Article

The role of cytokinin during Arabidopsis gynoecia and fruit morphogenesis and patterning

Departamento de Biotecnología y Bioquímica, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, km 9.6 Libramiento Norte, Carretera Irapuato-León, Irapuato, Guanajuato, Mexico, Departamento de Ingeniería Genética, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Irapuato, Guanajuato, Mexico, and Laboratorio Nacional de Genómica para la Biodiversidad (Langebio), Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, km 9.6 Libramiento Norte, Carretera Irapuato-León, Irapuato, Guanajuato, Mexico.
The Plant Journal (Impact Factor: 5.97). 05/2012; 72(2):222-234. DOI: 10.1111/j.1365-313X.2012.05062.x
Source: PubMed

ABSTRACT

Cytokinins have many essential roles in embryonic and post-embryonic growth and development, but their role in fruit morphogenesis is currently not really known. Moreover, information about the spatio-temporal localization pattern of cytokinin signaling in gynoecia and fruits is lacking. Therefore, the synthetic reporter line TCS::GFP was used to visualize cytokinin signaling during gynoecium and fruit development. Fluorescence was detected at medial regions of developing gynoecia, and, unexpectedly, at the valve margin in developing fruits, and was severely altered in mutants that lack or ectopically acquire valve margin identity. Comparison to developing gynoecia and fruits in a DR5rev::GFP line showed that the transcriptional responses to cytokinin and auxin are frequently present in complementary patterns. Moreover, cytokinin treatments in early gynoecia produced conspicuous changes, and treatment of valve margin mutant fruits restored this tissue. The results suggest that the phytohormone cytokinin is important in gynoecium and fruit patterning and morphogenesis, playing at least two roles: an early proliferation-inducing role at the medial tissues of the developing gynoecia, and a late role in fruit patterning and morphogenesis at the valve margin of developing fruits.

Full-text

Available from: Stefan De Folter
The role of cytokinin during Arabidopsis gynoecia and fruit
morphogenesis and patterning
Nayelli Marsch-Martı
´
nez
1,2,*
, Daniela Ramos-Cruz
1,3
, J. Irepan Reyes-Olalde
3
, Paulina Lozano-Sotomayor
3
,
Victor M. Zu
´
n
˜
iga-Mayo
3
and Stefan de Folter
3,
*
1
Departamento de Biotecnologı
´
a y Bioquı
´
mica, Centro de Investigacio
´
n y de Estudios Avanzados del Instituto Polite
´
cnico
Nacional, km 9.6 Libramiento Norte, Carretera Irapuato-Leo
´
n, Irapuato, Guanajuato, Mexico,
2
Departamento de Ingenierı
´
a Gene
´
tica, Centro de Investigacio
´
n y de Estudios Avanzados del Instituto Polite
´
cnico Nacional,
Irapuato, Guanajuato, Mexico, and
3
Laboratorio Nacional de Geno
´
mica para la Biodiversidad (Langebio), Centro de Investigacio
´
n y de Estudios Avanzados del
Instituto Polite
´
cnico Nacional, km 9.6 Libramiento Norte, Carretera Irapuato-Leo
´
n, Irapuato, Guanajuato, Mexico
Received 9 September 2011; revised 15 May 2012; accepted 23 May 2012; published online 7 August 2012.
*For correspondence (e-mail nmarsch@ira.cinvestav.mx or sdfolter@langebio.cinvestav.mx).
SUMMARY
Cytokinins have many essential roles in embryonic and post-embryonic growth and development, but their
role in fruit morphogenesis is currently not really known. Moreover, information about the spatio-temporal
localization pattern of cytokinin signaling in gynoecia and fruits is lacking. Therefore, the synthetic reporter
line TCS::GFP was used to visualize cytokinin signaling during gynoecium and fruit development. Fluorescence
was detected at medial regions of developing gynoecia, and, unexpectedly, at the valve margin in developing
fruits, and was severely altered in mutants that lack or ectopically acquire valve margin identity. Comparison to
developing gynoecia and fruits in a DR5rev::GFP line showed that the transcriptional responses to cytokinin
and auxin are frequently present in complementary patterns. Moreover, cytokinin treatments in early gynoecia
produced conspicuous changes, and treatment of valve margin mutant fruits restored this tissue. The results
suggest that the phytohormone cytokinin is important in gynoecium and fruit patterning and morphogenesis,
playing at least two roles: an early proliferation-inducing role at the medial tissues of the developing gynoecia,
and a late role in fruit patterning and morphogenesis at the valve margin of developing fruits.
Keywords: cytokinins, Arabidopsis gynoecium and fruit development, valve margin, cell proliferation,
TCS::GFP, auxin.
INTRODUCTION
Flower development is a key process for all living angio-
sperms, and is essential for sexual reproduction. Fruits
develop from the female reproductive part of the flower, which
is also referred to as the gynoecium and consists of one or
more ovule-bearing leaf-like structures, the carpels. Fruits are
important for seed dispersal and have a high nutritional value.
In Arabidopsis, the gynoecium starts developing as a
hollow tube from where the medial tissues initiate as two
internal ridges that grow towards each other to form the
septum (inside) and replum (outside). After fusion, ovules
start to develop from the internal tissue (placenta). Mean-
while, cell proliferation at the apical region of medial tissues
gives rise to the style, closing the hollow tube. The style is
then crowned by stigmatic papillae, where pollen tubes
germinate and grow through the transmitting tract to reach
the ovules in a mature gynoecium (Bowman et al., 1999;
Alvarez and Smyth, 2002; Roeder and Yanofsky, 2006; Sund-
berg and Ferra
´
ndiz, 2009). After fertilization, the fruit elon-
gates synchronically as the seed develops. Stigmatic papillae
degenerate, and the valve margin, which is located between
the valve and the replum, matures, involving lignification of
special cells (including the valves), finally leading to dehis-
cense and seed release (pod shattering) (Ferrandiz, 2002).
Some transcription factors involved in proper valve margin
development are INDEHISCENT (IND) and SHATTERPROOF1
and 2 (SHP1/2), and their absence results in indehiscent fruits
(Liljegren et al., 2000, 2004). Expression of the IND and SHP1/
2 genes is repressed in the valve by FRUITFULL (FUL). This
transcription factor is required to prevent conversion of the
valve into valve margin tissue, and valves acquire valve
margin identity and fail to elongate in ful mutant fruits
(Ferrandiz et al., 2000; Liljegren et al., 2000, 2004).
222 ª 2012 The Authors
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Page 1
Hormones play key roles in diverse plant processes
(Wolters and Jurgens, 2009), such as fruit development
and patterning (Balanza et al., 2006; Alabadi et al., 2009;
Sundberg and Ostergaard, 2009). Auxin has received special
attention. Alterations in biosynthesis, signaling or transport
components can severely affect gynoecium development
and patterning (Sta
˚
ldal and Sundberg, 2009; Sundberg and
Ferra
´
ndiz, 2009; Sundberg and Ostergaard, 2009). Auxin
gradients in the root specify the position of the root meristem
(Sabatini et al., 1999), and auxin has also been proposed to
act as an apical–basal gradient in fruit development (Nem-
hauser et al., 2000). Moreover, auxin biosynthesis genes are
regulated by transcription factors guiding fruit patterning
(Alvarez et al., 2009; Trigueros et al., 2009; Eklund et al.,
2010). In contrast, it was recently shown that lack of auxin but
the presence of gibberellins is required for correct valve
margin formation (Sorefan et al., 2009; Arnaud et al., 2010).
Cytokinins are signaling molecules derived from adenine,
and they have many essential roles in embryonic and post-
embryonic growth and development (Muller and Sheen,
2007, 2008; Werner and Schmulling, 2009; Argueso et al.,
2010). However, their role in gynoecium and fruit develop-
ment is just starting to be explored. Recently, an important
role for cytokinins for placental growth and ovule number
has been uncovered by mutations in CYTOKININ OXIDASE/
DEHYDROGENASE (CKX) genes. CKX enzymes are respon-
sible for cytokinin breakdown, and mutations in some of
them result in increased seed yield (Ashikari et al., 2005;
Bartrina et al., 2011). Bartrina et al. (2011) have shown that
cytokinins delay the differentiation of cells in the reproduc-
tive meristems, and regulate the activity of the ovule-
forming placenta (Bartrina et al., 2011). Furthermore, it has
been reported that exogenous cytokinin application can
cause parthenocarpic fruit growth (Vivian-Smith and Koltu-
now, 1999) and ectopic trichome formation on carpels in
transgenic plants expressing the cytokinin biosynthetic gene
ISOPENTENYL TRANSFERASE (IPT) under the control of a
carpel-specific promoter (Greenboim-Wainberg et al., 2005).
However, it is still not well known whether cytokinins are
important for the process of fruit patterning and morphol-
ogy. Moreover, detailed information about the spatio-tem-
poral localization pattern in gynoecia is lacking, and could
help to uncover further roles of cytokinins at later stages of
gynoecia and fruit development or patterning.
Here we report on analyses of the cytokinin signaling
pattern during various gynoecia and fruit developmental
stages in wild-type plants and mutant backgrounds. Fur-
thermore, we compared cytokinin signaling with auxin
signaling, and investigated the effects of endogenous and
exogenous cytokinin alterations during gynoecium and fruit
development. The results strongly suggest that cytokinins
play important roles in fruit patterning and morphogenesis,
including a previously unexpected role in valve margin
formation in fruits.
RESULTS
The TCS::GFP cytokinin reporter line reveals a dynamic
fluorescence pattern in developing gynoecia and fruits
To uncover as yet unknown roles of cytokinin in fruit
development, we sought to visualize cytokinin signaling
during fruit development in vivo. We employed the synthetic
reporter TCS::GFP (two-component output sensor), which
contains six direct repeats of the cytokinin-induced B-type
Arabidopsis response regulator binding motif (Muller and
Sheen, 2008). Gynoecia and fruits of TCS::GFP transgenic
plants at progressive floral developmental stages (according
to Smyth et al., 1990) were analyzed using confocal laser
scanning microscopy, and the optical sections obtained are
shown in Figure 1.
The gynoecium forms at the center of the floral meristem
at stage 6. At stage 7, the gynoecium grows as a hollow
tube, with two inner ridges, which will later give rise to the
medial tissues of the gynoecium and fruit, growing towards
each other. These incipient medial tissues show fluores-
cence, with the highest intensity from the bottom to the
middle (Figure 1a). Although low, fluorescence is also found
at the center of the top, as observed in the transverse picture
from above (Figure 2a). At stage 8, the tube increases in size
and the inner ridges keep on growing towards each other.
Fluorescence can now be detected all along the developing
gynoecium, from the bottom to the top, at the center of the
medial region, and the contact zone of the internal ridges
(Figures 1b and 2a). Furthermore, the flanks of the ridges
along the whole gynoecia also show fluorescence, except at
the top (Figures 1a and 2b). The medial ridges fuse at
stage 9, at which the septum originates. The medial region
shows fluorescence all along the gynoecium (longitudinal
view, Figure 1c), and at the edges of the internal ridges that
contact each other (top transverse view, Figure 2b). At
stage 10, after fusing, the ridges grow to the sides and
begin to form ovule primordia, arranged as interlocking
projections. On top of the developing gynoecia, stigmatic
papillae start forming. In the longitudinal axis, the abaxial or
external face of the gynoecium shows fluorescence as two
blurry lines at the sides of the incipient replum (the abaxial
medial tissue), along the ovary (Figure 1d). A transverse
section view reveals fluorescence at the center of the medial
region (Figure 2c). At this stage, the transmitting tract,
through which pollen tubes grow to reach the ovules, is
differentiating in this region. The fluorescence signal con-
tinues during stage 11, when developing ovules initiate
inner and outer integuments, and, at the top of the gynoe-
cium, the style is covered by stigmatic papillae. An abaxial
longitudinal view reveals fluorescence as two blurry lines at
the valve–replum junction along the ovary (Figure 1e), and a
transverse section shows strong fluorescence at the center
of the medial region that coincides with the position of the
transmitting tract (Figure 2d). It also shows fluorescence as
Cytokinins in Arabidopsis fruit 223
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Page 2
two faint lines at the lateral edges of the inner medial region
(Figure 2d). At stage 12, various tissues in the gynoecium
(valves, valve margins, replum and style) start to adopt their
specific morphological characteristics, and the gynoecium is
mature. An abaxial longitudinal view shows two fluorescent
lines along the valve–replum junction, and slight fluores-
cence in the funiculi (Figure 1f). A transverse section
(Figure 2e) shows fluorescence at the center of the medial
region and the funiculus, and two well-defined lines at the
valve–replum junction (the position where the valve margin
will be in the future fruit). At stage 13 of anthesis (opening of
the flower), a longitudinal abaxial image shows localized
fluorescence along the valve margin (Figure 1g). From this
stage on, we only analyzed fluorescence in the abaxial face
in the longitudinal axis of the gynoecium to follow this
unexpected localization. At stages 14–15, the fertilized fruit
starts to elongate. The fluorescence pattern is similar to that
at stage 13, with two well-defined fluorescent lines at the
valve–replum junction (the position of the valve margin) all
along the valves (Figure 1h). To determine whether this
precise localization of cytokinin signaling at the incipient
valve margin at the abaxial side of the gynoecium was
promoted by fertilization, we emasculated TCS::GFP floral
buds and compared fluorescence of pollinated and unpol-
linated pistils 24 h after pollination. No difference in the
abaxial pattern was observed (Figure 1j,k). Finally, at stages
16–17, when floral organs start to fall, leaving only the fruit
attached to the pedicel, the abaxial localization of the
fluorescence remains similar to the previous stages (Fig-
ure 1i). A close-up view revealed that fluorescence is
observed at the valve margin (Figure 1l).
In summary, the cytokinin signaling pattern changed from
early to later developmental stages, starting mainly at the
incipient medial region of young gynoecia, formed by
internal medial ridges, and later detected at the valve
margins of maturing fruits.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)
(j)
(k)
(l)
Figure 1. Fluorescence detection in single opti-
cal sections of gynoecia and fruits of the the
cytokinin signaling marker line TCS::GFP at con-
secutive floral developmental stages.
(a–i) The fluorescence signal is observed in
developing gynoecia and fruits at stages 7–8
(a), 8–9 (b), 9–10 (c), 10 (d), 11 (e), 12 (f), early 13
(g), 14 (h) and 17 (i). Stages are according to
Smyth et al. (1990).
(j, k) TCS::GFP fluorescence detection in non-
pollinated (j) and pollinated (k) gynoecia of
emasculated flowers, 24 h after emasculation
and manual pollination.
(l) Closer view of a stage 16–17 fruit showing
localized cytokinin signaling fluorescence at the
junction between replum and valves. Arrow-
heads in (a), (b), (g), (i) and (l) indicate the
location of fluorescence in those optical sections.
vm, valve margin.
224 Nayelli Marsch-Martı
´
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ª 2012 The Authors
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Page 3
Contrasting cytokinin and auxin localization patterns in
gynoecia and fruits
Cytokinin and auxin together regulate many key plant pro-
cesses, from growth and development to responses to the
environment, and they also actively regulate each other
through homeostatic feedback loops (Moubayidin et al.,
2009; Bishopp et al., 2011a; Su et al., 2011). Auxin plays a
very important role in fruit development (Sundberg and
Ferra
´
ndiz, 2009; Sundberg and Ostergaard, 2009). For in-
stance, at the valve margin, an ‘auxin minimum’ is required
for proper development of this region (Sorefan et al., 2009).
In order to visualize the relationship between the
hormones in the context of gynoecium and fruit develop-
ment in more detail, the fluorescence pattern of the auxin
reporter DR5rev::GFP (Benkova et al., 2003) in developing
gynoecia and fruits was observed using confocal laser
scanning microscopy, and compared to the TCS::GFP
pattern (Figure 2).
Opposite patterns were observed for these hormones in
specific tissues and stages of development. Images taken
from the top of young developing gynoecia showed cyto-
kinin signaling inside the developing structure, especially at
the growing ridges of the medial region (Figure 2a). In
contrast, the reporter line for auxin signaling showed
fluorescence outside this region, as a circle around the
developing ‘tube’ (Figure 2f). Longitudinal imaging of these
gynoecia revealed a circle of strong DR5 fluorescence at the
top (Figure 3a–e). Below the top, auxin reporter fluores-
cence was only observed at the incipient vasculature
(Figure 3a–c), which probably is unrelated to gynoecia and
fruit patterning and unrelated to cytokinin interaction. At
stages 10–12, gynoecia of the auxin marker line still showed
fluorescence at the vasculature and at the top (Figure 3d–f
and Figure S1), which was full of developing stigmatic
papillae, and where no fluorescence was observed for the
cytokinin marker line (Figure 1).
Stage 13 DR5 gynoecia kept showing fluorescence at the
base of stigmatic papillae and the vasculature (Figure 3g).
At stage 17, close observation of the auxin marker line
(Figure 3i) showed fluorescence at replum and valve cells,
and diminished fluorescence at the valve margins, as
previously reported (Sorefan et al., 2009). In contrast, the
cytokinin marker only showed fluorescence at the valve
margins (Figure 1i,l).
Taken together, these observations indicate that auxin
and cytokinin transcriptional responses frequently occur in
complementary patterns during gynoecia and fruit develop-
ment.
Alterations in endogenous cytokinin levels affect medial
region development in fruits
After observing the fluorescence patterns of the cytokinin
reporter line, we tested the effects of altering endogenous
cytokinin levels in fruit development. For this, the lac- and
Gal4-based transactivation system (pOp · LhG4) was used
to drive fruit expression of enzymes involved in cytokinin
biosynthesis or degradation (Moore et al., 1998; Shani et al.,
2010).
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)
(j)
Figure 2. Fluorescence detection and comparison of fluorescence patterns in
single optical transverse sections of gynoecia of the cytokinin signaling
marker line TCS::GFP and the auxin signaling marker line DR5rev::GFP during
development. (a–e) Cytokinin marker TCS::GFP gynoecia showing fluores-
cence in the medial tissues at various stages of development as indicated in
the drawings. Stages are according to Smyth et al. (1990). (f–j) Auxin marker
DR5rev::GFP fluorescence signal in developing gynoecia. The fluorescence
signal is observed as a circle around the top of the gynoecium at stage 7 (f).
Transverse sections showing the inner tissues of developing gynoecia at
further stages reveal fluorescence mainly at the vasculature (g–j). The images
shown in (a) and (f) were taken from the top of the gynoecium, and images (b–
e) and (g–j) are transverse sections showing the inner tissues of the
gynoecium. mr, medial region; o, ovule primordium/ovule; s, septum; r,
replum; v, valve; vm, valve margin; tt, transmitting tract. Arrowheads indicate
the valve margin, which is indicated by thin lines in the drawing.
Cytokinins in Arabidopsis fruit 225
ª 2012 The Authors
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Page 4
The driver lines used were pFUL::LhG4 and pSHP2::LhG4
(Y. Eshed, Department of Plant Sciences, Weizmann Insti-
tute, Israel, personal communication). These driver lines
were crossed to the operator lines Op::IPT7 (ISOPENTENYL-
TRANSFERASE7) and Op::CKX3 (CYTOKININ OXIDASE3)
(Werner and Schmulling, 2009; Shani et al., 2010). To
visualize the expression patterns directed by the promoters,
crosses to an Op::GUS line were included (Shani et al., 2010),
which showed GUS expression in fruits (Figure 4g,h). The
regulatory regions used lack introns that have been shown to
be essential for a correct spatial expression pattern for other
MADS box genes (Sieburth and Meyerowitz, 1997; Kooiker
et al., 2005; de Folter et al., 2007); however, although vari-
ations in intensity and pattern were observed in different
lines and fruits, in general, the pSHP2::LhG4 line drove GUS
expression in the valve margin, while pFUL::LhG4 drove
GUS expression at the valves (Figure 4g,h). Interestingly,
both promoters (driver lines) produced very similar results.
However, opposite effects in plant and replum development
were observed for crosses with the cytokinin biosynthesis
(IPT7) or degradation (CKX3) operator lines (Figure 4a–f).
The transactivation lines, including the Op::IPT7 genotype,
also showed clear alterations in other tissues, resembling
cytokinin-treated plants, as they showed wider stems and
serrated cauline leaves (Figure 4i–n), while transactivation
lines including Op::CKX3 had narrow stems.
The pSHP2 >>CKX3 and pFUL>>CKX3 fruits showed a
reduction in replum width in comparison to wild-type fruits
(Figure 4a–c,f). In contrast, pSHP2 >>IPT7 and pFUL>>IPT7
fruits showed an increase in the size of their repla
(Figure 4d–f). The pFUL::LhG4 promoter showed variations
in the intensity of GUS staining when crossed to the
Op::GUS line, and moderate to large increases in replum
width when crossed to the Op::IPT7 line. Remarkably, the
most altered repla had double the width of wild-type repla
and contained stomata, a type of cells not observed in wild-
type repla, but present in the style and valves of wild-type
fruits, and in tissues such as leaves or stems (Figure 4e). In
conclusion, alterations in the endogenous levels of cyto
kinins were able to affect the growth of the replum, an
external medial tissue.
Application of exogenous cytokinins severely alters gynoe-
cium morphology
The expression of enzymes altering endogenous cytokinin
levels in fruits affected the development of their repla.
However, internal cytokinin levels can be modulated by
feedback regulatory mechanisms that act upon other
enzymes in cytokinin metabolism. To reduce the influence
of these feedback loops, we tested the effects of contin-
uous exogenous application of this hormone in develop-
ing gynoecia. Flowering wild-type Arabidopsis Col plants
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)
Figure 3. Fluorescence signal detection in auxin
marker DR5rev::GFP gynoecia and fruits during
development (longitudinal sections). Fluores-
cence signal was observed in developing gynoe-
cia and fruits of stages 7–8 (a), 8–9 (b), 9–10 (c), 10
(d), 11 (e), 12 (f), early 13 (g) and 14 (h), and a
close-up of a fruit at stage 17 (i). White arrow-
heads in (b) indicate localized DR5rev::GFP fluo-
rescence at the top of the young gynoecium. The
fluorescence signal that forms a circle at the top
is observed as two dots in this optical section
along the gynoecium. White arrowheads in (i)
indicate the location where the cytokinin signal-
ing marker TCS::GFP fruits show the most
intense fluorescence, which coincides with the
lowest fluorescence signal in the auxin marker
DR5rev::GFP fruits (i). Stages are according to
Smyth et al. (1990). vm, valve margin.
226 Nayelli Marsch-Martı
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Page 5
were sprayed 5 days a week with a 100 l
M
benzylamin-
opurine (BAP) solution or a mock solution. The treatment
was initiated 1 week after bolting to avoid detrimental
effects on development during the vegetative phase. After
3–4 weeks of treatment, the plants showed clear known
effects of cytokinin application, such as short thick stems
and serrated cauline leaves. Remarkably, treated gynoecia
showed a conspicuous overgrowth of green tissue,
crowned by colorless tissue (Figure 5b,c). Close observa-
tion revealed that the colorless tissue at the top resem-
bled stigmatic papillae (Figure 5c), and transverse sections
of these fruits showed that the growth arose from the
replum (Figure 5e). Other aerial tissues did not show a
comparable over-proliferation response and extreme
morphological change. Fruit valves of treated plants
showed short cells of variable sizes (Figure 5c), contrast-
ing with the elongated, regularly sized cells of untreated
valves. Petals and cauline leaves showed serrations at
their edges, and anthers became shorter, but no other
striking effect comparable to conspicuous growth of the
replum was observed.
The developmental stage of the gynoecium at the start of
the treatment determined the severity of the outgrowth
effect. Treatment of the earliest floral buds (stages 6–8)
produced the most severe outgrowth, which was observed
in all gynoecia. The treatment of flowers at subsequent
stages resulted in a reduction of severity (stages 8–10,
intermediate; stages 10–12, slight). The treatment of fruits
(stages 13) did not produce any detectable outgrowths
after 4 weeks.
The effects suggest that cytokinins are also able to
severely alter fruit morphology, in addition to altering organ
number (Venglat and Sawhney, 1996; Lindsay et al., 2006;
Gordon et al., 2009), fruit size and yield (Ashikari et al., 2005;
Bartrina et al., 2011), inducing trichome formation in valves
(Greenboim-Wainberg et al., 2005) or triggering partheno-
carpy (Vivian-Smith and Koltunow, 1999), as previously
reported.
Cytokinin treatments of hormone signaling reporters and
fruit patterning mutants show altered responses
To better understand the striking outgrowth phenotype in
developing gynoecia sprayed with BAP (Figure 5), we
treated the cytokinin and auxin marker lines, and mutants
that lack various fruit tissues. First, TCS::GFP inflorescences
were sprayed to obtain an indication of the cytokinin
(a)
(i) (j) (k) (l)
(m)
(n)
(f)
(g)
(h)
(b) (c) (d) (e)
Figure 4. Alterations in endogenous cytokinin
levels affect replum width.
(a–e) Representative scanning electron micro-
graphs of a wild-type replum (a), compared with
repla of reduced size observed in the transacti-
vation lines pSHP2 >>CKX3 (b) and pFUL>>CKX3
(c), and repla of increased size in the transacti-
vation lines pSHP2 >>IPT7 (d) and pFUL>>IPT7
(e).
(f) Comparison of mean replum size measure-
ments in fruits of various transactivation lines
compared to wild-type fruits (n = 19). Error bars
indicate standard deviation.
(g, h) GUS staining of pSHP2 >>GUS (g) and
pFUL>>IPT7 (h) fruits. (i–n) The transactivation
lines including the Op::IPT7 genotype showed
alterations in other tissues, resembling cytoki-
nin-treated plants, i.e. wider stems, serrated
cauline leaves and altered fruit shape when
compared to wild-type plants.
(i–k) Whole-plant phenotype of wild-type (i),
pSHP2 > >IPT7 (j) and pFUL>>IPT7 (k) transacti-
vation lines.
(l) Siliques (from left to right) of wild-type,
pSHP2 >>IPT7 and pFUL>>IPT7 plants.
(m, n) From left to right, rosette and cauline
pSHP2 >>IPT7 (m) and pFUL>>IPT7 (n) leaves
followed by rosette and cauline wild-type leaves.
Scale bars = 200 lm (a–i).
Cytokinins in Arabidopsis fruit 227
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Page 6
transcriptional response after treatment. Intriguingly, an in-
creased fluorescence signal was only observed at the inter-
nal medial tissues of the treated gynoecia, but no
fluorescence was detected at the external proliferating tissue
(Figure 6a,c,d). As the cytokinin and auxin pathways interact
in various tissues, we investigated the auxin marker in
cytokinin-induced ectopic tissues. DR5rev::GFP plants were
subjected to the same cytokinin treatment, and, remarkably,
a clear fluorescence signal was detected at the tip of the
growing protuberances (Figure 6b).
Furthermore, various genotypes affected in the develop-
ment of diverse fruit tissues were sprayed with BAP. The
treated genotypes were ful, in which valves acquire valve
margin identity, shp1 shp2 and ind mutants, which lack the
valve margin, and 35S::FUL, in which valve margin and
replum are absent (Ferrandiz et al., 2000; Liljegren et al.,
2000, 2004). The ful, shp1 shp2 and ind gynoecia and fruits
were still able to moderately form ectopic proliferating
tissues (Figure 6g–i). However, 35S::FUL fruits that lack a
replum and valve margin did not show any external
proliferations, confirming that the external proliferation
originated mainly from the replum (Figure 6f). Interest-
ingly, 35S::FUL and shp1 shp2 fruits became wider, sug-
gesting that proliferation was occurring internally
(Figure 6f,h).
Together, the results of these experiments indicate that
the internal tissues of developing gynoecia respond to
external application of cytokinins by triggering proliferation,
mainly observed at the external medial tissue (replum).
External application of cytokinins also resulted in auxin
presence, detected as a fluorescence signal of the
DR5rev::GFP marker at the tips of the proliferating tissue.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(i)
(h)
Figure 6. Cytokinin treatment of hormone signaling reporters and mutants affected in fruit patterning.
(a–d) Gynoecia of hormone signaling reporter lines.
(a, b) Treated (sprayed with BAP) TCS::GFP gynoecia (a) and DR5rev::GFP gynoecia (b).
(c, d) Untreated (c) and treated (sprayed with BAP) (d) TCS::GFP gynoecia.
(e–i) Gynoecia of various genotypes sprayed with BAP: Wild-type (e), 35S::FUL (f), ful (g), shp1 shp2 (h) and ind (i). Scale bars = 1 mm (e–i).
(a)
(d) (e)
(b) (c)
Figure 5. Exogenous cytokinin treatments induce extensive proliferation at
the external medial region in developing gynoecia.
(a, b) Fruit of wild-type plants treated with a mock solution (a) or with 100 l
M
BAP (b).
(c) Scanning electron microscopy observations of a gynoecium of BAP-
treated wild-type plants.
(d, e) Transverse sections show clear differences in the development of a
gynoecium of a non-treated plant (d) compared to a gynoecium of a BAP-
treated plant, in which extensive outer proliferation is observed (indicated by
arrowheads) (e). Scale bars = 2 mm (a, b), 200 lm (c), 150 lm (d) and 300 lm
(e). o, ovule, r, replum, v, valve, vm, valve margin.
228 Nayelli Marsch-Martı
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Page 7
Alterations in valve margin identity modify the
cytokinin localization pattern
The cytokinin marker revealed fluorescence at the region
between valves and repla in mature gynoecia and develop-
ing fruits, and, in order to explore the biological significance
of this unexpected localization, we analyzed the cytokinin
marker in fruits that lacked a functional valve margin.
TCS::GFP fluorescence was analyzed in the ind mutant and
in the shp1 shp2 double mutant background. Interestingly,
while TCS::GFP fruits showing wild-type phenotypes pre-
sented a very well-defined fluorescent line in the region
between valves and replum, no fluorescence was detected in
this region in homozygous ind or shp1 shp2 mutants lacking
a dehiscence zone (Figure 7b,c). This demonstrates that
functional IND and SHP1/2 are required for cytokinin accu-
mulation at the valve–replum junction.
Conversely, in TCS::GFP ful mutant fruits, in which valves
acquire valve margin identity (Ferrandiz et al., 2000; Lilje-
gren et al., 2000, 2004), the whole valves showed very
intense fluorescence (Figure 7d). This was not observed in
wild-type valves, and further suggests that cytokinins and
valve margin identity are indeed connected.
Cytokinin application restores valve margin formation and
dehiscence in the shp1 shp2 and ind mutants
After observing the changes in the pattern of cytokinin sig-
naling in the three mutants affected in valve margin identity,
and to test a possible functional role for cytokinins in this
tissue, BAP was applied to valve margin mutants. In fruits of
the shp1 shp2 and ind mutants, the valve margin cannot be
easily distinguished at the abaxial face, and the fruits fail to
dehisce (Figure 8a,c). However, when BAP was locally
applied to shp1 shp2 and ind developing fruits (4 days after
pollination), the characteristic abaxial morphology of the
valve margin was recovered in maturing fruits (Figure 8b,d).
Moreover, when dry, these fruits showed increased
dehiscence when compared to mock-treated controls
(a)
(c)
(d)
(b)
Figure 7. Cytokinin localization is severely altered in mutants that lack valve
margins or that have ectopic valve margin identity in the valves. Cytokinin
signaling marker TCS::GFP fluorescence signal is observed in the valve
margins (indicated by arrowheads) of a segregating gynoecium with wild-
type phenotype (a). However, no signal is detected in the ind (b) or shp1 shp2
(c) mutants that lack valve margins. In the ful mutant, where valves acquire
ectopic valve margin identity, intense fluorescence signal is detected in the
whole valves (d). vm, valve margin, v, valve.
(a)
(b)
(c)
(e)
(f)
(g)
(h)
(d)
Figure 8. Local application of cytokinin restores dehiscense in valve margin
mutants.
(a–d) Scanning electron micrographs of fruits of the valve margin mutants
shp1 shp2 (a, b) and ind (c, d) painted with a mock solution (a, c) or a solution
containing BAP (b, d).
(e–h) Fruits of the valve margin mutants shp1 shp2 (e, f) and ind (g, h) to which
a mock treatment (e, g) or a mixture of lanolin with BAP (f, h) had been
applied. The solution was painted on, and the lanolin was applied to the
external medial region of developing mutant fruits. Scale bars = 100 lm (a–d)
and 1 mm (e–h).
Cytokinins in Arabidopsis fruit 229
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Page 8
(Figure 8e–h). This suggests that cytokinins have a func-
tional role in the process of valve margin formation that
leads to proper dehiscence in fruits.
DISCUSSION
Cytokinins may play different roles depending on the con-
text. For example, at the shoot apical meristem, they pro-
mote cell proliferation (Leibfried et al., 2005; Lindsay et al.,
2006; Gordon et al., 2009), but they play the opposite role at
the root apical meristem, where they promote cell differen-
tiation (Werner et al., 2003; Dello Ioio et al., 2008; Muller and
Sheen, 2008). Ectopic cytokinin at the apical tissues activates
shoot stem-cell genes such as CLAVATA1 and WUSCHEL,
while cytokinin at the basal cell in the early embryo results in
failed root-stem cell determination (Werner et al., 2003;
Lindsay et al., 2006; Dello Ioio et al., 2008; Muller and Sheen,
2008; Gordon et al., 2009). Other roles include delay of
morphogenetic activity at the leaf edge of species that form
compound leaves (Shani et al., 2010), and, together with
gibberellins, trichome formation (Gan et al., 2007).
Here, we investigated the role of cytokinins in gynoecium
and fruit development. For this, we first used the TCS::GFP
synthetic reporter (Muller and Sheen, 2008) to visualize
cytokinin output in vivo. As shown in Figure 9, the pattern of
cytokinin output changed from early to late developmental
stages, suggesting that cytokinins play at least two roles: an
early proliferation-inducing role at the medial region of the
developing gynoecia, and an unexpected, late role during
formation of fruit valve margins.
The fluorescence signal was first observed at the internal
tissues of developing gynoecia. Earlier observations in rice
and Arabidopsis ckx mutants, presumably containing higher
cytokinin levels and producing increased numbers of ovules
and seeds, indicated that cytokinins play an important role in
placental development (Ashikari et al., 2005; Bartrina et al.,
2011). Medial tissues (which include the placenta, septum
and replum, and from which the style and stigma develop)
are considered to be quasi-meristems, as they possess
characteristics of shoot apical meristems (Balanza et al.,
2006; Alonso-Cantabrana et al., 2007; Girin et al., 2009).
Cytokinins promote cell proliferation at shoot apical meris-
tems, and appear to perform this function also in the internal
tissues of developing gynoecia (Leibfried et al., 2005; Lind-
say et al., 2006; Gordon et al., 2009; Bartrina et al., 2011). In
this work, we observed a clear effect of increased or
decreased cytokinin levels on the size of the replum (the
external medial tissue) by using a transactivation system to
drive IPT7 or CKX3 expression from fruit promoters. Inter-
estingly, the two promoters used produced similar results,
but using a third promoter that did not show expression at
the valves, valve margin or replum did not produce signif-
icant replum size changes. An explanation for the similar
effect of the two promoters in the replum could be that, as
cytokinins are able to regulate meristem size through a non-
Figure 9. Schematic representations of cytokinin localization and working
model of interactions with fruit patterning genes and hormones.
(a) Schematic representation of the fluorescence pattern observed in the
cytokinin marker line TCS::GFP during progressive gynoecium and fruit
developmental stages.
(b) Schematic representation of two contrasting fluorescence patterns
observed in gynoecium and fruit internal and external tissues between
cytokinin TCS::GFP and auxin DR5rev::GFP marker lines. Left, drawings
representing the top apical view of stage 7–10 gynoecia. Cytokinin-induced
fluorescence can be observed at the inner medial tissues, while auxin-induced
fluorescence is observed as a circle at the top of the gynoecium. Right,
drawings representing the abaxial view of a region at the medio-lateral axis of
the ovary of a stage 12 (and onwards) gynoecium and fruit. Cytokinin-induced
fluorescence is strongly detected at the valve margins, while auxin-induced
fluorescence is undetectable in this tissue.
(c) Schematic model proposed for the interactions of cytokinins with the genes
IND, SHP1/2 and FUL, and the hormones auxin and gibberellin in the context of
valve margin development. Cytokinin signaling is repressed by FUL at the
valves, and promoted by SHP1/2 and IND at the valve margins. Auxin signaling
is absent from this tissue, while cytokinin signaling is strongly detected as well-
defined TCS::GFP fluorescence. Components of the auxin and cytokinin
pathways (including biosynthetic enzymes, transporters and/or signaling
and response components) may interact with each other and reinforce this
pattern. On the other hand, the combined presence of gibberellins and
cytokinins may promote valve margin development in this region.
230 Nayelli Marsch-Martı
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The Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), 72, 222–234
Page 9
cell-autonomous mechanism, according to data reported by
Bartrina et al. (2011), a similar mechanism could operate for
quasi-meristems.
Furthermore, in the experiment in which cytokinins were
exogenously applied to very young developing flowers,
cytokinins dramatically enhanced the proliferative activity of
the replum, and, although other tissues were affected by the
treatment, none of them displayed such conspicuous over-
growth (Figure 5). Moreover, treated mutants lacking valves
or valve margins were still able to form ectopic external
tissue, while 35S::FUL fruits that lack a replum did not.
Intriguingly, when the TCS::GFP line was treated, increased
fluorescence was observed in the inner medial tissues, but
was not detected in the external ectopic tissues (Fig-
ure 6a,d), suggesting that externally applied cytokinins are
able to trigger internal cytokinin signaling that induces
external proliferation in a non-cell-autonomous manner.
Other studies have shown that application of BAP to
developing inflorescences results in changes in floral organ
identity and number, explained by extended meristematic
activity (Venglat and Sawhney, 1996; Blahut-Beatty et al.,
1998; Lindsay et al., 2006). Interestingly, BAP-treated mature
flowers and fruits did not produce ectopic tissue (this study),
suggesting that cytokinins have a proliferation-inducing
activity in young gynoecia only, where the medial tissues
show meristematic characteristics.
Remarkably, comparison of the cytokinin marker with the
synthetic reporter for auxin output, DR5rev::GFP (Benkova
et al., 2003), revealed contrasting patterns in specific tissues.
Cytokinins and auxins often play antagonistic roles in
different tissues (Skoog and Miller, 1957). They show
complex interactions through reciprocal regulation of the
biosynthesis, signaling and transport components of each
other’s pathways in different cells. Auxin down-regulates
cytokinin biosynthesis and induces cytokinin negative reg-
ulators (Arabidopsis response regulator type A) at various
stages and tissues, such as early root development, the post-
embryonic root and the shoot apical meristem (Muller and
Sheen, 2008; Zhao et al., 2010). On the other hand, cytokinin
induces auxin negative regulators (Aux/IAA) and affects PIN
auxin-efflux transporters (Laplaze et al., 2007; Dello Ioio
et al., 2008; Pernisova et al., 2009; Ruzicka et al., 2009; Jones
et al., 2010; Bishopp et al., 2011b). Due to these interactions,
opposite localization patterns of auxin and cytokinins have
been reported for various tissues (e.g. Muller and Sheen,
2008; Bishopp et al. , 2011b), and were also observed at
different stages and regions of developing gynoecia and
fruits (Figures 1–3 and 9). It would be interesting to inves-
tigate whether the same molecular mechanisms that con-
nect these pathways in other tissues are also responsible of
the contrasting patterns observed in gynoecia and fruits.
During development, auxin is localized at the apical part of
gynoecia, where stigmatic cells develop (Aloni et al., 2006;
Benkova et al., 2003; Figure 3). Cytokinins were not detected
at this region (Figure 1), but external application of cytoki-
nins to developing DR5rev::GFP flowers resulted in a fluo-
rescence signal at the apex of the ectopic tissue that
developed from the replum (Figure 6b), where stigmatic
cells also developed, resembling the natural localization of
auxin and stigma at the top of the fruit. Jones et al. (2010)
have shown that cytokinin application in young, developing
tissues leads to a rapid increase in auxin biosynthesis, and
cytokinins are able to regulate PIN auxin efflux transporters
in various tissues (Laplaze et al., 2007; Dello Ioio et al., 2008;
Pernisova et al., 2009; Ruzicka et al., 2009; Jones et al., 2010;
Bishopp et al., 2011b). Therefore, it is tempting to speculate
that, in the context of gynoecium development, cytokinins in
the medial tissue may stimulate auxin biosynthesis and/or
transport, resulting in auxin accumulation at the top of the
gynoecium, leading to stigma development.
The unexpected visualization of older TCS::GFP gynoecia
and fruits at the valve margin suggested that cytokinins may
not only play a role in early gynoecium medial tissue
proliferation, but may also participate later in development
(Figures 1–3 and 9). Auxin depletion is required for proper
valve margin development (Sorefan et al., 2009). The bHLH
transcription factor IND promotes localization of PIN3 in the
plasma membrane of valve margin cells such that auxin is
‘pumped out’ (Sorefan et al.
, 2009). IND itself is activated by
SHP MADS box transcription factors at the valve margin
(Liljegren et al., 2000, 2004). We detected a sharp fluores-
cence signal at the replum and valve junction in the cytokinin
reporter line, which disappeared in ind and shp1 shp2
mutant backgrounds, indicating that cytokinin signaling is
working downstream of these valve margin regulators.
As auxin has been shown to down-regulate cytokinin
biosynthesis (Nordstrom et al., 2004), one possible scenario
could be that auxin depletion by IND (Sorefan et al., 2009) is
required for cytokinin appearance. On the other hand, it
cannot be ruled out that the valve margin regulators directly
activate the cytokinin pathway. As cytokinin signaling reg-
ulates the radial localization pattern of the PIN auxin
transporters in the root vasculature (Bishopp et al., 2011b),
if a similar phenomenon occurs at the valve margin, cytoki-
nins may also contribute to auxin depletion in this tissue. The
model presented in Figure 9 shows both scenarios.
IND also activates the gibberellin biosynthesis gene
GA3ox1 at the valve margin of fruits (Arnaud et al., 2010).
This may appear to contradict the cytokinin observations, as
previous earlier work has shown antagonistic effects of
cytokinin and gibberellin (Ezura and Harberd, 1995; Brenner
et al., 2005; Greenboim-Wainberg et al., 2005; Jasinski et al.,
2005; Yanai et al., 2005). However, these hormones can also
act simultaneously upon transcription factors that stimulate
trichome initiation (Gan et al., 2007). As IND is also neces-
sary for cytokinin presence (this work), valve margin forma-
tion may also require the cooperative action of both
hormones.
Cytokinins in Arabidopsis fruit 231
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Page 10
On the other hand, proper valve development requires
repression of valve margin identity in the valve tissue. FUL is
a MADS box transcription factor that represses SHP1/2, IND
and other genes involved in valve margin identity in the valve
(Ferrandiz et al., 2000; Liljegren et al., 2000, 2004). Absence
of FUL results in conversion of valve cells into valve margin
cells, and TCS::GFP ful mutants showed intense fluorescence
signal at the valves (Figure 7d). These results strongly
indicate a relationship between cytokinins and valve margin
identity. Furthermore, local application of cytokinin in devel-
oping fruits restored valve margin formation and increased
dehiscence in shp1 shp2 and ind mutants, suggesting that
cytokinins play a functional role in valve margin formation.
However, if this is the case, it is not clear how this late role
of cytokinins in valve margin formation is related to the early
role of cytokinins in proliferation of cells in (quasi)meriste-
matic tissues. Further work is required to unravel the
molecular mechanisms by which cytokinins act in this tissue.
In conclusion, the results suggest that cytokinins play at
least two different roles in gynoecium and fruit patterning
and morphogenesis: an early role stimulating proliferation
of the medial tissues, and a late role in valve margin
formation, opening new paths for detailed studies about
cytokinins in these processes. As observed for other parts of
the plant, different tissues respond differently to cytokinins,
which may be able to work in a non-cell-autonomous
manner. Further studies will help to unravel the role of the
specific cytokinin species synthesized, modified and de-
graded through various enzymatic routes, and the role of
transport, signaling and response components in the dis-
tinct effects of cytokinins during fruit development. More-
over, cytokinins may act in concert with auxin, as contrasting
patterns were observed for developing gynoecia and fruits
of the cytokinin and the auxin marker lines, and, on the other
hand, auxin signaling was observed in proliferating tissue
growing in cytokinin-treated gynoecia. In the future, new
attempts to understand the molecular interactions that
relate cytokinins to gynoecium and fruit tissue identity, key
transcription factors and other hormones will shed light on
the processes that shape fruits.
EXPERIMENTAL PROCEDURES
Plant growth and plant materials
Plants were germinated in soil in a growth chamber at 22C under
long-day conditions (16 hrs light, 8 hrs dark), and further grown in
soil under standard greenhouse conditions (natural light condi-
tions, around 22–25C). The TCS::GFP line (Muller and Sheen, 2008)
and the DR5rev::GFP line (Benkova et al., 2003) are in the Col
background. Transactivation lines pFUL::LhG4 and pSHP2::LhG4
(Yuval Eshed, Department of Plant Sciences, Weizmann Institute,
Israel) and Op::GUS, Op::IPT7 and Op::CKX3 (Werner and Schmul-
ling, 2009; Shani et al., 2010) are in the Ler background. The ind-2,
ful-1 and shp1 shp2 mutants (Ferrandiz et al., 2000; Liljegren et al.,
2000, 2004) are in the Ler background. 35S::FUL (Ferrandiz et al.,
2000) is in the Col background.
Hormone treatments
Seeds were germinated in a growth chamber (long days at 22C),
and plants were grown in soil under standard greenhouse condi-
tions. One week after bolting, wild-type and mutant plants were
sprayed 5 days a week with 100 l
M
benzylaminopurine (BAP;
Duchefa Biochemie, http://www.duchefa.com), 0.01% Silwet L-77
(Lehle Seeds, http://www.arabidopsis.com) or a mock solution
(100 lM BAP and 0.01% Silwet together). All aerial tissues were
sprayed, and effects were evaluated after 2 weeks (Figure 6a,b,d) or
3–4 weeks (Figures 5 and 6e–i). Alternatively, for the valve margin
mutants, local application of cytokinins to the external medial
region of the ovary of developing fruits was performed either by
using a paintbrush to apply a solution of 0.01% Silwet L-77/100 l
M
BAP 5 days a week (Figure 8f,h), or by spreading once with a 250 l
M
BAP lanolin paste (Figure 8b,d). The paintbrush treatment started
1 day after pollination, and photographs were taken when the fruits
were brownish (3–4 weeks after initiation of the treatment). Treat-
ment with lanolin paste started 4 days day after pollination, and
photographs were taken when the fruits reached stage 17.
Histology
For GUS analysis, Arabidopsis tissues were incubated overnight at
37C with an X-Gluc solution (Gold Biotechnology, https://www.
goldbio.com) (Jefferson et al., 1987). Fruits of plants treated with
cytokinins and control fruits were fixed in formaldehyde/acetic acid/
alcohol solution, then dehydrated, embedded in Paraplast (Sigma-
Aldrich, http://www.sigmaaldr ich.com), and 10 l m sections were cut
as previously described (Zu
´
n
˜
iga-Mayo et al., 2012). Tissue sections
were stained to analyze the transmitting tract using a solution of
alcian blue and counterstained with a solution of neutral red (Sigma-
Aldrich) (Zu
´
n
˜
iga-Mayo et al., 2012). Tissue sections were observed
by optical microscopy (Zeiss Axio Observer, http://www.zeiss.com).
Microscopy
Fluorescent images were captured using an LSM 510 META
confocal scanning laser inverted microscope (Zeiss). GFP was
excited using a 488 nm line of an argon laser, and propidium iodide
was excited using a 514 laser line. GFP emission was filtered using a
BP 500–550 nm filter, and propidium iodide emission (including
autofluorescence) was filtered using an LP 575 nm filter. We noted
variations in the fluorescence intensity when TCS::GFP plants were
grown in different seasons (summer and winter). For scanning
electron microscopy, plant tissue was collected from plants and
directly observed in a Zeiss EVO40 scanning electron microscope
with a 20 kV beam, using the SE detector (Figure 4) or the BSD
detector (Figures 5 and 8). For measuring the replum, photographs
taken at 215· magnification were used. Photographs of treated
silique sections were taken using a Zeiss AxioCam MRc camera
installed on a Zeiss Observer.Z1 inverted microscope. Images
of treated siliques, transactivatio n GUS-stained tissues and
phenotypes were obtained using a Leica EZ4 D stereomicroscope
(Leica, http://www.leica-microsystems.com).
ACKNOWLEDGMENTS
The TCS::GFP line was kindly provided by Bruno Muller (Institute of
Plant Biology, University of Zu
¨
rich, Zu
¨
rich) and Jen Sheen
(Department of Molecular Biology, Massachusetts General Hospital,
Boston, MA, USA). We thank Naomi Ori (The Robert H. Smith
Institute of Plant Sciences and Genetics in Agriculture, and Hebrew
University, Israel), Thomas Schmulling (Department of Biology,
Chemistry, Pharmacy, Institute of Biology, Berlin, Germany) and
Eilon Shani (The Robert H. Smith Institute of Plant Sciences and
232 Nayelli Marsch-Martı
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ª 2012 The Authors
The Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), 72, 222–234
Page 11
Genetics in Agriculture, and Hebrew University, Israel) for kindly
sharing the Op::GUS, Op::IPT7 and Op::CKX3 lines. We also thank
Yuval Eshed (Department of Plant Sciences, Weizmann Institute,
Israel) for kindly sharing the pSHP2::LhG4 and pFUL::LhG4 lines.
The 35S::FUL line was kindly provided by Cristina Ferra
´
ndiz
(Instituto de Biologı
´a
Molecular y Celular de Plantas. CSIC-UPV,
Valencia, Spain). We also acknowledge the Arabidopsis Biological
Resource Center for provision of mutant seed. I.R.O., P.L.S. and
V.Z.M. were supported by Mexican National Council of Science and
Technology (CONACyT) fellowships (numbers 210085, 219883 and
210100, respect ively). The authors acknowledge initial support from
Langebio intramural funds. This work was financed by the CONA-
CyT grant 82826.
SUPPORTING INFORMATION
Additional Supporting Information may be found in the online
version of this article.
Figure S1. Confocal micrograph of a DR5rev::GFP gynoecium at
stages 9–10.
Please note: As a service to our authors and readers, this journal
provides supporting information supplied by the authors. Such
materials are peer-reviewed and may be re-organized for online
delivery, but are not copy-edited or typeset. Technical support
issues arising from supporting information (other than missing
files) should be addressed to the authors.
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  • Source
    • "2C and 2E, fluorescence signal is observed in images of transversely cut gynoecia (in the ovary region) mounted in agar. The TCS::GFP signal is observed in the carpel margin meristems (CMM) and septa primordia in the medial domain of stage 9 gynoecia (Fig. 2C) and in stage 12 gynoecia in the septum, funiculi, and the valve margins (Fig. 2E), as we have observed before with this method (Marsch-Martinez et al., 2012). In the Experimental Procedures a detailed description is given on how to obtain a good transverse sectioned gynoecium. "
    [Show abstract] [Hide abstract] ABSTRACT: The gynoecium is the female reproductive structure and probably the most complex plant structure. During its development different internal tissues and structures are formed. Insights in gene expression or hormone localization patterns are key to understanding gynoecium development from a molecular biology point of view. Imaging with a confocal laser scanning microscope (CLSM) is a widely used strategy; however, visualization of internal developmental expression patterns in the Arabidopsis gynoecium can be technically challenging. Here, we present a detailed protocol that allows the visualization of internal expression patterns at high resolution during gynoecium development. We demonstrate the applicability using a cytokinin response marker (TCS::GFP), an auxin response marker (DR5::VENUS), and a SEPALLATA3 marker (SEP3::SEP3:GFP). The detailed protocol presented here allows the visualization of fluorescence signals in internal structures during Arabidopsis gynoecium development. This protocol may also be adapted for imaging other challenging plant structures or organs. This article is protected by copyright. All rights reserved. © 2015 Wiley Periodicals, Inc.
    Full-text · Article · Jul 2015 · Developmental Dynamics
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    • "The differentiation of the DZ is under the control of intricate regulatory networks involving multiple transcription factors. Recent investigations in pod dehiscence regulation have uncovered another layer of the regulatory network that include phytohormones in specifying the DZs (Sorefan et al., 2009; Arnaud et al., 2010; Marsch-Martinez et al., 2012). "
    [Show abstract] [Hide abstract] ABSTRACT: Seed shattering (or pod dehiscence, or fruit shedding) is essential for the propagation of their offspring in wild plants but is a major cause of yield loss in crops. In the dicot model species, Arabidopsis thaliana, pod dehiscence necessitates a development of the abscission zones along the pod valve margins. In monocots, such as cereals, an abscission layer in the pedicle is required for the seed shattering process. In the past decade, great advances have been made in characterizing the genetic contributors that are involved in the complex regulatory network in the establishment of abscission cell identity. We summarize the recent burgeoning progress in the field of genetic regulation of pod dehiscence and fruit shedding, focusing mainly on the model species A. thaliana with its close relatives and the fleshy fruit species tomato, as well as the genetic basis responsible for the parallel loss of seed shattering in domesticated crops. This review shows how these individual genes are co-opted in the developmental process of the tissues that guarantee seed shattering. Research into the genetic mechanism underlying seed shattering provides a premier prerequisite for the future breeding program for harvest in crops.
    Full-text · Article · Jun 2015 · Frontiers in Plant Science
  • Source
    • "The Nemhauser model has been very useful to frame the role of different players in Arabidopsis carpel development, but conclusive proof of the proposed auxin gradient has never been obtained. Actually, detailed descriptions of auxin accumulation throughout gynoecium development using a DR5rev::GFP reporter have shown that auxin maxima are formed in the apical domain, first as isolated foci and later as a continuous apical ring, while the proposed gradient cannot be observed (Girin et al., 2011; Marsch-Martinez et al., 2012a; Larsson et al., 2013). In addition, several recent studies indicate that the dynamics of auxin accumulation, homeostasis and response within the developing gynoecium are highly complex and we are still far from fully comprehending how positional information is translated into developmental outputs in gynoecium differentiation (Sohlberg et al., 2006; Ståldal et al., 2008; Ståldal and Sundberg, 2009; Marsch-Martinez et al., 2012b). "
    [Show abstract] [Hide abstract] ABSTRACT: The four NGATHA genes (NGA) form a small subfamily within the large family of B3-domain transcription factors of Arabidopsis thaliana. NGA genes act redundantly to direct the development of the apical tissues of the gynoecium, the style, and the stigma. Previous studies indicate that NGA genes could exert this function at least partially by directing the synthesis of auxin at the distal end of the developing gynoecium through the upregulation of two different YUCCA genes, which encode flavin monooxygenases involved in auxin biosynthesis. We have compared three developing pistil transcriptome data sets from wildtype, nga quadruple mutants, and a 35S::NGA3 line. The differentially expressed genes showed a significant enrichment for auxin-related genes, supporting the idea of NGA genes as major regulators of auxin accumulation and distribution within the developing gynoecium. We have introduced reporter lines for several of these differentially expressed genes involved in synthesis, transport and response to auxin in NGA gain- and loss-of-function backgrounds. We present here a detailed map of the response of these reporters to NGA misregulation that could help to clarify the role of NGA in auxin-mediated gynoecium morphogenesis. Our data point to a very reduced auxin synthesis in the developing apical gynoecium of nga mutants, likely responsible for the lack of DR5rev::GFP reporter activity observed in these mutants. In addition, NGA altered activity affects the expression of protein kinases that regulate the cellular localization of auxin efflux regulators, and thus likely impact auxin transport. Finally, protein accumulation in pistils of several ARFs was differentially affected by nga mutations or NGA overexpression, suggesting that these accumulation patterns depend not only on auxin distribution but could be also regulated by transcriptional networks involving NGA factors.
    Full-text · Article · May 2014 · Frontiers in Plant Science
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