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Ethylene and Fruit Ripening


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The ripening of fleshy fruits represents the unique coordination of developmental and biochemical pathways leading to changes in color, texture, aroma, and nutritional quality of mature seed-bearing plant organs. The gaseous plant hormone ethylene plays a key regulatory role in ripening of many fruits, including some representing important contributors of nutrition and fiber to the diets of humans. Examples include banana, apple, pear, most stone fruits, melons, squash, and tomato. Molecular exploration of the role of ethylene in fruit ripening has led to the affirmation that mechanisms of ethylene perception and response defined in the model system Arabidopsis thaliana are largely conserved in fruit crop species, although sometimes with modifications in gene family size and regulation. Positional cloning of genes defined by ripening defect mutations in the model fruit system tomato have recently led to the identification of both novel components of ethylene signal transduction and unique transcription factor functions influencing ripening-related ethylene production. Here we summarize recent developments in the regulation of fruit ripening with an emphasis on the regulation of ethylene synthesis, perception, and response.
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Ethylene and Fruit Ripening
Cornelius S. Barry,
and James J. Giovannoni
Boyce Thompson Institute for Plant Research, Ithaca, New York 14853, USA;
United States Department of Agriculture – Agriculture
Research Service (USDA-ARS), Plant, Soil and Nutrition Laboratory, Cornell University Campus, Tower Road, Ithaca,
New York 14853, USA
The ripening of fleshy fruits represents the unique
coordination of developmental and biochemical
pathways leading to changes in color, texture, ar-
oma, and nutritional quality of mature seed-bearing
plant organs. The gaseous plant hormone ethylene
plays a key regulatory role in ripening of many
fruits, including some representing important con-
tributors of nutrition and fiber to the diets of hu-
mans. Examples include banana, apple, pear, most
stone fruits, melons, squash, and tomato. Molecular
exploration of the role of ethylene in fruit ripening
has led to the affirmation that mechanisms of eth-
ylene perception and response defined in the model
system Arabidopsis thaliana are largely conserved in
fruit crop species, although sometimes with modi-
fications in gene family size and regulation. Posi-
tional cloning of genes defined by ripening defect
mutations in the model fruit system tomato have
recently led to the identification of both novel
components of ethylene signal transduction and
unique transcription factor functions influencing
ripening-related ethylene production. Here we
summarize recent developments in the regulation of
fruit ripening with an emphasis on the regulation of
ethylene synthesis, perception, and response.
Key words: Ethylene; Fruit; Tomato; Ripening;
Climacteric; Signal transduction
Fruits of different plant species are highly diverse,
ranging from dry seed capsules that burst to allow
seed dispersal, to relatively large complex fleshy fruits
that have evolved bright colors and complex aromas
to attract seed-dispersing birds and animals. Fleshy
fruits in themselves are botanically diverse with some
such as tomato and grape being true berries derived
from the ovary and others such as strawberry, pine-
apple, and apple derived from the receptacle tissues
or from expansion of the sepals. Fleshy fruits also
come in a wide range of sizes, shapes, and colors, and
each species possesses its own very unique flavor
characteristics. Ripening programs can also be di-
verse. For example, avocado do not ripen until after
harvest, whereas the majority of studied fruits ripen
on the plant. Despite this great diversity, aspects of
the ripening of fleshy fruits are often conserved be-
tween species. For example, the onset of ripening is
often associated with color changes, altered sugar
metabolism, fruit softening and alterations in tex-
ture, the synthesis of aroma volatiles, and an in-
creased susceptibility to pathogen infection. These
common events suggest that the underlying genetic
mechanisms that regulate fruit ripening may well be
conserved between fruits of different species (Adams-
Phillips and others 2004a, b; Giovannoni 2004).
Received: 16 January 2007; accepted: 18 January 2007; Online publica-
tion: 6 June 2007
*Corresponding author; e-mail:
J Plant Growth Regul (2007) 26:143–159
DOI: 10.1007/s00344-007-9002-y
Tomato is the most genetically tractable plant
system for studying fruit ripening because it has
simple diploid genetics and a relatively short gener-
ation time and small habit compared to many other
fruit crop species that are either polyploids or trees.
The ripening phenotype is easy to score and there is a
large collection of germplasm resources, including
monogenic mutants with inhibited or altered ripen-
ing phenotypes (, There
is also a long history and a wealth of biochemical and
molecular data relative to the processes that are in-
volved during ripening, and a large platform of tools
for functional genomics is continually being devel-
oped, including an emerging genome sequence (Fei
and others 2006; Mueller and others 2005).
Differential screens, candidate gene analysis,
gene expression profiling, and digital gene expres-
sion analysis have led to the identification of hun-
dreds of genes whose expression profiles change
during the course of fruit development and ripening
(Alba and others 2005; Fei and others 2004; Picton
and others 1993a, b; Slater and others 1985; Ze-
gzouti and others 1999). Through a combination of
approaches many of the downstream components
that mediate the biochemical changes associated
with ripening have been defined. For example, cell
wall hydrolases, the enzymes involved in carotenoid
synthesis and sugar metabolism, and some of the
enzymes involved in the generation of flavor and
aroma compounds have been characterized (Chen
and others 2004a, b; Fridman and others 2004;
Hirschberg 2001; Rose and Bennett 1999; Tieman
and others 2006). The pathways that determine the
competency of a fruit to ripen or the signals that
initiate the ripening program are less well defined,
although the molecular identification of mutants
that are impaired in fruit ripening are beginning to
yield valuable insight into some of these genetic
pathways, and multiple hormones, including jasm-
onates, auxin, and brassinosteroids, have all been
implicated in the promotion of ripening in various
species (Fan and others 1998; Given and others
1988; Manning and others 2006; Symons and oth-
ers 2006; Vardhini and Rao 2002; Vrebalov and
others 2002). Signaling through the plant hormone
ethylene, however, remains the most well-defined
pathway that mediates the phenotypic changes that
occur during ripening. Treatment of various fruits
with inhibitors that block ethylene synthesis or ac-
tion or the manipulation of these processes by
transgenic or mutant approaches have revealed the
essential role of this hormone in regulating fruit
ripening (Hobson and others 1984; Klee and others
1991; Lanahan and others 1994; Oeller and others
1991; Picton and others 1993a, b; Yang and Hoff-
man 1984). In this review we summarize our cur-
rent understanding of ethylene biosynthesis and
signaling pathways in relation to fruit ripening.
Major emphasis is placed on knowledge obtained
using the tomato model system, although where
appropriate we highlight discoveries and novel
findings in other fruit crop species.
Fruits have classically been categorized based upon
their abilities to undergo a program of enhanced
ethylene production and an associated increase in
respiration rate at the onset of ripening. Fruits that
undergo this transition are referred to as climacteric
and include tomato, apple, peach, and banana,
whereas fruits that do not produce elevated levels of
ethylene are known as nonclimacteric and include
citrus, grape, and strawberry. However, these dis-
tinctions are not absolute, as closely related melon
and capsicum species can be both climacteric and
nonclimacteric (see below for further discussion)
and some so-called nonclimacteric fruits display
enhanced ripening phenotypes in response to
exogenous ethylene (see below for further discus-
sion). Nevertheless, increased ethylene synthesis at
the onset of ripening is required for the normal
ripening of many fruits.
Two systems of ethylene production have been
defined in plants. System 1 functions during normal
growth and development and during stress re-
sponses, whereas system 2 operates during floral
senescence and fruit ripening. System 1 is autoin-
hibitory, such that exogenous ethylene inhibits
synthesis, and inhibitors of ethylene action can
stimulate ethylene production (Figure 1). In con-
trast, system 2 is stimulated by ethylene and is
therefore autocatalytic, and inhibitors of ethylene
action inhibit ethylene production (McMurchie and
others 1972).
The biochemical features of the ethylene biosyn-
thesis pathway in higher plants are well defined and
have been reviewed previously (Bleecker and Kende
2000). Briefly, ethylene is synthesized from methi-
onine in three steps: (1) conversion of methionine to
S-adenosyl-L-methionine (SAM) catalyzed by the
enzyme SAM synthetase, (2) formation of 1-
aminocyclopropane-1-carboxylic acid (ACC) from
SAM via ACC synthase (ACS) activity, and (3) the
conversion of ACC to ethylene, which is catalyzed
by ACC oxidase (ACO). The formation of ACC also
leads to the production of 5¢-methylthioadenosine
144 Cornelius S. Barry and others
(MTA), which is recycled via the methionine cycle to
yield a new molecule of methionine. Increased res-
piration provides the ATP required for the methio-
nine cycle and can lead to high rates of ethylene
production without high levels of intracellular
methionine. SAM is an important methyl donor and
is involved in multiple aspects of cellular metabo-
lism. Consequently, the two committed steps in the
synthesis of ethylene are the formation of ACC and
its conversion to ethylene. The genes encoding ACS
and ACO have thus been studied in more detail than
other enzymes in the pathway, although there is
evidence that several other genes involved in
methionine synthesis and the methionine salvage
pathway are differentially expressed during ripening
and in response to ethylene (Alba and others 2005;
Zegzouti and others 1999).
ACS and ACO are encoded by multigene families
in higher plants, with tomato possessing at least
nine ACS (LEACS1A,LEACS1B, and LEACS2-8) and
five ACO (LEACO1-5) genes (Barry and others 1996;
Nakatsuka and others 1998; Oetiker and others
1997; Van-der-Hoeven and others 2002; Zarem-
binski and Theologis 1994). Expression analysis has
revealed that at least four ACS (LEACS1A,LEACS2,
LEACO4) genes are differentially expressed in to-
mato fruit (Barry and others 1996, 2000; Nakatsuka
and others 1998; Rottmann and others 1991). LE-
ACO1,LEACO3,andLEACO4 are expressed at low
levels in green fruit that are in a system 1 mode of
ethylene synthesis, but the transcripts of each in-
crease at the onset of ripening as the fruit transition
to system 2 ethylene production and response.
During ripening, LEACO1 and LEACO4 are sustained
in expression, whereas the increase in LEACO3
expression is transient (Barry and others 1996; Na-
katsuka and others 1998). In the case of LEACO1
and LEACO4, ripening-related increases in transcript
abundance are largely blocked by 1-MCP treatment,
indicating that these genes are positively regulated
by ethylene. The regulation of ACS gene expression
during fruit ripening has been investigated using a
combination of ethylene and inhibitor studies to-
gether with expression analysis in various ripening
mutants (Barry and others 2000; Nakatsuka and
others 1998). The ripening-inhibitor (rin)andnon-
ripening (nor) mutants fail to undergo the typical
ripening-related increase in ethylene synthesis
(system 2) and respiration that occurs in wild-type
fruit and, as such, maintain a low-level system 1-
type ethylene production as they mature (Tig-
chelaar and others 1978). LEACS6 is expressed in
wild-type green fruit but rapidly declines at the
onset of ripening during the transition to system 2
ethylene synthesis. In contrast, LEACS6 transcripts
persist throughout development and ripening in the
rin mutant (Barry and others 2000). Ethylene and
1-MCP treatments indicated that this ripening-re-
lated decline was mediated by ethylene, suggesting
that LEACS6 is responsible for low-level ethylene
production in preclimacteric fruit (Barry and others
2000; Nakatsuka and others 1998). LEACS1A is also
expressed in preclimacteric fruits and declines upon
ethylene treatment, but transcripts show a transient
increase at the onset of ripening that is rin depen-
dent, suggesting that this gene may be important in
regulating ethylene synthesis during the transition
from system 1 to system 2 ethylene synthesis (Barry
and others 2000). LEACS4 is not expressed in green
fruit but is induced at the onset of ripening. This
induction is dependent on rin and is stimulated by
ethylene. LEACS2 expression is also induced at the
onset of ripening; this induction requires ethylene
but is independent of rin (Barry and others 2000;
Nakatsuka and others 1998). Therefore, it seems
likely that LEACS1A and LEACS4 are responsible for
initiating system 2 ethylene synthesis and that this
is maintained by a combination of LEACS2 and LE-
ACS4. The specific transcription factors that mediate
the changes in ACS and ACO gene expression at the
onset of ripening remain to be determined. In a
recent study, a 40-bp promoter fragment of LEACS2
was identified that is required for ethylene-induc-
ing-xylanase (EIX) responsiveness. Both in vitro and
in vivo studies indicated that a novel cysteine pro-
tease, designated LeCP, was bound to this region and
was capable of inducing LEACS2 expression when
Figure 1. Differential expression of ACS and ACO genes
associated with system 1 and system 2 ethylene synthesis
during fruit development and ripening in tomato. Au-
toinhibition of ethylene synthesis during system 1 ethyl-
ene production is mediated by a reduction in LeACS1A and
6expression. Autocatalytic ethylene synthesis at the onset
of fruit ripening is mediated through ethylene-stimulated
expression of LeACS2 and 4and LeACO1 and 4(see text for
Ethylene and Fruit Ripening 145
overexpressed in tomato leaves, suggesting that this
protein may possibly have dual functions as a pro-
tease and a transcriptional regulator (Matarasso and
others 2005). However, it remains to be determined
whether the same promoter region of LEACS2 and
LeCP are required for ethylene-regulated ripening
induction. Although regulation of ACS at the level
of expression and transcript abundance is clearly
important for ripening-related ethylene synthesis,
there is considerable evidence that regulation of
ACS activity, through protein phosphorylation and
turnover, also plays a critical role in the function of
this enzyme (for review, see Argueso and others,
this issue).
The physiologic and molecular pathways that act
to initiate the transition from a system 1 to a system
2 mode of ethylene synthesis at the onset of rip-
ening remain undefined. However, a recent study
performed on detached persimmon (Diospyros kaki
Thunb.) fruit indicated that ripening-related ethyl-
ene synthesis in the fruit was initiated by a burst of
drought-induced ethylene synthesis from the fruit
calyx following harvest (Nakano and others 2003).
Detached persimmon fruit initiated ethylene pro-
duction and associated loss of firmness within two
days after harvest. Treatment of fruit parts with the
ethylene action inhibitor 1-methylcyclopropene (1-
MCP) inhibited ethylene synthesis in all tissues
except the calyx, indicating that the ethylene pro-
duced from the calyx was independent of ethylene
itself. Induction of an ACC synthase gene, DkACS2,
correlated with ethylene synthesis and was unaf-
fected by 1-MCP. The calyx of fruits stored at high
humidity initiated ethylene production and DkACS2
expression four days later than fruits stored at low
humidity. Similar delays were also observed in the
pulp under high-humidity conditions. These data
clearly show a role for water loss in regulating the
onset of ethylene synthesis in detached persimmon
fruit. Although this mechanism has been described
only in persimmon fruit to date, certain fruits,
including avocado and the wild species of tomato
Solanum chilense and Solanum peruvianum, are
known to initiate ripening once abscission has oc-
curred from the parent plant. It is possible that
water loss from these fruits following detachment
could be a possible mechanism to initiate ethylene
synthesis and ripening (Grumet and others 1981;
Dopico and others 1993).
With the exception of ethylene, the signaling
pathways that regulate fruit ripening remain largely
undefined. In tomato, three pleiotropic nonripening
mutants, ripening-inhibitor (rin), non-ripening (nor),
and Colorless non-ripening (Cnr), have been described
in which virtually all aspects of the ripening process
are inhibited, including ethylene synthesis, in-
creased respiration, carotenoid accumulation, soft-
ening, and aroma production (Thompson and
others 1999; Tigchelaar and others 1978) (Figure 2).
In these three mutants, the typical ripening-associ-
ated rise in autocatalytic ethylene synthesis is
blocked due to abnormal regulation of ACS expres-
sion (see above). Although ethylene synthesis is
blocked in these mutants, studies using rin and nor
fruits have indicated that they retain the capacity to
synthesize wound ethylene, indicating that the
mutations are not simply the result of a general
block in ethylene synthesis (Lincoln and Fischer
1988; Yokotani and others 2004). Similarly, exog-
enous ethylene does not restore ripening in these
mutants, although ethylene-regulated gene
expression can be partially restored, indicating that
rin,nor, and Cnr fruits do not display ethylene
insensitivity (Barry and others 2000; Griffiths and
Figure 2. Fruit ripening mutants of tomato. From left to right, ripe fruit of wild type (cultivar Ailsa Craig) and near
isogenic lines homozygous for the ripening-inhibitor (rin), non-ripening (nor), Never-ripe (Nr), and Green-ripe (Gr) loci. Note
association of the macrocalyx (mc) (large sepal) phenotype with the rin mutation. The rin and nor loci act upstream in the
ripening regulatory pathway and are required for system 2 ethylene synthesis during fruit ripening. The nonripening
phenotypes of Nr and Gr are caused by reduced ethylene responsiveness (see text for details).
146 Cornelius S. Barry and others
others 1999; Thompson and others 1999; Yen and
others 1995; Yokotani and others 2004). Together
these data suggest that rin,nor, and Cnr act up-
stream of ethylene in the ripening cascade and
determine the competency of the fruit to ripen.
The molecular identities of the rin and Cnr loci
have been determined using positional cloning
strategies, and both encode different classes of
transcription factor (Manning and others 2006;
Vrebalov and others 2002). The rin locus harbors a
deletion occurring between two adjacent MADS-
box genes. Genetic complementation and antisense
experiments confirmed that one of these genes,
termed LEMADS-RIN, was responsible for conferring
the nonripening phenotype of the rin mutant,
whereas the associated macrocalyx (mc) phenotype
was the result of a promoter deletion in a second
gene, termed LEMADS-MC (Vrebalov and others
2002). RIN is a member of the SEPALLATA sub-
family of MADS-box genes, whereas MC is a mem-
ber of the APETALA 1 subfamily (Litt and Irish 2003;
Malcomber and Kellogg 2005). As MADS-box pro-
teins have been shown to act together in multimeric
complexes, it is possible that other MADS-box genes
act together with RIN to regulate fruit ripening in
tomato. Indeed, expression analysis and data min-
ing of EST collections have revealed several possible
candidates to fulfill this role (Fei and others 2004;
Giovannoni 2004). The Cnr mutation is the result of
an epigenetic mutation that causes hypermethyla-
tion and reduced expression of a SQUAMOSA PRO-
and others 2006). SBP-box proteins have been
shown to directly regulate the expression of MADS-
box genes, raising the possibility that CNR may act
to directly influence the expression of RIN or other
MADS-box genes during fruit ripening. The char-
acterization of the RIN and CNR transcription factors
is currently the subject of intense research. It will be
particularly interesting to identify the direct targets
of these proteins and determine how they are able
to regulate ethylene synthesis during ripening.
Much of our knowledge concerning the mode of
action of ethylene in plants has been generated
from the use of the triple-response screen in Ara-
bidopsis to identify mutants that are either insensi-
tive to ethylene or show enhanced ethylene
responses in the absence of exogenous ethylene.
The power of Arabidopsis molecular genetics has
facilitated the rapid identification of many compo-
nents of the signaling pathway from an initial mu-
tant phenotype. The components of ethylene
signaling and their mechanisms of action in Ara-
bidopsis are the subject of two additional reviews by
Hall and others and Li and Guo, in this special issue.
We focus our discussions on ethylene signaling re-
search in fruit crop species, primarily reviewing re-
search on tomato and how findings differ from the
Arabidopsis model.
The development of the triple-response screen in
Arabidopsis (Bleecker and others 1988; Guzman and
Ecker 1990), together with the identification of
ETR1 as an ethylene receptor (Chang and others
1993), led directly to the identification of an eth-
ylene-insensitive mutant of tomato and the cloning
of the family of ethylene receptors to which it be-
longed. The Never-ripe (Nr) mutant was initially
described as a nonripening mutant 50 years ago
(Rick 1956). Although Nr clearly displayed a dra-
matic inhibition of fruit ripening (Figure 2), other
phenotypes associated with Nr had been over-
looked. However, in light of the findings from Ara-
bidopsis, Lanahan and coworkers (1994) showed
that Nr displayed a range of phenotypes that could
be directly attributed to reduced ethylene sensitiv-
ity. The semidominant ethylene-insensitive pheno-
type of Nr is reminiscent of the etr1 ethylene
receptor mutant, and with the availability of the
ETR1 gene for use as a heterologous probe, several
ethylene receptor homologs were identified in to-
mato, one of which was found to cosegregate with
the Nr phenotype on chromosome 9 (Yen and
others 1995). Subsequent molecular analysis re-
vealed a single C > T base change that results in
conversion of a conserved proline residue into leu-
cine (Wilkinson and others 1995). This proline
residue lies within the N-terminal ethylene binding
domain of the NR protein at a similar location to
dominant ethylene-insensitive alleles of ETR1 that
disrupt ethylene binding (Chang and others 1993;
Hall and others 1999).
To date, a total of six ethylene receptors have
been identified in tomato: LeETR1,LeETR2,NR (also
referred to as LeETR3), and LeETR4,5, and 6(Klee
2004; Lashbrook and others 1998; Payton and others
1996; Tieman and Klee 1999; Wilkinson and others
1995; Zhou and others 1996). Based on structural
similarity, the Arabidopsis ethylene receptors have
been classified into two subfamilies (Guo and Ecker
2004). Subfamily-1 consists of ETR1 and ERS1
that share three N-terminal membrane-spanning
Ethylene and Fruit Ripening 147
domains and a conserved carboxy terminus histidine
(His) kinase domain. LeETR1,LeETR2,andNR pos-
sess a structure that is consistent with the subfamily-
1 receptors. Subfamily-2 receptors in Arabidopsis
(ERS2, ETR2, and EIN4) lack a complete His kinase
domain and possess an additional transmembrane-
spanning domain at the N terminus. The tomato
receptors LeETR4,5, and 6can be classified as sub-
family-2 receptors. In addition, receptor structure
differs with regard to the presence or absence of a
receiver domain at the carboxy terminus. Arabidopsis
ETR1, ETR2, and EIN4 all possess a receiver domain,
as do all of the tomato receptors with the exception
of NR. Thus, the receptor complement varies slightly
between Arabidopsis and tomato. Tomato contains
an additional subfamily-1 receptor compared with
Arabidopsis, but contains only a single receptor
lacking a receiver domain (NR), whereas Arabidopsis
has two receptors (ERS1 and ERS2) that lack this
domain. It is evident that the ethylene receptor
family shows a high degree of structural divergence
in plants, but despite this diversity all of the recep-
tors thus far examined have the capacity to bind
ethylene when expressed in yeast (OÕMalley and
others 2005).
The tomato ethylene receptors are differentially
expressed in organs and tissues at various stages of
development and in response to exogenous stimuli
(Ciardi and others 2001; Lashbrook and others
1998; Moeder and others 2002; Tieman and Klee
1999). The changes in receptor profiles appear to be
quantitative rather than qualitative, with expres-
sion of receptors detected in all tissues so far
examined, implying that all tissues have the po-
tential to respond to ethylene. However, specific
receptors appear to be more prevalent in certain
tissues; for example, NR and LeETR4 are highly ex-
pressed in reproductive tissues and transcript
abundance is enhanced during fruit ripening (Ti-
eman and Klee 1999; Wilkinson and others 1995).
Different expression levels of receptors may poten-
tially lead to different pools of ethylene receptors
that may act to regulate specific responses.
Characterization of the individual functions of
members of the ethylene receptor gene family is
subject to ongoing investigation. Experiments de-
signed to downregulate specific receptor isoforms
using antisense suppression have been reported for
LeETR1,NR,andLeETR4 (Hackett and others 2000;
Tieman and others 2000; Whitelaw and others
2002). Downregulation of LeETR1 expression in
transgenic plants did not alter fruit ripening but
resulted in plants with shorter internodes and re-
duced rates of floral abscission (Whitelaw and oth-
ers 2002). Downregulation of NR expression in a
wild-type background did not result in any dramatic
phenotypes but did result in subtle changes indica-
tive of slightly delayed fruit ripening, that is, re-
duced rates of ethylene synthesis and slower
carotenoid accumulation (Tieman and others 2000).
Elevated expression of LeETR4 was detected in the
NR antisense lines, suggesting that this receptor may
compensate for loss of NR. Reduction of LeETR4
expression using an antisense transgene resulted in
plants with enhanced ethylene sensitivity mani-
fested through extreme epinasty, increased floral
abscission, enhanced triple response, and acceler-
ated fruit ripening, confirming that LeETR4 acts as a
negative regulator of ethylene responses in tomato
(Tieman and others 2000). Interestingly, these
phenotypes could be complemented by overex-
pression of a NR transgene, indicating that these two
receptors are functionally redundant, a phenome-
non that is unexpected when one considers that
they are extremely divergent (see above). Although
studies of individual receptor function in tomato
requires additional experimentation, an obvious
difference between the tomato and Arabidopsis sys-
tems is evident. Reduction of the subfamily-2
receptor LeETR4 in transgenic plants leads to strong
phenotypic effects throughout the plant, whereas
single loss-of-function mutants in type 2 receptors
of Arabidopsis do not show dramatic phenotypic
changes (Hua and Meyerowitz 1998).
The dominant Green-ripe (Gr) and Never-ripe 2 (Nr-2)
mutants of tomato fail to fully ripen and possess a
fruit phenotype very similar to that of Nr (Jarret and
others 1984; Kerr 1958, 1982) (Figure 2). This
similarity prompted an examination of the ethylene
physiology of Gr and Nr-2. Examination of ethylene
synthesis and responses in Gr and Nr-2 fruits indi-
cated that reduced ethylene responsiveness was the
basis for ripening inhibition in these mutants (Barry
and others 2005). However, unlike Nr, which shows
reduced ethylene sensitivity throughout the whole
plant, Gr and Nr-2 show reduced ethylene sensi-
tivity predominantly in fruit and floral tissues with
weak ethylene insensitivity evident in roots. Dark-
grown Gr and Nr2 hypocotyls and petioles maintain
normal ethylene responsiveness. High-resolution
genetic and physical mapping of the Gr and Nr-2 loci
revealed that they were both linked to a 38-kb
interval of the long arm of chromosome 1, sug-
gesting that they may be allelic. This hypothesis was
confirmed when sequence analysis of this region
148 Cornelius S. Barry and others
identified a 334-bp deletion in both Gr and Nr-2
mutants compared to wild type. The deletion occurs
at the junction between the 5¢-UTR and the pro-
moter of a gene of unknown function and causes
ectopic expression of the gene in Gr and Nr-2 mu-
tant backgrounds, consistent with a dominant gain-
of-function mutation (Barry and Giovannoni 2006).
To avoid confusion with the NR ethylene receptor,
the gene residing at the Gr and Nr-2 locus was
designated GREEN-RIPE (GR).
Ectopic expression of GR under the control of the
CaMV35S promoter recreated the Gr mutant phe-
notype but did not lead to plants that displayed
whole-plant ethylene insensitivity despite high
levels of transgene expression. This suggests that GR
is able to selectively modify ethylene responsiveness
in a tissue-dependent manner indicating that com-
ponents of the ethylene signaling pathway must be
distinct in different tissues of tomato (Barry and
Giovannoni 2006). These differences have yet to be
defined, but in a separate study a homolog of GR,
was identified as a specific suppressor of ethylene
insensitivity mediated by the etr1-2 mutant allele of
Arabidopsis, suggesting that RTE1 and, therefore,
possibly GR act at the level of the ethylene receptors
(Resnick and others 2006). It is tempting to specu-
late that the specificity of GR action in tomato may
be linked to different pools of ethylene receptors
that are present in different tissues in tomato
(Lashbrook and others 1998; Tieman and Klee
GR and RTE1 encode predicted transmembrane
proteins of unknown function that are conserved in
plants, animals, and protists but are not present in
bacterial or fungal genomes. Higher plants typically
contain two or three GR homologs, whereas animal
and protist genomes possess a single copy of this
gene. In plants there appears to be two phyloge-
netically distinct clades. One clade contains GR,
RTE1, and a closely related tomato gene designated
GREEN-RIPE LIKE 1 (GRL1). The second clade
contains distant homologs of GR and RTE1 desig-
nated GRL2 and RTE1 HOMOLOG (RTH). GR and
RTE1 have clear impacts on ethylene responses in
plants, but it remains to be determined if GRL1,
GRL2, and RTH also function in the ethylene re-
sponse pathway or some other aspect of cellular
metabolism. The second scenario seems more likely,
particularly for the more distinct GRL2 and RTH
genes and in light of the fact that animals and
protists are not known to signal using ethylene. The
cloning of GR and RTE1 has identified new proteins
that can influence the ethylene response pathway
in plants; however, determining how these proteins
interact and function within the context of other
pathway components is required.
The constitutive triple response (ctr1) mutant of Ara-
bidopsis was identified in a genetic screen designed
to identify dark-grown seedlings that possess the
triple-response phenotype in the absence of exoge-
nous ethylene (Guzman and Ecker 1990). The CTR1
gene encodes a protein with high similarity to the
mammalian RAF serine/threonine MAP kinase ki-
nase kinase (MAP3K) and acts as a negative regu-
lator of ethylene responses (Kieber and others
1993). CTR1 localizes to the endoplasmic reticulum,
the same location as the ETR1 ethylene receptor,
and interacts preferentially with the type I ethylene
receptors ETR1 and ERS1 (Clark and others 1998;
Gao and others 2003). The identification of a
MAP3K that functions within the ethylene signaling
pathway has led to the speculation that a MAP ki-
nase cascade may mediate ethylene responses in
Arabidopsis, although this hypothesis awaits experi-
mental validation.
To date, four CTR1 homologs have been isolated
from tomato: tCTR1 (also known as ER50), tCTR2,
tCTR3, and tCTR4 (Adams-Phillips and others 2004a,
b; Leclercq and others 2002; Lin and others 1998;
Zegzouti and others 1999). These genes were iden-
tified either using differential display (ER50) or
through heterologous hybridization using the Ara-
bidopsis CTR1 gene as a probe. Phylogenetic analysis
has indicated that tCTR1,tCTR3, and tCTR4 are clo-
sely related to Arabidopsis CTR1. In addition, these
three genes are all able to complement, at least
partially, the weak ctr1-8 allele of Arabidopsis, sug-
gesting that the tomato genes are functionally
equivalent to CTR1. Interestingly, the efficacy of
complementation follows the phylogenetic rela-
tionship of the tomato genes to Arabidopsis CTR1
such that tCTR3 is able to fully complement ctr1-8,
whereas tCTR1 and tCTR4 display only partial com-
plementation (Adams-Phillips and others 2004a, b;
Leclercq and others 2002). This may be indicative of
slightly different signaling specificities of these pro-
teins. tCTR1,3,and4show differential expression in
various plant tissues, but as in the case of receptor
gene expression, all tissues express CTR-like genes
(Adams-Phillips and others 2004a, b; Leclercq and
others 2002). tCTR2 is more divergent and is the
likely ortholog of the ENHANCED DISEASE RESIS-
TANCE 1 (EDR1) gene of Arabidopsis that has been
implicated in disease resistance, stress responses,
Ethylene and Fruit Ripening 149
and ethylene-induced leaf senescence and may act
at the interface between the ethylene and salicylic
acid signaling pathways (Adams-Phillips and others
2004a, b; Frye and others 2001; Tang and others
These data indicate that tomato possesses at least
three CTR genes, whereas Arabidopsis possesses only
a single gene. Subsequent analysis of EST and
genomic sequence repositories has uncovered evi-
dence for multiple CTR-like genes in a number of
species, suggesting that the single gene found in
Arabidopsis may be the exception (Adams-Phillips
and others 2004a, b). The presence of multiple CTRs
raises questions as to the individual functions of
these genes and the level of redundancy that
operates within this gene family. These questions
will need to be addressed through the use of RNAi
targeted at individual members of the family and by
gain-of-function analysis. However, virus-induced
gene silencing (VIGS) of a generic tCTR sequence
led to tomato plants that showed severe epinasty
and upregulation of ethylene-induced gene
expression, indicating that silencing of tCTR genes
can mimic the Arabidopsis ctr1 mutant phenotype
(Liu and others 2002). Because CTR1 is able to
interact with at least some of the Arabidopsis ethyl-
ene receptors, it is likely that the tCTR proteins will
also interact with the tomato ethylene receptors.
The presence of larger gene families for both CTRs
and the ethylene receptors in tomato compared to
Arabidopsis indicates that the possible interactions
between these two families is potentially more
complex and that localization experiments will need
to be performed together with interaction studies to
confirm that any interactions are physiologically
In Arabidopsis, downstream ethylene responses are
mediated by two classes of transcription factors en-
coded by EIN3 and ERF gene families. ETHYLENE-
INSENSITIVE 3 (EIN3) also includes the EIN3-LIKE
(EIL) family members and the ETHYLENE RE-
SPONSE FACTOR (ERF) family is inclusive of genes
BINDING PROTEIN (EREBP). EIN3 is a positive reg-
ulator of ethylene responses with loss of function
resulting in ethylene insensitivity, whereas over-
expression results in a constitutive triple-response
phenotype (Chao and others 1997). Emerging evi-
dence suggests that the ethylene response pathway
is regulated at least in part by turnover of the EIN3
protein. In the absence of ethylene, two partially
redundant F-box proteins, EIN3-binding F-box 1
and 2 (EBF1 and EBF2), target EIN3 for degrada-
tion. A negative feedback loop exists whereby EIN3
is self-regulating through directly influencing the
accumulation of EBF1 and EBF2 (Gagne and others
2004; Guo and Ecker 2003; Potuschak and others
2003; Yanagisawa and others 2003). In a recent
study, the EIN5 protein was identified as the XRN4
5¢fi3¢exoribonuclease (Olmedo and others 2006).
Both EBF1 and EBF2 mRNAs are significantly more
abundant in the ein5 mutant background than in
wild type, suggesting that the wild-type function of
EIN5 is to regulate the accumulation of EIN3 via
turnover of EBF1/2 transcripts.
EIN3 binds a conserved motif known as the pri-
mary ethylene responsive element (PERE) that is
present within the promoters of ERF1 and several
senescence and ripening-related genes, including
E4,GST1,andLeACO1 (Solano and others 1998).
ERF1 expression is rapidly induced by ethylene, and
overexpression of ERF1 confers a constitutive eth-
ylene response phenotype. ERFs in turn bind to the
GCC-box that is present in the promoters of several
stress- and pathogen-responsive genes, including
chitinases and PDF1.2 (Solano and others 1998).
ERF1 and related genes form a subgroup of the large
APETALA2 (AP2) family of DNA-binding proteins
that consists of 145 members in Arabidopsis (Gutt-
erson and Reuber 2004). These transcription factors
act to both positively and negatively regulate tran-
scription and are involved in a wide range of
developmental processes, responses to environ-
mental challenges, and pathogen infections.
To date, four EIN3-LIKE genes have been de-
scribed in tomato, LeEIL1-4 (Tieman and others
2001; Yokotani and others 2003). LeEIL1-3 are each
able to complement the Arabidopsisein3-1 mutant
allele, indicating that they are able to function in
the ethylene signaling pathway. Antisense sup-
pression of EIL1-3 in tomato revealed that this gene
family is functionally redundant (Tieman and oth-
ers 2001). However, overexpression of a GFP-tagged
EIL1 in the Nr mutant restored normal fruit ripen-
ing and the expression of a subset of ethylene-
inducible genes in transgenic fruit. In addition,
petiole epinasty was restored in the 35S:EIL1:GFP
transgenic lines, but seedling responses remained
unaltered, suggesting that individual members of
the tomato EIL family may perform specific func-
tions in vivo (Chen and others 2004a, b). The
observation that sequences related to the PERE are
contained within the promoters of the ethylene-
regulated and ripening-related genes E4 and LeACO1
150 Cornelius S. Barry and others
suggests that EIN3 proteins may directly regulate
their transcription and that of other coregulated
genes (Solano and others 1998).
Thirty-six ERF1-like genes have been built into an
Arabidopsis and rice phylogeny and it is likely that as
more ESTs and tomato genomic sequence become
available, this number will increase (Gutterson and
Reuber 2004). Given the high copy number of this
gene family, it is not surprising that several mem-
bers have been isolated from fruit and show differ-
ential expression patterns during fruit development
and ripening (Alba and others 2005; Tournier and
others 2003). Because of the large size of this gene
family, assigning functions to individual family
members will be a daunting task, although the use
of phylogeny coupled to phenotypic information
from Arabidopsis may help guide studies in tomato
(Gutterson and Reuber 2004). Furthermore, the
promoters of many of the genes that have been
associated with ripening do not contain the GCC-
box that forms the binding site of the ERF protein,
suggesting that these factors may have a limited role
in the regulation of ripening. Interestingly, a num-
ber of stress- and defense-associated genes whose
expression can be enhanced by ethylene have been
shown to be induced during fruit ripening (Alba and
others 2005; Picton and others 1993a, b; Zegzouti
and others 1999). Stress-related genes often contain
the GCC-box within their promoter regions that can
be directly targeted by ERF proteins. Therefore, it is
possible that the ERF proteins that are present in
tomato fruit may regulate the expression of this
subset of ripening-related genes, a hypothesis that
will become open to testing as the tomato genome
sequence becomes available in the next few years
(Mueller and others 2005).
Clearly finding the immediate targets and in vivo
function of both the EIN3-LIKE and ERF protein
families during ripening will be important if a
transcriptional network of ethylene-regulated gene
expression is to be elucidated. A recent study
examining the comparative transcriptome of tomato
fruit during development and ripening in wild type
and the Nr mutant revealed that 37% of the gene
expression changes observed were influenced by
ethylene (Alba and others 2005). Furthermore,
clustering of gene expression profiles revealed that
many of these changes were coordinated, implying
that large groups of genes are coregulated. Although
this approach addressed only steady-state mRNA
levels and therefore did not distinguish between
transcriptional and post-transcriptional events, it is
highly likely that a large proportion of these chan-
ges involve at least partial ethylene-regulated tran-
scriptional control. Dissecting specific signaling
modules within this vast sea of ethylene-regulated
events will be challenging. One approach that may
be useful in this regard is a scaled-up version of the
experimental system used by Chen and others
(2004a, b) to study EIL1 function in tomato (de-
scribed above). A series of EIL or ERF constructs
under the control of an inducible promoter could be
introduced into the Nr mutant, possibly using
transient expression systems, and early changes in
gene expression could be monitored by microarrays
following induction. This approach could reveal the
identities of subsets of coregulated genes or path-
ways and provide information about potential tar-
gets of EILs and ERFs in tomato that could then be
addressed by in vitro and in vivo DNA-binding
Fruits have been classically categorized into cli-
macteric and nonclimacteric based on increased
ethylene synthesis and a concomitant rise in the
rate of respiration during ripening (Lelievre and
others 1997). The role of ethylene as the ‘‘ripening
hormone’’ in climacteric fruits such as tomato, ap-
ple, and banana has been firmly established. How-
ever, there is an increasing body of experimental
evidence that implicates ethylene in the ripening of
fruits that have been classically thought of as non-
climacteric. There are also a number of species in
which the fruits of different varieties and cultivars
exhibit both climacteric and nonclimacteric behav-
A Role for Ethylene in the Ripening of
‘‘Nonclimacteric’’ Fruit
The highly sensitive technique of laser photoacou-
stic spectroscopy coupled with the development of
specific apparatus to determine in planta ethylene
production in fruits and flowers of strawberry dur-
ing development and ripening has revealed an in-
crease in ethylene production and a concomitant
rise in respiration rate in red ripe strawberry fruits
(Iannetta and others 2006). Furthermore, experi-
ments with the ethylene action inhibitor silver
thiosulfate revealed that this increased ethylene
production was under the control of a positive
feedback mechanism in ripe fruits, suggesting that a
form of autocatalytic ethylene production is opera-
tional during ripening in strawberry. The timing of
Ethylene and Fruit Ripening 151
this ripening-related increase in ethylene produc-
tion is distinct from the patterns of ethylene pro-
duction typically associated with the ripening of
fruits such as tomato. For example, ethylene pro-
duction increases at the onset of ripening in tomato,
and ripening is severely disrupted in transgenic fruit
where this phenomenon is blocked (Oeller and
others 1991). In contrast, the increase observed in
strawberry was not detected until 24 h after the
fruits had developed full red pigmentation. There-
fore, the physiologic role of the increased ethylene
synthesis in strawberry remains to be determined.
Nevertheless, in support of a role for ethylene in the
ripening of strawberry, a body of molecular evi-
dence concerning ethylene-related changes in gene
expression is becoming available. For example, in-
creased expression of cystathionine-gamma-syn-
thase (CGS) and ACC oxidase genes has been
reported during ripening of strawberry (Aharoni
and others 2002; Marty and others 2000; Trainotti
and others 2005). Similarly, increased expression of
ethylene receptor homologs in strawberry fruit was
also observed with increasing ripeness. In particular,
increased expression of FaETR2, a type II receptor
homolog that is closely related to the ripening-re-
lated LeETR4, was associated with ripening (Train-
otti and others 2005). In addition, the isolation and
characterization of a peptide methionine sulfoxide
reductase (PMSR) gene that is expressed late in
strawberry ripening were also recently described
(Pedraza-Lopez and others 2006). This gene is
homologous to the tomato ripening-related gene E4,
whose expression is regulated by ethylene in to-
mato and may be involved in the methionine sal-
vage pathway that operates during increased
ethylene synthesis (Lincoln and others 1987). Al-
though currently untested, it may be pertinent to
examine the relationship of ethylene and the
expression of FaPMSR and other genes whose
expression is induced at late stages of ripening of
strawberry fruit. The emerging data could be con-
sistent with either a regulatory role for ethylene in
as-yet defined aspects of ripening in strawberry fruit
or a response to the dramatic cellular changes
associated with ripening and the concomitant loss in
cellular integrity and senescence characteristic of
this developmental process. Either answer would be
interesting because the former would demonstrate a
fundamental role of ethylene in ripening of most if
not all fruits and the other might point to a more
primal developmental response that may indeed
have been recruited through evolution as a signal-
ing system to catalyze the ripening process in a
subset of species. The creation of transgenic straw-
berry with reduced ethylene responsiveness, possi-
bly by expression of a dominant mutant allele of an
ethylene receptor, may help clarify this role.
Small but significant increases in ethylene syn-
thesis at the onset of ripening have also been de-
tected in grape berries. Chervin and others (2004)
demonstrated the presence of a transient peak of
ethylene production in grapes just prior to the onset
of ripening, and experiments with 1-MCP indicated
that ethylene was required at this stage for the onset
of anthocyanin accumulation, fruit swelling, and
the decrease in acidity that is associated with rip-
ening. Concomitant with an ethylene-stimulated
rise in anthocyanin production, the abundance of
four transcripts encoding enzymes involved in
anthocyanin synthesis also increased following
ethylene treatment (El-Kereamy and others 2003).
In an additional study, 1-MCP treatment of grape
berries was found to partially repress the ripening-
induced expression of the VvADH2 gene that en-
codes an alcohol dehydrogenase (Tesniere and
others 2004). These studies suggest that ethylene
may influence multiple aspects of ripening in grape.
A similar strategy as described for strawberry above
could clarify the role of ethylene in grape berry
Citrus is also classified as a nonclimacteric fruit,
but studies with inhibitors of ethylene action re-
vealed that ripening-related color changes in the
flavedo are regulated by endogenous ethylene and
that ethylene treatments can stimulate both chlo-
rophyll breakdown and carotenoid accumulation
(Goldschmidt and others 1993; Purvis and Barmore
1981; Stewart and Wheaton 1972). Furthermore,
studies have shown some genes, including chloro-
phyllase, to be ethylene regulated in citrus fruit
(Alonso and others 1995; Jacob-Wilk and others
1999). Recently, as in the case of strawberry, auto-
catalytic ethylene production has also been de-
scribed in citrus fruit, but again with altered timing
relative to typical ripening climacteric fruit.
Whereas mature fruits exhibited no increased eth-
ylene production associated with ripening, har-
vested immature fruits produce high levels of
ethylene that can be further stimulated by ethylene
and propylene treatments and inhibited by 1-MCP,
indicating the autocatalytic nature of this phenom-
enon (Katz and others 2004). This study clearly
showed that citrus fruit have the capability to pro-
duce autocatalytic ethylene, although there may be
little significance of this phenomenon in relation to
the ripening process. Citrus and other fruit trees
undergo a continual process of fruit drop or selective
abscission to ensure that resources are available to
allow the development of mature fruits. It is possible
that the climacteric behavior of young citrus fruitlets
152 Cornelius S. Barry and others
may be linked to this abscission process or may be
the result of stress during detachment as was wit-
nessed in persimmon fruit (see above).
Climacteric and Nonclimacteric:
Ethylene-Mediated Ripening Inhibition
Through Natural Allelic Variation
Apple cultivars are highly heterozygous and their
pedigrees are often poorly defined. In addition, they
possess different ripening rates leading to varied
storage properties ranging from rapid postharvest
deterioration to cultivars that can be stored for up to
a year under optimal conditions. A clear positive
correlation exists between ethylene production
during storage and softening, which in turn is
tightly associated with postharvest deterioration
(Gussman and others 1993). Sunako and others
(1999) identified two allelic forms of the Malus
domestica ACS1 gene from the Golden Delicious
cultivar, MdACS1-1 and MdACS1-2.MdACS1-1 is
highly expressed at the onset of apple fruit ripening,
whereas MdACS1-2 transcripts are absent from rip-
ening fruits. Sequence analysis of these alleles re-
vealed only seven nucleotide substitutions within
the protein-coding region of MdACS1 and six of
these encoded silent mutations. However, more
sequence divergence was evident in the 5¢-flanking
region, including the presence of a short inter-
spersed DNA element (SINE) in the MdACS1-2 allele.
Thirty-five apple cultivars were tested for the pres-
ence or the absence of the SINE element and results
indicated that cultivars were either homozygous for
either MdACS1-1 or MdACS1-2 or heterozygous for
each allele. In subsequent studies, cultivars that
were homozygous for the MdACS1-2 allele had sig-
nificantly lower internal ethylene concentrations
than MdACS1-1 homozygotes or MdACS1-1/MdACS1-
2heterozygous individuals and possessed enhanced
storage capabilities and reduced rates of fruit drop
(Harada and others 2000; Oraguzie and others 2004;
Sato and others 2004). These data indicate that the
insertion of a SINE into the promoter of the MdACS1
gene is directly responsible for reduced gene
expression, lower ethylene production, and en-
hanced storage properties of certain apple cultivars.
This discovery provides a useful tool for selecting
new apple varieties with optimal storage properties.
Reduction in ACS gene expression and ethylene
production also seems to be responsible for the
nonripening phenotype in peach cultivars that carry
the recessive stony hard (hd) mutation. hd fruit fail to
soften on the tree or postharvest, although other
ripening traits such as color development, soluble
solids, and flavor characteristics are fairly normal
(Haji and others 2001). Furthermore, the pheno-
type of hd can be reversed by ethylene treatment
(Haji and others 2003). Expression of a ripening-
related ACS gene, PpACS1, is eliminated in hd
backgrounds during ripening, although this gene
remains wound-inducible in both leaves and fruits
(Tatsuki and others 2006). The mechanism of the
reduction of PpACS1 expression in hd is currently
unknown. Southern analysis failed to reveal any
significant structural differences in PpACS1 between
hd and a normal-ripening cultivar, indicating that
disruption of the promoter by a transposable ele-
ment, as in the case of the MdACS1-2 allele, is un-
likely. It is possible that a SNP or small deletion or
insertion may disrupt a ripening-specific transcrip-
tion factor binding site in the PpACS1promoter or
that the hd phenotype may be caused by a mutation
in a ripening-specific transcription factor.
Melon is also a fruit crop that exhibits great phe-
notypic diversity. Fruits of the cantaloupe type often
have netted skin and orange flesh, are susceptible to
abscission, and produce large quantities of ethylene.
In contrast, melons of the ‘‘honey dew’’ type are of-
ten smooth skinned, have green or yellow flesh, do
not abscise, and produce little or no ethylene during
ripening. Therefore, melons behave as both climac-
teric and nonclimacteric fruit, and the level of eth-
ylene production produced by melon fruit is directly
proportional to postharvest rates of decay (Zheng and
Wolff 2000). Perin and others (2002) examined the
ethylene physiology of a smooth-fruited, nonabscis-
ing melon designated PI161375. They found that
exogenous ethylene failed to induce abscission, fruit
ethylene production, or the expression of ethylene-
regulated genes, suggesting that PI161375 fruit is
ethylene insensitive. However, the seedling triple
response in this line was normal. Genetic analysis of a
recombinant inbred population generated from a
charentais (cantaloupe) ·PI161375 cross indicated
that fruit abscission and ethylene production were
controlled by two independent loci designated
Abscission layer (Al)-3 and Al-4. F
progeny of the
charentais ·PI161375 cross produced fruits that
abscised and produced a climacteric ethylene peak,
indicating that the alleles from the PI161375 parent
are recessive. The fruit and abscission zone-specific
responses in PI161375 share similarities to, but are
distinct from, tomato mutants that display inhibited
fruit ripening. For example, like the PI161375 line,
the rin,nor, and Cnr mutants can be classified as
nonclimacteric; however, they retain the capacity to
respond to exogenous ethylene at the level of gene
expression (Tigchelaar and others 1978; Thompson
and others 1999; Yen and others 1995; Yokotani and
others 2004). In contrast, the Gr mutant of tomato
Ethylene and Fruit Ripening 153
displays tissue-specific ethylene insensitivity associ-
ated with fruit ripening and abscission, but unlike
PI161375, Gr fruit are capable of synthesizing large
quantities of ethylene (Barry and others 2005).
Pepper fruit also exhibits a wide range of ethylene
production rates and respiratory behavior during
ripening (Villavicencio and others 1999). Although
a role for endogenous ethylene in mediating the
phenotypic changes associated with ripening in
pepper has not been determined, exogenous ethyl-
ene treatments can lead to enhanced carotenoid
accumulation (Fox and others 2005). From the data
generated by studies on apple and melon, it seems
that large differences in fruit ethylene production
appear to be controlled by one or two genetic loci.
Although still unproven, it is possible that a similar
situation may be occurring between closely related
pepper species. QTL analysis on segregating popu-
lations generated from crosses between high and
low ethylene-producing parents should be able to
resolve these issues and address whether differing
ripening mechanisms truly exist within these spe-
cies or (as is more likely) nonclimacteric varieties
represent allelic variants in ethylene synthesis, re-
sponse, or more general ripening genes within what
are normally climacteric species.
Clearly, ethylene is required for the ripening of many
fruits and in its absence the ripening process fails to
proceed to completion, rendering the product
unpalatable. However, once initiated, ripening is a
one-way process and the beneficial aspects of ethyl-
ene for generating a high-quality product can soon be
outweighed by its propensity to stimulate over-rip-
ening and decay. This is particularly true under
postharvest storage conditions where considerable
effort is expended to control ethylene effects not only
in fruits but also in vegetables and ornamental crops.
Depending on the commodity, specialized harvest-
ing, packaging, shipping, temperature, and con-
trolled atmospheres may be required, adding to the
cost of production through additional labor and en-
ergy use. Control of ethylene responses is therefore
important for the agricultural and food industries.
Chemical Control of Ethylene Responsiveness
Several compounds have been developed that can
block ethylene action in fruits, vegetables, and floral
crops and these are thought to act through binding
to the ethylene receptors (Sisler 2006). 1-Methyl-
cyclopropene (1-MCP) is a potent inhibitor of eth-
ylene responses and under the tradenames
and SmartFreshit has been approved
for commercial use on ornamental and edible hor-
ticultural products, respectively. A whole research
field has evolved to test the efficacy and physiologic
effects of 1-MCP on fruit crops and a wide range of
effects are observed that vary between species and
even between cultivars (Watkins 2006). It appears
that this compound does have limitations in many
species, but major successes have been reported for
prolonging the storage life of apples leading to
widespread commercial utilization within the apple
Genetic Control of Ethylene Synthesis and
The role of ethylene in regulating fruit ripening in
tomato has been unambiguously determined
through the genetic manipulation of ethylene syn-
thesis and perception (Klee and others 1991; Oeller
and others 1991; Picton and others 1993a, b; Wil-
kinson and others 1997). Transgenic manipulation
of ethylene synthesis through downregulation of
ACS and ACO expression has also been achieved in
melon and apple (Ayub and others 1996; Dandekar
and others 2004). Transgenic melon expressing an
ACO antisense gene displayed inhibition of ripening,
including a reduction in external pigmentation and
fruit softening. The transgenic lines also failed to
develop a peduncular abscission zone and therefore
did not abscise from the plant at maturity, leading to
an enhanced accumulation of sugars (Guis and
others 1997). The production of volatile esters that
represent important flavor components in ripe
melon fruit are also greatly inhibited in the ACO
antisense lines (Bauchot and others 1998; Flores
and others 2002). In addition, fruit of the antisense
lines are less susceptible to chilling injury than those
of wild type (Flores and others 2004). Similarly,
ethylene-suppressed apples were firmer, had an
extended shelf life, altered sugar and organic acid
profiles, and reduced volatile ester formation that
was accompanied by ethylene-dependent reduc-
tions in alcohol acyl-CoA transferase (AAT) activity
(Dandekar and others 2004; Defilippi and others
2004, 2005).
The natural variation seen in ethylene production
and responsiveness in apples, peaches, melons, and
peppers indicate that there is considerable scope for
the generation of new cultivars within these species
with differing ethylene physiology and ripening
characteristics. The development of introgression
lines of tomato harboring defined chromosomal
154 Cornelius S. Barry and others
segments of wild species within the cultivated
Solanum lycopersicum genome is providing unprece-
dented quantities of information about loci that
control fruit quality traits (Gur and others 2004; Liu
and others 2003; Schauer and others 2006). Con-
siderable variation in ethylene synthesis and rip-
ening characteristics of wild species of tomato have
been reported (Grumet and others 1981). It may be
possible to harness the power of the introgression
lines for uncovering QTL that alter ethylene-related
phenotypes during fruit development and ripening.
From utilizing the wealth of information generated
using the Arabidopsis model and from many funda-
mental studies on fruit crop species, we now have a
basic framework of ethylene biosynthesis and sig-
naling during fruit ripening. This knowledge has
increased our ability to modify ethylene synthesis
and responses in fruit crops to lessen the effects of
postharvest deterioration. However many funda-
mental biological questions remain to be addressed:
How does the transition from system 1 to system
2 modes of ethylene synthesis occur at the onset of
ripening, what is the involvement of the rin,nor,
and Cnr loci in mediating this transition, and how is
the system 2 ethylene synthesis perpetuated?
What are the functions of the individual ethylene
signaling components and how is this network of
proteins assembled in vivo? Is the expansion of gene
families in tomato, and perhaps other fleshy fruit-
bearing species, linked to special ethylene signaling
requirements in these species? What are the
downstream targets of the EILs and ERFs in ripening
fruits and how do these transcription factors activate
or repress specific ripening-related pathways? How
are tissue-specific ethylene signaling effects, such as
those mediated by the GR protein and the Al3 and
Al4 loci, achieved?
What role does ethylene play in the ripening of
nonclimacteric fruits? What is the significance, if
any, of small transient increases in ethylene pro-
duction in species such as grape and strawberry? In
nonclimacteric fruits, do changes in ethylene sen-
sitivity rather than ethylene synthesis mediate
physiologic changes during ripening? Could non-
climacteric fruits carry mutations within compo-
nents of the ethylene synthesis or signaling
pathways? Within closely related species such as
capsicums and melons, what is the molecular
identity of loci that control climacteric ethylene
production, how widespread is this natural varia-
tion in fruit crop species, and can it be harnessed for
generating new varieties with broad consumer ap-
peal but with enhanced shipping and storage capa-
Clearly there is still much to discover within the
field of ethylene biology as it relates to both the
biological and practical aspects of fruit ripening. The
questions outlined above are complex and will re-
quire multidisciplinary experimental approaches to
resolve but will ultimately provide important
information regarding the mechanism by which this
hormone functions to regulate fruit ripening.
Work highlighted in this review, which was per-
formed by C.B. and others in the Giovannoni
laboratory, was supported by the USDA-NCGRI
(2002-35304-12530), National Science Foundation
(DBI-0501778, DBI- 0605659), and the United
States Department of Agriculture – Agricultural
Research Service.
Adams-Phillips L, Barry C, Giovannoni J. 2004a. Signal trans-
duction systems regulating fruit ripening. Trends Plant Sci
Adams-Phillips L, Barry C, Kannan P, Leclercq J, Bouzayen M,
Giovannoni J. 2004b. Evidence that CTR1-mediated ethylene
signal transduction in tomato is encoded by a multigene family
whose members display distinct regulatory features. Plant Mol
Biol 54:387–404.
Aharoni A, Keizer LC, van-den-Broeck HC, Blanco-Portales R,
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Ethylene and Fruit Ripening 159
... Following harvest, an apple's firm and crunchy texture deteriorates during long-term storage [1]. Texture deterioration occurs because apples are climacteric fruit and therefore experience a peak in respiration after fruit abscission accompanied by a characteristic increase in the production of ethylene [2]. Ripening results in the apple becoming sweeter due to the hydrolysis of starch into sugars and also increases volatile organic compound (VOC) production due to the role of ethylene as a plant growth regulator [2]. ...
... Texture deterioration occurs because apples are climacteric fruit and therefore experience a peak in respiration after fruit abscission accompanied by a characteristic increase in the production of ethylene [2]. Ripening results in the apple becoming sweeter due to the hydrolysis of starch into sugars and also increases volatile organic compound (VOC) production due to the role of ethylene as a plant growth regulator [2]. Postharvest treatments have traditionally focused on maintaining apple texture and prolonging their desirable sensorial characteristics [1,3]. ...
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The impact of the ethylene inhibitor, 1-methylcyclopropene (1-MCP), on four apple cultivars (Braeburn, Fuji, Jazz and Golden Delicious) over 150 days of storage at 2 °C was assessed. Proton transfer reaction quadrupole mass spectrometry (PTR-QUAD-MS) was used to monitor changes in VOC composition, while texture analysis and X-ray microcomputer tomography (µ-CT) scanning were used to study microstructural changes. The application of 1-MCP on apples reduced VOC emissions, concurrently maintaining a firmer texture compared to the untreated apples at each time point. The µ-CT scanning revealed how changes in specific morphological characteristics such as anisotropy, connectivity and porosity, size and shape, as well as the interconnectivity of intracellular spaces (IS) influenced texture even when porosity was similar. Additionally, this study showed that the porosity and connectivity of IS were associated with VOC emission and increased simultaneously. This study highlights how the morphological parameters of an apple can help explain their ripening process during long-term storage and how their microstructure can influence the release of VOCs.
... Early work has discovered several genes in the ACS family in different species and their differential expression during plant development. There are 12 ACS genes (ACS1 to ACS12) in Arabidopsis, and all of them display different expression patterns throughout growth and development [1,13] . In tomato, nine genes encoding ACS (LeACS1A, LeACS1B, and LeACS2-8) have been reported [1] , with LeACS1A, LeACS2, LeACS4, and LeACS6 are expressed in different stage of fruit development [14,15] . ...
... There are 12 ACS genes (ACS1 to ACS12) in Arabidopsis, and all of them display different expression patterns throughout growth and development [1,13] . In tomato, nine genes encoding ACS (LeACS1A, LeACS1B, and LeACS2-8) have been reported [1] , with LeACS1A, LeACS2, LeACS4, and LeACS6 are expressed in different stage of fruit development [14,15] . Three of the genes, LeACS1A, LeACS2, and LeACS4 showed a ripening-related increase in expression, while LeACS6 transcripts were present in mature green fruit but declined as ripening was initiated [15] . ...
... Once the ripening cascade has been initiated, it will complete its course without any contribution from the mother plant; this allows growers to harvest climacteric fruits at early ripening stages, which are easier to handle and transport, and can be stored for longer than ripe fruits. Tomato is considered the model species for the study of fleshy fruit development and climacteric ripening, and the current knowledge on tomato ripening is quite extensive (Barry and Giovannoni, 2007). Nevertheless, many pieces of this challenging puzzle are still missing. ...
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This article comments on: Zhao W, Wang S, Li W, Shan X, Naeem M, Zhang L, Zhao L. 2023. The transcription factor EMB1444-like affects tomato fruit ripening by regulating YELLOW-FRUITED TOMATO 1, a core component of ethylene signaling transduction. Journal of Experimental Botany 74, 6563–6574.
... 40 Water stress induces ethylene production, the key player in climacteric (apple, banana, tomato etc.) and nonclimacteric fruit (strawberry, orange, grapes, etc.) ripening. 41 Under controlled water management, qualities like total soluble solids and titratable acidity increased in orange fruits and saved water up to 1000 m 3 (m 3 ) per hectare. 42 These studies point out evidently that water management or irrigation practices need to be optimized for superior fruit quality and are perhaps essential for improving the quality attributes of fleshy fruits without any detrimental effects on the overall yields. ...
... THYLENE is an important plant hormone that acts as a regulator for various physiological processes in plants, such as fruit ripening, flowering, leaf senescence [1]- [3], and responses to environmental stresses [4]- [6]. For example, ethylene biosynthesis is triggered by a variety of abiotic stress conditions, including heat, shade, low nutrient availability, and water deficiency [7]- [9]. ...
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This paper presents a flexible tattoo sensor for real-time monitoring of gaseous ethylene released from plant leaves. To the best of our knowledge, this is the first reported plant tattoo sensor that can measure and monitor a plant hormone in situ. The tattoo sensor can be easily installed on leaves and removed when required using a stick-and-peel mechanism. Furthermore, temperature and humidity sensors were incorporated to correct the ethylene measurements based on the specific microclimate of the leaf. All sensors were chemiresistive in nature, causing variations in resistance as a result of changes in temperature, humidity, or ethylene. The ethylene sensor demonstrated a sensitivity of 0.6295 kΩ/ppm and a limit of detection of 0.13 ppm. The measurements were processed by an onboard data logger, transmitted wirelessly to the cloud, and accessed on a smartphone. Plants were subjected to multiple stressors, including water, temperature, and dark stressors, and the real-time ethylene measurements were recorded with the tattoo sensor. A statistically significant difference, characterized by p values less than 0.05, was observed between different types of stresses. Roll-to-roll fabrication of these tattoos will enable their large-scale implementation in greenhouses and agricultural fields. Index Terms-abiotic stress, electrochemical sensor, ethylene, plant hormone, plant wearables, tattoo sensor.
... Experimental evidence supporting the physiological function of ethylene in the initiation of fruit ripening is vast, and several excellent reviews are available on the subject [10,11,25]. However, until now, another suggestion about what the first signal initiating fruit ripening is has not been elaborated, to our knowledge. ...
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The paradigm that has prevailed for a long time sustains that ethylene is the first signal that initiates fruit ripening. However, in this manuscript, we present the hypothesis that a signal generated from the cuticle induces the synthesis of ethylene, and therefore, it is the initial signal that triggers the fruit-ripening phenomena. Among the experimental evidence supporting the hypothesis, we can mention that cuticle components released during the plant pathogenic attack can induce the synthesis of ethylene in plants. Also, it has been found that in fungi, a cuticle component can activate a transcription factor by phosphorylation, which induces the transcription of a gene encoding a cutinase. Besides, studies with plant tissues experiencing a high rate of cell expansion have shown that there is a careful synchronization between the demand of cuticle components and biosynthesis, which suggests that the plant cell can sense the moment in which the fruit would stop growing by cell expansion, and initiate the ripening phenomena. In this chapter, experimental evidences supporting the physiological role of the fruit cuticle in the fruit ripening phenomena will be presented and reviewed with the goal to show a possible role of the fruit cuticle in the onset of fruit ripening.
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Polygalacturonase (PG) is one of the largest families of hydrolytic enzymes in plants. It is involved in the breakdown of pectin in the plant cell wall and even contributes to peel cracks. Here, we characterize PGs and outline their expression profiles using the available reference genome and transcriptome of Akebia trifoliata. The average length and exon number of the 47 identified AktPGs, unevenly assigned on 14 chromosomes and two unassembled contigs, were 5399 bp and 7, respectively. The phylogenetic tree of 191 PGs, including 47, 57, 51, and 36 from A. trifoliata, Durio zibethinus, Actinidia chinensis, and Vitis vinifera, respectively, showed that AktPGs were distributed in all groups except group G and that 10 AktPGs in group E were older, while the remaining 37 AktPGs were younger. Evolutionarily, all AktPGs generally experienced whole-genome duplication (WGD)/segmental repeats and purifying selection. Additionally, the origin of conserved domain III was possibly associated with a histidine residue (H) substitute in motif 8. The results of both the phylogenetic tree and expression profiling indicated that five AktPGs, especially AktPG25, could be associated with the cracking process. Detailed information and data on the PG family are beneficial for further study of the postharvest biology of A. trifoliata.
Gasotransmitters and gaseous-signaling molecules are hugely important for controlling cell function and especially so during stress challenges. Past research has concentrated on molecules such as nitric oxide (NO) and hydrogen sulfide (H2S), although others such as ethylene and carbon monoxide (CO) are also important. Here, molecular hydrogen (H2) is added to the mix. H2 has been shown to ameliorate responses to a range of stressors in plants, including exposure to heavy metals, salinity, extreme temperatures, and UV radiation. Clearly, H2 is an important gas, which may be useful for enhancing plant growth and food security in the future. Exogenous treatments with H2 are easy in the form of hydrogen-rich water (HRW), but there are still issues with its wide-spread use. Furthermore, the molecular basis of the action of H2 in cells is still not clear. Here, aspects of the use and the action of H2 in plants are discussed, along with what might be learnt from other species.
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The global healthcare market in the post-pandemic era emphasizes a constant pursuit of therapeutic, adaptogenic, and immune booster drugs. Medicinal plants are the only natural resource to meet this by supplying an array of bioactive secondary metabolites in an economic, greener and sustainable manner. Driven by the thrust in demand for natural immunity imparting nutraceutical and life-saving plant-derived drugs, the acreage for commercial cultivation of medicinal plants has dramatically increased in recent years. Limited resources of land and water, low productivity, poor soil fertility coupled with climate change, and biotic (bacteria, fungi, insects, viruses, nematodes) and abiotic (temperature, drought, salinity, waterlogging, and metal toxicity) stress necessitate medicinal plant productivity enhancement through sustainable strategies. Plants evolved intricate physiological (membrane integrity, organelle structural changes, osmotic adjustments, cell and tissue survival, reclamation, increased root-shoot ratio, antibiosis, hypersensitivity, etc.), biochemical (phytohormones synthesis, proline, protein levels, antioxidant enzymes accumulation, ion exclusion, generation of heat-shock proteins, synthesis of allelochemicals. etc.), and cellular (sensing of stress signals, signaling pathways, modulating expression of stress-responsive genes and proteins, etc.) mechanisms to combat stresses. Endophytes, colonizing in different plant tissues, synthesize novel bioactive compounds that medicinal plants can harness to mitigate environmental cues, thus making the agroecosystems self-sufficient toward green and sustainable approaches. Medicinal plants with a host set of metabolites and endophytes with another set of secondary metabolites interact in a highly complex manner involving adaptive mechanisms, including appropriate cellular responses triggered by stimuli received from the sensors situated on the cytoplasm and transmitting signals to the transcriptional machinery in the nucleus to withstand a stressful environment effectively. Signaling pathways serve as a crucial nexus for sensing stress and establishing Frontiers in Plant Science CITATION Pandey P, Tripathi A, Dwivedi S, Lal K and Jhang T (2023) Deciphering the mechanisms, hormonal signaling, and potential applications of endophytic microbes to mediate stress tolerance in medicinal plants.
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The fruit ripening behavior of the green ripe mutant ( Gr ) of tomato ( Lycopersicon esculentum Mill.) was examined. Green ripe fruit are climacteric and evolve increasing amounts of ethylene after harvest; however, the time course for these events is dramatically altered in comparison with ‘Rutgers’. Maximal rates of C 2 H 4 evolution from Gr fruit were achieved 20 days after the initial increase, and 7 to 10 days prior to maximal respiratory rates. Fruit age at harvest did not affect either the rate or the magnitude of these processes. Wavelength scans of pigment extracts from 60 day postharvest Gr fruit indicated low levels of carotenoids. Mutant fruit also remain firm a long time after harvest. Polygalacturonase activity in Gr fruit increases with fruit age, but reaches only 3% to 5% of the total activity in ‘Rutgers’. PG activity was only slightly reduced when extracts were heated to 65°C for 5 min, suggesting that a heat stable isoenzyme of PG is predominant in mature mutant fruit in contrast to ‘Rutgers’ in which 90% to 95% of PG activity in ripe fruit is heat labile under these conditions. When heterozygous, the Gr mutation is dominant in its effects on total PG activity and on the time course from the initiation of C 2 H 4 and respiratory increases to their maximal rates of evolution. The magnitude of other changes in heterozygous mutant fruit was intermediate between normal and homozygous Gr .
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Transgenic Cantaloupe Charentais melons (Cucumis melo var. cantalupensis Naud. 'Vedrantais') exhibiting strong inhibition of ethylene production were used as a model to discriminate between ethylene-regulated and ethylene-independent ripening pathways. Compared to wild-type fruit, transgenic fruit did not undergo significant yellowing of the rind and softening of the flesh. However, these effects were completely reversed by treating transgenic fruit with 50 μL·L-1 exogenous ethylene. Pigmentation of the flesh occurred early before the onset of the climacteric and was thus unaffected by ethylene inhibition in transgenic fruit. Total soluble solids accumulated at the same rate in both types of fruit until 38 days after pollination when wild-type fruit abscissed. However, as ethylene-inhibited fruit failed to develop a peduncular abscission zone, they remained attached to the plant and accumulated higher amounts of sugars, mainly sucrose. Harvesting transgenic fruit resulted in a small but significant increase of internal ethylene associated with softening of the flesh.
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Ripening is a dramatic event in the development of many fleshy fruits. Tomato ripening involves a number of chemical and physical changes which convert the fruit from a relatively inedible state to one of optimal quality (2, 19). These changes appear to be highly synchronized, as evidenced by the fact that respiratory patterns, rate of ethylene production, carotene development, and flavor and textural changes normally associated with the ripening process, occur in close succession during the relatively short period in which the fruit ripens (2, 22). The association of these changes with seed maturation supports the popular view that ripening is of adaptive significance in seed dispersal by rendering fruit attractive to animals responsible for dispersal.
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Greenhouse-grown bell pepper (Capsicum annuum L. 'Robusta') were harvested at five stages of maturation (10% red to full red) in early winter 2002 (Expt. 1) and at two stages (10% red and full red) in early Spring 2002 (Expt. 2). The fruit were subsequently stored at 20°C in a continuous-flow chamber consisting of either 100 μL·L-1 ethylene (balance air) or air-only (control) at 90% relative humidity (RH). Individual fruit were removed from the chambers upon reaching full red color, and stored at -30°C until physicochemical analyses were conducted. Harvest maturity, and ethylene exposure had no appreciable effect on pulp soluble solids content, total titratable acidity or pH. Exposure to ethylene hastened ripening time compared to the air control but was independent of fruit maturity at harvest. Fruit exposed to ethylene reached full-red color 6.4 days (Expt. 1) and 4 days (Expt. 2) earlier than air-only fruit, respectively. There were no significant phytochemical and antioxidant differences noted for total carotenoids, total ascorbic acid, and soluble phenolics at various maturity stages due to ethylene exposure. Appreciable differences were observed between the two experiments for phytochemicals and antioxidants, as bell peppers from the latter experiment contained at least twice the concentrations of phytochemicals and antioxidant capacity as those from the first experiment. Differences in these parameters between experiments were attributed to environmental factors such as average temperature, day length, and light intensity. Ethylene was demonstrated to be an effective postharvest treatment for accelerating color change in this bell pepper cultivar, permitting earlier harvest without altering phytochemical synthesis rates.
Ethylene influences a number of developmental processes and responses to stress in higher plants. The molecular basis for the action of ethylene was investigated in mutants of Arabidopsis thaliana that have altered responses to ethylene. One mutant line, which has a dominant mutation at a locus designated etr, lacks a number of responses to ethylene that are present in the wild-type plant. These include inhibition of cell elongation, promotion of seed germination, enhancement of peroxidase activity, acceleration of leaf senescence, and feedback suppression of ethylene synthesis by ethylene. These diverse responses, which occur in different tissues of Arabidopsis, appear to share some common element in their transduction pathways—for example, a single receptor for ethylene. Results of ethylene binding experiments in vivo indicate that this receptor may be affected by the etr mutation.
The onset of apple [Malus sylvestris (L.) Mill. Var. domestica (Borkh.)Mansf.] fruit maturity is preceded by the production of ethylene, the ripening hormone, which induces fruit ripening. The amount of ethylene produced by the fruit correlates with the level of transcription of the ripening-specific 1-aminocyclopropane-1-carboxylate (ACC) synthase genes. We have found that an allele (MdACS1-2), which contains an inserted retroposon-like sequence at the 5′-flanking region, is transcribed at a lower level than the wild-type (MdACS1-1). MdACS1-2/2 homozygous fruit produce a lower level of ethylene at the climacteric stage than do the wild type fruit. We have also found that the preharvest drop rates of apple cultivars and strains of MdACS1-2/2 trees have less fruit drop than the MdACS1-1/1 or MdACS1-1/2 trees. Treatment of the MdACS1-1/2 trees with 1-MCP, an ethylene receptor blocker, further decreased fruit drop. Analysis of commercial apple cultivars for the presence of the MdACS1-2/2 allele may help in the early detection of apple cultivars with a low fruit drop rate.
Ethylene production and fruit softening during postharvest storage of several apple ( Malus domestica Borkh.) ripening variants were compared with two standard cultivars. PA14-238 and D101-110 produced only low levels of ethylene (<10 μl·kg –1 ·hour –1 ) at harvest and throughout most of 86 days of storage at 4C, whereas `Red Chief Delicious' and `Golden Delicious' fruit produced >100 μl ethylene/kg per hour during the same time period. PA14-238 and D101-110 flesh disks converted aminocyclopropane-1-carboxylic acid (ACC) but not methionine (MET) to ethylene. `Red Chief Delicious' readily converted both MET and ACC to ethylene at the end of cold storage. PA14-238 fruit were the firmest and did not soften during postharvest storage; however, D101-110 softened appreciably. NJ55 did not produce ethylene at harvest, but produced a significant amount of ethylene (90 μl·kg –1 ·hour –1 ) during storage. Despite its high capacity to produce ethylene, NJ55 remained nearly as firm as PA14-238 at the end of cold storage.
Bell peppers (Capsicum annuum L.) are classified as nonclimacteric fruits while some hot peppers have been reported as climacteric. Responses of peppers to exogenously applied ethylene-releasing compounds suggest ethylene involvement in the ripening process. Ethylene production and respiration rates in 13 cultivars of pepper: 'Camelot', 'Cherry Bomb', 'Chiltepin', 'Cubanelle', 'Banana Supreme', 'Habanero', 'Hungarian Wax', 'Mesilla', 'Mitla', 'Savory', 'Sure Fire', 'Tabasco', and 'King Arthur' were studied under greenhouse and field conditions. Fruit from each cultivar were harvested at different maturity stages determined by color, ranging from mature-green to full red-ripe. Carbon dioxide and ethylene production were measured by gas chromatography. Both variables were significantly different among maturity stages for all cultivars. Respiration rates were between 16.5 and 440.3 mg·kg-1·h-1 CO2. Ethylene production ranged from 0.002 to 1.1 μL·kg-1h-1. Two patterns of CO2 production were identified: higher CO2 production for mature-green fruit with successive decreases for the rest of the maturity stages or lower respiration rates for mature-green fruit with an increase in CO2 production either when fruit were changing color or once fruit were almost totally red. A rise in CO2 production was present for most cultivars. Ethylene evolution increased significantly at maturity or before maturity in all cultivars except 'Cubanelle' and 'Hungarian Wax'. Respiration rates and ethylene production were significantly different among cultivars at the mature-green and red stages.