In plants, abscisic acid-, stress-, and ripening-induced (ASR) proteins have been shown to impart tolerance to multiple abiotic stresses such as drought and salinity.

However, their roles in metal stress tolerance are poorly understood. To screen plant Cd-tolerance genes, the yeast-based gene hunting method which aimed to screen Cd-tolerance colonies from maize leaf cDNA library hosted in yeast was carried out. Here, maize ZmASR1 was identified to be putative Cd-tolerant through this survival screening strategy. In silico analysis of the functional domain organization, phylogenetic classification and tissue-specific expression patterns revealed that maize ASR1 to ASR5 are typical ASRswith considerable expression in leaves. Further, four of them were cloned for testifying Cd tolerance using yeast complementation assay. The results indicated that they all confer Cd tolerance in Cd-sensitive yeast. Then they were transiently expressed in tobacco leaves for subcellular localization analysis and for Cd-challenged lesion assay, continuously. The results demonstrated that all 4 maize ASRs tested are localized to the cell nucleus and cytoplasm in tobacco leaves. Moreover, they were confirmed to be Cd-tolerance genes in planta through lesion analysis in Cd-infiltrated leaves transiently expressing them. Taken together, our results demonstrate that maize ASRs play important roles in Cd tolerance, and they could be used as promising candidate genes for further functional studies toward improving the Cd tolerance in plants.





Figure 1. Phylogenetic relationships and conserved domains of maize ASRs. (A) Phylogenetic relationships of the maize ASRs and their homologs in grass species. Multiple alignments of ASR protein sequences were carried out with ClustalW utility embedded in MEGA7 software [40]. Phylogenetic trees were constructed by MEGA7 software using the neighbor-joining (NJ) method with 1000 bootstrap replicates, and tomato SlASR1 was used as the outgroup. Maize ASR1 was marked with one red dot, while other three maize ASRs for further experimental investigation were prefixed with triangles, and the left maize ASRs were marked with red diamonds. The ASRs in other plant species are prefixed as follows: Gm for soybean, Glycine max; Hv for barley, Hordium vulgare; Ma for banana, Musa acuminate; Os for rice, Oryza sativa; Sl for Tomato, Solanum lycopersicum; Ta for common wheat, Triticum aestivum; Tt for durum wheat, Triticum turgidum; Vv for grape, Vitis vinifera. (B) Domain organization of ZmASRs. The zinc-binding region, ABA/WDS domain, and the putative nuclear targeting signal were indicated in different color.