Environmental conditions predetermine innate antioxidants pool in sea oat (Uniola paniculata L.) seeds

Secondary metabolites such as antioxidants are critical components that protect seeds from stress damage during seed development, desiccation, and ex-situ storage. Antioxidants are essential determinants of seed quality, longevity, and persistence. Understanding the environmental factors that regulate the accumulation, content, and function of antioxidant pools in sea oat seeds is critical for gene banking and understanding the environmental impacts on seed quality. Germination, viability, and Trolox Equivalent Antioxidant Capacity (TEAC) were analyzed on seeds from 18 sea oat populations from the US Atlantic and Gulf of Mexico coasts. We �rst assessed baseline TEAC, followed by TEAC performed on imbibed seeds for 36 hours at 35/25 °C during the day and night, respectively. Then, we analyzed the relationship of the antioxidant pools from these 18 sea oat populations to sampling site environmental classi�cations. Higher baseline antioxidants were common in regions with extreme environmental conditions corresponding to sea oat populations growing at latitudinal extremes characterized by warmer and colder temperatures. Baseline antioxidants did not correlate with prevailing seed germination. However, higher concentrations of antioxidants following imbibition were associated with poor seed germination in warmer conditions. Our results indicate that climatic conditions and environmental components associated with temperatures and precipitation may largely in�uence the innate pool of antioxidants in sea oat seeds. Also, a high amount of antioxidants following sea oat seeds imbibition suggest seed damage or poor viability in�uenced by environmental stress conditions during seed development.


Introduction
Uniola paniculata L. (sea oats [Poaceae]) is an ecological keystone species occurring in coastal dune systems throughout the Yucatan peninsula, Cuba, the Bahamas, and southeastern US coastlines (Subudhi et al., 2005;Lonard et al., 2011;Hodel and Gonzales, 2013).Sea oats are essential for their ecological services, such as sand accretion, preventing erosion, and forming and stabilizing coastal dunes.These services largely buffer the effect of extreme events such as storms and hurricanes prevalent along those coastlines (Lonard et al., 2011;Hacker et al., 2019).Consequently, sea oats are in high demand for habitat restoration and re-vegetation programs.However, the spatial extent of sea oats populations has diminished greatly due to habitat destruction, conversion, and fragmentation.Protecting remaining sea oats populations is therefore crucial for maintaining donor populations for seed-based conservation and restoration programs.
Sea oats face diverse extreme conditions in the coastal dune habitat including osmotic stress from aerosolized salts and saline soils, hypoxia due to ooding, high and low-temperature stress, high humidity stress, and physical damage from tidal and wave over-wash or winds caused by storms and hurricanes.Sea oats also face burial from shifting dune sands.Therefore, sea oats and their seeds need highly functional stress-relief systems, especially antioxidants, to survive such conditions.However, there is limited knowledge on the presence, environmental in uence, and functional role of antioxidants in plants from coastal dune systems exposed to such wide-ranging stressors.
Antioxidants are a category of secondary metabolites synthesized by living organisms that have been associated with preventing oxidative damage to cells and tissues.These molecules function at relatively low concentrations during optimal and stress conditions (Halliwell et al., 1995;Begcy et al., 2012) directly impacting complex aspects of seed quality including nutritional components, seed size, water potential and dry matter content (Bailly, 2004(Bailly, , 2019;;Corbineau, 2012).In seeds, antioxidants are synthesized throughout the developmental program (i.e., histodifferentiation, reserve accumulation, and late maturation) and are thought to protect against stresses by quenching reactive oxygen species (ROS) and free radicals (Bailly, 2004;Chen et al., 2016;Adetunji et al., 2021).For instance, antioxidants balance ROS and free radicals generated in hydration-associated active metabolism from the initiation of embryogenesis through germination by acting as free radical scavengers (Pehlivan, 2017).Seed-based antioxidants also in uence the hydration process through the action of seed coat phenolics, which may offer a barrier to rapid imbibition (Ross et al., 2010).They also regulate cellular redox balance preventing oxidative damage during the imbibition process (Bailly, 2004).Antioxidants also substantially buffer the damage from ROS and other radicles generated by desiccation stress and lipid oxidation during seed storage (Groot et al., 2015;Carta et al., 2018;Adetunji et al., 2021;Stegner et al., 2022), Furthermore, antioxidants may be linked to maintaining seed persistence and viability in the soil seed bank (Long et al., 2015).
At an ecological level, plants adapt to prevailing environmental conditions such as precipitation, relative humidity, solar radiation, and temperatures, allowing normal plant development and reproductive success (Saatkamp et al., 2019).Such adaptions result in the development of tness-related traits vital to surviving harsh conditions such as drought and cold stress (Capblancq et al., 2022;Eckert and Neale, 2022).Studies have indicated a strong correlation between environmental conditions and plant traits.For example, the in uence of geographical location on seed germination has been observed in sea oats (Pérez and Kane, 2017).At the same time, environmental conditions during plant growth, owering, and seed development has been found to in uence subsequent seed germination and seedling response to oxidative stress (Nguyen et al., 2021).
Seeds traits including seed dormancy and vigor as well as resilience to environmental stresses have been suggested to vary broadly across a latitudinal gradient ( Gulf Coastlines, we investigated the functional dynamics of antioxidant pools in sea oats seeds collected from a continental-scale spatial distribution.First, we measured their antioxidant levels hypothesizing possible interactive links of the environmental conditions to the antioxidants levels and seed quality.We compared antioxidant levels from each population to 10-year climate data (precipitation, and temperature) and various ecological classi cation systems of the study sites.We further tested the potential role of the antioxidant pool on seed quality by correlating antioxidant levels to germination ability.

Plant material collection and seed lot processing
We collected one to three sea oats (Uniola paniculata) panicles from no less than 30 widely spaced plants across 18 sites along the US Atlantic and Gulf of Mexico (referred to hereafter as Gulf) coasts in October 2019 (Fig. 1A, Table 1).We spread panicles in a single layer on tarps within a non-climatecontrolled warehouse and circulated air over the panicles with an electric fan.We allowed panicles to dry for three days, then hand-stripped spikelets and threshed caryopses (referred to hereafter as seeds) from spikelets with a laboratory debearder (Heavy Duty Batch Debearder, DB3001-600, Mater Seed Equipment, Corvallis, OR).We conditioned seeds further by passing all lots through an air-screen cleaner (Clipper O ce Tester, A.T. Ferrell Co. Inc., Corvallis, OR) then an air-column separator (Oregon Seed Blower, Hoffman Manufacturing Inc., Corvallis, OR).We stored remaining seeds in the lab for two and half years (~ 23°C, 30-50% RH).Illumination coincided with the day temperature.We allowed seeds to imbibe under these conditions for 36 h, after which we froze the seeds in liquid nitrogen and stored them at -80 o C for use in the determination of Trolox Equivalent Antioxidant Capacity (TEAC) as described below.
We used three to four samples of 25 seeds in our germination experiments that were conducted under the same conditions described for the imbibition experiments.However, germination assays continued for 28 days.We inspected the germination boxes daily and counted germination when seeds exhibited 2 mm of radicle and coleoptile emergence (Begcy et al., 2018).We removed germinated seeds and any seeds showing signs of fungal contamination.We assessed the viability of any remaining, non-germinated seeds after day 28 using a tetrazolium stain (Peters, J. and Lanham, B., 2000).We analyzed embryo staining patterns at 2.5 to 6.4× and report germination on a viable seed basis (Supplementary Fig. S1A-B).

Seed water relations
We measured post-storage seed water potential, using three technical replicates, with a dewpoint potentiometer (WP4C, Decagon Devices, Pullman, WA).The potentiometer was calibrated before each measurement day according to manufacturer recommendations.We measured seed water potential at 20°C in precise mode.In this procedure, we used about 200 seeds to cover the bottom of the cuvette completely.Seed moisture content was calculated by recording the fresh mass on 20 seeds in ve replicates for each of the 18 populations studied.We then dried these seeds in a forced-air oven (Blue M, Illinois, USA) at 103 o C for 17 hours and report water content on a dry mass basis (g H 2 O • g dry seed mass − 1 [g g − 1 ]).

Climate and ecological data
We obtained climate data for the ten years before our seed sampling (2009-2019) using the PRISM application (Hart and Bell, 2015).The climate data used in this study included minimum (Tmin), mean (Tmean) and maximum (Tmax) temperatures, mean dew point temperature (Tdmean), total precipitation (ppt), and daily maximum vapor pressure de cit (Vpdmax).All data obtained were ltered at 4 km resolution using the location coordinates for sites corresponding to each of the 18 sea oat populations (Fig. 1A, Supplementary Table S1).Data were extracted using the prism R package (Hart and Bell, 2015).
First, we explored seasonal precipitation and temperature patterns along the latitudinal gradient where the sea oats populations were collected.We organized our analysis into groups of three months, roughly representing the four seasons (Winter, Spring, Summer, and Autumn).
Ecological descriptors comprised the: U.S Environmental Protection Agency (US EPA) ecoregion zoning (EPA3 and EPA4) (Supplementary Table S2), U.S. Department of Agriculture-Natural Resource Conservation Service (USDA-NRCs) and Food and Agriculture Organization of the United Nations (FAO) soil classi cation (Supplementary Table S3), American Horticulture Society (AHS) heat zoning (Supplementary Table S4), USDA plant hardiness zoning (Supplementary Table S5).We also used sea oat phylo-geography data as population grouping factors (Franks et al. 2004 Supplementary Table S6).

Antioxidant analysis
We estimated the levels of Trolox Equivalent Antioxidant Capacity (TEAC) in seeds using the modi ed ABTS/TEAC assay (Re et al., 1999).ABTS/TEAC determines the TEAC per gram of sample.ABTS (2,2'-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) reagent was prepared by dissolving 2.74 mg of ABTS in 1 mL of sterile, distilled H 2 O for each reaction, including the standards.Then, 300 mg of MnO 2 was added and mixed using a magnetic stirrer for 20 minutes.The mixture was lter sterilized through a 0.45 µm syringe lter.For calculations, a spectrophotometer (Scienti c Genesys 10S UV-VIS Spectrophotometer, Thermo Scienti c, Madison, USA) was zeroed at 734 nm with 100 µL PBS added to 1 mL of sterile distilled H 2 O. Absorbance readings were adjusted with 5 mM PBS to obtain a value of 0.700.We added 6.26 mg of Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) to 50 mL of 5mM PBS then mixed the reagents by sonication for 20 minutes to obtain 0.5 mM Trolox standard solution.Then, 1 mL of aliquots were prepared in cryovials and stored in a freezer at -20°C.During the assay, a 1 mL cryovial containing 0.5 mM Trolox was obtained from the freezer and allowed to thaw.We re-dissolved the solution by sonication for 10 minutes.Then, dilutions for ve triplicate standards were obtained at 0, 11, 22, 33, and 44 µl of 5mM Trolox corresponding to TEAC concentrations of 0, 5, 10, 15, and 20 µmol/L, respectively.
Antioxidants for TEAC estimation were extracted from seeds using a modi ed hydrophilic method (Re et al., 1999).Samples were nely ground using liquid nitrogen.Approximately 10 mg/sample was placed into 10×75 mm reaction vials.Triplicates were used per sample per reaction.TEAC was extracted by adding 5 mL of 75% aqueous methanol, followed by stirring under a nitrogen gas stream of 2.0 Pascals at 30°C for 60 minutes.Then, the obtained product was centrifugated at 2000 × g for 5 minutes to get a supernatant containing the extracts.A second extraction step was conducted by adding 2 mL of 75% aqueous methanol and covering the reaction vials.Then, each vial was vortexed and centrifugated at 2000 × g for 15 minutes.Supernatants of the rst and second reactions were combined.Concurrently, 10-20 mg of nely ground samples were prepared in oven-drying foil pans and dried at 103°C for 17 hours to obtain dry mass.
Extracts were re-suspended into 10 mL of 75% methanol.Each 100 µL of sample extract was mixed with 1 mL of 5 mM ABTS solution, vortexed for two mins, and placed in the spectrophotometer cuvette before reading the absorbance at 734 nm.Three biological replicates and three technical replicates were used.

Data analysis
We subjected data on seed water potential, moisture content, TEAC, and germination percentage to an analysis of variance (ANOVA) to estimate differences among populations.We utilized linear and polynomial regression to evaluate seed germination and TEAC levels against geographical, climatic, and ecological patterns.We also calculated Cohen's f 2 to standardize parameter effect size and used the following guidelines for interpretation: f 2 ≥ 0.02 represents a small effect; f 2 ≥ 0.15 medium effect; and f 2 ≥ 0.35 large effect (Cohen, 1988).Statistical analyses were performed using the R software/environment 1.3.Differences were considered statistically signi cant at p-value < 0.05 (Kim et al., 2021).

Results
Impact of the latitudinal gradient on seed physiology in sea oat populations along the US Atlantic and

Gulf Coastlines
To test the hypothesis of seed related traits variation across a wide latitudinal gradient ( eighteen sea oat populations distributed across the US Atlantic and Gulf Coastlines (Fig. 1A).Seed germination ranged from 20% (CIVA) to 95% (HIFL) (Fig. 1B).Seeds from 39% of the populations registered germination > 80%, most of the populations displaying this level of germination were from Florida (Fig. 1B).However, there were instances of poor germination (i.e., < 50%) for other populations, especially from the northern latitudes of the Atlantic coastline (Fig. 1B).Interestingly, seeds collected from populations in the southern US displayed germination percentages > 55% (Fig. 1B).
To further understand differences in seed physiology that may in uence variation of germination, we quanti ed seed moisture content and water potential (Fig. 2A-B).Seed moisture content was relatively similar across the latitudinal gradient and ranged from 0.1064 to 0.1129 g g − 1 .Seeds with lower moisture content (< 0.1100 g g − 1 ) comprised KIVA, FFNC, SIGA, GRFL, NPFL, and HBFL (Fig. 2A).While seeds collected from LTFL and CIVA displayed the highest water contents observed of 0.1129 and 0.1130 g g − 1 , respectively.The seeds with the lowest moisture content were collected from Florida.They included GRFL and NPFL at 0.1066 and 0.1064 g g − 1 , respectively (Fig. 2A).Sea oats seed water potential was also variable and ranged from − 89.01 to -100.84 MPa (Fig. 2B).Patterns of water potential did not necessarily track with patterns of water content as some populations displaying lower water content expressed higher water potential values.Conversely, other populations displaying higher water contents displayed lower water potentials (Fig. 1B and 2A-B).However, differences in seed water relations could not explain germination variation across populations.
To characterize the extent to which location and environmental conditions in uenced germination of sea oats seeds, we examined how germination related with latitude and temperature zoning (Fig. 3; Supplementary Tables S4 and 5).We observed a latitudinal pattern of germination response, with regions closer to the equator having higher germination while populations located further from the equator displayed reduced germination capacity (R 2 = 0.50), indicating a signi cant latitudinal in uence on germination ability (Fig. 3A).In addition, germination was lower in zones with fewer heat days above 30°C but increased as the number of heat days increased (Fig. 3B).Similarly, germination was lowest in zones with lower average minimum winter temperatures but increased as this average rose (Fig. 3C).We also calculated Cohen's f 2 to standardize parameter effect sizes and found a large effect of latitude and temperature zoning on germination capacity (Fig. 3).

Weather characteristics of sea oat seeds collection sites along the US Atlantic and Gulf Coastlines
To explain which factors could have contributed to the observed differences in seed traits across all sea oats populations (Fig. 1-3), we rst looked at precipitation and seasonal mean temperature patterns for sea oats collection sites along the US Atlantic and Gulf coastlines in a 10-year period prior to seed sampling (2009-2019).Precipitation patterns across the latitudinal gradient were similar in winter and spring, averaging 50 to 150 mm (Fig. 4A).However, in summer, southerly latitudes displayed higher precipitation (> 150 mm) than northerly latitudes (< 150 mm).In Autumn, the southerly latitudes maintained higher precipitation in September and October (> 100 mm) before dropping below 100 mm in November.Alternatively, precipitation increased considerably in northern latitudes to > ca.100 mm in September and October but remained lower (< 100 mm) in November (Fig. 4A).
Temperature patterns were similar across all seasons (Fig. 4B).However, higher temperatures were evident in southern latitudes compared to the northern ones during the Winter, Spring, and Autumn (Fig. 4B).Summer temperatures varied less across all the latitudes in which the sea oats populations were analyzed (Fig. 4B).Mean temperatures across all latitudes remained above 22°C throughout June, July, and August (Fig. 4B).Both the maximum vapor pressure de cit and the mean dew point had a similar pattern, with higher values in the southern latitudes and lower values in the northern latitudes in most seasons (Winter, Spring, and Autumn), except in summer season in which there were no differences between southern and north latitudes (Supplementary Fig. S2-3).

Latitude-dependent environmental conditions in uence TEAC concentration in sea oats seeds
Since our climate data pointed to precipitation and temperature as main drivers of germination differences across a latitudinal gradient, we used antioxidant levels in sea oats seeds as a proxy to explore a possible link of latitude-dependent environmental conditions with antioxidant accumulation and germination.First, we de ned baseline TEAC values obtained from mature ungerminated seeds before imbibition across all sea oats populations.We found that TEAC levels from our collected seeds ranged from 0 to 12 µmol/g DW (Fig. 5A).For instance, while seeds collected in CMNJ, CIVA, KIVA, VEFL, BBFL, HIFL DWFL and CHFL showed TEAC levels higher than 4 µmol/g, the remining populations showed lower values than that (Fig. 5A).Interestingly, in general, seeds from sea oat populations collected either from the most northern or southern locations showed higher antioxidant capacity than the ones collected in intermediate latitudes (Fig. 5A).These results suggest an in uence by extreme conditions experienced at northern or southern US latitudes.To further explore whether antioxidant capacity was maintained during the initiation of germination, we imbibed seeds for 36 h and quanti ed their TEAC levels (Fig. 5B).Contrary to the earlier patterns of baseline antioxidant levels, there was wider population variability across the geographical distribution of sea oats (Fig. 5A-B).The change in TEAC levels between dry and imbibed seeds showed considerable variation (Fig. 5C).For instance, seeds from some latitudes displayed a drastic drop in TEAC levels, especially CMNJ and KIVA (Fig. 5C), and those with signi cantly minimal changes comprised KIVA, FPFL, BBFL, DWFL, HBFL, and CHFL (Fig. 5C).However, some seeds registered a signi cantly positive change in TEAC > 5 µmol/g DW comprised of FLVA, FFNC, HISC, SIGA, FCFL, LTFL, GRFL, NPFL and VEFL (Fig. 5C).

Geographic and environmental patterns in relation to seed traits in sea oats from US Atlantic and Gulf coastlines
To assess the impact of latitude on antioxidant capacity, we used baseline seed TEAC concentrations and compared these with the latitudinal range where seeds were collected.In general, antioxidant levels in dry seeds displayed a concave parabolic pattern when evaluated against latitude, heat, and plant hardiness zones (Fig. 6A-C).Then, we compared antioxidant capacity levels obtained 36 h after imbibition with the latitudinal zones (Fig. 6D).We observed higher TEAC levels near the equator and lower levels in northern latitudes.Surprisingly, relationships between TEAC and AHS heat or USDA plant hardiness classi cations showed similar patterns when TEAC data obtained from seeds imbibed after 36h (Fig. 6E-F).Zones with a greater number of hot days (Fig. 6F) and less freezing conditions (Fig. 6E) displayed higher TEAC values.We found large effect on TEAC obtained from dry seeds compared against latitude (Fig. 6A) and temperature zoning (AHS heat) (Fig. 6B) as well as on temperature zoning in 36 h imbibed seeds (Fig. 6E).We found medium effect size when TEAC obtained from dry and imbibed seeds were compared against USDA plant hardiness (Fig. 6C, F) and imbibed seeds against latitude (Fig. 6C) Role of the environment and seed traits on the baseline germination and antioxidants in sea oats from the US Atlantic and Gulf coastlines No clear relationships between germination and baseline TEAC or TEAC after 36 h of imbibition were evident when considering the geographical range of sea oats used in this study (Supplementary Fig. S4A-B).Therefore, we analyzed relationships between germination and TEAC using subsets of data representing the extreme temperature regions where high TEAC levels had been observed.Similarly, no clear relationships existed between germination and TEAC after 36 h of imbibition for seeds originating from colder regions (> 33N) (Fig. 7A-B).However, seeds from warmer regions (< 30N), where high seed quality was expected, exhibited a signi cant negative linear relationship of TEAC levels in imbibed seeds after 36 h showing large effect sizes according to Cohen's f 2 coe cient (0.56, 0.64, Fig. 7B-D, respectively).
Finally, we compared the in uence of latitude and temperature patterns on TEAC levels on dry seeds and seeds imbibed for 36 h (Fig. 8).We observed that the minimum and maximum temperatures during the seed development period exhibited high TEAC levels in seeds collected from northern or south latitudes (Fig. 8A).As such, the minimum and maximum temperatures exhibited latitudinal patterns of distribution (R 2 of 0.22 and 0.20, respectively).Contrary, 36 h of imbibition resulted in lower TEAC levels on seeds collected on northern sites (Fig. 8B).

Discussion
Sea oats are the primary native dune grass used to restore and stabilize beaches and dunes across the southeastern United States.Commercial nurseries have propagated sea oats using eld-collected seeds to retain the genetic integrity of source populations.We investigated the effect of climate conditions on sea oats harvested across a wide latitudinal range that spanned more than 13 ° of latitude (Table 1).Our results show that sea oats seed germination was higher from the sites closer to the equator but decreased as the distance from the equator increased (Fig. 1B).The observations of comparative germination with respect to distance from the equator indicated that the sea oat seeds are of high quality in terms of germination percentage from warmer tropical regions compared to seeds from colder areas.Similar patterns of seed germination were observed between populations located in Southern Italy in comparison with a Spanish core population of Lavandula multi da L., a plant species present in the Western Mediterranean Basin (Panuccio et al., 2018).
Environmental and climate in uences on seed quality, antioxidants, and implications on seed germination Antioxidant activity plays a key role in various events of seed life.Antioxidants are produced throughout the seed developmental program and during the germination process.These molecules occur within metabolically active cells, but also in dry tissues, with physiological activity depending on seed moisture status (Vertucci and Farrant, 1995;Walters et al., 2002;Ballesteros et al., 2020).Water potential measurements of dry seeds tested in this study suggest that seeds from all populations fall into the same hydration level (i.e., Hydration level II, ca.-15 to 190 MPa (Vertucci and Farrant, 1995;Walters et al., 2002Walters et al., , 2005) ) which was like previous studies (Pérez and Kane, 2017).Hydration level II represents a cellular physiological state characterized by enzyme-mediated catabolic reactions, oxidative and peroxidative processes, and formation of free radicals and reactive oxygen species.Existing pools of molecular antioxidants synthesized during the seed developmental program are thought to play a protective role by quenching reactive oxygen species and free radicals in dry seeds (Vertucci and Farrant, 1995;Walters et al., 2002Walters et al., , 2005)).We did not observe a clear relationship between TEAC concentration and baseline seed water potential in this study.Nonetheless, seeds from all populations display antioxidant capacity that may be su cient to mediate aging associated deteriorative reactions to some degree during storage under non-optimal conditions of temperature and relative humidity (Walters et al. 2005).Moreover, it is well documented that environmental conditions directly impact seed quality as well as their desiccation tolerance, nutrient level, storage potential and the accumulation of antioxidants and other related secondary metabolites (Bewley et al., 2013;Pehlivan, 2017;Ellis, 2019;Malovichko et al., 2021).We observed differences in the accumulation of antioxidant levels in dry seeds compared to imbibed seeds across all populations (Fig. 5-6).Increased antioxidant activity during seed germination is vital in signaling and ROS homeostasis (Bailly, 2004; El-Maarouf-Bouteau and Bailly, 2008).High germination capacity associated with moderate changes in TEAC levels after initial hours of imbibition is expected to be a characteristic of good-quality seeds because it is linked to low lipid peroxidation (Rogozhin et al., 2001).Interestingly, in our study, seeds from warmer regions exhibited limited uctuations in TEAC levels after imbibition.Therefore, sharp increases in TEAC levels for seeds collected from populations located in warmer regions might be an adaptative response to climate conditions that directly impact their germination potential.These results agree with the elevated antioxidant levels (αtocopherol and tocotrienols) found in ax (Linum usitatissimum) (Oomah et al., 1997) and in barley (Hordeum vulgare) seeds after elevated temperatures (Roach et al., 2018).Similarly, a negative correlation between antioxidants (α-tocopherol and β-tocopherols) was associated with poor longevity for rice seeds from temperate regions (Lee et al., 2019).Therefore, the antioxidant variation may have been conditioned by environmental conditions as reported in other plant species.

Latitudinal gradients with antioxidant accumulation
We aimed to answer whether latitudinal gradients have an impact on seed trait performance.Our results showed variation on germination and antioxidant accumulation along the latitudinal gradient (Fig. 1B and 6).Interestingly, the latitude-associated geographic distribution of antioxidants in leaves of Bilberry (Vaccinium myrtillus L.) was linked to adaptions to prevailing conditions and prevailing abiotic stress associated with a given geographic location (Martz et al., 2010).Similarly, secondary metabolites such as anthocyanidin are strongly controlled by the latitude and geographic origin in V. myrtillus fruits (Åkerström et al., 2010).A similar case was reported for blueberries (Vaccinium ashei cv.'Brightwell') in higher altitudes with higher concentrations of avonoids, phenols, proanthocyanidins, and anthocyanins, among other compounds (Zeng et al., 2020).Our data shows that latitude in uences seed germination.
However, this in uence might be as the combinatorial result of the climate conditions.Therefore, further evidence needs to be provided to separate those variables.
In summary, our results show a signi cant in uence on seed physiological performance along a latitudinal gradient in sea oats.Combined with the environmental variables, latitude directly dictates antioxidant accumulation in ungerminated seeds and after imbibition.Antioxidants are among the seed storage components critical for persistence, longevity, storage quality, and viability (Long et al., 2015).Therefore, the involvement of the environment in accumulating these secondary metabolites alongside other storage reserves is critical in seed quality.Knowledge of the interaction of associated environmental factors and their interaction is crucial for plants.It can be helpful in guiding the collection of seed materials and seed quality control for storage and use in conservation efforts.
Baskin and Baskin 2014; Pérez and Kane 2017; Moreira et al. 2020; Zhou et al. 2021).Using sea oat populations distributed across the US Atlantic and

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Table 1
Sea oats seed collections sites from the USA Atlantic and Gulf Coastlines.