Plant Biotechnology Vol.33 No.1


Original Papers

An MYB transcription factor regulating specialized metabolisms in Ophiorrhiza pumila

Emelda Rosseleena Rohani, Motoaki Chiba, Miki Kawaharada, Takashi Asano, Yoshimi Oshima, Nobutaka Mitsuda, Masaru Ohme-Takagi, Atsushi Fukushima, Amit Rai, Kazuki Saito, Mami Yamazaki. . . . . . . 1 [PDF]

Camptothecin is a plant-derived alkaloid and important precursor of clinically used anti-tumor drugs, but little is known about regulatory mechanism of camptothecin production in plants. We show here that a MYB transcription factor, OpMYB1, isolated from Ophiorrhiza pumila is a regulator of camptothecin biosynthesis. OpMYB1 has an EAR-like motif and exhibits a transcriptional repression activity in an in vivo assay using Arabidopsis thaliana leaves. Overexpression of OpMYB1 in hairy roots of O. pumila resulted in reduced production of camptothecin and reduced expression of OpTDC encoding triptophane decarboxylase catalyzing the earliest step in camptothecin biosynthesis. From the deep transcriptome analysis, GO enrichment in secondary (specialized) metabolisms, especially in phenylpropanoid pathway was observed in the hairy roots over-expressing OpMYB1. Furthuremore, gene suppression by OpMYB1 was revealed in biosynthetic pathways of seco-iridoids, monoterpene indole alkaloids, anthraquinone and chlorogenic acid. These results suggested that OpMYB1 is a negative regulator to fine-tune the general specialized metabolisms in O. pumila.

Quantification of stimulus-induced callose spots on plant materials

Masaki Kohari, Kohei Yashima, Yoshitake Desaki, Naoto Shibuya. . . . . . . 11 [PDF]

Plants are known to synthesize and accumulate callose, a β-1,3-linked glucan, in response to pathogen infection, elicitor treatment and wounding. Although callose deposition has often been used as an index of defense responses in plant, its quantification is laborious and difficult. We have developed a simple computer program to quantify callose spots on microscopic images based on multiple Rotational Morphological Processing (RMP), which is suitable for the detection of biological spots with intricate morphology. The program developed in this study is useful for the detection of callose spots on various biological backgrounds, making it possible to compare the responses of plant materials with different genetic background or different stimuli. Validity of this program was supported by comparing the results obtained by the new method and conventional manual counting. This program can be applicable to wide range of images taken by various microscopic systems and also in variable formats.

An efficient Agrobacterium-mediated transformation method for switchgrass genotypes using Type I callus

Yoichi Ogawa, Masaho Honda, Yasuhiro Kondo, Ikuko Hara-Nishimura. . . . . . . 19 [PDF]

Switchgrass (Panicum virgatum L.) is an important bioenergy crop. A reliable and efficient transformation method is required to assist with molecular breeding of this crop. In this study, we established a simple and efficient Agrobacterium-mediated transformation method for caryopsis-derived Type I callus by optimizing the cocultivation (7 days at 22°C on medium supplemented with 10 g l−1 glucose and 100 μM acetosyringone) and preculture (2 weeks on medium supplemented with 5 g l−1 casamino acids after cell straining) conditions without the need for time-consuming treatments before and after cocultivation. The present transformation method was successfully applied to different genotypes of switchgrass including a recalcitrant lowland cultivar. The transformation efficiencies of callus lines of the lowland cultivars ‘Alamo’ and ‘Kanlow’ were 12.5–59% and 6.3–20%, respectively. An upland cultivar ‘Trailblazer’ formed Type I calli and one of three tested callus lines produced transgenic plants at relatively high efficiency (7.5%). In contrast, Type I callus formation was unsuccessful for two other upland cultivars, ‘Blackwell’ and ‘Cave-in-Rock.’ This simple and efficient transformation method is suitable for routine and large-scale experiments due to its ready availability of caryopses, prevalence of Type I callus formation, and longevity in the regeneration ability of the callus.

The Lotus intrinsic ethylene receptor regulates both symbiotic and non-symbiotic responses

Kana Miyata, Tomomi Nakagawa. . . . . . . 27 [PDF]

The phytohormone ethylene regulates plant growth, development, and responses to both biotic and abiotic stresses. Ethylene also negatively regulates rhizobial symbiosis in legumes, although the intrinsic ethylene signaling components in legumes are still largely unclear. We report a novel ethylene insensitive mutant named Ljetr1 from the model legume Lotus japonicus. Ljetr1 showed growth tolerance to high concentrations of 1-amino-cyclopropane-carboxylic acid, the biosynthetic precursor of ethylene. Petal senescence and abscission were delayed and number of nodules was slightly increased compared to wild-type. Mapping analysis and genome sequencing showed that Ljetr1 bears a mutation in the ethylene-binding domain of the Arabidopsis ETR1 homolog. Our results suggest that the Lotus intrinsic ethylene receptor LjETR1 regulates the ethylene signaling pathway in both non-symbiotic and legume-specific symbiotic responses.

Short Communication

Zinc accumulation and vegetation ecology in the allotetraploid, Arabidopsis kamchatica ssp. kawasakiana

Aki Kosugi, Chiaki Nishizawa, Akira Kawabe, Emiko Harada. . . . . . . 33 [PDF]

Arabidopsis kamchatica ssp. kawasakiana, a member of the family Brassicaceae, is an endangered winter annual species that grows on sandy coasts and lakesides. A. kamchatica is an allotetraploid plant produced by the hybridization of two closely related diploid taxa, the Zn/Cd hyperaccumulator A. halleri and the non-accumulator A. lyrata. The heavy metal accumulation and vegetation ecology of A. k. ssp. kawasakiana were investigated by collecting leaves and rhizosphere soil samples in three natural habitats on the shore of Lake Biwa in Japan. Leaf Zn contents in almost all plants were above the level required by hyperaccumulators. Plants from one habitat preferred to grow on soils with topical high Zn levels, whereas rhizosphere soils from other populations contained basal levels of Zn suggesting that plant vegetation is more affected by soil disturbance and by soil Zn contents. A. k. ssp. kawasakiana plants were also found to be tolerant to Zn and Cd, in contrast to the nontolerant species A. thaliana. These findings indicated that A. k. ssp. kawasakiana is a facultative Zn hyperaccumulator, inheriting the trait from its parent A. halleri. Furthermore, A. k. ssp. kawasakiana is a self-compatible plant and Arabidopsis floral dip transformation might be applicable to this plant. Considering their natural diversity, A. k. ssp. kawasakiana will help the determination of the molecular mechanisms by which plants accumulate and tolerate heavy metals.

Isolation of gametes from Brachypodium distachyon

Takao Matsumura, Takashi Okamoto. . . . . . . 39 [PDF]

Isolated gametes from maize, rice, Arabidopsis, wheat and tobacco have been used for investigations for mechanisms in reproductive or developmental processes, such as gamete differentiation, gamete fusion, and early zygotic development. In addition, the isolated gametes from maize and rice can also be applied for in vitro fertilization to analyze postfertilization events. In the last decade, Brachypodium distachyon (Brachypodium) has emerged as an effective model for wheat, since wheat with its hexaploid nature shows some analytical difficulties. In this study, to take advantages of this new model monocot plant for investigations using isolated gametes, procedures for the isolation of Brachypodium gametes were established as the first step. Ovaries were first harvested from mature and unpollinated Brachypodium flowers. Thereafter, a transverse incision was placed at the bottom region of the ovary, resulting in direct access to the embryo sac, and egg cells, which were released from the dissected ovaries, were isolated. For sperm cell isolation, when pollen grains were immersed in mannitol solution, sperm cells were successfully released from pollen grains.


Production of chrysanthemum periclinal chimeras through shoot regeneration from leaf explants

Ryutaro Aida, Katsutomo Sasaki, Norihiro Ohtsubo. . . . . . . 45 [PDF]

Periclinal chimeras play important roles in vegetatively propagated plants such as chrysanthemum (Chrysanthemum morifolium). For example, periclinal chimerism causes flower color variation in chrysanthemums. In this study, a method for periclinal chimera production in chrysanthemum was examined. A wild-type plant of chrysanthemum ‘Taihei’ and its transgenic plant carrying a yellowish-green fluorescent protein gene from the marine plankton Chiridius poppei (CpYGFP) were used as plant materials. The cut faces of the leaf explants of both materials were partially attached and then were detached for further culture. Mosaic calli consisted of transgenic and wild-type cells formed on the detached faces of the explants. We examined 996 regenerated shoots from 4,120 explants and found only a single chimeric shoot that appeared to show mericlinal chimerism. Repeated axillary bud elongation from the nodes of the mericlinal chimera produced one L1-fluorescent and one L3-fluorescent chimeric plant. The L1 chimera showed fluorescence in the epidermal cells and trichomes of leaf and stem. The L3 chimera showed fluorescence in the cells of the central parts of stem and leaf, as well as in the whole root tissues. In summary, we obtained chrysanthemum periclinal chimeras through regeneration from leaf explants using the fluorescent protein transgene as a selection marker.

Induction of tetraploid hardy rubber tree, Eucommia ulmoides, and phenotypic differences from diploid

Yuji Tokumoto, Hiroyuki Kajiura, Shinya Takeno, Yoko Harada, Nobuaki Suzuki, Takenori Hosaka, Koichiro Gyokusen, Yoshihisa Nakazawa. . . . . . . 51 [PDF]

Polyploid plants tend to have larger organs than diploids due to the doubling of their chromosome number. However, the enlarged regions, whether cells, tissues, or individuals differ depending on the plant species or taxon, as have metabolic changes. Eucommia ulmoides is a deciduous dioecious plant natively distributed in China that accumulates trans-1,4-polyisoprene (TPI) in laticifers throughout its tissues. To induce tetraploids of E. ulmoides, colchicine treatment time and concentration were assessed for ability to obtain tetraploids, and the resulting plants were analyzed for phenotypic differences from diploids with respect to individual size, 16 leaf attributes, sizes of three cell types (epidermis, laticifer, and parenchyma), and TPI content and molecular weight. Effective conditions for obtaining tetraploids were 0.05% colchicine for 48 h, which led to obtaining a total of 9 tetraploids. The tetraploids showed larger stomata size but lower stomata density than diploids. Significant differences in the mean values of other attributes including leaf size, cell size, and TPI content of tetraploids could not be detected between tetraploids and diploids, suggesting that E. ulmoides is less affected by gene doubling than other plant species, and that tetraploid individuals might not have a tendency to increase their biomass.