5. Korea

Molecular characterization of high level of VitE accumulating rice mutant induced
by in vitro mutagenesis
J.E. Hwang, D.S. Kim, S.-Y. Kang
Radiation Breeding Research Team, Advanced Radiation Research Institute,Korea Atomic Energy Research Institute

5.1 Introduction
Vitamin E is an important constituent of the human diet (Evans and Bishop, 1922). Vitamin E is an
essential lipid-soluble nutrient that consists of eight tocochromanols in nature, which can beseparated into two groups, α-, β, -γ- and δ- tocopherols and four corresponding unsaturatedderivatives, α-, β-, γ- and δ-tocotrienols. Tocopherols help maintain membrane structure and integrity(Falk and Munne Bosch, 2010), act as antioxidants and free radical scavengers (Tappel, 1962; Behland Moosmann, 2002), and perform other nonantioxidant functions related to signaling andtranscriptional regulation (Ricciarelli et al., 2002). Both tocopherols and tocotrienols occur in plants,
as well as in photosynthetic microbes such as Synechocystis, with the highest concentration in seeds.
The seeds of most plants have significant amounts of γ- tocopherol, whereas leaves havepredominately α- tocopherol. Tocotrienols, however, are not as prevalent in plants as are tocopherols,
but they are the primary form of vitamin E in the seeds of most monocot species (Padley et al., 1994)
and fruits of some dicots (Aitzetmüller, 1997).
In the case of tocochromanol biosynthesis pathway, seven genes coding for tocochromanol
biosynthesis enzymes have been identified and characterized in Arabidopsis and rice (Mene -Saffraneand DellaPenna, 2010). This pathway diverges at the step where polyprenyl side chain is attached tohomogentisic acid (HGA) by homogentisic acid phytyl transferase (VTE2) or homogentisic acidsolanesyl transferase (HST) (Mene-Saffrane and DellaPenna, 2010). The first committed step in thebiosynthetic pathway is the prenylation of homogentisic acid with phytyldiphosphate to form
2-methyl-6-phytylbenzoquinol (MPBQ). The overall tocopherol composition is determined by thecombined activities and substrate specificities of the homogentisate prenyltransferase (VTE2),
tocopherol cyclase (VTE1), and two methyltransferase enzymes (VTE3 and VTE4) present in a given
tissue (Jo and Hyun, 2011). The action of the cyclase directly on MPBQ leads to the formation of
δ-tocopherol. By contrast, ring methylation at position R 2 by the first methyltransferase, MPBQ
methyltransferase (VTE3), leads to 2,3-dimethyl-5-phytylbenzoquinol and the subsequent action oftocopherol cyclase leads to the formation of γ-tocopherol. The second methyltransferase, γ-tocopherol methyltransferase (VTE4), performs ring methylation at R 3, converting the γ – and δisoformstotheα-andβ-isoforms,respectively.

Tocotrienols

areformedwhengeranylgeranyldiphosphate is used in place of phytyldiphosphate as the side chain precursor.
In this study, T1001-1 was isolated from in vitro mutagenized population by ionizing radiation andshown to have increased VitE contents. To study the molecular mechanism of VitE biosynthesis, weidentified rice genome encodes seven VitE biosynthetic enzyme and we analyzed its expression
patterns. The results of DNA sequencing analysis demonstrate that the OsVTE2 promoter and- 38 -genomic sequences contain mutated region. In addition, we showed that the mutant confers retarded
seedling growth during the early seedling growth stage in rice. These observations su ggest that themutation of the OsVTE2 might affect a Vitamin E biosynthesis.

5.2 Materials and methods
5.2.1 Plant materials and growth conditions
The wild-type rice cv. Dongan embryo culture had been irradiated with gamma rays of 30–120 Gy
generated from a 60Co gamma irradiator (150 TBq of capacity; ACEL, Nordion, Ottawa, ON,Canada) at the Korea Atomic Energy Research Institute. The mutant cell lines were regenerated on
medium containing 0.5 mM 5MT. Regenerated plants from 5MT-resistant calli were geneticallystabilized through consecutive generations of self-pollination and the homozygous M10 progenies
were obtained as reported elsewhere (Kim et al., 2004).
For seedling growth test, seeds were sown on 1/2 MS medium containing Murashige and Skoog salts(Duchefa, Haarlem, Netherlands), 3% Sucrose and 0.7% phyta-agar (Duchefa, Haarlem, Netherlands)
under 16 h light/8 h dark cycle at 24 oC.
5.2.2 HPLC analysis
Tocopherols and tocotrienols were determined using HPLC (Sykam system S1211). Resolution ofvitamin E species was achieved using an Agilent Eclipse LC -Si column (4.6 mm length, 5 μm) and asolvent system consisting of methanol:water (95:5, v/v) with a flow rate of 1.5 ml min -1 . Sample
components were detected and quantified by fluorescence with excitation at 292 nm and emission at
330 nm. A sample volume of 10 μl was injected for the chromatographic analysis. Peaks of
α-tocopherol,

β-tocopherol,

γ-tocopherol,

α-tocotrienol,

β-tocotrienolandδ

γ-tocotrienol,δ-tocotrienol were identified by comparing their retention times with commercially available
authentic standards. Pure tocopherols and tocotrienols were obtained from Sigma -Aldrich (St. Louis,MO, USA) and Cayman Chemical (Ann Arbor, MI, USA), respectively. The content of each form wascalculated based on standard curves of external standards.
5.2.3 Semi quantitative RT-PCR analysis
Total RNA was isolated using Trizol reagents according to the manufacturer’s recommended
protocols (GibcoBRL, Cleveland, OH). 1 μg of total RNA was reverse transcribed in Power cDNASynthesis Kit (Intron Biotech Inc., Sungnam, Korea) for 60min at 42°C using 1 μg oligo(dT)15primers. 1 μl of cDNA was used for PCR amplification with each gene speci fic primer (Table 1). The
resultant RT-PCR products were electrophoresed and analyzed on a 2.0% (w/v) agarose gel afterstaining with ethidium bromide.
5.2.4 Western-blot analysis
The OsVTE2 expression pattern was determined by western blot analysis using the anti-OsVTE2
polyclonal antibody (1:5,000 dilution), followed by the addition of peroxidase - conjugated goatanti-rabbit IgG (1:5,000 dilution) according to the manufacturer’s guidelines. Hybridization toprotein bands was detected using the SuperSignal West Pico Trial Kit (Thermo, USA)- 39 -

5.3 Results and Discussion
5.3.1 HPLC detection of tocopherols and tocotrienols in rice grains
Rice mutant lines in the M 10 generation and the control (Dongan byeo) were grown in the same filed
under normal cultural conditions, and mature seeds were harvested for determination of vitamin Ecomposition. Normally, the β- and δ-isomers usually appear in trace amounts, while α-tocopherol andγ-tocotrienol are the most abundant isomers. In our experiments, we also found t hat it was difficult todetect δ-tocopherol. Therefore, this isomer was not included. In rice seeds, the major tocopherol
found is α-tocopherol, and amount of β-tocopherol and γ-tocopherol are relatively low (Fig. 1). As aresult, the contents of α-tocopherol increased to 42% in T1001-1 mutant seeds than that of thecontrols. HPLC analysis showed that α-tocotrienol and γ-tocotrienol were the major tocotrienols
present in seeds of mutants and control. However, we found that there was no significant change inthe content of each tocotrienol (Fig. 1). In the T1001-1 C mutant line, the γ-tocotrienol content wasincreased to 14% than in the control. The content of VitE (total of tocopherol and tocotrienol) wasincreased to 26% in T1001-1 c mutant seeds. Tocotrienols, like tocopherols, are also potent
antioxidants (Serbinova et al., 1991), but they are less readily absorbed than α -tocopherol(Kamal-Eldin and Appelqvist, 1996). Thus, high accumulation of α-tocopherolin T1001-1 mutants
provides effective protection against oxidative damage.

Figure 1. Total and individual tocochromanol accumulation in seed of the Dongan and T1001 -1.

Tocopherol and tocotrienol composition of the seed of rice was determined by HPLC. The total VitE
content was 26% increased in the T1001-1 mutant seeds compared with Dongan.
5.3.2 Phenotypic analysis of T1001-1 mutants
As T1001-1 mutants seedling grew slowly, we compared seedling growth rates in mutant and control
plants (Fig. 2B). The primary root elongation pattern of T1001 -1 mutant was not different from thatof control (Fig. 2C). Sattler et al. (2004) reported that vte2 mutants exhibited seedling growth inArabidopsis. Thus, these T1001-1 mutant phenotypes have possibility by increasing of VTE2
expression.
- 40 -Figure 2. Comparison of seedling growth in mutant and control plants .

(A)The early seedling growth differences between the control (Dongan) and mutant lines (T1001 -1)
for 7-days were determined by measuring the shoot and root lengths. (B) Quantitative analysis ofshoot lengths of 7-day-old seedlings from each mutant line. (C) Primary root length of 7-day-oldcontrol and mutant line seedlings.
5.3.3 Expression of tocochromanol biosynthesis genes
To study the molecular mechanism of VitE biosynthesis, we identified rice genome encodes seven
VitE biosynthetic enzyme and we analyzed its expression patterns. Semi quantitative reverse
transcription analysis revealed that seven VitE biosynthesis genes were expressed at different levels
in four T1001-1 mutant lines. Interestingly, VTE2 transcripts were highly accumulated in theT1001-1 C (Fig. 3), whereas the expression of VTE3 was decreased in T1001-1 lines (Fig. 3). This
result indicates that accumulation of VtiE in T1001-1 mutants is controlled by different expression ofVTE2 and VTE3 transcript.

Figure 3. Transcript levels of VitE biosynthesis genes in rice plants.

Expression profiles of 7 rice VitE biosynthesis genes in Dongan (Cont.) and mutant lines (T1001 -1).
Actin gene was used as loading control.
- 41 -5.3.4 Sequence analysis of VTE2 and VTE3
Fig. 2 showed dramatic change of VTE2 and VTE3 expression in T1001-1 C mutant. Therefore, VTE2and VTE3 genes were sequenced in both the control and T1001-1 C mutant. The A nucleotide atposition -245 is exchanged by a G nucleotide and the C nucleotide at position -106 is changed by Tof the VTE2 promoter in the T1001-1C mutant compared to the wild-type sequence (Fig. 4). The
position -245 mutation region was reported ZFP-TFs (zinc finger transcription factors) binding siteand -106 mutation region existence-box-cis element. ZFP-TFs were reported to upregulate theexpression of the endogenous Arabidopsis γ-tocopherol methyltansferage (GMT) gene (VanEenenaam et al., 2004). The presence of these motifs indicates that VTE2 may be regulated byZFP-TFs binding cis-acting elements. Genomic sequence of VTE2 was exchanged in only intron
region, therefore, there was no effect at amino acid sequence. In the T1001 -1 C mutant line, weobserved a G to A base change at position 662 and an added C of VTE3 promoter but not foundimportant cis-elements (Fig. 4). Sequencing of the VTE3 genomic DNA sequence was not different.
These results indicated that increasing of VTE2 transcript expression was controlled by VTE2
promoter sequence mutation in accumulation of tocochromanol.

Figure 4. Comparison of the promoter and genomic DNA sequences from Dongan (DA) and T1001 -1
mutant with the related region of VTE2 (A) and VTE3 (B).

Number of the DNA sequence is shown in the upper margin. The empty region at sequences indicatesthe different sequence in DA and T1001- 1C mutant.

- 42 -5.3.5 Protein expression of VTE2
The VTE2 expression pattern was determined by western blot analysis using the prediction 4 ofanti-VTE2 polyclonal antibody. To precisely define the expression pattern of VTE2, we studied theexpression levels of VTE2 protein by western blot analysis (Fig. 5). These results are similar with theexpression patterns of VTE2 transcript that was accumulated more extensively in T1001 -1 mutant
than Dongan byoe in protein level.

Figure 5. Western blot analysis of VTE2 in T1001-1 mutants and Dongan byeo .

Total proteins from seedling of Dongan or T1001-1 mutants were isolated and subjected to western
blot analysis.

- 43 -5.4 References
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