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Gibberellins (GAs) are diterpenoid hormones, control various physiological developments in plants. The role of gibberellins on morphology and secondary metabolite production was examined in Artemisia annua, a medicinal plant that has been acknowledged as a source of artemisinin, an antimalarial compound. Subsequently, the GA20ox gene from Torenia fournieri (TfGA20ox2) was transferred to A. annua by Agrobacterium-mediated transformation. Compared with wild type plants, all nine transgenic plants showed significantly higher plant heights and artemisinin contents. The highest artemisinin content and yield in TfGA20ox2-overexpressing plants was around two-fold higher than wild type. Moreover, transgenic plants had higher numbers of branches (52.4%) and greater branch lengths (60–203%), but smaller leaf size (77.6%). Interestingly, relative to wild type the number and size of glandular trichomes in transgenic leaves was about 30 and 35% higher, respectively. From GC–MS analysis, the proportion of diterpenes in transgenic plant extracts was 1.5-fold lower than those noticed in wild type, while the proportion of sesquiterpenes was increased about 1.6 times when compared to wild type. However, the content proportion of monoterpenes showed a slightly increase, whereas the level of triterpenes showed no variation. In addition, two monoterpenes (eucalyptol and borneol), four sesquiterpenes (α-caryophyllene, β-guaiene, δ-cadinene and β-cubebene) and one triterpenes (isomultiflorenone) were detected only in transgenic extract, whereas d-α-tocopherol, a diterpenoid compound was found only in wild type but not transgenic plant. These results suggested that gibberellins play a significant role in regards to morphology, trichome formation and terpenoid metabolite production in A. annua.

Torenia fournieri Lind. ex Fourn. (wishbone flower, bluewings) is a popular summer bedding plant in Louisiana. Clown Mixture cultivars are available in garden centers in March and April. Transplants of cultivar Clown Rose were purchased, transplanted to larger pots, and maintained in a greenhouse. A powdery mildew was observed on these plants in March and all plants (six) were severely diseased by May. Symptoms included leaf distortion and yellowing. Powdery mildew was not present on transplants and none was found in later checks of garden centers. An Oidium sp. was observed sporulating on both leaf surfaces of infected plants. Conidia were ellipsoid, produced in chains, lacked fibrosin bodies, and averaged 41 × 22 μm in dimensions. No sexual stage was observed. Healthy plants of Clown Mixture cultivars were obtained and inoculated by brushing conidia from infected plant leaves to leaves of healthy plants. Plants were maintained in a greenhouse where temperatures ranged from 16 to 26°C. Hyphal growth appeared on inoculated plants after 5 days and the reproductive structures formed later appeared the same as those on originally infected plants. Uninoculated plants remained healthy. No previous reports of powdery mildew diseases of T. fournieri in the United States were found. Other powdery mildew pathogens reported on T. fournieri are Sphaerotheca fuliginea (Schlechtend.:Fr.) Pollacci in Finland and Japan and an Erysiphe sp. in Japan (1). Reference: (1) K. Amano. Host Range and Geographical Distribution of the Powdery Mildew Fungi. Japan Scientific Press, Tokyo, 1986.

Torenia fournieri (Linderniaceae) is a common ornamental plant in China. It is also an important Chinese medicinal herb for its heart clearing and toxin removal properties. In October 2013, severe powdery mildew infections were observed on T. fournieri in Baihuayuan Garden (125.35°E, 43.88°N), China. Voucher specimens were deposited in the Herbarium of Mycology of Jilin Agricultural University under the accession number HMJAU02176. Whitish colonies covered the surface of leaves, petioles, sepals, and stems. The infected leaves became yellow and necrotic by advanced stages of the infection. Chasmothecia with a diameter between 63.5 and 95 μm were present singly or in groups, and bore dark brown mycelioid. The appendages were 0.5 to 4 times as long as the chasmothecial diameter, brown at the base and paler toward the apex. Asci were 2 to 6 per chasmothecium, short-stalked or sessile, 50 to 62 × 30 to 50.5 μm, and 2- to 4-spored. Ascospores were pale brown, oval to ellipsoid, 27 to 43 × 13 to 17 μm. Hyphae were flexuous to straight, branched, and septate. Appressoria were well-developed, lobed, and solitary or in opposite pairs. Conidiophores were unbranched, cylindrical, and 94 to 185 × 9 to 15 μm. Foot-cells were straight, cylindrical, 19 to 40 μm long, and followed by 1 to 3 cells shorter or nearly equal to the foot-cell. Conidia were singly produced, cylindrical or oval, 34 to 44 × 16 to 20 μm, and without distinct fibrosin bodies. Lobed germ tubes were produced at the tip of conidia. The morphological characteristics of sexual and asexual structures were consistent with Erysiphe macleayae (1,2). To confirm the identification, the complete internal transcribed spacer (ITS) region of rDNA of the pathogen was amplified with the primers ITS1/ITS4 and sequenced (3). The resulting 574-bp sequence (KJ600796) showed 100% similarity with E. macleayae (KF856294) and Oidium sp. isolated from Chelidonium majus (HQ286645 to 46) and one base different from M. microcarpa (JQ681217). Koch's postulate was completed by gently pressing a diseased leaf onto three young excised leaves of asymptomatic seedlings. Three non-inoculated leaves were used as controls. Inoculated leaves were incubated in separate petri dishes in a greenhouse at 20 to 25°C. Symptoms developed 5 days after inoculation, whereas the control leaves remained symptomless (voucher specimens HMJAU02176I). The morphology of the fungus on the inoculated leaves was identical to that observed on the originally diseased leaves. Powdery mildew on T. fournieri has only been reported as Golovinomyces sp. in Hungary (4). To our knowledge, this is the first report of powdery mildew caused by E. macleayae on T. fournieri worldwide. Infection of Torenia by E. macleayae, a common powdery mildew on various hosts of the Papaveraceae, is very unusual and unexpected. It demonstrates a wider host range of this species beyond the limits of the Papaveraceae. References: (1) U. Braun and R. T. A. Cook. Taxonomic manual of the Erysiphales (Powdery Mildews), CBS Biodiversity Series 11. CBS, Utrecht, the Netherlands, 2012. (2) M. J. Park et al. Plant Dis. 96:1376, 2012. (3) S. Takamatsu et al. Mycol. Res. 113:117, 2009. (4) P. Vági et al. Eur. J. Plant Pathol. 117:89, 2007.

Successful fertilization in flowering plants depends on the precise directional growth control of pollen tube through the female pistil tissue toward the female gametophyte contained in the ovule for delivery of nonmotile sperm cells. Cys-rich peptides LUREs secreted from the synergid cells on either side of the egg cell act as ovular attractants of pollen tubes. Competency control by the pistil is crucial for the response of pollen tubes to these ovular attractants. We recently reported that ovular 4-O-methyl-glucuronosyl arabinogalactan (AMOR) induces competency of the pollen tube to respond to ovular attractant LURE peptides in Torenia fournieri. The beta isomer of the terminal disaccharide 4-O-methyl-glucuronosyl galactose was essential and sufficient for the competency induction. However, critical and noncritical structures in the disaccharide have not been dissected deeply. Herein, we report the synthesis of new AMOR analogs and the structure-activity relationships for AMOR activity in the presence of these synthesized analogs. Removal of 4-O-methyl group or –COOH from the glucuronosyl residue of the disaccharide dramatically reduces AMOR activity. The pyranose backbone of the second sugar of disaccharide is essential for the activity but not hydroxy groups. The role of beta isomer of the disaccharide 4-Me-GlcA-β(1,6)-Gal is very specific for competency control, as there was no difference in effect among the sugar analogs tested for pollen germination. This study represents the first structure-activity relationship study, to our knowledge, of a sugar molecule involved in plant reproduction, which opens a way for modification of the molecule without loss of activity.

Precise directional control of pollen-tube growth by pistil tissue is critical for successful fertilization of flowering plants [1-3]. Ovular attractant peptides, which are secreted from two synergid cells on the side of the egg cell, have been identified [4-6]. Emerging evidence suggests that the ovular directional cue is not sufficient for successful guidance but that competency control by the pistil is critical for the response of pollen tubes to the attraction signal [1, 3, 7]. However, the female molecule for this competency induction has not been reported. Here we report that ovular methyl-glucuronosyl arabinogalactan (AMOR) induces competency of the pollen tube to respond to ovular attractant LURE peptides in Torenia fournieri. We developed a method for assaying the response capability of a pollen tube by micromanipulating an ovule. Using this method, we showed that pollen tubes growing through a cut style acquired a response capability in the medium by receiving a sufficient amount of a factor derived from mature ovules of Torenia. This factor, named AMOR, was identified as an arabinogalactan polysaccharide, the terminal 4-O-methyl-glucuronosyl residue of which was necessary for its activity. Moreover, a chemically synthesized disaccharide, the β isomer of methyl-glucuronosyl galactose (4-Me-GlcA-β-(1→6)-Gal), showed AMOR activity. No specific sugar-chain structure of plant extracellular matrix has been identified as a bioactive molecule involved in intercellular communication. We suggest that the AMOR sugar chain in the ovary renders the pollen tube competent to the chemotropic response prior to final guidance by LURE peptides.

Torenia fournieri E. Fourn., known as torenia or clown flower, is an annual plant in the Linderniaceae. In October 2014, typical powdery mildew symptoms were observed on torenia plants of an unknown cultivar in a private garden in Seongnam (37°26′50″N; 127°10′02″E), Korea. In August 2019, dozens of torenia of an unknown cultivar planted in shady borders of a roadside garden in Seoul (37°34′46″N; 127°01′25″E), Korea, were found to be infected with a powdery mildew with 100% disease incidence. Signs first appeared as circular to irregular white patches, which subsequently coalesced to develop into abundant hyphal growth on both sides of the leaves and also on flower stalks and calyxes. As the disease developed, affected leaves became wilted. Two voucher specimens have been deposited in the Korea University Herbarium (KUS-F28526 and F31142). Hyphal appressoria were nipple shaped or indistinct. Conidiophores were cylindrical, 110 to 240 × 10 to 12 µm, and had cylindrical foot cells, somewhat curved at the base, and 50 to 100 μm long, followed by two short cells and two to six immature conidia in chains with a sinuate outline. Conidia were hyaline, ellipsoid to barrel shaped, 26 to 38 × 16 to 22 μm (length/width ratio = 1.4 to 2.0), and devoid of distinct fibrosin bodies. Germ tubes were perihilar on the conidia. These morphological characteristics are consistent with those of Golovinomyces bolayi (Schwein.) U. Braun & R.T.A. Cook (Braun and Cook 2012). No chasmothecia were found during the growing season. To confirm identification, the internal transcribed spacer region of rDNA of KUS-F28526 was amplified with primers ITS1/ITS4 and sequenced. The resulting sequence was deposited in GenBank (accession no. MN420661). A GenBank BLAST search revealed a sequence similarity of 99.7% to G. bolayi ex Torenia fournieri (DQ538345). A pathogenicity test was performed by gently dusting conidia onto leaves of five healthy potted plants bought in a flower market. Five noninoculated plants served as controls. Powdery mildew colonies developed on all inoculated leaves after 7 to 10 days, whereas the control plants remained symptomless. The fungus present on the inoculated leaves was identical morphologically to that observed on the originally diseased leaves. The pathogenicity test was performed twice with similar results. Previously, G. bolayi on T. fournieri was known from Hungary (Braun et al. 2019). Two previous records of Oidium sp. on T. fournieri from Japan (Wada and Hirata 1977, figure plates uploaded on November 2008 and shown at www.naro.affrc.go.jp) and the United States (Holcomb 1999) are likely to be Golovinomyces, probably G. bolayi. To our knowledge, this is the first report of powdery mildew caused by G. bolayi on T. fournieri in Korea. Because powdery mildew infection detracts from the beauty and health of green leaves, control measures should be developed.

This study was conducted to examine the role of the transcription factors (TFs), RsMYB1 and mPAP1 together with B-Peru (mPAP1 + B-Peru), in regulating anthocyanin biosynthesis in the ornamental torenia (Torenia fournieri) cultivar Kauai Rose using Agrobacterium-mediated transformation. Expression levels of RsMYB1 were the highest in the lines RS5 and RS3, followed by RS4, RS2, and RS1, while transcript levels of mPAP1 + B-Peru increased in the order of PB-6 > PB-5 > PB-7 > PB-8 > PB-2. Moreover, transcript levels of the anthocyanin structural genes in transgenic lines were significantly higher than those in wild-type (WT) plants. Anthocyanin structural gene expression was specifically altered by TF overexpression: the highest expression of anthocyanidin synthase (ANS) was observed in transgenic lines with RsMYB1, while expression of dihydroflavonol-4-reductase (DFR) was the highest in lines with mPAP1 + B-Peru. We expect that enhanced expression of these anthocyanin structural genes will improve anthocyanin content in the flowers of transgenic torenia. Moreover, these results indicate that RsMYB1 and mPAP1 + B-Peru can be exploited as anthocyanin regulatory TFs to enhance anthocyanin content in other horticultural plants. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13205-018-1505-7) contains supplementary material, which is available to authorized users.

We present a semi-in vitro chemotropism assay that can be used to evaluate the chemoattractant effect of diffusible plant signaling molecules on growing pollen tubes. We constructed an array of microslit channels in a microfluidic device that prevented the passage of randomly growing pollen tubes but permitted ones that are responsive to the chemoattractant. Depending on the microslit channel size, 80%–100% of the randomly growing Torenia fournieri pollen tubes were excluded from reaching the source of the attractant. Thus, the selection of pollen tubes that are capable of responding to chemoattractants from a mixed population can be realized using this platform.

Background ALOG (Arabidopsis LSH1 and Oryza G1) family with a conserved domain widely exists in plants. A handful of ALOG members have been functionally characterized, suggesting their roles as key developmental regulators. However, the evolutionary scenario of this gene family during the diversification of plant species remains largely unclear. Methods Here, we isolated seven ALOG genes from Torenia fournieri and phylogenetically analyzed them with different ALOG members from representative plants in major taxonomic clades. We further examined their gene expression patterns by RT-PCR, and regarding the protein subcellular localization, we co-expressed the candidates with a nuclear marker. Finally, we explored the functional diversification of two ALOG members, TfALOG1 in euALOG1 and TfALOG2 in euALOG4 sub-clades by obtaining the transgenic T. fournieri plants. Results The ALOG gene family can be divided into different lineages, indicating that extensive duplication events occurred within eudicots, grasses and bryophytes, respectively. In T. fournieri, seven TfALOG genes from four sub-clades exhibit distinct expression patterns. TfALOG1–6 YFP-fused proteins were accumulated in the nuclear region, while TfALOG7-YFP was localized both in nuclear and cytoplasm, suggesting potentially functional diversification. In the 35S:TfALOG1 transgenic lines, normal development of petal epidermal cells was disrupted, accompanied with changes in the expression of MIXTA-like genes. In 35S:TfALOG2 transgenic lines, the leaf mesophyll cells development was abnormal, favoring functional differences between the two homologous proteins. Unfortunately, we failed to observe any phenotypical changes in the TfALOG1 knock-out mutants, which might be due to functional redundancy as the case in Arabidopsis. Conclusion Our results unraveled the evolutionary history of ALOG gene family, supporting the idea that changes occurred in the cis regulatory and/or nonconserved coding regions of ALOG genes may result in new functions during the establishment of plant architecture.

We developed a fertilization method in which pollen tubes entered into embryo sacs without any need to contact surrounding female sporophytic cells by using Torenia fournieri (Torenia) plants under the condition of hindering movement of the virus from a stigma, which is the first infection site leading to systemic infection. When RBDV-infected Torenia pollen grains were used for the developed fertilization method, the virus was transmitted to the seeds by pollen tubes germinating from them. On the other hand, no seeds were infected with the virus when Torenia plants were pollinated with healthy Torenia pollen grains in combination with RBDV-infected raspberry pollen grains, which caused the virus infection in the stigma by penetration of their pollen tubes arrested in its style. Our results indicate that vertical transmission of RBDV by pollen occurs in the transport of the virus into embryo sacs by pollen tubes reaching the embryo sacs.

The female gametophytes of many flowering plants contain one egg cell, one central cell, two synergid cells and three antipodal cells with respective morphological characteristics and functions. These cells are formed by cellularization of a multinuclear female gametophyte. However, the dynamics and mechanisms of female gametophyte development remain largely unknown due to the lack of a system to visualize directly and manipulate female gametophytes in living material. Here, we established an in vitro ovule culture system to examine female gametophyte development in Torenia fournieri, a unique plant species with a protruding female gametophyte. The four-nucleate female gametophyte became eight nucleate by the final (third) mitosis and successively cellularized and matured to attract a pollen tube. The duration of final mitosis was 28 ± 6.5 min, and cellularization was completed in 54 ± 20 min after the end of the third mitosis. Fusion of polar nuclei in the central cell occurred in 13.1 ± 1.1 h, and onset of expression of LURE2, a pollen tube attractant gene, was visualized by a green fluorescent protein reporter 10.7 ± 2.3 h after cellularization. Laser disruption analysis demonstrated that the egg and central cells were required for synergid cells to acquire the pollen tube attraction function. Moreover, aberrant nuclear positioning and down-regulation of LURE2 were observed in one of the two synergid cells after disrupting an immature egg cell, suggesting that cell specification was affected. Our system provides insights into the precise dynamics and mechanisms of female gametophyte development in T. fournieri.

The experiment was conducted in winter – spring 2016 – 2017 in Thua Thien Hue to identify suitable level of foliar fertilizer for Torenia fournieri Linden having good growth and development, beautiful color and long lifetime under local conditions. The experiment consisted of four treatments with three forliar fertilizers such: Dau Trau MK 30-10-5, Gibberellin 25-10-10, and HVP. The control treatment (I) was using sterilized water without foliar fertilizer. The results showed that all the foliar fertilizers well influenced on growth and development of Torenia fournieri Linden. Dau Trau MK 30-10-5 helped Torenia fournieri Linden having high quality and high value/cost ratio than the others.

Although chimeric repressors such as the Arabidopsis TCP3 repressor are known to have significant effects on flower morphology and color, their cellular-level effects on flower petals are not understood. The promoter sequences of the genes expressed in the flowers of cyclamen, a representative potted flower grown during the winter season, are also unknown. Here, we isolated eight promoters from cyclamen genes that are reportedly expressed in the petals. These promoters were then fused to four chimeric repressors and introduced into the model flower torenia to screen for effective combinations of promoters and repressors for flower breeding. As expected, some of the constructs altered flower phenotypes upon transformation. We further analyzed the effects of chimeric repressors at the cellular level. We observed that complicated petal and leaf serrations were accompanied by excessive vascular branching. Dichromatism in purple anthocyanin was inferred to result in bluish flowers, and imbalanced cell proliferation appeared to result in epinastic flowers. Thus, the genetic constructs and phenotypic changes described in this report will benefit the future breeding and characterization of ornamental flowers.

In flowering plants, pollen tube guidance is an enigmatic process involving multiple cell-to-cell communications. Several important signal molecules associated with this process have been previously identified, such as LUREs, which are pollen tube attractants derived from Torenia fournieri synergid cells. To identify pollen tube receptors of ovule-secreted attractant signals, we constructed an Arabidopsis semi-in vivo pollen tube guidance system and used microarray analysis to characterize the specific transcriptome. A distinct gene expression profile was observed for pollen tubes that had grown through stigma and style tissues of a pistil and communicated with ovules. Genes involved in signal transduction, transcription, and pollen tube growth were overrepresented, with many genes newly detected as expressed in pollen tubes. The roles of some candidate receptor-like kinase genes in micropylar pollen tube guidance were revealed by reverse genetics analysis. Our results provide valuable clues for research on cell-to-cell interactions during fertilization.

Excised placentae with ovules of Primula pubescens, P. auricula, Scopolia carniolica, Digitalis purpurea, Torenia fournieri and Chionodoxa luciliae were self pollinated in vitro and the development of seeds was observed. The same method was used for obtaining hybrid globular embryos from crosses between: P. pubescens x P. auricula, Scopolia carniolica x Physochlaina praealta, Melandrium album x Silene saxifraga and M. album x Arenaria pungens.

Gibberellins (GAs) are the plant hormones that control many aspects of plant growth and development, including stem elongation. Genes encoding enzymes related to the GA biosynthetic and metabolic pathway have been isolated and characterized in many plant species. Gibberellin 2-oxidase (GA2ox) catalyzes bioactive GAs or their immediate precursors to inactive forms; therefore, playing a direct role in determining the levels of bioactive GAs. In the present study, we produced transgenic plants of the liliaceous monocotyledon Tricyrtis sp. overexpressing the GA2ox gene from the linderniaceous dicotyledon Torenia fournieri (TfGA2ox2). All six transgenic plants exhibited dwarf phenotypes, and they could be classified into two classes according to the degree of dwarfism: three plants were moderately dwarf and three were severely dwarf. All of the transgenic plants had small or no flowers, and smaller, rounder and darker green leaves. Quantitative real-time reverse transcription-polymerase chain reaction (PCR) analysis showed that the TfGA2ox2 expression level generally correlated with the degree of dwarfism. The endogenous levels of bioactive GAs, GA1 and GA4, largely decreased in transgenic plants as shown by liquid chromatography-mass spectrometry (LC-MS) analysis, and the level also correlated with the degree of dwarfism. Exogenous treatment of transgenic plants with gibberellic acid (GA3) resulted in an increased shoot length, indicating that the GA signaling pathway might normally function in transgenic plants. Thus, morphological changes in transgenic plants may result from a decrease in the endogenous levels of bioactive GAs. Finally, a possibility of molecular breeding for plant form alteration in liliaceous ornamental plants by genetically engineering the GA metabolic pathway is discussed.

Flower-specific promoters can enable transgenic enhancement of valuable ornamental traits, including flower shape and color. However, the identification of strong, tissue-specific promoters remains a limiting factor. To obtain enhanced flower-specific promoters, we constructed four chimeric promoters (p35S-PCHS-Ω, p35S-LCHS-Ω, pOCS-PCHS-Ω and pOCS-LCHS-Ω) combining the 35S or OCS enhancer fused to a 302 bp CHSA core promoter fragment from petunia (PCHS) or a 307 bp CHS core promoter fragment from lily (LCHS), and also containing an omega element (Ω). Each promoter was fused to the β-glucuronidase (GUS) reporter gene, and we examined the levels and tissue specificity of GUS expression in transgenic Torenia fournieri. p35S-PCHS-Ω and p35S-LCHS-Ω drove strong, constitutive GUS expression in all tissues, especially in colored corollas (p35S-PCHS-Ω) or in colored corollas and roots (p35S-LCHS-Ω). pOCS-PCHS-Ω drove stronger GUS expression in colored corollas than in other tissues but expression was weaker than that of p35S-PCHS-Ω. pOCS-LCHS-Ω drove GUS in colored corollas but also in roots. Among the four chimeric promoters, pOCS-PCHS-Ω exhibited stronger activity only in colored corollas, making it useful for transgenic enhancement of floral traits, such as expressing ‘blue genes’ in lily to produce new lines with blue flowers.

Pollen tube guidance is controlled by multiple complex interactions with the female tissues. Here, we show that pollen tubes of Torenia fournieri are regulated by a stylar tissue in a length-dependent manner to receive and respond to attractant LURE peptides secreted from synergid cells. We developed an immunostaining method to visualize LURE peptides bound at the plasma membrane of the tip region of the pollen tube. Using this method, we found that LURE peptides bound specifically to pollen tubes growing through a cut style. The peptides also bound to pollen tubes growing through a shorter style, which were not competent to respond to these peptides. These observations suggested a possibility that acquisition of the LURE peptide reception ability and acquisition of full competency are separable processes. RNA-Seq suggested that the transcription profile of pollen tubes was affected by both the length of the style and the cultivation period, consistently with physiological changes in binding activity and LURE response ability. The database generated from de novo RNA-Seq of Torenia pollen tubes was shown to be useful to identify pollen tube proteins by mass spectrometry. Our studies provide insight and an effective platform for protein identification to understand pollen tube guidance.

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.

We identified a Torenia fournieri Lind. mutant (no. 252) that exhibited a sepaloid phenotype in which the second whorls were changed to sepal-like organs. This mutant had no stamens, and the floral organs consisted of sepals and carpels. Although the expression of a torenia class B MADS-box gene, GLOBOSA (TfGLO), was abolished in the 252 mutant, no mutation of TfGLO was found. Among torenia homologs such as APETALA1 (AP1), LEAFY (LFY), and UNUSUAL FLORAL ORGANS (UFO), which regulate expression of class B genes in Arabidopsis, only accumulation of the TfUFO transcript was diminished in the 252 mutant. Furthermore, a missense mutation was found in the coding region of the mutant TfUFO. Intact TfUFO complemented the mutant phenotype whereas mutated TfUFO did not; in addition, the transgenic phenotype of TfUFO-knockdown torenias coincided with the mutant phenotype. Yeast two-hybrid analysis revealed that the mutated TfUFO lost its ability to interact with TfLFY protein. In situ hybridization analysis indicated that the transcripts of TfUFO and TfLFY were partially accumulated in the same region. These results clearly demonstrate that the defect in TfUFO caused the sepaloid phenotype in the 252 mutant due to the loss of interaction with TfLFY.

SummaryA major goal of plant reproduction research is to understand and overcome hybridization barriers so that the gene pool of crop plants can be increased and improved upon. After successful pollen germination on a receptive stigma, the nonmotile sperm cells of flowering plants are transported via the pollen tube (PT) to the egg apparatus for the achievement of double fertilization. The PT path is controlled by various hybridization mechanisms probably involving a larger number of species-specific molecular interactions [1, 2]. The egg-apparatus-secreted polymorphic peptides ZmEA1 in maize [3] and LURE1 and LURE2 in Torenia fournieri [4] as well as TcCRP1 in T. concolor [5] were shown to be required for micropylar PT guidance, the last step of the PT journey. We report here that ZmEA1 attracts maize PTs in vitro and arrests their growth at higher concentrations. Furthermore, it binds to the subapical region of maize PT tips in a species-preferential manner. To overcome hybridization barriers at the level of gametophytic PT guidance, we expressed ZmEA1 in Arabidopsis synergid cells. Secreted ZmEA1 enabled Arabidopsis ovules to guide maize PT in vitro in a species-preferential manner to the micropylar opening of the ovule. These results demonstrate that the egg-apparatus-controlled reproductive-isolation barrier of PT guidance can be overcome even between unrelated plant families.

miR156/157 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 Arabidopsis MIR157b (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.

Plants under herbivore attack emit mixtures of volatiles (herbivore-induced plant volatiles, HIPVs) that can attract predators of the herbivores. Although the composition of HIPVs should be critical for the attraction, most studies of transgenic plant-emitted volatiles have simply addressed the effect of trans-volatiles without embedding in other endogenous plant volatiles. We investigated the abilities of transgenic wishbone flower plants (Torenia hybrida and Torenia fournieri) infested with spider mites, emitting a trans-volatile ((E)-β-ocimene) in the presence or absence of endogenous volatiles (natural HIPVs and/or floral volatiles), to attract predatory mites (Phytoseiulus persimilis). In both olfactory- and glasshouse-based assays, P. persimilis females were attracted to natural HIPVs from infested wildtype (wt) plants of T. hybrida but not to those of T. fournieri. The trans-volatile enhanced the ability to attract P. persimilis only when added to an active HIPV blend from the infested transgenic T. hybrida plants, in comparison with the attraction by infested wt plants. Intriguingly, floral volatiles abolished the enhanced attractive ability of T. hybrida transformants, although floral volatiles themselves did not elicit any attraction or avoidance behavior. Predator responses to trans-volatiles were found to depend on various background volatiles (e.g. natural HIPVs and floral volatiles) endogenously emitted by the transgenic plants.

Application of forchlorfenuron (CPPU) to flower buds induces morphologically different paracorollas, i.e., wide and narrow type, in torenia (Torenia fournieri L.). We investigated the morphological properties and the role of floral homeotic genes in the formation of these two types of paracorolla. The morphology of epidermal cells and distribution pattern of vascular bundles in the wide paracorolla was the same as in the petal; however, in the narrow paracorolla, the morphology of epidermal cells was either petal-like or stamen-like, and the distribution pattern of vascular bundles was stamen-like. In situ hybridization analysis of the floral homeotic genes showed that a class A gene, T. fournieri SQUAMOSA (TfSQUA), and class B genes, TfDEFICIENS (TfDEF) and TfGLOBOSA (TfGLO), were expressed in the broad part of the primordia of the wide paracorolla, as in the petal. Class C genes, TfPLENA1 (TfPLE1) and TfFARINELLI (TfFAR), were only expressed at the margin of the primordia. However, in the primordia of the narrow paracorolla, TfSQUA was only expressed at the margin, but the class B genes and one of the class C genes (TfPLE1) were expressed in a broad section, as in the primordia of the wide paracorolla. This expression pattern in the narrow paracorolla was intermediate between that of the petal and the stamen. In later developmental stages, quantitative real-time PCR analyses showed that, in the wide paracorolla, TfSQUA and class B genes were highly expressed but the expression of class C genes, as in the petal, was low. In the narrow paracorolla, class B genes were also highly expressed as in the petal; however, the expression of both TfSQUA and the class C genes was low, as in the stamen and the petal, respectively. This expression pattern probably reflects the unstable floral organ identity of the narrow paracorolla, and the expression pattern in paracorollas is determined by the site where the paracorolla is formed.

A new leaf yellowing disease was observed on periwinkle (Catharanthus roseus) in Taiwan in 2005. Sequence analysis of a 1.8 kb region of 16S rDNA, 16S-23S intergenic spacer region and partial 23S rDNA revealed that the periwinkle leaf yellowing (PLY) disease was associated with a phytoplasma from the 16SrI group. In addition, five cultivated plants including chrysanthemum (Chrysanthemum sp.), cosmos (Cosmos bipinnatus), torenia (Torenia fournieri), Persian violet (Exacum affine), and cucumber (Cucumis sativus), and one weed, goosegrass (Eleusine indica) were identified as host plants of this phytoplasma. Leafhoppers, Macrosteles orientalis, Cicadulina bipunctella, Phlogotettix cyclops and Balclutha sp., were identified as potential insect vectors due to the positive results in PCR detection. The name, “Candidatus Phytoplasma asteris” strain PLY, is proposed for this new phytoplasma. The in silico restriction fragment length polymorphism analysis of the Fn2R2 region sequence showed that the periwinkle leaf yellowing phytoplasma is closest to the 16SrI-B subgroup and may be a new 16SrI subgroup. Transmission experiments with field-collected M. orientalis and C. bipunctella indicated that both leafhoppers can transmit the PLY phytoplasma to healthy periwinkle plants. This work is the first description of a new 16SrI subgroup phytoplasma in Taiwan.

BACKGROUND AND AIMS During sexual reproduction in higher angiosperms, the pollen tubes are directed to the ovules in the pistil to deliver sperm cells. This pollen tube attraction is highly species specific, and a group of small secreted proteins, TfCRPs, are necessary for this process in Torenia fournieri. METHODS A candidate pollen tube attractant protein in Torenia concolor, a related species of T. fournieri, was isolated and the attractant abilities between them were compared. KEY RESULTS TcCRP1, an orthologous gene of TfCRP1 from T. concolor, is expressed predominantly in the synergid cell. The gene product attracted pollen tubes in a concentration-dependent manner, but attracted fewer pollen tubes from the other species. CONCLUSIONS The results indicated that this class of CRP proteins is a common pollen tube attractant in Torenia species. The sequence diversity of these proteins is important for species-specific pollen tube attraction.

While torenia (Torenia fournieri Lind.) is a useful model flower for molecular biological studies of floral architecture, the maintenance of plant materials and resulting transgenic plants requires vegetative propagation due to its heterozygous nature. Reduction of labor and costs for maintaining thousands of in vitro torenia cultures is therefore a critical issue. We found that substituting trehalose for sucrose drastically extended the culture period to 70 days, which is more than twice as long as for the common, sucrose-based medium, without reduction in plant viability. Comparative measurement of the plant mass indicated that the increased survival benefit of the trehalose-based medium might be on account of improvement in the rhizosphere environment through reduction of root density in the culture, rather than by reduced plant growth. No harmful effects arising from the trehalose-based medium were observed in 1,800 laboratory lines during the bimonthly subculture for over 12 months, except for a wilting on the first transfer to the trehalose-based medium. In conjunction with the use of the commercial food additive, Okome-ni-TREHA® rather than reagent-grade trehalose, we have succeeded in reducing the costs and labor associated with the culture medium to less than one third of those for the sucrose-based system.

The mutated carnation ethylene receptor gene Dc-ETR1nr was introduced into Torenia fournieri Lind. This gene contains a missense mutation causing conversion of a Pro36 residue of the carnation ethylene receptor protein Dc-ETR1 to Leu36, as occurs with the tomato mutant gene Never-ripe (Nr). Agrobacterium transformation of Torenia was performed, and four putative transgenic plants with Dc-ETR1nr were obtained. Real-time RT-PCR analysis confirmed Dc-ETR1nr mRNA expression in all transgenic plants. Unlike wild-type plants and 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) transgenic plants, none of the transgenic plants showed flower abscission in response to ethylene treatment. Flower life in all Dc-ETR1nr transgenic plants following wounding or ethylene treatment was longer than that in wild-type plants. Levels of autocatalytic ethylene production in all transgenic plants following wounding or pollination treatment were lower than those in wild-type plants. These results indicate that transgenic plants expressing Dc-ETR1nr have reduced ethylene sensitivity, resulting in inhibition of autocatalytic ethylene production and flower senescence.

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.

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.

Molecular breeding with genetic modification enables the production of novel floral traits in floricultural plants that could not be obtained by traditional breeding. To facilitate novel flower production, we collectively introduced 2 sets of 42 and 50 chimeric repressors of Arabidopsis transcription factors into Agrobacterium and then used these to co-transform torenia (Torenia fournieri). We generated 750 transgenic torenias, and identification of the transgenes revealed that more than 80% of the transgenic torenias had a single transgene. A total of 264 plants showed phenotypic modification, and 91.2% displayed modified flower colors and/or shapes, such as altered color patterns, curled petal margins, and wavy petals. These results indicated that the collective transformation system can be applied to molecular breeding of flowers. Detailed analysis of the phenotypes revealed that PETAL LOSS could control blotch sizes and that modification of cell shape could change the texture of petals. We found that the chimeric repressors of functionally unknown transcription factors also induced novel floral traits, and therefore, the transgenic torenias provide an understanding of the functions of transcription factors that could not be revealed by previous studies in Arabidopsis.

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. Electronic supplementary material The online version of this article (doi:10.1007/s00438-010-0574-z) contains supplementary material, which is available to authorized users.

The process of pollen germination and tube growth in the pistil involves a series of cell-cell interactions, some facilitating fertilization while others prohibiting pollen tube access to the female gametophyte, either because of incompatibility or as a result of mechanisms to avert polyspermy and to ensure reproductive success. Understanding pollen tube growth and guidance to the female gametophyte has long been a pursuit among plant biologists, and observations indicate that diverse strategies may be adopted by different plant species. Recent studies in Arabidopsis, maize, and Torenia fournieri suggest that low molecular weight secretory molecules probably play major roles in the short-range attraction of pollen tubes to the female gametophyte. The process of pollen tube growth in the pistil occurs beneath several cell layers so much of the information that conveys the intimate partnership between penetrating pollen tubes and the female tissues has come from fixed samples and observations of in vitro pollen tube growth responses to female factors. A unique glimpse of the in vivo pollen germination and tube growth process is provided here by intra-vital two-photon excitation (TPE) microscopy of pollinated Arabidopsis pistils that remained on intact plants. Further discoveries of critical factors of male or female origins and how they control the pollen tube growth and fertilization process will broaden our understanding of the common themes and diverse strategies that plants have evolved to ensure reproductive success. The advancement of imaging technology to monitor pollination and fertilization and the development of probes to monitor various aspects of the pollen tube growth process, including pollen intracellular dynamics, will allow us to superimpose details obtained from studying pollen tube growth in culture conditions to interpret and understand the in vivo events.