Flower color modification in Torenia fournieri by genetic engineering of betacyanin pigments.

BACKGROUND: Betalains are reddish and yellow pigments that accumulate in a few plant species of the order Caryophyllales. These pigments have antioxidant and medicinal properties and can be used as functional foods. They also enhance resistance to stress or disease in crops. Several plant species belonging to other orders have been genetically engineered to express betalain pigments. Betalains can also be used for flower color modification in ornamental plants, as they confer vivid colors, like red and yellow. To date, betalain engineering to modify the color of Torenia fournieri-or wishbone flower-a popular ornamental plant, has not been attempted. RESULTS: We report the production of purple-reddish-flowered torenia plants from the purple torenia cultivar "Crown Violet."  Three betalain-biosynthetic genes encoding CYP76AD1, dihydroxyphenylalanine (DOPA) 4,5-dioxygenase (DOD), and cyclo-DOPA 5-O-glucosyltransferase (5GT) were constitutively ectopically expressed under the cauliflower mosaic virus (CaMV) 35S promoter, and their expression was confirmed by quantitative real-time PCR (qRT-PCR) analysis. The color traits, measured by spectrophotometric colorimeter and spectral absorbance of fresh petal extracts, revealed a successful flower color modification from purple to reddish. Red pigmentation was also observed in whole plants. LC-DAD-MS and HPLC analyses confirmed that the additional accumulated pigments were betacyanins-mainly betanin (betanidin 5-O-glucoside) and, to a lesser extent, isobetanin (isobetanidin 5-O-glucoside). The five endogenous anthocyanins in torenia flower petals were also detected. CONCLUSIONS: This study demonstrates the possibility of foreign betacyanin accumulation in addition to native pigments in torenia, a popular garden bedding plant. To our knowledge, this is the first report presenting engineered expression of betalain pigments in the family Linderniaceae. Genetic engineering of betalains would be valuable in increasing the flower color variation in future breeding programs for torenia.

Torenia sp. Extracts Contain Multiple Potent Antitumor Compounds with Nematocidal Activity, Triggering an Activated DNA Damage Checkpoint and Defective Meiotic Progression.

Previously, we analyzed 316 herbal extracts to evaluate their potential nematocidal properties in Caenorhabditis elegans. In this study, our attention was directed towards Torenia sp., resulting in reduced survival and heightened larval arrest/lethality, alongside a noticeable decrease in DAPI-stained bivalent structures and disrupted meiotic progression, thus disrupting developmental processes. Notably, Torenia sp. extracts activated a DNA damage checkpoint response via the ATM/ATR and CHK-1 pathways, hindering germline development. LC-MS analysis revealed 13 compounds in the Torenia sp. extracts, including flavonoids, terpenoids, tanshinones, an analog of resveratrol, iridoids, carotenoids, fatty acids, and alkaloids. Of these, 10 are known for their antitumor activity, suggesting the potential of Torenia species beyond traditional gardening, extending into pharmaceutical and therapeutic applications.

Comparative analyses of Linderniaceae plastomes, with implications for its phylogeny and evolution.

Introduction: The recently established Linderniaceae, separated from the traditionally defined Scrophulariaceae, is a taxonomically complicated family. Although previous phylogenetic studies based on a few short DNA markers have made great contributions to the taxonomy of Linderniaceae, limited sampling and low resolution of the phylogenetic tree have failed to resolve controversies between some generic circumscriptions. The plastid genome exhibits a powerful ability to solve phylogenetic relationships ranging from shallow to deep taxonomic levels. To date, no plastid phylogenomic studies have been carried out in Linderniaceae. Methods: In this study, we newly sequenced 26 plastid genomes of Linderniaceae, including eight genera and 25 species, to explore the phylogenetic relationships and genome evolution of the family through plastid phylogenomic and comparative genomic analyses. Results: The plastid genome size of Linderniaceae ranged from 152,386 bp to 154,402 bp, exhibiting a typical quartile structure. All plastomes encoded 114 unique genes, comprising 80 protein-coding genes, 30 tRNA genes, and four rRNA genes. The inverted repeat regions were more conserved compared with the single-copy regions. A total of 1803 microsatellites and 1909 long sequence repeats were identified, and five hypervariable regions (petN-psbM, rps16-trnQ, rpl32-trnL, rpl32, and ycf1) were screened out. Most protein-coding genes were relatively conserved, with only the ycf2 gene found under positive selection in a few species. Phylogenomic analyses confirmed that Linderniaceae was a distinctive lineage and revealed that the presently circumscribed Vandellia and Torenia were non-monophyletic. Discussion: Comparative analyses showed the Linderniaceae plastomes were highly conservative in terms of structure, gene order, and gene content. Combining morphological and molecular evidence, we supported the newly established Yamazakia separating from Vandellia and the monotypic Picria as a separate genus. These findings provide further evidence to recognize the phylogenetic relationships among Linderniaceae and new insights into the evolution of the plastid genomes.

Biopolymer Based Multifunctional Films Loaded with Anthocyanin Rich Floral Extract and ZnO Nano Particles for Smart Packaging and Wound Healing Applications.

There are significant societal repercussions from our excessive use of plastic products derived from petroleum. In response to the increasing environmental implications of plastic wastes, biodegradable materials have been proven to be an effective means of mitigating environmental issues. Therefore, protein- and polysaccharide-based polymers have gained widespread attention recently. In our study, for increasing the strength of a biopolymer (Starch), we used ZnO dispersed nanoparticles (NPs), which resulted in the enhancement of other functional properties of the polymer. The synthesized NPs were characterized using SEM, XRD, and Zeta potential values. The preparation techniques are completely green, with no hazardous chemicals employed. The floral extract employed in this study is Torenia fournieri (TFE), which is prepared using a mixture of ethanol and water and possesses diverse bioactive features and pH-sensitive characteristics. The prepared films were characterized using SEM, XRD, FTIR, contact angle and TGA. The incorporation of TFE and ZnO (SEZ) NPs was found to increase the overall nature of the control film. The results obtained from this study confirmed that the developed material is suitable for wound healing and can also be used as a smart packaging material.

Enhanced Resistance to Fungal and Bacterial Diseases Due to Overexpression of BSR1, a Rice RLCK, in Sugarcane, Tomato, and Torenia.

Sugarcane smut caused by Sporisorium scitamineum is one of the most devastating sugarcane diseases. Furthermore, Rhizoctonia solani causes severe diseases in various crops including rice, tomato, potato, sugar beet, tobacco, and torenia. However, effective disease-resistant genes against these pathogens have not been identified in target crops. Therefore, the transgenic approach can be used since conventional cross-breeding is not applicable. Herein, the overexpression of BROAD-SPECTRUM RESISTANCE 1 (BSR1), a rice receptor-like cytoplasmic kinase, was conducted in sugarcane, tomato and torenia. BSR1-overexpressing tomatoes exhibited resistance to the bacteria Pseudomonas syringae pv. tomato DC3000 and the fungus R. solani, whereas BSR1-overexpressing torenia showed resistance to R. solani in the growth room. Additionally, BSR1 overexpression conferred resistance to sugarcane smut in the greenhouse. These three BSR1-overexpressing crops exhibited normal growth and morphologies except in the case of exceedingly high levels of overexpression. These results indicate that BSR1 overexpression is a simple and effective tool for conferring broad-spectrum disease resistance to many crops.

Whole-mount RNA in situ hybridization technique in Torenia ovules.

The expression pattern of an interested gene at a cellular level provides strong evidence for its functions. RNA in situ hybridization has been proved to be a powerful tool in detecting the spatial-temporal expression pattern of a gene in various organisms. However, classical RNA in situ hybridization (ISH) technique is time-consuming and requires sophisticated sectioning skills. Therefore, we developed a method for whole-mount in situ hybridization (WISH) on ovules of Torenia fournieri, which is a model species in the study of plant reproduction. T. fournieri possesses ovules with protruding embryo sacs, making it easy to be observed and imaged through simple manipulation. To determine the effect of classical ISH and our newly established WISH, we detected the expression of a D-class gene, TfSTK3, using both methods. The expression patterns of TfSTK3 are similar in classical ISH and WISH, confirming reliability of the WISH method. Compared with WISH, classical ISH always leads to distorted embryo sacs, hence difficult to distinguish signals within the female gametophyte. To understand whether our WISH protocol also works well in detecting genes expressed within embryo sacs, we further examined the expression of a synergid-enriched candidate, TfPMEI1, and clearly observed specific signals within two synergid cells. To summarize, our WISH technique allows to visualize gene expression patterns in ovules of T. fournieri within one week and will benefit the field of plant reproduction in the future.

Molecular Mechanisms of Plant Regeneration from Differentiated Cells: Approaches from Historical Tissue Culture Systems.

Plants can exert remarkable capacity for cell reprogramming even from differentiated cells. This ability allows plants to regenerate tissues/organs and even individuals in nature and in vitro. In recent decades, Arabidopsis research has uncovered molecular mechanisms of plant regeneration; however, our understanding of how plant cells retain both differentiated status and developmental plasticity is still obscure. In this review, we first provide a brief outlook of the representative modes of plant regeneration and key factors revealed by Arabidopsis research. We then re-examine historical tissue culture systems that enable us to investigate the molecular details of cell reprogramming in differentiated cells and discuss the different approaches, specifically highlighting our recent progress in shoot regeneration from the epidermal cell of Torenia fournieri.

The complete chloroplast genome of Torenia violacea (Azaola) Pennell 1943 (Linderniaceae).

Torenia violacea (Azaola) Pennell 1943 as a traditional Chinese medicine plant, has been used to treat multiple diseases. In this study, we sequenced, assembled, and characterized the complete plastome of T. violacea. The plastome (OQ167784) exhibited a typical quadripartite structure, with a total length of 154,007 bp, comprising a pair of inverted repeats (IRs; 24,809 bp) separated by a large single-copy (LSC) region (85,559 bp) and a small single-copy (SSC) region (18,830 bp). Through genome annotation, we identified 133 genes, including 87 protein-coding genes, eight rRNA genes, and 38 tRNA genes. Phylogenetic analysis revealed a close relationship between T. violacea and T. fournieri. The research results provide basic genetic resources for the development of species identification and investigation of phylogenetic relationships in the Torenia genus.

Optimization of an Efficient Protoplast Transformation System for Transient Expression Analysis Using Leaves of Torenia fournieri.

Torenia fournieri (T. fournieri) is one of the most widely used horticultural flowers and is considered a potential model plant for the genetic investigation of ornamental traits. In this study, we optimized an efficient protocol for high efficiency preparation and transformation of T. fournieri protoplast. The transformation rate reached ~75% when a 35S:GFP construct was used for the transformation. Using this system, we characterized the subcellular localization of several TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) transcription factors (TFs), and found a distinct localization pattern between the CIN and CYC classes of TCP TFs. Furthermore, we also demonstrated the feasibility of the expression of dual luciferase assay system in T. fournieri protoplasts for the measurement of the activity of cis-regulatory elements. Taken together, a well-optimized transient expression system in T. fournieri protoplasts would be crucial for rapid exploration of the gene function or cis-regulatory elements.

Overexpression of Rice BSR2 Confers Disease Resistance and Induces Enlarged Flowers in Torenia fournieri Lind.

Plant pathogens evade basal defense systems and attack different organs and tissues of plants. Genetic engineering of plants with genes that confer resistance against pathogens is very effective in pathogen control. Conventional breeding for disease resistance in ornamental crops is difficult and lagging relative to that in non-ornamental crops due to an inadequate number of disease-resistant genes. Therefore, genetic engineering of these plants with defense-conferring genes is a practical approach. We used rice BSR2 encoding CYP78A15 for developing transgenic Torenia fournieri Lind. lines. The overexpression of BSR2 conferred resistance against two devastating fungal pathogens, Rhizoctonia solani and Botrytis cinerea. In addition, BSR2 overexpression resulted in enlarged flowers with enlarged floral organs. Histological observation of the petal cells suggested that the enlargement in the floral organs could be due to the elongation and expansion of the cells. Therefore, the overexpression of BSR2 confers broad-spectrum disease resistance and induces the production of enlarged flowers simultaneously. Therefore, this could be an effective strategy for developing ornamental crops that are disease-resistant and economically more valuable.

Ectopic expression of AtNF-YA6-VP16 in petals results in a novel petal phenotype in Torenia fournieri.

MAIN CONCLUSION: A novel Torenia phenotype having separate petals was obtained by the combination of NF-YA6-VP16 with a floral organ-specific promoter. Genetic engineering techniques helped in obtaining novel flower colors and shapes, in particular, by introducing functionally modified transcription factors (TFs) to ornamental flower species. Herein, we used functionally modified Arabidopsis TFs fused with the repression domain SRDX and the activation domain VP16 to screen for novel floral traits in Torenia fournieri Lind (torenia). We avoided undesired phenotypes unrelated to flowers by expressing these TFs through a floral organ-specific promoter belonging to the class-B genes, GLOBOSA (TfGLO). Fourteen constructs were produced to express functionally modified Arabidopsis TFs in which each of SRDX and VP16 was fused into 7 TFs that were used for the collective transformation of Torenia plants. Among the obtained transgenic plants, phenotypes with novel floral traits reflected in separate petals within normally gamopetalous flower lines. Sequencing analysis revealed that the transgenic plants contained nuclear factor-YA6 (NF-YA6) fused with the VP16. In the margin between the lips of the petals and tube in the TfGLOp:NF-YA6-VP16 plants, staminoid organs have been developed to separate petals. In the petals of the TfGLOp:NF-YA6-VP16 plants, the expression of a Torenia class C gene, PLENA (TfPLE), was found to be ectopically increased. Moreover, expression of TfPLE-VP16 under the control of the TfGLO promoter brought a similar staminoid phenotype observed in the TfGLOp:NF-YA6-VP16 plants. These results suggest that the introduction of the TfGLOp:NF-YA6-VP16 induced TfPLE expression, resulting in the formation of staminoid petals and separation of them.

Transcriptome Dynamics of Epidermal Reprogramming during Direct Shoot Regeneration in Torenia fournieri.

Shoot regeneration involves reprogramming of somatic cells and de novo organization of shoot apical meristems (SAMs). In the best-studied model system of shoot regeneration using Arabidopsis, regeneration is mediated by the auxin-responsive pluripotent callus formation from pericycle or pericycle-like tissues according to the lateral root development pathway. In contrast, shoot regeneration can be induced directly from fully differentiated epidermal cells of stem explants of Torenia fournieri (Torenia), without intervening the callus mass formation in culture with cytokinin; yet, its molecular mechanisms remain unaddressed. Here, we characterized this direct shoot regeneration by cytological observation and transcriptome analyses. The results showed that the gene expression profile rapidly changes upon culture to acquire a mixed signature of multiple organs/tissues, possibly associated with epidermal reprogramming. Comparison of transcriptomes between three different callus-inducing cultures (callus induction by auxin, callus induction by wounding and protoplast culture) of Arabidopsis and the Torenia stem culture identified genes upregulated in all the four culture systems as candidates of common factors of cell reprogramming. These initial changes proceeded independently of cytokinin, followed by cytokinin-dependent, transcriptional activations of nucleolar development and cell cycle. Later, SAM regulatory genes became highly expressed, leading to SAM organization in the foci of proliferating cells in the epidermal layer. Our findings revealed three distinct phases with different transcriptomic and regulatory features during direct shoot regeneration from the epidermis in Torenia, which provides a basis for further investigation of shoot regeneration in this unique culture system.

Complete chloroplast genome sequence and phylogenetic analysis of Torenia fournieri.

Torenia fournieri belongs to the genus Torenia in the family Linderniaceae. The complete chloroplast genome of T. fournieri was sequenced and analyzed by Illumina sequencing in this study. The full length of the complete chloroplast genome is 153,938 bp, containing a pair of inverted repeat regions of 24,805 bp (IRa and IRb) separated by a large single copy region (LSC) of 85,498 bp and a small single copy region (SSC) of 18,830 bp. The T. fournieri chloroplast genome encodes 131 genes, comprising 87 protein-coding genes, 36 tRNA genes, 8 rRNA genes, without pseudogene. Phylogenetic analysis showed that T. fournieri was closely related to T. benthamiana and T. concolor within the genus Torenia in family Linderniaceae.

Ectopic expression of Torenia fournieri TCP8 and TCP13 alters the leaf and petal phenotypes in Arabidopsis thaliana.

Teosinte branched1/cycloidea/proliferating cell factor (TCP) transcription factors (TFs) are essential for regulating plant developmental processes, which is still largely unknown in Torenia fournieri (T. fournieri), a widely used horticultural flower. In this study, we used a de novo transcriptome assembly method to predict the TCP transcription factors in T. fournieri. In total, 15 out of 21 predicted T. fournieri TCPs (TfTCPs) were isolated and verified with Sanger sequencing. Phylogenetic analysis showed that these 15 TfTCPs could be classified into two major classes. Most of these TfTCPs were expressed in floral buds, flowers, or leaves, suggesting an important role in developmental regulation in these tissues. Moreover, TfTCP8 and TfTCP13, the homologues of the Arabidopsis thaliana TCP5-like transcription factor, were able to bind to the conserved Class II TCP binding motifs and are localized to the nucleus, indicating that TfTCP8 and TfTCP13 act as transcriptional regulators. In agreement with the overexpression phenotype of AtTCP5, ectopic expression of TfTCP8 and TfTCP13 resulted in narrow leaves and the small petal phenotype in Arabidopsis, suggesting that these two TfTCPs potentially regulate leaf or flower shape in T. fournieri.

Production of multi-petaled Torenia fournieri flowers by functional disruption of two class-C MADS-box genes.

MAIN CONCLUSION: Simultaneous knockdown or knockout of Torenia fournieri PLENA (TfPLE) and FALINELLI (TfFAR) genes with RNAi or genome-editing technologies generated a multi-petal phenotype in torenia. The MADS-box gene AGAMOUS (AG) is well known to play important roles in the development of stamens and carpels in Arabidopsis. Mutations in AG cause the morphological transformation of stamens and carpels into petaloid organs. In contrast, torenia (Torenia fournieri Lind.) has two types of class-C MADS-box genes, PLENA (PLE) and FALINELLI (FAR); however, their functions were previously undetermined. To examine the function of TfPLE and TfFAR in torenia, we used RNAi to knockdown expression of these two genes. TfPLE and TfFAR double-knockdown transgenic torenia plants had morphologically altered stamens and carpels that developed into petaloid organs. TfPLE knockdown transgenic plants also exhibited morphological transformations that included shortened styles, enlarged ovaries, and absent stigmata. Furthermore, simultaneous disruption of TfPLE and TfFAR genes by CRISPR/Cas9-mediated genome editing also resulted in the conversion of stamens and carpels into petaloid organs as was observed in the double-knockdown transgenic plants mediated by RNAi. In addition, the carpels of one TfPLE knockout mutant had the same morphological abnormalities as TfPLE knockdown transgenic plants. TfFAR knockdown genome-edited mutants had no morphological changes in their floral organs. These results clearly show that TfPLE and TfFAR cooperatively play important roles in the development of stamens and carpels. Simultaneous disruption of TfPLE and TfFAR functions caused a multi-petal phenotype, which is expected to be a highly valuable commercial floral trait in horticultural flowers.

Endothelial Nitric Oxide Mediates the Anti-Atherosclerotic Action of Torenia concolor Lindley var. Formosama Yamazaki.

Torenia concolor Lindley var. formosama Yamazaki ethanolic extract (TCEE) is reported to have anti-inflammatory and anti-obesity properties. However, the effects of TCEE and its underlying mechanisms in the activation of endothelial nitric oxide synthase (eNOS) have not yet been investigated. Increasing the endothelium-derived nitric oxide (NO) production has been known to be beneficial against the development of cardiovascular diseases. In this study, we investigated the effect of TCEE on eNOS activation and NO-related endothelial function and inflammation by using an in vitro system. In endothelial cells (ECs), TCEE increased NO production in a concentration-dependent manner without affecting the expression of eNOS. In addition, TCEE increased the phosphorylation of eNOS at serine 635 residue (Ser635) and Ser1179, Akt at Ser473, calmodulin kinase II (CaMKII) at threonine residue 286 (Thr286), and AMP-activated protein kinase (AMPK) at Thr172. Moreover, TCEE-induced NO production, and EC proliferation, migration, and tube formation were diminished by pretreatment with LY294002 (an Akt inhibitor), KN62 (a CaMKII inhibitor), and compound C (an AMPK inhibitor). Additionally, TCEE attenuated the tumor necrosis factor-α-induced inflammatory response as evidenced by the expression of adhesion molecules in ECs and monocyte adhesion onto ECs. These inflammatory effects of TCEE were abolished by L-NG-nitroarginine methyl ester (an NOS inhibitor). Moreover, chronic treatment with TCEE attenuated hyperlipidemia, systemic and aortic inflammatory response, and the atherosclerotic lesions in apolipoprotein E-deficient mice. Collectively, our findings suggest that TCEE may confer protection from atherosclerosis by preventing endothelial dysfunction.

A homolog of the ALOG family controls corolla tube differentiation in Torenia fournieri.

Flowers of honey plants (Torenia) face various abiotic stressors, including rain, that can damage pollens and dilute nectar. Many Torenia species are thought to have evolved a modified corolla base termed the corolla neck to prevent raindrops from contacting the nectar. Although this hypothesis was postulated long ago, direct validation is lacking. Here, we have evaluated Torenia fournieri, the corolla tube of which differentiates into distinct regions: a conical tube above that connects to an inflated base through a constriction. This constriction and inflated base are collectively referred to as the corolla neck. Using transcriptomic sequencing and genome-editing approaches, we have characterized an ALOG gene, TfALOG3, that is involved in formation of the corolla neck. TfALOG3 was found expressed in the epidermis of the corolla neck. Cells in the corolla bottom differentiated and expanded in wild-type T. fournieri, whereas such cells in TfALOG3 loss-of-function mutants failed to develop into a corolla neck. Water easily contacted the nectary in the absence of the corolla neck. Taken together, our study unveils a novel gene that controls corolla tube differentiation and demonstrates a hypothetical property of the corolla neck.

Complete plastome sequence of Torenia benthamiana Hance (Linderniaceae), an endemic herb in South China.

Torenia benthamiana Hance is an endemic plant species distributed in southern China. In this study, we report and characterize the complete plastid genome sequence of T. benthamiana in order to provide genomic resources helpful for promoting its conservation and garden utilization. The complete plastome is 153,526 bp in length and contains the typical quadripartite structure of angiosperm plastome, including two inverted repeat (IR) regions of 24,638 bp, a large single-copy (LSC) region of 85,417 bp, and a small single-copy (SSC) region of 18,833 bp. The plastome contains 114 genes, consisting of 80 unique protein-coding genes, 30 unique tRNA gene, and 4 unique rRNA genes. The overall A/T content in the plastome of T. benthamiana is 62.40%. The complete plastome sequence of T. benthamiana will provide a useful resource for the conservation and garden utilization of this species as well as for the phylogenetic studies of Linderniaceae.

Complete plastome sequence of Torenia concolor Lindley (Linderniaceae): an ornamental herb.

In this study we firstly reported the complete chloroplast genome of Torenia concolor, a species of Linderniaceae. The complete chloroplast genome of T. concolor is 153,853 bp in length with a typical quadripartite structure, consisting of a large single-copy region (LSC, 85,446 bp), a single-copy region (SSC, 18,837 bp), and a pair of inverted repeats (IRs, 24,785 bp). There are 114 genes annotated, including 80 unique protein-coding genes, 4 unique ribosomal RNA genes, and 30 transfer RNA genes. To investigate the evolution status of T. concolor, as well as Linderniaceae, we constructed a phylogenetic tree with T. concolor and other 16 species based on their complete chloroplast genomes. According to the phylogenetic topologies, T. concolor was closely related to Pedicularis hallaisanensis.