Calcium-dependent protein kinase; degenerate primer; ; nutrient; polymerase chain reaction; starvation
Among the downstream targets of calcium in plants, calcium-dependent protein kinases (CDPKs) form an interesting class of kinases which are activated by calcium binding. They have been implicated in a diverse array of responses to hormonal and environmental stimuli. In order to dissect the role of CDPKs in the mossFunaria hygrometrica, a polymerase chain reaction (PCR)-based approach was adopted to clone the gene. Using degenerate PCR primers against conserved regions of CDPKs, a 900 bp amplicon was obtained from the genomic DNA ofFunaria. Southern hybridization under low stringency conditions indicated the presence of several CDPK related sequences in theFunaria genome. This observation is consistent with reports of multigene families of CDPKs in other plants. The 900 bp fragment was subsequently used to isolate a 2.2 kb partial genomic clone of the CDPK gene fromFunaria. The genomic clone encodes an open reading frame (ORF) of 518 amino acids. Interestingly, unlike other CDPK genes from plants, the entire 1.5 kb ORF is not interrupted by introns. The deduced amino acid sequence of theFunaria gene shows extensive homology with CDPKs from higher plants, 73% identity with theFragaria CDPK and 71% identity with CDPK isoform 7 ofArabidopsis. Phylogenetic analysis revealed that theFunaria CDPK is closer to the CDPKs from higher plants like strawberry andArabidopsis as compared to those from lower plants such as the liverwortMarchantia, the green algaChlamydomonas or another mossTortula. Northern analysis shows enhanced expression of the CDPK transcript within 24–48 h of starvation for nitrogen, phosphorus or sulphur. So far the only other kinase which is known to be induced by nutrient starvation in plants is the wpk 4 which is a snf-1 related kinase (SnRKs). To our knowledge this is the first report that implicates a CDPK in the starvation response.