MOLECULAR CHARACTERIZATION OF PgiC IN A TETRAPLOID PLANT AND ITS DIPLOID RELATIVES

Abstract

Allotetraploid plant species combine the genomes of related diploid species, but little is known about whether homologous genes from the diploid genomes are expressed, how they interact, or whether they evolve differently when in a common tetraploid nucleus. Polyploidy may lead to gene silencing, but few molecular characterizations of silenced genes encoding enzymes in polyploids and related diploids have been reported. Here we describe the PgiC genes in the tetraploid Clarkia gracilis and related diploid species, which are native from California to southern Washington. PgiC encodes the cytosolic isozyme of phosphoglucose isomerase (PGIC; EC 5.3.1.9). The gene was duplicated in the basal stock of Clarkia and now both genes, PgiC1 and PgiC2, are active in about half of the diploid species, whereas only PgiC1 is active in the others. Clarkia gracilis was found to have three PgiC genes: two PgiC1s and a PgiC2. Reverse-transcriptase-polymerase chain reaction (RT-PCR) experiments, starting with mRNAs prepared from seedling leaves of C. gracilis, showed that the three genes are expressed. Analysis of their sequences showed they are evolving at similar rates to their homologues and that they have the same intron-exon structure. The presence of an expressed PgiC2 in C. gracilis was unexpected because all related diploid species, including one identified as a parent, have only active PgiC1s. The donor of the PgiC2 is now presumed extinct, but parsimony analysis identified its phylogenetic position. None of the PgiC genes that were active when C. gracilis arose were silenced. A possible example of gene conversion involving a 300-nuclectide region of one PgiC1 and PgiC2 was identified, but it probably occurred in the diploid parental species rather than in C. gracilis. PgiC2 is the first known example of an active locus in a tetraploid plant species that is no longer expressed in its diploid relatives.

The legacy of diploid progenitors in allopolyploid gene expression patterns

“…Ford & Gottlieb [15] showed that one of the four gene copies of PgiC had been disrupted in both of the allotetraploids, with different mutational events occurring in each one. However, they argued that this silencing could not be proved to be due to tetraploidy per se and 'may have more to do with the peculiar properties and history' ( [15], p. 706) of the PgiC loci: one paralogue had also been silenced in some diploids and retained in duplicate in allotetraploid C. gracilis (see above [14]). Though the true sequence of events remains unknown, this emphasizes the importance that Gottlieb placed on parental legacies.…”

“…huntiana and the other parental species was extinct. Ford & Gottlieb [14] subsequently studied the expression of cytosolic phosphoglucose isomerase (PgiC) in this allotetraploid using reverse-transcription polymerase chain reactions and sequencing and found that two copies of PgiC1 were expressed, but only one copy of the paralogue PgiC2 was expressed. While it might have been tempting to assume silencing of one homoeologue of PgiC2 subsequent to allotetraploidization, Ford and Gottlieb found that this gene was commonly silenced in the diploid relatives of the allotetraploid and concluded: 'C.…”

“…After his initial application of this technique to diploid speciation and phylogenetics [8,9], he turned his attention to protein expression in allopolyploids, first in Stephanomeria elata [10], then in Tragopogon miscellus [11,12] and later in Clarkia gracilis [13][14][15].…”

Single Mutations Silence Pgic2 Genes in Two Very Recent Allotetraploid Species of Clarkia

“…Sequence analysis of a small family of receptor-like kinase genes in hexaploid wheat showed independent evolution of homoeologous genes and identified several sequences with stop codons, suggesting they are not expressed (Feuillet et al 2001). PgiC genes in allotetraploid Clarkia gracilis were found to be evolving at similar rates to their orthologues in related diploid species, and there was no evidence of silencing (Ford and Gottlieb 1999). Significant numbers of genes were silenced in experimentally synthesized allotetraploids involving Arabidopsis thaliana, apparently by epigenetic instabilities, because at least some of them could be reactivated .…”

“…Genomic DNAs were prepared from seedling leaves of single individuals as described by Ford and Gottlieb (1999). Templates for genomic sequencing were obtained by PCR using primers directed against various combinations of exons, a conserved region (LP) about 400 nucleotides 5Ј of the start codon (Gottlieb and Ford 1996) and a conserved region (3P) upstream of the poly(A) cleavage signal.…”