scholarly journals Identification and Functional Characterization of Tissue-Specific Terpene Synthases in Stevia rebaudiana

2020 ◽  
Vol 21 (22) ◽  
pp. 8566
Author(s):  
Savitha Dhandapani ◽  
Mi Jung Kim ◽  
Hui Jun Chin ◽  
Sing Hui Leong ◽  
In-Cheol Jang
Keyword(s):  
Functional Characterization ◽  
Stevia Rebaudiana ◽  
Gas Chromatography Mass Spectrometry ◽  
Steviol Glycosides ◽  
Terpene Synthases ◽  
Tissue Specific ◽  
Wide Range ◽  
Volatile Organic ◽  
Specific Manner

In addition to the well-known diterpenoid steviol glycosides, Stevia rebaudiana (Stevia) produces many labdane-type diterpenoids and a wide range of mono- and sesquiterpenoids. However, biosynthesis of mono- and sesquiterpenoids in Stevia remains unknown. Here we analyzed the extracts of Stevia leaves, flowers, stems, and roots by Gas Chromatography–Mass Spectrometry and putatively identified a total of 69 volatile organic compounds, most of which were terpenoids with considerably varied quantities among the four tissues of Stevia. Using Stevia transcriptomes, we identified and functionally characterized five terpene synthases (TPSs) that produced major mono- and sesquiterpenoids in Stevia. Transcript levels of these Stevia TPSs and levels of corresponding terpenoids correlated well in Stevia tissues. Particularly, the root-specific SrTPS4 and SrTPS5 catalyzed the formation of γ-curcumene/zingiberene/β-sesquiphellandrene and α-longipinene/β-himachalene/himachalol as multifunctional sesqui-TPSs, respectively. Most of the SrTPSs were highly responsive to various environmental stresses in a tissue-specific manner. Taken together, our results provide new insights into how Stevia produces diverse terpenoids to confer differential responses to various environmental factors in each tissue.

scholarly journals Development of screening methods for functional characterization of UGTs from Stevia rebaudiana

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Eva Petit ◽  
Monique Berger ◽  
Laurent Camborde ◽  
Veronica Vallejo ◽  
Jean Daydé ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Transient Gene Expression ◽  
Stevia Rebaudiana ◽  
Expression Vectors ◽  
Screening Methods ◽  
Steviol Glycosides ◽  
Hydroxyl Groups ◽  
Uridine Diphosphate ◽  
Combined Use

Abstract Glycosylation is a key modification that contributes to determine bioactivity and bioavailability of plant natural products, including that of terpenoids and steviol glycosides (SVglys). It is mediated by uridine-diphosphate glycosyltransferases (UGTs), that achieve their activity by transferring sugars on small molecules. Thus, the diversity of SVglys is due to the number, the position and the nature of glycosylations on the hydroxyl groups in C-13 and C-19 of steviol. Despite the intense sweetener property of SVglys and the numerous studies conducted, the SVglys biosynthetic pathway remains largely unknown. More than 60 SVglys and 68 putative UGTs have been identified in Stevia rebaudiana. This study aims to provide methods to characterize UGTs putatively involved in SVglys biosynthesis. After agroinfiltration-based transient gene expression in Nicotiana benthamiana, functionality of the recombinant UGT can be tested simply and directly in plants expressing it or from a crude extract. The combined use of binary vectors from pGWBs series to produce expression vectors containing the stevia's UGT, enables functionality testing with many substrates as well as other applications for further analysis, including subcellular localization.

Designing, Construction and Functional Characterization of Tissue-specific Synthetic Promoter in Rice

2017 ◽  
Vol 43 (6) ◽  
pp. 789
Author(s):  
Rui WANG ◽  
Meng-Lin ZHU ◽  
Fang-Yuan GAO ◽  
Juan-Sheng REN ◽  
Xian-Jun LU ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Synthetic Promoter ◽  
Tissue Specific

Functional characterization of terpene synthases and chemotypic variation in three lavender species of section Stoechas

2014 ◽  
Vol 153 (1) ◽  
pp. 43-57 ◽  
Author(s):  
Tarek Benabdelkader ◽  
Yann Guitton ◽  
Bernard Pasquier ◽  
Jean Louis Magnard ◽  
Frédéric Jullien ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Terpene Synthases

Functional characterization of an adhesive component from the embryonic chick neural retina

1979 ◽  
Vol 36 (1) ◽  
pp. 323-342
Author(s):  
R. Rutz ◽  
J. Lilien
Keyword(s):  
Conditioned Medium ◽  
Ph Dependence ◽  
Functional Characterization ◽  
Surface Binding ◽  
Cell Agglutination ◽  
Tissue Specific ◽  
A Cell ◽  
Surface Ligand ◽  
Fixed Cell

We have developed a quantitative assay for tissue-specific adhesive components which is based on the agglutination of glutaraldehyde-fixed cells. At least 2 components are required for fixed-cell agglutination: a cell-surface ligand which is obtained from tissue culture-conditioned medium, and a soluble ‘agglutinin’ which accumulates in conditioned medium from monolayer cultures. Our results suggest that the surface-binding ligand and the agglutinin interact directly, resulting in tissue-specific agglutination of cells. The agglutination reaction exhibits divalent cation, temperature, and pH dependence. Several models of cell adhesion are described; the simplest of these which can account for the data is a multicomponent model in which the 2 adhesive components have structural roles.

scholarly journals Plugging Small RNAs into the Network

mSystems ◽  
2020 ◽  
Vol 5 (3) ◽  
Author(s):  
Lars Barquist
Keyword(s):  
Small Rnas ◽  
Posttranscriptional Regulation ◽  
Network Inference ◽  
Functional Characterization ◽  
The State ◽  
Diverse Range ◽  
Regulatory Interactions ◽  
Link Type ◽  
Wide Range

ABSTRACT Small RNAs (sRNAs) have been discovered in every bacterium examined and have been shown to play important roles in the regulation of a diverse range of behaviors, from metabolism to infection. However, despite a wide range of available techniques for discovering and validating sRNA regulatory interactions, only a minority of these molecules have been well characterized. In part, this is due to the nature of posttranscriptional regulation: the activity of an sRNA depends on the state of the transcriptome as a whole, so characterization is best carried out under the conditions in which it is naturally active. In this issue of mSystems, Arrieta-Ortiz and colleagues (M. L. Arrieta-Ortiz, C. Hafemeister, B. Shuster, N. S. Baliga, et al., mSystems 5:e00057-20, 2020, https://doi.org/10.1128/mSystems.00057-20) present a network inference approach based on estimating sRNA activity across transcriptomic compendia. This shows promise not only for identifying new sRNA regulatory interactions but also for pinpointing the conditions in which these interactions occur, providing a new avenue toward functional characterization of sRNAs.

scholarly journals Evaluation of Headspace Solid-Phase Microextraction Gas Chromatography–Mass Spectrometry for the Characterization of Volatile Organic Compounds from Melon (Cucumis melo L.) Flowers

2018 ◽  
Vol 81 (8) ◽  
pp. 1231-1239 ◽  
Author(s):  
Francisca Aliny Nunes Silva ◽  
Alexander Alves da Silva ◽  
Nayanny de Sousa Fernandes ◽  
Tigressa Helena Soares Rodrigues ◽  
Kirley Marques Canuto ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Gas Chromatography ◽  
Organic Compounds ◽  
Cucumis Melo ◽  
Solid Phase Microextraction ◽  
Solid Phase ◽  
Gas Chromatography Mass Spectrometry ◽  
Volatile Organic ◽  
Cucumis Melo L

scholarly journals Molecular characterization of a low-molecular-mass matrix metalloproteinase secreted by glomerular mesangial cells as PUMP-1

1992 ◽  
Vol 285 (3) ◽  
pp. 899-905 ◽  
Author(s):  
H P Marti ◽  
L McNeil ◽  
G Thomas ◽  
M Davies ◽  
D H Lovett
Keyword(s):  
Mesangial Cells ◽  
Polyclonal Antibodies ◽  
Mass Matrix ◽  
Interleukin 1 ◽  
Acute Glomerulonephritis ◽  
Glomerular Mesangial Cells ◽  
Tissue Specific ◽  
Specific Manner ◽  
Necrosis Factor

A polymerase chain reaction (PCR)-based homology cloning strategy was used to define the spectrum of stromelysin-like matrix metalloproteinases (MMPs) synthesized by cultured glomerular mesangial cells (MC). Using this technique, cDNAs encoding an unusual, truncated member of the MMP family, punctuated (putative) metalloproteinase (PUMP-1), were exclusively isolated. Incubation with the cytokines interleukin 1 and tumour necrosis factor increased the abundance of PUMP-1 mRNA in mesangial cells. The mesangial PUMP-1 mRNA is processed in a tissue-specific manner, yielding a transcript containing repeated 3′-untranslated region ATTTA motifs commonly found in cytokines with limited mRNA stability. Polyclonal antibodies prepared against the C-terminal region of the PUMP-1 protein documented release of this enzyme by cultures of cytokine-stimulated MC and permitted identification of PUMP-1-expressing mesangial cells within clinical biopsy specimens of acute glomerulonephritis. These findings represent new molecular and clinical evidence that non-malignant cells process and secrete this unusual member of the MMP family in a cytokine-mediated, tissue-specific manner. Mesangial synthesis of PUMP-1 may contribute to the progression of injury during glomerular inflammatory states.

scholarly journals Proteomics Strategy for Identifying Candidate Bioactive Proteins in Complex Mixtures: Application to the Platelet Releasate

2010 ◽  
Vol 2010 ◽  
pp. 1-12 ◽  
Author(s):  
Roisin O'Connor ◽  
Lorna M. Cryan ◽  
Kieran Wynne ◽  
Andreas de Stefani ◽  
Desmond Fitzgerald ◽  
...  
Keyword(s):  
Ion Exchange ◽  
Functional Characterization ◽  
Biological Tissues ◽  
Biochemical Properties ◽  
Human Platelets ◽  
Intact Proteins ◽  
Large Numbers ◽  
Wide Range ◽  
Platelet Releasate

Proteomic approaches have proven powerful at identifying large numbers of proteins, but there are fewer reports of functional characterization of proteins in biological tissues. Here, we describe an experimental approach that fractionates proteins released from human platelets, linking bioassay activity to identity. We used consecutive orthogonal separation platforms to ensure sensitive detection: (a) ion-exchange of intact proteins, (b) SDS-PAGE separation of ion-exchange fractions and (c) HPLC separation of tryptic digests coupled to electrospray tandem mass spectrometry. Migration of THP-1 monocytes in response to complete or fractionated platelet releasate was assessed and located to just one of the forty-nine ion-exchange fractions. Over 300 proteins were identified in the releasate, with a wide range of annotated biophysical and biochemical properties, in particular platelet activation, adhesion, and wound healing. The presence of PEDF and involucrin, two proteins not previously reported in platelet releasate, was confirmed by western blotting. Proteins identified within the fraction with monocyte promigratory activity and not in other inactive fractions included vimentin, PEDF, and TIMP-1. We conclude that this analytical platform is effective for the characterization of complex bioactive samples.

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