Introduction

During seed development, fat accumulates in storage tissues such as cotyledons, endosperm or shield (Huang 1996; Murphy and Vance 1999), which percentage varies widely between species, ranging from 1 to 60% (w/w of total dry weight) (Ohlrogge and Browse 1995). Some researchers found that lipids in oil-seeds accumulated to comparatively high levels from early to late stages of seed development (Archana et al. 1999; Neuhaus and Emes 2000; Özcan 2013; Yadav and Singh 2003). And some exist in the form of triacyglycerides in fat or oil bodies (OBs) for seed germination and seedling growth (Chapman and Ohlroggen 2012; Chapman et al. 2012), others may distribute in more complex structures such as membrane phospholipids. Furthermore, the sizes of the OBs are related to the content of triacylglycerol (TAG) (Tzen et al. 1993), and most of it may be the result of the fusion of small OBs and coalescences of smaller ones (Ariotti et al. 2012). Concerning the type of fatty acids (FAs) produced, Carvalho et al. (2010) reported that the three most abundant FAs in seeds of thirteen species of Artemisia were palmitic acid, linoleic acid and α-linolenic acid. In particularly, these latter ones unsaturated FAs (UFAs) are predominated in all Artemisia species. Rao et al. (2008) suggested that the high level of α-linolenic acid may largely be controlled by the level of some gene expression.

The deciduous tree Styrax tonkinensis, a member of the Styracaceae family, is characterized by rapid growth, a straight trunk and white fragrant flowers (Xu and Yu 2015). Its wood is suitable for the manufacture of handicrafts, pulp and paper and so on (Luo et al. 2007). It also produces benzoin which is a resin composed of fragrant fatty acids or triterpenoids that is utilized as a source of a complex spice or as a medicine (Wang et al. 2006; Burger et al. 2016). Its seeds have high oil content (aprox. 54.86% w/w, Wu et al. 2019) and nutritional components (oil, soluble sugar, starch, free amino-acid and soluble protein) that can be used as biofuel due to its excellent fatty acid composition and good fuel properties (Xiao et al. 2005; Liu et al. 2007; Zhang et al. 2017; Wu et al. 2019). Therefore, the species has high potential commercial value and can make an important contribution to the local economy (Ounekham 2009).

Many studies on S. tonkinensis have focused mostly on ornamental value (Xu and Yu 2015), rules of seed oil accumulation (Zhang et al. 2017, 2018; Wu et al. 2020), benzoin resin production (Wang et al. 2006) and so on. However, little is known about relatively comprehensive research of accumulation of fatty acids, oil body and ultrastructural changes during seed development. The objective of the present study was to monitor the process of lipid accumulation using transmission electron microscopy (TEM), and to measure the oil content and composition in seeds of S. tonkinensis during their development and maturation, with the intent to provide data for directional breeding programs aimed at improving oil content and desired FAs composition in S. tonkinensis seeds.

Materials and Methods

Plant material

The S. tonkinensis trees were grown under natural conditions within the breeding collection of Jiangsu Guoxing Biotechnology Limited, which is located in the Hewang Lake Community, Ma’an street, Luhe District, Nanjing City, Jiangsu Province, China (32°32′ N, 118°50′E). Peak flowering occurred on May 21, 2013. Therefore, data regarding temperature, total precipitation and evaporation at Luhe District from May 21^st to October 10^th, 2013 were recorded (Fig. 1). In early May, 2013, 10 robust trees (4 years old) that reached at peak fruiting were tagged for sampling. Seed harvesting was collected randomly from June 10, 2013 that was 20 days after flowering (DAF), and thereafter continued every 20–21 days until October 10, 2013 resulting in a total of seven sampling events. Then, fruits were peeled, mixed evenly and stored in a refrigerator (SANYO, Japan) at -70℃ before analysis.

Anatomical observation

In order to examine the process of material accumulation (especially lipid) in endosperm and embryo during development stages of S. tonkinensis seeds, some samples were observed by stereomicroscopy (Olympus Corporation, Tokyo, Japan) and TEM (JEOL, Tokyo, Japan). To keep the description simple, only one picture was selected for each phase.

After being cleaned with distilled water and drying, samples were selected from the endosperm near the radicle and embryo (hypocotyl cells near the radicle) for TEM analysis. Samples were fixed in 4% glutaraldehyde, cleaned with phosphate buffer (0.1 M, pH 7.2), and fixed again in 1% osmic acid (0.1 M pH 7.2 phosphate buffer). Before sectioning, the fixed samples were cleaned with phosphoric acid buffer solution and dehydrated in a graded series of ethanol (30-50-70-90-100%) solutions. Then, they were infiltrated, embedded polymerized in Epon812 epoxy resin bought from Electron Microscopy Sciences (E.M.S., USA), and then repaired, sliced semi-thinly using LKB-5 ultra-microtome (LKB Instruments Inc., Lucerne, Switzerland), positioned, sectioned ultra-thinly (50 nm) and semi-thin section (2 μm) for three times, stained with uranyl acetate and lead citrate, and finally observed and photographed in a JEM-1400 TEM (JEOL, Tokyo, Japan). The TEM images were viewed for measuring in Nano Measurer 1.2 (Fudan University, Shanghai, China, 2008) and calculated the size of oil body diameter (OBD) (Zhang et al. 2018). The images are shown in Fig. 1S a–h.

Oil content

A Soxhlet extraction method (GB/T14772-2008) was used to analyze the crude oil content in seeds. At each developmental stage, 0.5 g of finely ground seed sample was placed into aluminum boxes and dried at 105℃ for 2 h. The powdered samples were subsequently placed into individual Soxhlet extractor bottles and 200 mL of petroleum ether (boiling point 60–90℃) was added. The samples were repeatedly extracted at 85℃ in a water bath until all of the crude oil was removed. The filter paper bags were then baked at 105℃ to completely volatize the petroleum ether, placed into a dryer, allowed to cool, and then weighed until a constant weight was obtained. Each assay was repeated a total of 3 times for each sample.

Lipid analysis

Extraction and esterification of oil

The samples of extracted seed oil, obtained from the different aged seeds, were dried by using a rotary evaporator (RE-3002, Shanghai, China). Then, 4 mL mixture of benzene and petroleum ether (volume ratio 1:1), and 1 mL KOH-CH ₃ OH solution (0.4 mol/L) were added to each sample, vortexed and left to stand for 15 min. Subsequently, 8 mL distilled water was added, and each sample was sufficiently vortexed until the solution was clear and stratified. The resultant supernatant was collected, which was yellow, clear and emitted a mild oil odor, evaporated by decompressing, dissolved in 5 mL hexane, and then analyzed by gas chromatography and mass spectrometry (GC-MS) for precise determination of both lipid content and fatty acid composition following some reported methods (Sardesai and Manning 1968; Zhang et al. 2017).

Determination of fatty acid composition

Chromatographic analyses were conducted by a TRACE-DSQ GC-MS (Thermo Electron, U.S.A.) using a DB23 column. Injector temperature was 250℃, oven temperature program was from the initial temperature of 40℃ (for 1 min) to 195℃ (for 2 min), to 205℃ (for 2 min) with an increasing temperature rate of 2℃/min and finally raised to 230℃ at a rate of 8℃/min, which was maintained for 1 min. The carrier gas was helium with a flow rate of 1.0 mL/min. The amount of sample injected was 1 µL and the split ratio was 50:1. The time of solvent delay was 6 min and the ion source was electron impact ion source (EI). The temperature of the source was 230℃, the emission current was 34.6 µA, the ionization voltage was 70 eV, the interface temperature was 250℃, the scanning speed was 2.72 amu/s and the scanning range was 12–550 amu.

Data analysis

The seed oil components were determined by a comparison of the obtained mass spectra with a mass spectral library retrieved from NIST06. The spectrum analysis was also combined with an analysis of the related literature using the data processing system of the MSD workstation (Agilent Technologies 689OGC/5973MSD). The relative percentage of the components (FAs) was calculated using the area normalization method and the following formula:

% FA = (FA/total peak area of FAs) ×100

Where FA is linoleic acid, oleic acid, palmitic acid or stearic acid, and the total peak area of FAs was calculated by summing their area.

The unsaturated index and degree of FA was calculated as described by Zhou et al. (2013), that is

Index of unsaturated fatty acid (IUFA) = 

In the formula “Si” is the relative content of unsaturated fatty acid (UFA) and “Ti” is the number of unsaturated double bonds in the UFA.

Fig. 1: The records of the highest and lowest temperatures (a), and monthly total precipitation and evaporation (b) in Luhe District, Nanjing City, Jiangsu Province, China from May 21^st to October 10^th, 2013 (data from the public service website of Chinese Meteorological Administration at http: //www.weather.com.cn/)

Instead, the degree of UFA= UFAs relative content /saturated fatty acids (SFAs) relative content.

The above data were averaged by three independent experiments, and figures were computed in Microsoft excel. The correlation of fatty acids and OBD with environmental factors was analyzed by S.P.S.S. 19.0 (I.B.M., Armonk, NY, USA) using Pearson’s correlation analysis.

Results

Anatomy structural observations of endosperm and embryo

Some characteristics during different development stages of S. tonkinensis seeds observed by stereomicroscopy showed that seed developed gradually, the color changed from green to yellow, the substance in the seed altered from liquid state to semi-solid and solid state, and the development of embryo was later than that of endosperm (Table 1). Preliminary differentiation of embryo and dicotyledon are not obviously visible until 100 and 120 DAF, respectively. Therefore, in our study, the observation of the micro-structure of endosperm near the embryo was monitored by TEM from 0 to 140 DAF (Fig. 2a–g) and embryo from 100 to 140 DAF (Fig. 2 h–j).

Ultrastructural observations of endosperm cells indicated the formation and accumulation of some substances especially lipids during seed development. At 20 DAF (Fig. 2a), nucleus and nucleolus were obvious in some endosperm cells, and there were more mitochondria providing energy for endosperm cell division, and annulate lamellas in different periods. At 40 DAF (Fig. 2b), there were many high electron dense materials near the cell membrane, vesicles along with a few starch grains. At 60 DAF (Fig. 2c), large vacuole and vesicles appeared with high electron dense materials and a small amount of cytoplasm were distributed close to cell membrane, and more mitochondria occurred. At 80 DAF (Fig. 2d), large vacuole, more mitochondria and endoplasmic reticulum occurred and extracellular space was big. At 100 DAF, many different sizes of gradually accumulated OBs and a lot of high electron dense materials could be obviously observed (Fig. 2e). At 120 DAF (Fig. 2f), in endosperm cells, there were also many different sizes of vesicles with high electron dense materials and gradually grew OBs.

Table 1: Seed characteristics during different developmental stages of S. tonkinensis 

Fig. 2: TEM images of endosperm and embryo of S. tonkinensis. Endosperm at 20 (a), 40 (b), 60 (c), 80 (d), 100 (e), 120 (f) and 140 (g) DAF. Embryo at 100,120 and 140 DAF, respectively (h-j). CN: cell nucleus, CW: cell wall, ER: endoplasmic reticulum, ES: extracellular space, L: oil body or lipid body, LV: large vacuoles, M: mitochondria, N: nucleolus, O: osmiophilic globule

At 140 DAF (Fig. 2g), there were many large and small OBs and some high electron dense materials in endosperm cells.

In embryo cells, at 100 DAF (Fig. 2h), there were many high electron dense materials, mitochondria and some lipids except large nuclei and nucleoli. At 120 DAF (Fig. 2i), in addition to the larger cell nucleus and nucleolus, the quantity of OBs appeared more than those at 100 DAF and the sizes were also larger. At 140 DAF (Fig. 2j), nuclei and nucleoli were still observed; different sizes and shapes of high electron dense materials were found; and there were some cavity bubbles, annulate lamellas (not shown), and the dense larger oil body which distributed surrounding organelles in the cells.

Fig. 3: Changes of oi body diameter (OBD) in endosperm (a) and embryo (b) cells of S. tonkinensis.

Fig. 4: Changes of crude fat contents during seed development of S. tonkinensis.

(Mean of 3 determinations, bars correspond to the standard deviation.)

In endosperm cells, the mean value of OBs was 2.08 μm at 120 DAF (Fig. 3a). At 140 DAF, the largest diameter of the spherical bodies was about up to 5.41 μm, the smallest one 3.59 μm and the mean value increased up to 4.20 μm (Fig. 3a), indicating that the value of OBD increased rapidly during the stage of seed ripening at which fruit pulp became from green to yellow and endosperm structure was whole and the sizes of fruit and endosperm did not change.

During the embryo development, the OBD mean value at 100 DAF was 0.48 μm (Fig. 3b). And the value at 120 DAF ranged from 0.37 to 1.18 μm and the mean value was 0.73 μm. And the mean value at 140 DAF was 0.78 μm (Fig. 3b), larger than those at 100 and 120 DAF.

Changes in crude oil content during seed development

Table 2: Correlation analysis among some index in developing seeds of S. tonkinensis (Pearson coefficients, two-tailed, *0.01<P < 0.05, **P < 0.01)

Fig. 5: Content changes of the main fatty acids from S. tonkinensis seeds during development. Mean of 3 determinations, bars correspond to the standard deviation

Fig. 6: Changes of unsaturated degree of fatty acids and unsaturated index of fatty acids during seed development of S. tonkinensis (Mean of 3 determinations, bars correspond to the standard deviation)

Crude oil accumulation in developing seeds of S. tonkinensis showed a biphasic pattern, which could be characterized by four stages fast-slow-fast-slow (Fig. 4). The first and the second rapid stage occurred during the period of June 11–30 and August 10–30, 2013 and the crude oil content rose from 1.20 to 15.75% and from 22.28 to 64.14%, respectively. The highest level of oil content was observed at the last sampling time, October 10, 2013, when oil content reached 68%.

Changes in fatty acid composition during seed development

The composition of each sample of oil obtained from the S. tonkinensis seeds at different DAF was very complex and it changed with the different development stages, as shown by numerous peaks observed in the total ion flow chromatograms of methyl esterified oil samples (Fig. 2S a–g). The polyunsaturated linoleic acid was the most abundant FA during seed development, followed by, in the order, oleic acid, palmitic acid and stearic acid (Fig. 5). Interestingly, the variation of linoleic acid and of oleic acid showed an opposite trend after 40 DAF. Furthermore, linoleic acid reached the highest relative content at 80 DAF.

As illustrated in Fig. 6, fluctuations occurred also in the unsaturated index and unsaturated degree of fatty acid during seed development. The two indices rapidly increased during the first 60 days after flowering, reaching maximum values of 10.6 and 153.9 for the unsaturated degree and index, respectively. Following this time point, the unsaturated degree rapidly decreased to 4.4 at 100 DAF then gradually to 3.9 at 140 DAF; while the unsaturated index gradually decreased to the minimum values of 123.7 at 120 DAF.

Correlation analysis

The Pearson’s correlation analysis between fatty acids and OB ultra-structural variables during development stages indicated that the content of linoleic acid was greatly correlated with monthly mean maximum and minimum temperatures (Table 2), while negatively with palmitic acid (R= -0.840, P < 0.05) and OBD (R= -0.997, P < 0.05). The degree of unsaturated FAs was positively correlated with monthly average total evaporation, while index of unsaturated FAs negatively correlated with OBD. The content of stearic acid was positively correlated with palmitic acid, while negatively correlated with relative humidity of soil at 20 cm depth (R= -0.797, P < 0.05). The content of oleic acid was positively correlated with relative humidity of soil at 10 cm depth.

Discussion

Oil is mainly stored in the oil body in the form of TAG in subcellular structure of seed embryo or endosperm (Huang 1992; Durrett et al. 2008). The present finding indicates that the seed endosperm changes obviously from liquid to gel and finally becomes solid (Zhao and Wu 2020), both endosperm and embryo have storage function of nutrients (especially lipids). In 2013, the mean values of OBD in embryo and endosperm remarkably increased and at 140 DAF the OBD was 3.2- and 1.6-fold larger than at 100 DAF in endosperm and embryo, respectively. Interestingly, the OBD in endosperm was larger than the one in embryo which was consistent with the findings of Zhang et al. (2018), who reported about two different ways for lipid accumulation in seeds. Furthermore, it is worth pointing out that the number of OBs might be decreased during late mature stage of seeds as a result of small OB fusion, which determined the formation of larger OBs (Miquel et al. 2014).

In present study, the structural changes of OBs exactly coincided with dynamic changes of oil content, which could be explained by previous findings (Jolivet et al. 2011; Miquel et al. 2014) that the oleosins studied have specific functions in the dynamics of lipid accumulation. As well known, the various components of the oil may vary considerably from one year to another one (Salvador et al. 2003). These results may be explained by gene expression pattern, proteomics analysis and protein immunodetection (Jolivet et al. 2011). Although some researchers have reported about S. tonkinensis seed oil (Liu et al. 2007; Fu et al. 2014), to the best of our knowledge, the changes of its oil content during seed development have not been explored. Herein, the oil content of S. tonkinensis seeds, up to 68% of the total dry weight at 140 DAF (Fig. 4) which may reach the maximum oil yield (Zhang et al. 2017; Wu et al. 2020), was higher than the findings of Wu et al. (2019).

Fatty acids (FAs) composition during seed development is also a crucial parameter to monitor for evaluating the best period of seed harvesting (Kaushik et al. 2010). It also seemed that the compounds present in the oil and the synthetic rate of FAs changed during early stages of seed development were similar with the report of Yadav and Singh (2003). The percentage of UFAs in the seeds was much higher than those records for most oil-rich seeds, such as Telfaria occidentalis, Jatropha curcas (Esuoso et al. 1998; Adebowale and Adedire 2006). The relative content of UFAs was higher than the saturated ones and changed during all stages of seed development. The main UFAs present in the seeds of S. tonkinensis were linoleic acid and oleic acid, both of which are essential FAs for humans (Aguilera et al. 2000). Nevertheless, because of a high content of UFAs, when exposed to air it might be susceptible to oxidation, which may have a negative impact on the stability of biodiesel (Mohibbe et al. 2005). On the contrary, the high degree of unsaturation will be an advantage to make S. tonkinensis oil interesting for edible purposes (Adebowale and Adedire 2006; Liu et al. 2007; Shi et al. 2014). In addition, palmitic acid was the main SFAs similar with the oil from Pistacia lentiscus, even though the total FA composition was different (Charf et al. 2008).

The fluctuation of the degree and index of UFAs can reflect to some extent different membrane fluidity in the endosperm, as suggested by the fact that, at early stages of seed development, endosperm was liquid. It is well known that oil content of seeds varies significantly by genotype and environmental factors (Dwivedi et al. 1993). It is also reported that OBs microstructure and suspension stability may be affected by temperature, pH medium and salt presence, and so on (Jolivet et al. 2013). Our observed correlations indicated that the FAs composition (e.g., the degree and index of UFA) and OBD were strongly affected by temperature (Wolf et al. 1982), total evaporation and relative humidity of soil. Thus, the mechanism of environmental impact on FA synthesis in S. tonkinensis seeds needs further study.

Conclusion

In short, the study here reported that both endosperm and embryo in S. tonkinensis seeds have the function of storing fat. The changes of oil content, fatty composition and OBD were dynamic that indicate the best period for seed harvesting to obtain an oil with desired characteristics in terms of unsaturation and relative content of linoleic acid. This study provides a scientific basis for the development and utilization of such woody oil plants.

Acknowledgements

We would like to thank Jia Yu of Advanced Analysis & Testing Center of Nanjing Forestry University. We are also grateful to Dr. Zihan Zhang for helpful data process. This work was supported by “Yili Normal University's start-up fund for talent introduction (2019)”,“Xinjiang Uygur Autonomous Region Innovation Environment (talent, base) construction special item in 2020- Natural Science Plan (Natural Science Foundation) joint fund(267)”, “The Priority Academic Program Development of Jiangsu Higher Education Institutions” and “Jiangsu Three-New Agricultural engineering (SZX2014S0020)”.

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