Ultrastructural studies of seed coat and cotyledon during rapeseed maturation

CAO Jian-bo,HE Li-min,Chinedu Charles NWAFOR,QIN Li-hong,ZHANG Chun-yu,SONG Yantun,HAO Rong

1 Key Laboratory of Arable Land Conservation (Middle and Lower Reaches of Yangtse River),Ministry of Agriculture and Rural Affairs/Hubei Key Laboratory of Soil Environment and Pollution Remediation/College of Resources and Environment,Huazhong Agricultural University,Wuhan 430070,P.R.China

2 Public Laboratory of Electron Microscopy, Huazhong Agricultural University, Wuhan 430070, P.R.China

3 National Key Laboratory of Crop Genetic Improvement/College of Plant Science and Technology,Huazhong Agricultural University, Wuhan 430070, P.R.China

Abstract Brassica napus L.(B.napus) is an important oil crop worldwide and it rapidly accumulates oil at late stage of seed maturation.However,little is known about the cellular mechanism of oil accumulation and seed color changes during the late stage of rapeseed development.Here,we analyzed the ultrastructure of seed coat,aleurone and cotyledon in embryos of B. napus from 25 to 70 days after flowering (DAF).The pigments,which were deposited on the cell wall of palisade cells in seed coat,determined dark black color of rapeseed.The chloroplasts degenerated into non-photosynthetic plastids which caused the green cotyledon to turn into yellow.The chloroplasts in aleurone and cotyledon cells respectively degenerated into remnants without inner and outer envelope membranes and ecoplasts with intact inner and outer envelope membranes.From 40 to 70 DAF,there were degraded chloroplasts without thylakoid,oil bodies contacting with plastids or protein bodies,big starch deposits of chloroplasts degrading into small particles then disappearing,and small endoplasmic reticulum (ER)in aleurone and cotyledon cells.Additionally,there were decreases of chlorophyll content and dramatic increases of oil content in rapeseed.These results suggested that the rapid oil accumulation was independent on the NADPH synthesized by photosynthesis of chloroplasts and probably utilized other sources of reductant,such as the oxidative pentose phosphate pathway during the late stage of rapeseed development.The triacylglycerol assembly presumably utilizes the enzymes in the plastid,cytosol or oil body of cotyledon and aleurone cells.

Keywords:rapeseed,seed coat,plastid development,oil synthesis,ultrastructure

1.Introduction

Rapeseed is the third most important source of edible vegetable oils and the second most abundant protein supplement for animal feeds in the world (Huet al.2013;Carteaet al.2019).Rapeseed consists of high-quality nutritional compounds that are beneficial for food and human health application (Carteaet al.2019).The storage oil (mainly triacylglycerol,TAG) synthesis and accumulation is associated with the different cellular organelles during seed development in oilseeds of crops (Heleneet al.2003).The TAG synthesis is accomplished in multiple subcellular compartments by coordinated action of multiple pathways which esterify glycerol and fatty acids (FA) (Chapman and Ohlrogge 2012).

In most seeds,the TAG synthesis happens in different cellular organelles by the following steps.Firstly,the carbon sources of FA synthesis are hexose phosphate,triose phosphate,phosphoenolpyruvate (PEP) and/or pyruvate from glycolysis pathway of sucrose (Chapman and Ohlrogge 2012).Secondly,these carbon sources are transferred into the stroma of chloroplasts in green tissues or proplastids (or leucoplasts) in non-green tissues,then they are converted by enzymes to produce the FA precursors-Acetyl-CoA and Malonyl-CoA (Bateset al.2013).Thirdly,the FA precursors and the reductants-NADPH or NADH are transformed by fatty acid synthases,stearoyl-acyl carrier protein (ACP)desaturases (SAD),and acyl-ACP thioesterases to produce C16:0,C18:0and C18:1FA (Xu and Shanklin 2016).The NADPH/NADH and ATP are mainly generated from photosynthesis in the chloroplast in green seeds (Chapman and Ohlrogge 2012).Alternatively,the NADPH/NADH can be produced from the oxidative pentose phosphate pathway in plastids and ATP can come from the pyruvate phosphorylation in proplastids or is imported from cytosol in non-green seed(Chapman and Ohlrogge 2012).Fourthly,the FA-CoAs,which are linked by long chain acyl-CoA synthetase in the chloroplast outer envelope membrane,are exported to endoplasmic reticulum (ER) for TAG synthesis (Chapman and Ohlrogge 2012).Finally,the long chain FA-CoA are incorporated into glycerol-3-phosphate to form TAG through conventional Kennedy pathway in ER (Xu and Shanklin 2016).However,the enzymes involved in Kennedy pathway are also localized in other cell structures,i.e.,mitochondria,cytoplasm and lipid droplets (Maraschinet al.2019).

In seeds of oil crops,TAG accumulation parallels with the seed development ofArabidopsis,Brassica napusL.(B.napus) and other oil crops (Heleneet al.2003).The fatty acid accumulation inArabidopsisseed positively correlates with the increase of seed dry weight and represents a dramatical rise from 2 to 18 days after pollination (DAP) (approximately 9 μg/seed of oil content)then a slight reduction from 18 to 20 DAP (Baud and Lepiniec 2009).The accumulation of oil content in rapeseed is characterized by the high rates from 15 to 45 days after flowering (DAF) (approximately 500 μg/seed of oil content)(Vigeolaset al.2003).The accumulation of TAG in seeds of safflower and castor also occurs at high rates from 12 to 30 DAF (approximately 12 μg/seed-safflower,40%-castor)(Ichihara and Noda 1980;Velascoet al.2005).The sharp rise of oil content mainly happens in the late stage of seed development,especially the rapid oil accumulation of rapeseed which occurs after 30 DAF (Heleneet al.2003).

The seeds ofArabidopsisandB.napusconsist of three major components-embryo,seed coat and endosperm.Those components have different biological roles and morphogenesis:(1) embryos ofArabidopsisandB.napus,which are composed of cotyledons,hypocotyl and radicle,represent globular,green heart,torpedo,walking stick,curled cotyledon,and green cotyledons during seed development(He and Wu 2009).(2) Seed coat ofArabidopsisis differentiated from cells in five layers of ovule integuments(Haughn and Chaudhury 2005).Finally,the cells of the fifth layer of ovule integument synthesize proanthocyanidin(PA) flavonoid compounds and impart a brown color to seed coat (Haughn and Chaudhury 2005).Cells of the third and fourth layers of ovule integuments do not differentiate and are crushed together (Haughn and Chaudhury 2005).Cells of the second layer of ovule integuments (sub-epidermal layer) develop into palisade cells with thickened cell wall(Haughn and Chaudhury 2005).Cells of epidermal layer(layer 1) of ovule integuments synthesize a lot of mucilage (a pectinaceous carbohydrate) and form the columella (Haughn and Chaudhury 2005).(3) Endosperms ofArabidopsisandB.napusdegenerate to a layer of living aleurone cells and a thin layer of crushed parenchyma tightly spaced with the embryo from torpedo stage to the curled cotyledon stage(Haughn and Chaudhury 2005;Bethkeet al.2007).At the torpedo stage,the suspensor is degenerated and unable to provide food for embryo development (Schwartzet al.1994).The chloroplast in the embryo from heart stage to green cotyledons stage may provide ATP and NADPH for the fatty acid synthesis and storage in oil body of cotyledon cells(He and Wu 2009).In seeds ofArabidopsisandB.napus,the cotyledons store almost 90% oil contents and nourishes the germinating embryos (Groot and Vancaeseele 1993).However,the morphogenesis of seed and embryo are mainly focused on the early stage of seed development inArabidopsisandB.napus(He and Wu 2009).Therefore,the morphogenesis of rapeseed at the late stage corresponding to rapid oil accumulation needs to be further investigated.

In this study,we analyzed the contents of chlorophyll and fatty acid,morphogenesis of cotyledon,seed coat and aleurone at rapid oil accumulation stage of rapeseed,i.e.,from 25 to 70 DAF.The ultrastructural changes of seed coat possibly imply the mechanism of rapeseed color changes.The ultrastructural morphogenesis of chloroplast,protein body and oleosome in cotyledon and aleurone at this stage suggest that the compartments of NAPDH/ATP generation and ATG assembly are different from those at early stages of rapeseed development.

2.Materials and methods

2.1.Plant materials

The Chinese variety of oilseed rape (B.napus) Huashuang 4 was grown on the experimental farms at Wuhan (114.31°N,30.52°E),Hubei Province,China.The first flower opening was counted as the 1 DAF in the plant of oilseed rape.Three seeds were collected from the silique of second branch in each plant at 25–35,40–50 and 60–70 DAF.The seed coat and cotyledon of rapeseed at the above DAF were photographed by stereoscopic microscope (SMZ800,Nikon,Japan).The rapeseeds from three replicate plants were used for chlorophyll and oil contents analysis and ultrastructural observation.

2.2.Oil and chlorophyll content quantification

The seeds ofB.napusat 25–35 DAF,40–50 DAF and 60–70 DAF were used for oil and chlorophyll content estimation.For oil content analysis,gas chromatography with flame ionization detector (GC-FID) was used for fatty acid quantification according to the protocol as described by Shahidet al.(2019) with minor modifications.GC was programmed as described in Shahidet al.(2019).Fatty acids were identified and determined by comparing retention times to standards (C17:0).For estimation of the total chlorophyll content,the method for spectrophotometric determination of chlorophyll concentrations described by Zhanget al.(2015) was used.Briefly,chlorophyll was extracted after freshly harvested seeds were weighed and homogenized in liquid nitrogen,and subsequently extracted in three volumes of 80% (v/v) acetone containing 1 μmol L–1KOH.After centrifugation for 2 min at 16 000×g,the supernatant was used for spectrophotometric analysis.

2.3.Transmission electron microscopy

During 25–35,40–50 and 60–70 DAF,the seed coat and cotyledon were collected from seeds ofB.napusin the same patches of chlorophyll and oil content analysis.The ultrastructure of seed coat and cotyledon were studied by transmission electron microscopy (TEM).The seed coat and cotyledon were cut into 1 mm×1 mm×1 mm blocks and fixed in 2.5% (w/v) glutaraldehyde in 0.1 mol L–1phosphate buffer solution (PBS) (pH 7.2) at 4°C overnight.The fixed tissues were washed in PBS three times for 30 min each at room temperature (20–25°C),post-fixed for 2 h in 1%osmium tetroxide,dehydrated in graded series of acetone,infiltrated with Spurr resin (SPI,SPI Chem,West Chester,USA),and polymerized at 65°C for 48 h.The samples were cut into ultrathin sections (60–70 nm thick),stained with 2%uranyl acetate,and examined with a Hitachi transmission electron microscope (H-7650;Hitachi,Japan) at 80 kV.Each sample had three biological replicates with each replicate having at least three ultrathin sections observed under the electron microscope.To quantify the number of plastids,protein bodies and oil bodies in aleurone cells and cotyledon cells,30 aleurone cells or 30 cotyledon cells were observed from two independent biological replicates.

2.4.Statistical analysis

The significant differences of the contents of chlorophylla/band oil and the numbers of plastids,protein body and oil body in aleurone and cotyledon cells were assessed by using pairwise Student’st-test in Excel (Microsoft,http://www.microsoftstore.com).

3.Results

3.1.Color phenotypes of seed coat and cotyledon during the changes of chlorophyll and fatty acid contents in rapeseed

The seed coat color appeared dark green,brown mottled with green and dark black at 25–35 DAF,40–50 DAF and 60–70 DAF,respectively (Fig.1-A).Meanwhile,the color of cotyledon presented as dark green,light green and bright yellow at 25–35 DAF,40–50 DAF and 60–70 DAF,respectively (Fig.1-A).At 25–35 DAF,40–50 DAF and 60–70 DAF,the chlorophyllaandbcontents were gradually decreased from 0.11 and 0.24 mg g–1,0.10 and 0.20 mg g–1,to 0.05 and 0.08 mg g–1,respectively (Fig.1-B).In contrast,the content of fatty acid rapidly increased from 6 to 22%,and the fatty acid content was significantly higher at 60–70 DAF than that at 25–35 DAF (Fig.1-C).

3.2.Ultrastructure of differentiated seed coat and aleurone in rapeseed

There were four layers of seed coat including epidermis layers,palisade layer,outer epidermis of inner integument(Oeii) layer,and aleurone layer at the ultrastructural level(Fig.2).Epidermis layers containing three layers of cells,palisade layer containing one layer of cells with secondary cell wall thickening,Oeii layer containing one layer of cells and aleurone layer containing one layer of cells with dense cytoplasm were found in seed coat at 25–35 DAF and 40–50 DAF (Fig.2-A and B).Epidermis and Oeii layers all became a layer of dead cells without cytoplasm,palisade cells only with thickening cell wall and aleurone cells with dense cytoplasm were observed at 60–70 DAF (Fig.2-C).The cell wall of palisade cells was dramatically thickened and was deposited by many electron-dense materials(white arrow indicated) from 25–35 DAF to 60–70 DAF(Fig.2-A–C).

3.3.Ultrastructural morphologies of cotyledon cell and aleurone cell in rapeseed

The cotyledon is a major component of rapeseed,and aleurone layer is the only live tissue in the seed coat of mature rapeseed (Huet al.2013).To further investigate the color changes of seed coat and cotyledon,we analyzed the ultrastructure of cotyledon cells and aleurone cells at 25–35 DAF,40–50 DAF and 60–70 DAF (Fig.3).At 25–35 DAF,many chloroplasts,large oil bodies and small protein bodies (PBs),vacuoles were observed in aleurone cells and cotyledon cells;at 40–50 DAF,many PBs with irregular shapes and some non-photosynthetic plastids with starch grain appeared in aleurone cells and cotyledon cells;at 60–70 DAF,there were numerous nonphotosynthesis plastids without starch grain,and PBs and oil bodies becoming small round spheres in aleurone cell and cotyledon cell (Fig.3-A and B).The number of chloroplasts in aleurone cells and cotyledon cells significantly decreased from 25–35 DAF to 60–70 DAF(Fig.3-C).The number of non-photosynthesis plastids,PBs and oil bodies in cotyledon cells and the number of oil bodies in aleurone cells were significantly increased from 25–35 DAF to 60–70 DAF (Fig.3-C).However,the number of non-photosynthetic plastids and PBs in aleurone cells increased then decreased from 25–35 DAF to 60–70 DAF(Fig.3-C).There were not abundant ERs in aleurone and cotyledon cells (Fig.3-A and B).Only a few ERs were localized around the nucleus of aleurone and cotyledon cells at 25–35 DAF and 40–50 DAF,but there were few ERs in aleurone and cotyledon cells at 60–70 DAF (Appendix A).

3.4.Differential ultrastructural morphogenesis of differentiated chloroplast in aleurone cell and cotyledon cell

In aleurone cells,the chloroplast with starch grain surrounded by thylakoid,plastoglobules and the inner and outer envelope membrane was observed at 25–35 DAF;the differentiated chloroplast with starch grain and plastoglobules became non-photosynthetic plastid characterized by loss of intact thylakoid at 40–50 DAF;the non-photosynthetic plastid lost the inner and outer envelope membrane and only many plastoglobules remained at 60–70 DAF (Fig.4-A).In cotyledon cells,the chloroplast with starch grains had thylakoid and plastoglobules inside at 25–35 DAF,then chloroplast lost intact thylakoid and became non-photosynthetic plastid with starch grain at 40–50 DAF;the non-photosynthetic plastid turned into eoplast,which was a small sphere with many plastoglobules and intact inner and outer envelope membranes (black arrow indicated),and another type of non-photosynthetic plastid looked like eoplasts but did not have intact inner and outer envelope membranes at 60–70 DAF (Fig.4-B).

3.5.Differential morphological developments of protein body and the interaction between oil body and plastid in aleurone and cotyledon cells

In aleurone cells,there were many dispersed high electron-dense materials in spherical protein body Ӏ (PBӀ)at 25–35 DAF;the spherical PBӀ and dumbdell-shaped PBӀ with compact high electron-dense materials and some plastoglobule-like particles (black arrow indicated) appeared at 40–50 DAF;the small spherical PBӀs had numerous plastoglobule-like particles in slightly low electron-dense matrix at 60–70 DAF (Fig.5-A).In cotyledon cells,there were PBӀ with many small starch-like (very low electrondense and reflective) particles (black arrowhead indicated)and PBII with low electron-dense unconsolidated matrix at 25–35 DAF;there was one small starch-like particle in PBӀs with irregular shape and PBII with low electron-dense compact matrix at 40–50 DAF;the inside matrix of PBII became compact and the PBӀ with many plastoglobule-like particles did not have the starch-like particles at 60–70 DAF(Fig.5-B).The oil bodies were orderly arranged around protein body in aleurone cells and cotyledon cells at 60–70 DAF (Fig.5-A and B).The outer membranes of PBӀ,which contained some plastoglobule-like particles,were intruded by oil bodies (white diamond arrow indicated)(Fig.5-C).The PBӀ melted with the inner and outer envelope membrane of non-photosynthetic plastid (white arrow indicated) in aleurone cells at 25–35 DAF and 40–50 DAF(Fig.5-C).The two terminals of oil body respectively melted with non-photosynthetic plastid and PBӀ,and one oil body even penetrated into the starch grain in chloroplast (black diamond arrow indicated) in cotyledon cells at 25–35 DAF;in cotyledon cells at 40–50 DAF,the PBӀs were contacting and melting with the oil bodies (black diamond arrow indicated)and the starch-like particles (black diamond arrow indicated);some oil bodies even surrounded the starch-like particles(Fig.5-D).

3.6.Ultrastructural morphologies of oil bodies in aleurone and cotyledon cells

In both aleurone cells and cotyledon cells,the diameter of many oil bodies exceeded 1 μm at 25–35 DAF;the large oil bodies of more than 1 μm diameter divided into many small oil bodies of less than 0.5 μm diameter through the high electron-dense (black) lines formation in large oil body(sphere arrow indicated) at 40–50 DAF (Fig.6-A and B).At 60–70 DAF,the oil bodies of approximately 0.3 μm diameter were more intensively arranged in cotyledon cells than the sparse arrangement of oil bodies of approximately 0.3 μm diameter in aleurone cells (Fig.6-A and B).

4.Discussion

4.1.Pigment deposition on cell wall of palisade cells and chloroplast development lead to the color changes of rapeseed

The initially colorless proanthocyanidin (PA) is a class of polymeric compound arising from the flavonoid pathway(Marles and Gruber 2004).The colorful complexes,which come from the PA oxidation with polysaccharides and other phenolics in cell wall,lead to the brown or red-brown pigmentation in the seed coat (Marles and Gruber 2004).The pigments are accumulated in the inner integument layer 1 in tight contact with aleurone layer and responsible for the brown seed color ofArabidopsisseed (Beeckmanet al.2000).However,the pigments are mainly deposited in the palisade cell layer ofB.napusseed coat (Weiet al.2009).There are wider palisade tissues and more pigment in black rapeseed than those in yellow rapeseed (Li and Chen 1998).In our study,the green seed coat ofB.napusbecame black (Fig.1),and the cell wall of palisade cells became thickened and had a lot of high electron-dense material deposition (white arrow indicated) at late stage of rapeseed (Fig.2-A–C).The palisade cell layer belonged to the fourth layer of integument and was localized in the middle of inner integument layer 2 and aleurone layer (Fig.2-A–C).During the late stage of rapeseed,the gradually decreasing chloroplasts in aleurone and cotyledon cells were associated with the reduced chlorophylla/bcontents in rapeseed(Figs.1-B and 3-C).Those results indicate that the pigments accumulate on different cell layers of integument betweenArabidopsisandB.napusduring seed coat development.Meanwhile,the pigments accumulating on the cell wall of palisade cells in seed coat and the chlorophylls fading away in embryo cause the rapeseed to become black during the late stage of rapeseed maturation.The oxidized complexes of PA combining with the components of cell wall are possibly the major constituents of black pigment in seed coat of mature rapeseed.

4.2.Different development modes of plastids in cotyledon and aleurone cells

The cotyledon and aleurone development originate from the fertilization in the ovule ofArabidopsisandB.napus(Haughn and Chaudhury 2005).But the cotyledon is a component of embryo which develops from the diploid zygote by the egg and sperm nuclei fusion,while the aleurone originates from degenerated triploid endosperm which devlops from the sperm cell fusing with the diploid central cell (Haughn and Chaudhury 2005).The plastid biogenesis and homeostasis are closely coordinated with embryo and endosperm developments to make sure the cells of organs in correct functions (Jarvis and Lopez-Juez 2013).In embryos of most oilseeds,the proplastids,which are inherited maternally as undifferentiated and small plastids,develop into chloroplasts in an intermediate photosynthetically active period then dedifferentiate into non-photosynthetic plastids in a subsequent desiccation phase (Lieberset al.2017).DuringArabidopsisembryogenesis,the chloroplasts which originate from proplastids and are inherited by paternal or biparental means,can differentiate into other types of plastids (Lieberset al.2017).There is a thylakoid membrane system with green chlorophyll pigments,stroma with a conspicuous lipid structure of so-called plastoglobule inside to serve as lipid reservoirs for thylakoid membranes,inner envelope membrane,intermembrane space and outer envelope membrane in chloroplasts to fulfill photosynthesis (Lieberset al.2017).After desiccation stage,chloroplasts dedifferentiate into non-photosynthetic plastids characterized by only loss of photosynthesis function,then differentiate into eoplasts characterized by loss of thylakoid and only retaining plastoglubule (Lieberset al.2017).The eoplasts within the cotyledons differentiate directly into chloroplasts during seed germination (Lieberset al.2017).The chloroplast supports NADPH and ATP for the multiplication of oil body and protein body inArabidopsisembryo from green heart stage to green cotyledons stage (He and Wu 2009).In cotyledon and aleurone cells of rapeseed,there were also many chloroplasts in green seeds during 25–35 DAF;then the developed chloroplasts turned into non-photosynthetic plastids characterized by internally fragmented thylakoids in brown mottled green seeds and light green embryos during 40–50 DAF (Figs.1,3 and 4).At 60–70 DAF,most non-photosynthetic plastids de-differentiated into eoplasts characterized by small spheres with the intact inner and outer envelope membranes and many plastoglobules inside in cotyledon cells of bright yellow embryos (Figs.1,3 and 4).However,the non-photosynthetic plastids lost the inner and outer envelope membranes accompanied by many plastoglobule-like particles in protein bodies in aleurone cells at 60–70 DAF (Figs.3–5).Therefore,these findings indicate that the most chloroplasts de-differentiated into ecoplasts in cotyledon cells,while the chloroplasts were degraded into the remnants such as plastoglobules released into protein bodies in aleurone cells during the late stage of rapeseed development.The different genetic backgrounds of cotyledon and aleurone maybe lead to the differences in plastid development in rapeseed.

4.3.Oil accumulation and assembly are respectively independent of chloroplast photosynthesis and the enzymes of ER at late stage of rapeseed maturation

Seed oil biosynthesis begins in the plastids (chloroplasts in green tissue or non-photosynthesis plastids in non-green tissue) where the fatty acid synthesis is localized (Bateset al.2013).Meanwhile,the assembly of ATG molecule is associated with both ER and oil body (Bateset al.2013).The green embryos can utilize light reactions to generate NADPH and ATP as reductants and energy to support oil biosynthesis (He and Wu 2009;Bateset al.2013).During the development of rapeseed,the green embryo starts to provide NADPH and ATP for the oil biosynthesis at the heart stage (He and Wu 2009).However,in non-green oilseeds such as castor seed and sunflower seed,the majority of NADPH is produced by the oxidative pentose phosphate pathway in plastids,where three molecules of glucose-6-phosphate enter the pathway and are oxidized to three molecules of ribulose-5-phosphate and CO2,to form six NADPH molecules (Houstonet al.2009;Chapman and Ohlrogge 2012).In addition,a minor amount of NADPH is supplied by decarboxylation of malate in plastids (Houstonet al.2009),where the NADP-malic enzyme catalyzes the malate and NADP to form pyruvate,NADPH and CO2(Pleiteet al.2005).In this study,the late stage of rapeseed maturation was accompanied by a gradual decrease in chlorophyll content (Fig.1-B).Further analysis showed that many chloroplasts with thylakoid and large starch grain on 25–35 DAF degenerated into non-photosynthetic plastids and ecoplasts without thylakoid and starch grain in cotyledon and aleurone cells from 40 to 70 DAF (Figs.3 and 4).Furthermore,the color of seed coat in rapeseed turned to brown from 40 to 70 DAF (Fig.1-A) which possibly blocked the light shining into the cotyledon.Those findings indicate that the plastids do not have photosynthetic functions to generate NADPH and ATP in cotyledon of rapeseed at the late stage of seed maturation.However,the oil content dramatically increased from 15 to 24% in rapeseed from 40 to 70 DAF (Fig.1-C).Therefore,the oil synthesis of rapeseed might not be dependent on the NAPDH and ATP produced by photosynthesis of chloroplasts in cotyledon.The oil bodies in cotyledon were in contact with nonphotosynthetic plastids and protein body I,meanwhile,the large starch grain of plastid degraded into small starch particles at 40–50 DAF (Fig.5-D).The small starch particles were even released into cytosol and protein body Ӏ where the small starch particles merged with the oil bodies (Fig.5-D).Finally,the small starch particles disappeared in plastid and protein body of cotyledon cells on 60–70 DAF (Figs.3–5).Those findings imply that the glucose-6-phosphate degraded from starch grains (Sharkey and Weise 2016) possibly enters into the oxidative pentose phosphate pathway to provide NADPH for oil synthesis in cotyledon at the late stage of rapeseed maturation.

Most TAG biosynthesis occurs in the endoplasmic reticulum (ER) (Maraschinet al.2019).However,in rapeseed,the ER was not abundant around the nucleus of cotyledon and aleurone cells at 25–35 DAF and were seldom observed in cotyledon and aleurone cells at 40–70 DAF(Fig.2;Appendix A).But the large oil bodies still divided into small oil bodies in cotyledon and aleurone cells of rapeseed accompanied by the increase of oil content from 35 to 70 DAF (Figs.1 and 6).Except for ER containing most enzymes of TAG biosynthesis,the cytosol,plastid and oil body also contain the enzymes of TAG assembly in plant cells (Maraschinet al.2019).Therefore,the TAG assembly presumably utilizes the enzymes in the plastid,cytosol or oil body of cotyledon and aleurone cells during the late stage of rapeseed maturation.

5.Conclusion

The rapeseed turned to dark black color during the late stage of rapeseed development because of the pigment deposition on the cell wall of palisade cells in seed coat.At the same time,the chloroplast degenerated into different plastids and totally disappeared thylakoid to lost photosynthesis in aleurone and cotyledon cells,respectively.These ultrastructural results indicated the rapid oil accumulation of rapeseed was independent on the NADPH synthesized by photosynthesis of chloroplast and probably utilized other sources of reductant and the enzymes in plastid,cytosol or oil body in cotyledon and aleurone cells during late stage of rapeseed development.

Acknowledgements

This work was supported by grants from the National Natural Science Foundations of China (41877528,41471432 and 31500977).

Declaration of competing interest

The authors declare that they have no conflict of interest.

Appendixassociated with this paper is available on http://www.ChinaAgriSci.com/V2/En/appendix.htm