Angiosperm ovules consist of three proximal-distal domains – the nucellus, chalaza and funiculus – demarcated by developmental fate and specific gene expression. Mutation in three paralogous class III homeodomain leucine zipper (HD-ZIPIII) genes leads to aberrations in ovule integument development. Expression of WUSCHEL (WUS) is normally confined to the nucellar domain, but in this triple mutant expression expands into the chalaza. MicroRNA-induced suppression of this expansion partially suppresses the effects of the HD-ZIPIII mutations on ovule development, implicating ectopic WUS expression as a component of the mutant phenotype. bell1 (bel1) mutants produce aberrant structures in place of the integuments and WUS is ectopically expressed in these structures. Combination of bel1 with the HD-ZIPIII triple mutant leads to a striking phenotype in which ectopic ovules emerge from nodes of ectopic WUS expression along the funiculi of the primary ovules. The synergistic phenotype indicates that BEL1 and the HD-ZIPIII genes act in at least partial independence in confining WUS expression to the nucellus and maintaining ovule morphology. The branching ovules of the mutant resemble those of some fossil gymnosperms, implicating BEL1 and HD-ZIPIII genes as players in the evolution of the unbranched ovule form in extant angiosperms.

Ovules are the developmental precursors of seeds. In angiosperms, the ovule consists of three developmental domains; the nucellus, chalaza and funiculus (Fig. 1A) (Balasubramanian and Schneitz, 2000). The nucellus contains a megasporangium, where the female gametophyte develops. The inner and outer integuments form from the chalaza and enclose the nucellus. The funiculus is a stalk-like structure that connects the ovule to the ovary wall. For correct development of the ovules, it is essential to establish this proximal-distal patterning; hence, a shift in the boundary between domains results in aberrant ovule morphology (e.g. Balasubramanian and Schneitz, 2000; Gross-Hardt et al., 2002).

WUSCHEL (WUS) is a homeobox gene that characterizes the nucellus by its restricted expression in this domain, and this nucellar expression is necessary for initiation of the two integuments that develop from the chalaza (Gross-Hardt et al., 2002). WUS promotes the expression of an auxin efflux facilitator, PIN-FORMED 1 (PIN1), via transcriptional activation of SPOROCYTELESS [SPL; also known as NOZZLE (NZZ)], and this mechanism is necessary for nucellus formation (Bencivenga et al., 2012). Extension of the integuments is incomplete when WUS expression is driven in the chalzal domain by the AINTEGUMENTA promoter (pANT) (Gross-Hardt et al., 2002). In pANTWUS plants additional putative outer integuments also emerge just below the extended WUS expression area (Gross-Hardt et al., 2002; Sieber et al., 2004), as if a new boundary is established between the nucellus and chalaza. These results indicate that WUS regulation is key to defining the nucellus-chalaza boundary.

A recent study showed that externally applied cytokinin expands WUS expression into the chalaza (Bencivenga et al., 2012). WUS expression is also altered in bell1 (bel1) (Bencivenga et al., 2012; Brambilla et al., 2007) (see Fig. S2), but the change in the expression is not as profound, suggesting that other factors play roles in WUS regulation.

Class III homeodomain leucine zipper (HD-ZIPIII) genes establish the identity of the adaxial tissue of lateral organs (Emery et al., 2003; McConnell et al., 2001), as well as regulating WUS expression in shoot and floral apices in cooperation with CLAVATA3 or ERECTA (ER) homologs (Green et al., 2005; Landau et al., 2015; Lee and Clark, 2015; Mandel et al., 2014). In ovules, HD-ZIPIII genes are involved in the development of the integuments; thus, in most ovules of loss-of-function HD-ZIPIII gene mutants [i.e. corona phabulosa phavoluta (cna phb phv)] the integuments are absent, reduced or malformed (Kelley et al., 2009). The mechanism of these effects remains unclear.

Here, we show that CNA, PHB and PHV cooperatively repress WUS expression in the chalaza, and that this repression is important for ovule development. A combination of these mutations with bel1 leads to additional misexpression of WUS outside of the nucellus and a novel phenotype not seen in either class of mutant. Thus, we identify transcription factors necessary for boundary demarcation between the nucellus and chalaza and for patterning of WUS expression.

WUS expression extends into the chalaza in cna phb phv

In cna phb phv carpels with ovules at stage 2 (for stages, see Schneitz et al., 1995), WUS expression was ∼1.8-fold higher than in wild-type (WT) carpels at the same stage (Fig. S1). Since gynoecium formation is almost completed in cna phb phv at this stage, we hypothesize that the increase results from elevated WUS expression in developing ovules.

In contrast to WT, where WUS expression was confined to the nucellus (Fig. S2), in most cna phb phv ovules at stage 2-II, WUS expression extended into the chalaza, including the integuments (Fig. 1B). Misexpression of WUS in the chalaza was still observed in ovules at stage 2-III, whereas expression in the nucellus decreased (Fig. 1C). A subset of ovules did, however, exhibit WT-like expression of WUS (Fig. 1D). In cna phb phv some ovules do develop normally, although most exhibit aberrant integument development (Kelley et al., 2009). Coexistence of ovules with normal and abnormal WUS expression is thus consistent with the ovule phenotypes.

In summary, CNA, PHB and PHV are required for preventing WUS expression in the chalaza. However, the sporadic WT-like phenotype in cna phb phv ovules implies that other gene(s) are also involved in the regulation of WUS.

Ovule defects in cna phb phv are suppressed by pCNA:amiRWUS

Growth of the integuments was disturbed in pANTWUS plants (Gross-Hardt et al., 2002). Thus, the misexpression of WUS could account for the aberrant shape of the integuments in cna phb phv.

The ectopically expressed WUS in cna phb phv was knocked down using an artificial microRNA for WUS (amiRWUS). Since the CNA promoter (pCNA) drives gene transcription throughout the chalaza, including the two integuments, we utilized pCNA as the driver of amiRWUS expression (Fig. S1). In gynoecia of cna phb phv pCNA:amiRWUS at stage 2, amiRWUS reduced WUS expression to a level that averaged 60% of that observed in cna phb phv (Fig. 2A). Suppression of WUS expression was also evaluated using a gWUS-GFP3 transgene (Tucker et al., 2008) (Fig. S2). This gene showed an expanded GFP signal in cna phb phv, but the expanded signal was absent from the chalaza in almost all ovules of cna phb phv pCNA:amiRWUS (compare Fig. 2C,E with 2D,F; Fig. S2).

Development of the integuments was restored in most ovules of cna phb phv pCNA:amiRWUS (compare Fig. 2G,I with 2H,J). The percentage of normal ovules per carpel significantly increased in cna phb phv pCNA:amiRWUS as compared with cna phb phv (Fig. 2B). Therefore, the misexpression of WUS partly accounts for the aberrant shape of the integuments in cna phb phv.

Similar to phenotypes of loss-of-function cna phb phv mutants, ovules in HD-ZIPIII gain-of-function mutants, such as phb-1d or phv-1d, have aberrant integuments (Kelley et al., 2009). However, in phb-1d, WUS expression did not differ from WT (Sieber et al., 2004), suggesting that PHB has effects on integument growth that are independent of WUS repression.

As in lateral organ primordia, CNA, PHB and PHV are expressed in the adaxial tissue of the inner integument. Thus, it is suggested that polarity establishment is also required for inner integument expansion (Kelley et al., 2009). The aberrant ovules in cna phb phv pCNA:amiRWUS suggest that CNA, PHB and PHV promote integument growth by other mechanisms, such as establishing adaxial-abaxial polarity, as well as by repressing WUS. Alternatively, these aberrant ovules might be attributed to variability in expression of the amiRWUS transgene.

HD-ZIPIII expression is not sufficient to repress WUS

Seeds are formed even when either CNA, PHB or PHV is constitutively expressed by the CaMV 35S promoter (Prigge et al., 2005), in contrast to the seedless phenotype of wus (Gross-Hardt et al., 2002), suggesting that HD-ZIPIII factors do not directly repress WUS expression. We drove expression of CNA under the control of the WUS promoter to corroborate these results. Since HD-ZIPIII transcripts are post-transcriptionally targeted for degradation by microRNA (miR) 165/166 (Emery et al., 2003), we used CNA-δmiRNA, in which the miRNA binding site is modified to be insensitive to miR165/166, in addition to WT CNA.

Both in pWUS:CNA-δmiRNA (Fig. 2L,O) and pWUS:CNA (Fig. 2M,P), ovules did not obviously differ from those of WT (Fig. 2K,N), suggesting that CNA requires other factors to regulate WUS in the chalaza.

Pattern of cytokinin regulation is not altered in cna phb phv

WUS expression extends into the chalazal domain when cytokinin is externally applied to the gynoecium (Bencivenga et al., 2012). Therefore, misexpression of WUS in cna phb phv might result from cytokinin upregulation. We compared cytokinin responses between WT and cna phb phv ovules using a TCS:GFP marker (Müller and Sheen, 2008).

In WT ovules, cytokinin response is observed in the chalaza and funiculus at stage 2-III and later (Fig. 3A,B) (Bencivenga et al., 2012). The same pattern is observed in cna phb phv ovules with aberrant (Fig. 3C,D) or normal (Fig. 3E,F) integuments, but expression levels are reduced, compared with WT (Fig. 3G). These data indicate that CNA, PHB and PHV regulation of WUS expression is not by means of cytokinin upregulation. In addition, ovule morphology is not affected when cytokinin degradation is promoted by constitutive expression of Cytokinin oxidase/dehydrogenase genes (Werner et al., 2003), supporting the conclusion that the cna phb phv phenotype is independent of cytokinin regulation.

bel1 and cna phb phv exhibit synergistic phenotypes

In bel1 an amorphous structure forms in place of the integuments and can convert to a carpelloid organ (e.g. Robinson-Beers et al., 1992; Ray et al., 1994). In bel1 ovules WUS expression is shifted downward into the boundary region between the nucellus and chalaza (Bencivenga et al., 2012) (Fig. S2). Thus, we crossed cna phb phv with bel1 to evaluate the interaction of the genes.

bel1 cna phb phv ovules form the amorphous structure seen in bel1, but the primary ovulate axis was observed to branch through the formation of ectopic ovule primordia (Fig. 3H,I). These extra primordia were formed directly on the funiculus below the chalaza, in contrast to the extra putative nucelli of bel1, which are born on the adaxial side of the amorphous organ (Robinson-Beers et al., 1992). The ectopic ovules form amorphous organs typical of bel1 mutants in place of integuments (Fig. 3I). Formation of ectopic ovules is preceded by nodes of ectopic WUS expression in the funiculus (Fig. 3J,K). WUS is expressed in the nucellus and chalaza of bel1 cna phb phv ovules (Fig. 3J), as in cna phb phv ovules (Fig. 1B,C). However, stronger expression was detected in the nucelli of the ectopic ovules at the same stage in their development (Fig. 3J). As the amorphous organ enlarged, WUS expression decreased in the nucellus and chalaza of the primary ovulate axis, whereas expression persisted in the ectopic ovules (Fig. 3K).

The ovulate axis does not branch in cna phb phv (Kelley et al., 2009) nor in bel1 (Robinson-Beers et al., 1992). Consistent with the novel phenotype of bel1 cna phb phv, the WUS expression pattern in this quadruple mutant is different from that observed in bel1 (Fig. S2) or in cna phb phv (Fig. 1B,C). These synergistic phenotypes suggest that CNA, PHB and PHV act in a different pathway from BEL1 to regulate WUS. The combination of the two classes of mutations appears to allow for WUS expression further down the ovule axis than is observed in either single class. The formation of ectopic ovules from the funiculus implies that cells in the funiculus manifest placenta-like properties in the quadruple mutant.

CNA, PHB and PHV establish the boundary between nucellus and chalaza in Arabidopsis ovules

We show that CNA, PHB and PHV play a major role in the negative regulation of WUS in the chalaza. This regulation contributes to the establishment of the boundary between the nucellus and chalaza and promotes the proper development of two integuments. CNA, PHB and PHV repress WUS expression independently from BEL1 or cytokinin upregulation (Fig. 4A).

Integument growth is normal in cna, phb and phv single mutants (Kelley et al., 2009), suggesting that they redundantly repress WUS expression. In stage 2 ovules, expression of CNA, PHB and PHV does not completely overlap (Kelley et al., 2009; Sieber et al., 2004) (Fig. 4B), whereas WUS is misexpressed throughout the chalaza in cna phb phv, a much broader area than the sum of the CNA, PHB and PHV expression areas. This discrepancy would imply that CNA, PHB and PHV regulate the expression of WUS through their repression of a diffusing factor or an action of the factor (X in Fig. 4). Auxin is a possible candidate for this factor because it is predicted to flow through the chalaza (Bencivenga et al., 2012; Kelley et al., 2012), and alterations in the pattern of auxin response correlate with ectopic WUS expression seen in bel1 mutants (Bencivenga et al., 2012). Alternatively, CNA or PHV might have broader expression areas in stage I ovules, as actually reported for PHB (Sieber et al., 2004), and failure in suppression at this stage could affect the expression patterns of WUS at subsequent stages.

The striking synergistic branched ovule phenotype observed in bel1 cna phv phb mutants implies that the two classes of genes act in relative independence in their suppression of WUS activity outside of the nucellus. Angiosperm ovules are hypothesized to be homologous to the cupulate organs of some extinct gymnosperms, each of which were born on the apices of a branched axis (e.g. Doyle, 2006), but such branched structures are not observed in extant angiosperms. If this hypothesis is correct, then BEL1 and HD-ZIPIIIs could have played a role in the evolution of the unbranched ovule form seen in all extant angiosperms.

Growth conditions and plant materials

Arabidopsis plants were grown under continuous light at 23°C on soil. Seeds were sourced as described in the supplementary Materials and Methods.

Construction of transgenic lines

The amiRWUS fragment was synthesized following Web MicroRNA Designer 3 (http://wmd3.weigelworld.org/cgi-bin/webapp.cgi). amiRWUS binds 607 to 627 nucleotides of WUS (Fig. S1). amiRWUS, as well as pCNA (−4046 to −319), were inserted into pMLBarT (Fig. S1) using the GeneArt Seamless PLUS Cloning and Assembly Kit (Life Technologies). bel1-6/+ cna-2 phb-13 phv-11 er-2 plants were transformed with the construct by the floral dip method (Clough and Bent, 1998).

To generate pWUS:CNA-δmiRNA or pWUS:CNA, the WUS promoter and CNA cDNA sequences were obtained from Col-0 genomic DNA or cDNA by PCR, respectively. CNA-δmiRNA sequence was synthesized by overlap PCR as previously described (Emery et al., 2003). These fragments were cloned into pMLBarT as described above.

Further details of constructs and genotyping are given in the supplementary Materials and Methods and Table S1.

In situ hybridization and GUS staining

Fixation, embedding of tissue and in situ hybridization were performed as previously described (Mayer et al., 1998). GUS staining of pWUS>>uidA plants is described in the supplementary Materials and Methods.

Microscopy

For scanning electron microscopy (SEM), ovules were fixed (McAbee et al., 2006) or epoxy molds were made (Williams et al., 1987). Fluorescence images of GFP were taken with excitation and emission wavelengths of 470/20 nm and 505-530 nm, respectively, using an Axio Scope 2 Plus (Carl Zeiss).

qRT-PCR analysis

Total RNAs were extracted from gynoecia containing stage 2 ovules and contaminating DNA was digested with DNase. qRT-PCR analyses were performed using the One Step SYBR PrimeScript RT-PCR Kit (Takara). WUS and GFP expression levels were normalized to those of PP2AA3 (At1g13320) (Czechowski et al., 2005). For further details, see the supplementary Materials and Methods.

We thank John Bowman, Steven Clark, Shinobu Takada and Nottingham Arabidopsis Stock Centre (NASC) for seeds; and Debra Skinner and Marissa Simon for helpful comments.

Author contributions

T.Y. and C.S.G. designed the study. T.Y. and Y.S. performed most experiments. K.H. and K.N. made constructs. T.Y. and C.S.G. wrote the manuscript. All authors commented on the manuscript.

Funding

Supported by a Japan Society for the Promotion of Science (JSPS) grant (Kakenhi) [24570098 to T.Y.]; and a US National Science Foundation (NSF) grant [IOS1354014 to C.S.G.].

Balasubramanian
,
S.
and
Schneitz
,
K.
(
2000
).
NOZZLE regulates proximal- distal pattern formation, cell proliferation and early sporogenesis during ovule development in Arabidopsis thaliana
.
Development
127
,
4227
-
4238
.
Bencivenga
,
S.
,
Simonini
,
S.
,
Benková
,
E.
and
Colomboa
,
L.
(
2012
).
The transcription factors BEL1 and SPL are required for cytokinin and auxin signaling during ovule development in Arabidopsis
.
Plant Cell
24
,
2886
-
2897
.
Brambilla
,
V.
,
Battaglia
,
R.
,
Colombo
,
M.
,
Masiero
,
S.
,
Bencivenga
,
S.
,
Kater
,
M. M.
and
Colombo
,
L.
(
2007
).
Genetic and molecular interactions between BELL1 and MADS box factors support ovule development in Arabidopsis
.
Plant Cell
19
,
2544
-
2556
.
Clough
,
S. J.
and
Bent
,
A. F.
(
1998
).
Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana
.
Plant J.
16
,
735
-
743
.
Czechowski
,
T.
,
Stitt
,
M.
,
Altmann
,
T.
,
Udvardi
,
M. K.
and
Scheible
,
W.-R.
(
2005
).
Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis
.
Plant Physiol.
139
,
5
-
17
.
Doyle
,
J. A.
(
2006
).
Seed ferns and the origin of angiosperms
.
J. Torrey Bot. Soc.
133
,
169
-
209
.
Emery
,
J. F.
,
Floyd
,
S. K.
,
Alvarez
,
J.
,
Eshed
,
Y.
,
Hawker
,
N. P.
,
Izhaki
,
A.
,
Baum
,
S. F.
and
Bowman
,
J. L.
(
2003
).
Radial patterning of Arabidopsis shoots by class III HD-ZIP and KANADI genes
.
Curr. Biol.
13
,
1768
-
1774
.
Green
,
K. A.
,
Prigge
,
M. J.
,
Katzman
,
R. B.
and
Clark
,
S. E.
(
2005
).
CORONA, a member of the Class III Homeodomain Leucine Zipper gene family in Arabidopsis, regulates stem cell specification and organogenesis
.
Plant Cell
17
,
691
-
704
.
Gross-Hardt
,
R.
,
Lenhard
,
M.
and
Laux
,
T.
(
2002
).
WUSCHEL signaling functions in interregional communication during Arabidopsis ovule development
.
Genes Dev.
16
,
1129
-
1138
.
Kelley
,
D. R.
,
Skinner
,
D. J.
and
Gasser
,
C. S.
(
2009
).
Roles of polarity determinants in ovule development
.
Plant J.
57
,
1054
-
1064
.
Kelley
,
D. R.
,
Arreola
,
A.
,
Gallagher
,
T. L.
and
Gasser
,
C. S.
(
2012
).
ETTIN (ARF3) physically interacts with KANADI proteins to form a functional complex essential for integument development and polarity determination in Arabidopsis
.
Development
139
,
1105
-
1109
.
Landau
,
U.
,
Asis
,
L.
and
Williams
,
L. E.
(
2015
).
The ERECTA, CLAVATA and class III HD-ZIP pathways display synergistic interactions in regulating floral meristem activities
.
PLoS ONE
10
,
e0125408
.
Lee
,
C.
and
Clark
,
S. E.
(
2015
).
A WUSCHEL-independent stem cell specification pathway is repressed by PHB, PHV and CNA in Arabidopsis
.
PLoS ONE
10
,
e0126006
.
Mandel
,
T.
,
Moreau
,
F.
,
Kutsher
,
Y.
,
Fletcher
,
J. C.
,
Carles
,
C. C.
and
Williams
,
L. E.
(
2014
).
The ERECTA receptor kinase regulates Arabidopsis shoot apical meristem size, phyllotaxy and floral meristem identity
.
Development
141
,
830
-
841
.
Mayer
,
K. F. X.
,
Schoof
,
H.
,
Haecker
,
A.
,
Lenhard
,
M.
,
Jürgens
,
G.
and
Laux
,
T.
(
1998
).
Role of WUSCHEL in regulating stem cell fate in the Arabidopsis shoot meristem
.
Cell
95
,
805
-
815
.
McAbee
,
J. M.
,
Hill
,
T. A.
,
Skinner
,
D. J.
,
Izhaki
,
A.
,
Hauser
,
B. A.
,
Meister
,
R. J.
,
Venugopala Reddy
,
G.
,
Meyerowitz
,
E. M.
,
Bowman
,
J. L.
and
Gasser
,
C. S.
(
2006
).
ABERRANT TESTA SHAPE encodes a KANADI family member, linking polarity determination to separation and growth of Arabidopsis ovule integuments
.
Plant J.
46
,
522
-
531
.
McConnell
,
J. R.
,
Emery
,
J.
,
Eshed
,
Y.
,
Bao
,
N.
,
Bowman
,
J.
and
Barton
,
M. K.
(
2001
).
Role of PHABULOSA and PHAVOLUTA in determining radial patterning in shoots
.
Nature
411
,
709
-
713
.
Müller
,
B.
and
Sheen
,
J.
(
2008
).
Cytokinin and auxin interaction in root stem-cell specification during early embryogenesis
.
Nature
453
,
1094
-
1098
.
Prigge
,
M. J.
,
Otsuga
,
D.
,
Alonso
,
J. M.
,
Ecker
,
J. R.
,
Drews
,
G. N.
and
Clark
,
S. E.
(
2005
).
Class III homeodomain-leucine zipper gene family members have overlapping, antagonistic, and distinct roles in Arabidopsis development
.
Plant Cell
17
,
61
-
76
.
Ray
,
A.
,
Robinson-Beers
,
K.
,
Ray
,
S.
,
Baker
,
S. C.
,
Lang
,
J. D.
,
Preuss
,
D.
,
Milligan
,
S. B.
and
Gasser
,
C. S.
(
1994
).
The Arabidopsis floral homeotic gene BELL (BEL1) controls ovule development through negative regulation of AGAMOUS gene (AG)
.
Proc. Natl. Acad. Sci. USA
91
,
5761
-
5765
.
Robinson-Beers
,
K.
,
Pruitt
,
R. E.
and
Gasser
,
C. S.
(
1992
).
Ovule development in wild-type Arabidopsis and two female-sterile mutants
.
Plant Cell
4
,
1237
-
1249
.
Schneitz
,
K.
,
Hulskamp
,
M.
and
Pruitt
,
R. E.
(
1995
).
Wild-type ovule development in Arabidopsis thaliana: a light microscope study of cleared whole-mount tissue
.
Plant J.
7
,
731
-
749
.
Sieber
,
P.
,
Gheyselinck
,
J.
,
Gross-Hardt
,
R.
,
Laux
,
T.
,
Grossniklaus
,
U.
and
Schneitz
,
K.
(
2004
).
Pattern formation during early ovule development in Arabidopsis thaliana
.
Dev. Biol.
273
,
321
-
334
.
Tucker
,
M. R.
,
Hinze
,
A.
,
Tucker
,
E. J.
,
Takada
,
S.
,
Jürgens
,
G.
and
Laux
,
T.
(
2008
).
Vascular signalling mediated by ZWILLE potentiates WUSCHEL function during shoot meristem stem cell development in the Arabidopsis embryo
.
Development
135
,
2839
-
2843
.
Werner
,
T.
,
Motyka
,
V.
,
Laucou
,
V.
,
Smets
,
R.
,
Van Onckelen
,
H.
and
Schmülling
,
T.
(
2003
).
Cytokinin-deficient transgenic Arabidopsis plants show multiple developmental alterations indicating opposite functions of cytokinin in regulation of shoot and root meristem activity
.
Plant Cell
15
,
2532
-
2550
.
Williams
,
M. H.
,
Vesk
,
M.
and
Mullins
,
M. G.
(
1987
).
Tissue preparation for scanning electron microscopy of fruit surfaces: comparison of fresh and cryopreserved specimens and replicas of banana peel
.
Micron. Microsc. Acta
18
,
27
-
31
.

Competing interests

The authors declare no competing or financial interests.

Supplementary information