Litchi chinensis fatty acid diversity: occurrence of ... - Semantic Scholar

Biochemical Society Transactions (2000) Volume 28, part 6

~

~~

Litchi chinensis fatty acid diversity: occurrence of the unusual cyclopropanoic fatty acids E. Gontier*', N. Boussouel*, C. Terrasse*, M. Jannoyert, M. Menardf, B. Thomassetf and F. Bourgaud* *Laboratore Agronornie & Environnernent, ENSAIA-INRA, 2 av. de la Foret de Haye, BP 172, 54505 Vandoeuvre les Nancy, France, tCIRAD-FLHOR, Station de Bassin plat, BP 180, 974 I0 Saint Pierre Cedex, la Reunion, France, and fUMR 6022 du CNRS, Univenite de Technologie de Cornpiegne, BP 20529, 60205 Compiegne Cedex, France

[1,3,13-171 are of the highest interest for future developments. In order to evaluate the diversity of Litchi chinensis fatty acids, 36 different groups of seeds were analysed as methyl ester profiles by G C . These groups corresponded to 28 cultivars from two different sites on Reunion Island (Indian Ocean), and were collected on two different dates.

Abstract Litchi chinensis (Sapindaceae) is a tree that originates from China and is cultivated for its sweet fruits all over the world in warm climates. Unusual fatty acids such as cyclopropanoic fatty acids have been identified in the seeds of Litchi. Because of their potential value for industry (as inks, cosmetics, detergents, lubricants, etc.), the variability in the relative levels of unusual fatty acids in the seeds of 28 different Litchi varieties was analysed at two locations (on Reunion Island in the Indian Ocean) and on two different harvest dates. Except for one variety, all the seeds contained cis-9,10methylene-octadecanoic acid (C,,CA) at a relative level of 35-48 O 0 . T h e only variety that contained no or only traces of C,,CA was Groff, seeds of which were significantly much smaller than those from all other varieties.

Experimental Plant materials T h e total collection of C I R A D consisted of more than 40 L . chinensis ecotypes originating from different countries (China, Australia, Hawaii, Mauritius, Seychelles, India and Reunion Island). Our study was conducted on 28 of these ecotypes. Among these, some were located at Bassin Martin, at Bassin Plat or at both sites. Therefore this experiment had 36 types of sample. Each sample was represented by three trees (three replicates) and 30 fruits were harvested per tree. After weighing, the seeds were dried at 80 "C for 48 h.

Introduction Plant oils are largely used for food and industrial non-food applications [ 13. T h e physical properties of such oils are essentially due to their fatty acid composition [2]. Saturated fatty acids (palmitic, stearic) have high melting points but unsaturated (oleic) and polyunsaturated (linoleic, linolenic) fatty acids are easily oxidized [1,3]. For future industrial uses (as inks, detergents, paints, coatings, cosmetics, lubricants, etc.), new sources of fatty acids are needed. From this point of view, unusual fatty acids are studied largely because of their potential technical properties [4]. As an example, cyclopropanoic fatty acids have been isolated from different plants [4-91, such as Litchi chinensis [ 10-1 21. These unusual fatty acids could be used for the synthesis of new fat products, and thus plant sources and also gene sources ~

Fatty acid methyl ester (FAME) profile analysis Three seeds of each occurrence (cultivar, site, date) were ground and lipids from 0.1 mg of powder were extracted for 5 min with 1 ml of hexane (containing 10 mg/l of nonadecanoic F A M E as an internal standard). After centrifugation, 5 pl of trimethylammonium hydroxide was added (vortexed for 2 min) before injection of the hexane phase (1 pl) in G C (derived from [18]). Analysis was performed using a Chrompack CP9003 gas chromatograph in splitless mode, equiped with a 10-m Chrompack Cpsil5CB column (temperature from 90 to 300 "C at a 10 "C/min rate) and detection was performed with an F I D (flame ionization detector). FAME identification and quantification were achieved by comparison with standards from Sigma. Results are expressed as fatty acid relative content ('XI). Each occurrence was analysed in triplicate for further statistical analysis (ANOVA).

~~

Key words: cis-7.8-rnethylene-hexadecanoicacid, cis-9,I O-methylene-octadecanoic acid. Abbreviations used: FAME, fatty acid methyl ester; C,,CA, cis9, I 0-rnethylene-octadenoic acid: C l,CA. cis-7,8-methylenehexadecanoic acid. 'To whom correspondence should be addressed (e-rnail eric.gontier(a)ensaia.inpl-nancy.fr).

0 2000 Biochemical Society

578

Lipid Analysis

d

c 0

v)

L

0

U

579


Biochemical Society Transactions (2000) Volume 28, part 6

this hypothesis. O n the basis of the present results, we can conclude that the different occurrences tested were relatively homogeneous with regard to their seed fatty acid profile. T h e triacylglycerol levels were evaluated (reas described sults not shown) to be lower than 5 previously [10-12]. T h u s the varieties of Litchi tested cannot be considered as a potential source of unusual fatty acids, and extracting the seed oil would not be economically valuable. A further selection scheme could be envisaged, but because Litchi are trees (with slow growth) there would not be any rapid success in improving the oil level of the seeds. Nevertheless, the present study can be considered as a basis for further experiments on gene isolation, especially by using differentialdisplay techniques.

Results and discussion Among all the Litchi seed occurrences tested for their FAME profile, almost all contained palmitoleic (ClfiJ , palmitic (Clfi),cis-7,8-methylenehexadecanoic (C,,CA), linoleic (CISJ, linolenic (CIS&, oleic (C18 stearic (CIS)and cis-9,lO-methyleneoctadenoic (C,,CA) fatty acids in their fat fractions (Figure 1). In terms of fatty acid relative content, the statistical analysis revealed relative homogeneity for all the occurrences except for Groff, which presented a significantly different profile. Regarding palmitoleic and stearic contents, no statistical differences were determined by the NewmanKeuls test. All the values were in the range 0.13-1.21 '/o and 0.1-10.5 %,, respectively. For palmitic acid relative content, the highest level was detected in Groff (42.9 '1") which was significantly different (Newman-Keuls test) from all the other occurrences, which that were between 6.8 and 14.7 O d , . For C,,CA fatty acid content three groups were isolated (group A, Kwai Mi Hawai; group B, Violet, Thailand, b3b, g f l , Groff, e7 and b3; and group AB for the rest of the occurrences). Relative levels varied from 10.45 (Kwai Mi Hawai) to 1.5 O o (b3). T h e highest linoleic and linolenic contents were measured for Groff, which was significantly different from the others, which can be considered as a homogeneous group (Newman-Keuls test). Finally, C,,CA content showed the highest variability with group A (Kwai Mi Pink), group ABC (Violet), group BC (Haak ip, Lissel and Dehradum), group C (e7) and group D (Groff). In conclusion, regarding more specifically the cyclopropanoic fatty acids, the highest levels were determined in Kwai Mi Pink (469,,), Mauritus (45.1 '$) and Picard2 (45'4). In contrast, the lowest level was measured for e7, which contained only 23.3 O & . No C,,CA could be found in Groff. Compared with the other occurrences, Groff seeds were very small and embryos were aborted, and this may explain the results obtained here. In fact, cyclopropanoic fatty acid synthesis seems to be due to a cyclopropanoic fatty acid synthase that is responsible for the addition of a methylene group on to the double bond of oleic acid phosphatidic acid ester. If the corresponding gene is only expressed within the embryo, this would explain the absence of C,,CA in Groff seeds. Further experiments will thus have to be conducted to determine the expression profile of this gene to test


'