- Research article
- Open Access
Molecular and phylogenetic characterization of the sieve element occlusion gene family in Fabaceae and non-Fabaceaeplants
- Boris Rüping†1, 2,
- Antonia M Ernst†1, 2,
- Stephan B Jekat1, 2,
- Steffen Nordzieke1,
- Anna R Reineke3,
- Boje Müller1, 2,
- Erich Bornberg-Bauer3,
- Dirk Prüfer1, 2Email author and
- Gundula A Noll1
© Rüping et al; licensee BioMed Central Ltd. 2010
- Received: 15 January 2010
- Accepted: 8 October 2010
- Published: 8 October 2010
The phloem of dicotyledonous plants contains specialized P-proteins (phloem proteins) that accumulate during sieve element differentiation and remain parietally associated with the cisternae of the endoplasmic reticulum in mature sieve elements. Wounding causes P-protein filaments to accumulate at the sieve plates and block the translocation of photosynthate. Specialized, spindle-shaped P-proteins known as forisomes that undergo reversible calcium-dependent conformational changes have evolved exclusively in the Fabaceae. Recently, the molecular characterization of three genes encoding forisome components in the model legume Medicago truncatula (MtSEO1, MtSEO2 and MtSEO3; SEO = sieve element occlusion) was reported, but little is known about the molecular characteristics of P-proteins in non-Fabaceae.
We performed a comprehensive genome-wide comparative analysis by screening the M. truncatula, Glycine max, Arabidopsis thaliana, Vitis vinifera and Solanum phureja genomes, and a Malus domestica EST library for homologs of MtSEO1, MtSEO2 and MtSEO3 and identified numerous novel SEO genes in Fabaceae and even non-Fabaceae plants, which do not possess forisomes. Even in Fabaceae some SEO genes appear to not encode forisome components. All SEO genes have a similar exon-intron structure and are expressed predominantly in the phloem. Phylogenetic analysis revealed the presence of several subgroups with Fabaceae-specific subgroups containing all of the known as well as newly identified forisome component proteins. We constructed Hidden Markov Models that identified three conserved protein domains, which characterize SEO proteins when present in combination. In addition, one common and three subgroup specific protein motifs were found in the amino acid sequences of SEO proteins. SEO genes are organized in genomic clusters and the conserved synteny allowed us to identify several M. truncatula vs G. max orthologs as well as paralogs within the G. max genome.
The unexpected occurrence of forisome-like genes in non-Fabaceae plants may indicate that these proteins encode species-specific P-proteins, which is backed up by the phloem-specific expression profiles. The conservation of gene structure, the presence of specific motifs and domains and the genomic synteny argue for a common phylogenetic origin of forisomes and other P-proteins.
- Sieve Element
- Conserve Domain Database
- Genome Duplication Event
- Sieve Plate
- Genomic Synteny
In vascular plants, photoassimilates are transported through differentiated sieve elements (SEs) in the phloem forming a network of sieve tubes throughout the plant . The pressure-driven mass flow  requires a high degree of functional specialization of the phloem during development. In order to enable efficient translocation of photoassimilates, SEs loose most of their organelles and thus the ability to perform protein biosynthesis . Mature SEs are therefore dependent on adjacent, metabolically-active companion cells, which are connected to SEs by so-called pore-plasmodesm units . The pressure within the sieve tubes can reach 30 bar , so rapid and efficient protection against wounding is essential and favoured the evolution of a plugging mechanism based on specialized phloem proteins (P-proteins) . These structural proteins accumulate in the cytoplasm of metabolically-active, undifferentiated SEs, but are anchored to the plasma membrane when SEs mature . After wounding, they detach from their parietal location and plug downstream sieve plates by forming a gel-like mass, thereby preventing the loss of photoassimilates . This occurs in all the dicotyledonous plant families that have been studied, and P-proteins have also been identified in certain monocotyledonous plants . There is currently no standardized classification for P-proteins although different types were often distinguished by their tubular, fibrillar, granular or crystalline ultrastructure, which may represent different developmental or conformational states of the same protein subunits rather than evolutionarily-distinct families . Nevertheless, Fabaceae plants possess a special type of elongated crystalline P-protein bodies , which show a unique type of reactivity. The spindle shaped protein bodies, also known as forisomes ("gate bodies") , are able to undergo a reversible, calcium-induced conformational change and can consequently plug and open the sieve elements after wounding and regeneration. Three SEO (sieve element occlusion) proteins named MtSEO1, MtSEO2 and MtSEO3 have been identified in the model legume Medicago truncatula, and their role in forisome structure and assembly has been confirmed by immunological and GFP-fusion studies [13–15]. Comprehensive promoter analyses in M. truncatula roots and Nicotiana tabacum plants demonstrated a restricted expression of the corresponding MtSEO genes in immature sieve elements [14, 16], indicating a highly conserved regulation of promoter activities among diverse plant species, including non-Fabaceae lacking forisomes.
Although genes encoding forisome components of Fabaceae have recently been isolated and characterized, little is known about the genetic basis of structural P-proteins in other plant families. The only P-protein to be characterized thus far is phloem protein 1 (PP1) from Cucurbita maxima . Immunological studies identified this filamentous protein in SE slime plugs and P-protein bodies, although the corresponding mRNA was shown to accumulate in companion cells .
In this study, we report the identification of several new SEO genes in Fabaceae and, most interestingly, also in non-Fabaceae plants (which do not possess forisomes). The unexpected occurrence of SEO genes in plant families lacking forisomes may argue that these genes encode other structural P-proteins, which implies a common phylogenetic origin. To further characterize the newly-identified SEO genes, we analyzed their gene structure and genomic synteny using bioinformatics and studied their expression by RT-PCR. Selected SEO genes were also studied by promoter analysis in transgenic plants.
The SEO gene family in Fabaceae
Next, we set out to establish whether any of the newly-identified SEO proteins were present in the forisomes of either M. truncatula or G. max. Purified forisomes from each species were separated by SDS-PAGE and peptide sequences were generated for the 75-kDa protein band by ESI-MS/MS following established protocols [12–14]. Peptide sequences derived from the M. truncatula forisomes (Additional file 2) indicated the presence of one further SEO protein in addition to the known components MtSEO1-3 [13–15]. Similarly, four of the 26 G. max SEO proteins were identified in G. max forisomes, where no components had been previously identified (Additional file 2). These results suggest that many of the SEO genes do not encode forisome component proteins, although it is possible that they are present but at levels below our detection threshold. In order to distinguish SEO proteins known to encode forisome components from other SEO proteins, we propose that MtSEO1-3 should be renamed MtSEO-F1, MtSEO-F2 and MtSEO-F3, and that the newly identified component should be named MtSEO-F4 (SEO-F, Sieve Element Occlusion by Forisomes). The four SEO proteins found in purified G. max forisomes should similarly be designated GmSEO-F1-F4. For all other Fabaceae SEO proteins (and SEO proteins from other plant families, see below) whose function is currently unknown, we recommend the temporary assignment of a lower case letter (SEOa, SEOb, etc) until their presence in the forisome can be confirmed (in which case they will be assigned an SEO-F number) or another function is determined (in which case additional functional categories can be introduced).
The SEO gene family in non-Fabaceae
Using the Fabaceae SEO sequences described above, we screened the genomes of several dicotyledonous non-Fabaceae plants, i.e. Arabidopsis thaliana (Brassicaceae), Vitis vinifera (Vitaceae) and Solanum phureja (Solanaceae) as well as an EST collection for Malus domestica (Rosaceae). This identified three A. thaliana genes (designated AtSEOa-c), 13 V. vinifera genes (designated VvSEOa-m) and three S. phureja genes (designated SpSEOa-c) (Additional file 1). Two full-length SEO genes, designated MdSEOa and b (Additional file 1), and further partial fragments were found in the M. domestica EST collection. Potential SEO gene fragments of several other angiosperm plants could also be identified by BLAST search in NCBI GenBank (data not shown). In contrast, no SEO genes were identified in the yet sequenced genomes of the monocotyledons Oryza sativa, Brachypodium distachyon, Zea mays and Sorghum bicolor nor in the moss Physcomitrella patens. RT-PCR confirmed that all the A. thaliana, S. phureja and M. domestica genes were expressed with the exception of AtSEOc, which appears to be a pseudogene. The expression profiles of VvSEOa-m were not determined due to the lack of the sequenced genotype in our laboratories.
Phylogenetic relationships among SEO genes from Fabaceae and non-Fabaceaeplants
The phylogenetic relationships among the SEO genes were calculated by creating a maximum likelihood tree from an alignment of all SEO protein sequences. To provide further support for the tree topology we clustered the proteins into subgroups with OrthoMCL (Figure 1). The recently reported forisome protein VfSEO-F1 (formerly known as VfFOR1) from Vicia faba  and the potential forisome proteins CgSEOa (formerly known as CgFOR1) from Canavalia gladiata  and PsSEOa (formerly known as PsSEO1) from Pisum sativum  were also included in the phylogenetic tree. With the exception of MtSEO-F3, all SEO-F proteins clustered in subgroup 1. In addition, several potential pseudogenes as well as one GmSEO protein (GmSEOu) clustered within this group. It should be noted that GmSEOu and GmSEO-F2 share several identical forisome-specific peptide sequences, so GmSEOu is likely to be involved in the formation of forisomes. Subgroups 2 and 3, found on the same branch of the tree, contain predominantly G. max SEO proteins of yet unknown function (the only exception is MtSEO-F3). Thus, subgroups 1-3 appear to be Fabaceae-specific. Subgroup 4 contains nine Fabaceae SEO proteins and both SEO proteins (MdSEOa and b) from the closely related Rosaceae, whereas subgroup 5 contains SEO proteins from all plant families included in the study that have sequenced genomes. AtSEOa is the only member of subgroup 6, which is closely related to subgroup 5. Subgroup 7 contains SEO proteins from G. max, V. vinifera and A. thaliana.
Expression profiles and promoter activities of the SEOgenes
Domain analysis of SEO proteins
Motif search in SEO proteins
Next, we set out to identify motifs (shorter in length than the domains described above) that are specific for the SEO protein family. Therefore, highly-conserved regions were chosen from an alignment of SEO proteins and were used to construct Hidden Markov Model profiles which characterize the motifs. We identified numerous motifs that appeared to be unique to SEO proteins (Figure 6B) as they could not be detected in any other proteins from any of the other plants included in the analysis. The C-terminal M1 motif, containing several conserved cysteine residues, was representative of the entire SEO family, whereas M2 and M3 appeared to be specific for SEO subgroups, perhaps indicating structural and/or functional specialization. The M4 motif, which was 20-35 amino acids in length and matched an in-house database of disordered regions, was found in the N-terminal portion of SEO proteins in subgroups 5 and 6 (Figure 6B) and could reflect intrinsic disorder within this part of the protein. The individual position of the motifs is indicated in Figure 6A.
Finally, we investigated the organization of SEO genes in the M. truncatula, G. max, A. thaliana and V. vinifera genomes (M. domestica and S. phureja were excluded due to the lack of annotated genomic data). As shown in Additional file 5, most SEO genes appear to be organized in clusters. Seven of nine M. truncatula SEO genes (MtSEO-F1, 2, 4 and MtSEOa-d) are clustered in a 150-kbp segment of chromosome 1, in G. max, five SEO genes are found in a 50-kbp cluster on chromosome 10, while another six are found in a 55-kbp cluster on chromosome 20. Eight grapevine SEO genes are clustered in a 170-kbp segment of chromosome 14 and two of the three A. thaliana SEO genes are adjacent on chromosome 3.
Forisomes are specialized P-proteins found solely in the Fabaceae. Genes encoding forisome components were recently identified in M. truncatula, and are the founder members of the sieve element occlusion (SEO) gene family [13–16]. In this study, we identified many additional SEO genes in the genomes of two Fabaceae and, interestingly, also in several non-Fabaceae plant families (which do not possess forisomes). Even within the Fabaceae, it appears that only some of the newly-identified SEO genes encode forisome components. Phylogenetic analysis showed that all the known and novel SEO genes can be clustered in seven subgroups (Figure 1) and they have a similar exon-intron structure (Figure 2). Their expression is likely to be phloem-specific (Figure 3) most probably restricted to immature sieve elements (SEs) as shown for one Fabaceae and one non-Fabaceae SEO gene (Figure 4) and the proteins contain highly conserved domains and motifs (Figures 5 and 6).
Forisomes have been described as a special type of P-protein mainly based on their morphological characteristics. Like forisomes, P-proteins of various species were reported to accumulate in immature sieve elements  and in their differentiated state both types of P-protein share the same function of blocking sieve elements after phloem injury . Furthermore, forisomes and P-proteins show very similar ultrastructural characteristics in both the condensed and dispersed states [24, 25]. A calcium-induced reactivity, as known for forisomes, is also discussed for P-proteins [26, 27]. Although the dispersion process is similar in each case, the complete reversibility of the conformational switch is unique to forisomes . It therefore seems likely that the non-forisome SEO genes in the Fabaceae, and all the SEO genes in non-Fabaceae plant families, encode other (non-forisome) P-proteins. It should be noted that the Cucurbita maxima PP1 protein, which is the only non-forisome P-protein to be characterized thus far, shares neither significant sequence similarities nor any conserved domains with the SEO proteins described herein. For this reason, PP1 should not be assigned to the SEO gene family, despite its potential functional similarity. Additionally, we and others  have not identified any PP1 orthologs in the genomes of non-Cucurbitaceae plants, suggesting PP1 may play a unique role in the phloem of the Cucurbitaceae family.
We used a number of bioinformatics approaches to identify functional motifs and domains conserved in all SEO proteins or in particular phylogenetic clades. We identified a 'potential thioredoxin fold' domain common to all SEO proteins, which is also found in enzymes that catalyze disulfide bond formation . However, the canonical thioredoxin fold contains two central cysteine residues that are not present in the SEO proteins, indicating some functional divergence. Interestingly, thioredoxin folds lacking cysteines have also been found in other calcium-binding proteins such as calsequestrin . Calsequestrin has three such domains that condense to form an acidic platform for high-capacity but low-affinity calcium adsorption that is most likely non-specific . Calcium binding in forisomes is also weak and probably non-specific given that other divalent cations can also induce the typical conformational change [31, 12]. The core of the calsequestrin thioredoxin fold domain is a five-strand β-sheet sandwiched by four α-helices . A similar arrangement of α-helices and β-sheets is predicted within the thioredoxin domain of all SEO proteins analyzed with I-TASSER, which suggests that the modified thioredoxin fold could also be involved in calcium binding and the subsequent dispersion of forisomes and other P-proteins. Although thioredoxin folds are found in many different proteins, the presence of this domain together with the two PfamB domains we identified seems to be a unique characteristic of SEO proteins. Single PfamB domains were also identified in Fabaceae non-SEO proteins, but not in non-Fabaceae plants, which suggests the transfer of these domains from SEO to non-SEO genes by domain rearrangement [32, 33].
All the SEO proteins contain the conserved C-terminal motif M1, which is characterized by four spatially conserved cysteine residues (Figure 6B). Although these residues could form disulfide bridges, this requires an oxidizing environment and disulfide bridges are generally not present in cytosolic proteins . However, given the unique function of P-proteins and forisomes, it is possible disulfide bridges could form when the redox state of the cytosol is disrupted by cellular damage, stabilizing the dispersed state of SEO proteins and allowing them to seal off the injured sieve element. Indeed, forisome reactivity declines significantly in the presence of oxygen . We identified several other motifs that were representative of SEO protein subgroups, e.g. motif M4 with potential intrinsic disorder, found at the N-terminus of subgroups 5 and 6 (Figure 6B). Disordered regions do not have a fixed three-dimensional structure, but can be involved in a variety of different molecular processes such as DNA/RNA or protein binding . However, their precise role(s) in SEO proteins remains unclear.
The distribution and organization of SEO genes in M. truncatula and G. max genomes indicates that several gene duplication events have occurred during the evolution of the Fabaceae SEO-F genes. With one known exception, M. truncatula SEO genes are clustered on chromosome 1 (the location of MtSEO-F3 is unknown), suggesting proliferation through tandem duplication. There is evidence that most Fabaceae share a common whole genome duplication event, which occurred approximately 50-60 million years ago, before G. max and M. truncatula diverged from a common ancestor [36, 37]. Although this event cannot be verified by analyzing the position of SEO genes in the M. truncatula genome, it is possible that the evidence has been obscured by incidental gene loss events, since orthologs of several of the SEO genes present in G. max and other plants are missing in M. truncatula. For G. max, an additional whole genome duplication event is thought to have occurred ~15 million years ago [38, 39]. This is supported by the observed synteny in the SEO gene clusters on M. truncatula chromosome 1, G. max chromosome 10, and the paralogs on G. max chromosome 20 (Figure 7). Definitive G. max orthologs could be identified for five of the nine M. truncatula SEO genes, and most of them exist as paralogs in G. max, leading to the conclusion that they were present prior to the split of the two Fabaceae and therefore the duplication event in G. max. No equivalent to the SEO gene cluster in A. thaliana or the arrangement of SEO genes in V. vinifera could be identified in G. max or M. truncatula, as their orthologs are not organized in clusters in both Fabaceae. Therefore it seems likely that additional and independent gene duplication and reorganization events affected the SEO genes in A. thaliana and V. vinifera. We also identified a number of potential SEO pseudogenes, five of which are found in G. max, which probably could be maintained because of the functional redundancy created by gene duplication. Nevertheless, we cannot exclude the possibility that at least the expressed pseudogenes ('pot. ψe') have evolved to take on novel functions, such as the regulation of gene expression [40, 41]. SEO genes appear to be widespread in dicotyledonous plants and may therefore provide a powerful new tool to study the evolution of gene families in dicotyledons.
We provide evidence that SEO genes are widely distributed in non-Fabaceae species and most probably encode P-proteins. The strong conservation of the gene structure, protein motifs and domains, the phylogenetic profile and the genomic synteny indicate a common phylogenetic origin for all SEO genes. Numerous tandem gene and whole genome duplication events appear to have contributed to the evolution of forisome genes in Fabaceae. We identified a fourth M. truncatula gene encoding a forisome component and presented the first analysis of forisome genes in G. max.
Identification of gene family members
Protein models were obtained from the sequenced genomes of Medicago truncatula , Glycine max , Arabidopsis thaliana , Vitis vinifera , Oryza sativa , Sorghum bicolor , Zea mays , Brachypodium distachyon  and Physcomitrella patens . In addition, we used a Malus domestica EST collection from the "National Center for Biotechnology Information" and the not yet annotated genome sequence from Solanum phureja . With the previously published forisome proteins MtSEO1, MtSEO2 and MtSEO3 [14, 15] a BLASTP search was carried out against the protein annotations. Hits with significant similarities (E values lower than 1e-10) were analyzed for global similarity by aligning with the MtSEO proteins. Proteins with only local sequence similarities were not added to the SEO family. To identify further members the BLASTP search was repeated with the newly identified proteins. In addition and in case of missing protein annotations (EST data, non-annotated genomes), the full-length cDNA sequences of all identified SEO genes were used in a BLASTN search (threshold E < 0.001). Falsely-annotated SEO genes indicated e.g. by the presence of shortened open reading frames were re-annotated from genomic sequences with FGENESH or by aligning the genomic sequence with cDNA sequences of other SEO genes. For the identification of correctly spliced cDNA sequences, three independent cDNAs from total seedling RNA (see subheading "Expression analysis") were used for full-length amplification of the corresponding SEO genes using the oligonucleotides listed in Additional file 6. Sequence-verified SEO genes were deposited in GenBank (accession numbers are listed in Additional file 1). The exon-intron structure of the SEO genes was identified by comparing genomic and cDNA sequences. For MtSEO-F3, and the MdSEO and SpSEO genes, for which no or only preliminary genomic sequence data were available, genomic clones were amplified de novo by PCR.
Forisome isolation and peptide sequencing
Forisomes were isolated from purified M. truncatula and G. max phloem tissue (see next section) by density gradient centrifugation according to established protocols . After fractionation by SDS-PAGE the major 75-kDa protein band was excised from the gel matrix, purified and subsequently characterized by ESI-MS/MS as described [13, 14]. The resulting peptide masses were screened against a database containing the SEO proteins and only those peptides unique for a single SEO protein were considered for further analysis.
Phloem-enriched tissue was prepared from S. phureja and G. max cv. Williams 82 by scraping the inner side of peeled stem rinds with a scalpel. The remaining stem rind was used as phloem-deficient material. M. truncatula cv. Jemalong A17 phloem was enriched by cutting stems in half longitudinally, removing the pith and scraping off the cortex with a scalpel. The cortex of the stem was used as phloem-deficient material. For A. thaliana cv. Col-0 phloem-enriched tissue was obtained by cutting out midribs from young leaves. For control experiments with phloem-deficient material we used parts of leaves lacking visible veins.
Total RNA was isolated from tissues ground to powder under liquid nitrogen using the NucleoSpin RNA® Plant Kit (Macherey-Nagel, Düren, Germany). Total RNA was reverse transcribed with SuperScript II (Invitrogen, Karlsruhe, Germany) following the manufacturer's instructions. For all PCRs, partial but intron-spanning parts of the SEO genes were amplified using the oligonucleotides listed in Additional file 6. The integrity of all PCR products was verified by sequence analysis. If no products were generated, additional PCRs were performed using different combinations of primers on cDNA derived from young and old leaves, shoots, roots, buds and flowers. Only if no product was detected in this additional experiment were the corresponding SEO genes designated as potential pseudogenes. Expressed SEO genes containing frameshift mutations were designated as potential expressed pseudogenes. The expression of M. truncatula GAPDH , A. thaliana ACT2 , S. phureja GAPDH  and the G. max F-box gene  were used as positive controls.
The PAtSEOa-GFPER construct was generated by amplifying a 997-bp AtSEOa promoter-specific fragment (PAtSEOa) from A. thaliana genomic DNA, while the PGmSEO-F1-GFPER construct was generated by amplifying a 2500-bp GmSEO-F1 promoter-specific fragment (PGmSEO-F1) from G. max genomic DNA (oligonucleotides listed in Additional file 6). Both PCR products were digested with KpnI and XhoI and inserted into the corresponding restriction sites of pBSGFPER, containing the downstream ER-targeted GFP coding region . The promoter-GFPER constructs were then excised and transferred into the KpnI/HindIII sites of the binary vector pBIN19  to obtain pBPAtSEOa-GFPER and pBPGmSEO-F1-GFPER, respectively. The binary vector pBPAtSEOa-GFPER was introduced into Agrobacterium tumefaciens LBA4404  and transformation of A. thaliana was carried out by floral dip . Seeds were sterilized and germinated on Murashige and Skoog medium  supplemented with 50 μg/ml kanamycin for the selection of transgenic plants. GFPER expression was monitored in transverse and longitudinal stem sections by confocal laser scanning microscopy (CLSM; Leica TCS SP5 X, Wetzlar, Germany; excitation 488 nm, emission 500-600 nm). Sieve plates were stained with a 0.01% aniline-blue solution according to established protocols  and visualized by CLSM (excitation 364 nm, emission 470-530 nm). The binary vector pBPGmSEO-F1-GFPER was introduced into A. rhizogenes strain NCPPB2659 and transgenic G. max roots were obtained following established protocols . Longitudinal sections of the roots were analyzed as above.
The OrthoMCL program  was used to cluster SEO proteins into subgroups, with an inflation parameter of 3. The protein sequences were aligned with T-Coffee  and the alignment was end trimmed to start and end with the domains predicted for all SEO proteins (SEO-NTD and SEO-CTD). The optimal evolutionary model for the family was calculated from this alignment with ProtTest . RAxML  was used for tree building with the evolutionary model parameter JTT+F+I+G and a bootstrap of 1000. The best tree was visualized with FigTree .
Domain annotation was achieved by screening the NCBI Conserved Domain Database (v2.17)  in combination with CD-Search  and the PfamA and PfamB databases v23.0 , using a significance threshold of 1e-05. Domain arrangements were visualized using Jangstd . HMMER 3.0 beta 2 was used to construct Hidden Markov Models (HMMs) and carry out searches . Unique motifs in the SEO family were identified by extracting partial alignments to construct HMMs. Three dimensional protein structure prediction was carried out with I-TASSER . The resulting protein structures were compared to structures in the Protein Data Bank (PDB) using the program TM-align . Sequence logos of alignments were generated with WebLogo . To identify regions of disorder, an in-house database was created by scanning protein sequences from annotated plant genomes with VSL2B . Disordered sequences with a length of at least 20 amino acids were clustered with cd-hit , aligned and used to build HMMs. Disordered regions in the SEO family were predicted with this set of HMMs. All models were tested against SEO proteins as well as all available protein predictions for the plants included in our investigation.
The technical assistance of Ann-Christin Müller and the help of Sarah Weiss is gratefully acknowledged. This work was supported by grants from the VolkswagenStiftung (contract no. I/82 075) and the Fraunhofer Society. EBB and ARR acknowledge support from the Human Frontier Science Program.
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