Functional characterization of a tomato COBRA-likegene functioning in fruit development and ripening
© Cao et al.; licensee BioMed Central Ltd. 2012
Received: 25 November 2011
Accepted: 5 November 2012
Published: 10 November 2012
Extensive studies have demonstrated that the COBRA gene is critical for biosynthesis of cell wall constituents comprising structural tissues of roots, stalks, leaves and other vegetative organs, however, its role in fruit development and ripening remains largely unknown.
We identified a tomato gene (SlCOBRA-like) homologous to Arabidopsis COBRA, and determined its role in fleshy fruit biology. The SlCOBRA-like gene is highly expressed in vegetative organs and in early fruit development, but its expression in fruit declines dramatically during ripening stages, implying a primary role in early fruit development. Fruit-specific suppression of SlCOBRA-like resulted in impaired cell wall integrity and up-regulation of genes encoding proteins involved in cell wall degradation during early fruit development. In contrast, fruit-specific overexpression of SlCOBRA-like resulted in increased wall thickness of fruit epidermal cells, more collenchymatous cells beneath the epidermis, elevated levels of cellulose and reduced pectin solubilization in the pericarp cells of red ripe fruits. Moreover, transgenic tomato fruits overexpressing SlCOBRA-like exhibited desirable early development phenotypes including enhanced firmness and a prolonged shelf life.
Our results suggest that SlCOBRA-like plays an important role in fruit cell wall architecture and provides a potential genetic tool for extending the shelf life of tomato and potentially additional fruits.
The ripening of fleshy fruits involves a number of physiological processes including the production of aromatic compounds, nutrients, pigmentation, and softening of flesh to an edible texture [1, 2]. These processes have direct impacts not only on fruit firmness, color, flavor and nutritional content, but also on shelf life, consumer acceptability, processing qualities, in addition to pre- and postharvest disease resistance [1, 2]. Excessive fruit softening is the main factor contributing to damage during shipping, storage and post-harvest handling . Fruit firmness and texture also affect the integrity of chopped and diced fruit used for canning and fruit products . Because postharvest losses of fresh fruits due to excessive softening can account for as much as 30~40% of total production, considerable research had focused on mechanisms of fruit softening, often using tomato (Solanum lycopersicum) as a model system .
Fruit softening during the ripening process results in part from disassembly of the cell walls, leading to a reduction in intercellular adhesion, depolymerization and solubilization of pectins, depolymerization of hemicelluloses, and loss of pectic galactose side chains . Generally, the decline in fruit firmness due to softening is accompanied by elevated expression of numerous cell metabolism enzymes, including polygalacturonase (PG) [5, 6], pectin methylesterase (PME) , β-galactosidase , as well as cell wall loosening proteins such as expansin [9, 10]. Suppression of single genes encoding fruit PG [3, 11] or PME [7, 12] in transgenic tomato plants had limited impact on fruit softening during ripening, but conferred longer shelf life resulting from reduced susceptibility to postharvest pathogens. These results suggest that suppression of certain enzymes acting on cellulose, hemicellulose or pectin alone are not sufficient to prevent softening, likely due to functional redundancy of enzymes involved in what is likely a complex metabolic process . Nevertheless, a recent study has shown that down-regulation of genes encoding the N-glycan processing enzymes α-mannosidase and β-D-N-acetylhexosaminidase significantly increased fruit shelf life, which was attributed to decreased softening during ripening . These enzymes have been shown to break glycosidic bonds between carbohydrates, or between carbohydrates and noncarbohydrate structural molecules .
Expansins are cell wall-localized proteins faciliating wall loosening. They are involved in many aspects of cell wall modification during development through disruption of non-covalent bonds between matrix glycans and cellulose microfibrils [9, 10, 14, 15]. Transgenic silencing of the tomato expansin gene LeExp1 resulted in increased fruit firmness throughout ripening and improved fruit integrity during storage .
Molecular and genetic investigations have identified additional regulators of cell wall biosynthesis and regulation of cell expansion. One such activity is encoded by the COBRA gene previously reported in Arabidopsis, rice and maize [17–20]. The COBRA gene encodes a plant-specific glycosylphosphatidylinositol (GPI)-anchored protein with a ω-attachment site at the C terminus, a hydrophilic central region, a CCVS domain, a potential N-glycosylation site, an N-terminal secretion signal sequence, and a predicted cellulose binding site . It has been reported that COBRA localizes at the external plasma membrane leaflet through a glycosylphosphatidylinositol (GPI) moiety . Genetic impairment of the COBRA gene results in reduced levels and improper orientation of crystalline cellulose microfibrils in Arabidopsis and rice [17, 18, 22, 23]. Despite the many studies of the COBRA gene in several plant species, little has been learned concerning COBRA ortholog(s) in tomatoes, the model system for fleshy fruit development and ripening. Here we report functional characterization of a tomato COBRA gene (SlCOBRA-like). We specifically demonstrate its role in early fruit development and the potential for enhanced fruit firmness and shelf life by manipulating its expression in maturing transgenic tomato fruits.
COBRA gene family members in tomato
Tomato COBRA gene family in The SOL Genomics Network (SGN) and their sequence characteristics
Gene code (SGN)
N-terminal signal peptide
Potential ω- site
Expression of SlCOBRA-likein tomato
The full-length SlCOBRA-like cDNA was isolated from tomato seedlings by RT-PCR using gene-specific primers (Additional file 2: Table S1). The deduced SlCOBRA-like protein contains a central cysteine-rich domain (CCVS domain), a N-terminal secretion signal sequence for targeting to the endoplasmic reticulum, a highly hydrophobic C terminus and the ω-site required for processing the C-terminal  (Table 1). Moreover, several potential N-glycosylation sites frequently associated with GPI-anchored proteins and extracellular proteins, as well as one HMM-predicted putative cellulose binding domain II (E value =0.018) were observed in the SlCOBRA-like sequence (Additional file 3: Figure S2) [17, 21, 22]. Basic Local Alignment Search Tool (BLAST) analysis showed that SlCOBRA-like shares 63~80% similarity with other COBRA proteins from Arabidopsis, Oryza sativa, and Zea mays[17, 18, 20, 24]. Phylogenetic analysis revealed SlCOBRA-like shares the highest amino acid identity with AtCOBL1, thus localizing within in the same clade (Additional file 1: Figure S1).
Transgenic tomatoes whose endogenous SlCOBRA-likegene was repressed displayed abnormal fruit phenotypes
Because there are 17 COBRA members in tomato, it was necessary to verify the specificity of suppression of SlCOBRA-like in the 3 available CS lines. We examined the expression of SlCOBL1, 2 and 4 in CS fruits. There were two reasons we selected these 3 SlCOBLs: firstly they represent high (86.6% identical), medium (59.9% identical), and low (17.9% identical) similarity to SlCOBRA-like, respectively; secondly the fruit-derived ESTs of these 3 SlCOBLs were found in the SGN EST database, suggesting they are expressed in fruits, the tissues where phenotypes were most apparent. As shown in Figure 2B, the real-time RT-PCR assay indicated the expression of SlCOBL1, 2 and 4 was not affected in CS lines, suggesting that fruit cracking was caused specifically by repression of SlCOBRA-like expression.
Anatomical alterations of fruit pericarps in transgenic plants with altered SlCOBRA-likeexpression
To further compare the pericarp structure of fruits in WT and transgenic lines, detailed cytological analysis was performed. Anatomical paraffin sectioning and Calcofluor staining of pericarp tissue (MG stage, 35 DPA) revealed an increased number of collenchymatous cells beneath the epidermis in the TFM7-OE fruits compared to WT (Figure 3E, F, H, and I). In contrast, the TFM7-CS fruits displayed a "waviness" of the surface with an apparent lack of cuticle and an abnormal shape and size-distribution of the cells, particularly in the thin layer of elongated epidermal cells at the fruit surface. The small cells beneath the epidermis were almost absent in the TFM7-CS fruits and some collapsed parenchymatous cells in the mesocarp were observed in TFM7-CS (Figure 3D and G). Taken together, these results suggest that the SlCOBRA-like gene plays important roles in pericarp cell wall development of immature fruit.
Analysis of cell wall macromolecules by Fourier Transform Infrared (FTIR) spectroscopy
In addition, exploratory principal component analysis (PCA), a statistical method usually used for discriminant analysis of spectroscopic data , was performed for the complete set of 54 FTIR spectra collected from the WT and transgenic pericarp tissues (18 FTIR spectra for each population). Three principal component loadings (PCs) were extracted, among which PC1 accounted for 61.74% of the total variability, and with characteristic cellulose peaks (Figure 4D). The PCA scores of PC1-PC2 or PC1-PC3 revealing the difference between the WT and transgenic pericarp tissues were pronounced (Figure 4B and C): in this scatter plot, the cluster of TFM7-CS samples showed a clear separation from clusters of the WT or TFM7-OE samples, despite the fact that only subtle differences between the WT and TFM7-OE samples were observed, and in which slightly more positive scores were detected in TFM7-OE samples and more negative scores in WT samples (Figure 4B).
Altered cell wall composition of transgenic fruits with altered SlCOBRA-likeexpression at the red ripe (RR) stage
To quantify sugar composition in fruit cell walls, the trifluoroacetic acid (TFA)-soluble (noncellulosic) wall fraction was converted to alditol acetates and analyzed by gas chromatography (GC) . As shown in Figure 5C, the TFM7-OE fruit cell wall was found to possess more noncellulosic glucose (Glc) and cell wall galactosyl (Gal) residues. In contrast, the TFM7-CS fruit cell wall had less arabinose (Ara), Glc or Gal, but more xylose (Xyl).
Altered fruit texture and shelf life in transgenic fruits
Textural analyses of fresh intact fruits from wild-type (WT) and transgenic lines
Penetration massa(g) ±SE
Suppression of SlCOBRA-likeresults in up-regulation of cell wall-degradation and cell wall-based signalling genes
COBRA belongs to a multigene family consisting of 12 members in Arabidopsis, 11 in rice, and 9 in maize [18, 21, 33]. Arabidopsis COBRA, AtCOBL4, maize ZmBK2 as well as rice OsBC1 have been shown to be required for cellulose synthesis [17–20, 24], while Arabidopsis AtCOBL9 and maize ZmBk2L1 impact tip-directed growth during root hair development [34–37]. The newly released tomato genome sequence suggests there are at least 17 COBRA family members in tomato (Table 1). However, the role of COBRA in fruit development and texture has until now remained elusive. Our study addresses this question and provides a potential strategy for manipulation of fruit firmness and shelf life of tomatoes via modification of SlCOBRA-like expression.
The role of the SlCOBRA-likegene in fruit development
The SlCOBRA-like gene was highly expressed during the early stages of fruit development, but the expression level dramatically declined after the breaker stage (Figure 1A), indicating a role in early fruit development. This was further supported by phenotypic analysis of fruit–specific suppression of the SlCOBRA-like gene in the TFM7-CS transgenic tomato plants, which exhibited anatomical changes of fruits during early development. It is worth noting that there are 17 COBRA members in tomato, therefore it was necessary to verify the specificity of suppression of SlCOBRA-like in the 3 TFM7-CS lines to allow accurate interpretation of our results. RT-PCR showed that 3 SlCOBLs, representing the highest, medium and low similarity to SlCOBRA-like, were not repressed in the TFM7-CS fruits (Figure 2B), suggesting suppression of SlCOBRA-like is likely to be specific. However, it is possible that other SlCOBL members also play important roles in fruit development. Further characterization of other SlCOBL members, particularly SlCOBL1/4/9/14 that also contain all characteristic domains of COBRA (Table 1) and are expressed in fruit, will help to address this question.
Effect of the SlCOBRA-likegene on fruit cell wall biosynthesis and integrity
Overexpression of the SlCOBRA-like gene in transgenic fruits resulted in a significant increase of cellulose content (Figure 4A and 5A). On the other hand, more cell wall-bound Na2CO3-soluble pectin and cell wall galactosyl residues were found in the TFM7-OE RR fruit cell wall (Figure 5B, C), indicating that overexpression of SlCOBRA-like is responsible for less cell wall macromolecule solubilization/depolymerization during fruit ripening. These results, together with the cracking phenotype of the TFM7-CS immature fruits, suggest that the SlCOBRA-like protein is involved in not only regulating cellulose synthesis but also maintaining integrity of cell walls during processes of extension and assembly. Previous studies have also demonstrated the complexity of cell wall integrity. For example, the brittle phenotype, observed in rice bc1, Arabidopsis cobl4 and maize bk2, is not necessarily a result of cellulose deficiency because Arabidopsis cellulose synthases (cesAs) mutants with a reduction in cellulose content did not display the brittle phenotype .
SlCOBRA-like may also play an important role in fine-tuning the expression of several genes encoding enzymes involved in cell wall degradation and cell wall-based signalling. PG, TBG4 and LeExp1 mRNAs were elevated in immature TFM7-CS fruits (Figure 7A, B). These genes encode cell wall-degrading proteins and are normally induced at ripening (BR) and throughout later ripening [8, 9, 39]. In contrast, little if any change in expression was detected in PME and TBG6 (Figure 7B), whose expression usually declines rapidly as fruit begin to ripen [8, 40, 41]. It is also notable that the down-regulation of SlCOBRA-like led to elevated expression of genes encoding several receptor-like kinases (RLKs), which can relay a signal to the cytoplasm via the cytoplasmic kinase domain (Figure 7C) [42, 43]. Similar to COBRA proteins, arabinogalactan-proteins (AGPs) usually have an N-terminal GPI anchor site  and play important roles in cell expansion, proliferation and differentiation [44, 45], and signal transmission between the cell wall and cytoplasm . Interestingly, repression of SlCOBRA-like in TFM7-CS immature fruits repressed the mRNA accumulation of tomato LeAGP-S1 encoding the S1 subunit of AGP (Figure 7C), thus suggesting genetic interaction between COBRA and AGP in fruit cell wall development. However, the mechanism underlying this interaction remains to be elucidated.
Overexpression of SlCOBRA-likeenhances fruit firmness and shelf life
The plant cell wall is a highly organized fibrillar network providing mechanical support for cells, tissues, organs and the entire plant body . It was suggested that over 400 annotated proteins are localized in the cell wall (Arabidopsis Genome Initiative [AGI], 2000) and more than 1,000 genes in the genome are implicated in cell wall biogenesis and modification . Moreover, cell wall modifications have been implicated to be the major determinant of fruit softening, although changes in turgor pressure, anatomical characteristics, and cell wall integrity are also likely to play significant roles . In fact, transgenic manipulation of the activities of single cell wall-modifying enzymes in transgenic tomatoes had little impact on fruit softening during ripening . Here we show that although SlCOBRA-like is primarily expressed in early fruit development, it is required for normal fruit softening during ripening specifically through its reduced repression. Enhanced expression of SlCOBRA-like in transgenic TFM7-OE fruits conferred increased fruit firmness and extended postharvest shelf life (Table 2; Figure 6). Increased firmness might be due to both an increase in cellulose content also in addition to changes in pericarp anatomical structure, especially in the form of increased numbers of sub-epidermal collenchymatous cells. It has been shown that changes in pericarp architecture can have a profound impact on fruit firmness. Guillon and co-workers reported that suppression of the tomato DR12 gene (an auxin response factor) caused unusual pericarp cell division and a higher proportion of sub-epidermal collenchymatous cells, resulting in pleiotropic phenotypes including enhanced fruit firmness similar to what we report here for SlCOBRA-like overexpression .
We present data demonstrating that the SlCOBRA-like gene plays an important role in regulation of cell wall architecture during fleshy fruit development. Transgenic plants overexpressing SlCOBRA-like exhibited enhanced fruit firmness and prolonged shelf life. While aspects of regulation of SlCOBRA-like expression and cell wall modification in tomato fruit development remain open to further investigation, our study provides a potential strategy for genetic manipulation of improved fleshy fruit quality and shelf-life via altered COBRA expression.
Tomato plants (Lycopersicon esculentum cv. Alisa Craig) were grown in a greenhouse under natural light and irrigated manually every other day. For cytological, texture, cell wall composition and molecular analysis, fruits of WT and T1 generation transgenic lines were harvested at the immature green (15 DPA), mature green (MG, 35 DPA), Breaker (BR), and Red Ripe (RR, 7 days after BR) stages after tagging of flowers at anthesis.
Amino acid sequence analyses
Signal peptide and GPI modifications were predicted with SignalP Version 3.0 (http://www.cbs.dtu.dk/services/SignalP/)  and big-PI (http://mendel.imp.ac.at/gpi/gpi_server.html) , respectively. N-glycosylation site prediction was performed using NetNGlyc 1.0 (http://www.cbs.dtu.dk/services/NetNGlyc/). SUPER-FAMILY 1.69 (http://supfam.mrc-lmb.cam.ac.uk/SUPERFAMILY/hmm.html)  was used to predict cellulose-binding domains. Protein sequences were aligned using ClustalX  and the resulting alignments were used as input to generate a phylogenetic tree using MEGA2.1 . Statistical confidence of the nodes of the tree are based on 10,000 bootstrap replicates.
The full-length SlCOBRA-like cDNA was isolated by reverse transcription (RT)–PCR from tomato seedlings using PrimeSTAR HS DNA Polymerase (TaKaRa) and gene-specific primers (Additional file 2: Table S1). The cDNA was cloned into the modified binary vector pBI121 to generate an overexpression construct driven by the fruit-specific TFM7 promoter (Accession no.X95261) . Transgenic plants were generated by Agrobacterium tumefaciens-mediated transformation as described by Fillatti et al. . Transformed lines were selected on medium containing kanamycin (70 mg/L) and further confirmed by PCR for the presence of the NPTII (Kanr) marker gene (Additional file 2: Table S1). After RT-PCR analysis to verify SlCOBRA-like mRNA accumulation in the positive transgenic lines, three independent overexpressing lines (TFM7-OE) and three independent co-suppression lines (TFM7-CS) were identified from the primary transgenic (T0) population.
Gene expression analysis
Total RNAs were extracted using Trizol reagent following the protocol provided by the manufacturer (Invitrogen, Carlsbad, CA) and treated with DNase (TaKaRa, Dalian, China). About 1 μg of total RNA from each sample was used for first-strand cDNA synthesis. For real-time quantitative RT-PCR, the PCR reaction was performed using SyBR Green PCR Master Mix (Applied Biosystems) and gene-specific primers (Additional file 2: Table S1) on the iCycler PCR system (BIO-RAD, Hercules, California, USA). Each sample was amplified in triplicate. REST software  was used to quantify the mRNA levels of SlCOBRA-like and other selected genes with the UBI3 gene (Accession no.X58253) serving as the internal reference. Normalization was performed by the 2-Ct method. All primers used in this work are listed in Additional file 2: Table S1.
Histochemical staining and cytology
For fruit epidermis analysis, tissue was carefully isolated from the fruit surface with a razor blade, and the tissue-bound slide was rinsed twice in distilled water and mounted in 15% HCl under a cover slip and photographed using a Leica LDM 2500 microscope. Six exocarp slices from three different fruits per plant were isolated from identical positions of fruits at 15 DPA. For each exocarp slice, epidermal cell size (not including cell wall) and cell separation (spacing distance between cells) were measured at three different positions (10 cells each position) using the ImageProPlus software (IPP6.0, Media Cybernetics, Inc.). Values represent the means of 180 (6×30) cells.
For cytological assesment, three fruits per plant were collected at the MG stage. Fresh hand-cut pericarp sections (~0.1 mm thick) were incubated in a 0.005% aqueous solution of calcofluor (fluorescent brightener 28; Sigma) for 2 min  and visualized with a fluorescent microscope (Leica, Wetzlar, Germany). To examine pericarp cell wall structure, paraffin-embedded transverse sections (10 μm in thickness) were obtained using a Leica microtome (RM 2265, Meyer Instruments, Inc) and stained with safranin O (a cell wall-specific dye), followed by photography using a Leica microscope (LDM 2500).
Cell wall material (CWM) isolation and cell wall component analysis of RR fruit pericarp
Three RR fruits per plant were collected from non-transformed WT and three independent overexpression or co-suppression lines, respectively, and then their pericarp tissues were mixed, rapidly ground into fine powder in liquid nitrogen, and stored at −80°C until use. In each case, approximately 15 g frozen power was incubated with 70% ethanol for 90 min at 70°C to prevent autolytic activity. Insoluble material was washed sequentially with 95% ethanol, chloroform:methanol (1:1, v/v), and acetone. The dried pellets constituted crude cell wall extract/alcohol insoluble solids [AIRs] and were assayed for cellulose content using anthrone as a coloring agent using α-Cellulose (Sigma) as the standard, according to methods described previously .
The remaining AIRs were subsequently extracted with 90% (v/v) dimethyl-sulfoxide (DMSO) for 22 h at room temperature to solubilize starch, ending with two washes of the wall pellets with acetone and dessication in a vacuum oven . The pellets (the cell wall materials, CWM) were stored in a glass desiccator until use. To determine non-cellulosic sugar composition, about 5 mg CWMs was hydrolyzed with 2 M trifluoroacetic acid (TFA) containing 4 mM of myoinositol as an internal standard at 105°C for 3 h, and then the TFA-soluble fraction was converted to alditol acetates and analyzed by gas chromatography as described previously . Equimolar standards were also converted to alditol acetates to calculate response factors for quantitation of mol% relative to the myoinositol standard.
Pectin fractionation was carried out following the procedure of Rose et al. (1998) . About 100 mg CWMs was extracted with water, 50 mM CDTA in 50 mM sodium acetate (pH 6.0), 100 mM Na2CO3 containing 0.1% NaBH4, sequentially. The uronic acid (UA) content in the different pectin fractions was estimated colorimetrically using galacturonic acid as a calibration standard .
For FTIR spectra analysis, 6 RR fruits per plant from three independent overexpression, co-suppression or WT lines were collected, and the corresponding AIRs of pericarps from 54 (6×9) fruits were extracted as described above. In each case, AIR was spread thinly onto a barium fluoride window, dried on the window at 37°C for 20 min. An area of 50×50 μm was selected for analysis by FTIR microspectroscopy . All data sets were baseline-corrected and area-normalized before statistical analyses were applied. Exploratory PCA was carried out using PASW statistics software 18 (formerly known as SPSS Statistics, SPSS Inc.). Reference IR absorption spectra of cellulose were used for peak assignments [27, 58].
Textural and shelf-life analysis
Fruit firmness was determined based on compression mass and skin puncture strength of fresh intact fruits collected at MG (35 DPA) and RR (7 days after Break), using TA-XT Plus (Stable Microsystems Texture Analyser, UK). For the compression test, 15 fruits per plant were assayed at each stage. Each fruit was compressed to a 50% strain at the test speed of 2 mm s−1 with a 100 mm compression platen (P/100) and 10 g of applied force. Skin puncture strength and penetration distance of fresh intact fruits were measured by penetration using a 2mm Cylinder Probe (P/2N) with a trigger force of 5 g, loading at 2 mm s-1 to reach a 50% strain. Each fruit was tested three times at equidistant points along the equatorial plane of the fruit. 6 fruits per plant were taken at each stage. Values represent means ±SE (n=18).
For shelf life, fruits at the RR stage were detached and kept at room temperature (23~25°C and 55~60% relative humidity) for approximately 40 days. 6 replicates were taken for each individual plant. Average fresh weight loss was determined every 5 days until they lost their texture and structural integrity.
Statistical analysis was performed using PASW statistics software 18.0 (formerly known as SPSS Statistics, SPSS Inc.). For analyses of epidermal cells, cellulose content, pectin fractions, and sugar content, significance was calculated using the Student’s t test. For gene expression between WT and CS plants, a multiple comparison was performed by the LSD (Least significant difference) method.
Accession numbers for the SlCOBRA-like sequences reported in this article are BT013422 and JN398667. Other SlCOBL sequences were listed in Table 1. Sequence data in Figure 7 were listed in Additional file 2: Table S1. Other sequence data from this article can be found in GenBank under the following accession numbers:
Arabidopsis AtCOB(At5g60920), AtCOBL1 (At3g02210), AtCOBL2 (At3g29810), AtCOBL4 (At5g15630), AtCOBL5 (At5g60950), AtCOBL6 (At1g09790), AtCOBL7 (At4g16120), AtCOBL8 (At3g16860), AtCOBL9 (At5g49270), AtCOBL10 (At3g20580), AtCOBL11 (At4g27110); Zea mays ZmBK2 (ACF79122.1), ZmBK2L3 (NP_001104946), ZmBK2L6 (NP_001105970), ZmBK2L7 (NP_001105971.1), ZmCOBL4 (EU955798.1); Oryza sativa OsBC1 (Os03g0416200), OsCOBL2 (Os03g0416300), OsCOBL3 (Os05g0386800), OsBC1L6 (Os07g0604300); OsCOBL6 (Os07g0604400).
This work was supported by the National Science Fund for Distinguished Young Scholars (No. 30825030), National Natural Science Foundation of China (No. 31171179), the National Science and Technology Key Project of China (Nos. 2011CB100401, 2009ZX08009-072B and 2009ZX08001-011B) to YL, the University of Idaho internal funding to FX.
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