Elevation of seed α-tocopherol levels using plant-based transcription factors targeted to an endogenous locus

Abstract

Synthetic zinc finger transcription factors (ZFP-TFs) were designed to upregulate the expression of the endogenous Arabidopsis γ-tocopherol methyltransferase (GMT) gene. This gene encodes the enzyme responsible for the conversion of γ-tocopherol to α-tocopherol, the tocopherol species with the highest vitamin E activity. Five three-finger zinc finger protein (ZFP) DNA binding domains were constructed and proven to bind tightly to 9 bp DNA sequences located in either the promoter or coding region of the GMT gene. When these ZFPs were fused to a nuclear localization signal and the maize C1 activation domain, four of the five resulting ZFP-TFs were able to upregulate the expression of the GMT gene in leaf protoplast transient assays. Seed-specific expression of these ZFP-TFs in transgenic Arabidopsis produced several lines with a heritable elevation in seed α-tocopherol. These results demonstrate that engineered ZFP-TFs comprised of plant-derived elements are capable of modulating the expression of endogenous genes in plants.

Introduction

The tools of plant biotechnology allow the engineering of novel traits via introduction of foreign genes into plants and expression of these genes in a developmental or tissue specific manner using selected promoters. Desired traits can alternatively be obtained by regulating the expression of endogenous genes. One method to achieve this type of targeted gene regulation is through the use of engineered transcription factors. Transcription factors are trans-acting proteins that bind to specific cis-elements and regulate gene expression. Transcription factors are typically modular, consisting of a DNA-binding domain (DBD) and an effector domain (ED) that interacts with other regulatory proteins to either activate or repress transcription. The Cys₂–His₂ zinc finger proteins (ZFPs) are the most common DBDs in eukaryotes and over the past decade this motif has emerged as amenable to manipulations designed to achieve the specific recognition of a predetermined DNA sequence (Pabo et al., 2001, Segal and Barbas, 2001, Beerli and Barbas, 2002). These designer ZFPs have been fused to different EDs to create hybrid zinc finger protein transcription factors (ZFP-TFs) that have been used successfully in the regulation of endogenous chromosomal genes in both animal cell lines (Beerli et al., 2000; Zhang et al., 2000; Bartsevich and Juliano, 2000; Dreier et al., 2001 ; Liu et al., 2001, Ren et al., 2002) and more recently transgenic plants (Guan et al., 2002).

This study reports the production of ZFP-TFs comprised of plant-derived DNA sequences and engineered to activate the endogenous Arabidopsis γ-tocopherol methyltransferase (GMT) gene (GenBank Accession AF104220). GMT encodes the enzyme responsible for addition of a methyl group to ring carbon 5 of γ-tocopherol to form α-tocopherol (Fig. 1), the tocopherol isoform with the highest vitamin E activity (Bramley et al., 2000). GMT is frequently limiting in seed tissue and as a result the tocopherol composition of seed from many plant species is made up predominantly of γ-tocopherol (Sheppard et al., 1993). Transgenic overexpression of an Arabidopsis GMT cDNA was previously found to result in a large increase in α-tocopherol relative to control (Shintani and Della-Penna, 1998).

Five three-finger ZFPs were designed to bind to target 9 bp sequences found in the promoter or coding region of the endogenous Arabidopsis GMT gene. These ZFPs were each fused to the maize opaque-2 nuclear localization signal (GenBank Accession M29411) and the maize C1 (GenBank Accession TVZMMB) activation ED (Guyer et al., 1998, Goff et al., 1991) to make ZFP-TFs. Expression of these ZFP-TFs in transgenic Arabidopsis under the control of an embryo specific promoter (Kridl et al., 1991) resulted in several lines that had an elevated seed α-tocopherol percentage, with the best line demonstrating a heritable 20 fold increase in percent α-tocopherol relative to control seed. Transgenically modulating endogenous gene expression to achieve a desired phenotype using ZFP-TFs offers a powerful tool to plant biotechnologists (Guan et al., 2002, Ordiz et al., 2002). This is first time that ZFP-TFs derived wholly from plant sequences have been described and found to modulate expression of an endogenous target gene in a whole plant.