Plant Biotechnology Vol.32 No.2


Original Papers

Flower color modification in Rosa hybrida by expressing the S-adenosylmethionine: anthocyanin 3′,5′- O-methyltransferase gene from Torenia hybrida

Noriko Nakamura, Yukihisa Katsumoto, Filippa Brugliera, Linda Demelis, Daisuke Nakajima, Hideyuki Suzuki, Yoshikazu Tanaka. . . . . . . 109 [PDF]

We isolated a cDNA encoding S-adenosylmethionine: anthocyanin 3′,5′-O-methyltransferase (A3′5′OMT) from a cDNA library derived from Torenia hybrida petals that mainly accumulated malvidin type anthocyanins using the petunia A3′OMT cDNA as a probe. The torenia A3′5′OMT shared 52–72% amino acid sequence identity with previously reported AOMTs and belongs to the Group A1 methyltransferase family that also include caffeoyl CoA O-methyltransferase. The recombinant A3′5′OMT produced by Escherichia coli efficiently catalyzed methylation of the 3-glucoside and 3,5-diglucoside of delphinidin and cyanidin, but it did not catalyze the methylation of anthocyanidins, flavonols, or flavones. The torenia A35OMT gene was expressed in Nierembergia sp., the petals of which naturally accumulate anthocyanins derived from delphinidin. The resultant transgenic petals produced methylated anthocyanins, based on malvidin and petunidin, in addition to delphinidin, which indicated that the torenia A35OMT gene was functional in a heterologous plant. Rose petals rarely contain methylated anthocyanins. Transgenic rose petals expressing both a pansy flavonoid 3,5-hydroxylase (F35H) and the torenia A35OMT genes accumulated methylated anthocyanins based upon malvidin, petunidin, and peonidin, which comprised up to 88% of the total anthocyanidins, and their magenta color was more brilliant than that of the petals that accumulated delphinidin type anthocyanins by expressing the F35H gene alone. These results indicate that the torenia A35OMT gene is a useful molecular tool for altering and diversifying flower color.

Arabidopsis NAC domain proteins VND-INTERACTING1 and ANAC103 interact with multiple NAC domain proteins

Masatoshi Yamaguch, Isura Sumeda Priyadarshana Nagahage,Misato Ohtani, Toshiki Ishikawa, Hirofumi Uchimiya, Maki Kawai-Yamada,Taku Demura. . . . . . . 119 [PDF]

The Arabidopsis thaliana NAM, ATAF1/2 and CUC2 (NAC) domain transcription factor VND-INTERACTING1 (VNI1) was previously isolated as an interacting factor of VASCULAR-RELATED NAC-DOMAIN PROTEIN7 (VND7), a key regulator of xylem vessel differentiation, in a yeast two-hybrid screening. Here, we characterized VNI1 and its closest homolog, ANAC103, at the molecular level. Both VNI1 and ANAC103 interacted in vitro not only with VND proteins but also with other NAC domain proteins, such as NAC1 and CUC2. A transient expression assay showed that both VNI1 and ANAC103 are transcriptional activators. ANAC103 promoter activity was detected in vascular tissues, as well as in the trichomes, guard cells, and margins of young leaves. These data suggest that VNI1 and ANAC103 promote the differentiation of various types of cells by modulating the transcriptional activities of a wide range of NAC domain transcription factors.

The axhs1/dwf4 auxin-hypersensitive mutant of Arabidopsis thaliana defìnes a link for integration of auxin and brassinosteroid mediated root elongation

Taisuke Nagase, Hisabumi Takase, Jiro Sekiya, Rafael Prieto. . . . . . . 125 [PDF]

To understand the molecular mechanism of auxin-mediated root elongation, we carried out screening to isolate auxin-hypersensitive (axhs) mutants. The T-DNA insertional mutant axhs1 was selected on the basis of root phenotypes associated with auxin sensitivity. Results from brassinosteroid (BR) feeding experiments, Tail-PCR and genetic analyses indicate that AXHS1 encodes DWF4, which catalyzes the rate limiting step of BR biosynthesis. The axhs1 mutant shows increased sensitivity to indole-3-acetic acid (IAA), the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D), auxin transport inhibitors such as 2,3,5-triiodobenzoic acid (TIBA) and N-1-naphthylphthalamic acid (NPA), and the antiauxin p-chlorophenoxyisobutyric acid (PCIB) for the root elongation response. Analysis of the expression of the DR5:GUS and HS:AXR3NT-GUS reporter genes in wild type and axhs1 genotypes, and characterization of double mutants between axhs1 and mutants affected in auxin biosynthesis (wei2-1), auxin transport (aux1-7, eir1-1) and auxin signal transduction (tir1-1, axr1-3, axr2-1) indicate that auxin hypersensitivity in axhs1 is mediated by the auxin-signaling pathway and an AUX1, EIR1/ PIN2 dependent auxin uptake.

Characterization of hybrid seedlings from crosses of Nicotiana stocktonii Brandegee×N. tabacum L. and N. stocktonii×progenitors of N. tabacum

Naho Muraida, Wataru Marubashi. . . . . . . 139 [PDF]

Hybrid lethality is one of the sexual barriers preventing wide hybridization. In the genus Nicotiana, hybrid lethality is observed in some interspecific cross combinations. Interspesific hybridization of Nicotiana stocktonii×N. tabacum has not been carried out from the viewpoint of expression of lethality. N. tabacum has an S subgenome derived from N. sylvestris and a T subgenome derived from N. tomentosiformis. In this study, hybrid seedlings from the cross N. stocktonii×N. tabacum, were obtained by ovule culture. The hybrid seedlings were classified as normal seedlings, tumorous types and vitrified plants, and they did not express lethality. Hybrid seedlings from the cross N. stocktonii×N. sylvestris expressed lethality. Moreover, hybrid seedlings from N. stocktonii×N. tomentosiformis also expressed lethality. This suggests that both the S genome of N. sylvestris and the T genome of N. tomentosiformis have factors causing lethality in hybrids with N. stocktonii. However, hybrid seedlings from N. stocktonii×N. tabacum did not express lethal symptoms in this study. We suggested that the factors responsible for lethality in N. tabacum must have been lost or no longer expressed during the process of speciation, probably due to reorganization and modification of the genomes.

Screening RAPD primers to assess clonal fìdelity in somatic embryos of Sawara cypress (Chamaecyparis pisifera Sieb. et Zucc.) and fìeld performance of somatic embryo-derived trees

Yoshihisa Hosoi, Noritsugu Kuramoto, Tsuyoshi E. Maruyama. . . . . . . 149 [PDF]

In this study, 100 random amplified polymorphic DNA (RAPD) primers were screened to assess clonal fidelity in somatic embryos of Sawara cypress (Chamaecyparis pisifera Sieb. et Zucc.). Of the 100 primers tested, eight were selected based on their amplification products, which showed clear DNA fragmentation, generating 8.75 bands per primer in average.
The amplification products showed no genetic variation among the tested somatic embryos. In addition, the performance of somatic embryo-derived trees was monitored in the field, and their mean height growth was measured until the age of 10 years. Although no phenotypic differences were observed, the somatic embryo-derived trees showed lower growth compared with seedlings.

An upstream open reading frame represses expression of a tomato homologue of Arabidopsis ANAC096, a NAC domain transcription factor gene, in a peptide sequence-dependent manner

Abdul Latif Noh, Shun Watanabe, Hiro Takahashi, Satoshi Naito, Hitoshi Onouchi. . . . . . . 157 [PDF]

Many eukaryotic mRNAs contain one or more upstream open reading frames (uORFs) in their 5′ untranslated regions (5′-UTRs). Some uORFs encode regulatory peptides that repress translation of the main ORF. To comprehensively identify uORFs encoding regulatory peptides, genome-wide searches for uORFs with evolutionarily conserved amino acid sequences, referred to as conserved peptide uORFs (CPuORFs), have been conducted using bioinfomatic approaches. To date, more than 40 homology groups of CPuORFs have been identified in dicotyledonous plants. The Arabidopsis thaliana ANAC096 gene is one of the CPuORF-containing genes; however, the ANAC096 CPuORF exerts only little peptide sequence-dependent effect on expression of the main ORF. Here, we investigated the effect of the CPuORF sequence of a tomato ANAC096 homologue on expression of the main ORF, because it has a more highly conserved amino acid sequence than the ANAC096 CPuORF. Mutational analyses revealed that the CPuORF of the tomato ANAC096 homologue represses main ORF expression in a peptide sequence-dependent manner, and determined the critical amino acid residues of the CPuORF peptide responsible for the repression. This study identified a novel peptide sequence-dependent regulatory uORF and demonstrated that the level of uORF peptide-mediated repression can differ among closely related homologues.


Agrobacterium-mediated transformation and regeneration of Freesia×hybrida

Yohei Uwagaki, Eriko Matsuda, Masako Komaki, Minoru Murahama, Motoyasu Otani,Naoko Nishizawa, Tatsuro Hamada. . . . . . . 165 [PDF]

A method for Agrobacterium-mediated transformation of Freesia×hybrida is described. Cormlet-derived calli of two cultivars, ‘Mosera’ and ‘Ishikawa f3’ were co-cultivated with Agrobacterium tumefaciens strain EHA105 harboring the binary vector pIG121-Hm, which included hygromycin phosphotransferase gene and an intron-containing β-glucuronidase gene in the T-DNA region. Callus pieces were co-cultivated with A. tumefaciens on the callus proliferation medium [Murashige and Skoog (MS) medium containing 1 mg l−1 thidiazuron, 1 mg l−1 dicamba, 20 mg l−1 3′,5′-dimethoxy- 4′-hydroxyacetophenone, 1% (w/v) glucose, 3% (w/v) sucrose, and 0.2% (w/v) Gelrite]. Then, they were cultured on the callus proliferation medium containing 300 mg l−1 cefatoxime and 10 mg l−1 hygromycin B. Hygromycin-resistant lines of both cultivars regenerated into plantlets after transfer onto MS medium containing 2 mg l−1 3-indoleacetic acid and 3 mg l−1 6-benzyl aminopurine and/or plant growth regulator-free MS medium. Transgenic plants were identified by β-glucuronidase assay and verified by Southern blot analysis. Two transgenic plant lines were obtained from 475 callus pieces of ‘Mosera’, and one transgenic plant line was obtained from 290 callus pieces of ‘Ishikawa f3’. This is the first report of the genetic transformation of Freesia. This method will allow the genetic improvement of this horticulturally important flower.

Rapid evaluation of the frequency of gene targeting in rice via a convenient positive-negative selection method

Hiroaki Saika, Akiko Mori, Masaki Endo, Keishi Osakabe, Seiichi Toki. . . . . . . 169 [PDF]

Although gene targeting (GT) is a useful technology for precise mutagenesis of target sequences, its frequency is quite low. Establishing experimental procedures using a model system will enable us to improve this frequency and apply to GT as a universal system. Here, we propose a convenient system with which to evaluate the frequency of site- directed mutagenesis via GT using a positive-negative selection method. We constructed a GT vector harboring a partial rice acetolactate synthase gene with mutations conferring bispyribac sodium (BS) tolerance and a gene conferring blasticidin-S tolerance as a positive selection marker. In addition, diphtheria toxin A subunit gene was used as a negative selection marker to enrich GT cells. We regenerated GT candidate plants successfully at a frequency of 2.1 putative GT events/gram Agrobacterium-infected callus following dual selection on BS and blasticidin-S. Moreover, molecular analyses confirmed that GT events occurred in >80% of regenerated plants. Existing GT methods using positive-negative selection require that true putative GT events be verified by molecular analysis because of the growth of large numbers of cells in which partial GT vectors containing positive selection marker cassettes, but lacking the negative selection marker, have inserted at random loci. In contrast, the present method with dual selection on both BS and blasticidin-S allows direct enrichment of GT cells at high frequency without the need for further extensive molecular screening.

Transient expression of optimized and synthesized nattokinase gene in melon (Cucumis melo L.) fruit by agroinfìltration

Lan Han, Liquan Zhang, Jinglei Liu, Hong Li, Yingchun Wang, Agula Hasi. . . . . . . 175 [PDF]

A synthetic gene (sNK) encoding Nattokinase (NK) was constructed by modifying its sequence based on the codon usage in plants. A version (sNKi) incorporating the first intron from the tomato E8 gene was also constructed. The synthetic sNK and sNKi genes were transiently expressed in melon (Cucumis melo L.) fruit. Quantitative real-time reverse transcription PCR (qRT-PCR) analysis and fibrinolytic activity assays showed that the expression level of recombinant NK controlled by E8 promoter was higher than that controlled by 35S promoter, with the maximum fibrinolytic activity of 79.30 U ml−1. The intron could enhance the expression of sNK to an extent of 27.42%. Orthogonal tests determined the optimal agroinfiltration as: an acetosyringone concentration of 0.25 mM, an OD600=0.6 and harvesting 5 days after infiltration.