The Arabidopsis paraquat resistant1 mutant accumulates leucine upon dark treatment

Abstract

The Arabidopsis paraquat resistant1 (PAR1) was classified as L-type amino acid transporter 4 (LAT4) based on a phylogenetic analysis of selected genes from Saccharomyces cerevisiae, Arabidopsis thaliana (L.) Heynh, and Homo sapiens that clustered LAT4 with four other members as a LAT family in Arabidopsis. In silico analysis of the Arabidopsis LATs identified an amino acid permease domain and motifs that are common in amino acid transporters. However, their role in amino acid transport remained to be studied. A knockout mutant for PAR1/LAT4 gene, reported here as par1-5, showed significantly altered growth compared with wild type on leucine-containing growth medium. Mutant par1-5 seedlings showed reduced biomass compared with wild type on nitrate-containing Murashige and Skoog growth medium, which was further reduced when grown on medium containing nitrate and leucine. Radio-labelled leucine uptake studies using leaf protoplasts and seedlings showed increased accumulation of leucine in par1-5 mutants compared with wild type. Increased accumulation of leucine in par1-5 was detected when seedlings or protoplasts were treated in the dark prior to isotopic feeding. These studies suggest that the PAR1/LAT4 protein, in addition to its ability to mediate paraquat and polyamine transport, possess leucine transport activity that is regulated by physiological conditions such as dark induction.

The Arabidopsis L-Type Amino Acid Transporter 5 (LAT5/PUT5) Is Expressed in the Phloem and Alters Seed Nitrogen Content When Knocked Out

“…According to the in silico analysis, the LAT5/PUT5 is an integral membrane protein that has amino acid transporter and antiporter domains conserved in the sequence (Figure 1a,b) [11]. In human, the LATs conserve a cysteine residue in the second extra-cellular loop through which they form cys-cys di-sulfide bond with a glycoprotein subunit to function as exchangers [4][5][6]21].…”

“…Based on in silico analyses, the Arabidopsis LAT5/PUT5 is an integral membrane protein, with a molecular weight of 52.8 kDa, a theoretical isoelectric point of 5.3, and 12 transmembrane domains with both the n-and c-termini in the cytoplasmic side ( Figure 1a). In addition to the amino acid permease domain PF13520 reported to be conserved in all five Arabidopsis LATs [11], the LAT5/PUT5 putatively has PotE Amino Acid Transporter (COG0531), Amino Acid Permease (PRK11357), Arginine/Agmatine Antiporter (PRK10644), Arginine/Ornithine Antiporter (TIGR00905), and Glutamate/Gamma-Aminobutyrate Antiporter (TIGR00910) domains conserved in the amino acid sequence (Figure 1b). Human LATs have been demonstrated to form di-sulfide bridges with their glycoprotein subunits through a cysteine residue conserved between the third and fourth transmembrane domains [4].…”

“…(a) (b-e) (f) (g) Leucine was used to investigate the role of another member of the LAT family, LAT4/PAR1, in amino acid transport [11]. For the same reason, leucine was used to investigate the role of LAT5/PUT5 in this report.…”

“…All plants (unless otherwise mentioned) were grown on well-aerated fertilized soil in a growth chamber under a 16 h light-8 h dark regime under~170 µE•m −2 •s −1 light intensity at 23 • C daytime temperature. Growth condition and soil type were the same as described in Begam and Good, (2017) [11]. For phenotypic screening in the presence of leucine, nitrogen-free half-strength MS medium [28] was prepared using stock solutions of macronutrients, micronutrients, and vitamin mixture at a molar concentration described in the commercially available MS basal salt mixture (Catalog No.…”

“…The LATs in human function as heteromeric exchangers of large neutral amino acids at 1:1 stoichiometry [4][5][6]10]. However, the Arabidopsis LATs have been demonstrated to have roles in mediating the transport of a range of substrates including amino acid, polyamine, paraquat, and thiamine [11][12][13][14][15][16][17]. The LAT5 was previously studied for its role in developmental responses, such as flowering and senescence, where it was called PUT5 under the assumption that it is a polyamine uptake transporter [18].…”