Expression profiling of the maize flavonoid pathway genes controlled by estradiol-inducible transcription factors CRC and P

To determine the scope of gene expression controlled by the maize transcrip tion factors C1/R and P, which are responsible for activating flavonoid syn thesis, we used GeneCalling, an open-ended, gel-based, mRNA-profiling techn ology, to analyze cell suspension lines of the maize inbred Black Mexican S weet (BMS) that harbored estradiol-inducible versions of these factors. EMS cells were transformed with a continually expressed estrogen receptor/maiz e C1 activator domain fusion gene (ER-C1) and either a fusion of CI and R ( CRC), P, or luciferase genes regulated by a promoter containing four repeat s of an estrogen receptor binding site. Increasing amounts of luciferase ac tivity, anthocyanins, and flavan-4-ols were detected in the respective cell lines after the addition of estradiol, The expression of both known and no vel genes was detected simultaneously in these EMS lines by profiling the m RNA isolated from replicate samples at 0, 6, and 24 hr after estradiol trea tment. Numerous cDNA fragments were identified that showed a twofold or gre ater difference in abundance at 6 and 24 hr than at 0 hr, The cDNA fragment s from the known flavonoid genes, except chalcone isomerase (chi1), were in duced in the CRC-expressing line after hormone induction, whereas only the chalcone synthase (c2) and flavanone/dihydroflavonol reductase (a1) genes w ere induced in the P-expressing line, as was expected. Many novel cDNA frag ments were also induced or repressed by lines expressing CRC alone, P alone , or both transcription factors in unique temporal patterns. The temporal d ifferences and the evidence of repression indicate a more diverse set of re gulatory controls by CRC or P than originally expected. GeneCalling analysi s was successful in detecting members of complex metabolic pathways and unc overing novel genes that were either coincidentally regulated or directly i nvolved in such pathways.