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
1
Introduction
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.