Journal of Berry Research 8 (2018) 177–192 177

DOI:10.3233/JBR-180309
IOS Press

Metabolic and physiologic profile during

the fruit ripening of three blueberries
highbush (Vaccinium corymbosum) cultivars
M.L. Montecchiarinia , F. Bellob , M.F. Rivadeneirab , D. Vázquezb , F.E. Podestáa and K.E.J. Tripodia,∗
a Centro de Estudios Fotosintéticos y Bioquı́micos (CEFOBI), Facultad de Ciencias Bioquı́micas y Farmacéuticas,

Universidad Nacional de Rosario, Suipacha 531, 2000, Rosario, Argentina

b Estación
Experimental Concordia, Instituto Nacional de Tecnologı́a Agropecuaria (INTA), Colonia Yeruá,
Argentina

Received 23 April 2018; accepted 15 June 2018

Abstract.
BACKGROUND: Blueberry was introduced as a crop in Argentina about 30 years ago. Its harvesting period ranges from
September to December, during the northern hemisphere (its main export destination) fall season, which makes it a profitable
commercial crop. As most part of the production is exported fresh, the understanding of biochemical aspects connected with
fruit firmness is crucial to improve marketable conditions.
OBJECTIVE: The main purpose of this work is to explore the metabolic and physiologic changes in three highbush blueberry
cultivars during maturation and the possible association with their contrasting firmness features.
METHODS: Vaccinium corymbosum cv. ‘Emerald’, ‘Snowchaser’ and ‘O´Neal’, in order of decreasing firmness, were
collected at green and ripe stages. Metabolites were analyzed by gas chromatography-mass spectrometry (GC-MS) and HPLC.
Total phenolic compounds, pectin methyl esterase (PME) and ␤-galactosidase activities were quantified by colorimetric
assays.
RESULTS: Multivariate analysis of metabolites differentiated fruit regarding their maturation state in the first place. Malic,
citric and phosphoric acids, asparagine (Asn) and mannitol were more abundant in green fruits. Conversely, mature fruits were
distinguished by their higher content of citrulline and turanose. Other compounds were responsible for the differentiation
between varieties: histidine (His), valine (Val), arginine (Arg), methionine (Met) and sucrose where high in ripe Snowchaser,
while green and ripe Emerald had more tryptophane (Trp), glycine (Gly), phenylalanine (Phe), Trp, Gly and glucose. An
interesting finding is that Emerald, the firmer variety, had less xylose content at both stages, possibly owing to a minor degree
of cell wall degradation. Fold change of PME and ␤-galactosidase activity from green to ripe fruit demonstrated a divergent
tendency in Emerald and Snowchaser compared to O´Neal. A correlation study strongly and positively connected firmness
with citric acid and phenylalanine (Phe) content, while xylose, leucine (Leu) and shikimic acid were negatively related to
this attribute.
CONCLUSIONS: This study suggests that changes in the content of a few metabolite and activities of cell wall modifying
enzymes during maturation period could be correlated with the observed difference in firmness of the blueberries studied.
These findings may yield clues for improvements in fertilization protocols as well as to serve to the guided development of
new varieties based on biochemical quality traits.

Keywords: Blueberries, metabolomic, firmness, amino acids, pectin methyl esterase (PME) ␤-galactosidase

∗ Corresponding author: Dr. Karina E.J. Tripodi, CEFOBI, Suipacha 531, 2000 Rosario, Argentina. Tel./Fax: +54 341 4371955; E-mail:
tripodi@cefobi-conicet.gov.ar.

1878-5093/18/$35.00 © 2018 – IOS Press and the authors. All rights reserved
178 M.L. Montecchiarini et al. / Metabolic and physiologic profile during the fruit ripening

1. Introduction

Vaccinium corymbosum (blueberry) belongs to the Ericaceae family and is native from eastern of North
America. Regarding their growth size they can be classified in highbush (V. corymbosum), lowbush (V. angus-
tifolium) or rabbiteye (V. virgatum) [1]. Highbush blueberries comprise Southern and Northern varieties, with
low and high chilling requirement (number of hours at temperatures below 7◦ C between 200 and 400), respec-
tively. Southern highbush cultivars are thus well adapted to the mild climate of the Mediterranean region of
Argentina. The cultivated area of blueberries has expanded rapidly since it was initially introduced in the mid
1990 decade. Indeed, acreage has grown six times in the last ten years [https://inta.gob.ar/sites/default/files/script-
tmp-cadena arandano.pdf].
Most of the Argentinean berries production is exported to the northern hemisphere, during the boreal fall
season (September to December), when it might reach competitive prices due to lack of local production. Given
the relevance of this crop, many efforts have been directed to improve fruit attributes which may have influence
quality, transport and marketable conditions. One critical trait is firmness, since a soft fruit is perceived as over-
ripen and may impact negatively on the consumer´s choice. Loss of firmness is caused by several factors, such as
turgor, cell membranes damage, dehydration and cell wall dynamics [2–5]. At the same time, these components
are affected by climate, soil composition and varieties, among other aspects, causing inconsistent results in
terms of fertilization, pre- and postharvest treatments [6, 7]. In Argentine, as in many other countries, calcium
fertilization is a current practice, both by soil or foliar application [8–10], with fluctuating results. Research in
this field has been focused on the measure of several parameters related to cell wall metabolism after diverse
calcium treatments, like composition and methylation degree of pectin, activities of enzymes involved in cell
wall synthesis and degradation and/or calcium levels associated with wall components [9, 11, 12]. However,
there are not many studies intended to characterize different varieties under the same edaphic and fertilization
conditions that help to understand the intrinsic features of each cultivar that could be relevant for their general
field and postharvest behavior.
In this study, a metabolomic approach has been considered to evaluate three blueberry varieties broadly
cultivated in the eastern region of Argentina at two developmental stages. They were selected according to their
differences in firmness attributes and were characterized based on their metabolite content and physiological
features. They all are southern highbush cultivars, successfully adapted to mild winters in this region of South
America. ‘Emerald’ (U.S. Plant Patent 12165 P2) fruit is large, firm, exhibits a medium blue color, with good
flavor and is very productive. ‘Snowchaser’ (U.S. Plant Patent 19503 P3) is an early ripening variety, exhibits
a medium sized, good flavored fruit with a light blue color. ‘O´Neal’ is an early, public variety, with medium
size fruit and very sweet (http://www.fallcreeknursery.com/commercial-fruit-growers/varieties). The activity of
two enzymes linked to cell wall metabolism was also measured and the analysis were conducted to advance in
the understanding of metabolites/activities that could be considered as a biochemical signature in terms of fruit
quality. After these studies some basic differences were evident between developmental stages and varieties.
Furthermore, the abundance of some metabolites and relative variation of cell wall modifying enzymes could
be correlated with firmness. This knowledge may help to outline fertilization programs adapted to local climate
and soils. Likewise, it can assist agronomists with low-cost, reliable, methods for screening and selection of
varieties.

2. Methods

2.1. Fruit sampling

Blueberries from ‘Emerald’, ‘Snowchaser’ and ‘O´Neal’ cultivars were collected at local orchards in Concordia
(Entre Rı́os, Argentina) during the morning, in two consecutive seasons, 2015 and 2016. Mature bushes used
 M.L. Montecchiarini et al. / Metabolic and physiologic profile during the fruit ripening 179

for field experiments were located in commercial fields. The plants were grown on raised pine bark rows with
a plant density of 3333 plants/ha. Overhead sprinklers were used for frost protection. Standard agro-technical
procedures including winter pruning, fertirrigation, pest, diseases and weed control were performed during the
growing season. Flower phenology was monitored in each cultivar to determine the progression of fruit maturity.
The sampling dates in each cultivar were at 27 days after full bloom (DAFB) and 88 DAFB, corresponding
to initial green fruit (25% of the final size) and ripe fruit (full blue fruit), respectively. Snowchaser blooms in
June and harvest season last from September to November. Emerald blooms in July and harvest season spans
from October to December, while O´Neal blooms in late July and is harvested from November to December.
Thirty berries were collected from five different plants of each variety. After manual collection, epicarp and pulp
(meso and endocarp) were separated and frozen at –80◦ C until analysis, except for texture determinations that
were performed immediately after harvest. All the subsequent determinations were performed on the pulp. At
least three biological replicates were performed for metabolite measures. Each consisted of a pool with the same
weight of tissue from two different fruits.

2.2. Texture analysis

Compression measure was performed with a TA.TX Plus Texture Analyser (Stable Micro Systems Ltd, UK),
according to the following conditions: load: 5 kg; cylindrical plunger diameter: 75 mm; compression force at
10% of axial deformation; speed: 1 mm s−1 . Fifteen determinations were done for each sample.

2.3. Metabolite purification, derivatization and analysis

Samples were prepared as described by Perotti et al. [13]. Briefly, 300 mg of frozen tissue from 6 different
fruit (2 fruit per pool, 3 biological replicates) was powdered in a mortar with liquid nitrogen. After transfer to
glass tubes, 4.2 mL of cold methanol and 75 ␮g of ribitol (as internal standard) were added, to allow the relative
quantification of metabolites. Extracts were incubated at 70◦ C for 15 min. Afterwards, 1.5 mL of chloroform were
added, followed by incubation at 37◦ C for 5 min. Finally, after adding 3 mL of water, extracts were centrifuged for
15 min at 2200 × g and 4◦ C. The polar phase (450 ␮L) was dried in a vacuum centrifuge (CentriVap, Labconco)
until complete evaporation. For derivatization, 30 ␮L of 20 mg/mL methoxyamine in pyridine were added. Tubes
were shaken and incubated at 37◦ C for 90 min. Finally, 45 ␮L of N-methyl-N-trimethylsylil-trifluoroacetamide
(MSTFA) were added to each tube and incubated at 37◦ C for 30 min. Chromatographic runs were performed
by injecting 2 ␮L of derivatized sample in a 30 m long, 0.25 mm thick VSF GC/MS capillary column using an
automatic system (Varian Inc.) coupled to a ThermoQuest mass spectrometer. Data were collected and analyzed
using the Lab Solution software (Shimadzu). Spectra obtained were analyzed by comparing individual peak
areas for each metabolite relative to that of ribitol, the internal standard. Data were revised using the online
software Mass Spectra & Retention Time Index (MSRI) (The Comprehensive System Biology Project - CSB,
http://www.csbdb.de/index.html) from the Golm Metabolomic Institute (Germany) to confirm the identity of the
compounds.

2.4. Amino acid extraction, derivatization and analysis

Amino acids were extracted from 0.2 g of tissue of 6 different fruit (2 fruit per pool, 3 biological replicates)
that were powdered in a mortar with liquid nitrogen. After homogenization with 1.5 mL of HCl 0.1M, samples
were centrifuged and supernatants were deproteinized with TCA (10% final concentration) as described in
[14]. Derivatization was performed according to [15]. Samples were centrifuged, and supernatants were dried
with 50 ␮L of a methanol: water: triethylamine (2:2:1) mixture. Subsequently, 20 ␮L of a derivatizing mix of
methanol: water: triethylamine: phenyl isothiocyanate (7:1:1:1) were added to each sample. After incubation at
room temperature for 20 min, samples were filtered, resuspended in 500 ␮L of mobile phase and injected in a
180 M.L. Montecchiarini et al. / Metabolic and physiologic profile during the fruit ripening

250 mm × 4.6 mm i.d. 5 ␮m Luna™ C18 reversed phase column (Phenomenex, Torrance, CA, USA) at 40◦ C
and a flow rate of 1.0 mL/min following the protocol described in [16].

2.5. Total protein extraction and determination of protein concentration

Total protein extraction was carried out by grinding 0.6 g of tissue with liquid nitrogen and 1.5 mL of extraction
buffer (1 M NaCl, 12.5 mM citric acid, 50 mM Na2 HPO4 and PMSF 1:100 v/v, pH 6.5). Homogenates were
shaken for 1.5 h at 4◦ C, centrifuged at 10.000 × g for 20 min, and the recovered supernatants were maintained at
4◦ C for protein and enzyme activities measures. Protein concentration was determined using Bradford protein
assay method [17], using Bio-Rad protein assay reagent and bovine serum albumin as standard.

2.6. Pectin methyl esterase (PME) and β-galactosidase (β-gal) activity assays

PME activity was measured in berries using the pectoplate technique on a total protein and fresh weight basis
[18]. Each well contained 0.65 ug of total protein. After 16 hs of incubation at 37◦ C, plates were stained with
0.05% ruthenium red. After destain, the area of red haloes resulting from de-methylesterification of pectin was
registered in cm2 . One PME activity unit (U) is defined as the area of red halo/hour. ␤-galactosidase activity
was assayed in the same enzyme extracts as PME and was determined by measuring the hydrolysis rate of
4-nitrophenyl ␤-D-galacto-pyranoside (pNPG). Reaction mixtures were composed by 100 ␮L of extract, 500 ␮L
of 0.1 M HAc-NaAc buffer pH 4.5, 400 ␮L of BSA 0.1% and 400 ␮L of 13 mM pNPG substrate. After 25 min
at 37◦ C, 300 ␮L of reaction mixture aliquots were taken and reactions were stopped by the addition of 450 ␮L
of 0.2 M sodium carbonate. The amount of p-nitrophenol was measured at 415 nm using a molar absorption
coefficient of 18000 M–1 cm–1 . One unit of ␤- galactosidase was defined as the amount of extract used to release
1 nmol of p-nitrophenol/minute at 37◦ C [19]. Three biological replicates were analyzed, and measurements were
made in triplicate for both enzymes.

2.7. Total phenolics determination

Total phenolic content was assayed employing the Folin-Ciocalteu reagent [20]. Fifteen milligrams of endocarp
were extracted using 2 mL of buffer (80 % methanol and 1 % HCl) at room temperature for two hours in an
orbital shaker. The mixture was centrifuged at 2500 × g for 5 min and the supernatant was recovered. The pellets
were re-extracted by repeating the previous steps, the supernatants were mixed and used for phenolic content
determination. Combined supernatants (150 ␮L) were mixed with 100 ␮L of Folin-Ciocalteu reagent at room
temperature for 3 min. 500 ␮L of sodium bicarbonate (20%) were added and the reaction was allowed to stand
for 120 min at room temperature. Absorbance was measured at 730 nm and results were expressed as gallic acid
equivalents/g fresh weight. Determinations were made in triplicate.

2.8. Statistical procedures

Data presented was evaluated using t-Student test and analysis of variance (ANOVA). Significant differences
were calculated by the Bonferroni and Holm-Sidak test using the Sigma Stat Package (p < 0.05). In cases where
the normality test (Shapiro-Wilk) failed, Kruskal-Wallis ANOVA on Ranks was carried out and Dunn’s method
was used for all pairwise multiple comparison.
 M.L. Montecchiarini et al. / Metabolic and physiologic profile during the fruit ripening 181

3. Results and discussion

3.1. Firmness and total phenolic content of ripe blueberries

Since fruit from this three varieties were known to have dissimilar firmness [21, 22], assays were conducted to
ascertain the value of this parameter. Thus, considering the classification in soft (<1.6 N), medium (1.61–1.80 N)
and firm (>1.81 N) categories [23], O´Neal would be a soft variety, Emerald definitely firm and Snowchaser firm,
although very close to the medium firmness range.
Total phenolic content (TPC) in the pulp of blueberry samples was determined according to the Folin-Ciocalteu
colorimetric method in fully mature endocarps (Table 1). The TPC was similar in Snowchaser and Emerald, and
significantly lower in O’Neal. Phenolic compounds, that comprise flavonoids, anthocyanins and tannins, were
found to change in composition and accumulation degree in response to several growth conditions, such as
season, temperature, location or light incidence [24–26]. These compounds are related to plant defense and
possess antioxidant functions and, although usually concentrated in the epicarp, they are also present in fruit
pulp [25, 27]. In addition, the nutraceutical value of these substances for human health has been highlighted [28,
29]. One important fact about TPC, related to the purpose of this study, is that an inhibitory effect of phenolic
compounds on PME activity has been described [30].
Total free amino acid (FAA) levels, calculated from values acquired after HPLC analysis on a fresh weight
base, did not display variation between green and mature fruit of any variety (Fig. 1). This result is mainly a
consequence of the fact that concentrations of the two main contributors to total FAA content, proline (Pro) and
tryptophan (Trp) did not show statistically significant differences between stages in any variety. Emerald green
fruit contained a significantly higher FAA amount compared to O’Neal at the same stage, while Snowchaser
was not significantly different from either of the two other varieties at any stage (Fig. 1). The amino acids that
contributed the most to total FAA content were: glutamate (Glu) (12.0 %), Pro (46.4 %) and Trp (15 %) for
O’Neal green fruit, Pro (33.6 %), tyrosine (Tyr) (10.9 %) and Trp (16.4 %) for O’Neal ripe fruit, hydroxyproline
(OHPro) (9.4 %), Pro (44.1 %) and Trp (14.6 %) for Emerald green fruit, Pro (47.4 %) and Trp (13.2 %) for
Emerald ripe fruit, Pro (47.7 %) and Trp (9.9 %) for Snowchaser green fruit, and Pro (45.1 %), valine (Val)
(12.5 %) and Trp (12.2%) for Snowchaser ripe fruit (see Supplementary Table 1).
A few amino acids (Fig. 2) presented a particularly high abundance in some of the stages or varieties, in relation
to others. Those whose abundance was higher than 40 % of the total concentration in a particular variety and stage
were: Glu in O’Neal green fruit, aspartic acid (Asp) and glutamine (Gln) in Emerald green fruit, citrulline (Cit)
in Emerald ripe fruit, Asn and threonine (Thr) in Snowchaser green fruit, Val and His in Snowchaser ripe fruit
(Fig. 2, boxes). Gln relative content decreased with ripening in O’Neal and Emerald, Glu decreased in O’Neal,
His increased in O’Neal and Snowchaser but decreased in Emerald, Cit increased in O’Neal and Emerald but was
barely detected in Snowchaser and Thr increased in Emerald and decreased in Snowchaser (Fig. 2, asterisks).
The amount of each amino acid in ␮mol/g FW is given in Supplementary Table 2.

Table 1
Firmness and total phenolic content (TPC). Values represent means ± SDs. Different letters indicate statistically significant differences.
Data were tested using ANOVA. Significant differences were calculated by the Bonferroni and Holm-Sidak test using the Sigma Stat
Package (p < 0.05). In cases where the normality test (Shapiro-Wilk) failed, Kruskal-Wallis ANOVA on Ranks was carried out and Dunn’s
method was used for all pairwise multiple comparison

Variety Firmness (N) TPC (␮g gallic acid/mg fw)

Emerald 3.05 ± 0.43 a 0.36 ± 0.03 a
Snowchaser 1.91 ± 0.32 b 0.39 ± 0.03 a
O’Neal 1.53 ± 0.23 c 0.19 ± 0.01 b
182 M.L. Montecchiarini et al. / Metabolic and physiologic profile during the fruit ripening

Fig. 1. Total free amino acid (FAA) content at the green and ripe stages for each variety. Quantification data are presented as means ± standard
deviation of three biological replicates. Lower case letters indicate statistically significant differences between ripening stage in a variety.
Significant differences between varieties are represented by capital letters at green stage, and bold capital letters at ripe stage. Data was tested
using ANOVA and t-Student test. All pairwise multiple comparisons were performed by the Bonferroni and Holm-Sidak method using the
Sigma Stat Package (p < 0.05).

Fig. 2. Amino acid contribution in each variety and ripening stage. The amino acids whose levels were higher than 40% are enclosed in a
box, those with statistically significant difference in concentration between both maturation stages are indicated with asterisks (see the text
for more details).

FAA participate in several metabolic processes such as salinity response, protein turnover or nitrogen
metabolism and transport (especially for Glu, Gln and asparagine). Levels reported here were within the same
range of those informed for strawberries and tomatoes [31, 32] although in the latter total FAA content was lower
in green than in ripe fruit. In the blueberries analyzed here, total protein (in ␮g/g FW) was significantly lower in
Emerald at the green stage in comparison with other varieties (data not shown), suggesting that protein turnover
in green fruit is more relevant in this variety. The measure of enzymatic activities such as glutamate synthase,
 M.L. Montecchiarini et al. / Metabolic and physiologic profile during the fruit ripening 183

glutamine and asparagine synthetase could give information about the events linked to variation of these amino
acids. High FAA, especially for essential amino acids, may also indicate a better nutritional quality, though this
is not relevant in green fruit. A deeper analysis about the possible implications of these amino acids on berries
metabolism is considered in the context of other metabolite changes in section 3.2.

3.2. Metabolites in green and mature fruit: Sugars, sugar alcohols, amino acids, organic acids

The use of a metabolomic approach, aimed at comparing the natural variance of plants or to study the incidence
of maturation state, environment or other aspects in the plant metabolic content has been intense in the last years
[33–35]. Hence, by means of GC-MS and HPLC chromatographic techniques, changes in 40 metabolites of the
varieties under study were identified in green and mature pulp of fruit (see relative values of each metabolite in
Supplementary Table 3).
After data normalization, and multivariate statistical analysis, three principal components were able to explain
82.1 % of the total variance (Fig. 3). Principal component 1 (40.5 %) was able to distinguish green from ripe fruit.
The three varieties display higher levels of phosphoric acid (PA), galactose (Gal), melibiose (Mel), mannitol,
malic and citric acids, Asn, Asp, OHPro, Gln, Gly, Pro and isoleucine (Ile) at the green stage.
What this means for blueberry physiology is a multifaceted question since, as previously mentioned, FAA are
directly and indirectly involved in a number of biological processes. For instance, Pro, Arg, Met and Glu are
important in the regulation of plant responses to several environmental signals related to abiotic or biotic stress
[36, 37]. Others may have effects on fruit taste: Glu is responsible for the delicious (umami) taste but also has
taste-enhancing properties [38, 39]. His, Gly, lysine (Lys) and alanine (Ala) are highly correlated with sweetness,
as Val, Phe and Tyr are with bitterness [40]. Levels of most of the amino acid detected (see Supplementary Table 2)
were near or above their taste threshold [41]. Asn and Gln are involved in nitrogen transport and storage, being
important links between nitrogen and carbon metabolism. Additionally, essential amino acids (not synthesized
by mammals, e.g. methionine, cysteine, leucine, Lys, Ile, Val and aromatic amino acids) are appreciated by their
nutritional value. Leu and Ile are precursors of branched chain fatty acids, which increase membranes fluidity.
Pro, besides its role as osmoprotectant, is a component of an important family of glycosylated cell wall proteins,
along with OHPro [42]. Galactose is also a major component of these glycoproteins, followed by arabinose and
xylose [43]. From PCA analysis it is noticeable that in green fruit, several free amino acids with diverse functions
are abundant, suggesting that metabolic processes such as nitrogen transport and storage, protein turnover and
cell wall dynamic are very active at this stage.
As expected, malic and citric acid levels are more elevated in green fruit. These organic acids have sev-
eral roles in plant metabolism, as cytosolic pH regulators or as biosynthetic precursors, and are also key
contributors to taste in the mature fruit. Their level, controlled by degradation, synthesis and transport, fluc-
tuate with growth conditions, climate, maturation stage and variety [44]. Citric acid is also associated with
iron metabolism and availability. Mannitol and other polyalcohols are relevant osmolytes that protect tissues
from dehydration and help to maintain turgor pressure [45]. Mannitol is also linked to boron transport from
source to sink tissues through the phloem [46, 47], which may be very active during the green stage. Melibiose
(D-galactopyranosyl-alpha-1-6-D-glucopyranose), along with raffinose and stachyose, is part of the raffinose
family oligosaccharides (RFOs), metabolites known to accumulate under different environmental stresses [48].
Galactose is also associated with RFOs, with ascorbic acid synthesis (via galacturonic acid) and is a constituent
of pectin side-chains.
Ripe Emerald and O’Neal were connected by having higher citrulline (Cit) and turanose content with respect to
Snowchaser. Citrulline was first discovered in watermelon (Citrullus lanatus) and, although it is more abundant
in cucurbitaceous plants, it is an ubiquitous non-proteinogenic amino acid [49]. Studies carried out in watermelon
also showed increasing levels during fruit development [50]. Cit was not detected in Snowchaser at either maturity
stage. Cit is also related with Arg metabolism and, indirectly, with putrescine synthesis. Moreover, it is a potent
hydroxyl radical scavenger; it accumulates after drought stress, for which it has been ascribed a role as an
184 M.L. Montecchiarini et al. / Metabolic and physiologic profile during the fruit ripening

Fig. 3. Principal component (PC) analysis of metabolites from ripe and green fruit of the varieties under study. The percentage of variance
explained by each component is indicated between parentheses (A: PC1 + PC2, B: PC1 + PC3). Metabolites that contribute the most to
each component are indicated (the contribution data of all the variables is shown in supplementary table 4). Arg: arginine; Asn: asparagine;
Asp: aspartic acid; Cit: citrulline; CQA: caffeoyl quinic acid; Fru: fructose; Gal: galactose; Gln: glutamine; Gly: glycine; His: histidine;
Ile: isoleucine; Leu: leucine; Lys: lysine; Mel: melibiose; Met: methionine; MPG: mono palmitoyl glycerol; OHPro: hydroxy Proline; PA:
phosphoric acid; Phe: phenylalanine; Pro: proline; Raf: raffinose; Ser: serine; Suc: sucrose; Thr: threonine; Trp: tryptophan; Tur: turanose;
Val: valine; Xyl: xylose.
 M.L. Montecchiarini et al. / Metabolic and physiologic profile during the fruit ripening 185

osmoprotectant; and a role as a long- distance nitrogen transporter has also been suggested [49]. Turanose (3-O-
␣- D-glucopyranosyl-fructose) is an analog of sucrose with roles as a signaling molecule [51], able to activate
regulatory MAPK pathways independently from sucrose, and to greatly accelerate fruit ripening in strawberries
[52].
Notably, ripe Snowchaser fruit was separated from all other samples by principal component 2 (22.6%, Fig. 3
A), showing elevated contents of monopalmitoylglycerol (MPG), fructose, His, sucrose, Arg, Val and Met.
Meanwhile, glycerol and Thr were abundant in ripe Emerald, green Snowchaser and green O’Neal. An increased
content of MPG is produced by the action of lipases on diacylglycerols and may be indicative of a reorganization
of certain cellular membranes components. Sugars in general, besides being a source of energy in heterotrophic
tissues, have been connected to cold stress and osmoprotection or as ROS scavengers and protein stabilizers [53].
Sucrose is linked to a plethora of biological functions, and its signaling roles have been thoroughly reviewed
[54, 55]. Higher fructose levels contribute to a sweeter fruit, since its sweetening power is higher than that of
sucrose. As mentioned before, His is also associated with sweet taste, as Val is with bitterness.
A further distinction was hinted by principal component 3 (19.0 %, Fig. 3 B), since green and ripe Emerald
fruit formed a detached group characterized by higher contents of Phe, Trp, Lys and glucose, while Snowchaser
and O’Neal, held more serine (Ser), raffinose, caffeoyl-quinic acid (CQA), xylose and Leu levels at both maturity
stages.
Phenylalanine, as the other two aromatic amino acids, is synthesized in plants through the shikimate pathway
and is precursor of anthocyanins, flavonoids, lignin and other relevant phenolic compounds. Trp is a precursor
of auxin, melatonin, serotonin and niacin, having crucial hormonal and nutritional functions [56, 57]. It also
forms indole acetic (IAA) or jasmonic acid conjugates that thwart IAA hormonal responses [58]. CQA, esters
between quinic and caffeic acid, are part of a group of compounds also known as chlorogenic acids, molecules
with antioxidant properties [59]. Xylose has been identified as one the most abundant non-cellulosic sugars in
blueberry primary cell walls [60] and dicots in general [61]. It may arise from xyloglucan depolymerization,
which has a backbone of 1,4 ␤- linked glucose residues, as cellulose, but also holds short chains of xylose and
galactose. This glycan cross-links with cellulose, strengthening the wall, suggesting that its integrity may be
relevant during softening [62].

3.2.1. Summary of metabolite differences found between maturation stages and varieties
Analytical inspection of the differential metabolite abundance in blueberries made it possible to indicate in the
first place, a clear distinction between ripe and green fruit supported by the abundance of organic acids in green
fruit, which decrease later on during development. Other compounds, like galactose, Pro and OHPro, suggest
dynamic changes in cell wall metabolism, possibly related with cell expansion in this period. Secondly, a few
metabolites allowed further distinction between varieties: some of them linked with taste, aroma and nutritional
quality (sugars, free amino acids), while other were related with cell wall, hormone and antioxidant metabolisms
(xylose, Trp, Phe and CQA). Thus, ripe Snowchaser was characterized by high His, Val, Arg, Met and sucrose;
while green and ripe Emerald had more Trp, Gly, Phe and glucose.
An interesting finding is that Emerald had less free xylose content at both stages, which could indicate a lesser
degree of cell wall depolymerization than the other varieties, a fact that may contribute to the higher measured
firmness.

3.3. Cell wall modifying enzymes

As mentioned previously, events such as loss of turgor, degree of pectin methylation, solubilization, branching
and depolymerization, do not follow the same pattern in different species, or even in particular varieties from
the same species. The preceding section showed that variation in some metabolites levels point to a differential
change in cell wall metabolism depending on the variety and maturity stage. In order to increase our understanding
of these processes, the activities of two enzymes crucially involved in cell wall metabolism were measured. PME
186 M.L. Montecchiarini et al. / Metabolic and physiologic profile during the fruit ripening

Fig. 4. Activity of cell wall related enzymes in green versus ripe fruit. A. Pectin methyl esterase (PME) activity in the three varieties under
study. B. ␤-galactosidase (␤-gal) activity in the three varieties under study. C. Representative images of pectoplate experiment. Quantification
data are presented as means ± standard deviation of three biological replicates, independent replicates were carried out for each sample.
Lower case letters indicate statistically significant differences between ripening stage in a variety. Significant differences between varieties
are represented by capital letters at green stage, and bold capital letters at ripe stage. Data was tested using ANOVA and t-Student test. All
pairwise multiple comparisons were performed by the Bonferroni and Holm-Sidak method using the Sigma Stat Package (p < 0.05).

catalyzes the demethylation of pectin (polygalacturonic acid) rendering a more porous cell wall in which the
action of other pectinolytic enzymes, such as polygalacturonases, pectate lyases and ␤-galactosidases, is enabled
[63]. At the same time, PME activity is necessary to generate sites for calcium bridges (free carboxylates), which
turn the cell wall mechanically more resistant and less susceptible to hydrolysis [64]. ␤- galactosidases (␤-gal)
are present in plants as a family of glycosyl hydrolases that fulfill diverse roles: although mainly implicated in
cell wall metabolism, they are also able to modify glycoproteins and glycolipids [65]. Neutral sugars (galactose,
xylose, arabinose) could help to anchor pectins to the cell wall, thus as a consequence of ␤-gal activity on
xyloglucans and rhamnogalacturonan I, it becomes softer and free galactose levels increase [66, 67].
A significant lower PME activity was detected in Emerald mature fruit in contrast with the other two varieties,
which did not show substantial differences between them (Fig. 4 A and C). Additionally, a remarkable divergence
was exhibited by green fruits, where O’Neal presented a higher PME activity than the other varieties, followed by
Snowchaser and then by Emerald. Furthermore, PME activity increased in Emerald and Snowchaser, whereas in
O’Neal, it decreased in ripe versus green fruit. On the other hand, when ␤-gal activity was measured (Fig. 4 B),
Emerald green fruit showed the highest activity, with no significant difference between O’Neal and Snowchaser.
Considering the ripe stage, O’Neal had the highest ␤-gal activity while the level in the other varieties did not vary
considerably. With the intention to better understand the possible consequences on cell wall integrity, the fold
change between both activities at ripe versus green stage was calculated (Fig. 5). It was evident that Emerald and
Snowchaser, both firmer varieties, had the same tendency: higher PME and lower ␤-gal activity at ripe versus
green stage, while the opposite was observed in the less firm, O’Neal. However, the fold change was substantially
higher in Emerald (9.35) relative to Snowchaser (0.42) for PME, while for ␤-gal, levels decreased from green to
ripe in Emerald (–0.76) and Snowchaser (–0.20) at a comparable degree.
 M.L. Montecchiarini et al. / Metabolic and physiologic profile during the fruit ripening 187

Fig. 5. Enzyme activity fold change from green to ripe fruit. Fold change values for pectin methyl esterase (PME) and ␤-galactosidase
(␤-gal) were calculated as: (ripe value - green value)/green value.

A study indicates that pectin solubilization in berries is observed at early maturation stages and that ripening
is associated more with modifications in the hemicellulose-cellulose matrix than with pectin depolymerization
[60]. Several factors might influence pectin solubility, it is thought that a high methylation degree, associ-
ation with calcium and/or cross-linking with other cell wall components, turns it less soluble and difficult
to extract [66, 67]. In fact, loss of neutral sugars from side chains of rhamnogalacturonans increased pectin
solubilization in kiwi fruit [68]. Thus, one process that could promote its solubilization is demethylation con-
ducted by PME. Above mentioned results are in agreement with more active pectin de-methylation in green
O’Neal, that continue to be high in ripe stage. The important point here is that ␤-gal activity is higher in
the less firm variety at ripe stage, suggesting that a combination of a more soluble pectin in presence of
this high level of a hydrolytic enzyme could be in part the cause of a reduced firmness. On the contrary,
for Snowchaser and Emerald, pectin demethylation appears to increase during ripening but in concert with a
decrease in ␤-gal activity, more evident for Emerald, the firmer one. A similar observation, but with another
hydrolytic enzyme, polygalacturonase, was reported with grape berries [69]. However, it is worth to mention
that both activities might result from the contribution of distinct isoforms known to be present and differen-
tially expressed during development [65, 70]. Thus, which isoform is responsible for each activity needs further
research. The measure of other enzymatic activities, such as polygalacturonases, pectate lyases, as well as the
calcium content in cell wall is necessary to gain information about the metabolic process that is actually taking
place.

3.4. Firmness related metabolites

With the aim of gaining insight into the molecular processes that could be related more specifically with
fruit firmness, a correlation analysis between this factor, primary metabolites content, TPC, ␤-gal and PME
activities was performed. The correlation coefficient allowed to ascertain the linkage between these parameters,
independently of the variety. A total of 903 pairs were analyzed, from which, in mature fruit, 195 resulted in
significant correlation coefficients (p-value < 0.05, Fig. 6 A). Citric acid (0.94), Phe (0.75), xylose (–0.83), Leu
(–0.74) and shikimic acid (–0.73) demonstrated strong and significant positive and negative correlation with
firmness, respectively (Fig. 5 A). Other variables showed correlations that, although not statistically significant,
were strong, as for mannitol (0.77) and ␤-gal (–0.75).
As some metabolic changes that are known to be related to firmness in ripe fruit take place early in develop-
mental stages [71, 72], the same analysis was carried out in green fruit, in an attempt to relate them with observed
firmness later on in the ripe stage (Fig. 6 B). In this case, 185 pairs resulted in significant correlation coefficients
(p-value < 0.05). The following metabolites displayed a significant and strong correlation with firmness: sucrose
188 M.L. Montecchiarini et al. / Metabolic and physiologic profile during the fruit ripening

Fig. 6. Correlation matrix between primary metabolites and firmness in mature fruit (A) or green fruit (B). Circles represent Spearman’s
correlation coefficient value for a pair of metabolites. Positive correlations are displayed in light grey and negative correlations in dark grey.
Color intensity and size of the circle are proportional to the correlation coefficients. Significant correlations are indicated with a star inside
the corresponding circle (p-value < 0.5).

(0.96), malic acid (0.89), MPG (0.94), quinic acid (0.86), citric acid (0.86), ␤-gal (0.79), glucose (0.76), Met
(0.75), fructose (0.74), Thr (0.68), Val (0.73), Ala (0.76), Leu (–0.68), PME (–0.87), shikimic acid (–0.73),
xylose (–0.89) and citrulline (–0.84).
In plants, a biological marker has been defined as a characteristic that is objectively evaluated as predictor of
plant performance [73]. In this sense, a gene, transcript, metabolite or an enzyme activity, are subsets of these
biomarkers that could assist to different purposes, for instance, as diagnostic or breeding tools. One way of
validating a biomarker is to find out the correlation degree between the marker and the trait under study. In this
work, such a correlation analysis, performed between metabolites and enzymes levels in green and ripe fruit with
firmness observed at harvest date, helped us to infer that some metabolites can be used as markers of firmness.
Indeed, high levels of citric acid and Phe in ripe fruit, or sucrose, citric acid, malic acid, MPG, quinic acid or
␤-gal activity in green stage, are strongly associated with high firmness. Conversely, a high content of xylose,
Leu and shikimic acid in ripe fruit, or large amounts of xylose, PME activity, Cit and shikimic acid in green
stage, indicate reduced firmness.
Clarifying the mechanisms by which each of these components affect fruit firmness need extra research.
However, it is possible to deduce some clues about them. In tomato, malic acid content was highly correlated
with firmness and shelf-life [74], possibly by promoting a decrease in water loss by transpiration [75]. In the
present work, as in [12], the combination of high PME and ␤-gal activities in ripe fruit could indicate cell wall
solubilization and degradation, the last statement reinforced by high xylose content at both maturity stages in the
less firm variety. Regarding shikimic acid and Leu, precursors for phenylpropanoids and branched fatty acids,
respectively, the mechanistic relationships to reduced firmness are not clear at this time.
 M.L. Montecchiarini et al. / Metabolic and physiologic profile during the fruit ripening 189

4. Conclusion

Preliminary studies in blueberries, in the first place, helped to characterize changes between green and ripe
fruit, and in second place, made it possible to find out some specific differences between varieties. Another
relevant outcome is that these findings give some clues to further investigate the potential mechanisms involved
in the preservation of firmness in blueberries. Future studies of other development stages and the associated
proteome in each variety, as well as the activity of additional pectinolytic enzymes (like polygalacturonases
and pectate lyases), their activity modulators (i.e. PME and polygalacturonases inhibitors) and calcium levels
will probably contribute to a more complete understanding of changes during fruit growth. A strong correlation
between firmness and some metabolites or enzymes related with organic acids, carbohydrate, cell wall, cellular
membranes and protein metabolisms could be determined. Furthering our knowledge on how these changes are
associated with fruit quality in general, and with firmness in particular, will enhance our understanding about
suitable markers or standards useful for future comparison between fertilization treatments, variety selection and
local breeding programs.

Conflict of interest

The authors have no conflict of interest to report.

Acknowledgments

This work was supported by grants from by the Agencia Nacional de Promoción Cientı́fica y Tecnológica to
KEJT (PICT 2016-0091) and FEP (PICT 2015-1074). FEP and KEJT are members of the investigator Career
from the Consejo Nacional de Investigaciones Cientı́ficas y Técnicas (CONICET), MLM is a Doctoral Fellow
of CONICET. FB, MFR and DV are members of the Instituto Nacional de Tecnologı́a Agropecuaria (INTA).

Supplementary material

The supplementary material is available in the electronic version of this article: http://dx.doi.org/10.3233/JBR-
180309.

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