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A cell suspension based uptake method to study high affinity glucosinolate transporters

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Title A cell suspension based uptake method to study high affinity glucosinolate transporters
 
Creator Nambiar, Deepti M.
Kumari, Juhi
Arya, Gulab C.
Singh, Amarjeet K.
Bisht, Naveen C.
 
Subject Cotton cell suspensions
Secondary metabolite transporters
Glucosinolates
GTR transporters
Kinetic analysis
 
Description Accepted date: 15 May 2020
Background: Glucosinolates are an important class of secondary metabolites characteristic to the order Brassicales.
They are known to play a major role in plant defense and from the human perspective, can be anticarcinogenic or
antinutritive. GTRs are plasma-membrane localized high afnity glucosinolate transporters, which are important
components of the source (leaf ) to sink (seed) translocation of intact glucosinolates in members of Brassicaceae family. GTRs are identifed as major candidates for Brassica crop improvement, thus dictating a need for their functional
characterization. However, currently there are limitations in availability of heterologous assay systems for functional
characterization of plant secondary metabolite transporters. To date, the animal-based Xenopus oocyte system is the
best established heterologous system for functional characterization of these transporters. Inherent biochemical and
physiological attributes unique to the plant membranes necessitate the need for developing plant-based transporters
assay systems as well.
Methods: In this study, Agrobacterium mediated transformation was used to develop GTR expressing cotton cell lines
(CCL-1) for functional characterization of the Arabidopsis high afnity glucosinolate transporters, AtGTR1 and AtGTR2.
Following sub-cellular localization of AtGTRs, we standardized the glucosinolate uptake assays using cell suspension
cultures of AtGTR expressing CCL-1 its requirement of pH, salt, and time based glucosinolate uptake. Using the GTR
expressing CCL-1, we subsequently performed kinetic analysis of AtGTR1 and AtGTR2 for diferent glucosinolate substrates, sinigrin, gluconapin and sinalbin.
Results: Several clones expressing each of AtGTR1 and AtGTR2 were obtained showing high level of GTR expression
and were maintained through regular sub-culturing. Both AtGTR1 and AtGTR2 are predominantly plasma-localized
proteins when overexpressed in CCL-1 cells. Uptake assays were standardized, suggesting that glucosinolate uptake of
GTR expressing CCL-1 is robust within the physiological pH range 5–6, and at lower concentration of nitrate salts. GTR
expressing CCL-1 cells show increasing glucosinolate accumulation in time course experiment. Kinetic studies over a
wide glucosinolate concentrations (10–800 µM) revealed that our novel assay system displayed robust GTR-mediated
uptake of diferent glucosinolates and unambiguously helps elucidate the saturable kinetics of GTRs. Our system
confrms the high afnity of AtGTRs for both aliphatic and aromatic glucosinolates.
Conclusion: The transporter assay system described in this study holds potential for studying sub-functionalization
amongst GTR homologs present across Brassicaceae family. The fast growing CCL-1 cells, confer the benefts of an
in vitro system for quick assays and is plant based thus enabling optimal expression without sequence modifcations.
The efcient functioning of the GTR transporters in the heterologous CCL-1 opens the possibility of using this plant
cell suspension system for functional characterization of other metabolite transporters.
Research was supported by Department of Biotechnology-IYBA (BT/06/
IYBA/2012) project grants to NCB. DMN was supported from research fellowship of UGC (India) and NIPGR (New Delhi). We sincerely acknowledge Prof. Deepak Pental, CGMCP, Department of Genetics, UDSC, New Delhi, India for providing the CCL-1 culture. We also thank Ms.
Avni Mann and Mr. Vinod Kumar for their help in this study.
 
Date 2020-06-08T06:56:40Z
2020-06-08T06:56:40Z
2020
 
Type Article
 
Identifier Plant Methods, 16: 75
1746-4811
https://doi.org/10.1186/s13007-020-00618-0
https://plantmethods.biomedcentral.com/track/pdf/10.1186/s13007-020-00618-0
http://223.31.159.10:8080/jspui/handle/123456789/1067
 
Language en_US
 
Format application/pdf
 
Publisher BioMed Central Ltd