Stage-Specific Fatty Acid Fluxes Play a Regulatory Role in

Article pubs.acs.org/JAFC

Stage-Specific Fatty Acid Fluxes Play a Regulatory Role in Glycerolipid Metabolism during Seed Development in Jatropha curcas L. Bharatula Sri Krishna Chaitanya,† Sumit Kumar,† Shiva Shanker Kaki,§ Marrapu Balakrishna,§ Mallampalli Sri Lakshmi Karuna,§ Rachapudi Badari Narayana Prasad,§ Pidaparty Seshadri Sastry,† and Attipalli Ramachandra Reddy*,† †

Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Hyderabad 500046, India Council of Scientific and Industrial Research, Indian Institute of Chemical Technology, Hyderabad 500007, India

§

S Supporting Information *

ABSTRACT: The present study describes the changes in lipid profile as well as fatty acid fluxes during seed development in Jatropha curcas L. Endosperm from 34, 37, and 40 days after anthesis (DAA), incubated with [14C]acetate, showed significant synthesis of phosphatidylcholine (PC) at seed maturation. The fatty acid methyl ester profile showed PC from 34 DAA was rich in palmitic acid (16:0), whereas PC from 37 and 40 DAA was rich in oleic acid (18:1n-9). Molecular species analysis of diacylglycerol (DAG) indicated DAG (16:0/18:2n-6) was in abundance at 34 DAA, whereas DAG (18:1n-9/18:2n-6) was significantly high at 40 DAA. Triacylglycerol (TAG) analysis revealed TAG (16:0/18:2n-6/16:0) was abundant at 34 DAA, whereas TAG (18:1n-9/18:2n-6/18:1n-9) formed the majority at 40 DAA. Expression of two types of diacylglycerol acyltransferases varied with seed maturation. These data demonstrate stage-specific distinct pools of PC and DAG synthesis during storage TAG accumulation in Jatropha seed. KEYWORDS: acyl-edited DAG, 18:1-rich TAG, Jatropha curcas, phosphatidylcholine

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INTRODUCTION Seed oil is a major source of human nutrition, is used as feedstocks for nonfood uses such as soap and polymer production, and also serves as a high-energy biofuel.1 Triacylglycerols (TAG), produced by domesticated oilseed crops, are used primarily for nutritional applications. The fatty acyl chains of TAGs are chemically similar to aliphatic hydrocarbons found in petroleum. There are about 300 oilbearing species, of which about 60 hold promise for biodiesel production.2 Developed countries have been utilizing oleaginous plant species including soybean and rapeseed for the production of biodiesel, but developing countries have to focus their attention on nonedible oil-yielding plants species including Jatropha curcas, Pongamia pinnata, and Simarouba glauca for motor fuel production. The cultivation of these plants has bright prospects as it promotes rural development, generates additional income for farmers, and facilitates access to energy in poorer countries.3 Of these, J. curcas has shown great promise as a potential feedstock for biodiesel production.4 J. curcas L. (family Euphorbiaceae) is a perennial semi-evergreen plant that can be grown in varied soil conditions with minimal irrigation inputs. The seeds contain 40−45% of oil on dry weight basis, which appears to be an economically viable substitute for fossil fuel due to the presence of a large quantity of unsaturated fatty acids.5,6 J. curcas is drought tolerant, shows rapid growth, is easy to propagate, and has a short gestation period.7 Optimum plant size is an added advantage that makes seed collection convenient.8 Apart from its application as a biofuel, it also holds medicinal value, has a potential for carbon © 2015 American Chemical Society

sequestration and land restoration, has phytoremediation application, is a source for organic fertilizer, and can be used for the synthesis of industrial products.9,10 However, domestication of this plant under various conditions has given mixed results with respect to yield.11,12 To harness the complete potential of Jatropha as an economically sustainable and advantageous crop under various growth regimens, critically insightful measures for genetic diversity assessment with respect to yield optimization and postharvest management for further downstream processing should be implemented.13 Earlier studies have reported the physical properties of Jatropha seed oil. These studies report that palmitic (16:0), stearic (18:0), oleic (18:1n-9), and linoleic acid (18:2n-6) are the predominant fatty acids in mature Jatropha seeds, and unsaturation in molecular species of TAGs was favored during seed maturation.14,15 With the development of high-throughput sequence technology, J. curcas has been studied at the genomic and transcriptomic level to unravel the genes involved in oil biosynthesis.16,17 However, systematic studies on the biochemistry of oil synthesis and its storage as well as its regulation during the phase of rapid oil accumulation in the seeds are lacking. Hence, it becomes imperative to bridge the gap between biochemical regulation and molecular understanding of oil biosynthesis and accumulation for attaining successful Received: Revised: Accepted: Published: 10811

October 5, 2015 November 21, 2015 December 2, 2015 December 2, 2015 DOI: 10.1021/acs.jafc.5b04824 J. Agric. Food Chem. 2015, 63, 10811−10821

Article

Journal of Agricultural and Food Chemistry

Analysis of Lipids. Endosperm from 34, 37, and 40 DAA stages was carefully isolated from the respective seeds for lipid extraction.23 After extraction, total lipids were gravimetrically measured. The phospholipid and neutral lipid contents were estimated separately. Phospholipid content was determined by measuring phosphorus content in extracted total lipids.24 Total lipids were digested with sulfuric acid on a sand bath at 90 °C for 3 h to release phosphorus. Phospholipid content was derived by multiplying phosphorus by 25. Neutral lipid content was derived by subtracting phospholipid from total lipid. Uptake of 14C-Labeled Acetate into Lipids during Seed Development. Seeds of different stages were cut to isolate the endosperm. Slices (∼0.5 mm) were cut under 0.1 M MES buffer (pH 6.0) to prevent tissue oxidation. These slices were transferred to 25 mL conical flasks containing 2 mL of MES buffer, 10 mg mL−1 ampicillin, and 140 μmol of [14C]acetate. The flasks were incubated in a shaker incubator at 30 °C for 6, 12, and 18 h. Later the medium was aspirated, and the slices were washed thoroughly with cold MES wash buffer to remove adhering radioactive medium and ground to fine powder with liquid nitrogen to extract total lipids. The paper was airdried and suspended in 3 mL of liquid scintillating fluid. An aliquot of 10 μL of total lipid extract was spotted on a piece of Whatman no. 3 filter paper, washed thoroughly with Milli-Q water, driedn and suspended in 3 mL of 0.5% 2,5-diphenyloxazole (PPO) and 0.35% 1,4bis(5-phenyloxazol-2-yl)-8-benzene (POPOP) in toluene (w/v) a scintillating fluid. The radioactivity was determined using a liquid scintillation counter (PerkinElmer, Waltham, MA, USA). Total lipid amounting from 5 mg was applied as a spot at the origin of the TLC plate, and neutral lipid separation was carried out using hexane/ethyl acetate (90:10) as a solvent in the first dimension. The portion of the plates containing neutral lipid components (DAG and TAG) was separated from the origin while phospholipids were retained. These phospholipids were separated in the second dimension using chloroform/methanol/water (50:40:10) as a solvent. After separation, the TLC plates were exposed to X-ray film in a cassette with intensifiers for a period of 35 days to develop autoradiograms. To determine incorporation of label in components of lipids, TLC plates (used for neutral and phospholipid separation) were exposed to iodine vapors. The PC, DAG/ and TAG spots were marked and scraped off, and the radioactivity was determined. Fatty Acid Profile Analysis. Phospholipids were separated from the mixture of total lipids using a neutral alumina column. All neutral lipids were eluted from the neutral alumina column with ethyl acetate/ hexane (1:19). All other phospholipids except PC were eluted out with 30% chloroform in methanol, whereas PC was eluted out using methanol. TAGs from the neutral lipids mixture were separated on a silica column using ethyl acetate/hexane (1:49), and DAGs were eluted out using ethyl acetate/hexane (1:9). Fatty acid methyl esters were prepared by refluxing PC, TAG, and DAG obtained from 34, 37, and 40 DAA stage endosperm with 2% H2SO4 in methanol (w/v) for 6 h.25 The fatty acid composition of the samples was analyzed through gas chromatography (6890N series of Agilent Technologies, Palo Alto, CA, USA) using a DB 225 (inner diameter = 0.25 mm, length = 37 m, thickness = 0.25 μm) packed with 50% cyanopropylphenyldimethylpolysiloxane. The injector and flame ionization detectors were set at 250 and 270 °C, respectively. The oven temperature was programmed at 160 °C for 2 min and then increased to 230 °C at a rate of 5 °C min−1. Nitrogen was used as the carrier gas at a flow rate of 1.0 mL min−1. Molecular Species Analysis. Neutral lipids from 34, 37, and 40 DAA endosperm were separated from total lipids by eluting a silica column with ethyl acetate. Molecular species of TAGs and DAGs were analyzed by reverse phase HPLC (Agilent 1100 series, Palo Alto, CA, USA) equipped with an evaporative light scattering detector (Alltech ELSD-2000, Arcade, NY, USA).26 About 25 μL of the sample (1 mg mL−1) was injected in the Merck RP column [RP-18 (5 μm) 250-4], and the molecular species were eluted within 15 min using a mobile phase of 100% acetone at a flow rate of 1.5 mL min−1. The ELSD was operated with a drift tube temperature set at 45 °C with a nitrogen flow of 1.5 L min−1 (impactor on mode).

genetic improvement and metabolic engineering of Jatropha seeds for better quantity and quality of oil. Oilseeds incorporate the majority of their fatty acids into storage TAGs. The existence of different types of TAGs is attributed to the diversity in fatty acids. Previously, the highly conserved Kennedy pathway was thought to be responsible for the synthesis of all TAG species.18 However, the growing insights of lipidomics in the past decade have revealed the existence of mechanisms contributing to diversity in TAG molecules. One such mechanism that works in tandem with the Kennedy pathway was reported in castor, which revealed the existence of a PC-dependent acyl transferase contributing to the majority of TAG molecules.19 Recently, another pathway involving PC− DAG interconversion was found to be responsible for the generation of acyl-edited DAGs, which were utilized for TAG synthesis by the Kennedy pathway.20 The existence of such mechanisms has made it necessary to understand the major and minor routes of species-specific TAG synthesis and their regulatory aspects associated with the metabolic fluxes involved. Isotopic labeling appears to be a preferred approach for probing metabolism in individual tissues. It has been made more powerful with the progress in genetics and molecular biology coupled with the availability of advanced instrumentation techniques.21 This integrated approach can provide a dynamic description of operational fluxes in lipid metabolism occurring at a subcellular, tissue, and even organismal level. We took small steps in understanding the sequential TAG biosynthesis during seed development of Jatropha, which demonstrated the relevance of sugar fluxes among different seed tissues during Jatropha seed development.22 These preliminary studies on seed morphology and 14C-labeled sucrose and glucose incorporation at different developmental stages of Jatropha seed showed that the inner integument has the ability to utilize both sucrose and glucose as substrates for metabolism, whereas the endosperm selectively utilizes glucose for lipid biosynthesis.22 The present study describes the changes in lipid profile as well as metabolic reprogramming during the rapid phase of oil biosynthesis in the seeds of a potential biofuel tree species, J. curcas.

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MATERIALS AND METHODS

Plant Material and Growth Conditions. Seeds from mature plantations of a high-yielding J. curcas (variety, Chhattisgarh; planted in 2008) grown and maintained in the botanical gardens of University of Hyderabad (17.3°10′ N and 78°23′ E at an altitude of 542.6 m abovesea level), in Telangana state, India, were used for all experiments. The optimal flowering resulting in harvestable yields of fruits and seeds occurred at the advent of monsoon. Hence, we selected the developing fruits and seeds of various stages for all biochemical and metabolic experimental analyses during this season (July−October). Natural pollination resulted in transformation of the female flowers to fruits. The first visible appearance of fruit in each inflorescence bunch in each tree was regularly monitored and tagged accordingly. This stage was considered as 0 day after anthesis (DAA). In our preliminary analysis, we observed significant accretion in total lipid content in endosperm from 34 to 40 DAA and hence we selected seeds of these three stages for our metabolic and biochemical studies.23 Fruits of 34−40 DAA stages (number of fruits collected for each stage n = 60; minimum 10 fruits of each stage per tree) were collected and stored at −80 °C for further analysis. Regular monitoring of climatic features was performed with the help of a weather data logger, and important soil characteristics were measured at the experimental site during the course of growth of plants and are presented in Supplementary Table 1. 10812

DOI: 10.1021/acs.jafc.5b04824 J. Agric. Food Chem. 2015, 63, 10811−10821


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Molecular Weight Analysis Using Mass Spectrometry. Total lipids from 34, 37, and 40 DAA endosperm were extracted as described previously.23 DAG and TAG were separated using preparative TLC. About 100 mg of DAG and TAG was dissolved in 360 μL of chloroform separately. After this, 840 μL of working solution comprising chloroform/ethanol/00 μM ammonium acetate (3:6:0.3) was added to the above mixture for effective ionization of lipid molecules. Molecular species of DAGs/TAGs were separated by injecting lipid extracts into the ESI source on a triple-quadrupole mass spectrometer (API4000, Applied Biosystems, Foster City, CA, USA). Samples were infused at 30 μL min−1 with an autosampler (LC Mini PAL, CTC Analytics AG, Zwingen, Switzerland) fitted with an appropriate loop for the acquisition time. DAGs were detected as [M + Na]+,and TAGs were detected as [M + NH4]+ ions.27 Collision gas pressure was set at “low”, and the mass analyzers were adjusted to a resolution of 0.7 unit (full width at half-height). The source temperature (heated nebulizer) was set at 120 °C and the dissolution temperature at 350 °C. A voltage of 3.4 kV was applied to the electrospray capillary, the curtain gas was set at 20 units, and the two ion source gases were set at 50 μL mm−1. Regiospecific Analysis. DAG from total lipids at 34, 37, and 40 DAA endosperm was separated using preparative TLC. One hundred milligrams of DAG from each stage was dissolved in 5 mL of ethanol (95%) in a conical flask. Lipozyme RMIM (sn-1,3 specific lipase) was added to this reaction mixture and the flask incubated for 6 h with constant shaking.28 Naturally occurring 1,2-DAG molecules in solvent randomly get converted to 1,3-DAG molecules;29 hence, the incubation time was standardized to 6 h to minimize this conversion, thereby maximizing production of monoacylglycerols. Lipase was separated by centrifugation at 5000 rpm. The reaction was monitored by micro TLC using ethyl acetate/hexane (1:9) as mobile phase. After ensuring complete conversion, the mixture was purified on preparative TLC using ethyl acetate/hexane (1:9) as mobile phase, and the bands corresponding to ethyl esters and monoacylglycerols (MAG) were visualized with iodine vapors and scraped separately. Ethyl esters were directly analyzed by gas chromatography−flame ionization detector (GC-FID), whereas MAG were methylated using sodium methoxide in methanol. The FAMEs thus obtained were analyzed by GC-FID. Gene Expression Profiles Using Semiquantitative PCR. Total RNA was isolated from endosperm of 34, 37, and 40 DAA stages using Trizol reagent according to the manufacturer’s instructions (Sigma, USA). One microgram of RNA was used for cDNA synthesis by a Revert aid first-strand cDNA synthesis kit (Thermo Scientific, USA). Degenerate primers for endosperm specific genes (delta 12 desaturase, DGAT1, and DGAT2) were designed from available sequences in NCBI and used to obtain full-length DNA sequences from total cDNA. On the basis of these sequences, primers for 100 bp products for the above genes were designed. Delta 12 desaturase: forward primer, 5′-TCGTGGGAGGTCGGATATTAA-3′; reverse primer, 5′-CCCTGAGCGCTCGATGAG-3′. DGAT1: forward primer, 5′-TTGACCATATTCAAATCCAGAGAGAT-3′; reverse primer, 5′-GAAATGGATAGAGCCAAGCCATA-3′. DGAT2: forward primer, 5′-TTTTTGTAATGTGGCTGGACTTTG-3′; reverse primer, 5′-CCCGGTACCAGGGAAGTTTT-3′. Fresh cDNA from 34, 37, and 40 DAA endosperm was again synthesized. Stage-specific expression of the above genes with actin as an internal control was performed, and their expression profiles were recorded. Statistical Analysis. All biochemical, metabolic, and chromatographic experiments using seeds at various stages of development were performed in triplicates, and the results were presented as the mean of two seasons ± SD. Individual seeds of various stages collected randomly from the mature trees were considered as experimental units (n = 100). The mean values were compared using Student’s t test and ANOVA in Sigma Plot 11.0. Significant differences in the mean values of the tested variables, the P values, were Tukey corrected to reduce errors.

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RESULTS Lipid Content and Composition in the Endosperm during Seed Maturation. Total lipid content in endosperm at 32 DAA was minimal and could not be determined gravimetrically. However, between 34 and 40 DAA, the endosperm showed a progressive increase in total lipid from 5.4 to 42.7% of fresh weight, an increase of ∼8-fold (Figure 1A;

Figure 1. Total lipids, phospholipids, and neutral lipids accumulation during Jatropha seed development: (A) accumulation of total lipids occurring at 34, 37, and 40 DAA endosperm; (B) variations in phospholipid (black bars) and neutral lipid (gray bars) contents in the endosperm at 34, 37, and 40 DAA. Values are the mean of three independent experiments ± SD, and data were analyzed using ANOVA to test significant differences between the stages and among the variables. (∗)P < 0.05; (∗∗) P < 0.01; (∗∗∗) P < 0.001.

P < 0.001). During this period, the phospholipid content decreased from 81.3 to 3% (Figure 1B). Simultaneously, the neutral lipid content significantly (P < 0.001) increased from 18.7 to 97%, an increase of >5-fold (Figure 1B). Incorporation of [1,2-14C]Acetate into Lipids during Seed Development. The incorporation of [1,2- 14C]acetate into total lipids as well as into PC, DAG, and TAG was followed during different stages of seed development (Figures 2 and 3; Table 1). A high rate of incorporation of [14C]acetate was noted at all stages of development, which significantly increased with time of incubation. The incorporation of [14C]acetate in total lipids at 34 DAA increased ∼1.5-fold during 6−18 h, whereas its incorporation increased ∼2-fold at 37 and 40 DAA (Figure 2). To gain further insights into the precursor−product relationship among PC, DAG, and TAG, all three components were separated by two-dimensional TLC, and [14C]acetate incorporation into these components was recorded (Figure 3; Table 1). Percentage incorporation of [14C]acetate into PC, DAG, and TAG was calculated from Table 1. At 34 DAA, the 6 h incubation study showed incorporation of 64% 14C label in PC, 19% in DAG, and 17% in TAG. However, after 12 h of incubation, the incorporation of 14 C label was 3% in PC, 52% in DAG, and 45% in TAG, 10813


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37% label were observed in PC, DAG, and TAG, respectively. However, the incorporation at 18 h was 37, 20, and 49% in PC, DAG, and TAG, respectively. At 40 DAA, 6 h incubation studies showed 10% of 14C label in PC, 66% of label in DAG, and 24% in TAG. At 12 h, 38% of the label was in PC, 37% in DAG, and 40% in TAG, whereas at 18 h, 7% was in PC, 44% in DAG, and 49% in TAG (Table 1). The percentage incorporation of 14C label in PC, DAG, and TAG was calculated from Table 1, and the values were significant at P < 0.01. Fatty Acid Analysis of Lipid Components during Seed Development. The individual chromatograms showing separation of FAMEs of total lipids, PC, DAG, and TAG at 34, 37, and 40 DAA are presented in Supplementary Figure S1. The percentage composition of major fatty acids in total lipids, PC, DAG, and TAG at various stages of seed development was calculated from Supplementary Table 2 and is shown in Figure 4. Four major fatty acids including 16:0, 18:0, 18:1n-9, and 18:2n-6 were present in all stages of seed development. In total lipids, 16:0 content was reduced from 32% at 34 DAA to 14% at 40 DAA (Figure 4; P < 0.05). There was no significant change in 18:0 content, which was recorded to be about 6−8% at all stages, whereas the cumulative content of 18:1n-9 and 18:2n-6 increased from 59 to 85% as the seed developed (Figure 4; P < 0.05). However, at later stages, the total lipid extracts contained mainly DAG and TAG. PC contained mainly 16:0 (49.3%) at 34 DAA, which was reduced to 22 and 26% at 37 and 40 DAA, respectively. Simultaneously, 18:1n-9 content in PC showed a remarkable ∼4-fold increase from 37 to 40 DAA (Figure 4; P < 0.05). In contrast, both DAG and TAG

Figure 2. Incorporation of uniformly labeled [14C]acetate into total lipids during Jatropha seed development. Endosperm extracted from seeds at 34, 37, and 40 DAA stages was incubated with 14C-labeled acetate and incorporation followed over time. The incorporation into total lipids was measured after completion of 6 h (gray bar), 12 h (white bar), and 18 h (black bar) and expressed as nanomoles of acetate per gram of tissue. Values are the mean of three independent experiments ± SD, and the different letters indicate mean values significantly different at P < 0.01.

whereas after 18 h, 2% of label was in PC and 35 and 63% were in DAG and TAG, respectively (Table 1). At 37 DAA, the incorporation into PC, DAG, and TAG was 23, 39, and 38%, respectively, after 6 h of incubation, whereas at 12 h, 14, 56, and

Figure 3. Autoradiograms showing incorporation of 14C label from acetate into neutral lipids and phospholipids during seed development. TLC plates show the separation of (A) neutral lipid standards (DAG, FFA. and TAG) and (B) phospholipid standards (Lyso PC, PC, PI, and PE). Representative autoradiograms show variations in incorporation of 14C label into neutral lipid (C, E, and G) and phospholipid (D, F, and H) components respectively, extracted from endosperm of 34, 37, and 40 DAA after 6, 12, and 18 h of incubation. DAG, diacylglycerol; FFA, free fatty acid; Lyso PC, lysophosphatidylcholine; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; TAG, triacylglycerol. 10814


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Journal of Agricultural and Food Chemistry Table 1. Incorporation of 14C Label from Acetate into PC, DAG, and TAG from 34, 37, and 40 DAA Stages of Seed Development at 6, 12, and 18 h of Incubationa 6h

a

12 h

18 h

stage

PC

DAG

TAG

PC

DAG

TAG

PC

DAG

TAG

34 DAA 37 DAA 40 DAA

1.3 ± 0.04a 0.30 ± 0.02b 0.48 ± 0.01ab

0.39 ± 0.03a 0.51 ± 0.06b 3.02 ± 0.03c

0.35 ± 0.07a 0.51 ± 0.09b 1.12 ± 0.05c

0.01 ± 0.05a 0.32 ± 0.02b 2.52 ± 0.06c

0.1 ± 0.08a 1.32 ± 0.08b 2.00 ± 0.05c

0.1 ± 0.02a 0.67 ± 0.06b 2.18 ± 0.07c

0.01 ± 0.01a 1.91 ± 0.06b 0.07 ± 0.04ab

0.1 ± 0.03a 1.27 ± 0.08b 0.42 ± 0.03ab

0.1 ± 0.04a 3.17 ± 0.01b 0.49 ± 0.01c

The values presented are the mean ± SD (in nmol acetate g tissue−1). Different letters indicate that the values are significantly different at P < 0.01.

Figure 4. Graphical representations of FAME analysis of PC, DAG, and TAG at 34, 37, and 40 DAA: relative FAME profiles of PC (A, D, G), DAG (B, E, H), and TAG (C, F, I) at 34, 37, and 40 DAA during seed development. Values are the mean ± SD, and different letters indicate that the values are significantly different at P < 0.01.

showed high contents of 18:1n-9 at all stages and accounted for 46 and 55%, respectively, at 40 DAA (Figure 4; P < 0.05). It was also noted that at both 37 and 40 DAA, DAG and TAG contained significantly high amounts of 18:2n-6 (Figure 4; P < 0.05). Analysis of Molecular Species of DAG and TAG from 34, 37, and 40 DAA Endosperm. Individual chromatograms showing separations of DAG and TAG molecular species are presented in Supplementary Figure S2. These chromatograms were used to calculate percentage composition of the molecular species of DAG and TAG, which are shown in Tables 2 and 3, respectively. Separation of molecular species was based on effective carbon number (ECN) and theoretical carbon number (TCN). It was assumed that the sn-2 position in DAG and TAG was mainly occupied by fatty acids having greater numbers of double bonds, and this will restrict the number of molecular species arising from the combination of the four major fatty acids in DAG and TAG. Molecular species of DAG and TAG are shown as subsets of two or three fatty acids, respectively (Tables 2 and 3). With the above assumptions, 6 molecular species in DAG and 16 molecular species in TAG were characterized and their compositions at different stages of seed development were determined. Of the 6 molecular species of DAG, profound changes in 4 species, 16:0/18:2n-6, 18:0/ 18:1n-9, 18:1n-9/18:1n-9, and 18:1n- 9/18:2n-6, were observed during seed development (Table 2; P < 0.01). Higher contents of 16:0/18:2n-6, 18:0/18:1n-9, and 18:1n-9/18:1n-9, which were present substantially at 34 DAA (Table 2), were reduced as the seed reached maturity. Of these, 18:0/18:1n-9

Table 2. Molecular Species of DAG at 34, 37, and 40 DAA Stages of Seed Developmenta % composition molecular species 16:0, 16:0, 18:0, 18:1, 18:1, 18:2,

18:1 18:2 18:1 18:1 18:2 18:2

ECN

TCN

34 DAA

37 DAA

40 DAA

32 30 34 32 30 28

31.4 29.3 33.4 30.8 28.7 26.6

7.4 37.1 17.7 32 nd 5.6

11.7* 32.6ns 10.5* 16.1** 25.8** 3.2ns

13.2* 18.3** nd 13.8** 54.5*** 0.12*

a

Molecular species are represented as two fatty acids (no stereochemistry specified) and grouped on the basis of the individual fatty acid number of double bonds. Values are the mean ± SD, and data were analyzed using Student’s t test to test significant differences between the stages and among the variables. ECN, effective carbon number; TCN, theoretical carbon number; 16:0, palmitic acid; 18:0, stearic acid; 18:1, oleic acid; 18:2, linoleic acid; ns, not significant; nd, not detected. (*) P < 0.05; (**) P < 0.01; (***) P < 0.001.

could not be detected at 40 DAA. In contrast, 18:1n-9/18:2n-6, which was absent at 34 DAA, accounted for >50% of DAG species at 40 DAA (Table 2; P < 0.001). Two of three positions in TAG molecules at 34 DAA comprised 16:0 [(16:0/18:1n-9/ 16:0), (16:0/18:2n-6/16:0)], whereas the remaining position was occupied mostly by 18:1n-9 or 18:2n-6. These TAG species accounted for ∼25% of total TAG molecules. The second highest population of TAG species comprised 16:0/ 18:1n-9/18:0 (∼21%). Remaining TAG species were com10815


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and 40 DAA endosperm is depicted in Table 4. It was observed that of the four major fatty acids, 16:0 greatly favored the sn-1 position of DAGs at 34 DAA (Table 4), whereas DAGs from 37 and 40 DAA showed higher content of 18:1n-9 at sn-1 (Table 4). The sn-2 position of DAGs at 34 DAA preferred 18:1n-9 (Table 4), whereas a significant increase in 18:2n-6 content in the sn-2 position of DAGs was observed from 37 and 40 DAA (Table 4). Chromatograms showing the fatty acid compositions of sn-1 and sn-2 positions of DAG from 34, 37, and 40 DAA are shown in Supplementary Figure S3. Gene Expression Using Semiquantitative PCR. Stagespecific expression of endosperm-specific delta-12 desaturase, DGAT1, and DGAT2 with actin as an internal control was performed, and their mRNA expression levels are shown in Figure 7. DGAT2 expressed strongly at 34 DAA, and its expression gradually decreased with seed maturation (Figure 7A), whereas the expression of both DGAT1 and delta 12 desaturase was low at 34 DAA, and their expression was significantly enhanced at 37 and 40 DAA (Figure 7B,C).

Table 3. Molecular Species of TAG at 34, 37, and 40 DAA Stages of Seed Developmenta % composition molecular species

ECN

TCN

34 DAA

37 DAA

40 DAA

CN:ND

16:0, 18:1, 16:0 16:0, 18:1, 18:0 16:0, 18:1, 18:2 16:0, 18:2, 16:0 16:0, 18:2, 18:0 18:1, 18:1, 16:0 18:0, 18:1, 18:0 18:0, 18:2, 18:0 18:1, 18:1, 18:0 18:1, 18:1, 18:1 18:1, 18:2, 18:0 18:1, 18:2, 18:1 18:2, 18:2, 16:0 18:2,18:2,18:0 18:2, 18:2, 18:1 18:2, 18:2, 18:2

48 50 46 46 48 48 52 50 48 46 46 44 44 46 44 42

47.4 49.4 44.7 45.3 47.3 46.8 51.4 49.3 48.8 46.2 46.7 44.1 42.6 44.6 42 39.9

4 1.8 3 37 11 10 nd 1 11 2 nd 6 3 3 3 nd

5ns 2ns 11** 10** 2* 3** 4** 6** 8ns 17*** 22*** 12** nd 14*** 3ns 6**

nd 2ns 7* 9** 7* 4** 2* 4* 6* 12** 31*** 3* 3ns 3ns 21*** 3*

50:1 52:1 52:3 50:2 52:2 52:2 54:1 54:2 54:2 54:3 54:3 54:4 52:4 54:4 54:5 54:6

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DISCUSSION The highly conserved Kennedy pathway utilizes glycerol-3phosphate and fatty acyl-CoAs as substrates for the synthesis of both neutral and phospho glycerolipids in the endoplasmic reticulum (ER).18 More than 95% of the fatty acids including 16:0, 18:0, and 18:1n-9, which were synthesized in the plastids, are known to be exported to the ER in the form of acyl-CoAs for glycerolipid synthesis.30,31 Further unsaturation of 18:1n-9 occurs on PC to 18:2n-6 and subsequently to linolenic acid (18:3n-3) aided by oleate desaturase (FAD2) and linoleate desaturase (FAD3), respectively, located on the ER.32−34 Such reactions are believed to contribute to the variations in fatty acid composition of many oilseeds. Saturated fatty acids in glycerolipid molecules are usually confined to sn-1 and sn-3 positions of glycerol, whereas unsaturated fatty acids were acylated mainly at sn-2.35 Depending on the genus, a limited subset of these molecular species often predominates in the total TAG population.36−38 This diversity in TAG molecules can be attributed to multiple interconnected pathways integrated with differential metabolic fluxes as reported for some oilseeds.32 In this study, certain regulatory acyl-editing events involved in the synthesis of PC and TAG through DAG have been investigated in Jatropha seeds in a stage-specific manner with the help of tracer studies, gene expression analysis, and lipid profiling to understand the biosynthesis of oil during the ontogeny of Jatropha seed. Glycerolipid Synthesis through the de Novo Kennedy Pathway at 34 DAA. Lipid analysis showed that total lipid content was extremely low (∼5%; Figure 1A) at 34 DAA, of which phospholipid formed the bulk (∼79%) and the rest was neutral lipids (Figure 1B). This diminutive content of total lipids was evidenced by a reduced rate of fatty acid synthesis, which was demonstrated by [14C]acetate incorporation into total lipids (Figure 2). Earlier studies have shown the role of phospholipids in storage TAG synthesis.32 Hence, we followed the incorporation of [14C]acetate into PC, DAG, and TAG over time. Periodic variations in incorporation of 14C label into PC, DAG, and TAG indicated that syntheses of PC and TAG were independent of each other (Table 1; Figure 3). The majority of glycerolipids synthesized at this stage comprised 16:0, and most of it was found in PC (Figure 4A). This preferential synthesis of

a

Molecular species are represented as three fatty acids (no stereochemistry specified) and grouped on the basis of the individual fatty acid number of double bonds. Values are the mean ± SDm and data were analyzed using Student’s t test to test significant differences between the stages and among the variables. ECN, effective carbon number; TCN, theoretical carbon number; 16:0, palmitic acid; 18:0, stearic acid; 18:1, oleic acid; 18:2, linoleic acid; ns, not significant; (*) P < 0.05; (**) P < 0.01; (***) P < 0.001; CN:ND, carbon number:number of double bonds; nd, not detected.

posed of 18:1n-9/18:1n-9/18:2n-6, whereas 18:2n-6/18:2n-6/ 18:2n-6 was not detected at this stage (Table 3). 18:1n-9 and 18:2n-6, occupying sn-1 and sn-2 positions of TAGs, were the major species, which accounted for ∼40% of total TAG molecules at 37 DAA (Table 3; P < 0.001). Approximately 22% TAGs comprised 18:1n-9 occupying two positions, whereas the third position was occupied by 16:0. About ∼41% TAG species at 40 DAA were composed of 18:1n-9 and 18:2n-6 at sn-1 and sn-2 positions of TAG, respectively (Table 3; P < 0.001). Approximately 12% of TAG species comprised 18:2n-6 at two positions of three, whereas 16:0 was barely detected at this stage of seed development (Table 3). Molecular Mass Analysis of DAG and TAG Species during Seed Development Using Mass Spectrometry. Separation of DAG and TAG molecular species based on differences in molecular mass during seed development is shown in Figures 5 and 6. DAG (16:0/18:2n-6, m/z 617) was observed in abundance at 34 and 37 DAA (Figure 5A,B), which was absent at 40 DAA (Figure 5C). DAG (18:1n-9/18:2n-6, m/z 643) was significantly noted only at 40 DAA (Figure 5C). TAG (16:0/18:2n-6/16:0, m/z 851) was highly abundant at 34 DAA (Figure 6A), whereas C18-rich TAGs were not present at this stage. At 37 DAA, both C16- and C18-rich TAGs constitute the total TAG population (Figure 6B). C18-rich TAGs (18:1n-9/18:2n-6/18:1n-9, m/z 903; 18:1n-9/18:2n-6/ 18:0, m/z 905) constituted the majority of the TAGs at 40 DAA (Figure 6C). The mass of DAG with sodium ion adduct and TAG molecules with ammonium ion adduct is been shown in Supplementary Tables 3 and 4, respectively. Regional Distribution of Fatty Acids on sn-1 and sn-2 Positions in DAG from 34, 37, and 40 DAA Endosperm. Regiospecific distribution of fatty acids in DAGs from 34, 37, 10816


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Figure 5. Mass spectra of DAG (A−C) molecules during seed development. DAG molecules (m/z 550−700; between the dashed lines) adducted with Na+ ion ionized by electrospray at 34, 37, and 40 DAA are depicted.

involving acyl-edited PC molecules.40−42 Our results on 14C incorporation depict the most probable pathway responsible for incorporation of 18:2n-6 into DAG and TAG molecules. Time course studies showing a decline in 14C label from acetate in PC suggest the action of phospholipases on PC at 12 and 18 h to release 18:2n-6 and its acylation to glycerolipids. It was previously reported that phosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT) was responsible for synthesis of PUFA DAG species.40 Thus, it may be argued that (16:0/18:2n-6) DAG should have been derived through the action of PDCT involving turnover of both DAG and PC components by retaining the 14C label. In our study, it was interesting to note that at 34 DAA 14C label in PC was lost with time. Also, if PDCT was responsible for the production of (16:0/18:2n-6) DAG, then the 18:1n-9 content in PC should have been greater. However, the presence of (18:1n-9/18:1n-9) DAG species and the absence of (18:1n-9/18:2n-6) DAG

16:0-rich glycerolipids indicated plastidal regulation of fatty acid synthesis and release.39 Among the four major fatty acids in Jatropha, 18:2n-6 is the only acyl-edited product. The major fatty acids in PC were 16:0 and 18:2n-6 (Figure 4A), suggesting the (16:0/18:2n-6) PC molecule formed the majority of the total PC population. A low content of (16:0/18:1n-9) DAG (Table 2) implies that (16:0/ 18:1n-9) DAG synthesized at this stage was exclusively channeled for the production of (16:0/18:2n-6) PC. A low 18:1n-9 content in PC (Figure 4A) clearly suggests that the endosperm at 34 DAA selectively utilizes 16:0 (sn-1) containing DAG for PC production. DAGs from 34 DAA comprised mostly (16:0/18:2n-6) and (18:1n-9/18:1n-9) (Table 2), which were later utilized for synthesis of TAGs (Table 3; Figure 6A). The presence of 18:2n-6 in both DAG and TAG suggests its acylation to these components either through the de novo Kennedy pathway or any other auxiliary pathways 10817


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Figure 6. Mass spectra of TAG (A−C) molecules during seed development. TAG molecules with intact ammoniated ions of TAG molecules (m/z 800−1000) ionized by electrospray at 34, 37, and 40 DAA are depicted.

DAG for PC synthesis and the remaining DAG was utilized for the production of TAG. Initiation of Acyl-Edited DAG Synthesis at 37 DAA. Total lipid content in endosperm doubled at 37 DAA, and most of it was attributed to the increase in neutral lipid content (Figure 1A,B). The rate of fatty acid synthesis also increased significantly as evidenced by a metabolite labeling experiment with [14C]acetate (Figure 2). Periodic accretion of radioactivity in total lipids signifies that the endosperm is actively involved in the synthesis of fatty acids (Figure 2). Also, the rate at which PC synthesis occurred coincided with the rate of TAG synthesis as demonstrated by 14C incorporation studies (Figure 3; Table 1). FAME analysis revealed a decrease in 16:0 content followed by ∼4-fold increase of 18:1n-9 in PC, suggesting the synthesis of 18:1n-9-rich PC species at 37 DAA (Figure 4D). This

molecules rule out the participation of PDCT at this stage. Most of the TAG species in this stage composed of (18:1n-9/ 18:2n-6/18:1n-9) were in a combination of 18:1n-9/18:1n-9/ 18:2n-6 and 18:1n-9/18:2n-6/18:1n-9. Usually, fatty acid with the highest unsaturation should occupy the sn-2 position of glycerolipids, but our data showed that the earlier TAG molecules were derived from acylation of (18:1n-9/18:1n-9) DAG and the later from (18:1n-9/18:2n-6) DAG. The absence of (18:1n-9/18:2n-6) DAG confirms that 18:1n-9/18:1n-9/ 18:2n-6 is the TAG species produced at this stage (TAG molecule containing fatty acids of lesser unsaturation). 16:0rich TAG molecules synthesized at this stage can be attributed to the action of DGAT2, which expressed strongly at this stage (Figure 7A). Hence, it is likely that the endosperm at 34 DAA follows the de novo Kennedy pathway to synthesize most of its 10818


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Journal of Agricultural and Food Chemistry Table 4. Regional Distribution of Fatty Acids at sn-1 and sn-2 Positions in DAG from 34, 37, and 40 DAA Endosperma fatty acid methyl ester 16:0 18:0 18:1 18:2 16:0 18:0 18:1 18:2

34 DAA

37 DAA

sn-1 Position of DAG 48 20 21 11 sn-2 Position of DAG nd nd 56 44

DAGs were utilized for the production of remaining TAG molecules as also was evidenced by mass spectrometry analysis (Figure 6B). Expression of DGAT1 was significant at this stage with a simultaneous decrease in expression of DGAT2. Hence, it may be inferred that 16:0-rich TAGs were derived through the action of DGAT2, whereas C18-rich TAGs were through the action of DGAT1 (Figure 7A,B). These studies clearly demonstrate the metabolic reprogramming during TAG biosynthesis in Jatropha seed endosperm utilizing C18-rich PUFA DAG species at 37 DAA (Table 4), which were further acylated for the production of high 18:1n-9 containing TAG species (Figure 6B). Acyl-Edited DAGs Contribute to Synthesis of Storage TAGs at 40 DAA. The mature endosperm contained the highest amount of total lipid (Figure 1A), which is contributed by an exponential increase in neutral lipid content (Figure 1B). This was also demonstrated by an increase in incorporation of 14 C label into DAG and TAG with time (Figure 3). FAME analysis at 40 DAA showed that PC profile was almost similar to that of 37 DAA (Figure 4G). Hence, regulation of DAG-PC conversion was presumed to be similar to that at 37 DAA.19 The only fatty acid with significant accretion in TAG at this stage when compared to 37 DAA was 18:1n-9 (Figure 4I). Data on molecular species (Figure 6A) as well as regiospecific studies (Table 4) showed that (18:1n9/18:2n-6) DAG was exclusively utilized for the production of TAGs at this stage as evidenced by the presence of 18:1n-9 comprising ∼55% of TAG (Figure 4I). The data strongly suggest that acyl-edited (18:1n-9/18:2n6) DAG and 18:1n-9 CoA should be the major substrates for DGAT1 as it showed significantly higher expression (Figure 7C) at this stage for the production of 18:1n-9-rich TAG molecules (2 mol of 18:1n-9 per mol of TAG) (Figure 6C). The data in Table 2 also suggest that ∼70% of DAGs produced at this stage should be associated with acyl editing. Highly unsaturated 18:2n-6-rich TAGs accounted for 28% of the total TAG population at this stage. Because preferential synthesis of 18:1n-9-rich DAGs was observed to be associated with seed maturation (Table 2), it is most likely that acyl-edited DAGs synthesized at later stages were exclusively channelized toward bulk storage TAG synthesis during seed development in Jatropha (Figure 6C). The present study enabled us to establish a correlation between stage-specific biochemical changes in glycerolipid metabolism. Both Kennedy pathway and acyledited derived DAGs contribute to TAG biosynthesis, and this mechanistic glycerolipid lipome should be characteristic to Jatropha seeds.

40 DAA

23 14 36 27

10 8 53 29

nd nd 48 52

nd nd 45 55

a

16:0, palmitic acid; 18:0, stearic acid; 18:1, oleic acid; 18:2, linoleic acid; nd, not detected.

Figure 7. Semiquantitative PCR analysis of three endosperm-specific genes during seed development. Stage-specific expression of endosperm-specific (A) DGAT2, (B) DGAT1, and (C) delta 12 desaturase with actin as an internal control was performed at 34, 37, and 40 DAA.

indicates a shift in plastidal regulation at this stage accounting for the release of higher 18:1n-9 CoA when compared to that at 34 DAA. It may be implied that the endosperm at this stage has the ability to utilize 18:1n-9 (sn-1) containing DAGs for the synthesis of 18:1n-9-rich PC species as evidenced by our data showing a steep decline in (18:1n-1 9/18:1n-9) DAG molecules (Table 2). 18:1n-9-rich PC species are thus presumed to interact with Kennedy pathway-derived DAGs for de novo synthesis of highly unsaturated (18:1n-9/18:2n-6) DAG (Table 2). Increase in total lipid content (Figure 1A) in the endosperm was accounted for by a significant increment of neutral lipids (Figure 1B) at 37 DAA, suggesting that endosperm prioritizes itself for neutral lipid synthesis. This was further confirmed by an increase in 14C label in all products at all three time periods. The data in Table 2 showed that 16:0rich DAG molecular species account for ∼73% of the total DAG population, whereas acyl-edited C18-rich DAG species accounted for ∼26% of the total DAG population. This was further confirmed by the data in Table 3 indicating the relatively greater contribution of Kennedy pathway-derived TAGs (∼75% of the total TAG pool), whereas acyl-edited

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b04824. Supplementary Table 1; Supplementary Table 2; Supplementary Table 3; Supplementary Table 4; Supplementary Figure S1; Supplementary Figure S2; Supplementary Figure S3 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*(A.R.R.) Phone: + 91 4023134508. Fax: +91 4023010120. Email: [email protected]. 10819


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Journal of Agricultural and Food Chemistry Funding

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This work was supported by the Department of Science and Technology (DST), Government of India (Project DST/ISSTAC/CO2-SR-68/09) to A.R.R. B.S.K.C. and S.K. acknowledge fellowships from DST and UGC, New Delhi, respectively. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Facilities provided through a FIST grant to the department from DST, New Delhi, are gratefully acknowledged. We thank Enti Anjineyalu and Azmeera Thirupathi Naik (Indian Institute for Chemical Technology) for their help in mass spectrometry and gas chromatography work. We also thank Tree Oils India Ltd., Zaheerabad, for providing Jatropha seeds.

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ABBREVIATIONS USED DAA, days after anthesis; DAG, diacylglycerol; DGAT, diacylglycerol acyltransferase; ER, endoplasmic reticulum; FAME, fatty acid methyl esters; lyso-PC, lysophosphatidylcholine; PC, phosphatidylcholine; TAG, triacylglycerol; TLC, thin layer chromatogram

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