Relationships between substrate and the mobilization of reserve with temperature during seed germination of Ormosia coarctata Jack. Journal of Seed Science

: Seed germination studies provide essential information for biodiversity conservation and ecological restoration programs. This work aimed to investigate the relationship between the substrates and the mobilization of reserves during germination of Ormosia coarctata seeds under different temperatures. Samples were collected every 48 h for up to 240 h for quantification of lipids, soluble sugars, starch, and soluble proteins. The optimum temperature range for germination was 25 to 35 °C. The highest germination percentages were obtained using sand or paper roll. Carbohydrate, lipid, and protein contents decreased during germination, regardless of temperature. Galactose that monosaccharides in germinated


INTRODUCTION
was incubated with 80% (v/v) ethanol for 30 min at 75 °C, and the mixture was centrifuged at 10,000 g for 5 min. The supernatants were combined and taken to dryness. Then, the powder was resuspended in 1.0 mL of ultrapure water, and 0.5 mL of this solution was used to prepare an alditol acetate solution. Quantification was performed according to Englyst and Cummings (1985) on a Shimadzu GC 14-a gas chromatograph equipped with a flame ionization detector (FID) and a Shimadzu C-R8A Chromatopac integrator. A moderately polar column coated with 50% cyanopropylphenyl and 50% dimethylsiloxane was used. The gas flow was set at 0.25 mL.min −1 . The injector, detector, and column temperatures were respectively 2500, 2200, and 2750 °C. Samples were injected with a split ratio of 1/40; 1.0 μL of alditol acetate was used. Each treatment consisted of four replications.

Statistical analysis
The experiment was carried out in a 5 × 3 completely randomized factorial design, with five temperatures and three substrates. Treatments consisted of five replications of 20 seeds each, totaling 100 seeds per treatment. Germination percentages and GSI values were compared using Tukey's test. The level of significance was set at p < 0.05. Data were analyzed using R version 3.4.1 (R Core Team, 2017) and ExpDes package version 1.1.2 (Ferreira et al., 2013).

RESULTS AND DISCUSSION
The highest germination percentages were observed in O. coarctata seeds kept at 25, 30, or 35 °C in paper roll or sand; germination in these substrates did not differ significantly (p < 0.05) ( Figure 1A). Seeds germinated between papers had low germination percentage and GSI at all temperatures ( Figure 1B). GSI was highest at 30 and 35 °C ( Figure 1B). There was no interaction effect between temperature and substrate.
The highest germination percentages and GSIs were obtained using paper roll or sand as substrate ( Figures 1A and 1B), shows that their large contact area enabled O. coarctata seeds to absorb high amounts of water. A small contact area between substrate and seed might result in the rate of water loss being higher than the rate of absorption. The between paper method was unsuitable, probably because of the smaller area of contact between substrate and seed. When choosing the substrate, the shape and size of the seed must be taken into account (Brasil, 2009). M. brauna seeds , Inga laurina , Eugenia involucrate and Eugenia pyriformis (Gomes et al., 2016) presented higher percentages of germination in paper roll and sand. Thus, the contact area between the substrate and the seed influenced the greater water absorption and, consequently, higher germination values. Journal of Seed Science, v.42, e202042017, 2020 Seeds were not able to germinate at 15 °C during the 240 h experimental period. Seeds kept at 40 °C began to deteriorate and showed no signs of root protrusion after 96 h of imbibition. The theoretical optimal temperature was 27.8-27.9 °C, which is in agreement with the results.
The highest GSIs were achieved at 25, 30, and 35 °C. This temperature range is the same as that of the environment where seeds were collected. Oliveira et al. (2016) verified GSI values of Ormosia arborea seeds similar to those of the present study. According to the authors, 25-35 °C is the optimal germination temperature range for the species. Germination performance at a specific temperature range reflects the species adaptation to its native ecosystem, which in forest seeds may vary according to succession stage, biome, and environmental conditions (Wood and Prichard, 2003).
The rate of water uptake increased significantly with temperature, as shown by the fresh mass curves in Figure 2A. At 15 °C, imbibition was slow and continuous. At all temperatures, water content increased in the first 72 h, as water uptake at this stage is temperature independent. Imbibition occurs because of the difference in water potential between seed and substrate. In dry seeds, the matric potential is associated with the binding of water to the structural components of the cell wall and other macromolecules (Taiz et al., 2017). Imbibition initially results in the hydration of cell components, such as the cell wall and reserve polymers. The number of hydrated cells within the seed increases as imbibition proceeds (Nonogaki et al., 2010). Prior to hydration, the water potential of seed cells is more negative than that of the substrate. Upon contact, seeds rapidly absorb water.
The low and steady imbibition rate observed at 15 °C ( Figure 2) might be associated with the basic metabolism of the seed or with reserve degradation. Storage reserves are used during phase 2 of imbibition, increasing the osmotic potential. However, this phenomenon did not occur because water uptake was not sufficient. Low temperatures reduce imbibition rate, enzyme activity, and energy metabolism (Luo et al., 2019). It is possible that membrane-level changes are only part of the process. Most aquaporin plasma membrane intrinsic protein (PIP) genes are down-regulated in seeds under low-temperature stress (Luo et al., 2019). Reduction in the expression of these genes results in a low number of aquaporins, thereby affecting the water absorption rate of seeds.
Low temperatures affect the reorganization of cell membranes by making the process difficult and slow (Carvalho et al., 2009). The degree of fatty acid unsaturation of membrane phospholipids is altered at low temperature. This Figure 2. Imbibition curve of Ormosia coarctata seeds at different temperatures. Arrows indicate when radicle emergence occurred in 50% of the seeds.
Journal of Seed Science, v.42, e202042017, 2020 change affects the permeability of the membrane and its fluidity properties (Zheng et al., 2016;Noblet et al., 2017). In Zea mays seeds, low temperatures induce membrane disorganization, resulting in increased release of electrolytes into the medium and delayed germination (Noblet et al., 2017). A three-phase imbibition pattern was observed at 25, 30, and 35 °C, with rapid water absorption at the beginning, followed by a plateau and primary root growth. Radicle emergence occurred after 192 h at 25 °C and after 144 h at 30 and 35 °C (Figure 2).
Phase I occurs both in viable and nonviable seeds, as this physical process is independent of metabolic activity (Bewley et al., 2013) and temperature. During phase I, the energy metabolism is resumed, and the energy needed for enzyme activation and metabolite turnover is released (Nonogaki et al., 2010). Similar results were reported for seeds of Bowdichia virgilioides and D. nigra (Albuquerque et al., 2009;Ataíde et al., 2014). Little variation in fresh seed mass was observed from 72 to 144 h of imbibition at 25, 30, and 35 °C. This stabilization probably occurred because of the equilibrium between seed and substrate water potentials. Guimarães et al. (2008) reported that after tissues and organelles are hydrated, membranes and enzymes become functional (phase II) and storage reserves are broken down to be used for radicle elongation.
Seeds placed to germinate at 40 °C had a higher weight gain than seeds placed at other temperatures but were deteriorated after 96 h. The high temperature may have altered cell membrane permeability, leading to extravasation of cellular content. According to Badea and Basu (2009), high temperatures can modify components of the phospholipid bilayer. Microorganism contamination might be related to the loss of metabolites (Bewley et al., 2013), as exudates can serve as a substrate for microorganism development. In addition, the kinetic energy of particles is affected by the increase in temperature: particles become more accelerated and hydrogen bonds in macromolecules are weakened (Źróbek-Sokolnik, 2012). High temperatures can alter the tertiary structure of enzymes, which in turn reduces the rate of reactions (Źróbek-Sokolnik, 2012).
Mobilization of reserves was lowest at 15 °C because of the low metabolic activity of seeds. This condition results in low respiration rates, affecting the breakdown of seed reserves, especially of galactose, which is the main source of energy. Consequently, germination is affected, as it requires stable and functional mitochondria (Luo et al., 2019). Seeds at 40 °C had the highest consumption rate of reserves in 96 h of imbibition. Even though seeds germinated at other temperatures were imbibed for a longer period, 96 h of imbibition at 40 °C was sufficient for metabolic activation and reserve mobilization. However, these processes were interrupted at 96 h because seeds deteriorated. Changes in the respiratory activity of cells occur with seed deterioration, such as the release of high levels of energy (Horbach et al., 2018) caused by loss of metabolic control over the electron transport chain and oxidative phosphorylation.
Soluble sugar levels decreased during imbibition at all temperatures except 15 °C ( Figure 3A). The highest reductions occurred after 48 h of imbibition at 25, 30, and 35 °C. At 40 °C, the content of soluble sugars decreased by 50% in 96 h. The consumption of soluble sugars during germination processes is attributed to energy expenditure during respiration, energy generation, and supply of embryo growth (Koch, 2004;Souza et al., 2018). Moreover, sugar consumption is also due to its metabolic signaling role. Many sugars act as a signal for the synthesis of enzymes and phytohormones (Souza et al., 2018). Therefore, its consumption may be related to molecular signaling for the different stages of tissue development (Koch, 2004). The total content of soluble sugars and reducers from two sunflower cultivars decreased during the first 24 hours (Erbaş et al., 2016). According to the authors, these reserves are the first to be used as an energy source during the initial germination period.
Xylose levels did not change significantly during imbibition at 15 °C ( Figure 3C). At 25, 30, and 35 °C, xylose concentration decreased from 0.00126 to 0.00081 mg.g −1 dry matter after 48 h. At 40 °C, xylose was rapidly consumed. Galactose levels decreased at all temperatures, although at a slower rate at 15 and 40 °C ( Figure 3D). At 25, 30, and 35 °C, galactose was depleted after 144 h of imbibition. Seeds germinated at 25, 30, and 35 °C showed similar variation in the levels of xylose and galactose. Galactose was completely depleted toward the end of imbibition, whereas xylose was still detected. These results demonstrate that monosaccharides are preferentially broken down, regardless of the presence of oligosaccharides. Xylose and galactose were consumed or released at a higher rate in seeds imbibed at 40 °C, probably because of the degradation of cell membranes toward the end of imbibition. Thus, two processes occurred during hydration of O. coarctata seeds at 40 °C. First, seed reserves were broken down, as shown by the increase in metabolic activity; then, reserves were released to the medium as a result of thermal damage. Glucose was not detected at 25, 30, or 35 °C (data not shown). However, this monosaccharide was present in seeds imbibed for 144 h at 15 °C and for 48 h at 40 °C (data not shown). These results may be explained by the partial isomerization of galactose. At 15 °C, increased glucose levels were attributed to low metabolic activity to and, at 40 °C seed death. The not detection of glucose during the germination of O. coarctata seeds may be related to its use for the production of sucrose in embryonic tissues. In Cedrela fissilis, glucose was detected only during the early stages of Journal of Seed Science, v.42, e202042017, 2020 seedling development (Aragão et al., 2015). In Sebastina virgata seeds, glucose is formed during the development of the seedlings in relatively high amounts (Tonini et al., 2010). With the development of the primary root, the degradation of wall polysaccharides is released through the degradation of ABA, consequently sucrose and glucose increase (Tonini et al., 2010). Alternatively, monosaccharides may leave the embryo or cotyledons and are retained in the membrane or seed coat, as observed by Borges et al. (2002) in Platymiscium pubescens seeds.
The mobilization pattern of starch differed according to the germination temperature. Seeds imbibed at 15 °C showed no changes in starch concentration with time ( Figure 3B). At 40 °C, starch levels decreased rapidly after 48 h. The decrease in carbohydrate levels in seeds germinated at optimal temperatures (25-35 °C) indicates that there was a high mobilization of starch. Thus, soluble sugars together with starch reserves indicate degradation to obtain fast energy for embryo growtn.
Lipid levels varied with time of imbibition at all temperatures ( Figure 3E). O. coarctata seeds initially contained 8.2% lipids. Lipid mobilization was slower at 15 °C. At 25, 30, and 35 °C, lipid levels decreased by 2.6, 7.9, and 11.1%, respectively. At 40 °C, the lipid content decreased continuously as the cotyledons deteriorated. The oil stored in the seeds in the form of triacylglycerols plays an important role in the growth of the embryo, providing energy and carbon skeletons (Knauer et al., 2013). Most seeds consume first the reserves of lower metabolic expenditure for your use (Bewley et al. 2013). However, this behavior depends on the type of seed, the amount of reserves, and the speed of germination (Bewley et al. 2013).
Lipid mobilization was relatively lower than that of the other reserves. Variation in lipid levels was similar among the different germination temperatures. Mobilization of lipid reserves is more intense after germination (Hooks et al., 2010). Free fatty acids were probably used for germination instead of triglycerides. Low lipid mobilization can be associated with the low energy requirement during germination. To be converted into carbohydrates, lipids must first be broken down by lipases and then degraded through the β-oxidation and glyoxylate pathways (Taiz et al., 2017). Because they are highly reduced molecules, lipids have high energy density and can easily supply the energy needed in the early phases of seed hydration.
The initial protein content was 16.6%. Protein levels were affected by imbibition time and temperature ( Figure 3F). At 15 °C, the total protein content remained relatively stable for up to 72 h. After 96 h of imbibition, protein levels decreased by 2.3, 16.6, 17.2, and 17.5% in seeds soaked at 15, 25, 30, and 35 °C, respectively. At 40 °C, seeds showed a continuous reduction in protein content. The total protein level was 36.8% lower after 96 h of imbibition. During imbibition, stored proteins are proteolyzed by different enzymes (Bewley et al., 2013) resulting in free amino acids that are used for the synthesis of protein components and, therefore, essential for the development of the embryo (Erbaş et al., 2016).
Unfavorable temperature conditions can negatively affect reserve mobilization, thereby impairing germination (Mengarda et al., 2015). Ataíde et al. (2016), however, observed no differences in reserve mobilization in seeds germinated at 15 or 25 °C. The authors reported that soluble carbohydrates, starch, and protein levels decreased while lipid levels showed little variation.
O. coarctata seeds are composed mainly of cotyledons, as the embryo has a reduced size. Thus, we evaluated the levels of cotyledon reserves. Considering that the embryo has little demand for reserves at the beginning of germination, we assumed that the decrease in reserve levels was due to cotyledon respiration or exudation.

CONCLUSIONS
Paper roll and sand were the best substrates for O. coarctata seeds germination. The optimal temperature range was 25-35 °C. Carbohydrate, lipid, and protein reserves decreased at all temperatures. Mobilization patterns of soluble sugars, xylose, and galactose were similar in seeds germinated at 25, 30, and 35 °C. Glucose was detected in O. coarctata seeds at 15 and 40 °C, temperatures that are not optimal temperatures for germination. This monosaccharide was not present in seeds germinated at other temperatures.