Isolation and characterisation of sesquiterpene synthase from aromatic orchid Phalaenopsis bellina (Rchb.f.) Christenson.

BACKGROUND: Phalaenopsis bellina, an orchid native to Borneo, is renowned for its unique appearance. It releases distinct fragrances, which have been linked to the presence of terpenoids. However, the identification and study of sesquiterpene synthase in P. bellina remain limited. In this study, we examines the functional characterisation of terpene synthase (TPS) from P. bellina, known as PbTS, through recombinant protein expression and its manifestation in the flower. METHODS AND RESULTS: Gene annotation of PbTS revealed that the inferred peptide sequence of PbTS comprises 1,680 bp nucleotides encoding 559 amino acids with an estimated molecular mass of 65.2 kDa and a pI value of 5.4. A similarity search against GenBank showed that PbTS shares similarities with the previously published partial sequence of P. bellina (ABW98504.1) and Phalaenopsis equestris (XP_020597359.1 and ABW98503.1). Intriguingly, the phylogenetic analysis places the PbTS gene within the TPS-a group. In silico analysis of PbTS demonstrated stable interactions with farnesyl pyrophosphate (FPP), geranyl pyrophosphate (GPP), and geranylgeranyl pyrophosphate (GGPP). To verify this activity, an in vitro enzyme assay was performed on the PbTS recombinant protein, which successfully converted FPP, GPP, and GGPP into acyclic sesquiterpene ß-farnesene, yielding approximately 0.03 mg/L. Expressional analysis revealed that the PbTS transcript was highly expressed in P. bellina, but its level did not correlate with ß-farnesene levels across various flowering time points and stages. CONCLUSION: The insights gained from this study will enhance the understanding of terpenoid production in P. bellina and aid in the discovery of novel fragrance-related genes in other orchid species.

Genome-wide identification, expression pattern and interacting protein analysis of INDETERMINATE DOMAIN (IDD) gene family in Phalaenopsis equestris.

The plant-specific INDETERMINATE DOMAIN (IDD) gene family is important for plant growth and development. However, a comprehensive analysis of the IDD family in orchids is limited. Based on the genome data of Phalaenopsis equestris, the IDD gene family was identified and analyzed by bioinformatics methods in this study. Ten putative P. equestris IDD genes (PeIDDs) were characterized and phylogenetically classified into two groups according to their full amino acid sequences. Protein motifs analysis revealed that overall structures of PeIDDs in the same group were relatively conserved. Its promoter regions harbored a large number of responsive elements, including light responsive, abiotic stress responsive elements, and plant hormone cis-acting elements. The transcript level of PeIDD genes under cold and drought conditions, and by exogenous auxin (NAA) and abscisic acid (ABA) treatments further confirmed that most PeIDDs responded to various conditions and might play essential roles under abiotic stresses and hormone responses. In addition, distinct expression profiles in different tissues/organs suggested that PeIDDs might be involved in various development processes. Furthermore, the prediction of protein-protein interactions (PPIs) revealed some PeIDDs (PeIDD3 or PeIDD5) might function via cooperating with chromatin remodeling factors. The results of this study provided a reference for further understanding the function of PeIDDs.

Genetic profiles and phenotypic patterns in Taiwanese Phalaenopsis orchids: a two-step phenotype and genotype strategy using modified genetic distance algorithms.

This study establishes the first core collection (CC) for Taiwanese Phalaenopsis orchids to preserve genetic diversity and key traits essential for breeding and research, thereby enhancing breeding efficiency without the need for a large maintained parent population. We examined 207 commercial orchid cultivars from ten nurseries, characterized by two phenotypes and genotypic data from eight simple sequence repeat markers. Multiple imputation was applied to estimate missing phenotypes, minimizing potential uncertainties and ensuring the reliability of population structure analysis. Weighted k-means clustering identified seven distinct clusters, highlighting substantial genetic diversity. We proposed a two-step phenotype and genotype strategy and modified genetic distance algorithms to effectively preserve both phenotypic and genetic diversity while retaining key features. Consequently, 22 core accessions were selected, distributed across seven clusters, and representing the orchid germplasm collection. Our evaluation revealed significant diversity preservation, particularly in distinct characteristics and rare features, outperforming other methodologies. Pedigree background analysis further confirmed the representativeness of the CC in maintaining diverse genetic materials. We emphasized the importance of evaluating the CC by detailing the criteria and statistical analyses used to ensure the quality, representativeness, and effectiveness of the selected accessions. This study contributes to orchid breeding, conservation efforts, and sustainable agricultural practices by providing a valuable and comprehensive resource. In conclusion, our research establishes a groundbreaking CC, offering insights into the genetic landscape of Taiwanese Phalaenopsis orchids and highlighting potential advancements in breeding commercially desirable varieties.

Effects of Paclobutrazol on Reproductive and Vegetative Traits of Phalaenopsis Join Grace 'TH288-4'.

Phalaenopsis is the most popular potted plant worldwide. However, its typically long stalks often lead to increased shipping costs and risks. This study investigates the effectiveness of varying the concentration, timing, and frequency of paclobutrazol (PP333) applications on shortening the stalk of Phalaenopsis Join Grace 'TH288-4'. Concurrently, it also examines the potential for producing visually appealing and single-flower potted phalaenopsis products by means of truncation. Mature phalaenopsis plants were moved to a cool room in the seventh week to induce flowering. Four experimental groups were established based on different PP333 application schedules: the control (CK) group, with reverse osmosis water application in the second week; the T2 group, with a single application in the second week; the T2T3 group, with applications in both the second and third weeks; and the T7T8 group, with applications in the seventh and eighth weeks. The PP333 concentrations used were 250, 500, 750, and 1000 mg·L-1, applied as foliar sprays. The results showed that the shortest stalks, measured from the base to the first flower, were observed in the T2 group treated with PP333 at 750 mg·L-1 and in the T2T3 group with PP333 at 500, 750, and 1000 mg·L-1. These treatments resulted in stalk lengths of 19.18-22.17 cm, which are 67.2-71.6% shorter than the controls. PP333 application had minimal effect on the stalk diameter, pedicel length, flower width, length, and length/width ratio. However, root diameter was thicker in plants treated with PP333 compared with the control plants. For producing single-flower phalaenopsis, a foliar spray of 750 mg·L-1 PP333 is recommended approximately a month before moving the plants to cooler conditions, followed by truncation, retaining only the first flower. As a result, this study establishes a PP333 treatment protocol for phalaenopsis, offering a strategy to effectively shorten the stalks.

Perturbation of periodic spot-generation balance leads to diversified pigmentation patterning of harlequin Phalaenopsis orchids: in silico prediction.

BACKGROUND: A retrotransposon HORT1 in the promoter of the anthocyanin activator gene PeMYB11, microRNA858 (miR858) that targets PeMYB11, and a repressor PeMYBx have been implicated in pigmentation patterning diversity of harlequin Phalaenopsis orchids. However, the interrelationship among them remains to be elucidated. RESULTS: To understand how these factors interact to generate anthocyanin spots in Phalaenopsis, we successfully developed a mathematical model based on the known reaction-diffusion system to simulate their interplay and refined the conceptual biological model. Intriguingly, the expression of both PeMYBx and PeMYB11 were in phase for purple spot formation, even though they showed adverse effects on anthocyanin accumulations. An increase in the self-activation rate of PeMYB11 resulted in the increased size of purple spots, but no effects on spot fusion. Decreased degradation rate of miR858 in the purple regions, led to disruption of the formation of spotted pigmentation patterning and a full-red pigmentation pattern. Significantly, the reduced miR858 level promotes the fusion of large dark purple dots induced by the solo-LTR of HORT1, eventually generating the purple patches. In addition, the spatially heterogeneous insertion of HORT1 caused by the remnant solo-LTR of HORT1 derived from random homologous unequal recombination of HORT1 in individual cells of floral organs could explain the diverse pigmentation patterning of harlequin Phalaenopsis. CONCLUSIONS: This devised model explains how HORT1 and miR858 regulate the formation of the pigmentation patterning and holds great promise for developing efficient and innovative approaches to breeding harlequin Phalaenopsis orchids.

The HD-ZIP II gene PaHAT14 increases cuticle deposition by down-regulating ERF gene PaERF105 in Phalaenopsis.

To analyze the gene involved in orchid floral development, a HD-Zip II gene PaHAT14, which specifically and highly expressed in perianth during early flower development was identified from Phalaenopsis. Transgenic Arabidopsis plants expressing 35S::PaHAT14 and 35S::PaHAT14+SRDX (fused with the repressor motif SRDX) exhibited similar altered phenotypes, including small leaves, early flowering, and bending petals with increased cuticle production. This suggests that PaHAT14 acts as a repressor. In contrast, transgenic Arabidopsis plants expressing 35S::PaHAT14+VP16 (fused with the activation domain VP16) exhibited curled leaves, late flowering, and folded petals with decreased cuticle production within hardly opened flowers. Additionally, the expression of the ERF gene DEWAX2, which negatively regulates cuticular wax biosynthesis, was down-regulated in 35S::PaHAT14 and 35S::PaHAT14+SRDX transgenic Arabidopsis, while it was up-regulated in 35S::PaHAT14+VP16 transgenic Arabidopsis. Furthermore, transient overexpression of PaHAT14 in Phalaenopsis petal/sepal increased cuticle deposition due to the down-regulation of PaERF105, a Phalaenopsis DEWAX2 orthologue. On the other hand, transient overexpression of PaERF105 decreased cuticle deposition, whereas cuticle deposition increased and the rate of epidermal water loss was reduced in PaERF105 VIGS Phalaenopsis flowers. Moreover, ectopic expression of PaERF105 not only produced phenotypes similar to those in 35S::PaHAT14+VP16 Arabidopsis but also compensated for the altered phenotypes observed in 35S::PaHAT14 and 35S::PaHAT14+SRDX Arabidopsis. These results suggest that PaHAT14 promotes cuticle deposition by negatively regulating downstream gene PaERF105 in orchid flowers.

Identification of floral aroma components and molecular regulation mechanism of floral aroma formation in Phalaenopsis.

BACKGROUND: Most Phalaenopsis cultivars have almost no aroma, with a few exceptions. Phalaenopsis presents significant challenges in fragrance breeding due to its weak aroma and low fertility. It is therefore necessary to identify the aroma components and key regulatory genes in Phalaenopsis cultivars like 'Orange Beauty', 'Brother Sara Gold', 'Purple Martin', 'H026', 'SK16', 'SX098', and 'SH51', to improve the aroma of the common Phalaenopsis. RESULTS: Floral aroma components were tested on nine Phalaenopsis species, using smell identification and headspace gas chromatography-mass spectrometry. The result showed that alcohols, esters, and alkenes were the key specific components in the different species and cultivar aromas and the aroma intensity and component content of cultivars with different colors were different. The main components of the floral aromas in Phalaenopsis were alcohols (including eucalyptol, linalool, citronellol, and 1-hexanol), esters (including hexyl acetate, leaf acetate, and dibutyl phthalate), alkenes (including pinene and sabinene) and arenes (like fluorene). The transcriptome of flowers in the bud stage and bloom stage of P. 'SH51' was sequenced and 5999 differentially expressed genes were obtained. The contributions of the phenylpropionic acid/phenyl ring compound and the terpene compound to the aroma were greater. Sixteen genes related to phalaenopsis aroma were found. TC4M, PAL, CAD6, and HR were related to phenylpropanoid synthesis pathway. SLS, TS10, and P450 were related to the synthesis pathway of terpenes. TS10 and YUCCA 10 were involved in tryptophan metabolism. CONCLUSION: This is the first report on the floral aroma components and regulatory genes in Phalaenopsis. The proposed method and research data can provide technical support for Phalaenopsis breeding. © 2024 Society of Chemical Industry.

PaWOX3 and PaWOX3B Regulate Flower Number and the Lip Symmetry of Phalaenopsis.

The standout characteristic of the orchid perianth is the transformation of the upper median petal into a distinctively formed lip, which gives orchid flowers their typically zygomorphic symmetry and makes them the most popular ornamental plants worldwide. To study orchid flower development, two WUSCHEL-related homeobox (WOX) genes, PaWOX3 and PaWOX3B, were identified in Phalaenopsis. PaWOX3 and PaWOX3B mRNAs accumulate abundantly during early reproductive development and perianths of young buds, significantly decreasing in mature flowers and absent in vegetative leaves and roots. PaWOX3 and PaWOX3B virus-induced gene silencing (VIGS) knockdown in Phalaenopsis significantly reduces floral bud numbers, suggesting that PaWOX3/PaWOX3B may be involved in flower initiation. Transgenic Arabidopsis ectopically expressing repressor forms of PaWOX3/PaWOX3B and their Oncidium ortholog, OnPRS, exhibit lateral organ development defects, implicating these genes likely have function in regulating growth and differentiation for lateral organs. Neither PaWOX3, PaWOX3B single nor PaWOX3/PaWOX3B double VIGS Phalaenopsis altered the flower morphology. Interestingly, double silencing of PaWOX3 or PaWOX3B with OAGL6-2, which controlled the identity/formation of lips, altered the symmetry of 'BigLip' produced in OAGL6-2 VIGS. This result indicated that the levels of PaWOX3/PaWOX3B are still sufficient to maintain the symmetry for the OAGL6-2 VIGS 'BigLip'. However, the symmetry of the OAGL6-2 VIGS 'BigLip' cannot be maintained once the expression of PaWOX3 or PaWOX3B is further reduced. Thus, in addition to controlling lip identity, this study further found that OAGL6-2 could cooperate with functionally redundant PaWOX3/PaWOX3B in maintaining the symmetric axis of lip.

The Effect of Temperature on the Inflorescence Formation Model for Phalaenopsis.

Phalaenopsis orchids are a popular ornamental plant in the flower market. During some festivals, demand increases significantly. These mature orchids must be placed in cooling rooms for inflorescence formation at specific times to increase the financial return from their sale. The purpose of this study is to evaluate the effect of day and night temperatures on the inflorescence formation percentage using the proposed sigmoid model. Four varieties that are cultured in different vegetative temperature regimes are placed in a cooling room. An empirical inflorescence formation model is proposed as a management tool to predict the inflorescence formation percentage for Phalaenopsis. Some data sets from previous studies are used for comparison. The accumulation temperature is calculated using the day and night temperatures and is an index to predict the inflorescence formation percentage. The results show that there is a similar distribution of the inflorescence formation percentage and accumulation temperature for the four varieties. The proposed sigmoid model has a good fitting ability for the inflorescence formation percentage. This inflorescence formation model from the pooled data sets allows quantitative microclimate management of the vegetative and cooling room.

Genome-Wide Identification and Expression Pattern Analysis of TIFY Family Genes Reveal Their Potential Roles in Phalaenopsis aphrodite Flower Opening.

The TIFY gene family (formerly known as the zinc finger proteins expressed in inflorescence meristem (ZIM) family) not only functions in plant defense responses but also are widely involved in regulating plant growth and development. However, the identification and functional analysis of TIFY proteins remain unexplored in Orchidaceae. Here, we identified 19 putative TIFY genes in the Phalaenopsis aphrodite genome. The phylogenetic tree classified them into four subfamilies: 14 members from JAZ, 3 members from ZML, and 1 each from PPD and TIFY. Sequence analysis revealed that all Phalaenopsis TIFY proteins contained a TIFY domain. Exon-intron analysis showed that the intron number and length of Phalaenopsis TIFY genes varied, whereas the same subfamily and subgroup genes had similar exon or intron numbers and distributions. The most abundant cis-elements in the promoter regions of the 19 TIFY genes were associated with light responsiveness, followed by MeJA and ABA, indicating their potential regulation by light and phytohormones. The 13 candidate TIFY genes screened from the transcriptome data exhibited two types of expression trends, suggesting their different roles in cell proliferation and cell expansion of floral organ growth during Phalaenopsis flower opening. Overall, this study serves as a background for investigating the underlying roles of TIFY genes in floral organ growth in Phalaenopsis.

PeNAC67-PeKAN2-PeSCL23 and B-class MADS-box transcription factors synergistically regulate the specialization process from petal to lip in Phalaenopsis equestris.

The molecular basis of orchid flower development involves a specific regulatory program in which MADS-box transcription factors play a central role. The recent 'perianth code' model hypothesizes that two types of higher-order heterotetrameric complexes, namely SP complex and L complex, play pivotal roles in the orchid perianth organ formation. Therefore, we explored their roles and searched for other components of the regulatory network.Through the combined analysis for transposase-accessible chromatin with high-throughput sequencing and RNA sequencing of the lip-like petal and lip from Phalaenopsis equestris var.trilip, transcription factor-(TF) genes involved in lip development were revealed. PeNAC67 encoding a NAC-type TF and PeSCL23 encoding a GRAS-type TF were differentially expressed between the lip-like petal and the lip. PeNAC67 interacted with and stabilized PeMADS3, which positively regulated the development of lip-like petal to lip. PeSCL23 and PeNAC67 competitively bound with PeKAN2 and positively regulated the development of lip-like petal to petal by affecting the level of PeMADS3. PeKAN2 as an important TF that interacts with PeMADS3 and PeMADS9 can promote lip development. These results extend the 'perianth code' model and shed light on the complex regulation of orchid flower development.

Optimizing nutrient solution for vegetative growth of Dendrobium Tubtim Siam and Phalaenopsis Taisuco Swan through plant tissue nutrient balance estimation.

BACKGROUND: Orchids are grown without soil in many regions of the world, but there is a lack of studies to define the balanced and adequate nutrient solution for their cultivation, mainly in the vegetative growth phase. Therefore, this paper aims to evaluate the optimal concentration of the nutrient solution based on the proposal by Hoagland and Arnon (1950) in the vegetative growth phase capable of increasing the nutrient contents, growth, and dry matter production of Dendrobium Tubtim Siam and Phalaenopsis Taisuco Swan. In addition, this paper aims to estimate a new nutrient solution from the optimal nutrient contents in the dry matter of these orchid species to be used in the vegetative growth phase. RESULTS: Nutrient contents, growth, and dry matter production increased as the nutrient solution concentration increased up to an average concentration of 62 and 77% for D. Tubtim Siam and P. Taisuco Swan, respectively. We found that the Hoagland and Arnon solution presented a group of nutrients with concentrations above the requirement for P. Taisuco Swan (nitrogen, phosphor, calcium, and sulfur) and D. Tubtim Siam (phosphor, calcium, magnesium, and sulfur), while other nutrients in the solution did not meet the nutritional demand of these orchid species, inducing nutritional imbalance in the vegetative growth phase. CONCLUSION: We conclude that using a balanced nutrient solution created specifically for each orchid species in vegetative growth might favor their sustainable cultivation by optimizing the use of nutrients in the growing medium.

Identification of Viruses Infecting Phalaenopsis Orchids Using Nanopore Sequencing and Development of an RT-RPA-CRISPR/Cas12a for Rapid Visual Detection of Nerine Latent Virus.

Phalaenopsis orchids are one of the most popular ornamental plants. More than thirty orchid viruses have been reported, and virus-infected Phalaenopsis orchids significantly lose their commercial value. Therefore, the development of improved viral disease detection methods could be useful for quality control in orchid cultivation. In this study, we first utilized the MinION, a portable sequencing device based on Oxford Nanopore Technologies (ONT) to rapidly detect plant viruses in Phalaenopsis orchids. Nanopore sequencing revealed the presence of three plant viruses in Phalaenopsis orchids: odontoglossum ringspot virus, cymbidium mosaic virus, and nerine latent virus (NeLV). Furthermore, for the first time, we detected NeLV infection in Phalaenopsis orchids using nanopore sequencing and developed the reverse transcription-recombinase polymerase amplification (RT-RPA)-CRISPR/Cas12a method for rapid, instrument-flexible, and accurate diagnosis. The developed RT-RPA-CRISPR/Cas12a technique can confirm NeLV infection in less than 20 min and exhibits no cross-reactivity with other viruses. To determine the sensitivity of RT-RPA-CRISPR/Cas12a for NeLV, we compared it with RT-PCR using serially diluted transcripts and found a detection limit of 10 zg/µL, which is approximately 1000-fold more sensitive. Taken together, the ONT platform offers an efficient strategy for monitoring plant viral pathogens, and the RT-RPA-CRISPR/Cas12a method has great potential as a useful tool for the rapid and sensitive diagnosis of NeLV.

Orchid fruit and root movement analyzed using 2D photographs and a bioinformatics pipeline for processing sequential 3D scans.

Premise: Most studies of the movement of orchid fruits and roots during plant development have focused on morphological observations; however, further genetic analysis is required to understand the molecular mechanisms underlying this phenomenon. A precise tool is required to observe these movements and harvest tissue at the correct position and time for transcriptomics research. Methods: We utilized three-dimensional (3D) micro-computed tomography (CT) scans to capture the movement of fast-growing Erycina pusilla roots, and built an integrated bioinformatics pipeline to process 3D images into 3D time-lapse videos. To record the movement of slowly developing E. pusilla and Phalaenopsis equestris fruits, two-dimensional (2D) photographs were used. Results: The E. pusilla roots twisted and resupinated multiple times from early development. The first period occurred in the early developmental stage (77-84 days after germination [DAG]) and the subsequent period occurred later in development (140-154 DAG). While E. pusilla fruits twisted 45° from 56-63 days after pollination (DAP), the fruits of P. equestris only began to resupinate a week before dehiscence (133 DAP) and ended a week after dehiscence (161 DAP). Discussion: Our methods revealed that each orchid root and fruit had an independent direction and degree of torsion from the initial to the final position. Our innovative approaches produced detailed spatial and temporal information on the resupination of roots and fruits during orchid development.

Mechanism underlying the rapid growth of Phalaenopsis equestris induced by 60Co-γ-ray irradiation.

Gamma (γ)-ray irradiation is one of the important modern breeding methods. Gamma-ray irradiation can affect the growth rate and other characteristics of plants. Plant growth rate is crucial for plants. In horticultural crops, the growth rate of plants is closely related to the growth of leaves and flowering time, both of which have important ornamental value. In this study, 60Co-γ-ray was used to treat P. equestris plants. After irradiation, the plant's leaf growth rate increased, and sugar content and antioxidant enzyme activity increased. Therefore, we used RNA-seq technology to analyze the differential gene expression and pathways of control leaves and irradiated leaves. Through transcriptome analysis, we investigated the reasons for the rapid growth of P. equestris leaves after irradiation. In the analysis, genes related to cell wall relaxation and glucose metabolism showed differential expression. In addition, the expression level of genes encoding ROS scavenging enzyme synthesis regulatory genes increased after irradiation. We identified two genes related to P. equestris leaf growth using VIGS technology: PeNGA and PeEXPA10. The expression of PeEXPA10, a gene related to cell wall expansion, was down-regulated, cell wall expansion ability decreased, cell size decreased, and leaf growth rate slowed down. The TCP-NGATHA (NGA) molecular regulatory module plays a crucial role in cell proliferation. When the expression of the PeNGA gene decreases, the leaf growth rate increases, and the number of cells increases. After irradiation, PeNGA and PeEXPA10 affect the growth of P. equestris leaves by influencing cell proliferation and cell expansion, respectively. In addition, many genes in the plant hormone signaling pathway show differential expression after irradiation, indicating the crucial role of plant hormones in plant leaf growth. This provides a theoretical basis for future research on leaf development and biological breeding.

PeFtsH5 negatively regulates the biological stress response in Phalaenopsis equestris mitochondria.

Mitochondrial homeostasis plays a crucial role in determining cell fate by direct influence on cell apoptosis and autophagy. The ATP and Zn2+-dependent protease FtsH are of paramount importance in maintaining mitochondrial homeostasis. In Phalaenopsis equestris, three mitochondrial FtsH proteases were identified, one of which was encoded by the PeFtsH5 gene. This gene encoded a distinctive mitochondrial protein featuring a unique domain within the FtsH family. Down-regulating the expression of the PeFtsH5 homolog in Nicotiana benthamiana resulted in elevated expression levels of SA synthesis-related genes, leading to enhanced disease resistance. However, this down-regulation also caused cellular damage. Similarly, in P. equestris, the down-regulation of PeFtsH5 expression promoted the expression of defense response genes, leading to accelerated apoptosis and increased ROS levels. Nonetheless, this down-regulation also positively influenced plant resistance to biotic stress. Notably, the PeFtsH5 (i-AAA) protein, as revealed by dual membrane experiments, could form homopolymers exclusively, as it did not interact with the other two mitochondrial FtsH proteases. Consequently, this mitochondrial FtsH protease functioned as a homopolymer within P. equestris cells. The findings of this study elucidated the role of PeFtsH5 in responding to biological stress and provided new insights into its potential molecular mechanism. The result presented in this study hold promise for future research endeavors examining the regulatory effects of mitochondrial proteases on mitochondrial homeostasis and the development of stress-resistant P. equestris varieties through breeding programs.

Temperature-Regulated Flowering Locus T-Like Gene Coordinates the Spike Initiation in Phalaenopsis Orchid.

Phalaenopsis aphrodite can be induced to initiate spike growth and flowering by exposure to low ambient temperatures. However, the factors and mechanisms responsible for spike initiation in P. aphrodite remain largely unknown. In this study, we show that a repressor Flowing Locus T-like (FTL) gene, FTL, can act as a negative regulator of spike initiation in P. aphrodite. The mRNA transcripts of PaFTL are consistently high during high ambient temperature, thereby preventing premature spike initiation. However, during low ambient temperature, PaFTL expression falls while FT expression increases, allowing for spike initiation. Knock-down of PaFTL expression through virus-inducing gene silencing promoted spike initiation at 30/28°C. Moreover, PaFTL interacts with FLOWERING LOCUS D in a similar manner to FT to regulate downstream flowering initiation genes. Transgenic P. aphrodite plants exhibiting high expression of PaFTL do not undergo spike initiation, even when exposed to low ambient temperatures. These findings shed light on the flowering mechanisms in Phalaenopsis and provide new insights into how perennial plants govern spike initiation in response to temperature cues.

Genome-Wide Identification, Expression, and Molecular Characterization of the CONSTANS-like Gene Family in Seven Orchid Species.

The orchid is one of the most distinctive and highly valued flowering plants. Nevertheless, the CONSTANS-like (COL) gene family plays significant roles in the control of flowering, and its functions in Orchidaceae have been minimally explored. This research identified 68 potential COL genes within seven orchids' complete genome, divided into three groups (groups I, II, and III) via a phylogenetic tree. The modeled three-dimensional structure and the conserved domains exhibited a high degree of similarity among the orchid COL proteins. The selection pressure analysis showed that all orchid COLs suffered a strong purifying selection. Furthermore, the orchid COL genes exhibited functional and structural heterogeneity in terms of collinearity, gene structure, cis-acting elements within their promoters, and expression patterns. Moreover, we identified 50 genes in orchids with a homology to those involved in the COL transcriptional regulatory network in Arabidopsis. Additionally, the first overexpression of CsiCOL05 and CsiCOL09 in Cymbidium sinense protoplasts suggests that they may antagonize the regulation of flowering time and gynostemium development. Our study will undoubtedly provide new resources, ideas, and values for the modern breeding of orchids and other plants.

Characterization of NF-Y gene family and their expression and interaction analysis in Phalaenopsis orchid.

The complex of Nuclear Factor Ys (NF-Ys), a family of heterotrimeric transcription factors composed of three unique subunits (NF-YA, NF-YB, and NF-YC), binds to the CCAAT box of eukaryotic promoters to activate or repress transcription of the downstream genes involved into various biological processes in plants. However, the systematic characterization of NF-Y gene family has not been elucidated in Phalaenopsis. A total of 24 NF-Y subunits (4 NF-YA, 9 NF-YB, and 11 NF-YC subunits) were identified in Phalaenopsis genome, whose exon/intron structures were highly differentiated among the PhNF-Y subunits. The distribution of motifs between coding regions of PhNF-YA and PhNF-YB/C was distinct. Segmental and tandem duplication events among paralogous PhNF-Ys were occurred. Six pairs of orthologous NF-Ys from Phalaenopsis and Arabidopsis and five pairs of orthologous NF-Ys from Phalaenopsis and rice involved in the phylogenetic gene synteny were identified. The various cis-elements being responsive to low-temperature, drought and ABA were distributed in the promoters of PhNF-Ys. qRT-PCR analysis indicated all of PhNF-Ys displayed the spatial specificity of expression in different tissues. Moreover, the expression levels of multiple PhNF-Ys significantly changed responding to low-temperature and ABA treatment. Yeast two hybrid and bimolecular fluorescence complementation assays approved the interaction of PhNF-YA1/3 with PhNF-YB6/PhNF-YC7, respectively, as well as PhNF-YB6 with PhNF-YC7. PhNF-YA1/3, PhNF-YB6, and PhNF-YC7 proteins were all localized in the nucleus. Further, transient overexpression of PhNF-YB6 and PhNF-YC7 promoted PhFT3 and repressed PhSVP expression in Phalaenopsis. These findings will facilitate to explore the role of PhNF-Ys in floral transition in Phalaenopsis orchid.

PeCIN8 expression correlates with flower size and resistance to yellow leaf disease in Phalaenopsis orchids.

BACKGROUND: The orchid industry has seen a recent surge in export values due to the floral morphology and versatile applications of orchids in various markets for medicinal, food additive, and cosmetic usages. However, plant-related diseases, including the yellow leaf disease caused by Fusarium solani, have caused significant losses in the production value of Phalaenopsis (up to 30%). RESULTS: In this study, 203 Phalaenopsis cultivars were collected from 10 local orchid nurseries, and their disease severity index and correlation with flower size were evaluated. Larger flowers had weaker resistance to yellow leaf disease, and smaller flowers had stronger resistance. For the genetic relationship of disease resistance to flower size, the genetic background of all cultivars was assessed using OrchidWiz Orchid Database Software and principal component analysis. In addition, we identified the orthologous genes of BraTCP4, namely PeIN6, PeCIN7, and PeCIN8, which are involved in resistance to pathogens, and analyzed their gene expression. The expression of PeCIN8 was significantly higher in the most resistant cultivars (A7403, A11294, and A2945) relative to the most susceptible cultivars (A10670, A6390, and A10746). CONCLUSIONS: We identified a correlation between flower size and resistance to yellow leaf disease in Phalaenopsis orchids. The expression of PeCIN8 may regulate the two traits in the disease-resistant cultivars. These findings can be applied to Phalaenopsis breeding programs to develop resistant cultivars against yellow leaf disease.