opment (Singh 1998, Perry and Harwood 1993a). Incorporation ... labelling studies conducted by Perry and Harwood ..... Aach H., H. Frank and K.P. Heise 1997.
ACTA PHYSIOLOGIAE PLANTARUM Vol. 25, No. 1. 2003:77-82
A simple method for in vivo labelling of lipid fractions in developing seeds of Brassica campestris L. Niti Sharma, Archana, Sarla P Malhotra, Randhir Singh*
Plant Biochemistry and Molecular Biology Laboratory,.Department of Biochemistry, CCS Haryana Agricultural University, Hisar-125 004, India * corresponding author, tel.: 01662-237720-26-4500, fax: 01662-234952, e-mail: hau @ hau.ren.nic.in
Key words: Brassica campestris, developing seeds, lipid fractions, in vivo labelling
POPOP, 1,4-bis [5-phenyl-2-oxazolyl]-benzene; PPO, 2,5-diphenyloxazole.
An in vivo method of labelling lipid fractions in developing seeds of Brassica campestris using [ 1- 14C] acetate has been developed. The "wick" method for introducing label into the intact plant is quite effective, safe and easy to use. The results obtained were reproducible and comparable to those reported earlier for seeds procured from greenhouse grown plants. The labelling pattern showed that rapid oil deposition began around 20 days after anthesis (DAA) and continued until about 45 DAA. The proportion of label in polar lipids declined and that in non-polar lipids increased during the phase of active oil synthesis. Among phospholipids, the label was incorporated mainly in phosphatidyl choline (PC), which was found to be the major fraction of phospholipids. During development, the two galactolipids i.e. monogalactosyl diglyceride (MGDG) and digalactosyl diglyceride (DGDG) followed patterns exactly opposite to each other. The content of the label in MGDG decreased, while that in DGDG increased, indicating the conversion of MGDG to DGDG during maturation.
List of abbreviations: DAA, days after anthesis; DAG, diacylglycerol; DAGAT, diacylglycerol acyltransferase; TAG, triacylglycerol; MAG, monoacylglycerol; MGDG, monogalactocyl diacylglycerol; DGDG, digalactosyl diacylglycerol; PC, phosphotidyl choline;
Oil makes up about 29-54 % of the dry weight of Brassica seeds (Singh and Mehta 1992) and its syn-
thesis from sucrose constitutes one of the major metabolic activities of embryos during seed development (Singh 1998, Perry and Harwood 1993a). Incorporation of labelled precursors into fatty acids and glycerolipids has been followed as an index of biosynthetic capacity of the developing oilseeds (Ghosh and Sastry 1986, Perry and Harwood 1993b, Aach et al. 1997). Radiolabelled acetate is frequently used as a precursor for lipid biosynthesis because of its ready diffusion into the cells and organelles and its metabolic inertness in most situations. By incubating the cotyledons of soybean and sunflower with 14C-acetate and IH-glycerol (Slack et at. 1978); it was found that proportion of polar lipids declines and that of non-polar lipids increases during the phase of active oil synthesis. In groundnut seeds, the incorporation of [2-IH]-glycerol and 14C-acetate revealed triacylglycerol to increase progressively during the active period of lipid accumulation (Ghosh and Sastry 1986). Similarly, the
N. SHARMA, ARCHANA, S.P. MALHOTRA & R. SINGH
labelling studies conducted by Perry and Harwood (1993b) with Brassica napus showed diacylglycerols (DAGs), TAGs and polar lipids to be the major labelled classes during the period of rapid oil accumulation. Triki et al. (1999) examined the incorporation of label from [1-14C]-oleate and [1-14C] linoleate in microsomes isolated from developing sunflower cotyledons to elucidate the biochemical pathway involved in the formation of triacylglycerols rich in C18 polyunsaturated fatty acids. While studying the sources for fatty acid synthesis by castor bean endosperm, isolated leucoplasts showed much higher rates of fatty acid synthesis when incubated with radiolabelled malate rather than with pyruvate and acetate (Smith et al. 1992). In developing embryos isolated from oilseed rape (Brassica napus), lipid synthesis from [14C]-bicarbonate, [1-14C]-acetate and [U-14C]-glycerol was significantly driven by light (Aach et al. 1997). In most of these studies, lipids and lipid fractions were labelled with radiolabelled compounds under in vitro conditions by dipping the isolated seeds / embryos / cotyledons / microsomes/leucoplasts in radioactive solutions. In vitro labelling method suffers from several drawbacks including a change in the micro-environment of the tissue / organ upon detachment/isolation, which itself causes quantitative and qualitative changes in the synthesis of lipids and lipid fractions. Here we report a simple method for in vivo labelling of lipid fractions with [ 1-14C] acetate in the inflorescence of intact Brassica plants. The method would help the scientists to study lipid metabolism under in vivo conditions without involving any disturbance to the intact plant.
Material and Methods Plant material The crop of Brassica campestris L., cv. Toria, was raised in the pots filled with farm soil following the recommended agronomic practices in the green house of the department of Biochemistry, CCS Haryana Agricultural University, Hisar, India. Chemicals S o d i u m [1-14C]-acetate was purchased from Bhabha Atomic Research Centre (BARC), Bom-
bay. The solvents used were of analytical grade and all other chemicals used were of high purity and obtained from either BDH or Merck, India. Lipid standards for TLC, 2,5- diphenyloxazole (PPO) and 1,4-bis [5-phenyl-2-oxazolyl]-benzene (POPOP) were obtained from Sigma Chemical Co., St Louis, MO, USA. In vivo labelling Fifty ~tl of 0.02 M phosphate buffer (pH 7.0) containing 10 gCi of [1-14C]-acetate was fed into the intact plant through a wick of cotton thread passed just below the base of inflorescence (to ensure maximum translocation of label into the pods) with the help of a needle. The other end of the wick was passed through the base of a plastic Eppendorff microtube containing the radiolabelled acetate (Fig. 1). The base of the microtube was sealed with molten wax to avoid the escape of labelled acetate from any other place except the region covered by the cotton thread. The microtube was anchored to the plant with the help of a cello-tape. Sampling During active flowering phase, the fully opened flowers were tagged on the day of anthesis. The label was fed to the inflorescence at 15 DAA, as the active phase for oil synthesis starts from this period (Singh 1998, Archana et al. 1999). The pods from such flowers were sampled at 7 days interval starting from 21 DAA until complete maturity of the crop. Extraction and fractionation o f lipids
Total lipids were extracted using Folch method (Folch et al. 1957). The dried seeds (100 rag) were homogenized thoroughly in a pestle and mortar with 50 mg of anhydrous sodium sulphate and 2 ml of chloroform:methanol (2:1 v/v). The aqueous contaminants were partitioned into the upper aqueous phase following the addition of one-fifth volume of 1% NaC1. The upper and lower phases were separated using separatory funnel and washed separately with pure lower and upper phase solvents, respectively. The combined extracts from lower phase were dried under a stream of nitrogen gas and dissolved in a minimum volume of chloroform (1 ml). Fifty ~tl of the total lipid fraction were taken in
A SIMPLE METHOD FOR IN VIVO LABELLING ...
o v o x
>, o 3O E O.
_ _ Z1=
~ 28 OQys
Fig. 1. In vitro [1-]4C]-acetate labelling of lipid fractions in developing seeds of Brassica campestris.
the scintillation vial containing 5 ml of scintillation fluid [ethanol - toluene ; 40 : 1000 (v/v) + 8.4 g PPO + 0.14 g POPOP]. Remaining total lipids were separated into polar and non-polar lipids by solvent partition method (Nichols 1964). Non-polar lipids were eluted in petroleum ether (b. pt. 80-100 °C), while the polar lipids were eluted in methanol. Hundred pl each of polar and non-polar lipid fractions were added to scintillation vials containing the above scintillation fluid. Polar and non-polar lipids were further fractionated into individual components by preparative thin layer chromatography (TLC) on TLC plates (20 x 20 cm) coated with 0.5 mm thick layer of silica gel G (type 60; E. Merck). The solvent system used for the fractionation of non-polar lipids (Schwertner and Biale 1973) was n-hexane-diethyl ether-acetic acid; 80 : 20:1 (v/v/v). Glycolipids from the polar lipid fractions (Mackender and Leech 1974) were resolved into MGDG and DGDG by TLC using toluene-ethyl acetate-ethanol; 2 : 1 : 1 (v/v/v). Phospholipids remained at the origin in this solvent system and were separated by two dimensional TLC. The solvent system chloroform- methanol-water; 65 : 25 : 4 (v/v/v) was used in first dimension, while chloroform-acetone-methanol-acetic acid-water; 100 : 40 : 20: 20 : l0 (v/v/v/v/v) was used in the second dimension. The separated phospho- and glycolipids were identified by co-chromatography with authentic standards and by spray of specific reagents (arsenomolybdate and anthrone reagents for
Fig. 2. Incorporation of [1-14C]-acetate label in total (×), non-polar (©) and polar (zx)lipid fractions of developing seeds of Brassica campestris.
phosphorus and sugars, respectively). The spots obtained after TLC were scrapped off from the plates into the scintillation vials containing the scintillation fluid (5 ml). Various lipid fractions were counted for radioactivity on a LKB 1211 Wallac Rackbeta Scintillation Counter. All counts were corrected for background and also for quenching by the external standard ratio method. Each experiment was repeated twice and the samples thus collected were further analysed in duplicates. Hence, each point in the figures is the mean of four values +_ S.E.
Results and Discussion T h e method of in vivo labelling of lipids and lipid fractions gave high incorporation rates and produced labelling patterns from [ 1-[4C] acetate similar.to those obtained in studies conducted for determining lipid composition of the developing seeds of unlabelled green house grown crop of Brassica campestris (Archana et al. 1999). The incorporation of label into total lipids increased progressively from 21 DAA (10,470 cpm-g -1 dry wt) to 42 DAA (19,640 cpm.g -1 dry wt), attaining almost 100 % increase during this period. Hereafter, the incorporation further increased and attained a value of 22,840 cpm.g -1 dry wt at 56 DAA (Fig. 2). At the early stages of seed development, the incorporation of label into polar lipids was much higher (48% at 21 DAA), which later on decreased to 13 % at 56 DAA. However, the trend for the incorporation of
N. SHARMA, ARCHANA, S.P. MALHOTRA & R. S1NGH
6O a a
4C g 30
~ o =
Oays o f f e r
i 35 Doys
Fig. 4. Incorporation of [l-14C]-acetate label in phospholipid (©) and galactolipid (zx) fractions of polar lipids of developing seeds of Brassica campestris.
label into non-polar lipids was just the opposite to that obtained for polar lipids. Non-polar lipids were labelled predominantly at the later stages of seed development (75 %) rather than at earlier stages (30 %). These results are in agreement with those reported earlier by Perry and Harwood (1993a) and Archana et al. (1999). At earlier stages, membrane lipids are relatively more important, while at later stages, relative importance of storage lipids is more obvious. As in groundnut (Ghosh and Sastry 1986), castor bean (Canvin 1963) and Brassica napus (Perry and Harwood 1993a, Aach et al. 1997), there were three distinct phases of lipid accumulation in developing seeds of Brassica campestris also. During 0-15 DAA, very little non-polar lipids are formed but in the next period lasting from 21-42 DAA, there is rapid accumulation of lipids. During the third phase from 49-56 DAA, lipid synthesis continues but at a much reduced rate..
and then increased. However, significant changes occurred at 28 DAA and at 42 DAA. During this period, the incorporation of label showed opposite trend in these two fractions, i.e. incorporation increased in TAG fraction and decreased in DAG fraction, suggesting that the final acyl transfer step, diacylglycerol acyl transferase (DAGAT) may be limiting at times when there were high rates of lipid synthesis (Archana et al. 1999, Perry et al. 1999). The possibility that DAGAT activity may regulate the rate of TAG accumulation in oil tissues was inferred from experiments conducted by Griffiths and Harwood (1991). At no stage, incorporation of label in monoacyl glycerol (MAG) could be detected, indicating absence of any lipase activity during seed development ofBrassica campestris. It was contrary to the observations of Perry and Harwood (1993b), where they found lipase to be present in developing seeds of Brassica napus.
Incorporation of label in TAG followed a pattern similar to that of non-polar lipids (Fig. 3), amounting to about 30 % of total non-polar lipids at 21 DAA and 75 % at maturity. Active synthesis of TAGs starting at 20 DAA registered dramatic increase in subsequent stages, reaching maximum value at maturity (Archana et al. 1999). Since there was maximum incorporation of label into lipids during this phase, the dramatic increase in TAG during this period was eminent as TAG was the main fraction of storage lipids. The other non-polar lipid, in which the label was found to be incorporated, was DAG, which decreased up to 28 DAA
T h e Kennedy pathway for TAG synthesis (Perry et al. 1999, Harwood 1998) involves the participation of phospholipids. Phosphatidic acid and phosphatidyl choline play an important role in lipid accumulation in many oil storing seeds (Stymne and Stobart 1987). Keeping this in view, we examined the distribution of radioactivity into the individual polar lipid fractions also (i.e. phospholipids and glycolipids separately). The label was always present in both the polar lipid fractions. However, phospholipid fraction was more radiolabelled than the glycolipid fraction at all the stages of seed development (Fig. 3). These results bear close resem-
Fig. 3. Incorporation of [1-14C]-acetate label in TAG (o) and DAG (a) fractions of non-polar lipids of developing seeds of
A SIMPLE METHOD FOR IN VIVO LABELLING ...
"0_ 4o -&
~. 2o I
Ooys a f t e r
Fig. 5. Incorporation of [ 1-14C]-acetate label in phosphatidyl choline (o) phosphatidyl ethanolamine + phosphatidyl glycerol (zx) and phosphatidic acid (×) fractions of phospholipids of developing seeds of Brassica campestris.
blence to those reported earlier by Archana et al. (1999). Among the phospholipids, though the radioactivity could always be detected in different phospholipid fractions, its amount was very low in phosphatidic acid (Fig. 5), proving that the third stage of Kennedy pathway (phosphatidate phosphatase) exerts little flux control over the overall pathway. Among different fractions, phosphatidyl choline was maximally labelled, while the phosphatidyl ethanolamine and phosphatidyl glycerol were moderately labelled. Similar pattern of phospholipid deposition was observed earlier in seeds producing highly unsaturated oils such as safflower (Slack et al. 1978) and sunflower (Stymne and Stobart 1984a, 1984b), where phosphatidyl choline is involved in the production of linoleate- and linolenate-enriched TAGs. Phosphatidyl choline has now been shown to play a more general role in the movement of fatty acids during TAG biosynthesis (Schltz and Ohlrogge 2000). The significant accumulation of these phospholipids may also indicate cessation of membrane biogenesis at this stage. M G D G and DGDG were the two main glycolipids in which radioactivity could be detected throughout the period of seed development. However, the pattern of label incorporation was just the opposite in these fractions of glycolipids (Fig. 6). During early stages of seed development, MGDG was the fraction which was predominantly radiolabelled. The incorporation in this fraction decreased up to 42 DAA and then remained almost constant. In con-
Days a f t e r
Fig. 6. Incorporation of [l-14Cl-acetate label in MGDG (o) and DGDG (zx) fractions of galactolipids of developing seeds of Brassica carnpestris.
trast, in DGDG fraction, the radioactivity detected only in very small amounts during early stages, increased progressively up to 42 DAA and then remained almost constant. These results confirmed the earlier findings on Brassica campestris (Archana et al. 1999), where we argued that MGDG might be converted to DGDG during seed development. The results presented here are in close agreement, in all respects, with the results obtained for the overall changes in lipid composition during seed development of field/greenhouse grown Brassica campestris (Archana et al. 1999), indicating that the method of in vivo labelling reported here is quite safe, useful and effective in studying lipid metabolism during seed development in oil crops. Further, the method does not suffer from any drawback associated with in vitro studies which are usually conducted with detached tissues/organs and do not reflect the true picture of developmental changes occurring during seed maturation. As suggested by S ingh (1998), there may be developmental changes in the expression of metabolic pathways inside the plastids synthesizing seed reserves and in vitro studies carried out with detached tissues/organs or isolated plastids from these tissues suffer from many such limitations. It is, therefore, essential to obtain information on the role of these plastids in adjusting their metabolism with changing developmental stage of a tissue, which is possible only by use of a method involving in vivo labelling without causing any disturbance to the intact plant. Such studies would help us in delineating the details of
N. SHARMA, ARCHANA, S.P. MALHOTRA & R. SINGH
regulatory aspects of different metabolic pathways in developing seeds of crop plants. Acknowledgments
T h e financial assistance given by ICAR in the form of a research project entitled "Development of an in vitro liquid culture system for the study of lipid metabolism in Brassica campestris L." is thankfully acknowledged. Dr. O.K Hooda of Central Institute of Research in Buffalo, Hisar is thanked for providing the scintillation counter facilities. References Aach H., H. Frank and K.P. Heise 1997. Distribution of lipid radioactivity after fractionation of 14C-labelled zygotic rape embryos, J Plant Physiol, 151: 323-328. Archana, N. Sharma, S. P. Malhotra and R. Singh 1999. Changes in lipid composition during seed development of Brassica campestris, J Plant Biol, 26: 165-172. Canvin D. T. 1963. Formation of oil in Ricinus communis L., Can J Biochem Physiol, 41 : 1879-1885. Folch J., M. Lees and G. H. S. Stanley 1957. A simple method for the isolation and purification of total lipids from animal tissues, J Biol Chem, 226: 497-509. Ghosh S. and P. S. Sastry 1986. Triacylglycerol synthesis in developing seeds of groundnut (Arachis hypogaea): Lipid accumulation and incorporation of labelled precursors during seed maturation, Indian J Biochem Biophys, 23: 100-104. Griffiths G. and J. L. Harwood 1991. The regulation of triacylglycerol biosynthesis in cocoa (Theobroma cacao L.), Planta, 184: 279-283. Harwood J. L. 1998. Plant lipid biosynthesis: Fundamentals and agricultural applications. Cambridge University Press, Cambridge. Mackender R. O. and R. M. Leech, 1947. The galactolipid, phospholipid and fatty acid composition of the chloroplast envelope membranes of vicia faba L., Plant Physiol, 53: 496-502. Nichols B. W. 1964. Lipid separation by solvent partitioning, in New biochemical separations, edited by A T James & L J Morris (Van Nostrand Co., London) 321-325.
Perry H. J. and J. L. Harwood 1993a. Changes in lipid content of developing seeds of Brassica campestris, Phytochemistry, 32:1411-1415. Perry H. J. and J. L. Harwood 1993b. Radiolabelling studies of acyl lipids in developing seeds of Brassica napus: Use of [1-14C] acetate precursor, Phytochemistry, 33: 329-333. Perry H. J., R. Bligny, E. Gout and J. L. Harwood 1999. Changes in Kennedy pathway intermediates associated with increased triacylglycerol synthesis in oil seed rape, Phytochemistry, 52: 799-804. Schultz D. J. and J. B. Ohlrogge 2000. Biosynthesis of triacylglycerol in Thunbergia alata: Additional evidence for involvement of phosphatidyl choline in unusual monoenoic oil production, Plant Physiol Biochem, 38: 169-173. Schwertner H. A. and Y. B. Biale 1973. Lipid composition of plant mitochondria and chloroplasts, J Lipid Res, 14: 235-242. Singh R. 1998. Carbon and energy sources for fatty acid biosynthesis in non-photosynthetic plastids of higher plants, Proc Indian Natl Sci Acad, B 64: 335-354. Singh S. P. and S. L. Mehta 1992. Manipulation ofoil quality and quantity in annual oilseed crops, J. Oilseed Res, 9:97-118. Slack C. R., P. G. Roughan and N. Balasingham 1978. Labelling of glycerolioids in the cotyledons of developing oilseeds by [1-14C]-acetate and [2-3H]-glycerol, Biochem J, 170: 421-433. Smith R. G., D. A. Gautheir, D. T. Dennis and D. H. Turnip 1992. Malate and pyruvate dependent fatty acid synthesis in leucoplasts from developing castor endosperm, Plant Physiol, 98: 1233-1238. Stymne S. and A. K. Stobart 1984a. Evidence for the reversibility of the acyl-CoA lysophosphotidyl choline acyltransferase in the microsomes of developing safflower cotyledons and rat liver, Biochem J, 223: 305-310. Styrene S. and A. K. Stobart 1984b. The biosynthsis of triacylglycerols in microsomal preparations of developing cotyledons of sunflower (Helianthus annuus L.), Biochem J, 220:481-488. Stymne S. and A. K. Stobart 1987. Triacylglycerol biosynthesis, in Biochemistry of plants_edited by P K Stumpf & E E Conn (Academic Press, New York) 9: 175-214. Triki S., C. Demandre and P. Mazliak 1990. Biosynthesis of triacylglycerols by developing sunflower seed microsomes, Phytochemistry, 52: 55-62.
ReceivedJanuary 24, 2001; accepted October04, 2002