Tuber Oil Contents Differed Markedly Between Yellow and Purple Nutsedge
Under our experimental conditions, new shoots from seed tubers of the two nutsedges appeared above soil at 5–9 days after sowing on April 16, 2016. New tubers began to present from 6 to 8 weeks after shoot emergence. Both species lasted around five months for their growth and development.
Analyses of the proximate constituents of mature tubers (Fig. 1A) indicated that there are marked differences between two species in the contents of major storage reserves including starch, oil, sugar, and protein on a tuber dry weight basis (Fig. 1B), where starch was the component at highest level in the tubers. The most notable difference is that yellow nutsedge stored more than 25% oil of dry weight while purple nutsedge contained less than 3% oil in mature tuber, indicating that there is around 10-fold difference in oil content.
Analysis of fatty acid composition of oil from mature tubers showed that yellow nutsedge was predominated with oleic acid (C18:1) that accounted for more than 60% of total fatty acids, while purple nutsedge was presented with palmitic acid (16:0), C18:1, and linoleic acid (18:2) as major fatty acids, with concentrations ranging from 25–35% (Fig. 1C). In yellow nutsedge tuber, C16:0 and C18:2 were the second most, but each of them constituted less than 20% of total fatty acids. All these results indicated that significant difference also occurred in fatty acid composition of tuber oil between these two species, where purple nutsedge contained less oleic acid and more saturated fatty acids than yellow nutsedge.
To check whether there is also a difference of oil accumulation occurred in developing tubers, the changes in oil contents during tuber development were determined for two species. The oil accumulation patterns in the development period spanning around 110 days after tuber formation (DAF) was shown in Fig. 1D. The results showed that the oil accumulation in the two types of tubers continued to increase throughout the tuber development. In all developmental stages, however, yellow nutsedge tubers contained a significantly higher percentage of oil than the purple nutsedge tubers.
On a per dry tuber basis, both nutsedges had relatively slow oil accumulation at early stage (0–20 DAF) and late stage (50–110 DAF), much lower than that of middle stage (20–50 DAF) where oil was rapidly produced, suggesting that oil accumulation was directly related to the tuber development. The rate of oil accumulation of yellow nutsedge tubers in each of three development stages was significantly greater than that of purple nutsedge tubers, in which the accumulation rates with 0.11% oil⋅d− 1, 0.56% oil⋅d− 1, and 0.12% oil⋅d− 1 at earl-, mid- and later-stage, respectively, for yellow nutsedge is in sharp contrast to the corresponding values with 0.02% oil⋅d− 1, 0.05% oil⋅d− 1, and 0.01% oil⋅d− 1 for purple nutsedge.
Overall, the striking differences in oil content and the fatty acid composition were present between these two types of tubers, suggesting that there exists distinct transcriptional control of oil production for them.
Overall Level of Transcripts for Oil Production Is Higher in Yellow Nutsedge Than in Purple Nutsedge
To uncover the difference in the transcriptional control of oil production between the two species, we systematically conducted comparative transcriptome analyses of oil-related genes in developing tubers. Tuber samples at three different developmental stages (i.e. the early stage 20 DAF, the middle stage 50 DAF and the late stage 90 DAF) were used for comparative transcript analysis.
Oil production involves the conversion of sucrose up to TAG assembly or storage, primarily including cytosolic and plastidial carbon metabolism toward pyruvate generation in plastid, fatty acid (FA) synthesis in plastid, TAG synthesis in ER and TAG storage in oil body or lipid droplet. These four major metabolic processes are related to expression of more than 400 genes (Additional file 1: Table S2 and S3). In this study, transcript levels were represented by FPKM (fragments per kilobase of exon model per million mapped reads) per protein, where multiple transcripts for genes encoding for isoforms or subunits of the same protein family were summed. Among these metabolic pathways, more than 70% of the transcripts was associated with genes involved in TAG storage and only 8% and 3% with those in FA synthesis and TAG synthesis, respectively, in yellow nutsedge tuber (Fig. 2A). In contrast, the transcripts related to carbon metabolism were most abundant while those of TAG storage were the least in purple nutsedge tuber.
For every metabolic pathway, transcript levels were on average higher in yellow nutsedge than in purple nutsedge (Fig. 2B). The largest difference was noted for TAG storage, for which the transcript level was more than 25-fold higher in yellow nutsedge compared to purple nutsedge. Clear difference was also present for FA synthesis, where there is 2.5 times higher in yellow nutsedge than in purple nutsedge. Unexpectedly, there is no substantial difference of carbon metabolism and TAG synthesis between two species. A similar contrast between the two plants also occurred across the three developmental stages of tubers (Fig. 2C).
Notably, transcript patterns for other lipid-related metabolic pathways were comparable in yellow and purple nutsedge (Fig. 2D).
Overall, the results mentioned above suggest that transcriptional control of genes involved in TAG storage along with FA synthesis rather than TAG synthesis might be the major factors required for high oil accumulation of yellow nutsedge in relative to purple nutsedge.
Transcripts for Carbon Metabolism Toward Fatty Acid Synthesis Were Slightly Higher in Yellow Nutsedge Than Purple Nutsedge
The generation of plastid pyruvate for FA synthesis from sucrose primarily involves sucrose degradation in cytosol, hexose glycolysis and pentose phosphate pathway (PPP) occurring in both cytosol and plastid (Fig. 3A, B).
It was found that there were only 1.4-fold higher transcripts in yellow nutsedge compared to purple nutsedge for sucrose degradation pathway (Fig. 3B), which is catalyzed either by sucrose synthase (SUS) into uridine diphosphate glucose (UDP-Glu) and fructose (Fru), or by extracellular cell wall invertase (CWINV) or intracellular neutral invertase (CINV) into glucose (Glu) and Fru (Fig. 3A). SUS genes in both nutsedges were highly expressed at levels of over 2800 FPKM/protein that were at least 15-fold higher than CINV or CWINV (Fig. 3A, Additional file 1: Table S2), implying that SUS might play an important role as the preferred enzyme in initial sucrose metabolism in nutsedge tubers. This result could support the evidence that SUS activities in plant seeds or potato tuber were significantly higher than INV activities [13, 14, 15, 16].
Similar patterns of gene expresses involved in glycolysis were also present in the two tuber tissues (Fig. 3B). Transcripts for nearly all of glycolytic enzymes in cytosolic or plastidial compartments were at slightly higher or similar levels in yellow nutsedge compared with purple nutsedge (Fig. 3A). Only expression levels for cytosol isoforms of ATP-dependent phosphofructokinase (PFK) and fructose-bisphosphate aldolase (FBA), and plastid fructose kinase (FK), which were over 2.5-fold higher in yellow nutsedge than in purple nutsedge.
Comparing the transcripts for both plastid and cytosol glycolysis revealed some conserved features between the two tuber tissues. The transcript levels were much higher for almost all genes involved in cytosolic glycolysis than their counterparts for plastidial glycolysis (Fig. 3C), except for genes encoding for phosphoglucomutase (PGM) and glucose-6-phosphate isomerase (GPI) where transcripts were distributed in somewhat balanced manner between the two glycolytic pathways. For a complete glycolytic pathway in the two plant tubers, the glycolytic genes involved in late glycolysis associated with the seven downstream glycolytic steps (from fructose-bisphosphate aldolase (FBA) to pyruvate kinase (PK)) were overall more abundantly expressed in relative to those in early glycolysis. Notably among these genes, those encoding for cytoplasmic glyceraldehyde-3-phosphate dehydrogenase (GAPDH), FBA and enolase (ENO) were significantly highly expressed (> 1,800 FPKM). As a result, these data suggest that the cytosolic glycolysis metabolic pathway, particularly the late process, is highly active and might produce most of carbon precursors toward FA synthesis in the two tubers. The gene expression patterns of these two species resemble those observed in heterotrophic non-green oil seeds such as castor, safflower and sunflower [17], but differ from those in photoheterotrophic green seeds such as Arabidopsis, rapeseed and soybean [18] and oil-rich mesocarps of oil palm as well avocado that displayed more balanced distribution of transcripts between cytosol and plastid glycolysis [19, 20, 21].
Pentose phosphate pathway, a glycolysis bypass process, has been shown to provide carbon sources for pyruvate generation, which also occurred in both cytosol and plastid [22]. However, the overall transcripts for this pathway in cytosol or plastid is not much different between the two species (Fig. 3B). Only a significant increase in cytosolic ribose-5-phosphate isomerase (RPI) enzyme was observed in purple nutsedge compared with yellow nutsedge (Fig. 3A).
Significant Difference of Transcripts for Plastid Rubisco Bypass Is Present Between Two Species
Glycolysis is also bypassed by the production of 3-phosphoglycerate (3-PGA) catalyzed by ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), a key enzyme for fixing carbon dioxide. This process without full Calvin cycle is called Rubisco bypass or shunt [23]. Intriguingly, the expression of gene encoding for RubisCO ortholog was also appeared in two nutsedge tubers. In this study, six unigenes encoding for RubisCO small subunit (RbcS) and one for RubisCO large subunit (RbcL, ATCG00490) were detectable to express during tuber development, though RbcL was merely slightly transcribed (Fig. 4A; Additional file 1: Table S2). The presence of RubisCO genes, particularly RbcS in nutsedge tubers is surprising, since tubers are non-green and non-photosynthetic underground tissues that require little or no light for their development and growth. It was also found that RbcS homologous were expressed exclusively in other non-photosynthetic tissues including seeds, fruits, roots, and secretory organs such as trichomes, but seem to be almost absent in photosynthetic tissues [24, 25]. It is unclear why these RbcS proteins are maintained in the non-photosynthetic tissues including tuber, but one can expect that this type of RbcS may be evolved to accomplish different functions other than Calvin cycle occurred in photosynthetic tissues. It suggested that Rubisco, particularly RbcS, plays a specialized role in recycling of CO2 at high concentration environment generated by the metabolism in tissues that are less permeable to gas exchange [26].
Comparatively, the RbcS genes in purple nutsedge is poorly transcribed at low levels, while they were abundantly expressed in yellow nutsedge and displayed up-regulation patterns during tuber development (Fig. 4A), with the average transcript levels being more 12 times higher than in purple nutsedge (Fig. 3A). A recent study also showed that the ortholog of RbcS in potato starchy tuber displayed much more abundant transcripts in oil-accumulated transgenic lines expressing Arabidopsis WRI1 gene than in the control [27]. These results might reinforce the enzyme assay of the silique of rapeseed, which revealed that high oil content was associated closely with enhanced RbcS expression levels and its activities [28]. Evidence has shown that changes in the RbcS transcript abundance were directly correlated with the changes in Rubisco level [29, 30]. High expression of RbcS genes possibly reflected the high CO2 environment31 and was most likely associated with the ability of capturing CO2 resulting from the conversion of malate to pyruvate by the plastid NADP-dependent malic enzyme (ME) or pyruvate to acetyl-CoA by the plastid pyruvate dehydrogenase complex (PDHC) [15]. Previous studies demonstrated that RubisCO bypass or shunt skipping Calvin cycle for CO2 recapture in green oil-rich seeds brought about less loss of carbon as CO2 and produced more 3-PGA toward for FA synthesis, thus improving carbon conversion efficiency [23, 31, 32, 33, 34].
Whether the expression of RbcS gene is also related to high CO2 concentration and involved in recycling CO2 in tubers remains to be further studied. Nevertheless, the relatively high expression level of RbcS in yellow nutsedge compared to purple nutsedge indicated that RbcS might play a part role in carbon metabolism for oil production in oil tuber tissue.
Transcripts for Plastid Malate and Pyruvate Metabolism Are Strikingly Distinct Between Two Species
Pyruvate is the important carbon precursor required for plastid fatty acid synthesis. It can be generated either from phosphoenolpyruvate (PEP) catalyzed by PK or pyruvate phosphate dikinase (PPDK), or through the malate metabolism involved in malate dehydrogenase (MDH) and ME that catalyze the sequential production of malate and pyruvate from oxaloacetate (OAA) (Fig. 3A). In this study, the abundant expression of genes encoding for MDH, ME, and PK occurred in both cytosol and plastid, and transcript levels for MDH and ME were comparable to those for PK (Fig. 4B), implying that the malate metabolism is as important as PK in pyruvate generation and pyruvate is produced from both hexose via the glycolytic pathway and NADP-dependent malic enzyme for fatty acid synthesis in nutsedge tubers. Previous studies showed that malate was a major substrate for fatty acid synthesis in non-green seeds of safflower, castor bean, and sesame [35, 36, 37].
Higher expression of MDH, ME, and PK was noted in the cytosol than in the plastid (Fig. 4B), suggesting that the cytosolic pyruvate generation might be more prominent in two species, and malate and pyruvate produced in the cytosol could be transported to the plastid for subsequent pyruvate metabolism for fatty acid synthesis.
One interesting aspect from this comparison was that transcripts for cytosolic MDH, ME and PK were comparable in both nutsedge species (Fig. 4B). By contrast, transcript levels for plastidic counterpart and PPDK enzymes were more than two times higher in yellow nutsedge than in purple nutsedge. This suggests that plastid malate and pyruvate metabolism was more active and might produce more carbon source as well as reducing power (NADH/NADPH) required for fatty acid synthesis in yellow nutsedge relative to purple nutsedge. Therefore, our results imply that up-regulation of genes involved in plastid malate and pyruvate metabolism plays an important role in providing pyruvate for high oil synthesis.
Transcripts for Fatty Acid Synthesis Enzymes in Plastid Were More Abundant in Yellow Nutsedge than in Purple Nutsedge
The expressions of at least fourteen proteins required for de novo fatty acid synthesis in the plastid from pyruvate were all detectable in two species (Fig. 5A). Among these enzymes/proteins, PDHC, acetyl-CoA carboxylase (ACCase), and acyl-carrier protein (ACP) transcribed more abundant than any other enzymes (Fig. 5B), implicating the important roles of these proteins in fatty acid synthesis. The overall transcript levels for these three proteins accounted for over 50% of total for FA synthesis at each stage of tuber development. Similar phenomena were also observed in developing oil-rich seeds and mesocarps [17, 19, 21].
Almost all proteins were transcribed at comparatively higher levels in yellow nutsedge, coinciding with high oil accumulation in its tubers. Overall, transcript levels for these plastidial proteins were on average 2.5-fold higher in yellow nutsedge than in purple nutsedge (Fig. 2C). Significant individual differences were present for ACCase, hydroxyacyl-ACP dehydratase (HAD), stearoyl-ACP desaturases (SAD), and acyl-ACP thioesterase A (FATA), for which the transcripts were more than 3-fold higher in yellow nutsedge compared with purple nutsedge (Fig. 5A), suggesting that the metabolic pathways catalyzed by these enzymes perhaps were much more active in yellow nutsedge.
ACCase in the plastid is one of the main enzymes in regulating oil production, as it catalyzes the first truly committed step of the irreversible carboxylation of acetyl-CoA to malonyl-CoA with no other known metabolic fate, a rate-limiting pathway in the control of fatty acid synthesis [38]. The importance of ACCase in the control of plant lipid synthesis was confirmed by the evidence that about 55% of the total carbon flux for overall fatty acid synthesis was controlled by the ACCase in leaf tissues [39]. In this study, ACCase transcripts represented about 15% and 11% of the total levels for fatty acid synthesis enzymes in yellow nutsedge and purple nutsedge, respectively. Among the four subunits of heteromeric ACCase enzyme complex, three nuclear-coded subunits, biotin carboxylase (BC), followed by biotin carboxy-carrier protein (BCCP) with α-carboxyltransferase (α-CT), were most abundant in two species, while the plastid-coded subunit (β-carboxyltransferase, β-CT) was barely transcribed at very low levels (< 3 FPKM) (Fig. 6A). The relatively higher transcripts for nuclear-coded subunits against plastid-coded ones were also observed in oil-rich seeds and fruits [17, 19, 21], suggesting a conserved major role of nuclear-coded subunits for ACCase activity in diverse plants and tissues. In case of nuclear-coded three ACCase, transcripts for each subunit were 3.2 to 4.5 times higher in yellow nutsedge than in purple nutsedge. Previous study indicated that high ACCase mRNAs were consistent with actively synthesizing fatty acids and accumulating high levels of oil in seed [40]. Therefore, our results implied that the initial reaction of fatty acid synthesis catalyzed by ACCase, with the three ACCase subunits in a coordinated expression pattern, was more active and might provide more malonyl-CoA substrate needed for oil production in yellow nutsedge as to purple nutsedge.
HAD is responsible for the dehydration of 3-hydroxyacyl-ACP to generate trans-2-enoyl-ACP. In Arabidopsis, two genes (At2g22230 and At5g10160) have been identified to code for HAD, and they were highly expressed during fatty acid synthesis in developing seeds [41]. While the transcripts for the two HAD genes were detectable in most oil-rich plants [17, 19, 42, 43, 44], only one HAD ortholog (At5g10160) was represented in the two nutsedge tubers, similar to the case observed in other underground sink tissues such as potato [27], sweet potato [45], cassava [46] and sugar beet [47], suggesting its crucial role in fatty acid synthesis and a housekeeping function at least in underground storage tissues. A single HAD ortholog was also noted for other oil-rich plants, for example, castor bean and olive (Olea europaea) contain one At5g10160 ortholog [17, 48], while avocado include only At2g22230 ortholog [21]. These results indicated that the expressions of HAD orthologs are species- and tissue-specific, and different orthologs may have evolved to respond to fatty acid synthesis in different plants.
SAD and FATA are two important enzymes controlling levels of unsaturated fatty acid. High expression of SAD along with FATA in relative to FATB was found to occur in unsaturated fatty acid-rich oil tissues including seeds, fruits and tubers (Fig. 6B). During the tuber development, transcripts for SAD or FATA were much abundant in yellow nutsedge relative to purple nutsedge; particularly, FATA genes were up-regulated during development and transcribed at more than 10-fold higher levels in yellow nutsedge than in purple nutsedge at tuber maturation, associating with their fatty acid profiles of oil. It is noteworthy that in yellow nutsedge, transcripts for FATA were 3-fold more abundant as compared to FATB. In contrast, the FATA transcript levels were less than and comparable to FATB in purple nutsedge. Intriguingly, high ratio of FATA to FATB transcript was also observed in other plant oil storage tissues rich in oleic acid or its derivatives, such as oil seeds of rapeseed and castor bean [17] as well as hickory [49], and oil fruits of avocado [21] and olive [48, 50]. By contrast, the transcript ratio of FATA/FATB is only 0.1, 0.2, and 1.0, respectively, for seeds of Arabidopsis17 and soybean [42] and mesocarp of oil palm [19] that contain low levels of oleic acid. Altogether, these results revealed that the expression patterns of SAD and FATA genes in two tubers reflect the fatty acid composition, and the ratio of FATA to FATB expression is most likely correlated with the content of C18:1.
Transcripts for Most TAG Synthesis Genes in Endoplasmic Reticulum Were Similar or Less in Yellow Nutsedge Than in Purple Nutsedge
In contrast to transcript patterns for plastidial fatty acid genes, most TAG synthesis genes in endoplasmic reticulum were expressed at similar or less levels in yellow nutsedge as compared to purple nutsedge (Fig. 7A). For example, phosphatidic acid phosphohydrolase (PAP), phospholipid:diacylglycerol acyltransferase (PDAT), lysophosphatidylcholine acyltransferase (LPCAT) and Δ12-oleate desaturase (FAD2), an enzyme responsible for synthesis of C18:2 fatty acid in the endoplasmic reticulum, showed similar expression patterns between yellow and purple nutsedge, while lysophosphatidyl acyltransferase (LPAAT) and two enzymes that catalyze the exchanges of fatty acyl groups between phosphocholine (PC) and diacylglycerol (DAG), cytidine-5-diphosphocholine: diacylglycerol cholinephosphotransferase (CPT) and phosphatidylcholine: diacylglycerol cholinephosphotransferase (PDCT), had 2- to 5-fold lower transcripts in yellow nutsedge than in purple nutsedge .
Two exceptions were noted for glycerol-3-phosphate acyltransferase (GPAT9) and diacylglycerol acyltransferase (DGAT) that presented 2.5- and 3.4-fold higher transcripts in yellow nutsedge than in purple nutsedge, respectively (Fig. 7B). DGAT is the key enzyme that catalyzes the final step of acyl-CoA dependent TAG synthesis [51]. In the two species, transcripts were much higher for DGAT2 than for DGAT1, suggesting a more prominent role of DGAT2 than DGAT1, and DGAT2 may be a key mediator in tuber oil production. Our recent study of DGAT1 and DGAT2 functional analysis has provided an evidence to support this hypothesis [7].
It was noted that PDAT, an enzyme responsible for the transfer of fatty acyl moiety from PC to DAG destined to TAG synthesis, and an ortholog of Arabidopsis thaliana DGAT3 (At1g48300), the soluble cytosolic enzyme that might catalyze TAG synthesis using cytosolic acyl-CoA pool [52], were also detectable to have more abundant transcripts than DGAT1 during tuber maturation (Fig. 7C), but displayed similar expression patterns between the two species. Furthermore, transcripts for DGAT2, DGAT3 and PDAT enzymes in yellow nutsedge were all increased during tuber maturation, coinciding with tuber oil accumulation. Taken together, our data implicate the important roles of DGAT2, DGAT3, and PDAT rather than DGAT1 played in transcriptional regulation of TAG synthesis in the nutsedge tubers.
Great Transcriptional Divergence of TAG Storage Genes Between Two Species
Similar to oil seeds of plants [17], oil-tuber of yellow nutsedge were represented by the abundant expression of a large number of genes encoding for seed-like oil body proteins such as oleosin (OBO), caleosin (CALO), steroleosin (STERO), oil body associated protein (OBAP), and seed lipid droplet protein (SLDP), with the OBO transcript being the most abundant (Fig. 8A and 8B; Additional file 1: Table S3). In addition, their expression levels were increased during tuber maturation, consistent with the tuber oil accumulation. Large difference was noted for these proteins, for which their transcripts were over 6- to 160-fold higher in yellow nutsedge than in purple nutsedge. Similar contrast was also detectable across all the developmental stages. Therefore, all these results might imply the important roles of these oil body proteins particularly oleosin in stabilizing TAG and producing high oil content in yellow nutsedge. Previous studies have shown that oleosin is accumulated in a coincident manner with TAG accumulation and the abundant expression of oleosin is associated with high oil content in seeds [53, 54, 55, 56, 57, 58].
The high transcripts of oil body proteins in yellow nutsedge tuber are in sharp contrast to non-seed oily mesocarp tissues of olive, oil palm and avocado that showed lowly expressed transcripts for these structural proteins [19, 59, 60], which were considered less contribution to TAG storage or assembly in oil mesocarp tissues. Indeed, other lipid droplet protein such as lipid droplet-associated protein (LDAP) were found to display plentiful transcripts in these oil mesocarps [19, 21].
Intriguingly, a number of genes encoding for LDAP (At3g05500), LDAP-interacting protein (LDIP, At5g16550) and lipodystrophy protein (SEIPIN1, AT5G16460; SEIPIN2, AT1G29760) were also detectable to moderately express in developing tubers of two species (Fig. 8A and 8B; Additional file 1: Table S3). The transcript level for LDAP or LDIP were even higher than that of STERO. In addition, the temporal transcript patterns of the three proteins were similar to those of seed-like oil body proteins and were in accordance with oil accumulation during tuber development. However, transcripts for the three proteins showed no substantial differences between yellow and purple nutsedge.
It is noteworthy that among these eight proteins, OBO was transcribed most abundantly in yellow nutsedge, followed by CALO and LDAP in this descending order, similar to the case in oil seeds where they are predominantly expressed and the most abundant proteins on oil body [56]. By contrast, LDAP showed highest transcript level in purple nutsedge, which are 5 to 66 times higher than those of other oil body proteins.
Taken together, our results described above indicated that expression patterns of oil body proteins involved in TAG storage is positively associated with oil accumulation, but transcriptional control of these proteins is significantly different between the two species, most possibly tissue-specific.
WRI1 and ABI3 Show Much Higher Transcripts in Yellow Nutsedge Than in Purple Nutsedge
WRINKLED1 (WRI1) is an important transcriptional regulator in controlling oil accumulation in aboveground oil-rich seeds and fruits of plants, and has been well functionally characterized particularly in the model plant Arabidopsis thaliana [61, 62]. In the two nutsedge species, WRI1 and its multiple orthologs, WRI2 (AT2G41710), WRI3 (AT1G16060), and WRI4 (AT1G79700), belonging to the APETALA2-ethylene responsive element binding protein (AP2/EREBP) family, were all detectable to express in tubers, but they displayed low transcript levels (< 25 FPKM) (Fig. 9A). Particularly, WRI3 and WRI4 were only barely expressed, suggesting that they are unlikely to participate in the control of oil production in tubers. This may support the fact that in Arabidopsis, only WRI1 can activate fatty acid synthesis in oil seeds for oil production, and WRI3 and WRI4 are required for cutin synthesis in floral and stem tissues63. Although WRI2 transcript levels were relatively higher compared to WRI3 and WRI4, they were comparable between yellow and purple nutsedge. WRI2 was reported to was unlikely associated with fatty acid synthesis [63].
A significant difference between two species was noted for WRI1 ortholog that showed 14.3-fold on average higher transcripts in yellow nutsedge over purple nutsedge (Fig. 9A; Additional file 1: Table S3). A similar great contradiction was also observed between oil palm and date palm, in which oil palm mesocarp showed 57-fold higher expression of WRI1 orthologue19. In yellow nutsedge, the expression level of WRI1 was increased with tuber development (Fig. 9B), which is in accordance with oil accumulation pattern in tubers. Furthermore, the temporal expression pattern of WRI1 matched that of its potential targets such as PDH-β (AT2G34590), BCCP1 (AT5G16390), FAB2 (AT2G43710), FATA (AT3G25110) and FATB (AT1G08510).
Unlike oil seed tissues, the maturation master regulators that directly or indirectly control the expression of WRI1, such as LEAFY COTYLEDON (LEC1 (AT1G21970) and LEC2 (AT1G28300) and FUSCA3 (FUS3, AT3G26790) [22], were not detectable in two nutsedge tubers, as in oil mesocarps of oil palm and avocado [19, 21], supporting the previous report that FUS3-type proteins are present only in seed plants while LEC1- and LEC2-like proteins are appeared only in vascular and dicot plants, respectively [64]. Lack of these seed-like regulators suggested that the regulation of WRI1-related transcriptional network in non-seed tissues is different from that of oil seed tissues.
It was noteworthy that in yellow nutsedge tuber, an ortholog of ABSCISIC ACID INSENSITIVE 3 (ABI3, AT3G24650), the member of B3 domain superfamily, showed transcript levels comparable to WRI1 and quite similar temporal expression pattern (Fig. 9A and 9B). Similar to WRI1, ABI3 ortholog was up-regulated significantly in yellow nutsedge as compared to purple nutsedge. Genes that share similar expression patterns are likely to interact with each other and have regulatory relationships of functionally importance [65]. In this respective, however, it is unclear whether ABI3 is the upstream regulator of WRI1 and controls WRI1 expression in the nutsedge tubers as in plant oilseeds. Recent study indicated that ABI3 played an important role in regulating plant oil accumulation, which might be independent from WRI1 and LEC2 regulation networks occurred in oil-rich seed tissues [66].
To determine the relevant relations between WRI1 and ABI3 in regulating oil accumulation in oil tuber, a gene co-expression network was constructed and analyzed using the method of weighted gene co-expression network analysis (WGCNA) [67, 68]. The produced network indicated that these two transcriptional factors were classified as hub genes within a same cluster or module in the co-expression network (data not shown). An interesting finding from the co-expression analysis is that ABI3 and WRI1 are co-expressed in concert with the oil genes involved in carbon metabolism, fatty acid synthesis, TAG synthesis, and TAG storage pathways (Fig. 10A; Additional file 1: Table S4). Our analysis result of WRI1 in concerted regulation with the genes related to carbon metabolism, fatty acid synthesis, and TAG synthesis in yellow nutsedge tuber is similar to recent reports [69, 70]. These oil-related target genes are co-regulated by both of ABI3 and WRI1, indicating an overlapping set of targets for the two transcription factors. It was also shown that transcriptional regulation of ABI3 and WRI1 is closely interconnected and WRI1 is one of direct targets for ABI3, suggesting that ABI3 is the regulator of WRI1 in yellow nutsedge, in contrast to oil-rich mesocarps of oil palm and avocado where WRI1 is not under the control of ABI319,21. In oil palm, WRI1 transcriptional activation can be activated by three ABA-responsive transcription factors, NF-YA3, NF-YC2 and ABI5 [71].
It was noted that a large number of genes encoding for late embryogenesis abundant (LEA) proteins, which were charactered well as under control of ABI3 [72, 73, 74], are also identified as potential targets of ABI3 and WRI1, as shown in the co-expression network (Additional file 1: Table S4). Much higher transcripts for these LEA proteins in yellow nutsedge than in purple nutsedge reflect the enhanced desiccation tolerance of yellow nutsedge (Fig. 10B), which allow its tubers can be stored for long time11.
Collectively, these results reinforce the fact that WRI1 and ABI3, as the master regulators of oil accumulation, do not only regulate gene expressions involved in fatty acid synthesis, but also control the expression of genes encoding for seed-specific oil-body storage proteins [58, 66, 75, 76]. Therefore, WRI1-related regulation network of oil production in yellow nutsedge is most likely to differ either from oil seeds or oil fruits, possibly tuber-specific.