Ripening Mutants - Fruit Ripening: Physiology, Signalling and Genomics - page 260

can provide a range of activities from no
phenotypic effect through to complete loss
of function and, in the case of substitution
mutations, can provide valuable infor-
mation on the contribution of individual
amino acid residues to protein function.
Targeting-induced local lesions in
genomes (TILLING) has been utilized for
identifying allelic series of mutations within
genes of interest in ethyl methanesulfonate-
generated mutant populations (McCallum
et al. , 2000a,b; Colbert et al. , 2001).
TILLING platforms have been established
for several model species, including the
fl eshy fruit-bearing species of tomato and
melon (Gady et al. , 2009; Dahmani-Mardas
et al. , 2010; Piron et al. , 2010; Okabe et al. ,
2011; Gady et al. , 2012). A TILLING
population generated in the 'Micro-Tom'
cultivar was utilized for identifying
mutants in several ripening-related genes
including the family of six tomato ethylene
receptors (Okabe et al. , 2011). Char-
acterization of point mutations within the
N-terminal domain of the ETR1 ethylene
receptor identifi ed two mutants designated
Sletr1-1 and Sletr1-2 carrying the amino
acid substitutions P51L and V69D,
respectively, which resulted in dominant
ethylene insensitivity. The utility of
TILLING for recovering mutant alleles of
varying strengths was demonstrated, as the
Sletr1-1 mutant allele consistently resulted
in stronger ethylene-insensitive pheno-
types than observed with the Sletr1-2 allele
(Okabe et al. , 2011). Similarly, two
TILLING-derived alleles of varying severity
were identifi ed in PHYTOENE SYNTHASE
1 ( PSY1 ) of tomato, which controls sub-
strate fl ow into the carotenoid biosynthesis
pathway during fruit ripening (Gady et al. ,
2012). A TILLING platform has also been
established in melon and mutants in
several genes infl uencing fruit quality traits
recovered, including mutants within the
ACC oxidase ( ACO1 ) gene, with a G194D
substitution resulting in a delayed fruit-
ripening and extended shelf-life phenotype
(Dahmani-Mardas et al. , 2010). Together,
these studies demonstrate the utility of
TILLING as an approach to identify new
alleles in known ripening-related genes,

but the technology should also be applic-
able for investigating the function of
candidate genes identifi ed through expres-
sion analyses, protein-protein interaction
studies and other functional approaches.

15.8 Conclusions and Perspectives

Mutants that disrupt aspects of fruit
development and ripening have yielded
important insights into multiple aspects of
the ripening process and have played a
pivotal role in identifying genes involved
in ripening. Notably, the transcriptional
cascade involving RIN, NOR, CNR and
TAGL1 was defi ned in part through
analysis of the corresponding ripening
mutants. Chromatin immunoprecipitation
approaches are being utilized to identify the
targets of these transcription factors, and as
genome-wide approaches for determination
of transcription factor binding sites become
more routine, it is likely that a complete
transcriptional network required for
ripening will become available. This will
provide insights into the unique and
overlapping roles of each of these tran-
scription factors during ripening. Further-
more, as genomics-based methodologies
continue to develop, the ability to generate
molecular and metabolic phenotypes in-
creases, creating the potential to establish a
more complete understanding of altered
phenotypes associated with each ripening
mutant. These technologies will help to
create a systems-level understanding of
ripening-related networks that are likely to
have the genes identifi ed through char-
acterizing ripening mutants as the
principal nodes.
The underlying genes for the majority of
the known ripening mutants of tomato have
been cloned and characterized. Further-
more, given the logistics of performing
large-scale mutant screens to uncover fruit
ripening genes, it seems unlikely that
additional large genetic screens for ripening
mutants will be conducted, and it seems
more likely that reverse genetics-based
methods for determining gene function will
become more widespread. In addition to