Changes in sucrose metabolism gene expression.
Sucrose metabolism is controlled mainly by SPS, SS and invertases (Winter and Huber 2000; Wan et al., 2018; Stein and Granot, 2019). The reaction catalysed by SPS plays an important role in the regulatory steps controlling sucrose synthesis depending on the plant species considered (Huber and Huber, 1996; Do Nacismento et al., 1997; Winter and Huber 2000; Choudhury et al., 2010; Volkert et al., 2014). Further, SPS plays a crucial role in many physiological processes including the alteration of the cell wall ultrastructure (Geigenberger et al., 1999; Coleman et al., 2009). Present results showed that FvSPS1expression significantly increased in both FR and DR strawberries. Although a marked increase in sucrose was only seen in DR strawberries, we cannot rule out the possibility of post-translational modulation of its activity. Concerning both of the FvSS homologues analysed, we detected a slight increase in their expression. Although sucrose synthase is capable of both degrading and synthesizing sucrose, its primary function might be sucrose degradation (Nguyen-Quoc and Foyer, 2001). Whilst the role of each homologous remains to be elucidated, present results suggest that FvSS could play a role in sucrose cleavage in strawberries during ripening transition. In this sense, SS has been identified as an indicator of sink strength in growing tomato fruits (Sun et al., 1992) and as playing a crucial role in sucrose utilization in fruit development (Wang et al., 1993). Present data show increased FvCWINV1 and FvVINV1 expression in DR fruit compared to AR and FR fruit (∼2 and 3-fold respectively; Figures 3 C-D), whilst FvVINV2 expression was considerably decreased (∼6 fold). It is possible that whilst these two genes are from the same family, they have developed different functions. The possible involvement of these activities hydrolysing oligosaccharides containing fructose (Wan et al., 2018), seem not to be an option considering the levels of FOS and sucrose observed during ripening transition stage. Thus, the large sucrose increases detected in DR may be largely explained by the compensatory balance between the increase in FvSPS1 and the sharp decrease in FvVINV2, together with all of the activities involved in sucrose metabolism.
Regarding the gene expression analysed at LT and SL, no major changes in the FwSPS1 expressions were detected in CO2treatments. On the other hand, the slight increase of its expression observed at the end of CO2 treatment together with the maintenance of FwSPS1 expression in LTC match with the highest levels of sucrose detected. Considering all of these results together, the increase in sucrose, specifically in DR and in SLC, seems to not only be explained by changes in SPS expression. Thus, our results showed increased SS gene expression in strawberries stored at 0 ºC during early and prolonged storage at 0 ºC, with the lowest sucrose values being quantified in this work. In contrast, FvSS1expression in CO2-treated fruit was maintained, resembling levels observed immediately after harvest, and modulating the sharp increase of FvSS2 expression observed in un-treated samples. In fact, the low FvSS1 expression observed in LTA and LTC during SL could explain the fact that higher sucrose levels were observed in LTC which were similar to those observed in CO2 pre-treated fruit. As the SS pathway produces already phosphorylated glucose which does not, therefore, have to be phosphorylated at the expense of ATP in order to enter into the glycolytic pathway, the breakdown of sucrose into hexoses in the cytosol via SS is more energy-efficient. It has been reported that SS sustains glycolysis under hypoxic conditions (Germain et al., 1997; Ruan et al, 2012). In addition to its involvement in rapid sucrose degradation, SS has been linked to biosynthetic processes of cell wall polysaccharides by directly supplying UDP-glucose as a substrate for secondary cell wall formation (Salnikov et al., 2001; Li et al., 2019b; Coleman et al., 2009). Present results indicate that SS may play a role in modulating sucrose cleavage in strawberries stored in air at low temperature. It is possible that higher SS expression in strawberries stored at 0 ºC in air may contribute to sustaining glycolysis, whilst also providing UDP-glucose for the synthesis of RFOS. Present results indicate that low temperature cause increase in the levels of raffinose and estaquiose. These results are in line with the enhancement of RFOS in plants in response to different kind of stress. The sharp increase in FvCWINV1 expression observed in fruit stored in air at 0 ºC is also interesting. Present results show an apparent association between LT damage and up-regulation of cell wall invertases. In fact, increased cell wall invertase activities are typically associated with stress responses (Roitsch et al., 2003). Interestingly, the high FvCWINV1 expression observed here in addition to FvCWINV2 expression were controlled by high CO2 pre-treatment (Figure 5 C). The decrease observed in sucrose, glucose and fructose alongside the significant increase in expression of both FvCWINV and FvVINV in air-LT strawberries, suggests that storage at 0 ºC leads to a reprogramming of carbon metabolism with a preference for sucrose-consumption reactions. Sucrose breakdown at 0 ºC possibly occurs in fruit in order to meet demands for an increased substrate supply due to an increased respiration rate. In the same way, increased expressions ofFvVINV homologues in fruit stored at 0 ºC in air also suggests an association between LT damage and up-regulation of vacuolar invertase. Such up-regulation could possibly be integrated within the context of general damage caused by low temperature in the active transport systems of ions, soluble sugars and other metabolites. We suggest that the low utilization of hexoses in CO2 pre-treated fruits should contribute towards generating sufficient cell turgor, preventing sucrose degradation or rendering it unnecessary (Blanch et al., 2015b). It has been proposed that VINV plays a key role in regulating plant cell expansion through osmotic regulation (Sergeeva et al., 2006; Tang et al., 1999; Neumann et al., 2002; Wang and Ruan 2016; Wan et al., 2018). Distinct types of vacuoles with different functions have also been reported (Frigerio et al., 2008; Zouhar and Rojo, 2009; Marcos Lousa et al., 2012), some of which are synthesized at the end of the plant’s life cycle and are denominated senescence-associated vacuoles (Otegi et al., 2005). On the other hand, it is interesting to note that during SL,FvVINV expression observed in LTC remains very low compared to SLC, FvVINV2 expression remains particularly repressed following 2 days at 20 ºC in LTC samples, when considered relative to fruit stored in air (LTA) at low temperature. These results could explain the higher level of sucrose observed in LTC, with these samples being optimum for consumption. Thus, decreased expression of invertases in CO2-treated fruit could be integrated within the context of a general protective mechanism that controls total soluble sugar, with repression of this mechanism affecting postharvest transpirational water loss.