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Content Type. Release Date. We aimed to change cell wall properties by introducing the exogenous elastin motifs, as well as AGP motifs, into the wall through the synthetic extensin analogs . The cell wall properties of three different transgenic cell lines were all changed, but in different ways. One transgenic cell line was observed with a decreased cellulose crystallinity and increased wall xyloglucan content; the second transgenic cell line contained dramatically increased hydration capacity and notably increased cell wall biomass, increased di-isodityrosine, and increased protein content; the third transgenic cell line displayed wall phenotypes similar to wild type cells, except changed extractability of xyloglucan epitopes.
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These results suggest that wall extensin modification has potential as an approach to manipulate wall structure, in some instances resulting in greater sugar release for bioenergy production. Predicted polypeptide sequence of each glycomodule based on previous work on synthetic gene products. However, based on amino acid composition analysis, Pro residues in the elastin motifs were also hydroxylated. Size exclusion chromatograms of the three glycomodules. Each glycomodule was loaded and eluted on a Superose analytical size exclusion column. The retention time of was 28 min, while those of and were 26 min, which is consistent with the calculated molecular weights for Fifty nanograms of each above oligonucleotide were annealed in 1X ligase buffer and ligated to a pUC18 vector between the BbsI and BsmF1 restriction sites.
The 1. All gene sequences were confirmed by DNA sequencing. One hundred nanograms of above each constructed pBI plasmid were transformed into Agrobacterium tumefaciens strain LBA by the freeze-thaw method . The Kanamycin-selected cells were subcultured in SH culture media as described earlier . The culture media of day-old transgenic tobacco BY2 cells were filtered from the cultures, concentrated through rotary evaporation, and dialyzed against distilled deionized d.
Forty grams of above filtered fresh cells from each suspension culture were re-suspended in 80 ml of suspension buffer Tris-HCl, 50 mM, pH 7. The supernatant was discarded. The cell walls were washed with ml of 1 M NaCl and then with 2 L of d. H 2 O until the conductivity of the supernatant equal to that of d. The resulted cell walls were lyophilized. Cell walls used for glycosyl composition, IDT and di-IDT measurement, glycome profiling, sugar release, and solid state NMR analyses were prepared as follows  : Forty grams of filtered cells were washed with mM potassium phosphate pH 7.
The cell walls were washed in order with mM potassium phosphate pH 7. The mixture was incubated at room temperature for 1 hour followed by pelleting the precipitate in a microfuge. The absorbance of the resulting solution was measured at nm . The neutral monosaccharides were analyzed as alditol acetate derivatives by gas chromatography as described . Uronic acids were quantified by colorimetric method using m-hydroxydiphenyl .
The amino acid compositions of glycomodules were analyzed as reported . Hyp contents in cell walls were measured as described earlier . Hydrolysates were dried under nitrogen gas. The residues were separated on a polyhydroxyethyl A 10 nm, 9. In vitro cross-linking reactions of the glycomodules or fusion glycoproteins were carried out using the tomato pI 4. Suspension cultured cells were checked for green fluorescence under a Zeiss LSM laser-scanning confocal microscope, with excitation wavelength at nm and emission wavelength at nm.
The cells were inspected either in SH culture media or in 1 M mannitol for plasmolysis.
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Glycome profiling of wall glycans in the extracts using ELISA analysis to monitor the glycan epitopes were carried out as reported  , . ELISA assays were done on an equal sugar basis. Samples of hydrolysis liquid 0. The calibration of the system was performed with glucose standards. Bruker's TopSpin 2. Five readings were taken at each temperature. After selection via kanamycin resistance, at least two cell lines of each construct were cultured independently in liquid culture media.
The green fluorescent fusion glycoproteins were purified from the culture media as described earlier .
The resulting , , and hybrid glycomodules Fig. The retention times of the glycomodules on SEC were consistent with the estimated molecular weights of Amino acid composition analyses revealed that the mole percent of each amino acid in each glycomodule was close to the predicted composition, except for the higher content of Hyp and lower amount of Pro Table 1. This may have resulted from the hydroxylation of Pro residues in the elastin motifs Fig. The amino acid compositions indicated the glycomodules were the expected gene products and were consistent with earlier results obtained from expression of other extensin and AGP analogs in tobacco cells  —  , .
Glycosyl composition analyses demonstrated that , and all contained mainly arabinose, with lesser amounts of Gal, Rha, and uronic acid residues Table 2.
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Earlier work demonstrated that tomato pI 4. To test the cross-linking possibilities of , , and , each glycomodule was incubated with hydrogen peroxide and the pI 4.
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The reaction mixture was fractionated on an analytical Superose-6 size exclusion column to monitor the time-dependent decrease of , and monomers and the concomitant increase of multimers. The cross-linking rate was determined from calculating the loss of monomer during the reactions .
This result was consistent with our earlier results that larger extensin analogs cross-linked faster than smaller ones . Although elastins share an extended 3-dimensional structure, elastins fold to an ordered beta-spiral structure when the environmental temperature is higher than a certain point, the transition temperature . If the fused elastin motifs in , , and glycomodules would inherit this phase transition feature, the sizes of these glycomodules hence their possible stereo-structure in the walls would be temperature-dependent. We measured the radius of gyration of each glycomodule at different temperatures using dynamic light scattering .
Thus, only the glycomodule might exhibit an elastin-like transition in response to temperature change. Furthermore, the high production of EGFP led to partial separation of the plasma membrane from the walls of the EGFP cells when those cells were checked under normal culture conditions Fig. The Hechtian threads were evident under plasmolyzing conditions Fig.
Except panels D, F, H, and L were taken under normal light, other panels showed cells under excitation at nm and emission at nm. Panel A-H share the same scale bar, and panel I-L share the same scale bar. The deposition of the fusion proteins into the cell walls prompted us to further check the wall physical properties and chemical composition. One striking feature was the change of cell wall volume of EGFP cells, compared to that of wild type tobacco cells and walls isolated from the other transgenic lines, that arose when wall preparations were washed with NaCl solution and water see methods.
Specifically, after breaking 40 grams of filtered fresh cells by sonication, each preparation was centrifuged to pellet the crude walls. To further check the effect of extensin fusion protein deposition on the wall, glycome-profiling of cell wall extracts was used to compare the wall glycan epitopes between the transgenic versus wild type cells  , .
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The amount of material measured as dry weight released from the cell walls of each cell line by each extractant varied significantly Fig. The results, presented in a heatmap, showed that the epitope compositions and extractabilities of xylan, glucan, homogalacturonan HG backbone, and rhamnogalacturonan-I RG-I backbone did not change significantly across all the samples. However, two obvious altered patterns were observed after comparing the heatmaps of the transgenic cell lines versus WT.
The first altered pattern is that the oxalate and carbonate extracts from walls of the EGFP cells contained more arabinogalactan AG epitopes, including epitopes in the antibody groups AG1 to AG4 dotted white box, Fig. Labels at the bottom show reagents used for the different extraction steps.
The amounts of material extracted in each extraction step are indicated in the bar graphs at the tops of the heat maps. Extracts were ELISA-screened using plant cell wall glycan-directed monoclonal antibodies  , . Data are represented as heatmaps. The panel on the right of the heatmaps shows the antibodies that are grouped based on the principal cell wall glycans recognized  , . Strength of ELISA signal is indicated by black-blue-red-bright yellow scale with bright yellow depicting strongest binding and black indicating no binding.
These data support that EGFP fusion proteins were over-deposited into the walls. The second altered pattern is the dramatically increased presence of non-fucosylated xyloglucan epitopes in the chlorite extracts prepared from the EGFP or EGFP cell walls dotted green box, Fig. The modification of cell walls through extensin fusion protein overexpression was further corroborated by analyses of Hyp and protein contents in the walls.
This indicates that overexpression of EGFP significantly increased the protein content of the wall, which might increase the hydration capacity of these cell walls and hence cause the PCV increase.