Mitigating Salinity Stress in Sugar Beet Seedlings Through Exogenous Application of Putrescine and Salicylic Acid

Abstract

Salinity stress is a major constraint on the growth and productivity of sugar beet (Beta vulgaris L.). This study evaluated the potential of exogenously applied putrescine (Put) and salicylic acid (SA) to enhance salt stress tolerance. Thirty-day-old seedlings were grown for seven days under control conditions before being subjected to eight treatments for 10 days: (i) Control, (ii) Control + 0.6 mM Put, (iii) Control + 0.6 mM SA, (iv) Control + 0.6 mM Put + 0.6 mM SA, (v) Salinity (150 mM NaCl), (vi) Salinity + 0.6 mM Put, (vii) Salinity + 0.6 mM SA, and (viii) Salinity + 0.6 mM Put + 0.6 mM SA. Put and SA were applied once as a foliar spray at the onset of the treatments. Salt stress significantly reduced plant growth, biomass, chlorophyll content, and photosynthetic efficiency, while increasing reactive oxygen species (particularly H₂O₂) and lipid peroxidation. Foliar applications of Put and SA alleviated these adverse effects, either individually or in combination. Put primarily enhanced plant growth rate, shoot length, plant height, shoot and root biomass, leaf relative water content, respiration activity, and sucrose accumulation. SA improved root length, photosynthetic activity, water-use efficiency, and proline accumulation. When applied together, Put and SA combinedly increased growth rate, shoot length, plant height, shoot biomass, leaf relative water content, stomatal conductance, and the maximum quantum yield of PSII, while more prominently reducing malondialdehyde and H₂O₂ accumulation and enhancing antioxidant enzyme activities. These findings suggest that foliar application of Put and SA enhances salinity tolerance in sugar beet seedlings by improving antioxidant enzyme activities, osmolyte accumulation, and ion homeostasis, thereby mitigating oxidative stress under saline conditions. This outcome could contribute to potential applications in breeding programs and stress management in saline-prone regions.

1. Introduction

Salinity is one of the most important abiotic stress factors limiting the productivity of agricultural crops with adverse effects on germination, plant vigour and crop yield [1]. Currently, approximately 20% of total cropland and 33% of irrigated agricultural land are salinized because of poor agricultural practices and it is expected that by 2050, half of the croplands worldwide will become salinized [2].

Plant growth is affected by osmotic stress-specific ion toxicity, ion imbalance, and oxidative stress generated by salt stress [3]. It affects severely various morphological, physiological, and biochemical processes such as photosynthesis, accumulation of low molecular mass compounds, such as proline and glycine betaine and protein and lipid metabolisms [4]. The main adverse effect of salinity is the ionic stress resulting from an increment of sodium (Na⁺) and decrement of potassium (K⁺) uptakes, which leads to a reduction in the K⁺/Na⁺ ratio in plant cells [5]. Excessive sodium accumulation can markedly enhance the production of reactive oxygen species (ROS) such as O₂^•− (superoxide radical) and H₂O₂ (hydrogen peroxide), thereby disturbing cellular redox homeostasis. To counteract salinity-induced oxidative stress, plants activate a range of enzymatic and non-enzymatic defense systems. Augmentation in scavenging potential of ROS in salt-stressed safflower plants occurred through increasing the activities of catalase (CAT), superoxide dismutase (SOD) and peroxidase (POX), and enhancement of some non-enzymatic compounds such as phenolics, ascorbic acid, and α-tocopherol [6].

Salt stress restricts water uptake by plants, causing cellular dehydration along with reduced cell growth and division. To mitigate this, salt-exposed plants often accumulate higher levels of osmoprotectants such as soluble sugars, proline, and glycine betaine, which help sustain cellular water potential. This stress may also result in decreasing essential nutrients absorption, ion imbalance, and reduction in photosynthetic pigments and efficiency, leading to less growth and productivity of plants [7]. Several strategies, including the use of plant growth regulators, have been shown to enhance stress tolerance in diverse plant species [8,9].

Controlled foliar treatment with putrescine (Put) can activate physiological processes and promote the accumulation of osmolytes such as proline, total soluble sugars, and amino acids in plants [10]. Previous studies have shown that exogenous Put enhances salt tolerance through various mechanisms: by increasing osmolyte accumulation and modulating carbon fixation, electron transport, energy, and defense pathways in Cynodon dactylon; scavenging reactive oxygen species in Citrus aurantium; regulating photosynthetic pathways in Citrus reticulata × Citrus limetta; and affecting proteins related to photosynthesis and root growth in Cucumis sativa [11].

Moreover, salicylic acid (SA), a phenolic signaling molecule and plant growth regulator, has been reported to enhance plant tolerance to key abiotic stresses, including salinity and osmotic stress [12]. It plays a crucial role in regulating numerous physiological processes, including seed germination, plant growth and development, respiration, photosynthesis, transpiration, stomatal movement, flowering, disease resistance, enzyme activity, senescence, and nutrient uptake [13]. SA is synthesized through two distinct pathways: the phenylalanine ammonia-lyase (PAL) pathway, which is the predominant route, and the isochromatic (IC) pathway [14]. SA can be used either as a seed pretreatment or as a foliar spray to activate key abiotic stress-tolerance mechanisms [15,16]. Several studies have shown that low concentrations of SA enhance plant functions and resistance. For instance, foliar application of 1 mM SA improved plant growth and defense capacity while reducing ion toxicity under saline conditions [17]. However, inhibition of growth and induction of ROS production by a high concentration of SA has also been reported for many plant species [6]. Moreover, several molecular studies have confirmed that SA regulates multiple plant processes, thereby improving salt tolerance [18]. For this reason, it is expected that the combined foliar application of Put and SA would combinedly enhance salinity tolerance in sugar beet seedlings by improving their physiological and biochemical performance under saline conditions.

Typically, sugar beet is a biennial crop adapted to temperate regions, requiring adequate vernalization followed by exposure to long day lengths (>14 h) to initiate bolting [19,20]. The Republic of Korea, situated in the temperate zone within the mid-latitudes of the Northern Hemisphere, experienced average winter temperatures of 0.1–1.1 °C over the past 30 years. During spring (March–May), the day length ranges from 12 to 14 h, while in summer (June–August) it extends beyond 14 h [21]. Therefore, the climatic conditions of Korea may provide opportunities for introducing sugar beet breeding. On the other hand, despite having vast coastal areas with considerable potential for sugar beet cultivation, the high soil salinity and fluctuating environmental conditions in these regions of Korea pose major constraints to successful crop establishment and productivity [22]. Previous studies in other countries have reported that exogenous applications of Put and SA can alleviate abiotic stress-induced growth inhibition in sugar beet by enhancing osmotic adjustment, antioxidant defense, and photosynthetic performance [11,23,24,25,26,27]. However, to the best of our knowledge, no studies in Korea have addressed the role of sugar beet in mitigating salinity stress. Hence, this study was designed to evaluate the effects of Put and SA applications on growth, and quality traits of sugar beet under saline conditions, focusing on their physiological and biochemical responses for future breeding purposes.

2. Materials and Methods

2.1. Plant Materials and Salt Stress Treatment

Seedlings of sugar beet were raised in a semi-controlled greenhouse at the Department of Bio-Health Convergence, Kangwon National University, Chuncheon, Republic of Korea. The growth environment was maintained at 30/25 °C (day/night), 60–70% relative humidity, and a 12 h photoperiod. Seeds of the variety BSRI Sugar beet 2 were obtained from the Bangladesh Sugarcrop Research Institute (BSRI), Bangladesh. Prior to sowing, seeds were surface-sterilized with 70% (v/v) ethanol, 0.1% (w/v) HgCl₂, and 0.2% (w/v) thiram, and then sown in 16-cell plug trays (27 × 27 × 6 cm³) filled with commercial horticultural soil (Bio-soil No. 1, Heungnong Agricultural Materials Mart, Republic of Korea). For the first 15 days, seedlings were irrigated daily with tap water to field capacity. After this period, they were transplanted into pots (10.0 cm × 9.5 cm) containing 180 g of a substrate mixture composed of commercial horticultural soil and organic manure in a 3:1 ratio, and irrigated daily to field capacity. Thirty-day-old uniform seedlings were subsequently transferred into hydroponic containers with nutrient solution (

Table 1. Nutrient solution.

Chemical Name	A Tank (50 L) *	B Tank (50 L)
Ca(NO3)	1.5 kg
KNO3	3.79 kg	3.79 kg
(NH4)2HPO4		1.6 kg
MgSO4		4.3 kg
Fe-EDTA	460 g
MnSO4		30.8 g
H3BO3		57.2 g
ZnSO4		3.6 g
CuSO4		1.3 g
(NH4)6Mo7O24.4H2O		0.4 g

* Solutions from Tank A and Tank B were mixed to maintain an EC range of 1.2–1.7 dS m⁻¹ and a pH of 6.0.

). All seedlings were maintained under nutrient solution (control) conditions for seven days for adaptation and then transferred to separate hydroponic systems: one with nutrient solution only (control) and the other supplemented with 150 mM NaCl to impose salinity stress. After that, eight treatments, i.e., (i) Control, (ii) Control + 0.6 mM Put, (iii) Control + 0.6 mM SA, (iv) Control + 0.6 mM Put + 0.6 mM SA, (v) Salinity, (vi) Salinity + 0.6 mM Put, (vii) Salinity + 0.6 mM SA, and (viii) Salinity + 0.6 mM Put + 0.6 mM SA, were imposed for 10 days. At the onset of the treatment (day 1), Put and SA were applied once to both sides of the leaves using 1% (v/v) Tween-20 as a surfactant. The nutrient solution was renewed on the fifth day of the experiment to maintain consistent nutrient and salinity levels. The youngest completely formed leaves were collected from each treated seedling for further analysis.

2.2. Determination of Plant Height, Fresh Weight, and Dry Weight

On the 10th day after the initiation of salinity treatment, five seedlings from each treatment were randomly selected to measure the average shoot length, root length, plant height, and fresh weights of shoot, root, and whole plant. Subsequently, the plants were oven-dried at 60 °C for 48 h to determine shoot, root, and total plant dry weights. Afterward, plant growth rate was calculated on dry weight basis.

2.3. Leaf Gas Exchange Measurement

The average net photosynthetic rate (A, µmol m⁻² s⁻¹), transpiration rate (E, mmol m⁻² s⁻¹), and stomatal conductance (gs, mmol m⁻² s⁻¹) were measured on fully expanded leaves (third node from the top) of the five plants per treatment using an LCpro gas analyzer (ADC BioScientific Ltd., Hoddesdon, Herts EN11 ONT, UK). Measurements were conducted at ambient CO₂ concentration, an air temperature of 25–26 °C, and a photosynthetic photon flux density (PPFD) of 1000 µmol m⁻² s⁻¹ in the leaf chamber. Gas exchange recordings were performed at midday between 10:00 a.m. and 3:00 p.m.

2.4. Determination of SPAD Value and Photosystem II Quantum Yield

The SPAD value was determined using a Chlorophyll Meter SPAD-502Plus (KONICA MINOLTA INC., Osaka, Japan). Photosynthetic fluorescence parameters were assessed with a Fluor Pen FP 100 (Photon Systems Instruments, Drasov 470, 664 24 Drasov, Czech Republic) by recording the OJIP transient after a 20 min dark adaptation. Five seedlings from each treatment were randomly selected for average data collection.

2.5. Leaf Relative Water Content

Three fully expanded leaves were collected from each of three seedlings per treatment and immediately weighed to record fresh weight (FW). The samples were then submerged in deionized water for 48 h and reweighed to obtain turgid weight (TW). Finally, leaves were oven-dried at 60 °C for 48 h to determine dry weight (DW). Relative water content (RWC) was calculated using the following formula:

RWC (%) = [(FW − DW)/(TW − DW)] × 100

2.6. Estimation of Osmotic Adjustment Molecules

The three youngest fully expanded leaves were collected from each treated seedling (three seedlings per treatment) and immediately immersed in liquid nitrogen, followed by freeze-drying, before being subjected to further analyses.

Approximately 25 mg of freeze-dried plant material was used to quantify proline concentration. The analysis followed the method outlined by Bates [28]. Absorbance was measured at 520 nm using a UV–vis spectrophotometer (UV-1800 240 V, Shimadzu Corporation, Kyoto, Japan), and proline concentration was calculated based on a standard curve.

For the analysis of TSC and sucrose, a 25 mg freeze-dried sample was homogenized in 5 mL of 95% ethanol. The homogenate was then centrifuged at 5000 rpm for 10 min, and the supernatant was collected. This process was repeated using 70% ethanol, and the combined supernatant was stored at 4 °C for further analysis. TSC and TSS contents were determined following the methods described previously [29].

Ascorbic acid was extracted from 25 mg of freeze-dried leaf tissue using 10 mL of 6% trichloroacetic acid, following the method described previously [30]. A 4 mL aliquot of the extract was combined with 2 mL of 2% dinitrophenyl hydrazine and a drop of 10% thiourea solution in 70% ethanol. The mixture was heated in a water bath for 15 min, then allowed to cool to room temperature. Subsequently, 5 mL of 80% H₂SO₄ (v/v) was added at 0 °C. Absorbance was measured at 530 nm using a spectrophotometer (UV-1800 240 V, Shimadzu Corporation, Kyoto, Japan) and AsA content was determined by comparison with a standard curve ranging from 10–100 mg L⁻¹.

2.7. Determination of Malondialdehyde and H₂O₂ Content

Fresh leaves from three seedlings per treatment were collected, and a 200 mg sample was macerated in 5 mL of 0.1% trichloroacetic acid. The homogenate was then centrifuged at 12,000× g for 10 min at 4 °C, and the supernatant was stored at the same temperature for further analysis. Lipid peroxidation was evaluated by determining the malondialdehyde (MDA) content in sugar beet seedlings. The estimation of MDA and H₂O₂ was conducted following the procedure described in our previously published article [11]. All analyses were performed with three biological replicates.

2.8. Determination of Antioxidant Enzyme Activities

Leaf samples from each treatment were collected, quickly frozen in liquid nitrogen, and stored at −80 °C until analysis. A 200 mg portion of each sample was homogenized in 5 mL of 50 mM sodium phosphate buffer (pH 7.8) using a pre-chilled mortar and pestle, then centrifuged at 15,000× g for 20 min at 4 °C. The supernatant obtained was collected and stored at 4 °C for enzyme assays. Superoxide dismutase (SOD; EC 1.15.1.1) activity was determined as described previously [31], while guaiacol peroxidase (GPX; EC 1.11.1.7) and catalase (CAT; EC 1.11.1.6) activities were measured according to the method of Zhang [32].

2.9. Determination of Protein Content

For calculation of enzyme activities, protein content was determined spectrophotometrically at 595 nm by the method of Bradford [33].

2.10. Histochemical Confirmation of Reactive Oxygen Species

Hydrogen peroxide (H₂O₂) in sugar beet leaves was detected using the histochemical staining method described earlier [34]. Briefly, freshly plucked leaves were immersed in a 1 mg/mL diaminobenzidine (DAB) solution and incubated for 8 h. Following incubation, the leaves were transferred to absolute ethanol and boiled until deep brown spots, indicative of H₂O₂ accumulation, appeared on the leaf surfaces. After cooling, images of the stained leaves were captured using a Nikon D850 camera (Nikon Corporation, Ayutthaya, Thailand).

2.11. Statistical Analysis

All data are presented as mean ± SD. Statistical analyses were performed using SAS 9.4 (SAS Institute Inc., Cary, NC, USA), and mean differences were evaluated using Tukey’s post hoc multiple comparison test, with p < 0.05 considered statistically significant. Heatmaps and clustering analyses were generated from normalized mean values using MetaboAnalyst 4.0 (www.metaboanalyst.ca, accessed on 17 July 2025), with hierarchical clustering based on the Euclidean distance method. Principal component analysis (PCA) was performed using OriginLab 10.0 (OriginLab, Northampton, MA, USA).

3. Results and Discussion

3.1. Growth Parameters

Plant growth parameters varied significantly across treatments (

Table 2. Effect of application of putrescine (Put) and Salicylic acid (SA) on the plant morphological parameters of sugar beet grown under salinity stress.

Treatment	Plant GR on DW Basis (g day−1)	SL (cm)	RL (cm)	PH (cm)	SFW (g)	RFW (g)	PFW (g)	SDW (g)	RDW (g)	PDW (g)
Control	0.118 abc	26.33 ab	4.17 bc	30.50 abc	14.46 a	3.24 ab	17.70 a	1.16 ab	0.30 bcd	1.46 ab
C+P	0.158 abc	28.33 a	5.50 a	33.83 a	14.19 a	3.48 ab	17.67 a	1.41 ab	0.45 ab	1.86 ab
C+SA	0.166 ab	25.83 ab	5.17 ab	31.00 ab	15.58 a	4.10 a	19.67 a	1.45 ab	0.49 a	1.94 ab
C+P+SA	0.171 a	28.97 a	3.60 c	32.57 ab	15.50 a	3.45 ab	18.95 a	1.55 a	0.44 abc	1.99 a
Salinity	0.052 bc	16.33 c	3.73 c	20.07 d	5.42 b	1.25 c	6.67 b	0.62 b	0.17 d	0.80 b
S+P	0.077 abc	20.90 abc	3.73 c	24.63 bcd	7.18 b	2.32 bc	9.50 b	0.78 ab	0.27 cd	1.05 ab
S+SA	0.049 c	18.30 bc	4.27 bc	22.57 cd	5.69 b	1.17 c	6.86 b	0.60 b	0.17 d	0.77 b
S+P+SA	0.083 abc	21.13 abc	3.73 c	24.87 bcd	7.29 b	1.92 bc	9.21 b	0.84 ab	0.27 d	1.11 ab

Values are expressed as mean ± SD (n = 3). Different letters indicate significant differences (p < 0.05) among treatments within each parameter. GR, growth rate; SL, shoot length; RL, root length; PH, plant height; SFW, shoot fresh weight; SDW, shoot dry weight; RFW, root fresh weight; RDW, root dry weight.

). C+P+SA showed the highest growth rate (GR, 0.17 g day⁻¹), while salinity significantly (p < 0.05) reduced it to 0.05 g day⁻¹. Shoot length (SL) peaked at 28.97 cm in C+P+SA but significantly (p < 0.05) dropped 38% under salinity (16.33 cm). Root length (RL) decreased by 10.55% under salinity, with S+SA achieving the highest recovery (14.47%). Plant height (PH) reached 33.83 cm in C+P, but salinity significantly (p < 0.05) reduced it by 34.2% (20.07 cm). Shoot fresh weight (SFW) remained stable (14–15 g) in non-saline conditions but significantly (p < 0.05) fell to 5.42 g under salinity. Higher root fresh weight (RFW) was observed in C+SA, while under salinity it significantly (p < 0.05) dropped to 1.25 g. Higher plant fresh weight (PFW) was recorded in C+SA (19.67 g), where it significantly (p < 0.05) declined 62.3% (6.67 g) under salinity. Higher shoot dry weight (SDW) was found in C+SA (1.55 g), while it significantly (p < 0.05) dropped to 0.62 g under salinity. Higher root dry weight (RDW) was manifested by C+SA (0.49 g), while it significantly (p < 0.05) reduced to 0.17 g in salinity stress. Plant dry weight (PDW) was highest in C+P+SA (1.99 g) and significantly (p < 0.05) lower in salinity (0.80 g). On the other hand, despite substantial changes, no significant improvement was observed either by Put or SA treatments.

The present study demonstrated that several plant growth parameters were significantly (p < 0.05) influenced by different treatments, with salinity exerting a strong inhibitory effect. However, the application of Put (P) and SA alone or in combination mitigated salinity-induced stress, as reflected in various growth attributes. Salinity stress significantly (p < 0.05) reduced SL, PH, SFW, RFW, and PFW, consistent with previous studies [35,36]. The decline in plant growth under saline conditions is likely due to osmotic stress, ion toxicity, and nutrient imbalance, which impair cell division and elongation [37,38].

The application of SA and Put slightly improved several plant growth parameters under both saline and non-saline conditions. The highest growth rates under non-saline and saline conditions were observed in the C+P+SA treatment (0.17 g day⁻¹ and 0.083 g day⁻¹, respectively), suggesting that SA and Put enhance plant resilience under both normal and salinity stress conditions. Studies on bean and lettuce reported that Put application under both saline and normal conditions increased plant growth [13,39]. Another study reported that increasing salinity reduced plant height, fresh weight, dry weight, root length, and root collar diameter in lettuce, whereas the application of SA under salt stress enhanced these growth parameters [13]. SA is known to mediate antioxidant defense and osmotic adjustment, alleviating the effects of oxidative stress under salinity [40]. Similarly, Put, a polyamine, plays a key role in stabilizing membrane integrity and enhancing stress tolerance by modulating reactive oxygen species (ROS) levels [41,42].

Among all treatments, C+P+SA resulted in the highest SL, SFW, PDW and SDW under saline and non-saline condition, demonstrating the enhanced combined effect of Put and SA in mitigating salinity stress. The observed improvement in PH and RFW under the S+P+SA treatment (recovery rates of 34.5% to 58.8%) corroborates previous findings that polyamines and SA enhance plant growth by modulating hormonal balance and stress-responsive gene expression [43].

3.2. Changes in Photosynthesis

The photosynthetic rate was highest in the control plants (14.07 µmol m^˗2 s^˗1), with a significant (p < 0.05) decline observed under salinity stress (−71.78%) (Figure 1). Among the control treatments, the application of Put (C+P) and SA (C+SA) resulted in a moderate reduction in photosynthetic rate compared to the control, although the difference was not statistically significant (p < 0.05). On the other hand, exogenous application of Put (S+P) and SA (S+SA) led to a partial recovery of 31.40% and 67.59%, respectively.

Respiration rate followed a similar trend to photosynthesis, with the highest values recorded in the control plants (4.24 µmol m^˗2 s^˗1). The treatments C+P, C+SA, and C+P+SA did not significantly (p < 0.05) differ from the control. Salinity stress resulted in a significant (p < 0.05) reduction in respiration rate (−70.75%). Among the salinity-stressed treatments, S+P exhibited the highest recovery (77.96%).

Stomatal conductance was significantly (p < 0.05) higher in control plants compared to those subjected to salinity stress. The application of Put (C+P) and SA (C+SA) under control conditions caused a slight reduction in stomatal conductance. Salinity stress led to a significant (p < 0.05) decline (−85.88%), while the combined application of Put and SA (S+P+SA) induced a significant (p < 0.05) recovery (483.25%) in stomatal conductance.

The highest water-use efficiency (WUE) was observed in S+SA (5.88 µmol mol⁻¹ m^˗2 s^˗1), indicating an improved ability to sustain physiological functions under salinity stress. Under control conditions, WUE did not differ significantly among the treatments. Salinity stress led to an overall reduction in WUE, although S+SA significantly improved WUE compared to S+P and S+P+SA.

The results demonstrate the impact of salinity stress on Beta vulgaris L., particularly affecting photosynthetic rate, respiration rate, stomatal conductance, and water-use efficiency (WUE). The significant (p < 0.05) decline in photosynthetic rate under salinity stress (−71.78%) aligns with previous findings indicating that salt stress impairs photosynthetic machinery by reducing chlorophyll content, inhibiting CO₂ assimilation, and promoting oxidative damage [44]. However, the partial recovery observed with Put (S+P) and SA (S+SA) treatments, showing 31.40% and 67.59% improvement, respectively, suggests their role in mitigating stress effects. Put is known to stabilize membranes and enhance antioxidant defense, while SA has been reported to improve photosynthetic efficiency by modulating stomatal behavior and enhancing the antioxidant system [45,46].

The respiration rate followed a similar pattern, with a significant (p < 0.05) reduction under salinity stress (−70.75%) and a notable recovery (77.96%) in S+P. This supports the idea that Put enhances mitochondrial function and energy metabolism, helping plants maintain cellular respiration under adverse conditions [41,47,48]. The limited recovery with SA suggests it may prioritize photosynthetic processes over respiration pathways, as noted in earlier studies [49]. Stomatal conductance, which governs gas exchange, showed a significant (p < 0.05) decline (−85.88%) under salinity stress. This is consistent with research indicating that salinity-induced osmotic stress leads to stomatal closure to prevent water loss, albeit at the cost of reduced CO₂ uptake [50]. The remarkable increase in stomatal conductance (483.25%) with S+P+SA suggests an enhanced combined effect, potentially promoting stomatal reopening by balancing osmotic regulation and ROS accumulation [47].

Water-use efficiency emerged as a critical indicator of plant performance under stress. The highest WUE observed in S+SA (5.88 µmol^˗1 m^˗2 s^˗1) highlights role of SA in maintaining carbon assimilation while minimizing water loss. This aligns with previous studies suggesting that SA enhances WUE by optimizing stomatal aperture and improving osmotic adjustment [51]. The lesser improvement in WUE with S+P and S+P+SA implies that while Put supports cellular recovery, its role in enhancing water-use efficiency may be secondary to that of SA.

3.3. Changes in SPAD Value and Fluorescence Parameters

Data presented in

Table 3. Effect of application of putrescine on chlorophyll fluorescence of sugar beet seedlings grown under salinity stress condition.

Treatment	SPAD	F0	Fm	Fv/Fm	PI_abs	DIo/RC	LRWC (%)
Control	32.433 ab	7466 b	38,037 abc	0.8027 a	3.2050 a	0.3687 d	82.88 ab
C+P	32.550 ab	6838 b	29,964 cd	0.7633 ab	2.1810 ab	0.4407 cd	89.77 a
C+SA	35.183 a	8258 b	331,94 bcd	0.7507 b	1.7863 ab	0.5443 bcd	87.18 a
C+P+SA	32.583 ab	6692 b	24,698 d	0.7257 bc	1.4430 ab	0.5667 bc	86.51 a
Salinity	28.383 b	6903 b	27,461 cd	0.6960 c	2.2693 ab	0.6017 bc	58.23 c
S+P	34.783 a	6351 b	21,663 d	0.7320 bc	1.2407 ab	0.6410 b	61.73 bc
S+SA	34.483 a	13,676 a	46,024 ab	0.7030 c	0.7393 b	0.8817 a	58.22 c
S+P+SA	35.133 a	12,332 a	48,994 a	0.7487 b	1.2487 ab	0.7023 ab	61.94 bc

Values are expressed as mean ± SD (n = 3). Different letters indicate significant differences (p < 0.05) among treatments for each parameter. F0, fluorescence intensity at 50 µs; Fm, maximal fluorescence; Fv/Fm, maximum quantum yield of PSII; PI_abs, performance index on absorption basis; DIo/RC, dissipated energy flux; LRWC, relative water content.

showed that the highest SPAD value (35.18) was recorded under the C+SA treatment, while the lowest value (28.38) was observed under salinity stress conditions. Similarly, salinity stress reduced OJIP parameters, including fluorescence intensity at 50 µs (F0), maximal fluorescence (Fm), maximum quantum yield of PSII (Fv/Fm), and the photosynthetic performance index (PI_abs) in sugar beet leaves. On the other hand, under salinity stress conditions, the dissipated energy flux (DIo/RC) increased, with the highest value observed in the S+SA treatment (0.8817) compared to the control. Application of Put and SA increased the SPAD value, Fm, Fv/Fm and DIo/RC considerably, while PI_abs was decreased compared to salinity stress condition. Moreover, the highest leaf relative water content (LRWC; 89.77%) was recorded under the C+P treatment, while a significant decrease was observed under salinity stress (58.23%). In contrast, no significant changes were observed under either Put or SA treatments.

Salt stress adversely impacts the photosynthetic apparatus and its efficiency by affecting pigment content, stomatal function, gas exchange, thylakoid membrane integrity, electron transport, and the maximum quantum yield of photosystem II (Fv/Fm ) [52,53]. Salt stress may reduce chlorophyll content in plants by increasing chlorophyllase activity and destabilizing protein complexes [54,55].

In our study, SPAD values clearly declined in sugar beet leaves under salinity stress (Table 3). Salt-induced oxidative stress promotes ROS accumulation, which accelerates chlorophyll degradation through lipid peroxidation and destabilization of pigment–protein complexes in leaf tissues [26,56]. Exogenous application of Put enhances photosynthetic pigment content, and this effect has been well documented in previous studies [57,58]. In our study, foliar application of Put at different concentrations increased SPAD values in sugar beet leaves. Put plays a crucial role in protecting thylakoid membranes at the chlorophyll–protein complex sites, thereby positively influencing chlorophyll content [59]. Put-treated plants showed improved OJIP parameters along with increased chlorophyll content under salinity stress. These effects likely contributed to the enhanced growth of plants, probably by improving CO₂ fixation under salt stress. In this context, several studies have reported a correlation between abiotic stress tolerance and photosystem II efficiency [60,61]. In our study, salinity stress reduced Fv/Fm in sugar beet plants by 13.29%, whereas the combined S+P+SA treatment significantly restored it by 7.57%. Likewise, exogenous application of Put has been reported to enhance chlorophyll fluorescence parameters (F0, Fm, and Fv/Fm) in Iranian mandarin Bakraii (Citrus reticulata × Citrus limetta) under salinity stress [62]. Previous studies have identified higher Fv/Fm as an indicator of stress tolerance under conditions such as cold, salinity, and drought stress. PI_abs, a composite parameter from OJIP transient analysis, provides an integrated assessment of PSII performance by combining reaction center density, energy trapping efficiency, and electron transport capacity—key determinants of photosynthetic efficiency under stress. Higher PI_abs values indicate efficient light energy use, reduced photoinhibition, and sustained electron transport, contributing to better biomass accumulation under stress [63]. Its comprehensive nature accounts for its greater sensitivity to stress compared with the conventional Fv/Fm ratio [64]. Although, plants exhibiting both higher PI_abs and lower DIo/RC have reported enhanced tolerance to oxidative stress [11]. However, no significant improvement in PI_abs and DIo/RC were observed with either Put or SA treatments in our study, possibly due to the complex and sometimes inconsistent relationships between photosynthetic parameters and biomass accumulation as reported in earlier study [65]. Under abiotic stress, photosynthetic pigments in photosystems are often damaged, leading to reduced light absorption in PS I and PS II, which is a primary cause of decreased photosynthetic capacity [66]. Our results align with these findings, showing reduced total chlorophyll and photosynthetic quantum yield under salinity stress, which were improved by exogenous Put application.

SA application can further mitigate the salt-induced decline in photosynthetic efficiency, primarily by stimulating the ascorbate–glutathione pathway and enhancing glutathione (GSH) synthesis [12]. Leaf photosynthetic efficiency is closely correlated with the Fv/Fm ratio, where a decrease indicates photoinhibition and disruption of photon flux under environmental stress. Exogenous SA application largely alleviated the detrimental effects of salinity on photosynthetic efficiency. The Fv/Fm ratio, stomatal conductance, and net photosynthetic rate are significantly higher in leaves of SA-treated plants, resulting in better functioning of the photosynthetic machinery under saline conditions [6]. Application of SA significantly reduces the time to reach F₀ and Fₘ, while increasing variable fluorescence (Fv), PI_abs, and the efficiency of the water-splitting complex. This is achieved by enhancing leaf water content and the energy required for closure of reaction centers and the redox state of the primary quinone during the interval from F0 to Fm, ultimately improving photosynthetic function. Additionally, SA treatment may enhance photosynthetic activity by increasing leaf number and leaf area [53]. These findings reported by other researchers are in strong agreement with the results obtained in our study.

3.4. MDA and H₂O₂ Concentration

The effect of salinity on lipid peroxidation, indicated by MDA content, is presented in Figure 2A. Salinity increased MDA levels by 17.54%, indicating greater cellular damage. This was reduced with Put (13.99%), SA (19.86%), and the combination of Put and SA (26.37%) compared to salinity stress. Salinity stress also significantly increased hydrogen peroxide (H₂O₂) accumulation in sugar beet leaves by 78.33% (p < 0.05) compared to the control (Figure 2B). However, H₂O₂ levels were markedly reduced following treatments with Put (84.13%), SA (70.28%), and a combined application of Put and SA (84.12%) compared to the plants under salinity stress. Histochemical analysis further confirmed that salinity increased H₂O₂ accumulation in sugar beet leaves, with a visible reduction observed under Put and SA treatments, either individually or together (Figure 2C).

Plant cell membranes possess a highly intricate and delicate structure that plays a vital role in preserving cellular homeostasis. Under salt stress, disturbances in ion pumps and membrane channels—normally controlled by hormones and internal signals—can trigger electrolyte leakage [67]. When membranes are impaired, sodium ions may partially infiltrate the cells. Elevated sodium influx can, in turn, activate certain cellular enzymes, leading to the accumulation of hydrogen peroxide (H₂O₂) and other reactive oxygen species (ROS), which inflict oxidative injury. Excessive H₂O₂ formed as a result of sodium ion intrusion contributes to membrane disruption and impaired cellular functions. Hence, the role of H₂O₂ as a key mediator of salinity-induced stress has been widely investigated [68]. Among the markers studied, malondialdehyde (MDA) is of particular importance; it is a byproduct of lipid peroxidation, and its elevated levels serve as an indicator of oxidative stress and ROS-associated damage under salt stress [69].

In the present study, MDA levels increased under salt stress, reflecting reduced tolerance and membrane damage. Conversely, plants treated with Put displayed lower MDA accumulation, indicating protection of membranes against lipid peroxidation. This finding aligns with earlier results reported in 2022, which emphasized the role of Put in plant growth and development. As a polyamine with signaling functions, Put contributes to membrane stability by regulating enzyme and protein activities. Its protective mechanisms include maintaining ionic balance, enhancing the activity of antioxidants such as carotenoids, and modulating enzymes associated with cellular structure. Collectively, these actions strengthen membrane integrity, support normal cellular processes, and promote plant growth. Moreover, external application of polyamines, by stimulating Put metabolism, reduces MDA buildup through improved antioxidant defense [70,71]. Several previous studies have shown that exogenous application of Put regulates plant growth and the photosynthetic machinery, while also enhancing antioxidant capacity and modulating gene expression under various abiotic stresses [26,48,72].

3.5. Changes in Osmotic Adjustment Molecules

Figure 3 illustrates the changes in proline (Pro), ascorbic acid (AsA), total soluble carbohydrates (TSC), and sucrose content in sugar beet leaves under different treatments. Pro content increased dramatically by 1084% under salinity stress compared to the control, while no significant changes were observed in control plants treated with Put, SA, or their combination. Notably, proline levels further significantly (p < 0.05) increased with SA (37.84%) and Put + SA (26.44%) treatments compared to salinity alone, suggesting a potential protective role of these treatments in enhancing osmotic adjustment under stress.

AsA content remained relatively stable across the control and treatments with Put and SA. However, a noticeable decline (21.55%) was observed under salinity stress, suggesting oxidative stress-induced depletion, which was not fully recovered by the treatments. For total soluble carbohydrates (TSC), the highest levels were recorded under control conditions with the combined treatment of Put and SA (123.12 mg g⁻¹ DW), followed by Put (115.66 mg g⁻¹ DW) and SA (93.12 mg g⁻¹ DW). In contrast, salinity stress significantly (p < 0.05) reduced TSC content by 27.70%, with only minimal recovery observed across treatments, suggesting that carbohydrate metabolism was severely impaired under stress.

Sucrose content increased notably with Put treatment under salinity stress, showing a 25.33% rise compared to the control. This suggests Put plays a key role in supporting sugar partitioning and maintaining osmotic balance. Other treatments resulted in comparatively lower sucrose content, highlighting Put’s more prominent influence on sugar accumulation under stress.

Proline is one of the most important compatible solutes in plants, playing a crucial role in environmental stress adaptation [73]. Plants accumulate proline, a low-molecular-weight amino acid, under stress primarily via enhanced biosynthesis and reduced catabolism. While protein degradation may also contribute some amino acid substrates, the main driver appears to be altered metabolic flux toward proline [74,75,76]. In this study, a decrease in proline was observed in Put-treated plants, which could be attributed to the enhancement of the plant’s antioxidant system, leading to reduced protein breakdown. Additionally, in untreated plants, increased protein degradation and competition for glutamate as a precursor for proline synthesis may have contributed to the observed increase in proline levels. This elevated proline can also lead to increased osmotic pressure inside the cells, which may inhibit water absorption for chlorophyll synthesis, thereby potentially reducing chlorophyll content [67,77]. In contrast, the application of Put resulted in a decrease in proline, thereby promoting the development of resistance to salt stress.

The role of proline, TSC, sucrose, and total soluble proteins (TSP) in mitigating abiotic stress through osmotic adjustment, reactive oxygen species (ROS) detoxification, protein stabilization, and cell membrane protection has been described in previous studies [21,78,79]. TSC and sucrose, as key components of osmotic adjustment, are closely linked to the antioxidation system at the cellular level, actively participating in ROS detoxification [80]. In this study, a significant decrease in TSC was observed in leaves under salinity stress, with TSC levels increasing following the application of Put. A similar trend was reported in wheat under drought stress, where the influence of exogenous Put on proline, soluble, and insoluble sugar accumulation showed variability [81].

Additionally, the application of SA has been shown to enhance the synthesis of soluble sugars, proline, and glycine betaine in salt-stressed rapeseed plants, especially under 10 dS m⁻¹ NaCl. The strong correlation between endogenous SA levels and soluble sugars highlights SA’s role as a key regulator of osmo-protective mechanisms in stressed plants [17]. The increase in proline content due to SA application may result from the upregulation of pyrroline-5-carboxylate reductase and γ-glutamyl kinase activities, as well as a reduction in proline oxidase activity [25,82]. Moreover, SA application may also reduce proline content by enhancing chlorophyll synthesis, since both proline and chlorophyll share glutamate as a common precursor [7].

3.6. Changes in Antioxidant Enzyme Activity

The effects of salinity and the application of Put and SA on antioxidant enzyme activity are presented in Figure 4. Catalase (CAT) activity significantly (p < 0.05) declined under salinity stress (45.28%), compared to control. However, compared to salinity it increased significantly (p < 0.05), 29.90% and 79.08% when treated with SA and Put+SA treatment, respectively. Compared with control, superoxide dismutase (SOD) decreased significantly (p < 0.05) under salinity stress whereas it was further significantly increased by Put and Put+SA. On the other hand, higher guaiacol peroxidase (GPX) activity was observed when treated with Put and SA combinedly under nonsaline condition.

Salinity inevitably leads to oxidative stress through an increase in ROS, such as superoxide radical (O₂^•−), hydrogen peroxide (H₂O₂), and hydroxyl radicals (OH•) [4]. It is well established that cytotoxic ROS are major contributors to stress-induced damage of macromolecules and cellular structures [83]. Plants counteract this through enzymatic antioxidants such as SOD and CAT, which constitute an effective defense system against oxidative stress [84,85]. In the present study, activities of CAT, GPX, and SOD, along with proline content, were elevated in NaCl-stressed plants treated with Put and SA (Figure 4). Comparable results have been reported in chickpea, where exogenous Put alleviated NaCl stress by enhancing the activities of CAT, glutathione peroxidase, glutathione reductase, and SOD [86]. Notably, Put application consistently increased CAT activity across both salt concentrations, suggesting its role in sustaining or even stimulating antioxidant enzyme functions under stress. This protective effect likely arises from lowering ROS generation and thereby reducing the need for antioxidant consumption [71,87]. Furthermore, SOD activity exhibited concentration-dependent responses to Put, reinforcing its potential as a regulator of plant tolerance to salinity [24]. Our findings confirm the additive/enhanced response of Put in alleviating NaCl-induced oxidative stress, consistent with previous reports in Raphanus sativus under copper stress [88]. In this study, SA also contributed to stress mitigation by enhancing SOD activity under salinity conditions (Figure 4). Efficient detoxification of H₂O₂ primarily relies on CAT, GPX, and the ascorbate–glutathione cycle. In our results, CAT activity declined whereas GPX activity increased following NaCl treatment (Figure 4), a response commonly observed under various abiotic stresses. Notably, SA treatment elevated both CAT and GPX activities under salinity, which likely reduced H₂O₂ accumulation and thereby alleviated oxidative damage [23].

The alleviation of salinity stress by exogenous putrescine (Put) and salicylic acid (SA) in sugar beet appears to involve complex molecular and signaling mechanisms beyond the observed physiological and biochemical responses. Previous studies have shown that Put regulates the expression of stress-related genes involved in antioxidant defense (SOD, CAT, APX, POD), polyamine metabolism (ADC, ODC, SAMDC), and ion homeostasis, thereby improving ROS scavenging and maintaining cellular redox balance [89,90]. Enhanced expression of these genes contributes to stabilization of membranes, protection of photosynthetic pigments, and improved osmotic adjustment through the accumulation of compatible solutes. Similarly, SA acts as a crucial signaling molecule that activates multiple stress-responsive transcription factors, including WRKY, TGA, and NPR1, leading to induction of defense genes associated with antioxidant regulation and osmolyte biosynthesis [91,92].

In sugar beet, exogenous SA has been reported to enhance the expression of antioxidant and proline metabolism genes, resulting in improved tolerance to salinity-induced oxidative stress [93]. Moreover, SA interacts combinedly with polyamines, abscisic acid (ABA), and nitric oxide (NO) signaling to fine-tune stomatal regulation, ion transport, and osmoprotectant synthesis [47,94]. The combined application of Put and SA may therefore trigger crosstalk among these signaling networks, coordinating antioxidant activation and osmolyte accumulation that collectively sustain photosynthetic performance and growth under saline environments. Although the present study did not include gene expression data, the improved physiological and biochemical traits observed in Put- and SA-treated sugar beet seedlings are likely associated with upregulation of these molecular pathways as reported in earlier studies.

While the present study did not investigate gene expression patterns, the marked improvements in antioxidant enzyme activities, osmolyte levels, and photosynthetic traits under Put and SA treatments are presumably linked to the activation of stress-responsive genes and molecular signaling pathways documented in earlier research.

3.7. Hierarchical Clustering and PCA

The physiological and biochemical parameters measured across different treatments were used to generate a hierarchical clustering and heatmap (Figure 5). The analysis grouped the variables into three major clusters: cluster-A, cluster-B, and cluster-C. Cluster-A primarily comprised proline, water-use efficiency (WUE), DI₀/RC, and F₀, as revealed by the hierarchical clustering and heatmap. All the cluster-A variables showed minimal values at control condition but increased in saline condition, and it gradually increased when treated with SA or SA and Put. On the other hand, cluster-B represents some morphological parameters such as SL, PH, RDW, RFW, SDW, SFW, GR and osmolytes such as AsA, TSC and antioxidative enzyme such as CAT and GPX. All cluster-B variables showed maximum values at control condition treated with SA or SA and Put. This result indicates that Application of Put and SA increased the value of all parameters of cluster B considerably. Another group like cluster-C represents some photosynthetic attributes (A, E and gs) and some other parameters such as SPAD, Fv/Fm, Fm, Pi_Abs, H₂O₂, MDA and Root length. All the variables followed more or less similar pattern as like cluster B.

Principal component analysis (PCA) was performed to explore the relationships between different parameters and treatment groups (Figure 5). The first two principal components, PC1 and PC2, together explained 69.78% of the total variability. The control and salinity treatments exhibited opposite trends, and the PCA plot revealed the presence of two distinct groups across all treatment conditions. From the figure, the treatments salinity and salinity with Put and SA make one group, while control and control with Put and SA make up the other groups. All the variables of these particular groups have close association with each other. SPAD, F0, Fm, WUE, DIo/RC, SOD, Proline, and sucrose have an intimate relationship with salinity and salinity with Put and SA, where the rest of the variables are intimately associated with control and control with Put and SA.

The schematic diagram (Figure 6) depicts how salinity negatively impacts plants by lowering chlorophyll (Chl) content and leaf relative water content (LRWC), which in turn reduces photosynthetic rates and photosynthetic efficiency (Fv/Fm). Salinity stress increases the production of superoxide radical (O₂^•−), which in turn elevates hydrogen peroxide (H₂O₂) levels. This stress condition results in reduced levels of ascorbic acid (AsA) and total soluble carbohydrates (TSC), along with decreased activity of antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT), thereby further increasing H₂O₂ accumulation. The elevated H₂O₂ promotes excessive MDA production, which damages cell membranes and enhances lipid peroxidation.

Conversely, exogenous application of Put and SA mitigates oxidative damage and restores growth in salinity-stressed seedlings. These treatments enhance levels of proline (Pro), TSC, and sucrose, and stimulate the activity of antioxidant enzymes such as SOD, CAT, and guaiacol peroxidase (GPX), thereby maintaining reactive oxygen species (ROS) homeostasis. By reducing lipid peroxidation and preserving membrane integrity, Put and SA increase Chl content, LRWC, and Fv/Fm, ultimately improving photosynthesis and biomass accumulation. Although the study provides valuable insights into the protective roles of putrescine and salicylic acid under salinity stress, its scope was limited to a single cultivar, one salinity level, and short-term exposure; therefore, further multi-cultivar and long-term studies are needed to confirm these responses under field conditions.

4. Conclusions

Exogenous application of Put and SA effectively enhanced salt tolerance in sugar beet seedlings. Put primarily improved growth rate, shoot and root biomass, leaf relative water content, respiration, and sucrose accumulation, while SA enhanced root length, photosynthetic activity, water-use efficiency, and proline content. Their enhanced combined effect increased growth parameters, leaf relative water content, stomatal conductance, and PSII efficiency, while reducing H₂O₂ and MDA levels as a consequence of boosting antioxidant defenses. Foliar treatment with Put and SA also restored chlorophyll content and fluorescence under salinity, sustaining morpho-physiological functions. These results highlight Put and SA as promising tools for mitigating salt-induced damage, improving crop performance, and reducing yield losses in saline-prone areas. Further genetic and molecular studies are recommended to unravel the precise mechanisms underlying their protective effects. In the long term, integrating such physiological and biochemical approaches with breeding or biotechnological strategies could contribute to developing salt-resilient sugar beet cultivars and enhance sustainable crop production under increasing soil salinity stress.