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Plant Physiol. (1999) 120: 383-390
Molecular and Genetic Characterization of a Novel Pleiotropic
Tomato-Ripening Mutant1
Andrew J. Thompson,
Mahmut Tor,
Cornelius S. Barry,
Julia Vrebalov,
Caroline Orfila,
Michael C. Jarvis,
James J. Giovannoni,
Donald Grierson, and
Graham B. Seymour*
Horticulture Research International, Wellesbourne, Warwick CV35
9EF, United Kingdom (A.J.T., M.T., G.B.S.); Plant Science Division,
School of Biological Sciences, University of Nottingham, Sutton
Bonington Campus, Loughborough LE12 5RD, United Kingdom (C.S.B., D.G.); Department of Horticultural Sciences, Texas A&M University, College
Station, Texas 77843-2133 (J.V., J.J.G.); Centre for Plant
Biochemistry and Biotechnology, University of Leeds, Leeds LS12 9JT,
United Kingdom (C.O.); and Chemistry Department, University of
Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom (M.C.J.)
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ABSTRACT |
In this paper we describe a novel,
dominant pleiotropic tomato (Lycopersicon
esculentum)-ripening mutation, Cnr
(colorless nonripening). This
mutant occurred spontaneously in a commercial population.
Cnr has a phenotype that is quite distinct from that of
the other pleiotropic tomato-ripening mutants and is characterized by
fruit that show greatly reduced ethylene production, an inhibition of
softening, a yellow skin, and a nonpigmented pericarp. The ripening-related biosynthesis of carotenoid pigments was abolished in
the pericarp tissue. The pericarp also showed a significant reduction
in cell-to-cell adhesion, with cell separation occurring when blocks of
tissue were incubated in water alone. The mutant phenotype was not
reversed by exposure to exogenous ethylene. Crosses with other mutant
lines and the use of a restriction fragment length polymorphism marker
demonstrated that Cnr was not allelic with the
pleiotropic ripening mutants nor, alc, rin,
Nr, Gr, and Nr-2. The gene has been mapped to the top
of chromosome 2, also indicating that it is distinct from the other
pleiotropic ripening mutants. We undertook the molecular
characterization of Cnr by examining the expression of a
panel of ripening-related genes in the presence and absence of
exogenous ethylene. The pattern of gene expression in
Cnr was related to, but differed from, that of several
of the other well-characterized mutants. We discuss here the possible
relationships among nor, Cnr, and
rin in a putative ripening signal cascade.
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INTRODUCTION |
The ripening of a fruit imparts a variety of agronomically
important characteristics to an otherwise unpalatable product. These
include conversion of starch to sugars and changes in color, flavor,
and texture. Ripening is a tightly controlled and highly programmed
developmental event. Identifying the components of this developmental
switch is important not only for manipulating this key plant
process but also for understanding the regulation of plant
development.
The biochemical and molecular basis of ripening in both climacteric and
nonclimacteric fruit has been intensively studied. Genes involved
in cell wall degradation, color change, ethylene synthesis, and
perception have been cloned, and antisense techniques have been
developed to manipulate these aspects of ripening (Gray et al., 1994 ;
Wilkinson et al., 1995, 1997). Very little is known, however, about the
regulatory genes specifically associated with ripening. Several
single-gene mutations resulting in the reduction or almost complete
elimination of ripening are known in tomato (Lycopersicon
esculentum) fruit (Grierson, 1986). These mutant loci include
rin (ripening-inhibitor; Robinson
and Tomes, 1968), nor (nonripening;
Tigchelaar et al., 1973), Nr
(Never-ripe; Rick, 1956), Gr
(Green-ripe; Kerr, 1958), Nr-2
(Never-ripe 2; Kerr, 1982), and
alc (alcobaca; Kopeliovitch et al., 1981). These
pleiotropic mutations are extremely rare and are likely to encode
important regulatory genes. The Nr gene, which has been
cloned, encodes a protein with homology to the Arabidopsis ethylene
receptor ETR1 (Wilkinson et al., 1995), and the normal
alleles residing at rin and nor are the subject
of a map-based cloning program in one of the authors' laboratories
(J.J.G.). The aim is to eventually understand the structures of the
genes identified by these mutations and place their encoded proteins
into a framework that will describe the molecular regulation of fruit
ripening. It is unlikely that mutants are available for all of the
steps in such a regulatory pathway and new ripening mutants are
especially valuable. In the current paper we describe the molecular and
genetic characterization of a novel, dominant pleiotropic
tomato-ripening mutant, Cnr (colorless, nonripening).
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MATERIALS AND METHODS |
Tomato (Lycopersicon esculentum Mill. cvs Ailsa Craig
and Liberto) fruit and the mutant line Cnr derived from cv
Liberto were grown in a heated greenhouse using standard cultural
practices with regular additions of N,P,K fertilizer and
supplementary lighting when required. Plants were grown to three
trusses. Fruits were harvested at the following stages: mature
green (35 DPA), breaker, breaker plus 3 d, and breaker plus 7 d. Fruits of the Cnr mutant were also harvested at 35 DPA
and then at 49, 52, and 56 DPA, which are equivalent to the ripening
stages for the normal fruit. Seeds of Lycopersicon
cheesmanii (LA483), Gr (LA2453), and
Nr-2 (LA2455) were obtained from the Tomato
Genetic Resource Center (Davis, CA). The seeds for the rin,
nor, Nr, and y mutants were obtained from the
Glasshouse Crops Research Institute collection at Horticulture
Research International (Wellesbourne, UK).
Ethylene Production and Carotenoid Analysis
For measurement of ethylene production at each stage of ripening,
three fruit from each stage were placed in a gas-tight 1-L glass jar at
20°C for 1 h, after which time a 1-mL sample of headspace was
analyzed by GC (Ward et al., 1978). Each injection was repeated three
times. For analysis of total carotenoid levels, pericarp tissue was
freeze-dried and extracted with chloroform. Carotenoid content was then
determined by the method of Wellburn (1994). Carotenoids were extracted
from the pericarp of three individual Cnr and cv Ailsa Craig
fruit at each ripening stage.
Mechanical Tests for Fracture Energy and Analysis of Cell
Separation
Measurements of fracture energy were made by the procedure
described for potato tissue by Freeman et al. (1992). The force (F) required to propagate a preinitiated crack by driving a
0.28-mm stainless steel wire through 11-mm-diameter × 5-mm-thick
discs of tissue was measured and converted to gross fracture energy (E) by the equation E = F/d, where d is the diameter of
the disc.
For the cell-separation experiments, three individual Cnr,
cv Ailsa Craig, or rin fruit were selected at each of the
desired stages of development/ripeness, giving three replicates in each treatment. The fruits were peeled, and the pericarp was cut into 5-mm
cubes. These cubes, with an approximate volume of 3 mL, were suspended
in 9 mL of distilled water or 0.05 M CDTA,
adjusted to pH 6.5, or in 20 units of pectinase enzyme from
Aspergillus niger (Sigma) in 0.1 M
sodium acetate buffer, pH 4.0, for 3 h at room temperature with
gentle rocking. Cell separation was measured as the volume of separated
cells after removal of the remaining tissue and settling for 2 h
at 4°C. Cell separation in water and CDTA was calculated relative
to 100% cell separation obtained after pectinase treatment.
RFLP and Linkage Analysis
Genomic DNA was purified according to the method of Fulton et al.
(1995). Restriction digests, Southern blotting, and hybridization of
the labeled probes to isolated plant genomic DNA were carried out using
standard techniques (Sambrook et al., 1989). RFLP probes (provided by
Dr. S. Tanksley, Cornell University, Ithaca, NY) were radiolabeled with
32P using the Bioline oligolabeling system
(Bioline, London), according to the manufacturer's instructions. RFLP
markers were mapped relative to the mutant locus as two-point data
using the computer program MAPMAKER (Lander et al., 1987).
Radiolabeled Probes for RNA Analysis
Strand-specific, radiolabeled RNA probes for ACO1,
PG, PSY1, and E8 were synthesized from
linearized plasmid template DNA using either T3 or T7 RNA polymerase
(Promega) according to the manufacturer's instructions. Radiolabeled
ERT16 and 18S DNA probes were generated from a purified DNA insert with
random primers according to the procedures described by Feinberg and
Vogelstein (1983). A 189-bp fragment, corresponding to the
3 -untranslated region of the NR gene (Wilkinson et al.,
1995), was amplified by PCR using the primers NR5
(5-TAAATGACAAAAGGACAT-3) and NR3 (5-GTCAAAAGCTCGATGTAT-3).
The fragment was cloned using the TACloning kit (Invitrogen,
NV Leek, The Netherlands), and the resulting plasmid clone generated a
single-stranded, radiolabeled RNA probe, as described above. Details of
the these cDNA clones were described by Gray et al. (1994) and
Wilkinson et al. (1995).
RNA Gel-Blot Analysis and RNase Protection Assay
Total RNA was extracted from a pooled sample of pericarp from
three individual mutant or normal fruit, as described by Hamilton et
al. (1990). RNA gel-blot analysis was carried out using 10 µg of RNA
according to the method of John et al. (1995). RNase protection assays
were performed as described by Barry et al. (1996), with a few minor
modifications. Fifty micrograms of total RNA was hybridized with
radioactive probe in 50 µL of hybridization buffer. Digestion with 3 units of RNase ONE (Promega) for 3 h at 28°C removed the
unhybridized RNA.
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RESULTS |
Isolation of the Mutant and Phenotype
The mutant was isolated in 1993 as a single plant from a
commercial planting of the F1 hybrid cv Liberto.
It is characterized by fruit that fails to ripen, turns white when
mature (40-50 DPA), remains very firm, and then develops a yellow
skin. The underlying pericarp tissue remains completely
nonpigmented/white (Fig. 1, A and B).
Ripening was not restored by the application of exogenous ethylene at
100 µL L 1 to the mutant Cnr at 35 DPA (equivalent to mature-green fruit in cvs Ailsa Craig and Liberto)
continuously for 4 d at 19°C (Fig. 1C). However, germinating
seedlings showed the triple response in the presence of ethylene (Fig.
1D). To confirm that the yellow appearance of Cnr was due to
pigmentation in the skin, a double mutant with a colorless epidermis
(y) was generated. The fruit from this cross turned
white/cream in color (data not shown).
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| Figure 1.
A, Ripening of cv Ailsa Craig (left) and
homozygous Cnr fruit (right) in a cv Liberto background.
Fruit are shown at various DPA corresponding in the wild-type fruit to
mature-green, breaker, breaker-plus-3-d, and breaker-plus-7-d stages.
B, Skin peeled back on Cnr fruit 56 DPA (left) and
red-ripe cv Ailsa Craig (right) to reveal colorless flesh in the
mutant. C, Effect of exposure to 100 µL L1 ethylene for
4 d and then air alone for an additional 5 d at 19°C on the
appearance of cv Ailsa Craig (top) and Cnr (bottom)
fruit. D, Seedlings from (left to right) Cnr, cv Ailsa
Craig, and cv Liberto displaying sensitivity to ethylene, in contrast
to those of the Nr mutant.
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Allelism Tests with Other Pleiotropic Tomato-Ripening Mutants and
Genetic Mapping of Cnr
The original mutant was selfed to produce a homozygous
line in the F3 generation, which was crossed to
L. esculentum cv Ailsa Craig. We scored 99 of the
F2 plants for their ripening phenotype (Table
I). Mutant plants could not be separated
into heterozygous and homozygous classes based on the severity of the
phenotype. A  2 test for goodness-of-fit to a
ratio of 3:1 (mutant:wild type) gave a test statistic of 1.22, which is
not significant at the 5% level. Thus, we concluded that
Cnr is a dominant, nuclear-encoded mutation.
Additional crosses, including reciprocal crosses, with the pleiotropic
ripening mutants Nr, rin, Nr-2, and Gr, all
yielded wild-type plants in the F2 self- or
back-cross progeny (Table I), demonstrating that Cnr is
nonallelic to these loci and further supporting the dominant nuclear
nature of the mutation. In all cases, except for Cnr × rin, the ripening characteristics of the double mutants were
not visually distinct from Cnr alone. The phenotype of
Cnr was easily distinguished from the other mutants by the
lack of pigment in the pericarp. To test whether Cnr was allelic with the two additional pleiotropic ripening mutants
nor and alc, an RFLP analysis was performed.
Tigchelaar and Barman (1985) concluded that nor and
alc are allelic, although two tightly linked loci cannot be
excluded. The RFLP marker CT16, which maps 0.9 centimorgan from
nor (Giovannoni et al., 1995) was used to follow the
segregation pattern from a cross between Cnr (in L. esculentum background) and L. cheesmanii in an
F2 population. Of the 21 F2
plants analyzed, 13 showed recombination between Cnr and
CT16, indicating that these two loci segregate independently (Table
II). It follows that Cnr
cannot be allelic with nor or alc. Using the
F2 mapping population, we carried out additional experiments to map the Cnr gene. Initially, the least
ambiguous class of homozygous, wild-type F2
families was used to map the position of the gene. Subsequently, as
linked markers were identified, confirmation of the map position was
sought using heterozygous and homozygous mutant families, and linkage
was observed with molecular markers at the top of chromosome 2 (Fig.
2). Thus, Cnr was located in
an interval between the RFLP markers TG31 and CT106A. Nr,
rin, nor, alc, and Nr-2 all map to
chromosomes other than 2 (Gray et al., 1994). No map location could be
found in the literature for Gr.
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Table II.
Test for independent segregation of Cnr and CT16 in
the cross L. esculentum, (Cnr/Cnr) × L. cheesmanii, (+/+)
F2 self
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| Figure 2.
Linkage map of tomato chromosome 2 from the
analysis of F2 progeny from the cross L. esculentum (Cnr/Cnr) × L. cheesmannii (+/+). Rec Frac., Recombination
fraction; Dist, distance; cM, centimorgan.
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Biochemical and Molecular Characterization of Ripening Fruit
Ethylene production in the fruit of both cvs Liberto and Ailsa
Craig showed a climacteric rise as the fruit ripened. A much reduced
level of ethylene production was apparent in Cnr, as
illustrated in Figure 3. The ethylene
measurements were repeated on two separate occasions with different
batches of fruit, and similar results were obtained. A distinctive
feature of Cnr was the lack of pigmentation in the pericarp
tissue. Analysis of the total carotenoid content revealed that
ripening-related production of these compounds was absent in the
Cnr fruit. However, similar levels of total
colored carotenoids were present in the mature, unripe fruit and leaves of Cnr, in comparison with wild-type plants (Fig.
4). On handling the Cnr fruit,
we found that they were very firm in comparison with the wild type, and
mechanical tests demonstrated that they exhibited a higher fracture
energy (Table III), although the pericarp tissues often had a mealy appearance when cut. This apparent mealiness was reflected in altered cell-to-cell adhesion properties in
Cnr. Preliminary experiments showed that, unlike wild-type
fruit, significant cell separation occurred in the
Cnr pericarp tissue when it was left in water for a few
hours. To obtain a more accurate comparison of cell-to-cell adhesion in
Cnr and wild-type fruit, blocks of pericarp were floated in
water alone or in a solution containing the Ca chelator CDTA. The
degree of cell separation was then compared with the 100% cell
separation obtained after pectinase treatment. In unripe fruit there
was limited cell separation apparent in the Cnr and the
wild-type tissue either in water or in CDTA. However, blocks of
Cnr pericarp from 56-d-old fruit (equivalent to red-ripe, wild-type fruit) incubated in water alone showed extensive cell separation (Fig. 5). In contrast,
although the Ca chelator CDTA caused more extensive cell separation
than water in all of the fruit, the effects on Cnr were less
pronounced than on the wild-type material (Fig. 5B). In rin
fruit, even at 80 DPA, minimal cell separation occurred in water,
although loss of cell adhesion was enhanced by CDTA (Fig. 5B).
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| Figure 3.
Ethylene production by cv Ailsa Craig fruit at
mature-green (MG), breaker (B), breaker-plus-3-d (B+3), and
breaker-plus-7-d (B+7) stages and Cnr fruit at
equivalent ages postanthesis. Three fruit were placed in a gas-tight
jar, and ethylene was sampled in the headspace after 1 h. Values
shown are the means of three injections on the GC.
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| Figure 4.
Total carotenoid content of cv Ailsa Craig and
Cnr leaf, mature-green- (MG), breaker- (B), and
breaker-plus-7-d (B+7)-stage pericarp tissue. Vertical bars = SE; n = 3. DW, Dry weight.
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Table III.
Tissue strength in red-ripe fruit of cvs Ailsa
Craig and Liberto and Cnr fruit at an equivalent time postanthesis (65 DPA)
Means are six individual fruit (pericarp) or four individual fruit
(locule), with two replicates per fruit. Data analysis was by analysis
of variance after logarithmic transformation. For each column the same
letter denotes no significant difference at P < 0.01.
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| Figure 5.
Cell separation in pericarp tissue in unripe
(A) and ripe (B) cv Ailsa Craig and Cnr (from fruit of
an equivalent age) and from 60- and 80-DPA (dpa) rin
fruits incubated in water or CDTA for 3 h at room temperature.
Values were calculated relative to 100% cell separation obtained after
pectinase treatment. Vertical bars = SE;
n = 3.
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We followed changes in the expression of a panel of ripening-related
genes in cvs Ailsa Craig and Liberto and Cnr in the presence and absence of exogenous ethylene (Fig.
6). ACO (ACC
oxidase), PG
(polygalacturonase),
PSY1 (phytoene
synthase), E8, ERT16, a clone encoding an ABA stress-related protein, and NR (an
ethylene receptor) were chosen, because these messages are all
up-regulated or present during normal ripening. The pattern of
expression of these genes in Cnr could be grouped into four
broad categories: (a) no detectable message and expression not restored
by ethylene, e.g. PSY1, (b) transcripts detectable only
after exposure of the fruit to ethylene, e.g. PG, (c) low
levels of message in untreated fruit and enhanced expression in the
presence of ethylene, e.g. E8, NR, and ERT16; and
(d) low levels of message in untreated fruit that is not enhanced by
ethylene treatment, e.g. ACO.
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| Figure 6.
A, Ripening-related gene expression by RNA
gel-blot analysis. Total RNA was isolated from the fruit of cv Ailsa
Craig (AC), cv Liberto, and the mutant Cnr, and both
Cnr and cv Ailsa Craig after treatment with 100 µL/L
exogenous ethylene at mature-green (M), breaker (B), breaker-plus-3-d
(3), and breaker-plus-7-d (7) stages. Cnr fruit were
picked at equivalent DPA, as described in ``Materials and Methods''.
Ten micrograms of RNA was loaded per lane and hybridized with
previously characterized ripening-related genes (Gray et al., 1992, and
refs. therein; Picton et al., 1993). B, Analysis of NR
gene expression in Cnr by the RNase protection assay.
Fifty micrograms of total RNA from cvs Ailsa Craig, Liberto, and
Cnr from stages described in A was hybridized with a
radiolabeled NR gene fragment, and RNase protection
assay analysis was performed as described in ``Materials and Methods''.
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DISCUSSION |
Cnr is a spontaneous mutation isolated from a
commercial population of plants. The mutant has a phenotype that is
quite distinct from the other known pleiotropic mutants at both the
physiological and molecular levels. Our allelism tests and genetic
mapping experiments indicated that Cnr is a novel, dominant
pleiotropic ripening mutant.
The most readily apparent feature that distinguishes Cnr
from the other pleiotropic ripening mutants is its lack of pigment in
the pericarp tissue. The yellow color of the intact fruit comes from
pigmentation in the skin, as demonstrated by the cross between Cnr and the colorless fruit epidermis mutant y.
Analysis of the carotenoid composition of the Cnr fruit
pericarp indicated that the biosynthesis of these pigments is
completely inhibited in the pericarp of the mutant during ripening,
although unripe fruit and nonfruit tissues appeared to be unaffected by
the mutation. Other pleiotropic tomato-ripening mutants, e.g.
Nr, nor, and rin, all show some
carotenoid production in the pericarp, although the synthesis of
lycopene is reduced, delayed, or absent, depending on the mutation
(Tigchelaar et al., 1978). In Cnr,
PSY1 gene expression is absent, even in the presence of
ethylene (Fig. 6A), whereas in normal tomatoes it is up-regulated
during ripening (see Fray and Grierson, 1993, and refs. therein). The
protein product of PSY1 is phytoene synthase, which
catalyzes the formation of phytoene, the first C40 carotene
intermediate in carotenoid biosynthesis. This is an essential step in
the production of the carotenoids, which give the fruit its red color.
The product of PSY1 expression is responsible for the
formation of carotenoids in the fruit, whereas a functionally distinct
phytoene synthase is active in green tissues (Bramley et al., 1992;
Fray and Grierson, 1993; Fraser et al., 1994). The absence of
PSY1 expression in Cnr can probably explain the
lack of carotenoids in the pericarp of the fruit.
Another striking feature of Cnr fruit is their altered cell
adhesion, which is apparent when the fruit have reached an age equivalent to that of the red-ripe cvs Ailsa Craig and Liberto. The
gross energy needed for mechanical fracture of Cnr tissue was greater than that needed for the red-ripe wild type, but
Cnr pericarp cells appeared to separate more readily in
water, where turgor may provide the cell-separation forces (Jarvis,
1998). Experiments to compare the cell adhesion in Cnr with
wild-type pericarp tissue by incubation in water or the Ca chelator
CDTA (Fig. 5) showed that in unripe fruit these solutions had very limited ability to induce cell separation and therefore to solubilize the cell wall components responsible for cell adhesion. However, CDTA
was effective at inducing substantial cell separation in ripe wild-type
fruit, which indicates that Ca probably plays an important role in cell
adhesion in these fruit, most likely through its association with
pectic polysaccharides (Carpita and Gibeaut, 1993). In contrast, water
alone induced marked cell separation in Cnr fruit of an
equivalent age, indicating that a proportion of the wall components
involved in cell adhesion in the mutant are readily soluble. These
unusual physical properties of Cnr deserve further study,
and investigations are now underway on the type and nature of the
pectic components in the cell walls of this mutant (C. Orfila, G.B.
Seymour, A.J. Thompson, and J.P. Knox, unpublished data).
That wall hydrolase activity is altered in Cnr is indicated
from measurements of PG mRNA, which was detected only in the
fruit that was exposed to exogenous ethylene. Reduced levels of wall
hydrolases and modified tissue composition are likely to account for
the altered textural properties of the fruit.
Cnr fruit did not exhibit the characteristic climacteric
rise in ethylene production at the onset of ripening; these data are
consistent with reduced levels of ACO gene expression in
Cnr. Although ripening in Cnr is not restored by
the addition of exogenous ethylene, the mutant is not completely
insensitive to ethylene, as indicated by the characteristic triple
response in dark-grown seedlings and the accumulation of some ethylene-
and ripening-related mRNAs in the fruit after ethylene treatment
(Figs. 1 and 6). In these respects, Cnr very closely
resembles rin and nor (Knapp et al., 1989;
Lanahan et al., 1994; Yen et al., 1995) and does not possess the
ethylene-insensitive phenotype characteristic of Nr (Lanahan
et al., 1994; Yen et al., 1995). Therefore, based on these criteria,
Cnr, like rin and nor (Yen et al.,
1995), can be presumed to act upstream of ethylene biosynthesis during
the regulation of ripening.
Cnr, nor, and rin may act together in
a cascade or independently in a multibranched regulatory network to
control ripening, or they may represent developmental components
leading to fruit tissue correctly primed for ripening. To begin to
understand the relationship of Cnr to the other mutants, we
have examined the expression of a number of ripening-related genes in
the presence and absence of exogenous ethylene. A number of differences
in the molecular fingerprints of the mutants were observed. For
example, in mature nor fruit, PSY1 and
E8 mRNA are lacking and expression of these genes is not
restored by exposing the fruit to ethylene (Yen et al., 1995; J.J.
Giovannoni, unpublished data). In mature rin,
PSY1 and E8 are expressed at low levels, and the
transcripts are up-regulated by ethylene (Knapp et al., 1989). In
Cnr there is no evidence for PSY1 expression in
either the presence or absence of exogenous ethylene, but the
E8 message is partially restored by ethylene treatment.
Similarly, PG transcripts are absent in nor and
rin in the presence and absence of ethylene (Knapp et al.,
1989; Yen et al., 1995) but are partially restored by ethylene in
Cnr. ACO transcripts are enhanced by ethylene treatment in rin fruit (Knapp et al., 1989) but are unaffected by
ethylene in Cnr. These observations suggest that
nor may have a more global effect on
ethylene/ripening-related gene expression than Cnr and rin, whereas Cnr and rin may
differentially regulate different subsets of ripening genes in response
to ethylene. In this model nor is upstream of rin
and Cnr, because ethylene/ripening-related gene expression
appears to be more extensively inhibited in this mutant. However,
further work will almost certainly show that these genes act as part of
a complex, multibranched regulatory network. The real test of this and
other models awaits the cloning of the normal alleles for
nor, rin, and Cnr. The pleiotropic effects of the
Cnr mutation indicate that the gene has a regulatory
function, but its identity may be difficult to establish using only
biochemical and molecular assays. Current efforts are focused on
isolation of the Cnr gene by more precisely refining the map
position of this locus and by identifying tomato bacteria artificial
chromosome clones that hybridize to RFLP- or amplified restriction
fragment polymorphism-based flanking markers.
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FOOTNOTES |
1
This work was supported by the United Kingdom
Biotechnology and Biological Sciences Research Council.
*
Corresponding author; e-mail graham.seymour{at}hri.ac.uk; fax
44-1789-470552.
Received December 10, 1998;
accepted February 21, 1999.
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ABBREVIATIONS |
Abbreviations:
CDTA, cyclohexane diamine tetraacetic acid.
DPA, days postanthesis.
RFLP, restriction fragment-length polymorphism.
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ACKNOWLEDGMENTS |
We thank Rachel Edwards for expert technical assistance, John
Maxam-Smith (Practical Plant Genetics, Chichester, UK) for help with
performing the crosses, and Steve Tanksley (Cornell University, Ithaca,
NY) for the use of the RFLP markers. We are grateful to Peter Bramley
and Paul Fraser (University of London) and to Emma Schofield (Zeneca
Plant Science, Bracknell, Berkshire, UK) for help with the
carotenoid analysis and for useful discussions.
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LITERATURE CITED |
Barry CS,
Blume B,
Bouzayen M,
Cooper W,
Hamilton AJ,
Grierson D
(1996)
Differential expression of the 1-aminocyclopropane-1-carboxylate oxidase gene family of tomato.
Plant J
9:
525-535
[CrossRef][Web of Science][Medline]
Bramley PM,
Teulieres C,
Blain I,
Bird CR,
Schuch W
(1992)
Biochemical characterization of transgenic tomato plants in which carotenoid synthesis has been inhibited through expression of antisense RNA to pTom5.
Plant J
2:
343-349
[CrossRef]
Carpita NC,
Gibeaut DM
(1993)
Structural models of the primary walls in flowering plants: consistency of molecular structure with physical properties of walls during growth.
Plant J
3:
1-30
[CrossRef][Web of Science][Medline]
Feinberg AP,
Vogelstein B
(1983)
A technique for radiolabeling DNA restriction endonuclease fragments to high specific radioactivity.
Anal Biochem
132:
6-13
[CrossRef][Web of Science][Medline]
Fraser PD,
Truesdale M,
Bird CR,
Schuch W,
Bramley PM
(1994)
Carotenoid biosynthesis during tomato fruit development.
Plant Physiol
105:
405-413
[Abstract]
Fray R,
Grierson D
(1993)
Identification and genetic analysis of normal and mutant phytoene synthase genes of tomato by sequencing, complementation and co-suppression.
Plant Mol Biol
22:
589-602
[CrossRef][Web of Science][Medline]
Freeman M,
Jarvis MC,
Duncan HJ
(1992)
The textural analysis of cooked potato: three simple methods for determining texture.
Potato Res
35:
103-109
Fulton T,
Julapark C,
Tanksley S
(1995)
Miniprep protocol for extraction of DNA from tomato and other herbaceous plants.
Plant Mol Biol Rep
13:
207-209
[Web of Science]
Giovannoni JJ,
Noensie EN,
Ruezinsky DM,
Lu X,
Tracy SL,
Ganal MW,
Martin GB,
Pillen K,
Alpert K,
Tanksley SD
(1995)
Molecular genetic analysis of the ripening inhibitor and non-ripening loci of tomato: a first step in the genetic map-based cloning of fruit ripening genes.
Mol Gen Genet
248:
195-206
[CrossRef][Web of Science][Medline]
Gray JE,
Picton S,
Giovannoni JJ,
Grierson D
(1994)
The use of transgenic and naturally occurring mutants to understand and manipulate tomato fruit ripening.
Plant Cell Environ
17:
557-571
[CrossRef]
Grierson D
(1986)
Molecular biology of fruit ripening.
Oxf Surv Plant Mol Cell Biol
3:
363-383
Hamilton AJ,
Lycett GW,
Grierson D
(1990)
Antisense gene that inhibits synthesis of the hormone ethylene in transgenic plants.
Nature
346:
284-287
[CrossRef]
Jarvis MC
(1998)
Intercellular separation forces generated by intracellular pressure.
Plant Cell Environ
21:
1307-1310
[CrossRef]
John I,
Drake R,
Farrell A,
Cooper W,
Lee P,
Horton P,
Grierson D
(1995)
Delayed leaf senescence in ethylene-deficient ACC-oxidase antisense tomato plants: molecular and physiological analysis.
Plant J
7:
483-490
[CrossRef]
Kerr EA
(1958)
Mutations of chlorophyll retention in ripe fruit.
Rep Tomato Genet Coop
8:
22
Kerr EA
(1982)
Never ripe-2 (Nr-2) a slow ripening mutant resembling Nr and Gr.
Rep Tomato Genet Coop
32:
33
Knapp J,
Moureau P,
Schuch W,
Grierson D
(1989)
Organisation and expression of polygalacturonase and other ripening genes in Ailsa Craig `Neverripe ' and `ripening inhibitor' tomato mutants.
Plant Mol Biol
12:
105-116
Kopeliovitch E,
Rabinowitch HD,
Mizrahi Y,
Kedar N
(1981)
Mode of inheritance of alcobaca, a tomato ripening mutant.
Euphytica
30:
223-225
[CrossRef]
Lanahan MB,
Yen H-C,
Giovannoni JJ,
Klee HJ
(1994)
The Never ripe mutation blocks ethylene perception in tomato.
Plant Cell
6:
521-530
[Abstract]
Lander ER,
Green P,
Abrahamson J,
Barlow A,
Daly M,
Lincoln SE,
Newburg L
(1987)
MAPMAKER: an interactive package for constructing primary genetic linkage maps of experimental and natural populations.
Genomics
1:
174-181
[CrossRef][Medline]
Rick CM
(1956)
New mutants.
Rep Tomato Genet Coop
6:
22-23
Robinson R,
Tomes M
(1968)
Ripening inhibitor: a gene with multiple effects on ripening.
Rep Tomato Genet Coop
18:
36-37
Sambrook J,
Fritsch EF,
Maniatis T
(1989)
Molecular Cloning: A Laboratory Manual, Ed 2.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
Tigchelaar EC,
Barman RJ
(1985)
Allelism of the alcobaca ripening mutant and nor.
Rep Tomato Genet Coop
35:
20
Tigchelaar EC,
McGlasson WB,
Buescher RW
(1978)
Genetic regulation of tomato fruit ripening.
HortScience
13:
508-513
[Web of Science]
Tigchelaar EC,
Tomes M,
Kerr E,
Barman R
(1973)
A new fruit ripening mutant, non-ripening (nor).
Rep Tomato Genet Coop
23:
33
Ward TM,
Wright M,
Roberts JA,
Self R,
Osborne DJ
(1978)
Analytical procedures for the assay and identification of ethylene.
In
JR Hillman,
eds, Isolation of Plant Growth Substances, Vol 4.
Cambridge University Press, Cambridge, UK, pp 135-151
Wellburn AR
(1994)
The spectral determination of chlorophyll-A and chlorophyll-B, as well as total carotenoids using various solvents with spectrophotometers of different resolution.
J Plant Physiol
144:
301-313
Wilkinson JQ,
Lanahan MB,
Clark DG,
Bleeker AB,
Chang C,
Meyerowitz EM,
Klee HJ
(1997)
A dominant mutant receptor from Arabidopsis confers ethylene insensitivity in heterologous plants.
Nat Biotechnol
15:
444-447
[CrossRef][Web of Science][Medline]
Wilkinson JQ,
Lanahan MB,
Yen H-C,
Giovannoni JJ,
Klee HJ
(1995)
An ethylene-inducible component of signal transduction encoded by Never-ripe.
Science
270:
1807-1809
[Abstract/Free Full Text]
Yen H-C,
Lee S,
Tanksley SD,
Lanahan MB,
Klee HJ,
Giovannoni JJ
(1995)
The tomato Never-ripe locus regulates ethylene-inducible gene expression and is linked to a homolog of the Arabidopsis ETR1 gene.
Plant Physiol
107:
1343-1353
[Abstract]
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Top
Abstract
Introduction