Conservation of the abscission signaling peptide IDA during Angiosperm evolution: withstanding genome duplications and gain and loss of the receptors HAE/HSL2

Abstract

The peptide INFLORESCENCE DEFICIENT IN ABSCISSION (IDA), which signals through the leucine-rich repeat receptor-like kinases HAESA (HAE) and HAESA-LIKE2 (HSL2), controls different cell separation events in Arabidopsis thaliana. We hypothesize the involvement of this signaling module in abscission processes in other plant species even though they may shed other organs than A. thaliana. As the first step toward testing this hypothesis from an evolutionarily perspective we have identified genes encoding putative orthologs of IDA and its receptors by BLAST searches of publically available protein, nucleotide and genome databases for angiosperms. Genes encoding IDA or IDA-LIKE (IDL) peptides and HSL proteins were found in all investigated species, which were selected as to represent each angiosperm order with available genomic sequences. The 12 amino acids representing the bioactive peptide in A. thaliana have virtually been unchanged throughout the evolution of the angiosperms; however, the number of IDL and HSL genes varies between different orders and species. The phylogenetic analyses suggest that IDA, HSL2, and the related HSL1 gene, were present in the species that gave rise to the angiosperms. HAE has arisen from HSL1 after a genome duplication that took place after the monocot-eudicots split. HSL1 has also independently been duplicated in the monocots, while HSL2 has been lost in gingers (Zingiberales) and grasses (Poales). IDA has been duplicated in eudicots to give rise to functionally divergent IDL peptides. We postulate that the high number of IDL homologs present in the core eudicots is a result of multiple whole genome duplications (WGD). We substantiate the involvement of IDA and HAE/HSL2 homologs in abscission by providing gene expression data of different organ separation events from various species.

Keywords: LRR-RLK; Populus; fruit abscission; genome duplication; leaf abscission; oil palm; peptide signaling; phylogeny.

Figure 1

Phylogeny of HSL LRR-RLK evolution within Angiosperms. Phylogeny inferred from ML with 145 ingroup taxa and 784 amino acid characters. The phylogeny has been collapsed at different taxonomic levels and shows only bootstrap values >50. The expanded tree is presented in Supplementary Figure S1, which includes an overview of species comprising each order. The alignments used for the construction of this phylogeny are available as Supplementary Data Sheet 1 and 2.

Figure 2

Conserved amino acids in the LRR of HSL proteins. Heatmaps generated using Repeat Conservation Mapping (RCM) of LRR domains (http://144.92.198.58/main/main.php) reflecting degree of identity and similarity of aa residues in a given position (X axis) in a given repeat (Y-axis) on the surface of the LRR domain of HAE orthologs from the eudicots, HSL1 from eudicots, HSL1A and B from monocots, HSL2 from eudicot and from monocots. The heatmaps are generated using the same ortholog sequences as used for the phylogenetic analysis (Supplementary Figure S1). The alignments used for heatmap construction are available as Supplementary Data Sheet 2.

Figure 3

IDA and IDL peptides. (A) Structure of IDA and IDL prepropeptides. (B–D) Peptide consensus sequences as indicated. The alignment used for the construction of the peptide logos is available as Supplementary Data Sheet 3.

Figure 4

Expression of HAE and HSL2 promoter: GUS constructs at sites of ectopic abscission. (A–C) Enlargement of AZ and premature abscission of whole flowers and immature fruits compared to wild type silique (to the left in A) in A. thaliana plants overexpressing AtIDL1. (D) Enlarged vestigial AZ after abscission of a cauline leaf in A. thaliana overexpressing AtIDL1. (E,F) pHAE:GUS expression and (G) pHSL2:GUS expression in vestigial AZs at the bases of pedicels, branches, and cauline leaves.

Figure 5

Modes of abscission. (A) Abscission of (a) sepals, (b) petals, (c) stamen, and (d) carpels. (B) Abscission of leaves at the axil of the pedicel, and abscission of entire male inflorescence (catkin) in Populus spp. (C) Opening of valves in dehiscence zones of dry many-seeded capsules, and abscission of individual seeds. (D) Abscission of fleshy fruits at AZ on pedicel. (E) The oil palm drupe fruit are tightly arranged within spikelets and abscise one by one when ripe. (A–D) Image courtesy the private collection of Roy Winkelman. First published in Gray (1858) and Foster (1921). (E) Image courtesy Missouri Botanical Garden. http://www.botanicus.org.

Figure 6

Expression levels of genes encoding IDA ligands and HSL receptors in AZs. (A) qPCR detecting increased expression levels of Populus IDA genes, but not the PtHAE gene in axils of shade-treated leaves prone to abscise compared to axils in non-shaded, non-abscission aspen leaves. qPCR, averages and standard deviations of three biological replicates. Normalized to PtACTIN1 expression. ^*p < 0.05, t-test, non-shaded vs. shaded. (B) qPCR analysis of oil palm EgHSL1 and EgHSL2 expression in AZs from unripe (Sh1-U and Sh2-U) not actively abscising fruit and AZs of ripe (Sh1-R, Sh3-R) actively abscising fruit, as well as AZs from ripe fruit of a non-abscising tree (NSh1-R1, NSh1-R2). (C) qPCR analyses of oil palm EgHSL expression during ethylene-induced abscission in ripe 180 DAP fruits. Samples were taken after 0, 3, 6, and 9 h treatment with ethylene. Similar results were obtained when treating 145 DAP fruits (Supplementary Figure S3C). In both experiments fruit separated by 9 h of ethylene treatment. (D) qPCR analysis of oil palm EgIDA2 and EgIDA5 expression in the AZs of unripe (Sh1-U and Sh2-U) not actively abscising fruit and AZs of ripe (Sh1-R, Sh3-R) actively abscising fruit, as well as AZs from ripe fruit of a non-abscising tree (NSh1-R1, NSh1-R2). Expression levels of additional EgIDA genes are found in Supplementary Figure S3D. (E) qPCR analyses of oil palm EgIDA2 and EgIDA5 expression during ethylene-induced abscission in ripe 180 DAP fruits. Samples were taken after 0, 3, 6, and 9 h treatment with ethylene. Similar results were obtained when treating 145 DAP fruits (Supplementary Figure S3E). In both experiments fruit separated by 9 h of ethylene treatment.

Figure 7

Evolution of the IDA HAE/HSL2 signaling module. Phylogeny of Angiosperms adapted from Zhang et al. (2012), Vanneste et al. (2014), Zeng et al. (2014), and Dohm et al. (2014). Taxonomical levels are taken from Zeng et al. (2014). Numbers in superscript behind order names represent the number of species used in the analysis. Order names in bold represent those orders with at least one completely sequenced genome. The two possible evolutionary origins of IDA (PIP_Q) in monocots are illustrated.