Phytochemistry 98 (2014) 41–53
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Differential transcriptional activity of SAD, FAD2 and FAD3 desaturase genes in developing seeds of linseed contributes to varietal variation in a-linolenic acid content Ashwini V. Rajwade a, Narendra Y. Kadoo a, Sanjay P. Borikar b, Abhay M. Harsulkar c, Prakash B. Ghorpade d, Vidya S. Gupta a,⇑ a
Biochemical Sciences Division, CSIR-National Chemical Laboratory, Pune 411 008, India Organic Chemistry Division, CSIR-National Chemical Laboratory, Pune 411 008, India c Interactive Research School for Health Affairs, Bharati Vidyapeeth University, Pune 411 043, India d AICRP on Linseed, College of Agriculture, Nagpur 440 001, India b
a r t i c l e
i n f o
Article history: Received 19 August 2013 Received in revised form 15 November 2013 Available online 28 December 2013 Keywords: Linseed Flax ALA Fatty acid desaturases Fatty acids Gene expression
a b s t r a c t Linseed or ﬂax (Linum usitatissimum L.) varieties differ markedly in their seed a-linolenic acid (ALA) levels. Fatty acid desaturases play a key role in accumulating ALA in seed. We performed fatty acid (FA) proﬁling of various seed developmental stages of ten Indian linseed varieties including one mutant variety. Depending on their ALA contents, these varieties were grouped under high ALA and low ALA groups. Transcript proﬁling of six microsomal desaturase genes (SAD1, SAD2, FAD2, FAD2-2, FAD3A and FAD3B), which act sequentially in the fatty acid desaturation pathway, was performed using real-time PCR. We observed gene speciﬁc as well as temporal expression pattern for all the desaturases and their differential expression proﬁles corresponded well with the variation in FA accumulation in the two groups. Our study points to efﬁcient conversion of intermediate FAs [stearic (SA), oleic (OA) and linoleic acids (LA)] to the ﬁnal product, ALA, due to efﬁcient action of all the desaturases in high ALA group. While in the low ALA group, even though the initial conversion up to OA was efﬁcient, later conversions up to ALA seemed to be inefﬁcient, leading to higher accumulation of OA and LA instead of ALA. We sequenced the six desaturase genes from the ten varieties and observed that variation in the amino acid (AA) sequences of desaturases was not responsible for differential ALA accumulation, except in the mutant variety TL23 with very low (<2%) ALA content. In TL23, a point mutation in the FAD3A gene resulted into a premature stop codon generating a truncated protein with 291 AA. Ó 2013 Elsevier Ltd. All rights reserved.
Introduction Linseed or ﬂax (Linum usitatissimum L.) is a versatile crop grown since prehistoric times for its seeds and ﬁber. More recently, linseed oil has come into focus due to its fatty acid (FA) composition. It is the richest agricultural source of a-linolenic acid (ALA), an essential dietary polyunsaturated fatty acid of x-3 class (Morris, 2007). Its use in industrial as well as food and feed products including a wide variety of nutraceuticals and health foods has been reported. Many studies have established the health beneﬁts of ALA in prevention of cardiovascular diseases (Hu et al., 1999; Mori et al., 2000; Mozaffarian, 2005), cancer (Narisawa et al., 1994; Williams et al., 2007), neurodegenerative and inﬂammatory diseases (Joshi
⇑ Corresponding author. Tel.: +91 2025902237; fax: +91 2025902648. E-mail addresses: [email protected]
(A.V. Rajwade), [email protected]
(N.Y. Kadoo), [email protected]
(S.P. Borikar), [email protected]
(A.M. Harsulkar), [email protected]
(P.B. Ghorpade), [email protected]
(V.S. Gupta). 0031-9422/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.phytochem.2013.12.002
et al., 2006; Zhao et al., 2007), and also in lowering blood glucose and cholesterol levels (Pan et al., 2009; Pellizzon et al., 2007). Indian linseed germplasm consists of more than 3000 varieties with varying oil (30–40%) and ALA (40–50%) content. Varieties with less than 3% ALA were also developed and used in linseed breeding, targeting production of oil with improved tolerance to rancidity (Anonymous, 2010). Desaturation is an important biochemical process in the FA biosynthesis pathway. Fatty acid desaturases (FADs) are the key enzymes that convert saturated FAs with single bond between two carbon atoms (CAC) to unsaturated FA with double bond ([email protected]
) at a speciﬁc location in the fatty acyl chain. Desaturases are classiﬁed into two phylogenetically unrelated groups; the membrane bound fatty acid desaturases and the soluble desaturases (Shanklin and Cahoon, 1998; Sperling et al., 2003), although both are reported to be diiron-oxo enzymes (Fox et al., 1993; Shanklin et al., 2009). Soluble desaturases are acyl–acyl carrier protein (ACP) desaturases, represented by the stearoyl ACP desaturase (SAD or
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D9) (EC 188.8.131.52), which desaturatesstearoyl-ACP (an 18 carbon saturated FA, 18:0) to produce ACP-bound oleic acid (18:1) by introduction of the ﬁrst double bond at the 9th position from the carboxylic end (a end) of the fatty acyl chain. Membrane bound desaturases further introduce double bonds into fatty acids that are either esteriﬁed as acyl-CoA or bound to the glycerol moiety of glycerolipids (Los and Murata, 1998). For example, in the consensus pathway leading to the formation of ALA, further desaturation of oleic acid (OA, 18:1) to linolenic acid (LA, 18:2) is performed by membrane bound D12 desaturase (FAD2) by introduction of a double bond at the 12th position from the a end or 6th position from the x end (x-6). While, the third double bond is introduced at the 15th position from the a end (or x-3 position) of LA by D15 desaturase (FAD3) to form ALA (18:3) (Fulco, 1974; Heinz, 1993). The distribution of fatty acids and fatty acid desaturases is ubiquitous, observed in all aerobic organisms including algae, fungi, mosses, higher plants and mammals. They play key roles in maintaining proper structure and function of biological membranes (McConn and Browse, 1998; Ohlrogge and Browse, 1995; Schmid and Ohlrogge, 2002). Various studies have shown that the three desaturases, SAD (D9), FAD2 (D12) and FAD3 (D15), drive the poly-unsaturated fatty acids (PUFA) synthesis pathway and appear to exist in multiple forms and locations (plastidial or microsomal on the endoplasmic reticulum) within cells. These desaturases have been isolated and characterized from various plant systems like Arabidopsis, sunﬂower, soybean, Brassica etc. (Anai et al., 2005; Arondel et al., 1992; Bilyeu et al., 2003; Martinez-Rivas et al., 2001; Okuley et al., 1994; Schlueter et al., 2007; Serrano-Vega et al., 2003; Yadav et al., 1993). Microsomal FAD enzymes have been shown to be the major contributors to seed a-linolenic acid content in Arabidopsis (Yadav et al., 1993) and soybean (Bilyeu et al., 2003). In linseed, initially only one SAD gene was reported (Singh et al., 1994). Later, two isoforms of SAD gene (SAD1 and SAD2) were reported when isolating the promoter region of this gene (Jain et al., 1999). However, the expression proﬁle of only SAD1 gene isoform was analyzed during seed development in linseed variety AC McDuff, where the temporal expression pattern of the gene was discussed (Fofana et al., 2006). Further, two copies of FAD2 gene expressed in developing seeds were identiﬁed (Fofana et al., 2004). Though closely matching, the available sequences of these two genes were incomplete. Here also, expression proﬁles of only one FAD2 gene was studied during seed development in AC McDuff variety revealing a temporal expression pattern (Fofana et al., 2006). Later, two FAD2 genes with 85% amino acid (AA) sequence similarity were characterized from linseed (Khadake et al., 2009; Krasowska et al., 2007). Linseed has three microsomal D15 desaturase genes, viz., FAD3A, FAD3B (Vrinten et al., 2005) and FAD3C (Banik et al., 2011). Transcript proﬁling of these three genes during seed development in four Canadian linseed varieties varying in their ALA content was performed and the contribution of only FAD3A and FAD3B genes to ALA accumulation was shown by Banik et al. (2011). To best of our knowledge, till date, no reports providing a comprehensive transcriptional proﬁling of all the three desaturases in differential accumulation of ALA during seed development in high and low ALA containing varieties have been published. In the present study, we performed FA proﬁling of eight seed development stages of ten Indian linseed varieties, classifying them in high and low ALA groups based on their ALA content at the mature stage. Further, transcript proﬁling of all the six desaturase genes (SAD1 & SAD2, FAD2 & FAD2-2, FAD3A & FAD3B) was performed in the same stages of seed development of all the linseed varieties, using real time PCR, for their signiﬁcant contribution in fatty acids accumulation. To corroborate the association of a speciﬁc sequence variation with differential desaturase activity, the six desaturase
genes were isolated and sequence characterized from the ten linseed varieties.
Results Fatty acid proﬁling Six fatty acids [myristic (MA), palmitic (PA), stearic (SA), oleic (OA) linoleic acids (LA) and a-linolenic acids (ALA)] were proﬁled using gas chromatography (GC) (Fig. 1), of which myristic acid (MA; C14:0) was almost negligible throughout the developmental stages across the ten linseed varieties. The palmitic acid (C16:0) content was the highest at ﬂower stage in most of the varieties and gradually decreased through boll development to maturity. In case of the linseed varieties, NL260, NL97, JRF5 and Acc No. 4/ 47, however; it was maximum at 4 or 8 days after anthesis (DAA). The proportion of stearic acid (SA; C18:0) remained nearly constant in all the developing stages, irrespective of the varieties. The content of oleic acid (OA; C18:1) was low at the ﬂower stage, but showed an increasing trend up to 8–16 DAA and later remained steady or declined slowly up to maturity in most of the varieties. The linoleic acid (LA; C18:2) content was high at the ﬂower stage and gradually showed decline from 4 to 48 DAA in all the varieties. Conversely, ALA content was high at the ﬂower stage, then declined at 4 DAA and again increased from 8 or 12 DAA till maturity in all the varieties. However, there was a major difference observed in the accumulation trend of LA and ALA in TL23 where ALA content decreased steadily from 4 to 48 DAA; and at maturity, accounted only for 1.83% (±0.07). On the contrary, LA content was initially low and increased till maturity, accounting for 63.29% (±0.02) of the total FA composition. Based on the FA accumulation at 48 DAA, the nine varieties other than TL23, which showed higher proportion of ALA (35– 53%) than any other FA, were divided into two groups as high ALA (with >45% ALA) and low ALA (with <45% ALA) content. The difference in the mean ALA content between these two groups was statistically signiﬁcant (p < 0.002) based on the Student’s ttest. In the high ALA group, average ALA content was 48.76% ± 1.51 of the total FA composition; while for the low ALA group, the average ALA content was 39.53% ± 1.61 (Table 1). This indicated that there was a possibility of more efﬁcient conversion of intermediate fatty acids to ALA in the high ALA group than the low ALA group. However, in case of mutant variety TL23, there was efﬁcient conversion only up to LA and the ﬁnal conversion to ALA was inefﬁcient leading to its very low accumulation compared to LA content. Principal Component Analysis (PCA) was carried out to analyze the distribution of linseed varieties based on the contents of four major FAs according to the above results as well as the basic chemical nature, C18, of these FAs. Moreover, the SA is desaturated to OA, LA and ALA by the action of three desaturases, D9, D12 and D15, respectively in the linseed seed. Four principal components explained all the variation in the data and the ﬁrst two components accounted for 89% of the total observed variance (Fig. 2). PC1 had positive loading of SA, OA and ALA and negative loading of LA, while PC2 had positive loading of SA, OA and LA and negative loading of ALA. These loadings separated SA and OA in the 1st quadrant, LA in the 2nd quadrant and ALA in the 4th quadrant. In the score plot of principal components, all the nine varieties could be clearly separated into two groups as high and low ALA groups, while the variety TL23 lay in the 2nd quadrant away from both the groups. This separation was mainly across PC2 with low ALA group (Ayogi, ES44, JRF5 and Acc No. 4/47), having positive scores while high ALA group (NL260, EC9825, Padmini, NL97 and Surabhi) having negative scores. When the score plot and loading plot were compared
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Fig. 1. Fatty acid proﬁles of ten linseed varieties in eight seed developmental stages. Six fatty acids [Myristic (MA), Palmitic (PA), Stearic (SA), Oleic (OA), Linoleic (LA) and aLinolenic (ALA)] were analyzed from eight seed developmental stages from ﬂower to mature stage (48 DAA) in linseed.
Table 1 Fatty acid (FA) levels in variable ALA groups. Average content of six FAs (%) viz., Myristic (MA), Palmitic (PA), Stearic (SA), Oleic (OA), Linoleic (LA) and a-Linolenic (ALA) in high and low ALA groups of ﬂax varieties and TL23, a mutant variety.
High ALA group Low ALA group TL23
0.10 ± 0.03 0.05 ± 0.01 0.10 ± 0.01
6.54 ± 0.060 7.49 ± 0.26 7.82 ± 0.19
5.48 ± 0.33 7.14 ± 0.28 4.38 ± 0.07
25.56 ± 1.53 30.23 ± 1.66 22.57 ± 0.16
13.56 ± 0.61 15.56 ± 0.42 63.29 ± 0.02
48.76 ± 1.51 39.53 ± 1.61 1.83 ± 0.07
with each other, it was observed that OA and SA together separated the low ALA group in the 1st quadrant, while ALA separated the high ALA varieties mainly in the 4th quadrant. TL23 was placed in the 2nd quadrant because of biased very high LA content. Expression of desaturase genes The expression of SAD (SAD1 & SAD2), FAD2 (FAD2 & FAD2-2) and FAD3 (FAD3A & 3B) desaturase genes was quantiﬁed by real-time PCR in the ten linseed varieties at eight seed development stages from ﬂower to 48 DAA. A two-step reverse transcription method was used for relative quantiﬁcation of the gene expression as detailed in the Section 5. Expression level of a
target gene for each developmental stage of each genotype was calculated relative to the transcript accumulation of ETIF5a reference gene. Ampliﬁcation speciﬁcity of all the desaturase gene-speciﬁc and reference gene-speciﬁc primers was conﬁrmed by observing a single dissociation curve for each pair of the primers. Similarly, it was ensured that the ampliﬁcation efﬁciencies of the target and reference genes were similar (Livak and Schmittgen, 2001) so as to use the DCt method to express the results of the target genes relative to the reference gene. We used the DCt method of calculation as it allowed relative quantiﬁcation comparisons across several variables [ten varieties, eight developmental stages and six genes (two genes each for three desaturases)].
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Fig. 2. PCA plots. Score plot and loading plot (inset) of Principal Component Analysis (PCA) of four fatty acids viz., Stearic (SA), Oleic (OA), Linoleic (LA) and Linolenic (ALA) acids in the mature seeds of the ten linseed varieties.
SAD gene expression Though the levels of expression of the SAD1 and SAD2 genes were less compared to that of the ETIF5a reference gene in the ten varieties and eight developmental stages, they showed a deﬁnite pattern (Fig. 3A and B). In general, SAD1 expression was low at the ﬂower and 4 DAA stages. The expression increased from 8 DAA onwards in most of the varieties, reached its maxima at 12–30 DAA (0.015–0.371) and decreased as the seeds reached maturity. There was an average 7-fold increase in the relative expression from minimum to maximum expressing stage with Padmini and TL23 showing the highest fold increase (16 and 11-fold, respectively). The range of expression of SAD1 gene in groups of high and low ALA varieties and TL23 at their maximum expression stage is depicted in Fig. 4A. The average relative expression maxima of SAD1 in the same groups are given in Table 2, which indicated nearly 3-fold higher SAD1 expression in the group of high ALA varieties. The difference between the average gene expression levels was only marginally signiﬁcant (p = 0.098, Table 2), between the high and low ALA groups. The SAD2 expression pattern was quite similar to SAD1 expression with initial low expression in ﬂower and 4 DAA stages (Fig. 3C and D). The expression increased from 8 DAA, reached its maximum at 12–22 DAA and later decreased with maturity in the ten varieties. There was an average 7.5-fold increase in the relative expression from minimum to maximum expressing stages with ES44 and TL23 showing maximum (12 and 11-fold, respectively) fold variation. However, the range of expression of SAD2 gene as depicted in Fig. 4B and average relative expression maxima as given in Table 2 for high and low ALA groups, indicated only marginally higher SAD2 gene expression in the high ALA group. Statistically the relative expression level difference between high and low ALA groups was not signiﬁcant (p = 0.292, Table 2). TL23, on the contrary showed the highest expression of SAD2 among the groups. In general, the expression of SAD2 gene in all the developing seed stages was higher in the ten varieties as compared to SAD1 gene expression in the respective stages of respective varieties with the exception of Padmini, EC9825 and JRF5. In case of
JRF5, SAD2 expression was higher than SAD1 expression only at 22 DAA stage (Fig. 3). FAD2 gene expression The FAD2 gene transcripts in the ten linseed varieties were very low compared to the ETIF5a gene at ﬂower and 4 DAA stage (Fig. 5A and B). However, after this stage, the expression slowly increased to reach the peak levels by 16–30 DAA (in majority of the varieties at 22 DAA). Notably, there was an average 137-fold increase in the relative expression from minimum to maximum expressing stage with Padmini showing the highest fold (365-fold) increase. Further, Padmini and EC9825 were the only varieties, which showed higher transcript abundance relative to ETIF5a at their maximum expressing stage (22 DAA) and the increase was nearly three and two folds, respectively. It was also observed that in most of the varieties, the increase and later the decrease in the expression was sharp compared to the expression of other desaturase genes. The range of expression at their maximum expressing stage (Fig. 4C) and average relative expression maximum (Table 2) of FAD2 gene in groups of high and low ALA varieties indicated nearly 3.5-fold higher expression in high ALA group than in low ALA group. Statistically, the relative expression level difference between the high and low ALA groups was marginally signiﬁcant (p = 0.064, Table 2). FAD2-2 gene transcripts also showed a similar pattern as that of FAD2 gene. At the ﬂower stage, the FAD2-2 expression was 0.042– 0.272 fold compared to the reference gene (Fig. 5C and D). The expression marginally declined at 4 DAA and slowly increased from 8 to 22 DAA in most of the varieties. There was an average 5-fold increase in the relative expression from minimum to maximum expressing stage with ES44 showing the highest fold increase (9fold). The range of expression at their maximum expressing stage (Fig. 4D) and average relative expression maxima (Table 2) of FAD2-2 gene in groups of high and low ALA varieties and TL23 indicated nearly 2-fold higher expression in high ALA group than that in low ALA group varieties, while TL23 showed intermediate level of relative expression. Statistically, the relative gene expression level
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Fig. 3. SAD gene expression. Relative expression of SAD genes in seed developmental stages of high and low ALA linseed variety groups, (A) SAD1 gene expression in high ALA group, (B) SAD1 gene expression in low ALA group, (C) SAD2 gene expression in high ALA group, (D) SAD2 gene expression in low ALA group.
difference between the high and low ALA groups was highly signiﬁcant (p = 0.01, Table 2). Further, from Table 2 it can also be observed that FAD2 gene showed more than 2-fold higher relative expression compared to the FAD2-2 gene in high ALA group, whereas in low ALA group, the expression was higher by 1.3-folds. FAD3 gene expression The FAD3 (FAD3A and FAD3B) genes also showed a deﬁnite expression pattern in eight developing seed stages of the ten varieties, although there was much variation in their peak expression (Fig. 6). FAD3A transcripts were very low relative to reference gene in ﬂowers and at 4–8 DAA. The expression level then gradually increased till 16–30 DAA (in majority of the varieties at 22 DAA). There was an average 2000-fold increase in the relative expression from minimum to maximum expressing stage with Surabhi and Padmini showing the highest (nearly 4000-fold) increase (Fig. 6A and B). In most of the varieties the increase and then the decrease in the relative expression with the maturity was gradual. TL23 with the lowest ALA content (<2%) amongst the ten varieties, showed the lowest overall relative expression of FAD3A in all the eight developmental stages with expression maxima of 0.06-fold at 22 DAA. FAD3B expression was the lowest at 4 DAA and gradually increased till 16–22 DAA and later decreased up to maturity in both
high and low ALA groups (Fig. 6C and D). There was an average 500-fold increase in the relative expression from minimum to maximum expressing stage with EC9825 showing the highest fold increase (nearly 1500-fold). The linseed varieties, EC9825, Padmini, JRF5 and TL23 showed higher transcript accumulation relative to ETIF5a gene at their expression maxima as compared to the remaining six varieties. Further, the range of expression at their maximum expressing stage (Fig. 4E and F) and average relative expression maximum (Table 2) of FAD3A and FAD3B genes when compared in groups of high and low ALA varieties indicated nearly 5 and 3.5-fold higher expression in high ALA group, respectively. Statistically, for both FAD3A and FAD3B, the relative gene expression level difference between the high and low ALA groups was signiﬁcant at (p = 0.014 and 0.049, respectively; Table 2). Further, the overall expression of FAD3B gene in all the developing stages was much higher in the ten varieties as compared to FAD3A gene expression in the respective stages of all the ten varieties. Molecular characterization of SAD, FAD2 and FAD3 genes The SAD (SAD1 & SAD2), FAD2 (FAD2 & FAD2-2) and FAD3 (FAD3A & FAD3B) genes from the ten linseed varieties were ampliﬁed in 2– 5 overlapping PCR fragments. These gene fragments were directly sequenced and the sequences were assembled to obtain full-length
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Fig. 4. Expression range of six desaturase genes in high and low ALA groups and TL23. (A) Expression range of SAD1. (B) Expression range of SAD2. (C) Expression range of FAD2. (D) Expression range of FAD2-2. (E) Expression range of FAD3A. (F) Expression range of FAD3B.
Table 2 Relative expression (RQ) maxima of desaturase genes. Average RQ maxima (in folds) of desaturase genes in high and low ALA groups and TL23. Gene
High ALA group
Low ALA group
SAD1 SAD2 FAD2 FAD2-2 FAD3A FAD3B
0.137 ± 0.045 0.169 ± 0.017 1.104 ± 0.352 0.456 ± 0.065 0.773 ± 0.0.208 3.561 ± 1.067
0.047 ± 0.014 0.141 ± 0.022 0.319 ± 0.101 0.247 ± 0.019 0.151 ± 0.040 1.004 ± 0.338
0.098 0.292 0.064 0.01 0.014 0.049
0.016 ± 0.005 0.247 ± 0.094 0.356 ± 0.066 0.347 ± 0.001 0.061 ± 0.007 1.321 ± 0.045
a The difference between the mean RQ maxima between high and low ALA groups was tested using one way ANOVA with data on all the replicates.
gene sequences for each gene. Linseed SAD1 and SAD2 genes have three exons and the open reading frame (ORF) for both the genes is 1191 bases in length encoding 396 amino acids (Supplementary data 1 and 2). The deduced amino acid (AA) sequence of SAD1 gene was identical in the ten linseed varieties. Even in case of SAD2, the deduced AA sequence among the ten varieties was similar except for TL23 where a G282S substitution was observed. This AA substitution was similar to the SAD2 sequence in the Phytozome database (Lus10039241.g). Secondly, in the ten varieties P225S substitution compared to the SAD2 sequence from NCBI (AJ006958) was observed. This substitution was similar to the SAD2 sequence (Lus10039241.g) in the Phytozome database. When the respective DNA sequences of the genes were compared, a synonymous nucleotide substitution was also observed in SAD2 gene of TL23 (Supplementary data 3). AA sequences of both the desaturases also showed the presence of two consensus [(D/E) X2 H] domains, characteristic of soluble desaturases (Shanklin and Cahoon, 1998) (Supplementary data 1 and 2).
The two FAD2 genes (FAD2 & FAD2-2) in linseed are intronless and encode proteins of 378 and 382 AA in length, respectively (Supplementary data 4 and 5). The deduced AA sequence of FAD2 gene was identical in the ten linseed varieties; however, two AA substitutions (H53Y and D117V) were observed as compared to the reported FAD2 sequence in NCBI (DQ222824.1). These two AA substitutions were also reported by Khadake et al. (2009) in NL97 (EU660502.1), which is one of the ten varieties in our studies. We could obtain only partial sequence of the FAD2-2 gene from the ten linseed varieties from 106 to 1016 bp corresponding to AA position from 36 to 339 (length: 304 AA). A sequence of 294 AA from the ten linseed varieties was analyzed for the intervarietal comparison, which revealed that these partial AA sequences were identical in the ten varieties. However, at the nucleotide level, few synonymous substitutions were observed among the ten varieties in both the FAD2 and FAD2-2 genes (Supplementary data 3). All the sequences showed characteristic features of membrane-bound desaturases including the presence of three histidine boxes (Sankalin et al., 1994) and YNNKL motif (only in FAD2 sequences) at the C terminus of the protein, which has been reported to be necessary for ER localization of the enzyme (McCartney et al., 2004) (Supplementary data 4 and 5). Three FAD3 genes (FAD3A, FAD3B & FAD3C), have been reported in linseed; among which, FAD3C has been recently isolated and characterized (Banik et al., 2011). The FAD3A and FAD3B genes have six exons and encode proteins of size 392 and 391 AAs, respectively. In the present study, the deduced AA sequences of the FAD3A gene were identical in all the varieties, except the TL23 and the NL260 (Supplementary data 6). TL23 displayed a nonsense mutation (C–T) in the 5th exon causing a premature stop codon (TGA) that lead to a truncated protein with 291 AAs. NL260 showed two AA changes, T77A and H330P. Besides this, in three varieties
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Fig. 5. FAD2 gene expression. Relative expression of FAD2 genes in seed developmental stages of high and low ALA linseed variety groups, (A) FAD2 gene expression in high ALA group, (B) FAD2 gene expression in low ALA group, (C) FAD2-2 gene expression in high ALA group, (D) FAD2-2 gene expression in low ALA group.
(NL97, ACC No. 4/47 and TL23) an additional substitution (N299T) was observed. For FAD3B gene, the deduced AA sequences were identical in the ten varieties, except in TL23 where G283E change was observed. Similarly, in eight varieties except Padmini and NL260, I112S substitution was observed that was identical to the FAD3A sequence from NCBI (DQ116424.1) (Supplementary data 7). In addition to these few more synonymous mutations were also observed among the ten varieties in both the FAD3A and FAD3B genes (Supplementary data 3). Both the FAD3 AA sequences carried the three consensus His-rich motifs required to bind the di-iron active site and for catalysis (Shanklin and Cahoon, 1998; Teixeira et al., 2010). Besides these they also possessed a conserved dilysine ER-retrieval motif near the C-terminal end required for the steady state localization of the FADs in the ER (Arondel et al., 1992; McCartney et al., 2004) (Supplementary data 6 and 7).
Discussion The role of fatty acid desaturases in lipid biosynthesis pathway The acyl lipid metabolism has been well studied in various plants based on which it can be divided under following major
phases. The ﬁrst phase involves de novo fatty acid synthesis in the plastids using pool of acetyl-coenzyme A (Acetyl-CoA) as the carbon source (Post-Beittenmiller et al., 1991, 1992). Several enzymes such as acetyl-CoA carboxylase (ACCase), malonyl-CoA:ACP transacylase and ketoacyl synthesases (KAS) etc. are involved in this process leading to synthesis of 16- or 18-carbon product for transfer to glycerolipids or for export from the plastid. The second phase consists of termination of FA chain elongation and transport of these FA outside plastid to endoplasmic reticulum (ER). This takes place when the acyl group is removed from ACP by either of the two enzyme systems, an acyl-ACP thioesterase or acyltransferases in the plastid (Harwood, 1988; Ohlrogge and Browse, 1995). The last phase involves triacylglycerol (TAG) assembly. It takes place in ER and utilizes glycerol-3-phosphate (G3P) and acyl-CoA as primary substrates. Thus, FA composition in plant oils can be affected by many biochemical factors during lipid biosynthesis. FAs, mostly oleic acid (18:1) and a small amount of palmitic (16:0) and stearic acid (18:0), exported from plastids, are incorporated into phosphatidylcholine (PC), where they may be further desaturated by FAD2 and FAD3 enzymes to linoleic (18:2) and linolenic (18:3) acid respectively, or otherwise modiﬁed before entering TAG assembly (Ohlrogge and Browse, 1995). This desaturation produces the abundant polyunsaturated molecular class of PC.
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Fig. 6. FAD3 gene expression. Relative expression of FAD3 genes in seed developmental stages of high and low ALA linseed variety groups, (A) FAD3A gene expression in high ALA group, (B) FAD3A gene expression in low ALA group, (C) FAD3B gene expression in high ALA group, (D) FAD3B gene expression in low ALA group.
‘Acyl editing’ further makes available the pool of free fatty acids from the PC. This involves enzymes such as lyso-PC acyltransferase (LPCAT) and glycerophosphocholine acyltransferase (GPCAT) etc. The free fatty acids (FFA) are then activated to enter the acyl-CoA pool that can be used for phospholipid synthesis. This emphasizes the importance of acyl ﬂuxes through PC in determining fatty acid composition of TAG (Bates et al., 2009). Besides, multiple interconnected pathways leading to TAG biosynthesis have been described in a few maturing oilseeds (Bates et al., 2009; Baud and Lepiniec, 2010). TAG biosynthesis is governed by many fatty acid and position speciﬁc enzymes such as glycerol-3-phosphate acyltransferase (GPAT), lysophosphatidic acid acyltransferase (LPAAT), PA phosphatase (PAP) and diacylglycerol acyltransferase (DGAT) (Kennedy, 1961; Cao and Huang, 1986, 1987; Ohlrogge and Browse, 1995). The transcriptional regulation of the above genes involved in various phases of lipid metabolism and oil accumulation have been studied from various oil seeds (O’Hara et al., 2002; Saha et al., 2006; Baud and Lepiniec, 2009; Li-Beisson et al., 2010; Troncoso-Ponce et al., 2011; Jiang et al., 2012) and fruit crops (Tranbarger et al., 2011; Bourgis et al., 2011; Dussert et al., 2013). However, systematic comprehensive studies of the transcriptional regulation of FA synthesis and its accumulation in linseed are not available.
Hence, in the present study, we focused on understanding the transcriptional regulation of desaturation process in linseed, as it is the richest agricultural source of ALA and as it is known that sequential action of three microsomal fatty acid desaturases namely, SAD, FAD2 and FAD3, is responsible for the desaturation of stearic to a-linolenic acid through oleic and linoleic acid in the seeds (Fulco, 1974; Heinz, 1993). Variation in the content of these fatty acids in the seed might be a result of differential activity of one or more desaturase enzymes in this chain. However, except for the FAD3 gene (Banik et al., 2011), transcriptional correlation of these desaturases with ALA accumulation has not been studied in linseed varieties with varied ALA content. Expression dynamics of the six desaturase genes in linseed Real-time PCR is routinely used for temporal and spatial expression proﬁling of genes (Charrier et al., 2002; Czechowski et al., 2004; Jain et al., 2007). However, the accuracy in measurement of the transcript abundance depends on several factors such as RNA quality, cDNA quality and quantity, primer speciﬁcity and most important of all, the selection of a uniformly expressing stable reference gene for normalizing the transcript levels of the target genes (Gutierrez et al., 2006; Udvardi et al., 2008). In linseed,
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Huis et al. (2010) evaluated expression stabilities of 13 commonly used reference genes in 13 tissues of three developmental stages by computer algorithms geNorm and NormFinder. For all the tissues, geNorm identiﬁed three most stable genes as EF1a, ETIF5a, and UBI, whereas NormFinder predicted GAPDH as the stable gene. Accordingly, we initially evaluated the efﬁciency and stability of these three genes in the developing seed stages of the ten linseed varieties. Both EF1a and ETIF5a showed stable expression across varieties and seed developmental stages. We used ETIF5a as the reference gene for further studies as the ampliﬁcation efﬁciency of this gene was similar to that of the six target genes in order to use 2 DCt method for real-time PCR data analysis (Livak and Schmittgen, 2001; Pfafﬂ, 2005). This method gave us an advantage to make comparisons in the level of gene expression across developmental stages, varieties and genes. The SAD gene is of commercial interest for the manipulation of unsaturated fatty acids (Knutzon et al., 1991) and has been well characterized in many crop plants including linseed (Shanklin and Somerville, 1991; Singh et al., 1994). We studied the expressions of both SAD1 and SAD2 genes in the developing seeds and observed higher transcript levels of the SAD2 gene compared to the SAD1 gene in seven linseed varieties and also in average values for differential ALA containing variety groups under consideration (Fig. 3 and Table 2). Overall expression of the SAD2 gene appeared to be constitutive whereas the SAD1 gene expression appeared to be more temporal and genotype speciﬁc as indicated by differential expression in low and high ALA containing groups (Fig. 3). This observation has been supported with previous studies of SAD1 and SAD2 gene promoter activity in transgenic linseed (Jain et al., 1999). SAD1 promoter was reported to be weaker and spatial, whereas SAD2 promoter was stronger and constitutive in that analysis. The weaker activity of the SAD1 promoter was suggested to be because of 368-bp region in the SAD1 promoter containing negative regulatory elements and tissue speciﬁcity related elements. The D12 desaturase/FAD2 represents a diverse gene family in plants and is responsible for conversion of monounsaturated OA (18:1) to polyunsaturated fatty acid (PUFA), and is a major factor in determining the quality of plant oils. In our study, gene speciﬁc expression of FAD2and the FAD2-2, in linseed developing seeds has been analyzed for the ﬁrst time. In most of the varieties FAD2 expression increased sharply up to 22 DAA (i.e. during embryo development) to a level much higher than that at the ﬂower stage and slowly decreased as the bolls matured. These ﬁndings were similar to the earlier studies in linseed and Arabidopsis (Fofana et al., 2006; Ruuska et al., 2002). On the other hand, in most of the varieties, increase in the transcript level of FAD2-2 gene was gradual till 22 DAA followed by a sudden drop by 30 DAA. Further, FAD2-2 gene expression was higher at the early seed development stages (ﬂower to 16 DAA) than the FAD2 expression, while the FAD2 expression level at the maximum expression stage was higher than FAD2-2 expression. Thus, continuous higher expression of FAD2-2 gene in all the developing stages of linseed seeds up to 30 DAA in high ALA containing varieties than that in low ALA containing varieties indicated the role of FAD2-2 in differential LA accumulation; while relatively higher expression of FAD2 during 22–30 DAA than FAD2-2 indicates a surge of FAD2 activity for complete conversion of OA to LA. Contrary to this in case of TL23, expression of both the genes was nearly the same resulting in high accumulation of LA (Table 2). FAD3 or D15 is the last desaturase required in the series of desaturation reactions in deciding the level of ALA in the total FA content of the plant (Banik et al., 2011; Bilyeu et al., 2003; Vrinten et al., 2005; Yadav et al., 1993). In our study, the gene expression pattern for the FAD3A and the FAD3B in developing seed stages of the ten genotypes clearly showed signiﬁcantly higher FAD3 (both FAD3A and FAD3B) gene expression in high ALA group than the
expression in low ALA group (Table 2). Further, it was observed that the average expression maxima of the FAD3B (2.31 ± 1.0) was nearly 5-fold higher than the average expression maxima of the FAD3A (0.45 ± 0.2). These results were in accordance with the three times higher expression of FAD3B than FAD3A observed by Banik et al. (2011). Furthermore, our observation that 2000-fold increase in FAD3A expression from its minimum to maximum expression levels as compared to that of 500-fold difference in FAD3B expression levels suggested its probable help in complete conversion of available LA to ALA in the linseed seeds (Table 2). Differential expression of desaturase genes leading to variable ALA content The results of the expression analysis are in agreement with the FA data for the groups based on their ALA contents; where high ALA group showed higher expression of all the desaturase genes compared to that in the low ALA group; although, there were a few exceptions observed in individual varieties belonging to these groups. The FA values (Table 2) clearly indicated much efﬁcient conversion of all the intermediate FAs, namely SA, OA and LA to the ﬁnal ALA content due to efﬁcient action of all the three desaturases in case of the high ALA group. In case of the low ALA group, even though the initial conversion up to OA by the SAD2 was efﬁcient, the later conversions up to ALA by consecutive action of the FAD2 and FAD3 enzymes seemed to be inefﬁcient leading to higher accumulation of OA and LA instead of ALA. However, differential expression of the SAD1 in low and high ALA containing groups (although not highly signiﬁcant) might be partly responsible for high and low ALA accumulation in their respective groups in addition to inefﬁcient FAD2 and FAD3 activities in the low ALA group. These observations suggest us to further analyze the promoter region of all the three desaturase genes in the high and the low ALA containing linseed groups to understand the transcriptional regulation of expression of these genes. There are many earlier reports in various plant systems where promoter region analysis of a gene has revealed variation in transcription binding sites and corresponding variation in the transcription factors, leading to expression variation of the gene (de Meaux et al., 2005; McKhann et al., 2008; Yi et al., 2010). In the high ALA group, we observed that NL260 showed the highest ALA accumulation but moderate transcript abundance, while Padmini revealed the highest expression of all the desaturase genes but moderate ALA accumulation (Figs. 1, 3A, 3C, 5A, 5C, 6A and C). Similarly, though JRF5 belonged to low ALA group, high expression for most of the desaturases was observed. These three deviations pointed out that the desaturase activity might not be solely under the transcriptional control and there could be variety speciﬁc post-transcriptional, translational and post-translational regulations involved in deciding the total protein turnover or enzyme efﬁciency. In soybean, Tang et al. (2005) revealed the role of post-translational regulatory mechanisms in modulating the FAD2-1 enzyme activity. O’Quin et al. (2010) also reported the post-translational regulation of plant FAD3 desaturases via endoplasmic reticulum associated degradation pathway as a response to temperature variation. This however, is a physiological response, and it is not clear as yet if a similar way of controlling desaturases exist in plant seeds leading to synthesis of storage oils. Amino acid sequence comparison in desaturase genes across linseed varieties Full length gene sequences of all the six fatty acid desaturase genes (SAD1, SAD2, FAD2, FAD2-2, FAD3A and FAD3B genes) from each of the ten linseed varieties were compared with the respective reported gene sequences in NCBI as well as Phytozome
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databases. AA sequences were deduced from these sequences and variations were studied across the linseed varieties. Deduced AA sequences of all the desaturase genes were identical in the ten linseed varieties except for few variations observed in varieties TL23 (in SAD2, FAD3A and FAD3B) and NL260 (in FAD3A gene), which were not reported earlier. Further, in the ten varieties we observed that the soluble desaturase, SAD, showed the characteristic di-ironbinding motifs while the membrane bound desaturases, FAD2 and FAD3, showed the presence of three di-iron binding histidine boxes as well as speciﬁc motifs required for their steady state endoplasmic reticulum localization. The nucleotide sequence analysis of the FAD3A gene in TL23 showed the presence of a nonsense mutation creating a premature stop codon in the ﬁfth exon after amino acid 291. However, the FAD3B in TL23 was predicted to produce a full length FAD3B protein with single amino acid change at 283 AA (G to E transition) in the ﬁfth exon when compared with the FAD3B of the other genotypes. TL23, which was the mutant variety with only 1.83 ± 0.07% ALA, showed the lowest expression of the FAD3A gene throughout the seed developmental stages. This low accumulation of the FAD3A transcripts in TL23 could be attributed to the presence of premature translation termination codon (PTC) in the ﬁfth exon of the FAD3A gene. Similar low accumulation of gene transcripts and ALA was also observed by Vintren et al. (2005), where both FAD3A and 3B showed point mutation leading to PTC and by Banik et al. (2011), where only FAD3A showed presence of PTC. These PTCs signiﬁcantly reduced transcript levels of FAD3 genes in those mutant varieties. Degradation of mRNA containing PTCs by RNA surveillance system to prevent the accumulation of potentially detrimental truncated proteins has already been reported in various plant systems such as Arabidopsis (Arciga-Reyes et al., 2006; Kurihara et al., 2009; Yoine et al., 2006), Nicotiana (Wu et al., 2007), rice (Isshiki et al., 2001) etc. We also observed low expression of FAD3B in TL23, which might be responsible for the very low (1.83%) content of ALA in TL23. It is interesting to note that the haploid nuclear genome size of linseed variety CDC Bethun was estimated to be 373 Mb (Wang et al., 2012), while earlier reports determined the genome size ranging from 538 to 675 Mb (Evans et al., 1972; Ragupathy et al., 2011). Genome alterations in the ﬁrst generation progeny of linseed varieties (e.g. Stormont Cirrus) were also reported in response to speciﬁc environmental or growth conditions like different fertilizer or temperature regimes (Evans et al., 1966; Cullis, 2005). These changes in the genome size were attributed to the altered copy number of many types of genetic elements such as rRNA genes and insertional element named LIS-1 (Goldsbrough et al., 1981; Chen, 1999). Recently, the whole genome sequencing studies (WGS) predicted a large gene number and relatively high duplicated genes in linseed, which could be probably because of the recent (5–9 MYA) whole-genome duplication event in the lineage of L. usitatissimum (Wang et al., 2012). These studies indicate the dynamic nature of linseed genome. For last several years, breeding efforts in linseed have mostly focused on high oil and ALA content. Therefore, it would be essential to study the genes involved in lipid biosynthesis, with speciﬁc emphasis on desaturase genes, in context with their copy number and allelic combinations in linseed varieties with such varying genome content as well as ALA content.
Conclusions Considering the expression patterns of all the desaturase genes together, signiﬁcant contribution by the three desaturase genes (FAD2-2, FAD3A and FAD3B) for the differential ALA accumulation in high and low ALA groups of linseed was observed. While marginally signiﬁcant contribution was observed for the SAD1 and
FAD2 genes, the SAD2 gene did not contribute signiﬁcantly to ALA contents of the linseed varieties. These results suggested that the accumulation of ﬁnal product, ALA is not entirely dependent on the activity of one single desaturase gene, but is a cumulative result of the activities of all the three desaturases acting sequentially. Further, it was also seen that the variation in the AA sequences of desaturases in these varieties was not consistent with either of the groups and therefore, probably not responsible for the differential ALA accumulation in the groups except for the variety TL23. Further, transcriptional regulation analysis of various other genes involved in FA transport and storage lipid accumulation as well as post-transcriptional or post-translational regulation studies of all these genes might be essential to completely decipher variation in ALA content of linseed varieties grown under identical environmental conditions. Experimental Chemicals and Kits HPLC grade chloroform, methanol, hexane and other chemicals were procured from Merck (USA); while Butylatedhydroxytoluene (BHT), an anti-oxidant, procured from Sigma–Aldrich (USA) were used for preparing fatty acid methyl esters (FAMEs). Spectrum™ Plant Total RNA kit from Sigma–Aldrich, USA and MultiScribe™ Reverse Transcriptase from Applied Biosystems (USA) were used for RNA extraction and reverse transcription, respectively. RNase-free DNase was obtained from Promega (USA), while FastStart universal SYBR green master mix was from Roche (USA). Plant material High and low ALA containing varieties of linseed were selected based on earlier data obtained while studying the genetic diversity in linseed (Rajwade et al., 2010). The varieties with more than 45% ALA (NL260, EC9825, Padmini, NL97 and Surabhi) were considered under high ALA group; while those with less than 45% ALA were grouped under low ALA group (JRF5, Acc. No. 4/47, Ayogi and ES44). TL23, a mutant variety with less than 2% ALA was also included in the study. These varieties were grown at the ﬁelds of All India Coordinated Research Project on Linseed, College of Agriculture, Nagpur, India in triplicate sets. Young leaf tissue was collected from these varieties for genomic DNA extraction. For fatty acid proﬁling by gas chromatography (GC) analysis and expression studies, ﬂowers of each variety were individually tagged on the day of anthesis. Some of the open ﬂowers were collected on the same day and constituted the 0 days after anthesis (DAA) or Flower stage of the sample set. Further, developing bolls were collected on 4, 8, 12, 16, 22, 30 and 48 days after anthesis (DAA), with a total of eight time-points (including ﬂower stage). The tissues were immediately frozen in liquid nitrogen and stored at 80 °C till the extraction of genomic DNA or RNA. Fatty acid analysis Fatty acids extracted from each of the eight seed developmental stages of the ten linseed varieties were esteriﬁed to FAMEs as described by Rajwade et al. (2010). The extractions were performed twice with three bolls per replicate. 1 ll of chloroform reconstituted extracts were injected in 6890 N network GC system (Agilent Technologies, USA) with SP-2330 Supelco capillary column, 30 m long and 0.32 mm diameter. The GC was programmed for 150 °C for 10 min, followed by 10 °C rise/min up to 220 °C and steady for 10 min. Helium 1 ml/min was used as the carrier gas. The injector port was maintained at 240 °C and FID detector temperature
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was 275 °C. Fatty acid peaks were identiﬁed by comparing them with the proﬁles of standards procured from Sigma–Aldrich (USA). The area under the peak was expressed as percentage fatty acid content. FA proﬁling of each sample was repeated minimum twice. For obtaining the percent of FA content at each developmental stage, mean value of replicates was calculated and further averaging of the two replications was done. To analyze the distribution of ten linseed varieties based on the FA data, principal component analysis was carried out using Systat software (Version 11, Richmond, CA). Transcript proﬁling RNA from the eight seed developmental stages of ten linseed varieties was extracted using Spectrum™ Plant Total RNA kit and quantiﬁed using Nanodrop 1000 (Thermo scientiﬁc, USA). The RNA extractions were performed in triplicates (three biological replicates) and each replicate consisted of 3–5 bolls. The extracted total RNA was treated with RNase-free DNase and 1 lg of this was reverse transcribed using MultiScribe™ Reverse Transcriptase enzyme. Real-time PCR was performed on 7900HT Fast real-time PCR system (Applied Biosystems, USA) using FastStart universal SYBR green master mix and the gene-speciﬁc primers, designed using Primer3 (Rozen and Skaletsky, 2000) (Supplementary data 8). Real time PCR ampliﬁcation reactions were performed with following conditions: 95 °C desaturation for 10 min, followed by 40 cycles of 95 °C for 3 s, with primer annealing and extension at 60 °C for 30 s. Following ampliﬁcation, a melting dissociation curve was generated using a 60–95 °C ramp in order to monitor the speciﬁcity of each primer pair. Three linseed genes, viz. EF1a, ETIF5a and GAPDH, were initially evaluated as reference genes (Huis et al., 2010) and the ETIF5a gene was selected for further analysis based on the stability of expression of this gene across all the genotypes and developmental stages. The PCR efﬁciency of the target and reference genes was determined using the LinReg software (Ramakers et al., 2003; Ruijter et al., 2009). Only those primer pairs and c-DNA dilution combinations which showed similar efﬁciencies (between 1.92 and 2.05) and with R2 valueshigher than 0.998, were considered for further study. Three technical replicates of each of the three biological replicates were performed. The mean of these replicates was used for relative gene expression analysis by 2 DCt method (Livak and Schmittgen, 2001). The relative target gene expression was based on the variation in the number of thresh hold cycles (Ct) and was calculated in relation to reference gene (DCt) in each of developmental stages of all the ten varieties. One way analysis of variance (ANOVA) was performed to test the signiﬁcance of difference between the mean/average gene expression levels of high and low ALA groups. Sequencing the fatty acid desaturase genes from linseed Genomic DNA of the ten linseed varieties was extracted from the young leaf tissue of all the 10 linseed varieties using modiﬁed CTAB method as described by Ghosh et al. (2009). Desaturase gene speciﬁc primers were designed based on the cDNA (for FAD3: DQ116424, DQ116425) or full length genomic (for SAD: AJ006957, AJ006958 and FAD2: DQ222824, EU660501) sequences available in the NCBI database. Two to ﬁve overlapping primers spanning the entire gene sequence were designed for each gene (Supplementary data 9) using Primer3 software (Rozen and Skaletsky, 2000). For each primer pair, PCR conditions were optimized to yield single amplicon and amplicons were bidirectionally sequenced using MegaBACE 1000 DNA Analysis System (GE Healthcare, USA). Each PCR product was sequenced at least thrice and the sequences were blasted with the NCBI database to conﬁrm
their identity. Full-length sequences of the genes were assembled by aligning them using MEGA5 (Tamura et al., 2011). The sequence assembly was also validated using MIRA software (Chevreux et al., 2000). Sequences of each of the six desaturase genes from the ten varieties were aligned to identify haplotypes vis-à-vis reported respective desaturase gene sequences from the NCBI and Phytozome databases (Goodstein et al., 2012). Accession numbers The gene sequences obtained in this study were deposited at GenBank under accession number JQ963139–JQ963148 (SAD1), JQ963149–JQ963158 (SAD2), JQ963109–JQ963118 (FAD2), JQ963159–JQ963168 (FAD2-2), JQ963119–JQ963128 (FAD3A), and JQ963129–JQ963138 (FAD3B). Acknowledgments A.R. thanks Council of Scientiﬁc and Industrial Research (CSIR), India for Senior Research fellowship. The research was ﬁnancially supported by Department of Biotechnology, New Delhi, India (Project Code: GAP278426) and CSIR, India (Project Code: CSC0112) at CSIR-NCL, Pune. We acknowledge Rasika M. Bhagwat, Senior Research Fellow at CSIR-NCL, Pune for the technical support provided for the entire DNA sequencing work. Dr. Shobha V. Rao, Scientist G (Rtrd), Agharkar Research Institute, Pune, is gratefully acknowledged for the statistical analysis involved in the desaturase expression studies. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytochem. 2013.12.002. References Anai, T., Yamada, T., Kinoshita, T., Rahman, S.M., Takagi, Y., 2005. Identiﬁcation of corresponding genes for three low-alpha-linolenic acid mutants and elucidation of their contribution to fatty acid biosynthesis in soybean seed. Plant Sci. 168, 1615–1623. Anonymous, 2010. Linseed: Annual Report 2009–2010. Project Coordinating Unit (Linseed) C.S.A.U.A.T., Kanpur, UP, India. Arciga-Reyes, L., Wootton, L., Kieffer, M., Davies, B., 2006. UPF1 is required for nonsense-mediated mRNA decay (NMD) and RNAi in Arabidopsis. Plant J. 47, 480–489. Arondel, V., Lemieux, B., Hwang, I., Gibson, S., Goodman, H.M., Somerville, C.R., 1992. Map-based cloning of a gene controlling omega-3-fatty-acid desaturation in Arabidopsis. Science 258, 1353–1355. Banik, M., Duguid, S., Cloutier, S., 2011. Transcript proﬁling and gene characterization of three fatty acid desaturase genes in high, moderate, and low linolenic acid genotypes of ﬂax (Linum usitatissimum L.) and their role in linolenic acid accumulation. Genome 54, 471–483. Bates, P.D., Durrett, T.P., Ohlrogge, J.B., Pollard, M., 2009. Analysis of acyl ﬂuxes through multiple pathways of triacylglycerol synthesis in developing soybean embryos. Plant Physiol. 150, 55–72. Baud, S., Lepiniec, L., 2010. Physiological and developmental regulation of seed oil production. Prog. Lipid Res. 49 (3), 235–249. Bilyeu, K.D., Palavalli, L., Sleper, D.A., Beuselinck, P.R., 2003. Three microsomal omega-3 fatty-acid desaturase genes contribute to soybean linolenic acid levels. Crop Sci. 43, 1833–1838. Bourgis, F., Kilaru, A., Cao, X., Ngando-Ebongue, G., Drira, N., Ohlrogge, J.B., Arondel, V., 2011. Comparative transcriptome and metabolite analysis of oil palm and date palm mesocarp that differ dramatically in carbon partitioning. Proc. Natl. Acad. Sci. USA 108, 12527–12532. Cao, Y.Z., Huang, A.H.C., 1986. Diacylglycerol acyltransferase in maturing oil seeds of maize and other species. Plant Physiol. 82, 813–820. Cao, Y.Z., Huang, A.H.C., 1987. Acyl coenzyme A preference of diacylglycerol acyltransferase from the maturing seeds of Cuphea, maize, rapeseed and canola. Plant Physiol. 84, 762–765. Charrier, B., Champion, A., Henry, Y., Kreis, M., 2002. Expression proﬁling of the whole Arabidopsis Shaggy-like kinase multigene family by real-time reverse transcriptase-polymerase chain reaction. Plant Physiol. 130, 577–590.
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