Evolution of Terpene Synthases in Orchidaceae.

Terpenoids are the largest class of plant secondary metabolites and are one of the major emitted volatile compounds released to the atmosphere. They have functions of attracting pollinators or defense function, insecticidal properties, and are even used as pharmaceutical agents. Because of the importance of terpenoids, an increasing number of plants are required to investigate the function and evolution of terpene synthases (TPSs) that are the key enzymes in terpenoids biosynthesis. Orchidacea, containing more than 800 genera and 28,000 species, is one of the largest and most diverse families of flowering plants, and is widely distributed. Here, the diversification of the TPSs evolution in Orchidaceae is revealed. A characterization and phylogeny of TPSs from four different species with whole genome sequences is available. Phylogenetic analysis of orchid TPSs indicates these genes are divided into TPS-a, -b, -e/f, and g subfamilies, and their duplicated copies are increased in derived orchid species compared to that in the early divergence orchid, A. shenzhenica. The large increase of both TPS-a and TPS-b copies can probably be attributed to the pro-duction of different volatile compounds for attracting pollinators or generating chemical defenses in derived orchid lineages; while the duplications of TPS-g and TPS-e/f copies occurred in a species-dependent manner.

Antibacterial Prenylated p-Hydroxybenzoic Acid Derivatives from Oberonia myosurus and Identification of Putative Prenyltransferases.

Twelve hitherto unknown tandem prenylated p-hydroxybenzoic acid derivatives, namely, oberoniamyosurusins A-L, together with five known derivatives, were isolated from an EtOH extract of the whole parts of the plant Oberonia myosurus. Compounds 10, 13, and 17 exhibited moderate inhibitory activity against Staphylococcus aureus subsp. aureus ATCC29213 with MIC50 values ranging from 7.6 to 23 µg/mL. To determine the biosynthetic pathway of this class of tandem prenyl-substituted compounds, the full-length transcriptome of O. myosurus was sequenced, yielding 19.09 Gb of clean data and 10 949 nonredundant sequences. Two isoforms of p-hydroxybenzoic acid prenyltransferases were annotated and functionally characterized as the enzymes that might be involved in the biosynthesis of nervogenic acid (13) in Pichia pastoris.

Involvement of CsERF2 in leaf variegation of Cymbidium sinense 'Dharma'.

MAIN CONCLUSION: CsERF2, an ethylene response factor, plays a role in leaf variegation. Leaf variegation is a main ornamental characteristic in Cymbidium sinense (C. sinense). However, the mechanisms of leaf color variegation remain largely unclear. In the present study, we analyzed the cytological and physiological features, as well as molecular analyses of leaves from wild-type (WT) and leaf variegation mutants of Cymbidium sinense 'Dharma'. Chloroplasts with typical and functional structures were discovered in WT and green sectors of the mutants leaves (MG), but not in yellow sectors of the mutant leaves (MY). The activities of key enzymes involved in chlorophyll (Chl) degradation and their substrate contents were significantly increased in MY. Genes related to Chl degradation also showed a significant up-regulation in MY. Transcriptomic analysis showed that the expression of all identified ethylene response factors (ERFs) was significantly up-regulated, and the 1-aminocyclopropane-1-carboxylic acid (ACC) content in MY was significantly higher compared with MG. QRT-PCR analysis validated that the expression levels of genes related to Chl degradation could be positively affected by ethylene (ETH) treatment. Stable overexpression of CsERF2 in Nicotiana tabacum (N. tabacum) led to a decrease in Chl content and abnormal chloroplast. Transcriptomic analysis and qRT-PCR results showed that the KEGG pathway related to chloroplast development and function showed significant change in transgenic N. tabacum. Therefore, the leaf color formation of C. sinense was greatly affected by chloroplast development and Chl metabolism. CsERF2 played an important role in leaf variegation of C. sinense.

The decline in photosynthetic rate upon transfer from high to low light is linked to the slow kinetics of chloroplast ATP synthase in Bletilla striata.

Upon a sudden transition from high to low light, the rate of CO2 assimilation (AN) in some plants first decreases to a low level before gradually becoming stable. However, the underlying mechanisms remain controversial. The activity of chloroplast ATP synthase (gH+) is usually depressed under high light when compared with low light. Therefore, we hypothesize that upon a sudden transfer from high to low light, the relatively low gH+ restricts ATP synthesis and thus causes a reduction in AN. To test this hypothesis, we measured gas exchange, chlorophyll fluorescence, P700 redox state, and electrochromic shift signals in Bletilla striata (Orchidaceae). After the transition from saturating to lower irradiance, AN and ETRII decreased first to a low level and then gradually increased to a stable value. Within the first seconds after transfer from high to low light, gH+ was maintained at low levels. During further exposure to low light, gH+ gradually increased to a stable value. Interestingly, a tight positive relationship was found between gH+ and ETRII. These results suggested that upon a sudden transition from high to low light, AN was restricted by gH+ at the step of ATP synthesis. Taken together, we propose that the decline in AN upon sudden transfer from high to low light is linked to the slow kinetics of chloroplast ATP synthase.

Exploring molecular evolution of Rubisco in C3 and CAM Orchidaceae and Bromeliaceae.

BACKGROUND: The CO2-concentrating mechanism associated to Crassulacean acid metabolism (CAM) alters the catalytic context for Rubisco by increasing CO2 availability and provides an advantage in particular ecological conditions. We hypothesized about the existence of molecular changes linked to these particular adaptations in CAM Rubisco. We investigated molecular evolution of the Rubisco large (L-) subunit in 78 orchids and 144 bromeliads with C3 and CAM photosynthetic pathways. The sequence analyses were complemented with measurements of Rubisco kinetics in some species with contrasting photosynthetic mechanism and differing in the L-subunit sequence. RESULTS: We identified potential positively selected sites and residues with signatures of co-adaptation. The implementation of a decision tree model related Rubisco specific variable sites to the leaf carbon isotopic composition of the species. Differences in the Rubisco catalytic traits found among C3 orchids and between strong CAM and C3 bromeliads suggested Rubisco had evolved in response to differing CO2 concentration. CONCLUSIONS: The results revealed that the variability in the Rubisco L-subunit sequence in orchids and bromeliads is composed of coevolving sites under potential positive adaptive signal. The sequence variability was related to δ13C in orchids and bromeliads, however it could not be linked to the variability found in the kinetic properties of the studied species.

Decreased photosystem II activity facilitates acclimation to fluctuating light in the understory plant Paris polyphylla.

In forests, understory plants are usually exposed to sunflecks on timescales of seconds or minutes. However, it is unclear how understory plants acclimate to fluctuating light. In this study, we compared chlorophyll fluorescence, PSI redox state and the electrochromic shift signal under fluctuating light between an understory plant Paris polyphylla (Liliaceae) and a light-demanding plant Bletilla striata (Orchidaceae). Within the first seconds after transition from low to high light, PSI was highly oxidized in P. polyphylla but was highly reduced in B. striata, although both species could not generate a sufficient trans-thylakoid proton gradient (ΔpH). Furthermore, the outflow of electrons from PSI to O2 was not significant in P. polyphylla, as indicated by the P700 redox kinetics upon dark-to-light transition. Therefore, the different responses of PSI to fluctuating light between P. polyphylla and B. striata could not be explained by ΔpH formation or alternative electron transport. In contrast, upon a sudden transition from low to high light, electron flow from PSII was much lower in P. polyphylla than in B. striata, suggesting that the rapid oxidation of PSI in P. polyphylla was largely attributed to the lower PSII activity. We propose, for the first time, that down-regulation of PSII activity is an important strategy used by some understory angiosperms to cope with sunflecks.

Transcriptomic profiling of the flower scent biosynthesis pathway of Cymbidium faberi Rolfe and functional characterization of its jasmonic acid carboxyl methyltransferase gene.

BACKGROUND: Cymbidium faberi, one of the most famous oriental orchids, has a distinct flower scent, which increases its economic value. However, the molecular mechanism of the flower scent biosynthesis was unclear prior to this study. Methyl jasmonate (MeJA) is one of the main volatile organic compounds (VOC) produced by the flowers of C. faberi. In this study, unigene 79,363 from comparative transcriptome analysis was selected for further investigation. RESULTS: A transcriptome comparison between blooming and withered flowers of C. faberi yielded a total of 9409 differentially expressed genes (DEGs), 558 of which were assigned to 258 pathways. The top ten pathways included α-linolenic acid metabolism, pyruvate metabolism and fatty acid degradation, which contributed to the conversion of α-linolenic acid to MeJA. One of the DEGs, jasmonic acid carboxyl methyltransferase (CfJMT, Unigene 79,363) was highly expressed in the blooming flower of C. faberi, but was barely detected in leaves and roots. Although the ectopic expression of CfJMT in tomato could not increase the MeJA content, the expression levels of endogenous MeJA biosynthesis genes were influenced, especially in the wound treatment, indicating that CfJMT may participate in the response to abiotic stresses. CONCLUSION: This study provides a basis for elucidating the molecular mechanism of flower scent biosynthesis in C. faberi, which is beneficial for the genetically informed breeding of new cultivars of the economically valuable oriental orchids.

Enzymatic scarification of Anacamptis morio (Orchidaceae) seed facilitates lignin degradation, water uptake and germination.

The seed coat of many species contains hydrophobic lignins, and in soil the action of microbial ligninases may contribute to release from dormancy. Laboratory use of ligninases to stimulate germination is promising because of the specific action on the seed coat, whereas chemical scarification agents may also corrode the embryo. We hypothesised that exposure of Anacamptis morio (Orchidaceae) seeds to fungal laccase would stimulate germination, and that the mechanism involves lignin degradation and increased imbibition. Germination capacity in vitro was quantified with 1 U filter-sterilised laccase added to agar medium following autoclaving, compared to a 10% bleach solution (standard bleach surface sterilisation/scarification method used in orchid seed sowing). Lignin degradation was quantified using an optical method (phloroglucinol-HCl staining) combined with image analysis, following experimental pre-treatments involving immersion in laccase solution, distilled water (negative control) or bleach (positive control). Water uptake after experimental treatments was quantified as the proportion of seeds exhibiting visible uptake of an aqueous fluorochrome under UV excitation. Laccase stimulated a doubling of germination in vitro with respect to bleach surface sterilisation/scarification alone, from 23.7 to 49.8% (P = 0.007). Laccase and bleach methods both significantly decreased the optical signal of phloroglucinol (for laccase, to 79.9 ± 1.3% of controls; anova: F = 10.333, P = 0.002). Laccase resulted in a modest but highly significant (P < 0.0001) increase in water uptake with respect to the control (11.7%; cf 99.4% for bleach). Laccase scarification can stimulate germination of A. morio through a mechanism of targeted seed coat degradation. The results demonstrate the potential of this relatively non-invasive enzymatic scarification technique.

In silico characterization and transcriptional modulation of phenylalanine ammonia lyase (PAL) by abiotic stresses in the medicinal orchid Vanda coerulea Griff. ex Lindl.

Phenylalanine ammonia lyase (PAL) is the first enzyme of phenylpropanoid pathway. In the present study, a full-length PAL transcript from Vanda coerulea Griff. ex Lindl. (Family: Orchidaceae) was isolated and characterized. It was found that complete PAL transcript of V. coerulea (VcPAL; Gene Bank no. MG745168) contained 2175 bp with the open reading frame (ORF) of 2112 bp, encoding 703 amino acid residues. The multiple sequence alignment showed that VcPAL protein had 81% identity with that of the orchid, Bromheadia finlaysoniana. Phylogenetic analysis also disclosed that VcPAL shared the same evolutionary relationship with PAL proteins of other orchid species and to be closely related to that of other angiosperm species as well. The three-dimensional structure of VcPAL was found to be homo-tetrameric in nature consisting of four identical subunits with a molecular mass of 75 kDa per subunit. In silico characterization revealed the deduced protein to be a stable protein, comprising three major functional domains as reported in PAL proteins of other species. The transcription profiling of VcPAL exhibited the highest expression level to be present in the in vitro - raised leaf and root samples as compared to that of the ex vitro plant. The differential expression of VcPAL transcript was observed to be up-regulated by different types of abiotic stresses like wounding, cold, UV-B, salinity, and down-regulated by dark treatment. The study also exhibited that the VcPAL enzyme activity was directly proportional to the gene expression after the tissues were subjected to salinity and wounding stresses wherein a 1.7- fold increase in the enzyme activity was recorded in the leaf tissues exposed to salinity stress. A positive correlation could be found between the enzyme activity and the accumulation of phenylpropanoids such as total phenolic and flavonoid contents with R2 = 0.85 and 0.842 respectively.

Determination of endogenous IAA and carbohydrates during the induction and development of protocorm-like bodies of Cattleya tigrina A. Richard / Determinação de AIA e carboidratos endógenos durante a indução e desenvolvimento deestruturas semelhantes a protocormos de Cattleya tigrina A. Richard

Tissue culture techniques have been employed for orchid mass propagation by means of themorphogenetic route of protocorm like-bodies (PLBs). This study aimed to analyze and compare Indoleaceticacid (IAA) and sugar endogenous levels in protocorm-like bodies (PLBs) induction and developmentin Cattleya tigrina, in order to better understand this process and to optimize micropropagation procedureprotocols. Leaves grown on MS (Murashige and Skoog) culture medium, suplemented with 9 μM TDZfor PLBs induction and development were collected after 0, 2, 7, 14, 30, 60 and 100 days of cultivation, forfurther analysis. Increase of IAA and reduction of sugar levels are strongly related to morphogeneticresponse, that is, PLBs formation over the preexisting ones and leaf primordia formation. Sucrose, fructoseand glucose presence in this study is related to cell signaling. Thus, hormonal signals and carbohydratesalter metabolism, triggering PLBs initiation and development in C. tigrina.

Técnicas de cultura de tecidos têm sido empregadas para a propagação em massa de orquídeaspor meio da via morfogenética de estruturas semelhantes a protocormos (ESPs). O estudo teve comoobjetivo analisar e comparar os níveis endógenos de ácido indol-3-acético (AIA) e de açúcares na indução eno desenvolvimento de estruturas semelhantes a protocormos (ESPs) em Cattleya tigrina, visando à melhorcompreensão deste processo e à otimização de protocolos de micropropagação. Folhas cultivadas em meiode cultura MS (Murashige & Skoog), suplementadas com 9 μM de TDZ para indução e desenvolvimentode ESPs foram coletadas após 0, 2, 7, 14, 30, 60 e 100 dias de cultivo, para posterior análise. O aumento dosníveis de AIA e a redução dos níveis de açúcar estão fortemente relacionados à resposta morfogenética, ouseja, à formação de ESPs sobre os pré-existentes e à formação de primórdios foliares. A presença desacarose, frutose e glicose neste estudo está relacionada à sinalização celular. Assim, sinais hormonais ecarboidratos alteram o metabolismo, desencadeando a iniciação e o desenvolvimento de ESPs em C. tigrina.

Molecular cloning and expression analysis of an 1-aminocyclopropane-1-carboxylate synthase gene from Oncidium Gower Ramsey.

1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) is a rate-limiting enzyme in the biosynthesis of ethylene which regulates many aspects of the plant development and responses to biotic and abiotic stresses. In this study, a full-length cDNA of ACC synthase, OnACS2, was cloned from the senescing flower of Oncidium Gower Ramsey by RACE. The full-length cDNA of OnACS2 (GenBank accession no. JQ822087) was 1557 bp in length with an open reading frame (ORF) of 1308 bp encoding for a protein of 435 amino acid residues. The predicted OnACS2 protein had a molecular mass of 49.1 kDa with pI value of 7.51. Phylogenetic analysis indicated its evolutionary relationships with corresponding orthologous sequences in orchids, Hosta ventricosa and monocots. Real-time PCR assay demonstrated that OnACS2 was constitutively expressed in all tested organs with the highest transcript level in the gynandria. Differential expression pattern of OnACS2 gene correlated to the ethylene production and the subsequent occurrence of senescent symptoms in flower suggested that OnACS2 probably played an important role in the initiation of flower senescence.

Modification of flower colour by suppressing ß-ring carotene hydroxylase genes in Oncidium.

Oncidium 'Gower Ramsey' (Onc. GR) is a popular cut flower, but its colour is limited to bright yellow. The ß-ring carotene hydroxylase (BCH2) gene is involved in carotenoid biogenesis for pigment formation. However, the role of BCH2 in Onc. GR is poorly understood. Here, we investigated the functions of three BCH2 genes, BCH-A2, BCH-B2 and BCH-C2 isolated from Onc. GR, to analyse their roles in flower colour. RT-PCR expression profiling suggested that BCH2 was mainly expressed in flowers. The expression of BCH-B2 remained constant while that of BCH-A2 gradually decreased during flower development. Using Agrobacterium tumefaciens to introduce BCH2 RNA interference (RNAi), we created transgenic Oncidium plants with down-regulated BCH expression. In the transgenic plants, flower colour changed from the bright yellow of the wild type to light and white-yellow. BCH-A2 and BCH-B2 expression levels were significantly reduced in the transgenic flower lips, which make up the major portion of the Oncidium flower. Sectional magnification of the flower lip showed that the amount of pigmentation in the papillate cells of the adaxial epidermis was proportional to the intensity of yellow colouration. HPLC analyses of the carotenoid composition of the transgenic flowers suggested major reductions in neoxanthin and violaxanthin. In conclusion, BCH2 expression regulated the accumulation of yellow pigments in the Oncidium flower, and the down-regulation of BCH-A2 and BCH-B2 changed the flower colour from bright yellow to light and white-yellow.

Evolutionary history of PEPC genes in green plants: Implications for the evolution of CAM in orchids.

The phosphoenolpyruvate carboxylase (PEPC) gene is the key enzyme in CAM and C4 photosynthesis. A detailed phylogenetic analysis of the PEPC family was performed using sequences from 60 available published plant genomes, the Phalaenopsis equestris genome and RNA-Seq of 15 additional orchid species. The PEPC family consists of three distinct subfamilies, PPC-1, PPC-2, and PPC-3, all of which share a recent common ancestor in chlorophyte algae. The eudicot PPC-1 lineage separated into two clades due to whole genome duplication (WGD). Similarly, the monocot PPC-1 lineage also divided into PPC-1M1 and PPC-1M2 through an ancient duplication event. The monocot CAM- or C4-related PEPC originated from the clade PPC-1M1. WGD may not be the major driver for the performance of CAM function by PEPC, although it increased the number of copies of the PEPC gene. CAM may have evolved early in monocots, as the CAM-related PEPC of orchids originated from the monocot ancient duplication, and the earliest CAM-related PEPC may have evolved immediately after the diversification of monocots, with CAM developing prior to C4. Our results represent the most complete evolutionary history of PEPC genes in green plants to date and particularly elucidate the origin of PEPC in orchids.

Alternative translation initiation codons for the plastid maturase MatK: unraveling the pseudogene misconception in the Orchidaceae.

BACKGROUND: The plastid maturase MatK has been implicated as a possible model for the evolutionary "missing link" between prokaryotic and eukaryotic splicing machinery. This evolutionary implication has sparked investigations concerning the function of this unusual maturase. Intron targets of MatK activity suggest that this is an essential enzyme for plastid function. The matK gene, however, is described as a pseudogene in many photosynthetic orchid species due to presence of premature stop codons in translations, and its high rate of nucleotide and amino acid substitution. RESULTS: Sequence analysis of the matK gene from orchids identified an out-of-frame alternative AUG initiation codon upstream from the consensus initiation codon used for translation in other angiosperms. We demonstrate translation from the alternative initiation codon generates a conserved MatK reading frame. We confirm that MatK protein is expressed and functions in sample orchids currently described as having a matK pseudogene using immunodetection and reverse-transcription methods. We demonstrate using phylogenetic analysis that this alternative initiation codon emerged de novo within the Orchidaceae, with several reversal events at the basal lineage and deep in orchid history. CONCLUSION: These findings suggest a novel evolutionary shift for expression of matK in the Orchidaceae and support the function of MatK as a group II intron maturase in the plastid genome of land plants including the orchids.

Prolonged exposure to elevated temperature induces floral transition via up-regulation of cytosolic ascorbate peroxidase 1 and subsequent reduction of the ascorbate redox ratio in Oncidium hybrid orchid.

The bolting time of the Oncidium hybrid orchid is not season dependent and so it is a useful year-round model system to study thermal-induced flowering mechanisms in planta. Previously, we reported that a low ascorbate (AsA) content is essential for floral transition in Oncidium; however, the environmental factors governing initiation of the flowering process remained to be elucidated. The current study revealed that a prolonged elevated temperature treatment (30°C over a 14 d period) induces floral transition. This floral induction in response to thermal stress was associated with a significantly increased reactive oxygen species (ROS) level and a lowered AsA redox ratio, as well as prominently up-regulated expression of cytosolic ascorbate peroxidase (cytAPX1). Transcriptome analysis confirmed that increased temperature affected the differential expression of genes involved in antioxidant metabolism. Likewise, transgenic Arabidopsis ectopically overexpressing Oncidium cytAPX1 displayed an early-flowering phenotype and low AsA redox ratio under thermal stress, while cytAPX1 mutants, apx1-1 and apx1-2, exhibited a delayed-flowering phenotype and a high AsA redox ratio. Our present data illustrate that the floral transition response to thermal stress is mediated by the AsA redox ratio, and that CytAPX plays a pivotal role in modulating the AsA redox ratio in Oncidium hybrid orchid. Taken together, the results from this investigation of the thermal-induced flowering mechanism indicated that the AsA redox ratio is a master switch to mediate phase transition from the vegetative to reproductive stage.

Eugenol synthase genes in floral scent variation in Gymnadenia species.

Floral signaling, especially through floral scent, is often highly complex, and little is known about the molecular mechanisms and evolutionary causes of this complexity. In this study, we focused on the evolution of "floral scent genes" and the associated changes in their functions in three closely related orchid species of the genus Gymnadenia. We developed a benchmark repertoire of 2,571 expressed sequence tags (ESTs) in Gymnadenia odoratissima. For the functional characterization and evolutionary analysis, we focused on eugenol synthase, as eugenol is a widespread and important scent compound. We obtained complete coding complementary DNAs (cDNAs) of two copies of putative eugenol synthase genes in each of the three species. The proteins encoded by these cDNAs were characterized by expression and testing for activity in Escherichia coli. While G. odoratissima and Gymnadenia conopsea enzymes were found to catalyze the formation of eugenol only, the Gymnadenia densiflora proteins synthesize eugenol, as well as a smaller amount of isoeugenol. Finally, we showed that the eugenol and isoeugenol producing gene copies of G. densiflora are evolutionarily derived from the ancestral genes of the other species producing only eugenol. The evolutionary switch from production of one to two compounds evolved under relaxed purifying selection. In conclusion, our study shows the molecular bases of eugenol and isoeugenol production and suggests that an evolutionary transition in a single gene can lead to an increased complexity in floral scent emitted by plants.

Multiple isoforms of phosphoenolpyruvate carboxylase in the Orchidaceae (subtribe Oncidiinae): implications for the evolution of crassulacean acid metabolism.

Phosphoenolpyruvate carboxylase (PEPC) catalyses the initial fixation of atmospheric CO2 into oxaloacetate and subsequently malate. Nocturnal accumulation of malic acid within the vacuole of photosynthetic cells is a typical feature of plants that perform crassulacean acid metabolism (CAM). PEPC is a ubiquitous plant enzyme encoded by a small gene family, and each member encodes an isoform with specialized function. CAM-specific PEPC isoforms probably evolved from ancestral non-photosynthetic isoforms by gene duplication events and subsequent acquisition of transcriptional control elements that mediate increased leaf-specific or photosynthetic-tissue-specific mRNA expression. To understand the patterns of functional diversification related to the expression of CAM, ppc gene families and photosynthetic patterns were characterized in 11 closely related orchid species from the subtribe Oncidiinae with a range of photosynthetic pathways from C3 photosynthesis (Oncidium cheirophorum, Oncidium maduroi, Rossioglossum krameri, and Oncidium sotoanum) to weak CAM (Oncidium panamense, Oncidium sphacelatum, Gomesa flexuosa and Rossioglossum insleayi) and strong CAM (Rossioglossum ampliatum, Trichocentrum nanum, and Trichocentrum carthagenense). Phylogenetic analysis revealed the existence of two main ppc lineages in flowering plants, two main ppc lineages within the eudicots, and three ppc lineages within the Orchidaceae. Our results indicate that ppc gene family expansion within the Orchidaceae is likely to be the result of gene duplication events followed by adaptive sequence divergence. CAM-associated PEPC isoforms in the Orchidaceae probably evolved from several independent origins.

Molecular cloning and functional analysis of Three FLOWERING LOCUS T (FT) homologous genes from Chinese Cymbidium.

The FLOWERING LOCUS T (FT) gene plays crucial roles in regulating the transition from the vegetative to reproductive phase. To understand the molecular mechanism of reproduction, three homologous FT genes were isolated and characterized from Cymbidium sinense "Qi Jian Bai Mo", Cymbidium goeringii and Cymbidium ensifolium "Jin Si Ma Wei". The three genes contained 618-bp nucleotides with a 531-bp open reading frame (ORF) of encoding 176 amino acids (AAs). Alignment of the AA sequences revealed that CsFT, CgFT and CeFT contain a conserved domain, which is characteristic of the PEBP-RKIP superfamily, and which share high identity with FT of other plants in GenBank: 94% with OnFT from Oncidium Gower Ramsey, 79% with Hd3a from Oryza sativa, and 74% with FT from Arabidopsis thaliana. qRT-PCR analysis showed a diurnal expression pattern of CsFT, CgFT and CeFT following both long day (LD, 16-h light/8-h dark) and short day (SD, 8-h light/16-h dark) treatment. While the transcripts of both CsFT and CeFT under LD were significantly higher than under SD, those of CgFT were higher under SD. Ectopic expression of CgFT in transgenic Arabidopsis plants resulted in early flowering compared to wild-type plants and significant up-regulation of APETALA1 (AP1) expression. Our data indicates that CgFT is a putative phosphatidylethanolamine-binding protein gene in Cymbidium that may regulate the vegetative to reproductive transition in flowers, similar to its Arabidopsis ortholog.

Methylation effect on chalcone synthase gene expression determines anthocyanin pigmentation in floral tissues of two Oncidium orchid cultivars.

The anthocyanin-biosynthetic pathway was studied in flowers of Oncidium Gower Ramsey with yellow floral color and mosaic red anthocyanin in lip crests, sepals and petals, and compared with the anthocyanin biosynthesis in flowers of Oncidium Honey Dollp, a natural somatoclone derived from tissue culture of Gower Ramsey, with a yellow perianth without red anthocyanins in floral tissues. HPLC analysis revealed that the red anthocyanin in lip crests of the Gower Ramsey cultivar comprised peonidin-3-O-glucoside, delphinidin-3-O-glucoside and cyanidin-3-O-glucoside, whereas Honey Dollp was devoid of anthocyanin compounds. Among the five anthocyanin-biosynthetic genes, OgCHS was actively expressed in lip crests of Gower Ramsey flowers, but no transcripts of OgCHS were detected in Honey Dollp floral tissues. Transient expression of OgCHS by bombardment confirmed that recovery of the OgCHS gene expression completed the anthocyanin pathway and produced anthocyanin compounds in lip crests of Honey Dollp flowers. Transcription factor genes regulating anthocyanin biosynthesis showed no distinctive differences in the expression level of OgMYB1, OgbHLH and OgWD40 between the two cultivars. A methylation assay revealed that the promoter of OgCHS was not methylated in Gower Ramsey, while a positive methylation effect was present in the upstream promoter region of OgCHS in Honey Dollp. Overall, our results suggest that the failure of anthocyanin accumulation in Honey Dollp floral tissues may be attributed to inactivation of the OgCHS gene resulting from the epigenetic methylation of 5'-upstream promoter region.

Molecular characterization of a Cyrtochilum loxense Somatic Embryogenesis Receptor-like Kinase (SERK) gene expressed during somatic embryogenesis.

Somatic embryogenesis is crucial for the propagation of endangered Ecuadorian orchid species, among them Cyrtochilum loxense, in view of the fact that their number in nature or in collections is quite reduced. One of the genes expressed during somatic and zygotic embryogenesis is Somatic Embryogenesis Receptor-like Kinase (SERK). Despite the development of somatic embryogenesis protocols for orchids, no SERK genes have been isolated from this family. This is the first report on the isolation of a full-length orchid SERK sequence, namely that of Cyrtochilum loxense (ClSERK). The identity of ClSERK was inferred by the presence of all domains typical of SERK proteins: a signal peptide, a leucine zipper domain, five LRRs, a serine proline-rich domain, a transmembrane domain, a kinase domain, and the C-terminal region. We have observed that the ClSERK gene is highly expressed in embryogenic calluses generated from protocorms at the time of appearance of embryonic morphological features. At later stages when embryos become well visible on calluses, ClSERK gene expression decreases. Compared to early stages of embryo formation on calluses, the expression detected in leaf tissue is far lower, thus suggesting a role of this gene during development.