Comprehensive evaluation of fuel properties and complex regulation of intracellular transporters for high oil production in developing seeds of Prunus sibirica for woody biodiesel
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Based on our previous studies of 17 Prunus sibirica germplasms, one plus tree with high quality and quantity of seed oils has emerged as novel potential source of biodiesel. To better develop P. sibirica seed oils as woody biodiesel, a concurrent exploration of oil content, FA composition, biodiesel yield and fuel properties as well as prediction model construction for fuel properties was conducted on developing seeds to determine the optimal seed harvest time for producing high-quality biodiesel. Oil synthesis required supply of carbon source, energy and FA, but their transport mechanisms still remains enigmatic. Our recent 454 sequencing of P. sibirica could provide long-read sequences to identify membrane transporters for a better understanding of regulatory mechanism for high oil production in developing seeds.
To better develop the seed oils of P. sibirica as woody biodiesel, we firstly focused on a temporal and comparative evaluation of growth tendency, oil content, FA composition, biodiesel yield and fuel properties as well as model construction for biodiesel property prediction in different developing seeds from P. sibirica plus tree (accession AS-80), revealing that the oils from developing seeds harvested after 60 days after flowering (DAF) could be as novel potential feedstock for producing biodiesel with ideal fuel property. To gain new insight into membrane transport mechanism for high oil yield in developing seeds of P. sibirica, we presented a global analysis of transporter based on our recent 454 sequencing data of P. sibirica. We annotated a total of 116 genes for membrane-localized transporters at different organelles (plastid, endoplasmatic reticulum, tonoplast, mitochondria and peroxisome), of which some specific transporters were identified to be involved in carbon allocation, metabolite transport and energy supply for oil synthesis by both RT-PCR and qRT-PCR. Importantly, the transporter-mediated model was well established for high oil synthesis in developing P. sibirica seeds. Our findings could help to reveal molecular mechanism of increased oil production and may also present strategies for engineering oil accumulation in oilseed plants.
This study presents a temporal and comparative evaluation of developing P. sibirica seed oils as a potential feedstock for producing high-quality biodiesel and a global identification for membrane transporters was to gain better insights into regulatory mechanism of high oil production in developing seeds of P. sibirica. Our findings may present strategies for developing woody biodiesel resources and engineering oil accumulation.
KeywordsPrunus sibirica seeds Biodiesel fuel properties Oil synthesis Intracellular transporter Differential transcript profiles Transporter-mediated mechanism
adenine nucleotide carrier
nucleotide sugar transporter
pyruvate (PYR) carrier (bile acid: sodium symporter family protein)
adenine nucleotide uniporter
cold filter plugging point
days after flowering
ER membrane ATP transporter
FA methyl esters
glucose-6-phosphate (G6P) transporter
long-chain acyl-CoA synthase
lipid transfer proteins
malate (Mal) synthase
glyoxysomal NAD-Mal dehydrogenase
nucleoside triphosphate (NTP) transporter
oxidative pentose phosphate pathway
phosphoenolpyruvate (PEP) carboxykinase
phosphate (Pi) transporter
peroxisomal adenine nucleotide carrier
ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit
ribulose-5-phosphate (Ru5P) epimerase
quantitative real-time RT-PCR
sugar dependent 1
succinate (SUC)/fumarate carrier
tonoplast monosaccharide transporter
triose phosphate (TP) transporter
vacuolar glucose transporter
xylulose 5-phosphate (X5P)/phosphate transporter
Biodiesel, one of the renewable energy sources, has characterized as ecofriendly fuel with biodegradability and nontoxicity . In recent years, green diesel produced from seed oils of several woody plants has been shown with a notable advantage over conventional feedstock in China [2, 3, 4, 5, 6, 7, 8].
Siberian apricot (Prunus sibirica L.), small deciduous tree of the family Rosaceae and the genus Prunus, is widely distributed in China with the total area of about 1,700,000 ha, and the annual seed production is above 192,500 metric tons [9, 10, 11]. There are many germplasms of P. sibirica with different oil yield and quality in China, of which some accessions have been identified with rich oil content and high proportion of linoleic and oleic acid , and the mean oil content (50.5%) of mature dry seeds from 17 different germplasms was higher than that of Chinese traditional woody oil plants (such as Vernicia Montana, Sapium sebiferum, Pistacia chinensis, Lindera glauca, Jatropha curcas and Comus wilsoniana) [3, 7, 8, 13, 14, 15]. All these revealed that P. sibirica seed oils may be as a novel potential source of biodiesel feedstock in China. To provide a schedule for optimal harvest time of developing seeds with high-quality oils for better development of woody biodiesel, it is important to evaluate dynamic changes of oil content, FA composition, biodiesel yield and fuel property in developing P. sibirica seeds.
In oilseed plants, de novo FA synthesis and its elongation as well as triacylglycerol (TAG) assembly are known to occur in different subcellular organelles, but the end products of both plastidial FA synthesis and cytosolic FA elongation are transferred to the endoplasmic reticulum (ER) for TAG assembly [16, 17]. In general, sucrose as main source of carbon available for oil synthesis is stored in vacuole [17, 18, 19], which is needed to be transported into sink organs (such as fruit and seed), and then converted to key precursor (pyruvate) for FA synthesis in both cytosol and plastid [16, 20, 21]. In addition, de novo FA synthesis in plastid required energy (reducing power and ATP) from several metabolic pathways in different organelles . All these have revealed a complex network of carbon partitioning and energy provision for plant oil synthesis at subcellular level, thus requiring several specific membrane transporters [23, 24, 25]. In recent years, plastidial transporters have been ascribed a key role in carbon allocation and adenine nucleotide transport [26, 27, 28, 29, 30, 31, 32], and mitochondrial transporters implicated in ATP synthesis has been identified in plants [24, 33, 34, 35]. Also, several transport proteins have been shown to transport of lipid, FA or acyl-CoA [36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48], such as ATP-binding cassette (ABC) protein, acyl-CoA-binding protein (ACBP) and lipid transfer protein (LTP). Yet, the transporter-mediated mechanism of carbon source allocation and energy supply available for FA synthesis and oil accumulation still remains enigmatic. To develop the P. sibirica seed oils as woody biodiesel, our previous work focused on overall analyses of Illumina-sequencing data and lipid gene expression in developing seeds of P. sibirica, but we found that some short sequences were not so effective to get BLAST hits owing to lack of a characterized protein domain . Recently, we have performed 454 deep sequencing analysis of P. sibirica (SRX339392) , from which the provision of numerous longer database may make us possible to globally annotate transporters specific for oil synthesis in developing seeds of P. sibirica.
The aim of this sequential study was to better develop seed oils of P. sibirica as woody biodiesel. To this end, we selected one ideal germplasm (accession AS-80) with high quality and quantity of seed oils as experimental materials, and then focused on a temporal evaluation of growth tendency (size and weight), oil accumulation (content and composition), biodiesel yield and fuel properties of developing seeds from 10 DAF (immature stage) to 70 DAF (fully mature stage). Also, a triangular model was established for biodiesel property prediction of raw seed oils during development. Such assessment could provide vital information for selecting the optimal harvest time of developing seeds to obtain high-quality biodiesel. On the other hand, we performed a global analysis of membrane transporters as an attempt to gain new insights into molecular mechanism of high seed oil synthesis for development of woody biodiesel. Based on our previous 454 sequencing data of P. sibirica, we annotated the genes encoding for membrane transporters in various organelles (peroxisome, mitochondria, plastid, vacuole and ER). Finally, by both qRT-PCR and RT-PCR detections, some key transporters were identified to be involved in carbon source allocation and energy provision as well as metabolite transport for FA synthesis, TAG assembly and mobilization in developing seeds, with the aim of deriving a transporter-mediated metabolic model for high oil production. Our findings could provide a better understanding of oil accumulative molecular mechanism and also present new biotechnological targets to improve seed oil yields for the development of woody biodiesel.
Temporal analysis of growth tendency for developing seeds
Dynamic changes of oil accumulation and biodiesel yield of developing seeds
Dynamic changes of FA compositions and their relative proportions in developing seeds of P. sibirica
0.71 ± 0.01
19.92 ± 1.58
0.94 ± 0.09
2.51 ± 0.51
11.59 ± 0.91
57.95 ± 1.21
6.38 ± 0.42
11.30 ± 1.07
1.05 ± 0.11
3.01 ± 0.81
21.80 ± 1.05
52.79 ± 1.01
9.80 ± 0.63
0.11 ± 0.02
0.14 ± 0.05
5.02 ± 0.82
0.81 ± 0.04
4.97 ± 0.51
27.14 ± 1.02
51.62 ± 1.22
10.23 ± 0.71
0.10 ± 0.03
0.11 ± 0.01
4.11 ± 0.51
0.70 ± 0.05
1.50 ± 0.07
32.51 ± 1.03
50.49 ± 1.01
10.51 ± 0.61
0.08 ± 0.01
0.10 ± 0.02
3.92 ± 0.23
0.61 ± 0.01
1.24 ± 0.02
36.42 ± 1.81
47.14 ± 1.02
10.43 ± 0.54
0.13 ± 0.02
0.11 ± 0.01
2.91 ± 0.32
0.61 ± 0.02
1.07 ± 0.03
72.22 ± 2.01
22.47 ± 0.98
0.51 ± 0.03
0.09 ± 0.04
0.12 ± 0.04
2.86 ± 0.13
0.59 ± 0.01
1.01 ± 0.01
76.39 ± 3.02
18.49 ± 0.71
0.39 ± 0.01
0.14 ± 0.03
0.13 ± 0.02
Construction of prediction model for biodiesel fuel properties of raw oils from developing seeds
Temporal evaluation of biodiesel fuel properties of oils form developing seeds
Evaluation of biodiesel fuel properties of oils from different developing seeds of P. sibirica
Biodiesel fuel properties
IV (g/100 g)
KV (mm2 s−1, 40 °C)
D (kg/m−3, 15 °C)
147.72 ± 3.1
7.17 ± 0.8
47.24 ± 3.5
123.76 ± 2.5
− 2.18 ± 0.1
0.32 ± 0.1
2.32 ± 0.02
4.08 ± 0.09
882.75 ± 1.21
158.19 ± 2.6
4.67 ± 0.7
45.97 ± 3.8
130.75 ± 2.8
− 6.57 ± 0.1
− 3.99 ± 0.1
2.76 ± 0.03
4.14 ± 0.10
881.18 ± 1.05
162.13 ± 2.4
3.53 ± 0.2
45.49 ± 1.2
133.40 ± 1.5
− 8.58 ± 0.2
− 5.97 ± 0.2
2.90 ± 0.01
4.23 ± 0.11
879.39 ± 1.31
162.80 ± 2.2
1.89 ± 0.1
45.81 ± 0.9
133.38 ± 1.3
− 11.46 ± 0.3
− 8.79 ± 0.1
2.91 ± 0.01
4.28 ± 0.08
876.28 ± 0.89
165.29 ± 2.1
1.75 ± 0.3
45.11 ± 0.8
135.49 ± 1.1
− 11.71 ± 0.1
− 9.03 ± 0.2
2.94 ± 0.04
4.32 ± 0.11
878.45 ± 0.91
116.31 ± 1.5
1.30 ± 0.2
51.58 ± 0.7
105.65 ± 0.5
− 12.47 ± 0.1
− 9.81 ± 0.1
3.30 ± 0.01
4.36 ± 0.08
877.57 ± 0.97
115.63 ± 1.5
1.31 ± 0.3
51.71 ± 0.6
102.35 ± 1.5
− 12.48 ± 0.2
− 9.79 ± 0.1
3.33 ± 0.01
4.48 ± 0.10
876.09 ± 1.01
Genome-wide identification of genes for the transporters in developing seeds
In oilseed plants, de novo FA synthesis and its elongation as well as TAG assembly in multiple organelles required supply of energy, carbon source, or end product across membranes by selective transporters [16, 24, 36, 48], but the transport mechanism remains one of most interesting open challenges encountered in study of oil synthesis. Recently, the provision of numerous longer reads from our 454 deep sequencing of P. sibirica  may make us possible to globally annotate the genes for transporters by BLASTX against the public databases (Additional file 1: Table S1). A total of 116 genes were identified for membrane transporters in various organelles (plastid, vacuole, peroxisome, mitochondria and ER), mostly implicated in FA synthesis, TAG assembly and mobilization, including the interchange of glycolytic intermediate, transport of sucrose, acyl-CoA, adenine nucleotide and FA, and energy supply (ATP synthesis and TCA cycle) as well as oil mobilization (TAG hydrolysis, FA β-oxidation, glyoxylate cycle and gluconeogenesis) (Additional files 2, 3, 4, 5, 6, 7: Table S2, S3, S4, S5, S6, S7), implying a complex transporter-mediated mechanism of carbon allocation and energy supply for oil synthesis in developing seeds of P. sibirica. In the following, our work focused on the analysis of temporal transcript profiles for all annotated transporters by both qRT-PCR and RT-PCR during seed development to identify transporters specific for high oil production.
Temporal transcripts of plastidial transporters for carbon allocation and ATP transport in developing seeds
Transcript analysis of mitochondrial transporters for ATP synthesis and oil metabolism in developing seeds
By functional annotation, some orthologs of mitochondrial transporters were marked (Additional file 3: Table S3), including acetyl-carnitine/carnitine carrier (BOU), dicarboxylate/tricarboxylate carrier (DTC), adenine nucleotide transporter (ADNT1), ADP/ATP carrier (AAC1/3/5), phosphate carrier (PIC1/2/3), dicarboxylate carrier (DIC1/2), PYR carrier (MPC1) and succinate/fumarate carrier (SFC). Analyses of differential profiles showed that ACC3/PIC1 transcript was up-regulated during seed development, and ADNT1/DTC/ACC1/MPC1 transcript enhanced before 30 DAF, while ACC5 and PIC2/3 displayed low transcript (Fig. 3b; Additional file 8: Figure S1c), indicating that DTC MPC1, AAC1/3, ADNT1 and PIC1 may be as main transporters for PYR transport or ATP synthesis in developing seeds. In addition, the transporters for oil mobilization were detected with differential transcripts, of which SFC and DIC1 exhibited high transcript at 60–70 DAF, but a strong decline of transcript was observed for BOU (Fig. 3b), suggesting a specific contribution of DIC1/SFC to oil mobilization in late developing seeds.
Temporal transcripts for tonoplast transporters involved in sugar transport in developing seeds
Given vacuole as temporary reservoir for many metabolites [19, 54], we attempted to explore the possibility of tonoplast transporter in delivery them as carbon source for FA synthesis in developing seeds. Here, the genes for orthologs of glucose transporter (TMT1/2 and VGT1, cytosol glucose import), inositol transporter (INT1), malate transporter (ALMT9), dicarboxylate transporter (TDT) and sucrose symporter (SUT2/3/4, vacuole sucrose export) were annotated (Additional file 4: Table S4), implying a diversity of tonoplast metabolite transport in developing seeds. However, only SUT2/4 showed high transcript during development, and TMT2 increased transcript after 60 DAF (Additional file 8: Figure S1d; Fig. 3c), suggesting that SUT2/4 may be specific for export vacuole sucrose into cytosol during seed development, but TMT2-mediated glucose uptake into vacuole was at maturing stage.
Transcript analysis of ATP and nucleotide sugar transporters of ER membrane in developing seeds
It was shown that ER membrane-localized ATP transporter (ER-ANT1) and UDP-glucose transporter (AtUTr) may play a role for seed development and oil increase of Arabidopsis [55, 56]. Here, a combination of annotation and transcript detection indicated that ER-ANT1 transcript increased before 30 DAF and then maintained abundance toward mature stage (Additional file 5: Table S5; Additional file 8: Figure S1b; Fig. 3d), which was correlated with seed growth tendency during development (Fig. 1b, c), refeclting that ANT1-mediated ATP import into ER may be essential for seed growth. However, the finding of reduced AtUTr1 transcript (Fig. 3d) suggested its unimportance for the UDP-glucose transport during seed development.
Transcript analysis of transporters of peroxisomal membrane for TAG mobilization in developing seeds
Transcript analysis of transport proteins involved in FA transport for TAG synthesis in developing seeds
In this study, a concurrent evaluation of growth tendency, oil content, FA compositions, biodiesel yield and fuel properties as well as model construction for fuel property prediction was conducted on developing seeds from P. sibirica plus tree (germplasm accession AS-80) (Tables 1 and 2; Fig. 1b–d), revealing that the oils of developing seeds harvested after 60 DAF may be as novel potential feedstock for producing biodiesel with ideal fuel properties. However, current knowledge of how carbon or energy is channeled to oil accumulation in oilseed plants is still limited. To better develop the seed oils of developing P. sibirica as woody biodiesel, we performed a global analysis of transporters in developing seeds of P. sibirica at transcript level based on our recent 454 sequencing data , resulting in 116 genes to annotate for membrane transporters in different organelles. By qRT-PCR and RT-PCR, some key transporters related to carbon allocation, precursor transport and energy supply were identified for FA synthesis, TAG assembly and oil mobilization, which allowed us to unreal the transporter-mediated mechanism of high oil production in developing P. sibirica seeds for better development of woody biodiesel.
Transporters specifically for carbon allocation from sucrose to oil synthesis in developing seeds
In oilseed plants, photosynthetic sucrose stored in vacuole is transferred to sink organs as carbon source for oil synthesis via apoplastic pathway by sucrose transporter (SUT) [60, 61, 62]. Our finding of a close correlation between high transcriptional SUT2/4 and active oil synthesis of developing seeds (Figs. 1d, 3c; Additional file 8: Figure S1d) revealed an essentiality of SUT2/4 in sucrose import into cytosol as carbon source for FA synthesis in developing seeds of P. sibirica, as was noted in developing fruits of peach and pear [63, 64], which could support the idea of main carbon supply for fruit or seed as apoplastic sucrose [7, 17]. Given the fact of less transcript (Fig. 3c) for tonoplast transporters (VGT1 and TMT1) in relation to import hexose from cytosol into vacuolar , it could be speculated that differential transcripts between VGT1/TMT1 (down-regulation) and SUT2/4 (up-regulation) may contribute to provide cytosolic hexose for glycolysis during FA synthesis of developing P. sibirica seeds.
A major flux through glycolysis is expected to provide precursors responsible for high oil synthesis in oilseed plants [7, 11, 17, 20, 22, 30, 32, 66], all of which are needed to be imported into plastid by selective transporters. Our observations that both PPT1 (PEP transporter) and BASS2 (PYR carrier) showed high transcript and a positive correlation with active oil synthesis of developing seeds (Figs. 1d, 3a; Additional file 8: Figure S1a) revealed that the PPT1/BASS2-mediated unidirectional transport may contribute to provide PEP/PYR from cytosol into plastid glycolysis for FA synthesis destined to oil production in developing seeds of P. sibirica, which could be evidenced by the fact that overexpression of BASS2 or PPT1 increased oil content in Arabidopsis and tobacco seeds [67, 68]. In addition to glycolysis, FA synthesis was also fed by Rubisco shunt and OPPP [66, 69, 70, 71, 72]. The participation of such pathway in our work could be clearly demonstrated by rich transcription for G6P transporter (GPT1), plastidial OPPP and Calvin cycle-related enzymes (SBP, RBCS1B, GAPA/B and PRK) at 10–30 DAF (Figs. 3a and 4a, b), implying that a portion of GPT1-mediated G6P import into plastid may be essential for FA synthesis in early developing seeds, as was the case for other oilseed plants [66, 70, 71, 72].
Altogether, abundant transcripts of transporters (SUT2/4, GPT1, PPT1 and BASS2) were correlated with the increases of oil content and biodiesel yield of developing seeds (Figs. 1d and 3a, c), revealing its importance in allocation carbon source (sucrose) for high oil synthesis and biodiesel yield during P. sibirica seed development.
Transporters specifically involved in ATP provision for de novo FA synthesis in developing seeds
In addition to carbon supply, de novo FA synthesis in plastid requires ATP and reducing power, most of which are derived from other organelles by the transporters . Here, high transcripts were identified for both plastidial NTT2 relevant to import cytosolic ATP in exchange with ADP [27, 28, 29] and mitochondria ACC3 as ATP exporter in exchange with cytosolic ADP [33, 34] in developing seeds (Fig. 3a, b; Additional file 8: Figure S1a, c), paralleled to the active oil synthesis (Fig. 1d), emphasizing that coordinated transcripts of NTT2/ACC3 may provide a strong channeling of ATP into plastid for FA synthesis of developing seeds. Evidence for this conclusion was the finding (Fig. 3a, b) that PIC1 as transporter of cytosol Pi into mitochondria  showed high transcript, but less transcript was noted for PHT2.1/4.2/4.5 relevant to Pi import into plastid , implying its key role in supply mitochondrial Pi for ATP synthesis during seed development. It seems, therefore, that these bidirectional transporter-mediated exchange of adenine nucleotides (ATP, ADP and AMP) and Pi among plastid, cytosol and mitochondria could help to generate ATP available for FA synthesis in plastid, destined for high oil yield, as also noted in Arabidopsis .
Selective transport proteins involved in TAG synthesis destined for high-quality oil yield in developing seeds
Increasing TAG synthesis and oil yield would expand economic value for oilseed plants , but the mechanism of FA transport from plastid to ER for TAG assembly is still unknown. In recent years, some transport proteins of ABC and FAX have been shown for export plastid FA into ER for TAG synthesis [36, 37, 48, 75]. Of our identified 5 FAXs and 63 ABC transporters (Additional file 6: Table S6), only two plastid membrane-localized ABCB9/28 and FAX2 were highly expressed (Fig. 6b, c; Additional file 8: Figure S1e) and correlated with rapid oil synthesis (Fig. 1d) of developing seeds, so it was concluded that they may be as important transporters of FA export from plastid for TAG synthesis during seed development. This finding was contrasted with previous result of FAX1/ABCA9 as main supplier of plastid FA for TAG synthesis by both mutant and overexpression analyses [36, 48, 75]. We also noticed that ER-localized ACBP1, which in Arabidopsis transports acyl-CoA for TAG accumulation , increased transcript over the oil synthesis period of developing seeds (Figs. 1d, 6a), pointing to a role for ACBP1 as a key transporter of acyl-CoA substrate for ER TAG synthesis during seed development of P. sibirica.
Also of note was involvement of ACBP in control the partition of 18:1 between elongation and desaturation at ER . Our identification of high transcript for cytosolic ACPB4/6 before 50 DAF (Fig. 6a), the period at which polyunsaturated FA was actively synthesized (Fig. 2a; Table 1), implying that ACPB4/6-mediated transport may be crucial for polyunsaturated FA accumulation in developing P. sibirica seeds, which was evidenced by polyunsaturated FA increase in ACBP4/6-overexpressing Arabidopsis seeds [45, 46, 47]. Our previous works have indicated that raw seed oils for high-quality biodiesel were dependent on the ideal contents of polyunsaturated (< 60%), monounsaturated (> 30%) and saturated FAs (< 30%) , which was also shown in this study that the contents of polyunsaturated (18.9–23.0%), monounsaturated (73.0–77.1%) and saturated FAs (4.0–4.1%) were detected at 60–70 DAF (Fig. 2a; Table 1), during which the developing seeds were identified with high-quality oil, high-yield biodiesel and superior fuel properties (Fig. 1d; Table 2). This, integrated with high transcript for FA/acyl-CoA transport-related proteins (ABCB9/28, ACBP1 and FAX2) during TAG synthesis (Fig. 6a–c), revealed that coordinated transcripts of ABCB9/28, FAX2 and ACBP1/4/6 may contribute to affect FA composition and oil yield, destined to the eventual biodiesel yield increase and fuel property improvement in developing seeds of P. sibirica.
Transporter-mediated regulation for a limited loss of TAG breakdown during seed maturation
A decline in oil content during seed maturation has been reported in oilseed plants [76, 77, 78]. During storage oil mobilization, cytosolic FAs (from TAG lipolysis) and ATP are imported into peroxisome by the transporters of CTS and PNS for acyl-CoA formation and then degraded to acetyl-CoA via β-oxidation, which is further converted by glyoxylate cycle or TCA cycle [37, 38, 79]. Our findings that transcripts for PNC1 and CTS increased after 60 DAF, a temporal pattern also noted for TAGL2 and SDP1 (known for TAG breakdown) (Figs. 3d, 5a), revealed main contribution of them to TAG mobilization at seed maturity phase of P. sibirica. Also consistent with this conclusion, high transcript was characterized for mitochondria transporters of SFC and DIC1 (Fig. 3b) in relation to gluconeogenesis [35, 80], and enzymes of glyoxylate cycle (ACO3, CYS2, ICL and MLS), gluconeogenesis (PEPCK2) and β-oxidation (LACS6, MEP2 KAT2 and ACX2/3) after 60 DAF (Figs. 5b–d). Given that knockout of pnc1/2, cts, sfc or dic1 promoted oil increase in mature seeds of transgenic plants [78, 81, 82, 83, 84, 85, 86], together with our detection of only 2.1% decline of oil content for ripening seeds of P. sibirica (Fig. 1d), it was believed that the transporter-mediated TAG breakdown during seed maturity was limited, which could also be supported by lower transcript levels of transporters for TAG mobilization than for both FA synthesis and TAG assembly during seed development of P. sibirica (Figs. 3, 4, 5 and 6).
In this work, to better develop the seed oils of P. sibirica as woody biodiesel, one plus tree (germplasm accession AS-80) with high-quantity seed oils was selected as experimental material and then a subsequent exploration of dynamic patterns for oil accumulation, growth tendency and the biodiesel yield and fuel properties as well as the construction of prediction model for fuel properties was made during seed development, revealing that the oils from developing seed harvested at 60 DAF may be as potential raw material suitable for producing biodiesel with optimum fuel properties. Such assessment could provide a pivotal reference for exploitation of woody biodiesel plants. In an attempt to highlight the transport mechanism for oil synthesis during P. sibirica seed development, a global analysis of transporter was performed in developing P. sibirica seeds based on our recent 454 sequencing data, and a total of 116 genes for membrane intrinsic transporters in mitochondria, plastid, vacuole, peroxisome and ER were annotated. The differential transcript analysis by RT-PCR and qRT-PCR has led to the identification of some key transporters in carbon allocation and energy supply as well as intermediate transport responsible for FA synthesis and TAG assembly during seed development, including mitochondria transporter (ACC1/3, ADNT1, PIC1, MPC1 and DTC), plastid transporter (BASS2, GPT1, PPT1, NTT2, FAX2, PHT2.1/4.2/4.5, ABCB9/28 and BT1L), ER transporter (ER-ANT1) and tonoplast transporter (SUT2/4). We also marked some transporters (PNC1, DIC1, CTS and SFC) for TAG breakdown during seed maturity. Together, our findings and complex transporter-mediated metabolic model (Fig. 7) will provide a better insight into regulatory mechanism of high oil production and may also provide both a rich source of data and of considerable interest to those studying woody biodiesel plants.
Materials and methods
Collection of plant materials
The P. sibirica seeds from different developmental stages were collected from 10-year-old plus tree (germplasm accession AS-80) planting in Changping District of Beijing (E116°23′, N40°22′), China. The flowers of twenty trees with the same anthesis were marked and then one hundred fruits (5 fruits per tree) were harvested at 10 (immature stage), 20, 30, 40, 50, 60 and 70 DAF (full mature stage), respectively. After removing the sarcocarp, fresh seeds were immediately frozen in liquid nitrogen and stored at − 80 °C until use for qRT-PCR and RT-PCR assay. The fresh weight of seeds was measured with an electronic balance and the longitudinal and transverse diameter of seeds was determined with vernier caliper. All the determination was conducted in triplicate.
Seed oil extraction and trans-esterification
The fresh seeds from every developing stage were stored at room temperature for 1 week to dry until the samples attained uniform weight. About 30 g of dry seeds (3 samples per developing period) was crushed into an average particle size of 0.8 mm using domestic grinder and then the oils were extracted with petroleum ether using Soxhlet apparatus at 45–50 °C . After extraction for 6–8 h, the oil was separated from organic mixture by rotary evaporator (LABORTA 4000-efficient, Germany) and then dried at 105 °C in ventilated drying oven. The content of extracted oils from each developing seeds was calculated as the difference between the weights of dry seed sample before and after extraction and the results were expressed as the percentage of the extracted oil weight to dry seed weight (%, g/g). To analyze FA compositions, the oils from developing seeds were trans-esterified as previously described method . All analysis was performed in triplicate.
FA methyl ester analysis and biodiesel yield calculation
The FA methyl esters (FAMEs) obtained from each developmental seeds was analyzed for determining the FA compositions using an Agilent 6890 (California, USA) gas chromatograph equipped with a flame ionization detector (GC-FID) as previously described . The HP-INNOWax capillary column (inner diameter 0.32 mm, filmthickness 0.5 μm, split 1:20) was used and the temperature was programmed at 60 °C, with a rise of 4 °C min−1 to 220 °C and heated to 240 °C for 10 min. The carrier gas was helium with a flow rate of 1.0 mL min−1. The peaks of FAMEs were identified by comparing their retention time with that of the known standards, and peak integration was performed by applying HP3398A software. Each FAME analysis was run in triplicate and the data were presented as the mean. The biodiesel yield was calculated by the previous method , where the yield was expressed as the percentage (%, g/g) of the obtained total amount of FAMEs (g) to the used amount of raw oils (g).
Evaluation of biodiesel properties
The biodiesel fuel properties (IV, CN, CFPP, CP, OS, D and KV) were calculated from the FAME compositions according to the previously method [14, 50]. The determination was conducted in triplicate. The biodiesel fuel properties of seed oils were compared with the relevant specifications of EN 14214-2008 (European, 2008), ASTM D6751-2010 (USA, 2010), DIN V51606-1997 (Germany, 1997) and GB/T 20828-2007 (China, 2007).
Construction of prediction model for biodiesel fuel properties of raw seed oils
The triangular prediction model of seed oils from different developing stages was constructed according to the influence of the FAME compositions [7, 50]. To predict the biodiesel properties of oils from developing seeds, the percentages of saturated, monounsaturated and polyunsaturated FAMEs from different developing seeds were calculated to outline a triangular prediction graph, in which three angular points of the triangle meant the 100% of monounsaturated, polyunsaturated and saturated FAMEs, respectively. In triangular graph, the region existed at the far end of the polyunsaturated angular point (lower left vertex) and the saturated angular point (lower right vertex) was delineated to predict the biodiesel fuel properties, taking into account the CN, IV, CFPP and OS [7, 50].
Functional annotation of unigenes for the transporters in developing seeds
Based on our previous 454 pyrosequencing data (SRX339392) from different tissues of Siberian apricot , the unigenes were annotated to be involved in the transporters using BLASTX alignment against known protein databases of NCBI nonredundant, Arabidopsis proteome, SWISS-PROT, TREMBL, AP, CDD, PFAM and COG. Also, GO functional classifications were analyzed by GO terms (http://www.geneontology.org) using Blast2Go software, and KEGG pathway assignment was performed using the BLAST all against Kyoto Encyclopedia of Genes and Genomes database.
Gene expression analysis
The total RNA was extracted using RNeasy Plant Mini Kits (Qiagen, Inc., USA) and the obtained RNA was qualified and quantified using Nanodrop ND-1000 Spectrophotometer (N Wilmington, DE, USA). All the samples showed a 260/280 nm ratio from 1.9 to 2.1 and then was reverse transcribed using the Reverse Transcription System (Promega). The qRT-PCR was conducted on 7500 Real-Time PCR System using SYBR Premix Ex Taq Kit (TaKaRa) based on the manufacturer’s protocol. All the amplified primers (Additional file 10: Table S9) were designed by PrimerQuest (http://www.idtdna.com/PrimerQuest/Home/Index) software with melting temperatures at 62 °C, and the absence of secondary structures was verified by UNAFold program (http://eu.idtdna.com/UNAFold). The genes encoding for cyclophilin (CYP) and ubiquitin-conjugating enzyme (UBC) were used as inner references as our previously described . Negative controls consisting of nuclease-free water instead of template and reverse transcriptase controls prepared by substituting reverse transcriptase for nuclease-free water in cDNA synthesis step were included in all analyses for each primer pair. Additionally, the RT-PCR assay was conducted according to our previous study  and all gene-specific PCR products were confirmed by gel electrophoresis on 1.5% agarose gel and visualized after staining with ethidium bromide using the Gene Genius Imaging System and quantified with GeneTool software (Gene Company Limited). Three biological replicates with three technical repetitions each were performed for qRT-PCR and RT-PCR.
JW, WJL, SZL and SLC organized and planned the research, and drafted the manuscript. JYA, LLS and SBD carried out the RNA-Seq data analysis. LBW and HYY determined the biodiesel fuel properties the seed oils. ZXL and ZDY collected plant materials and analyzed dynamic patterns of seed growth (weight and size). SZL provided the funding, computational guidance, and was substantially involved in data analysis. All authors have read and approved the manuscript.
We thank the National Natural Sciences Foundation of China (No. 31570653) for financial support. We also like to thank the reviewers for positive criticism to improve the quality of the manuscript.
The authors declare that they have no competing interests.
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This research was supported by the National Natural Sciences Foundation of China (No. 31570653) and Beijing Advanced Innovation Center for Tree Breeding by Molecular Design.
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