4.2 PpINH1 interacts with PpVIN2, functioning as an inhibiter
VINs regulate cell growth (Wang et al., 2010), participate in fruit sucrose metabolism (Ohyama et al., 1995; Scholes et al.1996), and act as regulators in response to stress (Rausch & Greiner, 2004) as exemplified by the increased VIN activity in peach fruit during cold storage (He et al., 2018). Regulation of VIN by endogenous protein inhibitors occurs at both the transcriptional and translational level (Tauzin et al., 2014). In tomato, Qin et al. (2016) found that S1VIF, a tomato INH located in tomato vacuoles, mediates fruit maturation and interacts with VIN S1VI in Y2H system. As yet no studies have been conducted on the post-translational regulation of inhibitor-mediated VIN activity in peaches or other rosaceous plants. In our study, we found that in peaches only PpINH1 interacts with PpVIN2 in a Y2H screen. We also demonstrated this interaction in tobacco cells by BiFC (Figure 3 ).
Because peaches are difficult to stably transform and transgenic fruit requires 4-5 years to produce, we used the Agrobacteriumtransient transformation system to investigate biological functions of PpINH1. Our results showed that when PpINH1 is overexpressed in peach, VIN activity decreases (Figure 4e ), as would be expected if PpINH1 regulates VIN at the post-translational level. Zhang et al. (2015) also used the Agrobacterium system, introducingSly-INH tomato fruit. Sly-INH is an inhibitory protein that regulates tomato CWI activity at the post-translational level. Interestingly in our experiment, although PpINH1 expression increased substantially during cold storage, the decrease in VIN activity was not as dramatic. This disparity may be due to induction of plant defense responses by the Agrobacterium infection (Pitzschke, 2013), which would be expected to enhance VIN activity.
The interaction between PpINH1 and PpVIN2 was also shown in vitrousing recombinant proteins. The results showed that PpINH1 inhibition of PpVIN2 activity is pH dependent, with an optimum at pH 4.4, and temperature dependent, with an optimum at 35 °C (Figure 5i-l ). It is worth noting here that most plant vacuoles are acidic, and the interaction between PpINH1 and PpVIN2 could be regulated via pH variations within the vacuole (Tauzin et al., 2014).
In potato, the AI StvacINV1 , the INH StInvInh2B , and the sucrose non-fermentation-associated protein kinase 1 protein form a complex which regulates the activity of AI (Lin et al., 2015).Arabidopsis thaliana CWIN1 and tobacco inhibitor CIF interact to form a similar complex that has been analyzed structurally (Hothorn et al. 2010). Bioinformatics analysis indicates that PpINH1 has a four-helix bundle fold and four strictly conserved cysteine residues (Figure S3a ), while PpVIN2 is predicted to contain a five-bladed beta-propeller structure (Figure S3b ). As these structures are prerequisites for PpINH1and PpVIN2 to form an interaction complex (Hothorn et al. 2004ab), it is likely that PpINH1 forms a complex with PpVIN2 and mediates inhibition of PpVIN2 activity.