Available online at www.sciencedirect.
com
Targeting ripening regulators to develop fruit with high
quality and extended shelf life
Jaclyn A Adaskaveg and Barbara Blanco-Ulate
Fruit quality directly impacts fruit marketability and consumer
acceptance. Breeders have focused on fruit quality traits to
extend shelf life, primarily through fruit texture, but, in some
cases, have neglected other qualities such as flavor and
nutrition. In recent years, integrative biotechnology and
consumer-minded approaches have surfaced, aiding in the
development of flavorful, long-lasting fruit. Here, we discussed
how specific transcription factors and hormones involved in
fruit ripening can be targeted to generate high-quality fruit
through traditional breeding and bioengineering. We highlight
regulators that can be used to generate novel-colored fruit or
biofortify fresh produce with health-promoting nutrients, such
as vitamin C. Overall, we argue that addressing grower and
industry needs must be balanced with consumer-based traits.
Address
Department of Plant Sciences, University of California, Davis, Davis,
CA, USA
Corresponding author: Blanco-Ulate, Barbara (bblanco@ucdavis.edu)
Current Opinion in Biotechnology 2023, 79:102872
This review comes from a themed issue on Food Biotechnology
Edited by Max I. Teplitski and Jorge M. Fonseca
For complete overview of the section, please refer to the article
collection, “Food Biotechnology (2023)”
Available online 6 January 2023
https://doi.org/10.1016/j.copbio.2022.102872
0958-1669/© 2022 The Authors. Published by Elsevier Ltd. This is an
open access article under the CC BY license (http://
creativecommons.org/licenses/by/4.0/).
Introduction
Fruit are rich in essential nutrients, yet most people do
not consume the recommended amount of fresh produce
to sustain healthy diets and reduce disease risks (US
Department of Agriculture (USDA) ERS — Food
Availability and Consumption, URL: https://www.ers.
usda.gov). To promote consumption, expand access, and
reduce waste, fruit quality and shelf life need to be in­
creased through breeding and biotechnology, alongside
adequate harvest practices, transportation logistics, and
postharvest treatments. Commercial fruit crops are pri­
marily bred for high yield and extended shelf life to
meet the expectations for mass production and global
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markets, however, recently, there has been a shift of
focus toward developing new crop varieties that meet
consumer demands for better flavor and nutrition.
Balancing shelf life with consumer-based quality traits is
perhaps the biggest challenge breeders and researchers
face in the quest for better quality fruit, mainly because
these attributes appear to have negative genetic corre­
lations in many crops [1,2].
Quality (i.e. color, nutritional value, flavor, and texture)
peaks when fruit reach their optimum ripeness.
Many studies on fruit ripening of various plant species
have emerged in the past decade, helping to identify
genetic pathways and molecular regulators that can be
manipulated for crop improvement (Fig. 1). Moreover,
biotechnology advances have provided access to high-
quality genomic resources and tools, supporting
breeding strategies, genetic modification, and gene
editing in traditional and nontraditional fruit crops.
Here, we review current knowledge of the genetics of
fruit traits and argue that manipulating transcription
factors (TFs) is a promising approach to enhance fruit
quality. We discuss how pleiotropic effects could po­
tentially be avoided by targeting TFs that exclusively
regulate specific pathways (i.e. function-specific reg­
ulators) instead of master regulators. However, ripening
master regulators may remain useful if their effects on
gene expression can be fine-tuned (e.g. creating alleles
with different impact on gene expression). Similarly, the
timing and coordination of regulators need to be con­
sidered (e.g. using tissue- and stage-specific promoters)
to achieve desired effects on fruit traits [3]. Finally, we
consider the current climate surrounding consumer ac­
ceptance of genetically modified (GM) and gene-edited
fruit.
Increasing fruit pigmentation for visual appeal
Fruit color is a significant indicator of ripeness and nu­
tritional quality. It is often used as a harvest maturity
index and can predict shelf-life potential in certain crops
[8]. Fruit color is dependent on pigments that accumu­
late in the exocarp (skin) and mesocarp (flesh). As fruit
ripen, chlorophyll (green) is replaced by carotenoids
(yellow, orange, and red), anthocyanins (orange, red,
and blue), or betalains (yellow, violet) (Fig. 2). Fruit
gloss, influenced by the structure and composition of the
cuticle layer, also affects color perception [9].
Current Opinion in Biotechnology 2023, 79:102872
2 Food Biotechnology

pigment metabolism can be meticulous or have unin­

Text Box
Fruit quality and shelf-life attributes are inherently connected. Fruit
quality is defined by diverse attributes that give a product value,
including color, nutritional value, flavor, and texture, and requires
fruit to be undamaged and without signs of decay. Shelf life is a
multifaceted trait incorporating sensory attributes that make the fruit
look fresh and edible through time, such as firmness, moisture loss,
gloss, flavor life, and susceptibility to disease.
There have been multiple efforts to enhance pigmen­
tation in fruit crops by modulating genes in pigment
biosynthesis pathways [33,34]. Pink-flesh pineapples
have been engineered through the introduction of a
phytoene synthase gene (CrPSY) from tangerine in
combination with suppression via RNAi of two en­
dogenous lycopene genes (AcLYC-B and AcLYC-E) to
inhibit the conversion of lycopene into other carotenoids
[10]. This pineapple, known as Pinkglow™ is one of the
few bioengineered fruits available in the United States.
Yet, targeting or introducing single genes involved in
tended consequences on other metabolites. For ex­
ample, the overexpression of SlLYC-B or SlZDS in
tomato fruit increased carotenoid content but also in­
creased the production of abscisic acid (ABA), a deriva­
tive of this pathway, which affected the fruit’s normal
ripening progression [35,36].
A more effective means to manipulate pigment content
in fruit might be dissecting function-specific regulators,
particularly those that directly control the biosynthesis
pathway and act downstream of ripening regulatory
networks. This is the case of R2R3-MYB TFs involved
in regulating anthocyanin biosynthesis as first demon­
strated in apple and grape [37–39]. Wild and cultivated
tomatoes are red because of a mutation in the R2R3-
MYB TF SlAN2-like that suppresses anthocyanin bio­
synthesis. However, overexpressing the functional gene
in fruit turned on the pathway and produced antho­
cyanin-rich purple-fleshed tomatoes [11]. Purple toma­
toes with high anthocyanin content have also been
generated by introducing two TFs from snapdragon, the

Figure 1

Current Opinion in Biotechnology

Regulators of fruit ripening. Fruit ripening is a complex developmental program that determines the quality and shelf life of fresh produce [4]. The
activation and progression of ripening processes (pink box) are tightly controlled by multiple regulatory levels (orange box), which in turn are influenced
by environmental stimuli (brown box). Specific ripening regulators, such as plant hormones and signals (e.g. ethylene, ABA, sucrose, or NO2), may
have different roles depending on the fruit crop and whether the fruit is climacteric (e.g. tomato) or nonclimacteric (e.g. strawberry) [5,6]. TFs are central
in the control of ripening and generally act upstream of plant hormones (e.g. MYB; bHLH; NAM, ATAF and CUC, NAC; MCM1, AGAMOUS,
DEFICIENS, SRF, MADS; ethylene response factors, ERF). Ripening regulatory mechanisms involving noncoding RNAs (small RNA, sRNA; microRNA,
miRNA; long noncoding RNA, lncRNA) and epigenetic factors appear to be conserved across multiple plant species [7]. Ripening genes generally
encode enzymes in biochemical pathways that influence fruit quality traits. For example, pigment biosynthesis genes (e.g. phytoene synthase, PSY;
lycopene beta-cyclase, LYC-b), genes involved in the production of vitamins and antioxidants (e.g. vitamin-C biosynthesis gene L-galactono-1,4-
lactone dehydrogenase, GalLDH), genes encoding CWDEs (e.g. PL, PG, PME), genes involved in starch degradation (e.g. amylase, AMY) and sugar
transport (e.g. sucrose transport protein, SUC), and genes related with the production of aromatic VOC (e.g. alcohol acetyl transferase, AAT;
lipoxygenase, LOX).

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 Modulating ripening for high quality fruit Adaskaveg and Blanco-Ulate 3

Figure 2

Current Opinion in Biotechnology

Factors driving fruit quality traits and examples of ways to improve them. Fruit color, nutrition, flavor, and texture determine the overall quality and shelf
life of fruit. This table breaks up these categories of fruit quality (left colored blocks) into specific attributes. For example, color perception is comprised
of pigmentation and gloss of a fruit. The attributes are further defined by characteristic structures or metabolites. Examples of loci (genes and QTLs)
that can act as potential breeding or bioengineering targets are included. These targets are listed with their associated fruit crop and reference. The
orange font represents genes that are TF regulators, while the pink font represents ripening genes part of biochemical pathways. [10–30,5,31,32].

R2R3-MYB TF AmRos1 and the basic helix loop helix
(bHLH) TF AmDel under a fruit-specific promoter [40].
R2R3-MYBs also appear to be critical for the control of
betalain biosynthesis, uncovered through the recent as­
sembly of the pitaya genome [41]. A pitaya R2R3-MYB
TF (HuMYB1) involved in regulation was recently
identified and followed a very similar activation me­
chanism as with anthocyanins [12]. Because R2R3-MYB
TFs exist in diverse plant species, this class of TFs
could be easily edited with clustered regularly inter­
spaced short palindromic repeat (CRISPR) technologies
to engineer new fruit colors, for example, novel ‘white’
fruit such as the wild strawberry species Fragaria nil­
gerrensis, carrying a mutation in the FvMYB10 TF [42].
Pigmentation can also be controlled at higher-level reg­
ulatory points, but this usually comes with consequences
to other quality traits. For instance, exogenous applica­
tions of the hormone ABA induce anthocyanin

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4 Food Biotechnology
biosynthesis in table grapes, positively influencing color,
but negatively affected berry texture [43].
Biofortifying fruit to provide a reliable dietary
source of nutrients
Fruit are used as food source for macronutrients (e.g.
fiber, sugars, and lipids) and micronutrients (e.g. vita­
mins, minerals, and antioxidants) (Fig. 2). Macro­
nutrients are essential to provide energy and maintain
the body’s structure and functions. Vitamins are required
for various bodily functions and can only be found in
food sources such as fruit. Antioxidants inhibit cell da­
mage caused by oxidative agents. Carotenoids (pro-vi­
tamin A), and phenolics (anthocyanins and betalains),
reviewed above, are all antioxidants, as well as vitamin C
(ascorbic acid).
Vitamin-D deficiency is a global health problem due to
few dietary sources of this vitamin. Biofortification of
vitamin D in tomatoes has recently become possible by
engineering its biosynthesis from a pre-existing pathway
[13]. Owing to partial duplication of the pathway, a
single enzyme could be knocked out with
CRISPR–Cas9 to convert the precursor into vitamin D
without an expense to other metabolites. This discovery
has further implications for other Solanaceae plants.
Ascorbic acid, an important antioxidant and nutrient
for immune health and wound healing, has proven to
be less easily biofortified into fruit because increasing
biosynthesis also leads to activation of catabolic and
recycling pathways [44]. Post-transcriptional regula­
tion of an ascorbic acid biosynthesis enzyme from ki­
wifruit has been demonstrated using tobacco leaves
[14]. Removing the upstream open-reading frame
(uORF) that repressed translation increased ascorbic
acid concentration in the leaves. Function-specific
TFs can increase ascorbic acid in fruit without nega­
tive impact on quality as demonstrated in tomato.
SlHZ24, a bHLH TF, regulates ascorbic acid bio­
synthesis and catabolism genes, and its transient
overexpression has been reported to increase the ac­
cumulation of this vitamin [45]. Other TFs, SlNL33
and SlNFYA10, have been found to regulate the
pathway negatively, and silencing them also increased
ascorbic acid level [15,46]. Understanding regulatory
pathways governing nutrients can also facilitate tradi­
tional breeding programs. For example, a genome-
wide association study (GWAS) led to the discovery
and validation that alleles in SlbHLH59 determine
ascorbic acid content in tomato cultivars [16].
A less specific effect on nutrient accumulation can be
achieved with hormone applications. For example,
ethylene application in kiwifruit, a climacteric fruit, in­
creased ascorbic acid and other antioxidants during
Current Opinion in Biotechnology 2023, 79:102872
ripening [47]. Ascorbic acid also increased after nitric
oxide (NO2) application in sweet pepper, a non­
climacteric fruit [48].
Enhancing flavor to satisfy consumer
expectations
Fruit flavor is a critical quality trait for consumer ac­
ceptance. Flavor includes all sensations experienced
when eating, consisting of taste, aroma, and texture
(Fig. 2). In fruit, taste is mainly defined by a balance
between sweetness and acidity but can include bitter­
ness and umami. Fruit aroma comes from specific classes
and combinations of volatile organic compounds (VOCs).
For example, the unique kiwifruit flavor is associated
with esters, mainly ethyl butanoate and methyl bu­
tanoate [17].
Modulation of function-specific transcriptional and post-
transcriptional regulators offers an effective solution for
flavor improvement. A bHLH TF in banana
(MabHLH6) activates 11 starch-degrading genes ex­
pressed during fruit ripening and is a likely candidate for
increasing sugar and sweetness [18]. In strawberry,
editing the uORF of a bZIP TF that controls sucrose
biosynthesis led to its translational activation and higher
sugar content in fruit [19].
Combining datasets generated through genomic, tran­
scriptomic, metabolomic, and consumer panel studies
has proven to be an effective strategy for identifying
flavor-related genes and TFs. In tomato, metabolite
data, associated loci, and consumer panels were analyzed
across hundreds of varieties to determine key genes
contributing to flavor [20]. Coupling metabolites re­
levant to flavor with transcriptomic analyses can produce
gene networks and identify regulatory TFs involved in
flavor pathways. This approach was used in kiwifruit,
where the AcNAC4 TF regulating a key gene in ester
biosynthesis (AcAAT10) was validated [17,21]. Homologs
of these NAC TFs have been implicated in ester for­
mation in peach (PpNAC1) and apple (MdNAC5), sug­
gesting that their functions are conserved across diverse
families of climacteric fruit [22].
Flavor is impacted by postharvest handling. Chilling
during transportation and storage can alter fruit flavor.
Epigenetic factors (i.e. DNA methylation) have been
shown to regulate the suppression of VOC biosynthesis
in chilled tomatoes [49]. Most climacteric fruit are
picked unripe (but mature) and later treated with ethy­
lene to induce ripening. However, this practice has been
associated with poor flavor development. A study in off-
vine ripened tomatoes confirmed that the fruit presented
reduced VOC emission and a low sugar/acid ratio, due to
alterations in gene expression and decreased methyla­
tion of their promoters [50].
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 Modulating ripening for high quality fruit Adaskaveg and Blanco-Ulate 5
Genomic resources can help anticipate consumer pre­
ference and assist in breeding to select fruit with en­
hanced flavor. A population genomic study revealed that
distinct consumer preferences between eastern and
western countries drove selection for peach cultivars
with different acidity [51]. Fruit VOC profile data can
help predict consumer liking before performing sensory
panels, allowing for a more efficient selection of high-
flavor fruit, as seen in tomato and blueberry [52].
Moreover, the tomato pan-genome helped identify a rare
favorable allele selected against during domestication
that could be incorporated back into new cultivars to
improve fruit flavor [53].
Modifying fruit texture for longer shelf life
Fruit texture is associated with freshness, flavor, and
shelf-life potential. Texture involves many attributes,
such as firmness, juiciness, crispiness, and meltiness
(Fig. 2). Loss of firmness (i.e. fruit softening) is a hall­
mark of fruit ripening and negatively correlates with
shelf life. Fruit softening is mainly attributed to the re­
modeling and degradation of the polysaccharides in the
primary cell walls (CWs). The cuticle layer, deposited on
the CWs of epidermal tissue, also contributes to fruit
firmness by preventing water loss and maintaining cell
turgor pressure [9].
Traditional breeding has focused on creating firmer
fruit that withstand transportation and have longer
shelf life. This has been accomplished in tomato by
developing hybrid lines between elite varieties and
nonripening mutants such as ripening-inhibitor (rin) and
non-ripening (nor) [54]. These mutants have defects in
TFs considered master regulators of many ripening
processes, including the induction of cell wall-de­
grading enzymes (CWDEs) and changes in cuticle
composition [23,55]. Other efforts to improve firmness
and shelf life in climacteric fruit have taken advantage
of mutations affecting ethylene biosynthesis and per­
ception, as this hormone is also known to regulate
genes encoding CWDEs, among others [56,57].
However, as already discussed, modulating master
regulators or hormone pathways has numerous draw­
backs to other quality traits, such as color and flavor.
Thus, downstream TFs controlling specific CW en­
zymes, such as LOB TF (SlLOB1) in tomato, may
prove to be better targets [24]. Another possibility is to
leverage the availability of natural or induced allelic
variants in ripening master regulators to produce a
range of fruit phenotypes. For example, spontaneous
(i.e. alcobaca (alc) or equivalent delayed fruit dete­
rioration (dfd)) and CRISPR–Cas9-generated muta­
tions in the ripening regulator NOR can extend shelf
life with minimal impact on other fruit attributes
[25–27], compared with the canonical mutant nor (a
gain-of-function mutation) [23,58].
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RNAi and gene editing approaches have been used to
target ripening-specific CWDEs that influence fruit
firmness, such as polygalacturonase (PG), pectate lyase
(PL), and pectin methyl esterase (PME) [59]. The first
GM fruit product, the FLAVR SAVR™ tomato, was
engineered with antisense RNA against SlPG, however,
it did not show a phenotype for fruit firmness [60]. In
contrast, the CRISPR–Cas9 SlPL knockout in tomato
significantly improved fruit firmness and shelf life [28].
Similar observations were previously reported in straw­
berry using RNAi knockdowns of FvPL and FvPME
[29,61]. In addition to firmer fruit, tomato and strawberry
SlPL mutants have reduced fruit susceptibility to fungal
disease [61,62].
Breeding strategies and molecular studies have also fo­
cused on ameliorating textural defects such as fruit
mealiness caused by cold storage. Mealiness, considered
the opposite of juiciness, occurs when neighboring cells
lose adhesion and detach while remaining intact [63]. In
peach, quantitative trait loci (QTLs) associated with
cold-tolerant varieties have been identified to support
breeding for less mealy fruit [64]. Beyond QTLs, un­
derstanding the genetic mechanisms behind the trait
provides avenues for targeting breeding and genetic
modifications. For instance, peach mealiness appears to
be associated with increased DNA methylation, leading
to the downregulation and hypermethylation of mealy-
associated genes such as PpCYP82A3 [65]. Finding mo­
lecular approaches to avoid the deposition of methyl
groups in the promoters of key ripening genes in re­
sponse to cold storage can serve as a potential solution to
mealiness.
Looking ahead: toward the commercial
success of bioengineered fruit
The FLAVR SAVR™ tomato hit the market in the early
90s promising a product with longer shelf life. However,
this GM fruit had high production costs and was not well
accepted by consumers, which led to its removal from
the marketplace [30]. Since then, other bioengineered
crops with improved plant disease resistance or pro­
duction-related traits have become available worldwide
[66]. These products have not sparked much enthusiasm
mainly because they were not generated considering
consumer-based traits or due to public fear of GM or­
ganisms.
Recently, two fruits bioengineered for quality attributes
were approved by both US and Canadian regulatory
entities and are available to consumers. These are the
Pinkglow™ pineapple and the Arctic™ apple. The latter
was bioengineering to reduce oxidative browning in the
cut fruit [66]. These fruit are considered novelty items
because they are less available in the marketplace and
significantly more expensive than traditional cultivars.
Current Opinion in Biotechnology 2023, 79:102872
6 Food Biotechnology
Both fruits are primarily sold online and offered in lim­
ited supplies. The Pinkglow™ pineapple costs nearly
ten times more than a common yellow pineapple. De­
spite their limitations, these fruits were developed with
the consumer in mind, which may entice the public
more than previous GM products.
In 2022, the USDA deregulated the purple tomato de­
veloped with the expression of two snapdragon TFs
(discussed above, [40]). Novel fruit colors and potential
higher nutrition may draw consumers to a new emerging
category of bioengineered produce. These fruit will also
need to meet high consumer expectations of flavor, af­
fordability, and food safety to ensure their success. Gene
editing techniques such as CRISPR–Cas9 enable the
fine-tuning of quality traits in a variety of fruit crops and
may be more well received than previous GM products.
For example, Japan started selling the first Cas9-edited
fruit in the world in 2021, a health-promoting γ-amino­
butyric acid (GABA)-enriched tomato [67]. Gene-edited
fruit without foreign DNA have a more straightforward
regulatory path in the United States than GM products,
increasing the speed to market and reducing costs as­
sociated with authorization. However, this is not the case
in other parts of the world, such as the European Union.
In conclusion, researchers are armed with knowledge on
fruit ripening and tools to improve fruit quality and
generate greater access to fresh, flavorful, and nutritious
food. Demands for better-tasting, more sustainable fruit,
are in reach. We sit on the edge of an era where gene-
edited and bioengineered commodities can become a
new category in the market, if the consumer will allow it.
CRediT authorship contribution statement
Jaclyn A. Adaskaveg: Writing – original draft,
Visualization; Barbara Blanco-Ulate: Writing – review &
editing, Visualization, Supervision.
Conflict of interest statement
The authors declare that they have no known competing
financial interests or personal relationships that could
have appeared to influence the work reported in this
paper.
Data availability
No data were used for the research described in the ar­
ticle.
Acknowledgements
This work was supported by start-up funds from the College of Agriculture
and Environmental Sciences and the Department of Plant Sciences at
University of California, Davis (UC Davis) to BB-U. Funding for JAA was
partially provided by the Department of Plant Sciences Graduate Student
Researcher Award (UC Davis). We would like to acknowledge Amanda
Blanco-Ulate for creating the drawings in all figures. We thank Isabel B.
Current Opinion in Biotechnology 2023, 79:102872
Ortega-Salazar and Lara O’Shea-Farren for their feedback on the paper
narrative.
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