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.

Polyketide synthases (PKSs) of secondary metabolism: in silico identification and characterization in orchids.

Type III polyketide synthases (PKSs) catalyse the formation of an array of polyketides with diverse structures that play an important role in secondary metabolism in plants. This group of enzymes is encoded by a multigene family, the Type III polyketide synthase (PKS) gene family. Vast reserves of secondary metabolites in orchids make these plants suitable candidates for research in the area. In this study, genome-wide searches lead to the identification of five PeqPKS, eight DcaPKS and six AshPKS genes in Phalaenopsis equestris, Dendrobium catenatum and Apostasia shenzhenica, respectively. All the members showed the presence of two characteristic conserved domains (Chal_sti_synt_N and Chal_sti_synt_C) and were generally localised in the cytoplasm. The phylogenetic analysis led to the classification of these proteins into two groups: CHS (chalcone synthase (CHS) and non-CHS. A single protein in P. equestris and two proteins each in D. catenatum and A. shenzhenica clustered within the CHS clade. The majority of the genes exhibited similar structural patterns with a single intron. Expression profiling revealed the tissue-specific expression of these genes with high expression in reproductive tissues for most genes. A number of stress-responsive cis-regulatory elements were predicted, noteworthy amongst these are, ABRE and CGTCA that are chiefly responsible for responding to abscisic acid and methyl jasmonate, respectively. Our study provides a reference framework for future studies involving functional elucidation of PKS genes and biotechnological production of polyketides.Communicated by Ramaswamy H. Sarma.

Identification of five PeqPKS, eight DcaPKS and six AshPKS genes in Phalaenopsis equestris, Dendrobium catenatum and Apostasia shenzhenica based on genome-wide analysisPresence of characteristic conserved domains (Chal_sti_synt_N, Chal_sti_synt_C) with cytological localisationPhylogenetic clustering into two groups, CHS chalcone synthase (CHS) and non-CHSExpression profiling revealing high expression in reproductive tissuesPrediction of stress-responsive cis-regulatory elements like ABRE and CGTCA.

A Walk Through the Maze of Secondary Metabolism in Orchids: A Transcriptomic Approach.

Orchids have a huge reservoir of secondary metabolites making these plants of immense therapeutic importance. Their potential as curatives has been realized since times immemorial and are extensively studied for their medicinal properties. Secondary metabolism is under stringent genetic control in plants and several molecular factors are involved in regulating the production of the metabolites. However, due to the complex molecular networks, a complete understanding of the specific molecular cues is lacking. High-throughput omics technologies have the potential to fill up this lacuna. The present study deals with comparative analysis of high-throughput transcript data involving gene identification, functional annotation, and differential expression in more than 30 orchid transcriptome data sets, with a focus to elucidate the role of various factors in alkaloid and flavonoid biosynthesis. Comprehensive analysis of the mevalonate (MVA) pathway, methyl-d-erythritol 4-phosphate (MEP) pathway, and phenylpropanoid pathway provide specific insights to the potential gene targets for drug discovery. It is envisaged that a positive stimulation of these pathways through regulation of pivotal genes and alteration of specific gene expression, could facilitate the production of secondary metabolites and enable efficient tapping of the therapeutic potential of orchids. This further would lay the foundation for developing strategies for genetic and epigenetic improvement of these plants for development of therapeutic products.

Genome-wide characterization and expression profiling of the Phospholipase C (PLC) gene family in three orchids of economic importance.

BACKGROUND: Phospholipases hydrolyze glycerophospholipids and generate diverse lipid-derived molecules with secondary messenger activity. Out of these, phospholipase C (PLC) specifically cleaves the phospholipids at ester linkages and yields diacylglycerol (DAG) and phosphorylated head groups. PLCs are classified further as phosphatidylinositol-specific PLCs (PI-PLCs) and non-specific PLCs with biased specificity for phosphatidylcholine (NPC/PC-PLC). RESULTS: In the present report, we identified and characterized PLC genes in the genomes of three orchids, Phalaenopsis equestris (seven PePLCs), Dendrobium catenatum (eight DcPLCs), and Apostasia shenzhenica (seven AsPLCs). Multiple sequence alignment analysis confirmed the presence of conserved X and Y catalytic domains, calcium/lipid-binding domain (C2 domain) at the C terminal region, and EF-hand at the N-terminal region in PI-PLC proteins and esterase domain in PC-PLC. Systematic phylogenetic analysis established the relationship of the PLC protein sequences and clustered them into two groups (PI-PLC and PC-PLC) along with those of Arabidopsis thaliana and Oryza sativa. Gene architecture studies showed the presence of nine exons in all PI-PLC genes while the number varied from one to five in PC-PLCs. RNA-seq-based spatio-temporal expression profile for PLC genes was generated, which showed that PePC-PLC1, PePC-PLC2A, DcPC-PLC1A, DcPC-PLC1B, DcPC-PLC2, DcPC-PLC1B, and AsPC-PLC1 had significant expression in all reproductive and vegetative tissues. The expression profile is matched to their upstream cis-regulatory promoter elements, which indicates that PLC genes have a role in various growth and development processes and during stress responses. CONCLUSIONS: The present study unwrapped the opportunity for functional characterization of selected PLC genes in planta for plant improvement.

Genome wide characterization of the SERK/SERL gene family in Phalaenopsis equestris, Dendrobium catenatum and Apostasia shenzhenica (Orchidaceae).

Somatic embryogenesis receptor kinases (SERKs) play a significant role in morphogenesis, stress/defense and signal transduction. In the present study, we have identified two SERK and 11 SERK-like (SERL) genes in Phalaenopsis equestris, two SERK and 11 SERL genes in Dendrobium catenatum, and one SERK and eight SERL genes in Apostasia shenzhenica genome. Characterization of the SERK proteins revealed the presence of a signal peptide, a leucine zipper, five leucine-rich repeats (LRRs), a serine proline proline (SPP) motif, a transmembrane region, a kinase domain, and a C-terminus. Most of the SERK/SERL proteins were characterized with similar physicochemical properties. The presence of transmembrane region predicted their membranous localization. Tertiary structure prediction of all the five identified SERK proteins had sequence identity with BAK1 protein of Arabidopsis thaliana. Generally, all the SERK/SERL genes shared similar gene architecture and intron phasing. Gene ontology analysis indicated the role of SERKs in receptor and ATP binding, signal transduction, and protein phosphorylation. Phylogenetic analysis revealed the clustering of SERKs and SERLs in distinct clades. Expression of SERKs in reproductive tissues like floral bud, floral stalk, whole flower and pollen was reported to be higher than their expression in vegetative tissues with an exception of PeSERK1 and DcSERK1 which showed higher expression in leaves and roots, respectively. Likewise, a higher expression of AsSERK1 was observed in tubers. However, lower expression of SERLs was observed in majority of tissues studied irrespective of their vegetative or reproductive origin. This work paves way for future studies involving functional characterization of SERK/SERLs and their potential role in embryogenesis/organogenesis as an aid to regeneration and multiplication of endangered orchids.

Gene architecture and expression analyses provide insights into the role of glutathione peroxidases (GPXs) in bread wheat (Triticum aestivum L.).

Glutathione peroxidases (GPXs) are redox sensor proteins that maintain a steady-state of H2O2 in plant cells. They exhibit distinct sub-cellular localization and have diverse functionality in response to different stimuli. In this study, a total of 14 TaGPX genes and three splice variants were identified in the genome of Triticum aestivum and evaluated for various physicochemical properties. The TaGPX genes were scattered on the various chromosomes of the A, B, and D sub-genomes and clustered into five homeologous groups based on high sequence homology. The majority of genes were derived from the B sub-genome and localized on chromosome 2. The intron-exon organization, motif and domain architecture, and phylogenetic analyses revealed the conserved nature of TaGPXs. The occurrence of both development-related and stress-responsive cis-acting elements in the promoter region, the differential expression of these genes during various developmental stages, and the modulation of expression in the presence of biotic and abiotic stresses suggested their diverse role in T. aestivum. The majority of TaGPX genes showed higher expression in various leaf developmental stages. However, TaGPX1-A1 was upregulated in the presence of each abiotic stress treatment. A co-expression analysis revealed the interaction of TaGPXs with numerous development and stress-related genes, which indicated their vital role in numerous biological processes. Our study revealed the opportunities for further characterization of individual TaGPX proteins, which might be useful in designing future crop improvement strategies.