Effects of Salt Stress on the Physiology and Biochemistry of Six Poplar Germplasms and Evaluation of Their Salt Tolerance

Abstract

Global soil salinization is accelerating. As the dominant fast-growing plantation genus, Populus spp. largely determines the success of coastal and inland saline-land restoration and the associated carbon-sequestration benefits. Yet most current studies rely on single indicators and lack a multidimensional physiological framework for ranking salt tolerance. Here, six elite poplar cultivars were exposed to 0% (CK), 0.2% (ST1), 0.3% (ST2) and 0.4% NaCl (ST3) for 30 d. We quantified membrane-lipid peroxidation, photosynthetic performance, osmotic adjustment and antioxidant enzymatics, then integrated the data with a principal-component–membership-function model. With increasing NaCl, MDA and REC either rose continuously or peaked slightly below ST3. ‘YX2’ reached the highest MDA (71.3 µmol g⁻¹ FW) and REC (80.3%) under ST2. Pn and SPAD declined overall, but ‘YX3’ retained the greatest photosynthetic stability (6.1 µmol m⁻² s⁻¹ at ST3). Osmolytes accumulated differentially: soluble sugar in ‘PZ2’ rose 52% above CK at ST3; soluble protein in ‘YX2’ peaked at 12.7 mg g⁻¹ FW; proline exceeded 110 µg g⁻¹ FW in ‘YX2’, ‘PZ1’ and ‘PZ2’. Antioxidant enzymes were up-regulated with stress; ‘YX3’ CAT peaked at 69.7 U g⁻¹ FW under ST2, while SOD and POD remained highly active. Correlation analysis revealed that photosynthetic decline is tightly linked to membrane oxidative damage, while the coordinated enhancement of antioxidant enzymes and concurrent accumulation of osmolytes form a synergistic protection mechanism. PCA showed that PC1 (57.1%) integrated photosynthetic capacity, membrane integrity and antioxidant synergy, whereas PC2 (14.3%) represented osmotic and enzymatic protection. The combined D-value ranked cultivars as ‘YX2’ > ‘YX3’ > ‘PZ2’ > ‘PZ1’ > ‘ZX1’ > ‘YX1’. This multi-trait platform provides both a theoretical reference and a germplasm basis for saline-site afforestation and salt-tolerant poplar breeding.

1. Introduction

Soil salinization has become a global challenge that threatens the sustainable development of agriculture and forestry, affecting approximately 831 million hectares of soil resources worldwide [1]. Salt stress directly impairs plant growth and development, ultimately reducing crop yields [2], with annual economic losses in agriculture estimated as high as 27.3 billion US dollars [3]. The combined effects of climate warming, rising sea levels, and prolonged irrigation with highly mineralized groundwater exacerbate salt accumulation in surface soils, disrupting natural soil ecosystems [4]. Projections indicate that over 50% of the world’s arable land could become saline by 2050 [5]. Consequently, developing salt-tolerant plants has emerged as one of the most pressing ecological challenges requiring urgent solutions.

Typically, regional soil samples with electrical conductivity (EC) exceeding 4 dS m⁻¹ and exchangeable sodium percentage (ESP) above 15% are defined as saline-alkali soils [6]. High concentrations of Na⁺ and Cl⁻ in saline-alkali soils primarily hinder plant water and nutrient uptake [7]. Furthermore, persistent osmotic stress induces ionic imbalances within plant tissues, leading to ion toxicity, nutrient deficiencies, oxidative stress, altered cell membrane permeability, metabolic disruption, and accumulation of toxic substances [8]. To counteract salt stress, plants have evolved sophisticated mechanisms, including regulation of osmotic modulators and activation of antioxidant enzyme systems [9]. Understanding these physiological responses to salt stress provides essential insights for enhancing crop salt tolerance. However, plants exhibit varying capacities for tolerating high salinity, typically categorized into four groups: salt-sensitive, moderately salt-sensitive, moderately salt-tolerant, and salt-tolerant [10]. Salt-tolerant plants are considered valuable resources for identifying key loci and natural variants crucial to plant salt tolerance [8]. Comprehensive evaluation of salt stress performance across varieties using membership functions enables the identification and quantification of salt tolerance [11]. Therefore, clarifying physiological differences and establishing quantifiable evaluation systems are prerequisites for mining salt-tolerant germplasm and developing salt-tolerant cultivars.

Populus spp., as a flagship tree species for fast-growing timber and ecological protection in temperate regions, is urgently needed for vegetation restoration in saline-alkali soils and coastal shelterbelt construction. However, the varying salt tolerance among currently cultivated varieties severely limits their application scope. Current research on poplar salt tolerance lacks systematic comparisons across multiple genotypes, gradients, and indicators. This study utilized six major cultivated poplar varieties provided by the Henan Academy of Forestry Sciences. Four stress gradients were established: 0% (CK), 0.2% (ST1), 0.3% (ST2), and 0.4% NaCl (ST3). We systematically measured membrane stability, lipid peroxidation levels, photosynthetic efficiency, osmotic regulator content, and antioxidant enzyme activity across all varieties under these four NaCl concentrations. Correlation networks were then analyzed to reveal coupling relationships among these indicators under salt gradients. Principal component analysis (PCA) was subsequently employed to extract key salt-tolerance factors. Combined with the weighted membership function method, a comprehensive salt-tolerance evaluation model (D-value) was constructed to precisely quantify salt-tolerance thresholds across varieties and screen for extremely salt-tolerant germplasm. The findings aim to elucidate the patterns of salt tolerance variation among poplar germplasm, providing physiological basis and core materials for salt-tolerant poplar breeding, while offering methodological guidance for comprehensive salt tolerance evaluation in other woody plants.

2. Results

2.1. Changes in MDA and REC Content in Different Poplar Varieties Under Salt Stress

Salt stress induced a consistent rise in leaf MDA content across all six poplar cultivars, with concentrations peaking at the ST2 level before plateauing or declining slightly at ST3 (Figure 1a). At ST2, ‘YX2’ accumulated the highest MDA (71.3 µmol·g⁻¹ FW), exceeding the values recorded for ‘PZ1’, ‘YX1’ and ‘PZ2’ (54.9, 47.4 and 45.1 µmol·g⁻¹ FW, respectively), which did not differ significantly from one another. The lowest MDA concentrations were observed in ‘YX3’ and ‘ZX1’ (46.3 and 43.3 µmol·g⁻¹ FW). At ST3, MDA remained elevated in ‘YX2’ (76.9 µmol·g⁻¹ FW), whereas ‘PZ1’ exhibited a marked reduction to 39.5 µmol·g⁻¹ FW.

Relative electrolyte conductivity (REC) increased monotonically with salinity, mirroring the MDA trend (Figure 1b). Under control conditions, REC ranged from 23.1% to 29.7% without significant inter-cultivar differences. Exposure to ST1 triggered rapid increases; the largest increments were recorded for ‘PZ1’ and ‘ZX1’ (52.5% and 50.3%, respectively), while ‘YX2’ showed a more modest rise (45.1%). At ST2, ‘PZ1’ attained 80.3%, significantly exceeding the values for ‘YX1’ and ‘YX3’ (72.0% and 71.2%); ‘PZ2’, ‘ZX1’ and ‘YX2’ reached 63.7%, 58.5% and 56.3%, respectively. After ST3, REC exceeded 80% in all cultivars. ‘PZ1’, ‘YX1’ and ‘PZ2’ maintained the highest values (>86%), whereas ‘YX3’, ‘ZX1’ and ‘YX2’ were slightly lower (>82%), indicating a convergence of membrane injury at the highest salt load.

Figure 1. Changes in (a) malondialdehyde (MDA) content and (b) relative electrical conductivity (REC) of six poplar varieties under different NaCl treatments. The values are presented as the mean ± SE values of three independent biological replicates per treatment. Note: Different uppercase letters (A, B, C, D) indicate significant differences among varieties within the same treatment, whereas different lowercase letters (a, b, c, d) indicate significant differences among treatments within the same variety (p < 0.05) according to ANOVA followed by Duncan’s multiple range test. FW indicates fresh weight. Same notation as in Figure 2, Figure 3 and Figure 4.

Figure 1. Changes in (a) malondialdehyde (MDA) content and (b) relative electrical conductivity (REC) of six poplar varieties under different NaCl treatments. The values are presented as the mean ± SE values of three independent biological replicates per treatment. Note: Different uppercase letters (A, B, C, D) indicate significant differences among varieties within the same treatment, whereas different lowercase letters (a, b, c, d) indicate significant differences among treatments within the same variety (p < 0.05) according to ANOVA followed by Duncan’s multiple range test. FW indicates fresh weight. Same notation as in Figure 2, Figure 3 and Figure 4.

Figure 2. Changes in (a) net photosynthetic rate (Pn) content and (b) SPAD value of six poplar varieties under different NaCl treatments.

Figure 2. Changes in (a) net photosynthetic rate (Pn) content and (b) SPAD value of six poplar varieties under different NaCl treatments.

Figure 3. Changes in (a) soluble sugar (SS), (b) soluble protein (SP), and (c) proline (Pro) of six poplar varieties under different NaCl treatments.

Figure 3. Changes in (a) soluble sugar (SS), (b) soluble protein (SP), and (c) proline (Pro) of six poplar varieties under different NaCl treatments.

Figure 4. Changes in (a) catalase (CAT), (b) superoxide dismutase (SOD), and (c) peroxidase (POD) of six poplar varieties under different NaCl treatments.

Figure 4. Changes in (a) catalase (CAT), (b) superoxide dismutase (SOD), and (c) peroxidase (POD) of six poplar varieties under different NaCl treatments.

2.2. Changes in Pn and SPAD in Different Poplar Varieties Under Salt Stress

Salt stress suppressed net photosynthetic rate (Pn) in all six poplar cultivars in a concentration-dependent manner (Figure 2a). Under control conditions, ‘PZ2’ recorded the highest Pn (15.4 μmol·m⁻²·s⁻¹), significantly exceeding the other genotypes, whereas ‘PZ1’ exhibited the lowest value (10.7 μmol·m⁻²·s⁻¹). Exposure to ST1 reduced Pn universally, but the amplitude varied: ‘YX3’ displayed the smallest decline (−17.7%, 12.6 μmol·m⁻²·s⁻¹), maintaining a significantly higher rate than all others. ‘PZ2’ and ‘YX2’ followed (9.5 and 11.4 μmol·m⁻²·s⁻¹), while ‘PZ1’, ‘YX1’ and ‘ZX1’ dropped by 20–30% to 7.8–10.1 μmol·m⁻²·s⁻¹. At 0.3% NaCl (ST2), ‘YX3’ retained the highest Pn (13.5 μmol·m⁻²·s⁻¹), slightly above its ST1 value, whereas ‘PZ2’ remained stable at 12.1 μmol·m⁻²·s⁻¹; in contrast, ‘PZ1’ and ‘YX1’ fell further to 7.5 and 6.6 μmol·m⁻²·s⁻¹, respectively. After ST3, all genotypes registered Pn < 8.1 μmol·m⁻²·s⁻¹; ‘PZ2’ declined to the lowest value (3.9 μmol·m⁻²·s⁻¹), while ‘YX3’ maintained the relatively highest rate (6.1 μmol·m⁻²·s⁻¹), followed by ‘ZX1’ (8.0 μmol·m⁻²·s⁻¹).

SPAD values, an index of leaf chlorophyll content, decreased with increasing salinity, but the trajectory differed among cultivars (Figure 2b). Under control conditions, ‘PZ1’ exhibited the highest SPAD (50.4), followed by ‘YX2’ and ‘ZX1’ (48.4 and 45.5), whereas ‘PZ2’ and ‘YX3’ fell below 40. After ST1, SPAD in ‘PZ1’, ‘YX3’ and ‘ZX1’ dropped sharply by 25–30%, while ‘YX1’ remained unchanged (38.7), indicating greater chlorophyll stability. At ST2, ‘YX3’ and ‘PZ2’ declined to 27.1 and 33.2, respectively, representing the largest reductions; conversely, ‘PZ1’ rebounded to 41.5, possibly reflecting concentration effects or active chlorophyll synthesis, whereas ‘YX2’ retained a relatively high value (43.8). Under ST3, all genotypes registered SPAD < 35; ‘PZ2’ recorded the minimum (24.3) and ‘YX2’ the maximum (34.1). Values for ‘YX3’, ‘ZX1’ and ‘PZ1’ clustered between 28 and 30 without significant differences.

2.3. Changes in Osmolytes Content in Different Poplar Varieties Under Salt Stress

Salt stress consistently elevated leaf soluble sugar concentrations, although accumulation strategies differed markedly among the six poplar cultivars (Figure 3a). Under control conditions, ‘YX2’ contained the most soluble sugars (11.6 mg·g⁻¹ FW), whereas ‘PZ1’ contained the least (7.7 mg·g⁻¹ FW). Exposure to ST1 did not elicit a significant increase; several cultivars even registered minor declines. At ST2, ‘PZ2’ accumulated sharply to 12.5 mg·g⁻¹ FW, significantly outperforming all other genotypes; ‘ZX1’ and ‘YX3’ rose to 11.1 and 10.8 mg·g⁻¹ FW, respectively, while ‘PZ1’, ‘YX1’ and ‘YX2’ remained essentially unchanged. At ST3, soluble sugars continued to accrue. ‘PZ2’ reached the highest concentration (14.2 mg·g⁻¹ FW, +51.8% relative to the control), followed by ‘PZ1’ and ‘YX3’ (11.6 and 12.8 mg·g⁻¹ FW, respectively, both approximately +50%). In contrast, ‘YX2’ showed the smallest increment, attaining only 10.4 mg·g⁻¹ FW under ST3.

Soluble-protein content also increased with salinity, but the kinetic profiles diverged among cultivars (Figure 3b). Controls ranged from 5.5 mg·g⁻¹ FW in ‘YX1’ to 10.9 mg·g⁻¹ FW in ‘PZ1’. At ST1, ‘YX2’ essentially doubled its protein content (10.5 mg·g⁻¹ FW), becoming the highest-ranking genotype, whereas ‘YX1’ rose significantly to 8.3 mg·g⁻¹ FW; concurrently, ‘PZ1’ and ‘ZX1’ declined to 5.2–5.5 mg·g⁻¹ FW. During ST2, protein levels continued to rise. ‘YX2’ and ‘YX1’ peaked at 11.1 and 10.7 mg·g⁻¹ FW, respectively—significantly above the remaining genotypes—while ‘PZ2’ also increased markedly to 9.3 mg·g⁻¹ FW. Under ST3, ‘YX2’ maintained its lead (12.7 mg·g⁻¹ FW), and ‘PZ1’ registered the lowest content (8.5 mg·g⁻¹ FW).

Proline exhibited a near-monotonic increase with salt concentration, although absolute concentrations varied significantly among cultivars (Figure 3c). In the controls, ‘PZ1’ contained the most proline (82.3 µg·g⁻¹ FW) and ‘YX2’ the least (42.9 µg·g⁻¹ FW). ST1 evoked only modest increments, with ‘YX3’ and ‘YX2’ achieving the largest gains (both approximately 77.6 µg·g⁻¹ FW). At ST2, ‘YX2’ accumulated proline sharply, reaching 134.4 µg·g⁻¹ FW—the highest value recorded for any genotype at any treatment—whereas ‘PZ1’, ‘PZ2’ and ‘YX3’ rose to 88.7, 81.6 and 89.5 µg·g⁻¹ FW, respectively; smaller increases were observed in ‘YX1’ and ‘ZX1’. After ST3, proline continued to accumulate. ‘PZ1’, ‘PZ2’ and ‘YX3’ all exceeded 100 µg·g⁻¹ FW (112.9, 109.5 and 103.7 µg·g⁻¹ FW, respectively), while ‘YX2’ and ‘ZX1’ approached 110 µg·g⁻¹ FW. ‘YX1’ retained the lowest concentration (73.1 µg·g⁻¹ FW).

It is worth noting that comparative penetration strategies have emerged among six poplar germplasms. ‘PZ2’, ‘PZ1’ and ‘YX3’ relied predominantly on soluble-sugar build-up, whereas ‘YX2’ invested chiefly in proline, illustrating two functionally equivalent but chemically distinct routes for osmotic adjustment under salt stress.

2.4. Changes in Antioxidant Enzyme Activity in Different Poplar Varieties Under Salt Stress

Salt stress elicited cultivar-specific patterns of catalase (CAT) activity, with most genotypes showing an initial rise followed by a decline at the highest salinity, whereas others maintained an upward trajectory (Figure 4a). Under control conditions, ‘ZX1’ possessed the highest basal CAT activity (40.0 U·g⁻¹ FW), while the remaining five cultivars ranged from 3.3 to 10.7 U·g⁻¹ FW. After ST1, ‘YX3’ and ‘YX2’ surged to 35.6 and 26.0 U·g⁻¹ FW, respectively, representing 3- to 6-fold increases, whereas ‘PZ1’, ‘PZ2’ and ‘YX1’ registered only modest gains. At ST2, ‘YX3’ peaked at 69.7 U·g⁻¹ FW, significantly above all others; ‘PZ1’ and ‘YX1’ rose to 43.0 and 33.0 U·g⁻¹ FW, respectively, while ‘PZ2’ increased moderately to 14.3 U·g⁻¹ FW. Exposure to ST3 caused a sharp drop in ‘YX3’ (16.7 U·g⁻¹ FW), but ‘PZ2’ and ‘ZX1’ continued to climb to 40.0 and 61.7 U·g⁻¹ FW, respectively, and ‘YX2’ reached 55.7 U·g⁻¹ FW.

Superoxide dismutase (SOD) activity increased with salinity, although the rate of rise and the concentration at which maximum activity was achieved differed among cultivars (Figure 4b). Controls showed ‘YX3’ with the highest activity (54.7 U·g⁻¹ FW) and ‘ZX1’ with the lowest (14.3 U·g⁻¹ FW). Under ST1, ‘YX2’ displayed the greatest increment (67.9 U·g⁻¹ FW), while ‘PZ1’, ‘PZ2’, ‘YX1’ and ‘ZX1’ all rose significantly to 46–52 U·g⁻¹ FW; conversely, ‘YX3’ declined slightly to 37.1 U·g⁻¹ FW. At ST2, all cultivars except ‘PZ2’ exceeded 71.8 U·g⁻¹ FW, with ‘PZ1’ and ‘YX3’ achieving the highest values (78.9 U·g⁻¹ FW), although inter-cultivar differences were not statistically significant. After ST3, ‘PZ2’ and ‘YX3’ maintained upward trends and peaked at 79.0 and 79.1 U·g⁻¹ FW, respectively, whereas ‘YX2’ and ‘ZX1’ declined modestly to 77.0 and 56.5 U·g⁻¹ FW, and ‘PZ1’ and ‘YX1’ fell to 54.5 and 59.9 U·g⁻¹ FW.

Peroxidase (POD) activity rose continuously with increasing salt concentration, and inter-cultivar differences were highly significant at every treatment level (Figure 4c). Under control conditions, ‘YX1’ exhibited the highest POD activity (205.8 U·g⁻¹ FW), while ‘PZ1’ showed the lowest (96 U·g⁻¹ FW). ST1 elevated POD activity by 1–1.5-fold in all genotypes, with ‘PZ2’, ‘YX3’ and ‘PZ1’ registering the most pronounced increases (260–285 U·g⁻¹ FW). At ST2, ‘YX3’ and ‘YX1’ surged to 472.5 and 438.3 U·g⁻¹ FW, respectively, significantly higher than the others; ‘YX2’ also rose sharply to 448.3 U·g⁻¹ FW, while ‘PZ1’, ‘PZ2’ and ‘ZX1’ ranged from 215.8 to 324.2 U·g⁻¹ FW. Under ST3, ‘YX3’ continued to climb and maintained the highest activity (521.7 U·g⁻¹ FW), followed by ‘YX2’ (478.3 U·g⁻¹ FW) and ‘PZ2’ (416.7 U·g⁻¹ FW), whereas ‘PZ1’, ‘YX1’ and ‘ZX1’ clustered between 311.7 and 349.2 U·g⁻¹ FW.

2.5. Comparative Analysis of Comprehensive Salt Tolerance Among Different Poplar Varieties

2.5.1. Correlation Analysis of Various Indicators Under Different Salt Stress Conditions

Pearson correlation analysis of the ten physiological traits is shown in Figure 5. Pn was positively correlated with SPAD (p < 0.01) and negatively with REC, indicating that chlorophyll loss and membrane injury are primary constraints on photosynthesis. MDA content was positively correlated with REC, jointly reflecting the extent of lipid peroxidation. Within the antioxidant module, SOD activity was positively correlated with both POD and CAT, demonstrating synergistic ROS detoxification. Among osmolytes, SS, SP and Pro were positively inter-correlated, implying coordinated accumulation to counter osmotic stress. Overall, declining photosynthetic capacity under salt stress is closely linked to membrane oxidation, whereas enhanced antioxidant defense and osmotic adjustment act in concert to protect leaf function.

2.5.2. Principal Component Analysis of Indicators

Principal component analysis (PCA) was performed on various indicators across six varieties (Figure 6). Two main principal components (PC1 and PC2) cumulatively explained 71.4% of phenotypic variance across the first two principal components. PC1 explained 57.1% of total variance, primarily comprising SPAD, Pn, MDA, REC, SOD, and POD, reflecting photosynthetic maintenance capacity, membrane damage levels, and antioxidant capacity. PC2 explained 14.3% of the total variance, primarily influenced by SPAD, Pn, CAT, Pro, and SS, indicating the maintenance of photosynthetic capacity through antioxidant pathways and osmotic regulatory substances.

2.5.3. Comprehensive Evaluation of Salt Tolerance Among the Six Varieties of Populus

After converting the principal component scores (F1–F3) of each variety into membership function values (μ1–μ3), these were equally weighted and combined to derive the comprehensive salt tolerance index (D-value) (

Table 1. Ranking of comprehensive salt tolerance scores among the six varieties of Populus.

Variety Name	Principal Components			Membership Function			D-Value	Sort
	F1	F2	F3	μ1	μ2	μ3
‘PZ1’	−1.11	1.33	0.34	0.00	1.00	0.87	0.36	4
‘PZ2’	−0.19	−0.68	0.58	0.37	0.09	0.97	0.38	3
‘YX1’	−0.85	−0.49	0.54	0.11	0.17	0.95	0.23	6
‘YX2’	1.37	1.23	−0.19	1.00	0.96	0.67	0.95	1
‘YX3’	1.04	−0.87	0.67	0.87	0.00	1.00	0.67	2
‘ZX1’	−0.26	−0.52	−1.94	0.34	0.16	0.00	0.25	5

Notes: F1, F2, and F3 are principal component values corresponding to each trait; μ1, μ2, and μ3 are membership function values corresponding to each variety.

). Results showed that ‘YX2’ exhibited the highest D-value (0.95), significantly outperforming other genotypes and ranking first in salt tolerance; ‘YX3’ followed (0.67). ‘PZ2’ and ‘PZ1’ ranked third and fourth (0.38 and 0.36), with D-value in the moderate range. ‘ZX1’ and ‘YX1’ ranked fifth and sixth (0.25 and 0.23), respectively, with the lowest D-value. The overall ranking further confirmed that the evaluation system combining principal component analysis and membership functions can stably and quantitatively define the salt tolerance levels of different poplar varieties.

3. Discussion

3.1. Effects of Salt Stress on Cell Membrane Stability and Photosynthesis in Poplar

Under salt stress, plants suffer both primary injuries—plasma-membrane disruption that drives ion leakage and osmotic perturbation that stalls metabolism—and secondary injuries dominated by oxidative damage [12]. Reactive oxygen species (ROS) oxidise polyunsaturated membrane lipids, producing malondialdehyde (MDA); its concentration is therefore widely used as a proxy for lipid peroxidation [13,14]. Across six Populus genotypes we observed a pronounced, albeit biphasic, accumulation of MDA that rose with external NaCl but tapered at the highest dosage, indicating that the antioxidant machinery was ultimately overwhelmed. Parallelly, electrolyte leakage—manifest as increased relative electrical conductivity (REC)—escalated monotonically with stress intensity, corroborating the progressive loss of membrane integrity [15]. Since leaf Na⁺ and K⁺ concentrations were not measured here, REC and MDA serve as integrative indicators of salt stress damage. Future ionomics studies should distinguish osmotic from ion-specific effects. In addition, quantifying root morphology and leaf water potential across the same salt gradients will help link the shoot biochemical responses to whole-plant water status.

Carbon gain at the leaf level sets the upper limit for whole-plant productivity; net photosynthetic rate (Pn) therefore provides a direct read-out of carbon assimilation per unit leaf area [16], whereas SPAD chlorophyll index offers an instantaneous estimate of light-harvesting capacity and Photosystem II photochemical potential [17]. Previous work across a range of abiotic stresses—including heavy metals, drought and low temperature—has documented a synchronous decline in Pn and SPAD as stress intensifies [18]. Our data mirror this pattern: salt stress suppressed both Pn and SPAD in a dose-dependent manner, underscoring the impairment of photosynthetic machinery in salt-challenged poplar leaves.

3.2. Effects of Salt Stress on Osmolyte Accumulation in Poplar

When plants encounter adverse conditions, they activate inherent osmotic adjustment mechanisms to re-establish cellular water balance [19]. Under salt stress, soluble sugars and soluble proteins function similarly: both reduce intracellular water potential through their own accumulation, thereby buffering the osmotic pressure difference across the membrane and stabilizing the cell membrane [20]. Measurements of six poplar clones in this study showed that both solutes generally accumulated, but the allocation strategies differed significantly among varieties: ‘PZ2’, ‘PZ1’, and ‘YX3’ preferentially expanded the soluble sugar pool, whereas ‘YX2’, ‘YX3’, and ‘ZX1’ exhibited stronger protein synthesis or protection capacity under severe stress. This divergence pattern is consistent with reports in Secale cereale [21] and subtropical coastal tree species [22].

In addition, owing to its low molecular weight and strong hydrophilicity, proline can simultaneously adjust water potential and protect the conformation of proteins and nucleic acids, thereby alleviating cellular damage caused by salt stress [23]. Our data revealed that leaf proline content increased monotonically with NaCl concentration, with significant differences among varieties, indicating that poplar relies on proline accumulation to resist dehydration injury, a conclusion in line with previous findings [24].

3.3. Effects of Salt Stress on Antioxidant Enzyme Activities in Poplar

Among the secondary salt injuries triggered by salinity, ROS-driven oxidative stress is the most frequent and aggressive assault faced by plant cells [25]. To keep ROS below the toxicity threshold, poplar activates an enzymatic detoxification cascade: superoxide anions (O₂⁻) are rapidly dismutated by SOD to H₂O₂ and O₂, preventing direct attack on membrane lipids [26]. Subsequently, high-level H₂O₂ is mainly scavenged by CAT and APX, whereas lower concentrations are fine-tuned by POD [27]. Across six Populus genotypes we observed two temporal patterns for SOD, CAT and POD activities—either a “rise-then-fall” response or a sustained up-regulation—yet the amplitude and the timing of peak activity differed markedly among cultivars. This indicates that poplar mitigates salt stress by enhancing antioxidant flux to match ROS production. Notably, ‘YX3’, ‘YX2’ and ‘YX1’ retained exceptionally high POD capacity even under severe NaCl, efficiently draining the H₂O₂ pool generated by SOD and thereby complementing CAT; in contrast, ‘PZ1’ and ‘ZX1’ mounted a modest POD response, presumably relying on the SOD–CAT axis for bulk H₂O₂ removal. These divergent routing preferences within the SOD-H₂O₂-CAT/POD module constitute heritable variation that can be exploited to breed clones with superior antioxidant flux control under salt stress [28]. Significantly, in YX2, the MDA plateau at ST2 coincided with a transient CAT peak (Figure 4a), indicating a short-term ROS-scavenging surge that curtailed further lipid peroxidation; however, REC continued to rise through ST3 because Na⁺-induced micro-pores persisted even after ROS declined, reconciling the divergent kinetics of MDA and REC.

3.4. Screening of Salt-Tolerant Poplar Varieties in Response to Salt Stress

The strong negative Pn–REC correlation, together with the tight positive SOD–POD–CAT triangle, indicates that photosynthetic performance is controlled by membrane integrity, which is maintained by efficient ROS scavenging. Because PC1—accounting for 57% of the variance—integrates these traits, the combined response of membrane stability and antioxidant activity, rather than osmotic adjustment alone, represents the main mechanism underlying salinity tolerance in poplar. Both ‘YX2’ and ‘YX3’ loaded positively on PC1, yet followed contrasting strategies: ‘YX2’ combined high proline, superior POD/CAT activity and elevated REC—an energy-intensive protection mode—whereas ‘YX3’ maintained moderate antioxidants but preserved higher photosynthetic capacity, a low-damage/high-homeostasis tactic that secured second rank despite lower absolute osmolyte levels. Diverse traits and statistical frameworks have become the norm for screening stress-tolerant genotypes. Integrating multiple salt-stress indicators through membership-function analysis is now standard practice in soybean [29], sorghum [30] and rice [31]. Here, a combined ranking based on PCA scores and membership-function values ordered the six poplar clones as: ’YX2’ > ’YX3’ > ’PZ2’ > ’ZX1’ > ’YX1’ > ’PZ1’. ‘YX2’ proline-driven osmotic adjustment with sustained POD/CAT activity, conserving PSII efficiency (Pn 6.1 vs. 3.9 µmol·m⁻²·s⁻¹ in YX1 at ST3), unable to sustain antioxidants, undergoes run-away lipid peroxidation (MDA 76.9 vs. 45.1 µmol·g⁻¹ FW) and earlier photosynthetic collapse. High-antioxidant (YX3) or high-osmolyte (PZ2) strategies both enhance tolerance, but the balanced “osmotic + antioxidant + ion homeostasis” suite expressed by YX2 yielded the highest D-value, underscoring that functional redundancy outperforms single-trait extremes.‘YX2’ and ‘YX3’ maintained a “high-protection, low-damage” phenotype even under severe salinity and should be prioritized as tolerant parents in breeding programs. ‘ZX1’ and ‘PZ2’ occupy an intermediate niche and are suitable bridge materials for incremental improvement, whereas ‘YX1’ and ‘PZ1’ reached their tolerance threshold early and should either be excluded from saline-site afforestation or deployed only on marginal soils. Future work should involve long-term field trials to assess the salt tolerance and field performance of the selected germplasms. Due to limitations in greenhouse space and plant materials, this study used three biological replicates per cultivar in a completely randomized design; future field trials will apply a randomized complete block design with more replicates.

4. Materials and Methods

4.1. Experimental Materials and Site

The six Populus varieties tested— ‘74/76’ (‘PZ1’), ‘Zhonglin2025’ (‘PZ2’), ‘YUXIONG 1’ (‘YX1’), ‘YUXIONG 2’ (‘YX2’), ‘YUXIONG 3’ (‘YX3’), and ‘ZHOUXIONG 1’ (‘ZX1’)—were all sourced as one-year-old rooted cuttings. In May 2022, well-developed cuttings exhibiting uniform height and robust root systems were transplanted into plastic pots. These pots measured 21.5 cm in top diameter, 16.5 cm in bottom diameter, and 22 cm in height, with drainage holes at the base. The growing medium consisted of a mixture of yellow loam, yellow sand, humus, vermiculite, and perlite in a ratio of 5:1:2:1:1. Each pot received 6 kg of substrate and contained two cuttings. The potted seedlings were placed on nursery beds in a greenhouse and managed under consistent growing conditions. After one month of cultivation, salt stress experiments were conducted.

The experiment was carried out in the greenhouse of the Henan Academy of Forestry Sciences (Zhengzhou, Henan, China; 34°46′ N, 113°40′ E). The site is situated in the transition zone between the North subtropical and mid-subtropical monsoon climate, with an annual mean temperature of 14.7 °C, annual precipitation of 650 mm and 1 985 h of sunshine. Throughout the 2022 growing season (April–October) the greenhouse was maintained at 26–30 °C/19–23 °C (day/night), relative humidity 60–70%, and ambient photosynthetically active radiation (PAR) 950 ± 50 µmol m⁻² s⁻¹ during the 12 h photoperiod.

4.2. Experimental Design

Based on the dry soil mass per pot, target salt concentrations of 0, 2, 3, 4 g NaCl kg⁻¹ dry soil (equivalent to 0, 0.2, 0.3, 0.4% w/w) were established for CK, ST1, ST2, ST3, respectively. To impose the salt stress, the required NaCl was dissolved in 200 mL deionized water and applied in two equal splits on days 0 and 15; CK received the same volume of salt-free water on the same schedule. Immediately after each application, pots were irrigated to field capacity and placed in plastic trays; all leachate was returned within 24 h to prevent salt loss. Soil moisture was kept at 70% field capacity by daily weighing and replenishing with deionized water. The experiment ran for 30 days with no further salt additions. Each treatment comprised 20 seedlings arranged in three replicates.

Net photosynthetic rate and SPAD value was recorded on the 4th–6th leaves using a handheld chlorophyll meter at 30 d post-treatment. Meanwhile, fully expanded leaves were collected. After a brief rinse in ultrapure water and surface drying with lint-free paper, samples were immediately plunged into liquid nitrogen and transferred to −80 °C storage pending downstream analyses.

4.3. Experimental Indicators and Measurement Methods

Malondialdehyde (MDA): The content of MDA was determined by the thiobarbituric acid method [32].

Leaf relative electrolyte leakage (REC): REC was determined with a DDS-310 conductivity meter following a slightly modified Dexter method [33]. 0.2 g fresh leaves were immersed in 25 mL pure water, incubated at 25 °C on an orbital shaker for 6 h, and the initial conductivity (R₁) recorded. The same solution was then boiled for 30 min, cooled to 25 °C, and the final conductivity (R₂) measured. REC was calculated as (R₁/R₂) × 100%.

Net photosynthetic rate (Pn): Gas-exchange parameters were measured with a portable photosynthesis system (Hansatech Instruments, Pentney, UK) to obtain Pn.

Relative chlorophyll content (SPAD value): SPAD value was recorded using a handheld chlorophyll meter (SPAD-502; Konica Minolta, Osaka, Japan); three readings per leaf were averaged.

Osmolytes: Soluble sugar (Ss) was determined by the anthrone colorimetric assay [34]; soluble protein (Sp) by the Coomassie Brilliant Blue G-250 method [35]; and free proline (Pro) according to Bates et al. [36].

Antioxidant enzymes: Activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were measured following the standardized protocols of Ali et al. [37]. SOD activity was assessed using the NBT photoreduction method while POD activity was determined using the guaiacol colorimetric method. CAT activity was measured using the thiobarbituric acid colorimetric method.

4.4. Comprehensive Evaluation of Salt Tolerance

Principal component analysis (PCA) was first used to reduce variable dimensionality; the first P components whose cumulative contribution exceeded 85% were retained as integrated indicators. Component scores for each genotype were computed, membership-function standardization was applied to these scores, and a comprehensive index (D-value) was calculated. A higher D-value indicates superior overall salt tolerance and was used as the basis for final cultivar ranking.

4.5. Statistical Analysis

The data were arranged in Microsoft Excel 2018, and data were plotted using Origin 2025b software. Variance analysis, multiple comparisons, principal component analysis, Pearson correlation analysis was assessed by IBM SPSS 27.0 statistical software.

5. Conclusions

Salt stress elicited a genotype-specific cascade of oxidative damage, photosynthetic inhibition and osmolyte accumulation that allowed unambiguous ranking of poplar clones across NaCl regimes. The integrated PCA-membership model revealed that sustained antioxidant coordination and balanced carbon partitioning into osmotica are the cornerstones of superior salinity tolerance, providing a quantitative template for future germplasm evaluation. Our results underscore the need to move beyond single-trait selection and embed multi-physiological fingerprints into breeding pipelines. Looking forward, coupling the identified tolerance index with high-density genotyping and CRISPR-based validation of ROS-scavenging and osmoregulatory genes will accelerate the development of next-generation poplar cultivars tailored to increasingly saline marginal lands.