--John Bodeau and Virginia Walbot

We found that electroporating BMS protoplasts with gene constructs containing components of the maize anthocyanin genes is a simple and efficient technique for studying regulation of this set of genes. We tested promoter activity of a 700 bp upstream region of Bronze-2 using luciferase as a reporter gene, with the first intron from maize Adh1 inserted into the 5' untranslated region (Bz2-I-Luc). Similar constructs with the A1- and Bz1- promoters (A1-I-Luc and Bz1-I-Luc) were obtained from Michael Fromm, while constructs expressing the R and C1 regulatory genes from cDNAs expressed under the control of the CaMV 35S promoter were kindly provided by Susan Wessler and Steven Goff, respectively. As shown in Table 1, following electroporation of protoplasts the Bz2-promoter was activated by R and C1 over 640-fold, while a 33-fold and 150-fold activation of the A1- and Bz1- promoters occurred. No luciferase expression was observed from a construct containing the Bz2-promoter in reversed orientation, either with or without R and C1. Absolute induced levels of luciferase expressed from the Bz2 promoter were approximately twice those of a similar 35S-promoter construct, and approximately 100-fold above induced levels from Bz1- and A1- promoters. Similar results were also obtained using the particle gun to introduce these constructs into intact aleurones of mature seeds (data not shown).

Additionally, introduction of R and C1 into BMS protoplasts activated the endogenous anthocyanin structural genes, resulting in red protoplasts within 24 hours. By 48 hours, approximately 30% of surviving protoplasts appear pigmented.

RNA was isolated from electroporated protoplasts, and RNase protection demonstrated that induction levels of luciferase and visible pigment reflect differences in RNA message abundance of both introduced and endogenous genes. Both Bz1 and Bz2 messenger RNAs are undetectable in protoplasts not receiving R and C1, while cells receiving R and C1 accumulate high levels of endogenous Bz1 message, and a much lower level of Bz2 message. Luciferase message expressed from the Bz2-promoter is induced from undetectable to high levels comparable to the induced level of endogenous Bz1 message.

We are intrigued by the discrepancy between the high activity of the isolated Bz2-promoter, and the low accumulation of endogenous Bz2 message. Possible explanations for this difference include silencer elements in the endogenous gene which are not present in the 700 bp promoter fragment, and low stability of the Bz2 message.

Table 1.  Expression of anthocyanin gene promoter constructs in electroporated BMS cells.

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