Ripening Mutants - Fruit Ripening: Physiology, Signalling and Genomics - page 259

a 95-98% reduction in cutin content
compared with the wild type, and this is
accompanied by altered wax composition
(Isaacson et al. , 2009). The altered
chemical properties of the cd mutant
cuticles infl uence their physical properties,
leading to increased glossiness and altered
elasticity. The reduced cutin load of the
cd1 mutant correlated with an increase in
the rate of fruit water loss, leading to rapid
shrivelling of fruits following harvest.
However, the relationship between reduced
cutin load and increased rates of water loss
is not absolute, as the cd2 and cd3 mutants
do not exhibit increased rates of
postharvest water loss. In contrast, the
importance of the cuticle in reducing the
incidence of pathogen infection in tomato
fruit was highlighted by the observations
that cd1 , cd2 and cd3 each displayed an
increased susceptibility to postharvest
pathogens (Isaacson et al. , 2009). Positional
cloning revealed that CD2 encodes a
member of the class IV homeodomain-
leucine zipper (HD-Zip IV) gene family, a
class of transcription factors that have roles
in epidermal cell development, including
those reported to infl uence cutin and wax
biosynthesis in Arabidopsis and maize
(Javelle et al. , 2010; Wu et al. , 2011;
Nadakuduti et al. , 2012). Recent char-
acterization of the sticky peel ( pe ) mutant
of tomato indicated that it encodes an
allele of CD2 and possesses an altered fruit
phenotype identical to that of the cd2
mutant. However, a more in-depth char-
acterization of pe mutant phenotypes also
revealed cutin and wax defi ciencies in
leaves together with an associated increase
in cuticular permeability. In addition, the
pe mutant has a pale phenotype due to
reduced anthocyanin content, and its
leaves possess fewer type VI glandular
trichomes and consequently have reduced
volatile terpene emissions (Nadakuduti et
al. , 2012). These data reveal that, although
CD2 dramatically alters fruit cuticle
formation, it has a broader role in
regulating epidermal cell development and
specialized metabolism.
The wax composition, and in particular
the long-chain alkane (C28-C32) content of

cuticles, is typically associated with
cuticular permeability (Samuels et al. ,
2008). Mutants with reduced long-chain
alkane content often display enhanced
rates of water loss, and the importance of
alkanes in maintaining water balance is
further highlighted by the fi ndings that the
alkane content of cuticular waxes can
increase in response to water stress
(Riederer and Schreiber, 2001; Kosma et
al. , 2009; Buschhaus and Jetter, 2011; Wang
et al. , 2011). Tomato mutants defi cient in
alkane content in fruit cuticles have
increased cuticular permeability, which
can lead to enhanced rates of shrivelling
on or off the vine. This was observed in
a mutant of a very-long-chain fatty acid
E -ketoacyl-CoA synthase, designated
LeCER6 , and the positional sterile mutant
(Vogg et al. , 2004; Leide et al. , 2011).

15.7 TILLING and the Potential to
Identify Additional Mutations in
Ripening Genes

The majority of ripening mutants identifi ed
to date have strong visible phenotypes that
affect pleiotropic ripening processes, fruit
colour or cuticular properties. Further-
more, with the exception of the large
mutant collection developed in the M82
background (Menda et al. , 2004), ripening
mutants have not been identifi ed through
systematic screens, with the majority of the
classical ripening mutants occurring
spontaneously and identifi ed in grower
fi elds or during large breeding pro-
grammes. Consequently, mutants that alter
more subtle phenotypes associated with
the ripening process such as cell-wall
breakdown or aroma formation are
generally not available, and progress in
elucidating these ripening pathways have
generally been made through gene-
silencing approaches such as RNA inter-
ference. However, while gene-silencing
approaches can be powerful for deter-
mining gene function, they typically lead
to 'knockdown' phenotypes rather than
'knockout' phenotypes. In contrast, an
allelic series of mutations within a gene