Differential expression of IDA (INFLORESCENCE DEFICIENT IN ABSCISSION)-like genes in Nicotiana benthamiana during corolla abscission, stem growth and water stress

Background IDA (INFLORESCENCE DEFICIENT IN ABSCISSION)-like signaling peptides and the associated HAE (HAESA)-like family of receptor kinases were originally reported in the model plant Arabidopsis thaliana (Arabidopsis) to be deeply involved in the regulation of abscission. Actually, IDA peptides, as cell-to-cell communication elements, appear to be implicated in many developmental processes that rely on cell separation events, and even in the responses to abiotic stresses. However, the knowledge related to the molecular machinery regulating abscission in economically important crops is scarce. In this work, we determined the conservation and phylogeny of the IDA-like and HAE-like gene families in relevant species of the Solanaceae family and analyzed the expression of these genes in the allopolyploid Nicotiana benthamiana, in order to identify members involved in abscission, stem growth and in the response to drought conditions. Results The phylogenetic relationships among the IDA-like members of the Solanaceae studied, grouped the two pairs of NbenIDA1 and NbenIDA2 protein homeologs with the Arabidopsis prepropeptides related to abscission. Analysis of promoter regions searching for regulatory elements showed that these two pairs of homeologs contained both hormonal and drought response elements, although NbenIDA2A lacked the hormonal regulatory elements. Expression analyses showed that the pair of NbenIDA1 homeologs were upregulated during corolla abscission. NbenIDA1 and NbenIDA2 pairs showed tissue differential expression under water stress conditions, since NbenIDA1 homeologs were highly expressed in stressed leaves while NbenIDA2 homeologs, especially NbenIDA2B, were highly expressed in stressed roots. In non-stressed active growing plants, nodes and internodes were the tissues with the highest expression levels of all members of the IDA-like family and their putative HAE-like receptors. Conclusion Our results suggest that the pair of NbenIDA1 homeologs are involved in the natural process of corolla abscission while both pairs of NbenIDA1 and NbenIDA2 homeologs are implicated in the response to water stress. The data also suggest that IDA peptides may be important during stem growth and development. These results provide additional evidence that the functional module formed by IDA peptides and its receptor kinases, as defined in Arabidopsis, may also be conserved in Solanaceae.

both pairs of NbenIDA1 and NbenIDA2 homeologs are implicated in the response to water stress. The data also suggest that IDA peptides may be important during stem growth and development. These results provide additional evidence that the functional module formed by IDA peptides and its receptor kinases, as defined in Arabidopsis, may also be conserved in Solanaceae.

Background
The significance of the INFLORESCE DEFICIENT IN ABSCISSION ( IDA)-like gene family is primary associated with the observation that AtIDA was deeply involved in the regulation of the abscission of floral organs and cauline leaves in Arabidopsis thaliana (Arabidopsis) [1][2][3]. Abscission is an active, organized and highly coordinated cell separation process allowing the detachment of entire vegetative and reproductive organs through the modification of cell-to-cell adhesion and breakdown of cell walls at specific sites on the plant body known as abscission zones (AZs), a discrete group of functionally specialized cells (for a review, see [4]). From an evolutionary point of view, abscission is a very favorable process that has several advantages such as seed dispersal as well as the shedding of no longer needed, damaged or infected organs. In addition, the abscission process is related to other processes such as senescence, pathogen defense and drought stress tolerance [5]. Abscission of aerial organs, on the other hand, may become a major limiting factor of yield in an agricultural context. It is widely accepted that the control of abscission in Arabidopsis requires physical interaction of the hormonal peptide AtIDA, a pair of redundant receptor-like protein kinases, HAESA (HAE) and HAESA-LIKE2 (HSL2), and SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) co-receptors (for a review, see [6]). This interaction activates a signal transduction through a downstream MAP kinase cascade that leads to the expression of multiple abscission-related hydrolytic enzymes such as pectin-methylesterases, polygalacturonases, cellulases, xyloglucan-endotransglycosylase/hydrolases and expansins [7,8]. The release of this set of enzymes cause the disassembly of the cell wall and the dissolution of the middle lamella, resulting in the detachment of floral organs in Arabidopsis. Furthermore, in Arabidopsis ida mutants, petals remain indefinitely attached to the flower [1]. It has been also shown that synthetic IDA peptides were able to induce early floral abscission in Arabidopsis flowers [9].
The function of IDA peptides, as cell-to-cell communication elements, does not appear to be solely restricted to their roles in abscission. The activity of these peptides has also recently been involved in other developmental processes in Arabidopsis all of them settled on cell separation events such as the emergence of lateral roots and the root cap sloughing [10,11]. In addition, IDA peptides have recently been involved in the response to abiotic stresses. Expression analysis of Arabidopsis AtIDL6 and AtIDL7, for instance, revealed that these two genes are rapidly induced during various stress treatments [12].
It was subsequently determined experimentally that these peptides were involved in the stress response as modulators of reactive oxygen species (ROS) signaling [13].
Furthermore, treatments with AtIDL6 and AtIDL7 peptides caused downregulation of important stress-response key regulators like ZINC FINGER PROTEIN and WRKY transcription factors.
In addition to Arabidopsis, IDA-like genes have been identified in some crop species associated with organ abscission and the emergence of lateral roots [14] as well.
Solanaceae is a large plant family with approximately 90 genera comprising more than 3000 species found on almost all continents. Solanaceae is also one of the most economically important families worldwide. Some species of this family such as tomato (Solanum lycopersicum), potato (S. tuberosum), eggplant (aubergine; S. melongena) and pepper (Capsicum annum) are of great relevance as a human food source. Overall, more than 29 million hectares of these Solanaceae food species were cultivated globally in 2016, producing 644 million metric tons with a net production value of more than 146 billion US dollars (http://www.fao.org/faostat). Thus, in addition to being important in human nutrition, they are also relevant in economic and social terms. Other Solanaceae such as tobacco (Nicotiana spp.) have medical importance as a source of plant drugs while Nicotiana benthamiana is considered a relevant model organism for the study of plantmicrobe interactions and also in plant molecular research and biotechnology [22,23]. In this work, firstly, we determined the conservation and phylogeny of the IDA-like and HAElike gene families by taking advantage of the free availability of the diploid genome sequences of tomato, eggplant, pepper, N. Sylvestris, N. tomentosiformis, the allopolyploids N. tabacum and N. benthamiana, and the double haploid genome sequence of potato in the Solanaceae Genomic Network (SGN; https://solgenomics.net/).
Allopolyploidy is a type of whole genome duplication derived from hybridization of two or more diverged taxa, that primarily occurs through the fusion of unreduced (2n) gametes.
The result of this kind of genome merging is the occurrence of pairs of homologous genes from each of the diploid parents in the allopolyploid genome, called homeologs [24]. Therefore, in the allopolyploid genomes of N. tabacum and N. benthamiana we will likely find pairs of homeologs for many of the members of the gene families.
In order to identify and discriminate members involved in organ abscission, stem growth and in the response to drought conditions, we examined the expression of the homeolog genes of the N. benthamiana IDA-like and HAE-like families. The abscission of the corolla, the only organ that undergoes abscission in N. benthamiana, should be highly similar to that reported in N. tabacum [25]. The detachment of the corolla is due to the dissolution of the middle lamella and apparently to the disintegration of the parenchymal cells in its basal zone, a process that results in the detachment of the senescent corolla.

Results
The IDA-like gene family in the Solanaceae 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 [1].
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 [15] 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 [15], other prepropeptides of potato (StubIDA4), eggplant The third clade, clade III (shadowed in light gold), included AtIDL6 and AtIDL7, two IDAlike members of Arabidopsis that have been associated with processes different than cell separation, such as stress response [13]. 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.

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 IDAlike family in axillary buds, roots and leaves were determined ( Figure 4). While no differences in gene expression were found in axillary buds (see Additional file 5), 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.  (Table 1). This gene family was identified in a large number of Angiosperms [17] and their members contained a signal peptide targeting the protein to the apoplast through the secretory pathway and a conserved C-terminal part, the PIP motif (Additional file 4). The presence of a 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 [33].
The reduced size of the mature IDA-like peptides (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 is in line with this observation (Figure 1).
In contrast to tobacco, N. benthamiana has been described as an ancient allotetraploid whose polyploidy level (2n = 4x = 38) likely evolved through genome re-arrangements and fractionation giving rise to a remarkable descending dysploidy [35]. Parentals of N.
benthamiana are unknown, although it is believed that it comes from a hybridization event that occurred >10 Myr ago between species belonging to the Sylvestres and Noctiflorae sections of Nicotiana [35]. Two homeologs were identified for most of the analysed genes in our work, except for NbenIDA4. Genomic responses to polyploidy are complex in Nicotiana species, ranging from small to large genome re-sizing depending on the polyploid age and the similarity of parental genome donors [35]. Reduction in the number of chromosomes in N. benthamiana strongly suggests a considerable genome downsizing, probably as a consequence of being an older polyploid. The size reduction involves 1 Gb in length relative to tobacco (4.5 Gb genome size of N. tabacum, 3.5 Gb N. benthamiana).
Therefore, we suggest that gene loss might explain the absence of a second copy of NbenIDA4 in our data, rather than a misrepresentation in the draft assembly of the genome used for the analysis. Interestingly, several genetic studies estimated that the genome of N. tabacum had lost DNA from its progenitors since polyploidization and that this genomic loss was greater and biased towards the genome of the male parental N.
tomentosiformis [35][36][37][38]. Therefore, we also think that similar biased gene loss may have  [39,40]. In contrast, the presence of response elements to AUXs, ABA, MeJa and GAs in the promoter regions of the IDA-like family members of N. benthamiana and AtIDA and AtIDL1 suggests that these phytohormones might play a role in regulating 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 [41]. It has also been determined that ABA and MeJa have abscission-promoting effects, while the role of GAs is not entirely clear [6,42]. However, it has been shown in citrus that flower pollination increased bioactive gibberellin A1 (GA 1 ) levels and reduced ovary abscission and that the treatment of unpollinated ovaries with GA 3 also suppressed ovary abscission [43,44].  Figure 3C), that also increased during the last phases of the corolla abscission.
As described for IDA-like families in other species [12], 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 [45]. Moreover, all HAE-like genes analyzed also show higher expression levels in nodes and internodes, especially The occurrence of cis-acting elements related to the drought response in the two pairs of NbenIDA1 and NbenIDA2 homeologs (Figure 2) 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 homeologs was highly expressed in leaves from N.
benthamiana plants severely stressed while in roots, the genes that responded to water deficit were the members of the second pair. Furthermore, these two pairs of homeologs are phylogenetically close to AtIDA and AtIDL1, two Arabidopsis genes that are induced under abiotic stress conditions [12].
Our gene expression data showed that while most pairs of homeologs showed similar expression patterns, those of the NbenIDA2 pair exhibited high divergence (Figure 3). This might well be linked to the frequent observation that some duplicated genes, after a whole genome duplication event, evolve to undertake different functions or partition the function of the ancestral gene in a process of subfunctionalization. This process can include epigenetic, coding sequence or promoter modifications that alters regulatory mechanisms (e.g. silencing) and give rise for example to differential level of expression or tissue specificity. Subfunctionalization becomes more relevant when gene dosage is not an adaptive advantage for the polyploid [46]. Therefore, our gene expression data might be revealing a putative subfunctionalization of the pair of NbenIDA2 homeologs.
Interestingly, a noticeable different topology of cis-acting regulatory elements in the upstream 5'-UTR regions was found between NbenIDA2A and NbenIDA2B, compared for example to the NbenIDA1 pair. We took special care in primer specificity during qPCR assays in order to distinguish between both homeologs, since gene expression artifacts may be recurrent among genes derived from genome duplicated areas due to high sequence similarity.
It has been recently observed that IDA signaling peptides can certainly regulate important developmental processes as well as fundamental plant responses to environmental conditions [13]. Our data indicate that in the allopolyploid N. benthamiana, the two pairs suggest that the pair of NbenIDA1 homeologs are both involved in the natural process of corolla abscission. Interestingly, they also show specific differential expression under water stress conditions. NbenIDA1 homeologs are highly expressed in stressed leaves while NbenIDA2 homeologs, especially NbenIDA2B, are highly expressed in stressed roots.
In addition, nodes and internodes are the tissues with the highest expression of the IDAlike and HAE-like genes in normal active growing plants, suggesting that these peptides are also essential during stem growth and development. These results add new evidence that the functional module formed by IDA-like peptides and its receptor kinases as defined in Arabidopsis, may be conserved in Solanaceae.
Sequence alignments were performed through MEGA7 software [54] using the ClustalW algorithm with default parameters (DNA Data Bank of Japan, DDBJ; http://clustalw.ddbj.nig.ac.jp/). Phylogenetic trees were created using the Neighbor-Joining method [55] using 1000 bootstrap replicates. The trees are drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic trees. The evolutionary distances were computed using the Poisson correction method [56]

Availability of data and materials
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Competing interests
The authors declare that they have no competing interests