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.