An Arabidopsis cDNA Related to Animal Phosphoinositide-Specific Phospholipase C Genes

We have cloned an Arabidopsis thaliana cDNA and corresponding genomic clones exhibiting a high degree of sequence similarity to animal PI-PLC genes (Table I). A PCR strategy was designed to clone genes expressed in Arabidopsis shoots during floral induction. Arabidopsis plants grown under SD conditions (8 h of light/l6 h of dark) were transferred to LD conditions (16 h of light/8 h of dark) to induce flowering. Shoots were harvested during the first 3 d after the transfer and divided into expanded leaves and the remainder of the shoots. Vegetative shoots were also harvested before the transfer. Poly(A)+ RNA was isolated from each tissue, and the corresponding cDNAs were used as templates for PCR amplification. Degenerate primers deduced from conserved regions of CDC25 and SCD25 (Saccharomyces cerevisiae) and ste6 (Schizosacckaromyces pombe) were used. A 165-bp fragment that appeared specific to the shoot without leaves during the transition to flowering was isolated and used to identify Arabidopsis genomic clones. Six genomic clones were recovered, and a11 of them contained a HindIIJ fragment of approximately 700 bp that hybridized to the PCR product. The nucleotide sequence of the HindIII fragment did not exhibit any similarity to the CDC25 gene. Instead, it contained an ORF highly similar to animal PI-PLC genes. Approximately 100,000 clones of a primary cDNA library constructed from poly(A)+ RNA isolated from Arabidopsis shoots that had begun floral evocation were screened using the 703-bp genomic HindIII fragment as a probe, and one positive clone was recovered. Sequencing of the cDNA confirmed that it corresponded to the transcript from which the original PCR product was amplified, and the HindIII genomic fragment contained three exons of the gene. The cDNA is 1861 bp and contains an ORF of 533 amino acids and 20 A residues at the 3’ end. The ORF contains regions similar to two conserved domains of PIPLCs in animals, slime mold, and yeast (Berridge, 1993). Deletion and expression studies of mammalian PI-PLCs in Esckerichia coli and COS cells demonstrated that these domains are essential for the catalytic activity (Bristol et al.,