Norihiro Ohtsubo

MIXTA-Like Transcription Factors and WAX INDUCER1/SHINE1 Coordinately Regulate Cuticle Development in Arabidopsis and Torenia fournieri

MIXTA-like MYB transcription factors MYB106 and MYB16, which are known to be involved in epidermal cell shaping, regulate accumulation of cuticular waxes and nanoridges, as revealed by employing Arabidopsis and wishbone flower as model systems. MYB106 positively regulates the expression of cuticle biosynthesis genes and another cuticle regulator, WIN1/SHN1 transcription factor, as well. The waxy plant cuticle protects cells from dehydration, repels pathogen attack, and prevents organ fusion during development. The transcription factor WAX INDUCER1/SHINE1 (WIN1/SHN1) regulates the biosynthesis of waxy substances in Arabidopsis thaliana. Here, we show that the MIXTA-like MYB transcription factors MYB106 and MYB16, which regulate epidermal cell morphology, also regulate cuticle development coordinately with WIN1/SHN1 in Arabidopsis and Torenia fournieri. Expression of a MYB106 chimeric repressor fusion (35S:MYB106-SRDX) and knockout/down of MYB106 and MYB16 induced cuticle deficiencies characterized by organ adhesion and reduction of epicuticular wax crystals and cutin nanoridges. A similar organ fusion phenotype was produced by expression of a WIN1/SHN1 chimeric repressor. Conversely, the dominant active form of MYB106 (35S:MYB106-VP16) induced ectopic production of cutin nanoridges and increased expression of WIN1/SHN1 and wax biosynthetic genes. Microarray experiments revealed that MYB106 and WIN1/SHN1 regulate similar sets of genes, predominantly those involved in wax and cutin biosynthesis. Furthermore, WIN1/SHN1 expression was induced by MYB106-VP16 and repressed by MYB106-SRDX. These results indicate that the regulatory cascade of MIXTA-like proteins and WIN1/SHN1 coordinately regulate cutin biosynthesis and wax accumulation. This study reveals an additional key aspect of MIXTA-like protein function and suggests a unique relationship between cuticle development and epidermal cell differentiation.

Functional divergence within class B MADS-box genes TfGLO and TfDEF in Torenia fournieri Lind

Homeotic class B genes GLOBOSA (GLO)/PISTILLATA (PI) and DEFICIENS (DEF)/APETALA3 (AP3) are involved in the development of petals and stamens in Arabidopsis. However, functions of these genes in the development of floral organs in torenia are less well known. Here, we demonstrate the unique floral phenotypes of transgenic torenia formed due to the modification of class B genes, TfGLO and TfDEF. TfGLO-overexpressing plants showed purple-stained sepals that accumulated anthocyanins in a manner similar to that of petals. TfGLO-suppressed plants showed serrated petals and TfDEF-suppressed plants showed partially decolorized petals. In TfGLO-overexpressing plants, cell shapes on the surfaces of sepals were altered to petal-like cell shapes. Furthermore, TfGLO- and TfDEF-suppressed plants partially had sepal-like cells on the surfaces of their petals. We isolated putative class B gene-regulated genes and examined their expression in transgenic plants. Three xyloglucan endo-1,4-beta-d-glucanase genes were up-regulated in TfGLO- and TfDEF-overexpressing plants and down-regulated in TfGLO- and TfDEF-suppressed plants. In addition, 10 anthocyanin biosynthesis-related genes, including anthocyanin synthase and chalcone isomerase, were up-regulated in TfGLO-overexpressing plants and down-regulated in TfGLO-suppressed plants. The expression patterns of these 10 genes in TfDEF transgenic plants were diverse and classified into several groups. HPLC analysis indicated that sepals of TfGLO-overexpressing plants accumulate the same type of anthocyanins and flavones as wild-type plants. The difference in phenotypes and expression patterns of the 10 anthocyanin biosynthesis-related genes between TfGLO and TfDEF transgenic plants indicated that TfGLO and TfDEF have partial functional divergence, while they basically work synergistically in torenia.

Overexpression of Arabidopsis miR157b induces bushy architecture and delayed phase transition in Torenia fournieri

AbstractmiR156/157 is a small RNA molecule that is highly conserved among various plant species. Overexpression of miR156/157 has been reported to induce bushy architecture and delayed phase transition in several plant species. To investigate the effect of miR157 overexpression in a horticultural plant, and to explore the applicability of miRNA to molecular breeding, we introduced ArabidopsisMIR157b (AtMIR157b) into torenia (Torenia fournieri). The resulting 35S:AtMIR157b plants showed a high degree of branching along with small leaves, which resembled miR156/157-overexpressing plants of other species. We also isolated torenia SBP-box genes with target miR156/157 sequences and confirmed that their expression was selectively downregulated in 35S:AtMIR157b plants. The reduced accumulation of mRNA was probably due to sequence specificity. Moreover, expression of torenia homologs of the SBP-box protein-regulated genes TfLFY and TfMIR172 was also reduced by AtmiR157 overexpression. These findings suggest that the molecular mechanisms of miR156/157 regulation are conserved between Arabidopsis and torenia. The bushy architecture and small leaves of 35S:AtMIR157b torenia plants could be applied in molecular breeding of various horticultural plants as well as for increasing biomass and crop production.

Co-modification of class B genes TfDEF and TfGLO in Torenia fournieri Lind. alters both flower morphology and inflorescence architecture

The class B genes DEFICIENS (DEF)/APETALA3 (AP3) and GLOBOSA (GLO)/PISTILLATA (PI), encoding MADS-box transcription factors, and their functions in petal and stamen development have been intensely studied in Arabidopsis and Antirrhinum. However, the functions of class B genes in other plants, including ornamental species exhibiting floral morphology different from these model plants, have not received nearly as much attention. Here, we examine the cooperative functions of TfDEF and TfGLO on floral organ development in the ornamental plant torenia (Torenia fournieri Lind.). Torenia plants co-overexpressing TfDEF and TfGLO showed a morphological alteration of sepals to petaloid organs. Phenotypically, these petaloid sepals were nearly identical to petals but had no stamens or yellow patches like those of wild-type petals. Furthermore, the inflorescence architecture in the co-overexpressing torenias showed a characteristic change in which, unlike the wild-types, their flowers developed without peduncles. Evaluation of the petaloid sepals showed that these attained a petal-like nature in terms of floral organ phenotype, cell shape, pigment composition, and the expression patterns of anthocyanin biosynthesis-related genes. In contrast, torenias in which TfDEF and TfGLO were co-suppressed exhibited sepaloid petals in the second whorl. The sepaloid petals also attained a sepal-like nature, in the same way as the petaloid sepals. The results clearly demonstrate that TfDEF and TfGLO play important cooperative roles in petal development in torenia. Furthermore, the unique transgenic phenotypes produced create a valuable new way through which characteristics of petal development and inflorescence architecture can be investigated in torenia.

Generation of Novel Floral Traits Using a Combination of Floral Organ-Specific Promoters and a Chimeric Repressor in Torenia fournieri Lind.

In this study, we attempted to develop a new biotechnological method for the efficient modification of floral traits. Because transcription factors play an important role in determining floral traits, chimeric repressors, which are generated by attaching a short transcriptional repressor domain to transcription factors, have been widely used as effective tools for modifying floral traits in many plant species. However, the overexpression of these chimeric repressors by the Cauliflower mosaic virus 35S promoter sometimes causes undesirable morphological alterations to other organs. We attempted simultaneously to generate new floral traits and avoid such quality loss by examining five additional floral organ-specific promoters, one Arabidopsis thaliana promoter and four Torenia fournieri promoters, for the expression of the chimeric repressor of Arabidopsis TCP3 (AtTCP3), whose overexpression drastically alters floral traits but also generates dwarf phenotypes and deformed leaves. We found that the four torenia promoters exhibited particularly strong activity in the petals but not in the leaves, and that the combination of these floral organ-specific promoters with the chimeric repressor of AtTCP3 caused changes in the color, color patterns and cell shapes of petals, whilst avoiding other unfavorable phenotypes. Interestingly, each promoter that we used in this study generated characteristic and distinguishable floral traits. Thus, the use of different floral organ-specific promoters with different properties enables us to generate diverse floral traits using a single chimeric repressor without changing the phenotypes of other organs.

Efficient Modification of Floral Traits by Heavy-Ion Beam Irradiation on Transgenic Torenia

While heavy-ion beam irradiation is becoming popular technology for mutation breeding in Japan, the combination with genetic manipulation makes it more convenient to create greater variation in plant phenotypes. We have succeeded in producing over 200 varieties of transgenic torenia (Torenia fournieri Lind.) from over 2,400 regenerated plants by this procedure in only 2 years. Mutant phenotypes were observed mainly in flowers and showed wide variation in colour and shape. Higher mutation rates in the transgenics compared to those in wild type indicate the synergistic effect of genetic manipulation and heavy-ion beam irradiation, which might be advantageous to create greater variation in floral traits.

Faster, safer, and better DNA purification by ultracentrifugation using GelRed stain and development of mismatch oligo DNA for genome walking

Purification of plant DNA involves lengthy ultracentrifugation using ethidium bromide. Here, ultracentrifugation method is improved by staining with GelRed. The resulting method is faster, safer and of higher sensitivity. Purified DNA quality was confirmed by treatment with restriction enzymes and isolation of gene promoters. New type of long adaptor with mismatch sequence was also developed for promoter isolation.