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Isolation and Characterization of Two Persimmon Xyloglucan Endotransglycosylase/Hydrolase (XTH) Genes That Have Divergent Functions in Cell Wall Modification and Fruit Postharvest Softening.

Han Y, Ban Q, Hou Y, Meng K, Suo J, Rao J - Front Plant Sci (2016)

Bottom Line: We found that DkXTH6 protein was localized in cell wall by its signal peptide, while cytoplasmic DkXTH7 protein contained no signal peptide.When expressed in vitro, the recombinant proteins of both DkXTH6 and DkXTH7 exhibited strict xyloglucan endotransglycosylase (XET) activity but no xyloglucan endohydrolase (XEH) activity.The recombinant protein of DkXTH6 showed a higher affinity with small acceptor molecules than the recombinant DkXTH7.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Crop Stress Biology for Arid Areas, College of Horticulture, Northwest A&F University Yangling, China.

ABSTRACT
Fruit cell wall modification is the primary factor affecting fruit softening. Xyloglucan endotransglycosylase/hydrolase (XTH), a cell wall-modifying enzyme, is involved in fruit softening. In this study, two novel XTH genes (DkXTH6 and DkXTH7) were identified from persimmon fruit. Transcriptional profiles of both of the two genes were analyzed in different tissues of persimmon, and in response to multiple hormonal and environmental treatments [gibberellic acid (GA3), abscisic acid (ABA), propylene, and low temperature]. Expression of DkXTH6 was positively up-regulated during ethylene production and by propylene and ABA treatments, and suppressed by GA3 and cold treatment. In contrast, DkXTH7 exhibited its highest transcript levels in GA3-treated fruit and cold-treated fruit, which had higher fruit firmness. We found that DkXTH6 protein was localized in cell wall by its signal peptide, while cytoplasmic DkXTH7 protein contained no signal peptide. When expressed in vitro, the recombinant proteins of both DkXTH6 and DkXTH7 exhibited strict xyloglucan endotransglycosylase (XET) activity but no xyloglucan endohydrolase (XEH) activity. The recombinant protein of DkXTH6 showed a higher affinity with small acceptor molecules than the recombinant DkXTH7. Taken together with their opposing expression patterns and subcellular localizations, these results suggested that DkXTH6 might take part in cell wall restructuring and DkXTH7 was likely to be involved in cell wall assembly, indicating their special roles in persimmon fruit softening.

No MeSH data available.


Related in: MedlinePlus

Expression and activity of recombinant XTH proteins. (A) Proteins were separated on SDS–polyacrylamide gels and stained with Coomassie Blue. Lane 1, total protein (DkXTH6); lane 2, total protein (DkXTH7); lane 3, purified protein (DkXTH6); lane 4, purified protein (DkXTH7); and M, protein marks (Takara, Dalian, China). (B)In vitro XET assay of recombinant XTH proteins. The XET assay was performed by colorimetric method as described in Section Production and Purification of Recombinant XTH Proteins and Enzyme Activity Analysis. The empty vector pET32a (+) was used as the control. (C) The pH–rate profile of recombinant XTH proteins. (D) Dependence of XET activity of proteins on the concentration of XGOs. Vertical bars indicate standard errors of three replicates.
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Figure 7: Expression and activity of recombinant XTH proteins. (A) Proteins were separated on SDS–polyacrylamide gels and stained with Coomassie Blue. Lane 1, total protein (DkXTH6); lane 2, total protein (DkXTH7); lane 3, purified protein (DkXTH6); lane 4, purified protein (DkXTH7); and M, protein marks (Takara, Dalian, China). (B)In vitro XET assay of recombinant XTH proteins. The XET assay was performed by colorimetric method as described in Section Production and Purification of Recombinant XTH Proteins and Enzyme Activity Analysis. The empty vector pET32a (+) was used as the control. (C) The pH–rate profile of recombinant XTH proteins. (D) Dependence of XET activity of proteins on the concentration of XGOs. Vertical bars indicate standard errors of three replicates.

Mentions: To analyze the enzymatic properties of DkXTH6- and DkXTH7-encoded isoenzymes, recombinant XTH proteins (DkXTH6-RP and DkXTH7-RP) were obtained using prokaryotic expression. The crude proteins of DkXTH6-RP and DkXTH7-RP appeared mostly in the insoluble fraction. The recombined proteins were dissolved in 8 M urea buffer and purified using a Ni-NTA resin column and then were refolded using a reverse urea gradient (Figure 7A). Subsequently, the XET activity of DkXTH6-RP and DkXTH7-RP was measured by a colorimetric assay. Compared with the blank control, both DkXTH6-RP and DkXTH7-RP exhibited remarkably high XET activity (Figure 7B), indicating that the purified recombined proteins were active enzymes. The XEH activity of recombined proteins was also investigated by a viscometric assay, and T. reesei cellulase, which could depolymerize xyloglucan by hydrolysis activity, was used as a positive control. After treating xyloglucan with the recombined proteins for a set time, no evident decrease in viscosity of xyloglucan was observed (data not shown), suggesting that both DkXTH6-RP and DkXTH7-RP showed no XEH activity.


Isolation and Characterization of Two Persimmon Xyloglucan Endotransglycosylase/Hydrolase (XTH) Genes That Have Divergent Functions in Cell Wall Modification and Fruit Postharvest Softening.

Han Y, Ban Q, Hou Y, Meng K, Suo J, Rao J - Front Plant Sci (2016)

Expression and activity of recombinant XTH proteins. (A) Proteins were separated on SDS–polyacrylamide gels and stained with Coomassie Blue. Lane 1, total protein (DkXTH6); lane 2, total protein (DkXTH7); lane 3, purified protein (DkXTH6); lane 4, purified protein (DkXTH7); and M, protein marks (Takara, Dalian, China). (B)In vitro XET assay of recombinant XTH proteins. The XET assay was performed by colorimetric method as described in Section Production and Purification of Recombinant XTH Proteins and Enzyme Activity Analysis. The empty vector pET32a (+) was used as the control. (C) The pH–rate profile of recombinant XTH proteins. (D) Dependence of XET activity of proteins on the concentration of XGOs. Vertical bars indicate standard errors of three replicates.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4863071&req=5

Figure 7: Expression and activity of recombinant XTH proteins. (A) Proteins were separated on SDS–polyacrylamide gels and stained with Coomassie Blue. Lane 1, total protein (DkXTH6); lane 2, total protein (DkXTH7); lane 3, purified protein (DkXTH6); lane 4, purified protein (DkXTH7); and M, protein marks (Takara, Dalian, China). (B)In vitro XET assay of recombinant XTH proteins. The XET assay was performed by colorimetric method as described in Section Production and Purification of Recombinant XTH Proteins and Enzyme Activity Analysis. The empty vector pET32a (+) was used as the control. (C) The pH–rate profile of recombinant XTH proteins. (D) Dependence of XET activity of proteins on the concentration of XGOs. Vertical bars indicate standard errors of three replicates.
Mentions: To analyze the enzymatic properties of DkXTH6- and DkXTH7-encoded isoenzymes, recombinant XTH proteins (DkXTH6-RP and DkXTH7-RP) were obtained using prokaryotic expression. The crude proteins of DkXTH6-RP and DkXTH7-RP appeared mostly in the insoluble fraction. The recombined proteins were dissolved in 8 M urea buffer and purified using a Ni-NTA resin column and then were refolded using a reverse urea gradient (Figure 7A). Subsequently, the XET activity of DkXTH6-RP and DkXTH7-RP was measured by a colorimetric assay. Compared with the blank control, both DkXTH6-RP and DkXTH7-RP exhibited remarkably high XET activity (Figure 7B), indicating that the purified recombined proteins were active enzymes. The XEH activity of recombined proteins was also investigated by a viscometric assay, and T. reesei cellulase, which could depolymerize xyloglucan by hydrolysis activity, was used as a positive control. After treating xyloglucan with the recombined proteins for a set time, no evident decrease in viscosity of xyloglucan was observed (data not shown), suggesting that both DkXTH6-RP and DkXTH7-RP showed no XEH activity.

Bottom Line: We found that DkXTH6 protein was localized in cell wall by its signal peptide, while cytoplasmic DkXTH7 protein contained no signal peptide.When expressed in vitro, the recombinant proteins of both DkXTH6 and DkXTH7 exhibited strict xyloglucan endotransglycosylase (XET) activity but no xyloglucan endohydrolase (XEH) activity.The recombinant protein of DkXTH6 showed a higher affinity with small acceptor molecules than the recombinant DkXTH7.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Crop Stress Biology for Arid Areas, College of Horticulture, Northwest A&F University Yangling, China.

ABSTRACT
Fruit cell wall modification is the primary factor affecting fruit softening. Xyloglucan endotransglycosylase/hydrolase (XTH), a cell wall-modifying enzyme, is involved in fruit softening. In this study, two novel XTH genes (DkXTH6 and DkXTH7) were identified from persimmon fruit. Transcriptional profiles of both of the two genes were analyzed in different tissues of persimmon, and in response to multiple hormonal and environmental treatments [gibberellic acid (GA3), abscisic acid (ABA), propylene, and low temperature]. Expression of DkXTH6 was positively up-regulated during ethylene production and by propylene and ABA treatments, and suppressed by GA3 and cold treatment. In contrast, DkXTH7 exhibited its highest transcript levels in GA3-treated fruit and cold-treated fruit, which had higher fruit firmness. We found that DkXTH6 protein was localized in cell wall by its signal peptide, while cytoplasmic DkXTH7 protein contained no signal peptide. When expressed in vitro, the recombinant proteins of both DkXTH6 and DkXTH7 exhibited strict xyloglucan endotransglycosylase (XET) activity but no xyloglucan endohydrolase (XEH) activity. The recombinant protein of DkXTH6 showed a higher affinity with small acceptor molecules than the recombinant DkXTH7. Taken together with their opposing expression patterns and subcellular localizations, these results suggested that DkXTH6 might take part in cell wall restructuring and DkXTH7 was likely to be involved in cell wall assembly, indicating their special roles in persimmon fruit softening.

No MeSH data available.


Related in: MedlinePlus