THE LIPIDS OF GERMINATING WHEAT
B Y
F. G A R C Í A O L M E D O
Laboratorio de Química y Tecnología de Cereales. Instituto Nacional de Investigaciones Agronómicas. Madrid, Spain.
AN R. L. G L A S S
ABSTRACT.
The evolution of the fatty acids present in the lipíd classes of diffe-rent structural parts of the wheat kernel during germination in the dark has been investigated.
Lipid was extracted with chloroform: methanol ( 2 : 1), washed ac-cording to Folch et al., and fractionated by preparative T L C . Free fatty acids ( F F A ) , as well as those present in triglycerides ( T G F A ) , diglyce-rides (DGFA), monoglycediglyce-rides (MGFA), polar lipids ( P L F A ) , and the total lipid extract ( T F A ) have been determinated by GLC.
In all parts studied, the P L F A pattern is rather constant and, the rootlets excepted, so is their content in the tissues (% of dry matter). Observed changes in lipid content and composition are mainly due to the glyceride fractions.
In the degermed seed, lipid is lost at a faster relative rate than non-fat dry matter. The T G F A pattern shows a constant proportion of palmitic (C,6.0), stearic (C1 8 : 0), and linolenic (C81¡3), but that of linoleic
(C1 8 : 2), increases at the expense of oleic (C18;1), as germination proceeds.
A net loss of lipid followed by a net synthesis is observed in the germ during germination. A drastic change in T G F A pattern takes pla-ce during the first half of the propla-cess. The data suggests that the origi-nal triglycerides present in the germ are catabolized very rapidly while new triglycerides with a different fatty acid pattern are synthesized in the growing tissues.
INTRODUCTION ' '' !" ' ' ' .
302 ANALES DE INVESTIGACIONES AGRONÓMICAS
chloroform: ethanol ( 2 : 1 ) for iii) and iv); an chloroform: metha-nol: water ( 2 : 1 : 0 . 2 ) for v) and homologous fractions in separation B. The eluates were collected in glasse tubes, 2 cm. in diameter.
Quantitative analysis of fatty acids. — Determination of fatty acids present in the above fractions was carried out by GLC of their methyl esters. A methyllating reagent based in that of Glass et al. (14) was used: two internal standards, methyl undecanoate (170 mgr.) and methyl tri-decanoate (17 mgr.) were brought up to 100 mi. with a mixture of di-methyl carbonate-methanol-benzene ( 3 : 2 : 5). An exact volume of this reagent (0.5-2.0 mi.) was added to each fraction, the elution solvent being eliminated previously by adapting the ground glass tubes to a flash evaporator. The solution was made alkaline with 2N sodium methoxi-de, using phenolftalein as an indicator. After 20 minutes, 2N methanolic hydrochloric acid was added until acid p H and the mixture allowed to stand for another 45 minutes before chromatography.
Gas liquid Chromatography was carried out in a F & M Model 609 Gas Chromatograph with a ñame ionization detector. The 5 feet allu-minum colum was filled with diethylenglycol succinate over Diaport W (both F & M Scientific Corporation).
The sample (10-50 microliters) was injected at 110°C and, after 2 minutes, a 13° C./minute temperature increase was programmed to reach a máximum of 200° C. Identification of the methyl esters was achieved by comparison of their retention times with those of authentic standards (Hormel Institute) and by co-chromatography.
Quatitative determination was 'made by comparison of peak área of unknown with that of the internal standards. The tenfold difference in concentration of the later and the different amounts of methylating rea-gent added to each sample allowed the analysis of a wide range of con-centration. Only the major fatty acids of wheat, palmitic (C 16 : 0), stearic (C 1 8 : 0), oleic (C 1 8 : 1), linoleic (C 1 8 : 2) and linolenic acid (C 1 8 : 3) wrere studied. For the fatty acids present in the sterol esters fraction only
a máximum valué was obtained because due to other components also eluted with that fraction, there was considerable overlapping with uni-dentified peaks. These valúes were well under 1 % of the total fatty acids in all samples and have not been reported.
T H E LIPIDS OF GERMINATING WHEAT 303
RESUI/TS AND DISCUSSION
-Dry weight changes. — -Dry weifht changes in our experimental conditions are presented in table 1. Total dry weight loss was 9.6 % for the 9 days period, the 21,9 % loss due to endosperm degradation being partly compensated by the rapid growth of the germinating axis. Seed coat weight remained practically constant throughout.
T A B U Í 1. — D r y weight changes of structural parts of wheat Kernel in mg./lO seeds.
Germination stage
Dry seed Steeped (*)
36 hr. 72 hr. 108 hr. 144 hr. 180 hr. 218 hr.
Roots
— 1 6 8 10 12 13
Rest of germ
3 5 6 9 13 14 15
Seed coats
51 56 49 55 54 55 58
Endosperm
196 183 183 171 164 153 141
T O T A L
251 250 245 244 243 241 234 227
(*) No separation of the rootlets was attempted in this sample.
Changes in lipid extract and fatty acid content. — A! decrease in
the chloroform: methanol ( 2 : 1 ) extract and total fatty acid content (both as % of dry matter) of both the germ and the rest of the seed is observed during germination (fig. 1).
For the endosperm, which is being degraded, it means that the re-lative rate of lipid utilization is greater than that of non-fat dry matter. The opposite behaviour has been reported by Brown et al. (7) for the first 2 days of germination of soybean. The faster rate of fat utilization is due to the preferential degradation of triglyceride fatty acids (TGFA), the content (% of dry matter) of polar lipid fatty acids ( P L F A ) remaining constant and that of the free and partial glyceride fatty acids ( F F A , D G F A and MGFA) slightly increasing during the period of máximum metabolic activity (fig. 1, c).
304 ANALES DE INVESTIGACIONES AGRONÓMICAS
25 ,°A
h o u r s
Figure 1.—Changes in lipid extract and fatty acids content (%of dry matter) during germination. Germ minus rootlets (a); Rootlets (&); Degermed seed (c). Lipid extract (TL) according to Folch et al. (12); Fatty acids present in total extract (TFA); in Triglycerides (TGFA) in Diglycerides (DGFA); in Monoglycerides (MGFA); in Polar
T H E LIPIDS OF GERMINATING WHEAT 305
slowly during the second. Lipid degradation is faster than dry weight increase at first and slower afterwards, and so a net loss of lipid occurs in the first stages followed by a net synthesis. This does not seem to be the case for some oil bearing seeds where a constant total fat per seed-ling axis has been reported (4, 7). As in the endosperm, T G F A account for most of the lipid content decrease. As the seedling develops, P L F A level in the rootlets declines somewhat but this fraction soon becomes dominant due to the sharp disappearance of T G F A (fig. 1, b). In the rest of the germ P L F A content remains constant (fig. 1, a). The levéis of MGFA, D G F A and F F A are slightly higher during the first three days of germination when most of the germ T G F A degradation takes place.
It might be intersting to note that while F F A level is intermedíate berween D G F A and M G F A both in the endosperm and in the "rest of the germ", in the rootlets it becomes higher than either. This could be relatel to the fact that polar lipids metabolism is dominant in this part of the germ but no acceptable explanation can be offered.
The decreasing fatty acid content ( T F A ) of the lipid extracts (TL) of all seed parts studied can be explained, at least in part, by the increa-sing proportion of polar lipids in them, an increase in the insaponibiable not being excluded.
Changes of fatty acids pattern in lipid classes during germination. The overall fatty acid pattern ( T F A ) of the degermed seed (fig. 2) shows an increasing proportion of linoleic acid (C 1 8 : 2 ) and a decrea-sing one of oleic ( C 1 8 : l ) , while that of palmitic ( C 1 6 : 0 ) , stearic (C 18: 0) and linolenic (C 18: 3) cío not change. The P L F A pattern in practically constant, so the T G F A are mainly responsible of the above changes. The same trend can be observed in F F A and to a lesser degree in MGFA. It is to be noted that F F A , D G F A and MGFA show a grea-ter proportion of saturated fatty acids than either T G F A or P L F A . The analysis of two partially puré endosperm samples (steeped and 218 hr. germinated) gave superimposable patterns and trends for both T G F A but a still greater proportion of saturated fatty acids in F F A , M G F A and M G F A than that observed in the degermed samples (endosperm plus coats).
germi-3 0 6 ANALES DE INVESTIGACIONES AGRONÓMICAS
nating seeds by Crombie and Comber (4) and a preferential utilization of linoleic acid has been shown in Douglas fir seed (9) and cotton seed (8), while Brown et al. (7) have found that oleic acid degradation was
slo-70 60 20 10
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._.-°Cl82
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144 216 h o u r s S 7 2
,7oMGFA
- C , 8 . 2 6 0
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72 144
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144 216 h o u r s
° C l 8 2
c1 6 0 ° —
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— — o C i a i
7 2 144 216 h o u r s
Figure 2.—Changes of fatty acids pattern in lipid classes of degermed seed during germination. Palmitic acid (C16:0), Stearic acid (C18:0), Oleic acid (C18:l); linoleic
T H E LIPIDS OF GERMINATING WHEAT 307 60 50 AO 30 20fl 10 7, Cl8
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72 1AA 216 h o u r s 72 1AA 216 h o u r s
Figure 3.—Changes of fatty acids patterns in lipid classes of germ minus rootlets. Rootlets of steeped sample were not separated. D a t a for this sample is presented as
308 ANALES DE INVESTIGACIONES AGRONÓMICAS 5 0 4 0 30 20 10j| 0 5ol 4 0 30 20fl 10 0
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S 36 108 180 h o u r s
T H E LIPIDS OF GERMINATING WHEAT 309
wer then faster than that of the other fatty acids in the germination of soy bean. Although Mattson and Volpenheim (15) have reported that linoleic acid is mainly in the beta position in wheat triglycerides, this can hardly explain its accumulation during germination since no specificity for the alfa position has been shown in plant lipases.
The T F A pattern of both the rootlets and the rest of the germ (plu-mule plus scutellum) undergoes a drastic change during the first half of the germination period (fig. 3 and 4), the proportion of linolenic acid (C 1 8 : 3) increasing almost twofold and that of oleic (C 1 8 : 1) decrea-sing. Again the triglycerides can be made responsible for most of the change since the P L F A pattern suffers little alteration. The data in figu-res 1, 3 and 4 suggests that the original triglycerides pfigu-resent in the germ are used very rapidly in the first stages of germination while new trigly-cerides with a different fatty acid patern are synthesized in the growing tissues. In the second half of the germination period, when the synthetic process becomes dominant over degradation, both the T F A content of the germ (% of dry matter) and their pattern undergo little change. The F F A , D G F A and M G F A patterns seem to reflect the overlapping of the two processes, changing continuously as it is to be expected from their high turnover.
CONCXUSIONS.
Except for the rootlets, where some decrease in their P L F A level is observed, the evolution of this fraction follows rather closely that of non-fat dry matter. The P L F A pattern is different for each part
stu-died but remains constant throughout. '
The T G F A are mainly responsible for the changes in lipid content (% of dry matter) and fatty acid pattern observed in the different tissues.
In the endosperm, a preferential degradation of T G F A accounts for the faster utilization of fat over non-fat dry matter. Linoleic acid (C 1 8 : 2 ) depletion is slower and that of oleic acid (C 1 8 : 1 ) faster than the rest of the fatty acids.
310 ANALES DE INVESTIGACIONES AGRONÓMICAS
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