Enolase, a cadmium resistance related protein from hyperaccumulator plant Phytolacca americana, increase the tolerance of Escherichia coli to cadmium stress.

Phytolacca americana is a Cd hyperaccumulator plant that accumulates significant amounts of Cd in leaves, making it a valuable phytoremediation plant species. Our previous research found enolase (ENO) may play an important part in P. americana to cope with Cd stress. As a multifunctional enzyme, ENO was involved not only in glycolysis but also in the response of plants to various environmental stresses. However, there are few studies on the function of PaENO (P. americana enolase) in coping with Cd stress. In this study, the PaENO gene was isolated from P. americana, and the expression level of PaENO gene significantly increased after Cd treatment. The enzymatic activity analysis showed PaENO had typical ENO activity, and the 42-position serine was essential to the enzymatic activity of PaENO. The Cd resistance assay indicated the expression of PaENO remarkably enhanced the resistance of E. coli to Cd, which was achieved by reducing the Cd content in E. coli. Moreover, both the expression of inactive PaENO and PaMBP-1 (alternative translation product of PaENO) can improve the tolerance of E. coli to Cd. The results indicated PaENO may be alternatively translated into the transcription factor PaMBP-1 to participate in the response of P. americana to Cd stress.

The expression of the Cd resistance related protein PaENO can significantly increase the tolerance of E. coli to Cd, which was achieved by reducing the content of Cd in E. coli cells, and was independent of the enzymatic activity of PaENO. Moreover, PaENO may be alternatively translated into the transcription factor PaMBP-1 to participate in the response of P. americana to Cd stress.

Full-length transcriptome analysis of Phytolacca americana and its congener P. icosandra and gene expression normalization in three Phytolaccaceae species.

BACKGROUND: Phytolaccaceae species in China are not only ornamental plants but also perennial herbs that are closely related to human health. However, both large-scale full-length cDNA sequencing and reference gene validation of Phytolaccaceae members are still lacking. Therefore, single-molecule real-time sequencing technology was employed to generate full-length transcriptome in invasive Phytolacca americana and non-invasive exotic P. icosandra. Based on the transcriptome data, RT-qPCR was employed to evaluate the gene expression stability in the two plant species and another indigenous congener P. acinosa. RESULTS: Total of 19.96 Gb and 19.75 Gb clean reads of P. americana and P. icosandra were generated, including 200,857 and 208,865 full length non-chimeric (FLNC) reads, respectively. Transcript clustering analysis of FLNC reads identified 89,082 and 98,448 consensus isoforms, including 86,989 and 96,764 high-quality ones. After removing redundant reads, 46,369 and 50,220 transcripts were obtained. Based on structure analysis, total 1675 and 1908 alternative splicing variants, 25,641 and 31,800 simple sequence repeats (SSR) as well as 34,971 and 36,841 complete coding sequences were detected separately. Furthermore, 3574 and 3833 lncRNA were predicted and 41,676 and 45,050 transcripts were annotated respectively. Subsequently, seven reference genes in the two plant species and a native species P. acinosa were selected and evaluated by RT-qPCR for gene expression analysis. When tested in different tissues (leaves, stems, roots and flowers), 18S rRNA showed the highest stability in P. americana, whether infested by Spodoptera litura or not. EF2 had the most stable expression in P. icosandra, while EF1-α was the most appropriate one when attacked by S. litura. EF1-α showed the highest stability in P.acinosa, whereas GAPDH was recommended when infested by S. litura. Moreover, EF1-α was the most stable one among the three plant species whenever germinating seeds or flowers only were considered. CONCLUSION: Full-length transcriptome of P. americana and P. icosandra were produced individually. Based on the transcriptome data, the expression stability of seven candidate reference genes under different experimental conditions was evaluated. These results would facilitate further exploration of functional and comparative genomic studies in Phytolaccaceae and provide insights into invasion success of P. americana.

Prediction and Characterization of miRNA/Target Pairs in Non-Model Plants Using RNA-seq.

Plant microRNAs (miRNAs) are ∼20- to 24-nucleotide small RNAs that post-transcriptionally regulate gene expression of mRNA targets. Here, we present a workflow to characterize the miRNA transcriptome of a non-model plant, focusing on miRNAs and targets that are differentially expressed under one experimental treatment. We cover RNA-seq experimental design to create paired small RNA and mRNA libraries and perform quality control of raw data, de novo mRNA transcriptome assembly and annotation, miRNA prediction, differential expression, target identification, and functional enrichment analysis. Additionally, we include validation of differential expression and miRNA-induced target cleavage using qRT-PCR and modified RNA ligase-mediated 5' rapid amplification of cDNA ends, respectively. Our procedure relies on freely available software and web resources. It is intended for users that lack programming skills but can navigate a command-line interface. To enable an understanding of formatting requirements and anticipated results, we provide sample RNA-seq data and key input/output files for each stage. © 2019 The Authors. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

Isolation and expression analysis of cadmium-induced genes from Cd/Mn hyperaccumulator Phytolacca americana in response to high Cd exposure.

Phytolacca americana is recognised as a hyperaccumulator that accumulates cadmium (Cd) and manganese (Mn). Although most studies have provided abundant physiological evidence, little is known about the molecular mechanisms of Cd accumulation in P. americana. In this study, Cd-induced genes were isolated using suppression subtractive hybridisation (SSH) library construction, and gene expression patterns under Cd stress were quantified using real-time quantitative PCR. The functions of PaGST, PaMT and PaFe-SOD were confirmed in transformant yeast. Reactive oxygen species (ROS) formation and cell death in root tips were detected, and SOD and POD activities in leaf tissue were also analysed. There were about 447 expressed sequence tags (ESTs) identified and confirmed. GO analysis showed those genes were mainly involved in metabolism, cell stress and defence, transcription and translation, signal transduction, transport, energy and ion transport, which formed the basis for a molecular understanding of P. americana Cd tolerance mechanisms. Cd also stimulated ROS formation and modified the antioxidant systems. Taken together, our results indicate that ROS formation and Cd-induced gene expression favour P. americana tolerance by activating the defence system and permitting subsequent adaptation to Cd toxicity.

Diversity in cadmium accumulation and resistance associated with various metallothionein genes (type III) in Phytolacca americana L.

Metallothioneins (MTs) are known for their heavy metal deoxidation during phytoremediation. To estimate their roles in the cadmium (Cd) hyperaccumulator Phytolacca americana L., three MT genes, PaMT3-1, PaMT3-2 and PaMT3-3, belonging to the MT3 subfamily were cloned. They separately encoded 63, 65 and 65 amino acids, containing12, 10 and 11 cysteines (Cys), respectively. Each gene was individually transformed and expressed in Escherichia coli cells. A Cd-resistance assay showed that the recombinant strains had enhanced survival rates, especially those containing PaMT3-1 and PaMT3-3. Additionally, the recombinant strains were high Cd accumulators, with the recombinant PaMT3-1's maximum accumulation being 2.16 times that of the empty vector strains. The numbers of cysteines and the structures of MT proteins were associated with the Cd enrichment and resistance capabilities. PaMT3-1 could be an effective gene resource in future plant Cd remediation-related breeding programs.

Transcriptome analysis of Phytolacca americana L. in response to cadmium stress.

Phytolacca americana L. (pokeweed) has metal phytoremediation potential, but little is known about its metal accumulation-related genes. In this study, the de novo sequencing of total RNA produced 53.15 million reads covering 10.63 gigabases of transcriptome raw data in cadmium (Cd)-treated and untreated pokeweed. Of the 97,502 assembled unigenes, 42,197 had significant matches in a public database and were annotated accordingly. An expression level comparison between the samples revealed 1515 differentially expressed genes (DEGs), 923 down- and 592 up-regulated under Cd treatment. A KEGG pathway enrichment analysis of DEGs revealed that they were involved in 72 metabolism pathways, with photosynthesis, phenylalanine metabolism, ribosome, phenylpropanoid biosynthesis, flavonoid biosynthesis and carbon fixation in photosynthetic organisms containing 24, 18, 72, 14, 7 and 15 genes, respectively. Genes related to heavy metal tolerance, absorption, transport and accumulation were also identified, including 11 expansins, 8 nicotianamine synthases, 6 aquaporins, 4 ZRT/IRT-like proteins, 3 ABC transporters and 3 metallothioneins. The gene expression results of 12 randomly selected DEGs were validated using quantitative real-time PCR, and showed different response patterns to Cd in their roots, stems and leaves. These results may be helpful in increasing our understanding of heavy metal hyperaccumulators and in future phytoremediation applications.

A small RNA targets pokeweed antiviral protein transcript.

Ribosome-inactivating proteins (RIPs) are a class of plant defense proteins with N-glycosidase activity (EC 3.2.2.22). Pokeweed antiviral protein (PAP) is a Type I RIP isolated from the pokeweed plant, Phytolacca americana, thought to confer broad-spectrum virus resistance in this plant. Through a combination of standard molecular techniques and RNA sequencing analysis, we report here that a small RNA binds and cleaves the open reading frame of PAP mRNA. Additionally, sRNA targeting of PAP is dependent on jasmonic acid (JA), a plant hormone important for defense against pathogen infection and herbivory. Levels of small RNA increased with JA treatment, as did levels of PAP mRNA and protein, suggesting that the small RNA functions to moderate the expression of PAP in response to this hormone. The association between JA and PAP expression, mediated by sRNA299, situates PAP within a signaling pathway initiated by biotic stress. The consensus sequence of sRNA299 was obtained through bioinformatic analysis of pokeweed small RNA sequencing. To our knowledge, this is the first account of a sRNA targeting a RIP gene.

Synthesis of ε-Viniferin Glycosides by Glucosyltransferase from Phytolacca americana and their Inhibitory Activity on Histamine Release from Rat Peritoneal Mast Cells.

Glycosylation of (+)-ε-viniferin was investigated using glucosyltransferase from Phytolacca americana (PaGT3) as a biocatalyst. (+)-ε-Viniferin was converted by PaGT3 into its 4b- and 13b-ß-D-glucosides, the inhibitory activities on histamine release from rat peritoneal mast cells of which were higher than that of (+)-ε-viniferin.

Comparative analysis of two DOPA dioxygenases from Phytolacca Americana.

The comparative analysis of two Phytolacca americana DOPA dioxygenases (PaDOD1 and PaDOD2) that may be involved in betalain biosynthesis was carried out. The recombinant protein of PaDOD catalyzed the conversion of DOPA to betalamic acid, whereas DOD activity was not detected in PaDOD2 in vitro. The role of DOD genes is discussed in the evolutionary context using phylogenetic analysis, suggesting that DOD might have been duplicated early in evolution and that accumulation of base substitutions could have led to the different characteristics of DODs within the betalain-producing Caryophyllales.

Biochemical analysis of Phytolacca DOPA dioxygenase.

The biochemical analysis of Phytolacca americana DOPA dioxygenases (PaDOD1 and PaDOD2) was carried out. The recombinant protein of PaDOD1 catalyzed the conversion of DOPA to betalamic acid, whereas DOD activity was not detected in PaDOD2 in vitro. While the reported motif conserved in DODs from betalain-producing plants was found in PaDOD1, a single amino acid residue alteration was detected in PaDOD2. A mutated PaDOD1 protein with a change of 177 Asn to Gly showed reduced specific activity compared with PaDOD1, while DOPA dioxygenase activity was not observed for a mutated PaDOD2 protein which had its conserved motif replaced with that of PaDOD. A three-dimensional (3D) structural model of PaDOD1 and PaDOD2 showed that the conserved motif in DODs was located in the N-terminal side of a loop, which was found close to the putative active site. The difference in stability of the loop may affect the enzymatic activity of PaDOD2.

Pokeweed antiviral protein: its cytotoxicity mechanism and applications in plant disease resistance.

Pokeweed antiviral protein (PAP) is a 29 kDa type I ribosome inactivating protein (RIP) found in pokeweed plants. Pokeweed produces different forms of PAP. This review focuses on the spring form of PAP isolated from Phytolacca americana leaves. PAP exerts its cytotoxicity by removing a specific adenine from the α-sarcin/ricin loop of the large ribosomal RNA. Besides depurination of the rRNA, PAP has additional activities that contribute to its cytotoxicity. The mechanism of PAP cytotoxicity is summarized based on evidence from the analysis of transgenic plants and the yeast model system. PAP was initially found to be anti-viral when it was co-inoculated with plant viruses onto plants. Transgenic plants expressing PAP and non-toxic PAP mutants have displayed broad-spectrum resistance to both viral and fungal infection. The mechanism of PAP-induced disease resistance in transgenic plants is summarized.

Inhibition of pokeweed antiviral protein (PAP) by turnip mosaic virus genome-linked protein (VPg).

Pokeweed antiviral protein (PAP) from Phytolacca americana is a ribosome-inactivating protein (RIP) and an RNA N-glycosidase that removes specific purine residues from the sarcin/ricin loop of large rRNA, arresting protein synthesis at the translocation step. PAP is also a cap-binding protein and is a potent antiviral agent against many plant, animal, and human viruses. To elucidate the mechanism of RNA depurination, and to understand how PAP recognizes and targets various RNAs, the interactions between PAP and turnip mosaic virus genome-linked protein (VPg) were investigated. VPg can function as a cap analog in cap-independent translation and potentially target PAP to uncapped IRES-containing RNA. In this work, fluorescence spectroscopy and HPLC techniques were used to quantitatively describe PAP depurination activity and PAP-VPg interactions. PAP binds to VPg with high affinity (29.5 nm); the reaction is enthalpically driven and entropically favored. Further, VPg is a potent inhibitor of PAP depurination of RNA in wheat germ lysate and competes with structured RNA derived from tobacco etch virus for PAP binding. VPg may confer an evolutionary advantage by suppressing one of the plant defense mechanisms and also suggests the possible use of this protein against the cytotoxic activity of ribosome-inactivating proteins.

Regioselective glucosidation of trans-resveratrol in Escherichia coli expressing glucosyltransferase from Phytolacca americana.

A glucosyltransferase (GT) of Phytolacca americana (PaGT3) was expressed in Escherichia coli and purified for the synthesis of two O-ß-glucoside products of trans-resveratrol. The reaction was moderately regioselective with a ratio of 4'-O-ß-glucoside: 3-O-ß-glucoside at 10:3. We used not only the purified enzyme but also the E. coli cells containing the PaGT3 gene for the synthesis of glycoconjugates. E. coli cell cultures also have other advantages, such as a shorter incubation time compared with cultured plant cells, no need for the addition of exogenous glucosyl donor compounds such as UDP-glucose, and almost complete conversion of the aglycone to the glucoside products. Furthermore, a homology model of PaGT3 and mutagenesis studies suggested that His-20 would be a catalytically important residue.

Cd-induced changes in leaf proteome of the hyperaccumulator plant Phytolacca americana.

Cadmium (Cd) is highly toxic to all organisms. Soil contamination by Cd has become an increasing problem worldwide due to the intensive use of Cd-containing phosphate fertilizers and industrial zinc mining. Phytolacca americana L. is a Cd hyperaccumulator plant that can grow in Cd-polluted areas. However, the molecular basis for its remarkable Cd resistance is not known. In this study, the effects of Cd exposure on protein expression patterns in P.americana was investigated by 2-dimensional gel electrophoresis (2-DE). 2-DE profiles of leaf proteins from both control and Cd-treated (400µM, 48h) seedlings were compared quantitatively using ImageMaster software. In total, 32 differentially expressed protein spots were identified using MALDI-TOF/TOF mass spectrometry coupled to protein database search, corresponding to 25 unique gene products. Of those 14 were enhanced/induced while 11 reduced under Cd treatment. The alteration pattern of protein expression was verified for several key proteins involved in distinct metabolic pathways by immuno-blot analysis. Major changes were found for the proteins involved in photosynthetic pathways as well as in the sulfur- and GSH-related metabolisms. One-third of the up-regulated proteins were attributed to transcription, translation and molecular chaperones including a protein belonging to the calreticulin family. Other proteins include antioxidative enzymes such as 2-cys-peroxidase and oxidoreductases. The results of this proteomic analysis provide the first and primary information regarding the molecular basis of Cd hypertolerance in P. americana.

[Chromosome study on chronic lymphocytic leukemia using CpG-oligodeoxynucleotide as immunostimulant agent].

OBJECTIVE: To investigate whether CpG-oligodeoxynucleotide (CpG-ODN) can improve the detection rate of the karyotypic abnormalities in chronic lymphocytic leukemia (CLL). METHODS: The bone marrow (BM) or peripheral blood (PB) cells from 57 cases of CLL were collected and cultured with CpG-ODN DSP30+interleukin-2 (IL-2), phytohemagglutinin (PHA), pokeweed (PWM) or IL-2, respectively. Five days later cells were harvested for chromosome preparation. Karyotypic analysis was done using R banding technique. Panel fluorescence in situ hybridization (FISH) was carried out on 19 cases of CLL with normal karyotypes using the following probes: Cen12, D13S25, Rb1, ATM, p53, MYB and IgH. Genomic DNA from 21 cases of them was extracted from BM or PB leukocytes. The immunoglobulin variable heavy chain (IgVH) was amplified by polymerase chain reaction (PCR) and sequenced. CD38 and ZAP70 expressions in the leukemic cells were determined by flow cytometry (FCM). RESULTS: The detection rate of karyotypic abnormalities in the CpG-ODN+IL-2 group (43.85%) was obviously higher than that in the PHA (15.09%), PWM (17.31%) and IL-2 (3.13%) groups (P<0.01). Fifty-two types of karyotypic abnormalities were found. Among them, trisomy12 (+12) or +12 with other abnormalities were the most common, while translocations were the most frequent structural abnormalities including 3 unbalanced and 11 balanced translocations, among them 7 had rearrangements involving 14q32. Thirteen cases showed one or more abnormalities on FISH including trisomy 12 and p53 deletion each in one case, IgH rearrangement and partial deletion each in one case, 13q14.3 deletion in 11 cases of which 5 cases also had Rb1 deletion, 1 case had Rb1 partial deletion. No case with ATM or MYB deletions was found. PCR detected IgVH mutations in 10/21 cases. FCM showed 10/45 cases were CD38 positive, but 35 /45 were CD38 negative, 11/27 cases expressed ZAP70, but 16/27 did not. Among the 26 cases examined for CD38 and ZAP70 expressions simultaneously, 5 cases were CD38+ZAP70+, 13 were CD38-ZAP70-, 6 were CD38-ZAP70+, and 2 were CD38+ZAP70-, respectively. Statistic analysis showed a correlation between complex karyotype and IgVH without mutation, but no association between karyotype and CD38 or ZAP70 expression was observed. CONCLUSION: CpG-ODN immunostimulation can obviously raise the detection rate of abnormal karyotypes, especially translocations in CLL. FISH is an important complement to conventional karyotypic analysis. The combination of both methods can provide more comprehensive genetic information for CLL.

[Cloning and expression of pokeweed antiviral protein-II gene from the summer leaves of Phytolacca amercana].

The cDNA sequence encoding pokeweed antiviral protein-II was cloned from the fresh summer leaves of phytolacca amercana by RT-PCR. The recombinant PAP-II was subcloned into the expression vector pET-28a(+) and expressed in E. coli BL21 after IPTG induction. SDS-PAGE analysis showed that the expressed PAP-II existed in the form of inclusion bodies. The purified fusion protein was obtained after a series of steps including cell break, inclusion body solubilization, protein refolding and purification through BBST NTA resin column. The non-radioactive ELISA-based HIV-1 integrase assay showed that the recombinant pokeweed antiviral protein-II and RTA were able to inhibit HIV-1 integrase to some extent (IC50 = 303 microg/mL, 220 microg/mL respectively). MTT assay showed that cytotoxicity of pokeweed antiviral protein II for HEP-G2 cells and Hela cells was in a dose-dependent manner with IC50 s of 93 microg/mL and 102 microg/mL, respectively. The results suggested that pokeweed antiviral protein-II is a potent anti-tumor candidate. The finding of integrase inhibitory activity and the discovery of cytotoxicity provide more insights into the anti-HIV and the anti-tumor activities of PAP-II.

The hydrophobic surface of PaAMP from pokeweed seeds is essential to its interaction with fungal membrane lipids and the antifungal activity.

PaAMP is a small seed-specific antimicrobial protein from pokeweeds. It has a cysteine-knot fold with a positive patch and a hydrophobic surface. Site-specific mutagenesis was performed to study the roles of these two domains in antimicrobial activity and we found that the mutations in the hydrophobic surface had a more profound effect than that in the positive patch. A protein-membrane interaction was observed with the green fluorescence protein-PaAMP (GFP-AMP) fusion protein. The mutations that replace the amino acid residues forming hydrophobic surface with neutral residues abolished the interaction of PaAMP with the membrane and the binding of PaAMP to fungal sphingolipids while ergosterol enhanced the binding, suggesting that the hydrophobic surface was required for the interaction between PaAMP and fungal plasma membrane lipid raft.

Expression of pokeweed antiviral proteins in creeping bentgrass.

Fungal diseases of creeping bentgrass, an important amenity grass used extensively on golf courses, are a serious problem in golf course management. Transgenic approaches to improving disease resistance to fungal diseases are being explored in many species, and in some cases ribosome-inactivating proteins have been found to be effective. We have generated transgenic creeping bentgrass plants expressing three forms of ribosome-inactivating proteins from pokeweed, which are termed pokeweed antiviral proteins (PAP). PAP-Y and PAP-C are nontoxic mutants of PAP; PAPII is the native form of another ribosome-inactivating protein from pokeweed. In creeping bentgrass, PAP-C transformants did not accumulate the protein, suggesting that it is unstable, and in a field test these plants were not protected from infection by the fungal pathogen Sclerotinia homoeocarpa, the causal agent of dollar spot disease. PAPII transformants could accumulate stable levels of the protein but had symptoms of toxicity; one low-expressing line exhibited good disease resistance. PAP-Y transformants accumulated stable levels of protein, and under greenhouse conditions they appeared to be phenotypically normal.

Isolation and characterization of a novel ribosome-inactivating protein from root cultures of pokeweed and its mechanism of secretion from roots.

Ribosome-inactivating proteins are N-glycosidases that remove a specific adenine from the sarcin/ricin loop of the large rRNA, thus arresting protein synthesis at the translocation step. In the present study, a novel type I ribosome-inactivating protein, termed PAP-H, was purified from Agrobacterium rhizogenes-transformed hairy roots of pokeweed (Phytolacca americana). The protein was purified by anion- and cation-exchange chromatography. PAP-H has a molecular mass of 29.5 kD as detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its isoelectric point was determined to be 7.8. Yeast (Saccharomyces cerevisiae) ribosomes incubated with PAP-H released the 360-nucleotide diagnostic fragment from the 26S rRNA upon aniline treatment, an indication of its ribosome-inactivating activity. Using immunofluorescence microscopy, PAP-H was found to be located in the cell walls of hairy roots and root border cells. PAP-H was determined to be constitutively secreted as part of the root exudates, with its secretion enhanced by a mechanism mediated by ethylene induction. Purified PAP-H did not show in vitro antifungal activity against soil-borne fungi. In contrast, root exudates containing PAP-H as well as additional chitinase, beta-1,3-glucanase, and protease activities did inhibit the growth of soil-borne fungi. We found that PAP-H depurinates fungal ribosomes in vitro and in vivo, suggesting an additive mechanism that enables PAP-H to penetrate fungal cells.