Plant Biotechnology Vol.31 No.4


Original Papers

Diurnal expression of CONSTANS-like genes is independent of the function of cycling DOF factor (CDF)- like transcriptional repressors in Physcomitrella patens

Tetsuya Ishida, Takumi Sugiyama, Nobumitsu Tabei, Shuichi Yanagisawa. . . . . . . 293 [PDF]

The Dof-type transcriptional repressors CYCLING DOF FACTORS (CDFs) directly suppress the expression of CONSTANS (CO), which encodes a key regulator of photoperiodic gene expression, day-length perception and the floral transition in Arabidopsis thaliana. The genes encoding CDF-like (PpDof3 and PpDof4) and CO-like (PpCOL1-PpCOL3) proteins are present in the genome of the moss Physcomitrella patens, although P. patens lacks the genes encoding homologues of FLAVIN-BINDING KELCH REPEAT F-BOX1 (FKF1) and GIGANTEA (GI), which control the stability of CDFs in A. thaliana. In the current study, we investigated whether the functions of PpDof3 and PpDof4 are associated with the expression of PpCOL1-PpCOL3 in P. patens. We found that the diurnal expression patterns of PpDof3 and PpDof4 are similar to those of CDF genes and that like CDF1 from A. thaliana, PpDof3 and PpDof4 function as transcriptional repressors. However, targeted disruptions of PpDof3 and PpDof4 did not affect the expression of PpCOL1-PpCOL3,indicating that the expression of COLs is independent of the functions of PpDof3 and PpDof4 in P. patens.

In vitro shoot grafting on rootstock: An effective tool for Agrobacterium-mediated transformation of pigeonpea (Cajanus cajan (L.) Millsp.)

Gourab Ghosh, Arnab Purohit, Shreeparna Ganguly, Rituparna Kundu Chaudhuri,Dipankar Chakraborti. . . . . . . 301 [PDF]

Lack of reproducible in vitro transformation method in pigeonpea limits the application of biotechnological breeding approaches for its genetic improvement. The present study describes a transformation method using novel in vitro shoot grafting technique for two cultivars ICPL87 and ICPL87119. Modified Murashige and Skoog (MS) medium with1 mg l−1 6-benzylaminopurine and 0.2 mg l−1 α-naphthaleneacetic acid induced an average of 25 shoots from decapitated embryonic axis explants after six weeks of culture. These shoots were further elongated in a modified MS medium containing 0.5 mg l−1 6-benzylaminopurine along with 0.5 mg l−1 gibberellic acid for another four weeks. Grafting of pigeonpea shoots to seedling rootstock allowed 95% recovery of shoots. The whole regeneration process, starting from explant preparation to complete plant development, took 12–13 weeks. Further, the explants were infected with Agrobacterium tumefaciens harboring a binary vector pBI121. Transient and constitutive β-glucuronidase expressions were obtained in putative transgenic shoots selected at 100 mg l−1 kanamycin. The selected shoots were grafted on non-transgenic root stock to establish putative transformants. T0 and T1 transformants were confirmed through polymerase chain reaction for presence of neomycin phosphotransferase gene. An overall 9% of transformation efficiency was recorded in both cultivars.

Generation of fluorescent flowers exhibiting strong fluorescence by combination of fluorescent protein from marine plankton and recent genetic tools in Torenia fournieri Lind.

Katsutomo Sasaki, Ko Kato, Hiroshi Mishima, Makio Furuichi, Iwao Waga, Ken-ichi Takane,Hiroyasu Yamaguchi, Norihiro Ohtsubo. . . . . . . 309 [PDF]

Florescent proteins have been popularly used for studying genes and proteins of interest in various experiments at a cellular level, such as the analysis of intracellular localization and protein–protein interaction. However, the strength of fluorescence was insufficient for macro level observations of tissues or of the whole plant, and the fluorescent flowers that have been generated so far needed high-sensitive imaging equipment for the observation. Here we generated fluorescent Torenia flowers by the combined use of a high-performance fluorescent protein and the latest protein expression technologies, leading to the production of fluorescent proteins that can be easily and clearly observed. A coding sequence of a yellowish green fluorescent protein from the marine plankton Chiridius poppei (CpYGFP) was fused to the optimized sequences of the heat shock protein terminator and the 5′-untranslated region of the alcohol dehydrogenase gene of Arabidopsis to gain massive accumulation of the fluorescent protein. Strong fluorescence of CpYGFP was apparent in every part of the transgenic plant under the simple combination of a blue LED for excitation and an orange colored transparent acrylic filter for emission, while faint autofluorescence remained in the wild-type plants. By evaluating the combination of excitation wavelengths (excitation and emission filters) we were able to eliminate this undesired fluorescence. The fluorescent flowers could be used for ornamental purposes as well as for the analysis of fluorescent transgenic plants spatiotemporally in a nondestructive manner.

Overexpression of a CO homologue disrupts the rhythmic expression of clock gene LgLHYH1 in Lemna gibba

Kumiko Ito-Miwa, Masayuki Serikawa, Takao Kondo, Tokitaka Oyama. . . . . . . 319 [PDF]

In plants, the circadian clock is involved in the regulation of various physiological phenomena. The Arabidopsis CONSTANS gene (AtCO), a central component in photoperiodic flowering, as well as the CONSTANS-LIKE 1 gene (AtCOL1), which has been suggested to be involved in the light input pathway, belong to the CONSTANS-LIKE gene family (COL family). To investigate the function of the COL family in the circadian system, we characterized CO homologues in two Lemna plant species (LgCOH1 in L. gibba G3 and LaCOH1 in L. aequinoctialis 6746). The expression of LgCOH1 was upregulated by light, showing diurnal rhythmic expression with peak expression during daytime. We examined the effect of LgCOH1 overexpression (LgCOH1-ox) on a circadian bioluminescent reporter. LgCOH1-ox damped the circadian bioluminescence rhythm, suggesting that it disturbed the circadian system. The overproduction of the N-terminal region including the zinc finger regions, or the C-terminal region including the CCT domain of LgCOH1 damped the circadian bioluminescence rhythm, suggesting that both regions were involved in the phenotypic abnormalities found in the fulllength LgCOH1-ox mutant. The overexpression of AtCO also damped the circadian bioluminescence rhythm in our cotransfection assay using Lemna plants, while its effect on the circadian rhythm was weaker than that of LgCOH1-ox. Based on these results, we suggest that some COL family genes may function in the regulation of the circadian system including the light input pathway.


Regulation of xylanase elicitor-induced expression of defense-related genes involved in phytoalexin biosynthesis by a cation channel OsTPC1 in suspension-cultured rice cells

Haruyasu Hamada, Takamitsu Kurusu, Hiroshi Nokajima, Masahiro Kiyoduka, Kentaro Yano,Kazuyuki Kuchitsu. . . . . . . 329 [PDF]

Signal molecules derived from pathogens/microbes or plants (pathogen/microbe/damage-associated molecular patterns; PAMPs/MAMPs/DAMPs), elicitors, trigger changes in cytosolic free Ca2+ concentrations ([Ca2+]cyt) to activate plant immune responses. A rice two-pore channel 1 (OsTPC1) has been suggested to be involved in fungal xylanase elicitor (TvX)-induced defense responses including [Ca2+]cyt increase, phytoalexin production and hypersensitive cell death in suspension-cultured rice cells. However, little is known on the molecular links between [Ca2+]cyt rise and elicitor-induced gene expression. To gain insights on the possible roles of OsTPC1 in TvX-induced gene expression, we performed DNA microarray analysis using a rice 44K oligo-microarray system, and revealed that TvX induce expression of thousands of genes including WRKY-type transcription factors, a serine hydrolase involved in hypersensitive cell death, and diterpene cyclases required for phytoalexin biosynthesis, which are suppressed in the Ostpc1 knockout mutant. TvXinduced expression of genes involved in the methylerythritol phosphate (MEP) pathway, which is located upstream of the phytoalexin biosynthesis pathway, was also suppressed in Ostpc1 cells. Possible involvement of OsTPC1 in the regulation of gene expression and metabolism in cultured-rice cells is discussed.

Horticultural characterization of a tetraploid transgenic plant of Tricyrtis sp. carrying the gibberellin 2-oxidase gene

Masahiro Otani, Mitsuyo Ishibe, Phithak Inthima, Kanyaratt Supaibulwatana, Shiro Mori, Tomoya Niki, Takaaki Nishijima, Masaji Koshioka, Masaru Nakano. . . . . . . 335 [PDF]

The gibberellin 2-oxidase catalyzes the bioactive gibberellins or their immediate precursors to inactive forms. We have previously produced transgenic plants of the liliaceous plant Tricyrtis sp. containing the GA2ox gene from the linderniaceous plant Torenia fournieri. These transgenic plants showed dwarf phenotypes as expected but unfortunately had no flowers or only small, unopened flowers. Recently, one newly produced transgenic line (G2-55) formed fully opened flowers. G2-55 showed a moderately dwarf phenotype and the shoot length decreased to 63.4% of that of the control, non-transgenic plants. No significant differences in the number of flowers per shoot and in the flower size were observed between G2-55 and the control. Flow cytometry analysis and chromosome observation showed that G2-55 was tetraploid (2n=4x=52), whereas the other transgenic lines producing no or only small flowers were diploid (2n=2x=26) as the mother plant. Pollen fertility of G2-55 was 81.2% as determined by acetocarmine staining. The tetraploidy in G2-55 might be resulted from somaclonal variation of embryogenic calluses used as a target material for Agrobacterium-mediated transformation. The tetraploid transgenic plant G2-55 may be useable not only directly as a potted plant, but also as a material for further breeding of Tricyrtis spp.

In vitro regeneration through direct shoot organogenesis in Honey Orange (Citrus tangerina)

Yin Yin Nwe, Khin Thida Myint, Yuya Mochizuki, Mehran Vazirzanjani, Kouji Okayasu, Sakae Suzuki,Isao Ogiwara. . . . . . . 341 [PDF]

Rapid propagation of Honey orange (Citrus tangerina) was achieved by induction of shoots from epicotyl and cotyledonary node explants and rooting of cotyledonary node derived shoots. Significant explant differences were observed in the induction of direct shoots. Cotyledonary node explant is the most efficient in regeneration frequency followed by epicotyl explant. Cotyledonary node explants cultured on Murashige and Skoog (MS) medium supplemented with 8.88 μM N6-benzyladenine (BA) and 0.54 μM α-naphthaleneacetic acid (NAA) developed more than five shoots per explant. The isolated shoots transferred onto the MS medium supplemented with 5.4 μM NAA rooted 100% within 30 days.

Transcriptomic profiles of nodule senescence in Lotus japonicus and Mesorhizobium loti symbiosis

Sirinapa Chungopast, Hideki Hirakawa, Shusei Sato, Yoshihiro Handa, Katsuharu Saito,Masayoshi Kawaguchi, Shigeyuki Tajima, Mika Nomura. . . . . . . 345 [PDF]

Nodule senescence is a complex developmental process during which essential nutrients are recycled. In order to understand the regulatory mechanism, transcript-profiling analysis during nodule senescence was performed in the Lotus japonicus-Mesorhizobium loti symbiosis. Microarray data showed significantly up-regulated expressions in 641 genes out of a total of 20,165 genes during nodule senescence, and down-regulated expressions were observed in 416 genes. These up-regulated genes during senescence were related to cell wall/membrane/envelope biogenesis and extracellular structures. Down-regulated genes were mainly responsible for defense mechanisms. We classified senescence up-regulated genes in two clusters. Genes in cluster 1 were induced at senescence specific stage and those in cluster 2 were induced from nitrogen fixation stage and expressed until nodule senescence. The genes in cluster 1 included typical marker for senescence like gene for heat shock protein. Four hundred sixteen down-regulated genes during nodule senescence were also classified in two clusters, cluster 3 and cluster 4. These genes corresponded to metabolisms for amino acid and plant hormones which are necessary for growth and cell division during nodule development and nitrogen fixation. These results provide the comprehensive data source for investigation of molecular mechanisms underlying nodule senescence in Lotus japonicus-Mesorhizobium loti symbiosis.