CRYOBIOLOGY
14, 87-93 ( 1’377)
Changes
in Fatty Acid Composition of Winter during Frost Hardening ’
C. WILLEMOT,2 2 Agriculture 3 American
I-1. J. HOPE,’ 1~.J. WILLIAMS,”
Wheat
.\ND R. hlICII:iUD
2
Cnnadn Re.vearcll Station, Sfc-Fey, GIL’ 213, Quebec, Canada, and Bethcstla, Maryland 20014 Red Cross Blood Rcseurcla l&oratory,
From recent work on frost hardening it appears that varietal differences in frost hardiness might be related to ability to desaturate fatty acids at low temperature in alfalfa (7, 9), but not in wheat (4, 5, 13). Furthermore, alfalfa accumulates mainly linolcic acid at low temperature while wheat accumulates mainly linolcnic acid. Although these differences between alfalfa and wheat fatty acid composition after hardening would not bc surprising in two m~related genera, they might bc csplaincd in part by diffcrciices in the material used and its dc\rclopmcntal stage. Germinating wheat embryos, including shoot and root, W~TC used by dc la Roche et nl. (4, 5). The presence of shoot tissue might e\;plnin the accumulation of linolcnic acid, while the dcvclopmcntal changes occurring during germination might mask finer varietal differences related to frost hardc~ning. Grcnier and Willemot (9) used 3 wc>ck old alfalfa plants, which developed little during hardening, and anal>-zcd the root scparatcly from the shoot. We rcpeatcd the wheat cspcrimcnts of clc la Roche and his collcagucs under conditions more directly comparal)lc with tlic alfalfa cspcrimcllts pcrformcd in our Iaboratory. Twclvc day old pla~~tlcts wcrc hardened and the root and shoot lipids lvcrc Received Novcmlxr 25, 1975. 1 Contribution No. 82 from the Agriculture Canada Research Station, Ste-Foy.
Copyright 0 1957 by Academic Press. Inc. All rights of reproduction in any form reserved.
analyzed separately. The results obtained with embryos germinating at low temperature were confirmed in hardening root tissue. MATERIALS
AND METHODS
Winter wheat (Tliticfcnz aestiuunl, cvs Kharkov-frost hardy, and Champleinless hardy) were grown, hardened, and tcstcd for frost hardiness as described previously ( 1s). Five pots containing 15 plants each were frozen at each test temperature in a modified commercial frcezcr (17) fitted with a tcmpcratmc programmer ( Data-Tmk, model 5310, Research Inc., Minneapolis ) . A tcmpcratnre equilibration period of 12 hr at -4°C was included to freeze all water in the sand-vermiculite. Hardiness was fxprcsscd as temperature at which 50% of the plants died (killing temperature, LT30). After various times of hardening, lipids were cstractcd from about 2 g each of lcavcs and of roots (including crowns) separately, following a modification of the method of Bligh and Dyer (2, 18). The c&acts wcrc stored in a small vohmie of clilorofonrl-InctIranol (2 : 1, v/v) under nitrogcn at --20°C. Fatty acid nlctlr).l esters wcrc prepared by trcatiincnt of aliquots (l/10 and l/20 of the root and leaf CYtracts, respectively) with l-l?& borontrifluoride in methanol ( 12) and analyzed by glc (S). Phospholipids in the remainder of
1s.w
0011-2210
ss
ET AL.
WILLEMOT
portions: cllloroforrn-~nctliarlol-~~~tt(~r, 2:2: 1.8, v/v) and rccovcry of the chloroform phase. The fatty acid nrcthyl cstcrs rcslllting from mcthanoIj$s of the phospholipids were analyzed by glc. All extractions were made on five replicates. A similar experiment where the lipids from the five replicates were pooled gave essentially the same results. For statistical analyses, replicates were considered as subsamples of each treatment, Analysis of variance was carried out according to Steel and Torrie (16).
-2o-
Kharkov
-15-
0G 0
- IO
d,/,
f
f I’ ,’ I’
./ , x .-~~~~.~~~,.~~lx ,/ x .*‘ Champlein I: I’
,
-5 2
0
WEEKS
RESULTS
Both cultivars hardened rapidly initially (Fig. l), but Champlein approached maximum hardiness (killing temperature: -12.8”C) after 1 week. Kharkov maintained a high rate of hardening during the second week and reached a killing temperature of -20.2”C after 4 weeks of hardening. When expressed as microgram fatty acid per gram fresh weight, the results of glc analyses showed systematic variation. However, when these results were expressed as percentage of total fatty acids or phospho-
4
OF HARDENING
FIG. 1. Killing temperatures of two cultivars of winter wheat during controlled hardening.
the samples were separated from other lipids by preparative tic in acetone-acetic acid-water (100:2:1, v/v) (6) on precoated 1000 pm silica gel G plates (Analtech, Newark, Del. ) eluted with chloroform-methanol-water ( 1: 2: 0.8, v/v) and purified by the addition of 1 vol of chloroform and 1 vol of water (final proTABLE
1
Fatty ilcid Composition of Total Lipids from Winter Wheat Roots during Froai, IInrdeningz -___ __-. Cultivnr
I’atty
M’eehs
acidb~c 0
liharkov
Champlein
16:O IS:0 18:l 18:2 18:3 Total 16:O 18:O 1S:l 18:2 18:3 Tobal
190.7 * 5.5 f
9.-i I.1
3.5.0 f 1.7 481.1 z!z37.4 339.9 i 12.0 1072.2 z!c58.7 169.9 zt 6.7 f 33.Q f 437.4 f 317.2 zt 965.0 rt
of hardening
2
1
11.0 1.2 3.ls 3.5.2 15.6 48.8
223.0 f 1.3.3 II.9 It 2.8 fi9.6 f 13.2 507.3 zt 32.7 552.3 f 32.7 1364.1 i
87.3
226.5 f 1.5.7 11.0 f
1..5
56.4 f 3.8 516.2 f 42.3 530.9 h 36.7 1361.0 h 96..5
n rg/g fresh weight i SD. b Number of carbon atoms: nlunbcr of double bonds. c Traces of 16: 1 were present.
2XJ.7 6.6 62.0 437.7 642.8 13!%?
f It zt f f f
-I
30.3 1.!) 11.8
40.8 43.7 125.6
204.5 f7.7 2.r,rt I..: 2.7 39.8 It 422.5 A 24.7 573.7 r!z 20.8 1243 .I+ 46.1)
207.fi * .?.;I *
18.4
180.6 It t
20.9
1.4 30.0 * 8.S X8.9 f 30.7 623.1 f 73.0 1244.9 zt 130.7
23.8 z!z 3.2 301.1 f 48.4 514.7 zlz 64.8 1020.9 k 136.6
._ -
-----.
Kh:trkov
Chnpleili
__-
~~---~ ~.-~
..~---~
~~.
-. -
\~wknI)! tl:ll.df~llill~ 1 2 ,i::.i..i i
4li.S
.iliI 2 i
I ;;o.o
l(i:O IO: l-lu,tlnc IS:0 1x:1 IS:2 1s::: Total
429.S & 04.5 9.0 d * 1::.0 16.1 * 5.4 sti.4 It 1s.:: (i.x.:: zt sx.4 :3
14, 87-93 ( 1’377)
Changes
in Fatty Acid Composition of Winter during Frost Hardening ’
C. WILLEMOT,2 2 Agriculture 3 American
I-1. J. HOPE,’ 1~.J. WILLIAMS,”
Wheat
.\ND R. hlICII:iUD
2
Cnnadn Re.vearcll Station, Sfc-Fey, GIL’ 213, Quebec, Canada, and Bethcstla, Maryland 20014 Red Cross Blood Rcseurcla l&oratory,
From recent work on frost hardening it appears that varietal differences in frost hardiness might be related to ability to desaturate fatty acids at low temperature in alfalfa (7, 9), but not in wheat (4, 5, 13). Furthermore, alfalfa accumulates mainly linolcic acid at low temperature while wheat accumulates mainly linolcnic acid. Although these differences between alfalfa and wheat fatty acid composition after hardening would not bc surprising in two m~related genera, they might bc csplaincd in part by diffcrciices in the material used and its dc\rclopmcntal stage. Germinating wheat embryos, including shoot and root, W~TC used by dc la Roche et nl. (4, 5). The presence of shoot tissue might e\;plnin the accumulation of linolcnic acid, while the dcvclopmcntal changes occurring during germination might mask finer varietal differences related to frost hardc~ning. Grcnier and Willemot (9) used 3 wc>ck old alfalfa plants, which developed little during hardening, and anal>-zcd the root scparatcly from the shoot. We rcpeatcd the wheat cspcrimcnts of clc la Roche and his collcagucs under conditions more directly comparal)lc with tlic alfalfa cspcrimcllts pcrformcd in our Iaboratory. Twclvc day old pla~~tlcts wcrc hardened and the root and shoot lipids lvcrc Received Novcmlxr 25, 1975. 1 Contribution No. 82 from the Agriculture Canada Research Station, Ste-Foy.
Copyright 0 1957 by Academic Press. Inc. All rights of reproduction in any form reserved.
analyzed separately. The results obtained with embryos germinating at low temperature were confirmed in hardening root tissue. MATERIALS
AND METHODS
Winter wheat (Tliticfcnz aestiuunl, cvs Kharkov-frost hardy, and Champleinless hardy) were grown, hardened, and tcstcd for frost hardiness as described previously ( 1s). Five pots containing 15 plants each were frozen at each test temperature in a modified commercial frcezcr (17) fitted with a tcmpcratmc programmer ( Data-Tmk, model 5310, Research Inc., Minneapolis ) . A tcmpcratnre equilibration period of 12 hr at -4°C was included to freeze all water in the sand-vermiculite. Hardiness was fxprcsscd as temperature at which 50% of the plants died (killing temperature, LT30). After various times of hardening, lipids were cstractcd from about 2 g each of lcavcs and of roots (including crowns) separately, following a modification of the method of Bligh and Dyer (2, 18). The c&acts wcrc stored in a small vohmie of clilorofonrl-InctIranol (2 : 1, v/v) under nitrogcn at --20°C. Fatty acid nlctlr).l esters wcrc prepared by trcatiincnt of aliquots (l/10 and l/20 of the root and leaf CYtracts, respectively) with l-l?& borontrifluoride in methanol ( 12) and analyzed by glc (S). Phospholipids in the remainder of
1s.w
0011-2210
ss
ET AL.
WILLEMOT
portions: cllloroforrn-~nctliarlol-~~~tt(~r, 2:2: 1.8, v/v) and rccovcry of the chloroform phase. The fatty acid nrcthyl cstcrs rcslllting from mcthanoIj$s of the phospholipids were analyzed by glc. All extractions were made on five replicates. A similar experiment where the lipids from the five replicates were pooled gave essentially the same results. For statistical analyses, replicates were considered as subsamples of each treatment, Analysis of variance was carried out according to Steel and Torrie (16).
-2o-
Kharkov
-15-
0G 0
- IO
d,/,
f
f I’ ,’ I’
./ , x .-~~~~.~~~,.~~lx ,/ x .*‘ Champlein I: I’
,
-5 2
0
WEEKS
RESULTS
Both cultivars hardened rapidly initially (Fig. l), but Champlein approached maximum hardiness (killing temperature: -12.8”C) after 1 week. Kharkov maintained a high rate of hardening during the second week and reached a killing temperature of -20.2”C after 4 weeks of hardening. When expressed as microgram fatty acid per gram fresh weight, the results of glc analyses showed systematic variation. However, when these results were expressed as percentage of total fatty acids or phospho-
4
OF HARDENING
FIG. 1. Killing temperatures of two cultivars of winter wheat during controlled hardening.
the samples were separated from other lipids by preparative tic in acetone-acetic acid-water (100:2:1, v/v) (6) on precoated 1000 pm silica gel G plates (Analtech, Newark, Del. ) eluted with chloroform-methanol-water ( 1: 2: 0.8, v/v) and purified by the addition of 1 vol of chloroform and 1 vol of water (final proTABLE
1
Fatty ilcid Composition of Total Lipids from Winter Wheat Roots during Froai, IInrdeningz -___ __-. Cultivnr
I’atty
M’eehs
acidb~c 0
liharkov
Champlein
16:O IS:0 18:l 18:2 18:3 Total 16:O 18:O 1S:l 18:2 18:3 Tobal
190.7 * 5.5 f
9.-i I.1
3.5.0 f 1.7 481.1 z!z37.4 339.9 i 12.0 1072.2 z!c58.7 169.9 zt 6.7 f 33.Q f 437.4 f 317.2 zt 965.0 rt
of hardening
2
1
11.0 1.2 3.ls 3.5.2 15.6 48.8
223.0 f 1.3.3 II.9 It 2.8 fi9.6 f 13.2 507.3 zt 32.7 552.3 f 32.7 1364.1 i
87.3
226.5 f 1.5.7 11.0 f
1..5
56.4 f 3.8 516.2 f 42.3 530.9 h 36.7 1361.0 h 96..5
n rg/g fresh weight i SD. b Number of carbon atoms: nlunbcr of double bonds. c Traces of 16: 1 were present.
2XJ.7 6.6 62.0 437.7 642.8 13!%?
f It zt f f f
-I
30.3 1.!) 11.8
40.8 43.7 125.6
204.5 f7.7 2.r,rt I..: 2.7 39.8 It 422.5 A 24.7 573.7 r!z 20.8 1243 .I+ 46.1)
207.fi * .?.;I *
18.4
180.6 It t
20.9
1.4 30.0 * 8.S X8.9 f 30.7 623.1 f 73.0 1244.9 zt 130.7
23.8 z!z 3.2 301.1 f 48.4 514.7 zlz 64.8 1020.9 k 136.6
._ -
-----.
Kh:trkov
Chnpleili
__-
~~---~ ~.-~
..~---~
~~.
-. -
\~wknI)! tl:ll.df~llill~ 1 2 ,i::.i..i i
4li.S
.iliI 2 i
I ;;o.o
l(i:O IO: l-lu,tlnc IS:0 1x:1 IS:2 1s::: Total
429.S & 04.5 9.0 d * 1::.0 16.1 * 5.4 sti.4 It 1s.:: (i.x.:: zt sx.4 :3
