William B. Terzaghi2. Department of Biology, University of Utah, ... containing 50 mL of Leggett and Frere hydroponic medium. (LF medium; 8),modified by the ...
Plant Physiol. (1989) 91, 203-212 0032-0889/89/91/0203/1 0/$01 .00/0
Received for publication February 16, 1989 and in revised form May 11, 1989
Manipulating Membrane Fatty Acid Compositions of Whole Plants with Tween-Fatty Acid Esters1 William B. Terzaghi2 Department of Biology, University of Utah, Salt Lake City, Utah 84112, and Carnegie Institute of Washington, Department of Plant Biology, Stanford, California 94305 were transported inside plants. Plants of several different species incorporated exogenous fatty acids supplied as Tweenesters into membranes of both the tissues to which they were applied and tissues elsewhere in the plant.
ABSTRACT This paper describes a method for manipulating plant membrane fatty acid compositions without altering growth temperature or other conditions. Tween-fatty acid esters carrying specific fatty acids were synthesized and applied to various organs of plants growing axenically in glass jars. Treated plants incorporated large amounts of exogenous fatty acids into all acylated membrane lipids detected. Fatty acids were taken up by both roots and leaves. Fatty acids applied to roots were found in leaves, while fatty acids applied to leaves appeared in both leaves higher on the plant and in roots, indicating translocation (probably in the phloem). Foliar application was most effective; up to 20% of membrane fatty acids of leaves above the treated leaf and up to 40% of root membrane fatty acids were exogenously derived. Plants which took up exogenous fatty acids changed their pattems of fatty acid synthesis such that ratios of saturated to unsaturated fatty acids remained essentially unaltered. Fatty acid uptake was most extensively studied in soybean (Glycine max [L.] Merr.), but was also observed in other species, including maize (Zea mays L.), mung beans (Vigna radiata L.), peas (Pisum sativum L.), petunia (Petunia hybrida L.) and tomato (Lycopersicon esculentum Mill.). Potential applications of this system include studying intemal transport of fatty acids, regulation of fatty acid and membrane synthesis, and influences of membrane fatty acid composition on plant physiology.
MATERIALS AND METHODS Plant Materials Seeds of all species used were surface-sterilized by treatment with chlorine gas for 5 h, and germinated axenically. Except where indicated, seedlings were transferred to sterilized quart glass canning jars (covered with a 100 mm glass Petri dish lid) containing 50 mL of Leggett and Frere hydroponic medium (LF medium; 8), modified by the addition of Fe (as Sequestrene) to 90 gM. Plants were maintained at 25°C under continuous illumination (200 ,uE m-2 s-') from fluorescent bulbs (Sylvania cool-white), except where indicated in the text. Chemicals Tweens carrying specific fatty acids were synthesized as described ( 14). All solvents were reagent grade. Lipid Extraction and Analysis
Lipids were extracted and analysed as described (14) (WB Terzaghi, DC Fork, JA Berry, CB Field, unpublished data). Briefly, lipids extracted with hexanes:isopropanol (3:2, v/v) were usually fractionated by TLC and those comigrating with authentic Tweens were discarded (to prevent unmetabolized Tweens from contributing fatty acids to the analysis). Fatty acid methyl esters prepared from the remaining lipids by reaction with sodium methoxide in methanol were analyzed by GLC on a Varian 2100 gas chromatograph using 6-foot glass columns packed with 10% SP 2330 on Chromosorb 100/120 (Supelco). NL3 comigrate with Tweens in the TLC systems used, consequently fatty acids were prepared solely from PL and GL (i.e. membrane lipids). For radioactive analyses lipids were also extracted as described (14), except that the plant materials were ground with a mortar and pestle
Many aspects of plant fatty acid metabolism and influences of membrane fatty acid composition on plant physiology are poorly understood (1, 5, 7). To help study these problems a system for altering the membrane fatty acid composition of cultured soybean cells by adding Tween-fatty acid esters to their growth medium was developed (14, 15). Although this system is suitable for many studies related to membrane and lipid metabolism, its usefulness is limited by the requirement to work with cultured cells. This paper reports that Tween-fatty acid esters may also be used to modify membrane fatty acid compositions of whole plants, together with the unexpected result that fatty acids ' Supported by a grant from the NIES No. 01498 to Dr. Karl G. Lark, and by a grant from the Carnegie Institution of Washington to Dr. Christopher B. Field. CIW/DPB paper No. 1028. 2Supported by a Carnegie Institution of Washington postdoctoral Fellowship, by NSF predoctoral fellowship SPE 835-0132, and by a University of Utah graduate research fellowship. Present address: Plant Science Institute, Department of Biology, University of Pennsylvania, Philadelphia, PA, 19104.
3Abbreviations: NL, neutral lipids; PL, phospholipids; GL, glycolipids; SG, sterol glycosides; ESG, esterified sterol glycosides; FAME, fatty acid methyl esters; FFA, free fatty acids; MGDG, monogalactosyl diglyceride; DGDG, digalactosyldiglyceride; SL, sulfolipid; PC, phosphatidylcholine; PG, phosphatidylglycerol; PE, phosphatidylethanolamine; PI, phosphatidylinositol; LPC, lysophosphatidylcholine; CL cardiolipin; PA, phosphatidic acid. 203
Plant Physiol. Vol. 91,1989
TERZAGHI
204
Table I. Incorporation of 17:0 into Membrane Lipids Tween-1 7:0 was applied to the primary leaves or added to the nutrient solution (to 10 mm 17:0) of 10 d old soybean seedlings (cv Noir 1) growing axenically in glass jars. Eight d later lipids were extracted and analyzed as described in "Materials and Methods." Roots and shoots of untreated plants and those with Tween added to their medium were extracted separately. Roots, primary and trifoliolate leaves of those with Tweens applied to their primary leaves were extracted separately. Two plants were pooled for each sample. Fatty Acid Composition Treatment
Sample 16:0
17:0
16:1
18:0
18:1
18:2
18:3
sata
MO/%~~~~~~st
mol%
Untreated
Roots
25
NDb
ND
2
4
32
36
27
Leaves
16
ND
1
62
17
12
2
31
34
30
11
trP
2
2
3 4 3 1 1 1
17
16
1 ND
27
53
15
22 10 19
23 67 55
55 21 25
Roots Leaves Applied to 10 leaves Roots 10leaves Trifoliolates a Total saturated fatty acids (mol %). Added to medium
ND 3 16 36 1 1 10 10 tr 1 9 15 b C Trace. Not detected.
after boiling in isopropanol and the crude extract was filtered through GF/C filters. The filter cake (and filter) was then ground in more hexanes:isopropranol (3:2, v/v), and this extract was filtered through a second GF/C filter. The material left on this second filter is referred to as the filter cake; subsequent fractionation into lipid and aqueous phases and then of the lipid components was done as described (14). Radioactivity in the various fractions was determined by scintillation counting. For analysis of individual lipids crude extracts were separated into classes by column chromatography on silica gel (Baker 60/200) sequentially eluted with 10 column volumes each of chloroform (NL), acetone (GL), and methanol (PL). Each class was then fractionated by TLC and fatty acid methyl esters were prepared from individual lipids (identified with diagnostic sprays and by comigration with authentic standards); for radioactive analyses each lipid was scraped into vials and its radioactivity was determined by scintillation counting.
,15
10
5
£10 I
5
Liposome Preparation and Fluorescence Depolarization Fluorescence intensity and depolarization of trans-parinaric acid inserted into liposomes prepared from membrane PL was measured as described (16). Fo Chi Fluorescence
Fo Chl fluorescence as a function of temperature was measured as described ( 12). Tween Application Filter-sterilized Tweens were either painted onto leaves using sterilized metal spatulas, or added to the hydroponic solution as indicated. RESULTS Fatty Acid Uptake and Translocation Tweens carrying 17:0 (a fatty acid not found in soybeans) were synthesized and either applied directly to leaves or added
0
5
10
15
20
Days after imbibing
Figure 1. Kinetics of 17:0 incorporation by soybean (cv Noir I) seedlings. A, Surface-sterilized seeds were germinated in jars containing 20 mL LF medium supplemented with 10 mM 17:0 as Tween17:0. After 4 d the jars were transferred to constant light. At indicated times two plants were harvested, washed, separated into root and shoot, and lipids of each portion were extracted and analyzed as described in "Materials and Methods." B, Surface-sterilized seeds were treated as above, except that the medium was not supplemented with Tweens. Instead, Tween-1 7:0 was applied to the primary leaves once they were unfolded (10 d after imbibition). At indicated times two plants were harvested, washed, separated into roots, trifoliolate and primary leaves, and lipids of each portion were extracted and analyzed as described in "Materials and Methods." Note differences between panels in scale of vertical axes.
MANIPULATING PLANT MEMBRANE FATTY ACID COMPOSITIONS
nutrient solution of soybean seedlings growing axenically in glass jars (to avoid confusion due to microbial Tween metabolism). Plants treated with Tween-17:0 incorporated 17:0 into both roots and shoots regardless of application site, although foliar application was more effective (Table I). Larger amounts of 17:0 were observed in roots and in leaves younger than those treated with Tweens, indicating translocation of the exogenous fatty acids in some unknown form. Incorporation of 17:0 proceeded slowly, and maximal amounts of 17:0 in membrane lipids were not observed until up to 8 d after Tweens were applied to the plants (Fig. 1). Little incorporation was observed until the primary leaves appeared, even when Tweens were added to the medium (Fig. 1). In other studies, similar uptake kinetics were observed when Tweens were applied to older leaves, whereas little incorporation was observed when Tweens were applied to the cotyledons (W Terzaghi, unpublished data). Similar results were obtained with Tweens carrying other fatty acids, in soybeans, mung beans and maize (W Terzaghi, unpublished data). to the
205
tested incorporated exogenous fatty acids after 7 d of treatment, but largest amounts of incorporation were observed in soybean (cv Noir 1) and maize, which were therefore studied most extensively. Large differences were observed between soybean cultivars in 17:0 uptake. Seedlings of cv Noir 1 incorporated and translocated more 17:0 than seedlings of cv Minsoy when Tween-17:0 was applied to their primary leaves, although seedlings of cv Minsoy incorporated more 17:0 into roots when Tween-17:0 was added to their hydroponic medium (Table II). Differences between cultivars were not investigated in other species. Effects of Growth Conditions
Most plant species only tolerated foliar Tween application when grown under high relative humidity; either in sealed glass jars, humidified greenhouses or growth chambers, or when treated leaves were sealed inside plastic bags (under drier conditions treated leaves wilted and later abscised in all but a few species such as maize and alfalfa). Soybeans grown in sealed jars incorporated the most fatty acids; similar effects were observed in maize, although not as pronounced (Table IIIa). Maximal incorporation at application site and translocation of 17:0 was observed in both soybean and maize grown
Other Plant Species
Several plant species were tested for the ability to take up and transport exogenous fatty acids (Table II). Most plants
Table II. Plants Incorporating Exogenous Fatty Acids into Membrane Lipids Seeds of the indicated species were surface-sterilized, then germinated on sterile moistened filterpaper. Seedlings were transferred to sterile quart jars containing 20 mL LF medium, and were kept under constant light (200 IuE * m-2 s-1) at 260C. Tween-1 7:0 was either added to the medium (to 3 mM 17:0), or applied to the primary leaves once these were fully unfolded. Seven d after application plants were harvested, washed, and lipids were extracted separately from roots, primary leaves, and younger leaves as described in "Materials and Methods." [17:0] in Younger Treated Plant Leaves Organ [17:0] in Root [17:0] in Leaf
10
mol %
Glycine max cv Noir 1 cv Minsoy
Zea mays cv Trojan
Vigna radiata Unknown cv Pisum sativum cv Alaska
Lycopersicon esculentum cv Ace Medicago sativa L. Unknown cv
Root 10 leaf Root 10leaf
13 36 22 3
10 0 6
3 15 0 1
Root 10 leaf
11 15
2 8
3 10
Root 10leaf
10 17
17 5
No younger leaves 0
Root 10leaf
7 2
4 4
No younger leaves 0
Root 10leaf
7 0
0 3
No younger leaves 0
Root 10leaf
5 0
0 8
0 0
Root 10 leaf
2 0
1 4
0 0
Root 10leaf
4 2
0 3
0 0
1
Spinacea oleracea Unknown cv Petunia hybrida cv Mitchell
Plant Physiol. Vol. 91, 1989
TERZAGHI
206
at between 26 and 30°C; incorporation and translocation were reduced at higher or lower temperatures (Table IIlb). Nutrient concentrations also affected incorporation, and, to a lesser extent, translocation in soybeans (Table IMIc); maximal incor-
poration was observed in plants grown in 2x LF medium, which also gave best growth (W Terzaghi, unpublished data). Photoperiod had little effect on uptake (Table MId). Attempts to stimulate root uptake and translocation by growing plants hydroponically in the greenhouse in medium containing Tweens were unsuccessful (W Terzaghi, unpublished data). Distribution of Exogenous Fatty Acids between Lipids Plants used exogenous fatty acids to synthesize all membrane lipids detected, as illustrated by Figure 2, which compares lipids extracted from trifoliolate leaves of soybean plants whose primary leaves were treated with either [ 1-'4-C]acetate or Tween [1-'4-C] 18:1. Amounts of radioactivity incorporated into the lipid fractions are presented in Table IV.
Virtually all of the radioactivity applied as Tween was recovered from the plant, indicating very little net loss (e.g. from respiration), although some radioactivity was recovered from the medium, perhaps due to root exudation. Approximately 60% of the Tween radioactivity was recovered in the lipid fraction in all tissues; the remainder was about equally divided between the water soluble fraction and the filter cake. By contrast, distribution of acetate radioactivity varied substantially between tissues, and a much smaller proportion was translocated from the primary leaves (Table IVa). Essentially no radioactivity was incorporated into lipids by leaves which were extracted immediately after treatment with either substrate (W Terzaghi, unpublished data). Acetate- and Tween-treated plants also differed in their distribution of radioactivity between lipids: Tween-treated plants had more radioactivity in membrane lipids and less in NL (Table IVb). Distribution between membrane lipids was similar in both treatments, except that relatively little radioactivity was found in PG in Tween-treated plants. By contrast,
Table Ill. Effect of Growth Conditions on 17:0 Incorporation In part a) soybeans (cv Noir I) and corn (cv Trojan) were either grown in pots in a greenhouse, on a misting bench, in a growth chamber (day/night: 12/12 h, 30/200C, about 200 tE . m-2. s-1 PAR), or axenically in glass jars in the growth chamber. In b) plants were grown axenically in glass jars placed in growth chambers as in a) except that the temperature was either 15/1 00C, 30/200C or 40/300C. In c) plants were grown in the 30/200C growth chamber in jars containing 20 mL of either 1 x, 2x, 3x or 5x LF medium. In d) plants were grown axenically in glass jars either in the 30/200C growth chamber or under constant illumination at 260C. In all cases a), b), c), and d), Tween-1 7:0 was applied to the primary leaves once these were fully expanded. Seven d later plants were harvested, washed, and lipids were extracted and analyzed as described in "Materials and Methods." Roots, primary leaves, and younger leaves were extracted separately. Two plants were pooled for each sample. Treatment
Plant
Primary
[17:0] in bulk lipid Younger Rots
leaves
leaves mol %
a) Humidity Greenhouse
Greenhouse, bagged leaf Misting bench Growth chamber
Sealed jar
Corn Soybean Soybean Com Soybean Corn Soybean Corn
Soybean b) Temperature 1 5°Day/1 O0night
30°Day/200night 40°Day/30°night c) Nutrient concentration 1 x LF 2 x LF 3 x LF 5 x LF d) Photoperiod 12 h 24 h
Com Soybean Corn
12 9 8 8 17 2 5 8 9
2 7
10 3 tr
3 4 15 18
11
0 0 10 10 5 2 10 17 8 6 10 17 0 0
6 4 8 5 11 18
11 11 15
13 12 16 2
10 12 2
Soybean
7 9 8 4
Soybean Soybean
9 10
19 22
15 16
Soybean Corn Soybean Soybean Soybean Soybean
18 0 0
tr
-a
MANIPULATING PLANT MEMBRANE FATTY ACID COMPOSITIONS
and Pigents neutral lipids
steros
-
FFFA
SG "w
cerebrosides
'A
PI
p
4
~3G
c
0GOLP C
*Pigment ...%
7days after applying
40
..
A
..I
distribution between NL differed; in particular, plants treated with Tweens had more in waxes, and much less in sterols and pigments (Table IVb). Distribution of radioactivity between fatty acids in acetatetreated plants roughly mimicked their proportions in the bulk lipid; by contrast, 18:1 and 18:2 were relatively enriched whereas saturated fatty acids and 18:3 were relatively depleted in radioactivity in Tween-treated plants (Table IVc). The presence of radioactivity in sterols and saturated fatty acids in Tween-treated cells suggested some degradation of the [1-'4C] 18:1 and reutilization of the radioactive carbon (14). Distribution of 17:0 between individual membrane lipids was therefore also measured, as this would not arise from de novo synthesis and would therefore show how exogenous fatty acids were used. Similar results were obtained as with the radioactive fatty acids: 17:0 was found in all classes of membrane lipids, although with substantial variation between lipids (Table V). In particular, MGDG was relatively depleted in 17:0, and only trace amounts of 17:0 were detected in PG from leaves, either at the site of application or elsewhere in the plant. By contrast, root PG had about the same amount of 17:0 as other root phospholipids (Table V).
--j
Fatty Acid Modifications Radioactive 18:2 and 18:3 accumulated in membrane lipids of plants treated with radioactive 18:1, indicating desaturation (Table IVc). Longer saturated fatty acids accumulated in plants treated with shorter chain fatty acids; for example, 17:0 accumulated in plants treated with 15:0 (Table VI). However, as in cultured cells (14), no desaturation of exogenously applied odd-chain length saturated fatty acids was observed in membrane lipids. Modifications of even-chain saturated fatty acids and of fatty acids incorporated into NL were not studied.
en-(1-14C]18:1
_..
cerebrosides
207
_ F rev
Effects on Ratios of Saturated to Unsaturated Fatty Acids Tissues which took up substantial amounts of exogenous saturated fatty acids showed reduced amounts of 16:0 and 18:0, such that ratios of saturated to unsaturated fatty acids were essentially unaltered except in the roots of plants whose primary leaves were treated with Tween-1 7:0 (Tables I and VI).
LPW.
LPC
Pi
Pigment
*W.Y#
..w
w
7 days after applying Acetic Acid [1-
UC]
"-
.1 Or
Figure 2. Labeled trifoliolate leaf lipids of soybean (cv Noir I) seedlings whose primary leaves were treated with [1 -14C]acetic acid or Tween-[1-14C] 18:1. Surface-sterilized seedlings were grown 10 d under constant light in jars containing 20 mL LF medium. The primary leaves of two plants were treated with 20 nmol/leaf of [1 -'4C]acetate (Amersham, 56 mCi/mM); the primary leaves of two other plants were treated with 200 nmol/leaf of Tween-[1 -14C] 18:1 (4.5 mCi/mM; wtl). After 7 d plants were harvested, washed, separated into roots, trifoliolate and primary leaves, and lipids were extracted as described in "Materials and Methods." Aliquots of the crude trifoliolate lipid
Effects on Membrane Properties Lateral phase transitions were detected at higher temperatures in membrane phospholipids isolated from both leaves and roots of plants which incorporated exogenous saturated fatty acids (Fig. 3; Table VII). However, the Fo Chl fluorescence rise temperature, a physiological parameter thought to be influenced by membrane properties (12), was not significantly different in these plants (Table VII). extracts were separated by two-dimensional TLC on silica G and visualized by autoradiography. First dimension is chloroform:methanol:ammonium hydroxide (65:25:2, v/v); second is chloroform:methanol:acetic acid:water (85:15:10:3, v/v).
Table IV. Distribution of Radioactivity in Plants treated with [1-14C]Acetate or Tween-[1-14C] 18:1 Surface-sterilized soybean seedlings (cv Noir 1) were grown 10 d under constant light in jars containing 20 mL LF medium. The primary leaves of two plants were then treated with 20 nmol/leaf of [1 -14C]acetate (Amersham, 56 mCi/mM); the primary leaves of two other plants were treated with about 70 nmol/leaf of Tween-[1 _14C] 18:1 (4.5 mCi/mM; wtl). Seven d later plants were harvested, washed, and divided into roots, stems, trifoliolate, and primary leaves ("Medium," total radioactivity recovered in medium). Lipids were separately extracted from each part, and separated into "lipids," "water solubles," and "filter cake" as described in "Materials and Methods." Two plants were pooled for each sample. Treatment Treatment
Total
Organ
Lipid
CpM X 10O3
CpM X 10-3
cake
Water XSoluble 10-3
%
cpm x
%
CpM
a) Total Radioactivity Applied Root
[1-14C]acetate
Stem Primary leaf Trifoliolate
Tween-[1-14C] 18:1
Medium Recovered Applied Root Stem Primary leaf Trifoliolate Medium Recovered
Lipid
8000 287 706 4888 976 184 7041 2600 30 185 1176 1151 18 2560
4
9 61 12
43 225 2639 206
15 32 54 21
184 346 1526 541
65 49 31 55
60 134 723 229
20 19 15 24
18 102 715 725
60 56 61 63
6 35 279 176
20 19 26 15
6 47 182 250
20 25 15 22
2 (88%) 1 7 45 44 1
(98%)
[1-14C]acetate CpM X 10-3
Total
Class
557 179 132 115 44 38 30 12 8
64.3 20.6 15.2 13.2 5.1 4.4 3.5 1.4 0.9
100 32 24 21 8 7 5 2 1
106 63 1 12 3 8 2 4 13
31.7 18.8 0.2 3.6 1.0 2.3 0.7 1.1 4.0
100 59 1 11 3 7 2 3 13
202 133 49 11
23.3 15.3 5.6 1.2
10
1.1
100 66 24 5 5
149 95 28 5 20
44.7 28.5 8.5 1.6 6.1
100 64 19 4 14
108 35 25 16 6 6 4
12.4 4.0 2.9 1.8 0.7 0.7 0.5 0.1 1.2
100 32 23 15 6 6 4
79 42 5 13 3 1 2 2 11
23.7 12.7 1.6 3.8 0.9 0.4 0.7 0.5 3.2
100 54 7 16 4 2 3 2 14
Tween-[1X-14C]_ 8:1 CpM 10-TTotal
Class
b) Lipids (primary leaves only) NL Total Waxes
Chl Long-chain alcohols Other pigments, SG, ESG FAME Sterols Diglycerides FFA GL Total MGDG DGDG SL Unknowns PL Total PC PG PE Pi LPC CL PA Unknown Treatment
c) Fatty acids [1 -'4C]acetate
Tween-[1-14C] 18:1
1
10
1
10
Organ
CpM X 10-3
Saturateds
%
Primary leaf bulk lipid mol %
25
Trifoliolate bulk lipid mol %
5
Primary leaf bulk lipid mol %
5
Trifoliolate bulk lipid mol %
4
31 25 24 25 13 30 13 29
cm xx0-3 1o cpm
2 1
6 3
%
3 2 3 2 16 8 13 5
c 1802 X 10-3 CpM
8 2 14 7
%
CpM X 103
18:3
%
10 14 9 10 36 12 25 12
45
57 59 64 63 36 50 49 54
14 14
13
MANIPULATING PLANT MEMBRANE FATTY ACID COMPOSITIONS
209
Table V. Fatty Acid Compositions of Individual Lipids of Soybean Plants Treated with Tween-17:1 Soybean seedlings (cv Noir 1) were grown axenically in sealed quart glass jars under constant light (200 gE -m2 -s-1) at 240C until their primary leaves were fully unfolded, but before the first trifoliolate appeared. Tween-1 7:0 was then applied to the primary leaves, and 7 d later plants were washed, and lipids were extracted separately from trifoliolate leaves (which had emerged after Tween application), primary leaves, and roots. Individual lipids were prepared by column and thin layer chromatography, and their fatty acid compositions were determined as described in "Materials and Methods." Both control and treated lipids were pooled from 10 plants. Fatty Acid Composition Lipid Organ 16:0 16:1 17:0 18:0 18:1 18:2 18:3 z sat' mol %
Galactolipids MGDG
DGDG
Phospholipids CL
PG
PE
PC
Pi
a
Total
Control trifoliolate 17:0 trifoliolate Control 10 17:0 1 Control root 17:0 root Control trifoliolate 17:0 trifoliolate Control 10 17:0 10 Control root 17:0 root
Control trifoliolate 17:0 trifoliolate Control 10 17:0 10 Control root 17:0 root Control trifoliolate 17:0 trifoliolate Control 10 17:0 10 Control root 17:0 root Control trifoliolate 17:0 trifoliolate Control 10 17:0 10 Control root 17:0 root Control trifoliolate 17:0 trifoliolate Control 10 17:0 10 Control root 17:0 root Control trifoliolate 17:0 trifoliolate Control 10 17:0 10 Control root 17:0 root saturated fatty acids (mol %).
8 5
0 0
0 2
2 1
2 2 1 2
4
0
0
5 35 17 26 18 13 10 27 18
0 0 0 0 0 0 0 0 0
6 0 19 0 1 0 7 0 18
14
0
13
0
33 28 30 26 31 36 30 35 27 28 28 21
0 0 0 0
0 6 0 17
8 8 12 8
0
0
11
0 31 30 36 40 0 0 0 0
16 0 0 0 0 0 16 0 6
41 32 43
o
o
0
15
0
0
8 5 8 5 5 17 6 7 8 9 7 9
28 28 25 27 25
0 0 0 0 0 0 0 0 0 0 0 0 0
19 0 4 0 15 0 22 0 11 0 15 0
44
36
34
19 34
31 37 30 34 25
DISCUSSION This paper described a system for modifying membrane fatty acid compositions of plants of several different species without changing growth temperature or other conditions.
17
1
2 13 11 6 3 7 6
4
6 7 7 8 8 5 13 13 16 12 8 7
0 0
2 2 0
0
7
8 10 5
8 7 7
10 8 11
8
10 6 6 5
6 4 4 4
5
3 4 4
6 10
9 12 7
6 7
9 2 3 2 3 8 9
84 87 93 82 44 44 62 74 78 74 51 42
10 8 5 12 48 47 32 22 20 23 41 49
25 22 19 16 25 19 13 10 9 8 25 25 22 27 18 17 27 28 22 31 16 17 28 24 22 21 18 16 28 23
28 27 29 28 25 14 14 8 15 9 22 22 22 17 27 23 17 19 40 28 48 32 27 24 22 15 18 22 25 20
41 43 43 51 42 60 36 43 32 33 45 43 51 50 50 55 52 50 34 36 34 48 41 47 46 55 53 56 42 50
4 4 2 4 9
Plants treated with Tweens carrying specific fatty acids transferred these fatty acids from Tweens to all membrane lipids, both at the site of application and elsewhere in the plant (Fig. 1; Tables IV and V). Certain fatty acids were modified: unsaturated fatty acids were further unsaturated, and short-
210
TERZAGHI
chain fatty acids were elongated (Tables IV and VI). Phospholipids isolated from plants which had incorporated exogenous fatty acids had higher phase transition temperature (i.e. a minor component began freezing at higher temperatures, Fig. 3), but changes in a membrane-related process were not detected (Table VII). Uptake and incorporation of exogenous fatty acids by leaves at the treated site has been previously reported (2, 6, 16), but this is the first account of fatty acid translocation in plants. The appearance of exogenously supplied fatty acids throughout plants treated with Tweens indicated that translocation was probably in the phloem, as they were detected in tissues both above and below the treated tissue (Tables I, II, and VI). Moreover, tissues growing during the experiment (e.g. the 5.
60C
3
03
~~~~~~~1400
ZZ
~~~~~~~~~~~~~17:0 control 2.
3.1
3.2
3.3
3.5
3.4
li/T (OK
x
3.6
3.7
3.8
000)
Figure 3. Lateral phase transition temperatures of root phospholipids isolated from soybean plants (cv Noir 1) with altered root membrane compositions. Soybean seedlings (cv Noir 1) were grown axenically in sealed quart glass jar under constant light until their primary leaves fully unfolded, but before the first trifolioilate appeared. Tween-17:0 was then applied to the primary leaves, and 7 d later plants were washed, root lipids were extracted, and phospholipids were prepared by column chromatography as described in "Materials and Methods." Liposomes containing trans-parinarc acid were then prepared from these phospholipids and analyzed as described in "Materials and Methods." Both control and treated lipids were pooled from 10 plants. Fatty acid compositions are given in Table VIl.
Plant Physiol. Vol. 91,1989
trifoliolate leaves in Table I) frequently incorporated more exogenous fatty acids than the treated tissue, indicating transport to the most active sites of lipid synthesis. This was surprising, since lipids are not thought to be transported in plants (4). It will therefore be interesting to determine how this occurs and in what form the fatty acids are transported. One possibility is that the sugar or polyethylene glycol Tween moieties are specifically translocated and the fatty acids are carried with them. This might account for the greater uptake and translocation from leaves (Tables I and II), since leaves are the primary sites of carbohydrate synthesis. An alternative is a specific lipid transport mechanism such as the low- or high-density lipoproteins in humans (3); this could account for the large amounts of radioactivity recovered in the aqueous fractions of plants treated with radioactive Tweens (Table IV). Large differences were observed between species in amounts of exogenous fatty acids taken up and translocated (Table II). The variation between soybean cultivars suggests that these differences were due to variation in uptake ability, since these both grew comparably yet cv Noir I consistently took up and translocated more exogenous fatty acids than cv Minsoy under many conditions (W Terzaghi, unpublished data), although the alternative that conditions were not optimized cannot be ruled out. Cuticle thickness or properties may be responsible for these differences; an alternative is differences in the uptake or translocation pathways. The variation between cultivars shows that a screen of several cultivars would be worthwhile when adapting this system to other plants, and also suggests that a genetic study of the incorporation process may be possible. The most striking environmental factor affecting Tween uptake was humidity (Table III). At one extreme, Tween treatment under relatively dry conditions caused leaf mortality, presumably due to excessive water loss, perhaps through cuticle damage. Moreover, even under high humidity greatest uptake was observed in plants grown in glass jars, perhaps because this leads to less cuticle synthesis. Other optimal conditions for uptake were the same as for growth (Table III), indicating that plants take up more exogenous fatty acids when they are growing (relatively) rapidly. This has also been observed in tissue cultures (W Terzaghi, unpublished data); perhaps rapidly growing cells are less particular about the fatty
Table VI. Elongation of 15:0 by Soybean Plants Treated with Tween-15:0 Surface-sterilized soybean seeds (cv Noir 1) were germinated on sterile filter paper. Seedlings were transferred to sterile glass jars containing 20 mL LF medium and were maintained under constant light at 260C under constant light (200 E- m-1 -s-1). Medium of treated plants contained 3 mm 15:0 as Tween-1 5:0. After 7 d plants were washed, separated into roots and shoots, and lipids were extracted and analyzed as described in "Matenals and Methods." Fatty Acid Composition Treatment
Sample 15:0
16:0
16:1
0 0 7 4
31 16 19 11
0
17:0 18:0 18:1
18:2
18:3
sata
10
30 12 30
5
17
32 65 23 53
34 18 38 26
mo/ %
Control
Roots Leaves 3 mM 15:0 in medium Roots Leaves a Total saturated fatty acids (mol %).
1
0 1
0 0 8 6
3 2 4 5
4 3
MANIPULATING PLANT MEMBRANE FATTY ACID COMPOSITIONS
211
Table VIl. Lipid Compositions and Lateral Phase Transition Temperatures of Phospholipids Isolated from Soybean Plants with Altered Root Membrane Compositions, and Fo Chl Fluorescence Rise Temperatures Tween-17:0 was added to the medium or applied to the primary leaves, as indicated, of soybean seedlings (cv Noir 1) grown axenically in sealed quart glass jars under constant light. After 7 d plants were washed, root, primary, and trifoliolate lipids were extracted, and the phospholipids were prepared by column chromatography as described in "Materials and Methods." Liposomes containing transparinaric acid were then prepared from these phospholipids and analyzed as described in 'Materials and Methods." Fo Chl fluorescence as a function of temperature was measured on leaf discs cut from two plants of each treatment earlier on the day of harvest. Both control and treated lipids were pooled from 10 plants. Treatment
Organ
Tma
Fatty Acid Composition
Fo rise Tt' 16:0
17:0
18:0
18:1
18:2
18:3
z saP
mole %
60 Root 10 15° Trifoliolate 140 140 leaves Root on 17:0 10 150 Trifoliolate 200 140 17:0 in medium Root 10 140 Trifoliolate 150 a Phase separation temperature. d Not applicable. fatty acid content. Control
NAd 440 440
32 NDe 15 ND 18 ND NA 20 18 11 460 9 17 17 460 14 32 NA 14 2 450 440 1 19 b Fo Chi fluorescence rise e Not determined.
acids used for lipid synthesis because their fatty acid pools are depleted. Exogenous fatty acids appeared to be used for lipid synthesis essentially as if they were made by the plants: lipids such as MGDG which are normally highly unsaturated incorporated large amounts of 18:1 but relatively little 17:0, whereas the converse was true of relatively saturated lipids such as PI and PE (Tables IV and V). The striking anomaly is the very low levels of exogenous fatty acids in leaf (but not root) PG. This suggests that, as inferred with tissue culture cells (14), exogenous fatty acids are not converted to acyl-ACP thioesters, since in soybeans plastid PG is exclusively synthesized using acyl-ACP thioesters as substrates, and is the only lipid for which this is the case (1 1). The finding that exogenous oddchain length saturated fatty acids were elongated, but not unsaturated (Table VI), strengthens this interpretation, since these would also have to be converted to acyl-ACP thioesters to be desaturated (1 1). Again, as with tissue cultures (14), the questions of where, how, and why this elongation occurs remain unanswered. Further desaturation of unsaturated fatty acids can occur after these are attached to glycerol moieties (1 1), and presumably exogenous unsaturated fatty acids were desaturated by the same mechanism (Table IV). The failure to desaturate odd-chain length saturated fatty acids contrasts with the observation of Thompson et al. (16) that spinach leaves desaturated exogenous palmitic acid supplied as the free fatty acid. At present it is unclear whether this is due to differences between experimental organisms, mode of application, or fatty acid chain-lengths. There appeared to be some degradation and reutilization of the radioactive carbon of exogenous fatty acids, although
4 2 30 30 36 2 7 72 5 20 11 5 3 63 23 3 24 6 30 43 4 3 9 65 21 6 5 18 38 39 22 3 3 25 50 4 3 8 72 20 4 13 58 5 25 c Total saturated temperature.
little net loss from respiration (Table IV). Such recycling probably accounts for incorporation of radioactivity from [1"'C] 18:1 into such compounds as sterols, or saturated fatty acids (4). This contrasts with cultured cells, where very little degradation of exogenous fatty acids was observed (15). However, degradation and reutilization appeared to account for relatively little incorporated radioactivity, as most lipid radioactivity in cells treated with radioactive Tweens was found in the polar lipids rather than in NL, whereas NL were the major sink for exogenous radioactive acetate (Table IV). Indeed, even in the neutral lipid fraction most radioactivity was found in compounds such as waxes and long chain alcohols, which could be derived from exogenous fatty acids (4). Nonetheless, because of the degradation and reutilization, the data on fatty acid distribution between lipid classes obtained using 17:0 seems more reliable. Plants which incorporated exogenous fatty acids changed the proportions of fatty acids ordinarily present (i.e. synthesized by the plant) in ways which tended to counteract the imposed changes in composition (Tables I and VI). This was also observed in cultured soybean cells, where it was shown that this response was due to a change in patterns of fatty acid synthesis (15). It is therefore proposed, by analogy, that the observed changes in proportions of endogenous fatty acids in soybean plants were also generated by changes in their patterns of endogenous fatty acid synthesis. Changes in membrane composition increased the temperature at which lateral phase transitions were detected, but did not affect the temperature dependence of Fo Chl fluorescence (Fig. 3; Table VII). Similar results have been obtained with photosynthetic soybean cell cultures, where much larger
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TERZAGHI
changes in membrane composition can be generated, and the implications of these results will be discussed elsewhere (W Terzaghi, unpublished data). Fo Chl fluorescence rise temperatures also were not affected in Arabidopsis thaliana mutants with altered thylakoid membrane compositions (9). Results presented in this paper have certain implications for the common practice of adding Tweens as emulsifiers or wetting agents to solutions used to deliver other substances to plants. Some of the fatty acid moieties may also be taken up and translocated, where they may help stimulate growth (13), or affect endogenous lipid metabolism and membrane properties. They may also serve as elicitors of the phytoalexin or other responses (10). In conclusion, this paper described a system for modifying plant membrane compositions without altering growth temperature or other conditions. This system should therefore be useful for a variety of studies related to plant membranes and lipid metabolism, ranging from the incorporation and translocation process itself to influences of fatty acid composition on the temperature dependences of a variety of membranerelated processes. ACKNOWLEDGMENTS I wish to thank Dr. Christopher B. Field for his support and encouragement and for critical reading of the manuscript. I am also endebted to Dr. Karl G. Lark for his support and encouragement during the early stages of this work, and Dr. Pete D. Gardner for many helpful discussions and for the generous use of his laboratory for Tween synthesis and fatty acid analysis. I also wish to thank Dr. Joseph Berry for designing the apparatus used for the fluorescence depolarization measurements, for many helpful discussions, and for critical reading of the manuscript. I am also endebted to Dr. David C. Fork, Dr. William Eisinger, and Dr. J. T. B. Science for many helpful discussions and for critical reading of the manuscript. Finally, I wish to thank Dr. Reid Palmer for his gift of seeds of soybean cultivars Noir 1 and Minsoy.
2. 3.
4. 5.
6. 7.
8. 9.
10.
11.
12.
13. 14.
15.
16. LITERATURE CITED 1. Berry JA, Raison JK (1981) Responses of macrophytes to temperature. In OL Lange, PS Nobel, CB Osmond, H Ziegler, eds,
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Encyclopedia of Plant Physiology, New Series, Vol 12a. Springer-Verlag, Berlin, pp 277-338 Breidenbach RW, Waring AJ (1977) Response to chilling of tomato seedlings and cells in suspension cultures. Plant Physiol 60: 190-192 Fielding CJ, Fielding PE (1985) Metabolism of cholesterol and lipoproteins. In DE Vance, JE Vance, eds, Biochemistry of Lipids and Membranes. The Benjamin/Cummings Publishing Company, Menlo Park, CA, pp 404-474 Harwood JL, Russell NJ (1984) Lipids in Plants and Microbes. George Allen & Unwin, London Harwood ML (1988) Fatty acid metabolism. Annu Rev Plant Physiol Plant Mol Biol 139: 101-138 Hawke JC, Stumpf PK (1980) The incorporation of oleic acid linoleic acids and their desaturation products into the glycerolipids of maize leaves. Arch Biochem Biophys 203: 296-306 Kuiper PJC (1985) Environmental changes and lipid metabolism of higher plants. Physiol Plant 64: 118-122 Leggett JE, Frere MH (1971) Growth and nutrient uptake by soybean plants in nutrient solutions of graded concentrations. Plant Physiol 48: 457-460 McCourt P, Kunst P, Browse J, Somerville CR (1987) The effects of reduced amounts of lipid unsaturation on chloroplast ultrastructure and photosynthesis in a mutant of Arabidopsis. Plant Physiol 84: 353-360 Preisig LC, Kuc JA (1985) Arachidonic acid-related elicitors of the hypersensitive response in potatoe and enhancement of their activities by glucans from Phytophthora infestans [Mont.] de Bary. Arch Biochem Biophys 236: 379-389 Roughan PG, Slack CR (1984) Glycerolipid synthesis in leaves. TIBS 9: 383-386 Seeman JR, Berry JA, Downton WJS (1984) Photosynthetic response and adaptation to high temperature in desert plants. A comparison of gas exchange and fluorescent methods for studies of thermal tolerance. Plant Physiol 75: 364-368 Stowe BB, Obreiter JB (1962) Growth promotion in pea stem sections. II. By natural oils and isoprenoid vitamins. Plant Physiol 37: 158-164 Terzaghi WB (1986) A system for manipulating the membrane fatty acid composition of soybean cell cultures by adding Tween-fatty acid esters to their growth medium: basic parameters, and effects on cell growth. Plant Physiol 82: 771-779 Terzaghi WB (1986) Metabolism of Tween-fatty acid esters by cultured soybean cells: kinetics of incorporation into lipids, subsequent turnover, and associated changes in endogenous fatty acid synthesis. Plant Physiol 82: 780-786 Thompson GA Jr, Roughan PG, Browse JA, Slack CR, Gardiner SE (1986) Spinach leaves desaturate exogenous [14C]palmitate to hexadecatrienoate. Evidence that de novo glycerolipid synthesis in chloroplasts can utilize free fatty acids imported from other cellular compartments. Plant Physiol 82: 357-362
