Morphological, physiological and biochemical response of Lallemantia species to elevated temperature and light duration during seed development

Seed weight, storability, and germinability can depend on maternal plant's environment. However, there is slight information about the effect of light and temperature on seed quality of Lallemantia species. The purpose of this research was to determine the properties of physio-biochemical of maternal plant, seed quality, and seed chemical composition of Lallemantia species (Lallemantia iberica and Lallemantia royleana) under temperature (15 °C, 25 °C, and 35 °C) and photoperiod (8 hd-1, 16 hd-1, and 24 hd-1) maternal plants environment. Increasing temperature and photoperiod caused a reduction in leaf chlorophyll, stomatal movement, total soluble sugar, superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) enzymes activities, and an increment in malondialdehyde (MDA) and hydrogen peroxide (H2O2) content of seeds. However, the highest weight, germination, vigor index, and longevity, seed chemical compositions were obtained in offspring which matured under 25 °C for 16 hd-1. The highest germination, oil, and relative percentage of fatty acids (oleic acid (OA), linoleic acid (LA), and linolenic acid (LNA)) were obtained in L. iberica seeds. On the contrary, longevity, mucilage, and sucrose were more abundant in L. royleana seeds. Overall, this research has clearly shown that temperature and light quality and quantity of maternal plant's environment have an immensely effect on producing of seeds with high-quality. However, it is necessary to investigate the impact of the epigenetic mechanisms of the maternal plant on the offspring in future studies.


Introduction
Seed development is a crucial stage in the life cycle of plants and is ecologically and agronomically significant [1,2]. For the successful life cycle of crops in agricultural systems, high-quality seed is crucial to a rise in the production of seeds [3]. In other words, one of agriculture's primary concerns is the production of high-quality seeds [4]. Seed quality includes seed mass, storability, vigor, and germinability, and is used in agricultural systems to show the total value of a seed lot [5]. The main factor contributing to the importance of seed quality is that high-quality seeds have strong seed vigor, which can significantly increase germination rates and seedling establishment [3]. Some useful seed quality indices, such as seed weight, seed composition, germination, and seedling vigor can play a key role in the establishment of plants [6]. Some studies have revealed that there is a positive relationship between seed controlled chambers (BITEC-500, Shimadzu Corp., Kyoto, Japan) of a greenhouse at Shahed University, Tehran, Iran, in November 2020. Both Lallemantia species were grown under nine treatments constructed via combinations of temperature (15,25, and 35 • C) and photoperiod (8,16, and 24 h d -1 ) ( Table 2).
To do the potting experiment, 54 pots were used, and five seeds were sown in each plastic pot with a 20 cm diameter, which contained a mixture of soil (Table 3) and sand (3:2). From the planting stage to the start of the flowering stage ( Fig. 1(A-B)), the plants were grown under white fluorescent lamps (T5-28 W, Shanghai Flower and Biology Lighting Co., China), with a light intensity of 150 μmolm − 2 s − 1 , photoperiod 16 h d -1 , constant temperature of 23 • C, 70% relative humidity, and an 800 μmolmol − 1 CO 2 level in controlled chambers. After all the seeds were germinated, only three plants per treatment were selected to grow through the experimental period [20].
To preserve the moisture at 75% of the field capacity, pots were daily weighed, and distilled water was added to each replicate. When most plants had turned yellow (the physiological maturity stage), watering was stopped, and seven days later, the dried plants matured. At the end of the growing season, the seeds of L. iberica (moisture content: 7%) and L. royleana (seed moisture content: 5%) of each maternal plant's environment were harvested at the same time (February 8, 2021). In the following stage, seeds were stored at − 80C in screw-cap tubes until the start of trait measurements. The procedures described were carried out for each replication.

Physio-biochemical properties 2.3.1. Leaf chlorophyll
For the measurement of chlorophyll concentration (30 days after flowering), fresh leaves (0.5 g) were powdered using 10 ml acetone (80% v/v). Then, samples were centrifuged (3000 g) for 15 min; then, the absorption of extracts was read at 645 and 663 nm. Chlorophyll a and chlorophyll b concentration was estimated [39] using equations [1,2]:

Leaf stomata
About 30 days after flowering, three leaves were soaked in glutaraldehyde solution (4% w/v) for 24 h at 12 • C [40]. The stomata of leaves were measured by electron microscopy (CX41, Olympus Corporation, Tokyo, Japan).

Total soluble sugars
To quantify total soluble sugar content, 50 mg fresh leaves (after 30 days flowering) were mixed with sodium phosphate buffer (50 mM, pH 7.5) and centrifuged for 20 min at 12000 rpm [41]. The supernatants were used as the extract of total soluble sugar. Briefly, 0, 2 mL of supernatants was added to the anthrone reagent, then the mixture was placed at 80 • C for 40 min. The absorbance was read at 625 nm. Total soluble sugar was calculated using a standard curve of D-glucose [42].

Enzyme extraction and assay of enzyme activities
For the assay of superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX), about 30 days after flowering, 200 mg fresh leaves were powdered in an ice-cold condition in 50 mM sodium phosphate buffer (pH 7.0), a-dithiothreitol (2 mM), and EDTA (0.1 mM). The homogenates were centrifuged at 12000 rpm for 15 min at 4 • C. The activity of SOD was assayed using 40 mL enzyme extract, methionine (13 mM), p-nitroblue tetrazolium chloride (63 μm), riboflavin (1.5 μm), and 10 μM EDTA. The mixture was incubated for 10 min under florescent light with an intensity 50 μmol m − 2 s − 1 at 20 min. The reaction was monitored for 3 min at 560 nm. The amount of enzyme was expressed as Units mg − 1 protein [43]. The CAT reaction mixture included H 2 O 2 (500 μL 10 mM) and sodium phosphate buffer (1.400 μL 24 mM, pH 7.0). The consumption of H 2 O 2 was estimated at 240 nm for 3 min after the addition of enzyme extracts to the reaction mixture. CAT activity was estimated using molar extinction coefficient ε = 43.6 M − 1 cm − 1 [44]. The reaction of APX contained enzyme extract (50 μL), ascorbic acid (0.5 mM), sodium phosphate buffer (50 mM, pH 7.0), H 2 O 2 Table 2 Details of temperature and photoperiod treatments for Lallemantia specie from flowering stage to physiological maturity stage. (1 mM), EDTA (0.2 mM. The reaction was measured at 290 nm over 3 min. APX activity was calculated using molar extinction coefficient ε = 2.8 mM − 1 cm − 1 [45]. The protein content of the extracts was assayed using bovine serum albumin [46].

Hydrogen peroxide concentration
200 mg of fresh leaves (30 days after flowering) were powdered with trichloroacetic acid (5 ml of 0.1% (w/v)) in an ice bath. After centrifuging (13000 rpm for 20 min), phosphate buffer (0.5 mL of 5 Ml, pH 7.0) and potassium iodine (1 mL of 1 mM) was added along with 0.2 mL supernatant. The absorbance was read at 390 nm. Hydrogen peroxide was measured using a standard curve of H 2 O 2 [47].

Malondialdehyde concentration
Briefly, 200 mg of fresh leaves (30 days after flowering) were milled with 5 mL of 10% thiobarbituric acid (TBA) and centrifuged (16000 rpm for 20 min). Then, 1.0 mL of the supernatant was added to 1 mL of 0.5% TBA, heated at 100 • C for 15 min, and cooled in an ice bath. The absorbance was obtained at 532 nm and 600 nm. Malondialdehyde concentration was measured using the extinction coefficient of malondialdehyde at 532 nm (155 mM 1 cm − 1 ) [48].

Seed weight
The number of achenes per plant and the number of seeds per plant were measured. To obtain the mean seed weight, a batch of dry seeds was weighed and divided by the number of weighed seeds [49].

Seed germination
Seed germination was evaluated according to the standards of the International Seed Testing Association [50]. After that, in each  Petri dish, 50 seeds were placed on Whatman filter paper, and 10 ml of water was added to each Petri dish. A germination test was performed at 10 • C, 16/8 h (light/darkness), and 85% RH for 14 days. Seeds germinated were counted every day; afterward, the seed germination potential, mean germination time (MGT), and vigor index [51] were calculated according to the following equations [3][4][5]: Germination (%)=(germinated seeds number at initial stage(the fifth day)/total saples number) × 100 [3] MGT = Σ(Dn)/Σn [4] Where n is the number of germinated seeds on day D, and D is the number of days from the beginning of the germination test.

Seed longevity
The test of seed longevity was done by incubating seeds at 40 • C with 100% relative humidity in a closed tank with circulation for one day [50]. The seeds were then taken out, and their germination was counted.

Seed oil and fatty acid components
Dry seeds (1 g) were milled and wrapped in a thimble of extraction paper. Papers were placed inside the distillation flask in a soxhlet, and hexane solution (150 mL) was added. The solvent was placed at a range of boiling temperatures (40-60 • C) for 4 h to evaporate. The oil extraction was obtained by filtration and dehydration [52]. Eventually, the relative percentage of fatty acids was measured by a gas chromatograph system [53].

Seed mucilage
Dried seeds (10 g) were boiled at 100 • C for 30 min. After adding ethanol 80% (v/v) to the reaction, the samples were centrifuged at 30000 rpm for 40 min [54].

Seed sucrose
Dry seeds (0.3 g) were mixed with 80% ethanol (5 mL). The reaction was heated at 80 • C for 40 min and centrifuged at 100000 rpm for 10 min. The sucrose was estimated with a mixture of 0.15 mL supernatant and NaOH (0.15 mL 2 mM) at 100 • C for 5 min, which was then mixed together with HCL (2.1 mL 30%) and resorcinol (0.6 mL 0.1%) at 80 • C for 10 min. The reaction was read at 480 nm using sucrose as one standard [55].

Statistical analysis
The data were analyzed using SAS (SAS version 9.2, SAS Institute, Cary, NC, USA). Duncan's multiple range test (0.05%) was done to make a mean difference comparison. Origin Pro Software (OriginLab Corporation, Northampton, MA, USA) was used to run the Pearson correlation.

Results
An increase in chlorophyll a and b concentration was obtained in mother plants grown under standard temperature (25 • C). Furthermore, chlorophyll a and b concentrations increased in mother plants that were grown under the long day (16 h d -1 ). In comparison with L. royleana, a rise in chlorophyll a and b concentrations was observed in L. iberica leaves ( Table 4).
The most open stomata and the least closed stomata were obtained in maternal plants grown under standard temperature (25 • C). In addition, an increase in open stomata and a decline in closed stomata in both species were obtained in mother plants matured under long day (16 h d -1 ). It is evident that L. iberica had the greatest percentage of open stomata and the least percentage of closed stomata in comparison with L. royleana ( Table 4).
The otal soluble sugar content measured in both species' leaves were affected by temperature regimes and photoperiod. A decline in total soluble sugar content was observed in mother plants which were grown under high temperature (35 • C) and continuous light (24 h d -1 ). The total soluble sugar in the leaves of L. royleana was higher than that in L. iberica ( Table 4).
The SOD, CAT, and APX enzyme activities in both species of Lallemantia significantly increased as the temperature increased from 15 • C to 25 • C. There was an upward trend in SOD, CAT, and APX enzyme activities in maternal plants grown under long day (16 h d -1 ). Compared with L. iberica, the highest SOD, CAT, and APX enzyme activities were observed in L. royleana (Table 4)  Means comparison of physiological and biochemical indices, number achenes per plant, number seeds per plan and seed weight of Lallemantia species under different temperature and photoperiod treatments.

Plant species
Temperature SOD (Units/ mg protein) CAT (Units/ mg protein) APX (Units/ mg protein)  The most achenes, seeds per plant, and seed weight were observed in mother plants grown under high temperatures (25 • C) and long days (16 h d -1 ). Compared with L. royleana, the greatest achenes per plant, seeds per plant, and seed weight were observed in L. iberica (Table 4).
A rise in germination and vigor index were observed in seeds matured under high temperature (25 • C). The germination and vigor index were higher in seeds matured under long days (16 h d -1 ) than in other levels of photoperiod. The germination and vigor index in L. iberica were higher than those in L. royleana. MGT was found to be lowest in seeds matured at standard temperature (25 • C) and long day (16 h d -1 ). MGT in L. royleana seeds was lower compared with L. iberica (Table 5).
A reduction in longevity was indicated in seeds matured under high temperature (35 • C). However, the longevity of seeds matured under long day (16 h d -1 ) was more than that of other photoperiod treatments. Based on the results, an increase in longevity of L. royleana seeds was observed compared with L. iberica seeds (Table 5).
Seeds matured under standard temperature (25 • C) had a higher oil content and relative percentage of fatty acids. Furthermore, as day length increased, the oil content and fatty acids of LNA, LA, and OA in seeds matured decreased. Oil content and relative percentages of LNA, LA, and OA were higher in mature L. iberica seeds than in L. royleana seeds (Table 5). (Table 5).
Standard temperature (25 • C) had a significant effect on the increase in mucilage content of seeds compared with other temperature regimes. Results demonstrated that mucilage content increased from short day (8 h d -1 ) to long day (16 h d -1 ) in mature seeds. Compared with L. iberica seeds, the highest mucilage was obtained in L. royleana seeds ( Table 5).
The highest and lowest sucrose contents were observed in seeds matured under standard (25 • C) and high (35 • C) temperatures. Furthermore, seeds matured under long day (16 h d -1 ) conditions had higher sucrose content than those other photoperiod treatments. Compared with L. iberice seeds, the of seeds L. royleana had the greatest sucrose content ( Table 5).
The values of the Pearson correlation showed that seed weight had a positive correlation with open stomata, chlorophyll a, chlorophyll b, germination, and vigor index. It was clear that seed oil and seed mucilage had a positive correlation with seed weight, chlorophyll a, chlorophyll b, and open stomata. Besides, there was a positive correlation between seed longevity, seed mucilage, and seed sucrose content. Total soluble sugar, superoxidase dismutase, catalase, and ascorbate peroxidase activities were negatively correlated with MDA and H 2 O 2 . In addition, there was a negative correlation between MGT and germination, vigor index, and seed weight (Fig. 2).

Discussion
The maternal plant's environment during seed filling strongly influences seed quality, physiological and biochemical properties, and indices of seed quality [1,56]. An important photosynthetic pigment of the maternal plant is chlorophyll which determines the capacity for photosynthetic activity and the growth of the maternal plant [14]. The results suggested that standard temperature (25 • C) and long day (8 h d -1 ) conditions of the maternal plant's environment induced an increase in chlorophyll concentrations of both Lallemantia species which is most probably due to an increase in open stoma stomata [57]. An increase in open stomata allows for the intake of CO 2 which is required for the process of photosynthesis [58]. Therefore, an increase in CO 2 availability increases photosynthesis and drops the oxygenase activity of Rubisco [59]. Some studies have demonstrated that open and closed stomata on the leaves of the mother plant play a vital role in gas exchange, transpiration, and photosynthesis [14]. Higher chlorophyll concentration in L. iberica leaves than that in L. royleana can be due to an increase in open stomata [36].
Soluble sugar content could be a regulator in defense mechanisms, inducing the balancing of ROS in cells under different regimes of temperature and light [60]. This soluble sugar content acts as a scavenger of ROS and a membrane stabilizer against stress [61]. In this study, total soluble sugar content in the maternal plant leaves increased as it matured under standard temperature (25 • C) and long day (16 h d -1 ). The accumulation of soluble sugar in the mother plant's leaves appears to be caused by osmotic stress which is exacerbated by the high temperature and light of the maternal plant's environment [62]. Some studies have indicated that sugar acts as an antioxidant and causes the improvement of oxidative stress [63]. In comparison with L. iberica, further total soluble sugar in L. royleana can probably be attributed to the greater stability of the membrane against ROS [60].
Based on biochemical and molecular findings, it has been determined that decrease in membrane structure destruction prevents the production and accumulation of MDA and H 2 O 2 [64]. The reduction in MDA and H 2 O 2 concentration under standard temperature (25 • C) and long day (16 h d -1 ) can be associated with increasing SOD, CAT, and APX enzyme activities which act as the main line of defense [65]. It seems that the reduction in lipid peroxidation under standard temperature and long day during seed maturation indicates the reduction in membrane damage, and provides the essential carbon for the maintenance of raffinose and polysaccharides in the offspring [66]. According to recent research, sucrose, and raffinose are involved in the maintenance of cytoplasm glassy state and reduce lipid peroxidation [30,67]. In the current study, more SOD, CAT, and APX enzyme activities in L. royleana than that in L. iberica leaves were probably due to the limitated oil content, and reduction in the accumulation of H 2 O 2 and lipid peroxidation [64,68].
In our study, growth traits such as achenes per plant and seeds per plant, and seed weight decreased under high temperature (35 • C) and continuous light (24 h d -1 ), which can be attributed to a decline in chlorophyll concentrations and uptake of CO 2 availability for photosynthesis [69]. Numerous studies have shown a rise in the uptake of CO 2 during photosynthesis stimulates carbohydrates accumulation in leaves [38,70]. The transportation of carbohydrates from the source (leaf) to the sink (seed) has a crucial pattern in the growth and development of seeds [71]. On the other hand, it is probably the transgenerational epigenetic inheritance that supplies the mechanisms for the adaptation of maternal effects on offspring phenotypes such as seed weight, germination, seedling growth, and seed composition [72,73]. Our results showed that achenes per plant and seeds per plant, and seed weight were more in L. iberica than that in L. royleana, which can be attributed to the much higher seed weight of L. iberica [74,75].
An increase in germination and vigor index and decrease in MGT have been observed in some plants such as Brassica napus L. [76], and Orize sativa L [77]. under standard temperature and a long day. It has been reported that the increase in photosynthetic activity in the leaves of maternal plants can induce the production and accumulation of carbohydrates inside seeds [78]. In fact, carbohydrates are a crucial source of cell energy and assist the seeds in germination [79]. Starch is a strong carbohydrate that provides essential energy for the hydrolysis action of a and b amylase for the germination of aged seeds [80]. A rise in the germination and vigor index of L. iberica seeds compared with L. royleana seeds can be attributed to the increased seed weight [81]. On the other hand, the higher MGT of L. royleana seeds was probably due to the high mucilage around the seeds [40]. In other words, the presence of mucilage around the seed serves as a filter or sorbent under stress conditions and creates a hydrophilic property [82].
An increase in longevity of developed seeds under standard temperature (25 • C) and long day conditions can be attributed to high photosynthetic pigments of maternal plants which supply more carbohydrates for synthesizing starch, lipids, and proteins in seeds [83]. Some studies documented that carbohydrates, as the main reserves of seeds, induced the glassy state of the cell cytoplasm and inhibited the deterioration of seeds [84]. The glassy state raises the viscosity of the intracellular fluid and decreases metabolic activity in the cytoplasm [85]. Further seed longevity of L. royleana than that in L. iberica may be due to less oil in L. royleana seeds [86]. Hence, it can be inferred, high saturated fatty acids during seed storage induce a decline in the lipid fluidity of cell membranes and a rise in MDA and ROS accumulation [87]. In addition, the decline in the longevity of L. iberica may be due to the increase in oxidative damage in polyunsaturated lipids [88,89].
The highest oil content, relative percentage of fatty acids, and mucilage content were found in seeds that were matured under standard temperature (25 • C) and long day (16 hd-1). The reason could be attributed to increased chlorophyll content [90,91]. Obviously, photosynthesis activities in the leaves play an important role in the generation and transportation of carbohydrates into seeds in order to Ref. [92] produce the storage reserves such as lipid, protein, and starches [93]. The higher oil content and relative percentage of fatty acids in L. iberica seeds than in L. royleana seeds could be attributed to an increase in photosynthetic pigments and open stomata, which provide more carbohydrates for the biosynthesis of mucilage, oil, and fatty acids [38]. Furthermore, the increase in the oil and fatty acids of L. iberica seeds may be related to higher levels of nutrients such as nitrogen, phosphorus, potassium, calcium, and manganese [94,95]. Some studies have indicated that phosphorus in seeds has an important role in providing ATP and NADPH for biosynthetic fatty acids [38]. The results showed an increase in mucilage of L. royleana seeds in comparison with L. iberica, which may be due to epigenetic memory superseding that of maternal plants [23].
The reduction in sucrose content under standard temperature (25 • C) and long day (16 h d -1 ) conditions may be related to an rise in A. Paravar et al. chlorophyll content [96]. Chlorophyll is a crucial pigment that plays a role in the absorption, transmission, and transformation of light energy in photosynthesis [97]. The production of photosynthetic activity such as sucrose occurs in the carbon vertebrae, which provide energy for the metabolism and synthesis of amino acids in seeds [98]. Some studies have been indicated that maternal plants assimilate inorganic carbon via photosynthetic reactions in the forms of sucrose, starch, glucose, and fructose [99]. The carbon absorbed by mother plant is transferred to the offspring in the form of starch and a fraction of sugars [100]. Compared with L. iberica seeds, an upward trend in sucrose content of L. royleana seeds was observed, which can be related to the genetic differences between maternal plants [101].

Conclusion
The findings of this study revealed that the temperature and light of the maternal plant's environment had a strong influence not only on the physio-biochemical properties of maternal plants but also on the seed quality and chemical compositions of their offspring. The appropriate response of L. iberica and L. royleana maternal plants to the standard temperature (25 • C) and long day (16 h d -1 ) environment improved offspring weight, germination, vigor index, longevity, mucilage, oil, the relative percentage of fatty acids, and sucrose content. Furthermore, due to the higher dependency of L. iberica maternal plant on the temperature and light of the maternal plant's environment than L. royleana, the highest weight, germination, vigor, oil, and relative percentage of fatty acids were observed in the offspring of L. iberica. On the other hand, further longevity, mucilage, and sucrose observed in offspring of L. royleana compared with L. iberica was probably due to genetic reasons. Generally, this research highlighted how temperature and light in the environment could affect the maternal plants and offspring of L. iberica and L. royleana. However, it is more necessary to investigate the epigenetic mechanisms of maternal plants on the offspring of L. iberica and L. royleana in future research.

Author contribution statement
Arezoo Paravar: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.
Saeideh Maleki Farahani: Conceived and designed the experiments; Interpreted the data Contributed reagents, materials and analysis data.

Data availability statement
Data included in article/supplementary material/referenced in article.

Additional information
No additional information is available for this paper.

Acknowledgment
We kindly acknowledge the Shahed University of Tehran for their support of this research.