plants

Article
Abscisic Acid Synthesis and Signaling during the Ripening of
Raspberry (Rubus idaeus ‘Heritage’) Fruit
Fernanda Álvarez 1 , Mario Moya 2 , Claudia Rivera-Mora 2,3 , Paz E. Zúñiga 2,3 , Karla Jara-Cornejo 2,3 ,
Paula Muñoz 4 , Aníbal Ayala-Raso 5 , Sergi Munné-Bosch 4 , Carlos R. Figueroa 2,3 , Nicolás E. Figueroa 6 ,
Mónika Valdenegro 7 , Juan E. Alvaro 7 , Wilfried Schwab 6 , Bruno G. Defilippi 8 and Lida Fuentes 1, *

1 Centro Regional de Estudios en Alimentos Saludables (CREAS), CONICYT-Regional GORE Valparaíso

Proyecto R17A10001, Avenida Universidad 330, Placilla, Curauma, Valparaíso 2362696, Chile;
falvarez@creas.cl
2 Laboratory of Plant Molecular Physiology, Institute of Biological Sciences, Universidad de Talca,
Talca 3465548, Chile; mariomoyamo@gmail.com (M.M.); claudia.rivera@utalca.cl (C.R.-M.);
paz.zuniga@utalca.cl (P.E.Z.); karla.jara@utalca.cl (K.J.-C.); cfigueroa@utalca.cl (C.R.F.)
3 Millennium Nucleus for the Development of Super Adaptable Plants (MN-SAP), Santiago 8340755, Chile
4 Departament de Biologia Evolutiva, Ecología i Ciències Ambientals, Facultat de Biologia, Universitat de
Barcelona, Avinguda Diagonal, 645, E-08028 Barcelona, Spain; paula.munoz@ub.edu (P.M.);
smunne@ub.edu (S.M.-B.)
5 Instituto de Estadística, Facultad de Ciencias, Universidad de Valparaíso, Valparaíso 2360102, Chile;
anibal.ayala@postgrado.uv.cl
6 Biotechnology of Natural Products, Technical University Munich, D-85354 Freising, Germany;
ibv.nicfigueroa@gmail.com (N.E.F.); wilfried.schwab@tum.de (W.S.)
7 Escuela de Agronomía, Facultad de Ciencias Agronómicas y de los Alimentos, Pontificia Universidad Católica
de Valparaíso, Casilla 4-D, Quillota 2260000, Chile; monika.valdenegro@pucv.cl (M.V.);
juan-eugenio.alvaro@pucv.cl (J.E.A.)
8 Unidad de Postcosecha, INIA La Platina, Santa Rosa, Santiago 8820000, Chile; bdefilip@inia.cl
* Correspondence: lfuentes@creas.cl; Tel.: +56-32237286

Abstract: The raspberry (Rubus idaeus L.) fruit is characterized by its richness in functional molecules
Citation: Álvarez, F.; Moya, M.; and high nutritional value, but the high rate of fruit softening limits its quality during postharvest.
Rivera-Mora, C.; Zúñiga, P.E.; Raspberry drupelets have a particular ripening regulation, depending partially on the effect of
Jara-Cornejo, K.; Muñoz, P.; ethylene produced from the receptacle. However, the possible role of abscisic acid (ABA) in the
Ayala-Raso, A.; Munné-Bosch, S.; modulation of quality parameters during the ripening of raspberry is unclear. This study charac-
Figueroa, C.R.; Figueroa, N.E.; et al.
terized the fruit quality-associated parameters and hormonal contents during fruit development
Abscisic Acid Synthesis and
in two seasons. The quality parameters showed typical changes during ripening: a drastic loss
Signaling during the Ripening of
of firmness, increase in soluble solids content, loss of acidity, and turning to a red color from the
Raspberry (Rubus idaeus ‘Heritage’)
large green stage to fully ripe fruit in both seasons. A significant increase in the ABA content was
Fruit. Plants 2023, 12, 1882. https://
doi.org/10.3390/plants12091882 observed during the ripening of drupelets and receptacles, with the higher content in the receptacle
of ripe and overripe stages compared to the large green stage. Moreover, identification of ABA
Academic Editor: Tae-Hwan Kim
biosynthesis-(9-cis-epoxycarotenoid dioxygenase/NCED) and ABA receptor-related genes (PYRs-like
Received: 15 March 2023 receptors) showed three genes encoding RiNCEDs and nine genes for RiPYLs. The expression level
Revised: 30 April 2023 of these genes increased from the large green stage to the full-ripe stage, specifically characterized by
Accepted: 30 April 2023 a higher expression of RiNCED1 in the receptacle tissue. This study reports a consistent concomitant
Published: 5 May 2023 increase in the ABA content and the expression of RiNCED1, RiPYL1, and RiPYL8 during the ripening
of the raspberry fruit, thus supporting the role for ABA signaling in drupelets.

Keywords: ABA biosynthesis and signaling; ethylene; NCED and PYL genes; fruit quality parameters;
Copyright: © 2023 by the authors.
Rubus idaeus; softening
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
1. Introduction
creativecommons.org/licenses/by/ Raspberries have a unique flavor, an attractive color, and valuable health benefits (rich
4.0/). source of minerals and vitamins, dietary fiber, and various antioxidants) [1,2]. However,

Plants 2023, 12, 1882. https://doi.org/10.3390/plants12091882 https://www.mdpi.com/journal/plants

Plants 2023, 12, 1882 2 of 21

raspberries are soft fruit with a fast ripening rate, leading to a short postharvest shelf
life. In addition, it has been reported that fruit ripening in raspberry is characterized by a
progressive decrease in the firmness of the fruit, associated with cell wall modification and
expression of cell wall degrading-related genes; starting from the large green to overripe
stages of ripening [1,3–6].
To date, little is known about the hormonal regulation of raspberry ripening and
its impact on the final quality of this fruit. Raspberry is categorized as a non-climacteric
fruit [7], although ethylene production and respiratory peaks have been detected in the
receptacle from the white fruit stage until full maturity [5,8,9]. Therefore, ethylene has
been reported to partially regulate firmness loss during postharvest, but this regulation is
temperature-dependent [9]. In addition, this hormone would be responsible for the detach-
ment of the drupes to the receptacle [10]. Also, the indole-3-acetic acid (IAA) conjugation
with amino acids might play a central role in the onset of raspberry ripening by decreasing
the IAA-free content [11]. Recently, it has been reported that the maximum accumulation
of abscisic acid (ABA) takes place at the ripe stage [12]. The above finding is in agreement
with our previous de-novo assembly analysis during different fruit developmental stages,
which showed differentially expressed genes (DEGs) that included transcripts related to
the synthesis and response to ABA; content regulation, as well as the influx and efflux of
auxin; response to auxin; and ethylene biosynthesis and perception [13].
In grape and strawberry fruits, it has been pointed out that ABA is the main reg-
ulator of maturation, its biosynthesis is necessary for the onset of ripening, and its in-
crease is concomitantly with the progression of maturity [14–16]. In strawberry, ABA
treatment was reported to accelerate fruit softening and increase color and ethylene pro-
duction [17,18]. Also, the role of genes encoding enzymes related to ABA metabolism
such as 9-cis-epoxycarotenoid dioxygenase (NCED) and abscisic acid 8’-hydroxylase 1-like
in the grape and strawberry ripening has been demonstrated [15,19–21]. A remarkable
decrease in the ABA content and a significantly retarded ripening process resulted from the
down-regulation of FaNCED1 [15]. In turn, the pyrabactin resistance 1 (PYR1)/PYR1-like
(PYL) is an ABA receptor core of ABA signaling components [20,22,23]. The PYL family
members have been described in climacteric and non-climacteric fruit, increasing the ex-
pression of these genes during the ripening of the fruits of tomato [24], strawberry [22,25],
and grape [26].
Despite our understanding of the fundamental physiological and biochemical aspects
of non-climacteric fruit such as raspberry, many molecular mechanisms involved in the
raspberry fruit development and ripening, as well as their effect on quality parameters,
still need to be fully understood. In this study, we observed typical changes in quality
attributes during the ripening of raspberry in two seasons. We showed that ABA content
increases in parallel to raspberry ripening in both drupe and receptacle tissues. In addition,
ABA-related genes were identified, and marked differences were observed in the expression
profile of the RiNCED and ABA receptor genes (RiPYLs) depending on the fruit tissue.

2. Results
2.1. Fruit Quality Measurements
In this study, the fruit of raspberry (Rubus idaeus ‘Heritage’) was classified into five
developmental stages [9,27] during the 2020 and 2021 harvest seasons in Casablanca,
Valparaíso Region, Chile (33◦ 200 3900 S; 71◦ 220 1100 W; 247 masl) (Figure 1).
EER REVIEW 3 of 23

Plants 2023, 12, 1882 3 of 21

Figure 1. Developmental stages of raspberry (R. idaeus ‘Heritage’) fruit during 2020 and 2021 seasons.
The fruit was harvested and sorted by size and color as follows: large green (LG), white (W), pink (P),
red (R), and overripe (OR) fruit.
Figure 1. Developmental stages of raspberry (R. idaeus ‘Heritage’) fruit during 2020 and 2021
seasons. The fruit was harvested
During theand
2020 sorted by sizedecreased
season, firmness and color40%asfrom
follows: large
the W to green
P stages. (LG), white
Conversely, in
(W), pink (P), red (R),theand
2021 season, the
overripe firmness
(OR) fruit.drastically decreased 48% from the LG to W stages (Figure 2).
A significant increase in total soluble solids (TSS) was observed from the LG to W stages
and the W to P stages during both seasons, with a larger TSS content during the 2020 season
During the 2020 season,
compared firmness
to the decreased
2021 season 40%
(Figure 2). fromdecrease
A sharp the Wintotitratable
P stages. Conversely,
acidity (TA) was
in the 2021 season, observed
the firmness
from thedrastically decreased
LG to W stages (Figure 2),48%
with afrom
higherthe LGcontent
acidity to W stages (Figure
at the LG stage
of the 2021 season; however, the acidity of ripe stages was similar in both seasons.
2). A significant increase in total soluble solids (TSS) was observed from the LG to W stages
and the W to P stages during
2.2. ABA and ACC both seasons,
Dynamics with a larger
during Raspberry TSS content during the 2020
Fruit Development
season compared to the In 2021 season
this study, (Figure
the contents 2). A acid
of abscisic sharp decrease
(ABA) in titratable acidity (TA)
and 1-amino-cyclopropane-1-carboxylic
acid (ACC) were determined by ultrahigh-performance
was observed from the LG to W stages (Figure 2), with a higher acidity liquid chromatography
contentcoupled
at the toLG
electrospray ionization tandem mass spectrometry (UPLC/ESI-MS/MS).
stage of the 2021 season; Thehowever,
ABA content the
wasacidity
determinedof ripe stages and
in drupelets wasreceptacles
similar in both seasons.
of different fruit de-
velopmental stages and during two seasons (Figure 3). During the development of the
raspberry fruit, the ABA content increased in both tissues and seasons at the R and OR
stages compared to the LG stage (Figure 3). Furthermore, in both seasons, the ABA content
was higher (10,000–20,000 ng/gDW) in receptacles than in drupelets (6000–7500 ng/gDW)
at the R stage (Figure 3).
Previously, we showed the role of ethylene in partially regulating the firmness of
raspberry in a temperature-dependent manner during the postharvest season [9]. Therefore,
the content of ACC, the immediate ethylene precursor, was determined during ripening in
both seasons. The ACC content showed significant differences in receptacles between the
LG and R stages in the 2021 season only (Figure 3).
x FOR PEER REVIEW 4 of 23
Plants 2023, 12, 1882 4 of 21

Figure 2. Quality Figure 2. Quality

parameters during parameters during theofdevelopment
the development raspberry (R. of raspberry (R. idaeus fruit.
idaeus ‘Heritage’) ‘Heritage’) fruit.
◦
Firmness (N) was determined in the whole fruit, and total soluble solids content (TSS, °Brix)(TSS,
Firmness (N) was determined in the whole fruit, and total soluble solids content and Brix) and
titratable
titratable acidity (TA, %) wereacidity (TA, %) were
determined determined
in drupelets. in drupelets.
The The fruit was
fruit was harvested andharvested
sorted byandsize
sorted by size
and color as follows:andlarge
color green
as follows:
(LG),large green
white (LG),
(W), white
pink (W),
(P), redpink
(R),(P),
andredoverripe
(R), and overripe (OR)
(OR) fruit fruit stage. Data
stage.
Data represent therepresent
means the means
± S.D. ± S.D.
from from
five five sample
sample unitsunits
(each(each containingten
containing ten fruits)
fruits)bybythethe
development
development stage.stage. Significant
Significant differences
differences areare indicated(p(p≤≤0.05)
indicated 0.05)between
between developmental
developmental stagesstages as as
lowercase for
lowercase for 2020 and uppercase for 2021. Boxplots show each group’s distribution (developmentalstages), and
2020 and uppercase for 2021. Boxplots show each group’s distribution (developmental
the line
stages), and the black black(central
line (central
value value in the
in the box) box) indicates
indicates thethemedian
medianvalue
value for
for non-parametric
non-parametric analysis and
analysis and pointspoints
outsideoutside the boxplots
the boxplots showshow outlier
outlier values.
values.

2.2. ABA and ACC Dynamics during Raspberry Fruit Development

In this study, the contents of abscisic acid (ABA) and 1-amino-cyclopropane-1-
carboxylic acid (ACC) were determined by ultrahigh-performance liquid
chromatography coupled to electrospray ionization tandem mass spectrometry
(UPLC/ESI-MS/MS).
The ABA content was determined in drupelets and receptacles of different fruit
 raspberry fruit, the ABA content increased in both tissues and seasons at the R and OR
stages compared to the LG stage (Figure 3). Furthermore, in both seasons, the ABA content
Plants 2023, 12, 1882 was higher (10,000–20,000 ng/gDW) in receptacles than in drupelets (6000–7500 ng/gDW)
5 of 21
at the R stage (Figure 3).

Figure 3. ABA and

Figure and ACC
ACCcontents
contentsduring
duringthe development
the development of of
raspberry idaeus
(R. (R.
raspberry ‘Heritage’)
idaeus fruit.fruit.
‘Heritage’) The
fruitfruit
The was was
harvested in the
harvested in2020 and 2021
the 2020 seasons
and 2021 and sorted
seasons by size
and sorted byand
sizecolor
and as follows:
color large green
as follows: large
green (LG), (W),
(LG), white whitepink
(W),(P),
pink
red(P),
(R),red
and(R), and overripe
overripe (OR)
(OR) fruit. fruit.
ABA ABA
and ACCand ACC contents
contents (expressed
(expressed as ng/g
as ng/g DW) were determined in drupelets and receptacles from each stage using five
DW) were determined in drupelets and receptacles from each stage using five independent replicates independent
replicates (each containing
(each containing ten fruits).differences
ten fruits). Significant Significantare
differences
indicated are
(p ≤indicated (p ≤ developmental
0.05) between 0.05) between
developmental stages as lowercase for 2020 and uppercase for 2021. The boxplots show each group’s
stages as lowercase for 2020 and uppercase for 2021. The boxplots show each group’s distribution
(developmental stages), the black line (the central value in the box) indicates the median value for
non-parametric analysis, and the points outside the boxplots show outlier values. Otherwise, the
central value represents the mean value. DW: dry weight.
Plants 2023, 12, 1882 6 of 21

To determine the relationship of the ABA and ACC contents with the parameters of
the fruits’ quality, a correlation matrix analysis was performed between variables (Figure 4).
In the 2020 season, the standardized components describe principally a positive correlation
between the variables determined in drupelets, i.e., the TSS and ABA content (0.62), and a
negative correlation between firmness and ABA content (−0.66) and between the TA and
ABA content (−0.49) (Figure 4). In the 2021 season, a positive correlation was observed
between the TSS and ABA content (0.48), and negative correlations were observed between
Plants 2023, 12, x FOR PEER REVIEW 7 of 23
firmness and the ABA content (−0.66), and between the TA and ABA content (−0.61) in
drupelets (Figure 4).

Figure 4. Correlation matrix of variables in drupelets during the development of the raspberry (R.
idaeus ‘Heritage’) fruit in two seasons. The correlation was carried out by the Kendall method. ABA:
abscisic acid content; ACC: 1-aminocyclopropane-1-carboxylic acid content; TSS: total soluble solids
content; and TA: titratable acidity. Significant correlation values (p-value ≤ 0.05) are shown inside
the circles.
 Figure 4. Correlation matrix of variables in drupelets during the development of the raspberry (R.
idaeus ‘Heritage’) fruit in two seasons. The correlation was carried out by the Kendall method. ABA:
abscisic acid content; ACC: 1-aminocyclopropane-1-carboxylic acid content; TSS: total soluble solids
Plants 2023, 12, 1882 content; and TA: titratable acidity. Significant correlation values (p-value ≤ 0.05) are shown inside
7 of 21
the circles.

2.3. Identification of RiNCED and RiPYL Genes in the Raspberry Genome

2.3. Identification of RiNCED and RiPYL Genes in the Raspberry Genome
Since ABA increases significantly during ripening (P, R, and OR stages. Figure 3), we
Since
characterizedABAtheincreases
genessignificantly
related toduring
the ripening (P, R, and OR stages. Figure 3), we
ABA biosynthesis-(9-cis-epoxycarotenoid
characterized the genes related to the ABA biosynthesis-(9-cis-epoxycarotenoid
dioxygenase-like/RiNCED) and ABA receptor-related genes (PYRs-like receptors/RiPYL)dioxygenase-
like/RiNCED) and ABA receptor-related genes (PYRs-like receptors/RiPYL) identified
identified in the draft genome published by Wight et al., 2019 [28]. The study of gene in
the draft genome published by Wight et al., 2019 [28]. The study of gene families for each
families for each gene in the raspberry genome shows three and nine members for the
gene in the raspberry genome shows three and nine members for the RiNCED and RiPYL
RiNCED and RiPYL genes, respectively (Tables S1 and S2). The RiNCED sequence lengths
genes, respectively (Tables S1 and S2). The RiNCED sequence lengths ranged from 2005
ranged from 2005 to 2670 bp, and the deduced protein lengths were from 604 to 614 amino
to 2670 bp, and the deduced protein lengths were from 604 to 614 amino acid residues
acid residues (Table S1). However, the gene sequence lengths of the RiPYL gene family
(Table S1). However, the gene sequence lengths of the RiPYL gene family ranged from 750
ranged from 750 to 2973 bp, and the deduced protein length varied between 160 to 216
to 2973 bp, and the deduced protein length varied between 160 to 216 aminoacidic residues
aminoacidic residues (Table S2).
(Table S2).
2.4. Structure,
2.4. Structure, Conserved
Conserved Motifs
Motifs and Promoter Analysis
and Promoter Analysis ofof RiNCED
RiNCED and and RiPYL
RiPYL Genes
Genes
The gene structure and conserved motifs of the RiNCED
The gene structure and conserved motifs of the RiNCED and RiPYL genes and and RiPYL genes and
deduced
deducedidentified
proteins proteins identified in Rubus
in Rubus idaeus wereidaeus wereusing
explored explored using the AUGUSTUS
the AUGUSTUS Server
Server and MEME
and MEME Suite, respectively (Figures 5 and 6). The gene structure and
Suite, respectively (Figures 5 and 6). The gene structure and motif analysis showed a high motif analysis
showedofa conservation
degree high degree in of the
conservation
RiNCED gene in the RiNCED
family. gene RiNCED
The three family. The three RiNCED
sequences did not
show introns (Figure 5A). The results showed that the three RiNCED-deducedRiNCED-
sequences did not show introns (Figure 5A). The results showed that the three proteins
deduced six
exhibited proteins
highlyexhibited
conservedsix highly
motifs conserved
(Figure 5B). Eachmotifs (Figure 5B). Each
RiNCED-deduced RiNCED-
protein showed
deduced
six protein
conserved showed
motifs six conserved
similarly motifs
distributed similarly
in the distributed
three proteins in the indicating
identified, three proteins
the
identified,
low indicating
variability in this the
genelow variability in this gene family.
family.

Figure 5.
Figure 5. Gene
Gene structure
structure of
of RiNCEDs
RiNCEDs and
and conserved
conserved protein
protein motifs
motifs of
of deduced
deduced proteins.
proteins. (A) Gene
(A) Gene
structures for RiNCED. (B) Motif profile of RiNCED deduced proteins. The six motifs are displayed
structures for RiNCED. (B) Motif profile of RiNCED deduced proteins. The six motifs are displayed
in different colored boxes. CDS: coding sequence; UTR: untranslated region.
in different colored boxes. CDS: coding sequence; UTR: untranslated region.

However, the analysis of nine sequences of the RiPYL gene family showed that RiPYL1-
6 and RiPYL 9 have no introns; conversely, three introns were found for RiPYL7 and two in-
trons for the RiPYL8 gene (Figure 6A). The results showed that the nine RiPYL deduced
proteins exhibited three similarly distributed conserved motifs (Figure 6B) indicating the
low variability in the final protein sequence of this gene family.
The analysis of the promoter regions of the RiNCED and RiPYL genes for the iden-
tification of cis-elements for the phytohormone response, such as ABA, auxin, ethylene,
gibberellins, jasmonates, and salicylic acid was performed using the draft genome of
raspberry [28]. The analyses showed that most RiNCED and RiPYL sequences present
regulatory elements for ABA (Figure 7). Thus, RiNCED3 and RiPYL2, RiNCED1, and
RiPYL7 and RiPYL9 show 15, nine, and six ABA response cis-elements, respectively. It is
also shown that these promoter regions present cis-elements of the response to other hor-
mones, observing six ethylene cis-elements for the RiPYL5 promoter sequence. In addition,
most of the identified sequences showed cis-elements in the response to gibberellin (except
for RiNCED1), jasmonates (except for RiPYL8 and RiPYL9), and salicylic acid (except for
RiPYL9) (Figure 7).
 Plants 2023, 12, 1882 Plants 2023, 12, x FOR PEER REVIEW 8 of 21 9 of 23

Plants 2023, 12, x FOR PEER REVIEW 10 of 23

Figure 6. Gene structure of RiPYLs and conserved protein motifs of deduced proteins. (A) Exon-
RiPYL
intron structures forFigure genes. (B) Motif profile of RiPYL deduced proteins. The three motifs are
6. Gene structure of RiPYLs and conserved protein motifs of deduced proteins. (A) Exon-
displayed in different colored
intron boxes.
structures forCDS:
RiPYLcoding sequence;
genes. (B) UTR:ofuntranslated
Motif profile region.
RiPYL deduced proteins. The three motifs
are displayed in different colored boxes. CDS: coding sequence; UTR: untranslated region.

However, the analysis of nine sequences of the RiPYL gene family showed that
RiPYL1-6 and RiPYL 9 have no introns; conversely, three introns were found for RiPYL7
and two introns for the RiPYL8 gene (Figure 6A). The results showed that the nine RiPYL
deduced proteins exhibited three similarly distributed conserved motifs (Figure 6B)
indicating the low variability in the final protein sequence of this gene family.
The analysis of the promoter regions of the RiNCED and RiPYL genes for the
identification of cis-elements for the phytohormone response, such as ABA, auxin,
ethylene, gibberellins, jasmonates, and salicylic acid was performed using the draft
genome of raspberry [28]. The analyses showed that most RiNCED and RiPYL sequences
present regulatory elements for ABA (Figure 7). Thus, RiNCED3 and RiPYL2, RiNCED1,
and RiPYL7 and RiPYL9 show 15, nine, and six ABA response cis-elements, respectively.
It is also shown that these promoter regions present cis-elements of the response to other
hormones, observing six ethylene cis-elements for the RiPYL5 promoter sequence. In
addition, most of the identified sequences showed cis-elements in the response to
gibberellin (except for RiNCED1), jasmonates (except for RiPYL8 and RiPYL9), and
salicylic acid (except for RiPYL9) (Figure 7).

Figure 7. Promoter analysis of RiNCED and RiPYL genes identified in R. idaeus genome. The figure
represents
Figure the number
7. Promoter of of
analysis cis-elements
RiNCED andthat respond
RiPYL to identified
genes phytohormones in thegenome.
in R. idaeus promoter regions
The figurefor
represents the number
each sequence of cis-elements
identified thatdraft
in the genome respond to phytohormones in the promoter regions for
of raspberry.
each sequence identified in the genome draft of raspberry.

2.5. Phylogenetic Analysis and Classification of RiNCED and RiPYL Deduced Proteins
The phylogenetic analysis showed that each RiNCED family member belongs to the
three subfamilies—group I, II, and V—of the five NCED groups described in the plants
(Figure 8). RiPYL proteins were distributed within the three groups described in the plants
Plants 2023, 12, 1882 9 of 21

2.5. Phylogenetic Analysis and Classification of RiNCED and RiPYL Deduced Proteins
The phylogenetic analysis showed that each RiNCED family member belongs to the
three subfamilies—group I, II, and V—of the five NCED groups described in the plants
(Figure 8). RiPYL proteins were distributed within the three groups described in the plants
(Figure 8). The deduced protein sequences show a length of 614, 604, and 613 amino acid
residues for RiNCED1, RiNCED2, and RiNCED3, respectively (Table S1). The RiNCED1
predicted protein showed a low similarity with the other NCEDs described in the plant
species, with a similarity of 44% to CkNCED1 (ACU86971.1; Caragana korshinskii), PsNCED2
(BAC10550.1; Pisum sativum) and AhNCED1 (CAE00459.2; Arachis hypogaea). The RiNCED2
sequence showed a 100% similarity to FaNCED1 (ADU85829.1) described in Fragaria
x ananassa and 89% to MdNCED1 (AGQ03804.1) from Malus domestica. The RiNCED3
Plants 2023, 12, x FOR PEER REVIEW
showed low homology with the other sequences, showing a 37% = similarity to CcNCED611 of 23
(XP006420602.2) from Citrus clementina and CisNCED6 (XP006489810.2) from Citrus sinensis
(Figure 8).

Phylogenetic
Figure8.8.Phylogenetic
Figure tree
tree of of NCED
NCED deduced
deduced proteins
proteins from
from R. idaeus
R. idaeus and other
and other plantplant species.
species. The
The phylogenetic
phylogenetic tree tree
was was constructed
constructed based
based on on
thethe full-length
full-length sequencesofofthe
sequences theRiNCED-deduced
RiNCED-deduced
proteins using
proteins using the
theMEGA-X
MEGA-X software. Purple
software. dots dots
Purple are NCED deduced
are NCED protein identified
deduced in raspberry.
protein identified in
raspberry.
At the same time, the deduced protein sequences show a length of 210, 190, 216, 218,
174, At
160,
the192,
same184, and
time, 213
the amino protein
deduced acid residues for RiPYL1,
sequences RiPYL2,
show a length RiPYL3,
of 210, RiPYL4,
190, 216, 218,
RiPYL5,
174, 160, RiPYL6,
192, 184,RiPYL7,
and 213RiPYL8,
amino and
acidRiPYL9,
residuesrespectively
for RiPYL1, (Table S2). Most
RiPYL2, of the
RiPYL3, RiPYL
RiPYL4,
sequences
RiPYL5, identified
RiPYL6, in raspberry
RiPYL7, RiPYL8, andhadRiPYL9,
similarities with the(Table
respectively FvPYL sequences
S2). described
Most of the RiPYL
in Fragaria vesca (Figure 9). Thus, the RiPYL1, RiPYL2, RiPYL3, and RiPYL9,
sequences identified in raspberry had similarities with the FvPYL sequences described in RiPYL4,
RiPLY5,vesca
Fragaria RiPYL6, and9).
(Figure RiPYL8
Thus,sequences
the RiPYL1,showed a 67%,
RiPYL2, 99%,and
RiPYL3, 87%, 97%, 99%,
RiPYL9, 90%, RiPLY5,
RiPYL4, and 93%
similarity to FvPYL3 (XP004300241.1FvPYL2 (XP004291031.1),
RiPYL6, and RiPYL8 sequences showed a 67%, 99%, 87%, 97%, 99%, 90%, and 93% FvPYL4 (XP004302617.1),
FvPYL6 (XP_004308958.1),
similarity FvPYL13a (XP004293952.1),
to FvPYL3 (XP004300241.1FvPYL2 FvPYL13c
(XP004291031.1), (XP011460608.1),
FvPYL4 and
(XP004302617.1),
FvPYL7 (XP_004308958.1),
FvPYL6 of Fragaria (XP004293952.1),
(XP_004306686.1) FvPYL13a vesca, respectively. In turn, (XP011460608.1),
FvPYL13c the RiPYL7 sequenceand
FvPYL7 (XP_004306686.1) of Fragaria vesca, respectively. In turn, the RiPYL7 sequence
showed a 37% similarity to MdPYL3 (XP008380616.1) and MdPYL9 (XP008376697.2) from
M. domestica (Figure 9).
 Plants 2023, 12, 1882 10 of 21

x FOR PEER REVIEW 12 of 23

showed a 37% similarity to MdPYL3 (XP008380616.1) and MdPYL9 (XP008376697.2) from
M. domestica (Figure 9).

Figure 9. Phylogenetic
Figure 9. tree of PYL tree
Phylogenetic deduced proteinsproteins
of PYL deduced from R. idaeus
from andand
R. idaeus other plant
other plantspecies. The
species. The
phylogenetic tree was constructed based on the full-length sequences of the RiPYL deduced proteins
phylogenetic tree was constructed based on the full-length sequences of the RiPYL deduced proteins
using the MEGA-X
usingsoftware.
the MEGA-X Purple dotsPurple
software. are PYL
dots deduced protein
are PYL deduced identified
protein in in
identified raspberry.
raspberry.

2.6. Expression of RiNCED and RiPYL Genes

2.6. Expression of RiNCED and RiPYL Genes
Transcriptomic data were retrieved from the “Rubus idaeus Heritage transcripts v1.0”
Transcriptomic
databasedata
[13] were retrieved
available from the
in the genome data“Rubus
base for idaeus
Rosaceae Heritage transcripts
[29] to explore v1.0”
the differen-
tially expressed genes (DEGs) in the flower, green fruit,
database [13] available in the genome data base for Rosaceae [29] to explore theand pink fruit. The analysis of
expression patterns by FPKM showed upregulated DEGs at the
differentially expressed genes (DEGs) in the flower, green fruit, and pink fruit. The P stage (Figure 10). In this
transcriptomic database, RiNCED2 and RiPYL9 were not identified. Most of the identified
analysis of expression
genes havepatterns by FPKMinshowed
a higher expression upregulated
flowers and pink stage andDEGs at the P stage
low expression at the (Figure
G stage
10). In this transcriptomic
such as RiNCED1 database, RiNCED2
and RiPYL1-6, and RiPYL9
while RiNCED3 werepresent
and RiPYL8 not identified. Most atof
higher expression
the identified genes have
the P stage inacomparison
higher expression in flowers
to green fruits and(Figure
and flowers pink stage
10). and low expression
at the G stage such as RiNCED1 and RiPYL1-6, while RiNCED3 and RiPYL8 present higher
expression at the P stage in comparison to green fruits and flowers (Figure 10).
 analysis of expression patterns by FPKM showed upregulated DEGs at the P stage (Fig
10). In this transcriptomic database, RiNCED2 and RiPYL9 were not identified. Mos
the identified genes have a higher expression in flowers and pink stage and low express
Plants 2023, 12, 1882 at the G stage such as RiNCED1 and RiPYL1-6, while RiNCED3 and RiPYL811presentof 21 hig
expression at the P stage in comparison to green fruits and flowers (Figure 10).

Figure 10. RiNCED and RiPYL genes differentially expressed in database “R. idaeus Heritage tran-
scripts v1.0. NCED and PYL are included in the ABA biosynthesis and signaling pathways, respec-
tively. Gene expression was calculated as FPKM derived from flower (F), green fruit (G), and pink
fruit (P) RNA-Seq data [13,29]. ABA biosynthesis pathway modified from Han et al., (2004) [30]; ABA
signaling pathway modified from Yoon et al., (2020) [31].

The RT-qPCR analysis showed that RiNCED1, RiPYL1, and RiPYL8 transcripts in-
creased their expression during development, with the highest expression at the R and
OR stages (Figure 11). A significant increase in the RiNCED1 expression was observed
in drupelets at the OR stage in both seasons. In the receptacle, a significant increase in
RiNCED1 expression was observed at the OR stage for the 2020 season and at the R stage
for the 2021 season compared to the LG stage (Figure 11).
Although RiPYL1 expression tends to increase during ripening in both tissues and
seasons, a significantly higher RiPYL1 transcript level at the OR stage was observed in
drupelets in both seasons and receptacles in the 2021 season (Figure 11). Similar to the
RiPYL1 gene, the RiPYL8 gene expression shows a tendency to increase during ripening in
both tissues and seasons, showing a significant increase in drupelets at the R and OR stage
in both seasons and in receptacles at the OR stage for the 2021 season (Figure 11).
However, the standardized components describe a correlation between the ABA
content and gene expression variables (Figure 12). In the 2020 season, a low correlation was
observed, being positive for the ABA content and RiNCED1 expression in the receptacle
(0.43) and the ABA content and RiPYL8 expression in the drupelet (0.54). However, in
the 2021 season, the ABA content showed a positive correlation with the three studied
genes in both tissues, i.e., 0.67 with RiNCED1, 0.63 with RiNPYL1, and 0.67 with RiPYL8
in drupelets; and 0.63 with RiNCED1, 0.51 with RiNPYL1, and 0.47 with RiPYL8 in the
receptacle (Figure 12). The significant increase of the RiPYL1 and RiPYL8 gene expression
in drupelets at the R and OR stages suggest that the ABA signaling plays an important
role in the ripening of raspberry. In addition, the results suggest a correlation between the
ABA content and the expression of RiNCED1, RiPYL1, and RiPYL8 that depends on the
harvest season.
 increased their expression during development, with the highest expression at the R and
OR stages (Figure 11). A significant increase in the RiNCED1 expression was observed in
drupelets at the OR stage in both seasons. In the receptacle, a significant increase in
RiNCED1 expression was observed at the OR stage for the 2020 season and at the R stage
Plants 2023, 12, 1882 12 of 21
for the 2021 season compared to the LG stage (Figure 11).

Figure 11. Expression profiles of the RiNCED1, RiPYL1, and RiPYL8 genes during the development of
Figure 11. Expression profiles of the RiNCED1, RiPYL1, and RiPYL8 genes during the development
raspberry (R. idaeus ‘Heritage’) fruit. The fruit was harvested and sorted by size and color as follows:
of raspberry (R. idaeus ‘Heritage’) fruit. The fruit was harvested and sorted by size and color as
large green (LG), white (W), pink (P), red (R), and overripe (OR) fruit stages during the 2020 and 2021
follows: large green (LG), white (W), pink (P), red (R), and overripe (OR) fruit stages during the
seasons. Relative expression was determined in drupelets and receptacles from each stage using five
2020 and 2021 seasons. Relative expression was determined in drupelets and receptacles from each
independent sample extractions. Ri18S was used as the normalizer gene. Significant differences are
stage using five independent sample extractions. Ri18S was used as the normalizer gene. Significant
indicated (p ≤ 0.05) between the developmental stages as lowercase for 2020 and uppercase for 2021.
Boxplots show each group’s distribution (developmental stages), and the black line (central value
in the box) indicates the median value for non-parametric analysis and points outside the boxplots
show outlier values. Otherwise, the central value represents the mean value.
Plants 2023,12,
Plants2023, 12,1882
x FOR PEER REVIEW 13 of
15 of 21
23

Figure 12.Correlation
Figure12. Correlationmatrix
matrixof ofvariables
variablesduring
duringraspberry
raspberry(R.(R.idaeus
idaeus‘Heritage’)
‘Heritage’)fruit
fruitdevelopment
development
inintwo
twoseasons.
seasons.The
Thecorrelation
correlationwas
wascarried
carriedoutoutby
bythe
theKendall
Kendallmethod.
method.ABA:
ABA:abscisic
abscisicacid
acidcontent;
content;
ACC:
ACC:1-aminocyclopropane-1-carboxylate
1-aminocyclopropane-1-carboxylateacid; acid;RiNCED1:
RiNCED1:expression
expressionofofthe
theraspberry
raspberryNCED1
NCED1ABA ABA
biosynthesisgene;
biosynthesis gene; RiPYL1:
RiPYL1: expression
expression of theofraspberry
the raspberry
PYL1 ABA PYL1 ABA gene;
receptor receptor gene;
RiPYL8: RiPYL8:
expression
ofexpression of the
the raspberry raspberry
PYL8 PYL8 ABA
ABA receptor receptor
gene. gene.
Significant Significant
correlation correlation
values values
(p-value (p-value
≤ 0.05) ≤ 0.05)
are showed
are showed
inside inside the circles.
the circles.

3.3.Discussion
Discussion
The
Thefirst
firststep
stepto
totake
taketo
tounderstand
understandthe theregulatory
regulatoryrole
roleof
ofhormones
hormonesduringduringraspberry
raspberry
ripening
ripeningisis to
to determine its association
determine its association with
withthethequality
qualityparameters
parametersand andthe theexpression
expression of
of
thethekey
keyphytohormone-related
phytohormone-relatedgenes. genes.Here,
Here,weweshowed
showed that
that the
the ABA
ABA content
content and
and the
the
expression
expressionofofABA ABAbiosynthesis
biosynthesis(9-cis-epoxycarotenoid
(9-cis-epoxycarotenoiddioxygenase
dioxygenaselike/RiNCED)-
like/RiNCED)-and and
receptor
receptor (PYRs-like receptors/RiPYL)-related genes increase with the progressionof
(PYRs-like receptors/RiPYL)-related genes increase with the progression offruit
fruit
ripening,
ripening,irrespective
irrespectiveofofthe
theharvest
harvestseasons.
seasons.
During
During both seasons, thefruit
both seasons, the fruitquality
qualityparameters
parametersshow
showthethetypical
typicalchange
changeduring
during
raspberry
raspberry ripening, a decrease in firmness and acidity and an increase intotal
ripening, a decrease in firmness and acidity and an increase in totalsoluble
soluble
solids.
solids.Firmness
Firmnessdrastically
drasticallydecreased
decreasedduring
duringboth
bothseasons
seasons(Figure
(Figure2).2).Indeed,
Indeed,itithas
hasbeen
been
Plants 2023, 12, 1882 14 of 21

previously described that ‘Heritage’ shows a sharp decline in fruit firmness throughout the
ripening period compared to ‘Santa Clara’, ‘Santa Catalina’, and ‘Santa Teresa’ cultivars [32].
Ethylene has also been reported to be involved in some critical processes of non-
climacteric fruit ripening, e.g., coloration in grape [33], lignin accumulation in straw-
berry [34], and softening and detachment of raspberry drupelets [9,35–37]. In grape, the
ACC content reached 1000-fold higher levels than ethylene production, suggesting that
ACC biosynthesis was not limiting [33]. In raspberry, enhanced ethylene production
during ripening at various CO2 concentrations has been observed in the same location
during different seasons [5,9,11] with a partial delay in the decrease of firmness during the
postharvest treatment with the ethylene receptor inhibitor 1-methyl cyclopropene (1-MCP,
1600 ppb), with no effects on titratable acidity and total soluble solids [9]. However, in the
present study, ACC production was not correlated with the quality parameters (Figure 4).
This result observed during the preharvest together with the partial regulation of firmness
by ethylene during the postharvest season [9] suggest the involvement of other hormones
in the Heritage cultivar and possibly also in other raspberry varieties in general.
In non-climacteric fruit, ABA plays a crucial role during ripening [18,38]. Previously,
ABA was reported to be involved in the ripening of strawberry fruit [17,18,39] and has been
described as the principal regulator of the onset of ripening of the grape berry, including
softening, sugar accumulation, and coloration [40–43]. In both fruits, the ABA content has
been described as low during the early fruit development stages and high at the ripe fruit
stages [14,15]. In strawberry, ABA treatment was reported to accelerate fruit softening and
increase color and ethylene production [17].
Previous reports in raspberry show that ABA accumulation reaches the maximum
level at the ripe stage (26,920 ng/g), with the complete correspondence of a high expression
of RiNCED1 [12]. In the present study, the ABA content was near 7000 ng/g DW in the
drupelets at the R stage and was higher in the receptacle at the same stage (between 10,000
to 25,000 ng/g DW depending on the harvest seasons). Similar to the results reported by
Yang et al., 2020 [12], our results showed that the ABA content in drupelets is maintained or
decreased in the OR stage compared to the content in ripe fruits. On the contrary, the ABA
content in the receptacle continued increasing at the OR stage in both seasons (Figure 3). It
has been shown that postharvest application of exogenous ABA (1 mM) at the large green
stage promotes ABA biosynthesis, the accumulation of the soluble sugar and anthocyanin
contents, softening of fruit, and chlorophyll loss in the raspberry fruit [12]. The same
authors [12] showed that fluoridone, an ABA biosynthesis inhibitor, diminished the ABA
content, and consequently, delayed fruit softening, and reduced the chlorophyll content in
raspberry fruit. In this study, the correlation matrix shows a positive correlation between
the TSS and ABA content, a negative correlation between firmness and the ABA content,
as well as and between the TA and ABA content in both seasons (Figure 4). The previous
reports of Yang [12] and our results imply that ABA played an important role as a regulator
of raspberry ripening. However, treatments that inhibit ABA biosynthesis and signaling
should be carried out in fruit attached to the plant to accurately determine the role of
this hormone and its possible relationship with other compensatory mechanisms during
fruit development.
Regarding the ABA metabolism, the NCED gene encodes an enzyme that catalyzes the
conversion of violaxanthin or neoxanthin into xanthoxin and is a key regulatory component
for the ABA biosynthesis pathway, being widely reported during the ripening of climacteric
and non-climacteric fruit [15,19–21,44]. Although three NCED genes have been identified in
strawberry (i.e., FaNCED1, FaNCED2, and FaNCED3), FaNCED1 genes are closely related to
ABA biosynthesis and the strawberry ripening process [15,19]. In this study, three genes of
the RiNCED family (Figure 5; Table S1) were identified based on the genomic information
of Rubus idaeus [28]. The RiNCED family exhibits a high degree of conservation according to
their gene structure and the motif profile of the deduced proteins (Figure 5). The promoter
analysis of RiNCED genes shows the presence of 15 ABA cis-elements for RiNCED3, 9 for
RiNCED1, and 6 for RiNCED2. Also, the three promoter sequences present cis-elements in
Plants 2023, 12, 1882 15 of 21

response to auxin, jasmonates, and salicylic acid. Only the promoter of RiNCED1 shows cis-
elements that respond to ethylene; conversely, RiNCED2 and RiNCED3 promoters showed
no elements of the ethylene response (Figure 7). Although the analysis of the promoters in-
dicates a potential regulation of NCED expression by other phytohormones, further studies
must be carried out to demonstrate this relationship between both hormones in raspberry.
In peach (Prunus persica), a yeast one-hybrid assay suggested that the nuclear-localized
PpERF3 might bind to the promoters of PpNCED2/3 [44]. Also, promoter-GUS assays and
transient expression analyses of PpERF3 increased the expression of PpNCED2/3. Both
results suggested that ethylene promotes ABA biosynthesis by regulating the expression of
PpNCED2/3 mediated by PpERF3 [44]. In contrast, the jasmonate signaling pathway could
have an antagonistic effect on ABA biosynthesis in strawberry (Fragaria × ananassa), since
it has been reported that the ABA content decreased together with the downregulation of
FaNCED1 after methyl jasmonate (MeJA) treatment for five days in strawberry fruit [45].
In this study, the phylogenetic analysis showed that the RiNCED gene family com-
prised three subfamilies—group I, II, and V—of the five NCED groups described in plants
(Figure 8). RiNCED2 was the deduced protein sequence with a major homology to the
other NCED described in fruits, showing a 100% similarity to FaNCED1 (ADU85829.1)
described in F. × ananassa and 89% to MdNCED1 (AGQ03804.1) from M. domestica.
Regarding ABA signaling, the PYL ABA receptor gene family has been described in
fruits such as tomato [24], strawberry [25], and grape [26]. However, no information has
been reported in raspberry until now. In this study, the genomic information shows a total of
nine RiPYL members (Figure 6; Table S2) with differences in the exon and intron distribution
and content (Figure 6A). Likewise, nine members of FaPYL in F. × ananassa [25] have been
described. Although differences were observed in the presence of introns between the nine
copies of the RiPYL genes, the deduced proteins showed that six conserved motifs were
similarly distributed (Figure 6B), indicating the low variability in the final protein sequence
of this gene family. In apple, 13 PYL genes have been identified classified into four groups
according to the structural features of the amino acid residue sequence and they contained
at least three cis-elements related to the hormonal response and stress in their promoter
regions [46]. For example, the MdPYL7 promoter had cis-elements, including abscisic
acid-responsive elements (ABRE), salicylic acid-responsive elements (TCA-element), and
MeJA-responsive elements (CGTCA-motif, TGACG-motif). The MdPYL13 promoter region
also contained ethylene-responsive elements (ERE) [46]. Our phylogenetic analysis showed
that RiPYL genes were distributed within the three groups described in plants (Figure 9),
and most of the deduced proteins have a similarity of over 67%, with the FvPYL sequences
described in F. vesca. The main similarity (99%) was between the RiPYL2 and FvPYL2
sequences, and between the RiPYL5 and FvPYL13a sequences. In F. × ananassa, FaPYL2
showed the highest transcript level, and its transcript abundance increased rapidly at the
onset of red coloration [25]. However, a minor similarity (37%) was observed between the
RiPYL7 and MdPYL3/9 sequences described in apple (Figure 9). In this fruit, the MdPYL9
gene was significantly induced by drought treatment, and its over-expression conferred
enhanced tolerance to drought stress in transgenic apple plants [23].
In this study, most of the identified genes in the transcriptomic data have a higher
expression at the pink stage (Figure 10). The RT-qPCR analysis showed that the expression
of the RiNCED1, RiPYL1, and RiPYL8 transcripts increased during development, with the
highest expression levels at the R and OR stages (Figure 11). In grape and peach, VvNCED1
and PpNCED1 were highly expressed at the onset of the ripening of the fruit, preceding the
ABA accumulation, and were particularly necessary for the increase in ethylene production
during the ripening of peach [47]. The respective decrease and increase of the RiNCED1
expression after fluoridone and ABA application [12], together with our results, suggest
that ABA biosynthesis is necessary for the onset of the ripening and persists until the
senescence stages in the receptacle and drupelet tissues.
ABA signaling has been little studied in relation to fruit ripening thus far. In this study,
the expression of both genes related to ABA signaling, RiPYL1, and RiPYL8, increased
Plants 2023, 12, 1882 16 of 21

during ripening in both tissues and seasons, showing marked differences in drupelets
(Figure 11). In strawberry, it has been shown that during the de-greening from the large
green to white stages, the transcript levels of FaPYL2/4/8/9 decreased rapidly and increased
during coloration from the pink to red stages [25].
Our correlation analyses obtained with the data from two seasons indicated that the
feedback of ABA on the expression of the RiNCED1, RiPYL1, and RiPPYL8 genes could
depend on the season (Figure 12); however, it is important to highlight that the expression
of RiNCED1 maintains a positive correlation with the ABA content in the receptacle during
both seasons, as well as the expression of RiPYL8 in drupelets.

4. Materials and Methods

4.1. Plant Material
The raspberry (R. idaeus ‘Heritage’) fruit was collected from commercial orchards
located in Casa Blanca (33◦ 200 3900 S; 71◦ 220 1100 W; 247 masl), Chile, during two harvest
seasons in 2020 and 2021. Fruit attached to the receptacle and with peduncle were harvested
and sorted by size and color as large green (LG), white (W), pink (P), red (R), and overripe
fruit (OR) [9,27]. Immediately after harvest, half of the collected fruit was frozen in liquid
nitrogen for ABA and ACC determination, and RNA isolation. The other half was used to
determine the quality and physiological parameters.

4.2. Fruit Quality and Physiological Measurements

Five independent replicates of 15 intact fruit attached to the receptacle per develop-
mental stage were used for quality assessments. The firmness of the fruit was determined
using the Firm Tech II equipment (BioWorks Inc., Wamego, KS, USA) and expressed as
Newton (N) [9]. Eight grams of fruit tissue (drupelets) from each replicate were homoge-
nized in a mortar. The juice was analyzed for total soluble solids (TSS) using a refractometer
(ATAGO, Tokyo, Japan), expressed as ◦ Brix, and the titratable acidity (TA) was expressed
as the percentage of citric acid per 100 g of the fresh weight (FW) of fruit.

4.3. Determination of ABA and ACC Contents

The ABA and ACC contents during the ripening of raspberry were determined accord-
ing to Müller and Munné-Bosh (2011) [48]. First, fruit with a peduncle was collected and
immediately frozen in liquid nitrogen. Then, eight independent samples of four fruit were
freeze-dried, drupelets and receptacles were separated, and each tissue was powdered in a
mortar. Then, 100 mg of samples were extracted after adding internal standards. d6-ABA
and d4-ACC were added as the internal standards. After centrifugation (10,000 rpm for
15 min at 4 ◦ C), the supernatant was collected, the pellet was reextracted with 0.5 mL of
the extraction solvent, and the extraction was repeated three times. Then, supernatants
were combined and dried under a nitrogen stream, re-dissolved in 300 µL of methanol, cen-
trifuged (10,000 rpm for 5 min), and filtered through a 0.22 µm PTFE filter (Waters, Milford,
MA, USA). ABA and ACC were determined by UPLC/ESI-MS/ MS. The quantification was
carried out by creating a calibration curve including unlabeled analyte compounds (ABA
and ACC standards) ranging from 0.05 to 1000 ng ml−1 . Calibration curves for each analyte
were generated using the Analyst™ software (Applied Biosystems, Inc., California, USA).

4.4. Identification of RiNCED and RiPYL Genes in the Raspberry Genome

The RiNCED and RiPYL genes were identified in the draft genome published by
Wight et al., 2019 [28] using the Geneious Prime software [49]. Sequences of raspberry
RiNCED and RiPYL genes previously identified in the transcriptomes published by Travisany et al.,
2019 [13] and Wight et al., 2019 [28] were used as query sequences for a Blast search.
The obtained genomic sequences were analyzed with the Augustus server [50] for gene
prediction, intron-exon structure, coding sequence, and protein sequence.
Plants 2023, 12, 1882 17 of 21

4.5. Gene Structure and Promoter Analysis of RiNCED and RiPYL Genes
The intron-exon structure of the RiNCED and RiPYL genes was displayed by the Gene
Structure Display Server 2.0 [51,52]
Promoter analysis was performed using sequences of 2000 bp upstream of the tran-
scriptional start site obtained from the draft genome and analyzing them with the Plant-
CARE software [53] for the identification of the cis-elements related to the phytohormone response.

4.6. Characterization of RiNCED- and RiPYL-Deduced Protein Sequences

The phylogenetic analysis of RiNCED- and RiPYL-deduced proteins was carried out
using the Maximum Likelihood Method with a bootstrap value of 5000 replicates with the
MEGA-X software [54] using the deduced protein of other plant species (Tables S3 and S4).
The conserved motifs were identified by Multiple EM for the Motif Elicitation tool (MEME
version 5.4.1) [55,56].

4.7. Identification of the Differentially Expressed RiNCED and RiPYL Genes in the
Transcriptomic Data
To identify the differentially expressed RiNCED and RiPYL genes in the flower, green,
and pink fruit stages, a search was realized on a database of differentially expressed
genes (DEGs) “Rubus idaeus Heritage transcripts v1.0” [13] available in the Genome Data
Base for Rosaceae [29]. For the search, a Blastn was performed on the Genome Data
Base for Rosaceae page [57], and the coding sequences (CDS) of the RiNCED and RiPYL
genes were used as queries. Then, the values of fragments per kilobase of transcript per
million mapped fragments (FPKM) were determined for each identified sequence. These
values were expressed as log10 FPKM+1 to plot a heatmap later using the CummeRbund R
package [58].

4.8. RNA Isolation and Reverse Transcription Quatitative PCR (RT-qPCR) Analysis
Five fruits were powdered with liquid nitrogen in a mortal and 100 mg of powder was
used for the total RNA isolation from drupelets, and receptacle of each ripening stage using
the RNAqueous® Kit (ThermoFisher Scientific Inc. Waltham, MA, USA). DNase treatment
was performed using DNase I (Thermo Fisher Scientific Inc.). The first-strand cDNA was
generated using High-Capacity cDNA Reverse Transcription Kits (ThermoFisher Scientific
Inc.). Five individual extractions for each sampling were performed. Specific forward and
reverse primers were designed with high stringency using Primer3Plus [59]. The primers
for the housekeeping and candidate genes are detailed in Table S5. The expression of the
candidate genes was carried out using the KAPA SYBR® FAST qPCR Kit (Kapa Biosystems,
Wilmington, MA, USA) in the PikoReal® Real-Time PCR System (ThermoFisher Scientific
Inc.) according to Delgado et al., 2018 [60] with minor modifications. Each reaction was
performed in triplicate and normalized against the expression level of the Ri18S gene [13];
additionally, negative water control was included. The results were calculated using the
2−∆∆CT method [61] using fruit at the LG stage as the calibrator sample and assigned a
nominal value of 1.

4.9. Statistical Analysis

The data for each season were analyzed using the R Statistical Software [62]. For the
ABA and ACC content, firmness of TSS and TA, and relative expression, normality was
corroborated using the Shapiro-Wilk normality test. Based on this, it was decided which
comparison test should be used: the non-parametric Wilcoxon rank sum for one-sampled
data and Mann-Whitney for two-sampled data; otherwise the t-test was used. The resultant
p-values were adjusted using Bonferroni’s method. Also, the principal component analysis
was used to analyze the grouped data by year. The ABA and ACC content, firmness of TSS
and TA, and the relative expression values obtained during development were correlated
using the Kendall method. The data were visualized using the R package ggstatsplot [63]
and rstatix [64].
Plants 2023, 12, 1882 18 of 21

5. Conclusions
In this study, the ABA content showed a higher increase during the ripening of
raspberry, including the receptacle and drupelet tissues, during two seasons. Furthermore,
this increase was correlated with the change in the fruits’ quality parameters (i.e., firmness
lost, increase of soluble solids). In addition, we identified and characterized the main
ABA biosynthesis (RiNCED)- and ABA receptor (RiPYL)-genes, which showed a positive
correlation between their expression levels and the progress of the ripening of the raspberry
and the potential differential expression between fruit tissues. Further studies, including
the functional characterization of these key ABA pathway-related genes, could improve
the understanding of the role of this hormone in the raspberry fruit and the differences in
ABA signaling between drupelets and receptacles.

Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/plants12091882/s1. Table S1: RiNCED gene family identified
in Rubus idaeus genome; Table S2: RiPYL gene family identified in Rubus idaeus genome; Table S3:
Information about NCED protein sequences used for phylogenetic analysis; Table S4: Information
about PYL protein sequences used for phylogenetic analysis; Table S5: Information of qPCR primers.
Author Contributions: Conceptualization, L.F.; methodology, F.Á., C.R.-M., M.M., P.M., P.E.Z.,
K.J.-C., N.E.F., M.V. and L.F.; software, C.R.-M., P.E.Z., K.J.-C., A.A.-R. and L.F.; validation, C.R.-M.,
P.E.Z., K.J.-C., L.F. and A.A.-R.; formal analysis, C.R.-M., P.E.Z., K.J.-C., P.M., S.M.-B., L.F. and A.A.-R.;
investigation, C.R.-M., P.E.Z., K.J.-C. and L.F.; resources, L.F., C.R.F., M.V., S.M.-B., W.S., J.E.A., B.G.D.
and A.A.-R.; data curation, P.M., S.M.-B., C.R.-M., P.E.Z., K.J.-C., L.F. and A.A.-R.; writing—original
draft preparation L.F; writing—review and editing, L.F., J.E.A., S.M.-B., C.R.-M., P.E.Z., K.J.-C., A.A.-R.
and C.R.F.; visualization, C.R.-M., P.E.Z., K.J.-C., L.F. and A.A.-R.; supervision, L.F. and C.R.F.; project
administration, L.F.; funding acquisition, L.F. and S.M.-B. All authors have read and agreed to the
published version of the manuscript.
Funding: This research was funded by the FONDECYT 1201662 grant from ANID, Chile; S.M.-B.
laboratory was supported by ICREA Academia and 2021 SGR 00675 grants from Generalitat de
Catalunya and by CEX2021-001234-M support from Spanish Government.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: National Research and Development Agency (ANID, Chile)—Millennium Sci-
ence Initiative Program—NCN2021_010 and FONDECYT/Regular 1210941 to C.R.F.; NATIONAL
DOCTORATE Scholarship/2020—21201520, and 21201418; NATIONAL DOCTORATE/2019–21190862
to K.J.-C.
Conflicts of Interest: The authors declare no conflict of interest.

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