Figure 3.

Positional cloning and structure of the PHR1 gene. (A) PHR1 was first mapped between CAPS markers RPS2 and phra, and finally narrowed the physical localization between BACs F20O9 and F16A16. Within this region, a homolog to the PSR1 gene from C. reinhardtii (Wykoff et al. 1999) was identified (At4g28610). Sequencing of the region corresponding to the PSR1 homolog in the two alleles, phr1-1 and phr1-2 revealed that each had a mutation in this gene. The mutation in phr1-1 was a C-to-T transition, causing the introduction of a premature stop codon. The mutation in phr1-2 was also a G-to-A substitution, which impaired a GT splicing donor site. Nucleotides in the intron are shown in italics. The exon structure derived from comparison of the genomic and cDNA sequences is highlighted with boxes (empty, noncoding exons, or parts; full, coding exons, or parts). (B) Complementation of the phr1-1 mutant with plasmid pBIB∷PHR1, harboring the PHR1-coding region plus 2 kb upstream and 1.3 kb downstream sequences. T₂ progeny of a transgenic plant harboring a copy of the pBIB∷PHR1 T-DNA (middle), displays a 3:1 segregation of the colored phenotype when germinated directly in Pi starvation medium. Control progeny from wild-type and phr1-1 homozygous plants are shown at left and right, respectively. Scale bar, 0.5 cm. (C) Nucleotide and deduced amino acid sequence from the PHR1 cDNA. The two regions conserved between PHR1 and the C. reinhardtii PSR1 protein, corresponding to the MYB domain and to a predicted coiled–coil domain, are highlighted in reverse contrast and gray, respectively. A putative nuclear localization signal is shown (underlined).