A previously described D genome locus (Pln) that controls sterol esterification in the wheat kernel has been assigned to the short arm of chromosome 7 D by comparison of the steryl ester phenotype of euploid kernels of Triticum aestivum variety Chinese Spring with those of the compensated nulli-tetrasomic lines and the 7 D S ditelosomic. Palmitate is the predominant ester in all but the 7 D nullisomic combinations, which have linoleate as the main ester. These lines also show a marked decrease in sterol esterification and a two-fold increase in free sterol, indicating that chromosomes 7 A and 7 B do not compensate for the loss of esterification capacity associated with 7 D

Bennett

Bondjers

Bush

Carbonero

Dupéron

F. Garcia-Olmedo

García-Faure

García-Olmedo

Gilles

Grunwald

Hartman

J.V. Torres

Kemp

Méance

Skarsaune

English

García Olmedo, Francisco

Torres, V.

Archivo Digital UPM

Bjórkerud Anal.

Fd Agrie,

Michele Brillard and Paulette Dupéron,

Chromosomal location of a gene that controls sterol esterification in Triticum aestivum L.

Servicio de Coordinación de Bibliotecas de la Universidad Politécnica de Madrid

CHROMOSOMAL LOCATION OF A GENE THAT CONTROLS STEROL 
ESTERIFICATION IN TRITICUM AESTIVUM L. 
J.V. TORRES and F. GARCÍA-OLMEDO 
Department of Biochemistry, Politechnical University of Madrid, E.T.S. Ingenieros 
Agrónomos, Madrid 3 (Spain) 
SUMMARY 
A previously described D genome locus (Pin) that controls sterol esterifica-
tion in the wheat kernel has been assigned to the short arm of chromosome 
7 D by comparison of the steryl ester phenotype of euploid kernels of 
Triticum aestivum variety Chinese Spring with those of the compensated 
nulli-tetrasomic lines and the 7 D S ditelosomic. Palmitate is the predominant 
ester in all but the 7 D nullisomic combinations, which have linoleate as the 
main ester. These lines also show a marked decrease in sterol esterification 
and a two-fold increase in free sterol, indicating that chromosomes 7 A and 
7 B do not compénsate for the loss of esterification capacity associated with 
7D. 
INTRODUCTION 
It is widely acknowledged that the physiological functions of higher plant 
sterols have nowhere near been established [1—9]. Free sterols, steryl esters, 
steryl glycosides and acylated steryl glycosides have been isolated from differ-
ent tissues in a number of plant species. These sterol forms have been analyzed 
during development and differentiation processes, such as seed development 
and germination, senescence, etc., in order to find some clues to their func-
tions. Although such investigations have been largely inconclusive in this 
connection, they have at least resulted in the formulation of some hypotheses 
concerning the physiological roles of these compounds. 
It has been postulated that free sterols are integral parts of plant cell mem-
branes, where they influence cell permeability [3,4]. However, only cholesterol 
and campesterol have been shown to change permeability of excised barley 
roots, while the major higher plant sterols, /3-sitosterol and stigmasterol, were 
virtually ineffective [4]. 
Abbreviations: L, linoleate (pattern); PL, palmitate (pattern). 
Cholesterol and (3-sitosterol can act as precursors of C-21 plant steroids, 
but this function can hardly justify the high level of free sterol in most plant 
tissues [2]. 
Steryl esters have been variously implicated in storage [5], transport 
[1,3—5] and subtraction of free sterols from membranes in senescent cotyl-
edons [5]. 
Finally the glycosides, acylated and non-acylated, have been considered as 
reserved forms [7], or as structural elements [8], based on their evolution 
during germination. 
Experimental data obtained by different workers for different species 
are somewhat conflicting and do not make it possible to draw a general pie-
ture of some fundamental matters, such as intracellular distribution of sterol 
forms, their evolution in reserve and developing tissues, etc. 
In our opinión, some of the above hypotheses could be tested and some 
of the discrepancies resolved if intra-specific genetic variants affecting sterol 
metabolism could be compared in near-isogenic backgrounds and correlated 
with intra-specific physiological differences. Compensated nulli-tetrasomic 
lines of hexaploid wheat obtained by E.R. Sears can be suitable for such 
studies, provided that homoeologous loci affecting a given function do not 
produce very similar effects. This is the case of a previously described [10—12] 
D genome locus (Pin) that controls steryl ester pattern. We report here our 
investigation of nulli-tetrasomic lines of Triticum aestivum, variety Chinese 
Spring, in order to lócate this gene and to search for other possible non-
compensated homoeologous loci affecting sterol metabolism.. 
MATERIAL AND METHODS 
Nulli-tetrasomic lines of wheat, variety Chinese Spring, (T. aestivum L.) 
were obtained from E.R. Sears and from T. Mello-Sampayo, who also 
provided the 7 D S ditelosomic. 
Two kernels from each line were cut in in half with a razor blade. The 
halves distal from the embryo were used for biochemical analysis and those 
including the embryo for cytological control and multiplication. 
The half kernels were squashed between two polished metal plates and 
transferred to small tubes. Extraction was performed with diethyl ether 
( 5 + 5 vol.), first for 16 h, with four 1-min periods of sonication in a cleaning 
bath (Varían AG), then for 30 min, with 1 min sonication. It was previously 
found that extraction with chloroform:methanol (2:1) or water-saturated 
rc-butanol did not increase the yield of free and sterified sterol. 
The extraets were directly spotted under nitrogen in a Silicagel píate 
(0.2 mm thick) with the aid of a capillary tube. Two 18-cm runs with carbón 
tetrachloride and a 10-cm run with benzene : ethyl acétate (95:18) [13] 
resolved the individual esters and the free sterol in a single píate. Authentic 
samples of cholesteryl palmitate (Serva), oléate and linoleate (Merck AG) and 
j3-sitosterol (Serva) were also run in the TLC. 
Quantitation of the different sterol fractions was performed by a modifica-
tion of the fluorometric method of Bondjers and Bjórkerud [14]: Following 
chromatography, the plates were exposed to iodine vapor and the sterol spots 
were marked. After disappearance of iodine from the spots, these were 
scraped and twice extracted with chloroform. The combined extracts were 
brought up to 3.5 mi, then 0.25 mi of ZnCl2 reagent and 0.25 mi of acetyl 
chloride were added. After 40 min at 60° in a thermoblock, the tubes were 
cooled and the fluorescence measured in a Turner fluorometer (filters 7—60, 
365 mu, and 2 A, 415 mju). Two sets of standards were used: one was run 
through the complete procedure and the other was directly subjected to 
fluorometry. Recovery was higher than 95 %. 
RESULTS AND DISCUSSION 
Two steryl ester patterns, designated PL and L after their main esters, 
have been found in mature kernels from species of the Aegilops - Triticum 
group [12] and from other Gramineae (Agropyron, Sécale [10], Zea, Hordeum, 
Avena [15]). ThePLpattern is more widely distributed than the L in diploid 
species and shows dominance over it in synthetic and natural alloploids in 
which L and PL genomes coexist [12]. All tetraploid wheats included in our 
studies (T. durum, T. turgidum and T. polonicum, genomes AB, and T. 
timopheeui, AB) show the L pattern, while most T. aestivum (ABD) varieties 
and all D genome species (Ae. squarrosa, D, Ae. crassa, DMcr, Ae. uentricosa, 
DMV, Ae. cylindrica, DC, and Ae. juvenalis, DOM5) have the PL phenotype. 
Appropriate crosses between PL and L varieties indicated that steryl palmitate 
is inherited as though determined by a dominant alíele at a single locus, 
designedPto [12]. 
Free and sterified sterol patterns of the nulli-tetrasomic lines, the euploid 
and the 7 D S ditelosomic are shown in Fig. 1. Both nulli-7 D combinations 
show the L phenotype, while the euploid, the 7 D S ditelosomic and the 
remaining nulli-tetrasomics show the PL pattern. No other drastic change in 
the steryl esters of the nulli-tetrasomics is observed. We therefore conclude 
that gene Pin is located in the short arm of chromosome 7 D. 
An increased proportion of free sterol is associated with the decrease in 
steryl palmitate presented by lines nulli 7 D - tetra 7 A and 7 D - tetra 7 B. 
This is more clearly shown in Table I, where data for homoeologous chromo-
some group 7 are expressed on a dry matter basis. A reduction of 80—90 % 
in steryl palmitate net synthesis and a more than 100 % increment in free 
sterol indicate that chromosomes 7 A and 7 B do not compénsate for the 
loss of esterifying capacity associated with chromosome 7 D. On the other 
hand, tetra 7 D lines do not show an increase in steryl palmitate or in total 
esters, which is consistent with our previous observation of no dose effect 
for alíele Pin [12]. 
All diploid species, includingAe. squarrosa (D) and Ae. speltoides 
(potential B genome donor) also synthesize steryl linoleate. This observation 
WlOOg 
15 
30 
20 
10 
% i 
20 
10 
% 
eo 
50 
40 
30 
20 
10 
% 40 
_ _FREE _ + 
• STERIFIED 
LINOLEA TE 
33 
_ ,_ -OLÉATE 
^tr.J^^ . PALMITA TE 
M 1,1 I M M I I I I I I I I M I I I M I I I, ,1 I I I 
.^V-V^A^V-! # 3 _ FREE STEROL 
« u m - ÁA¿é¿¿fe¿¿¿ÁÁ¿¿¿¿¿¿¿¿ÁÁÁ¿¿¿ÁX¿¿¿MA¿¿¿ó i, 
TETRA — BDApABApABBDApABApABBDaDABBDApABBDADAB I — 
L D I T E L O 7DS 
EUPLOID 
HOMOEOLOGOUS CHROMOSOME GROUP No. 
Fig.l. Free and esterified sterol patterns in compensated nulli-tetrasomic, euploid, and 
7 D S ditelosomic lines of T. aestivum var. Chinese Spring. Free plus esterified sterol is 
expressed in mg/100 g dry matter. Sterol in each fraction is expressed as % of free plus 
esterified. 
TABLE I 
FREE AND ESTERIFIED STEROL IN COMPENSATED NULLI-TETRASOMICS OF 
GROUP 7 
Line 
Nulli 
7 A 
7 A 
7 B 
7 B 
7 D 
7 D 
Euploid 
Tetra 
7 B 
7 D 
7 A 
7 D 
7 A 
7 B 
mg sterol/100 g dry matter 
Free 
2.7 
2.2 
2.0 
2.3 
3.0 
3.0 
1.4 
Palmitate 
6.5 
5.8 
6.1 
6.8 
1.2 
0.7 
6.3 
Oléate 
2.2 
2.0 
3.2 
1.3 
2.2 
1.7 
1.5 
Linoleate 
3.1 
2.3 
1.8 
2.4 
2.8 
2.4 
2.8 
Free + esterified 
14.5 
12.3 
13.1 
12.8 
9.2 
7.8 
12.0 
and the fact that enzymes involved in steryl ester synthesis and degradation, 
characterized in other biological systems, are not specific for a single fatty 
acid, led us to the following tentative conclusions: (/) gene Pin is responsible 
for palmitate and linoleate synthesis and the L pattern is controlled by an 
alternative variant of gene Pin; (ii) if the B genome donor was a PL diploid, 
steryl ester synthetic capacity associated to the B genome was lost early in 
tetraploid wheat evolution, and (iii) part of the synthetic capacity for lino-
leate in T. aestivum (ABD) is associated with the D genome [12,16]. Since 
no significant changes of linoleate level are associated with any group 7 
chromosome, the above conclusions cannot be corroborated or discarded. 
The results in Table I are compatible with full compensation for linoleate 
synthesis between chromosomes of group 7, with lack of dose effect, or even 
with control of this synthesis by genes located in a different homoeologous 
group. 
Present availability of nulli-tetrasomics did not permit us to investígate 
their steryl glycosides or the anatomical and subcellular distribution of their 
free and sterified sterol. The above results have led us to a re-investigation 
of the action of gene Pin during kernel development to be published else-
where. Ten-fold intraspecific differences in endosperm free sterol have been 
found and their correlation with physiological differences is being investigated. 
ACKNOWLEDGEMENTS 
We thank Drs. E.R. Sears and T. Mello-Sampayo for the gift of samples. 
REFERENCES 
1 R.J. Kemp, L.J. Goad and E.J. Mercer, Phytochemistry, 6 (1967) 1609. 
2 R.D. Bennett, E. Heftmann and B.J. Winter, Phytochemistry, 8 (1969) 2325. 
3 C. Grunwald, Plant Physiol., 45 (1970) 663. 
4 C. Grunwald, Plant Physiol., 48 (1971) 653. 
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6 R. Dupéron, Michele Brillard and Paulette Dupéron, Compt. Rend., 274 (1972) 2321. 
7 P.B. Bush and C. Grunwald, Plant Physiol., 50 (1972) 69. 
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9 Marie-Andrée Hartman, Mina Ferne, C. Gigot, R. Brandt and P. Benveniste, Physiol. 
Vég., 11 (1973) 209. 
10 F. García-Olmedo, Bol. Inst. Nac. Invest. Agron., 25 (1965) 409. 
11 R. García-Faure, F. García-Olmedo and M. Vallejo, J. Sci. Fd Agrie, 19 (1968) 322. 
12 F. García-Olmedo, Nature, 220 (1968) 1144. 
13 S.K. Skarsaune, V.L. Youngs and K.A. Gilíes, Cereal Chem., 47 (1970) 533. 
14 G. Bondjersand Bjórkerud Anal. Biochem., 42 (1971) 363. 
15 K.A. Gilíes and V.L. Youngs, Cereal Chem., 41 (1964) 502. 
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Chromosomal location of a gene that controls sterol esterification in Triticum Aestivum L.

Chromosomal location of a gene that controls sterol esterification in Triticum aestivum L.

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