Content area
Full Text
Distinct Calcium Signaling Pathways Regulate Calmodulin Gene Expression in Tobacco1
Cold shock and wind stimuli initiate Ca2+ transients in transgenic tobacco (Nicotiana plumbaginifolia) seedlings (named MAQ 2.4) containing cytoplasmic aequorin. To investigate whether these stimuli initiate Ca2+ pathways that are spatially distinct, stressinduced nuclear and cytoplasmic Ca 2+ transients and the expression of a stress-induced calmodulin gene were compared. Tobacco seedlings were transformed with a construct that encodes a fusion protein between nucleoplasmin (a major oocyte nuclear protein) and aequorin. Immunocytochemical evidence indicated targeting of the fusion protein to the nucleus in these plants, which were named MAQ 7.11. Comparison between MAQ 7.11 and MAQ 2.4 seedlings confirmed that wind stimuli and cold shock invoke separate Ca 2+ signaling pathways. Partial cDNAs encoding two tobacco calmodulin genes, NpCaM-1 and NpCaM-2, were identified and shown to have distinct nucleotide sequences that encode identical polypeptides. Expression of NpCaM-1, but not NpCaM-2, responded to wind and cold shock stimulation. Comparison of the Ca 2+ dynamics with NpCaM-1 expression after stimulation suggested that wind-induced NpCaM-1 expression is regulated by a Ca 2+ signaling pathway operational predominantly in the nucleus. In contrast, expression of NpCaM-1 in response to cold shock is regulated by a pathway operational predominantly in the cytoplasm.
Calmodulin is highly conserved in eukaryotes and is considered to be a multifunctional protein because of its ability to interact and regulate the activity of a number of other proteins (Hepler and Wayne, 1985; Gilroy et al., 1993; Poovaiah and Reddy, 1993; Trewavas and Knight, 1994). In plant cells, calmodulin is considered to be the primary sensor for changes in cellular free Ca" levels ([Ca"li) (Roberts and Harmon, 1992). As [Ca2+] rises transiently after signaling, the combination of Ca 2+ with calmodulin leads to the activation of numerous target proteins initiating the physiological response.
Calmodulin has been purified and characterized from a number of plant species. Genomic and/or cDNA clones encoding calmodulin have been isolated and characterized from Arabidopsis (Ling et al., 1991; Perera and Zielinski, 1992), potato (Takezawa et al., 1995), and wheat (Yang et al., 1996). In all multicellular organisms in which it has been examined, genes encoding the different calmodulin isoforms are under the control of different promoters that exhibit distinct temporal and spatial expression...