Plant Biotechnology Vol.32 No.3

Contents

Original Papers

Peanut stunt virus-induced gene silencing in white lupin (Lupinus albus)

Masumi Yamagishi, Chikara Masuta, Masashi Suzuki, Osamu Netsu. . . . . . . 181 [PDF]

White lupin (Lupinus albus L.) plants develop cluster roots and have strong resistance to phosphorus starvation. Although many expressed sequences have been identified to explain the mechanisms used by white lupin to acquire phosphorus, the lack of a stable transformation technique has made it challenging to evaluate the functions of these expressed sequences. Virus-induced gene silencing (VIGS) is an attractive method for assaying gene function in species that are difficult to stably transform. Here, we show that the Peanut stunt virus (PSV) vector effectively induces silencing of endogenous genes in white lupin. It is unknown whether PSV is useful for VIGS; therefore, we first inoculated Nicotiana benthamiana plants with PSV harbouring fragments of the N. benthamiana phytoene desaturase gene (NbPDS). Two out of four distinct sequence fragments of NbPDS induced photo-bleaching in N. benthamiana, indicating that PSV can be used to knockdown endogenous gene sequences in a sequence-dependent manner. White lupin plants inoculated with PSV harbouring fragments of the L. albus PDS gene (LaPDS) developed photo-bleaching that was associated with a significant reduction in LaPDS mRNA accumulation. PSV spread systemically in leaves, roots, and cluster roots, and small interfering RNA of LaPDS was detected in these organs. This is the first study to demonstrate the use of VIGS by PSV, suggesting that this vector can be applied to supress endogenous gene expression in shoots and roots of white lupin and to clarify the mechanisms of phosphorus starvation resistance.

Biochemical and molecular characterization of orange- and tangerine-colored rice calli

Atsushi Ishihara, Kazuhiko Ohishi, Tetsuya Yamada, Mari Shibata-Hatta, Yuko Arai-Kichise, Satoru Watanabe, Hirofumi Yoshikawa, Kyo Wakasa. . . . . . . 193 [PDF]

We established an orange (L) and a tangerine (D) colored callus line by repeatedly subculturing rice callus line (N). The L line accumulated lycopene, β-carotene, and two lycopene isomers, while the D line accumulated much higher concentration of lycopene as well as a lycopene isomer than the L line. Genomic sequencing revealed the presence of SNP that results in the incomplete lycopene β-cyclase (β LCY) enzyme in the D line. We analyzed expression levels of carotenoid biosynthetic genes by qRT-PCR. Regarding the 2-C-methyl-D-erythritol-4-phosphate pathway genes, expression of the gene encoding geranylgeranyl diphosphate synthase 1 (GGPS1) was enhanced in both L and D lines as compared to the N line. Regarding the carotenoid pathway genes, the transcript amounts of the genes encoding phytoene synthase 2, phytoene desaturase, and ζ-carotene desaturase, were larger in the L line than in the N line; however, this pattern was not found in the D line. The expression levels of β LCY and lycopene ε-cyclase (ε LCY) gene in the D line and ε LCY in the L line were lower than in the N line. The transcript amounts of two carotenoid oxygenase genes were decreased both in L and D lines. We concluded that increased expression of GGPS1 and reduced expression of ε LCY, β LCY, and carotenoids oxygenase genes might allow an excess accumulation of carotenoids in L and D lines, and that the mutation in β LCY is responsible for the conspicuous phenotype in the D line as compared to the L line.

Functional expression of cytochrome P450 in Escherichia coli: An approach to functional analysis of uncharacterized enzymes for flavonoid biosynthesis

Kai Uchida, Tomoyoshi Akashi, Toshio Aoki. . . . . . . 205 [PDF]

Biochemical analyses of metabolic enzymes are performed using in vitro assays with enzymes and substrates. Enzyme samples are generally prepared from heterologous cell expression systems, which are also used as tools to obtain substrates that are not commercially available or cannot be easily isolated from natural sources. Cytochrome P450 (CYP) comprises a widely distributed family of monooxygenases that are commonly used for biosynthesis of natural products. Since CYP activity requires an electron transport system with membrane bound CYP reductase (CPR), it has been believed that CYP is not easily expressed in Escherichia coli, despite multiple advantages as a heterologous system compared with other systems that employ eukaryotic yeast and insect cells. In this study, we demonstrated simple and efficient methods for functional expression of CYPs in E. coli using commercially available vectors in which the transmembrane-domain truncated CYP was co-expressed with CPR as a discrete polypeptide, and we also used them to identify CYP81E11, CYP81E12, and CYP81E18 of soybean as isoflavone 2′-hydroxylase. Culture conditions were optimized for the bioconversion of I2′H, and the highest production was 161 mg l−1 of medium under optimized conditions. Subsequently, six other CYPs that are involved in flavonoid biosynthesis were tested for their applicability in the E. coli expression system. Establishment of the present method may facilitate functional expression of CYPs for preparation of CYP products, including substrates for enzymatic reactions, valuable natural products, and their unnatural derivatives.

The rice GRH2 and GRH4activate various defense responses to the green rice leafhopper and confer strong insect resistance

Takayuki Asano, Yasumori Tamura, Hiroe Yasui, Kouji Satoh, Makoto Hattori, Hideshi Yasui, Shoshi Kikuchi. . . . . . . 215 [PDF]

Rice GRH2 confers enhanced resistance to green rice leafhoppers (GRH), Nephotettix cincticeps Uhler. A pyramided line carrying GRH2 and GRH4 (TGRH29) showed strong resistance to GRH insects compared with a GRH2 near-isogenic line (TGRH11), although GRH4 alone did not confer any resistance to GRH. To explore the effects of GRH2 and GRH4 on GRH resistance, we investigated the transcriptional response of rice plants to GRH infestation using DNA microarray analysis. The expression of a large number of genes encoding pathogenesis-related proteins, lipoxygenases, terpene synthase (TPS) and WRKY transcription factor, was upregulated in response to GRH infestation in TGRH11 and TGRH29 compared with control plants. Quantitative RT-PCR revealed that expression of JAmyb and TPS was more strongly and more rapidly upregulated in TGRH29 compared with TGRH11 after GRH infestation. These results suggest that TGRH29 plants can more rapidly and strongly activate the defense response compared with plants carrying GRH2 alone. Furthermore, sesquiterpenes were emitted from TGRH29 plants in response to attack by GRH. The strong induction of sesquiterpene production in the TGRH29 line was correlated with the transcript levels of TPS genes. Our results suggest that GRH2 and GRH4 activate various defense responses and confer strong GRH insect resistance.

Agrobacterium-ediated transformation of protocorm-like bodies in Dendrobium Formidible ‘Ugusu’

Wasana Phlaetita, Dong Poh Chin, Ken Tokuhara, Ikuo Nakamura, Masahiro Mii. . . . . . . 225 [PDF]

Agrobacterium-mediated genetic transformation system was established in Dendrobium Formidible ‘Ugusu’ by inoculating PLBs with A. tumefaciens strain EHA101 (pIG121Hm) harboring hygromycin phosphotranferase (hpt) and neomycin phosphotranferase II (nptII) genes as selectable marker gene and β-glucuronidase (gus) gene as a reporter gene. For obtaining the optimum conditions for the transformation, several factors such as the stage of PLBs after subculture, bacterial concentrations, kind of inoculation medium, inoculation time and inoculation condition (with or without rotary shaking), were examined. After inoculation, PLBs were cocultivated for 3 days and then transferred for selection onto 2.5 g l−1 gellan gum-solidified ND medium containing 10 g l−1 maltose, 20 mg l−1 hygromycin and 20 mg l−1 meropenem. Hygromycin-resistant plantlets were regenerated from secondary PLBs after 4 months of selection. Transformation of these plants was confirmed by GUS histochemical assay, PCR and Southern blot analyses. The highest transformation efficiency of 18.5% was obtained when PLBs 3 weeks after subculture were inoculated with 1 : 10 diluted bacteria (OD600≈0.1) with liquid medium containing only 10 g l−1 maltose and 100 µM acetosyringone with shaking for 30 min.

Generation of transgenic tobacco plants with enhanced tocotrienol levels through the ectopic expression of rice homogentisate geranylgeranyl transferase

Hiroyuki Tanaka, Yukinori Yabuta, Masahiro Tamoi, Noriaki Tanabe, Shigeru Shigeoka. . . . . . . 233 [PDF]

Tocotrienols with three double bonds in the hydrocarbon tail are the major form of vitamin E in the seeds of most monocots and certain dicots. They have recently been attracting increasing attention for their various biological properties for human health. Homogentisate geranylgeranyl transferase (HGGT) catalyzes the committed step of tocotrienol biosynthesis. HGGT, except for enzyme from barley, has been not analyzed in detail, although cDNAs encoding HGGT have been isolated from barley, wheat, and rice. Since tocotrienol levels are higher in rice than in barley, rice HGGT (OsHGGT) may have superior enzymatic characteristics to those of barley HGGT. In the present study, we generated transgenic tobacco plants introducing OsHGGT cDNA into a nuclear genome and measured their tocopherol and tocotrienol levels. We demonstrated that the ectopic expression of OsHGGT enhanced tocotrienol levels without decreasing tocopherol levels in tobacco plants. The results of the present study may lead to a better understanding of the manipulation of vitamin E biosynthesis in leafy vegetables.

Short Communication

Long-term maintenance of high regeneration ability of switchgrass embryogenic callus

Yoichi Ogawa. . . . . . . 239 [PDF]

Switchgrass (Panicum virgatum L.) is one of the most important crops for forage and bioenergy, and embryogenic callus is an important material for molecular breeding of this species. In this study, the longevity of caryopsis-derived compact Type I callus of switchgrass was investigated during 24 months. The regeneration ability of the callus was gradually reduced after 18 months of subculture, but remained at a relatively high level after 24 months. In addition, albino formation was not induced throughout the 24-month subculture. Casamino acids improved the regeneration ability of embryogenic calli without apparent morphological change or albino induction, while proline induced friable Type II calli as well as albino shoots. Cell straining treatment coupled with medium containing casamino acids led to 4-fold higher regeneration ability. The ploidy levels of 24-month-old calli were similar to seedling explants. The present results indicate that caryopsis-derived Type I callus is stable and could be maintained long-term, and thus would be a useful source for genetic transformation of switchgrass.

Two B-type ATP-binding cassette (ABC) transporters localize to the plasma membrane in Thalictrum minus

Nobukazu Shitan, Kazuyoshi Terasaka, Hirobumi Yamamoto, Fumihiko Sato, Kazufumi Yazaki. . . . . . . 243 [PDF]

Alkaloids play important roles in plant defenses against herbivores and some alkaloids have medicinal uses. Medicinal alkaloids can be purified from plant tissues or produced axenically in cell culture systems. In culture, cells generally accumulate these toxic metabolites in the vacuole; however, treatment with benzyladenine (BA) induces cultured Thalictrum minus cells to produce the isoquinoline alkaloid berberine, which they release into the medium. A previous biochemical analysis suggested that B-type ATP-binding cassette (ABC) transporters participate in berberine efflux from cultured T. minus cells. In this study, we isolated full-length cDNAs of two novel B-type ABC transporter genes from T. minus, Tmabcb1 and Tmabcb2. The encoded transporters show significant amino acid sequence identity to the Coptis japonica berberine transporters CjABCB1 and CjABCB2. Real-time quantitative reverse transcription PCR analyses showed that BA induces an increase in Tmabcb1 and Tmabcb2 mRNA levels in cultured cells. Membrane separation and immunoblot analyses indicated that these proteins localize to the plasma membrane in T. minus cells. These data suggest that TmABCB1 and TmABCB2 participate in berberine transport in T. minus cells.

Dihydroflavonol 4-reductase activity is associated with the intensity of flower colors in delphinium

Natsuki Miyagawa, Taira Miyahara, Mitsutoshi Okamoto, Yukio Hirose, Kimitoshi Sakaguchi, Shoji Hatano, Yoshihiro Ozeki. . . . . . . 249 [PDF]

Flower color intensity is largely determined by the amount of accumulated anthocyanins. Delphinium flowers show a wide range of colors from pale pink to deep orange to red to dark blue. Here, we demonstrated that the level of anthocyanin accumulation in dark blue, orange and red varieties was higher than in pale blue and pale pink varieties. Since dihydroflavonol 4-reductase (DFR) is a key enzyme in anthocyanin biosynthesis and accumulation in plants, we investigated the relationship between flower color intensity and the level of DFR gene expression. Six delphinium varieties with different flower colors were analyzed. Varieties that accumulated relatively high levels of anthocyanin also had high levels of DFR expression and enzyme activity in crude protein extracts. By contrast, DFR expression and activity was low in varieties with low anthocyanin accumulation. Alignment of DFR amino acid sequences in the six varieties showed the presence of two types, termed DgDFR and DnDFR. Recombinant DgDFR and DnDFR proteins had similar substrate specificities, but the kinetic turnover rate of the DnDFR enzyme was higher than that of DgDFR. We conclude that DFR expression level is closely correlated with flower color intensity and that DFR is an important factor that determines anthocyanin accumulation and delphinium flower color intensity.

Note

Gene expression profile of Arabidopsis plants that overexpress ZEITLUPE/LOV KELCH PROTEIN1: up-regulation of auxin-inducible genes in hypocotyls

Aya Saitoh, Tomoyuki Takase, Hiroyuki Kitaki, Tomohiro Kiyosue. . . . . . . 257 [PDF]

ZEITLUPE (ZTL) is a blue-light photoreceptor with an F-box motif in Arabidopsis thaliana. The molecular mechanisms underlying the ZTL-dependent control of the circadian rhythm and repression of photoperiodic flowering are relatively well characterized. ZTL also positively regulates hypocotyl elongation under light, but the molecular mechanisms remain unknown. Using microarray analysis, we showed that 194 genes, including 17 SMALL AUXIN UP RNA (SAUR) genes (SAUR9, 15, 16, 19, 20, 22–24, 28, 29, 61–67) and two AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA) genes (IAA7, 29), were up-regulated and 283 genes were down-regulated in ZTL-overexpressing Arabidopsis seedlings. The results were confirmed for SAUR22, 23, and IAA29 by real-time quantitative reverse-transcription PCR. Application of the polar auxin transport inhibitor N-1-naphthylphthalamic acid (NPA), the auxin antagonist α-(phenyl ethyl-2-one)-indole-3-acetic acid (PEO-IAA), or the auxin biosynthesis inhibitor 5-(4-chlorophenyl)-4H-1,2,4-triazole-3-thiol (YUCASIN) inhibited hypocotyl elongation enhanced in ZTL-overexpressing seedlings. Our data suggest the involvement of auxin and auxin-inducible genes, including SAURs and IAAs, in hypocotyl elongation in ZTL-overexpressing seedlings.

Responses to intermittent exposure to green light during the dark period in Arabidopsis thaliana and possible involvement of cryptochrome 2

Masayuki Sato, Takumi Nishiuchi, Toshio Sakamoto. . . . . . . 263 [PDF]

The effects of green light treatment during the dark period were examined in Arabidopsis thaliana as a first step to understanding the mechanism of artificial green light effects in plants. Plants were grown under long-day conditions (an 18-h light and a 6-h dark cycle) and were intermittently exposed to green light during the dark period for 2 h 3 times a week. This green light treatment suppressed the elongation of roots and hypocotyls in wild-type plants. However, the green light-induced changes were not significant in the cry2 mutant that is deficient in the blue light receptor cryptochrome 2. The green light treatment elevated both jasmonic acid and salicylic acid levels in the wild-type plants but the elevation of the jasmonic acid level was impaired in the cry2 mutant plants. These results suggest that intermittent exposure to green light triggers artificial responses in Arabidopsis plants that do not occur in the natural environment, and that cryptochrome 2-dependent and jasmonic acid-mediated responses may be partly involved in the effect of green light on plants.

The metabolic profile of grape berry skin and a comparison of metabolomes before veraison and at harvest

Mami Suzuki, Ryo Nakabayashi, Tetsuya Mori, Kazuki Saito, Katsuhiro Shiratake. . . . . . . 267 [PDF]

Grape (Vitis vinifera L.) berry skin accumulates high amounts of secondary metabolites, such as catechin, resveratrol, and anthocyanin. Metabolome analysis using liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) was performed to profile and compare metabolites of grape berry skin before veraison and at harvest. Anthocyanines, such as marvidine 3-glucoside, were the most abundant phenolic compounds found in grape skins at harvest, whereas the amount of catechin was higher before veraison. Principal component analysis revealed seven stage-specific peaks of metabolites in grape berry skin. Two unidentified peaks were speculated by MS/MS databases searches, annotated or characterized as amino acids, with one annotated as arginine and the other characterized as neutral loss ion of glutamate and glutamine. Amino acids, including arginine, are important for grape berry taste and wine quality. Our metabolome analysis showed not only well known metabolites related to ripening of grape berry skin, including anthocyanines, but also speculated some amino acids, are important for grape berry quality.