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. 2017 Mar;29(3):461-473.
doi: 10.1105/tpc.16.00831. Epub 2017 Mar 14.

Evidence for Ancient Origins of Bowman-Birk Inhibitors From Selaginella moellendorffii

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Free PMC article

Evidence for Ancient Origins of Bowman-Birk Inhibitors From Selaginella moellendorffii

Amy M James et al. Plant Cell. .
Free PMC article

Abstract

Bowman-Birk Inhibitors (BBIs) are a well-known family of plant protease inhibitors first described 70 years ago. BBIs are known only in the legume (Fabaceae) and cereal (Poaceae) families, but peptides that mimic their trypsin-inhibitory loops exist in sunflowers (Helianthus annuus) and frogs. The disparate biosynthetic origins and distant phylogenetic distribution implies these loops evolved independently, but their structural similarity suggests a common ancestor. Targeted bioinformatic searches for the BBI inhibitory loop discovered highly divergent BBI-like sequences in the seedless, vascular spikemoss Selaginella moellendorffii Using de novo transcriptomics, we confirmed expression of five transcripts in S. moellendorffii whose encoded proteins share homology with BBI inhibitory loops. The most highly expressed, BBI3, encodes a protein that inhibits trypsin. We needed to mutate two lysine residues to abolish trypsin inhibition, suggesting BBI3's mechanism of double-headed inhibition is shared with BBIs from angiosperms. As Selaginella belongs to the lycopod plant lineage, which diverged ∼200 to 230 million years before the common ancestor of angiosperms, its BBI-like proteins imply there was a common ancestor for legume and cereal BBIs. Indeed, we discovered BBI sequences in six angiosperm families outside the Fabaceae and Poaceae. These findings provide the evolutionary missing links between the well-known legume and cereal BBI gene families.

Figures

Figure 1.
Figure 1.
The CTKSIPPIC Motif: Structures, Sequences, and Contexts. (A) Structural alignment of SFTI-1 (PDB code: 1JBL), a BBI from soybean (GmaBBIa; PDB code: 1BBI), and a BBI from barley (HvuBBIa; PDB code: 2FJ8). The trypsin inhibitory loop of all three sequences (boxed) share structural similarity. (B) BBIs are common in the phylogenetically separate cereals and legumes marked on an angiosperm phylogeny of rbcL sequences (Elliott et al., 2014). (C) Sequence alignment of SFTI-1, GmaBBI, and HvuBBI. The homologous inhibitory “heads” are indicated with dashed brackets. Inhibitory loops are indicated with Roman numerals and the corresponding loops are also labeled in (A). The soybean sequence exemplifies the “double-headed” structure, with two homologous inhibitory motifs, the second of which has been lost in cereal BBIs as a result of the loss of two Cys residues (inhibitory loop II). Many monocot BBIs have undergone internal gene duplications resulting in multiple inhibitory loops (e.g., inhibitory loop III).
Figure 2.
Figure 2.
S. moellendorffii BBI3 Is Predicted to Be a BBI Based on Sequence Similarity at the Inhibitory Motifs and Shared Primary Protein Architecture. (A) Boxshade alignment of S. moellendorffii BBI-like (SmoBBI) protein sequences with soybean BBI (GmaBBIb) and barley BBI (HvuBBIb) shows conservation of the trypsin inhibitory loop (gray box). All proteins, with the exception of SmoBBI4, share a similar protein architecture, with an ER signal (line above sequence) followed by Cys-rich sequence with two spatially distinct conserved inhibitory motifs. Cys residues that are conserved in all sequences are indicated with asterisks. (B) Protein homology model of SmoBBI346-116 aligned with Medicago scutellata BBI (PDB code: 1MVZ) is shown along with the sequence alignment. The conserved inhibitory loops are shown as opaque, while the remaining protein is translucent and the corresponding inhibitory motifs are boxed in the alignment. The side chains of the labeled P1 residues (R64, K67, R90, and L105) and disulfide bonds are displayed in stick format.
Figure 3.
Figure 3.
Trypsin Inhibition by Wild-Type and Mutant S. moellendorffii BBI3. (A) Full-length wild-type BBI3 showing predicted inhibitory residues (bold). (B) Partial BBI3 sequences showing mutations (black highlight). (C) Inhibitors were incubated with trypsin and a colorogenic substrate of trypsin whose activity is measurable as OD410. Error bars represent sd for three technical replicates in a single microtiter plate. Trypsin incubated with the colorogenic substrate but no inhibitor was used as a control (NIC).
Figure 4.
Figure 4.
Distribution of BBIs in Land Plants. The number of BBI-like sequences identified (#) in each plant genome searched. Accession numbers for all sequences are included in Supplemental Data Set 1. Predicted gene loss events are indicated with an X. The phylogeny of the plant genomes investigated is derived from species trees available from Cogepedia and APG III.
Figure 5.
Figure 5.
BBI-Like Sequences Identified from Quillwort and Banana. Alignment of translated sequences of genes cloned from genomic DNA from banana (M. acuminata; Mac) and cDNA from quillwort (I. drummondii, Idr). Sequences are aligned with a BBI encoded by a gene from soybean (GmaBBIb) to illustrate conserved regions including the inhibitory loops (gray boxes). Conserved Cys residues are indicated by asterisks.
Figure 6.
Figure 6.
Phylogeny of BBI-Like Sequences in Plants. Neighbor-joining tree from a protein distance matrix of 104 BBI-like sequences. Sequences were identified by searches within plant genome assemblies. Major clades are indicated and defined by branching patterns. The five S. moellendorffii sequences and the I. drummondii sequence were used to root the tree with the assumption they were evolutionarily most distant from all other sequences. Percentage bootstrap values from 1000 replicates of a distance neighbor-joining analysis are indicated at each node (only nodes with >50% bootstrap support are labeled). Identifiers and accession numbers are listed in Supplemental Data Set 1. The scale bar represents number of substitutions per site.

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