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

Abstract

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 affinity glucosinolate transporters, which are important components of the source (leaf) to sink (seed) translocation of intact glucosinolates in members of Brassicaceae family. GTRs are identified 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 affinity 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 different 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 different glucosinolates and unambiguously helps elucidate the saturable kinetics of GTRs. Our system confirms the high affinity 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 benefits of an in vitro system for quick assays and is plant based thus enabling optimal expression without sequence modifications. The efficient functioning of the GTR transporters in the heterologous CCL-1 opens the possibility of using this plant cell suspension system for fun...

GTR1 and GTR2 transporters differentially regulate tissue‐specific glucosinolate contents and defence responses in the oilseed crop Brassica juncea

“…In order to characterize multiple GTR s identified in Brassica species, we utilized a novel plant‐based transport assay system generated from non‐embryogenic calli of cotton cotyledons (CCL‐1), that can be maintained as homogenous cell suspension cultures (Nambiar et al, 2020; Singh & Pental, 2015). We decided to investigate any possible differences in specificity for different glucosinolate substrates among the GTR1 and GTR2 homologs of “A” subgenome, which constitutes half of the genome of two globally important oilseed crops, B. juncea (AABB) and B. napus (AACC).…”

“…"*", "**" and "***" denote p values less than 0.05, 0.01 and 0.001, respectively. Mean glucosinolate levels in different tissue types of the transgenic events are provided in supplementary Table S8, S9, S10 and S11, and statistical details are presented in supplementary Table S12 and S13 prerequisite, a novel cotton cell suspension transport assay system was established (Nambiar et al, 2020) and used to test the Brassica GTRs. Using the CCL-1 system, it was shown that all "A" subgenome GTR1 and GTR2 transporters were functional, displaying Km values for all the tested glucosinolates in the micromolar ranges and their transport activity conforming to the Michaelis-Menten kinetics (Figure 2 and Table 2).…”

“…Intrinsic biochemical differences between plant and animal plasma membranes, however, necessitate the development of plant‐based transport assay systems as well. As a prerequisite, a novel cotton cell suspension transport assay system was established (Nambiar et al, 2020) and used to test the Brassica GTRs. Using the CCL‐1 system, it was shown that all “A” subgenome GTR1 and GTR2 transporters were functional, displaying Km values for all the tested glucosinolates in the micromolar ranges and their transport activity conforming to the Michaelis–Menten kinetics (Figure 2 and Table 2).…”

“…The GTR over‐expression (OE) constructs were transformed into cotton cell line (CCL‐1) as described in Singh and Pental (2015). Glucosinolate uptake assays were performed for sinigrin (2‐propenyl glucosinolate; Merck, USA), gluconapin (3‐butenyl glucosinolate, isolated from B. rapa ) and sinalbin (4‐hydroxybenzyl glucosinolate; a kind gift from Dr. Michael Reichelt, Max Plank Institute of Chemical Ecology, Jena, Germany), using 7‐days‐old liquid suspension culture of the GTR‐transformed CCL‐1 calli as earlier described (Nambiar, Kumari, Arya, Singh, & Bisht, 2020). The detailed protocol is provided as Supplementary Method S1.…”

“…The GTR over-expression (OE) constructs were transformed into cotton cell line (CCL-1) as described in Singh and Pental (2015). Ecology, Jena, Germany), using 7-days-old liquid suspension culture of the GTR-transformed CCL-1 calli as earlier described (Nambiar, Kumari, Arya, Singh, & Bisht, 2020). The detailed protocol is provided as Supplementary Method S1.…”