ArticleInternational Journal of Bioscience and Biotechnology. 1(2):67-71  (2013)journal articl

Kubo,  Hiroyoshi

Nozue,  Masayuki

Yoshida,  Kiyoshi

International Journal of Biosciences and Biotechnology

English

Shinshu University Institutional Repository

UDAYANA UNIVERSITY BIOSCIENCES AND BIOTECHNOLOGY FORUM •  67CLONING OF FLOWERING GENES (WJFLC  AND WJFT) IN WASABI (JAPANESE HORSERADISH) AND MONITORING OF FLOWERING RESPONSE WITH THEIR EXPRESSION Masayuki Nozue1*, Hiroyoshi Kubo2 and Kiyoshi Yoshida31Division of Applied Biology, Faculty of Textile Science and Technology, Shinshu University, Tokida 3-15-1, Ueda 386-8567, Japan2Department of Biology, Faculty of Science, Shinshu University, Asahi 3-1-1, Matsumoto 390-8621, Japan3Marui Co. Ltd., Toyoshina 4932, Azumino 399-8205, Japan*Corresponding author: msnozue@shinshu-u.ac.jpABSTRACTWasabi (Wasabia japonica) is a commercially important crop in Japan. We isolated a FLC ortholog (WjFLC) and FT ortholog (WjFT) from wasabi. Predicted amino acid sequence encoded by WjFLC and WjFT showed 89% and 85% identities with FLC and FT of Arabidopsis, respectively. The expression of WjFLC was high in October and reduced in November when fl ower buds are formed in wasabi. On the other hand, expression of WjFT was not detected in October and was slightly detected in November. Thereafter, WjFT was highly expressed later in February. We examined the best condition for initiation of fl ower bud formation under various artifi cial environments by monitoring the fl owering response of wasabi using WjFLC and WjFT.Keywords: expression, fl owering genes, fl owering response, wasabiINTRODUCTIONWasabi (Wasabia japonica), a member of the Brassicaceae, is a commercially important crop in Japan. Wasabi is cultivated in restricted regions such a cool mountain streams or fi elds of highland because wasabi grows at relatively low temperature and low light. Growth conditions such light and temperature, have been examined from a horticultural point of view in wasabi (Tanaka et al., 2008; Tanaka et al., 2009). However, the physiological characters in fl owering are poorly understood. The expression patt ern of fl owering-related genes could be used as a monitor in order to determine the best condition for fl owering under artifi cial environment.  Flowering is controlled by various environmental factors, including low temperature and photoperiod (Jung and Müller, 2009; Amasino and Michaels, 2010). The molecular mechanism of the fl owering has been well examined in the winter-annual plant Arabidopsis. Vernalization induces fl owering by suppressing the expression of FLOWERING LOCUS C (FLC), which is a key repressor of fl owering (Michaels and Amasino, 1999; Sheldon et al., 2000). In Arabidopsis, CONSTANS (CO) is a key transcriptional regulator of the photoperiod pathway (Suárez-López et al., 2001). CO induces the expression of FLOWERING LOCUS T (FT), which is an integrator of fl oral-inductive signals from various genetic pathways (Helliwell et al., 2006). The expression of FT occurs in the leaf phloem, and FT protein moves to the meristem (Corbesier et al., 2007) and promotes fl oral transition through interaction with FLOWERING LOCUS D(FD) (Abe et al., 2005).Under natural conditions, diff erentiation of fl ower buds in wasabi occurs between October and January and ceases in February. Infl orescence starts to grow in early spring and fl owers open. After fl owering, wasabi grows vegetatively until the following autumn. This perennial feature of fl owering is distinct from that of annual and biennial Brassicaceae, which die after fl owering. Although fl owering is an interesting subject, there is litt le information on fl owering genes in wasabi. In this study, we cloned FLC and FT orthologs from wasabi and examined their expression in the plants grown in the conditions both fi eld and artifi cial environment. ARTICLEINTERNATIONAL JOURNAL OF BIOSCIENCES AND BIOTECHNOLOGY   •  VOL.  I  NO.  2  •  MARCH 2013 ISSN: 9 772303 33700868 • UDAYANA UNIVERSITY BIOSCIENCES AND BIOTECHNOLOGY FORUMMATERIALS AND METHODSRosett e leaves of wasabi plantswere harvested from a fi eld at Nakatou in Matsumoto, Japan on July 2, October 9, November 18, 2009 and February 13, 2010 and used for isolating RNA. The average temperature in early July, early October, middle of November and middle of February was 22°C, 14°C, 6°C and 0°C, respectively. Total RNA was isolated by the standard phenol-SDS methods.Cloning of FLC ortholog (WjFLC) and FT ortholog (WjFT) from wasabi carried out usingthe procedures described in previous papers (Kubo et al., 2011; 2012). Semi-quantitative RT-PCR was performed to detect the expression of WjFLC and WjFT (Kubo et al., 2011; 2012).For heterologous expression of WjFT in Arabidopsis, the cDNA fragment between the start codon and the stop codon was amplifi ed by PCR. The PCR product was inserted into a modifi ed version of binary vector pIG121-Hm. The plasmid was transferred to Agrobacterium tumefaciens C58C1 by electroporation method. Arabidopsis thaliana Columbia was grown as described previously (Kubo et al., 1999). Arabidopsis was transformed by vacuum infi ltration. Transformed plants were selected with an agar medium containing kanamycin and carbenicillin. T1 seedlings were grown in soil and used to count leaf numbers.RESULTS AND DISCUSSIONWe isolated a FLC ortholog (WjFLC) from wasabi (DDBJ/GenBank/EMBL accession no. ADK92387). The coding region was 594 bp, which encoded a protein with 198 amino acid residues. The amino acid sequence predicted from the cDNA showed 89% identity with AtFLC (Fig. 1). MADS box domain signature was found between 3rd and 57th amino acids by PROSITE search. All the amino acid residues in the MADS box domain were conserved between AtFLC and its ortholog in wasabi.A FT ortholog (WjFT) was also identifi ed in wasabi (DDBJ/GenBank/EMBL accession no. HQ667761). A cDNA encoding a protein with 175 amino acid residues was identifi ed. Eighty–fi ve percent of the amino acids was identical as between WjFT and AtFT.Flowering was promoted remarkably when 35S::WjFT was introduced into Arabidopsis (Fig. 3). In some transformed plants, infl orescence appeared when four rosett e leaves formed (Fig. 3A). The wild type plants never formed infl orescence at the same stage. The average number of rosett e leaves for the wild type was 12.4 under continuous light, but that of the T1 plants of the WjFT overexpressor (WjFTOX) was 5.4 (Fig. 3B). Strong expression of WjFT was observed in three representative WjFTOXlines (lines 25, 27 and 30) that showed the early-fl owering phenotype (Fig. 3C). This indicates that WjFT is a promoter of fl owering.Fig. 1. Comparison of amino acid sequences of WjFLC and AtFLC (Kubo et al., 2012). Identical and similar residues are indicated by asterisks and dots, respectively.Fig. 2. Comparison of deduced amino acid sequences of WjFT and AtFT (Kubo et al., 2011). Identical and similar residues are indicated by asterisks and dots, respectively.UDAYANA UNIVERSITY BIOSCIENCES AND BIOTECHNOLOGY FORUM •  69The expression of WjFLC was examined in various parts of plant. Wasabi was harvested on July 2 to extract RNA from root, apical part of lateral bud, young leaf and mature leaf. RNA was also extracted from young leaf, rhizome and root of plants grown in a growth chamber, too. WjFLC was expressed all the tissues examined (Fig. 4).The expressions of WjFT and WjFLC were examined in the fl owering process using wasabi grown in Nakatou area in Matsumoto, Japan. Wasabi grown in a fi eld usually started to form fl ower buds in October when the average temperature was lower than 15°C. Therefore, we expected that fl ower buds would be formed between October and November, because the average temperature around Nakatou area was 14°C in early October and 6°C in the middle of November. Rosett e leaves were harvested from the plant grown in the fi eld at Nakatou on October 9, 2009, November 18, 2009, and February 13, 2010 to examine the expression of WjFT and WjFLC (Fig. 5). The expression of WjFLC was high in October but reduced in November. In February, the expression of WjFLC could not be detected. Thus, the suppression of WjFLC expression seemed to be correlated with the induction of fl owering, indicating WjFLC functions as a repressor as shown in AtFLC. On the other hand, WjFT was not detected in October, but increase in November. In February, the expression of WjFT was high (Fig. 5). Fig. 3. Heterologous expression of WjFT in Arabidopsis (Kubo et al., 2011). A, This photograph shows a representative line that has two cotyledons, four rosett e leaves, and two cauline leaves. B, Average numbers of rosett e leaves (hollow box) and cauline leaves (solid box) were calculated from 32 wild type plants (Col) and 49 independent T1 plants of WjFToverexpressor (WjFTOX). Transformed plants selected with kanamycin (T1 seedlings) were grown in soil, and leaf numbers were counted. Bars indicate SD. C, The expression of WjFT in three WjFTOX lines (line 25, 27 and 30) and Col were examined by semi-quantitative RT-PCR. The expression of tubulin (AtTUB) was used as control.Fig. 4. Expression of WjFLC in various organs of wasabi (Kubo et al., 2012). Total RNA was extracted from root (Rt), apical region of lateral bud (M), young leaf (YL) and mature leaf (ML) of the plants grown in fi elds (Exp1), and root (Rt), rhizome (Rz) and young leaf (YL) of the plants grown in growth chamber (Exp2). The expression of WjFLC was examined by semi-quantitative RT-PCR. Tubulin (WjTUB) was used for control.Fig. 5. Expression of WjFT and WjFLC in fi eld grown wasabi plants (Kubo et al., 2011; 2012). Leaves were harvested on October 9 and November 18, 2009, and February 13, 2010. Diff erent plants were used in diff erent sampling dates. The expression of WjFT and WjFLC were examined by semi-quantitative RT-PCR. The expression of tubulin (WjTUB) was used as control. Experiments were triplicated from three diff erent plants.CLONING OF FLOWERING GENES WJFLC AND WJFT  IN WASABI  JAPANESE HORSERADISH  AND MONITORING OF FLOWERINGRESPONSE WITH THEIR EXPRESSION  | Masayuki Nozue, Hiroyoshi Kubo and Kiyoshi YoshidaINTERNATIONAL JOURNAL OF BIOSCIENCES AND BIOTECHNOLOGY   •  VOL.  I  NO.  2  •  MARCH 2013 ISSN: 9 772303 33700870 • UDAYANA UNIVERSITY BIOSCIENCES AND BIOTECHNOLOGY FORUMAtFLC has a central role in regulating the response to vernalization but several closely related homologs are identifi ed in Arabidopsis (Ratcliff e et al., 2003). It is not known whether wasabi has paralogous genes other than the WjFLC. Because WjFLC was the most similar to AtFLC than the other FLC homologs of Arabidopsis, WjFLC may be a counterpart of AtFLC and play a central role in regulating the response to vernalization.There is a possibility that wasabi has other WjFT paralogs. In a preliminary experiment, several putative FT homologs were identifi ed in wasabi when PCR was performed using genome DNA as template. However, WjFT may play a central role in fl owering because WjFT is the most similar to FT among the FT Fig. 6. Expression of WjFLC in wasabi plants grown under various artifi cial conditions in the closed growing system. Plants were grown for 2 months (A) and 6 months (B) at 18°C under 12hL:12hD after seeding, and then transferred to 18°C under long day (16hL:8hD) (18LD), 18°C under short day (8hL:16hD) (18SD), 10°C under long day (16hL:8hD) (10LD) and 10°C under short day (8hL:16hD) (10SD) for 1 to 4 months, respectively. Total RNA was extracted from leaves of four (A) or three diff erent plants (B). The expression of WjFLC was examined by semi-quantitative RT-PCR. The expression of tubulin (WjTUB) was used as control.homologs of Arabidopsis. Overexpression of WjFT remarkably promoted the fl owering of Arabidopsis. Furthermore, the expression of WjFT increased in the season when wasabi forms fl ower buds. As the environmental condition for fl owering has not been established in wasabi, WjFLC and WjFT could be useful in determining the best condition for fl owering. The expression of WjFLC was examined in 2-month–old and 6-month-old wasabi plants after seeding to determine the best condition for initiation of fl ower bud formation under artifi cial environment in the closed growing system. The suppression of WjFLC expression was not observed in 2-month-old plant grown at 10°C during 4 months (Fig. 6A), and no fl ower bud formation occurred. On the other hand, WjFLC Table 1. Number of Flower buds formed in wasabi under various artifi cial environmental conditions during 3 months.UDAYANA UNIVERSITY BIOSCIENCES AND BIOTECHNOLOGY FORUM •  71expression in 6-month-old plant was markedly reduced at 10°C (Fig. 6B), and fl ower buds were formed at 10°C under long day (16hL:8hD) rather than short day (8hL:16hD) (Table 1). These results indicated that wasabi plant must reach a certain developmental stage to sense low temperature for initiation of fl ower bud formation. Exposure to low temperature more than one month is essential to suppress the expression of WjFLC in the fl owering response in wasabi. The long day condition is also important for the expression of WjFT that is the strong promoter of fl owering.ACKNOWLEDGEMENTSThis research was supported by the Program for Fostering Regional Innovation in Nagano, granted by MEXT, Japan. This work was also supported by a Grants-Aid for Scientifi c Research (24580367) from MEXT, Japan. We are grateful to Shinshu University Research Center for Advanced Plant Factory (SU-PLAF), for supporting the present study.REFERENCESAbe M, Kobayashi Y, Yamamoto S, Daimon Y, Yamaguchi A, Ikeda Y, Ichinoki H, Notaguchi M, Goto K and Araki T. 2005. FD, a bZIP protein mediating signals from the fl oral pathway integrator FT at the shoot apex. Science 309: 1052-1056.Amasino RM and Michaels SD. 2010. The timing of fl owering. Plant Physiol. 154: 516-520.Corbesier LC, Vincent C, Jang S, Fornara F, Fan Q, Searle I, Giakountis A, Farrona S, Gissot L, Turnbull C and Coupland, G. 2007. FT protein movement contributes to long-distance signaling in fl oral induction of Arabidopsis. Science 316: 1030-1033.Helliwell CA, Wood CC, Robertson M, Peacock WJ and Dennis ES. 2006. The Arabidopsis FLC protein interacts directly in vivo with SOC1 and FT chromatin and is part of a high–molecular-weight protein complex. Plant J. 46: 183-192.Jung C and Müller AE. 2009. 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Agri. 21: 175-178.CLONING OF FLOWERING GENES WJFLC AND WJFT  IN WASABI  JAPANESE HORSERADISH  AND MONITORING OF FLOWERINGRESPONSE WITH THEIR EXPRESSION  | Masayuki Nozue, Hiroyoshi Kubo and Kiyoshi Yoshida

Cloning of flowering genes (WJFLC and WJFT) in wasabi (Japanese horseradish) and monitoring of flowering response with their expression

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Cloning of flowering genes (WJFLC and WJFT) in wasabi (Japanese horseradish) and monitoring of flowering response with their expression

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