- Research article
- Open Access
Characterization and analysis of the cotton cyclopropane fatty acid synthase family and their contribution to cyclopropane fatty acid synthesis
© Yu et al; licensee BioMed Central Ltd. 2011
- Received: 15 March 2011
- Accepted: 25 May 2011
- Published: 25 May 2011
Cyclopropane fatty acids (CPA) have been found in certain gymnosperms, Malvales, Litchi and other Sapindales. The presence of their unique strained ring structures confers physical and chemical properties characteristic of unsaturated fatty acids with the oxidative stability displayed by saturated fatty acids making them of considerable industrial interest. While cyclopropenoid fatty acids (CPE) are well-known inhibitors of fatty acid desaturation in animals, CPE can also inhibit the stearoyl-CoA desaturase and interfere with the maturation and reproduction of some insect species suggesting that in addition to their traditional role as storage lipids, CPE can contribute to the protection of plants from herbivory.
Three genes encoding cyclopropane synthase homologues GhCPS1, GhCPS2 and GhCPS3 were identified in cotton. Determination of gene transcript abundance revealed differences among the expression of GhCPS1, 2 and 3 showing high, intermediate and low levels, respectively, of transcripts in roots and stems; whereas GhCPS1 and 2 are both expressed at low levels in seeds. Analyses of fatty acid composition in different tissues indicate that the expression patterns of GhCPS1 and 2 correlate with cyclic fatty acid (CFA) distribution. Deletion of the N-terminal oxidase domain lowered GhCPS's ability to produce cyclopropane fatty acid by approximately 70%. GhCPS1 and 2, but not 3 resulted in the production of cyclopropane fatty acids upon heterologous expression in yeast, tobacco BY2 cell and Arabidopsis seed.
In cotton GhCPS1 and 2 gene expression correlates with the total CFA content in roots, stems and seeds. That GhCPS1 and 2 are expressed at a similar level in seed suggests both of them can be considered potential targets for gene silencing to reduce undesirable seed CPE accumulation. Because GhCPS1 is more active in yeast than the published Sterculia CPS and shows similar activity when expressed in model plant systems, it represents a strong candidate gene for CFA accumulation via heterologous expression in production plants.
- Cyclopropane Fatty Acid
- Cyclic Fatty Acid
- Sterculic Acid
Fatty acids containing three-carbon carbocyclic rings, especially cyclopropane fatty acids, occur infrequently in plants and their major plant producers include Malvaceae, Sterculiaceae, Bombaceae, Tilaceae, Gnetaceae and Sapindaceae [1–4]. They can represent a significant component of seed oils and accumulate to as much as 40% in Litchi chinensis [1, 5].
Cyclopropane synthases (CPSs) catalyze the cyclopropanation of unsaturated lipids in bacteria [6, 7], plants [8, 9] and parasites . There are two principle classes of bacterial cyclopropane synthases: the Escherichia coli cyclopropane synthase (ECPS) type that uses unsaturated phospholipids as substrates and Mycobacterium tuberculosis cyclopropane mycolic acid synthases (CMAs) that perform the introduction of cis-cyclopropane rings at proximal and distal positions of unsaturated mycolic acids [11–14]. Despite their different substrates the two classes of enzymes share up to 33% sequence identity suggesting a common fold and reaction mechanism. Moreover, a shared reaction mechanism is suggested by the fact that both E. coli CPS and M. tuberculosis CMA active site residues are almost completely conserved and harbor a bicarbonate ion in their active site [15, 16].
Although CPA had been identified in a few plant seeds as early as 1960s , the key gene responsible for their biochemical synthesis was not identified for more than three decades when Bao et al.  identified a cyclopropane synthase from S. foetida. The SfCPS is a microsomal-localized membrane enzyme, which catalyzes the addition of a methylene group derived from S-adenosyl-L-methionine across the double bond of oleic acid esterified to the sn-1 position of PC . The S. foetida enzyme is the first plant CPS that has been characterized, the other plant CPS has been reported to date is from Litchi sinensis (WO/2006087364).
E. coli CPS is thought to be involved in the long-term survival of non-growing cells and its expression can be associated with environmental stresses . Plant CPEs inhibit some insect stearoyl-CoA desaturases thereby interfering with their maturation and reproduction, suggesting that in addition to their role as storage lipids, CPE can also serve as protective agents. CPE are also strong inhibitors of a variety of fatty acid desaturases in animals [18–21], and feeding animals with CPE -containing oilseeds, such as cotton seed meal, leads to accumulation of hard fats and other physiological disorders [20, 22, 23]. For the same reasons vegetable oils that contain CPE must be treated with high temperature hydrogenation before human consumption. These treatments add to processing costs and also result in the accumulation of undesirable trans-fatty acids. Therefore, reducing the levels of CPE in cotton seed oil by gene-silencing or other techniques could reduce processing costs and the associated production of undesirable trans fatty acids as well as increasing the value of processed seed meal for food consumption (US2010/0115669).
Cyclic fatty acids, especially CPA such as dihydrosterculic acid, are desirable for numerous industrial applications and therefore it would be useful to identify candidate enzymes for heterologous expression in production plants with the goal of optimizing the accumulation of CPAs. CPAs have physical characteristics somewhere in between saturated and mono unsaturated fatty acids. The strained bond angles of the carbocyclic ring are responsible for their unique chemistry and physical properties. Hydrogenation results in ring opening to produce methyl-branched fatty acids. These branched fatty acids have the low temperature properties of unsaturated fatty acids, but unlike unsaturated fatty acids, their esters are not susceptible to oxidation and are therefore ideally suited for use in lubricant formulations  (WO 99/18217). Moreover, the methyl branched fatty acids are an alternative to isostearic acids that are used as cosmetics. Oils with high levels of cyclopropene fatty acids self-polymerize at elevated temperatures because the cyclopropene ring is highly strained and readily opens in an exothermic reaction. This property makes CPE particularly suitable for the productions of coatings and polymers. Sterculic acid (18-carbon cyclopropene) also has potential applications as a biocide in fatty acid soap formulation (US2008/0155714A1).
In this study, we identify three CPS isoforms from cotton and analyze their expression in different tissues to help define their physiological roles. We also present an analysis of the consequences of over-expressing cotton GhCPS1, 2 and 3 in yeast, tobacco suspension cells and Arabidopsis.
Plant growth conditions and transgenic analyses
Arabidopsis and camelina plants were grown in walk-in-growth chambers at 22°C for 16 h photoperiod, and cotton plants were grown in the greenhouse at 28°C for 16 h photoperiod. The full length cDNA corresponding to GhCPS1[GenBank:574036.1], GhCPS2 [GenBank:574037.1] and GhCPS3[GenBank:574038.1] genes were PCR amplified using gene specific primers with restriction site-encoding linkers and subsequently digested and cloned into pYES2 (Invitrogen) via corresponding SacI and EcoRI restriction sites. For Arabidopsis and camelina transformation, the genes were cloned downstream of the phaseolin seed-specific promoter in binary vector pDsRed . These binary vectors were introduced into Agrobacterium tumefaciens strain GV3101 by electroporation and were used to transform Arabidopsis via the floral dip method , and camelina through vacuum infiltration . Seeds of transformed plants were screened under fluorescence, emitted upon illumination with green light from a X5 LED flashlight (Inova) in conjunction with a 25A red camera filter . For tobacco Bright Yellow 2 (BY2) transformation, GhCPS1 was cloned into pBI121 using BamHI and SacI sites, and GhCPS2 and 3 were cloned into pBI121 using the XbaI and SacI restriction sites and transformed into BY2 cells. After 4-5 months kanamycin selection, stable transformed cell lines were collected 7 days after subculture and analyzed for fatty acid composition. The composition of pBI121-containing negative control lines were compared with lines transformed with SfCPS in pBI121.
Primers used in this study for:
GCPS1-Y2F: ACCGGAGCTCAcca t g g a a g t g g c c g t g a t c g
GCPS2-Y2F: ACCGGAGCTC Acca t g g a a g t g g c g g t g a t c g
GCPS1+2-R: CCGGAATTC t c a a t c a t c c a t g a a g g a a t a t g c
GCPS3-Y2F: ACCGGAGCTC AccATGGGTa t g a a a a t a g c a g t g a t a g g a g g a g
GCPS3-R: CCGGAATTC t t a a g a a g c t g a g g g g a a g t c t t t
GCPS1-5'PacI: tcccTTAATTAA a t g g a a g t g g c c g t g a t c g
GCPS2-5'PacI: tcccTTAATTAA a t g g a a g t g g c g g t g a t c g
GCPS1+2-3'XmaI: tcccCCCGGG t c a a t c a t c c a t g a a g g a a t a t g
SfCPS-5'PacI: tcccTTAATTAA a t g g g a g t g g c t g t g a t c g
SfCPS-3'XmaI: tcccCCCGGG t c a a t t a t c c g a g t a g g a a t a t g c
GCPS3-5'PacI: tcccTTAATTAA a t g a a a a t a g c a g t g a t a g g a g g a
GCPS3-3'XbaI: GCTCTAGA t t a a g a a g c t g a g g g g a a g t c t t t
Tobacco BY2 transformation
GCPS3-5'XbaI: GCTCTAGA a t g a a a a t a g c a g t g a t a g g a g g a
GCPS3-3'SacI: ACCGGAGCTC t t a a g a a g c t g a g g g g a a g t c t t t
GCPS2-5'XbaI: GCTCTAGA a t g g a a g t g g c g g t g a t c g
GCPS1+2-3'SacI: ACCGGAGCTC t c a a t c a t c c a t g a a g g a a t a t g
GCPS1-5'BamHI: CGCGGATCC a t g g a a g t g g c c g t g a t c g
SfCPS-5'XbaI: GCTCTAGA a t g g g a g t g g c t g t g a t c g
SfCPS-3'SacI: ACCGGAGCTC t c a a t t a t c c g a g t a g g a a t a t g c
Sequence homologous to the CPS sequences in lower case, flanking sequences in boldface, restriction site sequence underlined.
Phylogenetic and sequence analysis
Phylogenetic analysis of the cyclopropane fatty acid synthase (CPS) family was conducted by using full length protein sequences from cotton and cyclopropane fatty acid synthase from Sterculia, E. coli, Agrobacterium, Mycobacterium, and Arabidopsis. Full-length amino-acid sequences were first aligned by CLUSTALW version 2.0.12 (Additional file 1)  with default parameters (http://www.ebi.ac.uk/Tools/clustalw/), and imported into the Molecular Evolutionary Genetics Analysis (MEGA) package version 5.0 . Phylogenetic and molecular evolutionary analyses were conducted using the neighbor-joining (NJ) method  implemented in MEGA, with the pair-wise deletion option for handling alignment gaps, and the Poisson correction model for computing distance. The final tree graphic was generated using TreeView program .
RNA extraction, Reverse transcription and quantitative PCR analyses
RNA from cotton leaf, flower, stem and seeds at different development stages were extracted according to Wu et al.  and RNA from cotton root was isolated using Qiagen's plant Mini RNA kit. RNA quality and concentration were determined by gel electrophoresis and Nanodrop spectroscopy. Reverse transcription (RT) was carried out using Qiagen's QuanTect Reverse Transcription Kit. Quantitative PCR analyses were carried out using Bio-RAD iQ™ SYBR Green Supermix as described in Additional file . Primers ubiq7-1F (5'- GAAGGCATTCCACCTGACCAAC -3') and Ubiq7-1R (5'- CTTGACCTTCTTCTTCTTGTGCTTG -3') were used to amplify ubiquitin 7 as internal standard. Gene-specific primers used for qRT-PCR analysis were qGCPS-1F(5'- TTAAGTGGTCAACCGGCCATGCAA -3') and qGCPS-1R (5'-TTCTTTGGACTGGGCGGAACAGAA -3'), qGCPS2-1F (5'-ATATTCCCTGGAGGAACC CTG CTT-3') and qGCPS2-1R (5'-AAACCGGCAGCGCAGTAATCGAAA-3') for GhCPS2, and qGCPS3-1F (5'-ACTGGTTGCGAGGTGCATTCTGTT-3) and qGCPS3-1R (5'-TTGGAAAGCGCCAAGCACTGTTGA -3') for GhCPS3.
Fatty acid analyses
Yeast culture, expression induction, and fatty acid analyses were carried out as described . Lipids were extracted in methanol/chloroform (2:1) from 0.1 g of fresh weight cotton tissue and internal standard heptadecanoic acid was added. The isolated lipid was methylated in 1% sodium methoxide at 50°C for 1 hr and extracted with hexane. To analyze the fatty acids of single seeds, fatty acid methyl esters (FAMEs) were prepared by incubating the seeds with 35 μL 0.2M trimethylsulfonium hydroxide in methanol . For analysis of CFA in BY2 cell lines, FA were extracted from ~0.1 g of BY2 callus tissue, FAMEs were prepared as described above for cotton tissue, or FA dimethyloxazoline (DMOX) derivatives were prepared in a one-pot reaction in which FA are reacted with 2-amino-2-methyl-1-propanol in a nitrogen atmosphere at 190°C for 4 hours . Lipid profiles and acyl group identification were analyzed on a Hewlett Packard 6890 gas chromatograph equipped with a 5973 mass selective detector (GC/MS) and either Agilent J&W DB 23 capillary column (30 m × 0.25 μm × 0.25 μm) or SUPELCO SP-2340 (60 m × 0.25 μm × 0.20 μm) column. The injector was held at 225°C and the oven temperature was varied from 100-160°C at 25°C/min, then 10°C/min to 240°C. The percentage values were converted to mole percent and presented as means of at least three replicates.
The cotton cyclopropane fatty acid synthase family consists of three highly conserved members
The cotton CPSs have two enzymatic domains as reported for the Sterculia CPS ; the carboxy terminus encodes the CPS domain and catalyzes the synthesis of dihydrosterculate while the amino terminus encodes a distinct oxidase domain of unknown function. A sequence proposed to play a role in S-AdoMet binding, VL(E/D)xGxGxG [36, 37], was found in GhCPS3 as ILEIGCGWG and in a more degenerate form (DxGxGxG) in GhCPS1 and 2. Given that S-AdoMet binding and methyl group transfer is the only known function shared between CPS and other S-AdoMet binding enzymes, this segment of the GhCPSs seems very likely to be involved in binding this substrate. It should be noted that all CPS coding sequences lack the phenylalanine residue of the FxGxG, proposed by both Lauster  and Smith et al. . However, this motif has been found only in methyltransferases that act on nucleic acids .
Active site conservation
The crystallized M. tuberculosis CPS structure shows a bicarbonate ion hydrogen-bonded to five active-site residues , including two interactions via backbone amides. In E.coli CPS C139, I268, E239, H266, and Y317 are strictly conserved within all non-plant CPSs . In cotton, the amino acids corresponding to E. Coli I268, E239, and Y317 are conserved in all 3 GhCPS genes, i.e. I739, E710, Y794 for GhCPS1; I731, E702, Y786 for GhCPS2 and I736, E707, Y791 for GhCPS3. However, H266 has been substituted for Q in GhCPS (Q737, I729 and Q734 respectively). Interestingly, C139 remains the same in GhCPS3 as C602, but is substituted for S in the other two GhCPS (S602 in GhCPS1 and S595 in GhCPS2). An E. coli C139S mutant is less active than the wild-type enzyme (its catalytic efficiency is 31%), but addition of bicarbonate increases its Kcat, by a factor of two .
The GhCPS genes exhibit tissue-specific differences in their expression
Expression of GhCPS1 and 2 correlates with cyclic fatty acid accumulation levels
Tissue-specific FA composition of cotton tissues
22.6 ± 0.16
0.4 ± 0.02
16.2 ± 0.43
11.7 ± 0.07
18.9 ± 0.41
10.1 ± 0.32
1.0 ± 0.03
11.9 ± 0.21
6.6 ± 0.13
0.7 ± 0.12
20.7 ± 0.04
1.1 ± 0.01
7.1 ± 0.02
22.7 ± 0.01
20.3 ± 0.06
23.3 ± 0.13
0.8 ± 0.08
3.0 ± 0.02
0.9 ± 0.00
0.1 ± 0.09
23.9 ± 0.18
0.5 ± 0.05
11.0 ± 0.42
11.1 ± 0.43
36.5 ± 0.37
16.1 ± 0.34
0.5 ± 0.04
0.2 ± 0.02
0.4 ± 0.06
18.3 ± 0.24
0.3 ± 0.02
2.5 ± 0.04
19.6 ± 1.00
31.7 ± 0.37
26.8 ± 0.32
0.3 ± 0.06
0.3 ± 0.02
0.2 ± 0.02
20.1 ± 0.19
0.7 ± 0.04
4.8 ± 0.31
15.3 ± 0.72
26.8 ± 0.63
30.7 ± 0.50
0.5 ± 0.03
0.7 ± 0.01
0.4 ± 0.08
21.6 ± 0.43
0.6 ± 0.08
3.8 ± 0.21
13.9 ± 0.64
40.7 ± 0.52
18.4 ± 0.33
0.5 ± 0.17
0.3 ± 0.06
0.2 ± 0.06
20.3 ± 0.14
0.4 ± 0.02
2.2 ± 0.01
17.8 ± 0.05
53.3 ± 0.14
4.0 ± 0.07
0.4 ± 0.02
0.8 ± 0.01
0.3 ± 0.06
0.4 ± 0.08
21.0 ± 0.21
1.0 ± 0.03
2.5 ± 0.35
17.7 ± 0.12
54.4 ± 0.38
1.8 ± 0.25
0.5 ± 0.07
0.5 ± 0.07
0.3 ± 0.14
0.4 ± 0.09
In cotton tissues the abundance of GhCPS1 and 2 transcripts in different tissues is in general agreement with the cyclic fatty acid distribution, while GhCPS3 is expressed at very low levels in the root and stem tissues which are rich in the cyclic fatty acid. This suggests that GhCPS1 and 2 contribute to the CFA production in cotton. Cyclic fatty acids are synthesized very early in seed development, which was detected as early as 0 dpa in the ovule, and increased to peak at 40 dpa and then decreased a little at 50 dpa. This pattern is consistent with the expression pattern of GhCPS1, 2 and 3, but we cannot rule out GhCPS3 involvement in CFA production in the seed.
Expression of GhCPS1 and 2 in yeast demonstrates their physiological activity
Deletion of the N-terminal oxidase domain decreases CPS activity
Co-expression of the Agrobacterium oxidase with the E. Coli CPS resulted in lower CFA accumulation in yeast compared to expression of the CPS alone. A similar decrease was found when the Agrobacterium oxidase was fused with E. Coli CPS protein. Fusion of a plant CPS N-terminal oxidase to E.Coli CPS also inhibited its ability to produce CFA (data not shown). These results demonstrate that unlike the cotton CPSs, E.Coli CPS activity is not enhanced by the oxidase domain. We cloned the Agrobacterium, gene AGR-C-3599p (N-terminal homolog to plant CPS) and AGR-C-3601p (C-terminal homolog to plant CPS) that was located 802 bp upstream of AGR-C-3599p. We also failed to detect any CFA from the ACPS over expression in yeast, and neither co-expression of these two proteins in yeast nor the fusion of these two polypeptides into a single polypeptide yielded any cyclopropane fatty acids (data not shown).
Heterologous expression of GhCPS1 and 2 results in CFA accumulation in plants
When these genes were expressed in BY2 cell lines, ~1.0% of 19C cyclopropane was produced in GhCPS1 lines and SfCPS lines, and only a trace amount of CFA was detected in GhCPS2 transformed BY2 lines. No cyclopropane fatty acid was detected in lines transformed with GhCPS3, the chromatograms of which were indistinguishable from control pBI121-containing lines. In one of the 16 GhCPS2 I733T lines, 2.9% of CFA was detected.
These results demonstrate that GhCPS1 and 2 can cause the accumulation of CPA FA upon heterologous expression in plants.
Blast searching of the cotton genome database using the Sterculia CPS gene resulted in the identification of three cotton CPS homologous genes (GhCPS1, 2, 3). GhCPS1 and 2 show high similarities to the published SfCPS gene and their expression patterns correlate closely with CFA distributions in a variety of tissues. In addition, we confirmed their biochemical identity as cyclopropane synthases in yeast and plant.
Interestingly, GhCPS3's transcription level is relatively low in roots and stems where higher abundance of CFA is found. In addition, when heterologously expressed GhCPS3 did not result in detectable CFA accumulation in yeast, tobacco BY2 cell lines, or in Arabidopsis seeds. A number of plant sequences are related to GhCPS3; for instance, in Arabidopsis, 5 genes clustered in the same clade with GhCPS3 (Fig 1) and are more closely related to GhCPS3 than to SfCPS or to GhCPS 1 and 2. CPA and CPE have not been reported in Arabidopsis so far consistent with our analysis of Arabidopsis seeds and leaves in which we failed to identify any cyclopropane or cyclopropene fatty acids (data not shown). Since GhCPS3 and its Arabidopsis homologues are mainly expressed in leaf, it is possible that they catalyze the formation of other cyclopropanated products .
The role of the N-terminal oxidase portion of the plant-type CPS gene remains to be determined. From an evolutionary point of view, it is interesting to speculate on the origin of the cyclopropane synthases that contain the oxidase domain at the N-terminus. The oxidase gene and CPS gene are located adjacent to one another in the genomes of Agrobacterium and Mycobacteria; in plants the genes are fused to form a single product; taken together this suggests that the N-terminal domain in plants and its homologs in bacteria may play a role(s) related to cyclopropanation . There is a conserved FAD-binding motif in the first 21 aa of the plant oxidase domain. It is hard to envisage how a redox system such as a FAD-containing protein could be involved in the catalytic reaction of methylene addition. After removal of the oxidase portion, the C-terminal CPS portion of GhCPS1 still retains 30% of its activity, showing that the oxidase activity is not necessary for function but that an intact oxidase domain somehow enhances activity perhaps by conferring stability to the CPS polypeptide. Cotton CPS antibodies would be helpful in distinguishing whether the reduction of activity upon partial, or complete deletion, of the oxidase domain results from destabilization of the enzyme or from loss of catalytic activity. It is possible that the oxidase portion plays a potential role in either the desaturation of dihydrosterculic acid to produce sterculic acid or the α-oxidation of the product to form malvalic acid.
Malvalic acid is a predominant CPE in cotton. No chain-shortened CPA was found when GhCPSs are expressed in yeast, tobacco BY2 cell lines, or fad2/fae1 Arabidopsis seeds. The data also show that the presence of CPA was insufficient to induce α-oxidation in these systems. Since neither CPE nor α-oxidation products were observed, we conclude that additional gene products are required for these functions.
A variety of bacteria initiate the cyclopropanation of fatty acids in the stationary phase or upon exposure to low pH [6, 40, 41], osmotic stress [42, 43] and high temperature . The conversion of unsaturated fatty acids into the corresponding CFAs reduces the levels of unsaturated fatty acids in membranes and therefore contributes to a reduction of the membrane fluidity which renders lipid bilayers more rigid . Cyclization of fatty acid acyl chains is therefore generally regarded as a means to reduce membrane fluidity to adapt the cells for adverse conditions . The content of cyclopropane fatty acids with 25 carbon atoms is correlated with early growth in spring for Galanthus nivalis L. and Anthriscus silvestris L. . Lipids esterified with long chain cyclopropane fatty acids could contribute to the physiological adaptations of early spring plants and drought-tolerant plants by reducing membrane permeability to solvent .
CPE inhibited fatty acid desaturation in two fungi of interest to plant pathologists and CPE from Sterculia foetida affected U. maydis, the basidiomycete responsible for corn smut growth and morphology, suggesting that CPE serves as antifungal agent . Study of gene expression changes in Fusarium oxysporum f. sp. vasinfectum-infected cotton seedlings identified GhCPS2 (i.e., CD486555) as having increased expression in cotton roots at 3 days post-infection, together with a bacterially induced lipoxygenase. This makes GhCPS2 one of the few potential defense-related genes induced in infected roots and putative stress-related genes encoding proteins such as glutathione S-transferase (GST) 18 and nitropropane dioxygenase .
We have shown that both GhCPS1 and 2 contribute to CFA accumulation in cotton seeds; but GhCPS1 accounts for the majority of CFA accumulation in roots and stems. The information presented herein has potential uses for two distinct biotechnological applications. It is highly desirable to target both GhCPS1 and 2 for suppression to reduce the CPE content of cottonseed meal for use as animal feed. Conversely, to facilitate CPA accumulation for use as oleochemical feedstocks, our data suggests GhCPS1 to be the best choice for heterologous expression in a production plant.
We thank Dr. Carl Andre at Brookhaven National Laboratory for critical reading of our manuscript, Prof. John Ohlorogge at Michigan State University for SfCPS gene, Prof. Kent Chapman at the University of North Texas for providing us with the cotton seeds, and Mr. Kevin Lutke from Donald Danforth Plant Science Center who helped with the BY2 transformation. This work was supported by the Office of Basic Energy Sciences of the U.S. Department of Energy (JS), and by the National Science Foundation (Grant DBI 0701919) (RR and X-HY).
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