Complex structure of a maize Myb gene promoter: functional analysis in transgenic plants

Abstract

SummaryThe maize P gene encodes a Myb-like transcription factor that regulates synthesis of red¯avonoid pigments in¯oral organs. To study the transcriptional regulation of the P gene, candidate regulatory sequences of the P1-rr gene promoter were identi®ed by Ac insertional mutagenesis and subjected to functional testing in transgenic maize plants. The results indicate that a 561 bp fragment (Pb) encompassing the transcription start site (±235 to +326) supports weak expression of a GUS reporter gene in¯oral organs, including husk, silk, kernel pericarp, cob and male in¯orescence. Two other fragments, located approximately 1 and 5 kb 5¢ of the transcription start site, increased the levels of GUS activity in¯oral tissues and thus appear to contain enhancer elements. All of the tested constructs gave similar patterns of GUS expression, suggesting that the 561 bp Pb fragment that is common among the transgene constructs contains regulatory elements that promote activation in¯oral organs. The basal promoter and proximal enhancer fragments contain putative binding sites for bZip regulatory factors, and a complex arrangement of palindromes including a large inverted repeat of two tRNA-like genes. Possibly, interconversions between linear and cruciform conformations of the palindromes may affect protein/DNA interactions and thereby modulate P1-rr expression.

Gene Conversion Between Direct Noncoding Repeats Promotes Genetic and Phenotypic Diversity at a Regulatory Locus of Zea mays (L.)

“…According to the DNA gel blot patterns, the single-copy p1 alleles, including 6 P1-rw and 9 P1-rr alleles, can be classified into six allelic types: three P1-rr types and three P1-rw types (Table 1; Figure 1, A and B). The P1-rr4B2 and P1-rw1077 allelic types have been characterized and compared previously (Lechelt et al 1989;Sidorenko et al 2000;Zhang and Peterson 2005). Representative alleles from the other four allelic types (P1-rrCFS36, P1-rr1088, P1-rwCFS302, and P1-rwCFS342, phenotypes are shown in Figure 1A) were chosen for further analysis.…”

“…Structural comparisons of p1 alleles: Previous studies have shown that the coding regions of different p1 alleles share high sequence similarity; and DNA polymorphisms in noncoding regulatory regions other than in coding regions could be responsible for phenotypic variations of the p1 alleles (Lechelt et al 1989;Sidorenko et al 2000;Zhang and Peterson 2005). To examine sequence polymorphisms in noncoding regions of the single-copy p1 alleles studied here, we used genomic PCR with p1-specific primers to amplify both 59 and 39 noncoding regions of the P1-rr and P1-rw alleles.…”

“…In the 59 repeats, the most notable polymorphic regions overlap with the previously identified enhancer regions ( Figure 2). The distal enhancer region is located 5 kb upstream of the p1 transcription start site and includes the 405-bp fragment 15 and a 666-bp sequence termed fragment 14 (Sidorenko et al 2000;Zhang and Peterson 2005; Figure 2). Based on the copy number of fragment 15 and fragment 14 sequences in the enhancer-containing regions, three distinct structural types can be defined in six simplex p1 alleles: (i) 1 copy of fragment 15 and 0 copy of fragment 14, i.e., the enhancer-containing region contains only fragment 15, as shown in P1-rwCFS302 and P1-rwCFS342; (ii) 1.5 copies of fragment 15 and 1 copy of fragment 14, i.e., the enhancer-contaning region contains a partial copy of fragment 15 followed by fragment 14 and a full copy of fragment 15, as seen in P1-rw1077; (iii) 2 copies of fragment 15 and 1 copy of fragment 14, i.e., the enhancer-containing region contains a full copy of fragment 15 followed by fragment 14 and an additional full copy of fragment 15, as shown in P1-rr1088, P1-rrCFS36, and P1-rr4B2.…”

“…Gel electrophoresis and hybridization procedures were conducted as described in previous studies (Sidorenko et al 2000). The blot was probed with the p1-specific probe, genomic fragment 15 ( Figures 1B and 2).…”

“…Three distinct p1 alleles, P1-rr4B2, P1-rw1077, and P1-wr[w22], have been characterized and compared at the molecular level. All of these p1 alleles share highly similar coding regions, but differ in the flanking regulatory sequences (Chopra et al 1998;Sidorenko et al 2000). Our recent data have demonstrated that changes in the enhancer region, located 5 kb 59 of the transcription start site, result in phenotypic variations between P1-rr4B2 and P1-rw1077 (Zhang and Peterson 2005).…”