Degeneration of oil bodies by rough endoplasmic reticulum (rER)-associated protein during seed germination in Cannabis sativa L.
Institute of Cannabis Research, Colorado State University-Pueblo, 2200 Bonforte Blvd. Pueblo, CO 81001-4901, USA
Institute of Biological Resources, Chuncheon Bioindustry Foundation, 32, Soyanggang-ro, Chuncheon-si, Gangwon-do 24232, Republic of Korea
Department of Chemistry, Colorado State University-Pueblo, 2200 Bonforte Blvd. Pueblo, CO 81001-4901, USA
Corresponding author’s e-mail address: sanghyuck.park@csupueblo.eduAbstract
Abstract. Oil bodies serve as a vital energy source of embryos during germination and contribute to sustaining the initial growth of seedlings until photosynthesis initiation. Despite high stability in chemical properties, how oil bodies break down and go into the degradation process during germination is still unknown. This study provides a morphological understanding of the mobilization of stored compounds in the seed germination of Cannabis. The achenes of fibrous hemp cultivar (Cannabis sativa cv. ‘Chungsam’) were examined in this study using light microscopy, scanning electron microscopy and transmission electron microscopy. Oil bodies in Cannabis seeds appeared spherical and sporadically distributed in the cotyledonary cells. Protein bodies contained electron-dense globoid and heterogeneous protein matrices. During seed germination, rough endoplasmic reticulum (rER) and high electron-dense substances were present adjacent to the oil bodies. The border of the oil bodies became a dense cluster region and appeared as a sinuous outline. Later, irregular hyaline areas were distributed throughout oil bodies, showing the destabilized emulsification of oil bodies. Finally, the oil bodies lost their morphology and fused with each other. The storage proteins were concentrated in the centre of the protein body as a dense homogenous circular mass surrounded by a light heterogeneous area. Some storage proteins are considered emulsifying agents on the surface region of oil bodies, enabling them to remain stable and distinct within and outside cotyledon cells. At the early germination stage, rER appeared and dense substances aggregated adjacent to the oil bodies. Certain proteins were synthesized within the rER and then translocated into the oil bodies by crossing the half membrane of oil bodies. Our data suggest that rER-associated proteins function as enzymes to lyse the emulsifying proteins, thereby weakening the emulsifying agent on the surface of the oil bodies. This process plays a key role in the degeneration of oil bodies and induces coalescence during seed germination.
Teaser
Our study examines the microstructural characteristics of Cannabis seeds and the structural changes in cell storage organelles during germination using light microscopy, scanning and transmission electron microscopy. We provide detailed observations and experimental evidence of the localization and function of storage proteins and their associated enzymes during seed germination. The study also offers insights into the mechanisms underlying the degeneration and coalescence of oil bodies, which are vital processes in seed germination.
Introduction
Seed plants accumulate nutritional sources such as protein, lipids and carbohydrates in the endosperm or cotyledon for germination and post-germinative growth of the seedlings. Photosynthetic sugars are often polymerized into starch in amyloplasts or converted into lipids and stored as lipid droplets (LDs) in seeds. LDs are present in all plant cell types, ranging from a few LDs per cell in leaves to thousands of LDs per cell in seeds (Kang et al. 2022). LDs are often referred to in the literature by various terms, for example, lipid bodies, oil bodies, oleosomes or spherosomes depending on the characteristics of the species (Hsieh and Huang 2004; Purkrtova et al. 2008; Sánchez-Albarrán et al. 2019; Zienkiewicz and Zienkiewicz 2020; Nazari et al. 2022; Guzha et al. 2023). These organelles are surrounded by a half membrane and characteristically integrated with specific structural proteins (Tzen et al. 1993; Thiam et al. 2013; Huang 2018; Ischebeck et al. 2020).
LDs are not merely energy storage organelles, but also dynamic structures involved in diverse cellular metabolisms like membrane remodelling, regulation of energy homeostasis, stress responses and coordination between different organelles (Choi et al. 2022; Bouchnak et al. 2023). In addition, they play crucial roles at key sites in the freezing tolerance of seeds, engaging in direct interaction with glyoxysomes for seedling lipid degradation and producing antifungal compounds in leaves (Shimada et al. 2018; Olzmann and Carvalho 2019).
Lipids found in oilseeds are composed of a hydrophobic core filled with triacylglycerols (TAG), which are the most common storage lipids (Quettier and Eastmond 2009; Horn et al. 2013; Goold et al. 2015). The structural proteins of LDs commonly contain three membrane proteins known as oleosin, caleosin and steroleosin (Hsiao and Tzen 2011; Chapman et al. 2012; Huang 2018; Shimada et al. 2018). They are anchored in the phospholipid monolayer by a hydrophobic α-helical hairpin domain with a proline knot, and the C- and N-termini face of the cytosol (Alexander et al. 2002; Capuano et al. 2007; Purkrtova et al. 2008).
Oleosins are the most abundant integral membrane proteins of LDs in oilseeds. Particularly, the lipid droplet-associated proteins stabilize the LDs and prevent the coalescence or aggregation of this organelle in mature seeds (Huang 1992, 1994; Miquel et al. 2014). Hence, they are important regulators of LD dynamics; their ubiquitination, extraction and proteasomal degradation precede LD breakdown (Deruyffelaere et al. 2015; D’Andrea 2016).
Storage oil mobilization usually begins with seed germination. As a carbon or energy source in the germinating seeds, storage oil contributes to providing free-fatty-acids released from TAG by lipase or sugars through free-fatty-acid degradation by β-oxidation with subsequent gluconeogenesis (Graham 2008; Hielscher et al. 2017). The pathway of storage lipid conversion to sugars was examined in germinating lupin seeds (Borek and Ratajczak 2010; Borek et al. 2017). Subsequently, all the storage compounds are remobilized during post-germinative growth (Babazadeh et al. 2012; Miray et al. 2021).
Recently, some researchers reported on the LD degradation system in plants (Farquharson 2018; Kretzschmar et al. 2018; Traver and Bartel 2023). Since LDs are strongly associated with the endoplasmic reticulum (ER) (Suzuki 2017), this association has been observed at the electron microscopy level in many organisms (Fujimoto et al. 2013; Ohsaki et al. 2017; Sui et al. 2018; Renne et al. 2020; Kang et al. 2022; Zhang et al. 2022). During germination and seedling establishment, glyoxysomal enzymes degrade oil bodies to release storage lipids in seeds (Graham 2008; Shimada et al. 2018). Peroxisome contains the triacylglycerol lipase SUGAR-DEPENDENT1. This lipase is associated with the surface of the peroxisomes, and it is translocated to the oil body surface during seedling establishment (Thazar-Poulot et al. 2015).
Cannabis seeds contain approximately 18–30 % protein, 30–40 % oil and 25–34 % carbohydrate (Leonard et al. 2020; Vasantha Rupasinghe et al. 2020). Much of the knowledge of LD function in plants comes from studies of oilseeds (Laibach et al. 2015; Pyc et al. 2017b; Chen et al. 2023). Despite the importance of storing fats, oils and wax in seeds, our knowledge of the specificities of lipid metabolism remains uncertain.
This study represents a fundamental step towards the morphological elucidation of the mobilization mechanism of storage compounds in seeds. This research aimed to determine (i) the structural characteristics of storage compounds in the cotyledons of Cannabis, (ii) the degradation pathway leading to the β-oxidation of storage oil during seed germination, and (iii) the relationship between storage organelles such as oil bodies and protein bodies in oilseeds.
Materials and Methods
The achenes of fibre hemp cultivar (Cannabis sativa cv. ‘Chungsam’) obtained from Dangjin Agricultural Technology Centre (DATC), South Korea were used in this study. Dangjin area located in Chungcheong Province in South Korea (37°03ʹN, 126°51ʹE) provides favourable environmental conditions for high-quality hemp seeds. These achenes were collected from an approved farm by DATC 8 months prior. They were stored in a seed storage chamber of DATC at 4 °C. Twenty achenes were germinated for two days on sheets of filter paper moistened with sterile water in glass Petri dishes (150 mm × 20 mm) in an incubator with 65 % relative humidity and 20 °C under darkness (Blandinières et al. 2021). The embryo samples were obtained from the germinating seeds at various times in the growth phase; early (12h), middle (18 h) and late stage (24 h) after germination.
For light microscopy, the seeds were dissected with a razor under the stereoscopic microscope and fixed for 2 h in 2 % glutaraldehyde in 25 mM phosphate buffer, pH 7.2. After being rinsed with deionized water, they were post-fixed for 1 h in 2 % osmic acid and dehydrated with a graded ethanol series (50, 70, 80, 90, 95, 100 % ethanol). Then the samples were embedded in Spurr’s resin for 14 h and polymerized for 48 h at 60 °C. Semithin sections of 0.4 μm in thickness were cut on an ultramicrotome (Reichert Ultracut S, Leica, Germany) with glass knives and stained with toluidine blue-basic fuchsin. For the histochemical study, the fresh sections of the embryo tissue were touched on the slide glasses and stained with Sudan III, Alcian blue and Astra Blue. All the samples were observed and photographed using a light microscope (Axiophot II, Zeiss, Germany).
For scanning electron microscopy (SEM), the achenes were fixed in the same protocol described in the light microscopy sample preparation. Then the samples were transferred into isoamyl acetate. The samples were subjected to critical point drying with pressurized liquid carbon dioxide (Bioradical E3000, Bio-Rad, USA). The dried specimens were mounted on aluminium stubs, coated with gold-palladium in a sputter coater (JFC-1110E, JEOL, Japan), and photographed in a FE-SEM (JSM-6700F, JEOL, Japan) at 15 kV.
For TEM, the seeds were treated with the SEM fixation method described above. The materials were dehydrated with a graded ethanol series and replaced with propylene oxide. Subsequently, ultra-thin sections of 70 nm thickness were cut with a diamond knife (Micro Star SU-30, Ted Pella, USA) using an ultramicrotome (Reichert Ultracut S, Leica, Germany) and sections were collected on 300 mesh copper grids. The sections were stained for 20 min with 1 % uranyl acetate and for 10 min with 1 % lead citrate. Image acquisition was performed with a transmission electron microscope (JEM-2000 EX II, JOEL, Japan) at 80 kV.
Results
Cannabis achenes have a hard pericarp encasing a single seed. In this study, the achenes varied in length from 4 to 5 mm, and in diameter from 3 to 4 mm (Fig. 1A and C). The seed consisted of an endosperm and an embryo with two cotyledons and a radicle (Fig. 1B and D). The axis of the Cannabis embryo was curved and contained a U-shaped feature (Fig. 1B). The tip of the radicle and cotyledons were oriented toward the stylar end of the achene (Fig. 1B and D). When the germination began, the radicle emerged from the pericarp at the stylar end and split the seed coats into halves that were attached at the base (Fig. 1C). The scanning electron micrographs of the Cannabis seed showed that it consisted of endosperm, two distinctive cotyledons (outer, and inner cotyledon) and a radicle in a piece of the embryo (Fig. 2A). Specifically, the endosperm was confined to a peripheral region between the inner cotyledon and radicle in the mature seed (Fig. 2B). The deshelled seed was smooth and oval or orbicular in form as well as the enclosed seed (Fig. 3A). The epidermal cells of the embryo were rectangular in shape and arranged end to end in rows. They were equal approximately 15 µm in width but differed in length, the longer one being 60 µm and the shorter 14 µm. (Fig. 3B). The cotyledonary cells, functioning as storage, contained numerous oil bodies and protein bodies (Fig. 3C).
The cotyledons comprised several layers of parenchymatous cells (isodiametric cells) and two or more layers of palisade cells (Fig. 4A). TEM images revealed that the protein bodies of cotyledon cells measured between 2.5 and 3.5 µm in diameter (Figs. 4C, 5A–C). Large protein bodies are surrounded by many oil bodies ranging from 0.7 to 1.8 µm in diameter. However, the size of the extracted oil bodies varied from 0.1 to 2 µm (Fig. 5F). Oil bodies in Cannabis seed appeared spherical and were sporadically distributed in the cells (Fig. 5D and E). Isolated oil bodies were obtained by smearing small pieces of cotyledon onto a microscope slide and staining them with Sudan III (Fig. 5F).
Protein bodies in Cannabis seeds contained electron-dense globoids with a heterogenous protein matrix. The storage proteins were concentrated in the centre of the protein body as a dense homogenous circular mass surrounded by a light heterogeneous area (Fig. 6A and B). As the major seed storage organelles in Cannabis, protein bodies and oil bodies within the cotyledon cells underwent unique morphological changes throughout germination (Fig. 6B–D). The protein bodies and oil bodies gradually degenerated in the cells and were used as a primary energy source during germination. During germination, the rER was frequently present in all cotyledon cells (Fig. 7A and D). As ribosomes and rER began to increase, dense substances were also concentrated in the outer region of oil bodies (Fig. 7B and C). At the early stage of germination, dense substances aggregated adjacent to the oil bodies and associated with them (Fig. 8A and B). Later, the border of the oil bodies became a dense cluster and appeared as a sinuous outline. In addition, irregular hyaline areas were distributed throughout the oil bodies, reflecting the destabilized emulsification of oil bodies (Fig. 8C). Finally, the oil bodies fused with one another and had an irregularly contoured surface (Fig. 8D).
Discussion
Our fundamental data on seed germination provides insight into the understanding of the degradation mechanisms controlling the metabolism of storage proteins in the cotyledon of oilseeds. As small subcellular storage organelles, the protein bodies and oil bodies in the cotyledon cells of Cannabis seeds are gradually degenerated and used as a primary energy source during germination. Particularly, the biological function of storage proteins correlated with oil bodies stored in the cotyledon cells appears to be more diverse than simply constituting a source of carbon made available for the germinating seedling.
Morphology of achenes in Cannabis
Even though some researchers have reported, there is still a lack of comprehensive studies on the structure of Cannabis fruits and seeds as they relate to hempseed-based food products (Oseyko et al. 2019; Farinon et al. 2020). Small (1974) described that domesticated Cannabis plants have large achenes longer than 3.7 mm and lack an adhering of the perianth. The fruits of uncultivated plants are small and possess an adhering perianth. These wild types of morphological characteristics such as smaller fruits, adhering perianth and an elongated base are more adaptive in a wild environment.
Cannabis achene varies in size and shape depending on the varieties and cultivars, the average length of fruit is reported from 2 to 6 mm, with diameters from 2 to 4 mm depending on diverse varieties and cultivars (Clarke 1981). In dry seeds, the outer cotyledon is remote from the radicle, whereas the inner cotyledon is adjacent to the radicle. The former is about 50 % heavier than the latter in Cannabis (Small and Antle 2008). Our result showed that the fibre type of achene was large ranging mostly 5 mm in length and 4 mm in diameter, and the perianth partially remained at the base.
Indehiscent dry fruit contains a single seed encased in a pericarp or fruit husk. Observation of the longitudinal and transverse sections of Cannabis achene revealed that the embryo was encased by a multi-layered pericarp and seed coat casing as shown in Fig. 2. The U-shaped embryo was distributed unevenly in the seed, with higher concentrations in the dorsoventral regions and lower concentrations in the two lateral sides, the radicle and the chalaza region. Both embryo and endosperm are derived from individual fertilization processes and develop while embedded in maternal tissues that form the seed coats, an outer protective layer (Walker et al. 2011).
In some species of Brassicaceae and Solanaceae, the endosperm is confined to a peripheral aleurone-like cell layer in the mature seed (Pabon-Mora and Litt 2011; Lee et al. 2012). In particular, the structure of the Cannabis endosperm was like that of the plants. This type of endosperm acts as a mechanical barrier to inhibit embryonic growth, and as a nutrient reserve for seed germination and early seedling establishment (Yan et al. 2014). In the later stage of germination, endosperm rupture and radicle protrusion occur through the seed coat, completing the germination process (Weitbrecht et al. 2011; Rajjou et al. 2012). Therefore, the structure and strength of the seed coats and pericarp are critical to controlling seed germination in many plants.
Protein body and oil body in Cannabis seeds
The storage compounds are morphologically and biochemically remodelled extensively during germination (Waschatko et al. 2016). Protein bodies are vacuoles filled with storage proteins (Gunning and Steer 1996; Herman and Larkins 1999), and all storage proteins are initially synthesized on the rER (Bollini and Chrispeels 1979; Chrispeels 1991). Huang (1992, 1996) observed that lipase was newly synthesized de novo on free polysomes and bound specifically to the oil bodies during the germination of maize kernel. Additionally, the recognition signal on the oil bodies for lipase seemed to be captured by the oleosins.
In this study, the most prominent components found in Cannabis seeds were the protein bodies and oil bodies. A protein body contains a highly electron-dense globoid and a heterogeneous matrix and is surrounded by numerous oil bodies. Within the Cannabis seed, the large protein bodies are spherical or oval and ranged from 2.5 to 3.5 µm in diameter. Some researchers elucidated that 181 proteins were identified in hemp seeds with the main storage proteins globulin edestin in a concentration of 67–75 % and globular albumin that ranged from 25 to 37 % (Aiello et al. 2016; Aluko 2017). These proteins are antioxidants and act in a defensive role in germinating seeds when cleaved into fragments (Cattaneo et al. 2021). The protein content was within the range of 0.6 to 3.4 % (w/w) reported for oil bodies from seeds of various species (Tzen et al. 1993).
The most widespread sites for lipid accumulation in plant organs are seeds because high energy input is necessary for germination and seedling establishment (Penfield et al. 2004). Many plants store lipids in subcellular organelles, such as lipid droplets or oil bodies (Chapman et al. 2012; Song et al. 2017). LDs are surrounded by a monolayer and the surface-bound proteins are localized to the phospholipid monolayer (Walther et al. 2017). These organelles can protect the lipid reserves against oxidation and hydrolysis until seed germination and seedling establishment. Oil bodies are often considered to be spherical to ovoid, with diameters varying between species, ranging from 0.5 to 2.5 µm (Tzen et al. 1992; Wang et al. 2012). However, a study showed that the close packing of oil bodies in the cell matrix made them appear asymmetrical (Garcia et al. 2021).
In all plant seeds, oil bodies are found primarily in their cotyledons and radicles (Yoshida et al. 2003). They provide a source of energy for β-oxidation in neighbouring glyoxysomes during initial seed germination (Graham 2008; D’Andrea 2016). In Medicago truncatula oil bodies were aligned around the protein bodies (Song et al. 2017). However, many oil bodies were randomly filled with protein bodies in the cotyledon cells of Cannabis.
rER-associated proteins as a trigger for the degeneration of oil bodies
Oleosins are degraded prior to lipid mobilization from oil bodies via ubiquitination–proteasome pathway (Deruyffelaere et al. 2015). Molecular studies frequently reveal intimate connections between LDs with the ER (Brocard et al. 2017; Choi et al. 2022). Both oil bodies and protein bodies of cotyledonary cells rapidly undergo morphological changes during germination, as they are utilized as a primary energy source. This process involved the degeneration of the organelles and their fusion with one another, resulting in irregularly contoured surfaces.
Although peroxisomes or glyoxysomes were not observed in the cotyledon cells of this research, rER was frequently observed in all cotyledon cells when germination began. Moreover, highly electron-dense substances appeared adjacent to oil bodies and attached to them. Similarly, the peroxisome-associated lipase translocates to the oil body surface to break down the stored lipids during seedling establishment (Thazar-Poulot et al. 2015). Such different occurrences of specific organelles in germinating cells of seeds imply that the degradation pathway of storage compounds has diverse types depending on the species and its developmental process.
We have found that the substances surrounding the oil bodies play a critical role in facilitating their degeneration during seed germination. These substances condense the contents of the oil bodies, recruit them to the half-membranes of oil bodies, and significantly enhance their conjugating activity. We hypothesize that specific proteins are synthesized in the rER and exported into the cytoplasm near the oil bodies. These proteins then translocate into the oil bodies through the half-membrane of oil bodies. They may act as enzymes with active sites on the surface region of oil bodies, leading to the weakening of the half-membranes and inducing them to coalesce. Therefore, rER-associated proteins act as a trigger in the degeneration mechanism of oil bodies, the oil bodies lose their morphology and fuse with each other. Finally, irregular hyaline areas are distributed throughout oil bodies, reflecting the destabilization of the emulsification of the oil bodies. Further studies on the degeneration of oil bodies in other species will provide data on the seed germination mechanisms of oily seed plants.
Conclusions
As the storage organelles, protein bodies and oil bodies were packed in the cotyledon cells of Cannabis seeds. They remarkably changed the morphology at the early stage of germination. The storage proteins were concentrated in the centre of the protein body as a dense homogenous circular mass surrounded by a light heterogeneous area. Some of the storage proteins appear to act as emulsifying agents on the surface region of oil bodies. These proteins maintain the individuality and stability of oil bodies inside or outside the cotyledon cells. After rER appeared near the oil bodies, dense substances considered proteins rapidly aggregated adjacent to the oil bodies, resulting in the coalescence of oil bodies. We concluded that these rER-associated proteins play a key role in the degeneration of oil bodies by weakening the emulsifying agent on their non-polar surfaces and inducing the coalescence of oil bodies during seed germination. Finally, most oil bodies fused with one another and had an irregularly contoured surface at the late stage of germination.
Acknowledgements
We thank the colleagues at Institute of Cannabis Research (ICR), Colorado State University-Pueblo and Chuncheon Bioindustry Foundation (CBF) for providing material and experimental equipment.
Funding
This work was supported by the Institute of Cannabis Research, Colorado State University-Pueblo (ICR-FY21-Formula Grant-01) and the Korean government grant (MSIT 2021-DD-UP-0379).
Conflict of Interest
None declared.
Data Availability
The data underlying this article are available in the article.