3.2. Identification of sucrose metabolism genes in Fragaria vesca. Effect of ripening transition on sucrose metabolism gene expression.
In order to identify the putative homologues involved in sucrose metabolism in Fragaria vesca , data-mining approaches were performed using BLAST search queries targeting HMM profiles of individual protein domains. SPS (EC 2.4.1.14), SS (EC 2.4.1.13) and invertases (EC 3.2.1.26) (CWINV, UniProtKB Q9ZP42 and VINV, UniProtKB Q08IC1) are key enzymes involved in sucrose metabolism within plants. The number of individual enzyme families differs among the plant species, being found in plants encoded by different multigene families. The paralogues found were used alongside the closer Arabidopsisorthologues in order to form functional phylogenetic trees (Figure 2). Four homologues were found to cluster SPS into four distinct groups, with FvSPS1 being more similar than FvH4_2g28820 to AtSPS1F and AtSPS2F orthologues. On the other hand, FvH4_4g13520 clustered with AtSPS3F, whilst FvSPS2 was seen to cluster with AtSPS4F (Figure 2 A). Four homologues were also found to cluster SS into three groups. In this instance, FvSS1 and FvH4_1g07260 formed a group close to the cluster formed by AtSUS4 and AtSUS1, whilst FvSS2 clustered with AtSUS3 and FvH4_4g18710 clustered with AtSUS2 (Figure 2 B). Likewise, three homologues were found to cluster together for CWINV. Here it was found that FvCWINV1 and FvH4_6g33780 were closer than FvCWINV2. These were separated from 6-FEH of Beta vulgaris and Arabidopsiscluster formed by AtCWINV3, 1 and 5 (Figure 2 C). These in turn are seen to be significantly separated from the cluster formed by 1-FEH activities. However, two different soluble invertase VINV groups were found to cluster into two groups, with FvVINV2 being close to a cluster formed by AtVI2 and AtVI1 (figure 2 C), and qualitatively separated from clusters formed by 1-SST and 6-GFT activities.
Tissue expression pattern during berry ripening and development has been described by the fruitENCODE project (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA381300) and Fragaria vesca eFP Browser (Darwish et al. 2013; Kang et al. 2013; Hollender et al., 2014). This provides a basis from which we can select which genes to analyse. From this, genes with the greatest expression in fruit tissues or with significant changes in relative gene expression between different developmental and ripening stages were selected (see asterisk marked genes in Figure 2). These were then analysed using qPCR in ‘Mara des Bois’ strawberries at the three different ripening transition stages (Figure 3 E). FvH4_2g28820, FvH4_4g13520, FvH4_1g07260, FvH4_4g18710 and FvH4_6g33780 were not analysed as they demonstrated low expression in fruit.
Expression of FvSPS1 , which is involved in sucrose synthesis, significantly increased in both FR and DR strawberries (Figure 3 A), whilst no changes in FvSPS2 expression were observed. With regards to the expression of FvSS homologues involved mainly in sucrose breakdown, our results indicate that FvSS1 andFvSS2 are expressed at all of the analysed ripening stages. Specifically, FvSS1 was more abundantly expressed thanFvSS2 , with expression of both increasing up until the stage of ripeness (Figure 3 B). On the other hand, transcriptional analysis of the invertase gene families involved in sucrose cleavage shows a significant increase in FvCWINV1 and FvVINV1 expression at the end of the ripeness stage (DR, Figure 3 C y D). In contrast,FvCWINV2 and FvVINV2 expression significantly decreases in DR strawberries, in comparison to AR and FR fruit. In fact, a greater decrease in FvVINV2 expression (∼2.5-fold) was observed in DR relative to AR (Figure 3 D). These results seem to indicate that sucrose metabolism is highly active during the DR stage, with an increase inFvSPS1 and a decrease in FvVINV2 expression leading to the accumulation of sucrose.
3.3. Changes during early and late storage at 0 ºC with and without CO2.
3.3.1 Changes in sugar levels
We were interested to understand the way in which sugar levels are altered by postharvest environmental factors during storage. For this reason, we analysed sugar levels in strawberry fruits immediately after harvest (0d), stored in air at 0º C for 2 days (2d Air) or 7 days (7d Air), or stored in these same conditions but with an additional CO2 pre-treatment (2d CO2 and 2d CO2+5d Air). As shown in Figure 4 A, a significant decrease in the content of sucrose was quantified in fruit stored in air at the early stage of LT storage at 0 ºC (2d). The sucrose content decreased by 23 % respect to the initial value after 2 days at 0 ºC, whereas it was maintained at the end of CO2 treatment. Nonetheless, following transfer to regular air conditions the effects of high CO2 on retaining sucrose was progressively lost. Higher levels of sucrose were still recorded in comparison to air-stored fruit after 7 days at 0 ºC. Similarly, our results indicate that there were significant decreases in glucose and fructose concentration by day 2 at 0 ºC in fruit stored in air (2d Air, Figures 4 B-C) relative to freshly harvested fruit (0d). In contrast, a significant decrease in glucose and fructose was not observed in CO2-treated fruit. With regards to myo-inositol abundance, a significant increase (∼28%) in myo-inositol was quantified in CO2-treated fruit at LT storage (2d CO2 and 2d CO2+5d Air, Figure 4 D). In addition to a loss of hexoses and sucrose, there was a 42% decrease in trehalose concentration in strawberries stored at 0 ºC in air by day 2, compared with fruit at harvest (Figure 4 E). There was also a similar significant decrease in trehalose levels at the end of CO2- treated fruit. However, after 7 days at 0 ºC, CO2-treated fruit reached values similar to those quantified in fruit at harvest and 32 % greater than those found in air-stored fruit. With respect to FOS, only 1-kestose (DP3) showed significant changes during low temperature storage (Figure 4 F). Content of 1-kestose significantly increased at the end of CO2 treatment, while slightly decreased at the early stage of low temperature storage at 0 ºC (2d) compare to freshly harvested fruit. Opposite trends were observed following prolonged storage at 0 ºC (7d), with this variable increasing in air-stored fruit. With respect to 6G-kestose and FOS concentrations with higher DP, no clear trends were observed in changes during low temperature storage (Figures 4 G-I). In contrast, in the case of RFOS, significant increases were seen in the concentration of both raffinose (DP3) and estaquiose (DP4) in air-stored fruit, both during early storage and after prolonged storage at low temperature (Figures 4 J-K). Such increases were less pronounced in CO2-treated fruit. In the case of RFOS of DP4, only significant increases were quantified in air-stored fruit.
3.3.2 Changes in expression of genes involved in sucrose metabolism during LT
In order to verify changes to sucrose metabolic status during storage we analysed the expression of genes previously involved in the synthesis or breakdown of sucrose. With regards to synthesis, our results indicate a slight yet significant increase in FwSPS1 expression at the end of CO2 treatment. A similar increase was also observed after 7 days in air-stored fruit, with homologues being expressed to a greater extent that FwSPS2, which showed a minor decrease (Figure 5 A).
From the beginning of cold storage and following sucrose breakdown, air-stored fruit already showed an upregulation of FvSS1expression at low temperature and significantly higher transcription levels (Figure 5 B). However, application of high CO2prevented this increase in FvSS1 expression from occurring. In contrast, whilst FvSS2 expression was greatly up-regulated at low temperatures, CO2 treatment failed to maintain initial rates of FvSS2 expression. With regards to expression of putative cell-wall type invertase genes, transcription of the FvCWINV1gene occurred in time-dependent manner at low temperature, with greater homologue expression in comparison to FvCWINV2 (Figure 5 C). The increase in FvCWINV1 expression observed at the end of LT period (7d) is particularly striking. This was seen to be 8 times greater than its expression in freshly harvested fruits. Such a sharp increase inFvCWINV1 expression is largely dictated by high CO2 treatment, even after 5 days of transference to air. In a similar way, a significant increase in FvCWINV2 expression was only observed at the end of the LT period. Likewise, a similar pattern of FvVINV1 and FvVINV2 expression was observed. Expression of both was increased in air-stored fruit, particularly during late LT at 0 ºC, whilst high CO2 treatment prevented this rise and resulted in notable lower expression (Figure 5 D).