Calcium-dependent protein kinases (CDPKs) are major Ca²⁺ sensors in plants playing important roles in plant development and stress responses. In the present study, RT-PCR analysis revealed that VaCPK3a and VaCPK9 cDNAs lacking extensive regions in the kinase, autoinhibitory, and Ca²⁺-binding domains were numerous in probes derived from wild grapevine Vitis amurensis. Most of the VaCPK3a and VaCPK9-modified transcripts lacked canonical splice sites and possessed short direct repeated sequences (SDRs) instead. Three VaCPK9 transcript variants were generated using canonical 5'GT and 3'AG splice sites and lacked several subdomains in the kinase domain, including ATP-binding site, which is known to be indispensable for kinase activities. These observations indicate that post-transcriptional mRNA processing might lead to production of CDPKs with abolished phosphorylation activities. Recombinant proteins VaCPK3aSF2, lacking autoinhibitory and Ca²⁺-binding domain, and VaCPK3aSF3, lacking VIII–XI kinase subdomains and autoinhibitory domain, phosphorylated exogenous substrate in a Ca²⁺- independent manner. However, reverse transcription at +65°C using heat-stable reverse transcriptase (RT) markedly lowered abundance of the unusual VaCPK3a and VaCPK9 cDNAs with SDRs, while it did not eliminate VaCPK9 cDNAs generated using canonical splice sites. The results show that VaCPK9 gene undergoes unproductive alternative splicing mediated by canonical splice sites to generate three mRNA isoforms lacking important catalytic kinase subdomains. The unusual VaCPK3a and VaCPK9 transcripts with SDRs are likely to be false alternative transcripts generated by RT template switching in vitro. The data demonstrate that using non-thermostable RTs for studying alternative splicing are not appropriate.
Plastids are evolutionary descendants of cyanobacteria and retain many proteins of cyanobacterial origin including histidine kinases (HKs). Histidine kinases form a major group of protein kinases in cyanobacteria but a minor group in angiosperms; no HK has been detected in plant plastids so far. This raises the question: have higher plant plastids retained some cyanobacterial His/Asp regulatory systems or the latter have been replaced with Ser/Thr or Tyr protein kinases more typical for eukaryotes? To answer this question H1 antiserum against conservative phospho-acceptor motif of HKs was raised and applied for the analysis of different chloroplast fractions. In maize, three polypeptides with apparent molecular masses about 26, 27.5, and 28 kDa were revealed in thylakoid membranes. The results of in organello phosphorylation suggest that these proteins may be His-phosphorylated. Polypeptides with similar molecular masses were revealed in various mono- and dicotyledonous plants. Bioinformatic analysis demonstrated that in angiosperms these polypeptides might result from alternative transcription initiation and/or alternative processing of mRNAs of genes encoding well-known HKs. Besides, the genome of the moss Physcomitrella patens contains much more genes that could code for plastid HKs. The data obtained let us suppose that plastids of contemporary plants have HK(s) that could be involved in regulation of plastid gene expression.
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