A 235-bp region from a nutritionally regulated soybean seed-specific gene promoter can confer its sulfur and nitrogen response to a constitutive promoter in aerial tissues of Arabidopsis thaliana Motoko Awazuhara a,1 , Hoyeun Kim a,c , Derek B. Goto b , Akifusa Matsui a , Hiroaki Hayashi a , Mitsuo Chino a,2 , Sang-Gu Kim c , Satoshi Naito b , Toru Fujiwara a, * a Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan b Department of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan c School of Biological Sciences, Seoul National University, Seoul 151-742, Republic of Korea Received 7 December 2001; received in revised form 25 March 2002; accepted 25 March 2002 Abstract The seed-specific promoter of the b subunit gene of b-conglycinin, a major seed storage protein of soybean, is upregulated in response to both sulfur and nitrogen nutrition. This response was studied in non-seed tissues of transgenic Arabidopsis thaliana by fusing the b subunit promoter (Pb) downstream of an enhancer from the cauliflower mosaic virus 35S RNA promoter (P35S) and using the b-glucuronidase (GUS) reporter gene. The chimeric promoter was upregulated by sulfur deficiency (DS), indicating that the Pb is able to direct the DS response in non-seed tissues. Deletion analysis of the Pb revealed that, in seeds, the  /307  / /73 bp region is sufficient for the DS response. Insertion of the 235 bp region into the P35S conferred the DS response on this constitutive promoter in both seed and non-seed tissues. The extent of upregulation by DS was more evident when three of the 235 bp fragments were tandemly inserted into the P35S. The findings presented here suggest that a common mechanism exists for sulfur-regulated expression of the Pb in both seed and non-seed tissues and that this 235 bp region of the Pb is sufficient for regulation by both sulfur and nitrogen nutrition. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Arabidopsis thaliana ; b Subunit of b-conglycinin; Cauliflower mosaic virus 35S RNA promoter; Sulfur and nitrogen nutrition 1. Introduction Sulfur is mostly available to plants in the form of sulfate. Plants take up sulfate from the soil and transport it to leaves, the major site of sulfate reduction, after which it is assimilated into sulfur-containing compounds such as cysteine and methionine [1]. Sul- fur-containing compounds play a number of important roles in plant metabolism and defense responses. Glu- tathione, the most abundant form of reduced soluble sulfur in higher plants, is required for oxidative stress response and detoxification of xenobiotics [1]. In addi- tion, sulfur is also an important component for a number of plant secondary metabolites [1]. Despite the importance of sulfur-containing com- pounds in plants, sulfur supply from the environment is often below optimal levels. Plants respond to the sulfur-limiting environment by activating sulfate uptake and assimilation pathways [2]. We have been studying the mechanisms of sulfur regulation of gene expression using seed storage protein genes of soybean (Glycine max L. Merr) as a model system. Under limited levels of sulfur supply, plants generally accumulate lower levels of sulfur-rich proteins and higher levels of sulfur-poor proteins in seeds ([3], and references therein). Accord- ingly, accumulation of glycinin (11S globulin) is reduced in soybean under DS conditions, whereas that of b- * Corresponding author. Tel.:  /81-3-5841-5105; fax:  /81-3-5841- 8032 E-mail address: atorufu@mail.ecc.u-tokyo.ac.jp (T. Fujiwara). 1 Present address: Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 263-8522, Japan. 2 Present address: Department of Bio-Production Science, Faculty of Bioresource Science, Akita Prefectural University, Akita 010-0146, Japan. Plant Science 163 (2002) 75  /82 www.elsevier.com/locate/plantsci 0168-9452/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII:S0168-9452(02)00064-X