1. Introduction
Woodland strawberries (Fragaria vesca ) are characterised by a
delicious taste and an abundance of non-structural carbohydrates. During
development and ripening, strawberries undergo substantial changes to
multiple sensorial and biochemical attributes, acquiring good quality
and high commercial value at harvest (Roch et al., 2019). Being a
non-climacteric fruit, strawberries have to be harvested in an optimal
state for consumption since ripening does not continue once they have
been harvested. Thus, the harvest date has a decisive influence on
determining the duration of storage life and fruit quality. Soluble
sugar content is one of the most important quality traits since sugar
accumulation affects both fruit growth and quality (Hancock, 1999;
Schwieterman et al, 2014). At the same time the organoleptic quality of
the fruit that is largely established by its sweetness (Colquhoun et
al., 2012), depends on the content and composition of sugars since
different types of soluble sugars contribute differently to the relative
degree of sweetness (Kroger et al, 2006; Schwieterman et al 2014). Thus,
specific sugars or groups of sugars must be characterized and quantified
during ripening transition in the period around the anticipated optimum
harvest date, storage and shelf-life, in order to consider their
potential role as indicators of optimum harvest date.
Strawberries accumulate various types of soluble sugars which vary
throughout fruit development. The main sugars are sucrose, glucose and
fructose, followed by myo-inositol and trehalose (Zhang et al., 2011).
In immature fruit, changes during early fruit development mainly occur
in glucose and some polyalcohol sugars such as myo -inositol
(Ogiwara et al., 1998a, 1998b). During later fruit development, sucrose,
glucose and fructose become the predominant soluble sugars, with
galactose and some cell wall sugars also being observed in ripe fruit
(Menager et al., 2004; Fait et al., 2008; Basson et al., 2010; Zhang et
al., 2011). Despite this, little is known about the changes that take
place during different stages of ripening in the oligosaccharides levels
derived from sucrose and galactose, such as fructo-oligosaccharides
(FOS) and galactose-oligosaccharides (RFOS).
Sucrose content has been shown to be more responsible than any other
individual compound for a greater variation in sweetness intensity and
overall liking (Schwieterman et al, 2014). It is, therefore, essential
to investigate sucrose levels during ripening transition as this may
reflect high acceptance of strawberries at harvest. However, delaying
harvest day has a negative impact as it shortens postharvest life.
Consequently, adequate environmental conditions surrounding the fruit
are needed to control sucrose content and avoid, as much as possible,
rapid deterioration of the fruit following harvest. High
CO2 treatment has been used as coadjuvant technology to
alleviate physiological disorders caused by storage at severe low
temperature. Its application over only a short period of time at the
beginning of storage is a commercially available technology which is
used to reduce fungal decay and water loss. In addition to being
essential for sweetness and a substrate for FOS synthesis, sucrose has
an important role as a major osmotically active solute. It plays an
important role in regulating water within cells which join with other
major soluble carbohydrates such as glucose, fructose andmyo -inositol (Blanch et al., 2015b, Vimolmangkang et al. 2016).
FOS are olygomers that result from extended sucrose metabolism, where
fructosyl units are bound by β linkage to sucrose. This linkage favours
the formation of helical structures. FOS have different protective
effects against environmental stress in plants (Valluru and Van den
Ende, 2008; Hincha et al., 2000). We have previously reported the
implication of FOS on fruit water status in strawberries more than a
reserve carbohydrate, spite this fruit is a non-fructan accumulating
plant (Blanch et al., 2012). Their specific structure and biophysical
properties support the ability of these compounds to reorganize
water-hydrogen bonding networks which might be a factor contributing to
cellular water stabilization (Furiki, 2002). On the other hand, several
reports indicate an important role of RFOS in response to osmotic stress
(Ishitani et al., 1996; Loewus and Murthy, 2000; Sengupta et al., 2015).
Some of these oligosaccharides might act as osmoprotectants, protecting
against damage caused by the osmotic imbalance induced by several kinds
of stress (Hare et al., 1998; Verslues et al., 2006; Sperdouli and
Moustakas, 2012). This can function to stabilize cellular membranes by
replacing water molecules and thereby keeping membrane surfaces hydrated
(Verslues et al., 2006; Valluru and Van der Ende, 2008). In addition,
beneficial effects of FOS in the diet as a health-promoting food
ingredient have been recognized in humans (Sabater-Molina et al., 2009;
Closa-Monasterolo et al., 2013; Tousen et al., 2013; Yao et al.,2014;
Singh et al., 2017). Other sugars such as trehalose also act to protect
membranes and proteins from damage caused by different stress
conditions, including low temperatures (Fernandez et al. 2010, Delorge
et al., 2014, Lunn et al., 2014).
Low temperature and high CO2 are known to impact fruit
metabolism by reprogramming gene expression to involve numerous
transcription factors and activating abiotic stress genes (Rosales et
al., 2016; Romero et al 2016; Wang et al., 2017, Zhu et al 2018, Jin et
al. 2018, Li et al 2019a, Zhu et al 2019). However, the effect of high
CO2 on the expression of genes involved in sucrose
metabolism and underlying sucrose levels, during low temperature storage
and further shelf-life, remains unknown. In addition, although
information about sucrose metabolism gene expression during development
is available, little is known about the different stages of ripening
transition. During strawberry fruit development sucrose is imported from
photosynthetic tissues, through apoplast, to the berry, entering as
sucrose or being hydrolysed into glucose and fructose by cell wall
invertase (CWINV) (Koch, 2004; Fait et al., 2008; Basson et al. 2010).
Thus, cytoplasmatic sucrose can be reversibly cleaved by sucrose
synthase (SS) or irreversibly hydrolysed by invertases (Winter and
Huber, 2000; Koch, 2004). Different groups of intra or extracellular
invertases can be discerned. These include CWINV and soluble vacuolar
invertase (VINV), which have an acidic optimal pH, soluble cytoplasmic
invertase (NINV) that has a neutral to alkaline optimal pH, and soluble
apoplastic invertase (Roitsch et al., 2003). CWINV and VINV are closely
phylogenetically related through their activities, which are regulated
at both a transcriptional and post-translational level (Wan et al.,
2018). Invertases are highly homologous to fructan exo-hydrolases
involved in fructan degradation. However, in contrast to invertases,
fructan exo-hydrolases cannot use sucrose as a substrate (Van den Ende
et al 2004; Van den Ende 2013). Thus, FOS can be degraded by fructan
1-exohydrolases (1-FEHs), whilst plant FEHs lack invertase activity.
They are enzymes with a single function and probably evolved from CWINV,
serving only to degrade fructans. In contrast, FOS biosynthetic genes
from dicots, sucrose: sucrose 1-fructosyltransferase (1-SST) and
fructan: fructan 1-fructosyltransferase (1-FFT), evolved from VINV, use
sucrose and fructans, respectively, as preferential donor substrates
(Lasseur et al., 2009). On the other hand, RFOS is derived from
UDP-glucose which is simultaneously involved in sucrose synthesis
through the major enzyme, sucrose-phosphate synthase (SPS).
SPS reversibly catalyses sucrose-6-phosphate formation from
fructose-6-phosphate and UDP-glucose (Huber and Huber, 1996; Nguyen-Quoc
and Foyer, 2001). UDP-glucose can also join with glucose-6-phosphate
(G-6-P) to form trehalose-6-phosphate (trealose-6-P) and, subsequently,
trehalose (Ponnu et al., 2011). The importance of SPS in carbohydrate
metabolism and development has been confirmed in Arabidopsis and
rice mutant lines and muskmelon interference lines, and through
heterologous expression in tomato, tobacco and cotton plants (Worrell et
al., 1991; Galtier et al, 1993; Baxter et al., 2003; Haigler et al.,
2007; Tian et al., 2010; Volkert et al., 2014; Seger et al., 2014;
Hashida et al., 2016). Different potential benefits of SS over
invertases have also been previously reported (Zeng et al. 1999; Bologa
et al., 2003; Koch et al. 2000). The efficiency of SS in ATP net yield
has been extensively reported (Stitt and Steup, 1985; Sachs, 1994;
Stitt, 1998; Baroja-Fernández et al., 2009).
Whilst the SS pathway produces phosphorylated glucose, the
unidirectional invertase pathway releases glucose, which must then be
phosphorylated at the expense of ATP in order to enter the glycolytic
pathway. It has been reported that hypoxia caused by cellular oxygen
deficiency (Gibbs and Greenway, 2003; Greenway and Gibbs, 2003; Narsai,
et al., 2011), generally upregulates the expression of genes which code
enzymes involved in sugars, with the only exception being seen in
invertases.
The objective of the present work was firstly to analyse whether
sucrose, major water-soluble sugars and related oligosaccharides,
together with sucrose metabolism gene expression dynamically change
during ripening transition in attached strawberries. A second objective
was to explore the impact of environmental conditions on controlling
sucrose reserves and sucrose metabolism gene expression, after storage
and shelf-life. For this purpose, we analysed the accumulation of
different sugars and expression of the homologue genes involved in
sucrose metabolism in strawberries at three different ripening stages.
The effect of low temperature (0 ºC) and CO2pre-treatment (17% CO2 for 2 days) on sucrose retention
in strawberries, as well as the underlying molecular mechanisms, were
analysed during early and late phases of LT, and further SL.
Effectiveness of pre-treatment with high CO2 levels for
reducing weight loss and maintaining other major soluble carbohydrates
was determined. The effect of ripening stage, storage and shelf-life on
the accumulation of short-chain RFOS (raffinose, degree of
polymerization (DP) 3, and estaquiose, DP4) and short-chain FOS
(1-kestose and 6G-kestose, DP3, DP4, DP5) was also analysed.