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.

Stimulation of the cell cycle and maize transformation by disruption of the plant retinoblastoma pathway

“…In progeny, LIR:RepA Asp-700 , Ubi:moGFP, and 35S:bar segregated as a single locus that was followed by fluorescence and PCR (data not shown). Agrobacterium-mediated gene transfer was used to deliver Nos:CRC, a cell autonomous stimulator of anthocyanin production (28,29), to T 3 embryos harvested from segregating ears. Calli were screened for multicellular pigmented clusters in the absence of chemical selection.…”

“…For maize cobombardment experiments, a fusion between a maize-optimized phosphinothricin acetyl transferase (moPAT) gene and moGFP was generated (moPATϳmoGFP). In retransformation experiments, a fusion between domains of R and C1 (CRC) encoding the protein functions necessary to activate anthocyanin accumulation (28,29) was used as a marker to score transgenic calli, and the bar gene (30) was used for bialaphos selection. Promoters driving expression of visible marker genes included a doubleenhanced cauliflower mosaic virus (CaMV) 35S promoter (31), the nopaline synthase (Nos) promoter (32), and the maize ubiquitin (Ubi) promoter (33).…”

Future renewable resource needs: will genomics help?

“…A recent example is the improved understanding of regulation mechanisms for gene expression in the plant¯avonoid pathway. 10 Genomics is beginning to be applied to altered composition, through improved understanding of endogenous regulation within metabolic pathways, and to improve the nutritional and nutraceutical features of food plants. 11 However, to-date, very little has been done in terms of direct use of new genomics techniques within applications for renewable resources.…”

Application of high‐resolution capillary array electrophoresis with automated fraction collection for GeneCalling™ analysis of the yeast genomic DNA

“…One of the recently introduced techniques, referred to as GeneCalling , has been developed as a high-throughput, comprehensive and sensitive method for identification of differential expression of known as well as novel genes [5][6][7][8]. This method includes isolation of mRNA from tissue samples, conversion of mRNA into cDNA fragments, precise fragment size determination by capillary electrophoresis with sensitive laserinduced fluorescence detection, and identification and quantitation of expression differences.…”