scholarly journals Genome-Wide Identification and Functional Characterization of the Trans-Isopentenyl Diphosphate Synthases Gene Family in Cinnamomum camphora

2021 ◽  
Vol 12 ◽  
Author(s):  
Zerui Yang ◽  
Chunzhu Xie ◽  
Ting Zhan ◽  
Linhuan Li ◽  
Shanshan Liu ◽  
...  
Keyword(s):  
Essential Oil ◽  
Gene Family ◽  
Functional Characterization ◽  
Cinnamomum Camphora ◽  
Isopentenyl Diphosphate ◽  
Geranyl Diphosphate ◽  
Wide Range ◽  
Camphor Tree

Trans-isopentenyl diphosphate synthases (TIDSs) genes are known to be important determinants for terpene diversity and the accumulation of terpenoids. The essential oil of Cinnamomum camphora, which is rich in monoterpenes, sesquiterpenes, and other aromatic compounds, has a wide range of pharmacological activities and has therefore attracted considerable interest. However, the TIDS gene family, and its relationship to the camphor tree (C. camphora L. Presl.), has not yet been characterized. In this study, we identified 10 TIDS genes in the genome of the C. camphora borneol chemotype that were unevenly distributed on chromosomes. Synteny analysis revealed that the TIDS gene family in this species likely expanded through segmental duplication events. Furthermore, cis-element analyses demonstrated that C. camphora TIDS (CcTIDS) genes can respond to multiple abiotic stresses. Finally, functional characterization of eight putative short-chain TIDS proteins revealed that CcTIDS3 and CcTIDS9 exhibit farnesyl diphosphate synthase (FPPS) activity, while CcTIDS1 and CcTIDS2 encode geranylgeranyl diphosphate synthases (GGPPS). Although, CcTIDS8 and CcTIDS10 were found to be catalytically inactive alone, they were able to bind to each other to form a heterodimeric functional geranyl diphosphate synthase (GPPS) in vitro, and this interaction was confirmed using a yeast two-hybrid assay. Furthermore, transcriptome analysis revealed that the CcTIDS3, CcTIDS8, CcTIDS9, and CcTIDS10 genes were found to be more active in C. camphora roots as compared to stems and leaves, which were verified by quantitative real-time PCR (qRT-PCR). These novel results provide a foundation for further exploration of the role of the TIDS gene family in camphor trees, and also provide a potential mechanism by which the production of camphor tree essential oil could be increased for pharmacological purposes through metabolic engineering.

scholarly journals Functional Characterization of BrF3'H, Which Determines the Typical Flavonoid Profile of Purple Chinese Cabbage

2021 ◽  
Vol 12 ◽  
Author(s):  
Sangkyu Park ◽  
Hyo Lee ◽  
Myung Ki Min ◽  
Jihee Ha ◽  
Jaeeun Song ◽  
...  
Keyword(s):  
Chinese Cabbage ◽  
Expression Patterns ◽  
Functional Characterization ◽  
Phenylpropanoid Pathway ◽  
Amino Acid Sequences ◽  
In Planta ◽  
Normal Green ◽  
Wide Range

Flavonols and anthocyanins are the two major classes of flavonoids in Brassica rapa. To elucidate the flavonoid biosynthetic pathway in Chinese cabbage (B. rapa L. subsp. pekinensis), we analyzed flavonoid contents in two varieties of Chinese cabbage with normal green (5546) and purple (8267) leaves. The 8267 variety accumulates significantly higher levels of quercetin, isorhamnetin, and cyanidin than the 5546 variety, indicating that 3′-dihydroxylated flavonoids are more prevalent in the purple than in the green variety. Gene expression analysis showed that the expression patterns of most phenylpropanoid pathway genes did not correspond to the flavonoid accumulation patterns in 5546 and 8267 varieties, except for BrPAL1.2 while most early and late flavonoid biosynthetic genes are highly expressed in 8267 variety. In particular, the flavanone 3′-hydroxylase BrF3′H (Bra009312) is expressed almost exclusively in 8267. We isolated the coding sequences of BrF3′H from the two varieties and found that both sequences encode identical amino acid sequences and are highly conserved with F3'H genes from other species. An in vitro enzymatic assay demonstrated that the recombinant BrF3′H protein catalyzes the 3′-hydroxylation of a wide range of 4′-hydroxylated flavonoid substrates. Kinetic analysis showed that kaempferol is the most preferred substrate and dihydrokaempferol (DHK) is the poorest substrate for recombinant BrF3′H among those tested. Transient expression of BrF3′H in Nicotiana benthamiana followed by infiltration of naringenin and DHK as substrates resulted in eriodictyol and quercetin production in the infiltrated leaves, demonstrating the functionality of BrF3′H in planta. As the first functional characterization of BrF3′H, our study provides insight into the molecular mechanism underlying purple coloration in Chinese cabbage.

scholarly journals Identification and Characterization of trans-Isopentenyl Diphosphate Synthases Involved in Herbivory-Induced Volatile Terpene Formation in Populus trichocarpa

Molecules ◽  
2019 ◽  
Vol 24 (13) ◽  
pp. 2408 ◽  
Author(s):  
Nathalie D. Lackus ◽  
Nora P. Petersen ◽  
Raimund Nagel ◽  
Axel Schmidt ◽  
Sandra Irmisch ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Populus Trichocarpa ◽  
Insect Herbivory ◽  
Isopentenyl Diphosphate ◽  
Transcript Accumulation ◽  
Sequence Comparisons ◽  
Geranyl Diphosphate ◽  
Genes Encoding

In response to insect herbivory, poplar releases a blend of volatiles that plays important roles in plant defense. Although the volatile bouquet is highly complex and comprises several classes of compounds, it is dominated by mono- and sesquiterpenes. The most common precursors for mono- and sesquiterpenes, geranyl diphosphate (GPP) and (E,E)-farnesyl diphosphate (FPP), respectively, are in general produced by homodimeric or heterodimeric trans-isopentenyl diphosphate synthases (trans-IDSs) that belong to the family of prenyltransferases. To understand the molecular basis of herbivory-induced terpene formation in poplar, we investigated the trans-IDS gene family in the western balsam poplar Populus trichocarpa. Sequence comparisons suggested that this species possesses a single FPP synthase gene (PtFPPS1) and four genes encoding two large subunits (PtGPPS1.LSU and PtGPPS2.LSU) and two small subunits (PtGPPS.SSU1 and PtGPPS.SSU2) of GPP synthases. Transcript accumulation of PtGPPS1.LSU and PtGPPS.SSU1 was significantly upregulated upon leaf herbivory, while the expression of PtFPPS1, PtGPPS2.LSU, and PtGPPS.SSU2 was not influenced by the herbivore treatment. Heterologous expression and biochemical characterization of recombinant PtFPPS1, PtGPPS1.LSU, and PtGPPS2.LSU confirmed their respective IDS activities. Recombinant PtGPPS.SSU1 and PtGPPS.SSU2, however, had no enzymatic activity on their own, but PtGPPS.SSU1 enhanced the GPP synthase activities of PtGPPS1.LSU and PtGPPS2.LSU in vitro. Altogether, our data suggest that PtGPPS1.LSU and PtGPPS2.LSU in combination with PtGPPS.SSU1 may provide the substrate for herbivory-induced monoterpene formation in P. trichocarpa. The sole FPP synthase PtFPPS1 likely produces FPP for both primary and specialized metabolism in this plant species.

scholarly journals Functional identification of ygiP as a positive regulator of the ttdA-ttdB-ygjE operon

Microbiology ◽  
2006 ◽  
Vol 152 (7) ◽  
pp. 2129-2135 ◽  
Author(s):  
Taku Oshima ◽  
Francis Biville
Keyword(s):  
Functional Characterization ◽  
Anaerobic Growth ◽  
Functional Identification ◽  
New Members ◽  
E Coli ◽  
Inner Membrane Protein ◽  
Tartrate Dehydratase

Functional characterization of unknown genes is currently a major task in biology. The search for gene function involves a combination of various in silico, in vitro and in vivo approaches. Available knowledge from the study of more than 21 LysR-type regulators in Escherichia coli has facilitated the classification of new members of the family. From sequence similarities and its location on the E. coli chromosome, it is suggested that ygiP encodes a lysR regulator controlling the expression of a neighbouring operon; this operon encodes the two subunits of tartrate dehydratase (TtdA, TtdB) and YgiE, an integral inner-membrane protein possibly involved in tartrate uptake. Expression of tartrate dehydratase, which converts tartrate to oxaloacetate, is required for anaerobic growth on glycerol as carbon source in the presence of tartrate. Here, it has been demonstrated that disruption of ygiP, ttdA or ygjE abolishes tartrate-dependent anaerobic growth on glycerol. It has also been shown that tartrate-dependent induction of the ttdA-ttdB-ygjE operon requires a functional YgiP.

scholarly journals Functional Characterization of the mazEF Toxin-Antitoxin System in the Pathogenic Bacterium Agrobacterium tumefaciens

2021 ◽  
Vol 9 (5) ◽  
pp. 1107
Author(s):  
Wonho Choi ◽  
Yoshihiro Yamaguchi ◽  
Ji-Young Park ◽  
Sang-Hyun Park ◽  
Hyeok-Won Lee ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Physiological Function ◽  
Functional Characterization ◽  
Site Directed Mutagenesis ◽  
In Vitro Assays ◽  
Cell Growth Inhibition ◽  
The Right

Agrobacterium tumefaciens is a pathogen of various plants which transfers its own DNA (T-DNA) to the host plants. It is used for producing genetically modified plants with this ability. To control T-DNA transfer to the right place, toxin-antitoxin (TA) systems of A. tumefaciens were used to control the target site of transfer without any unintentional targeting. Here, we describe a toxin-antitoxin system, Atu0939 (mazE-at) and Atu0940 (mazF-at), in the chromosome of Agrobacterium tumefaciens. The toxin in the TA system has 33.3% identity and 45.5% similarity with MazF in Escherichia coli. The expression of MazF-at caused cell growth inhibition, while cells with MazF-at co-expressed with MazE-at grew normally. In vivo and in vitro assays revealed that MazF-at inhibited protein synthesis by decreasing the cellular mRNA stability. Moreover, the catalytic residue of MazF-at was determined to be the 24th glutamic acid using site-directed mutagenesis. From the results, we concluded that MazF-at is a type II toxin-antitoxin system and a ribosome-independent endoribonuclease. Here, we characterized a TA system in A. tumefaciens whose understanding might help to find its physiological function and to develop further applications.

scholarly journals NFB-01. FUNCTIONAL CHARACTERIZATION OF ATRX LOSS IN NF1-ASSOCIATED GLIOMA AND MPNST

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii417-iii418
Author(s):  
Ming Yuan ◽  
Karlyne Reilly ◽  
Christine Pratilas ◽  
Christopher Heaphy ◽  
Fausto Rodriguez
Keyword(s):  
Cell Lines ◽  
Functional Characterization ◽  
Tert Promoter ◽  
Aggressive Tumors ◽  
Sirna Knockdown ◽  
Nih 3T3 ◽  
Vitro Effect ◽  
Murine Glioma

Abstract To identify the biologic relevance of ATRX loss in NF1-associated gliomagenesis, we studied the effects of Atrx loss using four previously characterized Nf1+/-Trp53+/- murine glioma lines. Lines 130G#3 and 158D#8 (corresponding to grade IV and III gliomas, respectively) displayed preserved ATRX protein expression compared to NIH-3T3 cells. We studied the effects of Atrx knockdown in these two lines in the presence and absence of the TERT inhibitor, BIRBR1532. Using a telomere-specific FISH assay, we identified increased signal intensity after Atrx knockdown, only in the presence of the TERT inhibitor. These features are reminiscent of ALT, although there were no significant alterations in cell growth. Next, we studied the effect of ATRX loss in MPNST lines ST88-14, NF90-8, STS-26T. These cell lines all expressed ATRX and DAXX. However, STS-26T contained a TERT promoter mutation and ST88-14 had a known SNP in the TERT promoter, while NF90-8 had no alterations. ATRX siRNA knockdown showed no significant effects in cell proliferation or apoptosis. However, ATRX knockdown resulted in rare ultra-bright foci, indicative of ALT. Next, we studied the in vitro effect of the ATR inhibitor VE-821 in MPNST cell lines. Only NF90-8 (lacking TERT alterations) demonstrated a decrease in growth after ATRX knockdown and VE-821 treatment. However, ATRX knockdown alone did not affect sensitivity to carboplatin. Our findings further support a role for ATRX loss with subsequent ALT activation in a biologic subset of NF1-associated malignancies, thereby opening an opportunity for therapeutic targeting of these aggressive tumors using specific classes of drugs.

Functional Characterization of GABAA Receptor Ligands In Vitro

2004 ◽  
pp. 85-94
Author(s):  
Bjarke Ebert ◽  
Sally Anne Thompson ◽  
Signe Í. Stórustovu ◽  
Keith A. Wafford
Keyword(s):  
Gabaa Receptor ◽  
Functional Characterization ◽  
Receptor Ligands

scholarly journals Biochemical and Functional Characterization of Anthocyanidin Reductase (ANR) from Mangifera indica L.

Molecules ◽  
2018 ◽  
Vol 23 (11) ◽  
pp. 2876 ◽  
Author(s):  
Lin Tan ◽  
Mei Wang ◽  
Youfa Kang ◽  
Farrukh Azeem ◽  
Zhaoxi Zhou ◽  
...  
Keyword(s):  
Enzyme Assay ◽  
Molecular Mechanisms ◽  
Mangifera Indica ◽  
Functional Characterization ◽  
Citrate Buffer ◽  
Anthocyanidin Reductase ◽  
High Amino Acid ◽  
Optimum Ph

Mango (Mangifera indica L.) is abundant in proanthocyanidins (PAs) that are important for human health and plant response to abiotic stresses. However, the molecular mechanisms involved in PA biosynthesis still need to be elucidated. Anthocyanidin reductase (ANR) catalyzes a key step in PA biosynthesis. In this study, three ANR cDNAs (MiANR1-1,1-2,1-3) were isolated from mango, and expressed in Escherichia coli. In vitro enzyme assay showed MiANR proteins convert cyanidin to their corresponding flavan-3-ols, such as (−)-catechin and (−)-epicatechin. Despite high amino acid similarity, the recombinant ANR proteins exhibited differences in enzyme kinetics and cosubstrate preference. MiANR1-2 and MiANR1-3 have the same optimum pH of 4.0 in citrate buffer, while the optimum pH for MiANR1-1 is pH 3.0 in phosphate buffer. MiANR1-1 does not use either NADPH or NADH as co-substrate while MiANR1-2/1-3 use only NADPH as co-substrate. MiANR1-2 has the highest Km and Vmax for cyanidin, followed by MiANR1-3 and MiANR1-1. The overexpression of MiANRs in ban mutant reconstructed the biosynthetic pathway of PAs in the seed coat. These data demonstrate MiANRs can form the ANR pathway, leading to the formation of two types of isomeric flavan-3-ols and PAs in mango.

scholarly journals N-Acetylglucosamine-1-Phosphate Transferase, WecA, as a Validated Drug Target in Mycobacterium tuberculosis

2017 ◽  
Vol 61 (11) ◽  
Author(s):  
Stanislav Huszár ◽  
Vinayak Singh ◽  
Alica Polčicová ◽  
Peter Baráth ◽  
María Belén Barrio ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Target ◽  
Functional Characterization ◽  
Drug Candidate ◽  
Content Type ◽  
Chemical Libraries ◽  
Whole Cells ◽  
Encoding Gene

ABSTRACT The mycobacterial phosphoglycosyltransferase WecA, which initiates arabinogalactan biosynthesis in Mycobacterium tuberculosis, has been proposed as a target of the caprazamycin derivative CPZEN-45, a preclinical drug candidate for the treatment of tuberculosis. In this report, we describe the functional characterization of mycobacterial WecA and confirm the essentiality of its encoding gene in M. tuberculosis by demonstrating that the transcriptional silencing of wecA is bactericidal in vitro and in macrophages. Silencing wecA also conferred hypersensitivity of M. tuberculosis to the drug tunicamycin, confirming its target selectivity for WecA in whole cells. Simple radiometric assays performed with mycobacterial membranes and commercially available substrates allowed chemical validation of other putative WecA inhibitors and resolved their selectivity toward WecA versus another attractive cell wall target, translocase I, which catalyzes the first membrane step in the biosynthesis of peptidoglycan. These assays and the mutant strain described herein will be useful for identifying potential antitubercular leads by screening chemical libraries for novel WecA inhibitors.

scholarly journals Insecticidal and repellent activity of the essential oil of Cinnamomum camphora var. linaloolifera Y. Fujita (Ho-Sho) and Cinnamomum camphora (L.) J Presl. var. hosyo (Hon-Sho) on Sitophilus zeamaisMots. (Coleoptera, Curculionedae)

2015 ◽  
Vol 17 (4 suppl 1) ◽  
pp. 769-773 ◽  
Author(s):  
R.L. CANSIAN ◽  
V. ASTOLFI ◽  
R.I. CARDOSO ◽  
N. PAROUL ◽  
S.S. ROMAN ◽  
...  
Keyword(s):  
Essential Oil ◽  
Essential Oils ◽  
Insecticidal Activity ◽  
Sitophilus Zeamais ◽  
Repellent Activity ◽  
Cinnamomum Camphora ◽  
Preference Index ◽  
Lethal Doses ◽  
Repellency Activity

ABSTRACT The aim of this work was to evaluate the insecticidal and repellency activity of the essential oil of Cinnamomum camphora var. linaloolifera Y. Fujita (Ho-Sho) and Cinnamomumcamphora (L.) J Presl.var. hosyo (Hon-Sho), against the Sitophilus zeamais in maize grains. The essential oils were obtained by hydrodistillation and analyzed by GC-MS.The insecticidal activity was determined by the toxicity of different concentrations of essential oils during 24 hours of contact with the insects, in the absence of feed substrate. The Bioassays of repellency were conducted with lethal doses (LD50,LD25,and LD12.5) obtained from insecticidal bioassay. In order to compare the treatments the preference index (PI) was employed. The analysis of the essential oils of Cinnamomum camphora leaves indicated 68% of camphor and 9% of linalool for the variation Hon-Sho and 95% of linalool to the variation Ho-Sho. The variation Ho-Sho presented greatest insecticidal activity than the variation Hon-Sho against the Sitophiluszeamais, with LD50 of 0.35 μL/cm2, whereas in the variation Hon-Sho the ratewas 0.48 μL/cm2. However, considering only the concentrations of linalool and camphor of Ho-Sho and Hon-Sho, the lethal doses’ evaluation of these compounds were similar. The values of the preference index ranged from -0.3 to -0.8 for thevariation Ho-Sho and -0.2 to -0.7 for the variation Hon-Sho. The essential oils evaluated in this work showed repellent activity against Sitophiluszeamais in vitro and in trials performed in mini-silos.

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