© NIAB 2019.

This is an Open Access article, distributed Plant Genetic Resources: Characterization and Utilization (2019) 17(4); 375–378
under the terms of the Creative Commons Attribution licence doi:10.1017/S147926211900008X
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ISSN 1479-2621

Short Communication

A high proportion of beta-tocopherol in

vitamin E is associated with poor seed
longevity in rice produced under
temperate conditions
Jae-Sung Lee1, Jieun Kwak2, Jun-Hyeon Cho2, Dmytro Chebotarov1, Mi-Ra Yoon2,
Jeom-Sig Lee2, N. Ruaraidh Sackville Hamilton1 and Fiona R. Hay1,3*
1
International Rice Research Institute, Los Baños, College, Laguna 4031, Philippines, 2National Institute of Crop
Science, Rural Development Administration, Jeonju, Jeollabuk-do, Republic of Korea and 3Department of
Agroecology, Aarhus University, Forsøgsvej 1, 4200 Slagelse, Denmark

Received 29 November 2018; Accepted 21 February 2019 – First published online 10 April 2019

Abstract
Seeds of 15 diverse rice accessions, representing aus, indica, temperate japonica and tropical ja-
ponica subpopulations, were produced under temperate climate conditions in Korea and used for
vitamin E analysis and seed storage experiments at 45°C and 10.9% seed moisture content. High γ-
tocotrienol was significantly positively correlated with seed longevity. In addition, a high β-tocoph-
erol proportion relative to δ-tocopherol was significantly negatively correlated with seed longevity.
Using high-density single-nucleotide polymorphism marker data, DNA haplotype analysis showed
clear allelic variations in the region of two S-adenosylmethionine synthetase genes:
LOC_Os04g42095 and LOC_Os11g15410, which regulate the conversion of δ-tocopherol into β-toc-
opherol. Four indica accessions with rare and subpopulation-specific alleles showed a 2.3-fold
lower β-/δ-tocopherol ratio compared with accessions from other subpopulations.

Keywords: β-tocopherol, rice, seed longevity, vitamin E

Introduction indicated that the ratio of different vitamin E homologues

rather than total vitamin E content is crucial in extending
During seed storage, lipid peroxy radicals cause membrane seed longevity (Lee et al., 2017). In particular, a high pro-
breakdown and cell ageing. Vitamin E, a group of eight portion of γ-tocotrienol was correlated with high seed lon-
homologues (four tocopherols and four tocotrienols), is a gevity. A better understanding of the molecular basis of
strong antioxidant that scavenges lipid peroxy radicals, seed longevity may help improve longevity and/or identify
hence they play a role in seed longevity (Sattler et al., varieties that are short-lived in storage. We were therefore
2004; Chen et al., 2015; Lee et al., 2017). Our previous interested in understanding whether the relationship
study using a diverse rice panel grown in the Philippines between vitamin E homologue ratios and seed longevity
is still apparent when seeds are produced under different
environmental conditions. In this study, we used seeds pro-
duced under temperate conditions to assess the correlation
*Corresponding author. E-mail: [email protected] between seed longevity and vitamin E.
376 J.-S. Lee et al.

Table 1. Correlation coefficients (r) between seed longevity parameters and proportions of vitamin E homologues for 15 diverse
rice accessions.

Seed longevity Tocopherol Tocotrienol β-/δ-tocopherol

parameter ratio
α-T β-T γ-T δ-T α-T3 β-T3 γ-T3 δ-T3

Ki −0.387ns −0.533* 0.358ns 0.220ns −0.110ns 0.097ns 0.214ns −0.036ns −0.557*

Slope −0.131ns −0.046ns 0.209ns
0.425ns 0.163ns 0.384ns −0.162ns 0.201ns −0.279ns
p50 −0.477ns −0.740** 0.384ns
−0.020ns −0.406ns −0.152ns 0.516* −0.061ns −0.567*
* and **, significant at P < 0.05 and 0.01, respectively. ns, not significant (P > 0.05).

Experimental Hempstead, UK). Correlations between seed longevity

parameters and proportion of vitamin E homologues
Seeds of 20 Oryza sativa accessions held in the T.T. Chang were determined using STAR v2.0.1 (IRRI). Genomic infor-
Genetic Resources Center at the International Rice mation in the region of vitamin E-related genes were ex-
Research Institute (IRRI), representing the five subpopula- tracted from the 700k high-density single-nucleotide
tions of Indica and Japonica Variety Groups (Supplementary polymorphism (SNP) marker data (McCouch et al., 2016).
Table S1; IRRI, 2006a–q, 2007a–c)) were grown in the ex- The estimate for Ki (initial viability in normal equivalent
perimental field of the Rural Development Administration deviates (NED)) ranged between 1.10 and 10.90 NED
(RDA), Republic of Korea (N35° latitude and E128° longi- (mean 5.66 NED; Supplementary Table S1). The slope of
tude) in 2016. During seed production, mean temperature the survival curves, −1/σ, ranged between 0.18 and 0.44
and rainfall per day were 23.7°C and 6.3 mm, respectively. NED/d (mean 0.16 NED/d). Time for viability to fall to
After dormancy-breaking (50°C, 5 d), seeds were germi- 50%, p50, ranged between 5.73 and 34.58 d (mean 18.75
nated in seedling boxes and 30-d-old seedlings trans- d). Total vitamin E content varied between 9.53 and
planted on 5 June 2016, with 300 × 150 mm2 spacing. 21.66 mg/kg brown rice (mean 15.80 mg/kg). The propor-
Fertilizer, insect, disease and weed control were according tion of γ-tocotrienol was significantly positively correlated
to standard procedures. Five accessions did not flower. with p50 (r = 0.516; P < 0.05) (Table 1). In contrast, the pro-
Seeds of the other 15 accessions were harvested between portion of β-tocopherol was significantly negatively corre-
50 and 56 d after heading and dried at 25°C, 30–40% rela- lated with Ki (r = −0.533; P < 0.05) and p50 (r = −0.740;
tive humidity (RH). A sample of 300 g seeds of each acces- P < 0.01). There was no significant correlation between
sion were sent to IRRI for seed storage experiments; −1/σ and any of the vitamin E homologues. Using the pub-
remaining seeds were used for vitamin E analysis. Eight lic rice genomes database (Mansueto et al., 2017; Rice
vitamin E homologues (α-, β-, γ-, δ- tocopherol/tocotrienol) SNP-Seek Database: http://snp-seek.irri.org/, latest access
in lipid extracts of brown rice were analysed by ultra- on 26 January 2019), we determined the DNA haplotype
performance liquid chromatography (H-Class system, of 12 accessions in the region of six known
Waters, USA; Ko et al., 2003). Individual homologues S-adenosylmethionine synthetase (SAM) genes which
were determined at 298 nm excitation and 325 nm emis- regulate the conversion of δ-tocopherol into β-tocopherol
sion with a Lichrospher Si-60 column (250 × 4.6 mm i.d.; (Sattler et al., 2004). The association between haplotype
Merck Co. Germany) and quantified based on retention and β-/δ-tocopherol ratio was assessed. Clear allelic varia-
time and amounts of standards (Darmstadt, Germany; tions were observed in the region of two SAM genes:
Helios, Singapore). Seed longevity parameters were deter- LOC_Os04g42095 and LOC_Os11g15410. Four accessions
mined at IRRI through standard storage experiments (Hay belonging to the indica subpopulation with the favourable
et al., 2015, 2018). After moisture content (MC) equilibra- haplotype (shaded green in Fig. 1) showed a 2.3-fold lower
tion at 20°C, 60% RH, seeds were heat-sealed inside alu- β-/δ-tocopherol ratio (mean 0.49) compared with the eight
minium foil bags and placed at 45°C. Samples were taken accessions having the unfavourable haplotype (shaded
at 3-d intervals and tested for the ability to germinate on grey) (mean 1.11).
two layers of Whatman No. 1 filter paper in 90
mm-diameter Petri dishes. Additional samples were used
for monitoring MC: ground seeds were weighed before Discussion
and after 2 h oven-drying at 130°C (ISTA, 2018). The
mean MC was 10.3% (mean of all accessions and sample Consistent with Lee et al. (2017), the proportion of
times). Germination data were analysed by probit analysis γ-tocotrienol relative to other vitamin E homologues in the
in GenStat v. 18 (VSN International Ltd., Hemel caryopses was significantly positively correlated with seed
Beta-tocopherol and rice seed longevity 377

Fig. 1. Haplotype analysis on two S-adenosylmethionine synthetase genes regulating δ-tocopherol conversion into β-tocopherol
for 12 rice accessions (IRRI, 2006a-c, e-f, h-i, k, m, p-q; 2007, b). Green shading indicates the favourable haplotype, grey shading
the unfavourable haplotype; non-synonymous mutations are shown in red font. Three accessions, IRGC 117270, 117273 and
117277 (IRRI 2006g, j, n), were not included in the high-density SNP data set.

longevity (p50). Further, a high proportion of β-tocopherol other groups have the unfavourable haplotype. Further re-
relative to δ-tocopherol was significantly negatively corre- search such as transgenic experiments could be used to val-
lated with Ki and p50. Vitamin E α-/β- homologues have in- idate gene function. A better understanding of the
efficient molecular structures for scavenging free radicals molecular basis for seed longevity will facilitate screening
compared with γ–/δ- homologues (Jiang, 2014; Kim 2014; of genebank accessions for this trait and ultimately, ration-
Lee et al., 2017). LOC_Os11g15410 is annotated to alization of retest intervals.
S-adenosyl-L-methionine: benzoic acid/salicylic acid carb-
oxyl methyltransferase, but its network function has not
Supplementary material
been validated. In four indica accessions with high seed
longevity, there were two non-synonymous mutations (po-
The supplementary material for this article can be found at
sitions 8,732,644 and 8,733,700). An insertion at position
https://doi.org/10.1017/S147926211900008X.
8,733,744 and a deletion at position 8,733,747 may be
linked to the mutation at position 8,733,700 (Rice
SNP-Seek Database: http://snp-seek.irri.org/, last accessed Acknowledgements
26 January 2019). These functional changes might be re-
sponsible for inhibition of δ-/β-tocopherol conversion re- This research was funded by a grant from the Rural
sulting in less β-tocopherol and greater seed longevity. Development Administration, Korea (RDA) as RDA-IRRI
This hypothesis needs validating since the favourable cooperative research project (Grant Ref. PJ012723). We
haplotype was specific to the indica subpopulation and all thank Stephen Timple (IRRI) for technical support.
378 J.-S. Lee et al.

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