Figure legends
Figure 1. INH evolution in green plants. (a ) Phylogenetic tree constructed using INH proteins from 100 representative green plant species. (b ) INH gene copy number in 45 flowering plant species. (c ) Conservation of INH genes in 45 plants. Codons that encode the four highly conserved cysteine residues are shown in boxes, and the height of the logo indicates residue frequency.
Figure 2. Promoter analysis of PpINHs. Promoter regions are 3000 bp in length directly upstream from the first codon. Putative CBF/DREB (C-repeat binding factor/dehydration responsive element) binding sites are indicated by arrowheads.
Figure 3. Interaction analysis of PpVIN2 and PpINH1-5 in vivo . (a ) Interaction of five PpINH proteins and PpVIN2 analyzed using the Y2H system. The yeast strain was co-transformed with PpVIN2-pGBKT7 (BD) and PpINHx-pGADT7 (AD). BD-p53 and AD-T were used as positive controls, while BD-Lam and AD-T were used as negative controls. Yeast cells were spotted onto solid media (composition shown on left) and then incubated at 30 °C for 72 h. Columns A-E correspond to cells expressing PpINH1, 2, 3, 4, and 5, respectively. Interaction is detected only in column A (PpINH1). (b ) Subcellular localization was visualized for both proteins in tobacco leaves. The empty vector served as a negative control. Scale bars represent 10 μm to 20 μm. (c ) BiFC analysis of the interaction between PpINH1 and PpVIN2 in tobacco epidermal cells. Combinations of PpVIN2 -YN with YC,PpINH1 -YC with YN, and YN with YC served as negative controls. YC, C-termini of YFP; YN, N-termini of YFP. Scale bars represent 25 μm to 75 μm.
Figure 4. Overexpression (OE) of PpINH1 inhibits VIN activity in vivo . Peach fruit in the green ripening stage are shown at 0 h (a ) and 36 h (b ) after injection withAgrobacterium to obtain transient expression of PpINH1 . (c ) Histochemical GUS staining of peach tissue from fruits injected with pBI121-PpINH1 -GV3101 or pBI121-GV3101. Images were obtained 36 h after injection. Samples were photographed separately and the images were combined to create a collage. (d ande ) Effects of transiently overexpressed PpINH1 on the expression of PpINH1 (d ) and VIN activity (e ) in peach at 0, 24, 36, 48, and 72 h after injection withAgrobacterium . Values are means ± SE, n=3. Student’st -test was used to assess differences between treatment and control. “*”, “**”, and “***” indicate significant differences at p < 0.05, 0.01, and 0.001, respectively.
Figure 5.Expression, identification, and purification of PpINH1and PpVIN2 protein, and their interaction in vitro . SDS-PAGE (a ) and western blot analysis (b ) of PpINH1 detected in BL21(DE3) cell fractions. SDS-PAGE (c ) and western blot analysis (d ) of purified PpINH1. SDS-PAGE (e ) and western blot analysis (f ) of PpVIN2 expressed in X-33 cells. SDS-PAGE (g ) and western blot analysis (h ) of purified PpVIN2. Arrows indicate the position of the recombinant protein. (i ) Effect of PpINH1 on PpVIN2 activity at different pH levels. (j ) Effect of PpINH1 on PpVIN2 at different pH levels, expressed as inhibition rate (%). (k ) Effect of PpINH1 on PpVIN2 activity at different temperatures. (l ) Effect of PpINH1 on PpVIN2 at different temperatures, expressed as inhibition rate (%).
Figure 6. Effect of trehalose treatment on postharvest peach fruits. (a ) Representative images of CI in treated and untreated peaches after storage for 21 and 28 days at 5 °C. (b ) CI index, (c ) sucrose content, (d ) VIN activity, (e ) PpVIN2 expression, and (f ) PpINH1expression, in trehalose-treated and untreated peaches during cold storage. Values are means ± SE. Student’s t -test was used to assess differences between treatment and control groups. “*”, “*”, “**”, and “***” indicate significant differences at p < 0.05, 0.01, and 0.001, respectively.
Figure 7. A proposed model for inhibition of VIN activity by PpINH1. Upper panel: During cold storage, PpVIN2 expression and VIN activity both increase. VIN decomposes sucrose, a sugar that helps prevent chilling injury. Lower panel: PpINH1 inhibits VIN activity at the post-translational level. Trehalose treatment increases PpINH1 expression and thus indirectly increases sucrose levels, protecting against chilling injury.