Ripening Mutants - Fruit Ripening: Physiology, Signalling and Genomics - page 255

Thompson et al. , 1999; Kovacs et al. ,
2009). Furthermore, although the expres-
sion of ethylene-regulated genes can be
restored by ethylene treatment, the mutant
phenotypes are not reversed, suggesting
that these loci act upstream of ethylene to
control the ripening process (Tigchelaar et
al. , 1978; Lincoln and Fischer 1988;
Thompson et al. , 1999). The rin , nor and
Cnr loci have been isolated using
positional cloning strategies and each
encodes a transcription factor (Vrebalov et
al. , 2002; Manning et al. , 2006; Klee and
Giovannoni, 2011).
RIN encodes a MADS-box gene
designated MADS-RIN that is a member of
the SEPALLATA subfamily (Vrebalov et al. ,
2002). RIN infl uences the expression of
many ripening-related genes, and several
studies have identifi ed direct targets of the
RIN protein including the promoters of the
transcription factors RIN , NOR , CNR , TDR4
and HB1 , together with the promoters of
genes involved in ethylene biosynthesis,
aroma formation and cell-wall degradation
(Ito et al. , 2008; Fujisawa et al. , 2011;
Martel et al. , 2011; Osorio et al. , 2011).
RNA interference-mediated suppression of
a second MADS gene, designated TOMATO
AGAMOUS-LIKE 1 ( TAGL1 ), also results in
pleiotropic phenotypes including in-
hibition of fruit ripening and reduced
carpel expansion in tomato (Itkin et al. ,
2009; Vrebalov et al. , 2009). Furthermore,
suppression of MADS -box genes in
strawberry and bilberry also inhibit
ripening, suggesting a broad role for these
transcriptional regulators in the ripening
process (Jaakola et al. , 2010; Seymour et
al. , 2011). Overexpression of TAGL1 in
tomato causes a range of altered fl oral and
fruit phenotypes and most strikingly leads
to the development of fl eshy sepals that
undergo a form of ripening to accumulate
lycopene (Itkin et al. , 2009; Vrebalov et al. ,
2009). A T-DNA insertion mutant within
the 5 c untranslated region of TAGL1 ,
designated Arlequin ( Alq ), causes ectopic
expression of TAGL1 , also leading to the
development of fl eshy sepals (Gimenez et
al. , 2010). Interestingly, ectopic expression

of TAGL1 in the rin and nor mutant
backgrounds leads to partial ripening,
suggesting that TAGL1 acts downstream of
these regulatory loci (Gimenez et al ., 2010).
However, TAGL1 expression is not
dramatically reduced in the rin mutant
background (Vrebalov et al ., 2009), imply-
ing a more complex relationship between
these two transcriptional regulators.
The Cnr mutant is caused by an
epigenetic mutation that increases methyl-
ation in the promoter region of a
SQUAMOSA PROMOTER BINDING PRO-
TEIN homologue leading to reduced
expression of CNR and inhibition of
ripening (Manning et al. , 2006). The
expression of CNR is reduced in rin and
slightly elevated in TAGL1- silenced lines
(Vrebalov et al. , 2009; Martel et al. , 2011).
RIN binds the CNR promoter, but
chromatin immunoprecipitation experi-
ments in the Cnr mutant background
indicate that CNR or a protein regulated by
CNR is required for the promoter binding
activity of RIN (Martel et al. , 2011).
Overall, these data highlight the im-
portance of transcriptional regulators in
regulating ripening but also illustrate that
the transcriptional cascade probably
involves a complex network of interactions
rather than a linear chain of transcriptional
events.

15.3 Mutants that Disrupt Hormone
Biosynthesis and Signalling Impact on
Ripening

Plant hormones control many aspects of
plant growth and development together
with responses to diverse environmental
stimuli. Several of the major plant hor-
mones have been implicated in the
development and ripening of fl eshy fruit
and, for selection of these, their role has
been defi ned through the use of mutants
that disrupt either hormone biosynthesis or
perception (Srivastava and Handa, 2005;
Barry, 2010). Notably, the plant hormone
ethylene acts downstream of the ripening
regulators RIN , NOR and CNR to regulate