Genome-wide identification and expression analysis of the ADH gene family under diverse stresses in tobacco (Nicotiana tabacum L.)

Abstract

Background: Alcohol dehydrogenases (ADHs) are the crucial enzymes that can convert ethanol into acetaldehyde. In tobacco, members of ADH gene family are involved in various stresses tolerance reactions, lipid metabolism and pathways related to plant development. It will be of great application significance to analyze the ADH gene family and expression profile under various stresses in tobacco.

Results: A total of 53 ADH genes were identified in tobacco (Nicotiana tabacum L.) genome and were grouped into 6 subfamilies based on phylogenetic analysis. Gene structure (exon/intron) and protein motifs were highly conserved among the NtADH genes, especially the members within the same subfamily. A total of 5 gene pairs of tandem duplication, and 3 gene pairs of segmental duplication were identified based on the analysis of gene duplication events. Cis-regulatory elements of the NtADH promoters participated in cell development, plant hormones, environmental stress, and light responsiveness. The analysis of expression profile showed that NtADH genes were widely expressed in topping stress and leaf senescence. However, the expression patterns of different members appeared to be diverse. The qRT-PCR analysis of 13 NtADH genes displayed their differential expression pattern in response to the bacterial pathogen Ralstonia solanacearum L.

Infection: Metabolomics analysis revealed that NtADH genes were primarily associated with carbohydrate metabolism, and moreover, four NtADH genes (NtADH20/24/48/51) were notably involved in the pathway of alpha-linolenic acid metabolism which related to the up-regulation of 9-hydroxy-12-oxo-10(E), 15(Z)-octadecadienoic acid and 9-hydroxy-12-oxo-15(Z)-octadecenoic acid.

Conclusion: The genome-wide identification, evolutionary analysis, expression profiling, and exploration of related metabolites and metabolic pathways associated with NtADH genes have yielded valuable insights into the roles of these genes in response to various stresses. Our results could provide a basis for functional analysis of NtADH gene family under stressful conditions.

Keywords: Alcohol dehydrogenases (ADH); Gene expression; Nicotiana tobacum L.; Phylogenetic analysis; Stress.

Fig. 1

Chromosomal locations of NtADH genes. The scale on the left presents the length of chromosomes (Mb). Tandem duplicated gene pairs are displayed with boxes in yellow color, segmental duplicated gene pairs are connected by red lines

Fig. 2

Syntenic analysis of ADH genes among tobacco, Arabidopsis and tomato. The NtADH gene (NtADH8/12/18/38/52) and its orthologous syntenic genes in Arabidopsis and tomato are linked by the red line. The syntenic ADH gene pairs located in the synteny blocks between tobacco and tomato are linked by blue lines

Fig. 3

Gene structure and evolution of ADH family in Nicotiana tabacum L. A Phylogenetic relationships of NtADHs. Different subgroups were marked with different colors. B Intron–exon structure of NtADHs. Green boxes: UTR; Yellow boxes: CDS; spaces between the boxes: introns. The scale bar of bottom demonstrates the length of exons and introns

Fig. 4

Conserved motifs for NtADH proteins in Nicotiana tabacum L. Different motifs are showed with different colored boxes and numbers (1–20)

Fig. 5

Sequence logos of the conserved ADH_N and ADH_zinc_N repeats of the NtADH domain. A Sequence logo of Zn1 in ADH_N. B Sequence logo of Rossman fold in ADH_zinc_N

Fig. 6

Phylogenetic tree of Nicotiana tabacum, melon, tomato, mango, apricot, Arabidopsis and barley ADH genes. The phylogenetic relationships were generated by using MEGA-11 using the Maximum Likelihood (ML) method (1000 bootstrap replicates). The squares, five-pointed star, triangle, hexagon, circles and trapezium represent melon, tomato, mango, apricot, Arabidopsis and barley ADH proteins, respectively

Fig. 7

Predicted cis-elements in NtADHs promoters. Different shapes and colors represent the different types of cis-elements. Annotations of cis-elements were listed in Additional file 4: Table S4

Fig. 8

Gene expression profile under different condition A The expression of 53 NtADHs in tobacco leaves at five senescence stages. B The expression of 53 NtADH genes in response to topping. FPKM values for NtADH genes were transformed by log₁₀(n + 1)

Fig. 9

Disease symptoms in the cultivars Hongda at 0 h and 96 h by Ras. The basal parts of stems were magnified and shown in the circles. In 0 h (Left), and no symptoms were apparent. In 96 h (Right), leaves were withered and the basal part of stem was severely necrosis and turned to black

Fig. 10

Relative expression level of 13 NtADHs in response to inoculation of Ras. Error bars are standard deviations of three biological replicates

Fig. 11

Metabolic pathways in which the ADH gene is involved

Fig. 12

Overview of the alpha-linolenic acid pathways showing the changes of metabolite contents during curing process and the expression of NtADHs at the initial stage of curing