Expression Profiling of the Maize Flavonoid Pathway Genes Controlled by Estradiol-Inducible Transcription Factors CRC and P

Abstract

To determine the scope of gene expression controlled by the maize transcription factors C1/R and P, which are responsible for activating flavonoid synthesis, we used GeneCalling, an open-ended, gel-based, mRNA-profiling technology, to analyze cell suspension lines of the maize inbred Black Mexican Sweet (BMS) that harbored estradiol-inducible versions of these factors. BMS cells were transformed with a continually expressed estrogen receptor/maize C1 activator domain fusion gene (ER-C1) and either a fusion of C1 and R (CRC), P, or luciferase genes regulated by a promoter containing four repeats of an estrogen receptor binding site. Increasing amounts of luciferase activity, anthocyanins, and flavan-4-ols were detected in the respective cell lines after the addition of estradiol. The expression of both known and novel genes was detected simultaneously in these BMS lines by profiling the mRNA isolated from replicate samples at 0, 6, and 24 hr after estradiol treatment. Numerous cDNA fragments were identified that showed a twofold or greater difference in abundance at 6 and 24 hr than at 0 hr. The cDNA fragments from the known flavonoid genes, except chalcone isomerase (chi1), were induced in the CRC-expressing line after hormone induction, whereas only the chalcone synthase (c2) and flavanone/dihydroflavonol reductase (a1) genes were induced in the P-expressing line, as was expected. Many novel cDNA fragments were also induced or repressed by lines expressing CRC alone, P alone, or both transcription factors in unique temporal patterns. The temporal differences and the evidence of repression indicate a more diverse set of regulatory controls by CRC or P than originally expected. GeneCalling analysis was successful in detecting members of complex metabolic pathways and uncovering novel genes that were either coincidentally regulated or directly involved in such pathways.

A Multidrug Resistance–Associated Protein Involved in Anthocyanin Transport in Zea mays

“…Similarities between the anthocyanin biosynthesis and xenobiotic detoxification pathways, particularly the requirement for the activity of a GST to ensure proper anthocyanin localization, suggest that the ABC transporter responsible for anthocyanin sequestration is an MRP. Indeed, in a study of anthocyanin production in maize cell culture, two ABC transporters, one of which is an MRP, were identified as being upregulated in cells overexpressing the anthocyanin transcription factors R and C1 (Bruce et al, 2000). In this article, we identify that MRP, ZmMRP3, as coregulated with the anthocyanin pathway in planta, characterize the ZmMRP3 gene, and demonstrate the involvement of the corresponding protein in the transport of anthocyanin into the vacuole.…”

anthocyanin1 of Petunia Encodes a Basic Helix-Loop-Helix Protein That Directly Activates Transcription of Structural Anthocyanin Genes

“…In maize suspension cells, forced expression of P1 alone is sufficient to induce transcription of a1 (Grotewold et al, 1994;Bruce et al, 2000), and this induction cannot be enhanced by coexpression of R1; this indicates that P1, unlike C1, functions independent of R1-like bHLH factors (Grotewold, 1995;Sainz et al, 1997). Transcripts of an1, an2, jaf13, dfrA, and gapdh were detected by quantitative RT-PCR in the corolla and tube, anthers, ovaries, and sepals from flowers at various developmental stages and from leaves, stems, roots, and pistils of the wild-type line V30, as indicated above the lanes.…”

Development of a tightly regulated and highly inducible ecdysone receptor gene switch for plants through the use of retinoid X receptor chimeras

“…In order to achieve an optimal expression of transgenes with minimum undesirable effects, it is highly desirable to regulate the expression of transgenes in a controllable fashion. A number of chemical inducible gene regulation systems, or gene switches, have been developed based on a diverse collection of non-plant regulatory elements that respond to a variety of chemicals (Gatz et al 1992;Wilde et al 1992;Williams et al 1992;Mett et al 1993;Rieping et al 1994;Weinmann et al 1994;Aoyama and Chua 1997;Caddick et al 1998;Bohner et al 1999;Martinez et al 1999a, b;Bruce et al 2000;Padidam et al 2003). A chemical inducible gene regulation system that specifically regulates transgene expression in response to an exogenous inducer at a particular stage of plant development or in a specific organ is very valuable when using transgenes whose constitutive over expression is detrimental or lethal to the plant.…”

“…In an effort to find an optimal gene switch, several approaches have been tried (Ainley and Key 1990;Schena et al 1991;Gatz et al 1992;Mett et al 1993;Lloyd et al 1994;Weinmann et al 1994;Aoyama and Chua 1997;Bruce et al 2000;. However, most of the gene switches developed to date for use in plants are not suitable for field applications because of the impractical or incompatible characteristics of the chemical ligands.…”

“…However, most of the gene switches developed to date for use in plants are not suitable for field applications because of the impractical or incompatible characteristics of the chemical ligands. Furthermore, testing of these gene switches has been limited to one or two plant species such as Arabidopsis, tobacco or maize (Faiss et al 1997;Bruce et al 2000;Zuo et al 2001;Unger et al 2002;Chen et al 2003;Laufs et al 2003). None of the gene switches developed to date have most of the desirable properties (high specificity, low activity in the absence of ligand, robust and rapid response in the presence of ligand and functions in a wide variety of plant species) of an optimal gene regulation system (Lloyd et al 1994;Corlett et al 1996;Gatz 1996;Kang et al 1999;Ouwerkerk et al 2001;Andersen et al 2003;Tang and Newton 2004).…”