Distinct calcium signaling pathways regulate calmodulin gene expression in tobacco

Abstract

Cold shock and wind stimuli initiate Ca(2+) transients in transgenic tobacco (Nicotiana plumbaginifolia) seedlings (named MAQ 2.4) containing cytoplasmic aequorin. To investigate whether these stimuli initiate Ca(2+) pathways that are spatially distinct, stress-induced 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.

Figure 1

Targeting of aequorin to tobacco cell nuclei. Protoplasts of wild-type tobacco, MAQ 2.4, and MAQ 7.11 stained with DAPI are shown in A, B, and C, respectively. The same protoplasts treated with anti-apoaequorin and FITC-labeled secondary antibody are shown in D, E, and F. G shows a MAQ 7.11 protoplast treated with anti-apoaequorin and gold-labeled secondary antibody. Bar = 1 μm. C, Cytoplasm; N, nucleus.

Figure 2

Partial cDNA sequence of NpCaM-1 and NpCaM-2 showing nucleotide and predicted amino acid identities. A, Nucleotide sequence; B, amino acid sequence. Primers used for 3′-RACE and subsequent PCR are indicated in lowercase; stop codons are underlined. Homology is indicated with bars.

Figure 3

Expression kinetics of NpCaM-1 and NpCaM-2 determined by northern-blot analysis after stimulation by a single wind signal or a single cold shock. The 3′-UTRs of NpCaM-1 and NpCaM-2 were used as DNA hybridization probes to study the expression kinetics of NpCaM-1 and NpCaM-2. Water of room temperature was used as a control.

Figure 4

Wind- and cold-shock-induced changes in the cytosolic and nuclear free Ca²⁺ concentrations and the expression levels of NpCaM-1 and NpCaM-2. A, Ca²⁺ changes in cytoplasm (cyt) and nucleoplasm (nuc) after stimulation with 5 mL of air at t = 10 s. B, Ca²⁺ changes in cytoplasm (cyt) and nucleoplasm (nuc) after stimulation with 1 mL of ice-cold water at t = 10 s. C, Wind- (□ and ▪) and cold-shock (○ and ●)-induced changes in mRNA levels of NpCaM-1 and NpCaM-2 are indicated and are averages of three experiments. Data are shown as hybridization relative to non-induced mRNA levels (given a value of 1) and is plotted against time in minutes. ○, NpCaM-1; ●, NpCaM-2; □, NpCaM-1; ▪, NpCaM-2.

Figure 5

The effect of Ca²⁺ modulators on wind-induced changes in cytosolic and nuclear Ca²⁺ and NpCaM-1 and NpCaM-2 mRNA accumulation. Wind stimulation was applied by 5 mL of air at t = 10 s. The se for the peak values from eight experiments is indicated for the mean peak. CON, Control; THAP, thapsigargin; RR, ruthenium red; BA, BAPTA-AM. A, The effect of 200 μm thapsigargin. ○, CON NpCaM-1; ●, CON NpCaM-2; □, THAP NpCaM-1; ▪, THAP NpCaM-2. B, 50 μm Ruthenium red. ○, CON NpCaM-1; ●, CON NpCaM-2; □, RR NpCaM-1; ▪, RR NpCaM-2.; C, 1 mm BAPTA-AM; solvents were used as control. ○, CON NpCaM-1; ●, CON NpCaM-2; □, BA NpCaM-1; ▪, BA NpCaM-2.

Figure 6

The effect of Ca²⁺ modulators on cold shock-induced changes in cytosolic and nuclear Ca²⁺ and NpCaM-1 and NpCaM-2 mRNA accumulation. Cold shock stimulation was applied by a 1-mL injection of ice-cold water at t = 10 s. The se for the peak values from eight experiments is indicated for the mean peak. A, The effect of 10 mm LaCl₃ (LA). ○, CON NpCaM-1; ●, CON NpCaM-2; □, LA NpCaM-1; ▪, LA NpCaM-2; B, 20 mm GdCl₃ (GD); MgCl₂ concentrations of identical ionic strength were used as a control (CON). ○, CON NpCaM-1; ●, CON NpCaM-2; □, GD NpCaM-1; ▪, GD NpCaM-2.