New Phytologist - 2013 - Shahzad - Plant Defensin Type 1 PDF1 Protein Promiscuity and Expression Variation Within The

Research
Plant Defensin type 1 (PDF1): protein promiscuity and

expression variation within the Arabidopsis genus shed
light on zinc tolerance acquisition in Arabidopsis halleri
Zaigham Shahzad1, Vincent Ranwez2, Cecile Fizames1, Laurence Marques1, Benedicte Le Martret1,
Julien Alassimone1, Cecile Gode3, Eric Lacombe1, Teddy Castillo1, Pierre Saumitou-Laprade3,
Pierre Berthomieu1* and Francßoise Gosti1*
1
Biochimie et Physiologie Moleculaire des Plantes, Unite Mixte de Recherche Montpellier, SupAgro/CNRS/INRA/Universite Montpellier II, 2 Place Viala, F-34060, Montpellier Cedex 1,
France; 2Montpellier SupAgro, UMR AGAP, F-34060, Montpellier, France; 3Laboratoire de Genetique et Evolution des Populations Vegetales, UMR CNRS 8016, Universite des Sciences et
Technologies de Lille, Lille1, F-59655, Villeneuve d’Ascq Cedex, France
Summary
Author for correspondence: Plant defensins are recognized for their antifungal properties. However, a few type 1 defen-
Francßoise Gosti sins (PDF1s) were identified for their cellular zinc (Zn) tolerance properties after a study of the
Tel: +33 499 613 153
metal extremophile Arabidopsis halleri. In order to investigate whether different paralogues
Email: gosti@supagro.inra.fr
would display specialized functions, the A. halleri PDF1 family was characterized at the func-
Received: 18 March 2013 tional and genomic levels.
Accepted: 28 May 2013
Eleven PDF1s were isolated from A. halleri. Their ability to provide Zn tolerance in yeast
cells, their activity against Fusarium oxysporum f. sp. melonii, and their level of expression
New Phytologist (2013) 200: 820–833 in planta were compared with those of the seven A. thaliana PDF1s. The genomic organization
doi: 10.1111/nph.12396 of the PDF1 family was comparatively analysed within the Arabidopsis genus.
AhPDF1s and AtPDF1s were able to confer Zn tolerance and AhPDF1s also displayed anti-
Key words: Arabidopsis genus, Arabidopsis fungal activity. PDF1 transcripts were constitutively more abundant in A. halleri than in
halleri, biotic and abiotic stresses, compara- A. thaliana. Within the Arabidopsis genus, the PDF1 family is evolutionarily dynamic, in terms
tive genomics, gene duplication, orthologues, of gain and loss of gene copy.
paralogues, Plant Defensin type 1, zinc (Zn) Arabidopsis halleri PDF1s display no superior abilities to provide Zn tolerance. A constitu-
tolerance.
tive increase in AhPDF1 transcript accumulation is proposed to be an evolutionary innovation
co-opting the promiscuous PDF1 protein for its contribution to Zn tolerance in A. halleri.
in different plant species (Mergaert et al., 2003; Graham et al.,

Introduction
2004; Silverstein et al., 2005, 2007). Concomitant studies clearly
Defensins represent a large class of small peptides that are widely indicate that defensins are expressed in all plant tissues and that
distributed in Animalia and Plantae kingdoms. They are mem- they are involved in a wide range of numerous biological activities
bers of the antimicrobial peptide (AMP) superfamily (Thomma and physiological processes in response to different biotic and
et al., 2002; Ganz, 2003; Brown & Hancock, 2006), and present abiotic stresses. All of these characteristics have been largely docu-
the archetypal c-core signature motif of these membrane-active mented in recent reviews (Lay & Anderson, 2005; Wong et al.,
host defence polypeptides, which could be traced back to 2007; Carvalho & Gomes, 2009, 2011; De Coninck et al., 2013;
prokaryotic origins (Yount & Yeaman, 2004, 2006; Yeaman & Van der Weerden & Anderson, 2013). These data obtained in
Yount, 2007). Plant defensins, in particular, are characterized by different organisms locate defensins at the crossroads between
a cysteine-stabilized a-helix b-sheet three-dimensional structure biotic and abiotic plant responses, the outcome of which will ulti-
(CSab motif), also conserved over different species (Zhu et al., mately allow plants to adapt to environments that are challenging
2005). Early studies recognized plant defensins for their antifun- for their survival (Fujita et al., 2006; Atkinson & Urwin, 2012).
gal activity and abundant expression in seeds; hence they were Despite this increased knowledge and their functional impor-
considered to protect germination and seedling growth from soil- tance, defensins are still puzzling peptides. The mode of action
borne pathogens (Terras et al., 1995). Later on, a cumulative regarding defensin antifungal and antimicrobial activities remains
number of defensins and defensin-like proteins were discovered imprecise, with each studied defensin having specific characteris-
tics (Sagaram et al., 2012; Thevissen et al., 2012; De Coninck
*These authors contributed equally to this work. et al., 2013). The link between the different defensin activities is
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New
Phytologist Research 821
often established on the basis of sequence congruence and few orthologues in A. thaliana species is their higher constitutive tran-
studies show that the same defensin molecule supports different script accumulation. We propose that this evolutionary innova-
activities. This is an important point to address, because it could tion occurred independently of gene amplification in the
well be that different members of these large gene families ulti- A. halleri Zn-tolerant species and provided a means to enhance,
mately support different defensin activities, as gene duplications in this lineage, a PDF1 function that is shared among species of
are postulated to provide raw genetic material on which adaptive the Arabidopsis genus.
modification can occur (Ohno, 1970).
In order to gain insight into the functional diversity of these
Materials and Methods
bewildering peptides, the study of Plant Defensin type 1 genes
(PDF1s) offers a particularly interesting entry point. The PDF1
BAC clone screening, sequencing and annotation
multigenic family is well described in Arabidopsis thaliana and
includes seven members (Thomma et al., 2002), out of which A bacterial artificial chromosome (BAC) library constructed from
several encoded proteins have been purified and characterized for a single A. halleri plant (Lacombe et al., 2008) was screened by
antifungal activity (Terras et al., 1993; Penninckx et al., 1996; Southern hybridization as described in Lacombe et al. (2008)
Sels et al., 2007). Interestingly, PDF1s have also been shown to with three independent PDF1 probes. Probes were made from
play a role in zinc (Zn) tolerance (Mirouze et al., 2006) after a PCR products amplified from AhPDF1 cDNA (AY961376), and
study of the extremophile species Arabidopsis halleri, which dis- from AtPDF1.4 (gene ID: 838548) and AtPDF1.5 (gene ID:
plays high capacities to tolerate and hyperaccumulate Zn and 841943) genomic DNA using the primer pairs described in Sup-
cadmium. These characters are not present in the closest relatives, porting Information, Table S1. Further, restriction fragment
Arabidopsis lyrata and A. thaliana, from which A. halleri diverged length polymorphism (RFLP) analyses of BAC clones were per-
0.27–0.44 and 5.8–13 million yr ago (MYA), respectively (Koch & formed as described in (Shahzad et al., 2010). BAC clones 5I19,
Matschinger, 2007; Schranz et al., 2007; Beilstein et al., 2010; 5F13, 10J04, 3E16 and 9G01 were fully sequenced using the
Roux et al., 2011). The role of PDF1s in Zn tolerance was shotgun method followed by a Sanger-based finishing step
documented in wildtype yeast through functional heterologous (Genoscope, Evry, France). BAC clones 1N20 and 12A7 were
screening of a cDNA library of A. halleri that revealed four sequenced using the 454 Roche multiplexing technology (Tita-
AhPDF1 cDNAs (Mirouze et al., 2006), and in planta where nium kit) without a finishing step (CNRGV, Toulouse, France).
transgenic A. thaliana lines expressing one of the AhPDF1 para- For these two BAC clones, only the AhPDF1-harbouring contigs
logues exhibited increased Zn tolerance (Mirouze et al., 2006). were subsequently considered for analyses. The AhPDF1
Regarding Zn tolerance, PDF1s are novel because, according sequences present in BAC clones 8B03, 6D13 and 2M24 were
to their Gene Ontology annotation, they have no documented determined (GATC Biotech, Konstanz, Germany) after subclon-
roles in metal transport or chelation. In order to gain an insight ing DNA fragments in the pBSKS+ vector (Statageneâ; La Jolla,
into the functional diversity of defensins in relation to their role CA, USA). Genes were predicted using the FGENESH program
in Zn tolerance in planta, we wanted to track the evolution of (Salamov & Solovyev, 2000) under the SOFTBERRY software
PDF1s in A. halleri, compared with A. thaliana, in terms of (www.softberry.com) and were annotated based on BLAST simi-
PDF1 functional capacity, gene duplication and transcript accu- larity searches (Altschul et al., 1990). The presence of metal-
mulation. A limitation of this study was the lack of description of related cis-acting motifs in AhPDF1 putative promoter sequences
the whole PDF1 family in the A. halleri species, for it could well was searched using the last release of PLACE, SOFTBERRY
be that the four AhPDF1 cDNAs identified by functional screen- and PlantCARE databases (PLACE, http://www.dna.affrc.go.jp/
ing (Mirouze et al., 2006) represented neither the whole PDF1 PLACE/; Rombauts et al., 1999; PlantCARE, http://bioinforma
family in A. halleri nor their functional diversity. As PDF1 in tics.psb.ugent.be/webtools/plantcare/html/; Lescot et al., 2002;
A. thaliana exists as a multigenic family, the identification of or- SOFTBERRY).
thologous relationships linking PDF1s was a prerequisite. Only
then, it becomes possible to investigate functional changes occur-
Genetic mapping of AhPDF1 paralogues
ring between orthologous genes (i.e. genes related by a speciation
event at their most recent point of origin) and those occurring Using 62 published markers (Willems et al., 2007; Roosens et al.,
between paralogues (i.e. genes related from duplication events 2008b; Ruggiero et al., 2008; Frerot et al., 2010; Gode et al.,
(Fitch, 1970; Koonin, 2005; Kuzniar et al., 2008; Kristensen 2012) and three new microsatellite markers defined for this study
et al., 2011)). (Table S2), genotyping was performed using genomic DNAs of
The present study provides a genomic description of the PDF1 the parents of the A. halleri 9 A. lyrata petraea BC1 population
family in A. halleri and specifies the orthologous relationships and of 199 plants from this population. The new microsatellite
among PDF1s represented in the Arabidopsis genus. This high- markers were amplified in multiplex according to Gode et al.
lights the evolutionary dynamism of this family and opens the (2012). Mapping of AhPDF1-harbouring BAC clones was per-
possibility of PDF1 gene retention in the A. halleri lineage. Func- formed by PCR-dominant, cleaved amplified polymorphic
tional studies show that AhPDF1s and AtPDF1s are promiscuous sequence (CAPS) or simple sequence length polymorphism
proteins for antifungal and Zn tolerance properties and that the (SSLP) analysis, as described in (Willems et al., 2007) using the
major characteristic differentiating A. halleri PDF1s from their specific markers generated for this study (Table S3). Genotypes
No claim to original French government works New Phytologist (2013) 200: 820–833
New Phytologist Ó 2013 New Phytologist Trust www.newphytologist.com
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822 Research Phytologist
obtained in the BC1 population, for the BAC-derived markers,

Antifungal assays
were combined with the dataset used to construct the
A. halleri 9 A. lyrata petraea linkage map, as described in For all AhPDF1s, DNA fragments encoding the C-terminal
(Shahzad et al., 2010). mature part were cloned in the pET28(a+) vector (Novagen,
Darmstadt, Germany) using the primers listed in Table S6.
AhPDF1 proteins were produced as already described (Marques
In silico identification of A. lyrata PDF1s, sequence
et al., 2009), except that the final high-performance liquid chroma-
and phylogenetic analysis
tography (HPLC) purification step was omitted. The purity and
Arabidopsis lyrata PDF1s were identified by searching the whole mass of all the proteins were assessed by sodium dodecyl sulphate
genome shotgun (wgs) nucleotide sequence database using the polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-
tblastn program (Altschul et al., 1990) and the A. thaliana (TAIR, assisted laser desorption/ionization time-of-flight (MALDI-TOF)
The Arabidopsis information resource, http://www.arabidopsis. mass spectra. The protein concentration was measured using Brad-
org/; Lamesch et al., 2012) and A. halleri (this study) deduced ford assays (Bradford, 1976). Growth inhibition assays were
PDF1 protein sequences as queries (E-value < 1 9 e 20). Seven conducted against Fusarium oxysporum f. sp. melonii (INRA
homologous PDF1s (Table S4) were hence identified from the Avignon) as described in Marques et al. (2009). The minimal
A. lyrata subsp. lyrata (taxid:81972) genome (Hu et al., 2011). All inhibitory concentration (MIC) was determined as the lowest
AlPDF1 annotations were controlled manually based on overall defensin concentration that induced 100% reduction of growth
PDF1 nucleotide sequence alignments and the position of splicing compared with the control conditions.
sites. Consequently, this revealed the presence of a premature stop
codon after the 39th amino acid for the AlPDF1.2a gene (Notes
Zn tolerance assays in yeast
S1). The localizations of those AlPDF1s within the A. lyrata
genome were obtained using EnsEMBL Plant facilities (Kersey Entire coding sequences of A. halleri and A. thaliana PDF1s were
et al., 2010, 2012). cloned between EcoRI and XhoI restriction sites of the pYX212
Multiple sequence alignments were computed using MUSCLE yeast expression vector using the overlap extension (SOEing)
v3.8.31 (Edgar, 2004) and visualized with BOXSHADE 3.21 PCR-based method (Vallejo et al., 1994) and primers listed in
(http://www.ch.embnet.org/software/BOX_form.html) run through Table S7. The constitutive triose phosphate isomerase promoter
the Mobyle Portal (MOBYLE, http://mobyle.pasteur.fr/cgi-bin/ controlled PDF1 expression. Only the second exon of AhPDF1.6
portal.py#welcome; Neron et al., 2009). Gene tree inference was was cloned in the pYX212 vector, as only this part of the gene
done using the predicted protein sequences of the 25 Arabidopsis could be identified within the corresponding BAC clone. The
PDF1 homologues together with those of the six outgroup recombinant pYX212 vectors were introduced into the BY4741
A. thaliana PDF2 paralogues (TAIR; Notes S1). Those sequences Saccharomyces cerevisiae strain (MATa, his3Δ1, leu2Δ0, met15Δ0,
were aligned using MUSCLE v3.8.31 (Edgar, 2004) with default ura3Δ0) together with the pFL38H vector, which harbours a
options. Poorly aligned regions were removed from this align- functional HIS3 gene, using the lithium acetate/single-stranded
ment using the ‘automated1’ option of trimAl v1.3 (Capella- carrier DNA/polyethylene glycol method (Gietz & Woods,
Gutierrez et al., 2009), which is specifically designed to trim 2002). For drop assays, transformed yeast cells from overnight
alignments to be used in maximum-likelihood phylogenetic anal- cultures, were washed twice with ultrapure H2O and diluted to
yses. The phylogeny of the PDF1 family was then inferred by OD600nm = 1, 0.1 and 0.01. Ten microlitres of the yeast solutions
maximum likelihood, using RAxML v7.2.8 (Stamatakis, 2006). were dropped onto selective modified yeast nitrogen base (YNB)
Inference was done starting from 10 distinct randomly chosen medium (1.7 g l 1 YNB without amino acids without ammo-
maximum parsimony trees. The WAG protein model (Whelan nium sulfate (233520, Difco), 6.4 g l 1 NH4NO3, 2% (w/v)
& Goldman, 2001), using empirical base frequencies and a dis- D-glucose, 50 mM succinic acid-KOH, pH 4.5) supplemented
crete Gamma law with four categories to model heterogeneity of with ZnSO4 at various final concentrations, 1.4 lM for the con-
evolutionary rate among sites, was chosen. Branch supports were trol condition and 25 or 27.5 mM for Zn treatments, and grown
estimated through full bootstrap analyses, as opposed to the faster at 30°C. At least three independent clones were tested for each
RAxML bootstrap approximations. Identification of synteny con- construct. A MIC assay against the BY4741 S. cerevisiae clone
servation – that is ‘preserved co-localization of genetic loci and/or used was conducted and no inhibitory action was found for val-
genes on chromosomes and/or linkage group of different species’ ues up to 20 lM for all the 11 A. halleri defensins tested as previ-
(Abrouk et al., 2010) – within PDF1 regions was manually done at ously found for the AhPDF1.1b defensin (Marques et al., 2009).
the macroscopic (c. 20–50 kb) and microscopic (c. 1–5 kb) levels
after careful visual examination of the sequence similarity between
Quantification of transcript accumulation
genomic sequences bordering the PDF1 genomic sequences
(Table S5) using blastn with default parameters (Altschul et al., The SAF2 line of A. halleri used in this study was derived from
1990). When possible, synteny was also explored using the viewer an individual plant from the metallicolous Auby population
facilities offered by TAIR and BRAD (The genetics and genomics (France) through vegetative multiplication. In vitro micropropa-
database for Brassica plants, http://brassicadb.org/brad/index.php; gation and phenotypic analyses were performed as already
Cheng et al., 2012a) servers. described (Shahzad et al., 2010). The A. thaliana (Columbia)
New Phytologist (2013) 200: 820–833 No claim to original French government works
New
plants were grown from seeds in the same sterile hydroponic BAC clones (Table 1) were arranged into 10 groups depending
medium and harvested after 2 wk. All functional analyses were on both EcoRI and BamHI RFLP profiles (data not shown).
performed from plants cultured in sterile conditions. The pres- Sequences of BAC clones representing each of the 10 groups
ence of each AhPDF1 within the SAF2 plant genome was verified identified 13 AhPDF1s (Table 1) that all contained the charac-
and their sequences were checked (data not shown). Transcripts teristic PDF two-exon genomic structure (Silverstein et al.,
were quantified from five plant replicates treated independently. 2005), except for AhPDF1.6, which was missing the first exon
Roots and shoots were harvested separately and analysed inde- and intron and which we thus considered as a probable pseudo-
pendently. RNA extraction, cDNA synthesis and real-time gene. All the remaining AhPDF1s encoded 78- to 80-amino-
reverse transcription polymerase chain reaction (RT-PCR) were acid-long predicted proteins, as already reported for plant
performed essentially as described (Shahzad et al., 2010). Specific PDF1s (Carvalho & Gomes, 2011). AhPDF1.7 contains a four-
primer pairs were designed for each different PDF1 (Table S8). nucleotide-long insertion in its coding sequence, modifying the
Actin was considered as an internal control (Shahzad et al., reading frame and leading to a truncated protein as a result of a
2010). The PCR efficiency (E) of each PDF1 specific primer pair stop codon after the 54th amino acid (Notes S1). In some cases,
was determined after the analysis of five serial 1 : 10 dilutions of two PDF1s were present on the same BAC, such as AhPDF1.1a
BAC clone DNA for AhPDF1s and plasmid DNA for AtPDF1s and AhPDF1.1b, AhPDF1.2a and AhPDF1.2c, and finally
(Table S8), as previously described (Shahzad et al., 2010). The AhPDF1.8a and AhPDF.8b, being separated by a distance of
PCR efficiency of primer pairs used for Actin was determined on c. 3.4 kb, c. 13.6 kb and > 1.8 kb, respectively. On the basis of a
the gDNA for A. halleri as well as A. thaliana (Table S8). PCRs 100% sequence identity, three of the four AhPDF1 cDNAs pre-
were performed on cDNA samples in triplicate and PDF1s vs viously identified by yeast functional screening (Mirouze et al.,
Actin relative expression levels (RELs) were determined as previ- 2006) were also identified in this study (Table 1). No identity
ously described (Shahzad et al., 2010) and are listed in Table S9. was found for the fourth cDNA (AY961377). Using specific
Difference in transcript accumulation upon Zn addition was sta- primer pairs, no PCR amplicon could be generated for this gene
tistically validated by t-test at a 0.05 confidence threshold. when assayed on 34 A. halleri plants from the Auby population
(data not shown). We considered that the AY961377 PDF1 cor-
responded to a very rare PDF1 allele, or to a mutated version
Results
generated during the cDNA library construction. Hence, this
gene was removed from further analyses.
Identification of PDF1s in A. halleri and A. lyrata
Seven PDF1s were identified in the A. lyrata genome through
PDF1 genes present in the A. halleri genome (AhPDF1) were an in silico analysis (AlPDF1 and Table S4). In summary, 13
identified after a BAC library screening. A total of 26 identified AhPDF1 and seven AlPDF1 have been identified which reflect
Table 1 Identification of PDF1 paralogues in Arabidopsis halleri
Number of BAC clones

Locus containing PDF1 genes identified harbouring the AhPDF1 Name of the representative Accession
PDF1 genes in A. halleri paralogue BAC clone number
Locus 1 AhPDF1.1a1 4 8B03 HF545648

AhPDF1.1b2
Locus 2 AhPDF1.2a 4 1N20 HF545651
AhPDF1.2c
Locus 3 AhPDF1.2b3 2 5I19 HE601752
Locus 4 AhPDF1.4 2 6D13 HF545647
Locus 5 AhPDF1.54 1 5F135 HE601748
AhPDF1.5 2 2M24 HF545649
Locus 6 AhPDF1.6 2 10J04 HE601750
Locus 7 AhPDF1.7 2 3E16 HE601749
Locus 8 AhPDF1.8a6 3 12A077 HF545646
AhPDF1.8b8 HF563610
AhPDF1.8a 4 9G01 HE601751
–
Total 8 11 26 10
1
BAC, bacterial artificial chromosome. The cDNA of AhPDR1.1a is also known as AY961379 (Mirouze et al., 2006).
2
The cDNA of AhPDR1.1b is also known as AY961376 (Mirouze et al., 2006).
3
The cDNA of AhPDR1.2b is also known as AY961378 (Mirouze et al., 2006).
4
The AhPDR1.5 isolated from BAC clones 5F13 and 2Ms4 correspond to two allelic forms of the same gene.
5
The BAC clones 5F13 and 2M24 are allelic forms of the same region.
6
The AhPDF1.8a isolated from either BAC clones 12A07 or 9G01 correspond to two allelic forms of the same gene.
7
The BAC clones 12A07 and 9G01 are allelic forms of the same region.
8
The gene AhPDF1.8b was identified only from BAC clone 9G01.
New
the PDF1 genetic diversity described in the A. thaliana genome mapped at locus 5 as well as those mapped at locus 8 indeed corre-
(AtPDF1, TAIR). sponded to distinct allelic forms. Interestingly, the BAC clone
12A07 (mapped at locus 8) harboured two PDF1 copies (Ah-
PDF1.8a and AhPDF1.8b) while 9G01 (locus 8 also) harboured
Genetic mapping of PDF1 in A. halleri and orthologous
only one (AhPDF1.8a). Using a copy-specific marker,
relationships within the Arabidopsis genus
AhPDF1.8b was detected in only 59 of the 83 tested plants, indi-
The 10 representative AhPDF1-harbouring BAC clones were cating that this paralogue was thus probably unfixed in the
mapped at eight loci on the A. halleri 9 A. lyrata linkage map A. halleri Auby population.
(Fig. 1, Table 1). BAC clones 5F13 and 2M24 on one side, and Knowledge of the conserved genome macrosynteny existing
12A07 and 9G01 on another side, were colocalized at loci 5 and within the Arabidopsis genus (Kuittinen et al., 2004; Clauss &
8, respectively (Fig. 1). Interestingly, the AhPDF1s harboured by Koch, 2006; Schranz et al., 2006, 2007; Willems et al., 2007;
these BAC couples had high nucleotide sequence identity (99 Roosens et al., 2008a,b) permitted the association of ungapped
and 97%, respectively), even when considering noncoding regions of A. thaliana and A. lyrata genomes that showed con-
PDF1-flanking regions (data not shown). This suggested that the served local macrosynteny with A. halleri BAC sequences contain-
corresponding colocalized AhPDF1s could be allelic rather than ing PDF1s (Table S5, Fig. S1). When PDF1s were found within
closely linked loci. Using two different allele-specific markers these regions, they were grouped as orthologous. Then, when two
designed for every PDF1 gene present at loci 5 and 8 (Table S10), PDF1s paralogues were present at orthologous loci, the one-to-
the distribution of these genes was examined in a set of individual one orthologous relationships were established between the cop-
plants of the Auby population. A balanced distribution of these ies showing the strongest micro-synteny conservation (principle
PDF1 markers was observed (Table S11), indicating that PDF1s of parsimony) around and within the gene sequence, (Fig. S1).
LG1 LG2 LG4 LG7 LG8

0.0 1-00240 0.0 1-22940 0.0 2-09133 0.0 4-17925 0.0 5-17907(AhPDF1.2a &.2b)
2.1 1-01019 0.8 Ah75
3.7 1-01498 4.3 2-09533 4.2 4-17202 3.2 5-17648
6.0 2-09704 5.6 4-17540
7.1 1-22160(AhPDF1.8a &.8b) 7.1 2-09863
8.4 1-03095
11.1 4-16390
12.1 1-04266 11.9 4-15994
4-15470 12.9 5-19533
15.1 1-21141 12.6
15,8 1-04488 14.4 4-14965
1-04516 16.3 1-20637(AhPDF1.6) 16.6 2-10529
16.5
17.6 1-20517(AhPDF1.5) 19.9 2-11096
20.6 1-05768 19.7 1-24380 2-11087(AhPDF1.2b) 20.3 4-13275
22.0
23.0 1-06781(AhPDF1.4) 22.3 2-11066
24.4 1-24870 24.4 4-11410
25.4 4-12451 25.4 5-20841
26.5 1-08062
29.0 4-11409
28.9 1-25548
29.1 4-11384 29.9 5-21355
1-25687 31.4 2-11702
32.2
38.0 1-10858 38.6 4-09207

41.2 5-22729
46.1 1-26613
48.1 2-13171 47.1 5-23764
49.4 2-14018
51.3 (AhPDF1.7)
52.8 1-28728(AhPDF1.1a &.1b) 53.1 2-15786
54.1 5-24702
54.2 2-15997
55.4 5-26879
56.3 2-16299
58.6 1-28980
60.2 2-16779 59.5 Ah49
61.4 1-16487
63.3 2-17890 62.7 5-15022
64.2 1-17268 63.8 2-17894
67.6 2-18989
68.4 2-19245(AhMTP1A)
72.5 2-19245(AhMTP1C) 72.8 2-19531

75.3 5-16149
A. thal. Chr. 1 A. thal. Chr. 4 Unknownposition in A. thal.

A. thal. Chr. 2 A. thal. Chr. 5
Fig. 1 Genetic mapping of AhPDF1s on the Arabidopsis halleri 9 Arabidopsis lyrata petraea BC1 linkage map. The mapping was performed from an
analysis of 199 plants and the four parents of an A. halleri 9 A. lyrata petraea BC1 population using Joinmap 3.0. Only linkage groups (LGs) 1, 2, 4, 7 and
8 are shown. The AhPDF1s are indicated in red letters. The markers presented on the map are described in Supporting Information Table S2. They are
named according to the gene or bacterial artificial chromosome (BAC) to which the marker is mapped and are indicated after their approximate position in
the Arabidopsis thaliana genome on the right side of the LG bar (chromosome number-position in kb). The scale on the left of each LG represents
distances in cM. The bars representing the linkage groups are coloured in reference to the conserved synteny between the A. halleri and A. thaliana
genomes (Schranz et al., 2006). Regions showing no conserved synteny with A. thaliana are indicated in white.
New
From these analyses, AhPDF1s and AlPDF1s genes were named A. thaliana, grouped PDF1s together, as expected (Fig. 3b). This
in order to reflect the one-to-one PDF1 orthologous relationships confirmed that the collected sequences indeed encoded PDF1s
established within the Arabidopsis genus and in compliance with and that there have been no obvious annotation errors so far.
the PDF1 nomenclature already defined in A. thaliana (Fig. 2). Within the strongly supported clade of PDF1s (≥ 95% bootstrap
A consistent picture could thus be established in which the support), the evolutionary history was less clear. Indeed, only
A. thaliana, A. halleri and A. lyrata PDF1s are distributed on the four of the 23 clades were well supported and gathered PDF1s
basis of a one-to-one orthologous relationship over eight syntenic showing orthologous relationships within loci 4, 5, 6 and 7.
loci (Fig. 2). As each of those synthenic regions was present in the Owing to the poor resolution of the obtained PDF1 phylogeny,
three Arabidopsis spp., the most parsimonious scenario is that it was not possible to rely on reconciliation methods (Doyon
these eight regions were also present in the genome of the last et al., 2011) to draw up an evolutionary scenario on PDF1 gains
common ancestor of the Arabidopsis genus. Overall, except for and losses between species belonging to the Arabidopsis genus. We
loci 4 and 5, PDF1s were not similarly distributed over these could, however, infer the most parsimonious scenario for those
eight ancestral regions. This prompted us to investigate the evolu- events on the basis of orthologous relationships (Fig. 2). For
tionary dynamism of the PDF1 family. example, since PDF1s present at locus 8 are specific to A. halleri,
the most parsimonious scenario is that these PDF1s were gained
in this lineage. Moreover, as representatives of PDF1.2c and
Evolutionary history of the PDF1 family in the Arabidopsis
PDF1.2b are specifically missing in A. lyrata, these PDF1s were
genus
probably lost in this lineage. However, PDF1.3, which is only
This study gathered a dataset of 25 PDF1 coding sequences and a present in A. thaliana, could have been gained in this lineage or
corresponding dataset of their predicted amino acid translations lost in the last common ancestor of A. halleri and A. lyrata. Simi-
(Notes S1). Multiple alignment of the latter pinpointed 15 larly, since PDF1.1b, PDF1.6 and PDF1.7 were specifically
strictly conserved sites (Fig. 3a), eight of which were cysteine resi- absent in A. thaliana, they could have been lost in this lineage or
dues involved in the four disulphide bonds characterizing the gained in the last common ancestor of A. halleri and A. lyrata. In
CSab superfamily to which PDF1s belong (Zhu et al., 2005). order to distinguish between these two equally parsimonious sce-
The phylogeny of these 25 PDF1s and six PDF2s from narios, syntenic conservation in outgroup species was considered.
Hence, we took advantage of the complete sequenced genome
information available for the closest relatives, that is, Thellungiella
β parvula (Dassanayake et al., 2011; Cheng et al., 2012a,b) and
α Brassica rapa (Wang et al., 2011) to upscale our syntenic analysis
(Cheng et al., 2012a,b). Within locus 1, two PDF1 orthologous
were identified in T. parvula and B. rapa (Fig. S3 and data not
shown), thus making the loss of AtPDF1.1b in the A. thaliana
lineage the most parsimonious scenario. A clear synteny conserva-
tion was also identified at locus 4 where a single PDF1 was identi-
fied in both outgroup species (Fig. S2, and data not shown). For
the remaining PDF1 ancestral regions, no clear synteny conserva-
tion was identified between genomes of the Arabidopsis genus and
the T. parvula or B. rapa outgroup species (data not shown).
Hence, the evolutionary scenario underlying the gain or loss of
PDF1s in these regions within the Arabidopsis genus (loci 2, 3, 5,
6 and 7, see Fig. 5) remains unresolved.
In summary, this analysis led us to consider that the structure
of PDF1 family orthologous is evolutionarily dynamic within the
Arabidopsis genus and that PDF1s present at loci 1 and 4 have
likely been conserved throughout the Brassicaceae family suggest-
Fig. 2 Comparative genomic organization and orthologous relationship ing that PDF1s present at these loci could be founder from which
among PDF1s in the Arabidopsis genus. Within the Arabidopsis genus, the other PDF1 copies could have been duplicated.
25 PDF1 genes were arranged within each lane according to their one-to-
one orthologous relationship. The PDF1 grey shadowing indicates genes
likely to be nonfunctional as follows: the AlPDF1.2a gene had to be AhPDF1s are promiscuous, being antifungal and able
reannotated and the resulting predicted protein had a stop codon at the
to provide Zn tolerance
39th amino acid (Supporting Information Notes S1), the AhPDF1.6 gene
was considered to a pseudogene as the identified sequence lacked the first PDF1 proteins are structured with an N-terminal secretory signal
exon and subsequent intron parts, while the AhPDF1.7 gene had a four-
peptide that is cleaved to release the mature functional part
nucleotide insertion, resulting in the translation of a premature stop codon
after the 54th amino acid (Notes S1). The species tree is shown on the top. (Bendtsen et al., 2004) used to perform in vitro antifungal assays.
The a and b whole-genome duplications are indicated on the branches of This cleavage site was clearly located within all AhPDF1 pre-
the tree adapted from Beilstein et al. (2010). dicted proteins except for AhPDF1.4 and AhPDF1.5. In that
New
(a) γ core motif
Fig. 3 Phylogenetic analysis of the PDF1

family in the Arabidopsis genus. (a)
Comparative sequence alignment of
predicted amino acid sequences encoded by
25 PDF1 genes from Arabidopsis halleri,
Arabidopsis lyrata and Arabidopsis thaliana
species. Predicted protein sequences (Notes
S1) were aligned with MUSCLE3.8.31
software (Edgar, 2004) and visualized with
BOXSHADE 3.21 software. Names of the
PDF1 amino acid sequences are shadowed in
dark orange, light orange and white
according to the species of origin, that is,
A. halleri, A. lyrata and A. thaliana,
respectively. Stars indicate that the predicted
sequence of AlPDF1.2a and AhPDF1.7 genes
(b) was corrected to be aligned without taking
the premature stop codon into account (as
listed in Supporting Information Notes S1).
The identical (threshold: 0.55) and similar
residues are shadowed in blue and green
colours, respectively. Amino acids identical in
all PDF1s are shadowed in red and are copied
below the alignment under the identity lane.
The identity was refined by hand in order to
take into account the first two MA
(methionine, alanine) amino acids that are
missing in AhPDF1.6 and the fourth cysteine
residue which is conserved in all proteins
except AlPDF1.2a, which correspond here to
a reconstructed protein as mentioned earlier.
Based on the RsAFP1 structure (Fant et al.,
1998), the predicted cleavage site between
the N-terminal signal peptide and the mature
peptide is indicated by an arrow, while the
secondary structure elements (a-helix,
cylinder; b-strand, arrow) as well as the
disulphide bridge connectivity are shown
above and below the alignment, respectively.
(b) Phylogeny of the 25 proteins of the PDF1
family using six PDF2 proteins as outgroup.
The bootstrap value is indicated for each
clade. Each of the four most reliable clades of
the PDF1 family (whose bootstrap support is
≥ 95%) is indicated by a red rectangle.
case, the first amino acid of the mature peptide had to be deter- AtPDF1.8b mature part, this protein being the most divergent as
mined considering the alignment of the predicted protein compared to other PDF1s (Fig. 3a).
sequences (Fig. 3a). Each of the 11 AhPDF1 mature recombinant The comparative ability of AtPDF1s and AtPDF1s to induce
proteins was assayed in vitro for their ability to inhibit the growth cellular Zn tolerance was analysed upon expression in yeast. Thir-
of Fusarium oxysporum f. sp. melonii. Seven of the AhPDF1s teen of the 18 PDF1s similarly induced the highest Zn tolerance
showed the same 2.5 lM PDF1 minimal inhibitory concentra- activity (rectangle in Fig. 4). Lower Zn tolerance ability was
tion (MIC) (Table 2). With a MIC of 5 lM, AhPDF1.4 and Ah- observed for AhPDF1.4, AhPDF1.5 and for AtPDF1.5 (rounded
PDF1.6 presented lower antifungal activity. AhPDF1.8b and rectangle in Fig. 4). Meanwhile, AhPDF1.6 and AhPDF1.7 did
AhPDF1.7 were by far less active, with a MIC of 5–10 lM and not provide Zn tolerance (encircled in Fig. 4), which was not sur-
> 10 lM, respectively (Table 2). These latter results were likely prising since these genes encode a truncated version of PDF1
due: to the existence of a premature stop codon in the AhPDF1.7 missing the first exon (AhPDF1.6) – the translation of which has
coding sequence that removes half of the mature protein (Notes been shown to be mandatory for providing Zn tolerance (Oomen
S1); and to the numerous amino acid changes occurring in the et al., 2011) – or having a premature STOP codon (AhPDF1.7)
New
Table 2 Antifungal activity of AhPDF1s as tested against the fungus ZnSO4

Fusarium oxysporum f. sp. melonii Control 25 mM 27.5 mM
1 EV
Name of the PDF1 tested MIC (lM)
AhPDF1.2a
AhPDF1.1a 2.5
AhPDF1.2c
AhPDF1.1b 2.5
AhPDF1.2a 2.5 AhPDF1.2b
AhPDF1.2c 2.5
AhPDF1.1b
AhPDF1.2b 2.5
AhPDF1.5 2.5 AhPDF1.1a
AhPDF1.8a 2.5
AhPDF1.4 5.0 AhPDF1.8a
AhPDF1.6 5.0 AhPDF1.8b
AhPDF1.8b 10.0
AhPDF1.7 > 10.0 AhPDF1.6
1
Calculated minimal inhibitory concentrations (MICs) correspond to the AhPDF1.7
lowest protein concentrations causing 100% fungal growth inhibition. AhPDF1.5
AhPDF1.4
AtPDF1.1
(Notes S1). Interestingly, we noticed that AtPDF1.4 had a
slightly higher Zn tolerance than AhPDF1.4 although these two AtPDF1.2a
proteins differed at only two amino-acid positions. The Ala28 AtPDF1.2b
and Ser54 of AhPDF1.4 are changed to Gly and Arg in At-
AtPDF1.2c
PDF1.4, respectively. The Ala28Gly and Ser54Arg double substi-
tution was necessary to increase the degree of Zn tolerance AtPDF1.3
provided by AhPDF1.4 to that of AtPDF1.4 (Fig. S3). So this AtPDF1.4
study did not permit us to identify a specific motif or domain
AtPDF1.5
associated with Zn tolerance. 1 0.1 0.01 1 0.1 0.01 1 0.1 0.01
In summary, the overall AhPDF1 family showed antifungal OD600 nm
properties, and PDF1 family members coming from A. thaliana Fig. 4 In vitro functional characterization of the ability of Arabidopsis
or A. halleri showed similar in vitro cellular Zn-tolerance proper- halleri and Arabidopsis thaliana PDF1s to confer cellular zinc (Zn)
tolerance. Serial dilutions of the Saccharomyces cerevisiae BY4741 strain
ties. In most cases, PDF1 antifungal activity and Zn tolerance
expressing the pYX212 empty vector (EV) or a pYX212 harboring one of
were associated, thus giving promiscuous characteristics to this the A. halleri or A. thaliana PDF1s were spotted on medium supplemented
protein. Note, however, that AhPDF1.5 had standard antifungal with 1.4 lM (control), 25 mM or 27.5 mM ZnSO4, as indicated above the
activity but was not as efficient as other PDF1s for Zn tolerance. panels. Each spot was made with 10 ll of a yeast culture diluted at the
Conversely, AhPDF1.8b had lower antifungal activity but stan- OD600nm mentioned below the drops. Pictures were taken on day 2 for
control and day 11 for Zn treatments; they are representative of the three
dard Zn tolerance.
experiments, which have been performed with three independent yeast
transformants. Rectangles, ovals and rectangles with rounded corners
indicate the names of the PDF1 proteins presenting the standard,
PDF1 genes are constitutively more highly expressed
decreased and no-Zn-tolerance capacities, respectively.
in A. halleri than in A. thaliana
Expression of PDF1s was measured at the transcript level in roots
and shoots of A. halleri and A. thaliana plants grown in axenic cDNAs from the screening of a leaf cDNA library (see Table 1).
hydroponic conditions. Within each species, the transcript abun- Some AhPDF1s, however, displayed a different pattern:
dances varied by two to three orders of magnitude between differ- AhPDF1.4, AhPDF1.5 and AhPDF1.7 transcripts were accumu-
ent PDF1 paralogues (Fig. 5, Table S9). For a given PDF1, lated at similar abundances in roots and shoots, whereas
transcript abundances were, on average, higher in shoots than in AhPDF1.8a transcripts were c. 30 times more highly accumulated
roots. This was particularly obvious in A. thaliana, in which no in roots than in shoots. In response to increasing Zn concentra-
PDF1 transcripts could be detected in roots. This was also tions in the culture medium, the relative abundances of AhPDF1
observed in A. halleri where PDF1 transcripts were, on average, transcripts remained unchanged, apart from a significant increase
300 times more abundant in shoots than in roots (Table S9). In for AhPDF1.1a (92.5) and AhPDF1.2b (94), which was
A. halleri, the greatest organ differences were observed for PDF1 observed in shoots and roots. Overall, and most importantly,
transcripts located at locus 1 (AhPDF1.1b and AhPDF1.1a) and transcript analysis revealed that PDF1 transcripts were c. 1000
locus 3 (AhPDF1.2b). Transcripts of these PDF1s were the most times more highly accumulated in A. halleri than in A. thaliana
highly accumulated in shoots. Note that this observation is in (Fig. 5 and Table S9). Searches performed in the 2-kb-long
agreement with previous functional findings (Mirouze et al., regions upstream of the AhPDF1 coding sequences for putative
2006), which led to the cloning of the three corresponding metal cis-responsive elements or other particular motives, which
New
Shoots Roots
10
AhPDF1.1b
1
AhPDF1.2b*
AhPDF1.4 AhPDF1.7
AhPDF1.7
AhPDF1.1a*
Transcript level relative to actin 0.1 AhPDF1.4
AhPDF1.5 AhPDF1.5
AhPDF1.8a
0.01
AhPDF1.2a AhPDF1.1b
AhPDF1.8a AhPDF1.2b*
AtPDF1.4
0.001
AhPDF1.1a*
AtPDF1.5
0.0001
AtPDF1.2a
0.000 01
AtPDF1.2b
0.000 001
10 100 1000 10 100 1000
Zinc concentration in the media (μM)
Fig. 5 Accumulation of PDF1 transcripts in Arabidopsis halleri and Arabidopsis thaliana plants. Shoots and roots were collected from individual plants of
the A. halleri SAF2 genotype derived from the Auby accession that had been exposed to 10 (control), 100 or 1000 lM ZnSO4 for 4 d. Quantitative PCR
was performed using the gene copy specific primer pairs listed in Supporting Information Table S8. A. halleri and A. thaliana PDF1 transcript abundances
are expressed relative to Actin. Five biological replicates were analysed using three technical Q-PCR repeats. Error bars correspond to confidence intervals
at the 0.05 threshold. Stars indicate PDF1s for which a significant difference in transcript accumulation was observed upon zinc (Zn) addition in the media.
Regarding the A. halleri species, transcripts of AhPDF1.2c, AhPDF1.6 and AhPDF1.8b genes could not be detected in either shoots or roots. For the
A. thaliana species, transcripts of the AtPDF1.1, AtPDF1.2c and AtPDF1.3 genes could not be detected in shoots, and none of the AtPDF1 transcripts could
be detected in roots.
could potentially be correlated with the level of expression, were evolutionary walk towards Zn tolerance, the present study
unsuccessful (data not shown). focused on the roles played by PDF1s in antifungal activity and
cellular Zn tolerance. This is one of the few studies in which
multiple functional assays are conducted on the same defensin
Discussion
molecule. Indeed, in most cases, the diversity of defensin func-
Besides their involvement in plant responses to pathogen attack, tions is inferred indirectly from sequence similarity to a protein
the roles played by defensins in response to multiple abiotic mask typical of the large multigenic defensin family. The find-
stresses are diverse (Carvalho & Gomes, 2011). These include ings of the functional assays presented here showed that none of
the role played by PDF1s in providing cellular Zn tolerance, as the 11 identified A. halleri PDF1s provided a specific increase in
revealed through a study of Zn-tolerant and Zn-hyperaccumu- Zn tolerance as compared with the seven PDF1s originating
lating A. halleri species (Mirouze et al., 2006). Protein promiscu- from A. thaliana nontolerant species. In addition, the AhPDF1
ity is the phenomenon in which multiple functions may be antifungal activity and Zn-tolerance properties were globally
associated with a single peptide or protein structure (Nobeli correlated across the members of the AhPDF1 family. No obvi-
et al., 2009). It was initially proposed for defensins as an evolu- ous amino acid stretch has been found to be specifically associ-
tionary concept by Franco (2011), based on the idea that if pro- ated with one of the activities studied. So, from our results, we
tein and peptides possess a structure directly related to a single cannot correlate a function to a structure. Regarding the in
function it would hamper their ability to adapt and develop new planta data, we know that the overexpression of AhPDF1.1b
functions. This study describes the PDF1 family as represented provides A. thaliana with Zn tolerance (Mirouze et al., 2006);
by 25 members encoding fairly similar proteins and forming a and that the overexpression of AtPDF1.1 leads to a reduction in
quite homogeneous multigenic family within the Arabidopsis pathogen symptoms (De Coninck et al., 2010). Confronting the
genus (Fig. 3a). So far, PDF1 functional studies focused mainly in vitro assay and in planta phenotypic effects suggests that a sin-
on A. thaliana where three PDF1s (out of seven) were studied gle PDF1 molecule is likely responsible for both antifungal
and showed in vitro activity against different fungi (Terras et al., activity and cellular Zn tolerance. These two functions could be
1993; Penninckx et al., 1996; Sels et al., 2007, 2008). Having in assumed, for example, in different physiological or protein con-
mind that PDF1 promiscuity could contribute to A. halleri’s centration conditions as proposed in the case of pure protein
New
promiscuity (Franco, 2011). It should be noted that no informa- (IRT3), Ferric Reductase Deficient 3 (FRD3) and Zinc Iron
tion is currently available to enable us to determine whether the Permease 10 (ZIP10) (Talke et al., 2006). In other cases, such as
two PDF1 properties that are considered here (i.e. antifungal Heavy Metal ATPase 4 and MTP1, high constitutive transcript
activity as a response to biotic stress and Zn-tolerance capacity accumulation is associated with gene duplication in the A. halleri
as related to abiotic stress) originate from the same mode of lineage, as compared with a single orthologous gene represented
action. However, our current research is now focused on the in A. thaliana (Hanikenne et al., 2008; Shahzad et al., 2010). The
search for common properties that could explain their dual role. orthologous relationships documented here between PDF1s of
From sequences analyses, very few amino acids appear to be multigenic families occurring in the three species of the
common to all the PDF1s. Among these are the eight cysteines Arabidopsis genus highlight a new configuration in comparison to
supposedly involved in four disulphide bonds. One of our work- those mentioned earlier. Indeed, the high PDF1 transcript dereg-
ing hypotheses is that the common properties of defensins ulation is an evolutionary innovation, which most probably
would rely on these cysteines. As far as Zn tolerance is con- might have occurred in the A. halleri lineage independently on
cerned, we hypothesize that PDF1s could chelate Zn in some each PDF1 locus and on each harboured gene, thus contributing
special conditions in which the protein is not fully oxidized, as to the A. halleri adaptive walk towards Zn tolerance (Orr, 2005)
discussed previously (Marques & Oomen, 2011). The results of independently of PDF1 gene amplification in this lineage.
the exhaustive functional analysis presented here will be useful Indeed, although there are 11 PDF1s in A. Halleri, compared
for guiding future research on the PDF1 mode of action so as to with seven in A. thaliana or A. lyrata, only AhPDF1.8a and
elucidate how these two functional properties closely dovetail for AhPDF1.8b are specific to the A. halleri lineage (Fig. 2). Hereaf-
the benefit of plant responses to both biotic and abiotic stresses. ter, it is therefore unlikely that the 11 AhPDF1s are the result of
As there was no obvious functional discrimination in PDF1s specific gene expansion in A. halleri. Rather, the PDF1 family
originating from A. halleri as compared with A. thaliana, evolu- should be considered as evolutionarily dynamic. The orthologous
tionary processes undergone by PDF1s were investigated at the structure of the family might be a footprint of genome rearrange-
genomic level. Interspecies transcriptomic experiments per- ment in A. lyrata or might reflect genome reduction, which is
formed in comparison to A. thaliana revealed a global high con- known to have occurred in A. thaliana (Johnston et al., 2005;
stitutive accumulation of PDF1 transcripts in A. halleri (Talke Proost et al., 2011). The latter is more likely, as A. halleri and
et al., 2006). The analyses reported here provide a particularly A. lyrata are the two species that show the most similar organiza-
striking insight into each of the 11 AhPDF1s. The between- tion of PDF1s at the genome level, as expected for species that
species difference in PDF1 shoot transcript accumulation was diverged only c. 300 000 yr ago (Roux et al., 2011). It would thus
particularly high for specific AhPDF1s (AhPDF1.1a-AhPDF1.1b be interesting to investigate whether PDF1s harboured by loci 2,
and AhPDF1.2b) as compared with their orthologues in 3 and 8 were actually specifically retained in A. halleri to a greater
A. thaliana (AtPDF1.1 and AtPDF1.2b-AtPDF1.3; Figs 2, 5, extent than in the A. lyrata lineage. Interestingly, overall, PDF1
Table S9). Within A. halleri, these copies also showed the greatest transcripts are also constitutively highly accumulated in Noccaea
contrast in transcript accumulation between shoots and roots. As caerulescens (formerly Thlaspi caerulescens) as compared to the
an indication, when measured on the same A. halleri line, culti- A. thaliana model plant (Hammond et al., 2006; van de Mortel
vated in the same conditions and when quantified with respect to et al., 2006). When available, it would be very interesting to ana-
the same reference gene, the shoot relative transcript abundance lyse the N. caerulescens genome sequence in order to determine
of the highest accumulated PDF1s (AhPDF1.1b in Fig. 5) was in whether PDF1s have a similar genomic organization in this other
the same range as the shoot relative transcript abundance of metal-tolerant and hyperaccumulating species and whether dif-
another cellular Zn-tolerance gene, that is, the vacuolar Zn trans- ferent PDF1s have independently also been the focus of evolu-
porter Metal Tolerance Protein 1 (AhMTP1-As and AhMTP1-B in tionary transcriptional innovation.
fig. 7 of Shahzad et al., 2010). Gene duplication introduces genome plasticity, so that, upon
Higher constitutive transcript accumulation of metal homeo- evolution, paralogous sequences tend to diverge over time to per-
stasis-related genes is a hallmark of A. halleri evolutionary innova- form different functions via non-, sub- or neofunctionalization
tion, as compared with phylogenetically related species (i.e. routes, ultimately resulting in gain or loss of gene copies upon
A. thaliana and A. lyrata), which are Zn-nontolerant and the selection process (Ohno, 1970; Zhang, 2003; Lynch & Katju,
Zn-nonaccumulators (Talke et al., 2006; Hanikenne et al., 2008; 2004). As the strongest criterion differentiating PDF1 paralogues
Shahzad et al., 2010; Deinlein et al., 2012). This increase is con- is the wide variation in the range of constitutive expression levels,
sidered as a way of enhancing a function already present in the one might expect that this could be a criterion for discriminating
Arabidopsis ancestor (Hanikenne & Nouet, 2011). It occurs the genes playing bigger roles than others. In A. halleri, locus 8
between orthologous genes present in nontolerant species (Hani- specifically harbours PDF1s. However, these paralogues do not
kenne et al., 2008; Shahzad et al., 2010), but it could also be contribute mainly to the constitutive high transcript abundance,
linked to gene amplification in A. halleri spp., although this is which is reinforced for AhPDF1.8b as no transcripts were
not always the case (Talke et al., 2006). For instance, increased detected; it encodes proteins with lower antifungal activity, and
transcript accumulation seems to be the main evolutionary most importantly the gene copy is not fixed in the Auby popula-
change for genes that remain as a single copy throughout the tion. It seems also that Zn tolerance in A. halleri did not select for
Arabidopsis genus, for example, Iron-Regulated Transporter 3 the functional expression of PDF1 at loci 2, 6 and 7 based on the
New
low transcript accumulation and/or pseudogene structure favour of PDF1 amplification in A. halleri, but it paves the way
(AhPDF1.2a-AhPDF1.2c and AhPDF1.6) and the presence of a for possible PDF1 retention in this lineage. Finally, given that
premature stop codon (AhPDF1.7). Overall, out of the 11 PDF1s are at the crossroads of the plant response to biotic and
AhPDF1s identified, only three genes – AhPDF1.1a, AhPDF1.1b abiotic stresses, our results provide a strong basis for further
and AhPDF1.2a – located in loci 1 and 3, clearly retain fully the genetic diversity and evolutionary studies questioning, in particu-
functional and genomic characteristics to be completely opera- lar, the potential emergence of Zn tolerance as a defence mecha-
tional in A. halleri. They thus represent good candidates to deci- nism against biotic attack from pathogens or herbivores
pher the molecular mechanisms enabling PDF1s to provide Zn (Poschenrieder et al., 2006; Boyds & Martens, 2007; Rascio &
tolerance using overexpression and/or RNAi approaches. Navari-Izzo, 2011).
The response of A. thaliana PDF1s to Zn treatment has been
studied (Mirouze et al., 2006) after infiltration into the leaves of
Acknowledgements
plants grown in nonsterile conditions, but these findings cannot
really be compared with the present results obtained after Zn Z.S. was supported by a scholarship from the Higher Education
application in the culture medium of plants grown under axenic Commission, Islamabad-Pakistan. This work was partially sup-
conditions. In A. thaliana, the response to pathogens initially tar- ported by a French Agence Nationale de la Recherche grant ANR-
geted AtPDF1.2a, inducible in leaves following an ethylene/jasm- 10-BINF-01-02 ‘Ancestrome’. We are thankful to all BPMP
onate pathway (Penninckx et al., 1996, 1998; Manners et al., technical and administrative staff for their kind and generous
1998; De Coninck et al., 2010; Niu et al., 2011). Only recently, support. We are greatly indebted to Jer^ome Salse for pertinent
expression studies revealed similar up-regulation in the context of advice concerning synteny conservation searches. We are grateful
nonhost resistance for AtPDF1.2a-AtPDF1.2c and AtPDF1.2b- to Hatem Rouached for fruitful comments on the manuscript.
AtPDF1.3 (Hiruma & Takano, 2011). Hereafter, it would be We gratefully acknowledge each of the anonymous referees for
interesting to investigate the extent to which the regulation of dif- their helpful comments and constructive suggestions, which
ferent PDF1 transcripts is shared over, or specific to, different greatly improved the presentation of this article.
loci. For example, in A. lyrata locus 2 (Fig. 2), the AlPDF1.2c
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evolution of the CSalphabeta superfamily. Cellular and Molecular Life Sciences genus
62: 2257–2269.
Table S5 Identification of ungapped sequences where synteny

Supporting Information conservation is observed within the region harbouring PDF1s
Additional supporting information may be found in the online
Table S6 Gene-specific primer pairs used to clone the mature
version of this article.
part of AhPDF1 protein in pET28(a+)
Fig. S1 Example of syntenic conservation analysis performed at
Table S7 Primer sequences used for the amplification PDF1s
the macro- and microsyntenic scales for PDF1s present at locus 3.
coding DNA sequences from A. halleri and A. thaliana
Fig. S2 Orthologous relationship between A. thaliana and
Table S8 Gene-specific primer pairs used in real-time RT-PCR
T. parvula spp. in regions harbouring PDF1 genes present at locus
analyses
1 and locus 4.
Table S9 Relative accumulation of PDF1 transcripts in A. halleri
Fig. S3 In vitro functional characterization of amino-acid substi-
and A. thaliana spp.
tutions in AhPDF1.4 and AtPDF1.4 for zinc tolerance.
Table S10 Specific primers used to distinguish homologous
Notes S1 Sequence of the predicted PDF amino acid proteins
AhPDF1s
used for multiple sequence alignment and phylogenetic analysis,
provided in the form of the classical fasta files and organized
Table S11 Segregation of AhPDF1.5 and AhPDF1.8a markers
according to their orthologous relationships within inferred ances-
and analysis of AhPDF1.8b representation over A. halleri individ-
tral loci.
ual plants originating from the Auby population
Table S1 Specific primers used for the amplification of PDF1
Please note: Wiley-Blackwell are not responsible for the content
probes for the screening of the A. halleri BAC library
or functionality of any supporting information supplied by the
authors. Any queries (other than missing material) should be
Table S2 Markers used for the genotyping in the
directed to the New Phytologist Central Office.
A. halleri 9 A. lyrata petraea BC1 linkage map
Table S3 Markers used for genetic mapping of AhPDF1-har-

bouring BAC clones
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Plant Defensin type 1 (PDF1): protein promiscuity and

in different plant species (Mergaert et al., 2003; Graham et al.,

obtained in the BC1 population, for the BAC-derived markers,

Table 1 Identification of PDF1 paralogues in Arabidopsis halleri

Number of BAC clones

Locus 1 AhPDF1.1a1 4 8B03 HF545648

LG1 LG2 LG4 LG7 LG8

38.0 1-10858 38.6 4-09207

72.5 2-19245(AhMTP1C) 72.8 2-19531

A. thal. Chr. 1 A. thal. Chr. 4 Unknownposition in A. thal.

(a) γ core motif

Fig. 3 Phylogenetic analysis of the PDF1

Table 2 Antifungal activity of AhPDF1s as tested against the fungus ZnSO4

Table S5 Identification of ungapped sequences where synteny

Table S3 Markers used for genetic mapping of AhPDF1-har-

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