Generation of transgenic soybean plants with seed-specific knockdown of PLDα1
Soybean PLDα1KD transgenic plants were generated by stable transformation of soybean cultivar Jack with a soybean GmPLDα1 RNA interference (RNAi) construct under the control of the seed-specific promoter β-conglycinin (Fig. 1a, Additional file 1: Figure S1). T0 and T2 transgenic plants were screened with qRT-PCR and immunoblotting with an antibody against Arabidopsis PLDα1. This antibody specifically recognized the ~ 92 kDa GmPLDα1 at both leaf and seed tissues of soybean (Fig. 1). The transgenic and background cultivar Jack seeds displayed different levels of PLDα1 accumulation (Fig. 1). Immunoblotting screening for regenerated transgenic soybean lines showed that in the line #1020 (PLDα1KD2), PLDα1 proteins in the developing and mature seeds were almost completely diminished by expression of PLDα1RNAi (Fig. 1b), whereas in another regenerated transgenic soybean line #1048 (PLDα1KD1) displayed about 25% of that in wild-type soybean cultivar Jack (Fig. 1c). To confirm the seed specific expression of PLDα1RNAi and seed-specific suppression of soybean PLDα1, proteins extracted from leaves and seeds of two PLDα1KD lines and wild-type control (Jack) were immunoblotted for PLDα1, and PLDα1 proteins remained in leaves of both transgenic lines, PLDα1KD1 and PLDα1KD2, but was greatly diminished in their seeds (Fig. 1d). Assaying PLDα1 activity showed that PLD activity in PLDα1KD1 and PLDα1KD2 was 23% and 10% of that in wild- type, respectively (Fig. 1e).
Effects of PLDα1KD on the expression of other PLDs
The soybean PLD family has 23 members (Additional file 1: Figure S2a, Additional file 2: Table S2, Data S1). To evaluate the effect of PLDα1KD on the expression patterns of different PLDs, developing seeds of PLDα1KD from T3 and T5 generations of PLDα1KD transgenic lines were tested (Fig. 2). Three GmPLDαs are highly expressed (Additional file 2: Table S3) [32]. In PLDα1KD mutants, the expression of various PLDαs, including targeted PLDα1s, was reduced significantly compared with that of control at different developmental stages (Fig. 2, Additional file 1: Figure S2c, d). In control Jack seeds, GmPLDα3 is the most highly expressed, GmPLDα1 is the second highest, and GmPLDα4 is the third most highly expressed PLD gene in developing seeds (Fig. 2, Additional file 1: Figure S2b, Additional file 2: Table S4) [33]. When using a pair of primers that amplify all GmPLDαs, the total GmPLDα expression displayed a similar pattern as that for the above major GmPLDαs. GmPLDβ3 transcripts were lower in PLDα1KD mutants than in wild-type during early stages of seed development, but higher than in Jack at late stages of seed development (Additional file 1: Figure S2c). Among GmPLDβs, GmPLDβ4 showed the highest expression level in developing seeds, and then GmPLDβ1. Most other PLDβs were expressed at a low level in soybean developing seeds (Fig. 2, Additional file 1: Figure S2b, Additional file 2: Table S4) [33]. Among two major GmPLDδs expressed in developing seeds, GmPLDδ1 was lower at the stages of 4 and 5, and GmPLDδ2 was lower at the satges 2 and 5 than those in the wild-type (Fig. 2). The results indicate that GmPLDα1 RNAi also interfered with the transcripts of other PLD genes, likely due to the high sequence similarity among PLD genes.
PLDα1KD developing seeds had elevated levels of ROS-scavenging genes
Most plant tissues under abiotic stress conditions, such as drought, salinity, ozone, high temperature, and flooding, usually generate more reactive oxygen species (ROS), which is often associated with the synthesis of more enzymes involved in ROS-scavenging to reduce the oxidative damage [6, 34,35,36]. Under high temperature and humidity, wild-type developing seeds displayed increased expression levels of ROS-scavenging related genes, indicating that seeds might be faced with elevated ROS production (Additional file 1: Figure S3). The expression of several genes, such as glutathione S-transferases (GST23), peroxidase (POD), catalase (CAT1), superoxide dismutase (SOD1), ascorbic acid peroxidase (APX), as well as a heat shock protein STI [6, 35], were highly induced over the time during high temperature and humidity stress (Additional file 1: Figure S3). The level of GmPLDα1 transcript also increased by ninefold at 6 h after heat treatment, compared with seeds under normal temperature (Additional file 1: Figure S3). Most stress-responsive and ROS-scavenging genes, such as GST23, POD, CAT1, SOD1, STI, and APX, in PLDα1KD1 and PLDα1KD2 developing seeds displayed higher transcript levels than those in wild-type control under high temperature and humidity conditions (Fig. 3b).
Increased unsaturated fatty acids of TAG and phospholipids in PLDα1KD seeds
We examined changes of TAG contents and fatty acid composition of PLDα1KD and wild-type soybean lines at different developmental stages. Total fatty acid content steadily increased over the seed filling during maturation (Fig. 4, Additional file 1: Figure S4). In addition, clear differences between PLDα1KD and wild-type seeds in unsaturated fatty acids, 18:1, 18:2, and 18:3, were detected throughout the seed developmental stages. Wild-type soybean oil contains 13% palmitic acid (16:0), 4% stearic acid (18:0), 20% oleic acid (18:1), 55% linoleic acid (18:2), and 8% linolenic acid (18:3) (Fig. 4, Additional file 1: Figure S4). A higher content of unsaturated fatty acids in TAG was observed in PLDα1KD seeds than those in the wild-type Jack under normal conditions (Fig. 4, Additional file 1: Figure S4). The differences became bigger between PLDα1KD2 and wild-type than those between PLDα1KD1 and wild-type under stress conditions, suggesting that the degree of PLDα1 suppression may be proportional to the content of unsaturated fatty acids. To distinguish whether the difference in unsaturation resulted from fatty acids in TAG or phospholipids, we assayed fatty acid composition in TAG and phospholipids separated by TLC. The total TAG content in PLDα1KD mutant seeds was higher than that in wild-type seeds at most developmental stages (Fig. 4, Additional file 1: Figure S4). Correspondingly, the contents of total unsaturated fatty acids (mainly 18:1 and 18:2) in stage 5-seeds of PLDα1KD1 and 2 mutant lines were comparable. Both were about two and tenfold higher than those of wild-type seeds under normal growth conditions and the high temperature and humidity conditions, respectively (Fig. 4, Additional file 1: Figure S4). The total fatty acid content of WT under normal conditions was 1.5-fold higher than that under high temperature and humidity in stage-3 seeds. This result is consistent with a previous observation that high temperature decreased oil content of soybean seeds [6]. However, the total fatty acid content in developing GmPLDα1KD seeds was almost unchanged, which could mean that the knockdown of GmPLDα1 stabilizes the seed fatty acid contents under high temperature (Fig. 4, Additional file 1: Figure S4).
Up-regulation of FADs in PLDα1KD developing seeds as compared with wild-type
In the ER, FAD2 synthesizes linoleic acid from oleic acid and FAD3 catalyzes the conversion of linoleic acid into α-linolenic acid on PC (Fig. 5a). FAD2-2A (Glyma.19G147400), FAD2-2B (Glyma.19G147300), FAD2-2C (Glyma.15G195200), and GmFAD2-2D (Glyma.03G144500) were constitutively expressed in developing seeds and vegetative tissues of soybean [37,38,39] whereas FAD2-1A (Glyma.10G278000) and FAD2-1B (Glyma.20G111000) are specifically expressed in developing seeds, and play an essential role in controlling the oleic acid level in developing soybean seeds (Additional file 1: Figure S5) [32, 33]. The low-linolenic acid trait in soybean requires the combination of up to three different recessive alleles of FAD3 genes that encode omega-3 fatty acid desaturases [40, 41]. GmFAD3 includes GmFAD3A (Glyma.14G194300), GmFAD3B (Glyma.02G227200), and GmFAD3C (Glyma.18G062000).
Quantitative RT-PCR results showed that the expression of FAD2-1B was much higher in developing seeds of PLDα1KD than in wild-type under both normal and stress conditions (Fig. 5b). Microarray data showed that FAD2s were highly expressed in seeds during development [33]. FAD2-1B and FAD2-1A were mainly expressed at the late stages of developing seeds, and their transcripts were higher in PLDα1KD than wild-type under both conditions. The transcript levels of GmFAD3A, GmFAD3B and GmFAD3C were generally high in developing seeds (Fig. 5b, Additional file 1: Figure S5). Under high temperature, FAD3B expression levels increased to the highest level at the stage 4, and then decreased. However, under normal conditions, the FAD3B transcript level was higher at all developmental stages compared with that under stressed conditions. Furthermore, the FAD3B transcript level was much higher in PLDα1KD1 than wild-type seeds under normal conditions (Fig. 5b). The expression levels of the major FADs in seeds were higher under normal conditions than high temperature conditions, except for GmFAD2-2A and GmFAD3C, whose expression levels were generally low under normal conditions, and increased in response to elevated temperatures. The expression of chloroplast localized GmFAD6A was also higher in PLDα1KDs than in wild-type and up-regulated under stress conditions (Fig. 5b).
Increased contents of PC and PE in PLDα1KD seeds
The contents of PC and PE between wild-type and both PLDα1KDs lines were comparable under normal growth conditions (Fig. 6a), but they became different under high temperature and humidity conditions (Fig. 6b). To compare the unsaturation status of phospholipids in PLDα1KD seeds with wild-type at different developmental stages under stress conditions, we profiled phospholipids at developmental stages 2, 3, and 4 as PLDα1 expression was higher at these stages. The level of most phospholipids did not change substantially over these three stages (Fig. 6b). However, seeds from PLDα1KD1 and 2 plants had averagely 132% PC and 47% PE higher than those from wild-type in stage-3 seeds (Fig. 6b). The level of PC, particularly with unsaturated fatty acid acyl chains, was significantly higher in PLDα1KD seeds than wild-type. PLDα1KD seeds had higher levels of PCs and PEs with 36:5, 36:6, 36:4, 36:3, 36:2 34:3, 34:2 (total acyl carbons: total acyl double bonds) acyl chains (Fig. 6b). The PA level in PLDα1KD seeds at all three stages was much lower than that in wild-type, with reduction of more than 81% (Fig. 6b). In addition, total levels of LPC, lysophosphatidylethanolamine (LPE), and lysophosphatidylglycerol (LPG) were lower in PLDα1KD seeds than in wild-type seeds, with LPC and LPE being decreased by 90%, in stage-3 seeds (Fig. 7). Overall MGDG increased in both PLDα1KD seeds compared to wild-type seeds, whereas DGDG content kept unchanged. The difference of PC contents between GmPLDα1KD and wild-type seeds under high temperature and humidity was larger than those under normal conditions (Figs. 6, 7).
Decreased PA pools in PLDα1KD seeds
We further investigated the expression of genes involved in PA biosynthesis- and catabolism in PLDα1KD seeds (Fig. 8a). LPAATs that produce PA from lysoPA, PA hydrolases (PAHs) that dephosphorylate PA to yield DAG, and PLDs that produce PA from hydrolysis of phospholipids, all contribute to the changes of PA levels. The soybean genome contains multiple genes encoding LPAATs, corresponding to Arabidopsis AtLPAAT 1–5 that are essential enzymes for the de novo PA biosynthesis in both eukaryotic and prokaryotic pathways for glycerolipid biosynthesis [42, 43]. LPAAT function in TAG biosynthesis in soybean and Arabidopsis has been implicated [16]. In soybean developing seeds, the major GmLPAAT transcripts, including GmLPAAT2α1, GmLPAAT2α2, accumulated in similar patterns compared with DGAT, PDAT, or other TAG biosynthesis-related genes, which fluctuated in developing seeds (Figs. 8, 9, 10, Additional file 1: Figure S6) [33]. These GmLPAAT transcripts increased more than 21% in PLDα1KD seeds compared to wild-type at developmental stage 5 under both conditions. Both LPAAT2α1 and LPAAT2α2 genes were down-regulated in wild-type, and in both PLDα1KD lines, the transcript level of LPAAT2α2 was down-regulated but LPAAT2α1 remained high in response to high temperature and humidity stress (Fig. 8b).
Three PAH genes, homologous to AtPAH1 and AtPAH2, are present in the soybean genome. Two of them, Glyma.13G134500 (GmPAHβ1) and Glyma.10G046400 (GmPAHβ2), were highly expressed in developing seeds, in a trend coincident with seed oil accumulation (Additional file 1: Figure S7) [33]. The transcript levels for both GmPAHβ1 and GmPAHβ2 in PLDα1KD were one and twofold higher, respectively, than these in wild-type seeds at stages 4 under both conditions and the expression of GmPAHs was not affected at all by high temperature except GmPAHβ1 at stage 2 and GmPAHβ2 at stage 3 (Fig. 8b). The combined effects of suppressed PLDα1KD, and a markedly higher PAH expression level contributed to the decreased PA levels, which was confirmed by mature and developing seeds (Figs. 6, 7).
Altered transcript levels of DGATs and PDATs for TAG biosynthesis in PLDα1KD seeds
To explore how PLDα1KD affected TAG biosynthesis and phospholipid metabolism in soybean seeds, we examined several major genes involved in the Kennedy pathway (Fig. 8b). DGAT synthesizes TAG by transferring an acyl group to DAG from newly synthesized or recycled acyl-CoA (Fig. 8a). The DGAT family in the soybean genome has 10 members. Type 1 DGATs, Glyma.13G106100, Glyma.09G065300, and Glyma.17G053300, were highly expressed in seeds. Type 3 DGAT Glyma.17G041600 was also highly expressed in seeds. Compared with type 1 and type 3 DGATs, type 2 DGAT, such as Glyma.16G115700 and Glyma.09G195400, were expressed at a lower level in seeds [44]. The transcript level of these genes increased steadily over seed development (Additional file 1: Figure S8) [33]. PLDα1KD lines have lower transcript levels for several seed-specific DGATs, such as GmDGAT1A (Glyma.13G106100), GmDGAT1C (Glyma.09G065300) and GmDGAT3B (Glyma.17G041600), over all developmental stages under both conditions, indicating that PLDα1KD lines have reduced contributions through DGAT pathway towards TAG synthesis (Fig. 8b). Meanwhile, the expression of GmDGAT1A and GmDGAT3B was increased whereas the expression of GmDGAT1C was suppressed in PLDα1KD lines and wild-type under high temperature conditions compared with normal conditions.
PDAT transfers the sn-2 acyl group from phosphatidylcholine or phosphatidylethanolamine to DAG for TAG production in plants and yeast (Fig. 8a) [45, 46], and in soybean seeds, DAG from PC is primarily used for TAG biosynthesis. PDAT and DGAT were shown to have overlapping functions in TAG biosynthesis [47]. The soybean genome contains 6 putative PDAT genes, and among them, Glyma.12G084000, Glyma.11G190400, and Glyma.13G108100, as well as Glyma.07G036400, were highly expressed in seeds. Transcripts of these PDATs increased steadily during seed development except Glyma.07G036400 (Additional file 1: Figure S9) [33]. The transcript level of PDAT genes in PLDα1KD seeds was more than higher than those in wild-type seeds from stages 3 to 5 under both conditions, suggesting that PLDα1KD seeds have increased PDAT-mediated, DAG-PC dependent TAG biosynthesis (Fig. 8b). Meanwhile, high temperature and humidity suppressed the expression of GmPDAT1B in both PLDα1KD and wild-type seeds.
Enhanced PC–DAG conversion and acyl editing in PLDα1KD soybean seeds
To test whether the active PC–DAG–PDAT pathway contributed to more TAG biosynthesis in PLDα1KD than in wild-type, we compared the transcript levels of relevant genes. For PC synthesis, choline/ethanolamine kinase (CEK) produces phosphocholine that is used by CTP: phosphocholine cytidylyltransferase (CCT) to synthesize CDP- choline (Additional file 1: Figures S10, S11). DAG: cholinephosphotransferase (CPT) then transfers choline form CDP-choline to DAG to generate PC (Fig. 9a). The soybean genome has two CCT genes, CCT1 (Glyma.09G051200) and CCT2 (Glyma.15G157500), and their transcript levels fluctuated during seed development (Fig. 9b, Additional file 1: Figure S11) [33]. However, transcripts of CCTs in PLDα1KD lines were down-regulated by approximately 18% at stage 3 under both conditions. The expression of GmCCTs was suppressed in both PLDα1KD lines and wild-type seeds under high temperature and humidity conditions.
DAG-CPT and PDCT form an important PC–DAG exchange/conversion cycle to enforce the acyl editing of TAGs (Fig. 9a). In PLDα1KD developing seeds, DAG:CPTs (also called AAPTs), DAG:CPT1 and 2 (Glyma.12G081900 and Glyma.02G128300, respectively), were up-regulated as compared with those in developing seeds of wild-type under both conditions (Fig. 9b, Additional file 1: Figure S12). The soybean genome contains two PDCT genes, GmPDCT1 (Glyma.07G029800) and 2 (Glyma.08G213100). The two genes were highly expressed in developing soybean seeds (Additional file 1: Figure S13) [33]. The transcript of GmPDCT2 was up-regulated at early developmental stages and then decreased during late seed stages under both conditions. GmPDCT1 and 2 were significantly up-regulated at stages 2-3 in PLDα1KD developing seeds under both conditions (Fig. 9b). The expression of GmPDCT1 and GmDAG-CPT2 was suppressed at stages 3-5 in PLDα1KD developing seeds under high temperature and humidity conditions. Other two genes GmPDCT2 and GmDAG-CPT1 displayed complicated expression patterns in wild-type and PLDα1KD developing seeds in both environments. These data suggest that the activity of PC and DAG interconversion is increased when PLDα1 was suppressed in developing soybean seeds.
Reduced transcript levels of PLAs but increased levels of LPCAT in PLDα1KD seeds
Since pPLA affects TAG biosynthesis [15], we examined the expression of pPLAs that were either specifically or highly expressed in the developing soybean seeds. The soybean genome contains a large pPLA gene family, and several pPLAs were highly expressed in developing seeds, such as Glyma.08g028800, Glyma.11g036900, and Glyma.17G145900. The pPLAs, Glyma.18g251500 and Glyma.09g243100, were expressed only in seeds (Additional file 1: Figure S14) [32, 33]. The transcripts of pPLAs (Glyma01G.002400 and Glyma08G.028800) in PLDα1KD soybean seeds were, on average, 44% lower than those in wild-type seeds at stage-4 under both conditions, which was consistent with higher levels of PCs in PLDα1KD soybean seeds and lower levels of lysophospholipids (Figs. 6, 7, 10b). Meanwhile, high temperature and humidity suppressed the expression of GmPLA2s in both PLDα1KD lines and WT seeds. The down-regulation of both PLD and PLA expression may explain the higher level of PCs in PLDα1KD seeds.
As LPC and LPE content decreased significantly in PLDα1KD than wild-type seeds by more than tenfold at stage 3, we examined the expression of genes involved in the LPC and PC cycle. LPC acyltransferases (LPCAT) catalyzes the synthesis of PC from LPC using a new fatty acyl-CoA (Fig. 10a). AtLPCAT1 and 2 in Arabidopsis control the acyl editing process by acting as the main entry of unsaturated FAs into PC [16]. The lpcat1/lpcat2 mutant showed decreased PUFA in seed TAG [16, 17]. Soybean genome contains two LPCATs, Glyma.17G131500 (GmLPCAT1) and Glyma.05G049500 (GmLPCAT2). Transcript levels of two genes increased to the highest levels at middle stages and then decreased at the later stage of seed development in both PLDα1KD lines under stress conditions (Fig. 10b, Additional file 1: Figure S15) [33]. Both GmLPCAT transcripts were 17% higher in PLDα1KD seeds than in wild-type seeds at later stages under stress conditions (Fig. 10b). Meanwhile, Both genes were up-regulated in both PLDα1KD lines and wild-type under stress conditions. The down-regulation of GmPLAs and up-regulation of GmLPCATs in PLDα1KD soybean seeds could lead to reduced contents of LPC and LPE, as compared with wild-type (Figs. 6, 7, 10).
Higher germination rate of PLDα1KD seeds
To address the effect of PLDα1KD on soybean seed vigor after harvested from high temperature and humidity conditions, we tested the seed vigor and germination rate of these knockdown lines and wild-type after stored at high temperature and humidity for three months (28 ± 3 °C in the dark and ~ 50% humidity). PLDα1KD seeds displayed higher germination rates, than the wild-type seeds under high temperature and humidity conditions. Under stress conditions, the germination rates of wild-type, PLDα1KD2 and PLDα1KD1 seeds were 80%, 91%, and 95%, respectively, whereas they were 88%, 96%, and 96% under normal conditions (Fig. 11a, b). However, the germination rate of PLDα1KD1 was lower than wild-type at an early stage but then caught up and became higher than wild-type at later stages. We further analyzed hormone levels in those germination seeds at different days after imbibitions. Higher ABA contents in PLDα1KD seeds than in wild-type seeds were detected, suggesting that PLDα1KD seeds had deeper seed dormancy than wild-type seeds and less nutrient consumption in PLDα1KD than in wild-type seeds during storage. Meanwhile, PLDα1KD seeds had initially a lower level of indoleacetic acid (IAA), but later a higher IAA level than that did wild-type seeds (Fig. 11c). Similarly, the seeds of two PLDα1KD lines showed difference in jasmonate (JA) and Ile-conjugated JA level from wild-type (Fig. 11c). PLDα1KD line 1 (KD1) seeds had lower total JAs and a lower germination rate, whereas PLDα1KD line 2 had higher total JAs and a higher germination rate than wild-type (Fig. 11c). The content of MDA was decreased in PLDα1KD seeds germinating for 1 day and had no significant difference within 2–4 days compared with wild-type seeds. There was also no significant difference in the content of soluble sugar in both PLDα1KD and wild-type germinating seeds (Fig. 11d).