and significantly higher under the SS treatment than under the S treatment
SS treatments significantly up-regulated GmBAM1 in the radicles by 1.3–7.6-fold (S) and 7.9− 64.1-fold (SS) (Fig. 6c and d). Both treat­
compared with the control by 0.8–1.9-fold and 1.7− 6.9-fold, respec­ ments down-regulated GmA-INV activity, particularly in radicles where
tively (Fig. 5e and f). Overall, the relative expression levels of these three S caused 0.3–0.57-fold reduction and SS induced 0.5–0.8-fold suppres­
genes related to starch metabolism in the radicle were significantly sion, whereas cotyledons showed about 0.9-fold down-regulation under
greater under SS treatment as compared to S treatment. S and SS. The S and SS treatments significantly down-regulated GmA-
INV in cotyledons by about 0.9-fold, radicles by 0.3− 0.57-fold (S) and
0.5− 0.8-fold (SS) (Fig. 6e and f). However, salt stress significantly
3.6. Effect of different levels of salt stress on the expression of sucrose increased GmC-INV expression in the cotyledons of the three varieties by
metabolism-related genes 1.0–5.6-fold. The SS treatment significantly up-regulated GmC-INV in
radicles of HK68 by 0.9-fold, but did not significantly affect the
To understand the changes in the expression of genes related to su­ expression of GmC-INV in the other two varieties (Fig. 6g and h). These
crose metabolism in cotyledons and radicles of germinating soybean

Fig. 5. Effect of different levels of salt stress on the relative expression of genes related to starch metabolism in HK68, LK330, and MD46 soybean seeds at 4 days of
germination. (a) and (b), the relative expression levels of GmSS (Glyma.13G204700); (c) and (d), the relative expression levels of GmAMY3 (Glyma.08G296800); (e)
and (f), the relative expression levels of GmBAM1 (Glyma.16G217900). CK, control condition; S, 75 mmol L− 1 NaCl; SS, 150 mmol L− 1 NaCl. Data shown are the
means ± standard deviation of three independent samples. Different letters on vertical bars indicate significant differences between means at the P < 0.05 level.

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Z. Zhang et al. Plant Physiology and Biochemistry 225 (2025) 110050

Fig. 6. Effect of different levels of salt stress on the relative expression of genes related to sucrose metabolism in HK68, LK330 and MD46 soybean seeds at 4 days of
germination. (a) and (b), the relative expression levels of GmSPS (Glyma.14G029100); (c) and (d), the relative expression levels of GmSuSy (Glyma.03G216300); (e)
and (f), the relative expression levels of GmA-INV (Glyma.05G056300); (g) and (h), the relative expression levels of GmC-INV (Glyma.20G177200). CK, control
condition; S, 75 mmol L− 1 NaCl; SS, 150 mmol L− 1 NaCl. Data shown are the means ± standard deviation of three independent samples. Different letters on vertical
bars indicate significant differences between means at the P < 0.05 level.

results indicate molecular-level changes in sucrose metabolism occurred 3.7. Effect of different levels of salt stress on the expression of sucrose
in response to different levels of salt stress in germinating soybean. transporter protein-related genes in cotyledons and radicles

To understand the changes in sucrose transporter protein-related

gene expression during cotyledon and radicle germination in soybean

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Z. Zhang et al. Plant Physiology and Biochemistry 225 (2025) 110050

under different levels of salt stress, we determined the relative expres­ 4. Discussion
sion of GmSUC2, GmSWEET6, and GmSWEET15. Compared with the
control, salt stress significantly down-regulated GmSUC2 in the cotyle­ Excessive salt stress inhibits plant growth, but the inhibition rate
dons by 0.2− 0.9-fold. Although the S treatment did not significantly depends on the species owing to their differences in salt stress tolerance.
change GmSUC2 expression in the radicles of the three varieties, the SS Germination potential and rate have been widely used as basic in­
treatment significantly down-regulated GmSUC2 by about 0.5-fold dicators for salt tolerance at the germination stage. Additionally, these
(Fig. 7a and b). Compared with the control, the S treatment signifi­ two indicators have been used as direct germination evaluation in­
cantly down-regulated GmSWEET6 in the cotyledons by 0.5− 0.7-fold, dicators (Bao et al., 2020). In this study, salt stress, especially the SS
while the SS treatment did not significantly affect GmSWEET6 expres­ treatment, significantly inhibited the RL, RFW, and TFW of all varieties
sion in any of the varieties. Furthermore, the S and SS treatments compared with the control (Fig. 1). Before proceeding with a detailed
significantly up-regulated GmSWEET6 in the radicles of all varieties by discussion, it is critical to clarify whether the observed differences in
1.6–2.4-fold (S) and 3.6− 116.7-fold (SS) (Fig. 7c and d). Salt stress starch-sucrose metabolism under different levels of salt stress are a
significantly down-regulated GmSWEET15 in the cotyledons of all va­ consequence of divergent soybean germination outcomes or, conversely,
rieties by 0.3− 0.9-fold. Notably, the S and SS treatments up-regulated whether metabolic shifts drive the germination variations. Resolving
GmSWEET15 in radicles of three varieties, but the up-regulation was this causal relationship holds significant importance for interpreting the
significant under the SS treatment by 2.6–74.8-fold (Fig. 7e and f). underlying mechanisms. Previous studies have shown that the Arabi­
dopsis thaliana sweet11;12;15 triple mutants exhibit significantly
shorter root length than the wild type during the early seedling stage,
and this phenotype can be alleviated by exogenous application of 2 %

Fig. 7. Effect of different levels of salt stress on the relative expression of sucrose transporter-related genes in HK68, LK330, and MD46 soybean seeds at 4 days of
germination. (a) and (b), the relative expression levels of GmSUC2 (Glyma.16G157100); (c) and (d), the relative expression levels of GmSWEET6 (Glyma.04G198600);
(e) and (f), the relative expression levels of GmSWET15 (Glyma.06G166800). CK, control condition; S, 75 mmol L− 1 NaCl; SS, 150 mmol L− 1 NaCl. Data shown are the
means ± standard deviation of three independent samples. Different letters on vertical bars indicate significant differences between means at the P < 0.05 level.

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Z. Zhang et al. Plant Physiology and Biochemistry 225 (2025) 110050

sucrose (Chen et al., 2015). The absence of two major leaf SPS isoforms expression was increased in both shoot and root under NaCl treatment
(SPSA1 and SPSC) inhibited Arabidopsis growth. Under short-day con­ (Gong et al., 2015); In contrast, the SUC2 expression in Plantago major
ditions (6-week cultivation), the spsa1/spsc double mutant exhibited under NaCl treatment was similar to the results of this study, with a
significantly reduced SPS enzyme activity. Furthermore, under both decrease in expression occurring after 24 h of salt treatment
light and dark conditions, the sucrose accumulation in the phloem or (Pommerrenig et al., 2007). Excessive sucrose accumulation can trigger
efflux of the double mutant plants was significantly lower than that of “sugar damage,” causing a conversion imbalance between starch and
the wild type, and their rosette diameter was reduced by 50 % compared sucrose and a source–sink imbalance between cotyledons and radicles
to the wild type (Volkert et al., 2014). In this experiment, under salt (Dong and Beckles, 2019). The accumulated sucrose can be used for
stress conditions, soybean exhibited a reduced conversion of sucrose to energy metabolism, osmoregulation, and starch synthesis or is stored as
starch in the cotyledons (Fig. 2). However, sucrose transport to the reserve energy. The SS treatment also up-regulated GmSS and increased
radicles became more significant across different levels of salt stress starch accumulation in radicles (Figs. 2 and 7). Furthermore, other
(Fig. 7). Notably, radicle sucrose content increased with salt concen­ studies showed that starch could act as an ionic flocculant to prevent the
tration, yet radicle growth inhibition was significantly exacerbated systematic diffusion of excess Na+ ions in plants under salt stress, thus
(Fig. 1). Based on previous studies and our experimental findings, we disrupting the cellular osmotic balance (Kanai et al., 2007). Although
propose that a portion of the sucrose originally allocated for germination this may explain starch accumulation in soybean radicles under severe
and growth in soybean seeds is partially redirected to counteract salt salt stress, the role of starch-trapping ions in germinating soybean re­
stress. Potential mechanisms include: (1) enhanced sucrose accumula­ quires further investigation. These results indicate that severe salt stress
tion as an osmoprotectant, and (2) increased starch synthesis in limits the conversion of sucrose to starch in mobilized cotyledons of
salt-sensitive radicles under high-salinity conditions, possibly serving as germinating soybean reserves, which is then translocated to the radicle
binding agents for salt ions. These adaptive metabolic shifts likely for osmoregulation and energy metabolism. Excessive accumulation of
contribute to the restricted germination and growth observed in soy­ sucrose in the radicle may lead to “sugar damage”, and perhaps to
beans under salt stress. Furthermore, there was a significant difference prevent this, some of the sucrose is converted to starch. This suggests
in biomass accumulation of germinating soybeans under S and SS con­ that sprouting soybean under severe salt stress requires sucrose and
ditions, and perhaps differential performance in the accumulation of starch to work together to help it withstand adversity. The embryo root
starch and sucrose, which are involved in energy metabolism and maintains efficient starch-sucrose conversion under severe salt stress by
osmoregulation. accumulating more starch and sucrose than under moderate salt stress.
The starch-sucrose interconversion in the source reservoir tissues Similarly, starch content in Anacardium occidentale germinated nut
plays a key physiological role in regulating plant growth and physio­ under salt conditions (NaCl, EC = 18 dSm− 1, about 180 mmol L− 1 NaCl)
logical metabolism (Dong and Beckles, 2019). Sucrose can act as an was significantly lower and sugars contents were significantly higher
osmoregulatory substance in plant tissues to alleviate salt stress damage. compared to other lower electrical conductivity (Marques et al., 2013).
The experiments in this study showed that different levels of salt stress Mechanisms of differential regulation of starch-sucrose metabolic ho­
resulted in growth limitation of germinating soybeans and affected the meostasis in soybean reserve mobilization during germination under
metabolic homeostasis of cotyledons and starch in germinating soybean. different levels of salt stress set the stage for soybean germination and
The S treatment significantly decreased starch content in germinating even seedling establishment (Fig. 8). Then, whether there is a causal
soybean cotyledons but significantly increased sucrose content. relationship between the sucrose content in soybean seeds and
Furthermore, in radicles, the S treatment did not increase starch content salt-tolerance ability is the focus of future research work.
but also significantly increased sucrose accumulation (Fig. 2). Further­ Starch metabolism can inhibit the adverse effects of stress-induced
more, up-regulated GmSWEET6/15 in radicles and improved sucrose carbon depletion when carbohydrate assimilation is compromised in
translocation to the radicles (Fig. 7). Sucrose is translocated from source abiotic stress environments (Hare et al., 1998; Wanner and Junttila,
cells to phloem tissues and systemically distributed throughout the plant 1999; Krasavina et al., 2014). Salt stress decreases the α-amylase and
through sucrose transporter-mediated processes (Chen et al., 2012). The β-amylase activities of germinated seeds (Liu et al., 2018). Under
efficient transport of intercellular carbohydrates is mainly dependent on 100/200 mmol L− 1 NaCl treatment, sorghum exhibited a progressive
sucrose transporter proteins. Sucrose transporter proteins can integrate decline in both germination rate and root length, while starch content
the plant as a metabolic whole to keep the inter-tissue sugar levels increased with rising NaCl concentrations. Concurrently, the activity of
within an optimal range, thus improving plant growth or counteracting starch-metabolizing enzymes (e.g., α-amylase) and the expression of
adverse external factors (Dong and Beckles, 2019). These results indi­ associated genes (SS and α-Amy) were upregulated. However, the study
cate that soybean seeds under moderate salt stress limit the conversion lacked analysis of starch synthase activity, thereby failing to delineate a
of sucrose to starch in the mobilization of soybean reserves during complete starch metabolic pathway under salt stress (Punia et al., 2021).
germination and promote greater accumulation of sucrose to the radicle, In this study, none of the starch metabolizing enzyme activities in soy­
which in turn increases the overall sucrose content of soybean. It sug­ bean cotyledons under S treatment showed an increasing trend, and
gests that sprouting soybean needs sucrose more than starch under α-amylase activity was also significantly reduced in SS. In the radicle,
moderate salt stress to help it withstand the adversity. starch metabolizing enzyme activities were all decreased under S
The SS treatment significantly slowed the growth of soybean radicles treatment, but SSS, starch catabolism-related enzymes (α/β-amylase)
and decreased sucrose consumption compared to S treatment. As with activities and their key genes, GmAMY3 and GmBAM1, were overall
the S treatment, the SS treatment significantly reduced starch accumu­ increased in SS treatment (Fig. 3). The SS treatment up-regulated GmSS,
lation but significantly increased sucrose content in the cotyledons. thus promoting starch synthesis, increased starch accumulation in the
Furthermore, the SS treatment significantly increased starch and sucrose radicles (Figs. 3 and 5). Similarly, in Barley (Hordeum vulgare L.) seeds
accumulation in radicles (Fig. 2). Notably, the SS treatment rapidly with high starch content, seed α-amylase activity during germination
increased sucrose content in the radicle compared with the S treatment. under 250 mmol L− 1 NaCl treatment was reduced to reduce starch
Moreover, the SS treatment significantly up-regulated GmSWEET6/15 in mobilization efficiency and increase starch accumulation to withstand
radicles compared with the S treatment (Fig. 7), enabling the efficient salt stress (Xiong et al., 2024). This suggests that starch metabolism
flow of sucrose in radicles under severe salt stress. There was no mobilized by soybean reserves during germination becomes retarded
increased expression of GmSUC2 in cotyledons versus radicles under under moderate salt stress, which is also the case in cotyledons under
different levels of salt stress (Fig. 7), probably because the SUC2 in the severe salt stress, whereas starch metabolizing enzyme (synthesis and
soybean germination stage is more sensitive to salt stress and the catabolism) activities are partially increased in the radicle in response to
expression varies in different species or tissues. In Arabidopsis, the SUC2 severe salt stress.

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Z. Zhang et al. Plant Physiology and Biochemistry 225 (2025) 110050

Fig. 8. Homeostatic shifts of starch-sucrose metabolic in sprouting soybean reserve mobilization under different levels of salt stress. Salt stress stimulates a sugar-
mediated crosstalk response, Starch-sucrose accumulation in reserve mobilization of germinating soybean is exquisitely allocated to different levels of salt stress to
adapt to different adversities. These are achieved by germinating soybeans through the regulation of starch metabolizing enzymes, sucrose metabolizing enzyme
activities, and the expression of sugar transporter protein-related genes. The “↑” represents increased enzyme activity and the “↓” represents decreased enzyme
activity, both compared to the control treatment; SE//CC, Sieve element-companion cell complex.

Sucrose produces energy and hexose through catalysis by various suggests that soybean reserves mobilized during the emergence phase in
sucrose-metabolizing enzymes. Hexose is an essential substrate for the cotyledons reduce the conversion of sucrose to starch and accumu­
synthesizing cellulose, starch, fructans, proteins, and antioxidant com­ late more sucrose in response to increasing salt stress levels (Figs. 2 and
pounds. Furthermore, sucrose metabolism plays a central role in pro­ 4). Abiotic stresses (such as drought stress) increased the above- and
ducing carbohydrate nutrients required for plant growth and below-ground SPS activities and sucrose accumulation in soybean
development (Ruan, 2014). In the present study, salt stress significantly plants, thereby improving stress tolerance (Du et al., 2020a). Further­
enhanced the SPS and SuSy-S activities in the cotyledons while more, in the first treatment with 100/200 mmol L− 1 NaCl on tomato
decreasing SuSy-C and INV activities, as a whole, it seems that the ac­ young leaves, sucrose content gradually increased, while starch content
tivities of sucrose synthesis related enzymes increased and sucrose progressively decreased. Under 100 mmol L− 1 NaCl, SuSy activity was
catabolism related enzymes decreased under SS than S treatment. This elevated, and INV activity declined. Conversely, at 200 mmol L− 1 NaCl,

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Z. Zhang et al. Plant Physiology and Biochemistry 225 (2025) 110050

SPS activity rose, whereas AI activity was suppressed (Khelil et al., of GmAMY3 and GmBAM1 genes in response to the SS treatment. The SS
2007). Similarly, under different levels of salt stress (mild, moderate, treatment also significantly enhanced SPS, SuSy, and NI activities and
and severe), starch and sucrose content in the leaves subtending the up-regulated GmSWEET6/15 in the radicles. These results indicate that
cotton bolls of cotton plants progressively increased. This pattern might severe salt stress can enhance the starch-sucrose conversion efficiency of
be driven by reduced AI activity alongside elevated activities of SPS and soybean radicles and promote the accumulation and translocation of
SuSy (Ju et al., 2021). The excess hexose is metabolized into energy for starch and sucrose in radicles, thus effectively preventing SS damage.
growth and salt stress resistance (Janda et al., 2007). SuSy and INV
related genes induce the expression of sucrose transporter proteins by Author contributions
hydrolyzing sucrose into hexoses (glucose and fructose) for starch syn­
thesis (Wang et al., 2008; Jin et al., 2009). Consistent with our results, Jidao Du, Yanli Du and Zhaoning Zhang designed the experiments;
castor (Ricinus communis L.) seedlings exposed to 150 mmol L− 1 NaCl for Yu Cao and Guangda Wang provided the experimental methods;
6 days showed reduced biomass in both cotyledons and roots. While Zhaoning Zhang, Weiyu Wang and Ruiqi Feng completed the experi­
starch content in the roots remained unchanged, sucrose levels increased ments; Zhaoning Zhang and Yanli Du analyzed the data; Zhaoning Zhang
significantly. Notably, the activities of key sugar-metabolizing enzy­ completed the manuscript; Yanli Du and Qiang Zhao revised the
mes—SPS, SuSy, and α/β-amylase—exhibited no substantial enhance­ manuscript; All authors read and approved the final manuscript.
ment under these conditions. However, the contents of starch and
sucrose were increased in cotyledons, and the activities of sugar Funding and acknowledgment
metabolizing enzymes (SPS, SuSy and β-amylase) were enhanced. As a
whole, salt stress promoted sucrose biosynthesis and starch catabolism This work was supported by Heilongjiang Natural Science Founda­
in castor seedlings (Li et al., 2023). tion Joint Guidance Project (LH2024C077), the Application of Saline-
Radicles are reservoir organs that directly sense salt concentration. alkali Tolerant Soybean Germplasm and Demonstration of Saline-
Therefore, the differences in metabolic activity within the radicle tissues alkali Resistant Cultivation Technology (LJGXCG2022-111), the Na­
can reflect their adaptation to the external environment. Under different tional Key Research and Development Program (2021YFD1201603-3),
levels of salt stress in Schenkia spicata, root growth showed no significant the Talent Introduction Project of Heilongjiang Bayi Agricultural Uni­
change at 50 mmol L− 1 NaCl, but significantly decreased under 100/ versity (XYB202006) and the Heilongjiang Bayi Agricultural University
200 mmol L− 1 NaCl treatments. AI activity exhibited consistent changes Support Program for San Zong (ZRCQC202102).
with root growth patterns. Sucrose content remained statistically un­
changed at 50/100 mmol L− 1 NaCl, but showed a significant increase at Declaration of competing interest
200 mmol L− 1 NaCl (Mišić et al., 2012). In this study, the S treatment
significantly reduced the SuSy-C and INV activities in radicles, while SPS There is no conflict of interest in the submission of this manuscript,
and SuSy-S activities did not change significantly but up-regulated and the manuscript is published with the consent of all authors.
GmSWEET6/15, resulting in increased sucrose accumulation (Figs. 2, 4
and 7). This suggests that under moderate salt stress, sucrose slows Appendix A. Supplementary data
degradation and more translocation from soybean cotyledons to in­
crease sucrose content in the radicle as a defense against adversity. Supplementary data to this article can be found online at https://doi.
Furthermore, compared to the control and S treatment, radicles showed org/10.1016/j.plaphy.2025.110050.
promoted sucrose synthesis and catabolism under SS treatment by
enhancing the activities of key enzymes related to sucrose metabolism Data availability
(SPS, SuSy-S, SuSy-C, and NI) (Fig. 4). A high metabolic efficiency can
promote the production of large amounts of hexose from sucrose. Data will be made available on request.
Accumulated hexose can then promote starch production, accelerate
starch–sucrose conversion, and allow re-conversion of excess sucrose References
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