Original Papers

Cryopreservation of blueberry shoot tips derived from in vitro and current shoots using D cryo-plate technique

Sabitri Adhikari Dhungana, Hisato Kunitake, Takao Niino, Shin-ichi Yamamoto, Kuniaki Fukui, Daisuke Tanaka, Shinya Maki, Toshikazu Matsumoto. . . . . . . 1[PDF]

Cryopreservation is an important tool for long-term storage of plant germplasm that is currently used for plant germplasm storage at many institutes worldwide. Recently, novel cryogenic procedures (V and D cryo-plate methods) have been developed. In this study, the most suitable conditions for preserving blueberry shoot tips derived from in vitro and current shoots using the D cryo-plate method were investigated. The D cryo-plate method has advantages such as higher regrowth after cryopreservation and a more user-friendly process compared with conventional cryogenic methods. The optimum duration of desiccation for regrowth of shoot tips from each shoot type was 1 h. To induce dehydration tolerance for the shoot tips, the effects of two cryoprotection treatments (sucrose preculture and loading solution [LS] treatment) on shoot regrowth after cryopreservation were investigated. The combined effect of both treatments significantly increased percentage regrowth (approximately 90%). No regrowth of shoot tips was attained without the two treatments. Thus, preculture and LS treatment were effective to induce dehydration tolerance for cryopreservation of blueberry shoot tips. The optimized conditions for blueberry shoot tips using the D cryo-plate technique were: preculture with 0.3 M sucrose for 1 day, LS treatment (2 M glycerol +0.4–1.0 M sucrose) for 30 min, and air dehydration for 1 h. This optimized procedure was applied to additional blueberry cultivars shoot tips derived from in vitro shoots (regrowth 46.7–100%) and current shoots (regrowth 17.2–62.7%). Furthermore, in vitro shoot tips were suitable material for the D cryo-plate method in blueberry.

MYB-mediated upregulation of lignin biosynthesis in Oryza sativa towards biomass refinery

Taichi Koshiba, Naoki Yamamoto, Yuki Tobimatsu, Masaomi Yamamura, Shiro Suzuki, Takefumi Hattori, Mai Mukai, Soichiro Noda, Daisuke Shibata, Masahiro Sakamoto, Toshiaki Umezawa. . . . . . . 7[PDF]

Lignin encrusts lignocellulose polysaccharides, and has long been considered an obstacle for the efficient use of polysaccharides during processes such as pulping and bioethanol fermentation. However, lignin is also a potential feedstock for aromatic products and is an important by-product of polysaccharide utilization. Therefore, producing biomass plant species exhibiting enhanced lignin production is an important breeding objective. Herein, we describe the development of transgenic rice plants with increased lignin content. Five Arabidopsis thaliana (Arabidopsis) and one Oryza sativa (rice) MYB transcription factor genes that were implicated to be involved in lignin biosynthesis were transformed into rice (O. sativa L. ssp. japonica cv. Nipponbare). Among them, three Arabidopsis MYBs (AtMYB55, AtMYB61, and AtMYB63) in transgenic rice T1 lines resulted in culms with lignin content about 1.5-fold higher than that of control plants. Furthermore, lignin structures in AtMYB61-overexpressing rice plants were investigated by wet-chemistry and two-dimensional nuclear magnetic resonance spectroscopy approaches. Our data suggested that heterologous expression of AtMYB61 in rice increased lignin content mainly by enriching syringyl units as well as p-coumarate and tricin moieties in the lignin polymers. We contemplate that this strategy is also applicable to lignin upregulation in large-sized grass biomass plants, such as Sorghum, switchgrass, Miscanthus and Erianthus.

Biosynthesis of volatile terpenes that accumulate in the secretory cavities of young leaves of Japanese pepper (Zanthoxylum piperitum): Isolation and functional characterization of monoterpene and sesquiterpene synthase genes

Yoshiyuki Fujita, Takao Koeduka, Mitsuhiro Aida, Hideyuki Suzuki, Yoko Iijima, Kenji Matsui. . . . . . . 17[PDF] [Supplement1] [Supplement2] [Supplement3]

Volatile terpenes are ones of the characteristic aromas of Japanese pepper (Zanthoxylum piperitum). It has been hypothesized that the specialized epithelial cells surrounding the secretory cavities of Japanese pepper fruits and leaves are responsible for the synthesis of monoterpenes and sesquiterpenes, which are generally produced by terpene synthases (TPSs); however, direct evidence for the formation of terpenes in Japanese pepper remains elusive. Here we report that monoterpenes and sesquiterpenes accumulate inside the secretory cavities of Japanese pepper leaves, but not in other parts of leaf tissues that do not include secretory cavities. We have obtained cDNAs for ZpTPS1 and ZpTPS2, which are responsible for biosynthesis of the sesquiterpenes β-caryophyllene and germacrene D, respectively, in Japanese pepper. In addition, we also identified a cDNA for the monoterpene synthase ZpTPS3. Expression of ZpTPS3 in Escherichia coli in addition to Agrobacterium-mediated transient ZpTPS3 expression in Nicotiana benthamiana demonstrated the catalytic activity of ZpTPS3 to form β-phellandrene as the major product. In situ hybridization in Japanese pepper leaf tissue revealed that ZpTPS3 transcript specifically accumulated in the epithelial cells surrounding secretory cavities. Expression of ZpTPS3 in epithelial cells was only detectable during early stages of cavity development, whereas the formation of volatile terpenes occurred at a constant rate throughout the expansion of secretory cavities. Our studies have improved the understanding of the currently uncharacterized processes controlling volatile terpene biosynthesis in Japanese pepper leaves.

Expression of rice 45S rRNA promotes cell proliferation, leading to enhancement of growth in transgenic tobacco

So Makabe, Wataru Yamori, Kynet Kong, Hiroyuki Niimi, Ikuo Nakamura. . . . . . . 29[PDF] [Supplement]

An increase in plant biomass production is desired to reduce emission of carbon dioxide emissions and arrest global climate change because it will provide a more source of energy production than fossil fuels. Recently, we found that forced expression of the rice 45S rRNA gene increased aboveground growth by ca. 2-fold in the transgenic Arabidopsis plants. Here, we created transgenic tobacco plants harboring the rice 45S rRNA driven by the maize ubiquitin promoter (UbiP::Os45SrRNA) or cauliflower mosaic virus 35S promoter (35SP::Os45SrRNA). In 35SP::Os45SrRNA and UbiP::Os45SrRNA transgenic tobacco plants, the leaf length and size were increased compared with control plants, leading to an increase of aboveground growth (dry weight) up to 2-fold at the early stage of seedling development. Conversely, leaf physiological traits, such as photosynthetic capacity, stomatal characteristics, and chlorophylls and RuBisCO protein contents, were similar between the transgenic and control plants. Flow cytometry analysis indicated that the transgenic plants had enhanced cell-proliferation especially in seedling root and leaf primordia. Microarray analysis revealed that genes encoding transcription factors, such as GIGANTEA-like, were more than 2-fold up-regulated in the transgenic plants. Although the mechanism underlying the increased growth has yet to be elucidated, this strategy could be used to increase biomass production in cereals, vegetables, and bio-energy plants.


Sucrose supplementation suppressed the growth inhibition in polyhydroxyalkanoate-producing plants

Takeshi Yoshizumi, Miwa Yamada, Mieko Higuchi-Takeuchi, Ken’ichiro Matsumoto, Seiichi Taguchi, Minami Matsui, Keiji Numata. . . . . . . 39[PDF]

Polyhydroxyalkanoate (PHA) is a thermoplastic polymer with several advantageous properties, including biomass origin, biocompatibility, and biodegradability. PHA is synthesized in transgenic plants harboring 3 enzymatic genes: phaA, phaB, and phaC (collectively referred to as phaABC). PHA-producing plants exhibit severe growth inhibition that leads to extremely low PHA accumulation when these enzymes are localized in the cytosol. This growth inhibition could be attributed to the deleterious effects of the PHA biosynthetic pathway on endogenous essential metabolites or to PHA cytotoxicity itself. We performed precise morphological observations of phaABC-overexpressing Arabidopsis (ABC-ox), which displayed typical growth inhibition. On growth medium without sucrose, ABC-ox exhibited a pale green phenotype, dwarfism, including small cotyledons and true leaves, and short roots. ABC-ox partially recovered from this growth inhibition when the growth medium was supplemented with 1% sucrose. This recovery was reversed after ABC-ox grown on 1% sucrose medium was transferred to soil. ABC-ox grown on 1% sucrose medium not only demonstrated recovery from growth inhibition but were also the only examined plants with PHA accumulation, suggesting that growth inhibition was not caused by PHA cytotoxicity but rather by a lack of essential metabolites.

Isolation and molecular characterization of a Lotus japonicus R2R3-MYB subgroup 7 transcription factor gene

Satoru Kunihiro, Daigo Tanabe, Yuiko Niwa, Keisuke Kitamura, Jun Abe, Tetsuya Yamada. . . . . . . 45[PDF]

We isolated an ortholog (LjMYB12) of the Arabidopsis R2R3-MYB transcription factor (TF) gene from Lotus japonicus to investigate the regulation of flavonoid biosynthesis, which is driven by many paralogous genes in L. japonicus. We characterized the spatial and temporal expression of LjMYB12 in leaves, stems, roots, flowers, immature seeds, seedling leaves, and seedling roots. Expression was much higher in flowers than in other tissues. To verify the relationship between the expression of LjMYB12 and that of flavonoid biosynthesis genes, we generated transgenic L. japonicus plants overexpressing LjMYB12. Overexpression of LjMYB12 resulted in the upregulation of genes for a chalcone synthase paralog (CHS1), flavanone 3-hydroxylase, and flavonol synthase. Interestingly, LjMYB12 strongly activated CHS1 but did not activate other CHS paralogs. This result suggests differences in the spatial or temporal activation of CHS paralogs by R2R3-MYB TFs. Molecular characterization of R2R3-MYB TFs in L. japonicus will reveal the effects of gene duplication on the regulation of diverse flavonoid biosynthesis.

5′-non-transcribed flanking region and 5′-untranslated region play distinctive roles in sulfur deficiency induced expression of SULFATE TRANSPORTER 1;2 in Arabidopsis roots

Akiko Maruyama-Nakashita, Akiko Suyama, Hideki Takahashi. . . . . . . 51[PDF]

Plants increase sulfate uptake activity under sulfur deficiency (−S). In Arabidopsis, SULTR1;2 is the major high-affinity sulfate transporter induced in epidermis and cortex of roots for mediating sulfate uptake under −S. Though it is known that transcript levels of SULTR1;2 increase under −S largely due to the function of 5′-upstream region, contributions of 5′-non-transcribed flanking region and 5′-untranslated region (UTR) to transcriptional and post-transcriptional regulations have not yet been individually verified. To investigate the roles of 5′UTR of SULTR1;2 in −S responses, transcript levels and activities of firefly luciferase (Luc) were analyzed in transgenic plants expressing Luc under the control of the 2,160-bp long 5′-upstream region of SULTR1;2 with (PL2160) or without (PL2160ΔUTR) the 154-bp 5′UTR. Both transgenic plants expressed similar levels of Luc mRNAs that showed significant accumulations under −S relative to +S regardless of presence of the 5′UTR. In contrast, Luc activities were detected only in PL2160 plants, suggesting presence of 5′UTR of SULTR1;2 being necessary for translational initiation while its absence impairing translation of functional Luc protein in PL2160ΔUTR. These results indicate an essential role of the 5′-non-transcribed flanking region of SULTR1;2 at positions −2160 to −155 in −S-responsive transcriptional regulation.

Lipidomic analysis of soybean leaves revealed tissue-dependent difference in lipid remodeling under phosphorus-limited growth conditions

Yozo Okazaki, Kouji Takano, Kazuki Saito. . . . . . . 57[PDF] [Supplement]

Lipid remodeling in soybean under phosphorus (P)-limitation stress was investigated via lipidomic analysis. Principle component analysis of lipidome data from plants with 4 unfolded trifoliate leaves revealed that each leaf responded to P-limitation stress differently. Upon P limitation, a substantial decrease in phospholipids was observed particularly in the 1st and 2nd trifoliate leaves, while 3rd, and especially 4th, trifoliate leaves showed lipid profiles similar to those from control plants grown under P sufficiency. Under P-limited conditions, non-phosphorus glycoglycerolipid, glucuronosyldiacylglycerol (GlcADG), significantly increased in the 1st and 2nd trifoliate leaves. The levels of some other non-phosphorus glycoglycerolipids, including monogalactosyldiacylglycerol, digalactosyldiacylglycerol, and sulfoquinovosyldiacylglycerol (SQDG), were elevated under P-limited growth conditions, while there were only slight changes in the total levels of these lipid classes upon P limitation. These results indicate that the lipid metabolic pathway in tissues of soybean plants does not uniformly respond to P-limitation stress, where lipid remodeling is very active in older leaves and phosphate appears to be preferentially remobilized to the younger tissues under P-limited conditions.

Platform for “Chemical Metabolic Switching” to Increase Sesquiterpene Content in Plants

Keiko Kobayashi, Kanako Kobayashi, Haruhiko Yamaguchi, Yukino Miyagi Inoue, Keiji Takagi, Kazuhisa Fushihara, Hikaru Seki, Masashi Suzuki, Noriko Nagata, Toshiya Muranaka. . . . . . . 65[PDF]

The biosynthetic pathway of cytosolic isoprenoids bifurcates after farnesyl diphosphate into sesquiterpene and triterpene pathways. “Metabolic switching” has been used to increase sesquiterpene content in plants by suppressing the competitive triterpene pathway using transgenic technology. To develop “metabolic switching” without using transgenic technology, we developed a model system of “chemical metabolic switching” using inhibitors of the competitive pathway. Arabidopsis plants that overexpress the amorpha-4,11-diene synthase gene were treated with squalestatin, a squalene synthase inhibitor, or terbinafine, a squalene epoxidase inhibitor. We then analyzed total sterol content as major triterpenes and amorpha-4,11-diene in the plant. Plants treated with squalestatin showed decreased total sterol content and increased amorpha-4,11-diene content. In contrast, plants treated with terbinafine showed decreased total sterol content, but amorpha-4,11-diene accumulation was quite low. These results suggest that inhibition of the enzyme just below the branch point is more effective than inhibition of enzymes far from the branch point for “chemical metabolic switching”. In addition, the activity of 3-hydroxy-3-methylglutaryl-CoA reductase, the rate-limiting enzyme of the cytosolic isoprenoid biosynthetic pathway, was upregulated in plants treated with squalestatin, suggesting that feedback regulation of 3-hydroxy-3-methylglutaryl-CoA reductase may contribute to amorpha-4,11-diene production. Here we demonstrated the effectiveness of “chemical metabolic switching” in plants.