Chimeric Repressor Research Articles

Identification of Chimeric Repressors that Confer Salt and Osmotic Stress Tolerance in Arabidopsis

We produced transgenic Arabidopsis plants that express chimeric genes for transcription factors converted to dominant repressors, using Chimeric REpressor gene-Silencing Technology (CRES-T), and evaluated the salt tolerance of each line. The seeds of the CRES-T lines for ADA2b, Msantd, DDF1, DREB26, AtGeBP, and ATHB23 exhibited higher germination rates than Wild type (WT) and developed rosette plants under up to 200 mM NaCl or 400 mM mannitol. WT plants did not grow under these conditions. In these CRES-T lines, the expression patterns of stress-related genes such as RD29A, RD22, DREB1A, and P5CS differed from those in WT plants, suggesting the involvement of the six transcription factors identified here in the stress response pathways regulated by the products of these stress-related genes. Our results demonstrate additional proof that CRES-T is a superior tool for revealing the function of transcription factors.

Multi-petal cyclamen flowers produced by AGAMOUS chimeric repressor expression

Cyclamen persicum (cyclamen) is a commercially valuable, winter-blooming perennial plant. We cloned two cyclamen orthologues of AGAMOUS (AG), CpAG1 and CpAG2, which are mainly expressed in the stamen and carpel, respectively. Cyclamen flowers have 5 petals, but expression of a chimeric repressor of CpAG1 (CpAG1-SRDX) caused stamens to convert into petals, resulting in a flower with 10 petals. By contrast, CpAG2-SRDX only caused incomplete formation of stamens and carpels. Expression in Arabidopsis thaliana showed similar effects on flower organ specification. Simultaneous expression of CpAG1-SRDX and CpAG2-SRDX in cyclamen induced rose-like, multi-petal flowers, a potentially valuable trait in commercial ornamental varieties. Expression of CpAG2-SRDX in a cyclamen mutant lacking expression of CpAG1 more effectively produced multi-petal flowers. Here, we controlled the number of petals in cyclamen by simple genetic engineering with a chimeric repressor. This strategy may be applicable useful for other ornamental plants with two distinct AG orthologues.

Chimeric repressor of PtSND2 severely affects wood formation in transgenic Populus

NAC domain transcription factors are important regulators that activate the secondary wall biosynthesis in wood formation. In this work, we investigated the possible functions of an NAC family member SECONDARY WALL-ASSOCIATED NAC DOMAIN PROTEIN2 (PtSND2) using chimeric repressor silencing technology. Reverse transcription-polymerase chain reaction, subcellular localization and transcriptional activation analyses indicated that PtSND2 is a wood-associated transcriptional factor with the predicted transcriptional activation activity, which could be inhibited by the repression domain SUPERMAN REPRESSION DOMAIN X (SRDX) in yeast. Wood formation was severely repressed in transgenic poplar plants overexpressing PtSND2-SRDX. Meanwhile, the secondary cell wall thickness of xylem fibers was restrained, and the contents of cellulose and lignin were obviously decreased in the stems of transgenic plants. Further studies indicated that expressions of a number of wood-associated genes were down-regulated in the stems of transgenic plants. Our results suggest that PtSND2 may play important roles during the secondary growth of stems in poplar.

MIXTA-like transcription factors and WAX INDUCER1/SHINE1 coordinately regulate cuticle development in Arabidopsis and Torenia fournieri.

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.

A bHLH-Type Transcription Factor, ABA-INDUCIBLE BHLH-TYPE TRANSCRIPTION FACTOR/JA-ASSOCIATED MYC2-LIKE1, Acts as a Repressor to Negatively Regulate Jasmonate Signaling inArabidopsis

Jasmonates (JAs) are plant hormones that regulate the balance between plant growth and responses to biotic and abiotic stresses. Although recent studies have uncovered the mechanisms for JA-induced responses in Arabidopsis thaliana, the mechanisms by which plants attenuate the JA-induced responses remain elusive. Here, we report that a basic helix-loop-helix-type transcription factor, ABA-INDUCIBLE BHLH-TYPE TRANSCRIPTION FACTOR/JA-ASSOCIATED MYC2-LIKE1 (JAM1), acts as a transcriptional repressor and negatively regulates JA signaling. Gain-of-function transgenic plants expressing the chimeric repressor for JAM1 exhibited substantial reduction of JA responses, including JA-induced inhibition of root growth, accumulation of anthocyanin, and male fertility. These plants were also compromised in resistance to attack by the insect herbivore Spodoptera exigua. Conversely, jam1 loss-of-function mutants showed enhanced JA responsiveness, including increased resistance to insect attack. JAM1 and MYC2 competitively bind to the target sequence of MYC2, which likely provides the mechanism for negative regulation of JA signaling and suppression of MYC2 functions by JAM1. These results indicate that JAM1 negatively regulates JA signaling, thereby playing a pivotal role in fine-tuning of JA-mediated stress responses and plant growth.

Engineering the Oryza sativa cell wall with rice NAC transcription factors regulating secondary wall formation.

Plant tissues that require structural rigidity synthesize a thick, strong secondary cell wall of lignin, cellulose and hemicelluloses in a complicated bridged structure. Master regulators of secondary wall synthesis were identified in dicots, and orthologs of these regulators have been identified in monocots, but regulation of secondary cell wall formation in monocots has not been extensively studied. Here we demonstrate that the rice transcription factors SECONDARY WALL NAC DOMAIN PROTEINs (SWNs) can regulate secondary wall formation in rice (Oryza sativa) and are potentially useful for engineering the monocot cell wall. The OsSWN1 promoter is highly active in sclerenchymatous cells of the leaf blade and less active in xylem cells. By contrast, the OsSWN2 promoter is highly active in xylem cells and less active in sclerenchymatous cells. OsSWN2 splicing variants encode two proteins; the shorter protein (OsSWN2S) has very low transcriptional activation ability, but the longer protein (OsSWN2L) and OsSWN1 have strong transcriptional activation ability. In rice, expression of an OsSWN2S chimeric repressor, driven by the OsSWN2 promoter, resulted in stunted growth and para-wilting (leaf rolling and browning under normal water conditions) due to impaired vascular vessels. The same OsSWN2S chimeric repressor, driven by the OsSWN1 promoter, caused a reduction of cell wall thickening in sclerenchymatous cells, a drooping leaf phenotype, reduced lignin and xylose contents and increased digestibility as forage. These data suggest that OsSWNs regulate secondary wall formation in rice and manipulation of OsSWNs may enable improvements in monocotyledonous crops for forage or biofuel applications.

Verticillium Infection Triggers VASCULAR-RELATED NAC DOMAIN7–Dependent de Novo Xylem Formation and Enhances Drought Tolerance in Arabidopsis

The soilborne fungal plant pathogen Verticillium longisporum invades the roots of its Brassicaceae hosts and proliferates in the plant vascular system. Typical aboveground symptoms of Verticillium infection on Brassica napus and Arabidopsis thaliana are stunted growth, vein clearing, and leaf chloroses. Here, we provide evidence that vein clearing is caused by pathogen-induced transdifferentiation of chloroplast-containing bundle sheath cells to functional xylem elements. In addition, our findings suggest that reinitiation of cambial activity and transdifferentiation of xylem parenchyma cells results in xylem hyperplasia within the vasculature of Arabidopsis leaves, hypocotyls, and roots. The observed de novo xylem formation correlates with Verticillium-induced expression of the VASCULAR-RELATED NAC DOMAIN (VND) transcription factor gene VND7. Transgenic Arabidopsis plants expressing the chimeric repressor VND7-SRDX under control of a Verticillium infection-responsive promoter exhibit reduced de novo xylem formation. Interestingly, infected Arabidopsis wild-type plants show higher drought stress tolerance compared with noninfected plants, whereas this effect is attenuated by suppression of VND7 activity. Together, our results suggest that V. longisporum triggers a tissue-specific developmental plant program that compensates for compromised water transport and enhances the water storage capacity of infected Brassicaceae host plants. In conclusion, we provide evidence that this natural plant-fungus pathosystem has conditionally mutualistic features.

Rice phytochrome-interacting factor-like protein OsPIL1 functions as a key regulator of internode elongation and induces a morphological response to drought stress

The mechanisms for plant growth restriction during stress conditions remains unclear. Here, we demonstrate that a phytochrome-interacting factor-like protein, OsPIL1/OsPIL13, acts as a key regulator of reduced internode elongation in rice under drought conditions. The level of OsPIL1 mRNA in rice seedlings grown under nonstressed conditions with light/dark cycles oscillated in a circadian manner with peaks in the middle of the light period. Under drought stress conditions, OsPIL1 expression was inhibited during the light period. We found that OsPIL1 was highly expressed in the node portions of the stem using promoter-glucuronidase analysis. Overexpression of OsPIL1 in transgenic rice plants promoted internode elongation. In contrast, transgenic rice plants with a chimeric repressor resulted in short internode sections. Alteration of internode cell size was observed in OsPIL1 transgenic plants, indicating that differences in cell size cause the change in internode length. Oligoarray analysis revealed OsPIL1 downstream genes, which were enriched for cell wall-related genes responsible for cell elongation. These data suggest that OsPIL1 functions as a key regulatory factor of reduced plant height via cell wall-related genes in response to drought stress. This regulatory system may be important for morphological stress adaptation in rice under drought conditions.

Inducible and reversible regulation of endogenous gene in mouse

Methods for generating loss-of-function mutations, such as conventional or conditional gene knockout, are widely used in deciphering gene function in vivo. By contrast, inducible and reversible regulation of endogenous gene expression has not been well established. Using a mouse model, we demonstrate that a chimeric transcriptional repressor molecule (tTS) can reversibly inhibit the expression of an endogenous gene, Nmyc. In this system, a tetracycline response element (TRE) artificially inserted near the target gene’s promoter region turns the gene on and off in a tetracycline-inducible manner. NmycTRE mice were generated by inserting a TRE into the first intron of Nmyc by the knockin technique. NmycTRE mice were crossed to tTS transgenic mice to produce NmycTRE/TRE: tTS embryos. In these embryos, tTS blocked Nmyc expression, and embryonic lethality was observed at E11.5d. When the dam was exposed to drinking water containing doxycycline (dox), normal endogenous Nmyc expression was rescued, and the embryo survived to birth. This novel genetic modification strategy based on the tTS–dox system for inducible and reversible regulation of endogenous mouse genes will be a powerful tool to investigate target genes that cause embryonic lethality or other defects where reversible regulation or temporary shutdown of the target gene is needed.

Morphological changes in Ipomoea nil using chimeric repressors of Arabidopsis TCP3 and TCP5

Chimeric REpressor gene-Silencing Technology (CRES-T) is a reverse genetic method that converts transcriptional factors (TFs) to chimeric repressors by fusion with an ethylene-responsive element-binding factor (ERF)-associated amphiphilic repression domain. The plant expressing chimeric repressor is expected to show loss-of-function phenotype of the original TF even in the presence of other functionally redundant TFs. We used the CRES-T system for modification of flower shape in transgenic Ipomoea nil (formally Pharbitis nil). I. nil is emerging as a model plant for ornamental flowers because it has produced a wide variety of historical mutants, and it has been chosen to be a part of the National BioResource Project in Japan. We used cDNAs of TFs of Arabidopsis thaliana with the CRES-T system because A. thaliana TFs are well characterized compared with I. nil TFs. For this study, we selected two TCP (TEOSINTE BRANCHED1, CYCLOIDEA, and PCF) TFs, TCP3 and TCP5, because overexpression of these chimeric repressors TCP3SRDX and TCP5SRDX causes severe morphological alterations in A. thaliana. We found that these chimeric repressors cause morphological alterations, an undeveloped corolla and wavy petals in I. nil. In addition, the sympetalous corolla was easily disrupted to form choripetalous corolla. Although several TCP3SRDX transgenic ornamental flowers were reported, typical sympetalous flowers like I. nil have not yet been presented.

Targeted transcriptional repression using a chimeric TALE-SRDX repressor protein

Transcriptional activator-like effectors (TALEs) are proteins secreted by Xanthomonas bacteria when they infect plants. TALEs contain a modular DNA binding domain that can be easily engineered to bind any sequence of interest, and have been used to provide user-selected DNA-binding modules to generate chimeric nucleases and transcriptional activators in mammalian cells and plants. Here we report the use of TALEs to generate chimeric sequence-specific transcriptional repressors. The dHax3 TALE was used as a scaffold to provide a DNA-binding module fused to the EAR-repression domain (SRDX) to generate a chimeric repressor that targets the RD29A promoter. The dHax3.SRDX protein efficiently repressed the transcription of the RD29A::LUC transgene and endogenous RD29A gene in Arabidopsis. Genome wide expression profiling showed that the chimeric repressor also inhibited the expression of several other genes that contain the designer TALE-target sequence in their promoters. Our data suggest that TALEs can be used to generate chimeric repressors to specifically repress the transcription of genes of interest in plants. This sequence-specific transcriptional repression by direct on promoter effector technology is a powerful tool for functional genomics studies and biotechnological applications.Electronic supplementary materialThe online version of this article (doi:10.1007/s11103-011-9866-x) contains supplementary material, which is available to authorized users.

Class-C MADS-box転写因子のキメラリプレッサーと誘導型発現抑制システムを用いた八重アサガオの作出

Class-C MADS-box転写因子のキメラリプレッサーと誘導型発現抑制システムを用いた八重アサガオの作出

Male-sterile and cleistogamous phenotypes in tall fescue induced by chimeric repressors of SUPERWOMAN1 and OsMADS58

Since tall fescue ( Festuca arundinacea Schreb.) is an anemophilous (wind-pollinated) grass species, male sterility is strongly desired for transgenic tall fescue to prevent pollen dispersal. To create male-sterile tall fescue, we applied Chimeric REpressor gene-Silencing Technology (CRES-T) based on rice APETALA3 ( AP3) and AGAMOUS ( AG) orthologues that specify the formation of stamens. We fused the coding regions of rice AP3 orthologue SUPERWOMAN1 ( SPW1), and rice AG orthologues, Os12g0207000, Os01g0886200 and OsMADS58, respectively with the artificial sequence encoding the modified EAR-like motif repression domain (SRDX). We first introduced Os12g0207000SRDX, Os01g0886200SRDX and OsMADS58SRDX into rice for evaluation of their abilities to induce male sterility. The transgenic rice expressing OsMADS58SRDX had reiterated formation of lodicule-like organs instead of stamens and carpel, a typical phenotype of ag mutant. Thus, we found that OsMADS58SRDX was most suitable for our purpose. Next, we introduced SPW1SRDX and OsMADS58SRDX into tall fescue. Although the transgenic tall fescue did not have the stamen alterations seen in SPW1SRDX and OsMADS58SRDX rice, they either produced no pollen or produced immature pollen; thus, the anthers were not dehiscent and the plants were male-sterile. In addition to the male sterility, SPW1SRDX tall fescue showed a cleistogamous (closed) phenotype in which anthers were not observed outside the glumes, with thin, abnormally elongated lodicules. Some lines of OsMADS58SRDX tall fescue showed a cleistogamous phenotype in which the lodicules were homeotically transformed into lemma-like organs. In both cases, cleistogamous phenotype was associated with morphological changes to the lodicules. We also obtained a mild phenotype of OsMADS58SRDX tall fescue, which exhibited only the male sterility. In this study, we produced novel male-sterile phenotypes using chimeric repressors and thus suggest CRES-T as a tool for transgenic improvement of forage and turf grasses.

Aberrant vegetative and reproductive development by overexpression and lethality by silencing of OsHAP3E in rice

We generated transgenic rice plants overexpressing OsHAP3E which encodes a subunit of a CCAAT-motif binding HAP complex. The OsHAP3E-overexpressing plants showed various abnormal morphologies both in their vegetative and reproductive phases. The OsHAP3E-overexpressing plants were dwarf with erected leaves and similar to brassinosteroid mutants in the vegetative phase. In the reproductive phase, dense panicle was developed, and occasionally successive generation of lateral rachises and formation of double flowers were observed. These phenotypes indicate association of OsHAP3E with determination of floral meristem identity. On the other hand, repression of OsHAP3E by RNAi or by overexpressing chimeric repressor fusion constructs brought about lethality to transformed cells, and almost no transformant was obtained. This suggests that the OsHAP3E function is essential for rice cells. Altogether, our loss-of-function and gain-of-function analyses suggest that OsHAP3E plays important pleiotropic roles in vegetative and reproductive development or basic cellular processes in rice.

HsfA1d and HsfA1e Involved in the Transcriptional Regulation of HsfA2 Function as Key Regulators for the Hsf Signaling Network in Response to Environmental Stress

Heat shock transcription factor A2 (HsfA2) acts as a key component of the Hsf signaling network involved in cellular responses to various types of environmental stress. However, the mechanism governing the regulation of HsfA2 expression is still largely unknown. We demonstrated here that a heat shock element (HSE) cluster in the 5'-flanking region of the HsfA2 gene is involved in high light (HL)-inducible HsfA2 expression. Accordingly, to identify the Hsf regulating the expression of HsfA2, we analyzed the effect of loss-of-function mutations of class A Hsfs on the expression of HsfA2 in response to HL stress. Overexpression of an HsfA1d or HsfA1e chimeric repressor and double knockout of HsfA1d and HsfA1e Arabidopsis mutants (KO-HsfA1d/A1e) significantly suppressed the induction of HsfA2 expression in response to HL and heat shock (HS) stress. Transient reporter assays showed that HsfA1d and HsfA1e activate HsfA2 transcription through the HSEs in the 5'-flanking region of HsfA2. In the KO-HsfA1d/A1e mutants, 560 genes, including a number of stress-related genes and several Hsf genes, HsfA7a, HsfA7b, HsfB1 and HsfB2a, were down-regulated compared with those in the wild-type plants under HL stress. The PSII activity of KO-HsfA1d/A1e mutants decreased under HL stress, while the activity of wild-type plants remained high. Furthermore, double knockout of HsfA1d and HsfA1e impaired tolerance to HS stress. These findings indicated that HsfA1d and HsfA1e not only regulate HsfA2 expression but also function as key regulators of the Hsf signaling network in response to environmental stress.

Morphological changes of Rosa×hybrida by a chimeric repressor of Arabidopsis TCP3

Chimeric repressor gene-silencing technology is a useful tool for changing morphology of ornamental plants. It has previously been demonstrated that the chimeric repressor TCP3SRDX, which consists of Arabidopsis TCP3 and an ERF-associated amphiphilic repression motif repression domain, perturbs the marginal morphology of Arabidopsis leaves and flowers. To obtain new rose cultivars that have ornamental values, we attempted to alter the morphology of Rosa×hybrida cv. Lavande with TCP3SRDX. The TCP3SRDX transgenic rose plants showed interesting phenotypes: the number of leaflets and the size of leaf teeth increased, the petals were wavy, and the sepals were compound-leafy. We succeeded in altering rose morphology using Arabidopsis TCP3 without the sequence information of a TCP3 homologue in the target plant species.

The new FioreDB database provides comprehensive information on plant transcription factors and phenotypes induced by CRES-T in ornamental and model plants

FioreDB (http://www.cres-t.org/fiore/public_db/) is a database of phenotypes induced by Chimeric REpressor gene-Silencing Technology (CRES-T) in ornamental and model plants. CRES-T induces a loss-of-function phenotype of a transcription factor (TF) gene by expression of a chimeric repressor produced by fusion of a TF to the strong transcriptional repression domain (SRDX). The earlier version of FioreDB provided phenotypic information induced by many kinds of chimeric repressors in various plants including torenia, chrysanthemum, cyclamen, gentian, morning glory, lisianthus and Arabidopsis. Phenotypic information, however, was not linked with gene information. We report here the development of the new FioreDB that provides more than 300 phenotypic information of various plants, linked to more than 100 TFs. FioreDB also provides information about classification of TFs, putative repression motifs found in TFs and other proteins, and incorporates publicly available gene information such as sequences and microarray data for all Arabidopsis genes. The new FioreDB described here, will be a valuable resource for basic research of TFs and for the manipulation of traits of agronomically important plants by CRES-T, especially from the point of view of horticulture.

Arabidopsis chimeric TCP3 repressor produces novel floral traits in Torenia fournieri and Chrysanthemum morifolium

Chimeric REpressor gene-Silencing Technology (CRES-T) is a powerful gene-silencing tool to analyze the function of Arabidopsis transcription factors. To investigate whether CRES-T is also applicable to horticultural plants inadequate for genetic engineering because of their limited molecular biological characterization and polyploidy, we applied CRES-T to torenia and the hexaploid chrysanthemum and produced their transgenic plants expressing the chimeric repressor derived from the Arabidopsis TEOSINTE BRANCHED1, CYCLOIDEA, and PCF family transcription factor 3 (TCP3) fused with a plant-specific transcriptional repression domain named SRDX, consisting of 12 amino acids originated from the EAR-motif (TCP3-SRDX). Transgenic torenia and chrysanthemum expressing TCP3-SRDX exhibited fringed leaves and short pistils, while those expressing TCP3 fused with either the mutated repression domain (TCP3-mSRDX) or the overexpressor of TCP3 (TCP3-ox) did not exhibit phenotypic changes. In addition to fringed leaves, TCP3-SRDX transgenic torenia plants exhibited petals with fringed margins, distinctive color patterns, and reduced anthocyanin accumulation. In TCP3-SRDX transgenic chrysanthemum plants, floral organ development was suppressed as compared with the wild type. These results indicate that the Arabidopsis-derived TCP3-SRDX induced morphological changes in transgenic torenia and chrysanthemum although the observed phenotypes partially differ from each other. CRES-T may function in various plant species including polyploid species and modify their biological characteristics.

Production of picotee-type flowers in Japanese gentian by CRES-T

Chimeric repressor gene-silencing technology (CRES-T) is an efficient gene suppression system in a wide variety of dicots and monocots. In this study, we demonstrated that the CRES-T system functions in Japanese gentian. A chimeric repressor of the anthocyanin biosynthetic regulator gene GtMYB3, under the control of the Arabidopsis actin2 promoter, was introduced into blue-flowered gentian. Of 12 transgenic lines, 2 exhibited a picotee flower phenotype with a lack of pigmentation in the lower part of the petal. HPLC analysis showed that the petals of these lines contained less anthocyanin and more flavone than the wild-type, suggesting competitive accumulation of these two types of compounds. The expressions of ‘late’ flavonoid biosynthetic genes, including F3H, F3′5′H, DFR and ANS, were strongly suppressed in petals of these transgenic plants. In contrast, the ‘early’ flavonoid biosynthetic genes, such as CHS and FNSII, were not affected. Since FNSII is expressed more strongly in the lower part of petals than in the upper part, the absence of pigmentation in the lower parts might be induced by flavone synthesis. These results demonstrated that the suppression of anthocyanin biosynthetic genes by CRES-T was successfully applied to Japanese gentian to change petal color; therefore, this system could be useful for generating novel flower colors and patterns. Transgenic plants produced in this study might be utilized as elite materials in the breeding of Japanese gentian in the near future.

Utilization of a floral organ-expressing AP1 promoter for generation of new floral traits in Torenia fournieri Lind

To establish an efficient way to create novel floral traits in horticultural flowers, we have introduced many chimeric repressors of Arabidopsis transcription factors into torenia. Among them, we found a transgenic torenia exhibiting unopened flower buds and glossy dark green leaves with curled margins as a consequence of overexpression of Arabidopsis MYB24 with a transcriptional repression domain (MYB24-SRDX). Petals inside the flower buds exhibited a distinct coloration pattern. To bring out this favorable petal trait without inducing the unfavorable phenotypes due to the constitutive expression of chimeric repressors by the cauliflower mosaic virus 35S (35S) promoter, we tested the ability of a floral organ-specific Arabidopsis APETALA1 (AP1) promoter, which was found to be active in both petals and flower buds of torenia. As expected, AP1 pro:MYB24-SRDX transgenic torenias resulted in the opening of flowers and a normal leaf phenotype. Furthermore, these AP1 pro:MYB24-SRDX torenias exhibited wavy petals with a characteristic configuration. This is a good example of the utilization of a floral organ-specific promoter for creating distinct flower phenotypes without causing unfavorable morphological and physiological changes in other organs.