Natural variation in a polyamine transporter determines paraquat tolerance in Arabidopsis

Abstract

Polyamines (PAs) are ubiquitous, polycationic compounds that are essential for the growth and survival of all organisms. Although the PA-uptake system plays a key role in mammalian cancer and in plant survival, the underlying molecular mechanisms are not well understood. Here, we identified an Arabidopsis L-type amino acid transporter (LAT) family transporter, named RMV1 (resistant to methyl viologen 1), responsible for uptake of PA and its analog paraquat (PQ). The natural variation in PQ tolerance was determined in 22 Arabidopsis thaliana accessions based on the polymorphic variation of RMV1. An RMV1-GFP fusion protein localized to the plasma membrane in transformed cells. The Arabidopsis rmv1 mutant was highly resistant to PQ because of the reduction of PQ uptake activity. Uptake studies indicated that RMV1 mediates proton gradient-driven PQ transport. RMV1 overexpressing plants were hypersensitive to PA and PQ and showed elevated PA/PQ uptake activity, supporting the notion that PQ enters plant cells via a carrier system that inherently functions in PA transport. Furthermore, we demonstrated that polymorphic variation in RMV1 controls PA/PQ uptake activity. Our identification of a molecular entity for PA/PQ uptake and sensitivity provides an important clue for our understanding of the mechanism and biological significance of PA uptake.

Arabidopsis DPB3-1, a DREB2A Interactor, Specifically Enhances Heat Stress-Induced Gene Expression by Forming a Heat Stress-Specific Transcriptional Complex with NF-Y Subunits

“…For pGHX-NSF, first, the pGHX-NStrepII vector was generated from the pGHX vector (a hygromycin version of pGKX; Fujita et al, 2012) in the same way as pGKX-NMyc using the primer pair StrepII-N/SpeI and StrepII-C/XbaI. To construct the tandem tag vector pGHX-NSF, a DNA fragment containing the 33FLAG tag was amplified using PCR with the primer pair 33Flag-N/SpeI and 33Flag-C/XbaI; p3XFLAG-CMV-8 (Sigma-Aldrich) was used as a template.…”

Phloem-Specific Methionine Recycling Fuels Polyamine Biosynthesis in a Sulfur-Dependent Manner and Promotes Flower and Seed Development

“…Root application of PA has been previously shown to complement PA-associated growth defects (Waduwara-Jayabahu et al, 2012), and recently PA transporters have been identified from rice and Arabidopsis (Fujita et al, 2012;Mulangi et al, 2012aMulangi et al, , 2012b, suggesting uptake of polyamines by the root system. We added varying amounts of the non-Scontaining PA putrescine, spermidine, or spermine directly to the 10 mM sulfate-containing medium and observed that neither putrescine nor spermidine improved the growth of the mti1 and dep1 mutant plants (Supplemental Figs.…”

The Transcriptional Cascade in the Heat Stress Response of Arabidopsis Is Strictly Regulated at the Level of Transcription Factor Expression

“…Nucleotide substitutions in the CDS of HsfA1s were introduced via the megaprimer method (Sarkar and Sommer, 1990). For generation of GFP-fused genes, HsfA1s and its derivatives were amplified by PCR and inserted into the NotI-XhoI sites of the pGHX-NsGFP plasmid, a hygromycin version of pGKX-NsGFP Fujita et al, 2012). To generate the HsfA1d pro :sGFP-HsfA1d construct for the complementation assay, the 1142-bp promoter region upstream of the HsfA1d start codon was amplified and inserted into SacI-SpeI sites of the pGreen0129 (Hellens et al, 2000).…”

“…The coding sequences (CDSs) of HsfA1 genes were amplified by PCR and inserted into the NotI-XhoI sites of the pGHX plasmid, a hygromycin resistance-conferring version of pGKX Fujita et al, 2012). The CDS of HSC70-1 was amplified by PCR and inserted into the XbaISmaI sites of pGKX.…”