ADK is ubiquitous in the kingdoms Animalia and Plantae. ADK is found in the cytosol and also in many organelles such as mitochondria and chloroplasts. So far, ADK-encoding genes have been cloned from a wide variety of plant species [13]. However, genome-wide analysis of the ADK gene family has not been pursued in tomato, a model plant for studying plant fruit ripening. In the current study, 11 SlADKs in tomato were identified, and designated SlADK1-11 on the basis of their chromosomal location (Table 1). The phylogeny, motif, gene structure, chromosomal location, cis-elements and expression patterns in different tissues and under stress treatments were analyzed. This study provides comprehensive information on the SlADK family and will aid understanding of the function of SlADKs.
Previous research revealed seven ADK isoforms with high sequence homology in the Arabidopsis genome [14]. The lower number of ADK genes in Arabidopsis may be related to its small genome [38]. The SlADKs and AtADKs were classified into five groups, with genes in the same group showing a closer evolutionary relationship. For example, SlADK1, 4 and 9 belonged to group V; SlADK6 and 10 to group I; and SlADK8 and 11 to group IV. Much closer evolutionary relationships existed in the same subbranch, such as SlADK2 with 3, SlADK5 with 7, and SlADK1 with 9 (Fig. 1A). Subcellular localization prediction showed that SlADKs were distributed in mitochondria, chloroplast and other plastids in cells, with the greatest occurrence in chloroplasts (Table 1). This is consistent with previous reports that ADK activity in plants is mainly distributed in the chloroplast matrix and mitochondrial membrane space [39, 40, 41].
Motif analysis revealed a total of 10 motifs (Fig. 2A), with motifs 1, 2, and 4 shared by all SlADKs. In addition, motif 7 was unique to SlADK5 and 7 at the N-terminal, and motif 9 was unique to SlADK2 and 3 at the C-terminal (Fig. 2B). Common motifs imply functional redundancy and the specific motifs may contribute to the functional divergence [42]. For the evolution of multiple gene families, the model of gene organization is very important [34]. Gene structure analysis revealed 4–19 exons in each SlADK (Fig. 3A). All above mentioned genes in the same subbranch (Fig. 1A), such as SlADK1 and 9, SlADK2 and 3, and SlADK5 and 7 shared common motif compositions (Fig. 2B) and similar gene structure (Fig. 3A). This correlation between gene structure and motif arrangement further confirmed the classifications of the SlADKs.
The expression patterns of ADK genes in different tissues have been described in many species, including Arabidopsis [14] and rice [10]. In Arabidopsis, expression of AtADKs was detected in leaves, roots, and 16-d-old seedlings and AtADK1-5 were much more expressed than AtADK6, while AtADK7 was at the detection limit [14]. However, there was no uniform gene expression pattern for SlADKs in tomato. Our qPCR results for SlADKs in tomato were basically consistent with those predicted by the online TomExpress platform. For example, the prediction expression peak of SlADK2 and 11 was in bud of 3 mm and leaves, respectively, which was highly consistent with the qPCR result (Fig. 4A, B). Also, it was difficult to get satisfactory qPCR results for gene expression analysis of SlADK1, 2 and 11 due to quite low expression abundance and amplification efficiency, which was consistent with the software prediction that these three genes had very low expression levels in fruit (Fig. 4A, C). For qPCR detection, SlADK2, 3, 6, 7, 8 and 10 also showed similar expression patterns, which hint at similarities in structures, redundancies in functions as well as shared induction mechanisms (Fig. 4B).
Abiotic stresses, such as drought, high salinity, extreme temperature and flooding are major causes of crop loss worldwide, reducing average yields for most major crop plants by more than 50% [43]. In addition to regulation of growth and development, previous study showed that ADKs are widely involved in abiotic stress response in plants [16–19]. In our study, transcript profiles of the tomato ADK family were assayed under drought, salt and cold treatments. With the increased time of PEG6000 treatment, expression of most SlADKs was up-regulated, especially of SlADK1 and 11. Microarray data revealed that the expression of an ADK gene (SGN-U232826) in drought-tolerant tomato was induced by drought stress [18]. The sequence of SGN-U232826 was consistent with the SlADK10 identified in our study, which was induced during 24–72 h following PEG6000 treatment (Fig. 5). The maintenance of mitochondrial ATP synthesis during water stress is essential for preserving plastid function, and increased ADK gene expression may indicate the ability to provide more ATP for maintaining cellular activities under drought stress [18, 44]. Several enzymes, such as ADK and catalase, were specially induced by drought but repressed under salt stress in tomato [17]. In tomato, microarray analysis of genes revealed that an ADK homolog (U214214) was repressed in salt-treated tissues [17]. Also, in the present study, almost all SlADKs contained two expression peaks at 9 and 48 h; however, the two expression peaks for SlADK7 were at 6 and 24 h. Genes SlADK3 and 7 responded strongly to cold treatment, indicating that they may play a role in cold stress (Fig. 5).
In addition to the basic transcription elements TATA-box and CAAT-box distributed in multiple sites, most of the ADK family members also contain some specific cis-regulatory elements, such as ABRE, MYB, G-box and MYC (Table S2, Fig. 3C). When plants are exposed to abiotic stresses such as salt, drought or low temperature, ABA-dependent and -independent pathways are simultaneously activated [45, 46]. Genes involved in the ABA-dependent pathway not only induce ABA biosynthesis, but also regulate the expression of genes containing ABREs [47, 48]. The ABREs mainly occurred in SlADK3 and 6 and the G-box element was mostly distributed in SlADK3 (Table S2, Fig. 3C). In the barley HVA22 gene and the Lea gene promoter, the core sequence ACGT of G-box and other regulatory sequences (CE1 and CE3) constitute an ABA response complex to regulate the induced expression of ABA by these genes [49]. The MYB elements are found in the promoters of several stress-resistance genes in Arabidopsis [50]. Our results showed MYB elements distributed in all SlADKs, especially SlADK2 and 3 (Fig. 3C). The MYC element is a cis-acting element in response to drought and ABA, and exists in a variety of anti-stress gene promoters, with reports related to soybeans and Arabidopsis [51, 52]. Our results revealed that MYC existed in almost all SlADKs except SlADK1 and 7, and was distributed frequently in SlADK6 (Fig. 3C).
Previous evidence indicated that different hormones play important roles in cell responses and stress signal transduction [35–37]. For example, IAA is involved in almost all aspects of plant growth and development, from embryogenesis to senescence, from root tip to shoot tip [53]. Eth is a key regulator during fleshy fruit ripening [54]. ABA is a crucial phytohormone induced by biotic or abiotic stress, and plays important roles in plant tolerance to abiotic stresses [55]. MeJA play an important role in alleviating biotic (pathogens and insects) and abiotic stresses in plants [56]. Our study showed that the transcripts of these SlADKs were responsive to most hormone treatments (Fig. 6). In plants, many hormones need to cross function. For example, two plant hormones, ABA and Eth, play an important role in the complex story of abiotic stress and, consequently, cross-talk between these two has been reported [57]. Also, both Eth and SA play important roles in biotic stresses [58]. Notably, SlADK2 and 4 exhibited significant changes under these hormone treatments, suggesting that they have unique roles in hormone responsiveness (Fig. 6). So, understanding the response of SlADKs to hormones can lay a foundation for further elucidating their functions in plant growth and stress response.