Plant Science 145 (1999) 93–98 A soybean vegetative storage protein accumulates to high levels in various organs of transgenic tobacco plants Dana Guenoune a , Rachel Amir b , Bruria Ben-Dor a , Shmuel Wolf c , Shmuel Galili a, * a Department of Field and Garden Crops, Agriculture Research Organization (Volcani Center), P.O. Box 6, Bet -Dagan 50250, Israel b Department of Plant Physiology, Migal Technological Center, Kiryat Shmona, Israel c Department of Field Crops, Vegetables and Genetics and the Otto Warburg Center for Agricultural Biotechnology, The Hebrew Uniersity of Jerusalem, Faculty of Agriculture, Food and Enironmental Quality Sciences, Rehoot, Israel Received 3 February 1999; received in revised form 27 April 1999; accepted 6 May 1999 Abstract Soybean vegetative storage proteins (S-VSPs) are a group of high-lysine proteins. These proteins can accumulate up to 15% of the soluble leaf proteins in young shoots, as well as in shoots of depodded mature plants. Closely related proteins are found in forage crops, such as alfalfa. We have expressed the S-VSP gene, fused to the strong constitutive cauliflower mosaic virus 35S promoter, in transgenic tobacco as a model plant to study the potential of producing high levels of S-VSPs for the nutritional improvement of forage crops. S-VSP was detected in the soluble (albumin) fraction of the transgenic plants. The transgenic protein migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis similarly to the natural S-VSP of soybean, suggesting that it was correctly processed in tobacco and accumulated in the vacuoles. The S-VSP ranged between 2 and 6% of the soluble proteins in leaves of the transgenic plants and was present in various organs and in mature leaves. Our results suggest that S-VSPs can serve as an excellent protein source for improving the nutritional quality of crop plants, particularly cereal forage and grains, which contain limiting levels of lysine. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Vegetative storage proteins; Overexpression; Tobacco; Transgenic plants; Improved quality; Soybean www.elsevier.com/locate/plantsci 1. Introduction Human and monogastric animals are unable to synthesize the 10 essential amino acids, and must obtain them from their diet. Legumes and cereals supplied as a grains or as a forage are among the most important nutritional sources of protein for animal consumption. These crops, however, con- tain limiting levels of several essential amino acids. Legume proteins are deficient in the sulfur-con- taining amino acids, methionine and cysteine [1], while cereals are deficient mainly in lysine and tryptophan [2]. These deficiencies can be partly overcome by mixing meals from cereals and legumes. However, in intensively reared livestock diets, the entire requirement for these amino acids cannot be met by mixing alone and most feeds for poultry are supplemented with methionine, while lysine is added to the diet of both poultry and swine [3]. The demands to improve pasture and forage crops as sources for animal feeding are continuously increasing [4]. Genes encoding me- thionine-rich seed storage proteins, engineered for constitutive expression, have been used for im- proving the nutritional quality of forage crops, but in most cases these proteins did not exceed 1.3% of the soluble proteins in vegetative tissues of ho- mozygous plants [5 – 9]. Attempts to express lysine- rich seed storage proteins and synthetic genes in transgenic plants have also met with limited suc- cess [10,11]. Seed storage proteins are not the only natural protein source that can be used for improving nutritional quality. Several plant species contain * Corresponding author. Tel.: +972-3-9683872; fax: +972-3- 9669642. E-mail address: vclidg@vocani.agri.gov.il (S. Galili) 0168-9452/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII:S0168-9452(99)00079-5