Plant Molecular Biology 40: 687–698, 1999.
© 1999 Kluwer Academic Publishers. Printed in the Netherlands.
Phytoene synthase-2 enzyme activity in tomato does not contribute to
carotenoid synthesis in ripening fruit
Paul D. Fraser
, Wolfgang Schuch
and Peter M.
Division of Biochemistry, School of Biological Sciences, Royal Holloway University of London, Egham, Surrey,
TW20 0EX, UK (
author for correspondence);
Zeneca Plant Science, Jealott’s Hill Research Station, Bracknell,
Berkshire, RG42 6EY, UK;
present address: Schoolof Biological Sciences, University of Sussex, Falmer, Brighton,
Sussex, BN1 9QG, UK
Received 17 November 1998; accepted in revised form 1 April 1999
Key words: carotenoids, Lycopersicon esculentum, mutants, phytoene synthases
The characteristic yellow fruit phenotype of the r,r mutant and Psy-1 (phytoene synthase-1) antisense tomatoes
is due to a mutated or down-regulated phytoene synthase protein, respectively, resulting in the virtual absence of
carotenoids. Based on detailed carotenoid determinations Psy-1 appeared to barely contribute to the formation of
carotenoids in chloroplast-containing tissues. Despite the virtual absence of carotenoids in ripe fruit the formation
of phytoene in vitro was detected in fruit of both mutants. When [
C]isopentenylpyrophosphate(IPP) was used as
the substrate for phytoene synthase a reduction (e.g. r,r mutant, 5-fold) in the formation of phytoene was observed
with an accumulation (e.g. r,r mutant, 2-fold) of the immediate precursor geranylgeranyl pyrophosphate (GGPP).
Contrastingly, reduced phytoene synthase activity was not detected when [
H]GGPP was used as the substrate.
The proﬁle of phytoene formation during ripening was also different in the down-regulated mutants compared to
the wild-type. Using speciﬁc primers, RT-PCR analysis detected the presence of Psy-2 transcripts in the down-
regulated mutants and wild-type throughout fruit development and ripening. These data were supported by the
detection of phytoene synthase protein on western blots. Both GGPP formation and phytoene desaturation were
elevated in these mutants. Therefore, it appears that despite the absence of carotenoids in ripe fruit, both the
mutants have the enzymic capability to synthesize carotenoids in this tissue. Implications of the data with respect
to the regulation of carotenoid formation and the channelling of prenyl lipid precursors in tomato (and its potential
manipulation) are discussed.
Abbreviations: DMAPP, dimethylallyl pyrophosphate; d.p.b., days post-breaker; FPP, farnesyl pyrophosphate;
GGPP, geranylgeranylpyrophosphate;GPP, geranyl pyrophosphate;IPP, isopentenyl pyrophosphate; Mabs, mono-
clonal antibodies; Psy-l, phytoene synthase-1 gene; Pds, phytoene desaturase gene; RT-PCR, reverse transcriptase
polymerase chain reaction
Carotenoids are a widespread class of pigments found
in higher plants, algae, and some fungi and bac-
teria (Goodwin, 1980). In photosynthetic tissues
carotenoids are synthesized and located within the
chloroplast were they function as accessory pig-
ments and photoprotectants(Andersonand Robertson,
1960). In non-green plant tissues, carotenoids accu-
mulate in chromoplasts generating the characteristic
bright colours of ﬂowers and fruits.
Biosynthetically, carotenoids are isoprenoids de-
rived from the C
isoprenoid precursor geranylger-
anyl pyrophosphate (GGPP). Condensation of two
GGPP molecules to form phytoene (7,8,11,12,7
octahydro-ψ,ψ-carotene) is the ﬁrst committed step