Abstract
Screening for natural products which bind to proteins in planta has been used to identify ligands of the plant-specific glutathione transferase (GST) tau (U) and phi (F) classes, that are present in large gene families in crops and weeds, but have largely undefined functions. When expressed as recombinant proteins in Escherichia coli these proteins have been found to tightly bind a diverse range of natural product ligands, with fatty acid-and porphyrinogen-derivatives associated with GSTUs and a range of heterocyclic compounds with GSTFs. With an interest in detecting the natural binding partners of these proteins in planta, we have expressed the two best characterized GSTs from Arabidopsis thaliana (At), AtGSTF2 and AtGSTU19, as Strep-tagged fusion proteins in planta. Following transient and stable expression in Nicotiana and Arabidopsis, respectively, the GSTs were recovered using Strep-Tactin affinity chromatography and the bound ligands desorbed and characterized by LC-MS. AtGSTF2 predominantly bound phenolic derivatives including S-glutathionylated lignanamides and methylated variants of the flavonols kaempferol and quercetin. AtGSTU19 captured glutathionylated conjugates of oxylipins, indoles, and lignanamides. Whereas the flavonols and oxylipins appeared to be authentic in vivo ligands, the glutathione conjugates of the lignanamides and indoles were artifacts formed during extraction. When tested for their binding characteristics, the previously undescribed indole conjugates were found to be particularly potent inhibitors of AtGSTU19. Such ligand fishing has the potential to both give new insight into protein function in planta as well as identifying novel classes of natural product inhibitors of enzymes of biotechnological interest such as GSTs.
Highlights
The Arabidopsis genome encodes 55 members of the soluble glutathione transferase (GSTs; EC 2.5.1.18) superfamily, notably members of the plant specific phi (F), tau (U), lambda (L) and dehydroascorbate reductase (DHAR) classes, as well as theta (T), zeta (Z) enzymes (Dixon and Edwards, 2010)
We report on a directed and systematic search for natural products that selectively bind to the two best characterized and abundant GSTs in Arabidopsis, namely AtGSTU19 and AtGSTF2, when these proteins are expressed in living plants
AtGSTF2 and AtGSTU19 were individually transformed into the leaves of N. benthamiana through Agrobacterium infection using vacuum infiltration
Summary
The Arabidopsis genome encodes 55 members of the soluble glutathione transferase (GSTs; EC 2.5.1.18) superfamily, notably members of the plant specific phi (F), tau (U), lambda (L) and dehydroascorbate reductase (DHAR) classes, as well as theta (T), zeta (Z) enzymes (Dixon and Edwards, 2010) The majority of these genes are expressed in planta as the respective proteins and show both a complex regulation and sub-cellular localization (Dixon et al, 2009; Jiang et al, 2013; Labrou et al, 2015). AtGSTF12 is involved in the regulation of anthocyanin accumulation (Kitamura et al, 2004), while AtGSTU20 modulates responses to light reception (Chen et al, 2007) and AtGSTU17 plays a regulatory role in seedling development (Jiang et al, 2013)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.