BACKGROUND
legumes  are important economic crops that provide humans with food, livestock with feed and industry with raw materials  <cit> .
grain legume species, including pea , common bean , and lentil , account for over 33 % of human dietary protein.
other legumes, including clovers  and medics , are widely used as animal fodder  <cit> .
many legumes have been used in folk medicine, indicative of their bioactive chemical diversity  <cit> .
they play a critical role in natural agricultural and forest ecosystems because of their position in the nitrogen cycle  <cit> .
due to this nodal ecological position, pests, being nitrogen-limited feeders, are a major constraint to legume production.
they have consequently been involved in an evolutionary arms-race with legumes that defend themselves and their seeds through a wide array of chemical defenses and, remarkably, n-containing alkaloids, non-protein amino-acids and anti-nutritive peptides  <cit> .
the isolation of legume peptides found to be acutely toxic for insect pests in stored vegetables and crops, and non-toxic to other taxa  <cit> , has enlarged this defense arsenal, and, as a result, our possibilities for cereal grain protection  <cit> .
in plants as in animals, albumins  were defined by early biochemists as water soluble, moderately salt-soluble, and heat-denatured globular proteins.
plant albumins  <dig>  are a technology-defined salt-soluble fraction from legume seed proteins, subsequently shown to be restricted to leguminous species in which they constitute the main supply of sulfur amino acids  <cit> .
in pea seeds, the a <dig> gene, consisting of two exons and one intron, is transcribed as a single mrna encoding the secreted polypeptide pea albumin  <dig> .
the complex maturation of the latter finally leads to the release of two peptides, namely pa1b  and pa1a  .
to date, no function has been assigned to pa1a.
the insect toxicity of pa1b was discovered in  <dig> for weevils  <cit>  and subsequently extended to numerous other insects  <cit> .
in contrast to most animal venom toxins, it is active by ingestion, interacting with an intestinal binding site  <cit> , recently identified as a v-atpase proton pump  <cit> .
pa1b consists of  <dig> amino acids with six cysteines involved in three disulfide bonds, ensuring a compact and stable structure to the toxin  <cit> , and belongs to the knottin structural group  <cit> , the diversity of pa1b peptides within the same species was initially suggested by the work of higgins et al.
<cit> , which identified four functional genes that were present in the pea genome and expressed in pea seeds.
currently seven isoforms of pa1b have been isolated and biochemically characterized in the garden pea , indicating that these peptides belong to a multigenic family whose members have diverged slightly  <cit> .
more recently, a broad screen of more than  <dig> species scattered amongst the three major legume subfamilies identified ≈  <dig> pa1-like genes from numerous papilionoideae but none from caesalpinioideae or mimosoideae  <cit> .
thus, to date, the pa1b family seems to be strictly restricted to seeds of some legume sublineages and is the only one among more than  <dig> identified cysteine-rich families to have such a narrow distribution  <cit> .
this suggests that the family may be an important line of high-n seed defense against insects.
recently an interesting case of horizontal transfer to a parasitic broomrape has been documented but does not alter the overall picture  <cit> .fig.
<dig> peptide sequence features of the pa <dig> protein.
all original uniprot features of preproprotein pa <dig>  are displayed: signal peptide shown in green ; mature pa1b toxin and pa1a proprotein are displayed as red arrows; processed propeptides are in yellow boxes; cysteine-pairing is represented by the yellow arrows.
the β-strands are boxed in blue and the 3-
10-helix in red.
pa1b pertains to the albumin i  interpro family, which shows no relationship to other interpro families
although not the first plants to be subjected to genome sequencing, legumes are now included in genomic research specifically with soybean  and barrel medic  genome projects.
the complete analysis of the medicago genome for its rhizobial symbiotic features highlights this achievement  <cit> .
the recent release of a very significantly improved genome assembly prompted us to conduct a genomic exploration of pa1b homologues within legumes, with the major aims of deciphering the evolutionary history of albumins  <dig> and discovering new a1b variants with particular bioactivities.
the study was mainly devoted to the six legume species whose full genome sequencing/assembly had been completed and publicly available [ <dig> as of end 2014: medicago truncatula   <cit> , glycine max   <cit> , lotus japonicus   <cit> , cajanus cajan   <cit>  cicer arietinum   <cit>  and phaseolus vulgaris   <cit> , plus that of trifolium pratense   <cit> , still incompletely available.
a specific focus was drawn on the model legume m. truncatula  <cit> .
in this species, despite the fact that no pa1b peptide was biochemically detected in the seeds, we had previously identified the presence of high insecticidal toxicity and of homologous genes in its genome  <cit> .
RESULTS
specific expansion of the a1b family
the survey of the medicago truncatula genome  led to the identification of  <dig> a <dig> gene homologues.
<dig> genes were located on a m. truncatula chromosome , hence labeled medtrng, while eight genes were unassembled .
the detection of matching expressed sequence tags originating from mtr databases  showed that  <dig> of these genes are expressed .
finally, one est sequence homologous to pa <dig>  could not be associated to any pa <dig> gene and thus remains orphan, bringing the total a <dig> gene family to  <dig> members.
hmm profiles were constructed for both a1a and a1b families .
a sensitive search of protein databases with these hmms did not reveal significant relationships of these families outside the fabaceae.
even the closely related structural family of cyclotides, bearing a similar cysteine topology , cannot be phylogenetically related to the albumins i.fig.
<dig> organization of pa <dig> genes on medicago truncatula genome.
positions of genes are indicated on chromosomes .
the medicago truncatula physical scaffold map is that of the genome assembly version  <dig>  .
genes and their relative positions  on the chromosomes are those of assembly  <dig> ; a fictitious chromosome, called “0”, harbors the unplaced genes.
ac146565_ <dig> , ac146565_ <dig> , ac146565_ <dig>  and aj <dig>  are from genome version  <dig>  and are not present at 100 % match in assembly v <dig>  the orphan est “ta24778_3880” is also reported
the genomic organization and the structure of the mtr a <dig> genes have been studied next.
on the physical map, the  <dig> a1-encoding genes of m. truncatula are distributed on seven of the eight chromosomes, with an uneven distribution .
the length of all these genes varied between  <dig> and 1636 bp.
almost all genes  displayed a canonical organization with two exons and one intron.
the latter was systematically positioned in the sequence coding for the signal peptide and its length varied between  <dig> and 1199 bp .
out of the  <dig> members, six forms seemed not to be secreted .
medtr8g <dig>  was the only gene harboring the c-terminal a1a subunit alone, and consequently also had no signal peptide.
no trace of expression of this gene was found/published, questioning its functionality.
structural features of the medicago truncatula peptide sequences
the characteristics of the  <dig> a <dig> candidates  are presented in additional files.
all but  <dig> m. truncatula predicted peptides present a signal peptide 22– <dig> amino acids long, potentially leading the mature protein through the secretory/protein body pathway.
the multiple alignment of pa <dig> proteins showed an overall higher conservation of a1a subunit compared to a1b .
the location of the cysteine residues involved in the structural scaffolding of pa1b is globally conserved .
more precisely four different cysteine organizations were observed.
typical a1b knottin  are characterized by six cysteine residues in a strongly conserved topology with an antepenultimate c4xc <dig> motif.
<dig> mtr a1bs displayed this feature, whereas different patterns were observed for  <dig> a1b homologues .
cys <dig> was missing in the medtr3g <dig> encoded peptide, a1b from medtr6g <dig> and medtr3g <dig> harbored seven cysteines, and medtr3g <dig> and medtr3g <dig> held two additional cysteine residues after the cys <dig>
phylogenetic analysis of medicago truncatula a1bs
the bayesian  and maximum likelihood  unrooted phylogenic trees of the  <dig> mtr nucleotide sequences of pa <dig> were consistent and revealed six well-supported clusters labeled 1– <dig>  ≥ <dig>  and bootstrap values  ≥75 %, fig.
3).
the analysis of protein sequences provided similar results .
because these trees contained only mtr sequences, it was not possible to determine if the duplication events, which led to the expansion of pa <dig> in m. truncatula, occurred specifically in this lineage or if they were more ancient within papilionoidae .
to address this question, we searched for homologues in other representatives of the fabaceae for which genomic data were available.
this survey yielded  <dig> additional a1b sequences from different papilionoidae:  <dig> from cajanus cajan ,  <dig> from glycine max ,  <dig> from phaseolus vulgaris , and  <dig> from pisum sativum .
interestingly, while no a1b sequence was detected in the genome of lotus japonicus  and trifolium pratense , and only one in that of cicer arietinum , a toxic a1b sequence from the sophoreae styphnolobium japonicum, characterized by homologous pcr  <cit> , and not yet published , was included in the analysis; the cicer arietinum sequence was not included in the phylogeny due to the uncertainty on genome coverage  <cit> .
the bi and ml trees of the  <dig> pa <dig> nucleotide sequences were consistent but less resolved than those based on mtr sequences only due to the more restricted number of positions that could be kept for the analysis.
however, they were consistent with the currently accepted systematics of papilionoidae  <cit>  .
more precisely, a1b sequences from phaseoleae  formed a separate cluster , whereas pisum sativum and medicago truncatula sequences grouped together .
within phaseoleae, the  <dig> sequences from phaseolus vulgaris formed a monophyletic group , indicating a specific expansion of pa <dig> in this lineage likely through successive duplication events.
in contrast, the relationships among the multiple copies of pa <dig> observed in glycine max and cajanus cajan were not significantly supported , precluding any conclusion about the wealth of gene duplication events in these two phaseoleae lineages.
regarding medicago truncatula, the six clusters identified previously were recovered, and all but cluster  <dig> were again well supported .
the pisum sativum toxins formed a robust monophyletic group  related to mtr clusters , their exact relationships with the mtr clusters were not significantly supported.
the analysis of protein sequences provided similar trees .fig.
<dig> phylogeny, cxc pattern and tissue est expression of the medicago truncatula pa <dig> paralogues.
unrooted bayesian tree of medicago truncatula pa <dig> family .
the tree is presented according to rooted phylogeny shown on fig.
<dig>  numbers at branch correspond respectively to posterior probabilities calculated with mrbayes, and to bootstrap values estimated by phyml.
the scale bar represents the average number of substitutions per site.
six strongly supported clusters were boxed and highlighted with colored backgrounds.
the seven chemically and functionally synthetized sequences  are indicated on the tree.
among them, ag <dig>  eg <dig>  as <dig>  and as <dig> , and showed toxicity against insect cells, whereas gl <dig>  ds <dig> and qt <dig>  did not show toxicity to insect cells.
in the tissue expression part, the color-scale represent the expression value scales between  <dig> and > <dig> est per cluster.
two internal sub-clusters were defined for running site model in cluster  <dig> and  <dig>  and are named respectively 3a, 3b, and 6a, 6b .
red branches are those that were tested for positive selection
fig.
<dig> phylogeny of pa <dig> homologues identified in the complete genomes of fabaceae species.
bayesian tree of pa <dig> homologues identified in cajanus cajan , glycine max , phaseolus vulgaris , pisum sativum , and medicago truncatula  .
no homologues were detected in lotus corniculatus  and trifolium pratense , and only one in cicer arietinum , which was not included in this tree .
the tree was rooted with a sequence from styphnolobium japonicum , according to the current phylogeny of papilionoideae  <cit> .
numbers at branch correspond to posterior probabilities calculated with mrbayes and bootstrap values estimated by phyml.
the scale bar represents the average number of substitutions per site.
the six medicago truncatula sequence clusters identified previously  are recovered and indicated with the same colors.
labels on the left identify sustained nodes in the phylogeny of legumes, and labels on the right identify the protein species with substantial experimental data .
pea proteins are included as reference peptides
altogether, these two phylogenetic analyses suggested that in medicago truncatula i) cluster  <dig>  could have emerged from the legume toxin ancestor, ii) a1b-nodulins formed a distinct group  laying on two distinctive regions of chromosomes  <dig> and  <dig> , iii) the massive expansion of a1b on chromosome  <dig> resulted from specific successive duplications, and iv) according to functional data available from the literature and from this study , the phylogeny of a1b homologues suggested the toxin activity was ancestral in the m. truncatula lineage, and that non toxin a1b  emerged secondarily during the diversification of this gene family, v) the soybean leginsulin lies in an unresolved cluster composed of glycine and cajanus sequences.
bioactivity of synthetized peptides
starting from our phylogenetic analysis and taking into account the molecular requirements for bioactivity  <cit> , we selected and chemically synthesized eight peptide sequences, including the reference molecule pea albumin 1b.
we selected two sequences belonging to cluster  <dig> .
these were selected for their canonical crc  vs non-canonical cyc motif .
in addition, we selected several isoforms  scattered all over the whole tree but bearing the canonical crc pattern .
the activity of the peptides was assessed for their affinities for the pa1b-binding site, then for their cl <dig>  on cultured sf <dig> insect cells  <cit> .
the peptide activities are reported in table  <dig>  showing clearly that some sequences did not display toxicity, while others exhibited higher toxicity.
more precisely, ds <dig>  qt <dig> and gl <dig> peptides did not present binding and toxic abilities while as <dig>  ag <dig>  eg <dig> and as <dig> sequences revealed binding properties and toxicity.
four peptides  were folded with sufficient efficiency to yield the mg amounts needed for a standard sitophilus mortality assay  <cit> , and showed the expected toxicity .
interestingly, the presence of a tyrosine  instead of the canonical arginine r in the cxc motif in ds <dig> was correlated to an absence of binding and toxic properties.
sequence as <dig> led to biological properties similar to those of pea sequences.
the ag <dig> sequence displayed a very high toxicity, almost ten times superior to that of the original pea albumin 1b .
the toxicity results performed on sf <dig> cultured cells confirmed those obtained by binding affinity, with an overall good correlation between the two assays.table  <dig> affinity to the pa1b binding site and insect cell toxicity of synthetic peptides
scores a negative result
pa1b the referent molecule and ag <dig>  the mtr a1b with the highest toxicity were respectively highlighted in grey and pink.
the nomenclature for the mtr a1b  was arbitrarily defined as the first and last amino acid in the sequence and the total length.
cysteine architecture is highlighted in yellow.
sites under positive selection in their respective branches are reported as grey background
avariant amino-acids in ag <dig> compared to pa1b sequence are boldfaced
bthe ki of pa1b and the synthetic peptides was determined by ligand binding using 125i-pa1b, according to  <cit>
clc <dig> calculated from biological assays performed on cultured sf <dig> cells according to rahioui et al.
<cit>
ag <dig> acts as a potent blocker of insect cell membrane current
we further investigated the ag41’s biological activity by performing an electrophysiological experiment on cultured sf <dig> cells to check whether its cell-membrane ion transport alteration differed from that of pa1b, the model pea peptide  <cit> .
figure  <dig> shows effects of increasing concentrations of ag <dig>  and pa1b  on sf <dig> cell membrane current recorded in response to  <dig>  s duration voltage ramps applied from − <dig> to 90 mv.
at  <dig> nm, ag <dig> decreased by approximately 35 % the ramp current amplitude measured at +50 mv , while a similar concentration of  <dig>  nm pa1b had no effect .
the concentration/effect relationship was fitted to a hill equation and yielded ec <dig> values of  <dig>  ±  <dig>  nm  and  <dig>  ±  <dig>  nm  for ag <dig> and pa1b, respectively .
this experiment essentially showed that ag <dig> was much more efficient than pa1b to block ramp membrane current in sf <dig> cells , and the hill number was not significantly different between the two molecules/assays .fig.
<dig> electrophysiology of two a1b isoforms  on insect sf <dig> cells.
concentration dependent blockage of sf <dig> cells ramp membrane current by ag <dig> and pa1b.
a membrane currents recorded in response to voltage ramps of  <dig>  s duration applied from − <dig> to 90 mv in the absence  and presence of increasing concentrations of ag <dig>  and pa1b .
b mean concentration-response data for ag <dig>  and pa1b  inhibition of ramp membrane current, measured at +50 mv.
each point represents the mean ± s.e.
of n = 8– <dig> independent experiments.
hill equations were fitted to the data, with 100 % blockage taken as the fixed maximum effect, yielding ec <dig> values of  <dig>  ±  <dig>  nm  and  <dig>  ±  <dig>  nm  for ag <dig> and pa1b, respectively
molecular modeling of ag <dig>
the 3d structure model of ag <dig> protein  was built according to the procedure described in the materials and methods section.
as expected, the modeled 3d structure of ag <dig> adopted the knottin fold typical of pa1b  <cit>  .
to extend our comparison, we calculated the lipophilic potentials at the connolly surfaces of the proteins.
the 3-d structure delivers these molecular features which are correlated to protein functions.
the representation of the hydrophobic properties at the molecular surface of ag <dig> was typical of an amphipathic structure .
indeed, ag <dig> surface displayed a large hydrophobic face formed by the residues of the hydrophobic loop l2: val <dig>  leu <dig>  val <dig>  and ile <dig> but also by the facing residue phe <dig> of l <dig>  at the other pole of the molecule, the n and c-termini and a part of l <dig> defined the hydrophilic face with the polar residues ser <dig>  asn <dig>  thr <dig>  ser <dig>  asn <dig>  and ser <dig>  when hydrophobic potentials were calculated using the same hydrophobic scale, the surface of ag <dig>  appeared mainly hydrophobic, with a hydrophobic face significantly larger than that of pa1b , mainly due to the bulky aromatic residues trp <dig>  phe <dig>  phe <dig> that were only present in ag <dig> sequence .
the enhanced hydrophobicity of ag <dig>  together with the presence of a marked slit at the hydrophobic pole , could correlate with its increase insecticidal activities, since at least the hydrophobicity of the pole was pointed as a crucial determinant of insecticidal activity  <cit> .fig.
<dig> structures of two a1b isoforms .
a ribbon representation of pa1b .
b superposition of the backbones of pa1b  and ag <dig> .
c, d, e, f lipophilic potentials calculated with the molcad option of sybyl at the connolly surfaces of  pa1b and  ag <dig>  figures  are the same orientation as figures , using a common hydrophobic scale.
hydrophobic and hydrophilic areas are displayed in brown and blue, respectively.
green surfaces represent an intermediate hydrophobicity.
a 180 ° rotation according with respect to a vertical axis is applied from the upper  figures to the lower  figures
selective pressures on medicago truncatula albumin i sequences
an analysis of the selective pressures was conducted with the paml package on each of the six identified clusters in medicago truncatula.
branch models did not allow for the identification of branches with a significantly different evolutionary rate over the whole protein .
however, site and branch-site models enabled us to confirm previously identified sites under positive selection and even to identify some new.table  <dig> results of selection footprints analysis .
clusters are defined in the general medicago-only phylogenetic analysis described in fig.
<dig>  a further subdivision of cluster  <dig> and  <dig> into two internal sub-clusters  was defined for site models tests.
branch tested are colour-coded in red in fig.
<dig>  in each table cell are reported the significance of the model comparison , position and ω values of the amino-acid found to be under positive selection in the ‘site’ and ‘branch-site’ analyses after manual curation
p =  <dig>  10−4
p =  <dig>  10−13
p =  <dig>  10−4
p =  <dig>  10−3
p =  <dig>  10−6
p =  <dig>  10−44
p =  <dig>  10−14
p =  <dig>  10−10
p =  <dig>  10−15
probability associated with the lrt between the model m <dig> and the model m8a
probability associated with the lrt between the model for which branches in red are considered as foreground branches and the null model
ns not significant, no no sites validated after manual curation, - no partition tested
clusters  <dig>   <dig> and  <dig>  did not show any signature of positive selection.
by contrast, in the deeply-branching cluster  <dig>  the site model identified two sites under positive selection , site  <dig> falling within one of the three critical “spots” for insecticidal activity  <cit> , and site  <dig> located near the c-terminal ending of pa1a.
it is worth mentioning that site  <dig> was located in the important, and exposed, hydrophobic loop of pa1b and that a significant substitution was present at this site  for the two tested isoforms as <dig> and ds <dig>  this correlated well with the loss of insecticidal activity following the change to a bulky residue .
furthermore, in all insecticidal toxins tested so far, the  <dig>  residue was critical for the insecticidal activity  <cit>  and its neighboring  <dig> residue was a conserved glycine  residue.
sterical/hydrophobic constraints also seem to be crucial at that position.
the most curious feature in this cluster was, therefore, the absence of almost any trace of expression.
in cluster  <dig>  position  <dig> was a significant point of positive selection.
it did not lie on the a1b/toxin part of the protein, but rather at the precise position of the signal peptide intron.
this gathered the so-called a1b-nodulins, i.e.
showing nodule-induced expression  <cit> ; changes in the regulatory parts of the gene, including the gene’s canonical intron.
the other site under positive selection was at position  <dig>  again in the exposed loop , which was no longer hydrophobic within the whole nodulin group.
this corresponded to the loss of insecticidal activity observed in gl <dig> and contrasted with the basal conservation of this activity in isoforms ag <dig> and eg <dig> .
the last detected site in branch-site analysis within this cluster is at position  <dig>  a site almost adjacent to the critical cxc site located at positions .
the charge distribution within this central  site seemed to be an essential component of its activity.
in fact, in this cluster, there seemed to be a correlation between charges/residues at positions 74/ <dig>  possibly reminiscent of divergent sub-functionalization pressures on the nodulins and their signaling properties.
finally, the largest and late emerging cluster  <dig>  also has many sites which seemed to be subjected to positive selection: positions  <dig>   <dig> and  <dig> fall within three otherwise-conserved regions of the pa1a moiety .
position  <dig> pinpoints the n-terminus of the a1b peptide in one of the two sub-clusters, while position 92– <dig> marks the surrounding residues of a1b’s last cysteine, in the other sub-cluster.
both positions were in the hydrophilic part of the molecule, which was not implicated in the insecticidal activity.
finally, the most striking feature of positive selection was residue  <dig>  encompassing all of cluster  <dig>  this residue is located in the hyper-conserved cxc motif, for which an arginine residue is crucial for insecticidal activity .
in the whole cluster, the ratio of the non-synonymous on the synonymous substitutions at this position gives a clear signal of positive selection, which may result from an on-going process of neo-functionalization.
consistent with this interpretation, variations in expression patterns  were a clear characteristic of this peptide group.
interestingly, two sequences only retained the large-positive residue at this position , one of which confirms its insecticidal activity .
whether this was a reversion to, or a conservation of, the ancestral feature requires further analysis.fig.
<dig> heat map of all micro-array data available at mt-gene expression atlas.
all  a <dig> genes available in arrays were mapped against their tissue expression, and displayed as a heat-colored cells  superimposed with their individual data cloud .
original probeset data  were mapped to their corresponding gene in the v <dig> assembly; three genes are represented by more than one probeset .
full gene/probeset-id mapping is reported in additional file 6: table s1
expression analysis of the a <dig> gene family
est data
medicago expressed sequence tag repositories were carefully searched for all  <dig> mtr a <dig> genes.
a summary of the results are presented in additional file 4: table s <dig> and mapped on fig.
<dig>
the number of ests forming each ta varied from  <dig> to  <dig> .
all these ests were classified according to their expression in leaves, roots, seed or others.
almost all a <dig> genes were expressed in roots  .
roots seemed thus to be a privileged site for the expression of this molecule family in medicago.
in contrast, seed expression was exceptional .
in addition, 16 % were expressed in leaves and 28 % were found in other tissues/organs.
some genes  exhibited ubiquitous expression while others were organ-specific .
among the five genes expressed in seeds, the diversity of the cxc motif was of particular interest, where the typical insecticidal arginine residue was replaced by i, e or f suggesting the loss of insect-toxic abilities of theses peptides.
microarray data
extensive micro-array data for the species medicago truncatula were available at the gene expression atlas site mtgea .
we retrieved all data concerning the available a1b genes, resulting in a 25 ×  <dig> table .
the experiments were classified with a 13-tissue classification scheme and all data were mapped on a mixed cloud/heat-map outline, as shown in fig.
<dig>  the correlation between est and micro-array data was generally good, although some discrepancies were noted .
statistical artifacts arose with low-populated map cells , but major global features appeared, confirming the higher root expression .
multi-probe genes showed coherent patterns.
also, some expression correlations appeared between closely associated genes, with notable exceptions: medtr7g <dig> and medtr7g <dig> displayed very distinct profiles, and medtr3g <dig> could be easily differentiated from its neighbors.
the two latter genes  were, together with medtr3g <dig>  the highest expressors .
interestingly, medtr7g <dig> lay on the shorter branch of cluster  <dig> and should not be insecticidal, as is the case with medtr3g <dig>  which lay in an isolated long branch of cluster  <dig>  while medtr3g <dig> encoded an insecticidal peptide, and was branched basally in cluster  <dig> .
the est expression of the latter gene confirmed a large tissue distribution but with a low root-expression.
the main three a1b expressors were therefore likely to display very different functions in m. truncatula and only one of them retained its ancestral insecticidal function.
a1b-nodulins were not plotted in our heat-map  and they were low/conditional-expressors, which concurred with their proposed signaling functions.
finally, grouping genes according to their similar tissue-expression profiles resulted in the following organization : conductive organs  and flowers showed a quite similar expression of three genes among which medtr3g <dig>  seemed to be paradigmatic.
seed expressors, such as medtr7g <dig>  or a couple of medtr3g067nn genes , all encoded peptides lacking the crucial positively charged bulky residue  necessary for insecticidal activity.
seed expressors will also be highlighted in the next  section.
root expressors were more numerous and usually not predicted to be insecticidal, with the noticeable exception of medtr3g <dig> .
nodule expressors were, in general, like root expressors, with again the noticeable exception of the orphan expressed tag ta <dig>  which was also one of the few a1b isoforms expressed in the leaf.
its high insecticidal activity  was associated with this atypical expression profile .
proteomic data
previous data on mtr seed proteomics  <cit>  failed to reveal any expression of the two genes first identified from this species.
our present data clarified these results in that both genes identified by homology  have been shown to be silent or only expressed at very low levels, although quite conserved .
on the other hand, our raw proteomic data were reanalyzed with the transcriptomic and genomic data now available  and this resulted in the detection, in the seed extracts, of most of the a1b isoforms identified as seed expressors.
the hydrophobic peptide fraction, usually containing standard legume albumins a1b contained only traces of expression of medtr3g <dig>  <cit> , while the polar peptide fractions  <cit>  contained the newly identified peptides from v <dig> of the mtr assembly, distributed between the acidic polar fraction  and the basic polar fraction .
discussion
albumins i are more diverse in medicago truncatula than in any other legume
mtr-a <dig> peptides  are encoded by a multigenic family, currently comprising  <dig> members distributed along all but one of the eight medicago truncatula chromosomes.
although previously unsuspected for a1bs, this complexity seems to result from a lineage-specific gene expansion, maybe starting from the loci on chromosome  <dig> or  <dig>  through successive rounds of duplications.
in plant genomes, gene enrichment through duplication is commonplace, as occurs with the erf transcription-factors in cucumber  <cit> , and is mainly due to polyploidization, segmental and tandem duplications.
an example is the non-specific lipid transfer protein  in wheat vs rice and arabidopsis  <cit> .
recently, such mechanisms were also found to be implicated in domestication processes  <cit> .
tandem duplication events occurred repeatedly on chromosomes  <dig> and  <dig> .
the pattern of duplications also points to evolutionary links among sequences that lay on different chromosomes: chromosomes  <dig> and  <dig> , chromosomes  <dig> and  <dig> , and chromosomes  <dig> and  <dig> .
the expression pattern and selective pressure analyses shows that a number of sub- and neo-functionalization processes had occurred during the diversification of a1bs in m. truncatula.
this is not surprising for a single small molecule that has already been implicated in three independent biological functions/targets, namely insect-plant interactions, plant signaling/phosphorylation , and regulation of glycaemia in mammals  <cit> .
no parent peptide family was detected in other plant lineages
until now, the a <dig> gene family has been restricted to legume plants.
this concurs with a1bs being the only cysteine-rich peptide family, among more than  <dig> other known families, which was not present in any of the arabidopsis and rice genomes  <cit> .
interestingly, the genus clitoria  was recently shown to harbor a novel type of cyclotides, which was identified as a chimeric assemblage of a c-terminal pa1a and an n-terminal cyclotide  <cit> .
however, the evolutionary history of such chimeric molecules is not known but it strongly suggested that a recombination event has affected the pa <dig> coding gene present in the ancestor of phaseoleae, leading to the replacement of the pa1b domain by a cyclotide domain in the clitoria lineage.
another intriguing situation is the recent identification of an a1b gene acquired by horizontal gene transfer by phelipanche aegyptiaca and related species , not included in our dataset, which has likely conserved the insecticidal function  <cit> .
all these examples illustrate both the recombination properties of the two a <dig> functional modules  and the evolutionary stability of their respective signatures with the ability to persist in recipient organisms or genomic contexts long after the recombination event.
the insect toxin function seems to be ancestral in legume a1b evolution
the phylogenetic analysis of the pa <dig> family strongly suggested that the original legume gene present in the ancestor of the studied papilonoideae coded for a toxin aiming at protecting seed from insects.
s. japonicum is the most ancient and basal papilionoideae species for which experimental data of the presence of a1b peptides is available, initially through mass spectrometry and biological data  <cit> , and now through sequence information.
the cladrastis clade, to which s. japonicum belongs, is a basal legume tree group lying close to the swartzieae and ada clades, at the base of the papilionoidae  <cit> , and a sister group to the so-called 50 kb-inversion group  comprising most of the common papilionoidae.
therefore, s. japonicum  harbors the more divergent albumin i known to date.
consensus legume phylogeny therefore dates the a1b family back to the ancestor of s. japonicum and all other known families harbouring such peptides, especially the genistoid clade –genus lupinus; uniprot q <dig>  <cit> , with an estimated 56– <dig>  my/late paleocene origin  <cit> .
remarkably, multiple expansions of the pa <dig> family occurred during the diversification of phaseoleae and fabeae through successive gene duplications.
while, in some species  the resulting paralogues have kept the ancestral insecticidal function, in medicago truncatula and in glycine max functional diversification occurred .
it may be noted that the insect-toxin itself might not be mono-functional: the insecticidal homologue of pa1b in g. max , also named leginsulin, was first studied for its hormonal and signaling functions in soybean seeds .
diversified a1b expression and function in medicago: no longer a seed toxin
one of the most striking results of this genomic survey was that the standard situation prevailing in all other legumes studied so far, namely that albumins are toxic seed storage proteins, is not true in medicago truncatula.
instead, most genes that showed seed expression  are predicted not to be insect toxins, with the exception of eg41/medtr3g <dig> cluster  <dig>  moreover, we were unable to detect toxic peptides in m. truncatula seeds using our original homology-based genomic pcr strategy  <cit> , which was leading to the cluster_ <dig> members that had lost their expression.
in relation to their shift of expression out-of-seeds, medicago a1bs acquired an extremely diversified tissue distribution, dominated by root expression, which accounts for the presence of almost half of the array-detected transcripts .
this emerging pattern seems an alternation of loss/gain and conservation of function, linked to expression retargeting.
the current positive selection traces on two of the “functional hot spot” of the molecule  <cit>  is a clear indication of occurrence of discrete stepwise functional changes.
however, this assumption needs to be checked experimentally by functional studies.
nodulin and chromosome  <dig> expansion as recent neo-functionalization bursts
nodulins were defined by substractive expression methodologies as genes specifically expressed in legume rhizobial-associated nodules  <cit> .
early in this process, and after the identification of many transport-associated membrane proteins, some small proteins involved in signaling were identified   <cit> .
in this line of research, three nodulins mtn <dig> ,  <dig>  and  <dig> , were identified  <cit>  and these happened to be distant and short homologs of the albumins  <dig>  but their functions were not investigated further.
our work unveiled a group of  <dig> homologues to the first three described , that are all short and mostly 6-cysteine peptides .
they are grouped in cluster  <dig> and are basally related to a set of three more canonical sequences, two of which being readily insecticidal .
the so-called “nodulin” cluster is therefore arising from apparently standard toxins that have lost their pa1a moiety and have subsequently undergone significant sequence changes albeit retaining the cysteine scaffold .
traces of selection were detected at the basal branch of each of the nodulin sub-groups, namely the isolated outgroup medtr5g <dig> and the two sub-clusters 3a and 3b defined for the paml analysis.
in addition to the site features discussed in the results section, it is interesting to note that the loss of the pa1a domain is correlated with significant sequence changes, including that of the canonical hydrophobic loop and a total loss of insecticidal activity, even when the canonical crc stretch is retained .
in this putative “nodulin” cluster, it is likely that the structural constraints for knotted peptide folding are released, which would fit with the absence of a pa1a moiety, as this domain is now strongly suspected of serving as a chaperone for assisted co-translational folding of most canonical pa1b peptides  <cit> .
since knottins are not all difficult to fold  <cit> , the presence of a flanking pro-peptide chaperone is probably useful in a restricted part of the conformational space explored by the a1bs from medicago truncatula.
most cyclotides do not need chaperones either  <cit> .
insect toxins after all: one extinct and two active clusters?
the final issue deals with the expression of insect toxicity in medicago truncatula tissues.
we are well aware that our experimental data  is extremely partial, but we are now confident that the predictive power concerning insecticidal activity from sequence information is relatively good.
the first question pertains to cluster  <dig> genes, for which the expression data was almost non-existent.
the data was checked for two genes and it was confirmed with ngs data; genes medtr6g <dig>  and medtr6g <dig>  revealed no expression whatsoever .
as a control, we also checked two other est-orphan genes from the “nodulin” cluster: they were shown to be expressed, and induced, in nodules .
we are therefore confident that cluster  <dig> is globally composed of silent genes, although one of them  conserved its insecticidal activity.
apart from this silent gene set, our study detected two other clusters with interesting insecticidal activities: the nodulin-related cluster  <dig>  and the only crc-containing member from the chromosome  <dig> expansion cluster  <dig>  these two groups are expressed in roots and nodules, but ag <dig> shows an interesting and strong conditional expression in nodules , as well as a good expression in leaves.
from an ecological point of view, expressing a very potent insecticidal molecule in a high-value nitrogen source organ would not be fortuitous, as suggested by the fact that the nodule-specific  insects seem devoid of receptor binding sites, and therefore susceptibility to a1-type toxins  <cit> .
CONCLUSIONS
when viewing our survey as a search for protein innovation within a specific taxon , one may ask how unusual are a1b in this respect ?
a screen of the interpro  <cit>  and pfam  <cit>  databases for taxonomic boundaries of protein domain families retrieved only three families that are restricted to  the fabaceae/legumes: albumins  <dig>  nodule-specific glycine rich proteins, and the late nodulin family.
one may add the ncr expansion to this list  <cit> , which is not captured by a single protein family, and is distinct from a <dig> .
many other nodule-specific proteins were subsequently recovered in other plant taxa, such as enod hormones, and even the typically nodular leghemoglobin, related to the very ancient heme-binding globin family.
not surprisingly, these major protein novelties are somehow related to rhizobial symbiosis , but also concern small proteins.
this may illustrate how protein modules can be derived by a process of neofunctionalization of a previously existing scaffold .
in conclusion, the study of the multigenic insecticidal albumin <dig> family is of interest to the evolutionary history of legume-specific protein families, and to novel bioactive molecule discovery.
the exploration of our results may end up in new biopesticide leads, such as ag <dig>  for the control of a large array of insect pests  <cit> .

 legumes are important economic crops that provide humans with food, livestock with feed and industry with raw materials. grain legume species, including pea, common bean, and lentil, account for over 33 % of human dietary protein. other legumes, including clovers and medics, are widely used as animal fodder. many legumes have been used in folk medicine, indicative of their bioactive chemical diversity. they play a critical role in natural agricultural and forest ecosystems because of their position in the nitrogen cycle. due to this nodal ecological position, pests, being nitrogen-limited feeders, are a major constraint to legume production. they have consequently been involved in an evolutionary arms-race with legumes that defend themselves and their seeds through a wide array of chemical defenses and, remarkably, n-containing alkaloids, non-protein amino-acids and anti-nutritive peptides. the isolation of legume peptides found to be acutely toxic for insect pests in stored vegetables and crops, and non-toxic to other taxa, has enlarged this defense arsenal, and, as a result, our possibilities for cereal grain protection. in plants as in animals, albumins were defined by early biochemists as water soluble, moderately salt-soluble, and heat-denatured globular proteins. plant albumins are a technology-defined salt-soluble fraction from legume seed proteins, subsequently shown to be restricted to leguminous species in which they constitute the main supply of sulfur amino acids. in pea seeds, the a gene, consisting of two exons and one intron, is transcribed as a single mrna encoding the secreted polypeptide pea albumin. the complex maturation of the latter finally leads to the release of two peptides, namely pa1b and pa1a. to date, no function has been assigned to pa1a. the insect toxicity of pa1b was discovered in for weevils and subsequently extended to numerous other insects. in contrast to most animal venom toxins, it is active by ingestion, interacting with an intestinal binding site, recently identified as a v-atpase proton pump. pa1b consists of amino acids with six cysteines involved in three disulfide bonds, ensuring a compact and stable structure to the toxin, and belongs to the knottin structural group, the diversity of pa1b peptides within the same species was initially suggested by the work of higgins et al., which identified four functional genes that were present in the pea genome and expressed in pea seeds. currently seven isoforms of pa1b have been isolated and biochemically characterized in the garden pea, indicating that these peptides belong to a multigenic family whose members have diverged slightly. more recently, a broad screen of more than species scattered amongst the three major legume subfamilies identified ≈  pa1-like genes from numerous papilionoideae but none from caesalpinioideae or mimosoideae. thus, to date, the pa1b family seems to be strictly restricted to seeds of some legume sublineages and is the only one among more than identified cysteine-rich families to have such a narrow distribution. this suggests that the family may be an important line of high-n seed defense against insects. recently an interesting case of horizontal transfer to a parasitic broomrape has been documented but does not alter the overall picture.fig. peptide sequence features of the pa protein. all original uniprot features of preproprotein pa are displayed: signal peptide shown in green; mature pa1b toxin and pa1a proprotein are displayed as red arrows; processed propeptides are in yellow boxes; cysteine-pairing is represented by the yellow arrows. the β-strands are boxed in blue and the 3- 10-helix in red. pa1b pertains to the albumin i interpro family, which shows no relationship to other interpro families although not the first plants to be subjected to genome sequencing, legumes are now included in genomic research specifically with soybean and barrel medic genome projects. the complete analysis of the medicago genome for its rhizobial symbiotic features highlights this achievement. the recent release of a very significantly improved genome assembly prompted us to conduct a genomic exploration of pa1b homologues within legumes, with the major aims of deciphering the evolutionary history of albumins and discovering new a1b variants with particular bioactivities. the study was mainly devoted to the six legume species whose full genome sequencing/assembly had been completed and publicly available [ as of end 2014: medicago truncatula, glycine max, lotus japonicus, cajanus cajan cicer arietinum and phaseolus vulgaris, plus that of trifolium pratense, still incompletely available. a specific focus was drawn on the model legume m. truncatula. in this species, despite the fact that no pa1b peptide was biochemically detected in the seeds, we had previously identified the presence of high insecticidal toxicity and of homologous genes in its genome.  specific expansion of the a1b family the survey of the medicago truncatula genome led to the identification of a gene homologues. genes were located on a m. truncatula chromosome, hence labeled medtrng, while eight genes were unassembled. the detection of matching expressed sequence tags originating from mtr databases showed that of these genes are expressed. finally, one est sequence homologous to pa could not be associated to any pa gene and thus remains orphan, bringing the total a gene family to members. hmm profiles were constructed for both a1a and a1b families. a sensitive search of protein databases with these hmms did not reveal significant relationships of these families outside the fabaceae. even the closely related structural family of cyclotides, bearing a similar cysteine topology, cannot be phylogenetically related to the albumins i.fig. organization of pa genes on medicago truncatula genome. positions of genes are indicated on chromosomes. the medicago truncatula physical scaffold map is that of the genome assembly version. genes and their relative positions on the chromosomes are those of assembly; a fictitious chromosome, called “0”, harbors the unplaced genes. ac146565_, ac146565_, ac146565_ and aj are from genome version and are not present at 100 % match in assembly v the orphan est “ta24778_3880” is also reported the genomic organization and the structure of the mtr a genes have been studied next. on the physical map, the a1-encoding genes of m. truncatula are distributed on seven of the eight chromosomes, with an uneven distribution. the length of all these genes varied between and 1636 bp. almost all genes displayed a canonical organization with two exons and one intron. the latter was systematically positioned in the sequence coding for the signal peptide and its length varied between and 1199 bp. out of the members, six forms seemed not to be secreted. medtr8g was the only gene harboring the c-terminal a1a subunit alone, and consequently also had no signal peptide. no trace of expression of this gene was found/published, questioning its functionality. structural features of the medicago truncatula peptide sequences the characteristics of the a candidates are presented in additional files. all but m. truncatula predicted peptides present a signal peptide 22– amino acids long, potentially leading the mature protein through the secretory/protein body pathway. the multiple alignment of pa proteins showed an overall higher conservation of a1a subunit compared to a1b. the location of the cysteine residues involved in the structural scaffolding of pa1b is globally conserved. more precisely four different cysteine organizations were observed. typical a1b knottin are characterized by six cysteine residues in a strongly conserved topology with an antepenultimate c4xc motif. mtr a1bs displayed this feature, whereas different patterns were observed for a1b homologues. cys was missing in the medtr3g encoded peptide, a1b from medtr6g and medtr3g harbored seven cysteines, and medtr3g and medtr3g held two additional cysteine residues after the cys phylogenetic analysis of medicago truncatula a1bs the bayesian and maximum likelihood unrooted phylogenic trees of the mtr nucleotide sequences of pa were consistent and revealed six well-supported clusters labeled 1–  ≥ and bootstrap values  ≥75 %, fig. 3). the analysis of protein sequences provided similar results. because these trees contained only mtr sequences, it was not possible to determine if the duplication events, which led to the expansion of pa in m. truncatula, occurred specifically in this lineage or if they were more ancient within papilionoidae. to address this question, we searched for homologues in other representatives of the fabaceae for which genomic data were available. this survey yielded additional a1b sequences from different papilionoidae: from cajanus cajan, from glycine max, from phaseolus vulgaris, and from pisum sativum. interestingly, while no a1b sequence was detected in the genome of lotus japonicus and trifolium pratense, and only one in that of cicer arietinum, a toxic a1b sequence from the sophoreae styphnolobium japonicum, characterized by homologous pcr, and not yet published, was included in the analysis; the cicer arietinum sequence was not included in the phylogeny due to the uncertainty on genome coverage. the bi and ml trees of the pa nucleotide sequences were consistent but less resolved than those based on mtr sequences only due to the more restricted number of positions that could be kept for the analysis. however, they were consistent with the currently accepted systematics of papilionoidae. more precisely, a1b sequences from phaseoleae formed a separate cluster, whereas pisum sativum and medicago truncatula sequences grouped together. within phaseoleae, the sequences from phaseolus vulgaris formed a monophyletic group, indicating a specific expansion of pa in this lineage likely through successive duplication events. in contrast, the relationships among the multiple copies of pa observed in glycine max and cajanus cajan were not significantly supported, precluding any conclusion about the wealth of gene duplication events in these two phaseoleae lineages. regarding medicago truncatula, the six clusters identified previously were recovered, and all but cluster were again well supported. the pisum sativum toxins formed a robust monophyletic group related to mtr clusters, their exact relationships with the mtr clusters were not significantly supported. the analysis of protein sequences provided similar trees.fig. phylogeny, cxc pattern and tissue est expression of the medicago truncatula pa paralogues. unrooted bayesian tree of medicago truncatula pa family. the tree is presented according to rooted phylogeny shown on fig.  numbers at branch correspond respectively to posterior probabilities calculated with mrbayes, and to bootstrap values estimated by phyml. the scale bar represents the average number of substitutions per site. six strongly supported clusters were boxed and highlighted with colored backgrounds. the seven chemically and functionally synthetized sequences are indicated on the tree. among them, ag eg as and as, and showed toxicity against insect cells, whereas gl ds and qt did not show toxicity to insect cells. in the tissue expression part, the color-scale represent the expression value scales between and > est per cluster. two internal sub-clusters were defined for running site model in cluster and and are named respectively 3a, 3b, and 6a, 6b. red branches are those that were tested for positive selection fig. phylogeny of pa homologues identified in the complete genomes of fabaceae species. bayesian tree of pa homologues identified in cajanus cajan, glycine max, phaseolus vulgaris, pisum sativum, and medicago truncatula. no homologues were detected in lotus corniculatus and trifolium pratense, and only one in cicer arietinum, which was not included in this tree. the tree was rooted with a sequence from styphnolobium japonicum, according to the current phylogeny of papilionoideae. numbers at branch correspond to posterior probabilities calculated with mrbayes and bootstrap values estimated by phyml. the scale bar represents the average number of substitutions per site. the six medicago truncatula sequence clusters identified previously are recovered and indicated with the same colors. labels on the left identify sustained nodes in the phylogeny of legumes, and labels on the right identify the protein species with substantial experimental data. pea proteins are included as reference peptides altogether, these two phylogenetic analyses suggested that in medicago truncatula i) cluster could have emerged from the legume toxin ancestor, ii) a1b-nodulins formed a distinct group laying on two distinctive regions of chromosomes and, iii) the massive expansion of a1b on chromosome resulted from specific successive duplications, and iv) according to functional data available from the literature and from this study, the phylogeny of a1b homologues suggested the toxin activity was ancestral in the m. truncatula lineage, and that non toxin a1b emerged secondarily during the diversification of this gene family, v) the soybean leginsulin lies in an unresolved cluster composed of glycine and cajanus sequences. bioactivity of synthetized peptides starting from our phylogenetic analysis and taking into account the molecular requirements for bioactivity, we selected and chemically synthesized eight peptide sequences, including the reference molecule pea albumin 1b. we selected two sequences belonging to cluster. these were selected for their canonical crc vs non-canonical cyc motif. in addition, we selected several isoforms scattered all over the whole tree but bearing the canonical crc pattern. the activity of the peptides was assessed for their affinities for the pa1b-binding site, then for their cl on cultured sf insect cells. the peptide activities are reported in table  showing clearly that some sequences did not display toxicity, while others exhibited higher toxicity. more precisely, ds qt and gl peptides did not present binding and toxic abilities while as ag eg and as sequences revealed binding properties and toxicity. four peptides were folded with sufficient efficiency to yield the mg amounts needed for a standard sitophilus mortality assay, and showed the expected toxicity. interestingly, the presence of a tyrosine instead of the canonical arginine r in the cxc motif in ds was correlated to an absence of binding and toxic properties. sequence as led to biological properties similar to those of pea sequences. the ag sequence displayed a very high toxicity, almost ten times superior to that of the original pea albumin 1b. the toxicity results performed on sf cultured cells confirmed those obtained by binding affinity, with an overall good correlation between the two assays.table affinity to the pa1b binding site and insect cell toxicity of synthetic peptides scores a negative result pa1b the referent molecule and ag the mtr a1b with the highest toxicity were respectively highlighted in grey and pink. the nomenclature for the mtr a1b was arbitrarily defined as the first and last amino acid in the sequence and the total length. cysteine architecture is highlighted in yellow. sites under positive selection in their respective branches are reported as grey background avariant amino-acids in ag compared to pa1b sequence are boldfaced bthe ki of pa1b and the synthetic peptides was determined by ligand binding using 125i-pa1b, according to clc calculated from biological assays performed on cultured sf cells according to rahioui et al. ag acts as a potent blocker of insect cell membrane current we further investigated the ag41’s biological activity by performing an electrophysiological experiment on cultured sf cells to check whether its cell-membrane ion transport alteration differed from that of pa1b, the model pea peptide. figure  shows effects of increasing concentrations of ag and pa1b on sf cell membrane current recorded in response to  s duration voltage ramps applied from − to 90 mv. at nm, ag decreased by approximately 35 % the ramp current amplitude measured at +50 mv, while a similar concentration of nm pa1b had no effect. the concentration/effect relationship was fitted to a hill equation and yielded ec values of  ±  nm and  ±  nm for ag and pa1b, respectively. this experiment essentially showed that ag was much more efficient than pa1b to block ramp membrane current in sf cells, and the hill number was not significantly different between the two molecules/assays.fig. electrophysiology of two a1b isoforms on insect sf cells. concentration dependent blockage of sf cells ramp membrane current by ag and pa1b. a membrane currents recorded in response to voltage ramps of  s duration applied from − to 90 mv in the absence and presence of increasing concentrations of ag and pa1b. b mean concentration-response data for ag and pa1b inhibition of ramp membrane current, measured at +50 mv. each point represents the mean ± s.e. of n = 8– independent experiments. hill equations were fitted to the data, with 100 % blockage taken as the fixed maximum effect, yielding ec values of  ±  nm and  ±  nm for ag and pa1b, respectively molecular modeling of ag the 3d structure model of ag protein was built according to the procedure described in the materials and methods section. as expected, the modeled 3d structure of ag adopted the knottin fold typical of pa1b. to extend our comparison, we calculated the lipophilic potentials at the connolly surfaces of the proteins. the 3-d structure delivers these molecular features which are correlated to protein functions. the representation of the hydrophobic properties at the molecular surface of ag was typical of an amphipathic structure. indeed, ag surface displayed a large hydrophobic face formed by the residues of the hydrophobic loop l2: val leu val and ile but also by the facing residue phe of l at the other pole of the molecule, the n and c-termini and a part of l defined the hydrophilic face with the polar residues ser asn thr ser asn and ser when hydrophobic potentials were calculated using the same hydrophobic scale, the surface of ag appeared mainly hydrophobic, with a hydrophobic face significantly larger than that of pa1b, mainly due to the bulky aromatic residues trp phe phe that were only present in ag sequence. the enhanced hydrophobicity of ag together with the presence of a marked slit at the hydrophobic pole, could correlate with its increase insecticidal activities, since at least the hydrophobicity of the pole was pointed as a crucial determinant of insecticidal activity.fig. structures of two a1b isoforms. a ribbon representation of pa1b. b superposition of the backbones of pa1b and ag. c, d, e, f lipophilic potentials calculated with the molcad option of sybyl at the connolly surfaces of pa1b and ag figures are the same orientation as figures, using a common hydrophobic scale. hydrophobic and hydrophilic areas are displayed in brown and blue, respectively. green surfaces represent an intermediate hydrophobicity. a 180 ° rotation according with respect to a vertical axis is applied from the upper figures to the lower figures selective pressures on medicago truncatula albumin i sequences an analysis of the selective pressures was conducted with the paml package on each of the six identified clusters in medicago truncatula. branch models did not allow for the identification of branches with a significantly different evolutionary rate over the whole protein. however, site and branch-site models enabled us to confirm previously identified sites under positive selection and even to identify some new.table results of selection footprints analysis. clusters are defined in the general medicago-only phylogenetic analysis described in fig.  a further subdivision of cluster and into two internal sub-clusters was defined for site models tests. branch tested are colour-coded in red in fig.  in each table cell are reported the significance of the model comparison, position and ω values of the amino-acid found to be under positive selection in the ‘site’ and ‘branch-site’ analyses after manual curation p =  10−4 p =  10−13 p =  10−4 p =  10−3 p =  10−6 p =  10−44 p =  10−14 p =  10−10 p =  10−15 probability associated with the lrt between the model m and the model m8a probability associated with the lrt between the model for which branches in red are considered as foreground branches and the null model ns not significant, no no sites validated after manual curation, - no partition tested clusters and did not show any signature of positive selection. by contrast, in the deeply-branching cluster the site model identified two sites under positive selection, site falling within one of the three critical “spots” for insecticidal activity, and site located near the c-terminal ending of pa1a. it is worth mentioning that site was located in the important, and exposed, hydrophobic loop of pa1b and that a significant substitution was present at this site for the two tested isoforms as and ds this correlated well with the loss of insecticidal activity following the change to a bulky residue. furthermore, in all insecticidal toxins tested so far, the residue was critical for the insecticidal activity and its neighboring residue was a conserved glycine residue. sterical/hydrophobic constraints also seem to be crucial at that position. the most curious feature in this cluster was, therefore, the absence of almost any trace of expression. in cluster position was a significant point of positive selection. it did not lie on the a1b/toxin part of the protein, but rather at the precise position of the signal peptide intron. this gathered the so-called a1b-nodulins, i.e. showing nodule-induced expression; changes in the regulatory parts of the gene, including the gene’s canonical intron. the other site under positive selection was at position again in the exposed loop, which was no longer hydrophobic within the whole nodulin group. this corresponded to the loss of insecticidal activity observed in gl and contrasted with the basal conservation of this activity in isoforms ag and eg. the last detected site in branch-site analysis within this cluster is at position a site almost adjacent to the critical cxc site located at positions. the charge distribution within this central site seemed to be an essential component of its activity. in fact, in this cluster, there seemed to be a correlation between charges/residues at positions 74/ possibly reminiscent of divergent sub-functionalization pressures on the nodulins and their signaling properties. finally, the largest and late emerging cluster also has many sites which seemed to be subjected to positive selection: positions and fall within three otherwise-conserved regions of the pa1a moiety. position pinpoints the n-terminus of the a1b peptide in one of the two sub-clusters, while position 92– marks the surrounding residues of a1b’s last cysteine, in the other sub-cluster. both positions were in the hydrophilic part of the molecule, which was not implicated in the insecticidal activity. finally, the most striking feature of positive selection was residue encompassing all of cluster this residue is located in the hyper-conserved cxc motif, for which an arginine residue is crucial for insecticidal activity. in the whole cluster, the ratio of the non-synonymous on the synonymous substitutions at this position gives a clear signal of positive selection, which may result from an on-going process of neo-functionalization. consistent with this interpretation, variations in expression patterns were a clear characteristic of this peptide group. interestingly, two sequences only retained the large-positive residue at this position, one of which confirms its insecticidal activity. whether this was a reversion to, or a conservation of, the ancestral feature requires further analysis.fig. heat map of all micro-array data available at mt-gene expression atlas. all a genes available in arrays were mapped against their tissue expression, and displayed as a heat-colored cells superimposed with their individual data cloud. original probeset data were mapped to their corresponding gene in the v assembly; three genes are represented by more than one probeset. full gene/probeset-id mapping is reported in additional file 6: table s1 expression analysis of the a gene family est data medicago expressed sequence tag repositories were carefully searched for all mtr a genes. a summary of the results are presented in additional file 4: table s and mapped on fig.  the number of ests forming each ta varied from to. all these ests were classified according to their expression in leaves, roots, seed or others. almost all a genes were expressed in roots. roots seemed thus to be a privileged site for the expression of this molecule family in medicago. in contrast, seed expression was exceptional. in addition, 16 % were expressed in leaves and 28 % were found in other tissues/organs. some genes exhibited ubiquitous expression while others were organ-specific. among the five genes expressed in seeds, the diversity of the cxc motif was of particular interest, where the typical insecticidal arginine residue was replaced by i, e or f suggesting the loss of insect-toxic abilities of theses peptides. microarray data extensive micro-array data for the species medicago truncatula were available at the gene expression atlas site mtgea. we retrieved all data concerning the available a1b genes, resulting in a 25 ×  table. the experiments were classified with a 13-tissue classification scheme and all data were mapped on a mixed cloud/heat-map outline, as shown in fig.  the correlation between est and micro-array data was generally good, although some discrepancies were noted. statistical artifacts arose with low-populated map cells, but major global features appeared, confirming the higher root expression. multi-probe genes showed coherent patterns. also, some expression correlations appeared between closely associated genes, with notable exceptions: medtr7g and medtr7g displayed very distinct profiles, and medtr3g could be easily differentiated from its neighbors. the two latter genes were, together with medtr3g the highest expressors. interestingly, medtr7g lay on the shorter branch of cluster and should not be insecticidal, as is the case with medtr3g which lay in an isolated long branch of cluster while medtr3g encoded an insecticidal peptide, and was branched basally in cluster. the est expression of the latter gene confirmed a large tissue distribution but with a low root-expression. the main three a1b expressors were therefore likely to display very different functions in m. truncatula and only one of them retained its ancestral insecticidal function. a1b-nodulins were not plotted in our heat-map and they were low/conditional-expressors, which concurred with their proposed signaling functions. finally, grouping genes according to their similar tissue-expression profiles resulted in the following organization: conductive organs and flowers showed a quite similar expression of three genes among which medtr3g seemed to be paradigmatic. seed expressors, such as medtr7g or a couple of medtr3g067nn genes, all encoded peptides lacking the crucial positively charged bulky residue necessary for insecticidal activity. seed expressors will also be highlighted in the next section. root expressors were more numerous and usually not predicted to be insecticidal, with the noticeable exception of medtr3g. nodule expressors were, in general, like root expressors, with again the noticeable exception of the orphan expressed tag ta which was also one of the few a1b isoforms expressed in the leaf. its high insecticidal activity was associated with this atypical expression profile. proteomic data previous data on mtr seed proteomics failed to reveal any expression of the two genes first identified from this species. our present data clarified these results in that both genes identified by homology have been shown to be silent or only expressed at very low levels, although quite conserved. on the other hand, our raw proteomic data were reanalyzed with the transcriptomic and genomic data now available and this resulted in the detection, in the seed extracts, of most of the a1b isoforms identified as seed expressors. the hydrophobic peptide fraction, usually containing standard legume albumins a1b contained only traces of expression of medtr3g, while the polar peptide fractions contained the newly identified peptides from v of the mtr assembly, distributed between the acidic polar fraction and the basic polar fraction. discussion albumins i are more diverse in medicago truncatula than in any other legume mtr-a peptides are encoded by a multigenic family, currently comprising members distributed along all but one of the eight medicago truncatula chromosomes. although previously unsuspected for a1bs, this complexity seems to result from a lineage-specific gene expansion, maybe starting from the loci on chromosome or through successive rounds of duplications. in plant genomes, gene enrichment through duplication is commonplace, as occurs with the erf transcription-factors in cucumber, and is mainly due to polyploidization, segmental and tandem duplications. an example is the non-specific lipid transfer protein in wheat vs rice and arabidopsis. recently, such mechanisms were also found to be implicated in domestication processes. tandem duplication events occurred repeatedly on chromosomes and. the pattern of duplications also points to evolutionary links among sequences that lay on different chromosomes: chromosomes and, chromosomes and, and chromosomes and. the expression pattern and selective pressure analyses shows that a number of sub- and neo-functionalization processes had occurred during the diversification of a1bs in m. truncatula. this is not surprising for a single small molecule that has already been implicated in three independent biological functions/targets, namely insect-plant interactions, plant signaling/phosphorylation, and regulation of glycaemia in mammals. no parent peptide family was detected in other plant lineages until now, the a gene family has been restricted to legume plants. this concurs with a1bs being the only cysteine-rich peptide family, among more than other known families, which was not present in any of the arabidopsis and rice genomes. interestingly, the genus clitoria was recently shown to harbor a novel type of cyclotides, which was identified as a chimeric assemblage of a c-terminal pa1a and an n-terminal cyclotide. however, the evolutionary history of such chimeric molecules is not known but it strongly suggested that a recombination event has affected the pa coding gene present in the ancestor of phaseoleae, leading to the replacement of the pa1b domain by a cyclotide domain in the clitoria lineage. another intriguing situation is the recent identification of an a1b gene acquired by horizontal gene transfer by phelipanche aegyptiaca and related species, not included in our dataset, which has likely conserved the insecticidal function. all these examples illustrate both the recombination properties of the two a functional modules and the evolutionary stability of their respective signatures with the ability to persist in recipient organisms or genomic contexts long after the recombination event. the insect toxin function seems to be ancestral in legume a1b evolution the phylogenetic analysis of the pa family strongly suggested that the original legume gene present in the ancestor of the studied papilonoideae coded for a toxin aiming at protecting seed from insects. s. japonicum is the most ancient and basal papilionoideae species for which experimental data of the presence of a1b peptides is available, initially through mass spectrometry and biological data, and now through sequence information. the cladrastis clade, to which s. japonicum belongs, is a basal legume tree group lying close to the swartzieae and ada clades, at the base of the papilionoidae, and a sister group to the so-called 50 kb-inversion group comprising most of the common papilionoidae. therefore, s. japonicum harbors the more divergent albumin i known to date. consensus legume phylogeny therefore dates the a1b family back to the ancestor of s. japonicum and all other known families harbouring such peptides, especially the genistoid clade –genus lupinus; uniprot q, with an estimated 56– my/late paleocene origin. remarkably, multiple expansions of the pa family occurred during the diversification of phaseoleae and fabeae through successive gene duplications. while, in some species the resulting paralogues have kept the ancestral insecticidal function, in medicago truncatula and in glycine max functional diversification occurred. it may be noted that the insect-toxin itself might not be mono-functional: the insecticidal homologue of pa1b in g. max, also named leginsulin, was first studied for its hormonal and signaling functions in soybean seeds. diversified a1b expression and function in medicago: no longer a seed toxin one of the most striking results of this genomic survey was that the standard situation prevailing in all other legumes studied so far, namely that albumins are toxic seed storage proteins, is not true in medicago truncatula. instead, most genes that showed seed expression are predicted not to be insect toxins, with the exception of eg41/medtr3g cluster moreover, we were unable to detect toxic peptides in m. truncatula seeds using our original homology-based genomic pcr strategy, which was leading to the cluster_ members that had lost their expression. in relation to their shift of expression out-of-seeds, medicago a1bs acquired an extremely diversified tissue distribution, dominated by root expression, which accounts for the presence of almost half of the array-detected transcripts. this emerging pattern seems an alternation of loss/gain and conservation of function, linked to expression retargeting. the current positive selection traces on two of the “functional hot spot” of the molecule is a clear indication of occurrence of discrete stepwise functional changes. however, this assumption needs to be checked experimentally by functional studies. nodulin and chromosome expansion as recent neo-functionalization bursts nodulins were defined by substractive expression methodologies as genes specifically expressed in legume rhizobial-associated nodules. early in this process, and after the identification of many transport-associated membrane proteins, some small proteins involved in signaling were identified. in this line of research, three nodulins mtn, and, were identified and these happened to be distant and short homologs of the albumins but their functions were not investigated further. our work unveiled a group of homologues to the first three described, that are all short and mostly 6-cysteine peptides. they are grouped in cluster and are basally related to a set of three more canonical sequences, two of which being readily insecticidal. the so-called “nodulin” cluster is therefore arising from apparently standard toxins that have lost their pa1a moiety and have subsequently undergone significant sequence changes albeit retaining the cysteine scaffold. traces of selection were detected at the basal branch of each of the nodulin sub-groups, namely the isolated outgroup medtr5g and the two sub-clusters 3a and 3b defined for the paml analysis. in addition to the site features discussed in the results section, it is interesting to note that the loss of the pa1a domain is correlated with significant sequence changes, including that of the canonical hydrophobic loop and a total loss of insecticidal activity, even when the canonical crc stretch is retained. in this putative “nodulin” cluster, it is likely that the structural constraints for knotted peptide folding are released, which would fit with the absence of a pa1a moiety, as this domain is now strongly suspected of serving as a chaperone for assisted co-translational folding of most canonical pa1b peptides. since knottins are not all difficult to fold, the presence of a flanking pro-peptide chaperone is probably useful in a restricted part of the conformational space explored by the a1bs from medicago truncatula. most cyclotides do not need chaperones either. insect toxins after all: one extinct and two active clusters? the final issue deals with the expression of insect toxicity in medicago truncatula tissues. we are well aware that our experimental data is extremely partial, but we are now confident that the predictive power concerning insecticidal activity from sequence information is relatively good. the first question pertains to cluster genes, for which the expression data was almost non-existent. the data was checked for two genes and it was confirmed with ngs data; genes medtr6g and medtr6g revealed no expression whatsoever. as a control, we also checked two other est-orphan genes from the “nodulin” cluster: they were shown to be expressed, and induced, in nodules. we are therefore confident that cluster is globally composed of silent genes, although one of them conserved its insecticidal activity. apart from this silent gene set, our study detected two other clusters with interesting insecticidal activities: the nodulin-related cluster and the only crc-containing member from the chromosome expansion cluster these two groups are expressed in roots and nodules, but ag shows an interesting and strong conditional expression in nodules, as well as a good expression in leaves. from an ecological point of view, expressing a very potent insecticidal molecule in a high-value nitrogen source organ would not be fortuitous, as suggested by the fact that the nodule-specific insects seem devoid of receptor binding sites, and therefore susceptibility to a1-type toxins.  when viewing our survey as a search for protein innovation within a specific taxon, one may ask how unusual are a1b in this respect? a screen of the interpro and pfam databases for taxonomic boundaries of protein domain families retrieved only three families that are restricted to the fabaceae/legumes: albumins nodule-specific glycine rich proteins, and the late nodulin family. one may add the ncr expansion to this list, which is not captured by a single protein family, and is distinct from a. many other nodule-specific proteins were subsequently recovered in other plant taxa, such as enod hormones, and even the typically nodular leghemoglobin, related to the very ancient heme-binding globin family. not surprisingly, these major protein novelties are somehow related to rhizobial symbiosis, but also concern small proteins. this may illustrate how protein modules can be derived by a process of neofunctionalization of a previously existing scaffold. in conclusion, the study of the multigenic insecticidal albumin family is of interest to the evolutionary history of legume-specific protein families, and to novel bioactive molecule discovery. the exploration of our results may end up in new biopesticide leads, such as ag for the control of a large array of insect pests. 
evolutionary rate analyses highlighted branches and sites with positive selection signatures, including two sites shown to be critical for insecticidal activity. among these, ag, showed an unexpectedly high insecticidal activity. the availability of high-quality genomic resources for several fabaceae species, among which the model species medicago truncatula, allowed for a genomic analysis of this protein family aimed at i) deciphering the evolutionary history of a1b proteins and their links with a1b-nodulins that are short non-insecticidal disulfide-bonded peptides involved in root nodule signaling and ii) exploring the functional diversity of a1b for novel bioactive molecules. investigating the mtr genome revealed a remarkable expansion, mainly through tandem duplications, of albumin genes, retaining nearly all of the same canonical structure at both gene and protein levels. phylogenetic analysis revealed that the ancestral molecule was most probably insecticidal giving rise to, among others, a1b-nodulins. legumesinsecticidal proteininsect-plant interactioncystine-knot peptidesmultigenic protein family evolutioncnrsl / aufkarakianr investissement d'aveniranr-10-binf-01-01issue-copyright-statement© the author 2016
 albumin 1b peptides are small disulfide-knotted insecticidal peptides produced by fabaceae. to date, their diversity among this plant family has been essentially investigated through biochemical and pcr-based approaches. the availability of high-quality genomic resources for several fabaceae species, among which the model species medicago truncatula, allowed for a genomic analysis of this protein family aimed at i) deciphering the evolutionary history of a1b proteins and their links with a1b-nodulins that are short non-insecticidal disulfide-bonded peptides involved in root nodule signaling and ii) exploring the functional diversity of a1b for novel bioactive molecules.  investigating the mtr genome revealed a remarkable expansion, mainly through tandem duplications, of albumin genes, retaining nearly all of the same canonical structure at both gene and protein levels. phylogenetic analysis revealed that the ancestral molecule was most probably insecticidal giving rise to, among others, a1b-nodulins. expression meta-analysis revealed that many a1b coding genes are silent and a wide tissue distribution of the a transcripts/peptides within plant organs. evolutionary rate analyses highlighted branches and sites with positive selection signatures, including two sites shown to be critical for insecticidal activity. seven peptides were chemically synthesized and folded in vitro, then assayed for their biological activity. among these, ag, showed an unexpectedly high insecticidal activity. the study highlights the unique burst of diversity of a peptides within the medicago genus compared to the other taxa for which full-genomes are available: no a member in lotus, or in red clover to date, while only a few are present in chick pea, soybean or pigeon pea genomes.  the expansion of the a family in the medicago genus is reminiscent of the situation described for another disulfide-rich peptide family, the “nodule-specific cysteine-rich”, discovered within the same species. the oldest insecticidal a1b toxin was described from the sophorae, dating the birth of this seed-defense function to more than million years, and making this model of plant/insect toxin/receptor one of the oldest known. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords legumesinsecticidal proteininsect-plant interactioncystine-knot peptidesmultigenic protein family evolutioncnrsl / aufkarakianr investissement d'aveniranr-10-binf-01-01issue-copyright-statement© the author 2016 