Supplementary Materials Supplementary Data supp_66_13_3815__index. Overexpression of in mutant seedlings defective

Supplementary Materials Supplementary Data supp_66_13_3815__index. Overexpression of in mutant seedlings defective in PC production and in plants treated with l-buthionine sulphoximine (BSO), an inhibitor of PC biosynthesis, had no effect on Cd tolerance, suggesting that acts via PCs. In addition, overexpression of in mutant seedlings defective in the Cd transporters AtABCC1 and AtABCC2 complements the Cd sensitivity of double mutants, but not in the presence of BSO. Accordingly, the level of transcript in wild type seedlings was lower than that of and in the absence of Cd but higher after Cd exposure, and even higher in mutants. The results point to AtABCC3 as a transporter of PCCCd complexes, and suggest that its activity is regulated by Cd and is co-ordinated with the activity of AtABCC1/AtABCC2. is in the range of 2C11 (Grill (Ha genes were first NVP-BGJ398 distributor isolated from (Ha genes have been isolated from different plants such as (Heiss (Cobbett, 2000a). PCs are able to bind cytoplasmic Cd, forming stable PCCCd complexes, playing a major role in Cd detoxification: PC-deficient mutants of and overexpression leads to increased Cd tolerance (Vatamaniuk roots suggested that transport of PCCCd complexes is mediated by ATP-binding cassette (ABC)-type transporters (Salt and Rauser, 1995), ubiquitous transmembrane proteins that utilize ATP to translocate various substrates across membranes. ABC proteins have a characteristic modular structure consisting of a double set of two basic structural elements, a hydrophobic transmembrane domain (TMD) usually made up of six membrane-spanning -helices, and a cytosolic domain containing a nucleotide-binding domain (NBD) involved in ATP binding (Wanke and Kolukisaoglu, 2010); the two TMDs dimerize to form the substrate-binding cavity (Procko homologues have been identified in (Vatamaniuk (Sooksa-Nguan (Mendoza-Czatl increases Cd tolerance in seedlings (Song are able to complement the loss of YCF1, partially restoring Cd tolerance (Klein knockout mutants defective in overexpression in wild type, PC-deficient lines, and double mutants, combined with analysis of cellular Cd localization, and comparative analysis of Cd tolerance between and double mutants, it is shown that AtABCC3 is involved in the NVP-BGJ398 distributor vacuolar transport of PCCCd complexes. Material and methods Plant growth conditions and metal treatments Wild type, mutant lines (kindly provided by Markus Klein of Philip Morris International, Switzerland), (Song (Cobbett, 2000a; kindly provided by Chris Cobbett of University of Melbourne, Australia) AtPCSox-21, AtPCSox-20, AtPCSox-26, AtABCC3ox-was cloned into the strain GV3101 carrying the construct was used to transform wild type plants (ecotype Columbia) by standard floral dip transformation (Clough and Bent, 1998). Transformed plants were analysed by PCR with the following primers: LexA 4096 For 5-GCCATGTAATATGCTCGACT-3, MRP3 Rev 4467 5-GAGCTGACTTAAACCCAAAAT-3; and by real-time reverse tanscriptionCPCR (RTCPCR; see below). Homozygous T2 generations were obtained by self-fertilization of primary transformants and the seeds were grown as described below (Cecchetti (2013). The primers used to analyse transcript levels were: RTmrp3 For 3835 5-CTTCAGGTCCGATATGCTCCA-3, RTmrp3 Rev 3885 5-TGTTATTCCTCGCAACACAAGAG-3; ACTIN2 For 5-CCGATCCAGACACTGTACTTCCTT-3, ACTIN2 Rev 5-CTTGCACCAAGCAGCATGAA-3, and were designed as previously described (Cecchetti lines were used for crosses with homozygous AtABCC3ox-21 lines. F2 lines, homozygous for the AtABCC3ox construct and for the NVP-BGJ398 distributor mutation, were selected on hygromycin, and the mutation was verified by PCR with the following primers: For 5-TCAAGTATCCCCCTCACTGC-3; For 5-TCAAGTATCCCCCTCACTGG-3; and Rev 5-CGGGTTCTCTGTGTGGTCTA-3. Three independent homozygous lines named AtABCC3ox-20, AtABCC3ox-21, and AtABCC3ox-26 were used for subsequent Cd tolerance analysis. Statistical analysis Two-tailed and one-tailed Students plants after 9 d or 22 d of treatment, whereas they were prepared from AtABCC3ox lines after 5 d and 9 d of treatment. The enzymatic NVP-BGJ398 distributor digestion was carried according to Lindberg (2004). The same number of isolated protoplasts from wild type, mutants and enhanced in overexpressors To assess whether contributes to Cd tolerance, the growth of wild type and seedlings was analysed at Rabbit Polyclonal to MSK1 different Cd concentrations. In a previous study, it was shown that growth of seedlings is not affected at Cd concentrations up to 15 M, while it NVP-BGJ398 distributor is slightly reduced at 30 M and 60 M CdSO4, and severely inhibited at 90 M (Brunetti seedlings were grown in the presence of 0, 15, 30, 60, and 90 M CdSO4, and the fresh weight and root length were analysed after 9 d. As shown in Fig. 1,.