Download Molecular Analyses of MADS-Box Genes Trace

Molecular Analyses of MADS-Box Genes Trace Back to
Gymnosperms the Invention of Fleshy Fruits
Alessandro Lovisetto,1 Flavia Guzzo,2 Alice Tadiello,1 Ketti Toffali,2 Alessandro Favretto,1 and
Giorgio Casadoro1,*
1
Department of Biology, University of Padua, Padua, Italy
Department of Biotechnology, University of Verona, Verona, Italy
*Corresponding author: E-mail: [email protected], [email protected].
Associate editor: Neelima Sinha
2
Abstract
Key words: gymnosperm fruits, MADS-box genes, Ginkgo biloba, Taxus baccata.
Introduction
The seed habit is the most complex and evolutionarily
successful method of sexual reproduction found in vascular plants, and development of the seed habit represents
one of the most significant evolutionary events in the
history of vascular plants (quoted from Taylor et al.
2009). Its invention has boosted the success of seedproducing plants (i.e., Gymnosperms and Angiosperms)
owing to their increased possibility of spreading into
new habitats.
Also, the invention of fruits constitutes a remarkable
evolutionary advantage because they represent the instrument used by plants to disperse the seed into the environment. In the botanical jargon, a fruit derives from an ovary
following an event of fertilization. When an ovary develops
into a fruit, its wall becomes the pericarp which, on the
basis of its characteristics, can be distinguished into either
a parenchymatic fleshy type or a sclerenchymatic dry type
(Esaù 1965). Dry and fleshy fruits use different strategies to
disperse seeds. In particular, fleshy fruits are generally edible
and have attractive colors, so they direct the attention of
frugivorous animals toward their juicy pericarp and, by becoming a source of food, they entrust the animals with the
task of dispersing their seeds. Animals are mobile and are
therefore quite efficient in dispersing the seeds into the environment together with their own excrements.
Angiosperms are characterized by the production of
flowers; hence, they have ovaries that can be transformed
into fruits after fertilization. Having neither flowers nor ovaries to be transformed into a fruit, how do Gymnosperms
manage to disperse their seeds? Besides sclerenchymatic
strobili that open at maturity to release the seeds, many
Gymnosperms produce fleshy eatable structures around
the seed and use endozoochory for seed dispersal as Angiosperms do. The fleshy tissues that surround the seeds
of Gymnosperms are therefore functionally equivalent to
the angiosperm fruit (Herrera 1989).
All living orders of Gymnosperms (Cycadales, Ginkgoales,
Coniferales, and Gnetales) have species producing fruit-like
structures, with fleshy tissues that can originate from a variety of anatomical structures, depending on the species: seed
sarcotesta (Cycas and Ginkgo), aril (Taxus and Cephalotaxus),
bracts (Juniperus and Ephedra), and seed stalk (Podocarpus).
The origin of Gymnosperm fruit-like structures is considered
to be polyphyletic (Herrera 1989 and references herein) but
also Angiosperm fruits are considered to have a polyphyletic
origin in spite of the common carpellar derivation (Knapp
2002 and references herein). Interestingly, the habit of surrounding the seed with a fleshy tissue is not restricted to the
Gymnosperms since also some Angiosperms have maintained it. To the latter purpose, worth remembering are
the seeds of pomegranate, magnolia, peony, and others,
© The Author 2011. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please
e-mail: [email protected]
Mol. Biol. Evol. 29(1):409–419. 2012 doi:10.1093/molbev/msr244
Advance Access publication October 4, 2011
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Research article
Botanical fruits derive from ovaries and their most important function is to favor seed dispersal. Fleshy fruits do so by
attracting frugivorous animals that disperse seeds together with their own excrements (endozoochory). Gymnosperms
make seeds but have no ovaries to be transformed into fruits. Many species surround their seeds with fleshy structures and
use endozoochory to disperse them. Such structures are functionally fruits and can derive from different anatomical parts.
Ginkgo biloba and Taxus baccata fruit-like structures differ in their anatomical origin since the outer seed integument
becomes fleshy in Ginkgo, whereas in Taxus, the fleshy aril is formed de novo. The ripening characteristics are different,
with Ginkgo more rudimentary and Taxus more similar to angiosperm fruits. MADS-box genes are known to be necessary
for the formation of flowers and fruits in Angiosperms but also for making both male and female reproductive structures
in Gymnosperms. Here, a series of different MADS-box genes have been shown for the first time to be involved also in the
formation of gymnosperm fruit-like structures. Apparently, the same gene types have been recruited in phylogenetically
distant species to make fleshy structures that also have different anatomical origins. This finding indicates that the main
molecular networks operating in the development of fleshy fruits have independently appeared in distantly related
Gymnosperm taxa. Hence, the appearance of the seed habit and the accompanying necessity of seed dispersal has led to
the invention of the fruit habit that thus seems to have appeared independently of the presence of flowers.
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all of them surrounded by fleshy tissues that attract animals
when the fruit containing them is split open. Other Angiosperms, like strawberry- and pome-bearing species, produce
false fruits derived from nonovary tissues.
The activity of MADS-box transcription factors (TFs)
belonging to the C (i.e., AGAMOUS) and E (i.e., SEPALLATA)
types is necessary for the development of an ovary. (Pelaz
et al. 2000; Theissen and Saedler 2001; Krizek and Fletcher
2005). Recently, TFs of the same type have been shown to
be important also for the subsequent transformation of the
ovary into a fleshy fruit. In tomato, both TAGL1 (C type)
and RIN (E type) genes are involved in the development of
a ripe berry (Vrebalov et al. 2002, 2009; Itkin et al. 2009),
whereas MADS-box genes of the SEPALLATA type have
been shown to have a role in the development of the fleshy
strawberry tissues (Seymour et al. 2011). Finally, a C-type
gene from peach, when ectopically expressed in tomato,
caused the transformation of the normally leafy sepals into
ectopic fruits (Tadiello et al. 2009).
MADS-box genes are present also in Gymnosperms, and
similarly to what occurs in Angiosperms, some of them are
involved in the formation of the reproductive structures. In
particular, the interaction of B- and C-type proteins leads
to the development of the male strobili, whereas the sole
C-type proteins appear involved in the formation of the
female structures (Theissen and Melzer 2007; Wang
et al. 2010). The role played by these TFs has indirectly been
confirmed by Zhang et al. (2004) who were able to complement the agamous Arabidopsis mutant by using an
AGAMOUS gene from Cycas. Also, an AGAMOUS-like gene
from Picea exhibited C-type activity in Arabidopsis and
caused the homeotic transformation of sepals into
carpel-like structures (Tandre et al. 1998). As regards the
SEPALLATA genes, so far, they have not been found in extant Gymnosperms (Nam et al. 2003; Zahn et al. 2005),
however, in comprehensive phylogenetic trees, they form
a higher degree clade together with the AGL6 genes, and
Gymnosperms do have AGL6 genes that are expressed also
in their reproductive structures (Mouradov et al. 1998;
Winter et al. 1999).
Similarly to what occurs in Angiosperms, MADS-box
genes might have been employed by Gymnosperms also
to make their own fruit-like structures. Should this be
the case, the conclusion would be that the basic molecular
mechanisms necessary to make a fleshy fruit have actually
been invented by Gymnosperms. In this work, we have
investigated the involvement of various MADS-box genes
in the development of fleshy fruits in Ginkgo biloba and
Taxus baccata, species that represent two different developmental models owing to the fact that in Ginkgo, it is the
outermost seed tegument (sarcotesta) that grows and
becomes fleshy, whereas in Taxus, the fleshy aril is formed
de novo at the base of the ovule. Genes notoriously
involved in the formation of colors and in softening of
angiosperm fruits have also been studied in order to
understand whether the molecular mechanisms involved
in fruit ripening in Gymnosperms are like those present
in Angiosperms.
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Materials and Methods
Plant Material and RNA Extraction
Plant material came from the Botanic Garden of Padua.
In Ginkgo, initial samples consisted of whole ovules, then
the fleshy sarcotesta could be analyzed separately at various development stages indicated by dates in the figures.
In Taxus, the aril develops de novo as a collar at the base of
the ovule (supplementary data 1, Supplementary Material
online). Then it becomes visible as a green leafy structure
(LA), which grows thick and fleshy (FA) and starts to gradually develop a reddish color (Breaker: B1, B2) that becomes
bright and evenly distributed at the ripe stage (R). Total
RNA was extracted from different tissues according to
Chang et al. (1993). For each tissue, the extraction was
made using a pool of samples and was repeated at least
twice. RNA yield and purity were checked by means of ultraviolet (UV) absorption spectra, whereas RNA integrity
was ascertained by electrophoresis in agarose gel.
cDNA Isolation, Sequencing, and Analysis
All the cDNAs isolated de novo for this work were obtained
by reverse transcription polymerase chain reaction (RT-PCR)
using primers constructed following alignment of known
sequences. Primers are listed in supplementary data 2,
Supplementary Material online. In particular, for ripeningrelated genes, the template consisted of RNA extracted from
ripening pulp samples (Ginkgo: pulp of 19 July 2006; Taxus:
breaker-2 arils), whereas for MADS-box genes, the template
RNA was extracted from young ovules. The sequences obtained were registered in the Genebank database with the
following accession numbers: JF440955 (G. biloba expansin);
JF519740 (G. biloba pectate lyase [PL]); JF519741 (G. biloba
phytoene synthase); JF519743 (G. biloba ACC oxidase);
JF519744 (T. baccata PL); JF519745 (T. baccata expansin);
JF519747 (T. baccata phytoene synthase); JF519748 (T. baccata chalcone synthase); JF519750 (T. baccata ACO oxidase);
JF519751 (T. baccata ethylene receptor); JF519754 (T. baccata
AGAMOUS); JF519755 (T. baccata AGL6); and JF519756
(T. baccata TM8). DNA sequencing was performed at
BMR Genomics (Padua). Sequence manipulations, analyses,
and alignments were performed using the ‘‘Lasergene’’
software package (DNASTAR).
Construction of a MADS-Box Phylogenetic Tree
A set of MADS-box protein sequences downloaded from
GeneBank plus sequences obtained in this work were used
to construct a phylogenetic tree. The Genbank accession
numbers are AT4G18960 (Arabidopsis thaliana AG);
NM130127 (A. thaliana AGL6); NM115976 (A. thaliana
AGL13); PRU42400 (Pinus radiata PrMADS2); AB029470
(G. biloba GbMADS8); AB029463 (G. biloba GbMADS1);
U76726 (P. radiata PrMADS3); AB022665 (Gnetum parvifolium GpMADS3); L18924 (Zea mays ZAG1); AY114304
(G. biloba AG); AY295079 (Cycas edentata AG); AJ132215
(Gnetum gnemon GGM9); AJ132217 (G. gnemon GGM11);
AB359029 (Cryptomeria japonica AGL6); AB359027
(C. japonica M09 TM8); AB359028 (C. japonica O23
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TM8); AB029468 (G. biloba GbMADS6); AB029469
(G. biloba GbMADS7); AB029473 (G. biloba GbMADS11);
XM002283880 (Vitis vinifera TM8); X60760 (Lycopersicum
esculentum LeTM8); AB046596 (Cucumis sativus ERAF17);
X79280 (Picea abies DAL2); AB035567 (Chara globularis
CgMADS1); AB359031 (C. japonica AG); PMU69482 (Picea
mariana SAG1A); AJ132209 (G. gnemon GGM3); JF519754
(T. baccata TbAG); JF519755 (T. baccata TbAGL6); and
JF519756 (T. bacccata TbTM8). In particular, the tree
was constructed using the ‘‘MIK domain,’’ that is, the
MADS domain (60 aa) plus most of the I and K domains
(90 aa) similarly to Winter et al. (1999), Becker and Theissen
(2003), and Melzer et al. (2010). The amino acid sequences
were aligned with the CLUSTALW program, and the obtained alignments were used to construct the tree with
the MEGA 4.0.2 program. The tree was constructed with
the neighbor joining method (Saitou and Nei 1987) and
evaluated by bootstrap analysis.
Analysis of Gene Expression
This analysis was performed by standard real-time PCR.
Six micrograms of total RNA was pretreated with 2 U of
DNase I (Promega). The first-strand cDNA was synthesized
from 3 lg of the DNase I–treated RNA by means of the
High-Capacity cDNA Archive Kit (Applied Biosystems,
Foster City, CA), using random hexamers as primers. Primer
sequences for the selected genes are listed in supplementary data 2, Supplementary Material online. The internal
standards consisted of the internal transcribed spacer of
the ribosomal RNA sequences specific for each species
(supplementary data 2, Supplementary Material online).
PCR was carried out with the Gene Amp 7500 Sequence
Detection System (Applied Biosystems). The obtained CT
values were analyzed by means of the Q-gene software
by averaging three independently calculated normalized
expression values for each sample. Expression values are
given as the mean of the normalized expression values
of the triplicates, calculated according to equation (2) of
the Q-gene software (Muller et al. 2002).
In Situ Hybridization Analysis
Samples were fixed with 2% formaldehyde and 0.25% glutaraldehyde in phosphate-buffered saline buffer, pH 7.5. Clearing, infiltration, embedding, sectioning, prehybridization
treatments, and posthybridization washes were performed
according to Drews (1998), with minor modifications.
PCR fragments of C domain and/or 3#untranslated region
were isolated, cloned, and checked by sequencing. The list
of genes examined and the relative primers for the PCR
are listed in supplementary data 3, Supplementary Material
online. The PCR products were cloned and the sequence validated. The DIG-labeled RNA sense and antisense probes
were synthesized with the DIG RNA labeling Kit SP6/T7
(Roche diagnostics, Germany), the immunological detection
was performed with the anti-Digoxigenin-AP-Fab fragments
antibody (Roche Diagnostics), and the SIGMA FAST BCIP/
NBT tablets (Sigma) were used as phosphatase substrate, all
according to the manufacturer instructions.
Ethylene Treatments
Arils at the stage breaker-2 were either used immediately
after harvesting (control) or subjected to treatments with
exogenous ethylene (100 ll/l) in air as follows: a pool of
samples was left in air for 24 h, another pool was flushed
12 h with ethylene in an air-tight chamber and allowed to
stand in air for 12 more hours. A group of samples was kept
in air for 48 h while another pool was flushed 24 h with
ethylene and moved afterward to air for 24 more hours.
Results
MADS-Box Genes
In young ovules of both species, the expression of various
MADS-box genes was studied by in situ hybridization.
When fleshy tissues could be separated by the developing
seed, gene expression was studied by real-time PCR.
AGAMOUS-Like
AGAMOUS (AG) is necessary to make female reproductive
structures in Angiosperms (Rijpkema et al. 2010), and it
is also involved in the development of fleshy fruits like
tomato (Itkin et al. 2009; Vrebalov et al. 2009) and peach
(Tadiello et al. 2009). Since AGAMOUS has been shown to
be expressed also in Gymnosperm female reproductive
structures (Tandre et al. 1998; Zhang et al. 2004; Melzer
et al. 2010), it appeared an interesting candidate gene
for this study.
In particular, AGAMOUS had already been isolated in
Ginkgo (Jager et al. 2003), whereas no MADS-box gene
was known for Taxus, so an AG cDNA had to be isolated
by RT-PCR. Its sequence was used together with other AG
sequences to construct a phylogenetic tree, which showed
that it grouped with the other gymnosperm AG genes
(fig. 1). In particular it clustered with that of Cryptomeria
(Cupressaceae), probably because Taxaceae are phylogenetically closer to Cupressaceae than to Pinaceae (Judd et al.
2008).
In situ hybridization analysis revealed that in Ginkgo,
AGAMOUS was strongly expressed both in the nucellus
and in the not yet differentiated integuments of the young
ovules. Strong expression was also seen where the ovule
joins the peduncle, but the expression faded out moving
away from the ovule (fig. 2A and D). Upon differentiation
of the three zones that will give rise to the outer fleshy sarcotesta, to the sclerotesta, and to the thin innermost seed
coat, respectively, AG was still strongly expressed throughout the ovule (fig. 2C). This expression pattern was also
evident when a thick fleshy coat started to be visible
(fig. 2F).
In Taxus, AG was homogeneously expressed throughout
the female reproductive structure, including ovule, distal
portion of the peduncle and the outermost sterile scales
(fig. 2G). Later on the AG expression was particularly localized to the young developing aril, immediately below the
ovule (fig. 2I and K, arrows).
The expression of AGAMOUS obtained by real-time PCR
(fig. 3A and B) confirmed previous data for Ginkgo about
the presence of transcripts in male strobili and ovules (Jager
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FIG. 1. Phylogenetic tree showing subfamilies of the AGAMOUS,
AGL6, TM8 MIKC–type MADS-box proteins. Sequences relative to
the genes characterized in this work are marked by different arrows
according to subfamily type.
et al. 2003). As regards the fleshy sarcotesta, AG transcripts
reached very high levels during the phase of fast growth
(26 May), gradually decreased thereafter and, though
not much, increased again during the ripening stage
(29 August).
In Taxus, AGAMOUS was expressed in both male and female structures, although at much higher levels in the ovule.
As regards the aril, the expression was high in the green leafy
one, continued to slightly increase during the acquisition of
fleshyness, and reached a maximum at the start of ripening.
Thereafter, there was a little decrease but the transcripts remained high until the red ripe stage.
AGL6-Like
In G. gnemon, two different AGL6 genes are expressed in the
reproductive structures, although one gene seems to be
more ovule specific (Winter et al. 1999). Accordingly,
AGL6 genes are important for our study.
Two full-length cDNAs coding for two different AGL6 proteins (GbMADS1 and GbMADS8, respectively) in Ginkgo were
singled out by a search in public databases. As regards Taxus,
we had to clone ourselves the AGL6 cDNA. Yet, in spite of
considerable efforts, only one AGL6 cDNA could be isolated.
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The sequences from Ginkgo and from Taxus were used
together with other AGL6 sequences to construct a phylogenetic tree (fig. 1). The two Ginkgo AGL6 genes fell into
two separate clades, each together with another sequence
from P. radiata, whereas the sequence from Taxus formed
a separate clade with a sequence from C. japonica.
The in situ hybridization analysis showed that in young
Ginkgo ovules, both GbMADS1 and GbMADS8 were
strongly expressed in the integuments, nucellus, at the base
of the ovules, whereas a weaker signal was present in the
peduncle region adjoining the ovule (fig. 2P and Q). In
Taxus, the AGL6 gene was strongly expressed in the young
developing aril and in the ovule and less expressed in the
innermost scales and within the peduncle (fig. 2L). In the
latter region, the AGL6 expression faded out upon moving
away from the ovule.
The real-time PCR analysis of the Ginkgo AGL6 genes
showed important differences in their expression profile
(fig. 3C). In general, GbMADS8 had a much higher level
of expression than GbMADS1 and seemed to be more specific for the reproductive structures since GbMADS1 was
expressed also in young leaves. For both genes, the expression was significantly higher in ovules than in male strobili.
GbMADS1 showed very low and decreasing transcript
amounts in the pulp as to be barely detectable in the summer samples. Also the expression of GbMADS8 showed
a decreasing pattern until the last sampling when a marked
increase was measured concomitant with ripening.
The Taxus AGL6 gene was not expressed in leaves therefore, like the Ginkgo GbMADS8, it seems specific for the
reproductive structures (fig. 3D). In arils, the expression
was generally higher than in male strobili and in ovules.
In particular, a marked increase occurred during the transition from leafy to fleshy stages. Then, there was some decrease at the breaker-1 stage followed by a further increase
leading to maximum expression at the breaker-2 stage, that
is when arils are in full ripening process.
TM8-Like
Degenerate primers constructed following the protocol described in Hileman et al. (2006) were also used to try to
isolate a second AGL6 gene from Taxus. Instead of the long
sought AGL6 gene, an unexpected TM8-like gene was obtained. An informatic search was therefore done, and three
TM8-like sequences from Ginkgo could be retrieved from
public databases. The known TM8-like sequences were then
used to construct a phylogenetic tree (fig. 1). Interestingly,
angiosperms and gymnosperms sequences grouped together
into a general TM8 clade. Within such clade, three subgroups
could be distinguished that reflected the phylogenetic relationships among the species considered: one was formed by
the sole angiosperm sequences, the second groups contained
the sequences from Taxus and Cryptomeria (both of them
Coniferales), whereas the third group was formed by the
three Ginkgo sequences.
In situ hybridization analysis evidenced that in Ginkgo,
the three TM8-like genes had overlapping expression in
ovules. Strong GbMADS6 and GbMADS11 expression was
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FIG. 2. Expression of AGAMOUS (panels A, C, D, F, G, I, and K), AGL6-like (GbMADS1: panel P; GbMADS8: panel Q; and TbAGL6: panel L), and
TM8-like (Taxus TM8-like: panel N; GbMADS6: panels R; GbMADS11: panel S; and GbMADS7: panel T) analyzed by in situ hybridization in young
Ginkgo (A, C, D, F, P, Q, R, S, and T) and Taxus (G, I, K, L, and N) female reproductive structures. Panels B, E, H, J, M, O, and U: sense control in
Ginkgo (B, E, and U) and Taxus (H, J, M, and O). pc, pollen chamber; in, integuments; es, embryo sac; oi, outer integument; and ii, inner
integument. Arrows: developing arils. AG 5 AGAMOUS; AGL6 5 AGL6-like; TM8 5 TM8-like; s 5 sense probe; and a 5 antisense probe.
observed throughout the ovule, whereas a weaker signal
was visible in the peduncle region adjoining the ovule
(fig. 2R and S). GbMADS7 expression was very weak compared with the other two TM8-like genes but showed a similar pattern (fig. 2T). In Taxus, TM8 was strongly expressed
in the ovule and in the young developing arils and weakly
expressed in the distal part of the peduncle and in the more
apical sterile scales (fig. 2N).
Real-time PCR analysis revealed that of the three Ginkgo
TM8-like sequences, GbMADS7 was always expressed
at very low amounts compared with the other two
(GbMADS6 and GbMADS11). In particular, the highest expression level was observed in young leaves, followed by the
ovules, whereas in the pulp, its transcripts were always very
low (fig. 3E). GbMADS6 was the most highly expressed of
the three genes, and similarly to the other two, the maximum transcript amount was found in young leaves. Also
male strobili and ovules exhibited high transcript amounts
comparable to those present in the young sarcotesta undergoing a fast increase in thickness. Thereafter, in the latter tissue, the expression slightly decreased albeit remaining
quite high until the last sampling when ripening occurred.
The transcripts of the GbMADS11 gene were high in leaves,
ovules, and male strobili. In the fleshy sarcotesta, the
initially low expression (26 May) showed an increase
during the subsequent stage of fast growth (16 June), an
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FIG. 3. Relative expression profiles of Ginkgo biloba (Gb) and Taxus baccata (Tb) MADS-box genes: AG (AGAMOUS), AGL6, and TM8. Gb
samples: YL, young leaf; #, male strobili; $, young ovules; 26 May, 16 June, 19 July, 01 August, and 29 August sampling dates (2006 season) of
pulps at different developmental stages. Tb samples: YL, young leaf ; #, male strobili; $, young ovules; LA, leafy arils; FA, fleshy green arils; B1,
breaker-1 arils; B2, breaker-2 arils; and R, red ripe arils. Values (means of the normalized expression) have been obtained by RT-PCR analyses.
Bars are the standard deviations from the means.
unexpected drop in early summer (19/7) that was followed
by a significant increase to higher amounts when the fruit
was undergoing the ripening process.
Similarly to Ginkgo, also the Taxus TM8-like gene was
expressed in young leaves, male strobili, and ovules
(fig. 3F). However, contrary to Ginkgo, the highest expression was found in the arils and not in the leaves. In particular, there was a significant transcript amount during
growth of the leafy arils, followed by a further increment
that led to a maximum concomitant with the development
of fleshyness. Thereafter, the expression slowly decreased,
though remaining substantially high throughout the entire
ripening process.
Ripening Syndrome
The process of ripening transforms fleshy fruits into structures that are attractive for animals. In short, fruits become
more or less soft, their initially green color is substituted
by other colors that catch the eye of animals, while the
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production of characteristic aromas make them an appealing source of food. Such processes constitute the ripening
syndrome, and in this work, attention has been given to
softening and changes in color, both of them well known
at the molecular level in many angiosperm fruits. The formation of aroma has not been considered because it is
extremely variable and far less known.
Softening
In general, softening occurs as a consequence of pectin and
hemicellulose degradation, and different enzymes have
been shown to act on the different components (Brett
and Waldron 1996; Brummell and Harpster 2001). We have
chosen to analyze genes coding for PL and expansin whose
role has been shown in strawberry (Jiménez-Bermùdez et al.
2002) and tomato (Brummell et al. 1999), respectively.
Pectate Lyase. By aligning various known sequences, a
particularly conserved region was identified, and degenerate primers were prepared. RT-PCR experiments allowed
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the cloning of two cDNA fragments coding for PL in Ginkgo
and Taxus, respectively.
In Ginkgo, the gene was mostly expressed in growing tissues like young leaves, young reproductive structures (male
strobili and ovules), and young sarcotesta (fig. 4A). No measurable expression was observed during pulp ripening. On
the contrary, in Taxus, the PL gene showed a ripeningspecific expression pattern (fig. 4B). Transcripts started
to be measurable in green fleshy arils, then their amount
showed a great and continuous increase up to a maximum
in the breaker-2 arils and was finally followed by a slight
decrease at the red ripe stage.
Expansin (EXP). Expansins are known to cause cell wall loosening by breaking bonds between cellulose fibrils and matrix polysaccharides (Cosgrove 2000). Since cell wall
loosening occurs both during growth-related tissue expansion and during fruit softening, the activity of expansins can
be found in every type of cell separation events. The importance of a softening specific expansin has been shown
in tomato (Brummell et al. 1999). Degenerate primers for
RT-PCR experiments were prepared by aligning various
EXP sequences, and an EXP cDNA fragment was obtained
for both Ginkgo and Taxus.
The Ginkgo expansin had the highest expression in
young leaves (fig. 4C). Though much less, it was expressed
also in male strobili and ovules. As regards the pulp, the
gene was expressed mainly in the young growing sarcotesta
and decreased thereafter until the early summer samples,
then it showed a slight increase during ripening. In Taxus,
the EXP gene was expressed at low levels in young leaves
and ovules and almost 3-fold more in the male strobili
(fig. 4D). Much higher amounts of transcript were found
in the ripening arils where the gene reached its maximum
expression in the breaker-2 arils.
Change of Color
Ripening fruits usually lose their green color and accumulate other pigments, thus signaling to animals that they
have reached the eatable stage. But for betalains whose
presence seems limited to Caryophyllales, the nongreen
colors in plants, and particularly in fruits, are mostly due
to either carotenoids or anthocyanins/flavonoids (Tanaka
et al. 2008). The enzyme that specifically leads to the
synthesis of carotenoids is phytoene synthase (PSY).
Anthocyanins/flavonoids are phenols, and chalcone synthase (CHS) is an enzyme of the biosynthetic pathway that
acts upstream of all these colored molecules (Taiz and
Zeiger 2006).
Phytoene Synthase. Degenerate primers were prepared to be
used in RT-PCR experiments that led to the cloning of two
cDNA fragments encoding PSY in Ginkgo and in Taxus, respectively. In Ginkgo, high amounts of PSY transcripts were
found in young leaves (fig. 4E). Extremely, low levels of expression were observed in the yellowish male strobili, in the
green ovules, and in the young green sarcotesta. In Taxus,
some expression the PSY gene (fig. 4F) was observed in
the young leaves, but the highest transcript amount was
FIG. 4. Relative expression profiles of Ginkgo biloba (Gb) and Taxus
baccata (Tb) genes related to softening (A–D), color (E–H), and
ethylene biosynthesis (I, L). Samples are indicated as in figure 3.
Panel (M) shows relative expression of an ethylene receptor gene
(ETR) of T. baccata, whereas panel (N) shows the relative expression
of the yew ACO gene in arils either kept in air or treated with
exogenous ethylene (100 ll/l) in air (a: arils frozen immediately after
sampling ; b: arils kept 24 h in air; c: arils kept 12 h in ethylene
followed by 12 h in air; d: arils kept 48 h in air; and e: arils kept 24 h
in ethylene followed by 24 h in air). Values (means of the
normalized expression) have been obtained by RT-PCR analyses.
Bars are the standard deviations from the means.
415
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Lovisetto et al. · doi:10.1093/molbev/msr244
found in the arils where the gene expression showed a great
and continuous increase up to a maximum at the breaker-2
stage, followed by a decrease at the red ripe stage.
Chalcone Synthase. Degenerate primers to be used in RTPCR experiments were prepared by aligning several cDNA
sequences. One cDNA fragment coding for chalcone synthase was obtained for each species. In Ginkgo, the CHS
gene was highly expressed in male strobili and in ovules
(fig. 4G). Much lower amounts of transcript were visible
in young leaves and in young growing sarcotesta. All the
other samples showed extremely low levels of expression,
although a slightly increasing pattern was visible during ripening in the summer samples. In Taxus, the highest CHS
transcript amount was observed in the young leaves, followed by that present in the ovules. Arils had extremely
low amounts of CHS transcripts, but for the green leafy
ones that had a bit higher expression level (fig. 4H).
Ethylene-Related Genes
Angiosperm fruits may or may not have their ripening controlled by ethylene. In the first case, fruits are said to be
climacteric (e.g., tomato) and show a peak of ethylene production (climacteric ethylene) at the onset of ripening. The
synthesis of climacteric ethylene is autocatalytic; hence, the
hormone is able to stimulate its own synthesis, contrary to
what happens in young growing tissues where ethylene has
not such capacity. In the second case, fruits do not produce
any climacteric ethylene and are said to be nonclimacteric
(e.g., grape). ACC oxidase (ACO) is the enzyme that does
actually synthesize the hormone starting from the substrate ACC (1-aminocyclopropane-1-carboxylic acid). Since
a relation has been found between the expression of ACO
and the amount of measured ethylene, the expression of
ACO is often used as an indirect measure of ethylene
production.
ACC Oxidase. By using degenerate primers, two cDNA fragments were obtained that encoded a Ginkgo and a Taxus
ACO, respectively. In Ginkgo, the ACO gene was mostly expressed in young leaves and, at much lower levels, in male
strobili, whereas its expression was hardly detectable in all
the other samples (fig. 4I). Taxus behaved differently since
the ACO gene had a clear ripening-specific pattern of expression. The transcript amount was extremely low in all
the samples except in the arils that were becoming fleshy.
Particularly striking was the high expression increase
occurring during the late ripening stages (fig. 4L).
Ethylene Receptor. Based on the pattern of ACO expression,
ethylene seemed to be important for ripening in Taxus but
not in Ginkgo. Accordingly, only for Taxus, a cDNA was
cloned that codes for an ethylene receptor, and its expression analyzed in various tissues (fig. 4M). The ETR gene appeared to be expressed in all the examined tissues, and this
is in agreement with the fact that basal levels of ethylene
are made in plants by all living tissues (Taiz and Zeiger
2006). However, the expression of this gene showed
a marked and significant increase in breaker-2 and red arils
that paralleled the observed increases of ACO expression.
416
Treatment of Arils with Ethylene. The quasi parallel increasing expression of the ACO and ETR genes during ripening
suggested that the arils of Taxus might behave like climacteric fruits. Since ethylene can stimulate its own synthesis in
climacteric fruits, ethylene was used to treat arils at the
breaker-2 stage, that is, arils in which the synthesis of ethylene was apparently ‘‘on’’ but not yet at its maximum rate.
At the end of the experiment, the expression of the ACO
gene was measured in the different samples (fig. 4N). As
expected, the breaker-2 arils kept 24 h in air showed an
increased expression of ACO, but surprisingly, a comparable
increment was found also in the ethylene-treated arils. Similar results were obtained with the arils altogether treated
for 48 h, albeit in this case, the expression values were much
higher.
Discussion
Different Ripening Syndromes Are Present in
Ginkgo and Taxus Fruits
The two cell separation–related genes had a ripeningspecific expression in Taxus but not in Ginkgo. Actually,
albeit at very low levels, the Ginkgo expansin showed some
increasing expression in late stages of fleshy sarcotesta
development; therefore, it might be involved also in the
process of softening. We cannot however exclude that
other not yet cloned genes are involved in softening of
the Ginkgo fruit-like structures. Alternatively, the softening
in Ginkgo might occur, at least partly, by water loss through
the cuticle followed by a loss of tissue stiffness, the latter
a mechanism for softening suggested by Saladié et al.
(2007).
Also, the modality for changing color during ripening
turned out to be different in the two species. While in
the fleshy sarcotesta of Ginkgo, the phytoene synthase
(PSY) transcripts were barely detectable; in Taxus, the
PSY gene had a marked increasing expression in the ripening arils. Therefore, it can be concluded that carotenoids
are de novo accumulated in ripening Taxus arils. The latter
statement is also supported by the results of a chromatography where pigments extracted by means of diethyl ether
from both leafy green arils and red ripe ones were compared. Interestingly, the red berries contained newly
formed red carotenoids that were not present in the green
leafy arils (supplementary data 4, Supplementary Material
online).
The expression of the chalcone synthase (CHS) gene
seemed specific for ovules and young leaves in Taxus
and for ovules and male strobili in Ginkgo. In particular,
Ginkgo has deciduous leaves and forms its reproductive
structures when the new foliage is still poorly developed;
therefore, they might be easily damaged by UV radiations.
To the latter purpose, an Arabidopsis mutant, incapable to
make flavonoids because deprived of a functional CHS,
could survive only in the absence of UV radiation (Li
et al. 1993), thus evidencing the protective role performed
by flavonoids. Accordingly, the high levels of CHS expression observed in Ginkgo reproductive structures, but also in
Gymnosperm Fleshy Fruits · doi:10.1093/molbev/msr244
ovules and young leaves of Taxus, might be linked to the
formation of protective compounds. Therefore, most of the
yellowish color in the ripe and senescent Ginkgo fruit-like
structures might actually be due to photosynthetic carotenoids becoming visible after chlorophyll breakdown, as it
happens with senescent leaves. This idea seems supported
by the results of a chromatography where carotenoids from
either green or yellow pulps were compared and no apparent carotenoid difference was visible (supplementary data
4, Supplementary Material online).
In Ginkgo, the expression of an ACO gene suggests that
this gene is involved in the synthesis of growth-related hormone. In Taxus, the aril- and ripening-specific expression of
the ACO gene indicates that ethylene is involved in the ripening of yew berries. A quasi parallel increasing expression
of an ethylene receptor gene during ripening suggested
that the aril might be like a climacteric fruit. Yet, this idea
turned out to be untrue since treatments with exogenous
ethylene were unable to stimulate the autocatalytic hormone synthesis characteristic of the truly climacteric fruits.
Hence, it seems that the mechanism of climacteric fruit ripening has not yet appeared in Taxus.
In general, between the two gymnosperms, the ripening
of Taxus arils is the most similar to that of angiosperm
fruits. Ginkgophyta are considered to have appeared in
lower Permian (Zhou 2009), whereas Taxaceae appearance
has been situated in middle Jurassic (Taylor et al. 2009).
Accordingly, the apparently simpler ripening syndrome
of the Ginkgo fruit-like structures might reflect the greater
antiquity of Ginkgophyta compared with Taxaceae.
MADS-Box Genes of the Same Type Are Involved in
the Development of the Fleshy Fruit Habit in
Gymnosperms
The biochemical pathways that concur to establish the ripening syndrome of fleshy fruits are under the control of
different TFs. For instance, a MYB TF is involved in the
formation of colored molecules in ripening strawberries
(Aharoni et al. 2001). The synthesis of the bilberry fruit
pigments has been shown to be under the control of a
SQUAMOSA MADS-box TF (Jaakola et al. 2010). In tomato,
it has been shown that LeHB1, an HD-ZIP TF, can bind to
the promoter of an ACO gene, thus regulating its expression (Lin et al. 2008). The accumulation of pigments in the
tomato berry has been shown to be regulated by the TAGL1
MADS-box gene (Itkin et al. 2009; Vrebalov et al. 2009).
The various pathways that form the ripening syndrome
are situated at the end of the overall development of fleshy
fruits and, before they can be activated, a given anatomical
part must first undertake the developmental pathway leading to the fleshy fruit condition. Once the fleshy fruit habit
has been established, the ripening syndrome typical for
each species will be settled. This has been shown in tomato
where, by ectopically expressing a C type MADS-box gene
from peach, the normally green and leafy sepals acquired
the characteristic of carpels and were thus able to develop
as ectopic fruits that ripened like those derived from
the ovary (Tadiello et al. 2009). Therefore, of paramount
MBE
importance is the function of those genes whose activity
is able to switch on the fleshy fruit habit.
MADS-box genes are interesting candidate genes because
they are involved in the formation of reproductive structures
in both Gymnosperms and Angiosperms (Theissen et al.
2000). In ovary-derived fruits, MADS-box genes are further
used for the subsequent transformation of the carpel into
a fruit (Itkin et al. 2009; Tadiello et al. 2009; Vrebalov et al.
2009). In this work, MADS-box genes like AGAMOUS and
AGL6 have been shown to be involved also in the formation
of the gymnosperm fruit.
Only one AGAMOUS gene is known in Gymnosperms,
although a different situation is found regarding AGL6
genes. In various Gymnosperms, two AGL6 genes have been
evidenced; however, in Soltis et al. (2007), three such genes
were reported for Zamia fischeri and only one gene for
Welwitschia mirabilis. A southern analysis carried out by
us indicated that two AGL6 genes might be present also
in Taxus (supplementary data 5, Supplementary Material
online). However, in spite of considerable efforts, we were
able to clone only one AGL6 gene in this species, and such
a result is in agreement with the high levels of expression of
this gene in the arils. Therefore, though it appears that we
failed to clone a second AGL6 gene, the one characterized
in this work is certainly a gene highly involved in the
formation of the Taxus arils, and together with the results
obtained for the Ginkgo GbMADS8 AGL6 gene, it indicates
that genes belonging to the AGL6 group undoubtedly participate in the development and ripening of the fruit-like
structures of both gymnosperm species.
As summarized in Introduction, C- and E-type MADSbox genes are important for the development of angiosperm fruits. E-type (i.e., SEPALLATA) genes are not present
in Gymnosperms that have instead the closely related AGL6
genes. In Petunia, it was recently demonstrated that an
AGL6 gene had a SEPALLATA-like function in floral patterning (Rijpkema et al. 2009). Moreover, it was suggested in
Melzer et al. (2010) that the gymnosperm AGL6 genes
might carry out some functions performed in Angiosperms
by the E-type genes. Accordingly, some sort of ‘‘SEPALLATA
function’’ in gymnosperm fruits might be played at least by
the AGL6 gene whose transcripts are continuously present
in the developing fruits of both Ginkgo and Taxus.
Besides AGAMOUS and AGL6, a TM8-like cDNA for
Taxus and three cDNAs coding for the same gene type
in Ginkgo were also obtained. TM8 is a poorly known gene
isolated for the first time in tomato (Pnueli et al. 1991),
where it was found that in fruits, it is expressed throughout
development and ripening with an apparent maximum at
the ‘‘yellow’’ stage (Hileman et al. 2006).
Like in tomato, and in Ginkgo, the three TM8-like genes
were expressed in both vegetative and reproductive tissues,
and two of them were particularly expressed throughout development and ripening of the fleshy sarcotesta, thus indicating that they have a role in the formation of the Ginkgo
fruit-like structures. In Taxus, the expression pattern of the
TM8-like gene suggests that it might be more important for
the aril formation and ripening than in vegetative tissues.
417
Lovisetto et al. · doi:10.1093/molbev/msr244
AGAMOUS, AGL6, and TM8-like MADS-box genes
appear all to be involved in the development of the fleshy
structures in both Ginkgo and Taxus. Therefore, distantly
related taxa have recruited the same type of genes in order
to achieve the fleshy fruit habit, and this in spite of the
different anatomical structures actually employed for this
purpose. Nothing is known about Gnetales fleshy fruits, but
in Cycadales (C. edentata), it was evidenced that AGAMOUS was highly expressed also in the young sarcotesta
(Zhang et al. 2004). The fruit-like structures of Gymnosperms have in common a close vicinity to the developing
seed; hence, as observed in Ginkgo and in Taxus, genes normally involved in the development of ovule/seed have just
to spread their expression to close anatomical territories for
the development of a fleshy structure to be accomplished.
Therefore, as far as MADS-box genes are concerned, the
general molecular mechanism seems to be the same,
although it appears to have developed various times as
suggested by the different anatomical tissues toward which
such genes had to shift their expression for the fruit-like
structure to form.
Angiosperms use AGAMOUS, TM8, and SEPALLATA
genes, the latter absent in Gymnosperms, for the development of fleshy fruits. Given the phylogenetic vicinity
of AGL6 and SEPALLATA genes, it seems that the main
molecular networks underlying the formation of the fleshy
fruit habit have developed independently of the presence
of flowers and have basically been maintained in the
flower-producing Angiosperms. This idea is in agreement
with a recent finding (Chanderbali et al. 2010), which shows
that genetic programs operating in gymnosperm female
cones are also present in angiosperm carpels.
Supplementary Material
Supplementary data 1–5 are available at Molecular Biology
and Evolution online (http://www.mbe.oxfordjournals.org/).
Acknowledgments
Dr E. Negrisolo is thanked for helpful advice regarding
the construction of the phylogenetic tree. A. Pavanello
is thanked for skillful technical help. All plant material
used in this work was obtained by trees growing at the
Botanic Gardens of the University of Padua. This work
was supported by a grant from Ministero dell’Istruzione,
dell’Università e della Ricerca (MIUR), Italy, to G.C.
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