Raw oil contents in developing tiger nut tuber
To investigate the lipid accumulation during different plant tuber developing stages, we analyzed the oil content of different development stages of C.esculentus. Tiger nut tubers started to sprout after approximately 3-6 days upon seed sowing under regular growth conditions. The growth period of tubers took around 120 days, while their appearances turned from white to brown; the white appearance was observed during the initial 35 day after sowing (35 DAS) and early (50 DAS) stages, light brown on the middle stage (70 DAS), moderate brown on the late stage (90 DAS), and finally turned to dark brown in mature stage (120 DAS). The tuber inside is sustainable oyster white when the coat is removed (Figure 1A). During tuber development, the fresh weight and diameters of each tuber increased significantly following averages of 0.2g and 0.2mm per day, respectively (Figure 1B, 1C). We also determined the oil content during tuber development; it showed that raw oil content in tubers increased continuously from 4.6% to 22.2% throughout all developmental stages (Figure 1D). There was a rapid oil accumulation rate for the early and middle stages, which increased approximately on average 0.26% d-1 from 35 to 50 DAS and 0.33% d-1 from 50 to 70 DAS, respectively. That fascinating increase was followed by the late and mature stage, which increased approximately on average 0.25% d-1 from 70-90 DAS and 0.11% d-1 from 90-120 DAS, respectively.
The reserves of lipid droplets represent the main storage organelles for natural lipids in plants[27]. To evaluate the variation of lipid accumulation during the developmental process inside the tuber, we stained tubers of different stages with Nile Red to observe the structure and amount change of lipid droplets (LDs) (Figure 1E). The LDs showed irregular spherical structure and a constantly increasing amount with tuber development. The accumulation of neutral lipids in tubers was visually evident; this may indicate that the increase in the expression level of the TAG leads to an increase in the crude oil content; this classify TAG as a mojor contributor to the enhancement of oil accumulation in oil plants.
Lipid species analysis and variation of lipid content
To determine the changes in the overall lipid composition and distribution in C. esculentus tubers, we isolated metabolite from tubers and explored them by LC-MS analysis. Over 430 different lipid species in tubers, consisting of 133 triacylglycerols (TG) species (31.1%), 23 diacylglycerols (DG) species (5.4%), 21 phosphatidylglycerols (PGs) species (4.9%), and other lipid molecular species (Figure 2A). Neutral lipids (glycolipids and AcHex) composition was continuously increasing during all five different stages (Figure 2B). As the main neutral lipids component found in tubers, TG content continuously increased until the fully mature stage (Figure 2C). It implied that the increased proportion of neutral lipids is mainly justified by the continuous accumulation of TAG. Subsequently, the relative composition of polar lipids in the development of tiger nut tuber showed that expect neutral lipids, glycolipids, phospholipids, and sphingolipids main components of polar lipids (Figure 3A). During the phospholipid profiling analysis, the relative composition of PC was continuously decreased along the five different stages of tubers, the profiling of PE displayed a minor decrease at the five stages and only periodical increase at the late stage (Figure 3B). The decrease of PC, periodic decreased of PE and relative content increased of TAG suggests that during the development of tubers, PC is probably convert into TAG and PE is probably convert into TAG obviously on the early and middle stage. Other polar lipids profile changes were lack in regularity, only the Hexcer increased at all developing stages (Figure 3C, 3D), that the metabolism correlation with glycolipid is weak.
Molecular species dynamic changes during lipid accumulation
Rapid lipid accumulation was observed in both the early and middle stages of tuber development. Then, we analyzed the changes of molecular species during lipid accumulation stages in tubers. The most prominent variety of TAG at all time points was 16:0/16:1/18:1, followed by TAG species containing a single saturated fatty acid (16:0/16:1/18:1, 20:0/18:1/18:2, 26:0/18:1/18:1) and higher unsaturated species (16:1/18:2/18:2, 18:1/18:1/24:1, 18:1/18:2/24:1) (Figure 4A). The detected TAG molecular species are all shown in figure S1. The proportions of molecular species almost all showed an increasing trend compared with the initial stage (35DAS) for TAG. This trend of lipids increase explained the overall lipid composition and oil content of C. esculentus tubers. During the oil accumulation period a general shift towards more unsaturated TAG molecular species, leading to an increase of unsaturated fatty acid content in the lipids.
Major molecular species of DAG are displayed in figure 4B. The most abundant species in DAG at all stages of oil accumulation were 16:0/18:2 and 18:1/18:1, both of these two species increase in abundance during the developmental stage; the proportion ranged from 10% to 60% of the total DAG accumulation. Other significant molecular species of DAG were those carraying palmitoyl, oleoyl, or linolenyl (16:0/18:2, 18:3/18:2); the proportion of molecular species of 16:0/18:2 strongly decreased during lipid accumulation stages; finally, molecular species of 18:3/18:2 only increased in the last development stage. The complete analysis of all detected species in figure S2.
As immediate precursors of DAG in the Kennedy pathway, the main molecular species of PA are well displayed in Figure 5A. The major species of PA were 16:0/18:1, 18:0/18:2, and 18:3/18:2. Between 35 and 120 DAS, the abundance of 18:0/18:2 species significantly increased, while 18:3/18:2 undergo a constant decrease. The 16:0/18:1 species kept the increase in the previous four-time points and slightly decreased in the last time points. Other significant PA species were 16:0/16:0, 16:0/16:1, 16:0/18:3 and 24:0/18:2. In general, proportions of accumulation of the major PA species constantly increased during the whole process.
In oil crops, almost all fatty acyl in TAG flux through PC [28], so PC played a very important role during oil accumulation,. The main molecular species of PC during oil accumulation in C. esculentus tubers are shown in figure 5B, while a complete analysis of all detected species is deplicted in the figure S3. The main molecular species at 35 DAS were 16:0/18:3 and 18:2/18:2 but both showed a significantly decreased trend in all development stages. Molecular species 16:0/16:0 and 18:1/18:2 showed an increasing trend during developmental process. Some other molecules also presented a discontinuous trend of s proportion, molecular species 16:0/18:1 and 18:1/18:1, which increased between 35 and 70 DAS and slightly decreased in proportion between 90 and 120 DAS.
The DAG from the Kennedy pathway serves as a substrate for the synthesis of PC as well as PE, the two phospholipid have a strong correlation in the metabolism[29]. Therefore, we examined PE molecular species to compare with those of PC. The main molecular species of PE are shown in figure 5C with a complete breakdown of all species detected in figure S4. The pattern of molecular species is similar to that of PC, which is 16:0/18:3 and 18:2/18:2, showed significantly decreased tendency in proportion accordindly to the plant development. In figure 5C, the major species of PE showed the most significant were 16:1/18:1 and 26:0/18:2, which increased in proportion. In general, other molecular species maintained relatively steady proportions of PE during oil accumulation. This results demonstrate that the PE is synthesized through the same pathway as the PC, but have differential distribution in molecular species proportions.
Transcriptome analysis of C.esculentus during tuber development
Studies on the molecular mechanism of lipid biosynthesis during the development of tiger nuts are limited and the genetic resource of the tiger nut plant is insufficient. In this study, the expression analysis of relevant metabolic genes correlated with lipid metabolic (PAP2、PLD、FATA、SAD、DGAT etc.) was carried out by Illumina RNA-seq approach. More than 744 million clean reads and 150,153 transcripts were generated from five libraries. A total number of 8, 2,008, 3,967, and 7,345 different expressed genes corresponding to 50 VS 35, 70 VS 35, 90 Vs 35 and 120 VS 35 DAS were computed out, respectively. When comparing gene expressions across the five stages of tiger tubes development, it appeared that the proportion of the expressed genes during the developing stage simultaneously increase along with the plant growth.
Additionally, physiological analyses revealed different genes expression pattern of each developmental stage, which were mapped to the pathways in the biological pathways database of Kyoto Encyclopedia of Genes and Genomes (KEGG). These differentially expressed genes participated in various lipid metabolic related processes including linoleic acid metabolism, glycerophospholipid metabolism, etc. (Figure 6A). All the expressed genes involved in carbohydrate metabolic pathways such as the starch and sucrose metabolic and glycolysis were abundant during the late-developing stages. This suggesting that G6P and Acyl-CoA served as a precursor for the synthesis of lipid is of great abundant for supply in late developing stage.
The GO enrichment analysis of differentially expressed genes in tiger nut identified several biological processes, which probably represented specific or common conserved functions of those expressed genes throughout the plant development (Figure 6B). Various functions, such as RNA processing, DNA binding, and protein binding were significantly enriched in the genes with higher expression pattern, particularly at 90 and 120 DAS developing stage. Likewise, GO terms, response to oxidative stress, oxidoreductase activity and peroxidase activity were also significantly enriched for down-regulated genes at 90 and 120 DAS developing stage when compared with 35 DAS. In contrast, these terms were also significantly enriched for two gene sets exhibiting up-regulated expression pattern at 50 VS 35 DAS stages. Gene sets enriched on the metabolism process of macromolecular compounds like lipid metabolic process, lipid metabolic process, glycosphingolipid metabolic process, sphingolipid metabolic process, exhibited down-regulated expression pattern at 70, 90, and 120 when compared with 35 DAS stage. on the contrary, polysaccharide biosynthetic process indicated higher activity of lipids biosynthetic at initial developing stage. Interestingly, for 120 VS 35 DAS stage, we observed more significant enrichment of GO term in fatty acid metabolism, including the fatty acid derivatived from the metabolic process and unsaturated fatty acid metabolism, here these genes exhibited down-regulated patterns of tuber development. In addition, genes enriched on transferase activity and phospholipase C activity seemed to be less active at 120 DAS when compared with 35 DAS.
Majors genes are responsible for the molecular species composition of TAG
Plant oil quality usually is determined by oil content and fatty acid composition. The unsaturated fatty acyl group composition of TAG is an important factor in evaluating the oil quality. 12 candidate genes involved in fatty acid metabolism, elongation in the plastid, lipid desaturation, and synthesis mechanisms in the endoplasmic reticulum (ER) were characterized through the lipid metabolic pathway of maize[30](Figure 7). Among these candidate genes, most transcripts of GPAT1, DGAT, PLC, LPAAT, FATA, and SAD showed an abundant transcription profile in the early stage and showed dynamic decreased expression patterns during late and mature stages. Most transcripts of ACSL, PAP2, PLD and KAS have low abundance expression patterns during the initial early and middle stage and up-regulated expression patterns during the late and mature stage. Two of the PDAT transcripts were mainly expressed at a high level in 70 DAS; the other two had higher expression in the 90 DAS.
The comparative lipidomic analysis also revealed that the unsaturated linolenic acyl group (C18:3) carried by PC, and PE had a more significant decreased level in the mature stage compared with the initial stage, from 6.76% to 1.45% for PC and from 3.85% to 2.93% for PE, respectively. The composition rate of the oleic acyl group (C18:1) decreased by approximately 10.87% in PC and 11.47% in PE during the mature stage when compared with the initial stage (Figure S5). Higher transcriptional level of PDAT, PLC, and DGAT activity could therefore result from the reduced levels of monounsaturated and polyunsaturated phosphatidylcholine or phosphatidylethanolamines, which are substrates for the Flavin adenine dinucleotide FAD2 desaturase that responsible for the production of polyunsaturated fatty acyl groups in phospholipid. Furthermore, FAD2 had low transcriptional abundance during developing stages, except for the middle stage. The different expression patterns of FAD2 in ER during developmental stages could explain a portion of the downward trend of oleoyl composition for PC and PE, respectively. In addition, the flow of polyunsaturated acyl in PC or PE would partially influence the dynamic variation composition of molecular species in TAG.
Particle size analysis of lipid drop and expression profile of candidate oleosin genes
As intracellular storage organelles for neutral lipids, lipid droplet (LP) packed with natural lipids originated from the endoplasmic reticulum and mainly exist in a wide range of density distinct cytoplasm, together with starch and storage proteins [31,32]. We extracted lipid droplets and measured the particle size of the lipid droplet at different developing stages (figure 8A). Diameter sizes were range from 2,500 nm to 3,500 nm; diameters were undergoing of significant increase in70 DAS (Figure 8B). Lipid droplet associated proteins (LDAPs), which are uniformly expressed in all cells, facilitated lipid droplet biogenesis and prevented lipid droplets from aggregation; this activity is associated with the dynamic regulation of lipid droplet size, number, and distribution in response to cellular metabolism and lipid composition. As plant small molecular LDAPs, oleosin derived from green algae and universally in advanced plants[33]. Oleosins were helping to stabilize and protect neutral lipids by protecting against degradation. 15 putative oleosin-like (OLE) and two putative caleosin (CaLE) transcripts were characterized according to our transcriptome result of developing tubers. To determine the expression pattern of LDAP in developing tubers of tiger nut, the transcriptional levels of LDAPs were analyzed. Candidate oleosin transcripts were expressed comprehensively during tuber development. In particular, OLE9, OLE10, and OLE11, which are expressed predominantly in developing tubers, the three transcripts were exhibiting similar expression patterns and showed comparable decreased levels in 70 DAS (Figure 8C). However, the expression pattern of these three candidate oleosin transcripts was opposite correlated from LD size. This observation may indicate that these three candidate oleosin transcripts were probably involved in LD formation related processes, relevantly responsible for the raised particle size of LD 70 DAS stage.