Table 1.
Oligonucleotide sequences for primers used in this study.
Table 2.
Characteristics of XTHs isolated from persimmon.
Fig 1.
Phylogenetic analysis of deduced amino acid sequences of XTHs.
Five DkXTHs (triangle) and 23 XTH homologues from other species were used to generate the phylogenetic tree by MEGA version 5.1 with Bootstrap N-J Tree method (1000 bootstrap trials). Distance scale length of the tree was 0.05. PttXET16A and TmNXG1 (rotundity) were the first XET and XEH with three-dimensional structures, respectively. I, II, III-A, and III-B denote different clades of XTHs. GenBank accession numbers are indicated in the figure.
Fig 2.
Alignment of the five predicted persimmon XTH proteins.
Black shading represents identical amino acids, and grey shading identifies the residues shared by at least three of the XTHs. Gaps are indicated by dots to optimize the alignment. Putative catalytic domain, N-glycosylation site, and two cysteines are marked with “#,” “*,” and “+,” respectively. Straight lines identify loops 1–3 of DkXTH1 and DkXTH2. DNAMAN program was used to construct multiple alignments.
Fig 3.
Subcellular localization of DkXTH1 and DkXTH2 by transient expression in onion epidermal cells.
(a) Diagram of DkXTH1 and DkXTH2 constructs fused to GFP. (b) Panels 1, 2, 3, 4, and 5, transmission and fluorescence images of non-plasmolysed cells; panels 6 and 7, transmission and fluorescence images of plasmolysed cells. Plasmolysis was induced by 400 mM sucrose. CW, cell wall; N, nucleus; PR, protoplast. Scale bar = 50 μm.
Fig 4.
Largest diameter (a), growth rate (b), and firmness (c) during fruit development of Fuping Jianshi.
The largest diameter was measured using a Vernier caliper. Each time point was calculated from the means of three biological replicates, and each replicate included 15 fruits, which were from different trees. Vertical bars indicate standard errors of means.
Table 3.
Sugar content of hemicelluloses and xyloglucan (μg mg−1 cell wall dry weight) during Fuping Jianshi fruit development.
Fig 5.
Viscosity of xyloglucan (a) and XET activity (b) during fruit development of Fuping Jianshi.
The relative viscosity of xyloglucan was calculated using total xyloglucan extracted with 1 and 4 M KOH from Fuping Jianshi fruit at different stages of development. The XET assay was performed by colorimetric method as described in Section 2.6. Vertical bars indicate standard errors of three biological replicate assays.
Fig 6.
Expression analysis of DkXTH1-5 during Fuping Jianshi fruit development.
Expression level of gene at 140 DAFB was used as the control with a nominal value of 1. Vertical bars indicate the standard error of three biological replicate assays. Columns with different letters at each time point are significantly different (LSD, P = 0.05).
Fig 7.
Firmness, respiration rate, and ethylene production of FP-25°C, GMK-25°C, and FP-0°C.
FP-25°C indicated Fuping Jianshi fruit stored at 25°C; GMK-25°C indicated Ganmaokui fruit stored at 25°C; FP-0°C indicated Fuping Jianshi fruit stored at 0°C. Vertical bars indicate standard errors of three biological replicate assays.
Fig 8.
Expression analysis of DkXTH1-5 in FP-25°C, GMK-25°C, and FP-0°C during storage.
FP-25°C indicated Fuping Jianshi fruit stored at 25°C; GMK-25°C indicated Ganmaokui fruit stored at 25°C; FP-0°C indicated Fuping Jianshi fruit stored at 0°C. Expression of gene at 0 d was used as the control with a nominal value of 1. Vertical bars indicate the standard error of three biological replicate assays. Columns with different letters at each time point are significantly different (LSD, P = 0.05).
Fig 9.
Expression and activity of recombinant XTH proteins.
(a) Proteins were separated on SDS–polyacrylamide gels and stained with Coomassie Blue. Lane 1, pET32a(+) control protein; lane 2, unbound protein (DkXTH1); lane 3, total protein (DkXTH1); lane 4, purified protein (DkXTH1); lane 5, total protein(DkXTH2); lane 6, unbound protein (DkXTH2); lane 7, purified protein (DkXTH2); 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 2.7. The empty vector pET32a(+) was used as the control. Columns with different letters are significantly different (LSD, P = 0.05) (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.