Domain and motif analysis of the NtADH proteins
A total of 20 conserved motifs have been identified and designated as motif1 to motif20 (Fig. 4). The conserved motifs presented within the same subgroup exhibited similar composition, indicating that the NtADH members clustered in the same subgroup may share similar biological functions. Most of the NtADH proteins were found to contain approximately 10 motifs, and there was no discernible correlation between the number of motifs and the length of the protein. For instance, despite having the shortest protein length, NtADH37 did not have the lowest number of motifs. In addition, different subgroups usually possessed specific motifs. For example, motif5 was exclusive to subgroup A while motif16 was exclusive to subgroup B. Likewise, motif11, motif17, and motif18 were solely presented in subgroup E, and motif17 and motif11 tended to appear in pairs.
The protein sequences of the 20 motifs were uploaded to CDD program for domain analysis (Fig. 4, Additional file 2: Table S2). Motif1, motif6, and motif7 were annotated as components of the GroES-like (ADH_N) domain, motif2, motif12, motif14, and motif20 were annotated as components of the zinc binding (ADH_zinc_N) domain. In addition, motif13, motif9, motif4 and motif5 were annotated as components of ADH_zinc_N_2 domain, IPU_b_solenoid, TLV_coat domain, and PE family, respectively. No annotation information was obtained for the remaining motifs. The result indicated that all members possessed the conserved regions of GroES-like (ADH_N) and zinc binding (ADH_zinc_N) domains. To further investigate the conservative domain of the NtADH proteins, the conserved domain of (ADH_N) and zinc binding (ADH_zinc_N) sequence logos of the 53 NtADH protein were generated by WebLogo (Fig. 5). The analysis revealed that NtADH members possess typical characteristics of ADH conserved domains and all the members had a Zn1 binding feature [GHE (X)2G (X)5G (X)2V] (Fig. 5A) and a NADPH binding domain element [GXG (X)2G] (Fig. 5B). This result indicated that these proteins are likely to be zinc-dependent ADHs [23, 24].
Phylogenetic analysis of the NtADH gene family
To explore the evolution of the ADH gene family, a total of 84 ADH gene members from 7 species were selected for the construction of phylogenetic tree (Fig. 6), including melons (13), Arabidopsis thaliana (7), apricot (1), mangos (2), tomato (7), barley(1) and tobacco (53) (Additional file3: Table S3a). The ADH family members were clustered into 7 distinct subfamilies based on the phylogenetic tree, and the ADH members of tobacco were dispersed across 6 of these subfamilies, excluding subfamily A (short chain ADH protein). In addition, only NtADH members from tobacco were classified in subfamilies C, D and E. According to the phylogenetic tree, there were 31 sister pairs of homologous proteins, among which 4 pairs were orthologous genes and 27 pairs were paralogous (Additional file 3: Table S3b). Specifically, there were 21 paralogous pairs from tobacco, 2 pairs each from tomato and melon, and 1 pair each from mango and Arabidopsis thaliana. Previous studies have shown that CmADH1of melon [13], Mi-ADH of mango [17] and Le-ADH2 of tomato [19] are involved in the biosynthesis of fruit ripening and aroma volatiles, phylogenetic analysis showed that 13 NtADH genes (NtADH33/40/29/38/11/22/21/8/10/7/6/14/12) were grouped with these four genes (CmADH1, Mi-ADH1/2, Le-ADH2) suggesting that these NtADH genes may have a similar biological function and related to the biosynthesis of fruit ripening and aroma volatiles.
Cis ‑elements analysis of NtADHs
The cis-elements in promoter regions play a critical role in the initiation of gene expression. A total of 58 cis- elements were selected in the NtADHs promoter region (Fig. 7). Among them, the light-responsive elements were the most common in the NtADH gene promoters, accounting for the largest proportion (42.28%), including G-box, Box 4, GT1 motif, and TCT motif. Meanwhile, there were many cis-regulatory elements that associated with phytohormone-responsive were also present, such as CGTCA-motifs, TGACG-motif, and ABRE. In addition, cis-regulatory elements that associated with the response to external or environmental stresses were also present, including stress response elements, ARE (cis-acting regulatory element essential for the anaerobic induction), MBS (MYB binding site involved in drought-inducibility), LTR (low-temperature response elements) and defense response elements TC-rich repeats (cis-acting regulatory element involved in defense and stress responsiveness). The result indicates that the expression of these NtADH genes is likely regulated by light-responsiveness cis-elements, phytohormones, defense signaling transduction, and various abiotic stresses during growth and development of tobacco.
The expression patterns of NtADH genes under conditions of leaf senescence and abiotic stress
The FPKM values of NtADH genes at five senescence stages of tobacco leaves were obtained from our previous transcriptome data (Additional file5: Table S5). Finally, the expression profiles of 53 NtADH genes were analyzed. The results showed that the members of NtADH genes had differential expression pattern in tobacco leaves at different senescence stages (Fig. 8A), and these 53 NtADH genes were clustered into four groups (A ~ D). A total of 13 NtADH genes were included in group B, and these genes had high expression level at the five senescence stages of leaves, implying that these genes could play important roles during leaves senescence process, while 16 NtADH genes clustered in group A showed a low or no expression during the whole senescence process. Notably, the expression levels of NtADH7 genes increased gradually with the increasing of maturity, whereas those genes clustered in group D decreased in M5 stages except NtADH49. In terms of topping stress (Fig. 8B), the majority NtADH genes included in group B showed high expression levels at all stages, and some genes (NtADH41/8/10/1/45/29) had reached the peak expression level on the first and fourth days of topping, respectively. In contrast, the genes clustered in group A and C showed relative low expression level. These results indicated the functional diversity of tobacco NtADH members.
Expression analysis of the NtADHs in response to Ralstonia solanacearum
No obvious change was observed in the seedling at the initial stage after infected by Ralstonia solanacearum L. (Ras.), however, the primary symptoms induced by Ras. infection appeared in the seedling at 96 h (Fig. 9). At this stage, the seedling displayed leaf wilting and stem necrosis, while the roots turned yellowing and necrosis, whereas these symptoms were not apparent at 0 h (Fig. 9).
It was reported that HvADH1 in barley is an S gene and plays a pivotal role in regulating pathogen invasion [5]. To further explore the possible function of the ADH genes of tobacco, the expression patterns of NtADHs in response to pathogen infection were analyzed. (Fig. 10). A total 13 tobacco ADH genes that clustered with HvADH1 in B subgroups of the phylogenetic tree (Fig. 6) were selected for qRT-PCR analysis under Ras. infection. Most of the selected genes displayed a notable up-regulated expression in response to the infection. (Fig. 10). In comparison to the initial stage (0 h), a significant up-regulation was observed in 6 NtADH genes (NtADH14, NtADH7, NtADH12, NtADH11, NtADH40, NtADH8) at 12 h after inoculation. Specifically, NtADH40 exhibited a remarkable up-regulation, surpassing a 15-fold increase, while NtADH7 demonstrated an astonishing up-regulation of over 350-fold While the expression of NtADH6 and NtADH29 increase significantly at 24 h after inoculation. In addition, the expression of 3 genes (NtADH33, NtADH10 and NtADH22) displayed a gradual decrease in response to pathogen infection, followed by an increase. The expression patterns of the tobacco ADH genes in response to Ras. infection revealed distinct variations in both response speed and intensity among the different genes.
Changes of NtADH related-metabolomics during hypoxia and high-temperature curing process
The ADH gene plays a crucial role in multiple metabolic processes. Among the 53 NtADH genes identified in tobacco, a total of 41 genes were annotated in the KEGG database, including carbohydrate metabolism, lipid metabolism, and the biosynthesis of other secondary metabolites (Fig. 11). During the hypoxia and high-temperature curing process, a total of 1129 metabolites were identified at four comparison pairs (Additional file6: Table S6). Specifically, 137 differential metabolites were identified in the comparison of T1 VS T2, and 331 differential metabolites were identified in the comparison of T1 VS T3. In addition, 339 differential metabolites were identified in the comparison of T1 VS T4, and 322 differential metabolites were identified in the comparison of T1 VS T5. KEGG metabolite analysis indicated that these differential metabolites were enriched in the pathway of alpha-linolenic acid metabolism (ko00592), linoleic acid metabolism (ko00591), nucleotide metabolism (ko01232) and pyrimidine metabolism (ko00240), etc. (Additional file7: Fig. 1 ~ 4). Among them, alpha-linolenic acid metabolism is the pathway which belongs to lipid metabolism. According to the database of KEGG, four NtADH genes (NtADH20, NtADH24, NtADH48 and NtADH51) are involved in the pathway of alpha-linolenic acid metabolism, and the contents of 2 metabolites in this pathway, namely 9-hydroxy-12-oxo-10(E), 15(Z)-octadecadienoic acid and 9-hydroxy-12-oxo-15(Z)-octadecenoic acid were significantly up-regulated during the curing process. Based on the qRT-PR analysis, these 4 ADH genes (NtADH20, NtADH24, NtADH48 and NtADH51) exhibited significant different expression level at the initiation of the curing process (Fig. 12).