Plastoquinone and Ubiquinone in Plants: Biosynthesis, Physiological Function and Metabolic Engineering

Plastoquinone (PQ) and ubiquinone (UQ) are two important prenylquinones, functioning as electron transporters in the electron transport chain of oxygenic photosynthesis and the aerobic respiratory chain, respectively, and play indispensable roles in plant growth and development through participating in the biosynthesis and metabolism of important chemical compounds, acting as antioxidants, being involved in plant response to stress, and regulating gene expression and cell signal transduction. UQ, particularly UQ10, has also been widely used in people’s life. It is effective in treating cardiovascular diseases, chronic gingivitis and periodontitis, and shows favorable impact on cancer treatment and human reproductive health. PQ and UQ are made up of an active benzoquinone ring attached to a polyisoprenoid side chain. Biosynthesis of PQ and UQ is very complicated with more than thirty five enzymes involved. Their synthetic pathways can be generally divided into two stages. The first stage leads to the biosynthesis of precursors of benzene quinone ring and prenyl side chain. The benzene quinone ring for UQ is synthesized from tyrosine or phenylalanine, whereas the ring for PQ is derived from tyrosine. The prenyl side chains of PQ and UQ are derived from glyceraldehyde 3-phosphate and pyruvate through the 2-C-methyl-D-erythritol 4-phosphate pathway and/or acetyl-CoA and acetoacetyl-CoA through the mevalonate pathway. The second stage includes the condensation of ring and side chain and subsequent modification. Homogentisate solanesyltransferase, 4-hydroxybenzoate polyprenyl diphosphate transferase and a series of benzene quinone ring modification enzymes are involved in this stage. PQ exists in plants, while UQ widely presents in plants, animals and microbes. Many enzymes and their encoding genes involved in PQ and UQ biosynthesis have been intensively studied recently. Metabolic engineering of UQ10 in plants, such as rice and tobacco, has also been tested. In this review, we summarize and discuss recent research progresses in the biosynthetic pathways of PQ and UQ and enzymes and their encoding genes involved in side chain elongation and in the second stage of PQ and UQ biosynthesis. Physiological functions of PQ and UQ played in plants as well as the practical application and metabolic engineering of PQ and UQ are also included

Validation of Suitable Reference Genes for Quantitative Gene Expression Analysis in Panax ginseng

Reverse transcription-qPCR (RT-qPCR) has become a popular method for gene expression studies. Its results require data normalization by housekeeping genes. No single gene is proved to be stably expressed under all experimental conditions. Therefore, systematic evaluation of reference genes is necessary. With the aim to identify optimum reference genes for RT-qPCR analysis of gene expression in different tissues of Panax ginseng and the seedlings grown under heat stress, we investigated the expression stability of eight candidate reference genes, including elongation factor 1-beta (EF1-β), elongation factor 1-gamma (EF1-γ), eukaryotic translation initiation factor 3G (IF3G), eukaryotic translation initiation factor 3B (IF3B), actin (ACT), actin11 (ACT11), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and cyclophilin ABH-like protein (CYC), using four widely used computational programs: geNorm, Normfinder, BestKeeper, and the comparative ΔCt method. The results were then integrated using the web-based tool RefFinder. As a result, EF1-γ, IF3G and EF1-β were the three most stable genes in different tissues of P. ginseng, while IF3G, ACT11 and GAPDH were the top three-ranked genes in seedlings treated with heat. Using three better reference genes alone or in combination as internal control, we examined the expression profiles of MAR, a multiple function-associated mRNA-like non-coding RNA (mlncRNA) in P. ginseng. Taken together, we recommended EF1-γ/IF3G and IF3G/ACT11 as the suitable pair of reference genes for RT-qPCR analysis of gene expression in different tissues of P. ginseng and the seedlings grown under heat stress, respectively. The results serve as a foundation for future studies on P. ginseng functional genomics

Genome-wide analysis, molecular cloning and expression profiling reveal tissue-specifically expressed, feedback-regulated, stress-responsive and alternatively spliced novel genes involved in gibberellin metabolism in Salvia miltiorrhiza

Conserved domains of enzymes involved in gibberellin metabolism in S. miltiorrhiza. Conserved domains of enzymes involved in gibberellin metabolism in S. miltiorrhiza are shown. (DOC 183 kb

Comprehensive transcriptome analysis reveals novel genes involved in cardiac glycoside biosynthesis and mlncRNAs associated with secondary metabolism and stress response in Digitalis purpurea

<p>Abstract</p> <p>Background</p> <p><it>Digitalis purpurea </it>is an important ornamental and medicinal plant. There is considerable interest in exploring its transcriptome.</p> <p>Results</p> <p>Through high-throughput 454 sequencing and subsequent assembly, we obtained 23532 genes, of which 15626 encode conserved proteins. We determined 140 unigenes to be candidates involved in cardiac glycoside biosynthesis. It could be grouped into 30 families, of which 29 were identified for the first time in <it>D. purpurea</it>. We identified 2660 mRNA-like npcRNA (mlncRNA) candidates, an emerging class of regulators, using a computational mlncRNA identification pipeline and 13 microRNA-producing unigenes based on sequence conservation and hairpin structure-forming capability. Twenty five protein-coding unigenes were predicted to be targets of these microRNAs. Among the mlncRNA candidates, only 320 could be grouped into 140 families with at least two members in a family. The majority of <it>D. purpurea </it>mlncRNAs were species-specific and many of them showed tissue-specific expression and responded to cold and dehydration stresses. We identified 417 protein-coding genes with regions significantly homologous or complementary to 375 mlncRNAs. It includes five genes involved in secondary metabolism. A positive correlation was found in gene expression between protein-coding genes and the homologous mlncRNAs in response to cold and dehydration stresses, while the correlation was negative when protein-coding genes and mlncRNAs were complementary to each other.</p> <p>Conclusions</p> <p>Through comprehensive transcriptome analysis, we not only identified 29 novel gene families potentially involved in the biosynthesis of cardiac glycosides but also characterized a large number of mlncRNAs. Our results suggest the importance of mlncRNAs in secondary metabolism and stress response in <it>D. purpurea</it>.</p

Transcriptome-wide identification and characterization of miRNAs from Pinus densata

<p>Abstract</p> <p>Background</p> <p>MicroRNAs (miRNAs) play key roles in diverse developmental processes, nutrient homeostasis and responses to biotic and abiotic stresses. The biogenesis and regulatory functions of miRNAs have been intensively studied in model angiosperms, such as <it>Arabidopsis thaliana</it>, <it>Oryza sativa </it>and <it>Populus trichocarpa</it>. However, global identification of <it>Pinus densata </it>miRNAs has not been reported in previous research.</p> <p>Results</p> <p>Here, we report the identification of 34 conserved miRNAs belonging to 25 miRNA families from a <it>P. densata </it>mRNA transcriptome database using local BLAST and MIREAP programs. The primary and/or precursor sequences of 29 miRNAs were further confirmed by RT-PCR amplification and subsequent sequencing. The average value of the minimal folding free energy indexes of the 34 miRNA precursors was 0.92. Nineteen (58%) mature miRNAs began with a 5' terminal uridine residue. Analysis of miRNA precursors showed that 19 mature miRNAs were novel members of 14 conserved miRNA families, of which 17 miRNAs were further validated by subcloning and sequencing. Using real-time quantitative RT-PCR, we found that the expression levels of 7 miRNAs were more than 2-fold higher in needles than in stems. In addition, 72 <it>P. densata </it>mRNAs were predicted to be targets of 25 miRNA families. Four target genes, including a nodal modulator 1-like protein gene, two GRAS family transcription factor protein genes and one histone deacetylase gene, were experimentally verified to be the targets of 3 <it>P. densata </it>miRNAs, pde-miR162a, pde-miR171a and pde-miR482a, respectively.</p> <p>Conclusions</p> <p>This study led to the discovery of 34 conserved miRNAs comprising 25 miRNA families from <it>Pinus densata</it>. These results lay a solid foundation for further studying the regulative roles of miRNAs in the development, growth and responses to environmental stresses in <it>P. densata</it>.</p

Systematic characterization of gene families and functional analysis of PvRAS3 and PvRAS4 involved in rosmarinic acid biosynthesis in Prunella vulgaris

Prunella vulgaris is an important material for Chinese medicines with rosmarinic acid (RA) as its index component. Based on the chromosome-level genome assembly we obtained recently, 51 RA biosynthesis-related genes were identified. Sequence feature, gene expression pattern and phylogenetic relationship analyses showed that 17 of them could be involved in RA biosynthesis. In vitro enzymatic assay showed that PvRAS3 catalyzed the condensation of p-coumaroyl-CoA and caffeoyl-CoA with pHPL and DHPL. Its affinity toward p-coumaroyl-CoA was higher than caffeoyl-CoA. PvRAS4 catalyzed the condensation of p-coumaroyl-CoA with pHPL and DHPL. Its affinity toward p-coumaroyl-CoA was lower than PvRAS3. UPLC and LC-MS/MS analyses showed the existence of RA, 4-coumaroyl-3’,4’-dihydroxyphenyllactic acid, 4-coumaroyl-4’-hydroxyphenyllactic acid and caffeoyl-4’-hydroxyphenyllactic acid in P. vulgaris. Generation and analysis of pvras3 homozygous mutants showed significant decrease of RA, 4-coumaroyl-3’,4’-dihydroxyphenyllactic acid, 4-coumaroyl-4’-hydroxyphenyllactic acid and caffeoyl-4’-hydroxyphenyllactic acid and significant increase of DHPL and pHPL. It suggests that PvRAS3 is the main enzyme catalyzing the condensation of acyl donors and acceptors during RA biosynthesis. The role of PvRAS4 appears minor. The results provide significant information for quality control of P. vulgaris medicinal materials

Factors influencing householder self-evacuation in two Australian bushfires

The thesis investigated householder self-evacuation decision-making during bushfires in the Perth and Adelaide Hills in 2014 and 2015. It explored the factors that influenced householders&amp;rsquo; decisions to evacuate, identified factors that predict self-evacuation and established the characteristics of self-evacuators. The Protective Action Decision Model (PADM) provided a conceptual framework for the research. Its theoretical and analytical usefulness in an Australian context, was assessed. A mixed methods research strategy was used involving quantitative telephone surveys of 457 bushfire-affected participants and face-to-face interviews of 109 participants in 59 households. The study concluded that environmental and social cues and warnings and householders&amp;rsquo; perceptions of the threat, of hazard adjustments and of other stakeholders, influenced self-evacuation decision-making. Protective action perceptions, particularly the effectiveness of evacuating or not evacuating in protecting personal safety or property, were most important in predicting self-evacuation. Receipt of official warnings and the perception of likely impact of the bushfire on property were also important predictors. Undertaking long-run hazard adjustments, although not predictive of self-evacuation, was pivotal in shaping perceptions of the effectiveness of evacuating and remaining in protecting personal safety and property and indirectly influenced evacuation decisions. Seven archetypes that characterised householders&amp;rsquo; self-evacuation attitudes and behaviour were identified. These included Threat, and Responsibility Deniers, Dependent, and Considered Evacuators, Community Guided and Experienced Independents all who took different decisional &amp;lsquo;rules of thumb&amp;rsquo; and routes toward evacuating or remaining . The PADM needs to be split into two separate models to incorporate the influence of long-run hazard adjustments on protective action decision-making in an Australian bushfire. The findings suggest that future research on those who wait and see during a bushfire should take account of their decisional rules of thumb and that design and targeting of Australian bushfire safety policy should better account for self-evacuator characteristics

Genome-wide identification and characterization of novel genes involved in terpenoid biosynthesis in Salvia miltiorrhiza

Terpenoids are the largest class of plant secondary metabolites and have attracted widespread interest. Salvia miltiorrhiza, belonging to the largest and most widely distributed genus in the mint family, is a model medicinal plant with great economic and medicinal value. Diterpenoid tanshinones are the major lipophilic bioactive components in S. miltiorrhiza. Systematic analysis of genes involved in terpenoid biosynthesis has not been reported to date. Searching the recently available working draft of the S. miltiorrhiza genome, 40 terpenoid biosynthesis-related genes were identified, of which 27 are novel. These genes are members of 19 families, which encode all of the enzymes involved in the biosynthesis of the universal isoprene precursor isopentenyl diphosphate and its isomer dimethylallyl diphosphate, and two enzymes associated with the biosynthesis of labdane-related diterpenoids. Through a systematic analysis, it was found that 20 of the 40 genes could be involved in tanshinone biosynthesis. Using a comprehensive approach, the intron/exon structures and expression patterns of all identified genes and their responses to methyl jasmonate treatment were analysed. The conserved domains and phylogenetic relationships among the deduced S. miltiorrhiza proteins and their homologues isolated from other plant species were revealed. It was discovered that some of the key enzymes, such as 1-deoxy-D-xylulose 5-phosphate synthase, 4-hydroxy-3-methylbut-2-enyl diphosphate reductase, hydroxymethylglutaryl-CoA reductase, and geranylgeranyl diphosphate synthase, are encoded by multiple gene members with different expression patterns and subcellular localizations, and both homomeric and heteromeric geranyl diphosphate synthases exist in S. miltiorrhiza. The results suggest the complexity of terpenoid biosynthesis and the existence of metabolic channels for diverse terpenoids in S. miltiorrhiza and provide useful information for improving tanshinone production through genetic engineering

Identification, Molecular Cloning and Expression Analysis of Five RNA-Dependent RNA Polymerase Genes in Salvia miltiorrhiza

RNA-dependent RNA polymerases (RDRs) act as key components of the small RNA biogenesis pathways and play significant roles in post-transcriptional gene silencing (PTGS) and antiviral defense. However, there is no information about the RDR gene family in Salvia miltiorrhiza, an emerging model medicinal plant with great economic value. Through genome-wide predication and subsequent molecular cloning, five full-length S. miltiorrhiza RDR genes, termed SmRDR1–SmRDR5, were identified. The length of SmRDR cDNAs varies between 3,262 (SmRDR5) and 4,130 bp (SmRDR3). The intron number of SmRDR genes varies from 3 (SmRDR1, SmRDR3 and SmRDR4) to 17 (SmRDR5). All of the deduced SmRDR protein sequences contain the conserved RdRp domain. Moreover, SmRDR2 and SmRDR4 have an additional RRM domain. Based on the phylogenetic tree constructed with sixteen RDRs from Arabidopsis, rice and S. miltiorrhiza, plant RDRs may be divided into four groups (RDR1–RDR4). The RDR1 group contains an AtRDR and an OsRDR, while includes two SmRDRs. On the contrary, the RDR3 group contains three AtRDRs and two OsRDRs, but has only one SmRDR. SmRDRs were differentially expressed in flowers, leaves, stems and roots of S. miltiorrhiza and responsive to methyl jasmonate treatment and cucumber mosaic virus infection. The results suggest the involvement of RDRs in S. miltiorrhiza development and response to abiotic and biotic stresses. It provides a foundation for further studying the regulation and biological functions of SmRDRs and the biogenesi

Identification and characterization of the cytosine-5 DNA methyltransferase gene family in Salvia miltiorrhiza

Cytosine DNA methylation is highly conserved epigenetic modification involved in a wide range of biological processes in eukaryotes. It was established and maintained by cytosine-5 DNA methyltransferases (C5-MTases) in plants. Through genome-wide identification, eight putative SmC5-MTase genes were identified from the genome of Salvia miltiorrhiza, a well-known traditional Chinese medicine material and an emerging model medicinal plant. Based on conserved domains and phylogenetic analysis, eight SmC5-MTase genes were divided into four subfamilies, including MET, CMT, DRM and DNMT2. Genome-wide comparative analysis of the C5-MTase gene family in S. miltiorrhiza and Arabidopsis thaliana, including gene structure, sequence features, sequence alignment and conserved motifs, was carried out. The results showed conservation and divergence of the members of each subfamily in plants. The length of SmC5-MTase open reading frames ranges widely from 1,152 (SmDNMT2) to 5,034 bp (SmMET1). The intron number of SmC5-MTases varies between 7 (SmDRM1) and 20 (SmCMT1 and SmCMT2b). These features were similar to their counterparts from Arabidopsis. Sequence alignment and conserved motif analysis showed the existence of highly conserved and subfamily-specific motifs in the C5-MTases analyzed. Differential transcript abundance was detected for SmC5-MTases, implying genome-wide variance of DNA methylation in different organs and tissues. Transcriptome-wide analysis showed that the transcript levels of all SmC5-MTase genes was slightly changed under yeast extract and methyl jasmonate treatments. Six SmC5-MTases, including SmMET1, SmCMT1, SmCMT2a, SmCMT2b, SmCMT3 and SmDRM1, were salicylic acid-responsive, suggesting the involvement of SmC5-MTases in salicylic acid-dependent immunity. These results provide useful information for demonstrating the role of DNA methylation in bioactive compound biosynthesis and Dao-di herb formation in medicinal plants