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).