Evaluation of oil accumulation and biodiesel property of Lindera glauca fruits among different germplasms and revelation of high oil producing mechanism for developing biodiesel

doi:10.21203/rs.3.rs-1419380/v1

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Method Article

Evaluation of oil accumulation and biodiesel property of Lindera glauca fruits among different germplasms and revelation of high oil producing mechanism for developing biodiesel

https://doi.org/10.21203/rs.3.rs-1419380/v1

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published 25 Jan, 2023

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Background

Lindera glauca with rich resource and fruit oil has emerged as novel source of biodiesel in China, but different germplasms show a variation for fruit oil content and FA profile. To develop L. glauca fruit oils as biodiesel, a concurrent exploration of oil content, FA composition, biodiesel yield, fuel property and prediction model construction was conducted on the fruits from 8 plus germplasms to select superior genotype for ideal biodiesel production. Another vital focus was to highlight mechanism that govern the differences in oil content and FA profile of different germplasms. The cross-accessions comparisons associated with oil-synthesized gene transcriptional level and oil accumulative amount led to the identification of potential determinants (enzymes, transporters or transcription factors) and regulatory mechanisms responsible for high-quality oil accumulation.

Results

To select superior germplasm and unravel regulatory mechanism of high oil production for developing L. glauca fruit oils as biodiesel, 8 plus trees (accession LG01/02/03/04/05/06/07/08) with high-yield fruits were selected to evaluate the differences in oil content, FA profile, biodiesel yield and fuel property, and to construct fuel property prediction model, revealing a variation in the levels of fruit oil (45.12–60.95%), monounsaturated FA (52.43–78.46%) and polyunsaturated FA (17.69–38.73%), and biodiesel yield (80.12–98.71%) across different accessions. Of note, LG06 had a maximum yield of oil (60.95%) and biodiesel (98.71%), and ideal proportions of C18:1 (77.89%), C18:2 (14.16%) and C18:3 (1.55%), indicating that fruit oils from accession LG06 was the most suitable for high-quality biodiesel production. To highlight molecular mechanism that govern such differences in oil content and FA composition of different accessions, the quantitative relationship between oil-synthesized gene transcription and oil accumulative amount were conducted on different accessions to identify some vital determinants (enzymes, transporters or transcription factors) with a model of carbon metabolic regulatory for high-quality oil accumulation by an integrated analysis of our recent transcriptome data and qRT-PCR detection. Our findings may present strategies for developing L. glauca fruit oils as biodiesel feedstock and engineering its oil accumulation.

Conclusions

This study presents the cross-accessions evaluations of L. glauca fruit oils to determine superior accession for producing ideal biodiesel, and the associations of oil accumulative amount with oil-synthesized gene transcription was performed to identify some crucial determinants (enzymes, transporters or transcription factors) with metabolic regulation model established for governing high oil production. Our finding may provide molecular basis for new strategies of developing biodiesel resource and engineering oil accumulation.

woody biodiesel

fuel properties

oil accumulation

transcriptional regulation

provenances

Lindera glauca fruit

The global population is expected to hit 9.8 billion by 2050, at which the reserved fossil energy (oil and gas) is expected to run out [1, 2]. Thus, exploiting renewable and clean of energy source has become an imperative for sustainable economic development and global energy security. Biodiesel, known as FA methyl ester (FAME), is gradually gaining global attention as ecofriendly fuel for its nontoxicity and biodegradability [3], which has been widely used in several countries such as USA, Malaysia, Indonesia, Brazil, Netherlands, Germany and France [4]. In recent years, the Chinese government has designed a series of planning projects to support the development of renewable energy sources, and the oils from some woody plants (such as Prunus sibirica, Pistacia chinensis, Xanthoceras sorbifolia and Jatropha curcas) have been utilized as raw material for biodiesel production [5–10].

Lindera glauca, small tree species of the Lauraceae family, is widely distributed in the mountainous and lowland districts of China with the annual fruit production of above 120,000 metric tons, and the average yield of ripened fruits is about 11.5 metric ton/ha [11–15]. There are many germplasms of L. glauca with different oil content and FA compositions in China. Based on our previous studies on 102 fruit samples of L. glauca from 9 geographical provenances, some accessions have been identified with rich oil content and high proportion of oleic and linoleic acid, and notably, the oil content (42.0–53.0%) of ripen fruits [11, 12, 14] was higher than that of traditional woody oil plants [9, 16–20]. All these indicated that L. glauca fruit oils may be as potential source of biodiesel feedstock in China. Given the differences in fruit oil content and its FA compositions of different germplasms, it is vital to select superior germplasm with high-quality fruit oil for better development of woody biodiesel. However, an interesting challenge is the mechanism that governed such differences in oil content and FA composition of L. glauca fruits across different accessions. Therefore, understanding molecular basis of high oil production in L. glauca fruits has become another one imperative for developing woody biodiesel.

Plant oils are an important natural resource of carbon and energy to meet the increasing demands of food, biofuel, and industrial application [21]. In general, carbon source for oil synthesis of oil plants mostly as sucrose from photosynthetic tissues, which in non-green tissues (developing fruits and seeds) is mainly converted to pyruvate (PYR) via glycolysis or to glyceraldehyde 3-phosphate (GAP) via pentose phosphate pathway (PPP) in both cytosol and plastid, as crucial precursor for acetyl-CoA formation destined to de novo FA synthesis [14, 22]. It is known that oil biosynthetic process is composed of FA synthesis and triacylglycerol (TAG) assembly involved in several regulatory enzymes [21], of which de novo FA synthesis is initiated by carboxylation of acetyl-CoA to malonyl-CoA in plastid, and transferred to malonyl-ACP as two-carbon unit for FA elongation via a condensation cycle to elongate acyl chain by two-carbon units. The newly-synthesized free FAs are exported from plastid into cytosolic acyl-CoA and incorporated into phosphatidylcholine (PC) for desaturation in endoplasmic reticulum (ER). The traditional pathway to assemble FAs into triacylglycerol (TAG) via three sequential acylation of G3P with acyl-CoA to produce diacylglycerol (DAG) and subsequent TAG (known as Kennedy pathway) [23–25]. It is also noted that in oil plants, three potential mechanisms have shown to control FA flux via PC for TAG assembly, including esterification of nascent FA to PC, conversion of FA from PC to DAG for TAG assembly by PDAT, and direct utilization of PC-derived DAG as substrate for TAG synthesis by DGAT [21, 23–25]. All these revealed one complex metabolic regulatory network for oil synthesis at subcellular level. Yet, the mechanism of how carbon is channeled to FA synthesis and how they are assembled into TAG destined for oil accumulation in oil plants is still unclear. Recently, we performed transcriptome sequencing of developing L. glauca fruits to identify some enzymes and transcription factors relevant for oil biosynthesis [14], which could help us to gain new insight into the mechanism for controlling differences in oil content and FA composition among different germplasms.

The aim of this sequential study was to select ideal germplasm and to unravel regulatory mechanism of high oil production for better development of L. glauca fruit oil as potential feedstock for biodiesel, To this end, 8 selected plus tress (accessions LG01, LG02, LG03, LG04, LG05, LG06, LG07 and LG08) with high-yield fruits was used as material to assess the differences in oil accumulation (content and FA composition), biodiesel yield and fuel properties of fruit oils from all accessions. Also, a triangular model was constructed for biodiesel property prediction of raw fruit oils from different accessions. Such evaluation could help to select superior accession for developing biodiesel. Another focus of this work was to highlight mechanism that govern differences in fruit oil content and FA profile of different accessions, and to gain valuable information for engineering oil accumulation or molecular-assisted selection. In this regard, based on our recent transcriptomic result of developing L. glauca fruits, the comparative analysis of cross-accessions correlation of gene transcriptional level by qRT-PCR with oil accumulative amount was performed as an attempt to identify some crucial regulators (enzymes, transcription factors and transporters) involved specifically in carbon source supply (glycolysis, PPP and acetyl-CoA formation) and oil synthetic process (FA synthesis and TAG assembly) essential for high-quality oil accumulation in L. glauca fruits of all accessions. All these could facilitate the development of L. glauca fruit oils as biodiesel feedstock, and help to gain new insight into molecular regulatory mechanism of high-quality oil production for engineering oil accumulation in oilseed plants.

Variability in fruit oil content and biodiesel yield of 8 selected accessions

To develop L. glauca fruit oils as potential material for biodiesel, it was vital for us to determine ideal accession with high quantity and quality of fruit oils for gaining maximum economic benefits. Based on our recent studies of different germplasms [11, 12], eight plus trees (accessions LG01, LG02, LG03, LG04, LG05, LG06, LG07 and LG08) with high fruit yield were selected to investigate the variability in fruit oil content and biodiesel yield of different accessions. Here, the fruit oil contents varied from 44.12% (LG01) to 60.95% (LG06), followed by LG03 (46.48%), LG02 (47.39%), LG08 (48.52%), LG04 (49.09%), LG07 (51.81%) and LG05 (53.16%), of which the fruits of LG05, LG06 and LG07 had oil content more than 51.5% (Figure 1a). This allowed us to explore the differences in biodiesel yield from fruit oils across all accessions. The biodiesel yield from fruit oils varied among different accessions, ranging from 85.12% (LG01) to 98.71% (LG06) with an average value of 92.17% (Figure 1b), of which biodiesel yield of LG05 (97.26%), LG06 (98.71%) and LG07 (96.81%) was in the standard of EN 14214 (96.5%). These revealed a difference in fruit oil and biodiesel yield across different accessions, and three high oil-bearing accessions (LG05/06/07) with high-yield biodiesel could be valuable as source for developing biodiesel.

Variability in FA profiles of fruit oils from 8 selected accessions

Oil content and FA composition are known as two vital factors for determining whether oil plant can be suitable for biodiesel production. Here, 10 kinds of FA compositions were detected in L. glauca fruit oils of all accessions (Table 1), including capric acid (C10:0), lauric acid (C12:0), palmitic acid (C16:0), palmitoleic acid (C16:1), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), linolenic acid (C18:3), arachidic acid (C20:0) and eicosenoic acid (C20:1). The dominant compound was oleic acid (51.24-77.89%) with an average of 59.52%, followed by linoleic acid (16.14-33.87%), palmitic acid (2.65-5.26%) and linolenic acid (1.55-4.85%), but the other showed minor quantities (0.03-0.46 %), of which the maximum value of C18:1 (77.89%) and minimum value of C18:2 (16.14%) and C18:3 (1.55%) were all detected for accession LG06. Also, the total content of C18:1 and C18:2 in fruit oils varied from 84.29% to 94.04%, and three accessions (LG05, LG06 and LG07) had C18:1 content of more than 60 % (Table 1), implying that they were ideal raw material for biodiesel.

The ideal plant oils for high-quality biodiesel production generally contain a small amount of saturated FA (SFA), high amount of monounsaturated FA (MUFA), and low level of polyunsaturated FA (PUFA) [9, 26]. Here, the change analysis for SFA, MUFA and PUFA in fruit oils from different accessions showed that the contents of MUFA and PUFA varied from 52.43% to 78.46%, and from 17.69% to 38.73%, respectively, but the SFA content of all accessions was less than 9% (Figure 1c). Of note, LG06 had the highest content of MUFA (78.46%) and the lowest amounts of PUFA (17.69%) and SFA (3.85%), revealing that the fruit oils from accession LG06 with ideal FA compositions could meet the demand of high-quantity biodiesel production.

Table 1 The changes of FA compositions and relative proportions in L. glauca fruits of different accessions

Accessions	C10:0 (%)	C12:0 (%)	C16:0 (%)	C16:1 (%)	C18:0 (%)	C18:1 (%)	C18:2 (%)	C18:3 (%)	C20:0 (%)	C20:1 (%)
LG01	0.29±0.03	0.25±0.03	5.26±0.41	1.07±0.08	1.79±0.13	51.24±2.28	33.87±2.01	4.86±0.78	1.25±0.05	0.12±0.03
LG02	0.46±0.03	0.14±0.07	4.64±0.18	1.28±0.24	1.69±0.35	55.14±2.11	31.07±1.45	4.63±0.21	0.74±0.03	0.21±0.05
LG03	0.25±0.03	0.24±0.03	5.13±0.22	2.33±0.14	1.76±0.24	52.04±2.29	32.25±2.05	4.74±0.29	1.11±0.13	0.15±0.03
LG04	0.19±0.04	0.17±0.04	4.04±0.09	2.29±0.21	1.42±0.28	58.30±2.21	29.83±1.21	3.07±0.19	0.61±0.06	0.08±0.02
LG05	0.17±0.03	0.08±0.03	3.54±0.31	2.24±0.17	1.16±0.11	63.32±3.37	26.52±1.21	2.48±0.17	0.38±0.04	0.11±0.04
LG06	0.04±0.02	0.06±0.03	2.65±0.18	0.54±0.13	1.01±0.21	77.89±4.21	16.14±1.28	1.55±0.11	0.09±0.04	0.03±0.04
LG07	0.30±0.11	0.06±0.02	3.85±0.17	0.98±0.21	1.17±0.11	61.28±3.95	28.93±1.45	2.87±0.13	0.41±0.05	0.14±0.34
LG08	0.28±0.09	0.21±0.08	4.41±0.14	1.54±0.31	1.47±0.21	56.98±2.01	30.51±1.43	3.96±0.12	0.58±0.03	0.06±0.02

Error bars are standard deviations (SD) of three biological replicates.

Also of note was low ratio of PUFA/MUFA (especially C18:2/C18:1) or C20-24/C16-18 as key parameter for tribological property of plant oils for industrial application [27]. Here, the lowest ratios of PUFA/MUFA (0.225), C18:2/C18:1 (0.207) and C20-24/C16-18 (0.001) were noted for accession LG06, but the other accessions had a relative high ratios of PUFA/MUFA (0.442-0.739), C18:2/C18:1 (0.419-0.661) and C20-24/C16-18 (0.005-0.014) (Figure 1d-f), implying that the fruit oils from LG-06 may have ideal tribological properties.

These outcomes, together with the highest yield of oil and biodiesel for accession LG06, revealed that its fruit oil was the most suitable for biodiesel production.

Evaluation of biodiesel fuel properties of fruit oils from different accessions

Effective evaluation biodiesel fuel properties of iodine value (IV), cetane number (CN), oxidation stability (OS), cloud point (CP), cold filter plugging point (CFPP), density (D) and kinematic viscosity (KV), would provide a vital basis for exploiting biodiesel plants. Given a notable variation on oil content and total proportion of C18:1 and C18:2 in L. glauca fruits of different accessions (Table 1), it was important to assess fuel properties of biodiesel derived from fruit oils of different accessions for determining superior accession as biodiesel production.

CN, one key criteria for ignition delay time and combustion quality of biofuels, was detected to be varied from 48.81 to 51.15 in the FAMEs from fruit oils of all accessions, all of which could satisfy the standard of USA (ASTM D6751: CN>47), and most accessions (except LG01/03) was also in line with China standard (GB/T 20828: CN>49), while only LG06 (51.15) could meet Europe standard (EN 14214: 51<CN<65) (Table 2), implying that the biodiesels from fruit oils of all accessions had good ignition quality. Also, IV is a crucial index for assessing the unsaturated degree (DU) of FA and OS of biodiesel [28]. The IV values of all accessions varied from 102.15 to 115.09 (Table 2), all of which was less than the specified maximum limit (120) of ASTM D6751, EN 14214 and GB/T20828 standards. It is known that the values of both CN and IV are determined greatly by the DU [26]. The DU value presented here varied from 115.39 to 134.75 across all accessions, of which LG-06 had a minimum of 115.39, coincided with the highest CN value (51.15) and the lowest IV value (102.15) (Table 2).

KV is one key parameter for defining flow capability of biodiesel, used for estimating spray penetration and atomization of fuel [28]. The range of KV value (4.31-4.68 kg/m³) of biodiesels from all accessions could meet with the standards of ASTM D6751 (1.9<KV<6.0), EN 14214 (3.5<KV<5.0) and GB/T20828 (1.9<KV<6.0) (Table 2), revealing a good flow or spray capability of biodiesel fuel from L. glauca fruit oils of all accessions. Also, density (D) is one of vital fuel properties for assessing fuel transferred quantity by injection system for combustion [29]. The D value ranged from 871.54 to 887.14 mm/s of the biodiesels (Table 2), which satisfied with the standards of EN 14214 (860<D<900) and GB/T20828 (820<D<900), and thus concluded that the biodiesels from L. glauca fruit oils of all accessions possessed ideal combustion efficiency.

OS, as a crucial parameter, involves in the level and stability of biodiesel reaction with air [28]. In this study, the OS values of biodiesels from fruit oils of all accessions varied from 2.59 to 3.34 h (Table 2), all of which did not reach the minimum limit (6 h) specified in the standards of EN 14214 and GB/T 20828, but only accession LG06 (3.34 h) could meet the ASTM D6751 standard (OS>3.0 h), which was likely attributed to low content of PUFA (17.69%) in the fruit oils of LG06 compared with other accessions (29.01-38.73%) (Figure 1c).

CFPP and CP, two vital low-temperature parameters, are used to describe the maximum of filterability, but not limited by the standards of US and European. The CFPP valve ranged from -12.59°C to -9.74°C for biodiesels across all accessions (Table 2), less than the maximum limit (0°C) of Germany standard (DIN V51606) in summer, and this value for most accessions (except LG01/03) was lower than the minimum limit (-10.0°C) for spring and autumn, implying a good cold flow performance of biodiesel. Also, CP is one indicator for controlling fuel at low temperature [29], and its value ranged from -9.91°C to -7.71°C across all accessions (Table 2), which could meet ASTM D6751 standard (-12°C<CP<-3°C). This implied a better cold flow property of biodiesel from all accessions, especially LG06 with the minimum value of CFPP (-12.59°C) and CP (-9.91°C), which may be mostly attributed to high content of unsaturated FA (Figure 1c) and small amount of length chain saturated factor (LCSF) (Table 2).

Another was concerned about the content of C18:3 and the FAs with four double bonds in the FAMEs. Here, low amount (1.55-4.86 %) of C18:3 in fruit oils of all accessions and no four double-bond FAs (C18:4 and C20:4) in the FAMEs (Table 2) all satisfied the EN14214-2008 specification (< 12 % and 1 %, respectively).

Table 2 Evaluation of biodiesel fuel properties of fruit oils of L. glauca from different accessions

Accessions

Biodiesel fuel properties

LCSF

IV (g/100g)

CFPP (°C)

CP (°C)

OS (h)

KV (mm²s^-1, 40°C)

D (kg/m^-3, 15°C)

LG01

134.75±2.15

2.87±0.32

48.81±1.14

115.09±1.7

-9.74±0.14

-7.11±0.08

2.59±0.03

4.38±0.14

887.14±1.18

LG02

132.66±1.64

2.37±0.24

49.06±1.25

113.69±1.4

-10.61±0.15

-7.89±0.07

2.68±0.02

4.44±0.08

878.23±1.74

LG03

133.24±2.23

2.75±0.18

48.98±0.96

114.08±1.2

-9.94±0.13

-7.30±0.07

2.65±0.01

4.55±0.23

883.34±1.65

LG04

129.54±1.84

2.06±0.101

49.43±0.87

111.61±1.5

-11.16±0.13

-8.49±0.10

2.79±0.02

4.68±0.19

877.28±1.53

Lg05

126.15±1.65

1.70±0.12

49.84±0.68

109.34±1.2

-11.79±0.16

-9.12±0.09

2.93±0.02

4.36±0.07

876.35±1.09

LG06

115.39±1.24

1.24±0.09

51.15±1.01

102.15±0.9

-12.59±0.15

-9.91±0.09

3.34±0.03

4.31±0.06

871.54±0.94

LG07

128.87±1.91

1.79±0.12

49.52±0.93

111.16±1.1

-11.63±0.11

-8.96±0.09

2.82±0.01

4.48±0.11

876.09±1.09

LG08

131.48±1.72

2.17±0.14

49.20±0.79

112.90±1.3

-10.97±0.09

-8.31±0.08

2.71±0.02

4.58±0.12

885.13±1.91

DU, degree of unsaturation; LCSF, chain length saturated factor; CN, cetane number; IV, iodine value; CFPP, cold filter plugging point; CP, cloud point; OS, oxidation stability; KV, kinematic viscosity; D, density. Error bars are standard deviations (SD) of three biological replicates.

Construction of prediction model for biodiesel properties of raw fruit oils from different accessions

Determining biodiesel fuel property is very difficult because it takes a lot of time and cost and thus several attempts have been made to use effective methods to calculate or predict fuel property of biodiesel [26, 30, 31]. Recently, triangular predict model was constructed to effectively evaluate fuel properties of biodiesel based on FA compositions of raw oils from developing P. sibirica and L. glauca fruits [9, 14]. Such prediction as an attempt was performed here. To this end, the percentages of SFA, MUFA and PUFA in L. glauca fruit oils of all accessions (Figure 1c) were used as three angular points to establish a triangular graph (Figure 2), in which one specific region (marked in gray) was delineated to predict biodiesel fuel property for fruit oils from all accessions, taking into account the satisfactions of key fuel properties (CN, IN, CFPP, OS and CP). All accessions were presented in the gray area of our constructed triangular graph (Figure 2), of which accession LG06 was located at the far end of PMFA angular point (lower left vertex) and SFA angular point (lower right vertex), indicating that the fruit oils from all accessions (especially LG06) may be as potential material for biodiesel, coincided with our evaluated results of fuel properties for different accessions (Table 2). Thus, triangular predict model for fuel properties by FA compositions of raw oils could provide simple and effective selection of ideal plant resource biodiesel.

Together, the accession (LG05/06/07) with high fruit oil content and biodiesel yield, and ideal fuel property, could be suitable for high-quality biodiesel production. Yet, another question was the mechanism that governed such difference in oil content and FA composition of L. glauca fruits across all accessions (Figure 1a and Table 1). Effective increases in oil content and ideal FA profile require to unravel complex metabolic regulation network. Thus, in the following, our work focused on the identification of key regulators (enzymes, transcription factors and transporters) specific for carbon source supply and oil synthetic process (FA synthesis and TAG assembly) by the comparative analysis of cross-accessions association of fruit oil content with gene transcript level.

Transcript differences of carbon allocation specific for acetyl-CoA generation in fruits of different accessions

In oil plants, acetyl-CoA, one vital precursor for FA synthesis, is mainly derived from PYR via glycolysis or GAP via PPP in both cytosol and plastid by a series of regulatory enzymes [22, 32, 33]. Recently, our transcriptomic assay showed that oil accumulation of developing L. glauca fruits was regulated by differential transcripts of enzymes between plastidic and cytosolic glycolysis [14], including ATP-dependent phosphofructokinase (PFK), hexokinase (HXK), fructose-bisphosphate aldolase (FBA), phosphoglycerate kinase (PGK), triosephosphate isomerase (TPI), enolase (ENO), GAP dehydrogenase (GAPC) and pyruvate kinase (PK). To determine the relative flux of PYR from plastid or cytosol glycolysis destined to oil production in the fruits of different accessions, transcript differences of all enzymes of two glycolytic pathways were analyzed in the fruits of all accessions by qRT-PCR. The transcript levels of plastid glycolytic enzymes (HXK, PFK, FBA, TPI, GPI, GAPC, PGK, PGM, ENO1 and PK) increased with the increasing amount of fruit oils across all accessions, of which the richest transcripts were all noted for accession LG06 with the highest oil content (Figures 1a and 3a). Yet, all cytosolic isoforms showed less transcript across all accessions (Figure 3b). All these revealed a major role of plastid glycolysis in supply PYR for FA synthesis in the fruits of all accessions. Another note with regard to glycolysis was about transporter in interchange of glycolytic intermediate between cytosol and plastid [9, 34-36]. Given that the orthologs for triose phosphate transporter (TPT), G6P transporter (GPT1/2), phosphoenolpyruvate transporter (PPT1/2), xylulose 5-phosphate transporter (XPT), glycolipid transporter (GLT), and bile acid/sodium symporter (BASS2) was marked in developing L. glauca fruits by recent transcriptome analysis [14], it was crucial to explore which of them may contribute to transport glycolytic metabolite from cytosol into plastid for fruit FA synthesis of different accessions. Only GPT1, PPT1 and BASS2 showed high transcript and a high correlation to the amount of fruit oils across all accessions (Figures 1a and 3c), indicating that GPT1/PPT1/BASS2 may contribute to allocate cytosolic glycolytic metabolites (G6P, PEP or PYR) into plastid for FA synthesis destined to the eventual oil production in L. glauca fruits of all accessions.

Aside from glycolysis, FA synthesis was fed by GAP via PPP [9, 37, 38]. Our recent annotation of a complete plastidic PPP with differential transcripts in developing L. glauca fruits by transcriptome assay [14], allowed to address whether the fruit oil contents of different accessions were correlated to the increasing number of their transcripts. All enzymes of plastidic PPP, including 6-phosphogluconate dehydrogenase (6PGDH), transaldolase (TA), 6-phosphogluconolactonase (PGLS), G6P dehydrogenase (G6PDH), ribulose-5-phosphate (RP) epimerase (RPE), RP isomerase (RPI) and transketolase (TK), were detected with the abundantly coordinated transcripts of all accessions by qRT-PCR (Figure 3d), and their transcript levels were associated with the amount of fruit oils across all accessions (Figures 1a and 3d), pointing to a role of plastidic PPP in provision of GAP for FA synthesis.

Another was concerned about acetyl-CoA generation from PYR via acetyl-CoA synthetase (ACS), ATP-citrate lyase (ACL), or PYR dehydrogenase complex (PDC) [33, 39], all of which were marked with differential transcripts in developing L. glauca fruits by our recent transcriptome assay [14]. Hence, we performed the cross-accessions comparisons of transcripts to explore which of them may specifically devote to allocate PYR flux for acetyl-CoA formation destined for FA synthesis in all accession fruits. High transcript of plastidic PDC was closely consistent with the increase of fruit oil content across the accessions (Figures 1a and 4), while transcript level of cytosolic ACLB subunits (ACLB-1/-2) showed no notable up-regulation, and less transcript was detected for mitochondrial PDC, cytosolic ACLA and plastidic ACS (Figure 4), implying that plastid PDC may mostly contribute to acetyl-CoA formation for FA synthesis in all accession fruits. Also of note was the roles of cytosolic ACLB and mitochondrial PYR carrier (MPC), citrate synthase (CS) and dicarboxylate/tricarboxylate carrier (DTC) in cytosolic acetyl-CoA generation for FA elongation [9]. Here, low transcript of mitochondrial MPC, CS4 and DTC was similar to that of cytosolic ACLB and mitochondrial PDC (Figures 3c and 4), corresponding to small amount of C20:0 and C20:1 in fruit oils (Table 1), pointing to a role of them in providing cytosolic acetyl-CoA for FA elongation.

Transcript differences of enzymes and transporters for FA and TAG synthesis in fruits of different accessions

Increasing FA and TAG synthesis and storage oil yield would expand economic value for oil plants [9, 40]. Given differential transcripts of oil synthesis enzymes noted in developing L. glauca fruits by our recent transcriptome assay [14], it was necessary to explore quantitative relationship between the transcript levels of oil-synthesized genes and the amount of fruit oils from all accessions. By qRT-PCR, the abundantly coordinated transcripts were detected for FA synthetic enzymes, including acetyl-CoA carboxylase (ACC), malonyl-CoA-ACP transferase (MAT), fatty acyl-ACP thioesterase A/B (FATA/B), 3-ketoacyl ACP synthase I/II/III (KAS I/II/III), 3-ketoacyl ACP reductase (KAR), hydroxyacyl-ACP dehydrase (HAD), enoyl-ACP reductase (EAR) and 18:0-ACP desaturase 6 (SAD6) in the fruits of all accessions (Figure 5a), which was the case for the enzymes for de novo TAG assembly [acyl-CoA:G3P acyltransferase 9 (GPAT9), acyl-CoA:LPA acyltransferase 2 (LPAAT2), acyl-CoA:DAG acyltransferase 1 (DGAT1) and PA phosphatase 2 (PAP2)] (Figure 5b). A strong correlation of their transcript levels with fruit oil accumulation across all accessions (Figures 1a and 5) emphasized their importance for FA synthesis and TAG assembly. Also of note was the role of FA exporter (FAX) in FA import into ER for TAG synthesis and long chain acyl-CoA synthetase (LACS) in activating free FA to produce acyl-CoA pool [9, 41-43]. High transcript of FAX1/2 and LACS4 (Figures 3c and 5b) was comparable to that of FA-synthesized enzymes (Figures 5a), and showed a pattern that correlated with fruit oil content of all accessions (Figure 1a), and thus referred that both FAX1/2 and LACS4 may be pivotal for FA export from plastid into ER for acyl-CoA pool generation destined to TAG assembly.

ER-located FAD2 and 3 is known for desaturation of C18:1 to produce C18:2 and then C18:3, respectively. Here, FAD2 showed a relative high transcript, but less transcript was marked for FAD3 (Figure 5b), both of which were matched the level of C18:2 (high) and C18:3 (less) in fruit oils of all accessions (Table 1). High transcript similar to FAD2 was noted for acyl-CoA:lysophosphatidylcholine acyltransferase 2 (LPCAT2) (Figure 5b), involved in the transfer of C18:1 into PC for desaturation and release of PUFA into acyl-CoA pool for TAG assembly [23], all of which displayed a strong correlation to the amount of C18:2 in fruit oils across all accessions (Figure 1a), implying a specific contribution of FAD2 or LPCAT2 to PUFA production. Also, lower transcript was identified for PC:DAG cholinephosphotransferase(PDCT) and CDP-choline:DAG cholinephosphotransferase (CPT) (Figure 5b), two enzymes for interconversion of DAG and PC to produce PUFA-rich TAG [23, 44], indicating its unimportance for TAG synthesis. Another was concerned for DAG acyltransferase 1 (PDAT1) for the last assembly of TAG [45], and its low transcript showed no substantial difference across different accessions (Figure 5b), reflecting a very limited contribution of PDAT1 to TAG synthesis in the fruits of all accessions.

Transcript differences in transcription factors for regulating fruit oil accumulation of different accessions

Transcription factors (TFs) have shown to regulate oil biosynthesis of oil plants. Recently, differential transcripts were annotated for the TFs (ABI3, LEC1, WRI1, FUS3, AP2, GL2, HSI2, TT2, PKL and VAL2) in developing L. glauca fruits by our transcriptome analysis [14], which allowed us to explore the possible association of their transcript levels with accumulative amount of fruit oils across all accessions. Here, the transcripts of ABI3, LEC1 and WRI1 increased with the increase in fruit oil content of all accessions, while the transcripts of AP2 and GL2 showed a downtrend with the increase in fruit oil content, and less transcript was detected for TT2, FUS3, HSI2, VAL2 and PKL across all accessions (Figures 1a and 6), reflecting a complex transcriptional regulation for fruit oil synthesis of different accessions. Also, we performed the protein interaction analysis for the above TFs and oil-synthesis enzymes as an attempt to highlight TF-mediated regulation mechanism for fruit oil biosynthesis, and found that the enzymes for oil accumulation (carbon source supply, FA synthesis and TAG assembly) were highly associated with WRI1, and LEC1 showed a strong interaction with WRI1 (Figure 7), and thus considered that LEC1 and its targeted WRI1, located in the center position of interaction network, may contribute to regulate transcriptional expressions of enzymes relevant for fruit oil accumulation of all accessions.

L. glauca fruit oil as novel promising woody feedstock for biodiesel production

In this work, to determine ideal germplasm for developing L. glauca fruit oils as woody biodiesel, a concurrent evaluation of oil content, FA composition, biodiesel yield, and fuel properties and predicted model construction was performed for L. glauca fruits of 8 selected accessions (Tables 1 and 2; Figs. 1 and 2). Our results showed that the oil content (45.12–60.95%) of L. glauca fruits from different accessions (Fig. 1a) was higher than that of other traditional oil plants (11.68–40.28%) [9, 17–20, 33, 46–50], implying that L. glauca accessions, especially high oil-bearing LG05 (53.16%), LG06 (60.95%) and LG07 (51.81%), had great advantage as biodiesel feedstock. Indeed, high yield of biodiesel from the fruit oils of LG05 (97.3%), LG06 (98.7%) and LG07 (96.8%) (Figs. 1b) could meet EN 14214 standard (96.5%), as was higher than that of P. sibirica (88.7%) [6, 19], emphasizing that the L. glauca fruit oils of these accessions (LG-05/06/07) may be suitable for producing high yield of biodiesel.

The ideal oils for high-quality biodiesel production should contain suitable amount of SFA, high percentage of MUFA and low proportion of PUFA [9, 26, 27, 30]. Here, all accession fruits contained high content of MUFA (52.43–78.46%) and low contents of PUFA (17.69–38.73%) and SFA (3.85–8.84%) (Fig. 1c), and notably, the amount of C18:1 and C18:2 in all accessions accounted for more than 84% of fruit oils (Table 1), indicating that the fruit oils from all accession may be as raw material suitable for biodiesel, coincided with previous results of several traditional woody oil plants [8, 9, 51–54]. Given that accession LG06 had the highest content of MUFA, the lowest levels of PUFA and SFA and a small ratio of C18:2/C18:1 (Fig. 1d-f), integrated with ideal value of IN (102.157), CN (51.15), OS (3.34 h), CP (-9.91°C) and CFPP (-12.59°C) in biodiesel (Table 2), revealing that the fruit oils from LG06 with idea FA compositions could meet the demand of high-quantity biodiesel production.

Together, we identified 3 accessions (LG05, LG06 and LG07) that may be of interest for potential industrial applications, specifically LG06 with the maximum yield of oil and biodiesel, and superior fuel properties.

Strong funneling of carbon flux toward plastid acetyl-CoA for high FA production in fruits of all accessions

Several studies have shown that oil accumulation and FA composition vary considerably among different plants, provenances, tissues or developing stages [8, 9, 14, 21–23, 32, 33, 46, 55–57], but the mechanism for controlling these differences still remains enigmatic. Thus, it is vital to unravel regulators for high-quality oil accumulation.

In oil plants, the source of PYR for high oil production is largely controlled by glycolysis in cytosol or plastid [14, 22, 32–34, 56–59]. Here, transcript levels of glycolytic enzymes were much higher in plastid than in cytosol of all accessions (Fig. 3a, b), and notably, transcript abundance of plastid glycolytic enzymes was positively correlated with the increase of fruit oil content across all accessions (Figs. 1a and 3a), revealing a critical role of plastid glycolysis in providing PYR for FA synthesis, as also noted for several oil plants [9, 22, 32, 38, 56, 59]. In support of this finding, plastidic GPT1 (G6P transporter), PPT1 (PEP transporter) and BASS2 (PYR carrier) were transcriptionally coordinated with the increase of oil content in all accession fruits (Figs. 1a and 3c), implying a role of transporter-mediated import of G6P, PEP or PYR from cytosol into plastid glycolysis for FA synthesis in L. glauca fruits of all accessions, as could be evidenced by the fact that PPT1 or BASS2 overexpression promoted Arabidopsis seed oil increase [60, 61]. Yet, PPT1 transcript was on average 1.1- and 1.3-fold higher than that of GPT1 and BASS2 respectively (Fig. 3c), implying a major glycolytic carbon flux into plastid from cytosol at the level of PEP in L. glauca fruits, similar to other oil plants [22, 32, 37, 62, 63]. All these also clearly highlighted a strong coordinated funneling of glycolytic intermediate toward PYR in plastid, as could be further reflected by less transcript for mitochondrial MPC (PYR carrier) across all accessions (Figs. 3c). Aside from glycolysis, PPP as alternative pathway has shown to supply GAP for FA synthesis in oil plants [37, 38, 64]. Such pathway could be shown by our finding that the abundantly coordinated transcripts of plastid PPP and GPT1 displayed a close relation to the amount of fruit oils of all accessions (Figs. 1a and 3c, d), implying a portion of GPT1-mediated G6P import into plastid destined for FA synthesis. Yet, the fact that transcript level of plastid glycolytic enzymes was on average 2-fold higher than that of plastid PPP in all accessions (Fig. 3a, d) revealed that the necessity of carbon source for de novo FA synthesis in L. glauca fruits was mainly derived from plastidic glycolysis.

Acetyl-CoA, one vital precursor for de novo FA synthesis in plastid and its elongation in cytosol, was mostly derived from PYR via four alternative enzymes of PDC, ACL, ACS or CA [32, 39, 65, 66], all of which were shown with differential transcript in developing L. glauca fruits by our recent transcriptomic analysis [14]. Here, only plastidic PDC and cytosolic ACLB subunits (ACLB-1/-2) were highly expressed in all accessions (Fig. 4), while transcript level of plastidic PDC was on average 4-fold higher than that of cytosolic ACLB across all accessions, and showed a positive association with the increased fruit oil of all accessions (Fig. 1a), emphasizing that the availability of acetyl-CoA for FA synthesis in L. glauca fruits of all accessions was derived from glycolytic PYR via the action of PDC in plastid, as also noted for other oilseed plants [33, 38, 56, 66]. Also of note was the roles of cytosolic ACL and mitochondrial PDC in producing cytosolic acetyl-CoA for FA elongation, in which cytosolic PYR was imported into mitochondria by MPC (PYR transporter) for acetyl-CoA formation via mitochondrial PDC, as substrate for citrate formation by CS and then export by DTC transporter for cytosolic acetyl-CoA formation via ACL cleavage [9, 14, 32, 38, 67]. The fact that cytosolic ACLB-1/-2, and mitochondrial MPC, PDC and CS4 all showed low transcript (Figs. 3c and 4), coincided with small amount of C20:0 and C20:1 in fruit oils (Table 1), indicated that the production of cytosolic acetyl-CoA for FA elongation was coordinated at the transcript level.

Together, strongly increased plastid carbon supply via effective transport and glycolysis, and high action of plastid PDC, may provide great funneling of acetyl-CoA for high FA synthesis in L. glauca fruits of all accessions.

FA flux channeled into de novo TAG assembly for high-quality oil accumulation in fruits of all accessions

The PUFA (C18:2 and C18:3) content is a key factor affecting the quality of plant oils for human health, biofuel and other purposes [44]. In oil plants, C18:1, which was produced from C18:0 by SAD6 in plastid, is desaturated to C18:2 and then C18:3 by ER-localized FAD2 and FAD3, respectively. Here, transcript level of SAD6 in the fruits across all accessions was on average 1.3- and 3.5-fold higher than that of FAD2 and FAD3, respectively, which closely matched the amount of C18:1, C18:2 and C18:3 in fruit oils of all accessions (Table 1 and Fig. 5b), and thus considered that C18:1 as the richest component in fruit oils may be mostly attributed to their differential coordinate transcripts. This was in line with previous results in different tissues of oil palm [68] and developing seeds of P. sibirica [69], and also be supported by high amount of C18:1 in fad2/3 double mutant soybean [70].

The carbon chain length of FA and relative proportion of SFA and MUFA in the oils may be determined by de novo FA synthesis from acetyl-CoA in plastid [21]. Given a positive correlation between abundant transcript of FA-synthesized enzymes (ACC, MAT, KAR, HAD, KAS I/II/ III, EAR, FATA, FATB and SAD6) and accumulative amount of fruit oils across all accessions (Table 1 and Fig. 5a), it seems certain that the differences in fruit oil contents of different accessions may highly depend on the efficiency of FA synthesis in plastid, as also noted for oil palm [22]. Another difference across all accessions was transcript of ER-localized LACS4 crucial for activation of free FA to generate acyl-CoA pool for TAG assembly [21], and notably, its high transcript pattern (Fig. 5b) was closely related to fruit oil accumulative amount across all accessions (Table 1), indicating its importance in controlling plastid FA flux into acyl-CoA pool for TAG synthesis, as was evidenced by the increase of oil content in yeast mutant by over-expression of B. napus LACS4 [43]. Considering that FAX1/2 as plastid transporter for export FA into ER responsible for TAG synthesis [9, 41, 42] was highly expressed in all accession fruits (Fig. 3c), it was believed that the abundantly coordinated transcripts of FA-synthesized enzymes, LACS4 and FAX1/2 may provide a strong channeling of plastid FA flux into ER to generate acyl-CoA pool destined for TAG synthesis.

Plant oil synthesis involves a complex metabolic network for TAG assembly with distinct FA profiles, and de novo assembly of TAG from G3P and acyl-CoA can produce oils with rich C18:1 [23–25]. Yet, how acyl flux is channeled to produce idea oil composition in oil plants remains largely unclear. In this work, the accumulative amount of fruit oils with rich C18:1 was positively associated with high and increasing number of transcripts of crucial enzymes (GPAT9, LPAAT2 and PAP2) for de novo DAG synthesis via Kennedy pathway across all accessions (Table 1 and Figs. 5a), reflecting that direct utilization of de novo DAG for TAG synthesis may be as dominant route for L. glauca fruit oils with rich C18:1 of all accessions, as also noted in other oil plants [23, 71]. In general, C18:1 from plastid can be used directly for de novo DAG assembly by GPAT and LPAAT via Kennedy pathway, or incorporated into PC by LPCAT for desaturation by FAD2/3 [44]. Given that transcript level of LPCAT2 was on average 1.5-fold lower than that both GPAT9 and LPAAT2 for the first two acylation of TAG assembly in fruits of all accessions (Fig. 5), integrated with the fact of 18:1 as the richest component in fruit oils (Table 1), it seems certain that a major flux of 18:1 from acyl-CoA pool may be directly channeled into de novo DAG synthesis, and thus a small flux of 18:1 was used for desaturation. This conclusion could support earlier idea that LPCAT2 may transfer PC-derived PUFA directly into acyl-CoA pool for TAG synthesis via Kennedy pathway [72], and also was evidenced by the fact that lpcat1/lpcat2 mutant reduced PUFA content in Arabidopsis seed oils [24], but gpat9 mutant increased PUFA content and decreased seed oil content [73]. It was also noted that PUFA-rich TAG may be mostly attributed to PDCT or CPT [23, 44]. The fact that transcript levels of PDCT and CPT were on average 6.5-fold below that of both LPAAT and GPAT (Fig. 5), and showed no relation to variations for PUFA amount in fruit oils of all accessions (Fig. 5 and Table 1), indicated a negligible role of PDCT or CPT in PUFA production in fruit oils of all accessions, also noted in developing P. sibirica seeds [10]. This was in line with previous notion of low PUFA production in oil palm caused by less transcript of PDCT and CPT [68], but contrasted with a major contribution of PC-derived DAG to TAG synthesis in developing seeds of Arabidopsis and soybean [24, 44, 74].

Also of note was DGAT and PDAT vial for the final acylation of TAG synthesis [45], but it remains unknown what extent both enzymes devote to the last TAG assembly in oil plants. Here, PDAT1, similar to both PDCT and CPT, showed less transcript and no difference across all accessions (Fig. 5b), it appeared that PDAT1 was not a key contributor for TAG synthesis in L. glauca fruits of all accessions. This corresponded to earlier results in oil plants from transcriptomic or metabolic analysis [22, 68, 75], and also confirmed by no effect of PDAT1 mutant on seed oil content and FA profile [45, 76]. In support of this finding, transcript level of DGAT1 was on average 5-fold higher than that of PDAT1 across all accessions (Fig. 5b), and positively correlated with the amount of fruit oils (Table 1), revealing that DGAT1 may be a crucial regulator for oil synthesis in L. glauca fruits, coincided with metabolic and transcriptomic analyses of some oil plants [22, 33, 45, 56, 77], and also evidenced by the overexpression or mutant result of DGAT1 in several oil plants [76, 78–91].

Overall, FAX1/2-mediated plastidic FA export, LACS4-regulated effective generation of acyl-CoA pool in ER, and LPCAT2-mediated C18:1 flux into PC for desaturation and PUFA back into acyl-CoA pool, integrated with the abundantly coordinated transcripts for the enzymes of FA synthesis and de novo TAG assembly via Kennedy pathway, was crucial for the eventual high-quality oil accumulation in L. glauca fruits of all accessions (Fig. 8).

LEC1/WRI1-mediated regulatory network for oil accumulation in L. glauca fruits of all accessions

Plant FA and TAG biosynthesis, involving a series of gene expression, is highly regulated by several TFs [92–108]. Our findings that high transcript patterns of ABI3, LEC1 and WRI1 were positively correlated with the amount of fruit oils across all accessions, but the down-regulated profiles of GL2 and AP2 showed an inverse relationship with oil content (Figs. 1a and 6), indicated that ABI3, WRI1 and LEC1 may act as positive regulators for gene expression relevant for fruit oil synthesis of all accessions, but AP2 and GL2 may play negative regulatory factors, as was noted for developing P. sibirica seeds [10], and also compatible with our recent transcriptome result of developing L. glauca fruits [14]. Of note, WRI1, has been shown to control the expressions of its targeted genes involved in glycolysis, and FA and TAG biosynthesis during oilseed development [95, 99, 108–110]. This could be directly shown by the fact that the enzymes for carbon allocation (plastid glycolysis and acetyl-CoA formation), de novo FA synthesis and TAG assembly were transcriptionally coordinated with WRI1 across all accession fruits (Figs. 5 and 6), which in turn was closely related to the fruit oil content (Fig. 1a). Such strong relationship implied that these genes may be as the targets were activated by WRI1, in which carbon flux could be efficiently channeled into oil synthetic pathway for oil accumulation in the fruits of all accessions by collaborative manner, similar to the results of other oilseed plants [22, 56, 68, 94, 95, 110–113], and also evidenced by the increase in FA synthesis and oil accumulation by ectopic expression of WRI1 from many oilseed plants [92–97, 100, 101, 103, 105–107, 110–117]. WRI1 is known to be up-regulated by LEC1/2, ABI3 or FUS3 [93, 111, 118]. A similar profile of high transcript for ABI3, LEC1 and WRI1 in all accessions (Fig. 5) revealed WRI1 as a target gene of LEC1 or ABI3, as was also reported in A. thaliana and P. sibirica seeds [33, 93], but contrasted with the idea that WRI1 function might be independent of upstream TFs in controlling oil synthesis in oil palm [22]. LEC1 has been identified as a central regulator of oil synthesis in developing seeds [109, 119–121], and overexpression of LEC1 increased oil content by up-regulating several genes relevant for FA and oil synthesis in oilseed plants [96, 102, 109, 121, 122]. Overall, the abundantly coordinated transcripts were identified for the TFs (LEC1 and WRI1) and the enzymes of carbon flux allocation (plastid glycolysis and acetyl-CoA generation) and FA synthesis as well as TAG assembly (Figs. 4–6), showing a positive correlation with the accumulative amount of fruits oils across all accessions (Table 1), and thus concluded that LEC1/WRI1-mediated transcription regulatory network may play a major role in FA synthesis and oil accumulation in L. glauca fruits of different accessions (Fig. 7).

In this work, to select superior accession and unravel high oil-accumulative mechanism for developing L. glauca fruit oils as woody biodiesel, the comparative analyses of 8 plus accessions (LG01/02/03/04/05/06/07/08) were conducted on a concurrent exploration of oil content, FA profile, biodiesel yield, fuel property and its prediction model construction, revealing a difference of them across different accessions, of which three high oil-bearing accessions (LG05/06/07) would be valuable as promising feedstock for superior biodiesel production, especially LG06 with a minimum of oil and biodiesel yield, and superior fuel property. In an attempt to highlight molecular regulatory mechanism that govern such differences in oil content and FA composition of L. glauca fruits across different accessions, an integrated analysis for the associations between oil-synthesized gene transcription level and oil accumulative amount was conducted on different accessions to explore accessions-specific differences in central carbon allocation and to identify features specific for high oil accumulation. Importantly, some crucial regulators (enzymes, transcription factors and transporters) were identified to be involved specifically in carbon source supply (plastidic glycolysis and PPP, and acetyl-CoA generation) and oil biosynthetic process (FA synthesis and TAG assembly) destined for high-quality oil accumulation of L. glauca fruits of all accessions. Together, our findings and carbon metabolic regulation model (Fig. 8) could facilitate the development of L. glauca fruit oils as biodiesel feedstock, and may provide new insight into regulatory mechanism of high oil production destined for further metabolic engineering of oil accumulation in L. glauca and other oil plants.

Collection of fruit samples

A total of 8 plus L. glauca trees with high fruit yield (germplasm accessions LG01, LG02, LG03, LG04, LG05, LG06, LG07 and LG08) planting in JiGong Mountain National Natural Reserve (E114°06′, N32°125′) of Henan Province in China were selected, and the ripen fresh fruits were collected. One hundred and fifty fruits from 10 trees (15 fruits each tree) of each accession were selected each time. All samples were stored at -80 °C until use.

Oil extraction and trans-esterification

About 50 g of fresh fruit (3 samples per accession) were crushed into power, and then the oils were extracted with petroleum ether using Soxhlet apparatus at 45-50°C [14]. 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 the ventilated drying oven. The content of extracted oil from each accession fruit was calculated as the difference between the weights of fruit sample before and after extraction, and expressed as the percentage of the extracted oil weight to fresh fruit weight (%, g/g). To analyze FA compositions, the fruit oils from each accession were trans-esterified as previously described [14, 123]. All analysis was conducted on triplicate.

Analyses of FAMEs and biodiesel yield

The FA methyl esters (FAMEs) obtained from each accession fruit was used to determine FA compositions using Agilent 6890 (California, USA) gas chromatograph equipped with flame ionization detector (GC-FID) [14]. The HP-INNOWax capillary column (inner diameter 0.32mm, filmthickness 0.5μm, 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 assay was performed in triplicate, and the data were present as the mean. The biodiesel yield was calculated by the previous method [9], 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).

Quality of biodiesel fuel propery

The fuel properties (IV, CN, CP, CFPP, OS, D and KV) were calculated from the FAME compositions of fruit oils by our previous methods [9, 14, 20, 26]. All assay was performed for triplicate, and the biodiesel fuel properties of fruit oils were compared with the relevant standards of EN 14214 (European), ASTM D6751 (USA), DIN V51606 (Germany) and GB/T 20828 (China).

Construction of prediction model for biodiesel properties of raw fruit oils

Triangular prediction model of raw fruit oils from different accessions was constructed based on the influence of FA compositions [9, 14]. To predict biodiesel properties of fruit oils from each accession, the percentages of SAF, MUFA and PUFA from each accession fruit were calculated to outline triangular prediction graph, in which three angular points of the triangle meant the 100 % of SAF, MUFA and PUFA, 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 [9, 14].

qRT-PCR analysis

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. 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). All the qRT-PCR primers (Table S1) were designed by our recent transcriptome results of L. glauca fruits [14] using PrimerQuest (http://www.idtdna.com/PrimerQuest/Home/Index). The genes encoding large subunit ribosomal protein L32e and ubiquitin-conjugating enzyme (UBC) were used as inner references [14, 123]. The qRT-PCR was conducted on 7500 Real-Time PCR System by using SYBR Premix Ex Taq Kit (TaKaRa). The 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. Three biological replicates with three technical repetitions each were performed for qRT-PCR.

Protein interaction network analysis

The Search Tool for the Retrieval of Interacting Genes/Proteins (STRING version 9.0, http://string90.embl.de/) was employed to analyze the potential interactions between all our identified TFs and oil-synthesized enzymes or specific transporters. STRING analysis was conducted using high confidence (score 0.7), and cluster analysis was performed by using k-means with a value of k = 3.

ABI3, ABSCISIC ACID INSENSITIVE3; ACC, acetyl-CoA carboxylase; ACLA/B, ATP-citrate lyase subunit A/B; BASS, pyruvate (PYR) carrier (bile acid: sodium symporter); CFPP, cold filter plugging point; CN, cetane number; CP, cloud point; CPT, CDP-choline:DAG cholinephosphotransferase; DGAT, diacylglycerol (DAG) acyltransferase; DTC, dicarboxylate/tricarboxylate carrier; EAR, enoyl-ACP reductase; ENO, enolase; ER, endoplasmic reticulum; FAD, fatty acid (FA) desaturase; FAMEs, FA methyl esters; FATA, fatty acyl-ACP thioesterase A; FATB, fatty acyl-ACP thioesterase B; FAX, FA exporter; FBA, fructose-bisphosphate aldolase; FK, fructokinase; FUS3, FUSCA3; G6PDH, glucose-6-phosphate (G6P) dehydrogenase; GAPA, glyceraldehyde 3-phosphate (GAP) dehydrogenase subunit A; GLT, glycolipid transporter; GPAT, acyl-CoA:G3P acyltransferase; GPI, G6P isomerase; GPT, G6P transporter; HAD, hydroxyacyl-ACP dehydrase; HXK, hexokinase; INV, invertase; KAR, ketoacyl-ACP reductase; KAS, 3-ketoacyl ACP synthase; LACS, long-chain acyl-CoA synthase; LEC1, LEAFY COTYLEDON1; LPAAT, lysoPA acyltransferase; LPCAT, lysoPC acyltransferases; MAT, malonyl-CoA-ACP transferase; MPC, PYR carrier; 6PGDH, 6-phosphogluconate dehydrogenase; PAP, phosphatidic acid (PA) phosphohydrolases; PDAT, phospholipid:DAG acyltransferase; PDC, pyruvate (PYR) dehydrogenase complex; PDCT, PC:DAG cholinephosphotransferase; PFK, phosphofructokinase; PFP, pyrophosphate phosphofructokinase; PGK, phosphoglycerate kinase; PGLS, 6-phosphogluconolactonase; PGM, phosphoglycerate mutase; PK, PYR kinase; PPP, pentose phosphate pathway; PPT, PEP transporter; qRT-PCR, quantitative real time RT-PCR; RPE, ribulose-5-phosphate (Ru5P) epimerase; RPI, ribose 5-phosphate isomerase; TA, transaldolase; TAG, triacylglycerol; TK, transketolase; TPI, triose phosphate isomerase; TPT, triose phosphate transporters; WRI1, WRINKLED1; UGPG, UDP-glucose.

Ethical Approval and Consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was financially supported by The National Natural Science Foundation of China (Grant No. 31972952 and 31570653).

Authors’ contributions

ZXL, FC, SZL and YX organized and planned the research, and drafted the manuscript. LLS and ZXL carried out qRT-PCR detection and data analysis. HJW and JHH determined the fruit oils and biodiesel fuel property. FC and YX collected fruit materials of different germplasms. SZL provided the funding, computational guidance, and was substantially involved in data analysis. All authors have read and approved the manuscript.

Author details

¹Beijing Advanced Innovation Center for Tree Breeding by Molecular Design, College of Biological Sciences and Biotechnology, School of Soil and Water Conservation, National Engineering Laboratory for Tree Breeding, Beijing Forestry University, Beijing 100083, China.²Department of Biochemistry and Molecular Biology, Yanjing Medical College, Capital Medical University, Beijing 101300, China.

Acknowledgments

We thank the National Natural Sciences Foundation of China (Grant No. 31972952 and 31570653) for financial support. We also like to thank the reviewers for positive criticism to improve the quality of the manuscript.

Bora AP, Gupta DP, Durbha KS. Sewage sludge to bio-fuel: A review on the sustainable approach of transforming sewage waste to alternative fuel. Fuel. 2020, 259:116262.
Musa SD, Zhonghua T, Ibrahim AO, Habib M. China's energy status: A critical look at fossils and renewable options. Renewable and Sustainable Energy Reviews. 2018, 81:2281-90.
Knothe G, Razon LF. Biodiesel fuels. Progress in Energy and Combustion Science. 2017, 58:36-59.
Atabani AE, Silitonga AS, Badruddin IA, Mahlia TMI, Masjuki HH, Mekhilef S. A comprehensive review on biodiesel as an alternative energy resource and its characteristics. Renewable and Sustainable Energy Reviews. 2012, 16(4):2070-93.
Yu HY, Zhou S. Preparation of biodiesel from Xanthoceras sorblfolia Bunge seed oil. China Oils and Fats. 2009, 3:43-5.
Wang LB. Properties of Manchurian apricot (Prunus mandshurica Skv.) and Siberian apricot (Prunus sibirica L.) seed kernel oils and evaluation as biodiesel feedstocks. Ind Crop Prod. 2013, 50:838-43.
Li ZY, Xia F, Zhang LY, Fan CY, Wei GT. Research advances in biodiesel production on from Jatropha oil by chemical methods. China Science Paper. 2013, 8(3):221-4.
Ma Y, Wang S, Liu X, Yu H, Yu D, Li G, et al. Oil content, fatty acid composition and biodiesel properties among natural provenances of Siberian apricot (Prunus sibirica L.) from China. GCB Bioenergy. 2021, 13(1):112-32.
Wang J, Lin WJ, Yin ZD, Wang LB, Dong SB, An JY, et al. Comprehensive evaluation of fuel properties and complex regulation of intracellular transporters for high oil production in developing seeds of Prunus sibirica for woody biodiesel. Biotechnol Biofuels. 2019, 12(1):6.
Niu J, Hou XY, Fang CL, An JY, Ha DL, Qiu L, et al. Transcriptome analysis of distinct Lindera glauca tissues revealed the differences in the unigenes related to terpenoid biosynthesis. Gene. 2015, 599:22-30.
Qi J, Xiong B, Ju YX, Hao QZ, Zhang ZX. Study on fruit growth regularity and lipid accumulation of Lindera glauca. Chinese Agr Sci Bull. 2015, 31(4):29-33.
Xiong B, Qi J, Dong SB, Zhang LM, Zhang ZX, Ha DL, et al. Superior individual selection of Lindera glauca based on fruit and seed traits. Journal of Northeast Forestry University. 2016, 44(5):10-4.
Zhu B, Hou X, Niu J, Li P, Fang C, Qiu L, et al. Volatile constituents from the fruits of Lindera glauca (Sieb. et Zucc.) with different maturities. J Essent Oil-Bear Plants. 2016, 19(4):926-35.
Lin ZX, An JY, Wang J, Niu J, Ma C, Wang LB, et al. Integrated analysis of 454 and Illumina transcriptomic sequencing characterizes carbon flux and energy source for fatty acid synthesis in developing Lindera glauca fruits for woody biodiesel. Biotechnol Biofuels. 2017, 10(1):134.
Chen F, Miao X, Lin Z, Xiu Y, Shi L, Zhang Q, et al. Disruption of metabolic function and redox homeostasis as antibacterial mechanism of Lindera glauca fruit essential oil against Shigella flexneri. Food Control. 2021, 130:108282.
Wang R, Hanna MA, Zhou WW, Bhadury PS, Chen Q, Song BA, et al. Production and selected fuel properties of biodiesel from promising non-edible oils: Euphorbia lathyris L., Sapium sebiferum L. and Jatropha curcas L. Bioresour Technol. 2011, 102(2):1194-9.
Wang LB. Evaluation of Siberian Apricot (Prunus sibirica L.) germplasm variability for biodiesel properties. J Am Oil Chem Soc. 2012, 89(9):1743-7.
Chen YH, Chen JH, Chang CY, Chang CC. Biodiesel production from tung (Vernicia montana) oil and its blending properties in different fatty acid compositions. Bioresour Technol. 2010, 101(24):9521-6.
Wang L, Yu H. Biodiesel from Siberian apricot (Prunus sibirica L.) seed kernel oil. Bioresour Technol. 2012, 112:355-8.
Guo JY, Li HY, Fan SQ, Liang TY, Yu HY, Li JR, et al. Genetic variability of biodiesel properties in some Prunus L.(Rosaceae) species collected from Inner Mongolia, China. Ind Crop Prod. 2015, 76:244-8.
Bates PD, Stymne S, Ohlrogge J. Biochemical pathways in seed oil synthesis. Curr Opin Plant Biol. 2013, 16(3):358-64.
Bourgis F, Kilaru A, Cao X, Ngando-Ebongue G-F, Drira N, Ohlrogge JB, et al. Comparative transcriptome and metabolite analysis of oil palm and date palm mesocarp that differ dramatically in carbon partitioning. Proc Natl Acad Sci USA. 2011, 108(30):12527-32.
Bates PD, Browse J. The significance of different diacylgycerol synthesis pathways on plant oil composition and bioengineering. Frontiers in plant science. 2012, 3:147-.
Bates PD, Fatihi A, Snapp AR, Carlsson AS, Browse J, Lu C. Acyl editing and headgroup exchange are the major mechanisms that direct polyunsaturated fatty acid flux into triacylglycerols. Plant Physiol. 2012, 160(3):1530-9.
Bates PD. Understanding the control of acyl flux through the lipid metabolic network of plant oil biosynthesis. Biochimica et Biophysica Acta. 2016, 1861(9, Part B):1214-25.
Wang LB, Yu HY, He XH, Liu RY. Influence of fatty acid composition of woody biodiesel plants on the fuel properties. J Fuel Chem Technol. 2012, 40(4):397-404.
Yaşar F. Comparision of fuel properties of biodiesel fuels produced from different oils to determine the most suitable feedstock type. Fuel. 2020, 264:116817.
Singh D, Sharma D, Soni SL, Sharma S, Kumari D. Chemical compositions, properties, and standards for different generation biodiesels: A review. Fuel. 2019, 253:60-71.
Sakthivel R, Ramesh K, Purnachandran R, Mohamed Shameer P. A review on the properties, performance and emission aspects of the third generation biodiesels. Renewable and Sustainable Energy Reviews. 2018, 82:2970-92.
Ramos MJ, Fernández CM, Casas A, Rodríguez L, Pérez Á. Influence of fatty acid composition of raw materials on biodiesel properties. Bioresour Technol. 2009, 100(1):261-8.
Lin C-Y, Lin H-A, Hung L-B. Fuel structure and properties of biodiesel produced by the peroxidation process. Fuel. 2006, 85(12-13):1743-9.
Schwender J, Hebbelmann I, Heinzel N, Hildebrandt T, Rogers A, Naik D, et al. Quantitative multilevel analysis of central metabolism in developing oilseeds of oilseed rape during in vitro culture. Plant Physiol. 2015, 168(3):828-48.
Niu J, An JY, Wang LB, Fang CL, Ha DL, Fu CY, et al. Transcriptomic analysis revealed the mechanism of oil dynamic accumulation during developing Siberian apricot (Prunus sibirica L.) seed kernels for the development of woody biodiesel. Biotechnol Biofuels. 2015, 8(1):29.
Weber AP, Schwacke R, Flügge U-I. Solute transporters of the plastid envelope membrane. Annu Rev Plant Biol. 2005, 56:133-64.
Knappe S, Löttgert T, Schneider A, Voll L, Flügge UI, Fischer K. Characterization of two functional phosphoenolpyruvate/phosphate translocator (PPT) genes in Arabidopsis-AtPPT1 may be involved in the provision of signals for correct mesophyll development. Plant J. 2003, 36(3):411-20.
Eicks M, Maurino V, Knappe S, Flügge U-I, Fischer K. The plastidic pentose phosphate translocator represents a link between the cytosolic and the plastidic pentose phosphate pathways in plants. Plant Physiol. 2002, 128(2):512-22.
Andriotis VM, Kruger NJ, Pike MJ, Smith AM. Plastidial glycolysis in developing Arabidopsis embryos. New Phytol. 2010, 185(3):649-62.
Schwender J, Shachar-Hill Y, Ohlrogge JB. Mitochondrial metabolism in developing embryos of Brassica napus. J Biol Chem. 2006, 281(45):34040-7.
Fatland BL, Ke J, Anderson MD, Mentzen WI, Cui LW, Allred CC, et al. Molecular characterization of a heteromeric ATP-citrate lyase that generates cytosolic acetyl-coenzyme A in Arabidopsis. Plant Physiol. 2002, 130(2):740-56.
Graham IA. Seed storage oil mobilization. Annu Rev Plant Biol. 2008, 59:115-42.
Li N, Gügel IL, Giavalisco P, Zeisler V, Schreiber L, Soll J, et al. FAX1, a novel membrane protein mediating plastid fatty acid export. PLoS Biol. 2015, 13(2):e1002053.
Tian Y, Lv X, Xie G, Zhang J, Xu Y, Chen F. Seed-specific overexpression of AtFAX1 increases seed oil content in Arabidopsis. Biochem Biophys Res Commun. 2018, 500(2):370-5.
Tan X-l, Zheng X-f, Zhang Z-y, Wang Z, Xia H-c, Lu C, et al. Long chain acyl-coenzyme A synthetase 4 (BnLACS4) gene from Brassica napus enhances the yeast lipid contents. Journal of Integrative Agriculture. 2014, 13(1):54-62.
Lu CF, Xin ZG, Ren ZH, Miquel M. An enzyme regulating triacylglycerol composition is encoded by the ROD1 gene of Arabidopsis. Proc Natl Acad Sci USA. 2009, 106(44):18837-42.
Zhang M, Fan J, Taylor DC, Ohlrogge JB. DGAT1 and PDAT1 acyltransferases have overlapping functions in Arabidopsis triacylglycerol biosynthesis and are essential for normal pollen and seed development. Plant Cell. 2009, 21(12):3885-901.
Seiler GJ, Gulya T, Kong G, Thompson S, Mitchell J. Oil concentration and fatty-acid profile of naturalized Helianthus annuus populations from Australia. Genet Resour Crop Evol. 2018, 65(8):2215-29.
Kaushik N, Bhardwaj D. Screening of Jatropha curcas germplasm for oil content and fatty acid composition. Biomass Bioenerg. 2013, 58:210-8.
Hoseini SS, Najafi G, Ghobadian B, Mamat R, Ebadi MT, Yusaf T. Ailanthus altissima (tree of heaven) seed oil: Characterisation and optimisation of ultrasonication-assisted biodiesel production. Fuel. 2018, 220:621-30.
Amalfitano C, Golubkina NA, Del Vacchio L, Russo G, Cannoniero M, Somma S, et al. Yield, antioxidant components, oil content, and composition of onion seeds are influenced by planting time and density. Plants. 2019, 8(8).
Liu P, Zhang LN, Wang XS, Gao JY, Yi JP, Deng RX. Characterization of Paeonia ostii seed and oil sourced from different cultivation areas in China. Ind Crop Prod. 2019, 133:63-71.
Mohan MR, Jala RCR, Kaki SS, Prasad R, Rao B. Swietenia mahagoni seed oil: A new source for biodiesel production. Ind Crop Prod. 2016, 90:28-31.
Lovato L, Pelegrini BL, Rodrigues J, de Oliveira AJB, Ferreira ICP. Seed oil of Sapindus saponaria L.(Sapindaceae) as potential C16 to C22 fatty acids resource. Biomass Bioenerg. 2014, 60:247-51.
Yu HY, Fan SQ, Bi QX, Wang SX, Hu XY, Chen MY, et al. Seed morphology, oil content and fatty acid composition variability assessment in yellow horn (Xanthoceras sorbifolium Bunge) germplasm for optimum biodiesel production. Ind Crop Prod. 2017, 97:425-30.
Rodríguez-Rodríguez MF, Sánchez-García A, Salas JJ, Garcés R, Martínez-Force E. Characterization of the morphological changes and fatty acid profile of developing Camelina sativa seeds. Ind Crop Prod. 2013, 50:673-9.
Ma Y, Bi Q, Li G, Liu X, Fu G, Zhao Y, et al. Provenance variations in kernel oil content, fatty acid profile and biodiesel properties of Xanthoceras sorbifolium Bunge in northern China. Ind Crop Prod. 2020, 151:112487.
Troncoso-Ponce MA, Kilaru A, Cao X, Durrett TP, Fan J, Jensen JK, et al. Comparative deep transcriptional profiling of four developing oilseeds. Plant J. 2011, 68(6):1014-27.
Lu S, Sturtevant D, Aziz M, Jin C, Li Q, Chapman KD, et al. Spatial analysis of lipid metabolites and expressed genes reveals tissue-specific heterogeneity of lipid metabolism in high- and low-oil Brassica napus L. seeds. Plant J. 2018, 94(6):915-32.
Tao X, Fang Y, Xiao Y, Jin YL, Ma XR, Zhao Y, et al. Comparative transcriptome analysis to investigate the high starch accumulation of duckweed (Landoltia punctata) under nutrient starvation. Biotechnol Biofuels. 2013, 6(1):72.
Schwender J, Ohlrogge JB, Shachar-Hill Y. A flux model of glycolysis and the oxidative pentosephosphate pathway in developing Brassica napus embryos. J Biol Chem. 2003, 278(32):29442-53.
Fuchs J, Neuberger T, Rolletschek H, Schiebold S, Nguyen TH, Borisjuk N, et al. A non-invasive platform for imaging and quantifying oil storage in sub-millimetre tobacco seed. Plant Physiol. 2013, 161(2):583-93.
Lee E-J, Oh M, Hwang J-U, Li-Beisson Y, Nishida I, Lee Y. Seed-specific overexpression of the pyruvate transporter BASS2 increases oil content in Arabidopsis seeds. Frontiers in plant science. 2017, 8:194.
White JA, Todd J, Newman T, Focks N, Girke T, de Ilárduya OMn, et al. A new set of Arabidopsis expressed sequence tags from developing seeds. The metabolic pathway from carbohydrates to seed oil. Plant Physiol. 2000, 124(4):1582-94.
Ruuska SA, Girke T, Benning C, Ohlrogge JB. Contrapuntal networks of gene expression during Arabidopsis seed filling. Plant Cell. 2002, 14(6):1191-206.
Schwender J, Goffman F, Ohlrogge JB, Shachar-Hill Y. Rubisco without the Calvin cycle improves the carbon efficiency of developing green seeds. Nature. 2004, 432(7018):779-82.
Rawsthorne S. Carbon flux and fatty acid synthesis in plants. Prog Lipid Res. 2002, 41(2):182-96.
Ke J, Behal RH, Back SL, Nikolau BJ, Wurtele ES, Oliver DJ. The role of pyruvate dehydrogenase and acetyl-coenzyme A synthetase in fatty acid synthesis in developing Arabidopsis seeds. Plant Physiol. 2000, 123(2):497-508.
Hutchings D, Rawsthorne S, Emes MJ. Fatty acid synthesis and the oxidative pentose phosphate pathway in developing embryos of oilseed rape (Brassica napus L.). J Exp Bot. 2005, 56(412):577-85.
Dussert S, Guerin C, Andersson M, Joët T, Tranbarger TJ, Pizot M, et al. Comparative transcriptome analysis of three oil palm fruit and seed tissues that differ in oil content and fatty acid composition. Plant Physiol. 2013, 162(3):1337-58.
Mai Y, Huo K, Yu H, Zhou N, Shui L, Liu Y, et al. Using lipidomics to reveal details of lipid accumulation in developing Siberian apricot (Prunus sibirica L.) seed kernels. GCB Bioenergy. 2020, 12(7):539-52.
Pham A-T, Shannon JG, Bilyeu KD. Combinations of mutant FAD2 and FAD3 genes to produce high oleic acid and low linolenic acid soybean oil. Theor Appl Genet. 2012, 125(3):503-15.
Dyer JM, Stymne S, Green AG, Carlsson AS. High-value oils from plants. Plant J. 2008, 54(4):640-55.
Lager I, Yilmaz JL, Zhou XR, Jasieniecka K, Kazachkov M, Wang P, et al. Plant acyl-CoA:lysophosphatidylcholine acyltransferases (LPCATs) have different specificities in their forward and reverse reactions. J Biol Chem. 2013, 288(52):36902-14.
Shockey J, Regmi A, Cotton K, Adhikari N, Browse J, Bates PD. Identification of Arabidopsis GPAT9 (At5g60620) as an essential gene involved in triacylglycerol biosynthesis. Plant Physiol. 2016, 170(1):163-79.
Bates PD, Browse J. The pathway of triacylglycerol synthesis through phosphatidylcholine in Arabidopsis produces a bottleneck for the accumulation of unusual fatty acids in transgenic seeds. Plant J. 2011, 68(3):387-99.
Abdullah HM, Akbari P, Paulose B, Schnell D, Qi W, Park Y, et al. Transcriptome profiling of Camelina sativa to identify genes involved in triacylglycerol biosynthesis and accumulation in the developing seeds. Biotechnol Biofuels. 2016, 9(1):136.
Mhaske V, Beldjilali K, Ohlrogge J, Pollard M. Isolation and characterization of an Arabidopsis thaliana knockout line for phospholipid: diacylglycerol transacylase gene (At5g13640). Plant Physiol Biochem. 2005, 43(4):413-7.
Tang M, Guschina IA, O'Hara P, Slabas AR, Quant PA, Fawcett T, et al. Metabolic control analysis of developing oilseed rape (Brassica napus cv Westar) embryos shows that lipid assembly exerts significant control over oil accumulation. New Phytol. 2012, 196(2):414-26.
Ståhl U, Carlsson AS, Lenman M, Dahlqvist A, Huang B, Banas W, et al. Cloning and functional characterization of a phospholipid: diacylglycerol acyltransferase from Arabidopsis. Plant Physiol. 2004, 135(3):1324-35.
Xu J, Francis T, Mietkiewska E, Giblin EM, Barton DL, Zhang Y, et al. Cloning and characterization of an acyl-CoA-dependent diacylglycerol acyltransferase 1 (DGAT1) gene from Tropaeolum majus, and a study of the functional motifs of the DGAT protein using site-directed mutagenesis to modify enzyme activity and oil content. Plant Biotechnol J. 2008, 6(8):799-818.
Zhang TT, He H, Xu CJ, Fu Q, Tao YB, Xu R, et al. Overexpression of type 1 and 2 diacylglycerol acyltransferase genes (JcDGAT1 and JcDGAT2) enhances oil production in the woody perennial biofuel plant Jatropha curcas. Plants (Basel). 2021, 10(4).
Misra A, Khan K, Niranjan A, Nath P, Sane VA. Over-expression of JcDGAT1 from Jatropha curcas increases seed oil levels and alters oil quality in transgenic Arabidopsis thaliana. Phytochemistry. 2013, 96:37-45.
Xu R, Yang T, Wang R, Liu A. Characterisation of DGAT1 and DGAT2 from Jatropha curcas and their functions in storage lipid biosynthesis. Funct Plant Biol. 2014, 41(3):321-9.
Maravi DK, Kumar S, Sharma PK, Kobayashi Y, Goud VV, Sakurai N, et al. Ectopic expression of AtDGAT1, encoding diacylglycerol O-acyltransferase exclusively committed to TAG biosynthesis, enhances oil accumulation in seeds and leaves of Jatropha. Biotechnol Biofuels. 2016, 9:226.
Zhang FY, Yang MF, Xu YN. Silencing of DGAT1 in tobacco causes a reduction in seed oil content. Plant Sci. 2005, 169(4):689-94.
Zheng P, Allen WB, Roesler K, Williams ME, Zhang S, Li J, et al. A phenylalanine in DGAT is a key determinant of oil content and composition in maize. Nat Genet. 2008, 40(3):367-72.
Wang Z, Huang W, Chang J, Sebastian A, Li Y, Li H, et al. Overexpression of SiDGAT1, a gene encoding acyl-CoA:diacylglycerol acyltransferase from Sesamum indicum L. increases oil content in transgenic Arabidopsis and soybean. Plant Cell Tiss Org Cult. 2014, 119(2):399-410.
Jako C, Kumar A, Wei Y, Zou J, Barton DL, Giblin EM, et al. Seed-specific over-expression of an Arabidopsis cDNA encoding a diacylglycerol acyltransferase enhances seed oil content and seed weight. Plant Physiol. 2001, 126(2):861-74.
Zou J, Wei Y, Jako C, Kumar A, Selvaraj G, Taylor DC. The Arabidopsis thaliana TAG1 mutant has a mutation in a diacylglycerol acyltransferase gene. Plant J. 1999, 19(6):645-53.
Torabi S, Sukumaran A, Dhaubhadel S, Johnson SE, LaFayette P, Parrott WA, et al. Effects of type I Diacylglycerol O-acyltransferase (DGAT1) genes on soybean (Glycine max L.) seed composition. Scientific Reports. 2021, 11(1):2556.
Regmi A, Shockey J, Kotapati HK, Bates PD. Oil-producing metabolons containing DGAT1 use separate substrate pools from those containing DGAT2 or PDAT. Plant Physiol. 2020, 184(2):720-37.
Zhao J, Bi R, Li S, Zhou D, Bai Y, Jing G, et al. Genome-wide analysis and functional characterization of Acyl-CoA:diacylglycerol acyltransferase from soybean identify GmDGAT1A and 1B roles in oil synthesis in Arabidopsis seeds. J Plant Physiol. 2019, 242:153019.
Yeap Wc, Lee Fc, Shan S, Kumar D, Musa H, Appleton DR, et al. WRI1-1, ABI5, NF-YA3 and NF-YC2 increase oil biosynthesis in coordination with hormonal signaling during fruit development in oil palm. Plant J. 2017, 91(1):97-113.
Baud S, Mendoza MS, To A, Harscoët E, Lepiniec L, Dubreucq B. WRINKLED1 specifies the regulatory action of LEAFY COTYLEDON2 towards fatty acid metabolism during seed maturation in Arabidopsis. Plant J. 2007, 50(5):825-38.
Baud S, Wuillème S, To A, Rochat C, Lepiniec L. Role of WRINKLED1 in the transcriptional regulation of glycolytic and fatty acid biosynthetic genes in Arabidopsis. Plant J. 2009, 60(6):933-47.
Maeo K, Tokuda T, Ayame A, Mitsui N, Kawai T, Tsukagoshi H, et al. An AP2‐type transcription factor, WRINKLED1, of Arabidopsis thaliana binds to the AW‐box sequence conserved among proximal upstream regions of genes involved in fatty acid synthesis. Plant J. 2009, 60(3):476-87.
Shen B, Allen WB, Zheng P, Li C, Glassman K, Ranch J, et al. Expression of ZmLEC1 and ZmWRI1 increases seed oil production in maize. Plant Physiol. 2010, 153(3):980-7.
Pouvreau B, Baud S, Vernoud V, Morin V, Py C, Gendrot G, et al. Duplicate maize Wrinkled1 transcription factors activate target genes involved in seed oil biosynthesis. Plant Physiol. 2011, 156(2):674.
Tan HL, Yang XH, Zhang FX, Zheng X, Qu CM, Mu JY, et al. Enhanced seed oil production in canola by conditional expression of Brassica napus LEAFY COTYLEDON1 and LEC1-LIKE in developing seeds. Plant Physiol. 2011, 156(3):1577-88.
To A, Joubès J, Barthole G, Lécureuil A, Scagnelli A, Jasinski S, et al. WRINKLED transcription factors orchestrate tissue-specific regulation of fatty acid biosynthesis in Arabidopsis. Plant Cell. 2012, 24(12):5007.
Adhikari ND, Bates PD, Browse J. WRINKLED1 rescues feedback inhibition of fatty acid synthesis in hydroxylase-expressing seeds. Plant Physiol. 2016, 171(1):179-91.
Bhattacharya S, Das N, Maiti MK. Cumulative effect of heterologous AtWRI1 gene expression and endogenous BjAGPase gene silencing increases seed lipid content in Indian mustard Brassica juncea. Plant Physiol Biochem. 2016, 107:204-13.
Elahi N, Duncan RW, Stasolla C. Modification of oil and glucosinolate content in canola seeds with altered expression of Brassica napus LEAFY COTYLEDON1. Plant Physiol Biochem. 2016, 100:52-63.
Ivarson E, Leiva-Eriksson N, Ahlman A, Kanagarajan S, Bülow L, Zhu L-H. Effects of overexpression of WRI1 and hemoglobin genes on the seed oil content of Lepidium campestre. Frontiers in plant science. 2017, 7:2032.
Manan S, Ahmad MZ, Zhang G, Chen B, Haq BU, Yang J, et al. Soybean LEC2 regulates subsets of genes involved in controlling the biosynthesis and catabolism of seed storage substances and seed development. Frontiers in plant science. 2017, 8:1604.
Ye J, Wang C, Sun Y, Qu J, Mao H, Chua N-H. Overexpression of a transcription factor increases lipid content in a woody perennial Jatropha curcas. Frontiers in plant science. 2018, 9.
Vogel PA, Bayon de Noyer S, Park H, Nguyen H, Hou L, Changa T, et al. Expression of the Arabidopsis WRINKLED 1 transcription factor leads to higher accumulation of palmitate in soybean seed. Plant Biotechnol J. 2019, 10.
Cernac A, Benning C. WRINKLED1 encodes an AP2/EREB domain protein involved in the control of storage compound biosynthesis in Arabidopsis. Plant J. 2004, 40(4):575-85.
Jo L, Pelletier JM, Harada JJ. Central role of the LEAFY COTYLEDON1 transcription factor in seed development. Journal of Integrative Plant Biology. 2019, 61(5):564-80.
Pelletier JM, Kwong RW, Park S, Le BH, Baden R, Cagliari A, et al. LEC1 sequentially regulates the transcription of genes involved in diverse developmental processes during seed development. Proc Natl Acad Sci USA. 2017, 114(32):E6710-E9.
Chen B, Zhang G, Li P, Yang J, Guo L, Benning C, et al. Multiple GmWRI1s are redundantly involved in seed filling and nodulation by regulating plastidic glycolysis, lipid biosynthesis and hormone signalling in soybean (Glycine max). Plant Biotechnol J. 2020, 18(1):155-71.
Baud S, Lepiniec L. Physiological and developmental regulation of seed oil production. Prog Lipid Res. 2010, 49(3):235-49.
Deng S, Mai Y, Shui L, Niu J. WRINKLED1 transcription factor orchestrates the regulation of carbon partitioning for C18:1 (oleic acid) accumulation in Siberian apricot kernel. Scientific Reports. 2019, 9(1):2693.
Hofvander P, Ischebeck T, Turesson H, Kushwaha SK, Feussner I, Carlsson AS, et al. Potato tuber expression of Arabidopsis WRINKLED1 increase triacylglycerol and membrane lipids while affecting central carbohydrate metabolism. Plant Biotechnol J. 2016, 14(9):1883-98.
Sun R, Ye R, Gao L, Zhang L, Wang R, Mao T, et al. Characterization and ectopic expression of CoWRI1, an AP2/EREBP domain-containing transcription factor from coconut (Cocos nucifera L.) endosperm, changes the seeds oil content in transgenic Arabidopsis thaliana and rice (Oryza sativa L.). Frontiers in plant science. 2017, 8:63.
Chen L, Zheng Y, Dong Z, Meng F, Sun X, Fan X, et al. Soybean (Glycine max) WRINKLED1 transcription factor, GmWRI1a, positively regulates seed oil accumulation. Mol Genet Genomics. 2018, 293(2):401-15.
Liu J, Hua W, Zhan GM, Wei F, Wang XF, Liu GH, et al. Increasing seed mass and oil content in transgenic Arabidopsis by the overexpression of wri1-like gene from Brassica napus. Plant Physiol Biochem. 2010, 48(1):9-15.
Wu XL, Liu ZH, Hu ZH, Huang RZ. BnWRI1 coordinates fatty acid biosynthesis and photosynthesis pathways during oil accumulation in rapeseed. J Integr Plant Biol. 2014, 56(6):582-93.
Wang HY, Guo JH, Lambert KN, Lin Y. Developmental control of Arabidopsis seed oil biosynthesis. Planta. 2007, 226(3):773-83.
Lotan T, Ohto M-a, Yee KM, West MAL, Lo R, Kwong RW, et al. Arabidopsis LEAFY COTYLEDON1 is sufficient to induce embryo development in vegetative cells. Cell. 1998, 93(7):1195-205.
Jo L, Pelletier JM, Hsu S-W, Baden R, Goldberg RB, Harada JJ. Combinatorial interactions of the LEC1 transcription factor specify diverse developmental programs during soybean seed development. Proc Natl Acad Sci USA. 2020, 117(2):1223.
Mu J, Tan H, Zheng Q, Fu F, Liang Y, Zhang J, et al. LEAFY COTYLEDON1 is a key regulator of fatty acid biosynthesis in Arabidopsis. Plant Physiol. 2008, 148(2):1042.
Tang G, Xu P, Ma W, Wang F, Liu Z, Wan S, et al. Seed-specific expression of AtLEC1 increased oil content and altered fatty acid composition in seeds of peanut (Arachis hypogaea L.). Frontiers in Plant Science. 2018, 9:260.
Niu J, Chen Y, An J, Hou X, Cai J, Wang J, et al. Integrated transcriptome sequencing and dynamic analysis reveal carbon source partitioning between terpenoid and oil accumulation in developing Lindera glauca fruits. Scientific reports. 2015, 5:15017.

No competing interests reported.

TableS1.xls
Additional file 1: Table S1. The information of all primers used in this study for qRT-PCR analysis.

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Evaluation of oil accumulation and biodiesel property of Lindera glauca fruits among different germplasms and revelation of high oil producing mechanism for developing biodiesel

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