Integrative multivariate analysis reveals four distinct salinity tolerance strategies in Alstroemeria cultivars

Abstract

Salinity severely constrains the growth and ornamental quality of floricultural crops by disrupting plant morphology, photosynthetic performance, and redox homeostasis. This study investigated cultivar-specific morphophysiological and antioxidant responses of four Alstroemeria hybrida cultivars differing in growth habit (two dwarf: Inca Sweety and Dwarf Red; two tall: Orange Queen and Amatista) under increasing salinity levels (0, 20, 40, and 60 mM NaCl). Salinity induced distinct, cultivar-dependent response patterns across all measured traits. Dwarf cultivars exhibited superior structural stability, maintaining stem elongation, leaf area, and total chlorophyll content under moderate to severe salinity, whereas tall cultivars experienced pronounced reductions in biomass accumulation and pigment integrity. Inca Sweety displayed exceptional physiological homeostasis, characterized by stable chlorophyll indices, phenolic content, antioxidant capacity, and PAL activity, indicating effective prevention of oxidative damage rather than reactive detoxification. In contrast, Amatista showed high growth potential under non-stress conditions but exhibited sharp declines in growth, photosynthetic pigments, and antioxidant performance as salinity intensified. Antioxidant enzyme activities revealed mechanistic differences among cultivars: coordinated peaks of CAT and APX at moderate salinity in Amatista were followed by enzymatic collapse at high stress, while Inca Sweety exhibited delayed CAT induction but sustained activation of APX and GPX, suggesting a finely regulated H₂O₂-scavenging network. Overall, salinity tolerance in Alstroemeria was governed by the coordination, timing, and balance of antioxidant defenses rather than the magnitude of individual enzymatic responses. These findings highlight the importance of integrated redox regulation in determining cultivar performance under saline conditions and provide a mechanistic basis for selecting salt-resilient ornamental cultivars.

Introduction

Ornamental plants represent a major segment of global horticultural production, contributing substantially to international trade and greenhouse-based industries. The global cut flower market was valued at USD 39.08 billion in 2024 and is projected to reach over USD 51 billion by 2030¹. Among commercially important ornamentals, Alstroemeria stands out for its long vase life, vibrant floral colors, and suitability for year-round greenhouse cultivation. Native to South America, Alstroemeria hybrida is widely produced in countries such as the Netherlands, Chile, Colombia, and Ecuador, and its global demand has continued to expand in recent years².

Despite its commercial significance, Alstroemeria is highly sensitive to abiotic stresses, particularly salinity³. Salinity is one of the most widespread environmental constraints limiting the productivity of ornamental plants under greenhouse and soilless culture systems, as it can cause osmotic stress, ion toxicity, and nutrient imbalance even in controlled environments⁴. Saline soils are typically characterized by electrical conductivity (ECₑ) values exceeding 4 dS m⁻¹, corresponding to approximately 40 mM NaCl and an osmotic potential below − 0.2 MPa⁵. High salinity induces both osmotic and ionic stresses, restricting water uptake, disrupting nutrient balance, and generating oxidative stress through excess accumulation of Na⁺ and Cl⁻ ions⁶.

In ornamental species, salinity commonly leads to substantial reductions in visual quality — including leaf tip burn, chlorosis, and necrosis — which directly compromise marketability⁷. Growth-related traits such as plant height, leaf area, and biomass are negatively affected due to osmotic inhibition of cell expansion and ion toxicity⁸. Photosynthetic performance is also impaired, as salinity limits stomatal conductance, accelerates chlorophyll degradation, suppresses RuBisCO activity, and damages thylakoid membrane proteins⁹. Although direct studies on Alstroemeria are limited, in other ornamental species, increased nutrient solution electrical conductivity or saline irrigation has been shown to disrupt ion homeostasis, reduce chlorophyll content, and impair both flowering performance and overall plant vigor^10,11.

Although several studies have addressed salinity effects in individual ornamental species, comparative evaluations among different Alstroemeria cultivars remain limited. Understanding cultivar-specific variability is essential because genotypes may differ substantially in their physiological and morphological responses to salt stress, influencing their suitability for commercial production under suboptimal water quality. Furthermore, integrative multivariate approaches such as hierarchical clustering, correlation networks, heatmap visualization, and principal component analysis (PCA) provide powerful tools to identify response patterns and classify tolerance levels; yet these analyses have rarely been applied to Alstroemeria. Based on these considerations, we hypothesize that Alstroemeria cultivars exhibit distinct and quantifiable physiological and morphological responses to salinity stress, which manifest as differences in growth, photosynthetic efficiency, and aesthetic quality, and that multivariate analyses will reveal clear patterns of cultivar-specific tolerance. Therefore, the present study aims to evaluate the growth, physiological, and aesthetic responses of four Alstroemeria cultivars under varying salinity levels in greenhouse soilless culture, and to integrate these responses through PCA, heatmap, and correlation analyses in order to identify distinct tolerance groups, providing a practical framework for cultivar selection and management under saline conditions.

Materials and methods

Plant material

Alstroemeria plants (Alstroemeria hybrida) (Royal Van Zanten, The Netherlands) propagated from rhizomes were obtained from a commercial greenhouse. Four cultivars were used in this study: two dwarf cultivars, Inca Sweety with white flowers featuring pink centers and black spots, and Dwarf Red with red flowers and black spots; and two tall cultivars, Orange Queen with orange flowers and dark spots, and Amatista with purple flowers and black spots. Representative images of the four cultivars used in the experiment are shown in Fig. 1. Plants were transplanted into pots containing a mixture of perlite and cocopeat at a 1:3 ratio (v/v).

Fig. 1.

Representative flowers of the four Alstroemeria cultivars used in this study: Inca Sweety, Dwarf Red, Orange Queen, and Amatista.

Growth conditions

Plants were maintained in a greenhouse under controlled conditions. Rhizomes were planted in pots measuring 20 cm height and 21 cm diameter. The day/night temperature was maintained at 18–21 °C and 10–13 °C, respectively, with relative humidity between 60 and 70%. Photosynthetic photon flux density (PPFD) was approximately 400–500 µmol m⁻² s⁻¹. Pots were arranged randomly and positions were rotated weekly to minimize microclimatic variations.

Experimental design and treatments

The experiment was conducted as a factorial arrangement based on a completely randomized design (CRD) with four replications. The factors included four Alstroemeria cultivars (two dwarf and two tall) and four salinity levels (0, 20, 40, and 60 mM NaCl). Salinity treatments were initiated one month after plant establishment and continued for six weeks.

Irrigation and fertilization with salinity treatments

During the experiment, plants were irrigated with a complete nutrient solution prepared per 100 L of water using magnesium sulfate (10 g), potassium nitrate (32 g), ammonium nitrate (4 g), sodium molybdate (0.035 g), borax (0.03 g), monoammonium phosphate (5 g), manganese sulfate (0.2 g), zinc sulfate (0.15 g), potassium sulfate (8 g), Fe-EDDHA 6% (5 g), and calcium nitrate–ammonium nitrate (10 g). To impose salinity stress, NaCl was incorporated directly into the nutrient solution at concentrations of 0, 20, 40, or 60 mM, resulting in electrical conductivities (EC) of approximately 1.35, 3.8, 6.0, and 8.2 dS m⁻¹, respectively. Plants received the corresponding saline nutrient solutions throughout the experimental period. Irrigation was applied uniformly to maintain adequate substrate moisture, and to prevent excessive salt accumulation in the root zone, each irrigation was managed to produce a leaching fraction of approximately 10–15%, allowing part of the solution to drain freely. The total irrigation volume and leaching fraction were kept consistent across all treatments to ensure uniformity, and the electrical conductivity of both the nutrient solution and, periodically, the leachate was monitored to confirm that salt levels remained within the intended range for each salinity treatment.

Morphophysiological and biochemical analysis

Following the six-week salinity treatment, plants were evaluated for their morphophysiological and biochemical responses. Morphophysiological parameters included flowering stem length and diameter, fresh and dry weight of flowering stems, leaf number, chlorophyll index, leaf chlorosis, and leaf area. Biochemical assessments comprised chlorophyll a, chlorophyll b, total chlorophyll, carotenoids, total phenols, flavonoids, total antioxidant capacity, and the activities of antioxidant enzymes including catalase (CAT), ascorbate peroxidase (APX), and guaiacol peroxidase (GPX). All measurements were conducted following standard protocols to ensure reproducibility. Together, these evaluations provided a comprehensive assessment of the plants’ physiological and biochemical adaptations to the imposed salinity stress.

Morphophysiological measurements

Morphophysiological parameters of Alstroemeria plants were assessed after the salinity treatment. Flowering stem length was measured from the base of the stem near the crown to the tip of the uppermost flower, using a ruler, in millimeters (mm). Stem diameter was determined at three positions along each flowering stem (base, midpoint, and apex) with a digital caliper, taking two independent readings per position, and the average of the three positions was reported as the final stem diameter. Fresh and dry weight of flowering stems was recorded. Dry weight was obtained after oven-drying the stems at 70 °C until constant weight. Measurements were performed on three to five randomly selected flowering stems per plant under consistent environmental conditions to ensure accuracy and reproducibility.

Leaf number was counted on each plant, and the leaf area was quantified at the end of the growth period to compare untreated plants with those receiving the respective treatments. From each shoot, three fully expanded leaves were randomly collected from the mid-canopy region to represent the average leaf size of the plant. The surface area of each leaf was determined using a leaf area meter (AM200 Leaf Area Meter, ADC BioScientific Ltd., UK), which provides high-precision measurements based on optical scanning¹².

Chlorophyll index and pigment measurements

Chlorophyll index (SPAD)

The chlorophyll index of fully expanded leaves was measured using a SPAD-502 Plus chlorophyll meter (Konica Minolta, Japan). For each plant, four leaves were measured at three different points along the leaf blade, and the mean SPAD value per leaf was calculated. Measurements were performed on three to five randomly selected plants per treatment, with four replicates per treatment¹³.

Chlorophyll and carotenoid content

Leaf samples (0.1 g fresh weight) were collected from the same plants and homogenized in 80% acetone. The homogenate was centrifuged at 10,000 × g for 10 min at 4 °C, and the absorbance of the supernatant was recorded at 663, 645, and 470 nm using a spectrophotometer (HALODB-20, Dynamica). Chlorophyll a, chlorophyll b, total chlorophyll, and carotenoid contents were calculated according to Lichtenthaler¹⁴ and expressed on a fresh weight basis (mg g⁻¹ FW). All measurements were performed in four replicates per treatment.

Leaf chlorosis assessment

Leaf chlorosis was assessed visually following the modified method of Ao et al.¹⁵. At each evaluation time, two to three fully expanded leaves from the middle portion of each plant were selected, and their degree of yellowing was recorded using a five-point scale. In this scale, a score of 1 represented completely green, healthy leaves with no signs of yellowing; a score of 2 indicated mild chlorosis characterized by slight lightening of the leaf tissue; a score of 3 corresponded to moderate chlorosis with interveinal yellowing while the veins remained green; a score of 4 denoted severe chlorosis with noticeable vein paling and a marked reduction in overall greenness; and a score of 5 reflected extremely severe chlorosis associated with leaf reduction, necrotic spotting, desiccation, or growth cessation. The mean score of the evaluated leaves for each experimental unit was calculated for subsequent chlorosis analysis.

Extraction and quantification of total phenolics, flavonoids, and antioxidant capacity

Extraction of leaf and petal metabolites

For the preparation of methanolic extracts, 0.5 g of fresh leaf tissue was first frozen in liquid nitrogen and then finely powdered. The powder was homogenized in 5 mL of 85% methanol, subjected to ultrasonic-assisted extraction at 20 °C for 30 min, and centrifuged at 5000 rpm for 15 min. The clear supernatant was collected and stored at − 20 °C until analysis¹⁶.

Total phenolic compounds

Total phenols were determined using the Folin–Ciocalteu (F–C) assay according to Marinova et al.¹⁷. A 1-mL aliquot of the extract was mixed with 9 mL distilled water and 1 mL Folin–Ciocalteu reagent. After a 5-min reaction, 10 mL sodium carbonate solution was added, and samples were incubated at room temperature for 90 min. Absorbance was measured at 750 nm. Phenolic compounds were calculated from a gallic acid standard curve and expressed as mg gallic acid equivalents (GAE) g⁻¹ FW. It should be noted that PVPP treatment to remove interfering compounds was not applied; therefore, results should be interpreted with caution.

Total flavonoid content

Flavonoid concentration was determined following the aluminum chloride colorimetric assay¹⁸. Each reaction contained 500 µL extract, 1.5 mL of 80% methanol, 100 µL of 10% AlCl₃, 100 µL of 1 M potassium acetate, and 3.8 mL distilled water. Samples were incubated for 40 min at room temperature, and absorbance was recorded at 380 nm. Total flavonoids were quantified using a quercetin calibration curve and expressed as mg quercetin equivalents (QE) g⁻¹ FW.

Antioxidant capacity (DPPH assay)

Antioxidant capacity was assessed using the DPPH radical scavenging assay according to Nakajima et al.¹⁹. A mixture of 100 µL extract and 1.9 mL DPPH solution was incubated in the dark for 30 min at room temperature. Absorbance was measured at 517 nm, and scavenging activity was calculated as: Inhibition (%) = Ac-As/Ac × 100.

Where Ac and As denote the absorbance of the control and sample, respectively.

Determination of phenylalanine ammonia-lyase (PAL) activity

PAL activity was assayed following the method described by D’Cunha et al.²⁰ with minor modifications. The reaction mixture consisted of 1.0 mL of 50 mM potassium phosphate buffer (pH 7.0), 0.5 mL of 10 mM L-phenylalanine, 0.4 mL of distilled water, and 0.1 mL of the enzyme extract. The mixture was incubated at 37 °C for 1 h, after which the reaction was terminated by the addition of 0.5 mL of 6 M hydrochloric acid. The absorbance of the resulting solution was measured at 260 nm using a spectrophotometer. PAL activity was quantified based on a cinnamic acid standard curve and expressed as mg cinnamic acid g⁻¹ fresh weight.

Preparation of enzyme extracts

Enzyme extracts for antioxidant enzymes assays were prepared following Kang and Saltveit²¹ with minor adjustments to ensure optimal enzyme stability. Approximately 0.5 g of leaf tissue was ground in a pre-chilled mortar with 3 mL of ice-cold extraction buffer (50 mM Tris–HCl, pH 7.5) containing 3 mM MgCl₂ and 1 mM Na-EDTA. The homogenate was centrifuged at 4000 × g for 20 min at 4 °C, and the resulting supernatant was immediately used for all enzyme assays. For APX extraction, the buffer was supplemented with 0.2 mM ascorbate prior to pH adjustment.

Ascorbate peroxidase (APX) activity

APX activity was assayed following Nakano and Asada²², with minor modifications. Fresh leaf tissue (0.5 g) was frozen in liquid nitrogen and ground to a fine powder. The powder was homogenized in 5 mL of 50 mM phosphate buffer (pH 7.0) containing 1 mM EDTA and 1% (w/v) polyvinylpyrrolidone (PVP) to prevent phenolic interference. The homogenate was centrifuged at 12,000 × g for 15 min at 4 °C, and the supernatant was used as the enzyme extract.

The reaction mixture contained 2.5 mL of 50 mM phosphate buffer (pH 7.0), 0.2 mL of 1% H₂O₂, and 0.1 mL of enzyme extract. The decrease in absorbance at 290 nm was monitored for 1 min at room temperature. APX activity was calculated using an extinction coefficient of 2.8 mM⁻¹ cm⁻¹ and expressed as µmol ascorbate oxidized mg⁻¹ protein min⁻¹. Protein concentration in enzyme extracts was determined using the Bradford method²³.

Catalase (CAT) Activity

CAT activity was determined according to Aebi²⁴. The reaction mixture consisted of 2.5 mL of 50 mM phosphate buffer, 0.2 mL of 1% H₂O₂, and 0.3 mL of enzyme extract. The decline in absorbance at 240 nm was monitored for 1 min. Enzyme activity was calculated using an extinction coefficient of 43.6 mM⁻¹ cm⁻¹ and expressed as µmol H₂O₂ decomposed mg⁻¹ protein min⁻¹.

Guaiacol peroxidase (GPX) activity

GPX activity was measured following Upadhyaya et al.²⁵. The reaction mixture consisted of 1 mL of 1% guaiacol, 1 mL of 1% H₂O₂, 2.5 mL of 50 mM phosphate buffer (pH 7.5), and 0.1 mL of enzyme extract. The increase in absorbance at 420 nm was recorded for 1 min. Activity was calculated using an extinction coefficient of 26.6 mM⁻¹ cm⁻¹ and expressed as µmol guaiacol oxidized mg⁻¹ protein min⁻¹.

Measurement of oxidative stress markers

Malondialdehyde (MDA) Content

Lipid peroxidation was assessed by determining MDA content following the method of Horst and Cakmak²⁶. Fresh leaf tissue (0.2 g) was homogenized in 5 mL of 1% (w/v) trichloroacetic acid (TCA) and centrifuged at 8,000 × g for 10 min. One millilitre of the supernatant was mixed with 4 mL of a solution containing 20% (w/v) TCA and 0.5% (w/v) thiobarbituric acid (TBA).

The mixture was heated at 95 °C for 30 min and immediately cooled in an ice bath. After centrifugation at 8,000 × g for 5 min, the absorbance of the supernatant was recorded at 532 and 600 nm. MDA concentration was calculated using the equation below and expressed as µmol g⁻¹ fresh weight (FW):

Hydrogen peroxide (H₂O₂) Content

Hydrogen peroxide content was quantified according to Velikova et al.²⁷. Fresh leaf tissue (0.5 g) was homogenized in 3 mL of 0.1% (w/v) TCA and centrifuged at 12,000 × g for 15 min. The reaction mixture consisted of 0.5 mL of the supernatant, 0.5 mL of 10 mM potassium phosphate buffer (pH 7.0), and 1 mL of 1 M potassium iodide (KI). Samples were incubated in darkness at 35 °C for 1 h, and absorbance was measured at 390 nm. Hydrogen peroxide concentration was determined using a standard curve (Fig. 3) and calculated according to the following equation: Y = 0.0013X + 0.0356.

Fig. 3.

Effects of salinity (0, 20, 40, and 60 mM NaCl) on chlorophyll index (a), chlorosis index (b), chlorophyll a (c), chlorophyll b (d), total chlorophyll (e), and carotenoid (f) of four Alstroemeria cultivars (Dwarf Red, Inca Sweety, Amatista, and Orange Queen). Data represent mean ± standard error of four replicates. Different letters above the bars indicate significant differences among Treatment × Cultivar combinations (p < 0.05, Tukey’s test).

where Y represents the absorbance value and X indicates H₂O₂ concentration (µmol g⁻¹ FW).

Statistical analysis

All data were subjected to two-way ANOVA based on a factorial design with four Alstroemeria cultivars and four salinity levels, with four biological replicates per treatment combination. ANOVA modeling and mean separation were performed using SAS software (version 9.4; SAS Institute Inc., Cary, NC, USA). When the interaction between cultivar and salinity was significant, mean comparisons were conducted using Tukey’s test at p ≤ 0.05. For traits where the interaction was not significant, means were compared based on the main effects of cultivars or salinity as appropriate.

To comprehensively investigate multivariate relationships, interaction patterns, and treatment-dependent variation among the measured morphophysiological and biochemical traits under salinity stress, a suite of complementary analyses was performed using Python (version 3.11; Python Software Foundation, USA) and R (version 5.5; R Foundation for Statistical Computing, Vienna, Austria). Pairwise associations among traits were quantified using Pearson’s correlation coefficients, and only statistically significant correlations were retained for network construction. In the resulting networks, nodes represent individual traits, whereas edges indicate the strength and direction of correlations, enabling identification of key traits and interaction structures associated with distinct salinity tolerance strategies among the Alstroemeria cultivars.

Additionally, heat-map clustering was applied to visualize treatment-dependent patterns and assess similarity among cultivars and salinity levels. Principal component analysis (PCA) was performed to reduce dimensionality and determine the main components contributing to variance in the dataset, facilitating a comprehensive interpretation of overall response patterns to salinity stress.

Results

Morphological parameters

Salinity stress induced clear cultivar-dependent effects on morphological traits (Fig. 2). Stem length responses varied markedly among cultivars (Fig. 2a). Dwarf Red and Inca Sweety maintained relatively stable stem height across salinity treatments, whereas Amatista and Orange Queen exhibited significant reductions as NaCl concentration increased, with more pronounced declines observed in Orange Queen under higher salinity levels.

Fig. 2.

Effects of salinity (0, 20, 40, and 60 mM NaCl) on stem length (a), stem diameter (b), fresh and dry stem weight (c), leaf number (d), and leaf area (e) of four Alstroemeria cultivars (Dwarf Red, Inca Sweety, Amatista, and Orange Queen). Data represent mean ± standard error of four replicates. Different letters above the bars indicate significant differences among Treatment × Cultivar combinations (p < 0.05, Tukey’s test).

Stem diameter was more sensitive to salinity than stem length, but the magnitude of reduction varied among cultivars (Fig. 2b). In Dwarf Red, stem diameter decreased slightly under salinity, but these changes were not statistically significant, indicating relative stability. Inca Sweety exhibited a minor reduction at 20 mM NaCl, followed by a plateau between 20 and 40 mM, before declining again at 60 mM. In contrast, Amatista showed a continuous and statistically significant decline in stem diameter with increasing salinity. Orange Queen consistently exhibited the smallest stem diameter, with significant reductions at higher NaCl levels. Overall, dwarf cultivars maintained relatively stable stem diameters under salt stress, whereas taller cultivars were more susceptible to structural thinning.

Stem biomass accumulation was significantly affected by salinity in all cultivars (Fig. 2c). In Dwarf Red and Inca Sweety, fresh and dry stem weights decreased moderately with increasing NaCl, with reductions of 57–64% at 60 mM. Amatista exhibited the largest decline, with fresh and dry weights reduced by 31–70% under severe salinity, indicating higher sensitivity. In Orange Queen, a slight, non-significant increase (~ 43%) was observed at 20 mM NaCl, followed by reductions of approximately 50% at 60 mM, confirming a moderate susceptibility to high salinity. Overall, these results highlight cultivar-dependent differences in biomass responses under saline conditions.

Leaf-related traits exhibited clear cultivar-specific responses to salinity (Figs. 2d, e). In Dwarf Red and Inca Sweety, leaf number remained relatively stable under low to moderate salinity, with reductions only under severe stress, reflecting a threshold-dependent, short-term defense response. In contrast, Amatista showed a continuous decline in leaf number with increasing salinity, indicating higher sensitivity. Orange Queen maintained leaf number across all salinity treatments, suggesting a robust defense strategy that contributes to its resilience. Leaf area responses followed similar patterns: Inca Sweety preserved leaf area across the salinity gradient, while Amatista and Orange Queen exhibited marked reductions under high salinity. Dwarf Red showed only moderate changes in leaf area, primarily at the highest NaCl level.

Overall, morphological responses to salinity were primarily expressed through reductions in stem thickness, biomass accumulation, and leaf expansion, with the magnitude and pattern of these changes strongly dependent on cultivar identity (Fig. 2).

Photosynthetic pigments

Salinity stress induced clear but cultivar-dependent changes in photosynthetic pigments (Fig. 3). The SPAD chlorophyll index remained statistically stable in Inca Sweety across all salinity levels, whereas significant declines were observed in Dwarf Red, Amatista, and Orange Queen as salinity increased (Fig. 3a). In parallel, the chlorosis index increased progressively with rising NaCl concentration in all cultivars, showing a strong treatment-dependent response and minimal cultivar differentiation, particularly at moderate to high salinity levels (Fig. 3b). For some cultivars (e.g., Dwarf Red and Orange Queen), the inverse relationship between SPAD and chlorosis was less pronounced, likely due to limited SPAD readings per leaf or heterogeneous chlorophyll distribution across the leaf blade. Therefore, while SPAD provides a rapid, non-destructive estimate of chlorophyll status, it should be interpreted alongside detailed pigment quantification.

Chlorophyll a and b contents exhibited distinct genotypic patterns (Figs. 3c, d). In Inca Sweety and Dwarf Red, chlorophyll a remained statistically unchanged across salinity treatments, whereas significant reductions were recorded in Amatista and Orange Queen at higher salinity levels. Chlorophyll b responses were more variable, with transient increases under mild salinity in Inca Sweety, early reductions in Dwarf Red and Orange Queen, and a marked decline in Amatista beyond moderate salinity. Consequently, total chlorophyll content was maintained in Inca Sweety and Dwarf Red, while it decreased significantly in Amatista and Orange Queen under severe salt stress (Fig. 3e).

Carotenoid content also showed cultivar-specific responses to salinity (Fig. 3f). Inca Sweety exhibited a significant increase under mild salinity, followed by a gradual decline at higher NaCl levels. Dwarf Red maintained relatively stable carotenoid levels up to moderate salinity, whereas Amatista showed a continuous decrease with increasing stress. In Orange Queen, carotenoid content fluctuated across salinity treatments without a consistent declining trend. Genotype-dependent differences in leaf appearance, including chlorosis and necrosis severity, were consistent with the pigment responses observed under increasing salinity (Fig. 4).

Fig. 4.

Leaf morphological responses of four Alstroemeria cultivars (Dwarf Red, Inca Sweety, Amatista, and Orange Queen) exposed to four salinity levels (0, 20, 40, and 60 mM NaCl). Progressive changes in leaf color, chlorosis, and tissue damage illustrate the cultivar-specific sensitivity or tolerance to salinity stress.

Antioxidant parameters

Salinity induced clear cultivar-specific responses in antioxidant-related traits (Fig. 5). Total phenol content increased under mild to moderate salinity in Dwarf Red, Amatista, and Orange Queen, followed by a decline at the highest stress level in Dwarf Red and Amatista, whereas Orange Queen continued to show a steady increase. Inca Sweety maintained stable phenolic levels across all treatments. Petal responses mirrored those in leaves, exhibiting similar biphasic or stable patterns depending on the cultivar (Fig. 5a). It should be noted that total phenolic determinations were performed without PVPP treatment; therefore, these results should be interpreted as relative trends rather than absolute phenolic content.

Fig. 5.

Effects of salinity (0, 20, 40, and 60 mM NaCl) on total phenol (a), flavonoid (b), antioxidant capacity (c), PAL activity (d), CAT activity (e), APX activity (f), and GPX activity (g) of four Alstroemeria cultivars (Dwarf Red, Inca Sweety, Amatista, and Orange Queen). Data represent mean ± standard error of four replicates. Different letters above the bars indicate significant differences among Treatment × Cultivar combinations (p < 0.05, Tukey’s test).

Flavonoid content exhibited contrasting trends among cultivars (Fig. 5b). Inca Sweety remained low and unchanged under salinity. Dwarf Red showed a progressive decline with increasing NaCl, whereas Amatista and Orange Queen accumulated flavonoids progressively, reaching peak levels at 60 mM NaCl.

Antioxidant capacity measured via DPPH scavenging activity showed a relatively stable pattern in Inca Sweety and Orange Queen across treatments (Fig. 5c). In Dwarf Red, leaf antioxidant activity peaked at moderate salinity and declined at 60 mM, while petal activity remained largely unchanged. Amatista exhibited no significant change up to 40 mM, followed by a marked increase at the highest salinity level.

Salinity effects on enzyme activities also differed among cultivars (Figs. 5d–g). PAL activity remained stable in Inca Sweety and Dwarf Red, while Amatista showed minor increases at moderate salinity, and Orange Queen displayed significant variation at 60 mM. CAT activity increased under high salinity in Inca Sweety and Dwarf Red, peaked at moderate stress in Amatista, and gradually increased in Orange Queen. APX and GPX activities generally increased with rising NaCl, with cultivar-specific peak levels: Inca Sweety and Orange Queen showed a steady increase across the gradient, whereas Dwarf Red and Amatista peaked at intermediate salinity before further changes at 60 mM.

Collectively, these results indicate that antioxidant responses to salinity are both organ- and cultivar-dependent, with Inca Sweety maintaining stable chemical defenses, while other cultivars rely on variable activation of phenolic accumulation and enzymatic antioxidants to cope with increasing salt stress.

Oxidative parameters

Salinity stress induced progressive oxidative responses in all four Alstroemeria cultivars (Fig. 6). Leaf H₂O₂ content increased with rising NaCl concentrations, with the most pronounced accumulation observed at 40–60 mM. Dwarf Red consistently exhibited the highest H₂O₂ levels, while Amatista maintained the lowest values throughout the salinity gradient. Inca Sweety and Orange Queen showed intermediate responses.

Fig. 6.

Effects of salinity (0, 20, 40, and 60 mM NaCl) on on H₂O₂ accumulation (a) and MDA content (b) of four Alstroemeria cultivars (Dwarf Red, Inca Sweety, Amatista, and Orange Queen). Data represent mean ± standard error of four replicates. Different letters above the bars indicate significant differences among Treatment × Cultivar combinations (p < 0.05, Tukey’s test). Overall, oxidative stress responses were cultivar-dependent, with Dwarf Red and Orange Queen experiencing greater H₂O₂ accumulation and membrane lipid peroxidation under high salinity, while Inca Sweety and Amatista maintained relatively lower oxidative damage.

MDA content, an indicator of lipid peroxidation, also increased progressively under salinity (Fig. 6b). Dwarf Red and Orange Queen displayed the largest rises, whereas Inca Sweety exhibited moderate accumulation, and Amatista showed the lowest increase among all cultivars.

Multivariate analysis of morphophysiological and biochemical responses to salinity stress

Multivariate analyses revealed clear patterns of cultivar- and salinity-dependent responses in morphophysiological, biochemical, and oxidative traits of Alstroemeria (Fig. 7). Hierarchical clustering (heatmap, Fig. 7a) grouped traits into two main categories: growth- and photosynthesis-related parameters (leaf area, chlorophyll a and b, total chlorophyll, carotenoids, SPAD index, stem biomass, and leaf number), which were higher under control and mild salinity (0–20 mM NaCl), and stress- and defense-related traits (CAT, APX, GPX, PAL, total phenolics, flavonoids, antioxidant capacity, MDA, H₂O₂), which increased under moderate to severe salinity (40–60 mM NaCl). Sample clustering reflected salinity levels, with high-salinity treatments forming a distinct group, while control and low-salinity treatments clustered closely together. Cultivar differences were evident: Orange Queen and Inca Sweety maintained relatively higher growth- and pigment-related traits under moderate stress compared with Dwarf Red and Amatista.

Fig. 7.

Multivariate responses of Alstroemeria cultivars to salinity stress. The heatmap (a) shows separation of growth and antioxidant responses, with antioxidants increasing under higher salinity; values are log₂(x + 1) transformed for comparability and to avoid negative values. The correlation matrix (b) highlights the trade-off between growth and defense, while the PCA biplot (c) illustrates treatment- and cultivar-specific separation. Network analyses (d–g) reveal cultivar-specific trait interactions for Red Dwarf (d), Amatista (e), Orange Queen (f), and Inca Sweety (g), with nodes representing traits and edges indicating significant correlations; dense networks are observed in sensitive cultivars, whereas tolerant cultivars display more modular and resilient network structures.

Correlation analysis (Pearson matrix, Fig. 7b) supported these patterns. Growth- and photosynthesis-related traits were strongly positively correlated with one another, whereas antioxidant- and oxidative stress-related parameters were positively correlated within their group but negatively associated with growth traits. The chlorosis index showed positive correlations with MDA, H₂O₂, and antioxidant-related traits, and negative correlations with pigment content and biomass, consistent across salinity treatments.

PCA summarized the multivariate relationships (Fig. 7c), with the first two components explaining 57.6% of variance (PC1: 41.1%; PC2: 16.5%). PC1 primarily separated treatments based on salinity: high salinity was associated with elevated antioxidant enzyme activities, oxidative stress markers, phenolic and flavonoid contents, and chlorosis, whereas control and low-salinity treatments aligned with growth- and photosynthesis-related traits. PC2 captured cultivar- and flower-related biochemical variation, highlighting genotype-specific distributions in antioxidant capacity and secondary metabolites under stress.

Network analysis revealed cultivar-dependent connectivity of traits (Figs. 7d–g). Dwarf Red showed a dense network linking growth, pigment, antioxidant, and oxidative stress traits, with GPX and APX highly central. Amatista exhibited clearer separation of functional groups, with APX central and phenolic/flavonoid traits forming a distinct sub-network. Orange Queen displayed modular organization, linking antioxidant enzymes with pigment traits, while growth traits remained closely associated with pigments. Inca Sweety showed an intermediate structure, with moderate connectivity and integration of oxidative stress markers and antioxidant enzymes.

Overall, multivariate analyses confirmed that salinity stress induces coordinated adjustments of growth, pigment, antioxidant, and oxidative traits, with cultivar-specific strategies reflecting either stability in growth (Inca Sweety, Orange Queen) or stronger activation of defense mechanisms (Dwarf Red, Amatista) under increasing NaCl concentrations.

Discussion

The cultivar-specific morphological responses observed here reflect the interaction of two temporally distinct phases of salt stress: an initial osmotic phase that limits cell expansion and a later ionic phase causing Na⁺/Cl⁻ toxicity and nutrient imbalance²⁸. Stem elongation in Dwarf Red and Inca Sweety (Fig. 2a) remained relatively stable under increasing NaCl, indicating effective osmotic adjustment and ion regulation mechanisms that maintain turgor for cell expansion²⁹. In contrast, Amatista and Orange Queen exhibited sharp declines in stem length at intermediate–high salinity, consistent with limited osmotic adjustment and early ionic disruption^30,31.

Reductions in stem diameter and biomass (Fig. 2b–c) are indicative of growth inhibition under salinity stress and are consistent with the reduced carbon availability inferred from the overall decline in biomass accumulation. Based on previous studies, osmotic constraints at the leaf level may reduce stomatal conductance and photosynthetic carbon gain, while salinity-induced ionic imbalance—particularly Na⁺ interference with K⁺-dependent processes—could impair cell expansion and turgor-driven growth³². Although ion concentrations and anatomical traits were not directly measured in the present study, the observed biomass reduction, together with enhanced oxidative stress and antioxidant responses, supports the involvement of these mechanisms as plausible contributors to growth limitation under salinity³³.

Leaf number and area adjustments illustrate differential strategies among cultivars. Cultivars maintaining leaf count but reducing area, like Orange Queen, may prioritize light capture while minimizing water loss per leaf, whereas those reducing leaf number employ a conservative water-saving strategy^34,35.

Salinity also affected photosynthetic pigments and antioxidant defenses. Inca Sweety maintained SPAD index, total chlorophyll, and carotenoids across salinity levels (Fig. 3), reflecting stable photosynthetic machinery and effective ROS control through ionic regulation and enzymatic antioxidants^6,36,37. Dwarf Red, Amatista, and Orange Queen showed declines in chlorophyll a and b and fluctuations in carotenoids, consistent with pigment destabilization under ionic and oxidative stress^38–41.

Phenolic and flavonoid dynamics further revealed cultivar-specific defense mechanisms (Fig. 5a–b). Biphasic phenolic accumulation in Dwarf Red and Amatista suggests hormetic induction under moderate salinity followed by metabolic exhaustion at high stress⁴². Inca Sweety maintained stable phenolic levels, likely reflecting low ROS formation rather than inducible antioxidant demand. Orange Queen consistently increased phenolic content, suggesting delayed activation of non-enzymatic defenses⁴³. Flavonoid responses paralleled these trends, with late induction in sensitive cultivars indicating reliance on ROS scavenging as oxidative stress progressed^44–46.

Enzymatic antioxidants (CAT, APX, GPX) distinguished cultivar strategies (Fig. 5e–g). Inca Sweety demonstrated steady APX induction, supporting precise H₂O₂ detoxification and redox homeostasis⁴⁷. Dwarf Red showed threshold-dependent activation of CAT and GPX, with a more pronounced response under moderate salinity stress^48,49. However, both cultivars (Dwarf Red and Orange Queen) exhibited similar patterns in their enzyme activities, suggesting no significant differences in CAT behavior between them, as also reflected in the graph. Amatista displayed a transient antioxidant peak followed by decline at high NaCl, indicating susceptibility to oxidative stress^50,51. Orange Queen showed delayed but intensified antioxidant activity, consistent with reactive defense rather than preemptive protection^52,53.

Measurements of H₂O₂ and MDA (Fig. 6a–b) confirmed these oxidative patterns. Dwarf Red accumulated the highest oxidative markers under severe salinity, indicating greater lipid peroxidation and ROS burden. Orange Queen showed intermediate accumulation, whereas Inca Sweety and Amatista maintained lower levels, consistent with more effective control of oxidative stress. These biochemical indices corroborate morphological and pigment-based observations, demonstrating that tolerance emerges from the coordinated action of growth maintenance, pigment stability, and antioxidant defenses⁵⁴.

The differential salinity responses among Alstroemeria cultivars indicate that salt tolerance is not determined by a single trait but rather by the integration of multiple physiological and biochemical mechanisms. As illustrated in Fig. 8, Inca Sweety exhibited balanced growth and stable phenolic metabolism, maintained through efficient redox control, allowing it to cope effectively with salinity stress. In contrast, cultivars such as Amatista and Orange Queen faced oxidative challenges under high salinity conditions, which were reflected in the accumulation of reactive oxygen species (ROS), specifically hydrogen peroxide (H₂O₂) and malondialdehyde (MDA). These cultivars displayed limited antioxidant responses, which contributed to their reduced resilience under salinity stress. Dwarf Red, on the other hand, exhibited a threshold-dependent activation of antioxidant defenses, particularly catalase (CAT) and guaiacol peroxidase (GPX), which play a crucial role in mitigating oxidative stress under moderate salinity. While Dwarf Red may not maintain the same level of growth as Inca Sweety, its ability to activate oxidative stress detoxification mechanisms allows it to cope with salinity challenges more effectively than other cultivars like Amatista and Orange Queen. Despite the high oxidative pressures, Dwarf Red demonstrates a resilient mechanism for dealing with salinity, prioritizing enzymatic defenses over growth. Evaluating the PAL–phenol–flavonoid axis, in combination with enzymatic antioxidants (such as catalase (CAT), ascorbate peroxidase (APX), and guaiacol peroxidase (GPX)), and growth-related traits offers a robust mechanistic framework for understanding cultivar-specific resilience to salt stress. This multi-faceted approach enables the identification of key metabolic pathways that contribute to the adaptive response of each cultivar, thereby providing insights into their differential salt tolerance.

Fig. 8.

Salt tolerance strategies in Alstroemeria cultivars. The four cultivars—Inca Sweety, Dwarf Red, Orange Queen, and Amatista—demonstrate distinct responses to salinity stress. Inca Sweety maintains stable growth and phenolic metabolism, Dwarf Red prioritizes oxidative stress detoxification, Orange Queen exhibits limited defense, and Amatista shows oxidative damage under high salinity. This diagram summarizes the different adaptive strategies each cultivar uses to cope with salt stress.

Further research into the regulatory connections between ROS scavenging, secondary metabolite metabolism, and ionic homeostasis is essential for a more comprehensive understanding of the mechanisms underlying salinity tolerance in Alstroemeria. Investigating the temporal coordination of these defense responses will offer valuable insights and aid in the targeted selection of improved cultivars capable of thriving under saline conditions.

Conclusion

The present study demonstrates that salinity tolerance in Alstroemeria is a complex, genotype-specific phenomenon emerging from the coordinated regulation of growth, photosynthetic performance, and antioxidant defense. Inca Sweety exhibits a proactive and tightly integrated strategy, maintaining structural and physiological stability through balanced metabolic and enzymatic defenses. Dwarf Red relies on inducible enzymatic detoxification to mitigate oxidative stress, sacrificing biomass in the process, whereas Amatista shows a partially effective but ultimately unsustainable response under severe salinity. Orange Queen adopts a reactive and less coordinated defense strategy, resulting in compromised tolerance. Collectively, these findings highlight that resilience is governed not by individual traits but by the coherence and timing of systemic responses, with adaptive strategies reflecting a trade-off between growth potential and stress-defensive capacity. This framework provides mechanistic insight into cultivar-specific salinity tolerance and offers guidance for breeding and cultivation practices aimed at improving performance under saline conditions.

Author contributions

- **M.M.:** Conducted the experiment and performed the chemical analysis.- **Z.J.:** Provided assistance in designing the experiment, conducting chemical and statistical analysis, and writing the manuscript.

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Data availability

All data generated or analyzed during this study are included in this published article.

Declarations

Competing interests

The authors declare no competing interests.