Postharvest Biology and Technology 139 (2018) 20–30

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Postharvest Biology and Technology

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Ethylene regulation of sugar metabolism in climacteric and non-climacteric T

plums
⁎
Macarena Farcuha, Rosa M. Riverob, Avi Sadkac, Eduardo Blumwalda,
a
Dept of Plant Sciences, University of California, Davis, CA 95616, USA
b
CEBAS, CSIC, Murcia, Spain
c
Dept of Fruit Tree Sciences, ARO, The Volcani Center, Rishon LeZion, Israel

A R T I C L E I N F O A B S T R A C T

Keywords: We studied the effect of ethylene regulation on sugar metabolism in fruit of two Japanese plum (Prunus salicina
Japanese plums Lindl.) cultivars, the climacteric Santa Rosa and its non-climacteric bud mutant Sweet Miriam, throughout ri-
Sugar metabolism pening in postharvest storage. These cultivars share the same genetic background but due to bud mutations differ
Ethylene in their ripening behavior. We examined the responses to ethylene (propylene) and 1-methylcyclopropane (1-
Fruit
MCP) treatments on 11 key sugar metabolism-associated genes by integrating gene expression profiling and their
Non-climacteric
associated sugar contents. Our results demonstrated that ethylene was a crucial factor affecting overall sugar
Ripening
metabolism in both ripening types. More specifically, ethylene reduced sucrose catabolism and induced sucrose
biosynthesis but inversely, stimulated sorbitol breakdown and decrease sorbitol biosynthesis. Our analyses in-
dicated that glucose and fructose contents result from sorbitol and sucrose breakdown in climacteric and non-
climacteric fruit, respectively. In addition, a positive interaction was observed between ethylene and galactose
metabolism; while a negative effect of ethylene was reported on galactinol, raffinose, myo-inositol and trehalose,
which were higher in non-climacteric Sweet Miriam fruit and could contribute to increased fruit tolerance to-
wards the stress imposed by the ripening process per se and to withstand postharvest storage.

1. Introduction translocation of the sugar-alcohol sorbitol (Sor) occurs in addition to

sucrose (Suc) (Okie and Ramming, 1999). Suc synthesis results from the
Ethylene has been considered as the key ripening-related hormone enzymatic activities of sucrose phosphate synthase (SPS) (Yamaki,
(Burg and Burg, 1965). Fleshy fruit that present an increased respira- 1994), while Suc cleavage reactions are catalyzed by sucrose synthase
tion rate and a burst of ethylene biosynthesis during ripening are (SuSy) activity and cell wall, cytosolic and vacuolar invertases (CWINV,
classified as climacteric, whereas fruit that do not, are considered non- CytINV and VINV, respectively) (Klann et al., 1993; Li et al., 2012).
climacteric (Bapat et al., 2010; Biale, 1981; Brady, 1987; Giovannoni, Additionally, invertase inhibitors (INVINH) play roles as regulators of
2001). Nevertheless, regardless of their climacteric or non-climacteric invertases at the posttranscriptional level (Jin et al., 2009). Sor synth-
behavior, fleshy fruit undergo a complex and highly coordinated series esis is catalyzed by the enzyme sorbitol-6-phosphate-dehydrogenase
of events comprised in the developmental process of fruit ripening (S6PDH) that mediates the reduction of glucose-6-phosphate (G6P) to
(Grierson, 2013). These ripening-related changes determine the overall sorbitol-6-phosphate (Suzuki, 2015; Suzuki and Dandekar, 2014; Teo
final quality of fruit (Bouzayen et al., 2010; Klee and Giovannoni, et al., 2006). Sor breakdown is mediated by the activities of NAD+-
2011), including the modification of properties such as color, taste, dependent sorbitol dehydrogenase (NAD+-SDH) and sorbitol oxidase
texture and aroma (Giovannoni, 2004; Kumar et al., 2014; Seymour (SOX), which catabolize Sor into fructose (Fru) and glucose (Glu), re-
et al., 2013). Concerning taste, sweetness is of central importance and spectively (Teo et al., 2006). In addition to the major sugars Suc, Sor,
knowledge of the mechanisms involved in sugar metabolism, which Glu and Fru, fruit also contain sugars that are present in significantly
determine fruit sugar content and composition, are of crucial im- lower concentrations, including galactose (Gal), galactinol (Gol), raffi-
portance to develop cultivars that can meet consumer expectations nose (Raf), myo-inositol (Ino), and trehalose (Tre), among others. Gal
(Borsani et al., 2009; Desnoues et al., 2014; Singh and Khan, 2010). synthesis is mediated by the activities of alpha-galactosidase (AGAL),
In the Rosaceae family, which includes Japanese plums, the which hydrolases Raf to yield Gal and Suc, and beta-galactosidase

⁎
Corresponding author at: Department of Plant Sciences, University of California, 1 Shields Ave, Davis, CA 95616, USA.
E-mail addresses: mfarcuh@ucdavis.edu (M. Farcuh), rmrivero@cebas.csic.es (R.M. Rivero), vhasadka@volcani.agri.gov.il (A. Sadka), eblumwald@ucdavis.edu (E. Blumwald).

https://doi.org/10.1016/j.postharvbio.2018.01.012
Received 11 October 2017; Received in revised form 3 January 2018; Accepted 14 January 2018
0925-5214/ © 2018 Published by Elsevier B.V.
M. Farcuh et al. Postharvest Biology and Technology 139 (2018) 20–30

(BGAL), by cleaving galactosyl residues from cell wall polysaccharides; flow rate of 2 L min−1 in 330-L aluminum tanks completely sealed and
while free Gal is phosphorylated into galactose-1-phosphate by ga- connected to a flow-through system; fruit from the second group were
lactokinase (GALK) (Hubbard et al., 1989; Sozzi et al., 1998). The left to ripen under humidified, ethylene-free air containing 500 μL L−1
synthesis of Gol is mediated by the activity of galactinol synthase (GolS) of propylene (ethylene analogue, purchased from Praxair Inc., Danbury,
using UDP-galactose and Ino as substrates (Nishizawa et al., 2008). Gol, CT, US) at a flow rate of 2 L min−1 in 330-L aluminum tanks completely
together with Suc, are substrates used by raffinose synthase (RS) to sealed and connected to a flow-through system; while fruit from the
synthetize Raf, releasing Ino (Pillet et al., 2012). Finally, Tre catabolism third group, the controls, were left to ripen under humidified, ethylene-
is mediated by trehalase (TRE) (Ponnu et al., 2011). free air at a flow rate of 2 L min−1 in 330-L aluminum tanks completely
In our previous work (Farcuh et al., 2017) we identified and char- sealed and connected to a flow-through system. Humidified, ethylene-
acterized key mechanisms associated with sugar metabolism repro- free air was ensured by bubbling the gas mixture through distilled
gramming during ripening on-the-tree in a non-climacteric bud mutant water and by filtering atmospheric air through potassium permanga-
(Sweet Miriam) of a climacteric Japanese plum cultivar (Santa Rosa). nate (KMnO4), respectively.
We reported higher contents of Sor and lower contents of Suc, glucose Fruit from all groups were stored at 20 °C and 90% relative humidity
(Glu) and fructose (Fru) in the non-climacteric Sweet Miriam as com- for a maximum of 14 d. Evaluations were carried out at harvest (0) and
pared to the climacteric Santa Rosa cultivar. In addition, the content of after 1,3,5,7,10 and 14 d of storage. For each evaluation period, six
the minor sugars galactinol (Gol), raffinose (Raf), myo-inositol (Ino) and biological replications from each group were assessed. For each biolo-
trehalose (Tre) increased in Sweet Miriam, while galactose (Gal) con- gical replication, 6 fruit were used for the analysis of physicochemical
tents were higher in Santa Rosa. Although we were able to identify key parameters and ripening patterns, while 4 fruit were washed, peeled,
sugar metabolism-related genes and assess their roles using a Systems cut into small pieces, pooled together and frozen in liquid nitrogen in
Biology approach, information regarding the possible regulation of fruit order to be stored at − 80 °C for further analyses.
sugar metabolism by hormones is poorly characterized. It has been
reported that the suppression of ethylene biosynthesis or ethylene ac- 2.3. Fruit ripening patterns and physicochemical measurements
tion has no effect on fruit total soluble solids (TSS) and that the accu-
mulation of sugars in the fruit is an ethylene-independent event (Fan Fruit ripening patterns and physicochemical measurements were
et al., 1999; Knee, 1976; Menniti et al., 2004). Nevertheless, some data carried out as described previously in Kim et al. (2015a) and Farcuh
indicated that the interaction between sugars and ethylene could be et al. (2017). For each cultivar (Santa Rosa and Sweet Miriam), post-
sugar-type dependent (Li et al., 2016). Furthermore, a reciprocal cor- harvest storage stage (0,1,3,5,7,10 and 14 d of storage at 20 °C) and
relation between Sor and ethylene has been postulated. In the present group/treatment assayed (1-MCP, propylene, control), fruit ethylene
work, we treated fruit with propylene, an ethylene analogue (Burg and (C2H4 ng kg−1 s−1) and respiration production rate (CO2 μg kg−1 s−1)
Burg, 1967; Paul et al., 2012) and 1-methylcyclopropane (1-MCP), and as well as physicochemical properties including skin and flesh color,
inhibitor of ethylene binding to its receptors (Sisler and Serek, 1997; flesh firmness, soluble solids content (SSC), titratable acidity (TA), and
Watkins, 2006), and assessed gene expression profiling and fruit sugar pH were measured on six fruit from each biological replication.
analysis to characterize and compare the effect(s) of ethylene on the
regulation of sugar metabolism in Santa Rosa and Sweet Miriam Ja- 2.4. Sugar concentration quantification
panese plum fruit during postharvest storage.
2.4.1. NMR analyses
2. Materials and methods Six biological replicates of Santa Rosa and Sweet Miriam plum fruit
at each postharvest storage stage of evaluation (0,1,3,5,7,10 and 14 d of
2.1. Fruit material storage at 20 °C) and for each group/treatment assayed (1-MCP, pro-
pylene, control), were used to quantify the contents of Suc, Glu, Fru,
Fruit from the Japanese plum [Prunus salicina L.] cultivars Santa Sor, G6P, Gal, Raf, Ino, Tre and the cofactor NAD+. These metabolites
Rosa and Sweet Miriam were harvested from a commercial orchard were chosen for NMR analysis based on their biological significance, as
located in the California Central Valley production area (Parlier, CA, described in our previous work (Farcuh et al., 2017). The extraction of
USA) during two seasons as described in Farcuh et al. (2017). Fruit the metabolites and subsequent quantification was carried out as de-
growth and development patterns were monitored weekly (Kim et al., scribed in Farcuh et al. (2017) and all the results were expressed on dry
2015a). Using these data, but particularly fruit firmness as the maturity weight basis (g kg−1).
index, fruit were harvested at the ‘well-mature’ stage (Crisosto, 1994),
corresponding to a flesh fruit firmness of ∼37 N. This stage was 2.4.2. UHPLC-QTOF-MS/MS analyses
reached ∼112 d after full bloom (DAFB) in Santa Rosa and ∼170 DAFB Six biological replicates of Santa Rosa and Sweet Miriam plum fruit
in Sweet Miriam, just between the developmental stage S3/S4 (between at each postharvest storage stage of evaluation (0,1,3,5,7,10 and 14 d of
the end of the second exponential growth phase and the onset of ri- storage at 20 °C) and for each group/treatment assayed (1-MCP, pro-
pening) and S4-I (commercial harvest stage) stages described in Farcuh pylene, control), were used to quantify the contents of Gol. The che-
et al. (2017). In the case of Santa Rosa, due to its climacteric nature, the mical extraction, sugar separation and successive quantification was
‘well-mature’ stage also corresponded to the preclimacteric stage of this carried out as described in Farcuh et al. (2017) and all the results were
cultivar. Fruit with uniform size, absence of visual blemishes, bruises expressed on dry weight basis (g kg−1).
and/or diseases were chosen. After harvest, fruit were quickly trans-
ported to the laboratory. 2.5. Real-time quantitative RT-PCR analysis

2.2. Fruit postharvest storage and treatments RNA was isolated from each of the six biological replicates of Santa
Rosa and Sweet Miriam plum fruit at each postharvest storage stage of
A total of 1260 fruit were collected from Santa Rosa and Sweet evaluation (0,1,3,5,7,10 and 14 d of storage at 20 °C) and for each
Miriam cultivars. Fruit within each cultivar were randomized and di- group/treatment assayed (1-MCP, propylene, control), using the CTAB/
vided into 3 groups of 420 fruit each and commercially packed into NaCl method (Chang et al., 1993) with some modifications (Kim et al.,
cardboard boxes. Fruit from the first group were treated with 0.5 μL L−1 2015b). First-strand complementary DNA synthesis, primer design, and
1-MCP (SmartFresh™) at 20 °C for 24 h and immediately after the quantitative PCR were performed as described before (Kim et al.,
treatment were left to ripen under humidified, ethylene-free air at a 2015b). The sets of primers used for the amplification of the different

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target genes were previously described (Farcuh et al., 2017). Analysis of Physicochemical properties of the fruit were determined and are shown
the relative gene expression was performed according to the Com- on Fig. 1.
parative Cycle Threshold Method as described by Livak and Schmittgen Climacteric Santa Rosa control fruit ripening in postharvest storage,
(2001). The expression of the SAND protein-related trafficking protein maintained higher respiration rates than Sweet Miriam fruit, displaying
(MON) was used as a reference (Kim et al., 2015b). the typical respiratory burst and increased ethylene production rates
during ripening (Fig. 1A,B). Non-climacteric Sweet Miriam fruit dis-
2.6. Statistical analysis played constant and low ethylene production rates throughout post-
harvest storage (Fig. 1B). Upon propylene treatment, Santa Rosa fruit
The software package JMP® (ver.10.0, SAS Institute) was used for increased their respiration and ethylene production rates after 3 d of
the statistical analyses. Means of the six biological replications were storage, while Sweet Miriam fruit did not, supporting their non-cli-
submitted to three-way analysis of variance using Tukey’s test to macteric nature (Fig. 1A,B) (Minas et al., 2015). Following 1-MCP
compare between cultivars (Santa Rosa and Sweet Miriam), treatments treatment, Santa Rosa fruit dramatically decreased their respiration and
(1-MCP, propylene and control) and time in postharvest storage for ethylene production rates until 5 d of storage, reaching levels similar to
significant differences at P < 0.05 in all cases. those in Sweet Miriam fruit; while after 7 d of storage onwards, a re-
storation of the climacteric behavior in these fruit occurred (Fig. 1A,B).
3. Results Skin and flesh color hue values decreased in control fruit throughout
postharvest storage of both cultivars, with Santa Rosa displaying overall
3.1. Physicochemical properties and ripening patterns of climacteric Santa lower hue skin and flesh colors than Sweet Miriam (Fig. 1C,D). Fol-
Rosa and non-climacteric Sweet Miriam bud mutants throughout postharvest lowing propylene treatment, skin color hue values decreased dramati-
storage cally in Sweet Miriam fruit as compared to Sweet Miriam control fruit,
while flesh color hue values decreased faster in Santa Rosa treated fruit
Fruit from Santa Rosa and Sweet Miriam cultivars were harvested at (Fig. 1C,D). Upon 1-MCP treatment, Santa Rosa fruit flesh and skin
the “well-mature” stage (Crisosto, 1994), as mentioned previously and color exhibited higher hue values after 5 d of storage as compared to
were treated immediately after harvest with propylene and 1-MCP and control fruit and reached the same values as control fruit after 10 d of
kept in postharvest storage at 20 °C for a maximum of 14 d. postharvest (Fig. 1C,D).

Fig. 1. Fruit respiration, ethylene production rates and physicochemical properties of Santa Rosa (SR) and Sweet Miriam (SM) Japanese plum cultivars during ripening throughout
postharvest storage at 20 °C. Fruit respiration, ethylene production rates and physicochemical properties were determined in fruit from SR (left graph) and SM (right graph) cultivars
submitted to no treatment (control), 1-MCP treatment, and propylene treatment after 0,1,3,5,7,10 and 14 d at 20 °C. (A) Respiration production rates; (B) ethylene production rates; (C)
fruit skin color; (D) fruit flesh color; (E) fruit firmness values; (F) soluble solid contents (SSC); (G) fruit titratable acidity (TA) and (H) fruit pH. Values are means ± SE (n = 6). Different
letters indicate significant differences (p < 0.05) according to Tukey’s test.

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M. Farcuh et al. Postharvest Biology and Technology 139 (2018) 20–30

Flesh firmness during postharvest storage of Santa Rosa control and fruit, CWINV and CytINV transcript levels decreased throughout storage
propylene treated fruit reached the “ready-to-eat” stage (≤10 N) and yet VINV expression levels decreased in Santa Rosa and increased in
thus could not be further evaluated after 5 d of postharvest; while Sweet Sweet Miriam (Fig. 2). Sweet Miriam fruit displayed higher expression
Miriam control fruit displayed flesh firmness values of ∼25 N after 14 d of all assayed invertases as compared to Santa Rosa control fruit. After
of storage (Fig. 1E). Nevertheless, propylene treated Sweet Miriam fruit propylene treatments, Sweet Miriam fruit displayed a decrease in in-
were able to soften to the “ready-to-eat” stage after 10 d of postharvest vertases transcripts, while CWINV and VINV mRNA levels were higher
(Fig. 1E). Following 1-MCP treatment, Santa Rosa fruit decreased their in Sweet Miriam than in Santa Rosa propylene-treated fruit (Fig. 2). 1-
softening rate after 5 d of storage as compared to Santa Rosa control MCP treatments induced an increase in mRNA levels for the three in-
fruit and reached the “ready-to-eat” stage after 10 d of postharvest vertases in Santa Rosa fruit until 5 d of storage, with a dramatic de-
(Fig. 1E). crease afterwards due to the decrease in 1-MCP action. 1-MCP-treated
Sweet Miriam fruit presented higher SSC values at all stages Sweet Miriam fruit performed as Sweet Miriam control fruit, yet ex-
throughout ripening on postharvest storage and this parameter did not hibiting higher expression levels for all invertases with respect to 1-
respond to ethylene treatments (Fig. 1F). Titratable acidity values were MCP-treated Santa Rosa fruit. In addition, we assayed expression levels
lower in Sweet Miriam fruit and decreased in both cultivars throughout of INVINH due to their well-reported role as regulators of invertases at
ripening (Fig. 1G). Nevertheless, during postharvest storage Sweet the posttranscriptional level (Jin et al., 2009). Nevertheless, our results
Miriam TA values were constant (Fig. 1G). However, propylene treat- suggested that although both cultivars increased their INVINH mRNA
ment decreased TA values in Sweet Miriam fruit, while 1-MCP treated levels throughout postharvest storage, there was no effect of ethylene
fruit behaved as their respective controls in both cultivars (Fig. 1G). As on INVINH transcripts accumulation (Fig. 2).
expected, pH values were opposite to the TA values (Fig. 1H).
3.2.2. Sorbitol metabolism
3.2. Effects of ethylene on key sugar metabolism-associated genes and their In contrast to Suc, Sor contents decreased throughout postharvest
related metabolites in fruit of climacteric Santa Rosa and non-climacteric storage in Santa Rosa control fruit, but increased in Sweet Miriam fruit,
Sweet Miriam bud mutants throughout postharvest storage. with Sweet Miriam displaying a 3.5-fold higher Sor contents than Santa
Rosa fruit (Fig. 3). Propylene induced a decrease in Sor contents of
3.2.1. Sucrose, glucose and fructose metabolism Sweet Miriam fruit, although Sor contents remained higher in Sweet
Suc contents in Santa Rosa control fruit increased throughout Miriam than Santa Rosa fruit. While 1-MCP did not affect Sor contents
postharvest and were higher than those in Sweet Miriam control fruit, in Sweet Miriam fruit, a 2.5-fold increased Sor contents was observed in
which remained constant (Fig. 2). Upon propylene treatment, Sweet Santa Rosa fruit. This increase in Sor contents in Santa Rosa lasted until
Miriam fruit displayed increased Suc contents as compared to Sweet 5 d of storage and decreased afterwards, coincident with increased rates
Miriam control fruit, though lower than Santa Rosa control and pro- of ethylene production (Fig. 3).
pylene treated fruit after 3 and 5 d of storage. 1-MCP treatment in Santa Sor synthesis is mediated by the action of S6PDH (EC 1.1.1.200),
Rosa fruit decreased Suc contents with respect to control and propylene reducing G6P to Sor-6-phosphate (Suzuki and Dandekar, 2014). S6PDH
treatments, until 5 d of storage, period after which Suc concentration transcripts accumulation was higher in Sweet Miriam than in Santa
increased, coincident with increased rates of ethylene production; while Rosa fruit, although remained constant during postharvest storage and
Sweet Miriam fruit remained similar to control fruit. SPS (EC 2.4.1.14) was not affected either by propylene or 1-MCP treatments in either
transcript levels, associated with Suc synthesis (Yamaki, 1994), de- cultivar (Fig. 3). A decrease in G6P contents during postharvest storage
creased and remained constant throughout postharvest in Santa Rosa was observed in Sweet Miriam control fruit as well as upon propylene
and Sweet Miriam control fruit, respectively, although SPS expression and 1-MCP treatments, but not in Santa Rosa fruit. G6P concentrations
levels were higher in Santa Rosa fruit, in agreement with the higher Suc were higher in Sweet Miriam than in Santa Rosa fruit. G6P results from
contents in this cultivar (Fig. 2). Similar to what was observed for Suc, Glu phosphorylation mediated by HK (EC 2.7.1.1) (Li et al., 2012). HK
propylene treatments increased SPS transcript levels in Sweet Miriam mRNA levels were not affected by ethylene and remained constant in
fruit, while upon 1-MCP treatment, SPS expression levels in Santa Rosa both cultivars except after 7 d of storage in Sweet Miriam fruit, where a
fruit decreased until 5 d of storage, and increased thereafter (Fig. 2). decrease in transcripts was observed. HK transcripts were more abun-
Throughout storage, Glu contents decreased and Fru contents re- dant in Sweet Miriam than in Santa Rosa until 7 d of postharvest storage
mained constant in Santa Rosa control fruit, while both Glu and Fru and decreased afterwards (Fig. 3).
contents increased and remained higher in Sweet Miriam than in Santa Sor cleavage into Fru occurs via NAD+-SDH (EC 1.1.1.14), using
Rosa fruit (Fig. 2). Propylene treatments induced a decrease in Glu and NAD+ as a cofactor (Teo et al., 2006). NAD+-SDH transcript levels
Fru contents in Sweet Miriam fruit. On the other hand, Santa Rosa fruit increased dramatically throughout postharvest in Santa Rosa control
treated with 1-MCP exhibited an increase in Glu and Fru contents as and propylene treated fruit, while increased slightly in Sweet Miriam
compared to Santa Rosa control fruit (Fig. 2). fruit (Fig. 3). Santa Rosa fruit displayed a 2–3 fold higher NAD+-SDH
Suc breakdown into UDP-Glu and Fru is mediated by the action of expression levels and lower NAD+ contents than Sweet Miriam fruit.
SuSy activity (EC 2.4.1.13) (Klann et al., 1993). Only one Susy tran- Nevertheless, after propylene treatment Sweet Miriam fruit increased
script (ppa017606m.g) was identified among the 11 key sugar meta- NAD+-SDH expression levels with respect to Sweet Miriam control fruit
bolism-associated genes that were associated with the differences in with a corresponding decrease in NAD+ and Sor contents (Fig. 3).
sugar composition between the Santa Rosa and Sweet Miriam cultivars Following 1-MCP treatments, Santa Rosa fruit displayed significantly
(Farcuh et al., 2017). Transcript levels of SuSy decreased 5-fold in Santa lower NAD+-SDH transcripts accumulation as compared to Santa Rosa
Rosa control fruit throughout postharvest and were lower than Sweet control and propylene treated fruit until 5 d of storage, with an increase
Miriam control fruit, in agreement with the higher Suc contents in afterwards. The opposite trend was observed with NAD+ contents.
Santa Rosa (Fig. 2). Propylene treatment decreased SuSy transcripts Sweet Miriam fruit treated with 1-MCP displayed similar NAD+-SDH
accumulation in Sweet Miriam fruit as compared to control fruit. Upon expression levels as Sweet Miriam control fruit but lower than those of
1-MCP treatments, Santa Rosa fruit displayed about a 2-fold increase in Santa Rosa 1-MCP-treated fruit (Fig. 3).
SuSy mRNA levels with respect to Santa Rosa control fruit, until 5 d of
storage; while 1-MCP treatments had no effects on Sweet Miriam fruit. 3.2.3. Minor sugars metabolism
Suc catabolism into Glu and Fru is mediated by invertases (EC 3.2.1.26) Gal contents of Santa Rosa and Sweet Miriam control fruit increased
(including CWINV, CytINV and VINV) (Li et al., 2012), all of which throughout postharvest with Santa Rosa fruit displaying a 2.5-fold
were assayed in this study. In Santa Rosa and Sweet Miriam control higher Gal content than Sweet Miriam fruit (Fig. 4). Although

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Fig. 2. Sucrose metabolism-associated pathways in fruit of Santa Rosa (SR) and Sweet Miriam (SM) Japanese plum cultivars during ripening throughout postharvest storage at 20 °C.
Sucrose metabolism-associated pathways in fruit of Santa Rosa (SR; left graph) and Sweet Miriam (SM; right graph) Japanese plum cultivars submitted to no treatment (control), 1-MCP
treatment, and propylene treatment after 0,1,3,5,7,10 and 14 d of storage at 20 °C. Sugar contents are presented in graphs framed by dashed lines and are expressed as g kg−1 on a dry
weight basis. Relative gene expression levels were calculated and normalized using the SAND protein-related trafficking protein (MON) as reference gene. Values are means ± SE
(n = 6). The data were analyzed using three-way ANOVA followed by Tukey’s test. Different letters indicate significant differences (p < 0.05) according to Tukey’s test and are
comparing between both cultivars (graphs). Sucrose phosphate synthase (SPS); Sucrose phosphate synthase (SPS); Sucrose synthase (SuSy); cell wall invertase (CWINV); vacuolar
invertase (VINV); cytosolic invertase (CytINV); Invertase inhibitor (INVINH); sucrose (Suc); fructose (Fru); glucose (Glu); fructose-6-phosphate (F6P); UDP glucose (UDP-Glu).

propylene treatments did not affect Gal contents in Santa Rosa, it in- than in Sweet Miriam fruit (Fig. 5). Propylene treatment increased the
creased dramatically in Sweet Miriam fruit after 7 and 10 d of storage. GALK expression in Sweet Miriam fruit after 7 and 10 d of storage,
1-MCP treatments induced a decrease in Gal contents in both Santa while 1-MCP applications decreased GALK mRNA levels in Santa Rosa
Rosa and Sweet Miriam fruit (Fig. 4). Gal, together with Suc, are pro- until 5 d, consistent with the changes in AGAL and BGAL transcripts and
ducts of Raf catabolism via the action of AGAL (α-galactosidase) (EC Gal contents (Fig. 5).
3.2.1.22) (Hubbard et al., 1989). Gal synthesis is also the result of the Gol contents remained constant in Santa Rosa and Sweet Miriam
action of BGAL (β-galactosidase) (EC 3.2.1.23), mediating the catabo- control fruit throughout postharvest storage, although Gol contents
lism of galactosyl residues from cell wall polysaccharides (Sozzi et al., were higher in Sweet Miriam than in Santa Rosa fruit (Fig. 4). Propy-
1998). Santa Rosa fruit displayed a relatively constant AGAL expression lene induced a decrease in Gol contents in Sweet Miriam fruit, while 1-
during postharvest storage, while the expression of BGAL increased MCP treatments induced a notable increase in Gol contents in Santa
throughout storage (Fig. 5). AGAL and BGAL transcript accumulation Rosa fruit (Fig. 4). Furthermore, Gol synthesis, using UDP-Gal and Ino
remained constant throughout postharvest ripening in Sweet Miriam as substrates, is mediated by the action of GolS (galactinol synthase)
fruit (Fig. 5). AGAL and BGAL mRNA levels were 2 to 3-fold higher in (EC 2.4.1.123) (Nishizawa et al., 2008). GolS transcript levels were
Santa Rosa than Sweet Miriam control fruit, in agreement with the dramatically higher (8-fold) in Sweet Miriam than in Santa Rosa fruit
higher Gal contents displayed by Santa Rosa fruit. Interestingly, pro- throughout postharvest ripening, in agreement with the increased Gol
pylene treatments increased both AGAL and BGAL transcripts accu- contents observed in Sweet Miriam fruit (Figs. 4 and 5). Nevertheless,
mulation in Sweet Miriam fruit after 7 and 10 d of storage (Fig. 5), upon propylene treatment, Sweet Miriam fruit displayed a 3 to 4-fold
which correlated well with the increased Gal contents observed in decrease in GolS transcript accumulation with respect to Sweet Miriam
propylene-treated Sweet Miriam fruit (Fig. 4). Following 1-MCP treat- control fruit, although maintaining a higher GolS expression levels than
ment, Santa Rosa fruit displayed decreased AGAL and BGAL mRNA Santa Rosa propylene-treated fruit (Fig. 5). Following 1-MCP treat-
levels until 5 d of storage, but increased afterwards (with the restora- ments, Santa Rosa fruit increased their GolS mRNA levels with respect
tion of ethylene production) (Fig. 5). Gal can be phosphorylated into to Santa Rosa control and propylene-treated fruit until 5 d of post-
galactose-1-phosphate via GALK (galactokinase) EC 2.7.1.6) (Dai et al., harvest, supporting the high Gol contents in this cultivar (Fig. 5).
2006). GALK mRNA levels increased throughout postharvest ripening in Gol, together with Suc, are substrates used by RS (raffinose syn-
both cultivars, while GALK expression levels were higher in Santa Rosa thase) (EC 2.4.1.82) to synthetize Raf (Pillet et al., 2012). Raf contents

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Fig. 3. Sorbitol metabolism-associated pathways in fruit of Santa Rosa (SR) and Sweet Miriam (SM) Japanese plum cultivars during ripening throughout postharvest storage at 20 °C.
Sorbitol metabolism-associated pathways in fruit of Santa Rosa (SR; left graph) and Sweet Miriam (SM; right graph) Japanese plum cultivars submitted to no treatment (control), 1-MCP
treatment, and propylene treatment after 0,1,3,5,7,10 and 14 d of storage at 20 °C. Sugar contents are presented in graphs framed by dashed lines and are expressed as g kg−1 on a dry
weight basis. Relative gene expression levels were calculated and normalized using the SAND protein-related trafficking protein (MON) as reference gene. Values are means ± SE
(n = 6). The data were analyzed using three-way ANOVA followed by Tukey’s test. Different letters indicate significant differences (p < 0.05) according to Tukey’s test and are
comparing between both cultivars (graphs). Sorbitol-6-phosphate dehydrogenase (S6PDH); NAD+-dependent sorbitol dehydrogenase (NAD+-SDH); sorbitol oxidase (SOX); hexokinase
(HK); glucose-6-phosphate (G6P); sorbitol (Sor); Nicotinamide adenine dinucleotide(NAD+); fructose (Fru); glucose (Glu).

decreased throughout storage in Santa Rosa control fruit while re- Sweet Miriam fruit displayed 2-fold higher TRE mRNA levels than
mained constant in Sweet Miriam control fruit (Fig. 4). These results Sweet Miriam control fruit, consistent with the decrease in Tre contents
were consistent with RS transcript levels that were 2 to 3-fold higher in in Sweet Miriam propylene-treated fruit. 1-MCP treatments decreased
Sweet Miriam than in Santa Rosa fruit. Upon propylene treatments, TRE expression levels and increased Tre contents by 2-fold in Santa
Sweet Miriam fruit showed a 3 to 4-fold decreased Raf contents as well Rosa fruit with respect to Santa Rosa control and propylene-treated
as a decrease in RS mRNA levels with respect to Sweet Miriam control fruit until 5 d of storage, period after which ethylene levels were re-
fruit (Figs. 4 and 5). After 1-MCP treatments, Santa Rosa fruit displayed stored (Figs. 4 and 5).
increased Raf contents as well as RS transcripts with respect to Santa
Rosa control and propylene-treated fruit until 5 d of postharvest ri-
4. Discussion
pening, decreasing afterwards. Sweet Miriam 1-MCP-treated fruit be-
haved as Sweet Miriam control fruit (Figs. 4 and 5).
In the present work we compared profiles of sugar metabolism-re-
Ino contents, used by GolS and formed through the action of RS,
lated genes and metabolites of two Japanese plum cultivars under
increased throughout postharvest storage in both cultivars. Upon pro-
controlled conditions and under different ethylene and 1-MCP treat-
pylene treatment, Sweet Miriam fruit displayed lower Ino contents with
ments during postharvest storage. Our experimental system comprised
respect to Sweet Miriam control fruit; while after 1-MCP treatment,
Santa Rosa and Sweet Miriam plum cultivars, that share the same ge-
Santa Rosa fruit showed 2-fold higher Ino contents as compared to
netic background, but because of the bud-sport nature of the Sweet
Santa Rosa control and propylene-treated fruit until 3 d of storage
Miriam cultivar they display different ripening behaviors (i.e. climac-
(Fig. 4).
teric – Santa Rosa – and non-climacteric – Sweet Miriam –) (Farcuh
Regarding Tre, its breakdown occurs via TRE (trehalase) (EC
et al., 2017; Kim et al., 2015a; Minas et al., 2015). Although the main
3.2.1.28) (Ponnu et al., 2011). TRE transcript accumulation increased
sugar metabolism-related enzymes and their expression levels have
throughout postharvest in both Santa Rosa and Sweet Miriam fruit, yet
been identified and assayed in several climacteric and non-climacteric
were higher in Santa Rosa control fruit, in agreement with the lower Tre
fleshy fruit-types (Beauvoit et al., 2014; Borsani et al., 2009; Li et al.,
contents in this cultivar (Figs. 4 and 5). After propylene treatment,
2012; Moriguchi et al., 1992; Yamaki, 1986), little is known about their

25
M. Farcuh et al. Postharvest Biology and Technology 139 (2018) 20–30

Fig. 4. Minor sugar metabolism-associated pathways in fruit of Santa Rosa (SR) and Sweet Miriam (SM) Japanese plum cultivars during ripening throughout postharvest storage at 20 °C.
Galactose, galactinol, raffinose, myo-inositol and trehalose metabolism-associated pathways in fruit of Santa Rosa (SR; left graph) and Sweet Miriam (SM; right graph) Japanese plum
cultivars submitted to no treatment (control), 1-MCP treatment, and propylene treatment after 0,1,3,5,7,10 and 14 d of storage at 20 °C. Sugar contents are expressed as g kg−1 on a dry
weight basis. Values are means ± SE (n = 6). The data were analyzed using three-way ANOVA followed by Tukey’s test. Different letters indicate significant differences (p < 0.05)
according to Tukey’s test and are comparing between both cultivars (graphs). Galatokinase (GALK); Alpha-Galactosidase (α-GAL); Beta-Galactosidase (β-GAL); Galactinol synthase (GolS);
Raffinose synthase (RS); Trehalase (TRE); cell wall invertase (CWINV); vacuolar invertase (VINV); cytosolic invertase (CytINV); galactose (Gal); galactinol (Gol); raffinose (Raf); myo-
inositol (Ino); trehalose (Tre); fructose (Fru); glucose (Glu); sucrose (Suc); galactose-1-phosphate (Gal 1P); UDP- galactose (UDP-Gal).

regulation (Génard and Souty, 1996) during postharvest storage. exposure to exogenous ethylene throughout postharvest storage
Our previous study (Farcuh et al., 2017) demonstrated the repro- (Defilippi et al., 2004). Furthermore, in non-climacteric fruit such as
gramming of sugar metabolism in Sweet Miriam fruit type during ri- strawberries, Suc contents have been shown to accelerate the ripening
pening on-the-tree, identified genes associated with the differences in process (Jia et al., 2013a,b). In the case of grapes, fruit treated with 1-
sugar composition between both cultivars and suggested the possibility MCP reduced Suc accumulation with respect to control fruit (Chervin
of a link between ethylene action and overall fruit sugar homeostasis. et al., 2006); while low Suc contents were reported as one of the main
Here, we examined the responses to ethylene and 1-MCP treatments of factors associated with the late-ripening behavior of a spontaneous
these key sugar metabolism-related genes, throughout postharvest ri- sweet orange mutant as compared to its wild type (Zhang et al., 2014).
pening. We assayed gene expression levels and their associated sugar Thus, these above-mentioned studies support the higher Suc contents
contents and show that ethylene is a crucial factor affecting overall observed in Santa Rosa with respect to Sweet Miriam control fruit and
sugar metabolism in both ripening types. A summarized scheme of the the responses of both cultivars to ethylene and 1-MCP treatments and
overall results of this work is presented in Fig. 6. suggest a role for ethylene positively regulating Suc contents in cli-
macteric and non-climacteric fruit (Figs. 2 and 6). Regarding Suc me-
4.1. Ethylene reduces sucrose catabolism but hastens sorbitol breakdown tabolism, ethylene has been reported to strongly stimulate the expres-
during fruit ripening in postharvest storage sion of SPS, encoding the key Suc biosynthetic enzyme (Miron and
Schaffer, 1991) in fruit such as banana, peach and kiwifruit (Choudhury
Suc and Sor contents have been reported to vary dramatically et al., 2008; Langenkämper et al., 1998; Lombardo et al., 2011), in
among members of the Rosaceae family (Desnoues et al., 2014). For agreement with the results of this study in both cultivars (Figs. 2 and 6).
example, Suc contents are more abundant in climacteric peaches On the other hand, gene expression of Suc-breakdown related enzymes,
(Moriguchi et al., 1990), but Sor contents are higher in non-climacteric including invertases and SuSy, which catabolize Suc into hexoses
cherries (Gao et al., 2003). These results would support our findings on (Kleczkowski et al., 2010; Li et al., 2012), are negatively affected by
high Suc and high Sor contents in Santa Rosa and Sweet Miriam fruit, ethylene, as observed in tomato for invertases (Klann et al., 1996) and
respectively (Figs. 2, 3, 6). In addition, the contrasting ethylene pro- in the present study, supporting the higher Suc contents in Santa Rosa
duction rates between climacteric and non-climacteric fruit (Fig. 1B) and propylene-treated Sweet Miriam fruit (Figs. 2 and 6).
suggest the notion of ethylene being a key player in the regulation of In the case of Sor, although there is considerably less literature
sugar metabolism during ripening. available as compared to Suc, there seems to be an antagonistic inter-
In climacteric fruit such as tomatoes, Suc has been reported to act action between Sor and ethylene, as suggested in our previous work
synergistically with ethylene, hastening the ripening process (Farcuh et al., 2017) and as shown in this study (Figs. 3 and 6). In
throughout postharvest storage (Li et al., 2016). Likewise, in transgenic peach, Sor contents were rapidly consumed as the fruit ripened
apples, with a downregulation of ethylene biosynthesis, Suc contents throughout postharvest (Borsani et al., 2009; Lombardo et al., 2011);
were restored to the same concentrations as wild type apples only after while in apples treated with 1-MCP, an increased Sor accumulation was

26
M. Farcuh et al. Postharvest Biology and Technology 139 (2018) 20–30

Fig. 5. Relative gene expression levels of minor sugar metabolism-associated pathways in fruit of Santa Rosa (SR) and Sweet Miriam (SM) Japanese plum cultivars during ripening
throughout postharvest storage at 20 °C. Relative gene expression levels of galactose, galactinol, raffinose, and trehalose metabolism-associated pathways in fruit of Santa Rosa (SR; left
graph) and Sweet Miriam (SM; right graph) Japanese plum cultivars submitted to no treatment (control), 1-MCP treatment, and propylene treatment after 0,1,3,5,7,10 and 14 d of storage
at 20 °C. Relative gene expression levels were calculated and normalized using the SAND protein-related trafficking protein (MON) as reference gene. Values are means ± SE (n = 6). The
data were analyzed using three-way ANOVA followed by Tukey’s test. Different letters indicate significant differences (p < 0.05) according to Tukey’s test and are comparing between
both cultivars (graphs). (A) Alpha-Galactosidase (α-GAL); (B) Beta-Galactosidase (β-GAL); (C) Galatokinase (GALK); (D) Galactinol synthase (GolS); (E) Raffinose synthase (RS); (F)
Trehalase (TRE).

27
M. Farcuh et al. Postharvest Biology and Technology 139 (2018) 20–30

Fig. 6. Schematic summary of ethylene regulation of

sugar metabolism-related pathways in climacteric
and non-climacteric plum fruit during postharvest
storage at 20 °C. Sugars are presented in boxes that
when colored red or blue indicate that ethylene had
a positive or negative effect on that specific sugar
content, respectively. Thick upward red or blue ar-
rows indicate mRNA expression levels that were in-
duced or reduced by ethylene, respectively. Boxes
and arrows colored white indicate there was no ef-
fect of ethylene. Sucrose phosphate synthase (SPS);
Sucrose synthase (SuSy); Cell wall invertase
(CWINV); Vacuolar invertase (VINV); Cytosolic in-
vertase (CytINV); Invertase inhibitor (INVINH); sor-
bitol-6-phosphate dehydrogenase (S6PDH); NAD+-
dependent sorbitol dehydrogenase (NAD+-SDH);
sorbitol oxidase (SOX); hexokinase (HK);
Galactokinase (GALK); Alpha-Galactosidase (α-GAL);
Beta-Galactosidase (β-GAL); Galactinol synthase
(GolS); Raffinose synthase (RS); Trehalase (TRE);
sucrose (Suc); fructose (Fru); glucose (Glu); fructose-
6-phosphate (F6P); UDP glucose (UDP-Glu); glucose-
6-phosphate (G6P); sorbitol (Sor); Nicotinamide
adenine dinucleotide (NAD+); galactose (Gal); ga-
lactinol (Gol); raffinose (Raf); myo-inositol (Ino);
trehalose (Tre); galactose-1-phosphate (Gal 1P);
UDP- galactose (UDP-Gal). (For interpretation of the
references to color in this figure legend, the reader is
referred to the web version of this article.)

observed during storage (Lee et al., 2012), in agreement with our re- primary cell wall and a loss of Gal from the side chains of the polymers
sults in the climacteric Santa Rosa fruit (Figs. 3 and 6). Concerning Sor (Redgwell et al., 1997). The enzyme BGAL has been associated with the
metabolism, ethylene treatments in apples during postharvest storage cleavage of these galactosyl residues thus contributing to the free Gal
have been reported to decrease S6PDH protein levels, the main Sor- pool and to fruit softening (Ross et al., 1994; Sozzi et al., 1998). In
biosynthesis related enzyme (Zheng et al., 2013). While our results addition, AGAL also increases Gal contents by hydrolyzing Raf to yield
indicated higher S6PDH transcripts accumulation in Sweet Miriam with free Gal and Suc (Dai et al., 2006; Hubbard et al., 1989). Ethylene
respect to Santa Rosa fruit, a lack of ethylene effect was observed in promotes BGAL and AGAL transcript accumulation in Santa Rosa and
both cultivars (Figs. 3 and 6), what could be due to posttranscriptional Sweet Miriam fruit, thus supports the increase in Gal contents (Figs. 4,
modifications. Inversely, based on our results, Sor breakdown into Fru, 5A,B, 6) and contributes to the loss of fruit firmness throughout post-
catalyzed by NAD+-SDH (Teo et al., 2006) is suggested to be positively harvest displayed in Santa Rosa and propylene-treated Sweet Miriam
regulated by ethylene in both cultivars (Figs. 3 and 6). This is in fruit (Fig. 1E). On the other hand, free Gal has been reported to sti-
agreement with Begheldo (2008) that reported that propylene-treated mulate ethylene production and promote earlier ripening in tomatoes
peaches during storage increased their NAD+-SDH mRNA levels. (Gross, 1985) due to the capacity of Gal to promote the activity of 1-
Thus, our results suggested that there was an effect of ethylene on aminocyclopropane-1-carboxylic acid synthase (ACS), the rate-limiting
Suc and Sor metabolism and that the hexoses Glu and Fru in climacteric step in ethylene biosynthesis (Kim et al., 1987). This possible sy-
Santa Rosa and non-climacteric Sweet Miriam fruit resulted from Sor nergistic interaction between ethylene and free Gal is currently under
and Suc catabolism, respectively during ripening in postharvest storage investigation.
(Fig. 6). Nevertheless, why are there overall lower Glu and Fru contents Furthermore, our results suggest a negative effect of ethylene in Gol,
in Santa Rosa as compared to Sweet Miriam fruit? Possible explanations Raf, Ino and Tre contents (Fig. 6) due to the capacity of 1-MCP treat-
for this observations might be: (a) Glu and Fru have higher metabolic ments to increase Gol, Raf, Ino and Tre contents in Santa Rosa fruit and
accessibility to respiratory loss with respect to Suc and Sor, and re- the inverse effect in Sweet Miriam -propylene treated fruit (Figs. 4 and
spiration rates are dramatically higher in Santa Rosa than Sweet Miriam 6). In addition, higher Gol, Raf, Ino and Tre contents in Sweet Miriam,
fruit (Fig. 1A) and/or (b) the existence of an antagonistic interaction as compared to Santa Rosa fruit, were also observed in our previous
between Glu and ethylene that has been reported in Arabidopsis study during ripening on-the-tree (Farcuh et al., 2017).
(Yanagisawa et al., 2003) and is supported by our results in both cul- Increased contents of metabolites such as Gol, Raf, Ino and Tre have
tivars (Fig. 6). Furthermore, on our previous study, where we explored been reported to be key players in mitigating overall stress effects on
sugar metabolism reprogramming in Santa Rosa and Sweet Miriam plants due to their roles as osmoprotectans, cell membrane stabilizers,
cultivars during ripening on-the-tree (Farcuh et al., 2017), we observed and their high antioxidant capacities (Nishizawa et al., 2008; Sun et al.,
higher Glu and Fru contents in Santa Rosa than of Sweet Miriam fruit. 2013; Taji et al., 2002; Valluru and Van den Ende, 2011; Xue et al.,
The inverse results obtained between on-the-tree and postharvest ri- 2007). Dramatic increases in the contents of Gol and Raf were observed
pening sugar composition could be due to the effect of source-sink sugar in peaches submitted to cold and heat stresses throughout postharvest
transport occurring on-the-tree and which is not a critical factor during storage, and gene expression levels of the respective biosynthetic en-
ripening in storage. zymes GolS and RS paralleled this increase, rendering the fruit with
better capacity to withstand storage (Lauxmann et al., 2014). Thus, it
4.2. Ethylene positively interacts with galactose, but has negative effects on can be hypothesized that the higher contents of Gol, Raf, Ino and Tre in
galactinol, raffinose, myo-inositol and trehalose contents throughout Sweet Miriam fruit (Figs. 4 and 6) could be priming Sweet Miriam fruit
postharvest fruit ripening to better cope with stressful situations. This notion could support the
capacity of Sweet Miriam fruit to withstand almost three times longer
In general, fruit ripening comprises a number of biological pro- (14 d) in postharvest storage than Santa Rosa fruit (5 d), but needs to be
cesses, among them the solubilization of pectic polysaccharides of the further investigated.

28
M. Farcuh et al. Postharvest Biology and Technology 139 (2018) 20–30

5. Conclusion growth. J. Am. Soc. Hortic. Sci. 121, 1122–1131.

Gao, Z., Maurousset, L., Lemoine, R., Yoo, S.-D., Van Nocker, S., Loescher, W., 2003.
Cloning expression, and characterization of sorbitol transporters from developing
In conclusion, we used sugar related-gene expression and sugar sour cherry fruit and leaf sink tissues. Plant Physiol. 131, 1566–1575.
contents profiling in fruit of the climacteric Santa Rosa and its bud Giovannoni, J., 2001. Molecular biology of fruit maturation and ripening. Annu. Rev.
mutant, the non-climacteric Sweet Miriam cultivar, submitted to dif- Plant Biol. 52, 725–749.
Giovannoni, J.J., 2004. Genetic regulation of fruit development and ripening. Plant Cell
ferent ethylene and 1-MCP treatments throughout postharvest ripening. 16, S170–S180.
These treatments contributed to characterize the effect(s) of ethylene Grierson, D., 2013. Ethylene and the control of fruit ripening. Mol. Biol. Biochem. Fruit
on overall sugar metabolism in both ripening types as well as to com- Ripening 43–73.
Gross, K.C., 1985. Promotion of ethylene evolution and ripening of tomato fruit by ga-
pare between them. Ethylene seems to present contrasting effects on lactose. Plant Physiol. 79, 306–307.
Suc and Sor metabolism: it reduces Suc catabolism and induces Suc Hubbard, N.L., Huber, S.C., Pharr, D.M., 1989. Sucrose phosphate synthase and acid in-
biosynthesis but inversely tends to stimulate Sor breakdown and de- vertase as determinants of sucrose concentration in developing muskmelon (Cucumis
melo L.) fruits. Plant Physiol. 91, 1527–1534.
crease Sor biosynthesis. Furthermore, Glu and Fru contents are sug-
Jia, H., Li, C., Chai, Y., Xing, Y., Shen, Y., 2013a. Sucrose promotes strawberry fruit
gested to result from Sor and Suc breakdown in climacteric and non- ripening by stimulation of abscisic acid biosynthesis. Pak. J. Bot. 45, 169–176.
climacteric fruit, respectively. A positive interaction is shown between Jia, H., Wang, Y., Sun, M., Li, B., Han, Y., Zhao, Y., Li, X., Ding, N., Li, C., Ji, W., 2013b.
ethylene and Gal metabolism, since ethylene promoted an increase in Sucrose functions as a signal involved in the regulation of strawberry fruit develop-
ment and ripening. New Phytol. 198, 453–465.
free Gal contents. Finally, the negative effect of ethylene on Gal, Raf, Jin, Y., Ni, D.-A., Ruan, Y.-L., 2009. Posttranslational elevation of cell wall invertase
Ino and Tre contents, which were higher in non-climacteric Sweet activity by silencing its inhibitor in tomato delays leaf senescence and increases seed
Miriam fruit, could contribute to the increased capacity of this cultivar weight and fruit hexose level. Plant Cell 21, 2072–2089.
Kim, J., Gross, K.C., Solomos, T., 1987. Characterization of the stimulation of ethylene
to tolerate stresses associated with the ripening process per se and the production by galactose in tomato (Lycopersicon esculentum Mill.) fruit. Plant
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Kim, H.-Y., Farcuh, M., Cohen, Y., Crisosto, C., Sadka, A., Blumwald, E., 2015a. Non-
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Acknowledgements Kim, H.-Y., Saha, P., Farcuh, M., Li, B., Sadka, A., Blumwald, E., 2015b. RNA-Seq analysis
of spatiotemporal gene expression patterns during fruit development revealed re-
This research was supported by Will W. Lester Endowment of ference genes for transcript normalization in plums. Plant Mol. Biol. Rep. 33,
1634–1649.
University of California, Davis. M.F. was a fellowship recipient from Klann, E.M., Chetelat, R.T., Bennett, A.B., 1993. Expression of acid invertase gene con-
Programa Formacion de Capital Humano Avanzado, CONICYT, Chile trols sugar composition in tomato (Lycopersicon) fruit. Plant Physiol. 103, 863–870.
Klann, E.M., Hall, B., Bennett, A.B., 1996. Antisense acid invertase (TIV1) gene alters
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