Plant Biotechnology Vol.34 No.4


Original Papers

Overexpression of carotenogenic genes in the Japanese morning glory Ipomoea (Pharbitis) nil

Kenta Watanabe, Chihiro Oda-Yamamizo, Kimiyo Sage-Ono, Akemi Ohmiya, Michiyuki Ono . . . . . . . 177 [PDF]

Japanese morning glory, Ipomoea nil, has several coloured flowers except yellow, because it can accumulate only trace amounts of carotenoids in the petal. To make the petal yellow with carotenoids, we introduced five carotenogenic genes (geranylgeranyl pyrophosphate synthase, phytoene synthase, lycopene β-cyclase and β-ring hydroxylase from Ipomoea obscura var. lutea and bacterial phytoene desaturase from Pantoea ananatis) to white-flowered I. nil cv. AK77 with a petal-specific promoter by Rhizobium (Agrobacterium)-mediated transformation method. We succeeded to produce transgenic plants overexpressing carotenogenic genes. In the petal of the transgenic plants, mRNA levels of the carotenogenic genes were 10 to 1,000 times higher than those of non-transgenic control. The petal colour did not change visually; however, carotenoid concentration in the petal was increased up to about ten-fold relative to non-transgenic control. Moreover, the components of carotenoids in the petal were diversified, in particular, several β-carotene derivatives, such as zeaxanthin and neoxanthin, were newly synthesized. This is the first report, to our knowledge, of changing the component and increasing the amount of carotenoid in petals that lack ability to biosynthesize carotenoids.

Somatic embryogenesis from leaf explants of Tagetes erecta L.

Pablo Emilio Vanegas Espinoza, Israel Benítez-García, Annel Lizeth Leyva Peralta, Octavio Paredes-López, Alma Angélica Del Villar-Martínez . . . . . . . 187 [PDF]

Tagetes erecta is an asteraceous plant of industrial, ornamental and medicinal importance; its inflorescences have been used as a pigment source for food coloring, mainly for poultry skin and eggs. Nevertheless, there are few reports on plant regeneration or micropropagation, because unsuccesfull results in the plant’s reaction to the growth regulators, developing embryogenesis on Tagetes erecta. In this study, somatic embryogenesis was induced and plantlets of Tagetes erecta were regenerated. For induction of globular structures MS medium supplemented with 2,4-D (4.5 µM) and BAP (8.8 µM) was used; globular structures were transferred to MS medium with 45 g l-1 sucrose until the embryos maturation. Transmission electron microscopy showed characteristic subcellular structures of embryogenic callus. Somatic embryos were transferred to MS medium without plant growth regulators and whole plantlets were obtained. In vitro plants were successfully transplanted into a mixture of peat moss and vermiculite (1 : 1 v/v) under greenhouse conditions. In this study, somatic embryogenesis and plant regeneration system from foliar explants were established, an important requirement for performing genetic transformation events on Tagetes erecta.

Short Communications

Aromatic amino acid decarboxylase is involved in volatile phenylacetaldehyde production in loquat (Eriobotrya japonica) flowers

Takao Koeduka, Yoshiyuki Fujita, Takumi Furuta, Hideyuki Suzuki, Tomohiko Tsuge, Kenji Matsui . . . . . . . 193 [PDF]

Post anthesis, loquat flowers emit volatile benzenoids, including phenylacetaldehyde, phenylethyl alcohol, and 2-phenethyl benzoate. Previous studies have shown that pyridoxal phosphate-dependent aromatic L-amino acid decarboxylase (AADC) produces phenylacetaldehyde from L-phenylalanine. Here, two AADC genes (EjAADC1 and EjAADC2) were isolated from loquat (Eriobotrya japonica) flowers. The EjAADC1 and EjAADC2 proteins showed approximately 72% and 55% identity, respectively, to a rose AADC homolog that has phenylacetaldehyde synthase activity. Transcript analyses indicated that EjAADC1 was specifically expressed in petals, with the highest level of expression in fully opened flowers; the petals showed high levels of volatile benzenoids, including phenylacetaldehyde. In contrast, EjAADC2 was expressed at a lower level than EjAADC1 in all tested tissues, including leaves and developing flowers. Functional characterization of a recombinant EjAADC1 protein expressed in Escherichia coli showed that it catalyzes the formation of phenylacetaldehyde from L-phenylalanine in a pyridoxal phosphate-dependent manner. Our results suggest that EjAADC1 is mainly responsible for the biosynthesis of volatile benzenoids in loquat flowers.

In vitro bioassay of allelopathy of Arabidopsis thaliana by sandwich method and protoplast co-culture method with digital image analysis

Hamako Sasamoto, Yoshitaka Azumi, Makoto Shimizu, Yu-ki Hachinohe, Suechika Suzuki. . . . . . . 199 [PDF]

We examined the allelopathic activities of Arabidopsis thaliana, ecotype Columbia by two in vitro methods. The effect of dried leaves on the growth of recipient lettuce seedlings was examined by the sandwich method. The allelopathic activity on protoplast growth was examined by co-culture with recipient lettuce leaf protoplasts in 50 µl liquid medium using a 96-well culture plate. Non-spherically enlarged or dividing protoplasts of lettuce were counted under an inverted microscope. Inhibition of yellow accumulation during lettuce protoplast growth was quantitated by image analysis of scanned digital images of 96-well culture plates. The results were described as the percentages of control without A. thaliana. The results were compared with those obtained using several plants which had strong allelopathic activities on recipient lettuce using the same methods. The growth of lettuce protoplasts was inhibited at both 4 and 8 days of culture with protoplasts of A. thaliana. The results suggested the usefulness of the protoplast co-culture method for future studies on allelopathy.


Identification of novel factors that increase enzymatic saccharification efficiency in Arabidopsis wood cells

Misato Ohtani, Vasagi Ramachandran, Takuya Tokumoto, Arika Takebayashi, Ayumi Ihara, Tomoko Matsumoto, Ryoko Hiroyama, Nobuyuki Nishikubo, Taku Demura . . . . . . . 203 [PDF]

Developing methods to efficiently convert lignocellulosic polymers, i.e. cellulose, hemicellulose, and lignin into user-friendly carbon resources, such as fermentable sugars, is critical for improving plant biomass utilization. Here, we report the identification of genes that increase enzymatic saccharification efficiency in cultured Arabidopsis wood cells. We overexpressed a set of genes that were upregulated during the early stages of in vitro xylem vessel cell differentiation, including transcription factor and CAZYme genes, in Arabidopsis and tested their effects on enzymatic saccharification efficiency. Of the 96 transgenic seedlings sampled, 37 and 17 lines showed significant increases and decreases in glucose yields, respectively. Further analysis of 20 overexpression lines with high glucose yields in seedling samples indicated that compared to wild type, the glucose and xylose yields from inflorescence stem samples were higher in lines overexpressing genes encoding BETA-XYLOSIDASE 2, UDP-GLUCOSYL TRANSFERASE 88A1, AT3G15350 (a class GT14 glycosyltransferase protein), and the Dof-type transcription factor Dof4.6, whose detailed molecular functions have not yet been characterized. No apparent defect in growth or inflorescence stem structure was detected in these overexpression lines. Therefore, these four genes might represent novel factors that can be used to increase saccharification efficiency in wood tissues without negatively affecting total biomass production. Furthermore, our results confirm the validity of our screening strategy for isolating factors related to the saccharification efficiency of lignocellulosic biomass.t

Leaf age and time of inoculation contribute to nonhost resistance to Pyricularia oryzae in Arabidopsis thaliana

Yuri Yamauchi, Motoi Makihara, Atsushi Ishikawa . . . . . . . 207 [PDF]

The outcome of plant-pathogen interactions varies both with developmental stage and time of day. Rice blast caused by Pyricularia oryzae (syn. Magnaporthe oryzae) is a devastating disease of rice. The mechanisms of resistance to P. oryzae have been extensively studied and the rice-P. oryzae pathosystem has become a model system in plant-microbe interaction studies. However, the mechanisms of resistance to P. oryzae in nonhost remain poorly understood. To determine whether leaf age and time of inoculation would affect nonhost resistance (NHR) to P. oryzae in Arabidopsis thaliana, Columbia-0 (Col-0) and penetration2-1 (pen2-1) plants were inoculated with P. oryzae. The rate of entry of P. oryzae into Arabidopsis pen2-1 old leaves was significantly higher than that into young leaves after inoculation at dusk. However, there was no difference in the rates after inoculation at dawn. These results suggest that leaf age and time of inoculation are involved in nonhost resistance to P. oryzae in Arabidopsis.