Carrot AOX2a Transcript Profile Responds to Growth and Chilling Exposure

Abstract

Alternative oxidase (AOX) is a key enzyme of the alternative respiration, known to be involved in plant development and in response to various stresses. To verify the role of DcAOX1 and DcAOX2a genes in carrot tap root growth and in response to cold stress, their expression was analyzed in two experiments: during root growth for 13 weeks and in response to a cold challenge trial of 7 days, in both cases using different carrot cultivars. Carrot root growth is initially characterized by an increase in length, followed by a strong increase in weight. DcAOX2a presented the highest expression levels during the initial stages of root growth for all cultivars, but DcAOX1 showed no particular trend in expression. Cold stress had a negative impact on root growth, and generally up-regulated DcAOX2a with no consistent effect on DcAOX1. The identification of cis-acting regulatory elements (CAREs) located at the promoters of both genes showed putative sequences involved in cold stress responsiveness, as well as growth. However, DcAOX2a promoter presented more CAREs related to hormonal pathways, including abscisic acid and gibberellins synthesis, than DcAOX1. These results point to a dual role of DcAOX2a on carrot tap root secondary growth and cold stress response.

Keywords: Daucus carota; alternative oxidase; chilling stress; cis-elements; growth.

Figure 1

Measurements of carrot tap roots at 5, 7, 9, and 13 weeks post sowing. (a) Fresh weight (g) and (b) root length (cm) with a carrot scheme representing the general aspect of carrot tap roots at (A) 5, (B) 7, (C) 9, and (D) 13 weeks post sowing. The vertical bar in (b) indicates the part of the tap root used for length measurement.

Figure 2

Root fresh weight seven days after initiation of cold challenge experiment in carrot cultivars ’Nairobi’, ‘Newcastle’, ‘Nikki’, and ‘Norwich’. Plants from control were always grown at 25 °C and samples from cold treatment were grown at 5 °C during seven days. Significant difference in cultivar ‘Norwich’ is indicated by different letters (p < 0.05). Carrot plants are shown in Figure S1.

Figure 3

Expression box-plot and two-way ANOVA analysis for DcAOX1 during carrot root secondary growth in the cultivars (Cv) 203-1, 207-1, 699-1, 711-1 and ‘Rotin’. Transcript levels were determined by RT-qPCR. For each time point, four to six biological replicates were considered per cultivar. Significant differences in gene expression between time points are indicated by * (p < 0.05), ** (p < 0.01) or *** (p < 0.001). Boxplots show the distributions (median, spread and outliers) of the gene expression values.

Figure 4

Expression box-plot and two-way ANOVA analysis for DcAOX2a during carrot root secondary growth in the cultivars (Cv) 203-1, 207-1, 699-1, 711-1, and ‘Rotin’. Transcript levels were determined by RT-qPCR. For each time point, four to six biological replicates were considered per cultivar. Significant differences in gene expression between time points are indicated by * (p < 0.05), ** (p < 0.01), or *** (p < 0.001). Boxplots show the distributions (median, spread and outliers) of the gene expression values.

Figure 5

Expression box-plot and three-way ANOVA analysis for DcAOX1 during cold challenge (T1: 4 h, T2: 8 h, T3: 24 h, and T4: 7 days) in carrot cultivars (Cv) ’Nairobi’ (Nai), ‘Newcastle’ (New) ‘Nikki’ (Nik), and ‘Norwich’ (Nor). Transcript levels were determined by RT-qPCR. For each time point, four biological replicates were considered per temperature. Significant differences between temperatures for the same time point are indicated by * (p < 0.05), ** (p < 0.01) or *** (p < 0.001). Boxplots show the distributions (median, spread and outliers) of the gene expression values.

Figure 6

Expression box-plot and three-way ANOVA analysis for DcAOX2a during cold challenge (T1: 4 h, T2: 8 h, T3: 24 h and T4: 7 days), in carrot cultivars (Cv) ’Nairobi’ (Nai), ‘Newcastle’ (New) ‘Nikki’ (Nik), and ‘Norwich’ (Nor) eight weeks after sowing. Transcript levels were determined by RT-qPCR. For each time point, 10–12 biological replicates were considered per temperature. Significant differences between temperatures for the same time point are indicated by * (p < 0.05), ** (p < 0.01) or *** (p < 0.001). Boxplots show the distributions (median, spread and outliers) of the gene expression values.

Figure 7

Frequency of cis-regulatory motifs identified in 2.0 Kb promoter region of both DcAOX genes (DcAOX1 and DcAOX2a) using New PLACE and PlantCARE software’s.

Figure 8

Location of cis-regulatory elements within the DcAOX1 promoter, involved in cold-stress response (in light blue) in response to plant hormones: auxin (green), cytokinin (dark blue), abscisic acid (yellow), gibberellin (red), ethylene (orange), and calcium (grey).

Figure 9

Location of cis-regulatory elements within the DcAOX2a promoter’s, involved in cold-stress response (in light blue), in response to plant hormones: auxin (green), cytokinin (dark blue), abscisic acid (yellow), gibberellin (red), ethylene (orange), and calcium (grey).