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155М 0202-5493. МАСЛИЧНЫЕ КУЛЬТУРЫ. Научно-технический бюллетень Всероссийского научно-исследовательского института масличных культур. Вып. 2 (151-152), 2012
б) доклад
на международной конференции по подсолнечнику в Аргентине (27.02-01.03.2012)
TOCOPHEROL MUTATIONS IN SUNFLOWER SEEDS (a review report of invited speaker)
Ya.N. Demurin,
Doctor of biological sciences, professor VNIIMK
Russia, 350038, Krasnodar, Filatova st., 17
Tel.: (861) 274-55-94
E-mail: yakdemurin@yandex.ru
Keywords: antioxidant, lipids, isomer, genetics, vitamin E
UDK 633.854.78:575
Dear colleagues, I was kindly asked by the organizing committee of the 18th International Sunflower Conference (Mar del Plata, Argentina, 2012) to make a presentation covering the topic of tocopherols mutations in sunflower. My speech is considered to be a satellite to the Leonardo Velasco (Spain) report.The scope of my presentation includes a review on tocopherols mutations based mainly on classical genetics point of view.
Tocopherols are natural fat-soluble powerful antioxidants protecting lipids from autooxidation as the free radical scavengers. Four homologues a, P, y and 5 differ in the number and position of methyl groups on the aromatic part of the chromanol head. There is an inversion in vitamin E in vivo and antioxidant in vitro activities in the line of tocoph-erols where a-T has the highest vitamin and the lowest antioxidant properties [1].
Fig. 1
Chemical structure of tocopherols
Ri R2 Tocopherol
CH3 CH3 a (5,7,8-trimethyltocol)
CH3 H P (5,8-dimethyltocol)
H CH3 у (7,8-dimethyltocol)
H H б (8-methyltocol)
Tocopherols widely occur in plants but the form of these substances often differs in the leaves and seeds of the same species. а-T is typically the main form in leaves. The seeds of different plant species, however, can be enriched in any of the four forms. All oil crops can be divided into three groups. Sunflower, olive and safflower primarily contain а-T whereas flax, rape and soybean are enriched in y-T. Castor bean possesses high concentration of б-T. So, from the genetics point of view the high а-T profile is a wild type for sunflower species [2].
Fig.2
Tocopherols in oil crops
Crop Tocopherol composition, % Total content
а в Y б in oil, ppm
sunflower 94 2 Э 1 820
olive sc 0 10 0 160
safflower 85 Э 11 1 800
flax 0 0 sc 10 450
rape 29 Э 67 1 6Э0
soybean 10 2 62 26 1100
castorbean 6 6 2Э 65 480
phenotype had up to 100 % of y-T in LG17 line which was developed by VNIIMK. Later, two spontaneous mutations were obtained at Cordoba, Spain as a result of wide-scale germplasm screening. Inbred lines with increased P-T (T589) and high y-T (T2100) were developed. Another high y-T mutation was revealed with chemical EMS mutagenesis. Finally, in 2006 the tph3 or d cryptic modifier mutation was postulated by an American team of molecular geneticists to explain high content of P-T in tphl and high content of 5-T in double mutation [3].
Fig. 3
History of tocopherol mutations in sunflower
year
2006 ■ tph3 (d) ^ USA
2004 2003 - tph2 (EMS) - tphl, tph2 - tph2, high y - tphl, increased в
Spain, CSIC
1987 -1986 -
^Russia, VNIIMK
1966 - - no а-tocopherol ^Hungary
strains
Concerning genetic variability of tocoph-erols composition in sunflower seeds, it should be mentioned that the earliest data of changed tocopherols profile was obtained with two strains of OP variety planted in Hungary in 1966. These strains did not contain а-T, possessing only в- and y-isomers undivided by TLC. Unfortunately, these observations did not result in further investiga-tion.The first spontaneous mutation of tphl of tocopherol composition with about 50 % of increased content of в-T was revealed after selfing of a plant of OP variety VNIIMK 8931 in Krasnodar, Russia. It was done with half-seed technique on the basis of TLC followed by Emmerie-Engel reaction. An inbred line LG15 with increased в-T phenotype was developed. The second spontaneous mutation tph2 was found at VNIIMK in specimen № 44 of the VIR world germplasm collection. The mutant
The seed exchange of mutant lines between VNIIMK and Cordoba Institute allowed doing genetic identification. Allelic tests showed functional identity of both increased P-T lines carrying tphl and high y-T lines tph2 from different countries [4].
Fig. 4
Origin of tocopherol mutations
Mutation Type Line Origin Reference
tphl spontaneous spontaneous LG1S TS89 VNIIMK 8931 Peredovik Demurin (1986) Velasco et al. (2003)
tph2 spontaneous spontaneous EMS induced LG17 T2100 IAST-1 No.44 VIR Peredovik Peredovik Popov et al. (1987) Velasco et al. (2003) Velasco et al. (2004)
To compare tocopherol mutant phenotypes correctly it should be taken into account that the TLC profiles (indirect tocopherol determination) may differ from HPLC (direct determination) in the way of lowering the percentage of 5-T due to its relatively low levels of staining ability with Emmerie-Engel reaction. For example, the same oil showed higher to 15 % of 5-T determined with HPLC [5].
Fig. 5
The first picture of two tocopherol mutations included four genotypes followed by corresponding phenotypes. Obviously, tph2 has an epistatic effect on tphl. Recombinant double mutant homozygote produces new phenotype with increased content of 5-T [6].
Fig. 6
Tocopherol mutations in sunflower seeds
Line Mutation Dominance degree Tocopherol composition, %
a P y 6
VK 373 wild type 0.87 97 0 3 0
LG 15 tphl recessive 50 50 0 0
LG 17 tph2 recessive 5 0 95 0
LG 24 tphl, tph2 recessive 8 0 84 8
Expressivity of tocopherol mutations can be estimated with a range of phenotypic variability of constant inbred lines under influence of the modifier genes of genetic backgrounds, ontogenesis and environmental factors. The tphl mutation within different lines varies from 70 (max) to 30 (min) % of P-T content. The maximum value is reached due to action of tph3 enhancer modifier [7].
Fig. 7
The tph2 looks like a leaky one with maximum level of expressivity up to 100 % of y-T and nearly 20 % of y-T at minimum level. The inheritance analysis of this difference has shown the additive action of modifiers in F1 and F2 generations [8].
Fig. 8
Inbred lines with maximum expressivity of double homozygotes possess only y-T and 5-T, whereas minimum level of expressivity gives the unique phenotype which consists of four forms. The highest percentage of 5-T, up to 40 % (TLC), is believed to be reached due to action of tph3 allele [9].
Fig. 9
Genetic background influence on tphl + tph2
60
%
_ a
P
“V ■ 6
max min
expressivity
Seed maturation from 10 to 38 days after flowering influenced tocopherol composition for both normal and mutant genotypes in the way of increasing of a-T content. That can be explained by taking into account a-T as a final substance in biosynthesis [10].
Fig. 10
Seed maturation of tphl and tph2
a-tocopherol, %
120 i inn -
20
10 17 24 31 38 dAF
Mutations for tocopherol composition can be detected in different parts of a plant such
as roots, hypocotyl, leaves, pollen, and corresponding callus tissues. The only exception was the absence of tphl expressivity in the green leaves both for single and double mutations [11].
Fig. 11
Expressivity of tphl and tph2 in different parts of a plant
Mutation Part of a plant
seed / callus hypocotyl / callus leaf / callus root pollen
wild type -/ - -/ - -/ - - -
tphl + / + + / + -/ + + +
tph2 + / + + / + + / + + +
tphl, tph2 + / + + / + +* / + + +
* - as tph2 phenotype
An experiment with temperature influence showed that tphl increased by 9 % of a-T content under higher temperature. Both normal genotype and tph2 mutation were constant [12].
Fig. 12
Temperature influence on tphl and tph2
a - tocopherol, % 9n/1R°r
normal tph1 tph2
genotype
Molecular genetics approach revealed a modifier cryptic recessive mutation, designated d, which has no effect in a normal genotype but increases both P-T content in tphl homozygotes up to 70 % and 5-T in
tphl tph2 to 40 %. Three methyltransferase mutations thpl (m), tph2 (g) and tph3 (d) were mapped to linkage groups 1, 8 and 4 respectively. Probably, tph3 is a part of genetic background influencing mutation expressivity found earlier [13].
Fig. 13
Molecular genetics of tocopherols
(Hass et al., 2006; Tang et al., 2006;
Garcia-Moreno et al., 2006; Vera-Ruiz et al., 2006)
Mutation Linkage group
tphl ^ m 1
tph2 ^ g 8
tph3* d (tphl enhancer) 4
* Fernandez-Martinez et al., 2009
The combinations of three mutant alleles allow producing wide range of tocopherol profiles. A set of inbred lines of this genetic collection is the convenient tools for different types of research [14].
Fig. 14
Genotype-phenotype correspondence for tocopherol mutations (m)
nor one. Tphl mutation was recently shown to knockout the “5-carbon” type of methyltransferase and tph2 - the “7-type” of enzyme [15].
Fig. 15
Proposed genetic blocks in tocopherol biosynthesis
2-demethylphytylplastoquinol "7"-methylation/ cyclization
S tphl
phytylplastoq uinol 5 -tocopherol
cyclization I tph2{ "5"-methylation
Y -tocopherol p -tocopherol
\ tph2 tphl /
"5"-methylation \ / "7"-methylation
a - tocopherol
A few words about breeding application should be given. Tphl was used in the first commercial hybrid Krasnodarsky 917 with increased P-T content (registered in 1992). Moreover a new high oleic hybrid Oxy contains the triple tocopherol mutations which lead to high y/5-T content and enhanced oxidative stability of the oil [16].
Fig. 16
Tocopherol mutations in VNIIMK breeding program (Krasnodar, Russia)
Tphl Tph2 Tph3 Tocopherol composition (TLC), %
a p Y 5
+ + + 95 2 3 0
m + + 50 50 0 0
m + m 35 65 0 0
m m + 10 0 80 10
m m m 0 0 60 40
+ m + 5 0 95 0
+ m m 5 0 95 0
+ + m 95 2 3 0
There are two routes of a-T biosynthesis. The way from Y- to a-T is considered the main one and that from 5- via P- to a- a mi-
Hybrid State registration, year Mutation Tocopherol profile
Krasnodarsky 917 Oxy 1992 in trial tphl tphl, tph2 increased p (50 %) high y / 5 (60/40 %)
Conclusion includes: sunflower is the first plant with genes, identified by inheritance analysis, controlling tocopherol composition; there are three spontaneous recessive non-lethal non-seed specific mutations; there is
155М 0202-5493. МАСЛИЧНЫЕ КУЛЬТУРЫ. Научно-технический бюллетень Всероссийского научно-исследовательского института масличных культур. Вып. 2 (151-152), 2012
wide genotypic range of tocopherol profiles for a-, P-, y- and 5-forms.
Future direction will be focused at: a bridge between classical, molecular and biochemical genetics of tocopherols; we should clarify mechanisms of genetic blocks in tocopherol biosynthesis; breeding applications of new tocopherol profiles with enhanced oil quality will draw attention.
Acknowledgments: P.S. Popov (19282003) for a long time collaboration; J.F. Fer-nandez-Martinez (CSIC) for seed supply; financial support of research grant 11-0496522 from RFBR and VNIIMK; financial support of BASF (Russia) for conference participation.
