TYPE Original Research

PUBLISHED 11 January 2024

DOI 10.3389/fpls.2023.1324066

Differences in primary
OPEN ACCESS metabolism related to quality
EDITED BY
Ana Margarida Fortes,
University of Lisbon, Portugal
of raspberry (Rubus idaeus L.)
REVIEWED BY
Feng Xu,
fruit under open field and
Yangtze University, China
Andrew Breksa,
Agricultural Research Service (USDA),
protected soilless culture
United States

*CORRESPONDENCE
growing conditions
Lida Fuentes
lfuentes@creas.cl Claudia Fuentealba 1, Fernanda Álvarez 2, Excequel Ponce 1,
RECEIVED 18 October 2023 Sebastian Veas 1, Martina Salazar 2, Diego Romero 2,
ACCEPTED 07 December 2023
PUBLISHED 11 January 2024
Anibal Ayala-Raso 3, Juan E. Alvaro 1, Monika Valdenegro 1,
CITATION
Carlos R. Figueroa 4 and Lida Fuentes 2*
Fuentealba C, Álvarez F, Ponce E, Veas S, 1
Escuela de Agronomı´a, Facultad de Ciencias Agronómicas y de los Alimentos, Pontificia
Salazar M, Romero D, Ayala-Raso A,
Universidad Católica de Valparaı´so, Quillota, Chile, 2 Centro Regional de Estudios en Alimentos
Alvaro JE, Valdenegro M, Figueroa CR and
Saludables (CREAS), Valparaı´so, Chile, 3 Instituto de Estadı´stica, Facultad de Ciencias, Universidad
Fuentes L (2024) Differences in primary
de Valparaı´so, Valparaı´so, Chile, 4 Laboratory of Plant Molecular Physiology, Institute of Biological
metabolism related to quality of raspberry
Sciences, Universidad de Talca, Talca, Chile
(Rubus idaeus L.) fruit under open field and
protected soilless culture growing conditions.
Front. Plant Sci. 14:1324066.
doi: 10.3389/fpls.2023.1324066
Introduction: The raspberry (Rubus idaeus) fruit is characterized by good
COPYRIGHT
taste and high acceptability by consumers. Thus, the impact on the quality
© 2024 Fuentealba, Álvarez, Ponce, Veas,
Salazar, Romero, Ayala-Raso, Alvaro, attributes and metabolites related to raspberry taste should be evaluated in
Valdenegro, Figueroa and Fuentes. This is an crop alternatives such as the protected soilless culture. This study aimed to
open-access article distributed under the terms
of the Creative Commons Attribution License
evaluate the metabolic changes during fruit development and postharvest of
(CC BY). The use, distribution or reproduction raspberry grown in open field and protected soilless culture and their
in other forums is permitted, provided the relationship with quality parameters and sensory perception.
original author(s) and the copyright owner(s)
are credited and that the original publication
in this journal is cited, in accordance with Methods: In this study, the quality parameters and polar metabolites -sugar
accepted academic practice. No use, and amino acids- content were evaluated during raspberry ripening. In
distribution or reproduction is permitted
which does not comply with these terms. addition, ripe fruit was stored at 1 °C for five days, followed by one day of
shelf life at 20 °C.

Results: The physiological and quality parameters showed typical changes

during ripening in both growing conditions: a constant production of CO2, a
drastic loss of firmness, an increase in weight and soluble solids content, loss
of acidity, and a turning to red color from the green to fully ripe fruit stages in
both growing conditions. Fruit from the protected soilless culture had
significantly higher weight but a lower soluble solids content. The
metabolic analysis showed differences in primary metabolites content
during ripening and storage at 1 °C between both growing conditions. The
raspberries grown in the open field showed higher contents of sugars such as
D-glucose and D-fructose. On the contrary, the fruit from the protected
soilless culture showed higher contents of some amino acids such as L-
alanine, L-serine and L-valine, among others. The sensorial panel showed
significant differences in the perception of the sweetness, acidity, color and
firmness of ripe fruit from both growing conditions.

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Discussion: The present study provides interesting and useful results with
direct commercial application for this alternative growing system, mainly in
areas where soil and water scarcity are a reality.

KEYWORDS

metabolic pathways, fruit quality, sugars, softening, soilless culture

1 Introduction acids, varied when they were cultivated under the macro tunnel,
different substrates, and electrical conductivities of irrigation
Raspberries (Rubus idaeus L.) have an attractive color, unique (Akhatou et al., 2016). In summary, quality parameters are
flavor, and valuable health benefits. These include being a valuable subject to certain preharvest process or factors that could be
source of vitamins, minerals, dietary fiber, and antioxidant improved. These processes are as follows: the optimization of the
compounds (Graham and Simpson, 2018; Popović et al., 2022). interactions between cultivars, environmental conditions
During raspberry ripening, a progressive decrease in fruit firmness (temperature, light, concentration of CO2, and vapor pressure
can be observed, associated with cell wall modification and deficit (VPD)) and the composition and concentration of nutrient
increased sugar composition in the ripe fruit (Stewart et al., 2001; solutions, crop management, growing media together with the
Vicente et al., 2007; Graham and Simpson, 2018). It has been electrical conductivity threshold value. The use of adapting
reported that the onset of ripening is characterized by the fertigation to growing conditions and/or nutrient solutions of
accumulation of organic acids and soluble sugars, which moderately high conductivity seems promising in providing high
contribute to acquiring the fleshy trait associated with cell yields of remarkable quality while reducing the emission of
expansion (Dincheva et al., 2013). Once the fruit ripens, it nutrients to the environment in greenhouses (Moya et al., 2017;
reaches approximately 9% of soluble solids content, of which 5%– Rodrı́guez et al., 2019). However, to our knowledge, no studies
6% correspond to sugars (glucose, fructose, and a smaller amount of associate the changes in raspberry fruit quality and its acceptability
sucrose) (Graham and Simpson, 2018). In addition, the quality and with metabolic changes during ripening in protected soilless culture
marketability of raspberry is highly influenced by the concentration and open field.
of fructose, glucose, and sucrose as main soluble sugars (Kafkas The metabolomic profile has been an innovative tool for
et al., 2008; Dincheva et al., 2013). understanding the metabolic response of plants exposed to
The composition of fruits can depend on variety, maturity different growing and storage conditions. Ren et al. (2023) used
stage, and environmental and cultivation conditions (Kassim et al., widely targeted metabolome of raspberries to correlate the
2009; Akhatou et al., 2016; Marchi et al., 2019; Akšić et al., 2022; antioxidant capacity and metabolites of fruit grown under
Kotuła et al., 2022). Raspberries grown by conventional and organic different environments. Similarly, Durá n-Soria et al. (2021) used a
cultivation have been studied to evaluate the yield, quality, and multi-platform metabolomic approach to contrast the metabolite
phytochemical composition of the plant and its fruits (Anjos et al., profiles associated with flavor and nutrition in different raspberry
2020; Frı́as-Moreno et al., 2021; Kotuła et al., 2022). Moreover, cultivars. This study aimed to determine the change in the quality
covered tunnels and greenhouse soilless culture have become an and polar metabolites profile during the ripening and storage of
attractive alternative in places with scarce soil and water. In raspberry grown in the open field and protected soilless culture
addition, raspberries for dessert are primarily cultivated in growing conditions.
fertigated soilless culture under cover (Stojanov et al., 2019;
Balawejder et al., 2023). Pritts et al. (1999) reported that ripe
raspberry fruit produced in greenhouse conditions were larger, 2 Materials and methods
firmer, and much less prone to fruit rot than open field
production. Even more recently, Marchi et al. (2019) revealed 2.1 Plant material
that the yield of raspberry fruit grown under plastic cover in
Brazil was higher than non-covered but depends greatly on the Raspberry (R. idaeus ‘Heritage’) fruit was collected from
cultivar and the harvesting productive cycle (fall or spring). In commercial orchards located in Casablanca (longitude 33°20′39ʺS;
addition, Balawejder et al. (2023) improved the synthesis of latitude 71°22′07ʺW; 247 m.a.s.l.), Chile, and greenhouse located at
bioactive compounds through substrate modification in the the experimental station (Facultad de Ciencias Agronó micas y de
soilless cultivation of raspberries. Likewise, the primary los Alimentos, Pontificia Universidad Cató lica de Valparaı́so,
metabolites in strawberries, such as sugar and amino and organic located in Quillota (longitude 32°53′43ʺS; latitude 71°12′25ʺW,

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120 m.a.s.l). The protected soilless growing condition was in a parameters. In addition, a sensory panel was performed with ripe
greenhouse (polyethylene cover thickness, 200 mm) with a natural fruits (R stage).
ventilation system. The unit crop was a 10.5-L Projar Golden Grow Ripe fruits from each growing condition were detached from
Blend Medium Washed (Valencia, Spain) coir growth pot (235 × the receptacle and used for the postharvest assay. A total of 12
235 mm, H × W) with one bare root raspberry cane transplanted experimental units with four fruits (three units per block) were used
from the nursery at an approximate age of 3 months. The distance per each growing condition. The fruits were evaluated at harvest
between the unit crop corresponded to 0.4 m and between the rows (Day 0), after storage for 5 days at 1°C when the fruits were taken
to 1.6 m. Each unit crop was fertigated by one pressure out of cold storage [Day 5 (1°C)], and after cold storage and 1 day of
compensated dripper, with a nominal flowrate of 4.0 L·h−1 and shelf life at 20°C [Day 5 (1°C) + 1 (20°C)].
with four manifolds and their corresponding microtubes and spikes.
The raspberry plants were fertigated with a standard nutrient
solution adjusted to specific requirements and daily monitored 2.2 CO2 production and fruit
during the research. For the trial, two checkpoints were used for quality assessment
fertigation control: 1) a control dripper (4.0 L h−1) and 2) a drain
pan to monitor the supplied fertigation and its absorption response. The CO2 determination was performed according to Monsalve
The pH, volume of nutrient solution, and EC of the fertigation input et al. (2022). The samples of each independent unit for each assay
and drainage were measured at the control points. The nutrient were introduced into close tight chambers (390 mL) and were
solution EC was maintained at 1.7 mS cm−1 ± 0.1 and pH at 5.8 ± incubated at 20°C for 2 h. The needle of a CO2 detector (MAP
0.1. Each fertigation pulse was activated when 10% of the readily Headspace gas analyzer, Bridge Analyzers, USA) was injected into
available water in the substrate had been used and the volume the chambers, and the CO2 concentrations were recorded. The
necessary to produce between 20% and 30% drainage to avoid any results were expressed as milligrams of CO2 per kilogram per hour.
accumulation of salts. Pruning and other cultural management The individual weight of each fruit from the experimental units
practices were performed according to commercial practices. of developmental stages and postharvest assay per growing
A commercial orchard with traditional growing conditions was condition was measured using an analytical balance. The
selected for fruit grown in open field conditions. The distance moisture of ripe fruit was determinate according to AOAC
between the unit crop corresponded to 1 m and between the rows to protocols (AOAC (Association of Official Analytical Chemists),
2 m. The plants were watered for 2 h every 3 days by flooding 1997). The color of drupelets was determined in each fruit per
irrigation. During harvest time, the fertilization doses used were of developmental stage and after postharvest. The color was recorded
76, 76, 38, 19, 19, and 1 kg ha−1 year−1 for the nitrogen, potassium, using a chroma meter coupled with a Minolta DP-301 data
phosphorus, sulfur, magnesium, and calcium elements and 1.9 kg processor (model CR-300, Konica Minolta; Tokyo, Japan). The
ha−1 year−1 for the boron element, respectively (Hirzel, 2013). The measure was detailed as the CIELAB scale (L*, a*, b*, hue angle-h°,
Casablanca soil is classified as kaolinitic, fine, thermic Ultic and chroma-C). Fruit firmness was determined as the force
Palexeralf (Cauquenes Series), derived from granitic materials, necessary to deform the fruit in 1 mm using the FirmPro
subangular blocky structure, clay loam texture, reddish brown equipment (Happy Volt SPA, Santiago, Chile) and expressed as
(5YR4/4), slope 11.5%, moderate permeability and drainage, and Newton (N) (Monsalve et al., 2022). Six grams of drupelets from
rapid runoff (Quezada et al., 2014). each replicate were homogenized in a mortar. The juice was
The average minimum and maximum temperature for open analyzed for total soluble solids (TSS) using a refractometer
field (Casablanca) was downloaded from Red Agrometereoló gica (ATAGO, Tokyo, Japan), expressed as total soluble solid content
INIA data (available at: https://agrometeorologia.cl/). On the other TSS (brix degree), and the titratable acidity (TA) was analyzed using
hand, the average temperature and humidity for the protected a pH meter (model PL-500, Ezdo; Taipei, Taiwan) and expressed as
culture were registered using a HOBO Pro v2 Temperature/ the percentage of citric acid per 100 g of fresh weight (FW) of fruit.
Relative Humidity Recorder.
The fruits from both growing conditions were harvested in
January 2022. The experimental design consisted of four blocks of 2.3 Untargeted metabolomic analysis
six plants for each growing condition. For quality parameters, fruits
attached to the receptacle and with peduncle were harvested and The primary metabolites were evaluated at the W, P, and R
sorted by color and size as large green (LG), white (W), pink (P), red stages of development and during the postharvest trials. Extraction
or ripe (R), and over ripe (OR) stages (Monsalve et al., 2022; Á lvarez and derivatization of polar metabolites were performed according
et al., 2023). Four independent experimental units of 20 intact fruits to Fuentealba et al. (2017). A total of 100 mg of frozen drupelets
attached to the receptacle per developmental stages were used for powder was mixed with 500 µL of cold methanol and 20 µL of 3 mg
quality assessments. Immediately after harvest, half of the collected mL−1 phenyl b-D-glucopyranoside was used as internal standard.
fruit was frozen in liquid nitrogen for metabolite extraction. The The determination was done using an Agilent 7890B gas
other half was used to determine the CO2 production and quality chromatograph equipped with an HP-5 ms Ultra Inert column

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(30 m × 0.25 mm × 0.25 µm), a 5977A single quadrupole mass value < 0.5. During the sensory evaluation, the data obtained by the
detector and an electron impact ionization source (GC-MS) duo–trio and paired test was evaluated by t-test. The differences
(Agilent Technologies, Santa Clara, CA, USA). Chromatographic were established for a = 0.05 (*) considering seven hits of 10
peaks were deconvoluted and identified by comparing retention answers and a = 0.01 (**) considering eight hits of 10 answers in
times and mass spectra with a library constructed from commercial the three sessions, respectively (ISO, 2005; Iborra-Bernad
standards and the NIST14 library using Mass Hunter Quantitative et al., 2014).
software (Agilent Technologies, Santa Clara, CA, USA). The data
were expressed as relative abundance, which was obtained dividing
the peak area of each compound by the peak area of the internal 3 Results
standard (phenyl b-D-glucopyranoside), the fresh sample weight,
and quality control (QC) that consisted of a mixture of all samples. 3.1 Fruit quality during ripening
and postharvest

2.4 Sensory determinations The ripening stages (Figure 1) of raspberries harvested in open
field and protected soilless culture growing conditions were
The sensory differences in sweetness, acidity, firmness, and characterized by the evolution in CO2 production and quality
color of ripe fruit from protected soilless culture and open field parameters. A constant CO2 production was observed during
were determined in “Centro Regional de Estudios de Alimentos ripening in protected soilless growing conditions, and a
Saludables-CREAS” (Valparaiso, Chile). A total of 20 panelist was significant decrease in CO2 production during ripening was
trained with increased concentration of sucrose solutions (4, 6, 9, observed in open field growing conditions (Figure 1A). The
and 12 g L−1) and a fixed concentration of citric acid (0.43 g L−1) weight was the most remarkable difference between fruit
according to PN-ISO 3972:2016–07 normative (2016). After four harvested under open field and protected soilless culture growing
training sessions, 10 panelists (30 years’ average age), five women conditions. Even though the weight increased significantly in both
and five men, were chosen for the sensory determinations on growing conditions, the pattern of weight gain showed differences
raspberry. First, a duo–trio test (Mihafu et al., 2020) was carried in the fruit of the protected soilless condition. The ripe fruits from
out using fruit from open field conditions as the reference, and one the protected soilless culture showed higher weight in the OR stage
sample from the open field and another from protected soilless (3.9 ± 0.4 g) compared to those grown in the open field (2.6 ± 0.2 g),
growing conditions were used as coded samples. The panelists were and the fruit from the protected soilless culture continued to gain
asked to select the coded sample with the same sensorial perception weight after the white stage (Figure 1B). On the other hand, a
of the reference. Then, the sweetness discrimination of ripe fruit similar trend in color parameters was observed in both growing
from protected soilless culture and open field was done by a conditions, with a significant decrease in brightness (L*), hue angle-
sensorial paired comparison test without a reference sample. A h°, and b*, and a significant increase in red color (a*) parameter
pulp from a mix of fruits from the respective growing conditions from the large green (LG) stage to the pink (P) stage during
was used as the sample for the panelists to evaluate sweetness and ripening (Figure 1F).
acidity, and fruit was used to evaluate firmness and color. The tests In both growing conditions, firmness half decreased from the
were carried out in three sessions for each panelist. The results were LG to the W stages (Figure 1C). Although the average firmness for
expressed as the number of positive or negative hits per attribute. both conditions was similar (protected soilless, 0.9 ± 0.3 N; open
field, 0.82 ± 0.2 N), fruit with greater firmness was recorded in
greenhouse conditions (1.5N) compared to field conditions (1.1N).
2.5 Statistical analysis A significant increase in total soluble solids content (TSS) was
observed from the W stages for protected soilless culture and the R
The physiological and quality data for each growing condition stage for open field, with a higher TSS content in the ripe fruit of the
were analyzed using the ANOVA test and visualized using open field growing (11.1 ± 0.4 Brix degree) compared to protected
GraphPad Prism 8.0.2 (GraphPad Software, San Diego, CA, soilless condition (8.3 ± 0.5 Brix degree) (Figure 1D). The moisture
USA). During fruit development and postharvest, asterisks observed was 83.15% ± 2.79% and 85.17% ± 0.99% in the ripe fruit
indicate significant differences in physiological and quality of protected soilless and open field conditions, respectively,
parameters between ripening stages with LG stage, p ≤ 0.05 (*), p suggesting that TSS do not vary due to changes in humidity.
≤ 0.01 (**), p ≤ 0.001 (***), and p ≤ 0.0001 (****). Metabolomics A significant decrease in titratable acidity (TA) was observed in
data and correlation of response variables were analyzed by fruit from the LG to P stages of the protected culture and from the LG
principal component analysis (PCA) and partial least squares to W stages of the open field growing condition (Figure 1E) and similar
discriminant analysis (PLS-DA) using MetaboAnalyst 5.0 (https:// acidity for the ripe fruit from both growing conditions (protected
www.metaboanalyst.ca). The metabolites were associated with the condition: 2.1% ± 0.3%; open field condition: 2.3% ± 0.1%).
metabolic pathways using MetaboAnalyst 5.0. The correlation During the postharvest of ripe fruit (Figure 2), different CO2
between metabolite compounds was carried out by the Pearson production patterns were observed for the fruit of each growing
method. The criteria selection was with a p-value < 0.05 and an FDR condition, with a significant increase after cold storage [Day 5 (1°

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A B

C D

E F

FIGURE 1
Physiological and quality parameters during raspberry fruit development. Measurements were performed in fruits from protected soilless culture and
open field growing conditions. The upper panel shows the different fruit developmental stages in both seasons (bar = 1 cm). LG, large green; W,
white; P, pink; R, ripe fruit; and OR, overripe fruit stages. CO2 production (mg CO2 kg−1 h−1; A), weight (g; B), firmness (Newtons; C) and color
(Cielab scale; F) were determined in whole fruit. Total soluble solids content (TSS, °Brix; D) and titratable acidity (TA, %; E) were determined in
drupelets. Data represent the means ± S.D. from four sample units (each containing 20 fruits) in each developmental stage. Asterisks indicate
significant differences of physiological and quality parameters between ripening stages with LG stage, p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***), and
p ≤ 0.0001 (****).

C)] and shelf life [Day 5 (1°C) + 1 (20°C)] of raspberries harvested 3.2 Primary metabolites differences
in the open field compared to harvest day (Figure 2A). The decrease between soilless culture and open field
in weight of open field fruits during postharvest compared to fresh harvest fruit
ripe fruit was highly significant, unlike protected soilless fruit,
whose weight does not vary significantly during storage at 1°C For the metabolomic analysis, PCA and PLS-DA were
(Figure 2B). The fruits from both growing conditions showed a performed (Figures 3, 4). The relative abundance of untargeted
significant weight decrease after storage at 1°C, and the weight loss metabolites was used as response variables. The PCA showed that
significantly increased after the shelf life of fruit from both growing the first two components (PC1 and PC2) explain 68% of the
conditions (Figure 2C). After storage at 1°C, the weight loss was variability determined by all data analyzed, where the PC1
nearly 15% and increased significantly to 20% after shelf life at 20°C discriminates between growing conditions and PC2 between
for fruits from both growing conditions. The firmness showed no ripening stages and postharvest sampling (Figure 3A). When
significant decrease after cold storage, and the shelf life at 20°C for evaluating scores and loadings plots together, a higher
fruit from protected soilless culture and open field was similar concentration of sugars, including the main ones such as D-
(Figure 2D). The TSS content was significantly higher in open field glucose, D-fructose, and sucrose, were observed in the raspberries
growing conditions after cold storage and shelf life, without from the open field and (left side of both graphs in Figure 3). On the
significant differences in the fruit from the protected culture other hand, the samples from the protected soilless culture showed
(Figure 2E). The TA showed no significant differences during higher concentrations of all the amino acids detected (right side of
postharvest (Figure 2F). A significant decrease in the brightness both graphs in Figure 3). During fruit development and ripe fruit
(L*) and red color (a*) of fruit from both growing conditions was postharvest, a positive correlation was observed between sucrose
observed during postharvest, the loss of brightness of the ripe fruit and D-fructose (0.87), sucrose and D-glucose (0.89), and D-fructose
grown in the open field being higher (Figure 2G). and D-glucose (0.99) (Supplementary Figure 1).

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B C

D E

F G

FIGURE 2
Physiological and quality parameters during raspberry postharvest. Measurements were performed in fruits from protected soilless culture and open
field growing conditions. CO2 production (mg CO2 kg−1 h−1; A), weight and weight loss (g; B; C), firmness (Newtons; D), and color (Cielab scale; G)
were determined in whole fruit. Total soluble solids content (TSS, °Brix; E) and titratable acidity (TA, %; F) were determined in drupelets. Data
represent the means ± S.D. from five sample units (each containing ten fruits) in each developmental stage. Asterisks indicate significant differences
of physiological and quality parameters between ripening stages with LG stage, P ≤ 0.05 (*), P ≤ 0.01 (**), P ≤ 0.001 (***), and P ≤ 0.0001 (****).

Two independent PLS-DAs of each growing condition were the analysis of metabolic pathways revealed significant differences
conducted to observe the metabolic changes during ripening and in metabolites involved in the aspartate, alanine, and glutamate
fruit postharvest (Figure 4). Both growing conditions presented metabolism, and glycine, serine, and threonine metabolism during
high malic acid and galactinol contents at white and pink stages. shelf life (Figure 5). On the other hand, at the pink stage, the
The main difference between the open field (Figure 4A) and metabolites that showed differences were D-gluconic acid and
protected soilless culture (Figure 4B) was found in shelf-life, glucose-6-phosphate, which are related to the pentose phosphate
where the fruit from the protected soilless culture correlated to pathway and were more abundant in fruit grown in open field (data
amino acids and those from the open field to sugars. Furthermore, not shown).

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A B

FIGURE 3
Principal component analysis (PCA) of the metabolic profile of raspberries. (A) Scores plot. Raspberries grown in protected soilless culture are
represented in red and fruit grown in open field are represented in black. The different pre- and postharvest stages are represented as: White; Pink;
Ripe fruit; ColdSt: cold storage (5 days at 1°C); and S-life: shelf life (5 days at 1°C and 1 day at 20°C). (B) Loadings plot. The amino acids are
represented in black, organic acids in orange, sugars in green, and other compounds in blue.

A B

FIGURE 4
Partial least squares discriminant analysis (PLS-DA) of polar metabolites for raspberries grown in open field (A) and grown in protected soilless
culture (B). Red dots correspond to the development stages (White, Pink, and Ripe fruit) and postharvest time (ColdSt: cold storage (5 days at 1°C);
and S-life: shelf life (5 days at 1°C and 1 day at 20°C). The amino acids are represented in black, organic acids in orange, sugars in green, and other
compounds in blue.

A B

FIGURE 5
Metabolite pathway analysis during raspberry shelf life. (A) Pathways impact analysis. The metabolic pathways are depicted as circles based on their
enrichment scores (y-axis) and topology analyses (pathway impact, x-axis) conducted with MetaboAnalyst 5.0. The intensity of the circle colors (from
yellow to red) indicates the extent of significant metabolite changes in each respective pathway. (B) Hierarchical clustering analysis heatmap
performed using all metabolites during raspberries shelf life [Day 5 (1°C) + 1 (20°C)] between grown in the open field and protected soilless culture.

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3.3 Sensorial differences between conditions present a differential CO2 production during ripening.
protected soilless culture and open field These differences in CO2 production patterns are probably directly
harvest fruit related to temperature, since differences in CO2 production have
been observed for Heritage cultivar in the same location in a
The sensory differences in ripe fruit from protected soilless different season (Fuentes et al., 2015; Bernales et al., 2019).
culture and open field determined by a duo–trio test showed an During the fruit development of ‘Heritage’ raspberry, an
average of seven correct answers from the 10 panelists to correctly increased fruit weight and size from the small green to the white
identify the open field samples in sweetness, firmness, and color, stages has been reported. Then, when fruits reached 2 g average at
differentiating them from the greenhouse samples. Regarding the white stage, the weight increase was less pronounced from the
acidity, eight correct answers were recorded. The sensorial paired white to the ripe stages (Bernales et al., 2019). In the present study,
comparison test without a reference sample to determine the fruits grown in the protected soilless culture had a significantly
sweetness sample showed an average of the 10 panelists could higher weight than those grown in the open field, and the fruits
distinguish differences in the sweetness of raspberries (Figure 6B). grown in the protected soilless culture continued to gain weight
According to the statistical analyses, all sensory parameters show after the white stage (Figure 1B). The average weight obtained for
significant differences. Therefore, the significant differences in the the fruits at the mature (R) stage grown in the greenhouse was 4 g, a
total soluble solids content (Figure 1D) and at the level of primary value higher than what was previously reported (2.8 g) in the
metabolites (Figure 4) were discriminated in the sensory panel. Heritage cultivar under open field growing conditions (Bernales
et al., 2019). In addition to the higher weight that the ripe fruit
achieves growing in protected conditions, it is interesting to
4 Discussion highlight that the weight of this fruit does not vary significantly
during storage at 1°C (Figure 2B). In tomato, it has been reported
In the present research, we showed quality and metabolomic that fruit weight can be modified by the value of the electrical
changes in raspberries growing in two different conditions (i.e., conductivity of the nutrient solution (Moya et al., 2017). These
greenhouse and open field) during fruit development and results suggest that the major weight of raspberry fruit grown in the
postharvest. Here, we measured CO2 production, fruit quality protected soilless culture could be by nutrient solution (1.7 mS
parameters, primary metabolites, and sensory determinations in cm−1). However, strawberries grown in soilless conditions with
both cultivation conditions. different electrical conductivity levels (1.5, 2.5, 3.5, and 4.5 mS
Previous studies in different raspberry cultivars, including cm−1) showed the highest fruit average weight at 2.5 mS cm−1,
‘Heritage’, showed a variable trend of CO2 production of the fruit, which was not dependent on the increased electrical conductivity
although it coincides with the non-climacteric fruit behavior (D'Anna et al., 2003). Therefore, the effect of electrical conductivity
(Perkins-Veazie and Nonnecke, 1992; Contreras et al., 2021; on the size of raspberry fruits and the weight loss during fruit
Monsalve et al., 2022). In the present study, both cultivation postharvest should be further studied.

A B

FIGURE 6
The number of the success’s sensorial sample discrimination. (A) The sensory differences in sweetness, acidity, firmness, and color of ripe fruit from
protected soilless culture and open field were determined a duo–trio test using fruit from open field conditions as the reference, and samples from
each growing conditions as coded samples. (B) Then, the sweetness discrimination of ripe fruit from protected soilless culture and open field was
done by a sensorial paired comparison test without a reference sample. A pulp from a mix of fruits was to evaluate sweetness and acidity, and intact
fruits were used to evaluate firmness and color. The tests were carried out in three sessions for each panelist. The results were expressed as the
number of positive or negative hits per attribute. The differences were established for a = 0.05 (*) considering seven hits of ten answers, a = 0.01 (**)
considering eight hits of 10 answers, respectively.

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Independent of raspberry cultivar, ripening is associated with a field conditions (11.7°C–20.4°C; 62–92%) during ripening (January
decrease in fruit firmness and increased sugar composition (Stewart 2022). Therefore, it is not easy to establish differences in the content
et al., 2001; Vicente et al., 2007; Graham and Simpson, 2018; of soluble solids due to temperature.
Contreras et al., 2021; Monsalve et al., 2022; Á lvarez et al., 2023). Ripe raspberry is mainly composed of glucose and fructose
In the present study, a decrease in firmness and titratable acidity (Graham and Simpson, 2018), and fructose concentration is a
was observed during ripening, with similar average values at the crucial factor influencing the quality and marketability of
same developmental stage of both growing conditions. Despite the raspberries (Kafkas et al., 2008; Dincheva et al., 2013). In the
average firmness being similar at the ripe stage and during present study, the metabolomic analysis provides valuable insights
postharvest for both conditions, fruit grown in soilless conditions into the complex metabolic changes occurring in raspberries during
exhibited ripe fruit with greater firmness compared to those grown three stages of development (W, P, and R stages) and after
in field conditions. In fact, some panelists indicated that they postharvest storage of ripe fruit compared to two different
noticed greater firmness for the ripe fruit harvested in protected growing conditions. The PCA discriminated the samples from
conditions, which allowed them to differentiate it in the duo–trio both growing conditions (Figure 3). The fruit from Casablanca
test. The strawberry firmness significantly increased with increased (open field) correlated with the main sugars of raspberry (D-glucose
electrical conductivity, from 1.5 to 4.5 mS cm−1 (D'Anna et al., and D-fructose). In contrast, samples from Quillota (protected
2003). In this way, in the future, it would be interesting to evaluate soilless culture) exhibited a positive correlation with all the amino
the effect of increasing conductivity in protected cultivation acids detected by GC-MS, such as L-alanine, L-glutamic acid,
conditions since firmness is the crucial quality parameter in the glycine, L-serine, and L-valine. Durá n-Soria et al. (2021) reported
raspberry market, defining its sale as fresh, frozen, or pulp fruit. that fructose and glucose levels, together with citric acid (the main
In the present study, the ripening process exhibited consistent acid in raspberry fruits), were higher in raspberry fruits collected in
changes in color parameters across both growing conditions, Norway and Poland (the two coldest locations), which may, in part,
characterized by a reduction in brightness, hue angle, and yellow- be responsible for higher TSS and acidity. In the present study, the
blue coloration (b*), alongside increased redness (a*). This trend temperatures recorded in the protected culture are lower than the
was particularly evident in transitioning from the large green (LG) minimum temperatures reported for the open field, so the
to the pink (P) stages for both conditions (Figure 2G). However, correlations observed are likely due to the differences between
during postharvest, the fruit of open field conditions was observed maximum temperatures. Moreover, a positive correlation between
with a dark red color compared to protected conditions (Figure 2), sucrose and fructose, and sucrose and glucose were reported by
being differentiated by a panelist (Figure 6A), an interesting result other authors (Dincheva et al., 2013; Akšić et al., 2022), which
since markets prefer a brighter red color. agrees with our findings, observing a positive correlation between
We revealed that the growing conditions significantly influence these sugars (Supplementary Figure 1). Sensory evaluation
the total soluble solids content (TSS) of ripe fruit, compared to other confirmed the analytical results, with panelists able to discern
less influencing factors such as titratable acidity (TA) in fruit. Open sweetness differences, corroborating the significant variations in
field cultivation resulted in a higher TSS at the ripe stage than in TSS and primary metabolites. These findings emphasize the
protected soilless culture, indicating that fruits grown in the open influence of cultivation methods on the sensory and chemical
field may have a sweeter taste profile. Furthermore, the open field properties of raspberries, which can guide growers in optimizing
fruits maintained a higher TSS after postharvest storage and shelf fruit quality for consumer preferences.
life, suggesting better retention of sweetness over time, while TA Citric and malic acid are the primary organic acids found in
levels remained consistent postharvest for both cultivation raspberries that contribute to the characteristic tartness of the fruit
methods. These differences in soluble solids could be due to (Akšić et al., 2022). In our study, those organic acids did not show
differences in irrigation, temperature, or radiation. Pritts et al. significant differences between both growing conditions when all
(1999) showed that raspberry growing in the greenhouse tended stages were evaluated. Furthermore, the evolution of each
to be slightly less sweet and more acidic but well within the limits of metabolite during fruit developmental and postharvest was
acceptability. Previous reports on strawberry grown in soilless observed for both growing conditions through PLS-DA
conditions showed a scarcely influenced by different electrical (Figure 4). At the pink stage, the metabolites that showed
conductivity levels in the sugar content (D'Anna et al., 2003); differences were D-gluconic acid and glucose-6-phosphate, which
however, this effect should be studied in raspberry. On the other are related to the pentose phosphate pathway (PPP) and were more
hand, it has been reported that Heritage ripe raspberry from Alto do abundant in fruit grown in open field. The PPP serves as a
Rio Grande in Brazil, which experiences a temperate and semiarid respiratory pathway, playing a crucial role as a main supplier of
subtropical climate, exhibited higher acidity and TSS than those NADPH in plants. It is closely linked to plant growth, development,
from the cooler and wetter subtropical clime in the Mantiqueira and response to various environmental stresses (Tao et al., 2022).
Mountains. Conversely, the fruits of the Mantiqueira Mountains However, the main differences in primary metabolites were found
had a significantly higher total sugar content, although both in shelf-life stage, where the fruit from the open field besides
localities produced fruits with comparable moisture levels (Maro showing higher sugar contents also showed lower contents of
et al., 2014). In the present study, the protected soilless culture amino acids, malic acid, and a-ketoglutaric acid than the fruit
provided a wider range of temperature (6.5°C–39°C) and relative from the protected soilless culture (Figure 5B). These metabolites
humidity (23%–95%) (Supplementary Table S1) compared to open are involved in important metabolic pathways such as the alanine,

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Fuentealba et al. 10.3389/fpls.2023.1324066

aspartate, and glutamate metabolism, and glycine, serine, and review & editing. EP: Formal analysis, Methodology, Writing –
threonine metabolism, which showed a higher pathway impact review & editing. SV: Methodology, Writing – review & editing.
during 1 day of shelf life at 20°C (Figure 5A). Rodrı́guez et al. (2019) MS: Formal analysis, Methodology, Writing – review & editing.
reported that higher electrical conductivity of nutrient solution of DR: Formal analysis, Methodology, Writing – review & editing.
tomatoes grown during winter showed a higher concentration of AA: Formal analysis, Visualization, Writing – review & editing. JA:
amino acids, which may be due to an imbalance in the metabolic Conceptualization, Supervision, Writing – review & editing. MV:
processes associated with the flow of carbon and nitrogen. On the Conceptualization, Supervision, Writing – review & editing. CRF:
other hand, Ren et al. (2023) emphasized the role of amino acids Writing – review & editing. LF: Conceptualization, Methodology,
such as L-lysine and L-glutamine in contributing to the antioxidant Formal analysis, Writing – review & editing, Supervision,
capacity of raspberries, which should be further studied for both Project administration.
conditions. Therefore, the relationship of the metabolites with
essential pathways for plant growth and stress response
emphasizes the influence of growing conditions on the metabolic Funding
profile and quality of raspberries.
The author(s) declare financial support was received for the
research, authorship, and/or publication of this article. This study
5 Conclusions was supported by Agencia Nacional de Investigació n y Desarrollo
ANID-Chile, FONDECYT Regular 1201662 and Fondequip
Our results indicate that the protected soilless culture growing EQM140074 (Chile) grants.
conditions are a good alternative for cultivating raspberries in areas
with unsuitable soil and temperature fluctuations through the
utilization of adjusted continuous fertigation to growing Acknowledgments
conditions with nutrient solutions controlled by electrical
conductivity, achieving greater fruit weight and a potential The authors thank the panelists of the CREAS team.
firmness improvement. Although the lower content of soluble
solids and a difference in the metabolites related to sugars were
observed in the fruit of protected soilless conditions, which was Conflict of interest
perceived at the sensory level, these fruits show a better size and
good evolution of color and weight during cold storage. The present The authors declare that the research was conducted in the
study provides interesting and valuable results with a direct absence of any commercial or financial relationships that could be
commercial application for this alternative growing system, construed as a potential conflict of interest.
mainly in areas where soil and water scarcity are a reality. The author(s) declared that they were an editorial board
member of Frontiers, at the time of submission. This had no
impact on the peer review process and the final decision.
Data availability statement
The original contributions presented in the study are included Publisher’s note
in the article/Supplementary Material. Further inquiries can be
directed to the corresponding author. All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated
organizations, or those of the publisher, the editors and the
Ethics statement reviewers. Any product that may be evaluated in this article, or
claim that may be made by its manufacturer, is not guaranteed or
The studies involving humans were approved by Comité de endorsed by the publisher.
Bioseguridad, Bioé tica y É tica, Centro Regional de Estudios en
Alimentos Saludables-CREAS. The studies were conducted in
accordance with the local legislation and institutional Supplementary material
requirements. The participants provided their written informed
consent to participate in this study. The Supplementary Material for this article can be found online
at: https://www.frontiersin.org/articles/10.3389/fpls.2023.1324066/
full#supplementary-material
Author contributions SUPPLEMENTARY FIGURE 1
Metabolite correlation. The correlation between metabolite compounds was
CF: Conceptualization, Writing – review & editing, Formal carried out by the Pearson method using all data (fruit samples of
analysis, Supervision. FÁ : Formal analysis, Methodology, Writing – developmental stages and ripe fruit postharvest).

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Fuentealba et al. 10.3389/fpls.2023.1324066

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