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Harry J. Klee1 and James J. Giovannoni2
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1
University of Florida, Horticultural Sciences, Gainesville, Florida 32611;
email: hjklee@ifas.u.edu
2
Boyce Thompson Institute for Plant Research and United States Department of
Agriculture Robert W. Holley Center, Cornell University, Ithaca, New York 14853

Annu. Rev. Genet. 2011. 45:4159 Keywords

The Annual Review of Genetics is online at
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eshy fruits, seed dispersal, hormone signaling, MADS-box,
transcription factor
Abstract
Tomato ripening is a highly coordinated developmental process that
coincides with seed maturation. Regulated expression of thousands of
genes controls fruit softening as well as accumulation of pigments, sug-
ars, acids, and volatile compounds that increase attraction to animals.
A combination of molecular tools and ripening-affected mutants has
permitted researchers to establish a framework for the control of ripen-
ing. Tomato is a climacteric fruit, with an absolute requirement for the
phytohormone ethylene to ripen. This dependence upon ethylene has
established tomato fruit ripening as a model system for study of reg-
ulation of its synthesis and perception. In addition, several important
ripening mutants, including rin, nor, and Cnr, have provided novel in-
sights into the control of ripening processes. Here, we describe how
ethylene and the transcription factors associated with the ripening pro-
cess t together into a network controlling ripening.

41
 GE45CH03-Klee ARI 1 October 2011 15:38

INTRODUCTION ripening. Ethylene is not essential for ripening

of nonclimacteric fruits, and exogenous appli-
Ripening of eshy fruits is a complex and
cation of ethylene does not initiate ripening the
highly coordinated developmental process that
way it can in unripe climacteric fruits, although
coincides with seed maturation. The many
it can alter ripening characteristics in some
biochemical processes associated with ripening
species (e.g., color enhancement of citrus).
require changes in expression of hundreds
The most studied model of eshy fruit
to thousands of genes (2). Ripening involves
ripening is without doubt the tomato, Solanum
softening of the fruit tissues to facilitate seed
lycopersicum1 . Tomato is one of the most
dispersal, and the activities hypothesized
important horticultural crops and an important
to impact textural modications have been
source of nutrients worldwide. Indeed, in the
frequent targets of physiological and molecular
United States and many Western countries,
studies (37, 66). Although many fruits simply
although not the highest fruit or vegetable
decay and release seeds in the immediate
in vitamin A and C content, it is the major
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proximity of the parental plant, many other

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dietary source of these nutrients because

species have evolved to produce fruits that are
of high per capita consumption. Tomato
eshy, succulent, and attractive to the animals
production and consumption is increasing
that consume them, thus liberating the seeds
the world over (http://usda.mannlib.cornell.
from the protective fruit organ and dispersing
edu/MannUsda/viewDocumentInfo.do?
them over great distances. In addition to
documentID=1210). The tomato fruit is
softening, this latter class of fruits typically
climacteric, with an absolute requirement
exhibits increased accumulation of sugars,
for ethylene to ripen. This requirement has
acids, and volatile compounds that increase
made it an excellent model not only for the
interest and palatability to animals. In many
developmental control of ripening but also for
instances, especially for agriculturally impor-
study of ethylene synthesis and perception.
tant fruits, there is also a distinct color change
There are many well-characterized mutants
that acts as a signal to animals that the fruit is
altered in all aspects of fruit development
mature. Additional fruit attributes, including
and ripening, and for which many of the
early maturity, enhanced color, and increased
underlying genes have been cloned (reviewed
size, often reect the selection of so-called
in 28). There is also incredible diversity in
domestication traits, which have facilitated
fruit biochemistry, morphology, and ripening
current interest and use of fruits by humans.
behavior within the close relatives of tomato
Although we tend to think of ripe as a
that constitute Solanum sect. lycopersicon (57).
distinct developmental stage, it is actually an ar-
Most of these species produce fertile offspring
bitrary point of maximum palatability in a con-
when crossed with the domesticated tomato,
tinuous process leading to senescence of the en-
and the genetics of interspecies crosses has
tire fruit organ. Fleshy fruits are physiologically
been extensively exploited (21). Tomato was
classied as climacteric or nonclimacteric. Cli-
one the rst species to be genetically trans-
macteric fruits such as tomato, apple, banana,
formed by Agrobacterium tumefaciens (23). A
and avocado are characterized by an increase
high quality draft sequence of the 950 Mb
in respiration and concomitant increase in syn-
genome is available, as are sequences of related
thesis of the phytohormone ethylene upon ini-
species (http://solgenomics.net/).
tiation of ripening. Ethylene synthesis is essen-
tial for normal fruit ripening in these species,
and blocking either synthesis or perception pre-
1
Tomato has been recently renamed from Lycopersicon escu-
vents ripening. Nonclimacteric fruits such as lentum. This causes some confusion in gene nomenclature.
citrus, strawberry, and grape, in contrast, do Genes isolated prior to approximately 2007 generally con-
not exhibit increased respiration at the onset of tain an Le prex while genes isolated after the nomenclature
change have an Sl prex.

42 Klee Giovannoni
 GE45CH03-Klee ARI 1 October 2011 15:38

ETHYLENE AND TOMATO size the differences between the two systems as
FRUIT RIPENING they relate to the biology of fruit development.
Phytohormone action is generally mediated
at multiple levels. Synthesis and degradation Regulation of Ethylene Synthesis
of each hormone are highly regulated. Signal During Fruit Development
transduction is also a critical aspect of hormone
Ethylene is the simplest of plant hormones,
action and is generally regulated at multiple
consisting of just two carbons and four hydro-
levels. One of the emerging themes with regard
gens. It is an important hormone, mediating
to transduction of hormone signals in recent
many aspects of plant responses to biotic and
years is the importance of the ubiquitin protein
abiotic stresses. In response to most stresses,
degradation system (65). Most, if not all,
plants produce ethylene, which in turn slows
hormone signaling systems involve continuous
down plant growth. As a readily diffusible gas,
synthesis and degradation of one or more criti-
ethylene is an ideal stress hormone. While
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cal signaling intermediates. Hormone action is

Annu. Rev. Genet. 2011.45:41-59. Downloaded from www.annualreviews.org

under stress, plants produce it and when the

mediated by stabilization of these components
stress is removed, the hormone rapidly diffuses.
as opposed to de novo synthesis. This seemingly
Ethylene also mediates certain developmental
wasteful approach to signal transduction must
processes, most notably early fruit development
confer an evolutionary advantage. Because
and ripening in addition to organ abscission. As
plants cannot move, a rapid response to external
would be expected for such an important hor-
inputs is essential. Having the signaling compo-
mone, ethylene synthesis is highly regulated.
nents in place must facilitate signicantly faster
The biosynthetic pathway is simple, consisting
responses. With ATP being a relatively cheap
of only two enzymes. S-adenosylmethionine
commodity in plants, an ability to rapidly
is converted to 1-aminocyclopropane-1-
respond to external environmental stimuli
carboxylate (ACC) by ACC synthase (ACS).
balances the seemingly high metabolic cost
ACC is subsequently converted to ethylene by
associated with constant synthesis and degrada-
ACC oxidase (ACO). All available data support
tion of multiple proteins. Ethylene in tomato
a model in which formation of ACC is the rate-
follows the same basic principles as other
limiting step. ACO is not normally limiting for
hormones; synthesis and perception are highly
ethylene synthesis and although induced during
regulated events. Given that ethylene is a read-
ripening, substantial ACO activity is present
ily diffusible gas, degradation or modication
prior to ripening initiation. A major point of
of the hormone are not important regulatory
regulation for ethylene synthesis occurs at the
constraints.
level of ACS transcription (4, 52, 64). There
Because ethylene is such an agriculturally
are at least eight characterized ACS genes in
important hormone, tomato has been a useful
tomato and at least three more identied in the
model system for its study for many years, and
tomato genome sequence, and each one has a
we know as much about the regulation of its
distinctive pattern of tissue and stimulus speci-
synthesis in tomato as in any plant species. In
city. There are four characterized ACO genes
contrast, much of our knowledge concerning
in tomato with at least three additional genes
ethylene signal transduction is based on pio-
found in the genome sequence. Even though
neering work done in Arabidopsis (reviewed in
ACO activity is not limiting, certain ACO genes
13). Although there are very signicant differ-
are ethylene inducible, particularly in ripening
ences in the regulation of ethylene signaling in
fruits (5). An antisense gene targeting ACO1,
terms of gene family composition and expres-
the most highly induced ACO during ripening,
sion when comparing Arabidopsis and tomato,
prevents ethylene synthesis and ripening (32).
the fundamental genetic building blocks of the
During ripening, expression of two genes,
two systems are very similar. Here, we empha-
LeACS2 and LeACS4, is signicantly increased,

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ARI 1 October 2011 15:38
indicating that a major point of ripening regu-
lation is at the level of ACS transcription (5, 64).
An antisense gene targeting LeACS2 was highly
effective in preventing fruit ripening. The anti-
sense construct also effectively reduced LeACS4
(54). Recent work indicates another level of
ACS regulation (40). LeACS2 enzyme is sta-
bilized by phosphorylation and degraded after
dephosphorylation. LeACS2 protein level in-
uences cellular ACS activity, ACC content,
and ethylene production in fruit tissue. The re-
sults suggest that posttranslational regulation
by phosphorylation, mediated by a calcium-
dependent protein kinase, is an important regu-
latory point in the control of ethylene synthesis.
The essential nature of ethylene for fruit
ripening has been validated both by chemical
inhibitors of ethylene synthesis or perception
and by transgenic manipulation of ethylene
synthesis. Antisense genes targeting ACS (54)
and ACO (32) are highly effective in reducing
ethylene synthesis and delaying ripening. Sim-
ilarly, expression of a bacterial ACC deaminase
gene that metabolizes ACC to -ketoglutarate
also effectively reduces ethylene synthesis and
delays ripening of transgenic fruits (42).
THE ETHYLENE SIGNALING
PATHWAY IN TOMATO
Receptors
Plant ethylene receptors are ancestrally related
to bacterial two-component regulators. They
are endoplasmic reticulumassociated integral
membrane proteins with protein kinase ac-
tivities (Figure 1) (25, 51). Genetic analysis
in tomato and Arabidopsis has shown that
the receptors act as negative regulators of the
ethylene response pathway (35, 72). In the
absence of the hormone, receptors actively
suppress ethylene responses. Upon ethylene
binding, that suppression is removed and the
response occurs. The only single receptor
mutations that have observable phenotypes
confer dominant ethylene insensitivity. One
of the earliest known tomato fruit ripening
mutants is the dominant Never-ripe (Nr)
44 Klee Giovannoni
mutation (61). Nr fruits do not ripen, even
when exposed to ethylene. Flowers do not
senesce or abscise following fertilization nor do
seedlings respond to ethylene, indicating that
this mutation confers ethylene insensitivity
throughout the plant. Nr was originally isolated
as a spontaneous mutation in which a sector
within a single fruit in a commercial eld did
not ripen (C. Rick, personal communication).
The mutation was subsequently shown to be
the consequence of a single amino acid change
in the NR ethylene receptor (44). The lack
of Nr ripening conrms the essentiality of
ethylene perception for ripening.
In tomato, there are seven ethylene recep-
tor genes (LeETR1, LeETR2, NR, LeETR4,
LeETR5, LeETR6, and LeETR7) (45, 71, 81, 87).
Examination of the recently released tomato
genome sequence indicates that this is the en-
tire repertoire of tomato ethylene receptors.
Five of these receptors have been shown to bind
ethylene; two, LeETR6 and LeER7, were not
tested (53). Based on gene and protein struc-
tures, the ethylene receptors are divided into
subfamily 1 and subfamily 2. The subfamily
1 members have the highest similarity to his-
tidine kinases, whereas the subfamily 2 mem-
bers have diverged and acquired serine kinase
activities (51). Functional analyses have indi-
cated that no single Arabidopsis loss-of-function
mutation among its set of ve ethylene recep-
tors has a major effect on ethylene responses,
indicating a degree of functional redundancy.
However, subfamily 1 members are more crit-
ical for ethylene signaling, and they cannot be
functionally replaced by subfamily 2 members
(80). Based on knock-down antisense trans-
gene analyses, the tomato receptor system func-
tions very differently. Reduced expression of
either subfamily 2 receptor gene, LeETR4 or
LeETR6, results in substantially increased ethy-
lene sensitivity. Antisense plants with greatly
reduced expression of either of these two recep-
tors show phenotypes consistent with a consti-
tutive ethylene response, including signicantly
earlier fruit ripening (41, 72). This enhanced
ethylene sensitivity can be restored to wild type
by overexpression of the subfamily 1 receptor,
 GE45CH03-Klee ARI 1 October 2011 15:38

Ethylene binding
S S S S
S S S S Membrane E3
C2H4 C2H4
Cu Cu
Ubi

Histidine
P P
kinase

Receiver

Active suppression of
ethylene response

CTR1 EIN2
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EBF
?
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Ubi

Ethylene
EIL ERF responses

Figure 1
The ethylene signaling pathway. Ethylene receptors (upper left), shown as disulde-linked dimers, actively
suppress ethylene responses in the absence of ethylene. Upon ethylene binding, receptors undergo a physical
change that permits them to be targeted for degradation by an as yet unidentied E3 ligase. Although
receptors have protein kinase activity, it is not known whether the active or inactive form of the receptor is
phosphorylated. Given that ubiquitination is usually associated with phosphorylated forms of proteins, the
phosphorylated receptor is shown here as the form targeted for degradation. Receptors physically interact
with the negative regulator CTR1. Ein3-like transcription factors (EILs) either activate transcription of ERF
family members or are targeted for degradation by the EIN3 binding factor E3 ligases. Although EIN2 is
required for ethylene signal transduction, genetic analysis places it downstream of CTR1 and upstream of
the EIL family. Its mechanisms of action are undetermined.

NR. LeETR4, LeETR6, and NR expression in-
creases signicantly at the onset of fruit ripen-
ing, and these three receptor genes are by far
the most highly expressed in ripening fruits.
Loss-of-function for any of the other receptors
has no effect on ethylene sensitivity or ripening
behavior (41). Together these results indicate
that the subfamily 2 receptors have far more im-
portant functions in tomato than in Arabidopsis.
Nonetheless, generalized receptor function is
conserved across species. Reduction of receptor
content increases ethylene sensitivity (11, 31,
35, 41, 72), whereas increased receptor content
has the opposite effect (16).
Although the ethylene receptors all have
protein kinase activity, the mechanism whereby
signal transduction occurs has yet to be fully
determined. The best model proposes that the
receptors are in a functionally on state in the
absence of ethylene, consistent with the many
dominant mutations that confer constitutive
ethylene insensitivity. At least some of these
mutants fail to bind ethylene (67) and therefore
cannot be converted to the off state, per-
mitting ethylene signaling to proceed. Such a
model explains chemical inhibition of ethylene
action as well. Silver (I) ions can replace copper
(I) ions within the receptor complex, blocking
the ability of the receptor to be converted
to the off state (63). Similarly, the potent
ethylene analog, 1-methylcyclopropene, binds
irreversibly to the receptor complex, prevent-
ing ethylene binding and locking the receptor
in the on state (69). There is no conclusive

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ARI 1 October 2011 15:38
genetic evidence indicating precisely what the
on and off states of the receptors are at of the dominant etr11 receptor mutant. The
a molecular level. Work with tomato fruit
provides important information about the
receptors. Consistent with experimental evi-
dence, the negative regulation model predicts
that more receptor reduces and less receptor
increases ethylene sensitivity. It, therefore,
seems paradoxical that expression of multiple
receptor genes is very signicantly increased
at the onset of fruit ripening, a point when
ethylene action is absolutely essential. This
paradox was addressed by Kevany et al. (41).
Using antibodies against each of the three most
abundantly expressed receptors, these authors
showed that ethylene binding triggers receptor
protein degradation. Thus, although the genes
encoding the receptors are transcriptionally
activated during ripening, the actual levels of
receptor proteins signicantly drop, remaining
low throughout ripening. Thus, the receptor
off state may indeed be degradation of the
proteins following ethylene binding. As with
other hormone signaling systems, receptor
degradation is likely mediated by the 26S
proteasome-dependent pathway, although the
specic E3 protein(s) targeting degradation
have not yet been identied.
Green-Ripe. Green-Ripe (Gr) and its allele, Nr-
2, are also dominant nonripening mutants (4).
Their phenotypic similarity to Nr suggested
that they might be receptor mutants. However,
nonfruit tissues show only very slight or no
reduction in ethylene sensitivity. Interestingly,
the Gr mutation results from dominant over-
expression of the otherwise normal GR protein
in fruit, a tissue where this gene is not normally
highly expressed. GR is a member of a small
multigene family in tomato that includes GRL1
and GRL2, where GR has no apparent Arabidop-
sis ortholog (GRL1 and GRL2 do have Arabidop-
sis orthologs). Transgenic overexpression of
GRL1 or GRL2 does not impact fruit ripening
or ethylene responsiveness. At the same time
that Gr was being characterized an Arabidopsis
homolog, RTE1 (which is most closely related
to GRL1), was identied (60). This latter
46 Klee Giovannoni
mutant was identied in screens for revertants
evidence from both species suggests that GR
and RTE1 interact with ethylene receptors to
modify their action in an as yet unknown way,
although in the case of the Gr mutation the
GR protein may be interfering with normal
activity of a related protein in the fruit.
The CTR family. The single-copy CTR1
gene was rst identied in Arabidopsis. Loss-
of-function mutants exhibit a constitutive
ethylene response, indicating that its function
is to suppress the ethylene signaling pathway.
Unlike Arabidopsis, there are four genes whose
proteins exhibit signicant homology to
CTR1. Three of these genes (tCTR1, tCTR3,
and tCTR4) can functionally complement the
Arabidopsis ctr1 mutant, indicating a degree
of functional redundancy. Expression anal-
ysis suggests that tCTR1 is the most highly
expressed member of this family in the fruit
and its mRNA abundance is induced during
ripening and by ethylene (1, 46).
The ethylene-inducible transcription fac-
tors. At the bottom of the signaling path-
way are sets of transcription factors that acti-
vate ethylene-responsive genes. First, there is a
family of four Arabidopsis EIN3-related genes,
LeEIL14 (74, 84). Antisense knock-downs of
LeEIL1, LeEIL2, and LeEIL3 signicantly re-
duced ethylene sensitivity. All three of the genes
are expressed throughout the plant, and each
individual gene was capable of complementing
an Arabidopsis ein3 knockout. These results are
consistent with these three genes having redun-
dant function in tomato. However, Chen et al.
(14) reported differential restoration of ethy-
lene responsiveness between various tissues of
a Nr mutant upon introduction of an LeEIL1
overexpression construct.
In Arabidopsis, EIN3 protein levels are medi-
ated by a family of EIN3-binding factor (EBF)
F-box proteins that target them for degradation
by the ubiquitin/26S proteasome pathway (24,
30, 58). Two EBF genes, SlEBF1 and SlEBF2,
have been identied in tomato (83). The two
 GE45CH03-Klee ARI 1 October 2011 15:38
genes are functionally redundant. Reduced ex-
pression led to constitutive ethylene responses
and earlier fruit ripening, indicating that sta-
bilizing the LeEIL proteins activates ethylene
signaling.
At the bottom of the signaling cascade is a
family of transcription factors, the ethylene re-
sponse factors (ERFs), that are members of the
large AP2/ERF superfamily. Their expression
is activated by the EIN3-like EILs. Because this
is such a large family, it is assumed that there
will be a high degree of redundancy. No specic
functions have been assigned to any individual
family members in tomato.
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System 1 versus system 2 ethylene and
the transition to ripening. In tomato fruits,
ethylene has profoundly different effects de-
pending on the stage of development. There
is a distinct developmental switch that occurs
upon fruit maturation (reviewed in 27). Ex-
posure of immature fruits to ethylene results
in what has been termed system 1 ethylene
synthesis (82), characterized by induction of
a set of ethylene-regulated genes (2), autoin-
hibitory ethylene synthesis, and failure to ripen.
In contrast, mature fruits exhibit system 2 ethy-
lene synthesis characterized by induction of a
larger, partially overlapping set of genes (2),
autocatalytic ethylene synthesis, and ripening.
Most notably, system 2 involves induction of
the ripening-associated and ethylene-inducible
ACS2 gene, leading to autocatalytic ethylene
synthesis. Thus, both immature and mature
fruits respond to ethylene but in fundamentally
different ways. Although applied ethylene does
not initiate ripening in immature fruits, it does
signicantly hasten the onset of maturity (82);
the more ethylene to which an immature fruit
is exposed, the earlier it ripens. Similar effects
have been observed in banana, in which Burg &
Burg (10) demonstrated that treatment of im-
mature green banana fruits shortened the time
to ripening relative to untreated controls. This
hastening of the onset of ripening indicates
that there is a molecular mechanism whereby
fruits measure cumulative ethylene exposure
such that downstream development is altered.
This feature of ripening biology may be espe-
cially important for climacteric fruits that form
in clusters but where individual fruits initiate at
different times (such as banana and tomato) to
better synchronize maturity of all fruit on the
inorescence.
Consistent with the earlier ripening of fruits
exposed to exogenous ethylene, alterations in
ethylene signaling capacity also inuence the
time of ripening onset. Depletion of either
LeETR4 or LeETR6 expression has the effect
of increasing ethylene sensitivity and results
in earlier fruit ripening (41). Similarly, deple-
tion of SlEBF gene expression hastens the on-
set of ripening, presumably the consequence of
LeEIL accumulation (83). The observation that
ethylene binding triggers receptor degradation,
combined with the negative regulation model,
suggests that receptor levels might be critical
for modulating the timing of ripening. Imma-
ture fruits do synthesize a low basal level of
ethylene throughout development. It is tempt-
ing to postulate that depletion of receptors to
some critical threshold could trigger ripening.
Consistent with that model, exposure of imma-
ture fruits to ethylene does cause both recep-
tor depletion and earlier onset of ripening (41).
The results are consistent with a model in which
receptor levels modulate timing of the onset of
fruit ripening by measuring cumulative ethy-
lene exposure. Nevertheless, induction of sys-
tem 2 ethylene-responsive genes such as ACS2
suggests the presence of additional and specic
regulators that inuence and initiate the fruit
ripening process, including ethylene synthesis
genes.
TRANSCRIPTIONAL CONTROL
OF FRUIT RIPENING
The comprehensive molecular analyses of
ethylene synthesis and signal transduction dur-
ing ripening suggest the presence of additional
regulatory systems that coordinate the ethylene
burst and production of this necessary ripening
hormone. A number of interesting tomato
ripening mutants have been collected by
breeders and geneticists, and their subsequent
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ARI 1 October 2011 15:38
physiological characterization suggests that
they may represent defects in ripening regu-
latory systems. These mutations include the
ripening-inhibitor (rin), nonripening (nor), and
Colorless nonripening (Cnr) mutations that are
nonallelic but share common physiological
characteristics in that all (a) develop to the
mature green stage, where the fruit is full size
and the seeds are mature, yet do not advance to
ripening; (b) fail to undergo the climacteric rise
in respiration or produce ripening-associated
ethylene; (c) do not ripen in response to ex-
ogenous ethylene; yet (d ) respond to ethylene
in other tissues and fruit in which ethylene-
responsive genes are induced (20, 28, 50).
Together, these characteristics suggest that
all three mutations impact central ripening
phenomena necessary for induction of ethylene
in addition to activities for which ethylene
alone cannot compensate. In this latter regard,
it is a reasonable hypothesis that such genes
might represent conserved regulators impact-
ing ripening even in nonclimacteric fruits. All
three genes have been isolated by positional
cloning, and all encode transcription factors.
There is only one described rin mutation,
and it also displays a large sepal, or macrocalyx,
phenotype. The rin locus encodes a MADS-
box transcription factor termed RIN-MADS,
which is induced at the onset of ripening (78).
MADS-box genes are developmental regula-
tors conserved in eukaryotes and associated
with oral development in plants, including
members of the ABC model of oral devel-
opment (17). RIN-MADS is a member of the
SEPALLATA clade associated in Arabidopsis
with E function activities that determine the
growth and development of oral organs (86).
Phylogenetic analysis of the RIN gene and
examination of the tomato genome sequence
(http://solgenomics.net) indicate two ad-
ditional genes as compared to Arabidopsis,
including RIN-MADS, with the additional
gene having no transcriptional support in the
public tomato expressed sequence tag (EST)
collection. This suggests that it may have very
limited expression or be a pseudogene. The
RIN-MADS gene resides on chromosome 5
48 Klee Giovannoni
adjacent to the tomato APETALA1 ortholog,
MACROCALYX (MC). The rin mutation re-
sults from a spontaneous deletion that removes
the C-terminus of the RIN-MADS gene and
approximately 1 kb of the sequence separating
RIN-MADS from MC. The result is expression
of a chimeric RIN-MADS/MC mRNA that
has no apparent RIN-MADS or MC function.
The ripening and large sepal phenotypes of
the rin allele were recreated independently by
antisense repression of RIN-MADS and MC,
respectively (78). Screening of a strawberry
fruit cDNA library with a tomato RIN-MADS
cDNA yielded a homologous gene whose
expression was elevated during ripening. The
identication of a strawberry homolog of
RIN-MADS indicates that transcriptional
control of ripening is conserved among cli-
macteric and nonclimacteric species (67a, 78).
Similar banana genes associated with both peel
and pulp ripening have also been described,
although they remain to be functionally tested
(19). If these genes indeed control ripening
this would suggest that regulation of ripening
is a conserved function of the MADS-box
family preceding the divergence of dicots and
monocots. It is noteworthy that the original rin
allele is extensively used in tomato hybrid seed
production for heterozygous varieties char-
acterized by long shelf life and fruit rmness,
demonstrating through its practical utility
that RIN-MADS exerts broad control over
the ripening process (color, avor, texture).
RIN-MADS, as with many genes of this family,
has been shown to interact with CArG-box
elements in promoters of ACS2 and ACS4,
in addition to cell wall hydrolases, indicating
that it interacts with promoters of genes
representing a range of ripening activities (22).
CNR encodes a transcription factor of
the SQUAMOSA PROMOTER BINDING
PROTEIN family (SPBP), which is regulated
at least in part by changes in promoter methy-
lation in conjunction with ripening that do not
occur in the Cnr mutant (50). CNR expression
is reduced in the rin mutant, suggesting that
it may act downstream of RIN-MADS in the
regulatory hierarchy, although the relationship
 GE45CH03-Klee ARI 1 October 2011 15:38
among these regulators and ethylene is likely
not linear. SPBP targets include MADS-box
genes and the tomato ortholog of the Arabidop-
sis FRUITFUL (FUL) MADS-box gene, TDR4,
is substantially reduced in Cnr, suggesting that
it may be a CNR target and a regulator through
which CNR exerts its effects (50). Although
TDR4 repression was reported to yield no ob-
vious ripening phenotype in tomato, antisense
of an orthologous gene in billbery resulted in
reduced fruit pigmentation (39).
Most of the well-characterized tomato
ripening mutants have been cloned, but addi-
tional regulators certainly remain to be iden-
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tied. Transcriptional proling studies indi-
cate that hundreds of transcription factors show
differential expression during fruit develop-
ment, and many of these display expression
changes during ripening consistent with ripen-
ing functions (2). Some, such as LeHB-1, a HD-
zip homedomain protein that directly interacts
with and is necessary for ACO1 expression, may
regulate specic ripening processes. LeHB-1 re-
pression in transgenic plants results in reduced
ACO1 expression, a concomitant decrease in
ethylene synthesis, and delayed ripening (48).
TAGL1 (Tomato AGAMOUS-like 1) is a
member of the AGAMOUS clade of MADS-
box genes in tomato that also includes TAG1.
TAGL1 is induced both early in carpel devel-
opment and later at the onset of ripening. Re-
pression of TAGL1 via RNAi results in ripen-
ing inhibition and reduction in carpel thickness
due to fewer cell layers (38, 55, 79). The nor-
mal size and appearance of TAGL1-repressed
pericarp cells combined with TAGL1 expres-
sion in early fruit development suggests that this
effect results primarily from reduced cell divi-
sion. Fruits can be divided into dry and eshy
types, with the former dispersing their seeds via
wind, rain, attachment to animal fur, and dehis-
cence/shattering, and the latter through devel-
opment of succulent and attractive fruits that
are consumed by animal vectors of seed disper-
sal. Fleshy fruits therefore undergo a ripening
process that includes activities related to be-
coming attractive and palatable. TAGL1 repre-
sents a molecular link between these processes
of esh development and subsequent ripening.
In contrast to the SEPALLATA clade, which
may have expanded in tomato versus Arabidopsis
to facilitate eshy fruit ripening processes, the
AGAMOUS (AG) clade of MADS-box genes
is represented by four genes in both tomato
and Arabidopsis (33, 79). TAG1, the tomato or-
tholog of AG, was shown to confer a similar
homeotic function in carpel development to
its Arabidopsis counterpart (56). Interestingly,
TAG1 is expressed in ripening fruit and induced
when TAGL1 is repressed, suggesting a pos-
sible compensatory function during ripening.
The original TAG1 antisense experiments were
undertaken using the constitutively expressed
CaMV35s promoter and the resulting homeotic
conversion of carpels to petals left no fruit
for assessment of ripening function. However,
overexpression of TAG1 did result in eshy
expansion of, and lycopene accumulation in,
tomato sepals (56). In vitro growth conditions
that induced similar sepal phenotypes resulted
in TAG1 mRNA accumulation in sepals (8).
Transgenic overexpression of TAGL1 resulted
in sepal expansion and carotenoid accumula-
tion similar to that observed in TAG1 overex-
pression lines. It remains unclear whether both
genes play roles in expansion and/or ripening
or whether they are structurally similar enough
to confer similar phenotypes when ectopically
expressed. Repression of TAG1, specically, in
maturing fruit would address the function sug-
gested by these intriguing transgenic results and
its expression in ripening fruit.
The Arabidopsis orthologs of TAGL1 are the
paralogous SHATTERPROOF (SHP1, SHP2)
genes (49). Although TAGL1 activities are de-
velopmentally distinct from the role of the or-
thologous and parologous SHATTERPROOF
genes of Arabidopsis that control silique re-
plum development and shattering, the or-
thologous genes in both species regulate the
unique seed dispersal mechanisms of each fruit
type (i.e., eshy development and ripening
versus dehiscence). In contrast to the RIN-
MADS/SEPALLATA clade where gene family
expansion appears to have conferred a unique
function in terms of eshy fruit ripening, AG
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ARI 1 October 2011 15:38
clade genes have functionally differentiated be-
tween Arabidopsis and tomato to promote equiv-
alent functions in the context of distinct fruit
types.
The transcriptional regulators described
above represent positive effectors of the ripen-
ing pathways in that their absence through
mutation or transgenic intervention results
in reduced ripening phenotypes. A tomato
APETALA2 (AP2) gene family member,
SlAP2a has recently been shown to be a neg-
ative regulatory of ripening (15, 40a). SlAP2a is
a member of the AP2 subfamily of the AP2/ERF
superfamily, as it harbors two rather than one
conserved AP2 domain. SlAP2a is induced dur-
ing ripening, although its repression resulted
in accelerated ripening, elevated ethylene pro-
duction, and altered carotenoid accumulation.
All of these phenotypes were specic to the
fruit, suggesting SlAP2a is a fruit-specic and
ripening-related suppressor of ethylene pro-
duction and ripening.
In summary, it is clear that multiple tran-
scription factors inuence the ripening process
and that gene family expansion driven by poly-
plodization events contributed to the reservoir
of genes available for the present set of ripen-
ing regulators. The apparent conservation of
RIN-like MADS-box genes in ripening control
prior to the evolutionary split of monocots and
dicots suggests a conserved ripening function
in this important family of oral develop-
ment regulators. The contrast in activities of
AGAMOUS clade MADS-box genes suggests
other members of this family have diverged in
terms of specic function while retaining their
roles in seed dispersal via distinct activities in
very different (dry versus eshy) fruit types.
The fact RIN-MADS, NOR, CNR, and TAGL1
all play necessary roles in ripening indicates
that they are all involved either together or
separately in ripening control prior to ethylene.
The fact that ectopic expression of TAGL1
can apparently complement the rin and nor
mutations combined with its earlier expression
suggests a more primary position in this regu-
latory network (26), although it is curious that
the rin and nor mutants retain normal TAGL1
50 Klee Giovannoni
expression without ripening (79), suggesting
that the full complexity of this regulatory
mechanism remains to be uncovered. The
primary role of these regulators and their im-
pact on ethylene- and nonethylene-mediated
ripening activities is summarized in Figure 2.
COLOR, FLAVOR, AND
NUTRITION IN RIPE FRUITS
The Chloroplast to
Chromoplast Conversion
One of the most critical developmental acts
associated with ripening and palatability is the
conversion of chloroplasts to chromoplasts
(reviewed in 18). There are a number of
important structural changes that accompany
chromoplast conversion. These changes have
a major inuence on the nutrient and avor
composition of the fruit. The photosynthetic
capacity of the chloroplast is lost as the
thylakoid structures begin to disassemble.
Within the chromoplast, plastoglobules accu-
mulate. These are the sites for accumulation
of large quantities of carotenoids, principally
lycopene and -carotene, in the form of crystal
structures. Accumulation of these carotenoids
provides a visual indication that the fruit is
mature and suitable for consumption. These
carotenoids are also important human nutri-
ents, providing the precursor of Vitamin A as
well as abundant antioxidants to the diet.
The chromoplast and plastoglobule pro-
teomes have been examined in multiple
species. The tomato chromoplast is highly
metabolically active, and enzymes associated
with synthesis and metabolism of carotenoids,
amino acids, and fatty acids are detected (7). Al-
though the tomato plastoglobule proteome has
not been reported, that of the closely related
pepper has been determined (85). As is ex-
pected, the enzymes responsible for carotenoid
synthesis are detected within these structures.
The conversion of chloroplasts to chromo-
plasts and associated degradation of chlorophyll
and membrane structure/photosynthetic activ-
ity with concomitant carotenoid accumulation
 GE45CH03-Klee ARI 1 October 2011 15:38

Polyploidization events

Progenitor species Cultivated tomato

(Solanum lycopersicum)
Gene family expansion Functional divergence

RIN TAGL1

CNR TAG1
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NOR
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ACS, ACO genes AP2a HB-1 ACS, ACO genes

Nonethylene-regulated ripening genes Nonethylene-reguated ripening genes

SAM ACC C2H4
ACS ACO
Membrane
ETR
EIL
EILs CTR
Ethylene-regulated ripening genes
Ethylene signaling

Figure 2
Evolution and action of ripening transcription factors. Comparison of the tomato genome sequence to that
of grape reveals a history of both ancient and more recent polyploidization events that created a reservoir of
genes that would be permitted to evolve into fruit development and ripening functions. Within the
MADS-box family of transcription factors, for example, it appears that the SEPALLATA and CNR clades
(red ) expanded to include the RIN-MADS and CNR activities. In contrast, in the AGAMOUS MADS-box
clade it is more likely that functional divergence to promote seed dispersal in the context of a eshy ripening
fruit resulted without clade expansion (blue). The evolutionary origins of other ripening control genes
(orange) such as NOR and the SlAP2a and LeHB-1 ethylene synthesis regulators is less obvious, although they
and additional regulators such as RIN, CNR, and TAGL1 inuence ethylene synthesis genes, including
members of the ACS and ACO gene families. RIN, NOR, CNR, TAGL1, and possibly TAG1 are necessary
for expression of both ethylene-dependent and independent genes during ripening. As shown in Figure 1,
ethylene acts via conserved signal transduction machinery to modulate expression of the ethylene-regulated
subset of ripening genes.

are the main hallmarks of the ripening tran-
sition of tomato (and many fruits) and are
irreversible. The primary enzymatic regulator
of ux into the carotenoid pathway, phytoene
synthase (PSY), is under strong positive
ethylene control during ripening, indicating
a link between chromoplast development and
the primary ripening hormone. Also under
ethylene control is repression of the gene en-
coding the lycopene -cyclase (LYC) enzyme
that would otherwise convert red lycopene to
orange -carotene (2). Few regulatory genes
or activities specically involved in this process
beyond those involved in carotenoid synthesis
and chlorophyll degradation (chlorophyllase)
have been described. One gene clearly involved
in this transition is dened by the green-esh
( gf ) locus that encodes a STAY-GREEN
senescence-related regulator (6). The gf loss-
of-function mutations result in reduced plastid

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ARI 1 October 2011 15:38
conversion during ripening, yielding fruit
containing both chloroplasts and chromoplasts
and characterized by a brown or chocolate
color. Mutation in the orthologous pepper
gene has the same effect (6, 9), and such fruits
are used commercially because of their novel
appearance and avor characteristics.
Ripening and Flavor Chemistry
Most fruits, such as tomato, that are consumed
by humans have evolved to attract animal
seed dispersers. When tomato seeds are fully
mature, chemical changes occur in the fruit
to make it attractive to animals. Cell walls un-
dergo textural changes that result in softening.
As chloroplasts are converted to chromoplasts,
large amounts of carotenoids, principally
lycopene and -carotene, are synthesized, pro-
viding visual cues that the fruit is ripe. Ethylene
also induces changes in glycoalkaloid content
(36). High levels of -tomatine are present
in immature fruits, peaking at mature green.
Upon ripening, levels are signicantly reduced
in an ethylene-dependent manner (36). Given
that -tomatine is linked to bitter avor, the
effect is to increase palatability upon ripening
(62). Finally, certain chemicals that contribute
to taste begin to accumulate. In tomato, there
are three major classes of chemicals responsible
for avor: sugars, acids, and volatiles. Together
with textural and visual cues, these chemicals
stimulate the brain to produce a characteristic
tomato experience. Although sugars and acids
are absolutely essential for good taste, it is the
volatiles that really determine the unique avor
of a tomato (3). However, volatile synthesis and
regulation is the least studied aspect of avor.
Sugars and Acids
At the onset of ripening, starch that has ac-
cumulated throughout development is metab-
olized to glucose and fructose, the two main
sugars in a ripe fruit (34). These two sugars
can constitute 2%4% of the fresh weight of a
fruit. Organic acids, principally citric and malic
52 Klee Giovannoni
acids, are also abundant. The pH of a ripe fruit
is typically in the range of 4. Both the sugars
and acids are critical to good avor, although
different individuals have different preferences
for sweetness and acid balance. Interestingly,
recent work has shown that malic acid inu-
ences starch content via redox control of the en-
zyme responsible for starch synthesis, ADP glu-
cose pyrophosphorylase (12). Given that starch
synthesis is positively correlated with reducing
sugar content in ripe fruits, malic acid content
of the immature fruit is predicted to be inversely
correlated with reducing sugar content of the
ripe fruit. Indeed, we have validated this neg-
ative correlation in a large study of 80 differ-
ent heirloom tomato varieties (D.M. Tieman
& H.J. Klee, unpublished results).
Volatiles
A set of 2030 volatile chemicals positively
contribute to tomato avor; that is, they are
present in sufcient quantities to noticeably
stimulate the olfactory system. They are a
diverse set of chemicals derived from amino
acids, fatty acids, and carotenoids. Despite their
chemical diversity, they share one important
trait; each is derived from some chemical
that is essential for the human diet. Thus,
volatiles are derived from essential amino acids
(phenylalanine, leucine, and isoleucine) and
essential fatty acids (principally linolenic acid).
-carotene, the precursor of retinal (Vitamin
A) is the immediate precursor of one of the
most important avor volatiles, -ionone (29).
When we consider that there are approximately
1000 detectable metabolites in a tomato fruit
(36a), it is remarkable that virtually all of the
important avor volatiles are derived from
essential nutrients and is highly suggestive of
coevolution. Plants make the compounds that
are attractive to animals and animals, in turn,
have evolved to recognize and seek out the com-
pounds that indicate the presence of important
nutrients.
The content of most, although not all, of
the avor volatiles in a tomato increase at the
 GE45CH03-Klee ARI 1 October 2011 15:38

Table 1 Patterns of expression for a subset of the major flavor volatiles during fruit maturation. The precursors of the
volatiles are indicated: fatty acid (FA); branched chain amino acid (BCAA); phenylalanine (Phe). Levels of volatile
accumulation are normalized to mature green (MG) and shown for various ripening stages: breaker (Br), turning (Tu), fully
ripe (Red)
Volatile Precursor MG Br Tu Red
cis-2-penten-1-ol FA 1X 1X 2X 10X
trans-2-pentenal FA 1X 1X 1X 10X
cis-3-hexenal FA 1X 1X 1X 10X
trans-2-hexenal FA 1X 1X 2X 5X
trans-2-heptenal FA 1X 1X 2.5X 2.5X
-ionone Carotenoid 1X 1X 1X 10X
6-methyl-5-hepten-2-one Carotenoid 1X 1X 1X 10X
geranylacetone Carotenoid 1X 1X 1X 10X
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2-methylbutanal BCAA 1X 1.3X 1.5X 1.5X

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2-methyl-1-butanol BCAA 1X 1X 10X 10X

3-methylbutanal BCAA 1X 3X 5X 4X
3-methyl-1-butanol BCAA 1X 2X 10X 4X
isovaleronitrile BCAA 1X 1X 1X 10X
isobutyl acetate BCAA 1X 1X 1X 1X
isobutylthiazole BCAA 1X 1X 1X 1X
phenylacetaldehyde Phe 1X 1X 1X 1X
2-phenylethanol Phe 1X 1.3X 1.5X 1.5X
benzaldehyde Phe 1X 1X 1X 10X
methylbenzoate Phe 1X 1X 3X 2X

onset of ripening and peak either at or shortly
before full ripening (Table 1). This timing
suggests that synthesis of avor volatiles is
highly regulated. At least some part of the
regulation occurs at the level of transcription.
For example, the rate-limiting step for synthe-
sis of several Phe-derived volatiles, including
2-phenylethanol, phenylacetaldehyde, and
1-nitro-2-phenethane, is performed by a small
family of aromatic amino acid decarboxylases
(AADCs) (73). Expression of several of the
genes encoding these enzymes is upregulated
during ripening, and increased expression of
AADC enzymes results in increased metabolic
ux into this volatile synthesis pathway.
Synthesis of the volatiles that are upreg-
ulated during ripening is dependent upon
ethylene, as there are no ripening-associated
increases in the ethylene-insensitive Nr
mutant (G. Maloney & H.J. Klee, unpublished
results).
Regulation of several other volatile synthe-
sis pathways is less clear. For example, multiple
six-carbon volatiles, including cis-3-hexenal
and hexanal, are synthesized from linoleic and
linolenic acids. Three enzymes are known to
participate in their synthesis: 13-lipoxygenase
(LOXC), hydroperoxide lyase, and alcohol
dehydrogenase 2 (ADH2). These enzymes
are predicted to be plastid-targeted, consis-
tent with the abundance of the C18 fatty
acids in chloroplast membranes. The LOXC
and ADH2 RNAs increase during ripening,
roughly correlating with generation of the
associated six-carbon volatiles (43, 70). How-
ever, expression of the genes can be detected
in other tissues. Further, it is not clear whether
lipoxygenases can act on glycerol-attached
fatty acids or whether the action of a lipase is
an essential rst step in the pathway.
Regulation of the synthesis of apo-
carotenoid volatiles is not well understood. It

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is clear that a family of carotenoid cleavage
dioxygenases (CCDs) can generate the full
spectrum of apocarotenoid volatiles (76). In
tomato, there are two enzymes, CCD1A and
CCD1B, that can cleave multiple carotenoid
substrates to generate geranylacetone, pseu-
doionone, and -ionone (68). However,
although apocarotenoid volatile synthesis sig-
nicantly increases during ripening, there is no
corresponding increase in expression of either
gene. Further, antisense knock-downs of these
genes reduce, but do not eliminate, synthesis of
the volatiles. For these apocarotenoid volatiles,
there is good evidence that the carotenoid con-
tent of the fruit determines which volatiles are
synthesized (47, 77). There is a large increase in
lycopene and -carotene content in ripe fruits,
and the corresponding volatiles (6-methyl-
5-hepten-2-one and -ionone, respectively)
derived from those carotenoid precursors
increase proportionately (Table 1). A further
complication is that carotenoids are located
within the plastoglobules, but the CCD1
enzymes are not targeted to the plastids. There
is, however, another enzyme, CCD4, that has
not been fully characterized in tomato. The
CCD4 enzymes in several other species have
been shown to degrade carotenoids to release
volatiles. Given that apocarotenoid volatiles
are not normally produced at signicant levels
in green fruits and none of the CCD genes are
upregulated during ripening, it is possible that
physical changes in the plastids are responsible
for the ripening-associated increases in volatile
synthesis. Chromoplast differentiation is
associated with major structural alterations,
including the appearance of plastoglobules and
stromules (18, 59). Much as cell disruption
associated with wounding leads to synthesis of
C6 volatiles in leaves (75), synthesis of avor
SUMMARY POINTS
1. Ripening is concurrent with seed maturation and involves processes designed to release
the seed and attract seed-dispersing organisms.
54 Klee Giovannoni
volatiles may be associated with loss of physical
separation between enzymes and their natural
substrates during ripening.
To summarize, although synthesis of the a-
vor volatiles is clearly ripening-associated, the
regulatory mechanisms are not established. It is
likely that a large part of this regulation is medi-
ated by a subset of the many ripening-associated
transcription factors. But a part of the regula-
tion may be structural, i.e., subcellular physi-
cal changes that bring together substrates with
otherwise separated enzymes. In either case, the
regulated events are dependent upon ethylene
action.
CONCLUSIONS
The tomato fruit has emerged as the preemi-
nent model for study of fruit ripening and ethy-
lene control of developmental processes. A cas-
cade of transcription factors, many of which
have been characterized, initiates ripening. Al-
though the full complement and hierarchy of
transcription factor action are not entirely es-
tablished, signicant progress in identication
of a number of critical factors has been made.
Key to ripening is a poorly understood devel-
opmental switch in ethylene responsiveness of
the fruit organ that occurs upon maturation.
The ethylene response that denes a climac-
teric fruit coordinates expression of thousands
of genes that in turn control fruit softening as
well as accumulation of pigments, sugars, acids,
and volatiles. Together, this network of tran-
scriptional and hormonal regulators mediate
the physiological changes that make the fruit at-
tractive to seed-dispersing organisms, and with
the availability of a high quality tomato genome
sequence is the foundation for expansion and
renement of our understanding of the genetic
regulation of fruit development and ripening.
 GE45CH03-Klee ARI 1 October 2011 15:38

2. Ethylene is required for initiation of the ripening process. Fruits undergo a fundamental
shift in the way that they synthesize and respond to ethylene upon maturation.
3. The dependency upon ethylene for ripening has made tomato fruit ripening a model for
understanding regulation of its synthesis and perception.
4. Characterization of ripening mutations and functional analysis of putative regulatory
genes whose expression is associated with ripening reveal a network of transcription
factors that regulate ethylene synthesis and additional ripening processes. At least some
of these functions are likely to be widely conserved among fruit species.
5. Most, although not all, of the chemicals that contribute to avor are synthesized specif-
ically and accumulate to their highest levels at the ripe stage of development. For the
most part, regulation of volatile synthesis is not well understood.
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FUTURE ISSUES
1. What is the trigger that shifts the fruit from system 1 to system 2 ethylene synthesis? Is
this shift the cause or the consequence of a developmental signal to initiate ripening?
2. What additional transcriptional regulators remain to be identied, and what is the rela-
tionship of these regulators to each other?
3. Are there specic subregulators in terms of transcription factors or other genes that
specically regulate individual ripening pathways or processes distinct from the more
pleiotropic regulators dened to date (related to 4 below).
4. The mechanism(s) regulating volatile synthesis. What transcription factors mediate ex-
pression of the synthesis genes? What other structural factors regulate synthesis of
volatiles such as the apocarotenoids?

DISCLOSURE STATEMENT
The authors are not aware of any afliations, memberships, funding, or nancial holdings that
might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS
We wish to acknowledge the continuing support of the U.S. National Science Foundation to HJK
and JJG (IOS-0923312).

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Volume 45, 2011 Contents

Comparative Genetics and Genomics of Nematodes: Genome

Structure, Development, and Lifestyle
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Uncovering the Mystery of Gliding Motility in the Myxobacteria

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Genetics and Control of Tomato Fruit Ripening and Quality Attributes
Harry J. Klee and James J. Giovannoni p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p41
Toxin-Antitoxin Systems in Bacteria and Archaea
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Genetic and Epigenetic Networks in Intellectual Disabilities
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The Rules of Engagement in the Legume-Rhizobial Symbiosis
Giles E.D. Oldroyd, Jeremy D. Murray, Philip S. Poole, and J. Allan Downie p p p p p p p p 119
A Genetic Approach to the Transcriptional Regulation
of Hox Gene Clusters
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V(D)J Recombination: Mechanisms of Initiation
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Human Copy Number Variation and Complex Genetic Disease
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DNA Elimination in Ciliates: Transposon Domestication
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Double-Strand Break End Resection and Repair Pathway Choice
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vi
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CRISPR-Cas Systems in Bacteria and Archaea: Versatile Small RNAs

for Adaptive Defense and Regulation
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Human Mitochondrial tRNAs: Biogenesis, Function,
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The Genetics of Hybrid Incompatibilities
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Maternal and Zygotic Control of Zebrash Dorsoventral Axial
Patterning
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Genomic Imprinting: A Mammalian Epigenetic Discovery Model

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Sex in Fungi
Min Ni, Marianna Feretzaki, Sheng Sun, Xuying Wang, and Joseph Heitman p p p p p p 405
Genomic Analysis at the Single-Cell Level
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Uniting Germline and Stem Cells: The Function of Piwi Proteins
and the piRNA Pathway in Diverse Organisms
Celina Juliano, Jianquan Wang, and Haifan Lin p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 447

Errata

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