Allelic Heterogeneity and Trade-Off Shape Natural Variation for Response to Soil Micronutrient

Summary

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.

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) explaining 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 polymorphism. 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 phenotyped 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 micronutrient 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 complemented by either increasing soil pH with additional CaCO₃ mixed 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 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 (MOT1^Ler) used initially to reveal the gene's activity [13], [14] is functionally different from the MOT1^Sha defective allele. MOT1^Sha doesn't bear the promoter 53 bp-deletion as in Ler (Figure S1) and in fact is not showing MOT1^Ler-like transcriptional down-regulation compared to MOT1^Bay or MOT1^Col (Figure S3). Instead, MOT1^Sha seems defined by a single amino-acid change in the protein relative to Bay-0 and Col-0 (Figure S1), strongly suggesting that MOT1^Sha is 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 MOT1^Sha haplotype is indeed clearly defined solely by the D104Y amino-acid change, while the MOT1^Ler genotype is more complex and diverse (Table S1). All Sha-like and Ler-like accessions that have been phenotyped show that both MOT1^Sha and MOT1^Ler 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 in many organisms and is likely to help explain part of the missing heritability [17], [18].

Regarding MOT1 defective haplotypes, MOT1^Sha is confined to ‘West-Asia’ (including Russia) with a high frequency among these populations (Figure S6) and displays a very low polymorphism level (πSha = 0,00016) in comparison to other haplotype clusters, including the worldwide-distributed MOT1^Ler allele (πLer = 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 parameters 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 MOT1^Sha allele 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 MOT1^Sha) 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].

In summary, we have identified a new functional variant at MOT1 that contributes to explain most of the species' diversity in Mo homeostasis, and associated phenotypes that provide likely explanations 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, 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-0×Shahdara 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 insertion 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 20°C. Chemical complementations were achieved in the same condition but, either with 8 g CaCO₃ per liter of peatmoss to reach a pH∼6, or using 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 from ‘West-Asia’) and 5 A. halleri accessions (I-14, I-16, F-1, PL-22 and TZC; obtained from H. Frérot 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 π [37] and θ_w [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 calculated 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

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