Three complete chloroplast genomes from two north American Rhus species and phylogenomics of Anacardiaceae.

BACKGROUND: The suamc genus Rhus (sensu stricto) includes two subgenera, Lobadium (ca. 25 spp.) and Rhus (ca. 10 spp.). Their members, R. glabra and R. typhina (Rosanae: Sapindales: Anacardiaceae), are two economic important species. Chloroplast genome information is of great significance for the study of plant phylogeny and taxonomy. RESULTS: The three complete chloroplast genomes from two Rhus glabra and one R. typhina accessions were obtained with a total of each about 159k bp in length including a large single-copy region (LSC, about 88k bp), a small single-copy regions (SSC, about 19k bp) and a pair of inverted repeats regions (IRa/IRb, about 26k bp), to form a canonical quadripartite structure. Each genome contained 88 protein-coding genes, 37 transfer RNA genes, eight ribosomal RNA genes and two pseudogenes. The overall GC content of the three genomes all were same (37.8%), and RSCU values showed that they all had the same codon prefers, i.e., to use codon ended with A/U (93%) except termination codon. Three variable hotspots, i.e., ycf4-cemA, ndhF-rpl32-trnL and ccsA-ndhD, and a total of 152-156 simple sequence repeats (SSR) were identified. The nonsynonymous (Ka)/synonymous (Ks) ratio was calculated, and cemA and ycf2 genes are important indicators of gene evolution. The phylogenetic analyses of the family Anacardiaceae showed that the eight genera were grouped into three clusters, and supported the monophyly of the subfamilies and all the genera. The accessions of five Rhus species formed four clusters, while, one individual of R. typhina grouped with the R. glabra accessions instead of clustering into the two other individuals of R. typhina in the subgenus Rhus, which showed a paraphyletic relationship. CONCLUSIONS: Comparing the complete chloroplast genomes of the Rhus species, it was found that most SSRs were A/T rich and located in the intergenic spacer, and the nucleotide divergence exhibited higher levels in the non-coding region than in the coding region. The Ka/Ks ratio of cemA gene was > 1 for species collected in America, while it was < 1 for other species in China, which dedicated that the Rhus species from North America and East Asia have different evolutionary pressure. The phylogenetic analysis of the complete chloroplast genome clarified the Rhus placement and relationship. The results obtained in this study are expected to provide valuable genetic resources to perform species identification, molecular breeding, and intraspecific diversity of the Rhus species.

A chromosome-level genome assembly of the Rhus gall aphid Schlechtendalia chinensis provides insight into the endogenization of Parvovirus-like DNA sequences.

The Rhus gall aphid, Schlechtendalia chinensis, feeds on its primary host plant Rhus chinensis to induce galls, which have economic importance in medicines and the food industry. Rhus gall aphids have a unique life cycle and are economically beneficial but there is huge gap in genomic information about this group of aphids. Schlechtendalia chinensis induces rich-tannin galls on its host plant and is emerging as a model organism for both commercial applications and applied research in the context of gall production by insects. Here, we generated a high-quality chromosome-level assembly for the S. chinensis genome, enabling the comparison between S. chinensis and non-galling aphids. The final genome assembly is 344.59 Mb with 91.71% of the assembled sequences anchored into 13 chromosomes. We predicted 15,013 genes, of which 14,582 (97.13%) coding genes were annotated, and 99% of the predicted genes were anchored to the 13 chromosomes. This assembly reveals the endogenization of parvovirus-related DNA sequences (PRDs) in the S. chinensis genome, which could play a role in environmental adaptations. We demonstrated the characterization and classification of cytochrome P450s in the genome assembly, which are functionally crucial for sap-feeding insects and have roles in detoxification and insecticide resistance. This genome assembly also revealed the whole genome duplication events in S. chinensis, which can be considered in comparative evolutionary analysis. Our work represents a reference genome for gall-forming aphids that could be used for comparative genomic studies between galling and non-galling aphids and provides the first insight into the endogenization of PRDs in the genome of galling aphids. It also provides novel genetic information for future research on gall-formation and insect-plant interactions.

Comparative genetic diversity and structure of Rhus gall aphid Schlechtendalia chinensis and its host plant Rhus chinensis.

Investigating the population genetic structure of parasites and their host plants can provide valuable insights into their coevolutionary processes. In this study, we assessed and compared the population genetic diversity and structure of 12 Rhus gall aphid (Schlechtendalia chinensis) populations and their respective host plant (Rhus chinensis) using amplified fragment length polymorphism (AFLP) markers. Analysis of molecular variance (AMOVA) revealed that both the aphid and its host plant exhibited higher genetic variance within populations than among them, indicating that their coevolutionary history may have produced analogous patterns of population genetic structure. Additionally, we considered alternative factors that could contribute to this outcome, such as intraspecific gene flow, hybridization, or environmental influences. Our analysis did not reveal a significant correlation between genetic and geographic distances of either the aphid or host plant populations, leading us to reject the isolation-by-distance model as a plausible explanation for the demographic histories of these two species.

Genome-wide identification and characterization of the chemosensory relative protein genes in Rhus gall aphid Schlechtendalia chinensis.

BACKGROUND: The Rhus gall aphid Schlechtendalia chinensis specially uses the only species Rhus chinensis and certain moss species (Mniaceae) as its primary host plant and secondary host plants, respectively. Rhus galls are formed on the primary host by the sucking of aphids, and used in traditional medicine as well as other various areas due to their high tannin contents. Chemoreception is critical for insect behaviors such as host searching, location and identification of mates and reproductive behavior. The process of chemoreception is mediated by a series of protein gene families, including odorant-binding proteins (OBPs), chemosensory proteins (CSPs), olfactory receptors (ORs), gustatory receptors (GRs), ionotropic receptors (IRs), and sensory neuron membrane proteins (SNMPs). However, there have been no reports on the analysis of molecular components related to the chemoreception system of S. chinensis at the genome level. RESULTS: We examined the genes of eight OBPs, nine CSPs, 24 ORs, 16 GRs, 22 IRs, and five SNMPs in the S. chinensis genome using homological searches, and these chemosensory genes appeared mostly on chromosome 1. Phylogenetic and gene number analysis revealed that the gene families, e.g., ORs, GRs, CSPs and SNMPs in S. chinensis, have experienced major contractions by comparing to Myzus persicae, while the two gene families OBPs and IRs had slight expansion. The current results might be related to the broader host range of M. persicae versus the specialization of S. chinensis on only a host plant. There were 28 gene pairs between genomes of S. chinensis and Acyrthosiphon pisum in the chemoreceptor gene families by collinear comparison. Ka/Ks ratios (< 1) indicated that the genes of S. chinensis were mainly affected by purification selection during evolution. We also found the lower number and expression level of chemoreception genes in S. chinensis than in other 11 aphid species, such as ORs, GRs and IRs, which play an important role in host search. CONCLUSION: Our study firstly identified the genes of the different chemosensory protein gene families in the S. chinensis genome, and analyzed their general features and expression profile, demonstrating the importance of chemoreception in the aphid and providing new information for further functional research.

Relative quantification of phenolic compounds in exocarp-mesocarp and endocarp of sumac (Toxicodendron vernicifluum) combined with transcriptome analysis provides insights into glycosylation of flavonoids and biflavonoid biosynthesis.

The pericarp of fruit can be differentiated into endocarp, mesocarp, and exocarp. To explore the differences in gene expression and metabolites in different tissues of the pericarp, the fruits of sumac (Toxicodendron vernicifluum) were separated into endocarp and mesocarp-exocarp. The metabolites and transcriptome of exocarp-mesocarp and endocarp of Toxicodendron vernicifluum were analyzed by HPLC-QTOF-MS/MS and RNA sequencing, respectively. A total of 52 phenolic compounds were identified, including 3 phenylpropane derivatives, 10 urushiol compounds and 39 flavonoids. The exocarp-mesocarp contained more urushiol compounds and flavonoid glycosides while the endocarp contained more biflavonoids, such as rhusflavone and dihydromorelloflavone. The characteristic component of endocarp was rhusflavone and the characteristic component of exocarp-mesocarp was urushiol (triene). Most of the genes involved in flavonoid synthesis pathway were upregulated in endocarp compared with exocarp-mesocarp and positively correlated with the content of flavonoids. The candidate genes related to the synthesis of components of flavonoid glycosides and biflavonoids were screened. Metabolomic and transcriptomic analyses provide new insights into the synthesis and distribution of flavonoid glycosides and biflavonoids in the fruits of Toxicodendron vernicifluum.

Mobilome of the Rhus Gall Aphid Schlechtendalia chinensis Provides Insight into TE Insertion-Related Inactivation of Functional Genes.

Transposable elements (TEs) comprise a considerable proportion of insect genomic DNA; how they contribute to genome structure and organization is still poorly understood. Here, we present an analysis of the TE repertoire in the chromosome-level genome assembly of Rhus gall aphid Schlechtendalia chinensis. The TE fractions are composed of at least 32 different superfamilies and many TEs from different families were transcriptionally active in the S. chinensis genome. Furthermore, different types of transposase-derived proteins were also found in the S. chinensis genome. We also provide insight into the TEs related insertional inactivation, and exogenization of TEs in functional genes. We considered that the presence of TE fragments in the introns of functional genes could impact the activity of functional genes, and a large number of TE fragments in introns could lead to the indirect inactivation of functional genes. The present study will be beneficial in understanding the role and impact of TEs in genomic evolution of their hosts.

Variation among the Complete Chloroplast Genomes of the Sumac Species Rhus chinensis: Reannotation and Comparative Analysis.

The sumac Rhus chinensis Mill. is an economically and ecologically important shrub or tree species in the family of Anacardiaceae with a wide distribution in East to Southeast Asia. We assembled the complete chloroplast genome of 159,187 bp in length and the GC content of 37.8%. The genome encoded 132 genes, including 86 protein-coding genes, 37 tRNA genes, 8 rRNA genes, and 1 pseudogene, and 77 SSRs were identified as well as the interval regions, totaling 46,425 bp in length. The mauve alignment revealed one gene rearrangement among the Rhus species. All the SSRs were divided into five types, most of which consisted of mono- and tri- repeat motifs. Our genome exhibited the longest size and more annotated genes compared to the three other genomes of R. chinensis reported in GenBank. We also discovered some relatively highly variable regions in the complete chloroplast genomes of the Rhus species. The ML phylogenetic analysis of the available chloroplast sequences of the Anacardiaceae well supported the monophyly of each tribe and each genus; the tribe Rhoideae was close to the tribe Anacardiaceae with a high support of 100%, and they then grouped with the tribe Spondiadeae. R. chinensis was sister to R. potaninii, and they then grouped with the species R. typhina.

Detoxification Gene Families at the Genome-Wide Level of Rhus Gall Aphid Schlechtendalia chinensis.

The Rhus gall aphid Schlechtendalia chinensis uses the species Rhus chinensis as its primary host plant, on which galls are produced. The galls have medicinal properties and can be used in various situations due to their high tannin content. Detoxification enzymes play significant roles in the insect lifecycle. In this study, we focused on five detoxification gene families, i.e., glutathione-S-transferase (GST), ABC transporter (ABC), Carboxylesterase (CCE), cyto-chrome P450 (CYP), and UDP-glycosyltransferase (UDP), and manually annotated 144 detoxification genes of S. chinensis using genome-wide techniques. The detoxification genes appeared mostly on chromosome 1, where a total of two pair genes were identified to show tandem duplications. There were 38 gene pairs between genomes of S. chinensis and Acyrthosiphon pisum in the detoxification gene families by collinear comparison. Ka/Ks ratios showed that detoxification genes of S. chinensis were mainly affected by purification selection during evolution. The gene expression numbers of P450s and ABCs by transcriptome sequencing data were greater, while gene expression of CCEs was the highest, suggesting they might be important in the detoxification process. Our study has firstly identified the genes of the different detoxification gene families in the S. chinensis genome, and then analyzed their general features and expression, demonstrating the importance of the detoxification genes in the aphid and providing new information for further research.

Comparative mitochondrial genomes of the Rhus gall aphid Kaburagia rhusicola subspecies with variable gall shapes.

Rhus gall aphids (Hemiptera: Aphididae: Eriosomatinae) stimulate the formation of galls on their primary host plants (sumacs: Rhus spp., Anacardiaceae). The shapes of galls are often used as an extended phenotype to identify the aphid species and subspecies. We collected four Rhus galls with conspicuously different shapes formed by Kaburagia rhusicola aphids, whose sequences of the complete mitochondrial genomes (mitogenomes) were obtained by high-throughput sequencing. Each mitogenome was assembled into a circular molecule containing 13 protein-coding genes, two rRNAs, 22 tRNAs and one control region. All the protein-coding genes had a typical ATN initiation codon and TAA termination codon except for cox1 and nad4, which had a single T as stop codon. All the tRNAs could be folded as a typical clover-leaf secondary structure, except for trnS1 lacking a dihydrouridine (DHU) arm. The relative synonymous codon usage and ratio of nonsynonymous to synonymous substitution rates showed that the four K. rhusicola samples were highly similar to the subspecies K. r. ovogallis. The phylogenetic analyses grouped these samples with K. r. ovogallis in a clade sister to K. r. rhusicola. All these molecular analyses demonstrated that our current samples represented one subspecies of Kaburagia rhusicola, i.e., K. r. ovogallis, and the gall shape was variable even at the subspecies level in Kaburagia gall aphids.

Identification of reliable reference genes for quantitative real-time PCR analysis of the Rhus chinensis Mill. leaf response to temperature changes.

Rhus chinensis Mill. (RCM) is the host plant of Galla chinensis, which is valued in traditional medicine. Environmental temperature directly determines the probability of gallnut formation and RCM growth. At present, there is no experiment to systematically analyse the stability of internal reference gene (RG) expression in RCM. In this experiment, leaves that did not form gallnuts were used as the control group, while leaves that formed gallnuts were used as the experimental group. First, we conducted transcriptome experiments on RCM leaves to obtain 45 103 differential genes and functional enrichment annotations between the two groups. On this basis, this experiment established a transcriptional gene change model of leaves in the process of gallnut formation after being bitten by aphids, and RCM reference candidate genes were screened from RNA sequencing (RNA-seq) data. This study is based on RCM transcriptome data and evaluates the stability of 11 potential reference genes under cold stress (4 °C) and heat stress (34 °C), using three statistical algorithms (geNorm, NormFinder, and BestKeeper). The results show that GAPDH1 + PP2A2/UBQ are stable reference genes under heat stress, while GAPDH1 + ACT are the most stable under cold stress. This study is the first to screen candidate reference genes in RCM and could help guide future molecular studies in this genus.

Characterization of Mariner transposons in seven species of Rhus gall aphids.

Transposable elements (TEs), also known as jumping genes, are widely spread in the genomes of insects and play a considerable role in genomic evolution. Mariner/DD34D family belongs to class II transposable elements which is widely spread in the genomes of insects and have considerable role in genomic evolution. Mariner like elements (MLEs) were searched in the genomes of seven species of Rhus gall aphids belonging to six genera. In total, 121 MLEs were detected in the genomes of the seven investigated species of Rhus gall aphids, which showed a wide distribution in both close and distant related species. The sequences of MLEs ranged from 1 to 1.4 kb in length and the structural analysis of the MLEs showed that only five copies were potentially active with intact open reading frame (ORF) and terminal inverted repeats (TIRs). Phylogenetic analysis showed that all the 121 MLE sequences belonged to four subfamilies, i.e., Mauritiana, Drosophila, Vertumana and Irritans, among which Drosophila and Vertumana subfamilies were reported in aphids for the first time. Our present report revealed the diversity and distribution of MLEs in Rhus gall aphid genomes and expanded our understandings on the characterization of transposable elements in aphid genomes, which might be useful as genetic markers and tools and would play an important role in genomic evolution and adaptation of aphids.

Structure, Distribution, Chemical Composition, and Gene Expression Pattern of Glandular Trichomes on the Leaves of Rhus potaninii Maxim.

Rhus potaninii Maxim is an economically and medicinally important tree species in China. It produces galls (induced by aphids) with a high abundance of tannins. Here, we discuss the histology, cellular structures and their distribution, and the macromolecular components of secretive glandular trichomes on the leaves of R. potaninii. A variation in the density of glandular trichomes and tomenta was found between the adaxial and abaxial sides of a leaf in different regions and stages of the leaf. The glandular trichomes on R. potaninii trees comprise a stalk with no cellular structure and a head with 8-15 cells. Based on staining, we found that the secretion of glandular trichomes has many polysaccharides, phenolic compounds, and acidic lipids but very few neutral lipids. The dense glandular trichomes provide mechanical protection for young tissues; additionally, their secretion protects the young tissues from pathogens by a special chemical component. According to transcriptome analysis, we found enhanced biosynthetic and metabolism pathways of glycan, lipids, toxic amino acids, and phenylpropanoids. This shows a similar tendency to the staining. The numbers of differentially expressed genes were large or small; the averaged range of upregulated genes was greater than that of the downregulated genes in most subpathways. Some selectively expressed genes were found in glandular trichomes, responsible for the chitinase activity and pathogenesis-related proteins, which all have antibacterial activity and serve for plant defense. To our knowledge, this is the first study showing the components of the secretion from glandular trichomes on the leaf surface of R. potaninii.

Phytochemical characterization of some sumac (Rhus coriaria L.) genotypes from southern part of turkey.

The present research aimed to study the total phenolics, total anthocyanins, total antioxidants, aroma profile, organic acids, and carbohydrate contents of 15 sumac genotypes selected from Kahramanmaras province of Turkey. Total phenols and anthocyanins were spectrophotometrically assessed. The DPPH method was used to determine the antioxidant capacity of the genotypes. Volatile component profiles were identified by HS-SPME/GC-MS while organic acids and carbohydrates were assessed by HPLC techniques. Total phenolic content of the genotypes varied from 36.38 (46SMC02) to 58.66 mg/g dw (46SMC10). Total anthocyanin content ranged from 10.87 (46SMC12) to 119.74 mg/L (46SMC05). The total antioxidant capacity was in the range of 73.37 (46SMC07) and 77.00% (46SMC06). A total of 26 volatile compounds were distinctly detected from the genotypes: 11 volatile compounds were classified as alcohols, 7 as terpenes, 6 as aldehydes and 2 as ketones. l-ascorbic acid, oxalic acid, citric acid and malic acid volumes were detected in the genotypes and their quantity ranged from 2.13 to 40.3, 1.3 to 2.9, 49.8 to 95.1 and 1360 to 2800 respectively. Sucrose quantity was found to vary between 1.41 (46SMC14) and 5.85% (46SMC01), glucose between the detection limit (46SMC01, 46SMC13 and 46SMC15) and 0.73% (46SMC09), xylose between 8.53 (46SMC14) and 30.17% (46SMC09) and fructose between an undetected value (46SMC09, 46SMC10 and 46SMC11) and 1.93% (46SMC13). The results presented here indicate that sumac fruit is a good source of nutritious compounds that may be used directly as a food source or food additives.

Establishment of an efficient in vitro propagation protocol for Sumac (Rhus coriaria L.) and confirmation of the genetic homogeneity.

The conventional reproduction methods are not efficient for regeneration of Sumac (Rhus coriaria L.). The purpose of this work was to study the micropropagation of R. coriaria using lateral buds as explant in Murashige and Skoog (MS) medium with different concentrations of plant growth regulator (PGRs). Four concentrations of Benzylaminopurine (BAP) in combination with three concentrations of indol-3-butyric acid (IBA) and 1.0 mg/L gibberellic acid (GA3) were tested for establishment and shoot multiplication. For root induction, IBA was used at four levels combined with 0, 0.5 and 1 mg/L of naphthalene acetic acid (NAA) in full and half strength of MS medium. BAP at 2 mg/L with 1 mg/L IBA was best, with 88.88% of establishment. The highest shoot proliferation (12.30 ± 0.30) was obtained in medium fortified with 2 mg/L BAP plus 0.5 mg/L IBA and the highest shoot length (8.50 cm) was obtained at 3 mg/L BAP plus 1 mg/L IBA. The highest rooting (100%) was observed in 1/2-strength MS medium containing 1 mg/L IBA with 0.5 mg/L NAA. In conclusion, an efficient protocol with high rate of proliferation and rooting is described for R. coriaria, which can be used in massive propagation.

[Analysis of the chloroplast genome characteristics of Rhus chinensis by de novo sequencing].

Rhus chinensis is an important economic species, which could provide raw materials for pharmaceutical and industrial dyes. Rhus chinensis is famous for its resistance to drought, cold, and salt. It grows in temperate, warm temperate, and subtropical regions. We report here Rhus chinensis chloroplast genomes by de novo sequencing. The results show that the length of Rhus chinensis was 159 082 bp, exhibiting a typical four-part structure with two single-copy regions (long single copy [LSC] and short single copy [SSC] sections) separated by a pair of inverted repeats (IRs). The length of LSC and SSC was 85 394 bp and 18 663 bp, respectively. The genomes contained 126 genes, including 88 protein encoding genes, 8 rRNA and 30 tRNA genes. In the chloroplast genome, 61.97% of the sequence were gene coding region. In the sequence of gene encoding region, the vast majority of sequences were protein encoding region, accounting for 86.65%, followed by rRNA (10 620 bp, 10.77%) and tRNA (2 540 bp, 2.58%). In Rhus chinensis chloroplast genome, only 8 genes contain introns, all containing 1 intron except ycf3 gene (2 introns). The Rhus chinensis chloroplast genome contains 755 SSR locies. SSR mainly consists of dinucleotide and mononucleotide, accounting for 60% (453) and 28.74% (217) respectively. The clustering results show that Anacardiaceae were closest to Rhus chinensis, followed by Aceraceae and Sapindaceae. This study provides a molecular basis for the classification of Rhus chinensis.

[Cloning,expression and characterization of chalcone isomerase from medicinal plant Chinese sumac (Rhus chinensis)].

Flavonoids are a group of secondary metabolites found in plants. They have many pharmacological functions and play an important role in Chinese sumac( Rhus chinensis),which is a well-known traditional Chinese medicinal plant. Chalcone isomerase( CHI,EC 5. 5. 1. 6) is one of the key enzymes in the flavonoids biosynthesis pathway. In this paper,the full-length c DNA sequence encoding the chalcone isomerase from R. chinensis( designated as Rc CHI) was cloned by RT-PCR and rapid-amplification of c DNA Ends( RACE). The Rc CHI c DNA sequence was 1 058 bp and the open reading frame( ORF) was 738 bp. The ORF predicted to encode a 245-amino acid polypeptide. Rc CHI gene contained an intron and two exons. The sequence alignments revealed Rc CHI shared47. 1%-71. 6% identity with the homologues in other plants. Real-time PCR analysis showed that the total flavonoid levels were positively correlated with tissue-specific expressions of Rc CHI mRNA in different tissues. The recombinant protein was successfully expressed in an Escherichia coli strain with the p GEX-6 P-1 vector. In this paper,the CHI gene was cloned and characterized in the family of Anacardiaceae and will help us to obtain better knowledge of the flavonoids biosynthesis of the flavonoid compounds in R. chinensis.

Allergen­removed Rhus verniciflua Stokes suppresses invasion and migration of pancreatic cancer cells through downregulation of the JAK/STAT and Src/FAK signaling pathways.

Pancreatic cancer is a leading cause of mortality and morbidity worldwide. Due to drug resistance, and the high toxicity and adverse side effects of existing chemotherapeutic drugs, the current treatment of highly aggressive pancreatic cancer is considered inadequate. Allergen­removed Rhus verniciflua Stokes (aRVS) has a strong antiproliferative effect in various cancer cells, and due to its low toxicity, it has emerged as an attractive candidate for cancer treatment. However, the potential use of aRVS as a treatment for pancreatic cancer is relatively unexplored. The present study examined the effects of aRVS on the invasion and migration of pancreatic cancer cells, and identified the molecular mechanisms underlying its anticancer effects. aRVS inhibited the Janus kinase/signal transducer and activator of transcription pathway in pancreatic cancer cells, and decreased the protein expression of mucin 4. In addition, it inhibited the activation of focal adhesion kinase and Src signaling, and decreased the expression of matrix metalloproteinase 9, which may reduce the migration and invasion of pancreatic cancer cells. In conclusion, the present study suggested that aRVS may be a potential treatment for aggressive pancreatic cancer.

Molecular mechanisms of tannin accumulation in Rhus galls and genes involved in plant-insect interactions.

For galling aphids and their hosts, tannins are crucial for plant-insect interactions and for protecting the host plant from herbivory. Due to their peculiar chemical characteristics, tannins from plant galls have been used for medical and chemical purposes for more than 2000 years. In this study, hydrolyzable tannin concentrations in galls increased from gall initiation (38.34% on June 21) to maturation (74.79% on August 8), then decreased gradually thereafter (58.83% on October 12). We identified a total of 81 genes (named as GTS1-81) with putative roles in gallotannin biosynthesis and 22 genes (TS1-22) in condensed tannin biosynthesis. We determined the expression profiles of these genes by real-time PCR over the course of gall development. Multiple genes encoding 1-beta-D-glucosyl transferases were identified, which may play a vital role in gallotannin accumulation in plant galls. This study is the first attempt to examine the molecular basis for the regulation of tannin accumulation in insect gallnuts. The differentially expressed genes we identified may play important roles in both tannin biosynthesis and plant-insect interactions.

Cloning and characterisation of a phenylalanine ammonia-lyase gene from Rhus chinensis.

KEY MESSAGE: The gene and cDNA sequence encoding PAL from Chinese medicinal plant Rhus chinensis were cloned and analyzed, furthermore the biochemical properties, kinetic parameters, differential expression and key sites were studied. Rhus chinensis is a well-known Chinese medicinal plant. Phenylalanine ammonia-lyase (PAL) is the first enzyme of phenylpropanoid pathway. Several recent studies suggested that PAL also play an important role in plant-aphid interaction. In this study, both the cDNA and the genomic sequence encoding PAL from Rhus chinensis (designated as RcPAL) were cloned and analyzed. The 3,833 bp gene contained a 1,342 bp intron and two extrons. The ORF was 2,124 bp and predicted to encode a 707-amino acid polypeptide. The results of real-time PCR showed that RcPAL expressed in all tested tissues and followed the order: stems > young leaves > petioles > roots > seeds > mature leaves. RcPAL was successfully expressed in E. coli with the pET-28a-RcPAL recombinant vector. The recombinant protein exhibited a high level of PAL activity. Biochemical properties and kinetic parameters of recombinant RcPAL were further studied. The results showed that the optimal temperature and pH for RcPAL activity were 45 °C and 9.0, and the K m and K cat values were 7.90 mM and 52.31 s(-1), respectively. The active sites and substrate selectivity site were also investigated with site-directed mutagenesis methods, suggesting that Phe(126) is responsible for the substrate selectivity. To our knowledge, this was the first full-length PAL gene cloned and characterized from the family Anacardiaceae so far.

Kinetic and biochemical properties of high and low redox potential laccases from fungal and plant origin.

The electrochemical studies of laccase-mediator systems are aimed at understanding the mechanism of their redox transformation and their efficiency in both homogeneous and heterogeneous reactions; this topic has paramount application spanning from bleaching of paper pulp and the enzymatic degradation of lignin to the biosensors and biofuel cell development. In this paper four different laccases from Trametes hirsuta (ThL), Trametes versicolor (TvL), Melanocarpus albomyces (r-MaL) and Rhus vernicifera (RvL) were characterized from both biochemical and electrochemical points of view. Two of them (TvL and ThL) are high redox potential and two (RvL and r-MaL) are low redox potential laccases. The outline of this work is focused on the determination of catalytic and bioelectrochemical properties of these four enzymes in homogenous solution as well as immobilized onto electrode surface in the presence of a set of different redox mediators. The results measured in the homogenous reaction system correlated well with those measured with the immobilized enzymes. In addition, they are in good agreement with those reported with reference techniques, suggesting that the electrochemical methods employed in this work can be applied well in place of the traditional techniques commonly used for the kinetic characterization of laccases. These results are also discussed in terms of the known amino acid sequences and three-dimensional (3D) structures of the laccases.