The Effect of One-Year Seed Spaceflight Storage on Yield, Biochemical and Mineral Characteristics of Mature Leafy Vegetables Belonging to Brassicaceae, Apiaceae and Asteraceae Families

Abstract

Spaceflight is known to produce genetic changes in seeds, usually accelerating aging, though species and varietal differences have been poorly investigated. Comparisons were carried out in terms of yield, biochemical characteristics and mineral composition between mature plants grown from seeds subjected to a one-year spaceflight, belonging to Brassicaceae (Brassica juncea and Eruca sativa), Apiaceae (Anethus graveolens, and Coriandrum sativum), and Asteraceae (Lactuca sativa, six cultivars) families, and non-treated control. Among the studied species, only Brassica juncea and Eruca sativa demonstrated a growth stimulation effect caused by seed spaceflight, while significant growth inhibition was recorded in Apiaceae plants and three cultivars of Lactuca sativa L. No differences in the total antioxidant activity (AOA), polyphenol and ascorbic acid content were detected between ‘space-treated’ and control plants. On the contrary, significant decrease in proline accumulation and increase in malonic dialdehyde and photosynthetic pigments levels were shown by Brassicaceae species. The effect of long-term seed spaceflight on the mineral composition of mature plants was reflected in the inhibition of accumulation of all 24 elements analyzed in Apiaceae plants, except for Se, whose concentration was higher in all ‘space-treated’ plants compared to the control. Spaceflight seed storage increased V levels in lettuce and decreased Na accumulation in all the investigated species. The results reveal species-dependent changes in the accumulation of macro-, micro- and toxic elements in Apiaceae, Brassicacea, and Asteraceae representatives due to spaceflight seed storage. The detected differences in plant elemental composition between ‘space’ treatment and control partly explain the corresponding yield gap and suggest a relationship between mineral status and adaptability. The highest beneficial effect of spaceflight seed storage on yield was recorded in Eruca sativa cultivar, Rusalochka.

1. Introduction

The effects of environmental stresses on plant growth and development have constantly drawn attention of researchers due to the vital role of plants in human survival both on Earth and in long-term spaceflight. In the latter case, cosmic radiation, microgravity, decreased magnetic field, and circadian rhythms disturbances represent the main stress factors affecting both humans and plants [1]. Some studies investigated seed viability in conditions of long-term exposure to the harsh space environment without and with shielding against space radiation [2,3,4]. Previous investigations were conducted with seeds of rice [5], tomato [6,7], Eruca sativa [8], Arabidopsis, tobacco [3], licorice [9], Glycyrrhiza uralensis [10], Robina pseudoacazia [11], and Acer mono maxim [12]. Multiple investigations of seeds of more than 140 species [13,14,15] have allowed researchers to identify more than 200 promising varieties with premium quality, yield and disease resistance, suggesting great species and varietal differences in plant response to spaceflight seed storage [16]. A remarkable increase in germination rate was recorded in space-mutated seeds of wheat, maize, barley, triticale, soybean, sunflower, cucumber, tomato and cotton, while no significant differences were noted in rice, pea, millet, lettuce, sweet pepper and tobacco. Conversely, reduced seed germination rates were recorded in sorghum, watermelon, eggplant, radish and towel gourd [15,17]. Half-year spaceflight storage of lettuce rocket seeds led to reduced ‘space’ seed germination vigor, and a remarkable increase in seed aging-sensitivity [8]. Significant oxidant stress in tomato fruit was recorded due to half a year seed storage in ISS, reflected in high total antioxidant activity, polyphenol and ascorbic acid contents [6]. Investigations of the effect of a 15-day space flight revealed an acceleration of alfalfa seed germination and inhibition of the root growth due to chromosomal damage and abnormal mitosis induced by cosmic radiation [18]. Other results revealed reduced germination, lethality, sterility, and accelerated senescence [19,20,21]. Multiple studies conducted in ISS revealed significant changes in hormonal, amino acid, protein biosynthesis, and redox homeostasis in plants due to short-term spaceflight [5,22,23,24,25]. The latter phenomena suggest that the response variations to spaceflight seed storage are highly species and varietal dependent, and there are great prospects for quick space plant breeding to produce crops with shortened vegetation phases, high yields, and resistance to diseases [26,27,28].

The need to evaluate the patterns of plant protection in these conditions stimulates further investigations to reveal similarities in the response of different species to spaceflight seed storage. In this respect, the present study was aimed at assessing species and varietal differences in biochemical and mineral composition of mature plants belonging to Apiaceae, Asteraceae, and Brassicaceae families, caused by one-year seed spaceflight storage.

2. Material and Methods

2.1. Experimental Protocol and Growing Conditions

Research was carried out in a greenhouse at the Federal Scientific Vegetable Center, to assess yield, biochemical and mineral characteristics of green and spicy flavoring plants, i.e., 6 cultivars of Lactuca sativa L., Asteraceae family (cvs. Moskovsky parnikovy, Petrovich, Synthesis. Picnic, Cavalier and Bouquet), 2 species of Brassicaceae family (Brassica juncea, cv. Sudarushka, and Eruca sativa, cv. Rusalochka) and 2 representatives of Apiaceae family (Anethus graveolens, cv. Kulinar, and Coriandrum sativum, cv. Yubilar) grown from seeds subjected to the International Space Station (ISS) ‘Science’ module in sealed foil bags from 5 October 2021 to 10 October 2022 (Figure 1), in comparison with plants from untreated seeds.

Figure 1. Seed appearance of the tested species and cultivars.

A randomized complete block design was used for the treatment distribution in the greenhouse, with three replicates.

The air temperature on board during the spaceflight seed storage was 22–23 °C, with 25% humidity inside the seed sealed foil bags. After returning to Earth, the seeds were kept in a refrigerator at +7 °C before sowing. The control seeds were also stored in sealed foil bags at the same temperature and humidity conditions, during the whole experiment at the Federal Scientific Vegetable Center.

Sowing was carried out on 20 April 2023 in plastic containers, 7.5 L each, with 15 plants per pot, and were grown in the greenhouse. The harvests of Lactuca sativa, Brassica juncea and Eruca sativa took place on 11 May 2023, and those of Anethus graveolens and Coriandrum sativum on 15 May 2023 during the phase of commercial usefulness.

2.2. Sample Preparation

After harvesting, plants were cleaned with fresh water to remove any soil and dried with filter paper. Leaf area, plant total weight, and height were measured. The samples were homogenized and a fraction of them was used as fresh homogenates to determine ascorbic acid and nitrates; the other part was dried at 70 °C to constant weight and used to measure polyphenols, proline, malonic dialdehyde content, total antioxidant activity and mineral composition.

2.3. Dry Matter

The dry residue was assessed gravimetrically by drying the samples in an oven for 3 days at 70 °C until constant weight.

2.4. Mineral Composition

The Al, As, B, Ca, Cd, Co, Cr, Cu, Fe, I, K, Li, Mg, Mn, Mo, Na, Ni, P, Pb, Se, Si, Sr, V, and Zn contents in dried homogenized samples were assessed using ICP-MS on the quadruple mass-spectrometer Nexion 300D (Perkin Elmer Inc., Shelton, CT, USA), equipped with the seven-port FAST valve and an ESI SC DX4 autosampler (Elemental Scientific Inc., Omaha, NE, USA) at the Biotic Medicine Center (Moscow, Russia). Rhodium 103 Rh was used as an internal standard to eliminate instability during measurements. The Quantitation was performed using external standards (Merck IV, multi-element standard solution); Perkin– Elmer standard solutions for P, Si, and V, and all the standard curves were obtained at five different concentrations. For quality checking purposes, internal controls and reference materials were tested together with the samples daily. Microwave digestion of samples was carried out with sub-boiled HNO₃ diluted 1:150 with distilled deionized water (Fluka No. 02, 650 Sigma-Aldrich, Co., Saint Louis, MO, USA) in the Berghof SW-4 DAP-40 microwave system (Berghof Products + Instruments Gmb H, 72, 800 Eningen, Germany). The instrument conditions and acquisition parameters were: plasma power and argon flow, 1500 and 18 L min⁻¹, respectively; aux argon flow, 1.6 L min⁻¹; nebulizer argon flow, 0.98 L min⁻¹; sample introduction system, ESI ST PFA concentric nebulizer and ESI PFA cyclonic spray chamber (Elemental Scientific Inc., Omaha, NE, USA); sampler and slimmer cone material, platinum; injector, ESI Quartz 2.0 mm I.D.; sample flow, 637 L min⁻¹; internal standard flow, 84 L min⁻¹; dwell time and acquisition mode, 10–100 ms and peak hopping for all analytes; sweeps per reading, 1; reading per replicate, 10; replicate number, 3; DRC mode, 0.55 mL min⁻¹ ammonia (294993-Aldrich Sigma-Aldrich, Co., St. Louis, MO 63103, USA) for Ca, K, Na, Fe, Cr, V, optimized individually for RPa and RPq; STD mode, for the remainder of analytes at RPa = 0 and RPq = 0.25.

2.5. Ascorbic Acid

The ascorbic acid content was determined by visual titration of leaf extracts in 3% trichloroacetic acid with sodium 2,6-dichlorophenol indophenolate solution (Tillman’s reagent) [29].

2.6. Total Polyphenols (TP)

Total polyphenols were determined in 70% ethanol extract using the Folin–Ciocalteu colorimetric method as previously described [30]. A half gram of dry homogenates was extracted with 20 mL of 70% ethanol at 80 °C for 1 h. The mixture was cooled down and quantitatively transferred to a volumetric flask, and the volume was adjusted to 25 mL. The mixture was filtered through filter paper, and 1 mL of the resulting solution was transferred to a 25 mL volumetric flask, to which 2.5 mL of saturated Na₂CO₃ solution and 0.25 mL of diluted (1:1) Folin–Ciocalteu reagent were added. The volume was brought to 25 mL with distilled water. One hour later, the solutions were analyzed through a spectrophotometer (Unico 2804 UV, Suite E Dayton, NJ, USA), and the concentration of polyphenols was calculated according to the absorption of the reaction mixture at 730 nm. As an external standard, 0.02% gallic acid was used. The results were expressed as mg of Gallic Acid Equivalent per g of dry weight (mg GAE g⁻¹ d.w.).

2.7. Antioxidant Activity (AOA)

The antioxidant activity was assessed using a redox titration method [30] via titration of 0.01 N KMnO₄ solution with ethanolic extracts of dry samples, as described in the Section 2.6. The reduction of KMnO₄ to colorless Mn⁺² in this process reflects the quantity of antioxidants dissolvable in 70% ethanol. The values are expressed in mg Gallic Acid Equivalents (mg GAE g⁻¹ d.w.).

2.8. Proline

Proline concentration was determined according to [31], with slight modifications. About 50 mg of dry homogenized plant leaves were ground with 10 mL of 3% sulfur salicylic acid in a mortar. The mixture was filtered and 1 mL of the resulting filtrate, 2 mL of ninhydrin reagent and 2 mL of acetic acid were heated at 95 °C for 1 h. Proline concentration was assessed using the absorption value of the reaction mixture toluene extract at 505 nm, and a calibration curve with 5 different proline (Merck, Rahway, NJ, USA) concentrations.

2.9. Malonic Dialdehyde

Lipid peroxidation was measured by tracing malonic dialdehyde content using thiobarbituric acid method as described by Heath and Parker [32], with a small modification. About 0.1 g of dried samples were mixed with 10 mL of 0.5% thiobarbituric acid solution and heated at 95 °C for half an hour. The mixture was cooled down and the absorption at 232 nm was determined. MDA content was calculated using the extinction value equal to 155 mM cm⁻¹.

2.10. Photosynthetic Pigments

About 50 mg of fresh samples was homogenized in a porcelain mortar with 10 mL of 96% ethanol. The homogenized sample mixture was quantitatively transferred to a volumetric flask, bringing the volume to 25 mL, and then the mixture was filtered through filter paper. In the resulting solution, analyses of chlorophyll-a, chlorophyll-b and carotene were performed through a spectrophotometer (Unico 2804 UV, USA). The calculation of chlorophyll and carotene concentrations was achieved using appropriate equations [33]:

Ch-a = 13.36A₆₆₄ − 5.19A₆₄₉;

(1)

Ch-b = 27.43A₆₄₉ − 8.12A₆₆₄;

(2)

C c = (1000A₄₇₀ − 2.13 Ch-a − 87.63 Ch-b)/209;

(3)

where A = Absorbance, Ch-a = Chlorophyll a, Ch-b = Chlorophyll b and C c = Carotene.

2.11. Statistical Analysis

The data were processed by one-way analysis of variance and mean separations were performed through the Duncan’s multiple range test, with reference to a 0.05 probability level, using SPSS software version 28 (IBM, Armonk, NY, USA). Data expressed as a percentage were subjected to angular transformation before processing.

3. Results and Discussion

According to plant classification, the three studied plant families belong to Asterids and Rosits clades and differ in their seed dormancy type (Table 1). Indeed, the physiological dormancy type is predominant in seeds of Brassicaceae and Asteraceae species, whereas the morphological and morpho-physiological type of dormancy is more typical of Apiaceae seeds [34,35,36].

Table 1. Characteristics of Brassicaceae, Asteraceae and Apiaceae families.

Family	Dormancy Type	Clade	Ref.
Brassicaceae	Physiological, non-deep physiological	Asterids	[34]
Asteraceae	Non-deep physiological	Rosits Rosits	[35]
Apiaceae	Morphological, morpho-physiological		[36]

3.1. Yield and Biometrical Characteristics

The results of the present study indicate great differences between the examined families in mature plant response to long-term spaceflight seed storage. A negative effect of long-term spaceflight on the seed quality was recorded in the present work by the fact that seeds of only four Lactuca sativa cultivars germinated, while the seeds of cultivars Cavalier and Bouquet did not produce any seedlings. These results are consistent with the well-known observation that long-term exposure to microgravity inside spaceships is often associated with accelerated aging in humans and plants [37], and oxidation promotion of the most important molecules, such as proteins, lipids, and nucleic acids [38,39].

Nevertheless, the behavior of other plant species and cultivars demonstrated extremely high variability in response to spaceflight seed storage (Table 1). Indeed, the weight and height of control and experimental lettuce plants did not differ significantly for cultivars Petrovich and Picnic, and these parameters were slightly higher for cvs. Moskovsky parnikovy and Synthesis, grown from ‘space’ seeds, while variations in dry matter content were insignificant.

Indian mustard demonstrated dry matter increase due to seed spaceflight exposure, and a 23% weight increase.

The most impactful effects of seed long-term spaceflight storage on plant growth were recorded in the experimental plants of Eruca sativa, whose weight and height were 2.40 and 1.32 times higher than the corresponding parameters of the control plants (Table 1).

Interestingly, a previous investigation by Chandler et al. [8] demonstrated significant inhibition of Eruca sativa seedling growth as a result of one-year seed storage in ISS, contrary to the above results. Taking into account the existence of significant varietal differences in the response to the seed spaceflight storage recorded in lettuce cultivars (Table 1) and other crops [16], this may suggest a similar phenomenon for different Eruca sativa cultivars, though the latter hypothesis needs further investigations. Besides, seed germination and mature plant often demonstrate contradictory response to seeds spaceflight storage [16]. In any case, cultivar Rusalochka of Eruca sativa grown from spaceflight seeds seems the most promising.

Among the representatives of the three families investigated, the ‘space-treated’ plants of Apiaceae showed the most significant growth inhibition resulting in a decrease of 1.26 times in dill and 1.60 times in coriander weight (Table 2; Figure 2). Further investigations are needed to unveil if this phenomenon is associated with the morpho-physiological type of Apiaceae seed dormancy.

Table 2. Yield and biometrical characteristics of control and ‘space-treated seed’ plants.

Family	Cultivar	Seed Source	Dry Matter (%)	Height (cm)	Plant Weight (g)
Asteraceae (Lactuca sativa)	Moskovsky parnikovy	Control	5.8 a	18 b	3.81 a
		Space	5.5 a	22 a	4.43 a
	Petrovich	Control	4.7 a	23 a	6.1 a
		Space	4.7 a	23 a	5.5 a
	Synthesis	Control	4.9 a	23 b	3.34 b
		Space	4.7 a	27 a	4.60 a
	Picnic	Control	5.9 a	25 a	3.48 a
		Space	5.6 a	23 a	3.07 a
Brassicaceae	Brassica juncea Sudarushka	Control	8.1 b	28 a	1.48 b
		Space	9.1 a	28 a	1.82 a
	Eruca sativa Rusalochka	Control	7.6 a	19 b	1.03 b
		Space	7.7 a	25 a	2.41 a
Apiaceae	Anethum graveolens Kulinar	Control	6.9 b	30 a	1.72 a
		Space	8.0 a	29 a	1.36 b
	Coriandrum sativum Yubilar	Control	5.9 a	25 a	1.58 a
		Space	6.0 a	22 a	1.01 b

Within each column and plant family, values with the same letters do not differ statistically according to Duncan test at p < 0.05.

Figure 2. Differences in plant weight between ‘space-treated’ and control plants. Lactuca sativa: (1) cv. Moskovsky parnikovy; (2) cv. Petrovich; (3) cv. Synthesis; (4) cv. Picnic; Brassica juncea: (5) cv. Sudarushka; Eruca sativa: (6) cv. Rusalochka; Anethum graveolens: (7) cv. Kulinar; Coriandrum sativum: (8) cv. Yubilar. Within each family, values with the same letters (‘a’, ‘b’, ‘c’) do not differ statistically according to Duncan test at p < 0.05; ‘c’ means that weight differences between ‘space-treated’ and control plants are not significant at p > 0.05.

Table 2 data indicate that the Asteraceae species (Lactuca sativa) demonstrated significant varietal differences between the cultivars showing a total growth inhibition (cvs. Bouquet and Cavalier) or a slight growth stimulation, while a negative effect on plant growth was recorded for Apiaceae plants and a positive one for Brassicaceae species (Figure 2). In this respect, leaf area of Brassicaceae ‘space-treated’ plants showed significantly higher values compared to control plants (Figure 3). Conversely, the leaf area of lettuce cultivars (Synthesis, Petrovich and Moskovsky parnikovy, Picnic) did not differ between ‘space-treated’ and control plants (Figure 3).

Figure 3. Leaf area of ‘space-treated’ and control plants. Lactuca sativa: (1) cv. Moskovsky parnikovy, (2) cv. Petrovich, (3) cv. Synthesis, (4) cv. Picnic; Brassica juncea: (5) cv. Sudarushka; Eruca sativa: (6) cv. Rusalochka. Within each species and cultivar, values with the same letters do not differ statistically according to Duncan test at p < 0.05.

3.2. Photosynthetic Pigments

Changes in the intensity of plant growth due to ionized radiation and microgravity are closely related to the intensity of photosynthesis (Table 3). Among Lactuca sativa cultivars, only Picnic showed a significant decrease in the total chlorophyll and carotene content in leaves of ‘space-treated’ plants compared to control ones, while the opposite effect was revealed for Brassicaceae representatives. Indeed, the total chlorophyll and carotene content in leaves of Indian mustard was 1.31–1.33 times higher in plants grown from ‘space-treated’ seeds. These values reached 2.53 and 1.82 times increase in leaves of Eruca sativa, which is in accordance with the recorded growth stimulation of these plants (Table 2). Significant differences in carotene levels between leaves of ‘space-treated’ and the control plants suggest an important role for these substances in plant adaptation as well as disturbances in photopigments accumulation due to spaceflight seed storage. Indeed, carotenoids have an important role in light capture, antioxidant protection, phytohormone biosynthesis [40,41] and stabilization of photosynthetic apparatus [42]. The present results demonstrated a close relationship between chlorophyll and carotene accumulation in lettuce leaves (r = 0.959 at p < 0.001). The significant carotene decrease in ‘space-treated’ Apiaceae plant leaves, compared to leaves of control plants, relates to growth inhibition of Apiaceae crops and a tendency to chlorophyll level decrease.

Table 3. Leaf photosynthetic pigment levels in ‘space-treated seed’ and control plants (mg g⁻¹ f.w.).

Family	Cultivar	Seed Source	Chl a	Chl b	Total Chl	Car	Chl a/Chl b Ratio	Chl/Car Ratio
Asteraceae (Lactuca sativa)	Moskovsky parnikovy	Control	0.51 a	0.30 b	0.81 a	0.11 a	1.70	7.36
		Space	0.60 a	0.38 a	0.98 a	0.13 a	1.58	7.54
	Petrovich	Control	0.51 b	0.32 a	0.83 a	0.09 b	1.59	9.22
		Space	0.62 a	0.34 a	0.96 a	0.13 a	1.82	7.38
	Synthesis	Control	0.59 a	0.35 a	0.94 a	0.12 a	1.69	7.83
		Space	0.57 a	0.34 a	0.91 a	0.11 a	1.68	8.27
	Picnic	Control	0.82 a	0.45 a	1.27 a	0.21 a	1.82	5.05
		Space	0.62 b	0.37 b	0.99 b	0.12 b	1.68	8.25
Brassicaceae	Brassica juncea Sudarushka	Control	1.08 b	0.67 b	1.75 b	0.18 b	1.61	9.72
		Space	1.42 a	0.88 a	2.30 a	0.24 a	1.61	9.58
	Eruca sativa Rusalochka	Control	0.75 b	0.54 b	1.29 b	0.11 b	1.39	11.73
		Space	1.27 a	1.99 a	3.26 a	0.20 a	0.64	16.30
Apiaceae	Anethum graveolens Kulinar	Control	1.07 a	1.38 a	2.45 a	0.035 a	0.78	68.57
		Space	1.07 a	1.22 a	2.29 a	0.020 b	0.88	114.5
	Coriandrum sativum Yubilar	Control	1.00 a	1.22 a	2.22 a	0.019 a	0.82	246.67
		Space	0.91 a	1.06 a	1.97 a	0.017 a	0.86	115.88

Within each column, values with the same letters do not differ statistically according to Duncan test at p < 0.05.

3.3. Antioxidant Status

The data presented in Table 4 indicate that long-term spaceflight did not significantly affect the accumulation of ascorbic acid, total phenolics, and the level of total antioxidant activity (AOA) of mature plants. Low antioxidant status changes have been previously recorded also in tomato grown from half-year spaceflight stored seeds [6]. The stability of antioxidant parameters in the present experiment entails the existence of special adaptation mechanism of plants to unfavorable seed storage conditions.

Table 4. Leaf antioxidant status of ‘space-treated seed’ and control plants.

Family	Cultivar	Seed Source	AA (mg 100 g−1 f.w.)	AOA (mg GAE g−1 d.w.)	TP (mg GAE g−1 d.w.)	Pro (mg g−1 d.w.)	MDA (µM g−1 d.w.)
Asteraceae (Lactuca sativa)	Moskovsky parnikovy	Control	19.7 a	29.5 a	18.8 a	1.11 a	0.42 a
		Space	17.7 a	29.5 a	18.8 a	1.25 a	0.41 a
	Petrovich	Control	9.8 a	29 a	19.2 a	0.96 a	0.42 a
		Space	11.1 a	31.0 a	18.8 a	0.95 a	0.40 a
	Synthesis	Control	11.9 a	28.7 a	20.7 a	0.99 a	0.53 a
		Space	13.0 a	31.1 a	21.1 a	1.05 a	0.48 a
	Picnic	Control	11.2 a	26.8 a	17.5 a	0.85 a	0.46 a
		Space	9.1 a	27.4 a	18.2 a	0.96 a	0.48 a
Brassicaceae	Brassica juncea Sudarushka	Control	26.9 a	32.2 a	22.1 a	2.48 a	0.40 b
		Space	21.0 b	34.9 a	23.4 a	1.64 b	0.50 a
	Eruca sativa Rusalochka	Control	46.3 a	34.6 a	24.4 a	2.72 a	0.42 b
		Space	45.6 a	40.5 a	22.9 a	1.99 b	0.54 a
Apiaceae	Anethum graveolens Kulinar	Control	18.4 a	44.3 a	21.2 a	1.65 a	0.40 a
		Space	16.6 a	37.3 a	21.8 a	1.59 a	0.37 a
	Coriandrum sativum Yubilar	Control	33.3 a	51.3 a	17.1 a	1.89 a	0.43 a
		Space	27.7 b	53.7 a	17.7 a	1.61 a	0.45 a

AA: ascorbic acid; AOA: total antioxidant activity; TP: total content of polyphenols; Pro: proline; MDA: malonic dialdehyde. Within each column and cultivar, values with the same letters do not differ statistically according to Duncan test at p < 0.05.

Indeed, among the parameters tested, only proline content decreased significantly in Indian mustard and rocket of the Brassicaceae family with the corresponding enhanced accumulation of malonic dialdehyde (MDA). The decrease in the proline content and the increase of MDA accumulation in Brassicaceae plants due to ionizing radiation and microgravity indicate the existence of significant oxidant stress in these plants, causing lipids peroxidation, and suggesting the inability of plants to activate the synthesis of proline known to promote membrane stabilization, maintain cell turgor, and decrease the concentration of reactive oxygen species [43,44].

In this respect, the following paradox may be highlighted: despite the increase in oxidant stress caused by spaceflight storage of seeds and lack of significant changes in antioxidants accumulation, Brassicaceae plants demonstrated enhanced ability both to synthetize photosynthetic pigments (Table 3) and provide high yield (Table 2).

3.4. Mineral Composition

Viability of plants and protection against unfavorable environmental conditions are closely connected also with mineral nutrition and the ability to accumulate essential macro- and micro- elements. According to the present results, spaceflight seed storage did not significantly affect the concentration of nitrates in mature plants. Indeed, nitrate levels in lettuce leaves reached 0.60–0.71%, were in the range of 0.50–0.70% in Brassicaceae plants, and 0.71% and 0.82% in dill and coriander species, respectively.

Ash content showed decreased values in the ‘space-treated’ Brassicaceae and Apiaceae species with a rather controversial effect on lettuce cultivars (Figure 4). Indeed, among the cultivars tested, significantly decreased ash content in plants grown from spaceflight stored seeds was recorded only in cvs. Synthesis and Moskovsky parnikovy, while a tendency to higher ash levels was observed for cvs. Picnic and Petrovich (Figure 4). Generally, decreased ash content in plants relates to malnutrition and, in case of plants subjected to radiation and microgravity, to a possible disruption of macro-elements transport across plants [45]. Extremely scant information regarding the effect of ionizing radiation and microgravity on mineral composition of plants makes the present investigation relevant to elements profile of the tested crops (Table 5, Table 6 and Table 7).

Figure 4. Effect of seed spaceflight storage on ash content in plants belonging to Asteraceae (Lactuca sativa: 1—cv. Moskovsky parnikovy; 2—cv. Petrovich; 3—cv. Synthesis; 4—cv. Picnic), Brassicaceae (5—Brassica juncea: cv. Sudarushka; 6—Eruca sativa: cv. Rusalochka) and Apiaceae (7—A. graveolens: cv. Kulinar; 8—Coriandrum sativum: cv. Yunilar) families. Values with the same letters do not differ statistically according to Duncan test at p < 0.05.

Table 5. Mineral composition of Brassicaceae plants grown from spaceflight long-stored and control seeds (mg kg⁻¹ d.w.).

	Brassica juncea		Eruca sativa
	Control	Space	Control	Space
Ca	11,490 ± 1140 a	10,435 ± 1040 a	13,714 ± 1302 a	16,693 ± 1650 a
K	78,751 ± 7571 a	57,504 ± 5700 b	78,146 ± 7798 a	66,768 ± 6599 a
Mg	1804 ± 178 a	1384 ± 131 b	1866 ± 186 a	1798 ± 180 a
Na	853 ± 85 a	326 ± 32 b	580 ± 58 a	455 ± 45 b
P	6435 ± 649 a	5149 ± 510 b	4842 ± 481 a	5294 ± 530 a
B	22.6 ± 1.3 a	22.0 ± 2.1 a	14.3 ± 1.4 a	12.9 ± 1.3 a
Co	0.046 ± 0.010 a	0.056 ± 0.005 a	0.086 ± 0.008 a	0.097 ± 0.010 a
Cu	3.92 ± 0.32 a	3.50 ± 0.33 a	2.98 ± 0.30 a	3.65 ± 0.35 a
Fe	60.6 ± 6.0 a	75.9 ± 7.6 a	137 ± 13 a	130 ± 13 a
I	0.23 ± 0.02 a	0.21 ± 0.02 a	0.50 ± 0.05 a	0.38 ± 0.03 b
Li	0.65 ± 0.06 a	0.73 ± 0.07 a	1.42 ± 0.13 a	1.45 ± 0.13 a
Mn	10.0 ± 0.9 b	17.2 ± 1.7 a	15.8 ± 1.5 a	16.8 ± 1.6 a
Mo	10.6 ± 0.9 a	8.73 ± 0.85 a	13.7 ± 1.3 a	15.6 ± 1.5 a
Se	0.13 ± 0.01 b	0.25 ± 0.01 a	0.09 ± 0.01 c	0.29 ± 0.03 a
Si	74.1 ± 7.1 b	94.6 ± 9.2 a	186 ± 18.2 a	106 ± 10 b
Zn	39.1 ± 3.8 a	38.4 ± 3.8 a	30.8 ± 3.0 a	29.5 ± 2.9 a
Al	13.2 ± 1.3 b	17.0 ± 1.5 a	43.7 ± 4.3 a	22.3 ± 2.2 b
As	0.140 ± 0.010 a	0.055 ± 0.005 b	0.150 ± 0.01 a	0.120 ± 0.01 b
Cd	0.085 ± 0.010 a	0.077 ± 0.007 a	0.170 ± 0.010 a	0.160 ± 0.01 a
Cr	0.19 ± 0.02 b	0.47 ± 0.04 a	0.30 ± 0.03 a	0.22 ± 0.02 b
Ni	0.20 ± 0.02 b	0.32 ± 0.03 a	0.37 ± 0.03 b	0.58 ± 0.05 a
Pb	0.13 ± 0.01 b	0.20 ± 0.02 a	0.35 ± 0.03 a	0.27 ± 0.02 b
Sr	51.4 ± 5.0 a	38.9 ± 3.6 b	71.2 ± 7.0 a	57.0 ± 5.6 b
V	0.041 ± 0.001 b	0.071 ± 0.007 a	0.13 ± 0.01 a	0.10 ± 0.01 b
Ca/Sr	224	268	193	293
K/Na	92	176	135	147

Within each species and along each line, values with the same letters do not differ statistically according to Duncan test at p < 0.05.

Table 6. Mineral composition of Apiaceae plants, grown from spaceflight long-stored and control seeds.

	Anethum graveolens L.		Coriandrum sativum L.
	Control	Space	Control	Space
Ca	12,117 ± 1200 a	9587 ± 933 b	8321 ± 813 a	6199 ± 596 b
K	90,870 ± 9000 a	78,268 ± 7786 a	96,105 ± 9577 a	60,309 ± 6000 b
Mg	1634 ± 161 a	1397 ± 135 a	1572 ± 150 a	1107 ± 102 b
Na	744 ± 74 a	571 ± 57 b	570 ± 56 a	350 ± 33 b
P	7369 ± 724 a	5685 ± 553 b	9044 ± 900 a	5913 ± 590 b
B	19.7 ± 2.0 a	17.0 ± 1.7 a	24.1 ± 2.4 a	19.5 ± 1.9 b
Co	0.072 ± 0.006 a	0.045 ± 0.004 b	0.059 ± 0.005 a	0.039 ± 0.003 b
Cu	2.37 ± 0.20 a	1.61 ± 0.14 b	4.37 ± 0.44 a	3.38 ± 0.32 b
Fe	170 ± 17 a	111 ± 10 b	125 ± 12 a	83.3 ± 8.1 b
I	0.66 ± 0.06 a	0.34 ± 0.03 b	0.49 ± 0.04 a	0.48 ± 0.04 a
Li	0.83 ± 0.08 a	0.68 ± 0.06 b	0.83 ± 0.08 a	0.60 ± 0.06 b
Mn	22.4 ± 2.1 a	18.7 ± 1.9 a	27.9 ± 2.6 a	20.5 ± 2.0 b
Mo	3.94 ± 0.34 a	2.55 ± 0.22 b	3.9 ± 0.3 a	3.06 ± 0.30 b
Se	0.076 ± 0.01 b	0.109 ± 0.02	0.072 ± 0.01 b	0.116 ± 0.02 a
Si	40.9 ± 4.0 a	18.9 ± 1.8 b	147 ± 14.5 a	90.7 ± 9.0 b
Zn	13.1 ± 1.3 a	11.2 ± 1.1 a	27.9 ± 2.8 a	20.7 ± 2.0 b
Al	55.6 ± 5.4 a	18.9 ± 1.8 b	34.1 ± 3.4 a	15.8 ± 1.5 b
As	0.13 ± 0.01 a	0.08 ± 0.01 b	0.12 ± 0.01 a	0.11 ± 0.01 a
Cd	0.11 ± 0.01 a	0.10 ± 0.01 a	0.18 ± 0.02 a	0.16 ± 0.01 a
Cr	0.29 ± 0.02 a	0.18 ± 0.01 b	0.28 ± 0.02 a	0.11 ± 0.01 b
Ni	3.07 ± 0.3 a	2.96 ± 0.3 a	1.42 ± 0.12 a	1.04 ± 0.1 b
Pb	0.26 ± 0.02 a	0.21 ± 0.02 b	0.39 ± 0.03 a	0.37 ± 0.03 a
Sr	40.9 ± 4.0 a	28.0 ± 2.8 b	36.9 ± 3.6 a	31.8 ± 3.1 a
V	0.26 ± 0.02 a	0.12 ± 0.01 b	0.15 ± 0.01 a	0.10 ± 0.01 b
Ca/Sr	296	342	226	195
K/Na	122	137	169	172

Within each species and along each line, values with the same letters do not differ statistically according to Duncan test at p < 0.05.

Table 7. Mineral composition of Lactuca sativa plants, grown from spaceflight long-stored and control seeds.

	Petrovich		Synthesis		M. Parnikovy		Picnic
	Control	Space	Control	Space	Control	Space	Control	Space
Ca	7222 ± 698 a	8428 ± 816 a	5384 ± 522 a	5917 ± 579 a	10,725 ± 1000 a	8090 ± 800 b	5943 ± 588 a	5576 ± 550 a
K	64,394 ± 6003 a	73,598 ± 7160 a	69,683 ± 6943 a	49,991 ± 5000 b	60,939 ± 5890 a	58,027 ± 5723 a	74,870 ± 7367 a	81,600 ± 10,012 a
Mg	2288 ± 217 a	2264 ± 208 a	1698 ± 155 a	1521 ± 150 a	2897 ± 281 a	2009 ± 200 b	1937 ± 194 a	1756 ± 175 a
Na	608 ± 58 a	685 ± 622 a	745 ± 74 a	526 ± 53 b	787 ± 78 a	565 ± 56 b	639 ± 64 a	551 ± 55 a
P	9327 ± 897 a	9937 ± 967 a	8685 ± 845 a	7919 ± 786 a	8618 ± 658 a	7868 ± 680 a	5160 ± 510 a	5120 ± 510 a
B	15.7 ± 1.4 b	19.2 ± 1.8 a	15.2 ± 1.5 a	13.5 ± 1.3 a	17.6 ± 1.7 a	15.8 ± 1.5 a	16.3 ± 1.6 a	16.7 ± 1.6 a
Co	0.082 ± 0.007 a	0.096 ± 0.01 a	0.052 ± 0.005 a	0.061 ± 0.005 a	0.1 ± 0.01 a	0.072 ± 0.005 b	0.051 ± 0.005 a	0.054 ± 0.005 a
Cu	3.86 ± 0.31 b	4.73 ± 0.5 a	3.44 ± 0.31 a	3.43 ± 0.30 a	4.21 ± 0.41 a	3.16 ± 0.32 b	2.86 ± 0.26 a	3.32 ± 0.32 a
Fe	119 ± 10 b	171 ± 17 a	72.8 ± 7.3 b	90.2 ± 9.0 a	122 ± 12 a	143 ± 14 a	81.0 ± 8.0 a	91.1 ± 9.0 a
I	0.63 ± 0.05 b	1.21 ± 0.11 a	0.38 ± 0.03 b	0.59 ± 0.05 a	0.52 ± 0.05 a	0.59 ± 0.06 a	0.27 ± 0.03 a	0.31 ± 0.03 a
Li	0.58 ± 0.05 a	0.64 ± 0.06 a	0.53 ± 0.05 a	0.47 ± 0.04 a	0.81 ± 0.08 a	0.57 ± 0.05 b	0.67 ± 0.06 a	0.57 ± 0.05 a
Mn	14.9 ± 1.3 a	17.6 ± 1.6 a	11.1 ± 1.0 b	16.4 ± 1.6 a	18.4 ± 1.77 a	14.2 ± 1.4 b	21.4 ± 2.0 a	19.4 ± 1.9 a
Mo	2.09 ± 0.20 a	2.47 ± 0.23 a	1.66 ± 0.12 a	1.74 ± 0.17 a	3.05 ± 0.30 a	2.64 ± 0.25 a	1.65 ± 0.15 a	1.86 ± 1.7 a
Se	0.060 ± 0.006 b	0.078 ± 0.007 a	0.054 ± 0.004 b	0.081 ± 0.008 a	0.060 ± 0.06 b	0.090 ± 0.009 a	0.059 ± 0.006 b	0.128 ± 0.012 a
Si	162 ± 14 a	155 ± 15 a	109 ± 10 a	93.6 ± 9.0 a	111 ± 10 a	106 ± 10.0 a	117 ± 11 a	111 ± 10 a
Zn	26.2 ± 2.4 a	32.4 ± 3.1 a	25.4 ± 1.4 a	23.8 ± 2.3 a	35.9 ± 3.5 a	27.2 ± 2.7 b	28.2 ± 2.8 a	25.8 ± 2.5 a
Al	61.7 ± 6.0 b	125 ± 12.0 a	41.2 ± 4.0 b	58.8 ± 5.5 a	76.9 ± 7.6 a	71.7 ± 7.0 a	65.2 ± 6.5 a	58.4 ± 5.5 a
As	0.12 ± 0.01 b	0.15 ± 0.01 a	0.11 ± 0.01 a	0.079 ± 0.008 b	0.097 ± 0.01 a	0.12 ± 0.01 a	0.086 ± 0.008 b	0.13 ± 0.01 a
Cd	0.14 ± 0.01 a	0.15 ± 0.01 a	0.11 ± 0.01 a	0.10 ± 0.01 a	0.2 ± 0.02 a	0.14 ± 0.01 b	0.098 ± 0.01 a	0.098 ± 0.001 a
Cr	0.36 ± 0.03 b	0.51 ± 0.05 a	0.23 ± 0.02 b	0.35 ± 0.03 a	0.32 ± 0.03 a	0.34 ± 0.03 a	0.18 ± 0.02 b	0.24 ± 0.02 a
Ni	0.52 ± 0.05 a	0.46 ± 0.04 a	0.27 ± 0.02 b	0.42 ± 0.04 a	0.49 ± 0.04 b	0.71 ± 0.07 a	0.23 ± 0.02 a	0.27 ± 0.02 a
Pb	0.51 ± 0.05 a	0.59 ± 0.05 a	0.31 ± 0.03 a	0.38 ± 0.04 a	0.59 ± 0.05 a	0.44 ± 0.04 b	0.29 ± 0.02 a	0.34 ± 0.03 a
Sr	29.8 ± 2.6 b	43.5 ± 4.1 a	28.5 ± 2.7 a	30.5 ± 3.0 a	52.7 ± 5.2 a	39.4 ± 3.9 b	24.60 ± 2.40 a	24.9 ± 2.4 a
V	0.21 ± 0.02 b	0.39 ± 0.03 a	0.14 ± 0.01 b	0.2 ± 0.02 a	0.20 ± 0.02 b	0.26 ± 0.02 a	0.12 ± 0.01 b	0.24 ± 0.02 a
K/Na	106	107	93.5	95.0	77.4	102.7	117.2	184.4
Ca/Sr	242.3	193.7	188.9	325.1	203.5	205.3	241.6	223.9

Within each cultivar and line, values with the same letters do not differ statistically according to Duncan test at p < 0.05.

The results of the present research indicate the existence of great changes in macro-, micro- and toxic elements accumulation, due to one-year seed storage in ISS, in ‘space-treated’ and control plants belonging to Brassicaceae, Apiaceae, and Asteraceae families.

Both significant species and varietal differences arose from the comparison between the species belonging to the three families examined, in terms of mineral accumulation (Table 8). In this respect, the remarkable accumulation decrease of most macro- and micro-elements in dill and coriander due to long-term spaceflight seed storage was in agreement with the detected growth inhibition. The most significant increase in plant yield of rocket (more than twice) may be connected with the relative stability of macro-element content and evident decrease in accumulation of toxic elements, such as Al, As, Cr, Pb, Sr and V.

Table 8. Changes in macro-, micro- and toxic element accumulation in Brassicaceae, Apiaceae and Asteraceae species, as a result of long-term seed storage on ISS.

	Brassicaceae		Apiaceae		Asteraceae
	Mustard	Rocket	Dill	Coriander	M. Parnikovy	Petrovich	Synthesis	Picnic
Ca			−	−	−
K	−			−			−
Mg	−			−	−
Na	−	−	−	−	−		−
P	−		−	−
B				−		+
Co			−	−	−
Cu			−	−	−	+
Fe			−	−		+	+
I		−	−			+	+
Li			−	−	−
Mn	+			−	−		+
Mo			−	−
Se	+	+	+	+	+	+	+	+
Si	+	−	−	−
Zn				−	−
Al	+	−	−	−		+	+
As	−	−	−			+	−	+
Cd					−
Cr	+	−	−	−		+	+	+
Ni	+	+		−	+		+
Pb	+	−	−		−
Sr	−	−	−		−	+
V	+	−	−	−	+	+	+	+

‘−‘ corresponds to decreased element content in ‘space-treated’ plants compared to control; ‘+’ corresponds to increased element content compared to control.

The 23% yield increase of mustard due to utilization of ‘space’ seeds may reflect the ability of this plant to accumulate toxic elements without growth inhibition [46]. Indeed, besides the decreased contents of K, Mg, and P in ‘space-treated’ plants, Indian mustard showed increased levels of Mn, Se, and Si, known to participate in plant protection against stresses [47]. This plant is known to highly tolerate and accumulate heavy metals and is often used for phytoremediation of heavy metals such as Ni [48] and Pb [49]. Indian mustard demonstrates the ability to avoid metal toxicity via metal binding to the cell wall, or by reducing transport across the cell membrane, by chelation of the metal ions, and compartmentalization into the vacuole [50].

On the other hand, Al, Cr, Ni, b, and V may also have a beneficial effect on plant growth, like the stimulation of root formation elicited by low Cr concentration [51,52]. Moreover, aluminum is toxic to plants in acidic soil but demonstrates a growth stimulation effect and mitigates environmental stresses in neutral soil [53]. At low concentration, Ni is known to activate superoxide dismutase, stimulate respiration, and nitrogen fixation [54].

The growth stimulation effect of V was previously described in several plant species: pepper, maize, basil, and tomato [55]; this element is known to act as a redox catalyzer in electron transportation in photosystems I and II, depending on the environmental conditions [55,56].

Lettuce plants showed significant varietal differences in mineral accumulation: a remarkable decrease of macro-element content (cv. M. parnikovy), lack of significant changes in macro-element concentration (cvs. Picnic and Petrovich) and a decrease of potassium content in the cultivar Synthesis. Each cultivar was characterized by its own specific response: cv. Petrovich and Synthesis increased the accumulation of many important micro-elements (Co, Cu, Fe, I, Se) but also toxic elements (Al, As, Cr, Sr, and V). The cv. Moskovsky parnikovy demonstrated decreased levels of some macro- and micro-elements as well as of the toxic Cd, Pb, and Sr. The cultivar Picnic showed only the increase of As, Cr, and V.

Overall, based on the data presented in Table 8, several peculiarities in Brassicaceae, Apiaceae, and Asteraceae representatives may be highlighted.

3.4.1. Vanadium

Indeed, all Asteraceae species showed a significant increase in V accumulation (Figure 5).

Figure 5. Effect of seed spaceflight storage on V accumulation in lettuce cultivars. Values with the same letters do not differ statistically according to Duncan test at p < 0.05.

Positive or negative effects of V on plants primarily depend on its concentration [56]. High levels are known to reduce plant growth and disturb physiological status, while low concentrations show growth stimulation [56]. Among the four lettuce cultivars examined, only cv. Synthesis was characterized by significant yield increase (38%) due to spaceflight seed storage, while Petrovich and Picnic demonstrated a tendency to growth inhibition and Moskovsky parnikovy a slight yield improvement (16%) compared to control plants.

3.4.2. Selenium

Furthermore, the plants of the three families examined had an increase in Se levels due to spaceflight seed storage effect (Figure 6). The latter result reflects the ability of Se in plant protection and is of special interest taking into account the protective properties of this element against all forms of oxidant stresses, including heavy metals [57,58,59]. In this respect, the increased accumulation levels of heavy metals in Indian mustard and lettuce cultivars may demonstrate a decreased harmful effect due to high levels of Se accumulation.

Figure 6. Effect of seed spaceflight storage on Se accumulation in plants belonging to Asteraceae (Lactuca sativa: 1—cv. Moskovsky parnikovy; 2—cv. Petrovich; 3—cv. Synthesis; 4—cv. Picnic), Brassicaceae (Brassica juncea: 5—cv. Sudarushka; Eruca sativa: 6—cv. Rusalochka) and Apiaceae (A. graveolens: 7—cv. Kulinar; Coriandrum sativum: 8—cv. Yunilar) families. Values with the same letters do not differ statistically according to Duncan test at p < 0.05.

Furthermore, the comparison of nutritional profiles of the tested plant species suggests that the plants with ‘space’ growth stimulation effect (mustard and rocket) are characterized by seeds with the highest levels of protein and the lowest of carbohydrates (Figure 7). In this respect, rocket seeds rank first both in protein accumulation and ability to increase Se content in mature plants due to long-term spaceflight seed storage (Figure 6). Genetic changes in plant seeds subjected to cosmic radiation primarily affect the biosynthesis of amino acids and proteins [8,21], substances capable of replacing sulphur in methionine and cysteine with selenium [57].

Figure 7. Nutritional profile of the tested species. Within each quality parameter, values with the same letters do not differ statistically according to Duncan test at p < 0.05.

3.4.3. Sodium

Changes of Na accumulation due to spaceflight seed storage is another valuable peculiarity of ionizing radiation and microgravity effect. Figure 8 data indicate sodium concentration decrease in tissues of most plants with the highest effect on Indian mustard (Figure 8).

Figure 8. Effect of seed spaceflight storage on Na accumulation in plants belonging to Asteraceae (Lactuca sativa: 1—cv. Moskovsky parnikovy; 2—cv. Petrovich; 3—cv. Synthesis; 4—cv. Picnic), Brassicaceae (Brassica juncea: 5—cv. Sudarushka; Eruca sativa: 6—cv. Rusalochka) and Apiaceae (A. graveolens: 7—cv. Kulinar; Coriandrum sativum: 8—cv. Yunilar) families. Values with the same letters do not differ statistically according to Duncan test at p < 0.05.

The concentrations of sodium (Na⁺) and potassium (K⁺) in plant tissues are important determinants of stress tolerance and adaptability level [60]. In this respect, Indian mustard showed the highest K/Na increase (1.91 times higher than the control values) providing a beneficial growth stimulation effect on plants grown from ‘space-treated’ seeds.

4. Conclusions

The results of the present investigation revealed species and varietal differences in plant response to long-term seed spaceflight storage. Among Apiaceae, Asteraceae, and Brassicaceae families, the first one demonstrated significant plant growth inhibition due to long-term seed spaceflight storage, while the last one showed significant yield increase and stimulation of photosynthetic pigments biosynthesis in ‘space-treated’ plants. Dramatic decrease of macro- and micro-elements, except Se, was recorded in Apiaceae plants. The remarkable increase and decrease of Se and Na accumulation, respectively, in all plants tested, and the V increase in lettuce cultivars entail the important spaceflight effects on plant seeds.