Calmodulin Gene Expression in Response to Mechanical Wounding and Botrytis cinerea Infection in Tomato Fruit

Abstract

Calmodulin, a ubiquitous calcium sensor, plays an important role in decoding stress-triggered intracellular calcium changes and regulates the functions of numerous target proteins involved in various plant physiological responses. To determine the functions of calmodulin in fleshy fruit, expression studies were performed on a family of six calmodulin genes (SlCaMs) in mature-green stage tomato fruit in response to mechanical injury and Botrytis cinerea infection. Both wounding and pathogen inoculation triggered expression of all those genes, with SlCaM2 being the most responsive one to both treatments. Furthermore, all calmodulin genes were upregulated by salicylic acid and methyl jasmonate, two signaling molecules involved in plant immunity. In addition to SlCaM2, SlCaM1 was highly responsive to salicylic acid and methyl jasmonate. However, SlCaM2 exhibited a more rapid and stronger response than SlCaM1. Overexpression of SlCaM2 in tomato fruit enhanced resistance to Botrytis-induced decay, whereas reducing its expression resulted in increased lesion development. These results indicate that calmodulin is a positive regulator of plant defense in fruit by activating defense pathways including salicylate- and jasmonate-signaling pathways, and SlCaM2 is the major calmodulin gene responsible for this event.

Keywords: calcium signaling; jasmonic acid; plant defense; postharvest decay; salicylic acid.

Figure 1

Expression of SlCaMs in wounded tomato fruit. Transcription levels of SlCaMs were measured by RT-qPCR. Relative gene expression levels are shown following normalization with actin transcript values. Error bars represent standard error of the mean. For each gene, different letters indicate statistically significant differences among mean values (p < 0.05). The results are based on at least three repeats in three independent experiments.

Figure 2

Expression of SlCaMs in response to fungal infection. Tomato fruit at the mature-green stage were mechanically wounded and immediately treated with water (mock inoculation) or inoculated with Botrytis cinerea conidia. The wounded and wounded-plus-inoculated areas were excised after 0 to 48 h of incubation at 20 °C. Total RNA samples for RT-PCR were isolated from pericarp tissue and transcript levels of SlCaMs genes were determined by RT-qPCR. Relative gene expression levels are shown following normalization with actin transcript values. For each gene, different letters indicate statistically significant differences among mean values (p < 0.05). The results are based on at least three repeats in three independent experiments.

Figure 3

SlCaMs expression levels in response to salicylic acid treatment. Transcription levels of SlCaMs genes were investigated by quantitative real time RT-qPCR. Mature green stage fruit were treated with 4 mM salicylic acid for different time periods as indicated. Relative gene expression levels are shown following normalization with actin transcript values. Error bars represent the standard error of the mean. For each gene, different letters indicate statistically significant differences among the means (p < 0.05). The results are based on at least three repeats in three independent experiments.

Figure 4

SlCaMs gene expression in response to methyl jasmonate treatment. Transcription levels of SlCaMs genes were investigated by quantitative real time RT-qPCR. Mature green stage fruit were treated with 20 μM methyl jasmonate for different periods of time. Relative gene expression levels are shown following normalization with actin transcript values. Error bars represent standard error of the mean. For each gene, different letters indicate statistically significant differences among mean values (p < 0.05). The results are based on at least three repeats in three independent experiments.

Figure 5

Expression level of SlCaM2 in tomato fruit in relation to B. cinerea infection. Mature green stage fruits were agroinjected with A. tumefaciens carrying different constructs. pDF28F, empty vector (control); 35S:SlCaM2-S, pDL28F carrying SlCaM2 in the sense orientation; 35S:SlCaM2-A, pDL28F carrying SlCaM2 in the antisense orientation. (A) Examination of expression levels of SlCaM2 in different transgenic fruits. 48 h after agroinjection, a piece of pericarp tissue from each fruit was used for RT-qPCR. The results are based on at least three repeats in three independent experiments. For each gene, different letters indicate statistically significant differences among mean values (p < 0.05). (B) Pathogen resistance and susceptibility test shows that overexpression of SlCaM2 enhanced resistance to B. cinerea. The fruits agroinjected with different constructs were inoculated into a wound with water (mock) or B. cinerea conidia, and put in a moisture saturated container to observe decay development. The photos were taken 72 h post inoculation.