IDA-like genes were searched in relevant genera of the Solanaceae family including several species of Nicotiana (N. sylvestris, N. tomentosiformis, N. tabacum and N. benthamiana), and other crops of agronomic interest such as tomato (Solanum lycopersicum), potato (S. tuberosum), eggplant (S. melongena) and pepper (Capsicum annuum) (Table 1). This gene family was present in all species analyzed  and as expected, their members contained a signal peptide targeting the protein to the apoplast through the secretory pathway and a conserved C-terminal part of IDA-like peptides, the PIP or extended (E)PIP motif (Additional file 4). The presence of this signal peptide in the sequence of all identified genes suggests a mechanism of posttranslational maturation in the apoplast similar to that described in Arabidopsis, where the prepropeptide is proteolytically processed by subtilisin-like serine proteinases to yield a bioactive peptide . This cleavage that occurs between positions 6 and 7 of the EPIP motif , normally occupied by a K and a G respectively, leaves a bioactive peptide of 14 amino acids containing the PIP motif with G7 as N-terminal and N20 as C-terminal residues (Figure 2). It is accepted that the G7-N20 14mer constitutes the peptide signal controlling abscission in Arabidopsis  and that in the C-terminal part of many groups of this kind of hormonal peptides, the positions R/K18, H19 and N20 (Figure 2. A) constitute the recognition site for a co-receptor of the SERK family . Thus, it has been proposed that in Arabidopsis the interaction of the IDA ligand peptide with HAE/HSL2 RLKs could lead to the recruitment of a co-receptor of the SERK family starting the signal transduction resulting in floral organ abscission . The sequence logo of the EPIP motif of AtIDA and AtIDL1-5 (Figure 2, A) is very similar to those of the Solanaceae studied in here, since it holds the amino acids and characteristic domains described in Arabidopsis (Figure 2. C). Furthermore, the representation of the EPIP motif of all IDA peptides that have been associated so far with abscission, this is Arabidopsis AtIDA and AtIDL1 , citrus CitIDA3 , soybean GmIDA2a and GmIDA2b , Litchi LcIDL1 , oil palm EgIDA5 , Lupinus LlIDA  and tomato SlycIDA1  and its orthologs with very similar sequence detected in clade I of Figure 1, including StubIDA4, SmelIDA5, CaIDA4, NbenIDA1A and NbenIDA1B) suggests that the relevant positions of the EPIP motif are P11, S13A/G14P15S16 and R18H19N20 (Figure 2, D).
In spite of these similarities, there are other members of the IDA-like family in Arabidopsis (AtIDL6-8, Figure 2, B) recently associated with other processes than abscission, such as stress response . For instance, position 14 in the Solanaceae is occupied by an A rather than a G, as in these members of Arabidopsis, although both are small nonpolar aliphatic amino acids that should allow similar binding conditions of the mature peptide with its receptor. However, the general H19N20 motif observed in the Solanaceae and in the Arabidopsis members implicated in abscission responsible of the recruitment of a SERK co-receptor, is not conserved in these sequences.
The high level of conservation of the EPIP motif and the reduced size of the mature peptide (an alignment of the complete coding sequences of these genes can be seen in Additional file 4), precluded the study of the phylogenetic relationships based on these premises and, therefore, a circular phylogenetic tree was generated using complete sequences encoding prepropeptides including a signal peptide and a variable region (Figure 1). This tree shows that the Arabidopsis IDA-like gene family exhibits a higher degree of diversification than the Solanaceae genera studied, with the exception of Nicotiana. The two Arabidopsis prepropeptides putatively related to abscission, AtIDA and AtIDL1 [1, 27] nested in a small clade grouped with a more bigger clade including many IDA-like members from the different species of Solanaceae. This big clade was divided in two subgroups in one of which was nested the tomato SlycIDA1, associated with abscission , and its orthologs in S. melongena, C. annuum, S. tuberosum and in the four Nicotiana species studied. Interestingly, there were IDA-like paralogs genes in N. tabacum, named “A” and “B” members, that exhibited high similarity to the peptide pairs of N. sylvestris and N. tomentosiformis, respectively, supporting the hypothesis that N. tabacum (4.5 Gb, genome size) is an allotetraploid originated from the hybridization of N. sylvestris (2.6 Gb) and N. tomentosiformis (2.7 Gb) . This explains why the IDA-like family in tobacco contains twice as many members than those of their parental organisms (Table 1). N. benthamiana is also an allotetraploid that shows “A” and “B” IDA members, except for the second member of the NbenIDA4 pair that appears to be lost in this genome. The identification of N. sylvestris as one of the parental species of N. benthamiana, in contrast to N. tabacum, still needs additional confirmation .
The analyses of the cis-acting regulatory elements in the 5’-UTR regions of the N. benthamiana IDA-like family and Arabidopsis AtIDA and AtIDL1 (Figure 3) failed to identify ethylene response elements, in agreement with the idea that IDA-like genes regulating the abscission process are not directly dependent upon ethylene [36, 37]. It is well known, however, that ethylene promotes both abscission and the expression of IDA-like genes, as shown in soybean, oil palm and citrus, for instance [15, 17, 38]. Based on this observation, it may therefore be estimated that IDA-like genes could act downstream of the ethylene signaling pathway as recently proposed by Meir and co-workers . In contrast, the presence of response elements to AUXs, ABA, MeJa and GAs in the promoter regions of the genes examined was abundant, suggesting that these hormones may play a role in the regulation of the expression of these genes. The occurrence of functional indole-3-acetic acid (IAA) signaling in the abscission zone during organ separation, for instance, has been demonstrated by Basu and co-workers . It has also been determined that ABA and MeJa have abscission-promoting effects, while the role of GAs is not entirely clear, since GAs appear to have different effects depending on the concentration and mode of application [6, 41]. However, it has been shown in citrus that flower pollination increased gibberellin A1 (GA1) levels and reduced ovary abscission and that the treatment of unpollinated ovaries with GA3 also suppressed ovary abscission [42, 43].
The analyses also indicated that the coding and promoter sequences of NbenIDA1A and NbenIDA1B are highly similar and that the hormonal regulatory elements of both promoters share the same response elements in similar positions, in addition to the same drought response element. Furthermore, the pair of NbenIDA2 genes also contains drought stress response elements in their promoter regions (Figure 3). Likewise, the coding and promoter sequences of the IDA1 genes in N. attenuata, N. sylvestris, N. tomentosiformis, N. benthamiana and N. tabacum are very similar and have the same response elements in the same positions in their promoters, except N. attenuata (Additional file 1).
The results described above (Figure 1, 2 and 3) suggested that in N. benthamiana, NbenIDA1A and NbenIDA1B peptides may be involved in the abscission process. This suggestion is also supported by the gene expression patterns found at the corolla base of the flowers during the process of natural abscission. Thus, Figure 4.B shows that there is a positive correlation between the expression of NbenIDA1A and NbenIDA1B and the developmental stage of the corollas of the flowers, the only tissue susceptible to abscission in this species. As described in citrus styles , the accumulation of the CitIDA3 transcript increased with the progress of the abscission process, reaching the highest values during the late stages of development. The expression pattern of NbenIDA1B and especially of NbenIDA1A suggests that these genes are positive regulators of corolla abscission in N. benthamiana. Similarly, there seems to be a correlation between the expression of the IDA-like genes and that of their putative receptors of the HAE-like family, NbenHAE.1, NbenHAE.2 and NbenHSL.2.2 (Figure 4.C), that also increased during the last phases of the corolla abscission.
As described for IDA-like families in other species , the different members of the N. benthamiana family are also expressed in multiple plant tissues (Figure 4). This is not a surprise since the IDA-like signaling peptides, as cell-to-cell communication elements, function in several cell separation events, including lateral root emergence and root cap sloughing [10, 11]. Interestingly, in plants of N. benthamiana actively growing, the highest expression level of most members of the IDA-like family was found in nodes and internodes. It is worth mentioning that the promoter regions of NbenIDA2B, NbenIDA3A, NbenIDA4, NbenIDA5A and NbenIDA5B genes contain GAs response elements, and that these hormones are pivotal regulators of stem growth . Moreover, all HAE-like genes analyzed also show higher expression levels in nodes and internodes, especially NbenHSL2.1 and NbenHSL2.2, in parallel with the pattern observed for the IDA-like genes. These expression patterns might be linked to the formation of vascular bundles and to the cell elongation and division associated with the process of stem elongation implying cell wall remodeling. Thus, the results appear to suggest that the signaling module formed by the IDA-like peptides and its kinase receptors may be conserved in other plants such as N. benthamiana.
The occurrence of cis-acting elements related to the drought response in the gene pairs NbenIDA1A-NbenIDA1B and NbenIDA2A-NbenIDA2B (Figure 3) also suggested to test the response of the IDA-like genes to water stress conditions. In the experiment reported in Figure 5 it is clearly observed that the first pair of genes was highly expressed in leaves from N. benthamina plants severely stressed while in roots, the genes that responded to water deficit were the members of the second pair. Furthermore, these 4 genes are phylogenetically close to AtIDA and AtIDL1, two Arabidopsis genes that are induced under abiotic stress conditions .
Furthermore, NbenIDA2B shows basal or very low expression levels in all plant tissues analyzed (Figure 4. B), but its expression is highly increased in conditions of severe drought stress only in roots (Figure 5). Thus, this gene shows a very relevant tissue-specific differential expression, and although its function has not been elucidated in this work it can be suggested that might be related to the development of lateral roots, since this is a common response to drought stress also attributed to IDA-like peptides [10, 14, 46].
It has been recently observed that IDA signaling peptides can certainly regulate important developmental processes as well as fundamental plant responses to environmental conditions . Our data indicate that in the allotetraploid N. benthamiana, the NbenIDA1 and NbenIDA2 gene pairs are differentially involved in the responses to drought stress while only NbenIDA1 genes are apparently implicated in the natural process of corolla abscission. These data suggest that IDA-like signaling peptides can play different biological roles in various tissues and under distinct abiotic conditions.