Plant Science 150 (2000) 21–28 A modified 10 kD zein protein produces two morphologically distinct protein bodies in transgenic tobacco Jennifer Randall a , Suman Bagga a,b , Henry Adams c , John D. Kemp a, * a Department of Entomology, Plant Pathology and Weed Science, Gene Lab, New Mexico State Uniersity, Box 3GL, Las Cruces, NM 88003, USA b Department of Agronomy and Horticulture, New Mexico State Uniersity, Box 3GL, Las Cruces, NM 88003, USA c Department of Cell Biology, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX 77025, USA Received 10 March 1999; received in revised form 14 July 1999; accepted 15 July 1999 Abstract The 10 kD zein protein contains an N-terminal signal peptide that directs the protein into the endoplasmic reticulum (ER) of developing corn seeds. Subsequent to signal peptide removal, the mature protein is folded into its tertiary conformation and deposited into protein bodies. In transgenic tobacco leaves, the 10 kD zein protein accumulates and forms novel ER derived protein bodies (S. Bagga, H. Adams, F. Rodriquez, J.D. Kemp, C. Sengupta-Gopalan, Coexpression of the maize -zein and -zein genes results in stable accumulation of -zein in endoplasmic reticulum-derived protein bodies formed by -zein, The Plant Cell 9 (1997) 1683–1696). In this study, the amino acid sequence of the 10 kD zein signal peptide was modified to determine the effect on cleavage and accumulation patterns. The modified zein gene was constitutively expressed in tobacco where its protein accumulates in novel protein bodies in leaves. Amino acid sequencing of the accumulated protein indicates that the cleavage site for the signal peptide was altered so that the mature protein includes three additional amino acids on the N-terminus. Electron microscopy (EM) analysis of leaves from transgenic plants containing the modified gene indicates the presence of two morphologically distinct protein bodies. Furthermore, immunolocalization analysis shows that the modified protein is localized in both types of protein bodies, which are described as spherical and aggregate in this report. This is in contrast to the accumulation of unmodified 10 kD zein protein in transgenic leaves where only spherical protein bodies are observed. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Zein; Signal peptide; Targeting; Transgenic plants; Protein bodies www.elsevier.com/locate/plantsci 1. Introduction Zeins are alcohol soluble seed storage proteins found in the endosperm of maize. They are differ- entiated into four classes: , , , and  based upon their solubility and their mobility on a SDS- PAGE gel [2]. All zein proteins contain an N-ter- minal signal peptide that directs them into the endoplasmic reticulum (ER). Once translocation of the protein into the ER is complete, a signal peptidase cleaves the signal peptide from the ma- ture protein. The mature protein is then folded into its tertiary configuration and subsequently deposited into ER derived protein bodies. In the endosperm of maize seeds the production of zeins is developmentally regulated and each protein class is deposited in a specific manner within protein bodies [3]. The  and  zeins com- prise the core of the protein body with the beta and gamma zeins surrounding the periphery [4]. It is not clear how the zeins are retained in the ER. They do not contain a known ER retention signal such as the KDEL or the HDEL motif found in other ER resident proteins [5]. However, high levels of binding protein (BiP), an ER resident protein that acts as a chaperone, are found associ- ated with the protein bodies [6–8]. This associa- * Corresponding author: Tel.: +1-505-6465453; fax: +1-505- 6461302. E-mail address: jkemp@nmsu.edu (J.D. Kemp) 0168-9452/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII:S0168-9452(99)00149-1