The IDA-like gene family in the Solanaceae
Table 1 summarizes all IDA-like genes identified in our search in representative species of the genus Nicotiana such as N. sylvestris, N. tomentosiformis, N. tabacum and N. benthamiana in addition to other Solanaceae of agronomic interest such as tomato, potato, eggplant and pepper. All prepropeptides identified share two relevant characteristics, a signal peptide targeting the protein to the apoplast through the secretory pathway and a highly conserved C-terminal signature termed PIP motif typical of this gene family .
IDA-like families of the Nicotiana species N. sylvestris and N. tomentosiformis consisted of 5 members, while in the allopolyploids N. benthamiana and N. tabacum these families are formed by 5 pairs of homeologs, with one exception corresponding to NbenIDA4 whose homeolog pair was not found in the genomic screening. All IDA-like genes found in Nicotiana are new identifications, as the six members found in S. melongena and C. annuum and the seven members of the S. tuberosum family. In S. lycopersicum, five out of the eight IDA-like genes detected, members 1 to 5, were already described in  and named SlIDA1-5, while the other three peptides, SlycIDA6-8, are incorporated in the current work.
Phylogenetic relationship among IDA-like prepropeptides in Solanaceae
The phylogenetic relationships among the IDA-like members of the species of Solanaceae studied, in addition to those of Arabidopsis, are grouped in three major clades (Figure 1). Clade I (shadowed in green colors) was divided in two subclades. The subclade shadowed in green contained the two Arabidopsis prepropeptides involved in floral organ abscission, AtIDA and AtIDL1 [1, 9]. The largest subclade grouped members of all eight Solanaceae species studied, as well as AtIDL8, the most divergent IDA-like peptide from Arabidopsis. In this subclade, Solanaceae members are further divided in two major groups. The group shadowed in lime green contained SlIDA1, the IDA-like member of tomato that has been associated with leaf abscission , other prepropeptides of potato (StubIDA4), eggplant (SmelIDA5) and pepper (CaIDA4) as well as the IDA1 members of the Nicotiana species under study. These were NsylIDA1 and NtomIDA1 of the diploid species N. sylvestris and N. tomentosiformis, respectively, and the two pairs of NtabIDA1 and NbenIDA1 homeolog prepropeptides corresponding to N. tabacum and N. benthamiana. The 5’-UTR regions and the predicted CDSs of all these IDA1 genes from the genus Nicotiana showed high degree of conservation (see Additional files 1 and 3). The other group shadowed in light green included other prepropeptides from the Nicotiana, Solanum and Capsicum genera, with a small subdivision composed of AtIDL8 together with SlycIDA6, SlycIDA7 and StubIDA1 (Figure 1). A second clade, clade II (shadowed in light orange), appeared to be limited to the Solanaceae family. This clade included prepropeptides from the Nicotiana, Solanum and Capsicum genera, but none from Arabidopsis, an observation suggesting that it might have diverged before the irruption of the Brassicaceae family 40 million years ago . The third clade, clade III (shadowed in light gold), included AtIDL6 and AtIDL7, two IDA-like members of Arabidopsis that have been associated with processes different than cell separation, such as stress response . The topology of the clade showed that there was a great diversification in Arabidopsis that generated at least six members, AtIDL2-7. It also included prepropeptides from the Nicotiana and Solanum genera, but none from Capsicum.
Cis-acting regulatory elements in the promoter regions of the N. benthamiana IDA-like family
Figure 2 shows a schematic representation of the cis-acting regulatory elements along 1000 bp of the 5’-UTR region of the IDA-like family members of N. benthamiana and AtIDA and AtIDL1 of Arabidopsis. Searches for response elements to hormones related to ABA, methyl jasmonate (MeJa), AUXs or GAs, as well as response elements to biotic and abiotic stresses were performed. Interestingly, the pair of NbenIDA1 homeologs contained similar promoter regions carrying response elements to ABA, MeJa and AUX as AtIDA in similar locations; these phytohormones have been involved in the abscission process in different ways [1, 31, 32]. The pairs of NbenIDA1 and NbenIDA2 homeologs also carry drought response elements in their promoter regions. On the other hand, NbenIDA2B, NbenIDA3A, NbenIDA4, and the pair of NbenIDA5 homeologs are characterized by the occurrence of GA response elements (Figure 2).
Expression patterns of IDA-like and HAE-like genes in Nicotiana benthamiana during growth and abscission
Expression analysis of the family of IDA-like ligand peptides and their putative HAE-like receptors in different plant tissues of N. benthamiana are presented in Figure 3. HAE-like receptors were identified through the analyses of the phylogenetic relationships between the HAE-like receptor-like kinases (RLKs) of Arabidopsis and Nicotiana (see Additional files 2 and 3). The plant material selected for gene expression analysis is shown in the panel A of Figure 3. This plant material included different vegetative tissues of a plant in active growth (apical buds, young and mature leaves, nodes and internodes, and roots), as well as reproductive tissues (anthers, styles, stigmas, and fruits) at different developmental stages, including samples of the base of the flower corollas, a tissue that in tobacco (N. tabacum) has been shown to respond to the abscission process . Panel B in Figure 3 shows the expression of the N. benthamiana IDA-like and HAE-like homeologs in apical buds, nodes, internodes, the whole corolla, the ensemble formed by the stigma and the style and also in roots relative to the lowest expression level of each gene. Panel C in Figure 3 shows the expression pattern of each IDA-like and HAE-like homeolog in leaves, anthers and fruits relative to that at the earliest developmental stage in every organ and panel D shows the expression patterns of each homeolog gene at the corolla base in developmental stages 2, 4 and 5 relative to the corolla developmental stage 1. Virtually all members of the IDA-like family of N. benthamiana were mainly expressed in nodes and internodes, although NbenIDA1A expression levels were not especially high in internodes (Figure 3B). No changes in the expression patterns of IDA-like homeolog genes were observed in leaves and fruits (Figure 3C) but the expression level for all of them except NbenIDA3B and NbenIDA4 showed a tendency to increase between closed and dehiscent anthers (Figure 3C). Interestingly, expression of both NbenIDA1 homeologs at the base of the flower corolla increased with the stage of development of the tissue, in parallel to the progress of the abscission process (Figure 3D). The expression pattern of NbenIDA1B was similar to that detected in NbenIDA1A although at a much more modest level. The expression levels of the NbenIDA2 homeologs were transiently high in stage 2 when the corolla tube is fully elongated and the limb lobes are still closed to return later in stages 4 and 5 to almost the basal level of expression (Figure 3D).
The highest expressions levels of the putative receptors of the IDA-like peptides, NbenHAE.1, NbenHAE.2, NbenHSL2.1 and NbenHSL2.2, were also registered in nodes and internodes (Figure 3B). Additionally, their expression levels also showed a tendency to increase between closed and dehiscent anthers (Figure 3C) and a slight increase was observed at the corolla base associated with corolla development (Figure 3D).
Expression patterns of IDA-like genes in Nicotiana benthamiana during water stress
The presence of drought response elements in the promoter regions of some particular IDA-like members, e.g. NbenIDA1A, NbenIDA1B, NbenIDA2A and NbenIDA2B (Figure 2), suggested that their expression might be regulated by the water status of the plant. Therefore, we exposed actively growing plants of N. benthamiana to 6 (mild stress) and 8 (severe stress) days of water stress and the expression levels of all members of the IDA-like family in axillary buds, roots and leaves were determined (Figure 4). While no differences in gene expression were found in axillary buds, those of the pair of NbenIDA1 homeologs dramatically increased in leaf blades of plants subjected to severe water stress. In contrast, this condition resulted in higher increases in transcripts belonging of both NbenIDA2 homeologs in roots, indicating differential roles of this gene family in response to water stress. Changes in the expression of the rest of genes were of minor relevance although it is worth to mention that these members tended to repress their expression levels in roots of plants subjected to water stress, although NbenIDA5A expression was also reduced in stressed leaves.