Exogenous Proline Application Mitigates Salt Stress in Physalis ixocarpa Brot.: Morphophysiological, Spectroscopic, and Metabolomic Evidence

Abstract

Salt stress severely constrains agricultural productivity in arid and semi-arid regions. This study evaluated exogenous proline as an osmoprotector in Physalis ixocarpa Brot. (Mexican husk tomato) under salinity. Germination screening identified 75 mM NaCl as a threshold stress level, reducing germination by 38.9% while maintaining seedling viability. Proline pretreatment (30-min imbibition) at 8 mM restored germination to 78% and fresh weight to control levels under salt stress. In vitro experiments revealed that 8 mM proline enhanced chlorophyll content above salt-stressed controls while reducing root length from 9.72 to 5.08 cm, indicating resource reallocation toward photosynthetic protection. Infrared spectroscopy showed characteristic polysaccharide shifts and bands potentially associated with proline incorporation. Gas chromatography–mass spectrometry metabolomics of stem–leaf extracts revealed salt-induced synthesis of nitrogenous osmolytes (such as long-chain amines) and carbohydrate reorganization from α-D-glucopyranoside to β-D-riboside. Proline treatment restored the original carbohydrate profile while generating pyrrolidine derivatives (2.83%), evidence of active proline metabolism. Phenolic antioxidants (e.g., catechol) present in controls were absent under both salt stress and proline treatment, suggesting that proline’s protective mechanism may operate through metabolic regulation of osmolyte pathways and membrane stabilization rather than inducing phenolic antioxidant synthesis. These findings demonstrate proline’s multifaceted protective mechanisms and support its potential application for enhancing salt tolerance in this crop.

1. Introduction

Global climate change and intensive agricultural practices have led to increased soil salinization, affecting over 800 million hectares worldwide [1]. This phenomenon is particularly severe in arid and semi-arid regions, where high evapotranspiration rates combined with poor-quality irrigation water result in salt accumulation in agricultural soils [2]. Salinity stress represents one of the most detrimental abiotic factors limiting crop productivity, causing osmotic stress, ionic toxicity, and oxidative damage that collectively impair plant growth and yield [3,4].

The genus Physalis (Solanaceae) comprises over 100 species characterized by an inflated calyx that envelops and protects the fruit against herbivores and climatic adversities [5]. Among these, Physalis ixocarpa Brot., commonly known as Mexican husk tomato, “tomatillo”, or “tomate de cáscara”, holds significant economic and nutritional importance in Mexico [6]. The fruits are widely used in traditional Mexican cuisine as ingredients in salads, soups, stews, and sauces, representing a valuable food resource [7]. Beyond their nutritional content of vitamins C and B3, carbohydrates, proteins, and minerals [7], extracts from P. ixocarpa fruits have demonstrated antimicrobial activity against Staphylococcus aureus [8] and possess potential chemopreventive properties due to the presence of physalins [9], highlighting the species’ diverse biotechnological potential, widely recognized for other members of the genus [10,11].

Physalis ixocarpa holds considerable economic importance in Mexican agriculture, ranking among the country’s five most economically significant vegetable crops [9,12]. In 2020, Mexican tomatillo cultivation covered approximately 40,117 hectares across 30 states, with an average yield of 19.3 t/ha [13]. Production has grown substantially, reaching approximately 778,000 tons by 2018, representing 4.7% of national vegetable output [14]. The leading production state, Sinaloa, generated 164,500 tons valued at MX$501 million (≈US$22.8 million), with Zacatecas and Jalisco as other major producers [14]. The crop supports predominantly smallholder farmers, with most production occurring on plots smaller than one hectare [12]. Mexico’s tomatillo exports have shown remarkable growth, increasing from US$26 million in 2009 to US$82 million in 2018, primarily to the United States, though markets in the UK, UAE, France, Canada, and Belize are expanding [14].

Physalis ixocarpa cultivation is primarily established in semi-arid zones where climate change and drought events occur with increasing frequency [15]. Under water stress conditions, various biochemical mechanisms become compromised, including net photosynthesis, photosystem II quantum yield, electron transport rate, protein synthesis, lipid metabolism, and overall plant morphology [2]. The combination of salt accumulation from irrigation water and high evapotranspiration rates in these regions poses a significant challenge to sustainable production [16].

Plants have evolved various adaptive mechanisms to cope with salt stress, prominently featuring the synthesis and accumulation of compatible osmolytes. These organic solutes are biocompatible, low-molecular-weight compounds with neutral charges that do not interfere with cellular metabolic reactions [17,18,19]. Their accumulation in the cytoplasm increases hyperosmotic tolerance, balances water potential, and counteracts the ionic imbalance generated by vacuolar sequestration of Na⁺ and Cl⁻ ions. Among this diversity of osmolytes, proline stands out for its cryoprotective and osmoregulatory functions. Intracellular proline levels increase dramatically in response to adverse environmental conditions, far exceeding basal physiological concentrations [20]. This accumulation capacity serves as an adaptation marker to water stress, widely used as a selection criterion for identifying species with exceptional tolerance or resistance [21]. Recognition of proline’s protective mechanisms has led to its exogenous application as a crop management strategy, with demonstrated effectiveness across diverse plant species under various abiotic stresses, with mechanisms extending beyond simple osmotic adjustment to include enhancement of photosynthesis, antioxidant activity regulation, and maintenance of ion homeostasis [22].

While the protective role of proline under stress conditions has been documented in various crop species, its specific effects on P. ixocarpa and the underlying molecular mechanisms remain largely unexplored. Therefore, the objective of this study was to evaluate the osmoprotective action of exogenous proline application as a salt stress mitigator in P. ixocarpa through integrated morphophysiological, spectroscopic, and metabolomic analyses. We hypothesized that proline application would enhance salt tolerance by modulating osmotic adjustment, protecting photosynthetic machinery, and reorganizing metabolic pathways.

2. Materials and Methods

2.1. Plant Material and Reagents

Seeds of P. ixocarpa were obtained from plants cultivated at the Horto Florestal Unit of the State University of Feira de Santana (UEFS, Feira de Santana, Bahia, Brazil). Sucrose (CAS 57-50-1), agar (CAS 9002-18-0), Murashige and Skoog (MS) medium, sodium chloride (NaCl; CAS 7647-14-5), L-proline (CAS 147-85-3), and hydrochloric acid (HCl; CAS 7647-01-0) were supplied by Merck KGaA (Darmstadt, Germany). All reagents were of analytical grade.

2.2. Germination Experiments and Salinity Dose Determination

2.2.1. Salinity Tolerance Assessment

To determine the appropriate salt stress level, a screening was conducted using 5400 seeds over September–October 2024. Seeds were surface-sterilized with 70% ethanol for 30 s, followed by immersion in 2.5% sodium hypochlorite solution with two drops of neutral detergent for 10 min, and three rinses with sterile Milli-Q water. Seeds were placed in Petri dishes containing MS medium supplemented with agar (8 g/L) and sucrose (30 g/L), and different NaCl concentrations: 0 (control), 25, 50, 75, 100, 125, 150, 175, and 200 mM. pH was adjusted to 5.7 with HCl. Each treatment consisted of 100 seeds per replicate, with three replicates per experimental repeat and two independent experimental repeats (first repeat: 2–16 September 2024; second repeat: 23 September–7 October 2024), totaling 600 seeds per treatment across all 9 NaCl concentrations. Germination rates were assessed after 15 days at 25 °C under a 12-h photoperiod with artificial lighting.

2.2.2. Proline Pretreatment Under Salt Stress

Based on the previous experiments, 75 mM NaCl was selected as the stress condition. To evaluate the protective effects of exogenous proline against salt stress, experiments were conducted from October to December 2024. Six treatment groups were established: (1) Control—seeds imbibed in sterile distilled water for 30 min, then plated on MS medium without NaCl; (2) Salt stress—seeds imbibed in sterile distilled water for 30 min, then plated on culture medium with 75 mM NaCl; (3–6) Salt stress + proline—seeds imbibed in filter-sterilized proline solutions (4, 6, 8, or 10 mM, respectively) for 30 min at room temperature (25 °C) with gentle agitation, then plated on culture medium with 75 mM NaCl. Following imbibition, all seeds were air-dried in a laminar flow hood for 2.5 h before plating, with lids repositioned every 30 min to ensure uniform drying. This pretreatment protocol ensured that proline was absorbed by the seeds prior to salt stress exposure, mimicking a seed priming approach. Each treatment comprised 100 seeds/replicate, with three replicates and two independent experimental repeats (first repeat in October–November 2024, second repeat in November–December 2024). Germination percentage and fresh weight were recorded after 15 days.

2.3. In Vitro Culture Experiments

The in vitro culture experiments were performed from January to February 2025. The culture medium was similar to that used for the germination experiments. After heating to boiling with periodic homogenization, 15 mL aliquots were distributed into 210 test tubes (25 mm diameter × 150 mm length) and autoclaved (121–127 °C, 15 min). Seeds were surface-sterilized as described above and inoculated under aseptic conditions, one seed per tube.

After 15 days of growth, uniform seedlings were transferred to fresh tubes containing treatment media. The experimental design included six treatments: (1) control without salt stress, (2) 75 mM NaCl alone, and (3–6) 75 mM NaCl combined with 4, 6, 8, or 10 mM proline. Each treatment consisted of seven replicates with five plants each, totaling 35 plants per treatment. After an additional 15 days, plants were harvested for analysis.

2.4. Morphophysiological Analyses

The following parameters were measured: number of green leaves, number of senescent leaves, root length (cm), shoot length (cm), and dry weight of roots, leaves, and stems (g) after drying at 38 °C for 72 h. Chlorophyll content was determined spectrophotometrically following Lichtenthaler et al. [23]. Two leaf discs (5 mm diameter) from five plants per treatment were extracted in 10 mL of 95% ethanol for 24 h in darkness. Chlorophyll a and b contents were determined at 664 and 649 nm wavelengths, respectively, using a UV-Vis/NIR Jasco V-670 spectrophotometer (Jasco, Tokyo, Japan).

2.5. ATR-FTIR Spectroscopy of Plant Tissues

Dried composite samples, prepared separately for each tissue type (roots, stems, and leaves), from control, salt-stressed (75 mM NaCl), and combined treatment (75 mM NaCl + 8 mM proline) plants were analyzed using a Thermo Nicolet iS50 Fourier-transform infrared (FTIR) spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) equipped with an attenuated total reflection (ATR) accessory. Spectra were acquired in the mid-infrared region (4000–400 cm⁻¹) with 4 cm⁻¹ resolution and 64 scans per sample. Three replicates were analyzed for each plant part and treatment. Data were processed using OMNIC 9.3.32 software.

2.6. Extract Preparation and GC-MS Analysis

For extraction, composite samples of dried stem and leaf material were prepared for each treatment. Due to differences in biomass accumulation under stress, the total available material varied between treatments. To create a representative sample for each condition, all available leaf material was combined with a proportional amount of stem material. The final masses used for extraction were control—77 mg total (57 mg stem, 20 mg leaf); salt-stressed—97 mg total (82 mg stem, 15 mg leaf); and proline treatment (8 mM)—91 mg total (68 mg stem, 23 mg leaf). Each composite sample was extracted with 20 mL of a methanol:water (1:1 v/v) solution. The mixture was subjected to ultrasonic extraction using a probe-type sonicator (UP200Ht; Hielscher Ultrasonics, Teltow, Germany) at 40 W, 23.95 kHz for 10 min at 30 °C. Following sonication, the extract was transferred to centrifuge tubes and centrifuged at 5000 rpm for 10 min. After accounting for 2 mL evaporative loss during processing, 17 mL of the resulting 18 mL supernatant was freeze-dried. The resulting lyophilized powder was then dissolved in HPLC-grade methanol to a final concentration of 5 mg/mL for gas chromatography–mass spectrometry (GC-MS) analysis.

GC-MS analyses were performed using an Agilent (Santa Clara, CA, USA) 7890A gas chromatograph coupled to a 5975C mass spectrometer. The chromatographic conditions were as follows: 1 µL injection (splitless mode, 280 °C), oven program 60 °C (2 min) to 300 °C at 10 °C/min (held 15 min), HP-5MS UI column (30 m × 0.250 mm × 0.25 µm). The MS conditions were as follows: electron impact source 230 °C, quadrupole 150 °C, 70 eV. Compounds were identified via NIST11 database matching; only compounds with quality scores >70 were considered reliably identified.

2.7. Statistical Analysis

Data were analyzed using Sisvar version 5.8 Build 98 (DES/UFLA, Lavras, Minas Gerais, Brazil). Normality and homoscedasticity were verified using the Shapiro–Wilk and Levene tests, respectively. One-way ANOVA followed by Tukey’s HSD test (p = 0.05) was performed. Data are presented as means ± standard deviation (SD).

3. Results

3.1. Determination of Optimal Salt Stress Level

Germination response to NaCl showed progressive decline (

Table 1. Effect of NaCl concentration on Physalis ixocarpa seed germination after 15 days.

NaCl (mM)	Germination Rate (%)	Reduction from Control (%)
0	98.7 ± 1.2 a	-
25	73.0 ± 2.9 b	26.0
50	66.7 ± 1.2 c	32.4
75	60.3 ± 0.5 d	38.9
100	57.3 ± 1.2 d	42.0
125	47.0 ± 1.4 e	52.4
150	39.3 ± 0.5 f	60.2
175	34.3 ± 1.7 f	65.2
200	23.0 ± 1.6 g	76.7

Values: means ± SD (n = 6, 100 seeds/replicate). Different letters indicate significant differences (Tukey’s test, p < 0.05).

). Control seeds achieved 98.7% germination, while 75 mM NaCl reduced germination by 38.9%, representing moderate stress that maintains seedling viability. At 75 mM, seedlings developed expanded cotyledonary leaves similar to controls, whereas concentrations ≥100 mM arrested development at radicle protrusion.

3.2. Proline Pretreatment Effects on Salt-Stressed Seeds

Proline’s protective effects showed clear concentration dependency (

Table 2. Effects of proline pretreatment on germination under 75 mM NaCl stress.

Treatment	Germination Rate (%)	Fresh Weight (g)
Control (no salt)	98.0 ± 1.4 a	1.354 ± 0.026 a
75 mM NaCl	62.0 ± 0.8 d	0.642 ± 0.022 c
NaCl + 4 mM proline	70.7 ± 2.5 c	0.758 ± 0.015 b
NaCl + 6 mM proline	76.7 ± 0.9 b	0.799 ± 0.019 b
NaCl + 8 mM proline	78.0 ± 0.8 b	1.322 ± 0.022 a
NaCl + 10 mM proline	70.7 ± 2.1 c	0.664 ± 0.008 c

Values: means ± SD (n = 6, 100 seeds/replicate). Different letters within columns indicate significant differences (Tukey’s test, p < 0.05).

). At 4 mM, germination improved modestly to 70.7%, while 6 mM achieved 76.7%. The optimal concentration was 8 mM, restoring germination to 78% and recovering fresh weight to near-control levels (1.322 vs. 1.354 g), indicating not just improved germination but enhanced seedling vigor. However, at 10 mM, both parameters declined (70.7% germination, 0.664 g fresh weight), suggesting a biphasic response typical of osmolyte applications.

3.3. Morphophysiological Responses in In Vitro Culture

3.3.1. Vegetative Development and Biomass Distribution

Proline treatment differentially affected plant organs (

Table 3. Key morphophysiological and photosynthetic parameters of Physalis ixocarpa showing significant responses to salt stress and exogenous proline treatment.

Treatment	Green Leaves (Count)	Root Length (cm)	Root Dry Weight (g)	Stem Dry Weight (g)	Chlorophyll a (Abs at 664 nm)	Chlorophyll b (Abs at 649 nm)
Control	0.951 ± 0.961 ab	9.721 ± 2.459 c	0.072 ± 0.025 c	0.061 ± 0.020 ab	0.149 ± 0.037 bc	0.088 ± 0.017 ab
75 mM NaCl	0.404 ± 0.612 a	8.540 ± 1.253 bc	0.053 ± 0.020 bc	0.073 ± 0.021 b	0.051 ± 0.031 a	0.066 ± 0.020 a
NaCl + 4 mM Pro	1.176 ± 0.747 ab	5.809 ± 0.985 a	0.030 ± 0.014 ab	0.048 ± 0.017 ab	0.061 ± 0.019 ab	0.066 ± 0.017 a
NaCl + 6 mM Pro	1.394 ± 0.878 ab	6.562 ± 1.287 ab	0.023 ± 0.003 a	0.047 ± 0.011 ab	0.235 ± 0.022 c	0.127 ± 0.016 b
NaCl + 8 mM Pro	1.621 ± 0.449 ab	5.079 ± 1.458 a	0.019 ± 0.006 a	0.044 ± 0.013 a	0.363 ± 0.031 d	0.190 ± 0.017 c
NaCl + 10 mM Pro	2.186 ± 1.108 b	6.239 ± 1.109 ab	0.027 ± 0.010 a	0.051 ± 0.018 ab	0.234 ± 0.057 c	0.133 ± 0.027 b
Pr > F (model)	0.007	<0.0001	<0.0001	0.022	<0.0001	<0.0001

Values: means ± SD of 7 replicates with 5 plants per replicate (n = 35 plants per treatment). Different letters indicate significant differences (Tukey’s test, p < 0.05).

). Green leaf numbers increased with 10 mM proline (2.186 vs. 0.404 in salt-stressed controls). In contrast, both root and stem development were reduced by proline application. Root length decreased from 9.721 cm (control) to 5.079 cm (8 mM proline), with root dry weight falling from 0.072 to 0.019 g. Similarly, stem dry weight was lowest in the 8 mM proline treatment (0.044 g), significantly less than in the salt-stressed plants (0.073 g). Parameters such as senescent leaves (p = 0.172), shoot length (p = 0.101), and leaf dry weight (p = 0.324) showed no significant differences among treatments and are presented in Table S1.

The visual differences among treatments are evident in Figure 1, which shows representative plants after 30 days of in vitro culture. Control plants exhibited vigorous growth with expanded green leaves, while salt-stressed plants showed stunted growth and chlorotic symptoms. Proline treatment (particularly at 8 mM) partially restored plant vigor, with improved leaf coloration despite reduced root development.

3.3.2. Chlorophyll Content

Proline demonstrated pronounced protective effects on the photosynthetic apparatus (Table 3). The 8 mM treatment yielded the highest chlorophyll a (0.363 Abs at 664 nm) and b (0.190 Abs at 649 nm) contents, exceeding even non-stressed controls (0.149 and 0.088, respectively).

3.4. ATR-FTIR Analysis of Plant Tissues

Spectroscopic analysis was employed to identify changes in the functional groups [24,25,26,27,28] of chemical constituents in the leaves, stems, and roots of P. ixocarpa under control, salt stress (75 mM NaCl), and salt stress with proline (8 mM) conditions. The analysis revealed distinct spectral modifications indicative of a coordinated plant response to salinity and the mitigating effects of proline (Table S2).

Table 4. Key ATR-FTIR spectral changes (cm⁻¹) showing tissue-specific responses to treatments.

Family	Assignment	Control	75 mM NaCl	75 mM NaCl + 8 mM Proline	Salt Effect	Proline Effect
Leaf tissue
Carboxylates/ Carbonyls	C=O stretching; characteristic of the acetyl ester bond	ND	1739	1737	Appears	Unchanged vs. salt
Lignin/Aromatics	C-O-C vibrations in aromatic ether structures (as in lignins)	1145	ND	1147	Disappears	Similar to control
Other	symmetric stretching vibration of methyl groups (CH3)	ND	2877	ND	Appears	Similar to control
Other	stretching of C-O and C-N bonds, as well as N-H bending vibrations	ND	1233	1233	Appears	Unchanged vs. salt
Polysaccharides	O-H with different degrees of hydrogen bonding (cellulose, hemicelluloses, pectin...)	ND	3311	ND	Appears	Similar to control
Polysaccharides	O-H with different degrees of hydrogen bonding (cellulose, hemicelluloses, pectin...)	3274	3281	3274	Shift +7.0	Similar to control
Polysaccharides	asymmetric C-O-C stretching in polysaccharides	1101	1096	1102	Shift −5.0	Similar to control
Stem tissue
Carboxylates/ Carbonyls	C=O stretching; characteristic of the acetyl ester bond	1737	ND	1738	Disappears	Similar to control
Carboxylates/ Carbonyls	C=O stretching in esters, anhydrides, or lactones	1762	ND	1762	Disappears	Similar to control
Carboxylates/ Carbonyls	“Fermi band” of aldehydes	2733	ND	2731	Disappears	Similar to control
Lignin/Aromatics	C-O-C vibrations in aromatic ether structures (as in lignins)	ND	1176	1161	Appears	Shift vs. salt
Lignin/Aromatics	out-of-plane bending of aromatic C-H bonds	ND	740	ND	Appears	Similar to control
Lignin/Aromatics	typical of lignin	ND	1515	ND	Appears	Similar to control
Lignin/Aromatics	O-H deformation in carboxylic acids; syringyl units in lignins	1203	ND	1203	Disappears	Similar to control
Lignin/Aromatics	asymmetric stretching of carboxylates (COO−) and C=C stretching in aromatic rings	1586	ND	1586	Disappears	Similar to control
Lignin/Aromatics	C=O stretching in aldehydes, carboxylic acids, and phenols (kaempferol)	1683	ND	1683	Disappears	Similar to control
Lignin/Aromatics	guaiacyl structures in lignin	848	856	848	Shift +8.0	Similar to control
Lignin/Aromatics	C=C stretching in aromatic rings	1485	1474	1483	Shift −11.0	Similar to control
Other	asymmetric deformation of -CH2- groups and asymmetric deformation of -CH3 groups	ND	1450	1449	Appears	Shift vs. salt
Other	out-of-plane C-H deformation in structures with trans double bonds (=C-H)	ND	966	ND	Appears	Similar to control
Other	symmetric C-H bonds in methylene groups (-CH2-), typical of waxes	2851	ND	2850	Disappears	Similar to control
Other	O-H stretching, typical of carbohydrates	3403	ND	3402	Disappears	Similar to control
Polysaccharides	ring deformations in pyranose structures (sugar rings)	ND	1150	1131	Appears	Shift vs. salt
Polysaccharides	C-H and O-H bending; typical of cellulose; symmetric COO− stretching of proline	ND	1409	1408	Appears	Shift vs. salt
Polysaccharides	C-H bending (hemicelluloses)	ND	715	ND	Appears	Similar to control
Polysaccharides	symmetric C-O-C stretching in polysaccharides	ND	869	ND	Appears	Similar to control
Polysaccharides	C-O stretching in cellulose and other polysaccharides	ND	989	ND	Appears	Similar to control
Polysaccharides	C-H stretching in C-H groups of carbohydrates or cellulose	ND	2899	ND	Appears	Similar to control
Polysaccharides	O-H with different degrees of hydrogen bonding (cellulose, hemicelluloses, pectin...)	3212	ND	3210	Disappears	Similar to control
Polysaccharides	C-O stretching in polysaccharides (cellulose, hemicellulose, pectins); typical of glycosides	1051	1065	1051	Shift +14.0	Similar to control
Polysaccharides	asymmetric C-O-C stretching in polysaccharides	1101	1116	1102	Shift +15.0	Similar to control
Polysaccharides	C-H bending, characteristics of the pyranose ring in cellulose and hemicellulose	926	931	926	Shift +5.0	Similar to control
Polysaccharides	β-glycosidic bond between monosaccharides	897	887	897	Shift −10.0	Similar to control
Polysaccharides	symmetric deformation of CH2 groups in cellulose and hemicellulose; aromatic ring in phenols	1377	1371	1378	Shift −6.0	Similar to control
Root tissue
Lignin/Aromatics	out-of-plane deformation of aromatic C-H bonds (flavonoids and lignin)	ND	828	827	Appears	Shift vs. salt
Lignin/Aromatics	typical of lignin	ND	1513	1516	Appears	Shift vs. salt
Polysaccharides	O-H with different degrees of hydrogen bonding (cellulose, hemicelluloses, pectin...)	ND	3266	3281	Appears	Shift vs. salt

ND = not detected.

summarizes the most relevant spectral changes, selected based on their magnitude and interpretative significance. We focused on bands that either appeared de novo, completely disappeared, or showed substantial shifts (≥5 cm⁻¹) between treatments, as these changes indicate significant molecular reorganization rather than minor conformational adjustments. Minor spectral variations (<5 cm⁻¹) were excluded as they likely represent subtle structural modifications without major functional implications.

In the leaf tissue, salt stress prompted the appearance of several new absorption bands that were absent in the control samples. These included bands at 3311 cm⁻¹ (O-H bonds in polysaccharides), 2877 cm⁻¹ (symmetric stretching of CH₃ groups), 1739 cm⁻¹ (C=O stretching in acetyl esters), 1436 cm⁻¹ (asymmetric deformation of -CH₂- groups and asymmetric deformation of -CH₃ groups), and 1233 cm⁻¹ (stretching of C-O and C-N bonds, as well as N-H bending vibrations). Concurrently, a band at 1145 cm⁻¹, associated with C-O-C vibrations in aromatic ethers like lignin, disappeared under salt stress. The application of proline alongside salt stress reversed some of these changes, making the spectra more similar to the control. For instance, the bands at 2877 cm⁻¹ and 1145 cm⁻¹ returned to their control state (absent and present, respectively). However, the bands at 1739 cm⁻¹ and 1233 cm⁻¹ that appeared with salt stress remained unchanged by proline addition. A notable shift was observed in the band for O-H bonds in polysaccharides, which moved from 3274 cm⁻¹ in the control to 3281 cm⁻¹ under salt stress, a shift that was fully reversed by proline treatment.

The stem tissue exhibited the most extensive spectral alterations. Salt stress induced the disappearance of numerous bands present in the control, particularly those related to carbohydrates and lignin. These included signals for O-H stretching (3403 and 3212 cm⁻¹), C=O stretching in various compounds like aldehydes, esters, and phenols (1762, 1737, and 1683 cm⁻¹), carboxylate and aromatic ring vibrations (1586 cm⁻¹), and C-H bonds in waxes (2851 cm⁻¹). The application of proline restored all of these bands to a profile similar to the control. Conversely, salt stress led to the emergence of many new bands, such as those for lignin (1515 and 740 cm⁻¹), polysaccharides (2899, 1150, 989, 869, 715 cm⁻¹), and C-H deformations (1450 and 966 cm⁻¹). The proline treatment caused many of these newly appeared bands (2899, 1515, 989, 966, 869, 740, and 715 cm⁻¹) to disappear, again reflecting a reversion to the control profile. Several bands showed significant shifts under salt stress, which were later restored by proline. For example, a band for C=C stretching in aromatic rings shifted from 1485 cm⁻¹ to 1474 cm⁻¹, and a band for glycosidic bonds in polysaccharides shifted from 1051 cm⁻¹ to 1065 cm⁻¹; both returned to control values with proline.

In the root, the spectral changes were less numerous but still clear. Salt stress induced the appearance of new bands associated with lignin and aromatic compounds at 1513 cm⁻¹ and 828 cm⁻¹, as well as a polysaccharide-related O-H band at 3266 cm⁻¹. Unlike in other tissues, these bands did not disappear with proline treatment but instead showed a slight shift in their position, suggesting that while proline influenced these structures, it did not fully reverse the salt-induced changes in the roots.

3.5. GC-MS Metabolomic Analysis

The GC–MS profiles of P. ixocarpa leaf and stem extracts revealed marked compositional shifts between the control (non-saline), salt stress (75 mM NaCl), and salt stress with exogenous proline application (8 mM) treatments.

Under non-saline conditions (Table S3), the dominant peak was ethyl α-D-glucopyranoside (21.93%), accompanied by lipid monoesters such as hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester (6.96%), and octadecanoic acid, 2,3-dihydroxypropyl ester (4.86%). Saturated fatty acids were also abundant, including n-hexadecanoic acid (3.67%), octadecanoic acid (0.96%), (Z,Z,Z)-9,12,15-octadecatrienoic acid (1.20%), and (Z,Z)-9,12-octadecadienoic acid (0.91%), along with phenolics such as catechol (1.92%) and 4-ethylcatechol (1.29%), and nitrogenous heterocycles like 2-pyrrolidinone (2.00%). The control extract also contained L-proline, 5-oxo-, methyl ester (1.30%). These components suggest a baseline metabolic profile focused on energy storage, membrane integrity, and secondary metabolism in unstressed plants.

Saline stress induced notable shifts in the phytoconstituent composition (Table S4), with a reduction in the abundance of certain carbohydrate derivatives and an increase in amine and cyclic compounds potentially linked to osmotic adjustment and stress tolerance. The dominant compound shifted to ethyl β-D-riboside at 13.03%, replacing the higher abundance of ethyl α-D-glucopyranoside observed in the control. Lipid components remained important, with octadecanoic acid, 2,3-dihydroxypropyl ester (6.55%) and glycerol 1-palmitate (6.16%) among the main constituents, indicating enhanced glycerol-based lipid metabolism under salt exposure. Fatty acids remained significant, with n-hexadecanoic acid at 3.14%, (Z,Z)-9,12-octadecadienoic acid at 1.36%, octadecanoic acid at 0.99%, and (Z,Z,Z)-9,12,15-octadecatrienoic acid at 0.89%, but their relative proportions decreased compared to the control. Unique to the saline-stressed extract were compounds such as N,N-dimethyl-1-dodecanamine (4.89%), 2-dodecyl-5-methylpyrrolidine (4.07%), cyclodecane (2.86%), and oxalic acid, cyclohexyl ethyl ester (2.46%), suggesting upregulation of amine derivatives and cyclic structures that may contribute to ion homeostasis or antioxidant activity. Phenolic compounds like 2,4-bis(1,1-dimethylethyl)-phenol increased to 1.41%, while others, such as catechol, were absent, implying a reallocation of resources toward stress-responsive pathways. L-proline, 5-oxo-, methyl ester increased to 1.76%, and 2-pyrrolidinone persisted at a similar level (2.03%). Overall, saline stress appeared to diminish the abundance of glycosides and phenolics while promoting nitrogenous and lipid-related compounds, consistent with metabolic reprogramming to cope with osmotic and ionic imbalances.

With exogenous proline under salt stress, the profile (Table S5) reverted in part to the control-like pattern, with ethyl α-D-glucopyranoside again becoming the most abundant peak (21.40%). Lipid monoesters, including hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester (7.73%) and octadecanoic acid, 2,3-dihydroxypropyl ester (5.42%), approached control values. Fatty acids showed similar trends, with n-hexadecanoic acid elevated to 4.61%, octadecanoic acid at 1.18%, (Z,Z,Z)-9,12,15-octadecatrienoic acid at 1.11%, and (Z,Z)-9,12-octadecadienoic acid at 0.49%, reflecting partial recovery from stress-induced reductions. The proline-treated extract featured several proline derivatives and metabolism-related compounds absent or less abundant in the other profiles, including pyrrolidine (2.83%), 1-pyrrolidinylacetonitrile (2.73%), DL-proline, 5-oxo- (1.09%), 1-methyl-2-pyrrolidinone (0.54%), 5-methoxypyrrolidin-2-one (0.47%), 1-methyl-2-pyrrolidone-4-carboxamide (0.48%), and 1-[2-oxo-4-(1-pyrrolidinyl)butyl]-2-pyrrolidinone (0.47%), which are indicative of enhanced proline catabolism or derivatization. Compounds unique to this treatment, such as oxalic acid, monomorpholide, ethyl ester (1.59%), and 6-methyl-5-oxo-1,2,3,5-tetrahydro-imidazo(1,2-a)pyrimidine (0.87%), further highlight proline’s influence on nitrogen metabolism. Compared to saline stress alone, proline application reduced the prominence of stress-specific amines like N,N-dimethyl-1-dodecanamine, and increased heterocyclic diversity.

A side-by-side quantitative comparison of selected major compounds is shown in

Table 5. Quantitative comparison (% area) of selected major compounds in the GC-MS profiles of the control, salt-stressed, and salt-stressed with exogenous proline application stem + leaf extracts.

Compound	Control	75 mM NaCl	75 mM NaCl + 8 mM Proline
Ethyl α-D-glucopyranoside	21.93	ND	21.40
Ethyl β-D-riboside	ND	13.03	ND
Hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester	6.96	ND	7.73
Octadecanoic acid, 2,3-dihydroxypropyl ester	4.86	6.55	5.42
n-Hexadecanoic acid	3.67	3.14	4.61
2-Pyrrolidinone	2.00	2.03	1.83
Catechol	1.92	ND	ND
L-Proline, 5-oxo-, methyl ester	1.30	1.76	ND
DL-Proline, 5-oxo-	ND	ND	1.09
N,N-dimethyl-1-Dodecanamine	ND	4.89	ND
2-Dodecyl-5-methylpyrrolidine	ND	4.07	ND
Pyrrolidine	ND	ND	2.83
1-Pyrrolidinylacetonitrile	ND	ND	2.73

ND = not detected.

. This quantitative comparison highlights two clear trends. First, salt stress caused a shift in the dominant carbohydrate from ethyl α-D-glucopyranoside to ethyl β-D-riboside, while also introducing long-chain amines and alkyl-substituted pyrrolidines not detected in the control. Second, proline supplementation restored the carbohydrate profile toward the control state and was associated with the emergence of multiple pyrrolidine-based metabolites.

4. Discussion

4.1. Optimal Salt Stress Level and Proline Dosage

The selection of 75 mM NaCl as the experimental stress level was based on achieving moderate stress (38.9% germination reduction) while maintaining seedling viability. This threshold aligns with established screening protocols where 30–40% germination reduction represents sufficient stress to evaluate tolerance mechanisms without causing complete growth arrest [2,29]. The comparable threshold of 80.7 mM reported in artichoke (calculated from the −0.40 MPa osmotic potential using the Van’t Hoff equation) [30] and similar values in other solanaceous crops [31] validate this choice. Higher concentrations (≥100 mM) arrested development at radicle protrusion, preventing meaningful physiological assessments.

The optimal effect at 8 mM under salt stress reflects the compound’s concentration-dependent nature, falling within the 5–10 mM range reported as effective across multiple species [22], and consistent with observations in other Solanaceae where low to moderate proline levels enhance tolerance but higher doses may reduce benefits. Specifically, the reduced efficacy above 10 mM aligns with the findings of Heuer [32] in tomato and Kong et al. [33] in rice, where exogenous proline at 10 mM was toxic to the plants and reduced seed germination, respectively. According to Alfosea-Simón et al. [34], excessive proline may disrupt cellular homeostasis or trigger feedback inhibition of endogenous proline synthesis. Additionally, Rajasheker et al. [35] note that excessive osmolyte accumulation can function as a growth regulator, potentially explaining why higher concentrations become counterproductive despite their osmotic potential.

4.2. Resource Reallocation Strategy

The coordinated reduction in both root and stem biomass under proline treatment, coupled with enhanced chlorophyll content, strongly indicates a resource reallocation strategy. This parallels responses in pepper, where some varieties prioritize metabolic protection over structural growth under stress [36]. Under salt stress alone, plants exhibited the highest stem dry weight, suggesting a possible investment in structural reinforcement to cope with stress. However, with proline application, resources were diverted away from both root development (root length reduced from 9.72 to 5.08 cm) and stem growth (stem dry weight reduced from 0.073 to 0.044 g). Rather than representing stress damage, this trade-off suggests that exogenous proline provides sufficient osmotic protection, allowing resources normally invested in structural tissues to be redirected toward preserving and enhancing the photosynthetic machinery. This aligns with observations in tomato, where proline improved photosynthetic efficiency under salinity [22,37]. Lin et al. [38] also reported that exogenous application of proline markedly inhibited root growth of rice.

4.3. Proline’s Multifaceted Protective Mechanisms

The FTIR spectral data provide compelling evidence of the biochemical adjustments in P. ixocarpa in response to salt stress and demonstrate the protective role of exogenous proline.

The disappearance and shifting of bands in the 3400–3200 cm⁻¹ (O-H stretching) and 1200–900 cm⁻¹ (C-O, C-O-C stretching) regions, particularly in the stem, point to significant modifications in polysaccharides such as cellulose, hemicellulose, and pectins. Salinity is known to affect carbohydrate metabolism, and these spectral changes, including shifts in the β-glycosidic bond signal (897 cm⁻¹), suggest a reconfiguration of cell wall components and storage carbohydrates. The restoration of these carbohydrate-related bands to control-like states upon proline application is a key finding. It suggests that proline helps maintain carbohydrate balance, preventing the drastic salt-induced metabolic shifts. This is consistent with the GC-MS results, which show proline treatment restores the primary carbohydrate, ethyl α-D-glucopyranoside, to control levels after it was diminished by salt.

Changes related to lignin and other phenolic compounds were prominent. In the stem, multiple bands assigned to lignin and aromatics disappeared under stress, while in the root, new lignin-associated bands appeared. This suggests a tissue-specific response, potentially involving the degradation of certain phenolic structures in the stem while reinforcing the root cell walls through lignification to control ion uptake. This tissue-specific response aligns with observations in Solanaceae under salt stress, where proline application can modulate cell wall composition to reduce ion toxicity [35]. The restoration of several key structural bands demonstrates proline’s protective effects on cellular architecture. In stems, the return of the polysaccharide band from 1065 cm⁻¹ (salt-shifted) to its original position at 1051 cm⁻¹, along with the restoration of the 1101 cm⁻¹ band at 1102 cm⁻¹, indicates that proline helps maintain or restore normal polysaccharide conformation. The similar restoration of the guaiacyl lignin band from 856 to 848 cm⁻¹ suggests that proline also normalizes lignification patterns disrupted by salt stress, particularly in stems and leaves, though root changes were not fully reversed.

Finally, the appearance of bands related to C=O (1739 cm⁻¹) and C-N (1233 cm⁻¹) bonds in leaves under both salt and salt-plus-proline treatments suggests proline maintains certain adaptive modifications rather than reversing all stress-induced changes. These may correspond to the formation of new stress-related esters or nitrogenous compounds that become a stable part of the leaf’s metabolic profile under saline conditions.

Overall, the FTIR analysis corroborates the view of salinity as a significant stressor that remodels plant biochemistry and highlights the efficacy of proline in mitigating these effects, primarily by stabilizing carbohydrate profiles and participating directly in the metabolic stress response.

4.4. GC-MS Metabolomic Analysis

The GC–MS data demonstrate that P. ixocarpa undergoes substantial metabolic reprogramming under salt stress, with changes encompassing primary metabolites such as carbohydrates and fatty acids, as well as nitrogen-containing secondary metabolites. The shift from ethyl α-D-glucopyranoside dominance in the control to ethyl β-D-riboside under salt stress suggests a reallocation of carbon skeletons, consistent with the osmotic and salt stress-induced carbohydrate alterations reported by Kerepesi et al. [39]. Pentoses require less carbon investment per osmotic unit and may better stabilize cellular structures under dehydration stress, representing an energy-efficient osmotic adjustment strategy.

Salt stress also promoted the accumulation of nitrogen-rich compounds not prominent in the control, including long-chain dimethylamines and alkyl-substituted pyrrolidines. These molecules may act as osmoprotectants, membrane stabilizers, or signaling intermediates, reflecting a biochemical strategy to mitigate ionic toxicity. The concurrent disappearance of phenolic antioxidants, particularly catechol (1.92% in control, absent under salt stress), warrants careful interpretation. Rather than indicating compromised antioxidant capacity, this unexpected finding may reflect (1) a redirection of phenylpropanoid pathway flux toward lignification for structural reinforcement, as supported by our FTIR data showing tissue-specific alterations in lignin-associated bands, particularly the appearance of new lignin bands in roots (1513 cm⁻¹) while stem lignin bands disappeared, or (2) proline’s effectiveness in preventing oxidative stress formation through preemptive osmotic and membrane stabilization, thereby reducing the metabolic cost of phenolic antioxidant synthesis. The persistence of this phenolic absence under proline treatment supports the latter hypothesis, aligning with the energy-efficient stress tolerance strategy proposed by Rajasheker et al. [35], where osmolyte-mediated protection reduces the need for energy-expensive secondary metabolite production.

While our data strongly suggest that proline prevents oxidative damage through metabolic regulation and membrane stabilization, we acknowledge that direct measurement of oxidative stress markers would provide definitive evidence for this mechanism. Future studies should quantify malondialdehyde (MDA) and hydrogen peroxide (H₂O₂) levels to confirm whether proline-treated plants indeed experience lower oxidative damage despite reduced phenolic content.

Several alternative mechanisms may explain how proline provides protection without inducing phenolic synthesis. Exogenous proline has been shown to enhance enzymatic antioxidant systems, including superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX), which can compensate for reduced phenolic antioxidants [40,41]. Additionally, proline strengthens the ascorbate–glutathione (AsA-GSH) cycle, improving H₂O₂ detoxification through enhanced activities of dehydroascorbate reductase and glutathione reductase while maintaining favorable AsA/DHA and GSH/GSSG ratios [42,43]. Proline itself also functions as a direct radical scavenger and protein stabilizer, potentially reducing primary reactive oxygen species (ROS) generation in chloroplasts and mitochondria [20]. These mechanisms collectively provide plausible explanations for the observed depletion of phenolic antioxidants that are not restored by exogenous proline treatment.

The application of exogenous proline during salt stress induced a distinct compositional response. The restoration of ethyl α-D-glucopyranoside to levels comparable to the control, alongside increased monoacylglycerols and saturated fatty acids, points to a stabilization of membrane lipid composition and carbohydrate metabolism. Moreover, this treatment was characterized by the appearance and relative enrichment of several pyrrolidine-based metabolites (pyrrolidine, 1-pyrrolidinylacetonitrile, DL-proline, 5-oxo-, and various pyrrolidinones) that were absent or minor under the other conditions. These compounds are chemically consistent with proline-derived structures, and their emergence supports the interpretation that the supplied proline was at least partially metabolized into related cyclic and amide derivatives. Such transformations may contribute to osmotic adjustment and redox regulation, functions often attributed to proline and its catabolites [20,44]. The appearance of pyrrolidine derivatives (2.83%) alongside other nitrogenous metabolites suggests active proline catabolism and metabolic redirection, a pattern also observed in tomato, where exogenous proline enhanced nitrogenous compounds while reducing Na⁺ accumulation [32,37]. This metabolic reprogramming likely involves the abscisic acid (ABA)-dependent signaling pathways described by Rajasheker et al. [35], where proline catabolism through the proline–Δ¹-pyrroline-5-carboxylate (P5C) cycle [20] contributes to cellular redox homeostasis during stress recovery.

The pre-emptive protection mechanism is supported by multiple lines of evidence. Proline functions as an osmoprotectant that maintains cellular turgor before dehydration-induced damage occurs, with cellular proline concentrations increasing up to 100-fold within hours of stress initiation [20,44]. This rapid accumulation prevents the concentration of reactive species that would otherwise trigger oxidative cascades [45]. Furthermore, proline directly stabilizes membrane phospholipids through hydrogen bonding with both phosphate groups and fatty acid chains, preventing lipid peroxidation at its source [46,47]. The metabolic cost analysis reveals that proline synthesis requires only 2 adenosine triphosphate (ATP) and 2 nicotinamide adenine dinucleotide phosphate (NADPH) per molecule via the Δ¹-pyrroline-5-carboxylate synthetase (P5CS)-pyrroline-5-carboxylate reductase (P5CR) pathway [48], whereas phenolic biosynthesis through the shikimate and phenylpropanoid pathways demands substantially higher energy investment—approximately 15 ATP and 10 NADPH per lignin monomer unit [49,50]. This 5-fold difference in energy requirements explains why proline-mediated protection represents an evolutionarily advantageous strategy, allowing plants to maintain protection while preserving resources for growth and development [35].

Interestingly, the abundance of certain nitrogenous compounds elevated in the salt-stressed plants without proline (such as N,N-dimethyl-1-dodecanamine and 2-dodecyl-5-methylpyrrolidine) was reduced in the proline-supplemented plants. This shift suggests that exogenous proline may partially replace or downregulate alternative osmolyte pathways, redirecting nitrogen flux toward proline-derived metabolites instead of long-chain amines. This modulation of nitrogen partitioning could represent a more efficient osmoprotective strategy, leveraging the multifunctional role of proline in stress physiology.

Overall, the results indicate that salt stress alone reconfigures the metabolic profile toward nitrogen-rich osmolytes and altered carbohydrate identity, whereas the presence of exogenous proline not only restores control-like features but also enriches the profile with proline-related derivatives. These findings align with the dual role of proline as both a protective osmolyte and a precursor to nitrogen-containing metabolites with potential antioxidant and signaling functions [44]. This is consistent with reviews indicating that exogenous proline enhances salt tolerance in crops like pepper and tomato by modulating osmolyte accumulation and reducing oxidative stress [22,35].

4.5. Implications for Agricultural Practice

The findings suggest that exogenous proline application at 8 mM via seed pretreatment could improve germination and early growth of P. ixocarpa under moderate salinity, potentially extending cultivation in semi-arid regions where this crop is economically important. Given the crop’s importance in Mexican cuisine and its cultivation in semi-arid regions prone to soil salinization, this approach may help maintain productivity in marginal lands where irrigation water quality is poor. The pretreatment method is simple, requiring only brief imbibition and air-drying, making it feasible for small-scale farmers without specialized equipment. In related Solanaceae, field applications have shown promising but variable results. Kahlaoui et al. [37] demonstrated yield improvements in tomato through enhanced chlorophyll fluorescence and improved ion balance, though responses were cultivar-specific. Similarly, in pepper, Escalante Magaña [36] observed genotype-dependent effects, with some varieties showing enhanced tolerance while others showed limited response. These findings suggest that while P. ixocarpa shows a robust response to 8 mM proline in our study, field validation across different cultivars will be essential before commercial implementation. Implementation costs could be reduced through optimization of proline concentration for specific soil salinity levels and exploration of alternative proline sources or biosynthetic precursors.

4.6. Methodological Considerations

The integration of morphophysiological, spectroscopic, and metabolomic analyses proved essential for understanding proline’s complex effects, revealing mechanisms like carbohydrate restoration that single methods might miss. This aligns with studies in Solanaceae where integrated analyses have elucidated osmolyte roles in salt tolerance [35]. Limitations include the in vitro setup, which may not reflect field conditions such as microbial interactions or prolonged stress. Future work could quantify proline uptake to differentiate exogenous from endogenous contributions and include temporal metabolomics to track response dynamics, as suggested for abiotic stress research [22].

5. Conclusions

This study shows that exogenous proline application at 8 mM effectively mitigates salt stress in P. ixocarpa through multiple coordinated mechanisms. Rather than simply providing osmotic adjustment, proline induces fundamental metabolic reorganization that prioritizes photosynthetic protection over structural growth, as evidenced by an enhancement in chlorophyll content alongside reduced root and stem biomass. The restoration of normal carbohydrate profiles, despite the continued absence of phenolic antioxidants, reveals a protective strategy where proline mitigates oxidative damage through metabolic regulation of osmolyte pathways and membrane stabilization rather than through phenolic antioxidant induction, representing an energy-efficient alternative to conventional stress responses. The detection of multiple pyrrolidine derivatives indicates active proline metabolism extending beyond osmotic protection, with potential contributions to cellular redox balance and nitrogen metabolism. These findings advance understanding of proline beyond simple osmolyte function, revealing its role as a metabolic regulator that enables energy-efficient stress tolerance. The practical seed pretreatment protocol and mechanistic insights provide strategies for cultivating P. ixocarpa in saline soils, with potential to extend production into marginal lands.