DREB1 and DREB2 Genes in Garlic (Allium sativum L.): Genome-Wide Identification, Characterization, and Stress Response

Abstract

Dehydration-responsive element-binding (DREB) transcription factors (TFs) of the A1 and A2 subfamilies involved in plant stress responses have not yet been reported in Allium species. In this study, we used bioinformatics and comparative transcriptomics to identify and characterize DREB A1 and A2 genes redundant in garlic (Allium sativum L.) and analyze their expression in A. sativum cultivars differing in the sensitivity to cold and Fusarium infection. Eight A1 (AsaDREB1.1-1.8) and eight A2 (AsaDREB2.1-2.8) genes were identified. AsaDREB1.1-1.8 genes located in tandem on chromosome 1 had similar expression patterns, suggesting functional redundancy. AsaDREB2.1-2.8 were scattered on different chromosomes and had organ- and genotype-specific expressions. AsaDREB1 and AsaDREB2 promoters contained 7 and 9 hormone- and stress-responsive cis-regulatory elements, respectively, and 13 sites associated with TF binding and plant development. In both Fusarium-resistant and -sensitive cultivars, fungal infection upregulated the AsaDREB1.1-1.5, 1.8, 2.2, 2.6, and 2.8 genes and downregulated AsaDREB2.5, but the magnitude of response depended on the infection susceptibility of the cultivar. Cold exposure strongly upregulated the AsaDREB1 genes, but downregulated most AsaDREB2 genes. Our results provide the foundation for further functional analysis of the DREB TFs in Allium crops and could contribute to the breeding of stress-tolerant varieties.

Keywords: Allium sativum L.; DREB proteins; Fusarium; cold stress; gene expression; gene structure.

Figure 1

Distribution and structure of the AsaDREB genes identified in the A. sativum genome. (a) Chromosomal localization of AsaDREB1.1–1.8 (red) and AsaDREB2.1–2.8 (blue) genes. (b) Exon-intron composition. Chromosome lengths are based on the A. sativum cv. Ershuizao genome (PRJNA606385); chr, chromosome.

Figure 2

Evolutionary relationships between AsaDREB1 and AsaDREB2 proteins (red) and A. thaliana DREB TFs (black; NCBI ID and A-type are indicated). The unrooted dendrogram was constructed using the Neighbor-Joining method (bootstrap test: 1000 replicates) in MEGA 7.0.26; the evolutionary distances were computed using the JTT matrix-based method and are in the units of the number of amino acid substitutions per site.

Figure 3

Distribution of conserved motifs in A. sativum and A. thaliana DREB1 (a) and DREB2 (b) proteins. Analysis was performed using MEME 5.4.1; the length of each box corresponds to that of the motif.

Figure 4

Heatmap of AsaDREB1 and AsaDREB2 expression in A. sativum cv. Ershuizao (PRJNA607255). AsaDREB mRNA levels were analyzed in the roots, bulbs (stages 1–8 corresponding to 192-, 197-, 202-, 207-, 212-, 217-, 222-, and 227-day-old bulbs, respectively), leaves, pseudostems (ps.stem), buds, flowers, and sprouts. The color scheme indicates the gene expression gradient from low (red) to high (green).

Figure 5

Transcription of the AsaDREB1 and AsaDREB2 genes in A. sativum cv. Sarmat; ps.stems, pseudostems. The data were normalized to GAPDH and UBQ mRNA levels. The significance of differences in the gene expression between organ types was analyzed using one-way ANOVA; obtained p-values are given in the Supplementary Table S2 (p < 0.05 indicates significant gene expression difference between organ types).

Figure 6

Expression of the selected AsaDREB genes in the roots of A. sativum FBR-resistant cv. Sarmat and FBR-susceptible cv. Strelets in response to F. proliferatum infection. The plants were incubated with F. proliferatum conidia and analyzed for the transcription of the indicated genes 24 and 96 h post inoculation (hpi). The data were normalized to GAPDH and UBQ mRNA levels and presented as fold change (mean ± SE) of control (expression in cv. Sarmat at 24 hpi taken as 1); * p < 0.05 compared with the uninfected control. The significance of differences in gene expression between the control and experiment, as well as between the time-dependent control and experiment data, was analyzed using one-way ANOVA; obtained p-values are given in Supplementary Table S3.

Figure 7

Heatmap of the time-dependent AsaDREB gene expression in A. sativum winter-hardy cv. Strelets and medium winter-hardy cv. Sarmat after cold stress. Plants were exposed to cold (+4 °C) for 2, 4, 6, and 24 h and analyzed for the expression of the indicated AsaDREB genes in the leaves. The data were normalized to GAPDH and UBQ mRNA levels; transcriptions at 0 h (normal conditions before treatment) were taken as 1. The color gradient indicated expression decrease (blue) and increase (red) relative to the control. * p < 0.01 compared with the control (0 h).