DataSheet_1_Identification of key amino acid residues in AtUMAMIT29 for transport of glucosinolates.xlsx

Glucosinolates are key defense compounds of plants in Brassicales order, and their accumulation in seeds is essential for the protection of the next generation. Recently, members of the Usually Multiple Amino acids Move In and Out Transporter (UMAMIT) family were shown to be essential for facilitating transport of seed-bound glucosinolates from site of synthesis within the reproductive organ to seeds. Here, we set out to identify amino acid residues responsible for glucosinolate transport activity of the main seed glucosinolate exporter UMAMIT29 in Arabidopsis thaliana. Based on a predicted model of UMAMIT29, we propose that the substrate transporting cavity consists of 51 residues, of which four are highly conserved residues across all the analyzed homologs of UMAMIT29. A comparison of the putative substrate binding site of homologs within the brassicaceous-specific, glucosinolate-transporting clade with the non-brassicaceous-specific, non-glucosinolate-transporting UMAMIT32 clade identified 11 differentially conserved sites. When each of the 11 residues of UMAMIT29 was individually mutated into the corresponding residue in UMAMIT32, five mutant variants (UMAMIT29#V27F, UMAMIT29#M86V, UMAMIT29#L109V, UMAMIT29#Q263S, and UMAMIT29#T267Y) reduced glucosinolate transport activity over 75% compared to wild-type UMAMIT29. This suggests that these residues are key for UMAMIT29-mediated glucosinolate transport activity and thus potential targets for blocking the transport of glucosinolates to the seeds.

硫代葡萄糖苷（glucosinolates）是十字花目（Brassicales）植物的关键防御化合物，其在种子中的积累对下一代植株的保护至关重要。近期研究证实，通常多氨基酸进出转运蛋白（Usually Multiple Amino acids Move In and Out Transporter，简称UMAMIT）家族的成员，可介导种子结合态硫代葡萄糖苷从生殖器官内的合成位点向种子转运，是该过程的必需因子。本研究旨在鉴定拟南芥（Arabidopsis thaliana）中主要种子硫代葡萄糖苷转运蛋白UMAMIT29的硫代葡萄糖苷转运活性相关氨基酸残基。基于UMAMIT29的预测结构模型，我们提出其底物转运腔由51个氨基酸残基组成，其中4个为所有已分析的UMAMIT29同源物共有的高度保守残基。通过比对十字花科特异性、硫代葡萄糖苷转运进化枝与非十字花科特异性、非硫代葡萄糖苷转运的UMAMIT32进化枝的推定底物结合位点，共鉴定出11个差异保守位点。将UMAMIT29的这11个残基分别突变为UMAMIT32中的对应残基后，5个突变体变体（UMAMIT29#V27F、UMAMIT29#M86V、UMAMIT29#L109V、UMAMIT29#Q263S及UMAMIT29#T267Y）的硫代葡萄糖苷转运活性较野生型UMAMIT29降低了75%以上。上述结果表明，这些残基是UMAMIT29介导的硫代葡萄糖苷转运活性的关键位点，同时也是阻断硫代葡萄糖苷向种子转运的潜在靶点。