Spectral quality during pod development affects omega-6 desaturase activity in soybean seed endoplasmic reticulum

PHYSIOLOGIA PLANTARUM 91: 346-351. 1994

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Spectral quality during pod development affects omega-6 desaturase activity in soybean seed endoplasmic reticulum Marcia J. Holden, Helen A. Norman and Steven J. Britz

Holden, M. J., Norman, H. A. and Britz, S. J. 1994. Spectral quality during pod development affects omega-6 desaturase activity in soybean seed endoplasmic reticulum. - Physioi Plant. 91: 346-351. Polyonsaturated fatty acids (i.e. linoleic acid [18:2], linolenic acid [18:3]) in triacylglycerois (TAG) of soybean seeds increase more during reproductive development under simulated shadelight: i.e., light with reduced blue and/or increased far-red (Britz and Gavins 1993). Elevation of 18:2 and 18:3 is matched by corresponding reduction of oleic acid (18:1), consistent with observations that total seed oil remains constant. We therefore tested the hypothesis that spectral quality affects the activity of the omega-6 and/or omega-3 desaturases involved in TAG biosynthesis. Membranes were isolated from developing soybean cotj'ledons 24-31 days after flowering. Separate fractions, enriched for chloroplasts and endoplasmic reticulum (ER) respectively, were obtained by sucrose gradient ceotrifugation and incubated in an in vitro desatyrase as.say system containing "'C-18:l-CoA at room temperature. Omega-6 and omcga-3 desaturase activity was. calculated from the rate of formation of "'€-18:2 and '''C-18:3. Radioactive 18:2 and 18:3 were recovered only from phosphatidylcholine (PC) in both ER and chloropiast membranes, consistent with membrane-bound desaturases with specificity towards PC. The specific activity of omega-6 desaturase was high in ER membranes from seeds matured under light sources that promote a canopy shade response, but was reduced in membranes from seeds matured under lamps (high blue and low far-red emission) previously shown to reduce the level of 18:2 in seed oil by 50%. Chioroplast membranes possessed both omega-6 and omega-3 desaturases. Light appeared to have little or no effect on the activity of chioroplast desaturases. Key words - Chloropiast, omega-6 desaturase, endoplasmic reticulum, Glycine max, low pressure sodium lamps, soybean, spectral quality. M. J. Holden (corresponding author) and. S. J. Briiz. Climaie Stress Laboratory; H A. Norman, Weed Science Laboratory, USDA-ARS, Beltsvllle Area Research Center, Beltsville, MD, 20705, USA. This paper in part of the contributions to the European Symposionn Photomorphogenesis in Plants, held in Tirrenia, Pisa, Italy. 11-15 July, 1993.

- , . , . . Introduction A recent study has shown that light quality can have a profound effect on fatty acid desaturation in developing soybean seeds. Britz and Cavins (1993) investigated the specific effects of low pressure sodium (LPS) and cool white fluorescent (CWF) lamps on soybean seed development. While seed parameters such as dry matter, protein, and oil (as. % of seed dry matter) remained the same,

speciftc changes m the proporttons of some fatty actds ^ , ?,,. . • JMOI C J were observed. Htgher concentrations of 18:2 were found in seeds that developed tinder LPS (blue deficient) latnps cotnpared with those that developed under CWF (broad spectrum) lamps (53 vs 37%). Concurrent with the increase in 18:2 tittder LPS lamps was a proportional decrease in 18:1. Other fatty acids remained essentially the same (Britz and Cavins 1993). When plants were grown under CWF lamps supplemented with far red radiation.

Received 20 December, 1993 346

Physiol. Plane. 91. 1994

values for 18:1 and 18:2 were intermediate between those seen with CWF alone and those seen with LPS lamps (S. J. Britz, unpublished data). These effects, thought to be mediated by phytochrome and blue light photoreceptors, indicated that canopy shade can affect fatty acid content of soybean seeds. The low ratio of 18:1/18:2 in daylit plants could result from internal shading of developing seeds in green pod walls. Such shading would drastically lower the PP/PM in seeds. Not a lot is known about the regulation of fatty acid desaturation in triglycerides of seeds or the effect of environment on the process. Understanding of this regtilation is of practical interest. Alteration of the proportions of 18:! and 18:2 in favor of an increase in 18:1 is a desirable outcome in terms of cutxent dietary' recommendations and reducing oxidative damage during oil storage (Parthasarathy et al. 1990, Smith 1981). There are several metabolic steps between synthesis of 18:1 in the chioroplast and desaturation to 18:2 in the ER. These include hydrolysis of 18:l-acyl carrier protein (ACP), esterification to CoA, transport of 18:l-CoA across the chloropiast membranes, incorporation into lysophospholipid species in ER membranes and desaturation to 18:2 on phosphatidylcholine (PC; Jaworski 1987). Despite the multiple steps involved, there is precedence for the PC-linked oleate (omega-6) desaturase as a likely candidate for regulation by spectral quality. High 18:1 mutants of soybean and sunflower previously showed decreased omega-6 desaturase activity (Martin and Rinne 1986, Garces and Mancha 1991). In this study, the roles of blue and far-red radiation in the regulation of the activity of oleate desaturase (EC 1.3.1.35) were investigated. Plants were grown under (1) blue and far-red deficient LPS lamps, (2) broad spectrum CWF lamps alone, and (3) CWF plos supplemental farred radiation (CWF-(-FR). Abbreviations - CHAPS, 3-([3-cholamidopropyll-dimethylammonio)-l-propane-sulfonate; CWE, cool white fluorescent; FA, fatty acid; EAME, fatty acid methyl esters; LPS, low pressure sodium; PC, phosphatidyicholine; Pf^, far-red absorbing phytochrome; P|M. total phytochrome; Pf/P,,,,, phytochrome photostationarj' state; TAG, triacylglycerol; 18:1, oleic acid; 18:2, linoleic acid; 18:3, hnolenic acid.

Materials and methods Plant material and growth conditions A determinate, early flowering, strongly dwarfed soybean {Glycine max L. Merr.) was used (Britz and Cavins 1993). Plants were grown in vermiculite in eleven-1 pots watered daily with a modified half-strength Hoaglands solution in controlled environment chambers (EGC M-31, Chagrin Falls, OH, USA) at 27±1°C., 6 0 ± 5 % relative humidity, 400 (xl 1'' CO2 and 14 h of light per 2.4 h period from either CWF lamps (F96T12CWWHO; Sylvania, Danvers, MA, USA), LPS lamps (SOX 180 W; Philips N. A., Bloomington, NJ, USA), or CWF lamps Physiol. Plant. 91. t994

supplemented with incandescent lamps (100 W) and farred fluorescent lamps (F96T12/232A'HO; Sylvania). Photosynthetically active radiation (PAR) averaged 500600 for all of the light treatments as measured at the top of the plant canopy. Bed height was adjusted to compensate for the large differences in plant height resulting from differential spectral quality (Britz and Cavins 1993). Lamps were separated from the plant compartment by an acrylic barrier. Irradiances were determined either with a spectroradiometer (Model 740A, Optronics Laboratories, Orlando, FL, USA) as described elsewhere (Britz 1990) or with a quantum sensor (LiCor Corp., Lincoln, NE, USA) calibrated with a spectroradiometer for use with LPS lamps. Spectroradiometric measurements were used to characterize the spectral quality of the three light sources. Seeds were harvested at intervals up to and including maturity for oil analysis or were har\'ested about 24 and 31 days after flowering (DAP) for isolation of membranes and subsecjoent assay of fatty acid desaturase activity. Seeds came from relatively well-illuminated pods located on the upper half of the plant. Only two growtb chambers were utilized at any time. One chamber was set up with LPS lamps and the other with CWF or CWF + FR. Experiments were ruti concurrently in LPS and CWF equipped chambers or LPS and C W F - F F R equipped chambers. Seeds from LPS-grown plants were handled under LPS lamps during harvesting and the initial steps in membrane isolation (homogenization and centrifugation). Membrane isolation and assays Seeds (40 g) were homogenized using a mortar and pestle in a medium (80 ml) consisting of 250 mM sucrose, 15 mM HEPES, pH 7.5, 1 mM EDTA, 1 mM EGTA, 0.1% BSA, 0.5% polyvinylpyrrolidone (PVP), and 0.25 mM phenylmethylsulfonyl fluoride. The homogenate was filtered through nylon mesh cloth and centtifuged at 1 000 g for 10 min. The supetnatant (15 ml) was layered onto continuous sucrose gradients of 20% to 56% sucrose in 5 mM HEPES, 1 mM EDTA, ! mM MgCli, pH 7.5. The gradients were centrifoged at 60 000 g for 1.5 h and then fractionated into 1.5-ml fractions. Protein was determined by the dye-binding assay of Bradford (1976) and chlorophyll by the method of Vernon (1960). Antimycin A-insensitive, NADPH cytochrome c reductase and cytochrome c oxidase (enzyme markers for ER and mitochondria, respectively) were measured by the methods of Hodges and Leonard (1972). In vitro fatty acid desaturasc assay Membrane fractions, purified by sucrose density gradient centrifugation, were resuspended in incubation buffer (30 mM HEPES, pH 7.5, containing 330 n:iM sorbitol, 1 tnM EDTA, 1 mM BGTA, 8 mM ATP, 1 mM MgCU, 0.2% BSA, 2.7 mM NADH, 2.7 mM NADPH, 10 mg 347

ml-' ferredoxin, 0.5% PVP, 0.5% CHAPS and catalase [2000 units ml"', bovine liver, Sigma Chem. Co., St. Louis, MO, USA]; Norman et al. 1991). Membranes were incubated with '''C-18:l-CoA at room temperature for 45 min. Reactions were terminated with methanol, lipids were extracted and resolved into neutral lipid, galactolipid, and phospholipid classes using silica Sep-Paks, and phosphatidylcholine (PC) was recovered by TLC (Norman and St. John 1987) from total phospholipids. Fatty acid methyl esters (FAME) were prepared using BFjmethanol and resolved by HPLC with in-line radioactivity detection for determination of radiolabeled FAME and desaturation rates (Norman et al. 1991). FAME were resolved by HPLC employing a 25 cm x 4.6 mm i.d. Beckman Ultrasphere (5 jim) reversed-pbase C,8 column. The mobile phase was MeOH-MeCN-H^O (76:12:12) delivered at a flow rate of 1 ml min"'. For detemiination of acyl-CoA species at the end of the desaturation assay, the aqueous phase of the original lipid extraction was saponified at 80°C and then acidified. FAME were prepared with BFj-methanol, extracted with petroleum ether, and resolved by HPLC. Fatty acid analysis of soybean oil Seed samples (200 mg) were ground and treated with sodium methoxide (5 ml) for 45 min and acidified (1 ml of 10% [v/v] acetic acid). FAME were then extracted

from the aqueous mixture into heptane and analyzed by gas-liquid chromatography (Varian model 3700, 2 m x 2 mm column packed with 100/120 mesh GasChrom Q coated with 5% LAC-2R-446; AUtech Assoc, Deerfield, IL, USA) used as column support. Results and discussion The pattem of oil and fatty acid synthesis in developing soybean seeds grown under different light qualities was followed starting about 20 days after flowering (DAF). While oil accumulation occurred throughout the period of seed development from 24 to 50 days, biosynthetic activity was most active between 20 and 40 days after flowering (Fig. 1A,B). Seeds developing under LPS lamps showed a steady decrease in the proportion of 18:1 and an increase in 18;2 (Fig. ID) over the period of oil accumulation consistent with the relative amounts of the fatty acids observed previously in the mature seed (Britz and Cavins 1993). The proportion of 18:1 was generally higher than that of 18:2 in the seeds of the CWF-grown plants (Fig. 1A,C). Again, this reflects the values measured in the mature seed oil (Britz and Cavins 1993). Developing soybean seed was harvested between 24 and 32 DAF during the active phase of oil accumulation. Membrane fractions were prepared from seed homogenates using sucrose density gradients (Fig. 2). Fractions enriched for chloroplasts (e.g., fraction 5, Fig. 2) and eo

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Fig. 1. Developmental pattern of soybean fatty acid composition of seeds from plants grown under LPS or CWF lamps. In A and B, the data are expres.sed as mg oil seed"' or mg fatty acid seed"'. The same data are expressed as % (w/w) of total oil or fatty acid in C and D. Data are from one experiment represctitative of two. 348

Physiol. Plant. 9t. 1994

Fig. 2. Sucrose gradient centrifugation of post 1 000 g supernatant of soybean seed homogenate. Subcellular markers were chlorophyll for chloroplasts, cytochrome c oxidase for mitochondria, and NAD(P)H-cytochrome c reductase for endoplasmic reticulum. Enzyme activities are expressed as nmol Cyt c reduced or oxidized (mg protein)"' min"'. Chlorophyll is expressed as mg fraction"'. Data are from one experiment, representative of sixteen.

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Fraction number endoplasmic reticulum (e.g., fraction 8, Fig. 2) were assayed for omega-6 desaturase activity using '*C-18:1CoA as substrate. Under the conditions of this assay, desattirafion to 18:2 and 18:3 was measureable only when '•*C-18:1 was esterified to phosphatidylcholine. However, '*C-18:1 was found in the neutral, galacto-, and other phospholipid fractions, suggesting that there was acyltransferase activity with a variety of lipid substrates. No desaturation occurred in the absence of NADH or NADPH (data not shown). Plant grown under LPS lamps had higher levels of omega-6 desaturase activity (Fig. 3) and the mature seed oil contained higher levels of 18:2 (52.5% of total FA) as compared to seed 18:1 (26.8%; Britz and Cavins 1993). In contrast, plants grown under CWF lamps had higher levels of 18:1 (46% of total FA) as compared to 18:2 (36%; Britz and Cavins 1993), and concurrently lower Ptiysiol. Plant. 9t, 1994

levels of omega-6 desaturase activity (Fig. 3) during the most active periods of TAG synthesis in the developing seeds. The matttre seeds from plants grown under CWF supplemented with far red had percentages of 18:1 and 18:2 that were intermediate between those of seeds from the CWF and LPS grown plants (S. J. Britz and J. F Cavins, unpublished data). The desaturase activity of developing seeds from CWF-i-FR was also intermediate (Fig. 3) betvi'een the CWF seeds and the LPS seeds. Since the labeled 18:2 was found linked to PC and 18:l-CoA was the added substrate, it is clear that at least two steps were required in the process of desaturation. That is, the "C-IS:! tnust be esterified to lysopbospholipid in the endoplasmic reticulum by an acyl transfeiase and then desaturated. Thus it is possible that the acyl transferase could be a rate-limiting step in the process restricting the amount of "'C-18:1-PC that is available as 349

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Fig. 3. Phosphatidylcholine-linked omega-6 and omega-3 desaturase activities in membrane fractions fi'om developing soybean seeds grown under CWF, CWF (supplemented with FR) or LPS light sources. The fatty acid substrate for omega-6 desatnrase is 18:1 (oleic acid) and the substrate for the onaega-3' desaturase is 18:2 (linoleic acid). Data are averages of 4

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8 experiments for LPS. Developing seeds were harvested between 24 and 32 DAF. Rates are expressed as; nmol 18:2 or 18:3 synthesized (mg protein)"'

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