The pea light-independent photomorphogenesis1 mutant results from partial duplication of COP1 generating an internal promoter and producing two distinct transcripts

Abstract

The pea lip1 (light-independent photomorphogenesis1) mutant shows many of the characteristics of light-grown development when grown in continuous darkness. To investigate the identity of LIP1, cDNAs encoding the pea homolog of COP1, a repressor of photomorphogenesis identified in Arabidopsis, were isolated from wild-type and lip1 pea seedlings. lip1 seedlings contained a wild-type COP1 transcript as well as a larger COP1' transcript that contained an internal in-frame duplication of 894 bp. The COP1' transcript segregated with the lip1 phenotype in F(2) seedlings and could be translated in vitro to produce a protein of approximately 100 kD. The COP1 gene in lip1 peas contained a 7.5-kb duplication, consisting of exons 1 to 7 of the wild-type sequence, located 2.5 kb upstream of a region of genomic DNA identical to the wild-type COP1 DNA sequence. Transcription and splicing of the mutant COP1 gene was predicted to produce the COP1' transcript, whereas transcription from an internal promoter in the 2.5-kb region of DNA located between the duplicated regions of COP1 would produce the wild-type COP1 transcript. The presence of small quantities of wild-type COP1 transcripts may reduce the severity of the phenotype produced by the mutated COP1' protein. The genomic DNA sequences of the COP1 gene from wild-type and lip1 peas and the cDNA sequences of COP1 and COP1' transcripts have been submitted to the EMBL database under the EMBL accession numbers AJ276591, AJ276592, AJ289773, and AJ289774, respectively.

Figure 1.

RT-PCR of Wild-Type and lip1 Seedlings. Wild-type (WT) and lip1 seedlings were grown for 7 days in continuous darkness. Shoot tissue was excised from the seedlings, and the total RNA was extracted and used to produce first-strand cDNA by using oligo(dT) primers and reverse transcriptase. The resulting cDNA was used as template in PCR reactions using two sets of primers. (A) Primers PCCOPF and PCCOPR (see Methods), which are expected to amplify a 2016-bp fragment containing the entire pea COP1 coding region. (B) Primers FS1 and RS1 (see Methods), which are expected to amplify a 1227-bp region within the pea COP1 open reading frame. The PCR products produced were separated by electrophoresis in a 1% agarose gel. Numbers at left and right indicate the approximate sizes (in kilobases) of PCR products produced.

Figure 2.

Schematic Representation and Translation in Vitro of COP1 and COP1′ Transcripts. (A) Schematic representation of COP1 and COP1′ transcripts as determined from sequence analysis of the cDNAs amplified with PCCOPF and PCCOPR primers. The region in black indicates the region from +303 to +1197 (relative to start codon) duplicated in the COP1′ transcript. (B) Cloned RT-PCR products were transcribed in vitro using T7 RNA polymerase, and increasing amounts (0.4 to 1.6 μg) of RNA were translated by using wheat germ extract and ³⁵S-labeled methionine and cysteine. Translation products were separated by electrophoresis on an SDS–10% polyacrylamide gel and compared with markers of molecular mass, shown at left in kilodaltons. Arrowheads indicate major translation products of 100 and 70 kD.

Figure 3.

Segregation Analysis of Transcripts and Genomic DNA in F₂ Progeny. Wild-type (WT) and lip1 pea plants were crossed to produce F₁ plants, which were allowed to self-fertilize to produce F₂ seed. Parental wild-type, lip1, F₁, and F₂ seeds were germinated and grown for 7 days in darkness, at which time the lip1 phenotype was scored; asterisks indicate seedlings showing the lip1 phenotype. (A) Transcript analysis. Total RNA was extracted from the shoots of parental wild-type, lip1, F₁, and 12 F₂ seedlings. Top, RNA gel blot analysis using ³²P-labeled probe against pea COP1; bottom, RT-PCR products amplified by using primers FS1 and RS1 from first-strand cDNA produced from total RNA used for RNA gel blot analysis. (B) DNA gel blot analysis. Genomic DNA was extracted from parental wild-type, lip1, F₁, and 11 F₂ seedlings (different from those shown in [A]). Genomic DNA was digested overnight with DraI, fractionated by electrophoresis on a 0.7% agarose gel, blotted to GeneScreen Plus membrane, and hybridized with a ³²P-labeled COP1 probe. Markers at left in (A) and (B) indicate the positions of size markers in kilobases.

Figure 4.

Schematic Representation of COP1 Genes in Wild-Type and lip1 Peas and Predicted Pattern of Splicing. (A) COP1 gene in wild-type peas. A 9.5-kb pea genomic DNA fragment produced using the primers PCCOPF and PCCOPR on wild-type genomic DNA was inserted into the vector pTOPO-XL, and the DNA sequence was obtained by using a directed sequencing approach. Boxes indicate regions of genomic DNA that contain the COP1 coding region; the pattern of splicing is shown below. (B) COP1 gene in lip1 peas. A 20-kb region of overlapping DNA was obtained in PCR reactions on lip1 genomic DNA. A 9.5-kb fragment produced by use of the primers PCCOPF and PCCOR, a 3-kb fragment produced by the primers E7.F and E2.R, and an 8-kb fragment produced by the primers PCCOF and DREV2 were inserted into the vector pTOPOXL. The DNA sequence was obtained by using a directed sequencing approach, and a contig of overlapping DNA was produced. Boxes show regions of genomic DNA containing the COP1 coding region; hatched boxes indicate duplicated exon sequences. The 2.5-kb CPP region between exon 7′ and exon 1, used for subsequent promoter analysis, is also indicated by hatching. The pattern of splicing predicted from analysis of intron/exon boundaries is shown below. Exon 1 (black box) is skipped due to lack of recognized intron/exon boundary at the 5′ end. .

Figure 5.

Analysis of a Putative Promoter Region within COP1 in lip1 Peas. The 2.5-kb CPP region located between exon 7′ and exon 1 (see Figure 4) was used to create a transcriptional fusion with the uidA reporter gene and a nos terminator sequence. (A) GUS actively expressed from a CaMV 35S promoter in onion epidermal cells after microprojectile bombardment. (B) GUS activity from the CPP region. (C) GUS histochemical staining after bombardment with a promoterless construct. (D) GUS activity in a representative 7-day-old transgenic Arabidopsis seedling containing uidA fused to CPP. ; .

Figure 6.

Duplicated Motifs within the COP1′ Protein. (A) Motifs within Arabidopsis COP1 required for repression of photomorphogenic development in darkness (modified from that shown in Torii et al., 1998). COP1 contains a Zn binding RING domain (RING) and a coiled-coil domain (COIL) at the N terminus between which is found a cytoplasmic localization signal (CLS). A region containing at least five WD-40 repeats (WD-40) is found at the C terminus with a nuclear localization signal (NLS) located toward the middle of the protein. (B) Motifs within pea COP1′. The duplicated region within COP1′ contains part of the cytoplasmic localization signal, the coiled-coil domain, and the region containing the nuclear localization signal. COP1′ contains intact N- and C-terminal motifs. (+), position of NLS as shown in Figure 6A.