Compositional and transcriptional analyses of reduced zein kernels derived from the opaque2 mutation and RNAi suppression Alessandra Frizzi • Rico A. Caldo • James A. Morrell • Meng Wang • Linda L. Lutfiyya • Wayne E. Brown • Thomas M. Malvar • Shihshieh Huang Received: 18 August 2009 / Accepted: 28 April 2010 / Published online: 16 May 2010 Ó Springer Science+Business Media B.V. 2010 Abstract Corn protein is largely made up of a group of nutritionally limited storage proteins known as zein. The reduction of zein can be achieved by a transcriptional mutation, opaque2 (o2), or a transgene targeting zein through RNA interference (RNAi). Zein reduction results in an increase of more nutritionally balanced non-zein proteins, and therefore enhance the overall quality of corn protein. In this study, the composition of mature kernels and the transcriptional profile of developing kernels of these two types of zein reduced kernels were compared. Both zein reduced kernels contained higher levels of lysine and tryptophan and free amino acids were 10–20-folds more abundant than the wild-type counterpart. We also found that free lysine contributed partially to the increased lysine in o2 kernels while protein-bound lysine was mainly responsible for the increased lysine in transgenic zein reduction (TZR) kernels. Although they had relatively similar gene expression patterns in developing endosperm, o2 kernels had greater transcriptional changes than TZR kernels in general. A number of transcripts that were spe- cifically down-regulated in o2 were identified. Many pro- moter sequences of these transcripts contain putative O2 binding motifs, suggesting that their expression is directly regulated by O2. Keywords Zein Á High lysine corn Á Opaque2 Á QPM Á Zein reduction Introduction Lysine is the most deficient essential amino acid in corn for human consumption (Shewry 2007). To improve the lysine content of corn, two high lysine maize lines were identified in 1960s (Mertz et al. 1964; Nelson et al. 1965). These two maize mutants, opaque2 (o2) and floury2 (fl2), contain approximately double the amount of lysine compared to wild-type. In addition, they have considerable increases in tryptophan and methionine which are also deficient in corn, and a reduced level of leucine that is beneficial to animal nutrition (Harper et al. 1970). However, the soft, starchy endosperm phenotype often associated with these mutants must be overcome for them to be generally applicable. Over the last few decades, genetic modifiers have been used successfully to develop inbred varieties with normal endosperm derived from o2 mutant in developing countries (Krivanek et al. 2007; Prasanna et al. 2001). These varie- ties, known as Quality Protein Maize (QPM), have been shown to have the potential to improve the diet of corn- eating populations by several studies (FAO 1992). Although in limited scale, the hybrid versions of QPM were once commercially available in the United State Electronic supplementary material The online version of this article (doi:10.1007/s11103-010-9644-1) contains supplementary material, which is available to authorized users. A. Frizzi Á S. Huang (&) Calgene Campus, Monsanto Company, 1920 Fifth Street, Davis, CA 95616, USA e-mail: shihshieh.huang@monsanto.com R. A. Caldo Á J. A. Morrell Á L. L. Lutfiyya Á W. E. Brown Creve Coeur Campus, Monsanto Company, 800 North Lindbergh, Creve Coeur, MO 63167, USA M. Wang Chesterfield Campus, Monsanto Company, 700 Chesterfield Parkway West, Chesterfield, MO 63017, USA T. M. Malvar Mystic Research, Monsanto Company, 62 Maritime Drive, Mystic, CT 06355, USA 123 Plant Mol Biol (2010) 73:569–585 DOI 10.1007/s11103-010-9644-1