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.