Using RNAi to improve plant nutritional value: from mechanism to application Guiliang Tang 1 and Gad Galili 2 1 Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA 2 Department of Plant Science, The Weizmann Institute of Science, Rehovot 76100, Israel RNA interference (RNAi) is an ancient mechanism of gene suppression, whose machinery and biological functions are only partially understood. Intensive studies have focused on developing RNAi technologies for treating human diseases and for improving plant traits. Yet application of RNAi to improving the nutri- tional value of plants for human and animal nutrition, and development of the related RNAi technologies are still in their infancy. Here we discuss current knowledge of plant RNAi function, as well as concepts and strategies for the improvement of plant nutritional value through the development of plant RNAi technologies. Although total yield is still the first priority for both traditional plant breeding and contemporary plant genetic engineering in developing countries, the goal of improving the nutritional value of plants is receiving increasing attention [1,2]. Some major diseases, such as heart disease and cancer, can be prevented by dietary supplements of specific nutrients [3]. In particular, essential amino acids, minerals, fatty acids and vitamins are key factors for robust human health and growth [2], and a diet of plant foods rich in essential nutrients can significantly improve human health and life expectancy [4]. Traditional breeding has been tremendously successful in improving the nutritional value of food and feed [5]; however, this process is time-consuming and the limited genetic resources of most crops have left little room for continued improvement by these means. Over the past few decades, the possibilities for improvement have been broadened by extensive gene mapping and identification, whole-genome sequencing of model plants and crops, and the development of gene transfer technologies. Directed efforts are now underway to use genetic engineering of metabolic pathways to alter plant nutrients [2]. These efforts depend on a detailed understanding of plant metabolic pathways and their constituent enzymes. Currently, the principal strategy for transgenic enhancement of plant nutrients involves increasing the expression of anabolic biosynthetic genes. Unfortunately, the efficacy of this approach for crop engineering has been restricted by two main obstructions. First, the introduc- tion of extra gene copies can have the non-intuitive effect of decreasing expression from both the introduced and homologous endogenous loci – a phenomenon of gene silencing known as CO-SUPPRESSION (See Glossary) [6]. Second, feedback metabolic loops tend to maintain homeostatic levels of nutrients. For example, plants might respond to an increase in nutrient production induced by overexpression of biosynthetic genes by activating a degradation pathway that either negates nutrient accumulation or converts nutrients into undesir- able metabolites [7]. It is therefore necessary to consider schemes for metabolic engineering that decrease the levels of catabolic enzymes; effective and expedient methods to achieve this, however, have been wanting in the past. Ironically, the very phenomenon of co-suppression that plagues some overexpression efforts might be useful for realizing such a reduction in catabolic enzymes. Co- suppression has been recently recognized as a manifes- tation of RNA INTERFERENCE (RNAi) – an endogenous pathway of negative posttranscriptional regulation. RNAi has revolutionized the possibilities for creating customized ‘knock-down’ of gene activity. RNAi operates in both plants and animals and uses double-stranded RNA (dsRNA) as a trigger that targets homologous mRNAs for degradation. Methods that introduce dsRNA into plant and animal cells have been enormously successful in decreasing cognate gene expression in vivo [8–11]. In this review, we first examine current understanding of the endogenous RNAi pathway in plants. We then discuss strategies and applications of RNAi for improving plant nutritional value via the coordinated overexpression and suppression of genes in plants. Finally, we discuss the development of plant RNAi technologies. The RNAi pathway in plants The phenomenon of plant co-suppression was accidentally discovered during attempts to alter the pigmentation of commercial petunia flowers [6]. It was supposed that deeper flower colors might result from the overexpression of a chalcone synthase gene driven by the constitutive 35S promoter. Instead, both endogenous and transgenic chalcone synthase genes were silenced in these plants, Corresponding author: Guiliang Tang (guiliang.tang@umassmed.edu). Available online 29 July 2004 www.sciencedirect.com 0167-7799/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2004.07.009 Review TRENDS in Biotechnology Vol.22 No.9 September 2004