D. Takezawa

Calmodulin gene family in potato: developmental and touch-induced expression of the mRNA encoding a novel isoform

Eight genomic clones of potato calmodulin (PCM1 to 8) were isolated and characterized. Sequence comparisons of different genes revealed that the deduced amino acid sequence of PCM1 had several unique substitutions, especially in the fourth Ca2+-binding area. The expression patterns of different genes were studied by northern analysis using the 3′-untranslated regions as probes. The expression of PCM1, 5, and 8 was highest in the stolon tip and it decreased during tuber development. The expression of PCM6 did not vary much in the tissues tested, except in the leaves, where the expression was lower; whereas, the expression of PCM4 was very low in all the tissues. The expression of PCM2 and PCM3 was not detected in any of the tissues tested. Among these genes, only PCM1 showed increased expression following touch stimulation. To study the regulation of PCM1, transgenic potato plants carrying the PCM1 promoter fused to the β-glucuronidase (GUS) reporter gene were produced. GUS expression was found to be developmentally regulated and touch-responsive, indicating a positive correlation between the expression of PCM1 and GUS mRNAs. These results suggest that the 5′-flanking region of PCM1 controls developmental and touch-induced expression. X-Gluc staining patterns revealed that GUS localization is high in meristematic tissues such as the stem apex, stolon tip, and vascular regions.

A novel kinesin-like protein with a calmodulin-binding domain

Calcium regulates diverse developmental processes in plants through the action of calmodulin. A cDNA expression library from developing anthers of tobacco was screened with 35S-labeled calmodulin to isolate cDNAs encoding calmodulin-binding proteins. Among several clones isolated, a kinesin-like gene (TCK1) that encodes a calmodulin-binding kinesin-like protein was obtained. The TCK1 cDNA encodes a protein with 1265 amino acid residues. Its structural features are very similar to those of known kinesin heavy chains and kinesin-like proteins from plants and animals, with one distinct exception. Unlike other known kinesin-like proteins, TCK1 contains a calmodulin-binding domain which distinguishes it from all other known kinesin genes. Escherichia coli-expressed TCK1 binds calmodulin in a Ca2+-dependent manner. In addition to the presence of a calmodulin-binding domain at the carboxyl terminal, it also has a leucine zipper motif in the stalk region. The amino acid sequence at the carboxyl terminal of TCK1 has striking homology with the mechanochemical motor domain of kinesins. The motor domain has ATPase activity that is stimulated by microtubules. Southern blot analysis revealed that TCK1 is coded by a single gene. Expression studies indicated that TCK1 is expressed in all of the tissues tested. Its expression is highest in the stigma and anther, especially during the early stages of anther development. Our results suggest that Ca2+/calmodulin may play an important role in the function of this microtubule-associated motor protein and may be involved in the regulation of microtubule-based intracellular transport.

A Potato cDNA Encoding a Homologue of Mammalian Multidrug Resistant P-Glycoprotein

A homologue of the multidrug resistance (MDR) gene was obtained while screening a potato stolon tip cDNA expression library with35S-labeled calmodulin. The mammalian MDR gene codes for a membrane-bound P-glycoprotein (170–180 kDa) which imparts multidrug resistance to cancerous cells. The potato cDNA (PMDR1) codes for a polypeptide of 1313 amino acid residues (ca. 144 kDa) and its structural features are very similar to the MDR P-glycoprotein. The N-terminal half of the PMDR1-encoded protein shares striking homology with its C-terminal half, and each half contains a conserved ATP-binding site and six putative transmembrane domains. Southern blot analysis indicated that potato has one or two MDR-like genes. PMDR1 mRNA is constitutively expressed in all organs studied with higher expression in the stem and stolon tip. The PMDR1 expression was highest during tuber initiation and decreased during tuber development.

Isolation and characterization of two cDNAs that encode for calmodulin-binding proteins from corn root tips

Abstract A corn root tip cDNA expression library was screened with 35S-labeled calmodulin to isolate cDNAs that code for calmodulin-binding proteins (CBP). Several positive clones that bind to 35S-calmodulin were isolated. Two CBP clones (CBP-1 and CBP-5) that bind to 35S-labeled calmodulin as well as biotinylated calmodulin were sequenced and characterized. Comparison of the deduced amino acid sequence of both the clones showed one hundred percent consevation of a 34-amino acid stretch at their carboxy-terminal end. Whereas, less homology was observed in other regions of amino acid sequence. The highly conserved 34-amino acid stretch contained a putative calmodulin-binding domain, a basic amphiphilic alpha helix. The nucleotide and deduced amino acid sequence of both the clones did not show homology with any of the sequences in the nucleic acid and protein data bases. Northern analysis with both CBP-1 and CBP-5 clones indicated that the corresponding genes are expressed in different parts of the root although there was a difference in the extent of expression. Wind induced the expression of CBP-5, but it did not have any effect on CBP-1 mRNA level. Southern analysis indicated that CBP-1 and CBP-5 are coded, most likely, by one or two genes.

Regulated Expression of a Calmodulin Isoform Alters Growth and Development in Potato

A transgene approach was taken to study the consequences of altered expression of a calmodulin isoform on plant growth and development. Eight genomic clones of potato calmodulin (PCM1 to 8) have been isolated and characterized (Takezawa et al., 1995). Among the potato calmodulin isoforms studied, PCM1 differs from the other isoforms because of its unique amino acid substitutions. Transgenic potato plants were produced carrying sense construct of PCM1 fused to the CaMV 35S promoter. Transgenic plants showing a moderate increase in PCM1 mRNA exhibited strong apical dominance, produced elongated tubers, and were taller than the controls. Interestingly, the plants expressing the highest level of PCM1 mRNA did not form underground tubers. Instead, these transgenic plants produced aerial tubers when allowed to grow for longer periods. The expression of different calmodulin isoforms (PCM1, 5, 6, and 8) was studied in transgenic plants. Among the four potato calmodulin isoforms, only the expression of PCM1 mRNA was altered in transgenic plants, while the expression of other isoforms was not significantly altered. Western analysis revealed increased PCM1 protein in transgenic plants, indicating that the expression of both mRNA and protein are altered in transgenic plants. These results suggest that increasing the expression of PCM1 alters growth and development in potato plants.

Calcium-Dependent Protein Kinase Genes in Corn Roots

Two cDNAs encoding Ca(2+)-dependent protein kinases (CDPKs), CRPK1 and CRPK2 (corn root protein kinase 1 and 2), were isolated from the root tip library of corn (Zea mays L., cv. Merit) and their nucleotide sequences were determined. Deduced amino acid sequences of both the clones have features characteristic of plant CDPKs, including all 11 conserved serine/threonine kinase subdomains, a junction domain and a calmodulin-like domain with four Ca(2+)-binding sites. Northern analysis revealed that CRPK1 mRNA is preferentially expressed in roots, especially in the root tip; whereas, the expression of CRPK2 mRNA was very low in all the tissues tested. In situ hybridization experiments revealed that CRPK1 mRNA is highly expressed in the root apex, as compared to other parts of the root. Partially purified CDPK from the root tip phosphorylates syntide-2, a common peptide substrate for plant CDPKs, and the phosphorylation was stimulated 7-fold by the addition of Ca2+. Our results show that two CDPK isoforms are expressed in corn roots and they may be involved in the Ca(2+)-dependent signal transduction process.