Mol Gen Genet (1989) 219:225-234 MGG © Springer-Verlag 1989 Isolation and molecular analysis of the maize P locus Christa Lechelt 1' *, Thomas Peterson 2' **, Alan Laird 1' ***, Jychian Chen 3" ****, Steven L. Dellaporta 3 Elisabeth Dennis 2, W.J. Peacock 2, and Peter Starlinger 1 1 Institut ffir Genetik, Universit~it zu K61n, Weyertal 121, D-5000 K61n 41, Federal Republic of Germany 2 CSIRO division of Plant Industry, GPO Box 1600, Canberra ACT 2601, Australia 3 Department of Biology, Yale University, PO Box 6666, New Haven, Connecticut 06511, USA Summary. The maize P locus is involved in the synthesis of a red flavonoid pigment in the pericarp, cob and other floral tissues. The tissue-specific pattern of expression of certain P alleles suggests that P may be a complex locus, with more than one functional unit. The P- VV allele, which specifies variegated pericarp and variegated cob, however, shows that insertion and excision of the transposable ele- ment Ac affects both pericarp and cob expression as though cob and pericarp pigmentation are controlled by a single gene. Using Ac as a transposon tag, we have isolated 34 kb of genomic DNA from the P-VV and P-RR allele. The cloned DNA contains two 5.8 kb cross-hybridizing regions, in direct orientation relative to each other, separated by 6.6 kb of intervening DNA. A sequence motif of 250 bp is repeated at three locations within the cloned region: once within each of the 5.8 kb repeats, and once outside the 5.8 kb repeats. DNA fragments flanking the Ac element detect five transcripts in RNA from wild type (P-RR) that are absent from mutant (P- VV) tissues. To localize the tran- scribed sequences, DNA probes spanning the 34 kb of cloned DNA were used in Northern analysis of RNA from mutant and wild-type kernels. The results suggest the pres- ence of a single transcriptional unit located primarily within the DNA between the 5.8 kb repeats. The five RNAs tran- scribed from this region may be formed by alternative splic- ing. The size of the P gene derived from the length of the transcribed region seems much smaller than the gene size estimated from Ac-induced P-VV mutations. Key words: Zea mays - Ac P gene - Pericarp - Transpo- son Tagging Introduction The P locus of Zea mays L. is located on the short arm of chromosome 1 and is one of at least 14 characterized * Present address: Max Planck Institut ftir Zfichtungsforschung, Abteilung Biochemie, D-5000 K61n 30 (Vogelsang), Federal Re- public of Germany ** Present address: Cold Spring Harbor Laboratory, Box 100, Cold Spring Harbor, New York 11724, USA *** Present address: 3 Garrard Street, Spence ACT 2615, Austra- lia **** Present address: Institute of Molecular Biology, Academia Sinica, Nankang Taipei, 11529 Republic of China Offprint requests to ." C. Lechelt loci that are involved in the flavonoid biosynthetic pathway (Coe and Neuffer 1977). The P locus is involved in the synthesis of a red phlobaphene-like pigment found in cer- tain floral tissues, most notably the cob glumes and the pericarp. Comparison of flavonoid pigments occurring in differ- ent tissues of maize strains of defined genotypes indicates that the P gene controls a biosynthetic pathway very similar to the one leading to anthocyanidins (Styles and Ceska 1977). P controls the production of flavan-4-ol, while an- thocyanidins are derived from flavan-3,4-diol. In cob glumes and pericarp, flavan-4-ol is polymerized to phloba- phenes. In the absence of a functional P gene, no flavan-4-ol is formed, nor are the precursors of this molecule detected. These results suggest a regulatory role for the P gene in phlobaphene biosynthesis (Styles and Ceska 1977). The na- ture of this hypothetical regulation remains to be eluci- dated. Alleles of the P locus are commonly assigned a two- letter suffix which describes the expression of the particular P allele in the pericarp and cob glumes. Thus, P-RR speci- fies red pericarp and red cob glumes, P-WW specifies white (more accurately colorless) pericarp and cob, P-WR speci- fies white pericarp/red cob and P-R W specifies red pericarp/ white cob. The independent pigmentation of pericarp and cob specified by P-WR and P-RW might suggest that P is a multi-cistronic locus. Yet, no confirmed cases of recom- bination between the two hypothetical genes have been found in genetic experiments (Anderson 1924). Therefore, either the genes must be closely linked or there is in fact only one gene at the locus which can be differentially ex- pressed in pericarp and cob tissues. This latter explanation is supported by insertional mutations of P. For example, the P-VV allele, which specifies variegated pericarp and variegated cob (Emerson 1917), has the maize transposable element Ac inserted at the P locus (Barclay and Brink 1954). The instability of both pericarp and cob glume pigmenta- tion in the P-VV allele suggests that the pericarp and cob expression are not independently controlled by separate genes. The Ac element has been isolated and characterized from other unstable mutants in maize (Fedoroffet al. 1983; Behrens et al. 1984). Thus, Ac-specific sequences have been used to clone DNA probes from the P locus (Chen et al. 1987). We report here an extensive molecular cloning, struc- tural characterization, and transcriptional analysis of the P locus. Our results support the idea that P contains a single transcriptional unit.