Allelic heterogeneity and trade-off shape natural variation for response to soil micronutrient.

Abstract

As sessile organisms, plants have to cope with diverse environmental constraints that may vary through time and space, eventually leading to changes in the phenotype of populations through fixation of adaptive genetic variation. To fully comprehend the mechanisms of evolution and make sense of the extensive genotypic diversity currently revealed by new sequencing technologies, we are challenged with identifying the molecular basis of such adaptive variation. Here, we have identified a new variant of a molybdenum (Mo) transporter, MOT1, which is causal for fitness changes under artificial conditions of both Mo-deficiency and Mo-toxicity and in which allelic variation among West-Asian populations is strictly correlated with the concentration of available Mo in native soils. In addition, this association is accompanied at different scales with patterns of polymorphisms that are not consistent with neutral evolution and show signs of diversifying selection. Resolving such a case of allelic heterogeneity helps explain species-wide phenotypic variation for Mo homeostasis and potentially reveals trade-off effects, a finding still rarely linked to fitness.

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Allelic Heterogeneity and Trade-Off Shape Natural
Variation for Response to Soil Micronutrient
Seifollah Poormohammad Kiani1, Charlotte Trontin1, Matthew Andreatta2, Matthieu Simon1,
Thierry Robert3, David E. Salt2, Olivier Loudet1*
1INRA, UMR1318, Institut Jean-Pierre Bourgin, Versailles, France, 2Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, Indiana
United States of America, 3Laboratoire d’Ecologie, Syste ´matique, et Evolution, Universite ´ Paris-Sud XI, Orsay, France
Abstract
As sessile organisms, plants have to cope with diverse environmental constraints that may vary through time and space,
eventually leading to changes in the phenotype of populations through fixation of adaptive genetic variation. To fully
comprehend the mechanisms of evolution and make sense of the extensive genotypic diversity currently revealed by new
sequencing technologies, we are challenged with identifying the molecular basis of such adaptive variation. Here, we have
identified a new variant of a molybdenum (Mo) transporter, MOT1, which is causal for fitness changes under artificial
conditions of both Mo-deficiency and Mo-toxicity and in which allelic variation among West-Asian populations is strictly
correlated with the concentration of available Mo in native soils. In addition, this association is accompanied at different
scales with patterns of polymorphisms that are not consistent with neutral evolution and show signs of diversifying
selection. Resolving such a case of allelic heterogeneity helps explain species-wide phenotypic variation for Mo homeostasis
and potentially reveals trade-off effects, a finding still rarely linked to fitness.
Citation: Poormohammad Kiani S, Trontin C, Andreatta M, Simon M, Robert T, et al. (2012) Allelic Heterogeneity and Trade-Off Shape Natural Variation for
Response to Soil Micronutrient. PLoS Genet 8(7): e1002814. doi:10.1371/journal.pgen.1002814
Editor: Rodney Mauricio, University of Georgia, United States of America
Received April 6, 2012; Accepted May 21, 2012; Published July 12, 2012
Copyright: ? 2012 Poormohammad Kiani et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors received no specific funding for this work.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: Olivier.Loudet@versailles.inra.fr
Introduction
Some of the most important constraints that plants have to
adapt to are those related to soil properties [1,2]. These are also
possibly some of the least well studied constraints, because they are
spatially heterogeneous, thus not prone to typical geographic clines
[3,4], and require analysis at the local/population scale [5]. In this
context, quantitative genetics approaches hold great promise to
reveal the genetic basis of adaptation by enabling the identification
of the molecular origin of phenotypic differences between
populations or even between species [6,7]. One of the benefits of
identifying the causative polymorphism(s) and/or gene(s) explain-
ing natural phenotypic variation is that it allows direct testing for
correlations between environmental factors populations may be
responding to and the occurrence of the target genetic polymor-
phism. This contrasts with working indirectly through populations
phenotype, which may reflect contradictory patterns and trade-
offs, if not genetic drift [5,8]. Moreover, this approach also enables
direct testing for fitness advantages or potential cost of adaptation
(i.e. antagonistic pleiotropy, an expected argument for local
adaptation), that may be masked by linkage to deleterious
mutations or genetic drift in reciprocal transplant experiments
[9,10]. Molecularly identified examples of potentially-adaptive
variation are still largely lacking and the debate is open as to the
scale and rate of adaptive evolution [11,12].
In this work, we aimed at identifying the molecular bases of
natural variation in accumulation of an essential micronutrient
and understanding the ecological significance of this diversity. We
describe both the fitness trade-offs of this variation and its potential
adaptive advantage in the environment, revealing a system that is
unlikely to have remained neutral.
Results/Discussion
Bay-0 and Shahdara, two strains (accessions) derived from wild
populations of Arabidopsis thaliana, show contrasted growth
behavior when grown on acidic peatmoss substrate (Figure 1).
Using a segregating population derived from the cross of these two
accessions, we determined the Shahdara growth defect to be
segregating from a major-effect recessive locus on chromosome 2,
as confirmed by a near-isogenic line derived from a residual
heterozygous interval in one of the recombinant inbred lines
(HIF084; Figure 1). Additional recombinant lines were pheno-
typed and genotyped to pinpoint the causative interval to 80 kb
(Figure S1), covering 19 annotated genes. Among these, one gene
appeared as a good functional candidate: MOT1 was previously
linked to the transport and homeostasis of the essential micronu-
trient molybdenum (Mo) in the plant [13,14], an element which
availability is known to vary with soil pH [15]. Indeed, a T-DNA
insertion mutant in the MOT1 gene (mot1.1; Figure S1) shows a
phenotype similar to Shahdara on peatmoss and –contrary to the
Bay and Col alleles– the Sha allele is not able to restore wild-type
growth when combined with the mutant allele in F1 hybrids
(Figure 2). Moreover, we show that defective growth is comple-
mented by either increasing soil pH with additional CaCO3mixed
to the peatmoss (Figure S2) or increasing Mo availability (without
altering soil pH) by adding Mo in the watering solution (Figure 3).
Hence, genetic and chemical complementation shows that acidic
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soil pH is responsible for reducing Mo-bioavailability and that,
combined with a defective allele at MOT1, this results in the
typical Mo-deficiency syndromes of reduced leaf Mo contents,
strongly altered growth and development, necrosis [16]. These
observations of a significant phenotypic consequence of variation
at MOT1 provide a model for the potential adaptive significance of
this variation that goes beyond the simple variation in Mo content
revealed previously [13,14].
Although we find that Landsberg erecta (Ler) has a similar
behaviour than Shahdara in our conditions (Figure 3), this
defective allele (MOT1Ler) used initially to reveal the gene’s activity
[13,14] is functionally different from the MOT1Shadefective allele.
MOT1Shadoesn’t bear the promoter 53 bp-deletion as in Ler
(Figure S1) and in fact is not showing MOT1Ler-like transcriptional
down-regulation compared to MOT1Bayor MOT1Col(Figure S3).
Instead, MOT1Shaseems defined by a single amino-acid change in
the protein relative to Bay-0 and Col-0 (Figure S1), strongly
suggesting that MOT1Shais hypofunctional. However, the MOT1
protein produced from the Sha allele is still able to increase Mo
accumulation when heterologously expressed in yeast (Figure S4).
We then genotyped a random worldwide sample of ,300
accessions for the Sha-like amino-acid change and the Ler-like 53-
bp deletion and find that these alleles are both present at
intermediate frequencies (15–20%) among the populations.
Sequencing 102 of these accessions for the whole gene and
promoter region revealed that the very conserved MOT1Sha
haplotype is indeed clearly defined solely by the D104Y amino-
acid change, while the MOT1Lergenotype is more complex and
diverse (Table S1). All Sha-like and Ler-like accessions that have
been phenotyped show that both MOT1Sha
haplotypes are perfectly associated with defective growth under
acidic soil conditions (Table S1) and complementation crosses with
five additional Sha-like accessions confirm allelism to mot1.1
(Figure S5). Taking into account this allelic heterogeneity now
explains most of the species variation toward low-Mo contents
revealed in previous work [13] (http://www.ionomicshub.org/
arabidopsis/). This form of complexity –in addition to genetic
heterogeneity– is probably more frequent than previously thought
and MOT1Ler
in many organisms and is likely to help explain part of the missing
heritability [17,18].
Regarding MOT1 defective haplotypes, MOT1Shais confined to
‘West-Asia’ (including Russia) with a high frequency among these
populations (Figure S6) and displays a very low polymorphism
level (pSha=0,00016) in comparison to other haplotype clusters,
includingtheworldwide-distributed
(pLer=0,0017; Table S1). This may translate a recent and rapid
expansion of the Sha allele through ‘West-Asia’, which could be
due to neutral processes such as gene surfing associated with post-
glaciation recolonization events from Central Asia [19]. This may
also witness local positive selection events in favour of the Sha
allele. Indeed, patterns of nucleotide polymorphisms at MOT1 in
the sample of 102 accessions strongly deviate from the expectation
under the strict neutrality model, contrarily to two control loci, PI
and COI1 (Table S2). Negative values of Tajima’s D reveal an
excess of rare alleles at MOT1, suggesting the possible occurrence
of at least one past selective sweep that has targeted this locus.
Other well documented evolutionary processes such as population
expansion after the last glaciation event [20] and population
genetic structure [21] could also have contributed to the excess of
rare alleles observed at the genome-wide level [22], as well as at
the MOT1 locus in A. thaliana. Nevertheless, the HKA and
McDonald-Kreitman tests, which do not rely on the frequency
spectrum, support the hypothesis of diversifying selection at the
species level (Tables S2 and S3). The excess of within-species
polymorphisms relatively to inter-specific divergence and the
excess of non-synonymous polymorphisms observed at MOT1 may
result from the selection of different haplotypes at the worldwide
scale. Interestingly, this trend is also clear when considering only
accessions from ‘West Asia’, suggesting that the selection process
could happen at different geographical scales.
Our own documented collection of wild populations from
diverse regions in ‘West-Asia’ allowed us to investigate potential
relationships between MOT1 alleles and environmental parame-
ters described precisely at the population site, especially soil
properties. We saw no relationship with soil pH (indeed, none of
the described populations were facing acidic soil conditions), but
there was an obvious trend for populations with the defective
MOT1Shaallele to grow on soils with high water-extractable Mo
content (Figure 4). This may indicate that the defective Sha allele
is a protective response to Mo accumulation in environments with
excess Mo. Indeed, under such conditions in the laboratory, we
observe a strong decrease in fitness (through the total number of
seeds produced per plant) in all genotypes (Figure S7), indicating
that plants have to find the right balance between Mo deficiency
and Mo toxicity, a trade-off that could be resolved partly through
variation in function of the Mo transporter MOT1. Moreover, we
show that a defective MOT1 allele (either mot1.1 or MOT1Sha) is
accompanied by a slightly increased average seed mass (another
component of fitness) specifically under Mo-toxic conditions
(Figure S7), the outcome of which is difficult to estimate in nature
[23]. It is however worth noting that previous studies in A. thaliana
have shown positive effects of increased seed size for example on
subsequent root and shoot growth [24] or seedling survival under
limiting conditions [25].
Insummary, we haveidentified a newfunctional variant atMOT1
that contributes to explain most of the species’ diversity in Mo
homeostasis, and associated phenotypes that provide likely explana-
tions for its non neutral evolution and its correlation to native soil. It
is still a rare finding to be able to relate functional genetic variants to
fitness or trade-off effects [26–28], and even more to associate this
variation to the environment [3,7]. Our work indicates that
environmental parameters of importance, such as soil properties,
MOT1Ler
allele
Author Summary
Plants are studied for their ability to adapt to their
environment and especially to the physical constraints to
which they are subjected. It is expected that they evolve in
promoting genetic variants favorable under their native
conditions, which could lead to negative consequences in
other conditions. One approach to study the mechanisms
and dynamics of these adaptations is to discover genetic
variants that control potentially adaptive traits, and to
study directly these variants in wild populations to try to
reveal their evolutionary trajectory. We have identified a
new polymorphism in a gene coding for a transporter of
molybdenum (an essential micronutrient for the plant) in
Arabidopsis; we show that this variant has strong
phenotypic consequences at the level of plant growth
and reproductive value in specific conditions, and that it
explains a lot of the species diversity for these traits.
Especially, the variant is associated with a clear negative
effect under molybdenum-deficient conditions (caused by
soil acidity) and with a subtle positive effect under
molybdenum-plethoric conditions. Interestingly, the land-
scape distribution of the variant is not random among
Asian populations and correlates well with the availability
of molybdenum in the soil at the precise location where
the plants are growing in the wild.
Natural Variation for Response to Soil Nutrient
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may be heterogeneously distributed and therefore require local
description [5] and study of local adaptation [9,11], which is greatly
facilitated by the identification of the causative locus.
Materials and Methods
All accessions used and the Bay-06Shahdara RIL set [29] were
obtained from Versailles Arabidopsis Stock Centre (http://
dbsgap.versailles.inra.fr/vnat/). Heterogeneous Inbred Family
‘HIF084’ was derived from RIL084 (segregating for the region
of interest) as previously described [30]. New collections of A.
thaliana accessions were partly described previously [31] and are
shown at http://www.inra.fr/vast/collections.htm. T-DNA inser-
tion mutant mot1.1 corresponds to line SALK_118311 as described
[13,14]. Genetic complementation tests were performed on F1
plants issued from the cross of diverse accessions to mot1.1 or its
wild-type background.
Acidic soil assays were performed on ‘Floratorf’ peatmoss
(Floragard, Germany) mixed with CaCO3 (4 g per liter of dry
peatmoss) to maintain a soil pH,5, watered with classical nutrient
solution and grown under typical long-day conditions at 20uC.
Chemical complementations were achieved in the same condition but,
eitherwith8 gCaCO3perliterofpeatmosstoreachapH,6,orusing
a watering solution supplemented with 1 mM Na2MoO4. Mo toxicity
was tested on regular fertilised soil mix (pH=6) watered with nutrient
solution supplemented with 7 mM Na2MoO4, or not (control).
MOT1 sequencing, qPCR analysis of expression (normalised
against GAPDH and PP2A), functional characterisation in yeast
were performed as previously described [13]. Extractable Mo was
determined in soils by the method of Soltanpour and Schwab [32]
using ICP-MS as the detector.
A total of 102 A. thaliana accessions (including 48 accessions
known to maximize A. thaliana diversity [33] and 44 accessions
Figure 1. Acidic peatmoss substrate induces severe growth defect linked to the Shahdara allele. When grown on peatmoss at a pH close
to 5, Shahdara is subject to severe growth and developmental arrest, necrosis and death, contrary to Bay-0 which develops normally. In the cross
between these two strains, this phenotype is entirely controlled by a single locus (Figure S1), as confirmed by near-isogenic line ‘HIF084’ segregating
solely for a region of chromosome 2.
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from ‘West-Asia’) and 5 A. halleri accessions (I-14, I-16, F-1, PL-22
and TZC; obtained from H. Fre ´rot at Univ. Lille [34]) were
sequenced at MOT1 (including 1 kb upstream and 0.3 kb
downstream for A. thaliana accessions) and at two reference loci,
COI1 (At2g39940; 2,600 bp coding sequence) and PI (At5g20240;
2,150 bp coding sequence). Those genes were either used
previously as reference or shown to have a neutral pattern of
polymorphisms in A. thaliana [35,36]. Sequences were aligned
using Codoncode Aligner v3.7.1. and subsequent alignments were
improved visually.
Intraspecific analyses i.e. nucleotide diversity estimated by p [37]
and hw[38], and Tajima’s D statistics [39] were calculated using
DNAsp v5.10.01 on the whole region sequenced. Ten thousands
coalescent simulations under the strict Wright-Fisher neutral
model assuming no recombination and conditioning on S were
performed to estimate statistical significance of Tajima’s D.
For interspecific analyses, the orthologous of MOT1 and of the
two control loci in A. halleri were used. The McDonald-Kreitman
test [40] was performed by using DNAsp v5.10.01 in order to test
for possible excess or deficiency in replacement substitutions at
MOT1. Singletons were discarded for this analysis in order to
reduce the contribution of slightly deleterious mutations (expected
at very low frequencies and unlikely to become fixed). Neutral
index was calculated as previously described [41]. Divergence
between A. thaliana and A. halleri, defined as the average number of
nucleotide differences between populations per gene, was calcu-
lated using DNAsp v5.10.01 and used to perform HKA tests with
the multilocus HKA program available from J. Hey laboratory
(http://genfaculty.rutgers.edu/hey/software).
Supporting Information
Figure S1
a candidate gene. A. The physical region of chromosome 2 found
to be linked to the growth defect phenotype is shown (physical
positions are given in Mb). B. Zooming in the candidate region
highlights recombinants within the inbred lines that allow to fine-
map the causative locus, thanks to additional markers (vertical
dashed lines). Individual lines’ genotype are depicted in horizontal
coloured boxes (green=Bay allele; purple=Sha allele ; dashed=-
heterozygous) and their phenotype on peatmoss are indicated
(S=Sensitive; R=Resistant). C. This allows to narrow down the
causative region to 80 kb, a region containing 19 predicted genes
including the candidate At2g25680 (MOLYBDATE TRANSPORT-
ER 1). D. MOT1 has been sequenced in parental accessions and
polymorphisms between Col-0, Bay-0 and Shahdara are repre-
sented along the single-exon gene model, including an amino-acid
change specific to Shahdara (D104Y). The position of the insertion
of a T-DNA in the mot1.1 mutant (SALK_118311) is indicated.
(TIF)
Fine-mapping the causative locus identifies MOT1 as
Figure S2
soil pH. Increasing soil pH from ,5 (‘Control’) to ,6 (‘+CaCO3’)
by doubling the amount of CaCO3 mixed to the peatmoss
substrate rescues normal vegetative growth of the Shahdara strain.
(TIF)
Chemical complementation links growth defect with
Figure 2. Mutant analysis and allelic complementation con-
firms MOT1 as the likely causative gene. Peatmoss phenotype of
diverse genotypes is shown, including Bay-0 and Shahdara parental
lines, mot1.1 mutant and its wild-type genetic background (Col-0), and
F1 plants from complementation crosses between these genotypes.
Unlike other alleles, the Shahdara allele at MOT1 is not able to rescue
the mutant phenotype.
doi:10.1371/journal.pgen.1002814.g002
Figure 3. Chemical complementation links growth defect with Mo shortage. Accessions with potentially functional (Bay-0, Col-0) and
defective MOT1 alleles (Ler, Shahdara, mot1.1) were grown on peatmoss substrate watered with nutrient solution containing either traces of Mo
(‘Control’) as in Figure 1, or 1 mM Na2MoO4(‘+Na2MoO4’). pH was checked to remain unchanged across treatments at ,5.
doi:10.1371/journal.pgen.1002814.g003
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Figure S3
MOT1Shadefective allele. MOT1 transcript accumulation relative
to GAPDH and PP2A controls is shown from roots of diverse
genotypes as in Figure 3. Contrary to Ler and mot1.1, Shahdara
accumulates normal levels of transcript. Standard errors are
shown.
(TIF)
MOT1 transcript accumulation does not explain
Figure S4
allele of MOT1 was overexpressed in yeast heterologous system
(Sha/p416) and shown to lead to Mo accumulation compared to
the empty vector (p416, used as reference) or the yeast wild-type
strain (BY4741), to an extent not significantly different from the
Col MOT1 allele (Col/p416). Error bars represent interquantile
range of medians.
(TIF)
MOT1Shais able to transport Mo in yeast. The Sha
Figure S5
confirm the causative gene and polymorphism. Peatmoss pheno-
type of diverse genotypes is shown: 5 independent Sha-like
accessions (Stw-0, Kly-2, Sij-4, Kondara and Zal-3) and F1 plants
from complementation crosses between each of these accessions
and either the mot1.1 mutant or its wild-type genetic background
(Col-0). As in Figure 2, all accessions sharing the MOT1Sha
haplotype are both sensitive and unable to rescue the mutant
phenotype.
(TIF)
Multiple accessions sharing the MOT1Shahaplotype
Figure S6
alleles at MOT1. Original collection site and functional MOT1
haplotype (Sha-like in red dots, Ler-like in yellow, Col-like in blue)
is shown on a world map for the 102 accessions sequenced in
Table S1. Ler-like accessions are found across the whole species
known distribution range, while Sha-like accessions are restricted
to Asia and Russia (‘West-Asia’).
(TIF)
Worldwide distribution of functionally contrasted
Figure S7
number and weight- of contrasted MOT1 genotypes. The fitness
consequences of Mo toxicity was tested on regular (non-acidic) soil
mix with different nutrient solutions containing either traces of Mo
Effect of Mo toxicity on fitness components -seed
(‘Control’) or 7 mM Na2MoO4(‘Na2MoO4(7 mM)’). The assay
was performed to compare (A) the mot1.1 mutant and its wild-type
(‘WT’) genetic background, (B) the Bay and Sha allele in the
HIF084 background (‘HIF[Bay]’ vs ‘HIF[Sha]’). In both cases, the
defective MOT1 allele is represented with black bars. To avoid
heterogeneity/effects on descendance conveyed through the
maternal plant, the mutant assays were performed as a progeny
testing from a mother plant segregating for the T-DNA insertion.
Two fitness parameters are represented: the total number of seeds
produced per plant (on the left) and the weight of 1,000 seeds (on
the right). Error bars show 95% confidence interval of the mean.
For the weight of 1,000 seeds, there is no significant difference
between genotypes under ‘control’ treatment, while defective
MOT1 alleles have significantly larger seeds under Mo excess (t-
test; p,0.017 when comparing mot1.1 and WT; p,0.0018 when
comparing HIF084[Bay] and HIF084[Sha]).
(TIF)
Table S1
Polymorphisms detected across 102 A. thaliana accessions for the
MOT1 locus, including 1 kb upstream (promoter region) and
0.3 kb downstream of the coding region (between blue vertical
double-lines). The position (coordinates) of polymorphic bases
(regions) are indicated in bp from TAIR10 reference. Synonymous
polymorphisms are highlighted in light grey, non-synonymous
polymorphisms in medium grey and missing data in dark grey. A.
lyrata and A. halleri serve as outgroups. Accessions individually
phenotyped on acidic peatmoss substrate are indicated (S=Sen-
sitive; R=Resistant). The main functional haplotypes highlighted
with colours (Sha-like in red, Ler-like in yellow, Col-like in blue)
are those represented on Figure S6, including the Sha-like
haplotype defined by the ‘D104Y’ polymorphism, and the Ler-
like haplotype associated to the ‘‘53 bp-deletion.’’
(XLS)
Haplotype diversity at MOT1 among 102 accessions.
Table S2
two reference loci (COI1 and PI).
(XLS)
Genetic diversity and tests of selection at MOT1 and
Table S3
(XLS)
Detailed results of HKA simulations.
Figure 4. West-Asian populations highlight the correlation between MOT1Shaallele and high native soil Mo content. Soil parameters
(pH, extractable Mo) from the precise original collection site are represented for independent populations carrying (red dots) or not (black dots) the
MOT1Shaallele.
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