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Correlating SNP Genotype with the Phenotypic Response to Exposure to Cadmium in Populus spp. Marta Marmiroli,* Giovanna Visioli, Elena Maestri, and Nelson Marmiroli Division of Genetics and Environmental Biotechnologies, Department of Environmental Sciences, University of Parma, Viale G.P. Usberti 11/A, 43124 Parma, Italy.
bS Supporting Information ABSTRACT: Species within the genus Populus include potential phytoextractors of heavy metal ions from contaminated soils, and genetic markers predictive of performance would be a useful tool for selection and breeding. Here, we have identified sequence variation within seven target and three nontarget genes among a set of 11 Populus spp. clones. Sequence variants were present in both the coding and noncoding regions; the former can potentially affect the functionality of the target genes. At the same time, the effect of exposure of the clones to cadmium ions on the morphology and the distribution of various metal ions was investigated by scanning electron microscopy microanalysis. A positive correlation was established between genetic variation, cadmium accumulation, and its bioconcentration in the root.
’ INTRODUCTION The pollution of soil and water by heavy metal ions represents a potential health hazard. However, the ability of plants to absorb these ions offers an attractive means of decontamination, since the aerial parts of the plants can be readily harvested and then disposed of, thereby depleting the amount of contaminant in the soil.1 Since a key property of an efficient phytoextractor is its ability to rapidly amass biomass, the focus on identifying potential candidate species has been on fast growing woody species such as poplar (Populus spp.) and willow (Salix spp.), which have both proven to be effective as absorbers of heavy metal ions. A number of experiments have shown that both these species are capable of taking up heavy metal ions in the range of several parts per million of dry weight, of which 70% remains in the root while the remaining 30% is translocated to the aerial part of the plant.24 Populus spp. are widely used for the commercial production of paper and bioenergy (ref 5 and additional references in the Supporting Information (SI)), and the physiology, phenology and genetics of a number of species in this genus have been thoroughly investigated.6 The genus belongs to the clade “Eurosid I” in the Angiosperm Phylogeny Group (APG) system,7 and its various species are distributed widely throughout the Northern Hemisphere, ranging from the tropics to the Arctic Circle.8 The Western poplar (P. trichocarpa) in particular has been adopted as a model tree species,6,8 and its genome has been recently fully sequenced.9 The ecological benefits provided by r 2011 American Chemical Society
the genus include carbon sequestration, nutrient cycling, bioremediation, and biofiltration.11 Ionic cadmium (Cd) is toxic to humans, animals and insects. In plants it interferes with cellular homeostasis, and when present in sufficiently high concentrations can stunt growth and cause chlorosis.12 Phytoremediation of Cd contaminated soil has been attempted using a number of plant species, among which Populus spp. have given the most encouraging results ( ref 13 and SI). It is unknown at this stage whether intra- or interspecific genetic variation influences the ability to either tolerate and/or accumulate Cd; as a result, it is still unclear how best to select for the most efficient genotypes with respect to their phytoremediation capacity.14 It has proven particularly difficult to demonstrate the effectiveness of phenotypic selection as a means of increasing phytoremediation efficiency, and even selection based on a genetic marker approach has yet to pay dividends. Now with a greater understanding of the genetic control of Cd homeostasis, the opportunity exists to base a selection strategy on sequence variation within the key genes involved in this process.15 Recent literature advocates the use of target genes involving adaptive responses where coding regions and regulatory regions show the effect of selection.16 Received: November 3, 2010 Accepted: March 21, 2011 Revised: March 15, 2011 Published: April 14, 2011 4497
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Table 1. Target Genes Identified in Populus spp., Based on Sequences Identified in Arabidopsis thaliana genomic location sequence identifier in
gene used for
notes on
of the sequence in the genome of
portion considered
searching, from Arabidopsis thaliana
gene name in
poplar EST
poplar
P. trichocarpa
for analysis
genome
Arabidopsis
database
sequence
(JGI database)
(JGI database)
At4g23100
glutamate-
T097G11
from cDNA
scaffold
11969066
cysteine ligase
library of shoot
LG_III
11969454
GSH1 (AtECS1)
meristem
(2 distinct
11972462
(P. tremula P. tremuloides)
fragments)
11972749
scaffold LG_I
26199992
803 bp At4g39330
cinnamyl-alcohol
V014B11
from cDNA
dehydrogenase
library of male
(CAD1)
catkins
26200292
(P. trichocarpa) 384 bp At1g47240
Nramp2, metal transporter
V014E06
from cDNA library of male
(AtNramp2)
scaffold_205
207677 208739
scaffold
2612561
catkins (P. trichocarpa) 732 bp
At1g10970
ZIP4, metal
POPLAR.4432
from cDNA
transporter similar
cluster
library of senescing
to Zn and Cs
containing 6
leaves (P. tremula)
transporter of Thlaspi caerulescens
clones: est I063P34
547 bp
LG_XVIII
2612899
ZNT1 At4g29510
At5g20720
arginine
F062P06
from cDNA
methyltransferase
library of flower
(AtPRMT11)
buds (P. trichocarpa)
chloroplast
521 bp from cDNA
T074C12
chaperonin 10 Cpn21 (AtCPN21)
library of shoot meristem
scaffold_145
379279 379847
scaffold
674825
LG_XVIII
675142
(P. tremula P. tremuloides) 494 bp At3g59140
na
ABC-like,
Q004H06
from cDNA
member of MRP
library of dormant
subfamily
buds (P. tremula)
(AtMRP14) ribulose-
725 bp shoot 36 months
AJ775246
(P. euphratica)
1,5-bisphosphate
scaffold LG_XII
scaffold LG_XIII
951979 953190
8425069 8425200
carboxylase oxygenase large subunit At5g05290
alpha-expansin 2
DB899652
mixture of leaf, bud, stem, root
At1g18800
(nucleosome
(P. nigra) from terminal
DT493200
assembly protein)
vegetative buds
NAP1-related protein 2
(P. trichocarpa)
Variation within genic sequences between and among species can be readily assessed in plant populations.17 The aim of the
scaffold LG_XIII scaffold LG_XVII
12410950 12411072 4104739 4104905
present analysis was to identify such variation in both the coding and noncoding regions of a set of target and nontarget genes 4498
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Environmental Science & Technology among a panel of Populus spp. clones. The response of these same clones to Cd exposure was also assayed, using both de novo generated and already published data.18 We describe here the distribution of sequence variants in a set of target genes among the clones, and correlate these with the level of Cd present in the plant tissue. Such correlations can be informative for the design of a Cd phytoextraction ideotype, which would greatly simplify the task of breeding for more effective phytoremediators.
’ EXPERIMENTAL SECTION Plant Materials. Eleven clones of Populus spp. of diverse geographical origin were chosen from the collection maintained by the Institute of Agro-environmental and Forest Biology, National Research Council, Monterotondo Scalo, Rome, Italy; the panel comprised Populus trichocarpa Torr. & Gray Nisqually 1, three accessions of P. nigra L. (Poli, 58861 and Turkia), two of P. alba L. (14P11, 6K3), one of P. deltoides Bartram ex Marshall (Lux), three of P. euramericana (= P. deltoides P. nigra) (I-214, “L. Avanzo” and A4A), and one of P. interamericana (= P. deltoides P. trichocarpa) (11-5). Target Genes. Seven target genes were chosen for the identification of sequence variation (Table 1) on the basis of their involvement in Cd, Zn, Ni uptake, sequestration, storage, and translocation in hyperaccumulators such as Thlaspi caerulescens J&C Presl. and Arabidopsis halleri (L.) O'Kane & Al-Shehbaz, from outcomes of proteomic analysis in Populus spp. 19,20 and from earlier investigations of various Thlaspi spp. populations.21 These were GSH1 (involved in glutathione synthesis22,23), CAD1 (cell wall synthesis; 24,25), NRAMP2 and ZIP4 (metal transporters24,26), PAM1 (defense protein; 27), CPN21 (plastid chaperonin28), and ABCMRP14 (membrane transporter26). Further details concerning these genes are provided in the SI. Three nontarget genes (not associated with the response to heavy metal ions) were included for comparative purposes; these were RBC (ribulose-1,5-bisphosphate carboxylase oxygenase, large subunit), EXP2 (R-expansin 2) and NAP1 (nucleosome assembly protein related protein). DNA Isolation and Sequencing. DNA was isolated from 1 g fresh leaf taken from cuttings of the same age grown without any exposure to Cd, using a GenElute Plant Genomic DNA Miniprep Kit (Sigma-Aldrich, Milan, Italy). PCR primers were designed using “Primer Premier 5” software (www.premierbiosoft.com/ primerdesign/) based on genomic sequence identified in the JGI database (http://genome.jgi-psf.org/) (SI Table S1), and the resulting PCR products were purified from agarose gels with the Illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare, Milan, Italy) and then sequenced on a Beckman Coulter CEQ 8000 DNA Analysis Sequencer (Beckman Coulter, Fullerton, CA). In Silico Sequence Analysis. Sequences were aligned using ClustalW (ref 29, align.genome.jp/) mounted on the EMBL server (www.ebi.ac.uk/clustalw/). Their intron/exon structure was determined by comparing the amplified genomic sequence with available cDNA and/or EST sequence (www.popgenie.db. umu.se, Genome Browser). The presence of single nucleotide polymorphisms (SNPs) among the 11 clones was determined using MEGA software (ref 30, www.megasoftware.net/). Inferred amino acid sequences were similarly compared to identify substitutions. Phylogenies of the sequences were derived using the Neighbor Joining algorithm (www.ebi.ac.uk/clustalw/), and the Mantel test, as implemented in GenAlEx 6.2,31 was applied to
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compare genetic distance matrices. The inferred peptide sequence of each target gene within each clone was compared with its counterpart P. trichocarpa Nisqually 1. Divergence in amino acid composition was based on both the BLOSUM8032 and the Grantham33 matrices (the latter is based on chemical differences between amino acid residues, with higher scores being associated with substitutions involving dissimilar amino acids). Tajima’s test for neutrality was applied to both complete nucleotide sequences and coding regions only using DNAsp software.34 Phenotypic Response to Cd Exposure. Poplar cuttings were raised hydroponically for two months in 1/3 strength Hoagland’s nutrient solution (pH 6.5) under a controlled temperature and illumination regime, following methods described elsewhere.18,35 The nutrient solution was then adjusted to 50 μM CdSO4 and the plants (four replicates per clone) remained in culture for a further three weeks. Atomic absorption spectrometry (AAS)18 and scanning electron microscopic (SEM) microanalysis (SEM/ EDX) were used to quantify the presence of Cd and other elements in leaf and stem. Measurements were made of leaf area development, root elongation, leaf trichome density, and cuticular wax deposition on the leaf. For the SEM/EDX analysis, leaves were removed from the stem at the nodal intersection, while stems and roots were cut into 1 mm thick transverse sections. The material was prepared following methods described elsewhere.36 Three analyses were performed, one based on morphology, one on the quantification of Cd and other elements, and third involved mapping the distribution of Cd. The latter was obtained using a Jeol 6400 Scanning electron microscope (Jeol, Osaka, Japan) equipped with an Oxford X-ray detector (Oxford Instruments, Oxford, UK) and supported by INCA software (www.jeol.com). The following operating parameters were applied: 14 mm working distance, 20KeV electron beam energy and 1525% dead time for the acquisition of X-ray data. The element mapping resolution was standardized by the software, with the number of frames set at 3000 (giving an acquisition time of 2 h for each map). The magnification varied from 100 to 700, depending on the sample. Each clone was represented by three root and stem sections and three leaves, taken from each of the three replicates of both Cd treated and nontreated (control) samples. The quantification of metal ions was obtained by taking a measurement at 15 positions per stem section sited at various distances from the center, and at ten randomly chosen positions in both the leaf midrib and leaf blade. The content of each particular ion was expressed as a percentage of the total metal ion content, using the software package LINK ISIS in “Semiquant” mode (www.jeol.com). All one way analyses of variance and Student t tests were performed using SPSS Statistics 17.0 (www.spss.com/statistics/) and significance was declared at p < 0.05. The variance was tested for homogeneity (F-max) and the means compared using the Tukey test.
’ RESULTS AND DISCUSSION Genetic Analysis. All ten gene fragments were successfully amplified from each of the 11 Populus clones. The number of amplified fragments present in the amplicon and their sizes were uniform across the clones for eight of the ten genes (the exceptions were CPN21 and ABCMRP14). The placement of the SNPs identified within the target genes (SI Figure S1) indicated an even distribution across exons and introns. The phylogeny based on the target gene fragment sequence 4499
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Figure 1. Correlation between genotypic and phenotypic variation in a panel of Populus spp. clones. Top: Phylogeny of clones based on sequence variation within target genes. Bottom table: Cd accumulation and tolerance in plants exposed to 50 μM Cd (standard errors shown in parentheses). § indicates averaged data derived from combining the present results with those reported by Zacchini et al.18 BCF: bioconcentration factor. The leaf and stem Cd contents were estimated from SEM microanalysis (see SI Figure S2). For the leaf trichomes,“þþþ” and “þþ” indicate, respectively, 100% and 60% more trichomes on the leaves of Cd treated plants. For leaf wax, “þþþ” indicates 100% more wax on the leaf surface of Cd treated plants.
Table 2. SNP Occurrence in Target Gene Sequences Across a Panel of Populus spp. Clones, And the Prediction of Peptide Substitutions with Respect to the Standard Sequence of P. trichocarpa Nisqually 1a target genes SNP occurrence,
ABCMRP14 162/1043
CPN21
GSH1 bis
NRAMP
83/318
49/288
62/1055
CAD1 15/359
PAM 102/569
ZIP4 18/336
GSH1
total target
total non
genes
target genes
13/389
504/4612
36/422
10/221
285/2412
7/119
3/168
198/2061
27/255
whole sequence SNP occurrence, introns
92/728
19/89
33/202
47/755
SNP occurrence, exons
68/463
45/197
16/86
15/300
Average number of pairwise
n.a. 15/359
84/417 18/152
n.a. 18/336
59.42
20.71
15.25
20.07
4.87
26.47
6.44
3.89
0.111
1.286
0.416
0.246
0.215
1.185
0.213
0.540
19.64
2.94
differences (SNP) Tajima’s D (SNP) SNP leading to
8
4
2
5
7
844.44
286.09
201.55
159.18
102.00
1.82
1.45
2.34
0.53
0.892
1.420
0.503
0.812
4
na
na
13
3
46
7
99.91
79.82
0
na
na
0.28
0.66
0.24
0
na
na
0.265
0.529
0.346
na
na
na
synonymous codons average Grantham score for amino acid substitutions average Grantham scores relative to exon length Tajima’s D (NonSyn/Syn ratio) a
na, not applicable.
alignments (Figure 1) included a clade which linked the three P. nigra clones, the second clade capturing the two P. alba clones (14P11, 6K3) and a third which contained P. trichocarpa, P. deltoides and the hybrid 115. A similar phylogeny based on the three nontarget gene fragment sequences produced a
grouping where the three P. nigra clones were no longer clustered together, nor were the two P. alba ones (not shown). The location and number of SNPs within each of the gene fragment sequences are reported in Table 2 and SI Figure S1. In total, the 4612bp of target gene sequence contained 504 SNPs, 4500
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Figure 2. Morphological changes in Populus spp. clones exposed to 50 μM Cd. A: SEM images of the leaf surface, showing the midrib of treated and nontreated plants of clones Poli and A4A (100 magnification); 1 = upper leaf epidermis, 2 = midrib, 3 = trichomes, 4 = wax. B: SEM images of stem cross sections of treated and nontreated plants at 200 (whole cross section) and 700 (sclerenchymatous tissues) magnification, and SEM/EDX maps showing the distribution of Cd within the cross section. Clones A4A and I-214 are shown. x = xylem; s = sclerenchyma; p = periderm; e = epidermis. 4501
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Environmental Science & Technology equivalent to a mean frequency of one SNP per 9.2bp. The proportion of SNPs in introns was higher than the proportion of SNPs in exons, but this difference was not statistically significant. About 75% of these SNPs were of the transition type, consistent with the proportion estimated from a prior analysis of Populus ESTs.37 Within the nontarget sequences, 36 SNPs were identified over a 422bp stretch (one SNP per 11.7 bp). However, the much higher mean frequency of pairwise differences within the target as compared to the nontarget gene sequence (19.6 vs 2.9, significant at p < 0.05) among the clones indicated that the former sequences were considerably more polymorphic than the latter. The greatest difference in SNP frequency was between target and nontarget intronic sequence (11.8% vs 5.8%), whereas the proportions of polymorphic sites within the exonic sequence were very similar (9.6% vs 10.6%). The genes differed widely from one another with respect to the frequency of SNPs, as has also been noted within wild populations of P. trichocarpa.38 The genetic distance between the clones was estimated both at the nucleotide and the amino acid level, and Mantel correlations between these two distance matrices produced a determination coefficient (R2) of 0.009, signifying that only a very small proportion of the nucleotide variation was reflected in variability at the amino acid level. Amino acid substitutions were based on comparisons with the in silico translation products of P. trichocarpa Nisqually 1 whole genome sequence. The nucleotide sequences isolated de novo from Nisqually 1 were fully consistent with their equivalents in the whole genome sequence. Differences in the deduced peptide sequences were analyzed by both the BLOSUM 8032 and the Grantham33 matrices, and the resulting correlation between the scores obtained using the two matrices was 0.82, which indicates a high degree of agreement. The mean Grantham scores for amino acid substitutions differed among the target genes (Table 2). For instance, all the GSH1 SNPs produced synonymous codons, whereas the gene ABCMRP14 contained 34 possible amino acid substitutions. The highest frequency of nonsynonymous substitutions occurred within ABCMRP14 and CPN21. Selection effects on the various gene sequences were estimated at the both the nucleotide and amino acid level. When Tajima’s D was calculated from the nucleotide sequence, all but ZIP4 produced a negative value, indicating the prevalence of negative (purifying) selection. At the same time, the values of the D (NonSyn/Syn) ratios computed from the coding sequences were mostly
