Phalaenopsis Bellina Research Articles

Insights to Phenylalanine Ammonia Lyase (PAL) and Secondary Metabolism in Orchids: An in silico Approach.

The phenylalanine ammonia lyase (PAL) catalyses the first step of phenylpropanoid metabolic pathway which leads to the biosynthesis of a diverse group of secondary metabolites. Orchids serve as a rich source of metabolites and the availability of genome or transcriptome for selected orchid species provides an opportunity to analyse the PAL genes in orchids. In the present study, 21 PAL genes were characterized using bioinformatics tools in nine orchid species (Apostasia shenzhenica, Cypripedium formosanum, Dendrobium catenatum, Phalaenopsis aphrodite, Phalaenopsis bellina, Phalaenopsis equestris, Phalaenopsis lueddemanniana, Phalaenopsis modesta and Phalaenopsis schilleriana). Multiple sequence alignment confirmed the presence of PAL-specific conserved domains (N-terminal, MIO, core, shielding and C-terminal domain). All these proteins were predicted to be hydrophobic in nature and to have cytoplasmic localisation. Structural modelling depicted the presence of alpha helices, extended strands, beta turns and random coils in their structure. Ala-Ser-Gly triad known for substrate binding and catalysis of MIO-domain was found to be completely conserved in all the proteins. Phylogenetic study showed that the PALs of pteridophytes, gymnosperms and angiosperms clustered together in separate clades. Expression profiling showed tissue-specific expression for all the 21 PAL genes in the various reproductive and vegetative tissues which suggested their diverse role in growth and development. This study provides insights to the molecular characterization of PAL genes which may help in developing biotechnological strategies to enhance the synthesis of phenylpropanoids in orchids and other heterologous systems for pharmaceutical applications.

PbABCG1 and PbABCG2 transporters are required for the emission of floral monoterpenes in Phalaenopsis bellina.

Terrestrial plants emit volatiles into the atmosphere to attract both pollinators and the enemies of herbivores, for defense. Phalaenopsis bellina is a scented orchid species in which the main scent components are monoterpenes, including linalool and geraniol, and their derivatives. Here, we investigated whether ABC transporters are involved in floral scent emission. We carried out whole-genome identification of ABC transporter-related genes using four floral transcriptomics libraries of P. bellina. We identified 86 ABC subfamily G genes related to terpenoid transport. After comparing the gene expression patterns of P. bellina with that of Phalaenopsis aphrodite subsp. formosana, a scentless species, followed by gene-to-gene correlation analysis, PbABCG1 and PbABCG2 were selected. The temporal expression of both PbABCG1 and PbABCG2 was highly correlated with that of the key enzyme PbGDPS and the major transcription factor PbbHLH4 in monoterpene biosynthesis, with optimal expression on day5 post-anthesis. Spatial gene expression analysis showed that PbABCG1 was highly expressed in sepals, whereas PbABCG2 was expressed in the lip. Subcellular localization with a GFP fusion protein revealed that both PbABCG1 and PbABCG2 are cytoplasmic membrane proteins. Co-downregulation of PbABCG1 and PbABCG2 using both double-strand RNA interference and tobacco rattle virus-based gene silencing led to a significant decrease in monoterpene emission, accompanied by an increase in the internal monoterpene pools. Furthermore, ectopic expression of PbABCG1 and PbABCG2 in an ABC16- mutant yeast strain rescued its tolerance to geraniol. Altogether, our results indicate that PbABCG1 and PbABCG2 play substantial roles in monoterpene transport/emission in P. bellina floral scent.

Terpene Synthase-b and Terpene Synthase-e/f Genes Produce Monoterpenes for Phalaenopsis bellina Floral Scent.

Orchids are the most species-rich plants and highly interactive with pollinators via visual or olfactory cues. Biosynthesis and emission of volatile organic compounds (VOCs) to the atmosphere facilitate the olfactory cues and ensure successful pollination. Phalaenopsis bellina is a scented orchid with monoterpenes as major VOCs, comprising linalool, geraniol, and their derivatives. Comparative transcriptomics analysis identified four terpene synthase-b (TPS-b) genes and two TPS-e/f genes with differential gene expression between scented and scentless Phalaenopsis species. Here, we confirmed their differential expression between scented and scentless Phalaenopsis orchids and excluded one TPS-b candidate. We analyzed the temporal and spatial expression and functionally characterized these TPSs. Both TPS-b and TPS-e/f genes showed an increased expression on blooming day or 3 days post-anthesis (D + 3) before the optimal emission of floral scent on D + 5, with especially high expression of PbTPS5 and PbTPS10. The TPS-b genes are expressed exclusively in reproductive organs, whereas the TPS-e/f genes are expressed in both reproductive and vegetative organs. In planta functional characterization of both PbTPS5 and PbTPS10 in tobacco and scentless Phalaenopsis plants did not produce terpenoids. Further ectopic expression in scented Phalaenopsis cultivar P. I-Hsin Venus showed that linalool was the main product, with PbTPS10 displaying 3-fold higher activity than PbTPS5. On in vitro enzyme assay with purified recombinant TPS-b proteins ectopically expressed in Escherichia coli, geraniol was the product catalyzed by PbTPS5 and PbTPS9. PbTPS3 was a linalool/(β)-cis-ocimene synthase and PbTPS4 a linalool synthase. In conclusion, both TPS-b and TPS-e/f enzymes orchestrated floral monoterpene biosynthesis in P. bellina.

Total RNA Extraction from the Aromatic Phalaenopsis bellina, Endemic Orchid in Sabah, Borneo

Phalaenopsis bellina is an attractive orchid due to its unique appearance and distinctive floral fragrance. Many past studies on this plant focused on the plant at the molecular level; however, this requires sufficient quantities of high-quality P. bellina RNA. RNA is more delicate to manipulate than DNA due to its structural instability and its vulnerability to various secondary metabolites, such as polyphenols and polysaccharides. Therefore, in this study, 4 RNA isolation methods, a modified phenol-chloroform method and 3 commercial kits (Vivantis, Novogene, and Analytik Jena) were used on the leaves and flowers of P. bellina for comparison. The yield and purity of the total RNA were determined using spectrophotometry. The results showed that the total RNA isolated using the modified phenol-chloroform method had the highest yield (1223.75±68.51 ng/µL) and purity compared to the 3 commercial kits, with an OD260/280 value of 2.07 and an OD260/230 value of 2.26, respectively. In particular, the isolated RNA did not show any detectable genomic DNA contamination or other impurities. The RNA isolated using the phenol-chloroform method was also evaluated by electrophoresis, reverse transcription, and PCR. The results indicated that the phenol-chloroform method appears to be superior for total RNA extraction. Thus, this developed method is proven to be suitable for the RNA extraction of plants rich in polysaccharides and polyphenols and is amenable for future molecular studies on P. bellina.

The variation of volatile compounds emitted from aromatic orchid (Phalaenopsis bellina) at different timing and flowering stages

Phalaenopsis bellina, is an orchid widely known for their distinctive fragrance. Of late, fragrant orchids are getting more attention from Orchid breeders for their horticultural market value. Although volatile compounds have been identified in several orchid species, the floral scent emission of P. bellina is far from understood. Therefore, this study was carried out to investigate the influence of different timing and floral development stages on the volatile emissions from P. bellina flowers using Solid Phase Micro Extraction (SPME) and Gas Chromatography-Mass Spectrometry (GC-MS). The volatile compound was extracted during morning and afternoon sessions from three different floral developmental stages. The volatile compounds emitted from flowers from two different sessions were trapped with SPME fiber for 30 minutes before directly injected into GC-MS for identification. Results showed that approximately 79 volatile compounds were identified, with the terpenoid presented as the major compound class. P. bellina had the highest number of volatiles during the morning and full bloom (41), with 29.82% monoterpene and 23.33% sesquiterpenes accounted in total. α-farnesene (19.56%) was abundance during morning emission and remains as the highest volatile in afternoon emission (44.08%), even higher when compared to morning emission. However, a decrease in the volatile compound was observed in afternoon emission, in which only 34 volatile compounds detected. Meanwhile, partial bloom developmental stage revealed linalool as the major terpenoid volatile compounds (25.89%), with only 20 volatile compounds recorded. On the other hand, no volatile compounds were recorded and profiled from flower bud stage. The establishment of a floral scent study provides a brief overview of the regulation of fragrance in P. bellina, which can be continued through gene isolation or fragrance-related enzymes study. This information will provide necessary information on orchid floral scent research that useful in boosting horticultural trade of the scented orchids and their function in pollination ecology study

Development of an Efficient Particle Bombardment Transformation System for the Endemic Orchid, Phalaenopsis bellina

Phalaenopsis bellina is an important indigenous orchid with high commercial value. In this study, we established an efficient particle bombardment transformation system for P. bellina using the protocorm-like-bodies (PLBs) as target tissues. Leaf derived PLBs were proliferated on ½ strength Murashige and Skoog (MS) medium supplemented with 0.8 μM 2,4 dichlorophenoxyacetic acid (2, 4-D). Both physical and biological parameters affecting the transformation system were optimised using the green-fluorescent protein (GFP) and β-glucuronidase (GUS) as reporter systems. Optimal bombardment conditions with 6 cm target tissues distance, 1100 psi acceleration pressure, 1.0 μm gold particle size, 27 mmHg chamber vacuum pressure, single bombardment time, spermidine as DNA precipitation agent, 72 h post bombardment incubation time, 2 μg plasmid DNA in 0.15:0.12 pmol ratio (pSMCHS:p35SGFP) were successfully determined. Surviving PLBs transformants were successfully recovered from the hygromycin selection medium and verified using genomic PCR analysis. The established system is not only useful for a simple and reliable transient gene analysis but as well as generating stable transformants for selective traits improvement in orchids.

PbbHLH4 regulates floral monoterpene biosynthesis in Phalaenopsis orchids.

Floral scent is an important factor in attracting pollinators and repelling florivores. In Phalaenopsis bellina (Orchidaceae), the major floral scent components are monoterpenoids. Previously, we determined that expression of GERANYL DIPHOSPHATE SYNTHASE (PbGDPS) is highly correlated with monoterpene biosynthesis in Phalaenosis orchids. Here, we found that both cis- and trans-regulation were present on the GDPS promoters, with trans-regulation playing a key role. To investigate the regulation of biosynthesis of floral scent, we compared the transcriptomic data of two Phalaenopsis orchids with contrasting scent phenotypes. Eight transcription factors (TFs) that exhibited sequential elevation in abundance through floral development in P. bellina were identified, and their transcript levels were higher in the scented orchid than the scentless one. Five of these TFs transactivated several structural genes involved in monoterpene biosynthesis including PbbHLH4, PbbHLH6, PbbZIP4, PbERF1, and PbNAC1. Ectopic transient expression of each of these TFs in scentless orchids resulted in stimulation of terpenoid biosynthesis. PbbHLH4 most profoundly induced monoterpene biosynthesis, with a 950-fold increase of monoterpenoid production in the scentless orchid. In conclusion, we determined that biosynthesis of orchid floral monoterpenes was sequentially regulated, with PbbHLH4 playing a crucial role for monoterpene biosynthesis.

Highly Efficient Proliferation and Regeneration of Protocorm-like Bodies (PLBS) of the Threatened Orchid, Phalaenopsis bellina

Phalaenopsis bellina is an important indigenous fragrant orchid threatened with extinction. In this study, we evaluated the effect of medium strength, sucrose, nitrogen (NH4NO3) and potato extract on proliferation of P. bellina protocorm-like bodies (PLBs) to improve micropropagation in this species. Optimal treatment for PLBs proliferation rate with an average fresh weight (FW) of 0.97±0.16 g was obtained through culturing on half strength (½) MS medium containing 20 g/L sucrose, 15 mM NH4NO3 and 20% w/v potato extract supplemented with 0.8 μM 2,4 dichlorophenoxyacetic acid (2,4-D). The optimal treatment produced large, healthy and greenish PLBs with reduction in the occurrence of culture browning. In contrast, treatments with high potato extract (>20% w/v) or NH4NO3 (>30 mM) concentrations tend to have inhibitory effect and resulted in low PLBs proliferation rate, with an average FW of 0.77±0.15 g and 0.69±0.15 g, respectively. Plant regeneration of PLBs was achieved on plant growth regulator (PGR)-free ½ MS medium. In total, 60 healthy PLBs from the optimal treatment were successfully regenerated, acclimatized with 100% survival percentage and grew well in a mixture of soil, sand and vermicompost (8:4:2 (w/w/w)). With the optimal treatment, PLBs proliferation rate was enhanced by 27.63%. Our findings offer an improved micropropagation protocol of the endangered P. bellina for conservation and commercial production.

A Dual Repeat Cis-Element Determines Expression of GERANYL DIPHOSPHATE SYNTHASE for Monoterpene Production in Phalaenopsis Orchids.

Phalaenopsis bellina is a scented orchid emitting large amount of monoterpenes. GERANYL DIPHOSPHATE SYNTHASE (PbGDPS) is the key enzyme for monoterpene biosynthesis, and shows concomitant expression with the emission of monoterpenes during flower development in P. bellina. Here, we identified a dual repeat cis-element in the GDPS promoter that is critical for monoterpene biosynthesis in Phalaenopsis orchids. A strong correlation between the dual repeat and the monoterpene production was revealed by examination of the GDPS promoter fragments over 12 Phalaenopsis species. Serial-deletion of the 2-kb GDPS promoter fragments demonstrated that the integrity of the dual repeat was crucial for its promoter activities. By screening the Arabidopsis transcription factors (TFs) cDNA library using yeast one-hybrid assay, AtbZIP18, a member of group I of bZIP TFs, was identified to be able to bind the dual repeat. We then identified PbbZIP4 in the transcriptome of P. bellina, showing 83% identity in the DNA binding region with that of AtbZIP18, and the expression level of PbbZIP4 was higher in the scented orchids. In addition, PbbZIP4 transactivated the GDPS promoter fragment containing the dual repeat in dual luciferase assay. Furthermore, transient ectopic expression of PbbZIP4 induced a 10-fold production of monoterpenoids in the scentless orchid. In conclusion, these results indicate that the dual repeat is a real TF-bound cis-element significant for GDPS gene expression, and thus subsequent monoterpene biosynthesis in the scented Phalaenopsis orchids.

A Defined Culture Medium Suitable for Sensitive Phalaenopsis Species Seedlings

Half strength MS medium supplemented with peptone was found to inhibit growth of considerable number of Phalaenopsis species seedlings and this hamper conservation effort. It is therefore important to design a reproducible defined medium that is not selective and could support a wide range of genotypes. This could be achieved by adjusting the macronutrient concentrations. A new defined medium was designed with nitrate-nitrogen (NO3-N) to ammonium-nitrogen (NH4-N) ratio of 5 and N to S ratio of 15, buffered with 800 mg dm-3 KH2PO4 and NPK ratio of 2.0:0.8:3.1. Mean fresh weight, dry weight and root to shoot ratio was compared with control medium. In contrary to control medium that stunted growth of significant number of Phalaenopsis aphrodite, Phalaenopsis bellina and Phalaenopsis violacea seedlings, the new medium was found to significantly support growth of the seedlings and improved root development. In addition, the new medium was not selective against sensitive seedlings as the data from new medium pass normality test while data from control medium was skewed in P. bellina. This new medium is recommended for maintaining genetic diversity in conservation programs by improving seedling survival and growth in vitro.

Establishment of a short-term storage method via encapsulation of protocorm-like bodies in Phalaenopsis bellina (Rchb. f.) Christenson

Establishment of a short-term storage method via encapsulation of protocorm-like bodies in Phalaenopsis bellina (Rchb. f.) Christenson

In vitro induction and proliferation of protocorm-like bodies (PLBs) from leaf segments of Phalaenopsis bellina (Rchb.f.) Christenson

An in vitro culture procedure was established to induce protocorm-like bodies (PLBs) from leaf segments of the Phalaenopsis bellina (Rchb.f.) Christenson directly from epidermal cells without intervening callus on ½ strength modified Murashige and Skoog (MS) (in Physiol Plant 15:473–497, 1962) medium supplemented with 1-Naphthaleneacetic acid (NAA; 0, 0.1, 1 mg/l) and Thidiazuron (TDZ; 0, 0.1, 1, 3 mg/l). The best response was established at 3 mg/l TDZ which induced 78% of leaf segments to form a mean number of 14 PLBs per explant after 16 weeks of culture. No PLBs were found when leaf segments were cultured on ½ strength modified MS media supplemented with 0.1 and 1 mg/l NAA. The best induction percentage for auxin: cytokinin combination was at the combination of NAA and TDZ at 1.0 and 3.0 mg/l which gave 72% induction with 9 PLBs per explant. Semi-solid ½ strength MS and liquid Vacin and Went (VW) (in Bot Gaz 110:605–613, 1949) medium were used in order to find the highest survival and number of PLBs proliferation after 3 months in culture. Half strength MS showed an average of 9 PLBs in comparison with VW with an average of 5.3 PLBs per explants. Histological observations revealed that the regenerated PLBs were generally formed from the epidermal layers of the posterior regions of the leaf segments. Scanning electron micrograph of PLBs showed the origin of newly formed PLB from the peripheral region of leaf segments.

Cryopreservation of protocorm-like bodies (PLBs) of Phalaenopsis bellina (Rchb.f.) christenson by encapsulation-dehydration

Protocorm-like bodies (PLBs) of Phalaenopsis bellina were successfully cryopreserved by the encapsulation-dehydration approach. Various stages in obtaining successful cryopreservation using this method were optimized. Encapsulated PLBs precultured in half-strength MS medium supplemented with 0.75 M sucrose for 3 days exhibited the highest viability in terms of 2,3,5-triphenyltetrazoliumchloride (TTC) reduction. The amount of sucrose in the PLBs after incubation in different concentrations of sucrose for different periods of time determined by HPLC. The highest sucrose concentration was 7 mg/g of PLBs for the PLBs treated with 0.75 M sucrose for 3 days as compared to the control which had only 1 mg/g sucrose. After sucrose preculture, the PLBs were subjected to desiccation using one of two methods. Desiccation using silica gel was more efficient in reducing PLBs moisture content. After 6 h of desiccation, PLBs desiccated using laminar air flow had 43.5% moisture content while for those desiccated using silica gel had 32% moisture content. PLBs desiccated to different moisture contents were plunged into LN. After storage in LN the encapsulated PLBs were re-warmed. Two weeks after re-warming PLBs viability was determined by TTC reduction and re-growth assessed. Encapsulated PLBs precultured with 0.75 M sucrose for 3 days followed by desiccated using silica gel for 5 h resulting in a moisture content of 39% lead to the highest post re-warming viability in terms of TTC reduction (46.6% of control PLBs) and 30% re-growth.

Phylogenetics and Biogeography of the Phalaenopsis violacea (Orchidaceae) Species Complex Based on Nuclear and Plastid DNA

The Phalaenopsis violacea complex includes two species: P. violacea Witte and Phalaenopsis bellina (Rchb.f.) E. A. Christ. However, three forms of P. violacea have been found in different areas, including Sumatra, the Malay Peninsula, and Mentawai Island. The phylogenetic tree inferred from the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (nrDNA), the trnL intron, and the atpB-rbcL spacer of plastid DNA were used to clarify the phylogenetics and biogeography of the P. violacea complex. Analyses of the trnL intron sequences and of the atpB-rbcL spacer did not allow for apparent discrimination among these three species of the P. violacea complex. Based on the phylogenetic tree inferred from the ITS sequence, P. bellina cannot be separated from populations of P. violacea, with the exception of the population distributed on Mentawai Is., Indonesia. Based on morphological characteristics, P. violacea distributed on Mentawai Is. has a long and roundish rachis and is separate from the other groups of the P. violacea complex described by Christenson (Timber, Portland, OR, 2001). Therefore, the results of this study show a trend that supports the conclusion that the population of the P. violacea complex on Mentawai Is. is a separate species from P. violacea. Based on the biogeography of the P. violacea complex, Mentawai plants of this complex might be descended from those on the Sumatra/Malay Peninsula.

A NOVEL FUNCTIONAL GERANYL DIPHOSPHATE SYNTHASE FOR SCENT PRODUCTION IN PHALAENOPSIS BELLINA FLOWERS

Geranyl diphosphate is the precursor for monoterpenes, the major floral scent in Phalaenopsis bellina. The cDNA of P. bellina GDP synthase (PbGDPS) was cloned, and its sequence contained a glutamate-rich motif (Glu-rich) corresponding to the second aspartate-rich motif (SARM) but not any aspartate-rich motifs (Asp-rich). The recombinant PbGDPS enzyme revealed dual prenyltransferase activities by the production of both GDP and farnesyl diphosphate (FDP). Spatial and temporal expression analyses illustrated that the expression of PbGDPS was flower specific and concomitant with maximal emission of monoterpenes on day 5 post-anthesis. In addition, the expression was strongly related to the intensity of the emitted scent among different plant varieties. This is the first report of a functional GDPS without an Asp-rich motif functioning as a homodimer in a plant (P. bellina, Orchidaceae, monocot).

A novel homodimeric geranyl diphosphate synthase from the orchid Phalaenopsis bellina lacking a DD(X)2–4D motif

Geranyl diphosphate (GDP) is the precursor of monoterpenes, which are the major floral scent compounds in Phalaenopsis bellina. The cDNA of P. bellina GDP synthase (PbGDPS) was cloned, and its sequence corresponds to the second Asp-rich motif (SARM), but not to any aspartate-rich (Asp-rich) motif. The recombinant PbGDPS enzyme exhibits dual prenyltransferase activity, producing both GDP and farnesyl diphosphate (FDP), and a yeast two-hybrid assay and gel filtration revealed that PbGDPS was able to form a homodimer. Spatial and temporal expression analyses showed that the expression of PbGDPS was flower specific, and that maximal PbGDPS expression was concomitant with maximal emission of monoterpenes on day 5 post-anthesis. Homology modelling of PbGDPS indicated that the Glu-rich motif might provide a binding site for Mg(2+) and catalyze the formation of prenyl products in a similar way to SARM. Replacement of the key Glu residues with alanine totally abolished enzyme activity, whereas their mutation to Asp resulted in a mutant with two-thirds of the activity of the wild-type protein. Phylogenetic analysis indicated that plant GDPS proteins formed four clades: members of both GDPS-a and GDPS-b clades contain Asp-rich motifs, and function as homodimers. In contrast, proteins in the GDPS-c and GDPS-d clades do not contain Asp-rich motifs, but although members of the GDPS-c clade function as heterodimers, PbGDPS, which is more closely related to the GDPS-c clade proteins than to GDPS-a and GDPS-b proteins, and is currently the sole member of the GDPS-d clade, functions as a homodimer.