RESEARCH ARTICLE Copyright © 2018 American Scientific Publishers All rights reserved Printed in the United States of America Journal of Biobased Materials and Bioenergy Vol. 12, 1–9, 2018 Identification and Characterization of Four Nicotianamine Synthase Genes in Sedum alfredii Hance Shaoning Chen 1 2 , Zulfiqar Ali Sahito 1 , Min Zhang 3 , Ying Feng 1 , Qianying Yang 1 , and Xiaoe Yang 1 ∗ 1 Ministry of Education Key Laboratory of Environmental Remediation and Ecosystem Health, College of Environmental and Resources Science, Zhejiang University, Hangzhou 310058, China 2 College of Life Science, Zhejiang Sci-Tech University, Hangzhou 310018, China 3 Zhejiang Mariculture Research Institute, Wenzhou 325005, China Sedum alfredii Hance is known for its dynamic property of co-hyperaccumulation of Cadmium (Cd) and Zinc (Zn). Therefore, studies and investigations of fundamental molecular mechanisms under- lying Cd/Zn co-hyperaccumulation have been receiving a growing interest. Nicotianamine (NA) is a ubiquitous molecule found in almost all plant species, playing a key role in phytosiderophores biosynthesis and ion uptake. Nicotianamine Synthase (NAS) gene family is known for its cytaliz- ing synthesis of NA in trafficking and chelating of transition metals in plants. However, information of NAS genes in S. alfredii Hance remains limited. In this study, four NAS genes were isolated from S. alfredii Hance (SaNAS), based on the analysis of RNA-sequence data. The bioinformat- ics analysis implies that the four SaNAS genes were closely clustered with VvNAS1 genes and shared 67.5%–71.9% identity at protein level. Quantitative reverse transcript-PCR expression anal- ysis revealed that all those four SaNAS genes were highly expressed in root tissues, under Cd excessive conditions. SaNAS2, SaNAS3 and SaNAS4 expressions were up-regulated by Zn stress. Furthermore, yeast complementation assays further confirmed that overexpression of SaNAS3 gene proliferated Cd accumulation in yeast cells, while SaNAS2 enhanced the Cd tolerance of yeast cells. Moreover, subcellular localization of SaNAS also reveals that these SaNAS proteins are localized throughout the cell. This work provides comprehensive information about SaNAS genes in response to hyperaccumulation of Zn and Cd in S. alfredii species. These results may enlarge our knowledge of NAS genes in hyperaccumlator species and provide genetic resource for phytoremediation using genomic modified plants. Keywords: Nicotianamine Synthase Genes, Sedum alfredii Hance, Hyperaccumulation, Zinc, Cadmium. 1. INTRODUCTION Nicotianamine (NA) is a non-protein amino acid molecule that functions primarily in metal uptake, translocation and homeostasis in several higher plant species. 1 2 In gram- inaceous plants, NA serves as the precursor of mug- ineic acid (MA), a phytosiderophore of iron acquisition. 3 It chelates the ions of Fe(II) and other heavy metals. 4 In non-gramineous plants, NA serves as a metal chelator and forms complex network with ions of Fe(III), Fe(II), Mn(II), Zn(II), Ni, and Cu(II). 5–8 Synthesis of NA from three molecules of S-adenosyl- methionines (SAM) is catalyzed by nicotianamine synthase (NAS). 9 10 NAS genes were identified and characterized in both graminaceous and non-graminaceous ∗ Author to whom correspondence should be addressed. Emails: xeyang@zju.edu.cn, mrna@163.com plants including rice, maize, barley, Arabidopsis thaliana, Arabidopsis halleri. 11 Studies on NAS genes showed that NAS expression is regulated by several metals includ- ing iron, Zn, Mn, Ni and Cu. 12–14 It suggested that NAS expression regulation is involved in metal uptake, acquisition and homeostasis. In Zn/Cd hyperaccumulator A. halleri, higher expressions of NAS genes were found in comparison with A. thaliana. 15 Furthermore, A. halleri NAS2-RNAi plants showed reduced Cd accumulation. 16 Similarly, AtNAS4 gene was up-regulated in A. thaliana in response to Cd stress. 17 Although the role of NAS in heavy metal resistance is partly understood, the characterization of NAS genes involved in Cd tolerance in S. alfredii Hance remains limited. S. alfredii Hance has been identified as a Cd hyper- accumulator species from the Pb/Zn mining zones, south east of China. This plant can accumulate 9000 and J. Biobased Mater. Bioenergy 2018, Vol. 12, No. xx 1556-6560/2018/12/001/009 doi:10.1166/jbmb.2018.1803 1