Evolutionary analysis and role of invertase inhibitors in regulating sucrose metabolism in peach fruit under chilling stress

Xingxing Wang†, Yi Chen†, Shu Jiang, Feng Xu, Hongfei Wang, Yingying Wei*, Xingfeng Shao*

College of Food and Pharmaceutical Sciences, Ningbo University, Ningbo 315800, China

† co-first author

^* Corresponding author. E-mail address: weiyingying@nbu.edu.cn（Y. Wei）; shaoxingfeng@nbu.edu.cn (X. Shao).

Tel.: +86 574 87609573; fax: +86 574 87608347.

Running title: PpINH1 inhibits VIN activity in peach fruit.

Abstract:

Sucrose metabolism, particularly the decomposition of sucrose by invertase, plays a central role in plants’ response to cold stress. Invertases inhibitors (INHs) evolved with higher plants as essential regulators of sucrose metabolism. By limiting invertase activity, INHs keep cellular sugar levels elevated, which provides enhanced protection for plants under stress. As the only vacuolar invertase (VIN) gene in peaches sensitive to chilling temperatures, our results showed thatPpVIN2 expression increases significantly during cold storage, while VIN activity increases more modestly. We also found that peaches transiently overexpressing PpINH1 had decreased VIN activity. The interaction of PpINH1 and PpVIN2 was shown by yeast two-hybrid, bimolecular fluorescence complementation, and in vitro, with recombinant proteins. During cold storage, trehalose treated peaches had significantly increased PpINH1 expression, decreased VIN activity, and significantly higher sucrose content than untreated fruit. As a result, treated fruit had enhanced resistance to chilling injury. Collectively, our data show that the post-translational repression of VIN activity by PpINH1 helps maintain sucrose levels in peaches during cold storage, thereby improving resistance to chilling injury.

Keywords： invertase inhibitor; molecular evolution;Prunus persica ; chilling stress

Damage from low temperature stress is responsible for significant economic losses in commercial agriculture. Sugar plays a predominant role in protecting plants from damage due to low temperatures, because it regulates osmotic pressure, stabilizes membrane structures, eliminates reactive oxygen species, and functions as a signaling molecule (Keunen et al. 2013). Peaches (Prunus persica L. Batsh), like many fruits that are sensitive to low temperatures, develop chilling injuries after storage for 1 or 2 weeks at 2–5 °C (Lurie, & Crisosto, 2005). Sucrose, the major sugar in peach fruit, decreases after harvest, while reducing sugar content increases (Borsani et al., 2009). Sucrose decomposition and the continued demand for sugar in peaches at low temperatures affects their sensitivity to cold (Puig et al., 2015). Previously we found that sucrose degradation increases in peaches subjected to chilling stress and that maintenance of high sucrose levels improves membrane stability and resistance to cold stress (Wang et al., 2013; Yu et al., 2016).

Invertases are classified according to their pH optima as acid, alkaline, or neutral invertase. Acid invertase (AI) is thought to be the most important enzyme in fruit sucrose metabolism because it controls the composition of sugars and affects the response to stress (Zhang, Zhang, & Jiang, 2013; Tauzin et al., 2014). AI are further subdivided according to subcellular localization into cell wall-bound invertases (CWIN) and vacuolar acid invertases (VIN). The latter is also known as soluble AI. Multiple invertase genes are found in most plants, and peach contains 2 VIN and 5 CWIN genes (He et al, 2018). Evolutionary analyses show that CWINs exhibit more sequence variability than VINs, and suggest that they share a common VIN ancestor (Wan, Wu, Yang, Zhou, & Ruan, 2018).

VINs catalyze the irreversible decomposition of sucrose into fructose and glucose, thereby helping to establish and maintain cell osmotic potential and protect plants from environmental stresses (Wan et al, 2018). Generally, VINs are significantly up-regulated in plants under cold stress, but VIN activity does not increase proportionately with gene expression (Liu et al., 2013). In peach, only PpVIN2 expression is sensitive to low temperature (He et al., 2018). AlthoughPpVIN2 expression increases substantially at cold temperatures, VIN activity increases far less, approximately 2-fold (He et al., 2018). This result suggests that VIN activity is regulated by a post-transcriptional mechanism.

Invertase inhibitors (INHs) were first identified in the 1960s as endogenous proteins that inhibit invertase activity in potato (Solanum tuberosum ) (Schwimmer et al. , 1961). They have since been shown to interact with VINs and regulate their activity, thereby playing a vital role in sugar signaling and carbon allocation (Wan et al, 2018). INHs are members of the pectin methylesterase inhibitor-related protein family, and are classified as cell wall inhibitors (CIF) or vacuolar inhibitors (VIF) according to their subcellular location (Rausch, & Greiner, 2004). In contrast to model plants, little is known in peaches about INHs and their physiological significance, nor has the post-translational regulation of VIN by inhibitors been examined in peach fruit.

To investigate whether peach INH (PpINH) functions as a post-translational regulator of VIN activity in fruit, five PpINHgenes were cloned and their interactions with PpVIN2 were studied using the yeast two-hybrid (Y2H) system. Based on the result of Y2H assays, the interaction between PpINH1 and PpVIN2 was further determined by biomolecular fluorescence complementation (BiFC) in tobacoo. PpINH1 function was also investigated in peach fruit using anAgrobacterium -based transient expression system. Finally, PpINH1 and PpVIN2 proteins were prepared using heterologous expression systems, and interactions between the proteins were studied in vitro .

Trehalose is a non-reducing glucose disaccharide (α-D-glucopyranosyl-1,1-αD-glucopyranoside) that is synthesized during abiotic stress (Benaroudj et al. 2001; Elbein et al. 2003). Exogenous trehalose treatment has been used to enhance the cold tolerance of crops and harvested fruits (Kosar et al., 2019) and fresh-cut peppers (Ding & Wang, 2018). Here, we treated peaches with exogenous trehalose to investigate its effect on chilling injury (CI), sucrose content, VIN activity, PpVIN2 expression, and INH gene expression. The results demonstrated that the application of exogenous trehalose decreased sucrose metabolism and CI in peach fruit subjected to cold stress.